Liver Regeneration And Carcinogenesis: Molecular and Cellular Mechanisms

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Liver Regeneration a n d C a r c l' n o g e n e s l's

Molecular and Cellular Mechanisms

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L~ver Regeneration and Carcinogenesis Molecular and Cellular Mecbanisms

Edited by Randy L. Jirtle Departmentof Radiation Oncology Duke UniversityMedical Center Durham, North Carolina

AcademicPress San Diego New York Boston London Sydney Tokyo Toronto

Cover illustration: A Modification of Figure 1 from Chapter 4 of this volume.

This book is printed on acid-free paper.

Copyright 9 1995 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX Library of Congress Cataloging-in-Publication Data Liver regeneration and carcinogenesis : molecular and cellular mechanisms / edited by Randy L. Jirtle. p. cm. Includes index. ISBN 0-12-385355-9 (case : alk. paper) 1. Liver--Cancer. 2. Liver--Regeneration. 3. Pathology, Molecular. 4. Liver--Molecular aspects. I. Jirtle, Randy L. [DNLM: 1. Liver Regeneration. 2. Liver Neoplasms--genetics 702 L7848 1995] RC280.L5L587 1995 6516.3'62--dc20 DNLM/DLC for Library of Congress 95-14344 CIP PRINTED IN THE UNITED STATES OF AMERICA 95 96 97 98 99 00 QW 9 8 7 6

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This book is dedicated to my wife Nancy and my children, Bonnie and ]ames.

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Contents Contributors xvii Preface xxi

1 Liver Regeneration Then and Now Nancy L. R. Bucher

I. II. III. IV. V.

Landmarks 1 Normal Adult Rat Liver Liver Regeneration 3 Hepatocyte Priming 5 Regeneration Signals 7 A. Hormones 7 B. Growth Factors 12 C. Cytokines 17 D. Interactions 18 VI. Conclusions 19 References 19

2 Hepatocyte Growth Factor (HGF) and Its Receptor (Met) in Liver Regeneration, Neoplasia, and Disease George K. Michalopoulos

I. Introduction 27 II. Structural and Functional Aspects of HGF and the HGF Receptor 28 III. HGF Localization 30 A. In Liver 30 B. In Extra Hepatic Tissues 31

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32 IV. Liver and the Processing of HGF A. HGF and Liver Regeneration 33 V. HGF and the Early Proteolytic Events Following Partial Hepatectomy 37 40 VI. HGF Localization A. In Liver Embryogenesis 40 B. In Liver Disease 41 C. In Liver Carcinogenesis 42 VII. Summary 43 References 44

3 Structure and Functions of the HGF Receptor (c-Met) Paol0 M. Comogli0 Elisa Vigna

I. Hepatocyte Growth Factor and Scatter Factor 51 II. HGF Receptor 53 53 A. Encoding by the c - m e t Oncogene 54 B. Post-translational Modifications C. Positive and Negative Regulation 55 D. Signal Transduction 57 E. Tissue Distribution and Subcellular Localization 59 III. Regulation of c - m e t Expression 60 IV. Role of HGF in Tissue Regeneration and Embryogenesis 61 V. Role of c - m e t in Carcinogenesis 62 References 63

4 Expression and Function of Growth-Induced Genes during Liver Regeneration Rebecca Taub

I. Liver Regeneration: The Important Questions 71 II. Immediate-Early Gene Expression in Hepatic Cells 72 III. Modification of Preexisting Transcription Factors

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IV.

V.

VI.

VII. VIII.

IX.

X.

Immediately Following Partial Hepatectomy Turns on Immediate-Early Genes 76 Induction Patterns of 70 Genes Following Partial Hepatectomy Define the Temporal Course of Liver Regeneration 78 Transcription Factors Induced in the Regenerating Liver 80 A. The LRF-1/JunB Story 80 B. RNR-1, a Novel Nuclear Receptor That Acts through the NGFI-B Half-Site 82 Immediate-Early Genes Involved in Signal Transduction 84 A. PRL-1, a Member of a Novel Class of Protein-Tyrosine Phosphatases 84 Immediate-Early Genes That Are Secreted Proteins 86 Liver-Specific Immediate-Early Genes: Relationship to the Maintenance of Hepatocyte Differentiation and Metabolism 87 A. Identification of CL-6 88 Immediate-Early Genes in H35 Cells That Are Expressed as Delayed-Early Genes in Regenerating Liver 89 A. Delayed-Early Genes That Encode RNA Binding Proteins 89 Conclusions 91 References 93

5 Stem Cells and Hepatocarcinogenesis Snorri S. Th0rgeirss0n

I. Introduction 99 II. Cellular Biology of the Hepatic Stem Cell Compartment 101 A. Experimental Systems in Vivo 101 B. Experimental Systems in Vitro 102 III. Neoplastic Development in the Liver 104 A. Hepatic Stem Cells and Hepatocarcinogenesis 104 B. Transformation of Liver Derived Epithelial (Oval) Cells 106 IV. Conclusions 108 References 109

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6 Contributions of Hepadnavirus Research to Our Understanding of Hepatocarcinogenesis Charles E. R0gler Leslie E. R0gler Deyun Yang Silvana Breiteneder-Geleef Shih Gong Haiping Wang

I. General Overview of Hepadnavirus Animal Models and Hepatocarcinogenesis 113 II. Hepatitis B Virus Envelope Protein (HBsAg) Transgenic Mice 117 A. HBsAg (Line 50-4) Transgenic Model of Hepatocarcinogenesis 117 B. Noncytopathic HBsAg Transgenic Mice: Role of Cytokines in Gene Regulation 118 III. Woodchuck Hepatitis Virus (WHV) Model of Hepatocarcinogenesis 122 A. Toxic Oxygen Radicals and WHV Persistent Infection 122 B. Insertional Mutagenesis 122 C. Integration and Human HCC 125 D. Analysis of Precancerous Lesions and HCCs: The Case for a Role of IGF2 in Tumor Promotion IV. Hepadnavirus X Gene Encodes an Oncogenic Transcriptional Transactivator 129 A. Background 129 B. X Gene Transactivation Mechanism 131 C. In Vitro Assays for Hepadnavirus Transforming Activity 132 D. HBx Transgenic Mice Develop HCC 132 V. Conclusions 134 References 134

7 Apoptosis and Hepatocarcinogenesis R01f Schulte-Hermann Bettina Grasl-Kraupp Wilfried Bursch

I. Apoptosis and Other Types of Active Cell Death 141

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II. Active Cell Death in the Liver 145 A. Detection and Quantification 145 B. Models 146 C. Duration of Apoptosis in the Liver 151 III. Biochemical and Molecular Aspects of Apoptosis 152 A. Events Associated with Cell Killing 153 B. Events Associated with Preparation for Active Cell Death 154 C. Signal Factors 156 IV. Active Cell Death in the Stages of Hepatocarcinogenesis 158 A' Cancer Prestages in the Liver 158 B. Kinetic Aspects of Cell Proliferation and Death in Cancer Prestages 160 C. Active Cell Death and Initiation 161 D. Active Cell Death and Tumor Promotion 163 E. Active Cell Death and Tumors 165 V. Conclusions 166 References 167

8 Liver Tumor Promotion and the Suppression of p53-Dependent Cell C y c l e C h e c k p o i n t F u n c t i o n Yingchun Zhang Chia Chia0 Laura L. Byrd David G. Kaufman William K. Kaufmann I~ Introduction 179 II. Mechanisms of Cell Cycle Control 180 III. Cell Cycle Checkpoints, Lifespan Extension, and Genetic Instability 181 IV. Isolation of EL/EGV Hepatocytes and Promotion of Hepatocarcinogenesis in Vitro 183 V. Immortal Rat Hepatocytes Require PB for Clonal Expansion 186 VI. Mechanisms of Promotion of Hepatocarcinogenesis by Phenobarbital 191 VII. Conclusions 193 References 194

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9 Mechanisms of Liver Tumor Promotion JeremyJ. Mills RandyL.Jirtle IvanJ. Boyer I. Introduction 199 II. Stages of Liver Carcinogenesis 201 A. Initiation 201 B. Promotion 202 C. Progression 203 III. Cell Cycle Regulation and Liver Carcinogenesis 204 A. Cyclins and Cyclin-Dependent Kinases B. Rb Gene 206 C. p53 Gene 207 IV. TGF[3 and Liver Carcinogenesis 208 A. TGFI3 and TGFI3 Receptors 210 B. M6P/IGF2 Receptor 212 C. Experimental Results 214 V. Apoptosis and Liver Carcinogenesis 216 VI. Summary 217 References 218

10 Hypomethylation of DNA: An Epigenetic Mechanism That Can Facilitate the Aberrant Oncogene Expression Involved in Liver Carcinogenesis JenniferL. Counts JayI. Goodman 227 I~ Introduction II. Epigenetics 230 230 III. DNA Methylation IV. Working Hypothesis and Experimental Model 235 V. Liver Tumor Promotion: A Role for Hypomethylation of DNA 236 VI. Methyl Deficient Diets 238 VII. DNA Damage and Altered DNA Methylation 240

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VIII. DNA Methylation and Chemoprevention 241 IX. Differences in DNA Methylation between Rodents and Humans 243 A. Capacity to Maintain DNA Methylation 243 B. Methylation of the 5' Flanking Region in H a - r a s 244 X. Conclusions 245 A. DNA Methylation and Multistage Carcinogenesis 245 B. DNA Methylation and Risk Assessment 246 C. DNA Methylation and Chemoprevention 246 XI. Summary 246 References 247

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Transgenic Models of Hepatic Growth Regulation and Hepatocarcinogenesis Eric P. Sandgren I. Transgene-Based Strategies for Studying Liver Growth, Development, and Cancer 257 II. Oncogenic Transgenes and Hepatic Neoplasia 259 A. Viral Oncogenes 259 B. Cellular Oncogenes 264 C. Oncogene-Transformed Immortalized Cell Lines 267 D. Conclusions 267 III. Growth Factor Transgenes and Hepatic Neoplasia 268 A. Transforming Growth Factor Alpha 268 B. Insulin-like Growth Factors 270 C. Hepatocyte Growth Factor 271 D. Conclusions 271 IV. Hepatotoxic Transgenes and Liver Neoplasia 271 A. Hepatitis B Virus Surface Antigen 271 B. Urokinase-Type Plasminogen Activator 273 C. Alpha- 1-Antitrypsin 274 D. Conclusions 275

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276 V. Transgenes and Multistage Carcinogenesis A. Cooperating Events in Transgene-Induced Hepatocarcinogenesis 276 276 B. Coexpression of Multiple Transgenes 278 C. Transgenes and Chemical Carcinogens D. Transgene-Based Mutagenesis Assay Systems 280 E. Conclusions 281 282 VI. Transgenes and Hepatic Growth Regulation A. Liver Gene Expression 282 286 B. Fetal and Neonatal Liver Development C. Liver Regeneration 287 D. Conclusions 288 289 VII. Assessment and Future Directions References 290

12 Genetic Susceptibility to Liver Cancer Norman R. Drinkwater Gang-H0ngLee

I. Introduction 301 II. Genetics of Human Liver Cancer 302 A. Genetic Epidemiology 302 B. Genetic Diseases Associated with an Increased Risk for Liver Cancer 303 III. Genetics of Experimental Liver Cancer 305 A. Variation among Inbred Strains 305 B. Specific Mutations Affecting Hepatocarcinogenesis 313 IV. Conclusion 314 References 315

13 Surgical Treatment of Hepatic Tumors and Its Molecular Basis Ravi S. Chari R. Daniel Beauchamp

I. Introduction 323 II. Diagnosis of Surgical Liver Tumors 324 A. Evaluation of Asymptomatic Liver Mass

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III. Indications for Surgical Treatment of Liver Tumors 326 A. Cystic Liver Disease 326 B. Neoplasms of the Liver 328 IV. Surgical Anatomy 336 A. Surgical Resection and Transplantation V. Hepatic Regeneration after Resection and Transplantation: Current Clinical Concepts References 345

339 342

14 Gene Therapy for the Treatment of Inherited and Acquired Diseases of the Liver Brian E. Huber

I. Human Gene Therapy--A Definition 351 II. Strategies for Liver-Directed Gene Therapy 352 A. Gene Replacement (Repair) or Excision Therapy 352 B. Gene Addition Therapy 353 C. Gene Addition Therapy and the Hepatocyte 355 D. Gene Addition Therapy--Ex Vivo versus in Vivo Liver-Directed Gene Therapy 356 III. Gene Transfer Techniques for Liver-Directed Gene Therapy 361 A. Chemical Transfer Methods 362 B. Electroporation 362 C. Microinjection into the Nucleus . . . . D. Scrape Loading 364 E. Macroinjection 364 E Ballistic Barrage 364 G. Receptor-Mediated Gene Delivery 364 H. Liposomal Gene Delivery 366 I. Retroviruses 366 J. Adenoviruses 368 IV. Clinical Applications of Gene Therapy Directed to the Hepatic Compartment 369 A. Metabolic and Plasma Protein Disorders 369

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B. Primary and Metastatic Liver Cancer 374 C. Viral Diseases of the Liver 375 D. Hepatocellular Transplantation 376 V. Conclusions 377 References 378

Index

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Contributors Numbers in parentheses indicate the pages on which the authors" contributions begin.

R. Daniel Beauchamp (323) Department of Surgery, Vanderbilt University, Nashville, Tennessee 77550 Ivan J. Boyer (199) MITRE Corporation, Inc., McLean, Virginia 22102 Silvana Breiteneder-Geleff (113) Institute of Clinical Pathology, University of Vienna Medical School, A-1090 Vienna, Austria Nancy L. R. Bucher (1) Department of Pathology, Boston University School of Medicine, Boston, Massachusetts 02118

Wilfried Bursch (141) Institute ffir Tumor Biologie-Cancer Research, A-1090 Wien, Austria Laura L. Byrd (179) Department of Pathology, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Ravi S. Chari (323) Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710 Chia Chiao (179) Department of Pathology, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Paolo M. Comoglio (51) Institute for Cancer Research and Treatment (IRCC), University of Torino School of Medicine, 10126 Torino, Italy Jennifer L. Counts (227) Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824 Norman R. Drinkwater (301) McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53076 Shih Gong (113) Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461 Jay I. Goodman (227) Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824 xvii

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Contributors

Bettina Grasl-Kraupp (141) Institute ffir Tumor Biologie-Cancer Research, A- 1090 Wien, Austria Brian E. Huber (351) Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709 Randy L. Jirtle (199) Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710 David G. Kaufman (179) Department of Pathology, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

William K. Kaufmann (179) Department of Pathology, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Gang-Hong Lee (301) Department of Pathology, Asahikawa Medical College, Asahikawa 078, Japan George K. Michalopoulos (27) Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15261 Jeremy J. Mills (199) Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710 Charles E. Rogler (113) Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461 Leslie E. Rogler (113) Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461

Eric P. Sandgren (257) Department of Experimental Pathology, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53 706 Rolf Schulte-Hermann (141) Institute fiir Tumor Biologie-Cancer Research, A-1090 Wien, Austria Rebecca Taub (71) Department of Genetics and Medicine, Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Snorri S. Thorgeirsson (99) Laboratory of Experimental Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Elisa Vigna (51) Institute for Cancer Research and Treatment (IRCC), University of Torino School of Medicine, 10126 Torino, Italy Haiping Wang (113) Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461

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Deyun Yang (113) Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461 Yingchun Zhang (179) Department of Pathology, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

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Preface The fact that the liver regenerates following injury has been known since ancient times. According to Greek mythology, Prometheus was chained to a rock for defying Zeus by stealing fire from Mount Olympus. He was subjected to an eagle eating his liver during the day, and it regenerated by night. Because of this marked capacity to regenerate and the ability of chemical carcinogens and viruses to transform hepatocytes, the liver is used extensively as a model for investigating the molecular mechanisms of normal cell proliferation and carcinogenesis. Recently, striking advances have occurred in our understanding of hepatocyte growth regulation and how chemical agents and viruses alter these normal growth regulatory pathways during the genesis of liver tumors. Both in vitro and in vivo models of neoplastic transformation have established that the pathway to neoplasia has multiple steps. Aberrant expression of proto-oncogenes or the expression of mutant forms of these genes (i.e., oncogenes) can lead to neoplastic transformation. The loss of tumor suppressor gene function is also involved in the development of both rodent and human liver cancer. This book demonstrates that the determination of the factors involved in controlling the regeneration of normal liver has led to a clearer understanding of the mechanisms involved in the formation of various liver diseases, including hepatocellular carcinomas. The chapters in this book are grouped into three main research areas. The first section of the book covers the subject of liver regeneration. Liver regeneration has been investigated for many years, particularly since 1931 when Higgins and Anderson published a classic paper describing a partial hepatectomy technique in rats that reproducibly stimulates the liver to regeneration. An overview of liver regeneration research both then and now is described in the first chapter by one of the premier scientists in this field of investigation, Dr. Nancy Bucher. The growth factor that plays a central role in stimulating hepatocyte proliferation following liver injury, hepatocyte growth factor, and its receptor (c-met) are discussed in Chapters 2 and 3, respectively. The immediate-early response genes that are expressed in the early stages of liver regeneration are described in Chapter 4. The second section of this book deals with the subject of liver carcinogenesis. The topics were chosen to provide an overview of this immense subject. xxi

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Chapter 5 covers the role that hepatic stem cells play in both liver regeneration and carcinogenesis. It is well known that hepatitis virus infection is a major risk factor for the development of human hepatocellular carcinomas. The contribution of hepadnavirus research to our understanding of liver cancer is discussed in Chapter 6. Programmed cell death, apoptosis, has been increasingly shown to be mechanistically involved in liver tumor promotion and carcinogenesis as discussed in Chapter 7. Transforming growth factor 13and the suppression of p5 3-dependent cell cycle checkpoint function as they relate to liver tumor promotion are discussed in Chapters 8 and 9. In Chapter 10 it is postulated that hypomethylation of the DNA may represent an epigenetic mechanism for hepatocyte transformation. The use of transgenic animals to ascertain directly the role of growth factors, oncogenes, etc., in liver carcinogenesis is a powerful molecular technique thoroughly presented in Chapter 11. It is also important to realize that humans vary in their susceptibility to tumor formation. Chapter 12 describes the importance of genetics in the development of liver tumors in both humans and animals. The final section of this book deals with two vastly different techniques for treating liver cancer. Although hepatocellular carcinoma is one of the most common neoplasms in the world, primary cancer of the liver is relatively rare in the United States. Hepatocellular carcinomas are, however, still of significance in the United States because they are highly lethal; the 5-year survival rate is less than 10%. One method of treating primary and metastatic liver tumors is surgical resection. When the malignancy is not amenable to a simple resection, the whole liver can now be transplanted. These surgical procedures for the treatment of liver cancer are described in Chapter 13. As the genes involved in liver carcinogenesis are elucidated, it becomes more probable that this information will be used to develop successful molecular strategies for cancer treatment. The potential of using gene therapy approaches to treat liver tumors and genetic liver diseases is discussed in Chapter 14. In conclusion, the veritable explosion of scientific information that has occurred recently in liver biology encompasses many research disciplines. This book is an attempt to bring these diverse research results together in a coherent manner. Scientists who will benefit from this book include toxicologists, virologists, molecular biologists, cell biologists, cancer biologists, pharmacologists, pathologists, surgeons, and gastroenterologists who are interested in furthering their understanding of the molecular mechanisms controlling liver regeneration and hepatocellular carcinogenesis. I would like to thank Ms. Charlotte Brabants at Academic Press for her continued support and patience, the contributors of this book for writing their chapters when it is increasingly difficult to find the time for such

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scholarly endeavors, and Ms. Roxanne Scroggs for her secretarial assistance.

Randy L. Jirtle

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1 Liver Regeneration Then and Now N a n c y L. R. B u c h e r Department of Pathology Boston UniversitySchool of Medicine, Boston, Massachusetts02118

I. Landmarks As is well known to devotees of liver regeneration, according to the Prometheus myth the ancient Greeks were aware not only that the liver rapidly grows to restore lost tissue, but also that it continues to do so after repeated insults. After that, as far as we know, it was not until late in the nineteenth century that Canalis carried out the first scientifically motivated partial hepatectomy. By 1894 the phenomenon of regeneration had been investigated in rats, mice, rabbits, and dogs (Bresnick, 1971). Now, 100 years later we still do not fully understand the mechanisms involved despite accelerating progress. An early breakthrough was the report of Higgins and Anderson (1931) that detailed a simple surgical procedure for performing a partial hepatectomy in the rat, thereby opening the way for reproducible quantitative studies. The rat is a creature well designed for the study of liver regeneration. The structure of its liver is such that excision of the two main lobes consistently removes 68% of the whole organ; this percentage is independent of rat strain and animal age (Bucher and Swaffield, 1964). Moreover, although the liver regenerates in all Species that have been examined so far, the regrowth is the most rapid and dramatic in the rat; even in the mouse it is slower by about a day. Although most studies have been performed in rats and mice; dogs, and rabbits have also been used. Investigation of the regeneration of human liver is limited to clinical and cell culture studies, which have received

Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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recent impetus from the availability of hepatocytes from livers to be used for transplants (see Chapter 13). In 1936 Brues and colleagues (Brues et al., 1936) carried out the first really basic scientific studies on the regenerating liver, showing that liver mass begins to increase within a few hours after partial hepatectomy, whereas restoration of cell number starts a day later. In addition, they showed that in the regenerating liver the rate of DNA synthesis is greatly increased, as determined by labeling with 32pi, then newly available and heralding the modern era of radioisotopes (Brues et al., 1944). The advent of 14C and 3H soon thereafter was indeed a boon to those investigators who had of necessity until then depended entirely on counting mitotic figures to quantify growth rates. A third landmark, occurring in the late 1960s, was the development by Berry and Friend (1969) of the collagenase perfusion for rat hepatocyte isolation and culture, opening whole new horizons for liver research. This enzymatic process was latter modified for isolating human hepatocytes from surgically removed and discarded pieces of human liver (Strom et al., 1982). Thus, hepatocytes from various species can now be studied in a controlled environment, since they are released from growth regulatory influences imposed by the body. There are numerous review articles on liver regeneration that provide a chronological overview of the progression of this research. The following list includes many, but not all of these reviews: Fishback, 1929; Mann, 1944; Harkness, 1957; Weinbren, 1959; Leduc, 1964; Bresnick, 1971; Bucher and Malt, 1971; Hays, 1974; Lewan et al., 1977; Starzl and Terblanche, 1979; Bucher, 1963, 1967a,b, 1982, 1987; Alison, 1986; Fleig, 1988; Leffert et al., 1988; Fausto and Mead, 1989; Fausto, 1990; Michalopoulos, 1990; Fausto, 1991; Bucher, 1991; Andrus et al., 1991; Bucher and Strain, 1992; DuBois et al., 1994. The present review focuses on regeneration of rat liver in vivo.

II. N o r m a l A d u l t R a t Liver In normal adult rat liver 60% of the cells are hepatocytes and the remainder are various types of nonparenchymal cells. The hepatocytes, however, are much larger and constitute about 90% of the liver volume. Hepatocytes are not all alike. There are many well-studied functional differences between mature, differentiated hepatocytes which occur in a gradation of zones from the periportal to the pericentral regions of the liver lobule (Gumucio and Chianale, 1988; Gumucio and Berkowitz, 1992). Hepatocytes are also normally long lived, and the liver continues to grow as the animal ages, albeit at a diminishing rate. A peculiarity of rodent livers is that although at birth the

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Liver Regeneration Then and N o w

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hepatocytes are mononuclear and diploid, as the animals mature many cells become polyploid; in the rat about /30% become binucleate, 50 to 70% tetraploid, and I to 2% octaploid (Wilson and Leduc, 1948; Bucher, 1967a, b; Tamura et al., 1992). During liver regeneration the number of binucleate cells decreases, but the overall ploidy again increases, peaking at around 72 hr, and then gradually reverting to normal. Even in young animals all but a tiny fraction (0.01 to 0.05%) of the hepatocytes are noncycling G o phase cells, and, despite the polyploidy, they require a new round of DNA synthesis before they can divide. Although for most purposes these few proliferating cells can be ignored, in the early years careful counting of mitotic figures revealed a diurnal oscillation of activity, increasing 2- to 5-fold from a nadir at around 8 A M tO a peak at about 8 P M (Harkness, 1957; Bucher, 1963, 1967; DuBois et al., 1994). In contrast, in regenerating livers the mitotic activity increases 50- to 100-fold. So why is this minor oscillation worthy of note? The answer is that certain genes currently being investigated also exhibit diurnal periodicity, as illustrated by the wide swings in D binding protein (DBP) expression (Mueller et al., 1990). ICER, an isoform of the cyclic AMP-response element modulator (CREM) and the 3-hydroxy-3-methylglutaryl (HMG) CoA reductase genes behave similarly (Masquilier et al., 1993; Bach et al., 1969; Edwards et al., 1972). The diurnal periodicity program also carries over into the regenerating liver, so that although the interval from partial hepatectomy to the peak of DNA synthesis in young adult rats regularly occurs at 22 to 24 hr, its magnitude varies depending on the time of day that the liver samples are taken (Klinge and Mathyl, 1969). It is entrained by illumination and feeding patterns; rats are nocturnal and the feeding and activity programs can be reversed by reversing the light/dark cycle. Food intake is also a major determinant; if access to food is restricted to 2 to 4 hr at a constant time of day for 3 weeks followed by partial hepatectomy, the peak rate of DNA synthesis is increased approximately three-fold (Hopkins et al., 1973a,b; Bucher et al., 1978a).

III. Liver Regeneration It is generally thought that the liver regenerates in response to an excessive metabolic workload imposed by the body. The questions are what sort(s) of workload(s)? How is metabolic work translated into a growth stimulus? What starts the growth, and what stops it? What are the growth effectors? What are the molecular mechanisms? These questions are now being productively addressed in in vitro systems, but hepatocyte culture methodology was not developed until around 1970. Consequently, all of the earlier exper-

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iments were carried out in the whole animal. Whole animal studies were valuable for delineating the salient features of the growth process, are central to an understanding of hepatic growth regulation, and continue to deserve attention because they involve physiological aspects of the growth regulation that do not exist in cultures. The term "regeneration" is firmly embedded in the literature, but is inaccurate in that the excised lobes do not regrow. Instead, the remaining lobes enlarge, undergoing what is more accurately termed a compensatory hyperplasia. The histological architecture is preserved throughout this process. In the normal adult rat liver, the cells are highly differentiated and essentially all in a state of growth arrest, or G 0. They are induced to enter the cell cycle by cell loss or functional inadequacy, for example due to surgical resection, infectious, toxic or physical injury, or to metabolic imbalances caused by severe diabetes, to drastic changes in nutrients, or to pregnancy in rats and mice where multiple fetuses occur in a small animal. When the excess metabolic workload is removed, the liver shrinks back to normal size by apoptosis (See Chapter 7). Partial hepatectomy is widely used to induce liver regeneration, because it is fast and easy to perform, well tolerated, delivers a quantifiable stimulus and is free of the side effects and damage to surviving cells associated with carbon tetrachloride (CCI4) or other commonly used toxic agents. Following the standard 68% hepatectomy, the potentially interrelated growth and stress associated changes start almost immediately. The earliest documented events include monovalent cation fluxes, changes in intracellular pH, alterations in amino acid transport (Leffert et al., 1988), and activation of certain liver function specific and immediate-early genes (see Chapter 4) (Fausto, 1990; Mohn et al., 1991a,b). Early events also involve activation of second messengers and signaling pathways and further changes in gene expression and attendant alterations in the biochemistry of the cells as they undergo the transition from G Oto the G 1 phase of the cell cycle and continue through G 1 into the S phase. DNA synthesis, marking the S phase of the cell cycle, begins at about 14 to 16 hr following a partial hepatectomy in young adult rats, rises steeply to a sharp peak at 22 to 24 hr then falls and continues at a diminished level until the original liver complement is restored in a little more than a week. Mitosis follows DNA synthesis 6 to 8 hr later. DNA synthesis and mitosis are observed to occur first in hepatocytes in the periportal area, subsequently spreading centrally. During the first 72 hr, when the major regeneration has occurred, about 80% of the new hepatocytes are formed in the periportal region of the liver lobules. DNA synthesis in littoral and ductal cells follows that in hepatocytes and peaks 12 to 24 hr later (Harkness, 1957; Grisham, 1962). The regenerative process is most rapid in weanling animals and slowest in old animals (Bucher, 1967).

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In regenerating livers DNA synthesis first appears periportally, but progresses in time to involve the whole lobule with the possible exception of a very few cells close to the central vein (Grisham, 1962). The fact that all hepatocytes have proliferative potential has been shown in animals given [3H]thymidine repeatedly for several days, during which essentially all of the hepatocytes become labeled (Michalopoulos, 1990). When rats are subjected to partial hepatectomies at monthly intervals for a year, the livers regenerate repeatedly. Although at first the existing lobules enlarge, new lobules ultimately develop after additional hepatectomies (Ingle and Baker, 1957; Simpson and Finkh, 1963). The rate at which regrowth occurs is proportional to the extent of liver loss. If the deficit is small, the liver regrows more slowly, despite its high growth potential (Bucher, 1967; Bucher and Malt, 1971). If the liver resection is increased beyond the usual 68% up to 80 to 90%, however, the animals are severely stressed and DNA synthesis is not further increased (Caruana et al., 1986).

IV. Hepatocyte Priming Despite years of effort by numerous investigators, uncertainty still persists regarding the actual signals that initiate, maintain, regulate, and terminate liver regeneration. The answers are likely to remain equivocal until the growth process is clearly delineated at the molecular level; however, earlier work with animal models has brought this problem into clearer focus. Cross-circulation of blood between partially hepatectomized and normal rats, initially via capillaries (parabiosis) and later via arteriovenous shunting through polyethylene cannulas, demonstrates that blood from a hepatectomized partner will stimulate DNA synthesis in the intact liver of the normal partner. The stimulus is proportional to the amount of liver excised in the hepatectomized partner (Bucher et al., 1951; Moolten and Bucher, 1967; Fleig, 1988). Additional evidence of blood borne signaling comes from experiments in which autologous liver grafts and transplanted hepatocytes are stimulated to grow by partial resection of the liver of the host animal. Thus, although initially controversial, it is now well established that liver regeneration is tightly regulated by substances carried in the blood (Grisham et al., 1964; Jirtle and Michalopoulos, 1982; Bucher and Strain, 1992). In the cross-circulation experiments it was found necessary to maintain the blood exchange for at least 12 to 14 hr; shorter exchanges failed to stimulate DNA synthesis. However, if the exchange was interrupted for 2 to 4 hr during the early period of a total elapsed time of 14 hr, DNA synthesis still occurred in the hepatocytes of the intact partner (Bucher et al., 1969). It seemed that initiation of DNA synthesis must progress in a stepwise fashion,

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i.e., that the first step activates the process and a booster is needed to move the cells forward into the S phase. Further support for this notion came from the finding that although DNA synthesis following partial hepatectomy is constant at 22 to 24 hr in young adult rats, this interval could be shortened by about 8 hr if sham operations were performed from several hours to several days prior to the partial hepatectomy. Thus, regeneration seems to gain a head start by a surgical pretreatment, which by itself does not induce DNA synthesis. This finding suggests that response to stress may be an integral part of the liver regenerative process (Moolten et al., 1970; Fausto, 1990). This stepwise induction of liver growth within the animal has now been amply confirmed in cell culture, and found widely applicable to cell growth in general. The process of inducing hepatocytes to undergo transition from the G Oto the G 1 phase of the cell cycle is termed "priming." Further stimulation is required for progression through G 1 into the S phase. Activation of the heat shock protein gene, hsp 70, occurs during the prereplicative period of liver regeneration (Ohmori et al., 1990), and it may play a role in regulating the cell cycle (Schlesinger, 1990). It was shown in 1949 (Leduc) that when mice were changed from low to high protein diets, the liver exhibited a wave of mitotic activity, the peak occurring earlier when the protein enrichment was greater. Similarly, when rats were fed only 20% glucose for 3 days, followed by a balanced amino acid meal (casein hydrolysate by gavage), the ensuing peak of DNA synthesis appeared about 8 hr earlier than in normally fed hepatectomized controis, and was about 70% as high (Bucher et al., 1978a). The hepatocytes could thus be primed by means other than surgical stress. These earlier studies have been confirmed and extended by recent research. The protein-free diet induces the expression of mRNAs of several growth associated immediate-early genes, now recognized as evidence that priming has taken place. Prominent among immediate early genes commonly expressed following growth stimulation in most systems, including regenerating liver, are c-myc, c-jun and c-fos. In the protein deprivation model, however, c-fos mRNA is not expressed (Horikawa et al., 1986; Mead et al., 1990; Fausto, 1990). Are only particular ones of these genes required for liver growth? Do the various genes involved in priming and progression through G1 differ depending on the metabolic status of the cells or the nature of the stimulus? Do various pathways converge to control liver growth? These fundamental questions still need to be answered. Although pretreatments prior to partial (68 %) hepatectomy serve to head start the growth response, either extra stress or glucose administration at the time of the hepatectomy moderately depresses liver regeneration (Moolten et al., 1970; Carter et al., 1989; Holecek et al., 1991; Bucher and Strain, 1992). Following excision of 90% of the liver, survival is low and regen-

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Liver Regeneration Then and Now

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eration does not occur without supportive treatment. Administration of glucose permits survival and regeneration, but it is no greater than the diminished regeneration occurring in glucose treated 68% hepatectomized controls (Gaub and Iversen, 1984; Caruana et al., 1986). From the foregoing discussion, it is clear that stress and the metabolic status of the cells are involved in the priming process, so that they are then responsive to subsequent growth signaling; however, following liver loss, only the liver grows. Whether this tissue growth specificity is determined by a unique hepatic response to these seemingly nonspecific stimuli, or to more specific stimulation of previously primed cells at a later stage remains an unanswered question (see Chapter 2).

V. Regeneration Signals The signals that initiate regeneration are clearly derived from extrahepatic sources, since they are transmitted by the blood. The signaling molecules are generally thought to be combinations of interacting hormones, growth factors, and cytokines, although direct action of nutrients or metabolites has not been ruled out. Table 1 lists a number of the effectors postulated to be mechanistically involved in regulating liver regeneration. The complexity of this problem is compounded by the fact that the same growth factor can act both positively and negatively; transforming growth factor 13(TGFI3), hepatocyte growth factor (HGF), and glucagon are well-known examples (see Chapter 2) (Michalopoulos, 1990; Sporn and Roberts, 1990). Infusion of insulin and epidermal growth factor (EGF) individually into normal rats weakly stimulates DNA synthesis, but when combined, the stimulus is substantial. Addition of glucagon to this combination is inhibitory, but when insulin is omitted glucagon becomes weakly stimulatory. Similar effects are demonstrable in hepatocyte cultures. Thus, it appears that hormones and growth factors interact in synergistic or antagonistic combinations, and the same substance may act positively or negatively, depending on the circumstances. A. Hormones 1. Insulin and Glucagon

Early work pointed to the importance of a portal blood supply, and emphasized insulin as a major hepatocyte growth modulator (Hays, 1974; Starzl and Terblanche, 1979). In splanchnic eviscerated rats (i.e., ablation of the gastrointestinal tract, pancreas, and spleen), partial hepatectomy results in a delayed and weakened DNA synthesis--about 20% of the post partial

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Nancy L. R. Bucher

Table 1 Growth Effectors Proposed to Control Liver Regeneration I. HORMONES Insulin and glucagon Norepinephrine, vasopressin, angiotensin II, and neurotensin Growth hormone and insulin-likegrowth factors 1 and 2 (IGF1 and 2) Parathyroid hormone, calcitonin, and dihydroxycholecalciferol Glucocorticoids Thyroid hormones Prolactin Prostaglandins Estrogens and androgens II. GROWTH FACTORS A. Stimulators

Epidermal growth factor (EGF) Transforming growth factor oL(TGFa) Hepatocyte growth factor (HGF) Acidic fibroblast growth factor (aFGF) Hepatocyte stimulatory substance (HSS) B. Inhibitors

Transforming growth factor 13(TGF13) Activin Hepatocyte proliferation inhibitor (HPI) Platelet derived growth inhibitors a and 13(PDGI a and 13) HI. CYTOKINES Interleukins 1 and 6 (IL-1 and IL-6) Tumor necrosis factor el (TNFel)

hepatectomy response of control animals. Thus, in severely stressed animals, the liver still responds weakly to partial hepatectomy, even in the apparent absence of portal blood factors including insulin (Bucher and Swaffield, 1973). Treatment of these rats with insulin and glucagon separately has minimal effects, but in combination these two hormones dramatically restored the DNA synthesis to that in normal regenerating livers (Bucher and Swaffield, 1975). Administration of insulin and glucagon at the same doses to control rats, however, does not stimulate liver growth (Bucher and Strain, 1992). Taken together, these observations suggest that insulin and glucagon enhance the action of prior growth initiators. Other whole animal studies also point to modulating roles for these two hormones. For example, administration of neutralizing insulin antibodies diminishes but does not abolish liver regeneration. Whether under appropriate conditions insulin can serve as a prime mover in liver regeneration cannot be totally excluded. An example is severe diabetes, in which a brief administration of insulin potently stimulates hepatocyte proliferation (Bucher and Strain, 1992). Glucagon has also been reported to stimulate hepa-

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Liver Regeneration Then and N o w

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tocyte DNA synthesis (Hasegawa and Koga, 1977). In the experiments described, pharmacological doses of glucagon were administered, whereas insulin was used in the physiological range. Nevertheless, definitive evidence for insulin and glucagon being primary stimulators of liver growth is elusive, because ancillary actions of unknown growth effectors cannot be excluded in whole animal studies. 2. Norepinephrine, Vasopressin, Angiotensin II, and Neurotensin

Norepinephrine and vasopressin, which have similar effects on hepatocytes, also appear to be modulators of liver regeneration. Norepinephrine acts at the early stage of liver regeneration as evidenced by a reduction of DNA synthesis by prazosin, a highly specific c~-adrenergic antagonist. Moreover, adrenergic innervation is found to be substantially increased in regenerating liver, and the growth process is greatly reduced by surgical or chemical sympathetic denervation. Within 2 hr post partial hepatectomy plasma catecholamines increase. A shift from the largely ~1 type to the [3-type adrenergic receptors occurs after regeneration is underway, but 13-adrenoreceptor blocking agents fail to inhibit and [3-agonists fail to enhance regeneration. Similar observations have been made in EGF-stimulated hepatocyte cultures. In addition, the growth inhibitory action of TGF[31 is counteracted by the addition of norepinephrine (Michalopoulos, 1990; Bucher and Strain, 1992). Liver regeneration is also depressed in the hereditary vasopressin-deficient Brattleboro rat strain, and vasopressin administration largely restores the hepatic proliferative capability (Russell and Bucher, 1983). In the rat, vasopressin acts through the Ca 2+ mediated V-1 receptor, which is abundant in rat hepatocytes, whereas these high affinity receptors appear to be barely detectable in rabbit and human hepatocytes. Moreover, human hepatocyte cultures are unresponsive to this hormone (Bucher and Strain, 1992; DuBois et al., 1994). Possibly other hormonal substances carry out in human hepatocytes whatever interactive functions vasopressin fulfills in the rat. In EGF-stimulated hepatocyte cultures the effects of norepinephrine, vasopressin, angiotensin II, and neurotensin (Hasegawa et al., 1994) or other neurotransmitters resemble, to slightly differing extents, the effects of e~ladrenergic agents in vivo. These hormones also have similarities with regard to hepatic carbohydrate metabolism, suggesting that like insulin and glucagon they may function as regulators of nutrient availability and utilization in the regenerating livers. Epinephrine appears to be the most potent growth effector of this hormone group, at least in vitro.

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Nancy L. R. Bucher

3. Growth Hormone and Insulin-Like Growth Factors 1 and 2

Growth hormone amplifies DNA synthesis following partial hepatectomy and accelerates it by acting as a priming agent (Moolten et al., 1970). Conversely, hypophysectomy delays and diminishes the regenerative process (Bucher and Strain, 1992; Ekberg et al., 1992; DuBois et al., 1994). Most, but not all of the effects of growth hormone are mediated through the insulin-like growth factors, IGF1 and IGF2, which are mainly produced by the liver. Although hepatocytes have been reported to contain few IGF1 receptors (IGFlr), one of the most highly expressed immediate-early genes in regenerating liver is insulin-like growth factor binding protein-1 (IGFBP-1) (see Chapter 4). The IGFBP-1 peptide has been implicated in modulating the mitogenic effect of IGFs on tissues. It is not expressed in mitogen-stimulated fibroblasts and may be specific to regenerating liver (Mohn et al., 1991b); the mRNA level is unaffected by sham operation (Ghahary et al., 1992). Although the IGFBP-1 mRNA is found in both parenchymal and nonparenchymal cells following partial hepatectomy, the protein appears to be present only in the hepatocytes (Lee et al., 1994). High levels of the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r) are also found in hepatocytes during liver regeneration (Scott et al., 1990; Jirtle et al., 1991). IGF2 interacts with these receptors which may, in conjunction, with IGFBP-1, mediate possible actions of the IGFs in regenerating liver (Lee et al., 1994). The latent complex of TGFI31 also binds to the M6P/IGF2r, thereby facilitating the activation of TGFI31 (Dennis and Rifkin, 1991). Therefore, it appears that M6P/IGF2r is involved in the liver growth process, but its function has not been totally defined (Jirtle et al., 1991). 4. Parathyroid Hormone, Calcitonin, Calcium, and Dihydroxycholecalciferol

This group of hormones is also reported to influence the course of liver regeneration, but not to initiate it. Liver regeneration is diminished in parathyroidectomized rats, and is restored by calcium injection. Secretion of both calcitonin and parathyroid hormone increase soon after partial hepatectomy, and the calmodulin level also rises. Both hormones, as well as the vitamin D metabolite 1-oL,25-dihydroxycholecalciferol [1,25-(OH)2-D3], which is actually a steroid hormone, probably all exert their regulatory function through calcium ions. The 1,25-(OH)2-D 3, has been suggested to participate in the regulation of several DNA replication-linked genes (c-myc, c-myb, and histone H4) that are essential for cell proliferation

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Liver Regeneration Then and Now

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(Bucher and Strain, 1992). These hormones are of interest because of mounting evidence for the importance of calcium in early signaling events associated with activation of growth in various types of cells including hepatocytes. S. Other Classic Hormones

Through the years, on the basis of both in vivo and in vitro studies, most of the classic hormones have been put forward as having possible, though not clearly defined roles in regulating liver regeneration. However, with a few exceptions most of the evidence lacks firm underpinnings. Nevertheless, they cannot be excluded form participating in liver regeneration because of possible interactions with other effectors. a. Glucocorticoids In vivo studies on the possible role of glucocorticoids in liver regeneration are equivocal, and suggest that the glucocorticoids are relatively unimportant except under special conditions (Bucher and Strain, 1992; DuBois et al., 1994). In hepatocyte cultures, however, these hormones are widely used to promote cell survival and function; effects on growth vary with dosage and culture conditions, and these vary considerably among different laboratories. Consequently, the extent of their influence on the regeneration process remains unclear. b. Thyroid Hormones The effects of thyroid hormones on liver regeneration are likely to be primarily dependent on their effects on metabolism. c. Prolactin regeneration.

Prolactin lacks definitive support as a regulator of liver

d. Prostaglandins Prostaglandins (PG) may be involved in liver regeneration according to a few reports. PGE 2 reportedly undergoes a transient increase during liver regeneration that is inhibited by indomethacin, which inhibits DNA synthesis as well. Additional work with inhibitors of prostaglandin and thromboxane production in regenerating liver and with arachidonati, PGE2, and PGF2~ in cultures suggests that these hormones may act at a later stage in the cell cycle (Bucher, 1991; Bucher and Strain, 1992). e. Estrogens and Androgens On the basis of a number of somewhat discordant studies, estrogens and androgens appear not to play a major role in liver regeneration. Certain sexually dimorphic aspects of liver function are recognized, and estrogens rather then androgens seem to exert a modest influence (Bucher and Strain, 1992). In a recent report, estradiol has been

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Nancy L. R. Bucher

found to enhance the stimulatory effects of EGF, transforming growth factor oL (TGFcx), HGF, and acidic fibroblast growth factor (aFGF) by about two-fold in hepatocyte cultures (Ni and Yager, 1994), but long-term exposure of rats to ethinyl estradiol reduces the proliferative capacity of hepatocytes (see Chapter 9) (Yager et al., 1994). In general, hormones seem to be growth modulators. The hormones of most interest and most studied so far are insulin, glucagon, and norepinephrine. Both in vivo and in vitro, they substantially influence the activity of other growth effectors toward hepatocytes. Therapeutic aspects of liver transplantation have revived interest in these hormones. B. Growth Factors

"Cytokine" is used interchangeably with "growth factor" by some investigators, but it is restricted to products of the immune system by others. These substances are polypeptides which act at short range in several modes, designated as paracrine, autocrine, and juxtacrine. They are thereby distinguished from the classical endocrine hormones, which act at long range. Because of limited availability, most studies of these growth effectors largely focused on their molecular biology, and have been carried out primarily in cell culture. While this work has contributed measurably to our understanding of the mechanisms through which these substances operate, the studies are conducted on cells in artificial environments that influence cellular behavior. In vitro studies can tell us what cells can do under specific conditions, but not necessarily what they actually do in vivo. Consequently, they are largely beyond the scope of this review, which is concerned with hepatocyte performance within the animal. Among the generally positive acting growth factors, HGF and TGFcx are the most potent. The somewhat less effective factors are EGF and aFGF, which is probably the least potent. These comparative potencies are of necessity based on in vitro studies. Therefore, they are not necessarily meaningful, because under physiological conditions various growth effectors, including hormones, growth factors, cytokines, nutrients, and metabolites, as well as cell-cell and cell-extracellular matrix interactions are integrated to control and fine tune the liver regenerative process. HGF and aFGF, as well as a recently described form of EGF termed heparin binding EGF (HB-EGF) bind to heparin, and also to heparan sulfate proteoglycans (HSPGs) in the extracellular matrix, where they may be sequestered, and interact with other HSPGs as well as with specific receptors on the cell surface. In a preliminary report, HB-EGF stimulates rat hepatocyte growth in vitro as potently as HGF, and like HGF, its mRNA is expressed in the nonparenchymal cells but not in hepatocytes (Higashiyama et

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al., 1991, 1993; Ito et al., 1994). Of the generally negative acting growth factors, the most extensively studied is TGFI31. 1. Epidermal Growth Factor

EGF is an effective mitogen for a wide variety of cells. It is originally isolated from the submaxillary salivary gland of the male mouse where it is present in high abundance, but it occurs in various other tissues as well. It has high homology and essentially identical biological activity to urogastrone, which is now considered to be the human form of EGE Other members of the EGF superfamily include HB-EGF, and TGFa which bind to the EGF receptor. The biological effects of EGF and TGFa are similar but not identical (Carpenter and Wahl, 1990). EGF was the first growth effector found to be mitogenic for hepatocytes, demonstrated both in vivo and in vitro, and greatly augmented by combination with insulin (Bucher and Strain, 1992). In addition to initiating liver regeneration, EGF also promotes fat accumulation, glycogen synthesis, gluconeogenesis, and hepatocyte motility. Furthermore, it elicits additional biological responses that are unrelated to mitogenesis (St. Hilaire and Jones, 1982; Carpenter and Wahl, 1990; Carpenter and Cohen, 1992). The EGF/TGFa receptor decreases during the prereplicative period, and continues to decline through the initial peak of DNA synthesis, reaching a nadir at 36 to 48 hr (DuBois et al., 1994). Several studies have dealt with the uptake of EGF from the blood, its binding to the receptor, internalization, and subsequent intracellular processing of the receptor complex (Michalopoulos, 1990). Liver regeneration in sialoadenectomized mice is delayed and DNA synthesis reduced; it is restored to normal by EGF treatment (Noguchi et al., 1991). In a recent preliminary report, involving a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, EGF mRNA has been found to increase 30-fold within 15 min after partial hepatectomy, followed by a steep decline to subnormal levels in 4 to 8 hr. This occurs only in the hepatocyte population (Mullhaupt et al., 1993). Although EGF has been extensively studied in hepatocytes both in vivo and especially in vitro, there are many divergencies and thus its role in liver regeneration remains unclear (see Chapter 2) (Fausto, 1990, 1991; Michalopoulos, 1990; Selden and Hodgson, 1991; Bucher and Strain, 1992). 2. Transforming Growth Factor

In hepatocyte cultures, TGFa is more potent than EGF in stimulating DNA synthesis (Fausto, 1991; Webber et al., 1993). The precursors of EGF,

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Nancy L. R. Bucher

TGFcx, and certain other growth factors are transmembrane glycoproteins that are cleaved to yield soluble growth factors, which act locally in paracrine or autocrine modes. The membrane bound forms, however, are also active and can interact with receptors on the surface of adjacent cells, thereby sustaining cell-cell adhesion as well as direct cell-cell (juxtacrine) stimulation (Massagu6, 1990). TGFa appears to be a more likely physiological regulator of liver regeneration than EGF (Fausto, 1990, 1991; Michalopoulos, 1990; Selden and Hodgson, 1991; Bucher and Strain, 1992). TGF(x mRNA increases within the hepatocytes at 4 to 8 hr after partial hepatectomy, reaching a maximum of 8- to 10-fold above sham-hepatectomized controis by 12 to 24 hr. The mature form of the TGFcx peptide increases by 4 hr after partial hepatectomy, is maximal at 18 hr, and remains significantly increased at 48 hr. These changes slightly precede the DNA synthesis peak. In a later study, although the TGF(x content increased prior to the DNA synthesis peak, the mature form was not detected until 48 hr post partial hepatectomy, and was still present at 96 hr. These observations suggest that the membrane anchored precursor and the mature forms of TGF(x may have different functions in liver regeneration (Russell et al., 1993). 3. H e p a t o c y t e G r o w t h F a c t o r / S c a t t e r Factor

HGF activity was first detected with the use of hepatocyte cultures, and was shown to be present in serum, plasma, and platelets from normal and partially hepatectomized rats (see Chapter 2) (Strain et al., 1982; Michalopoulos et al., 1982; Russell et al., 1984a,b; Nakamura et al., 1984), and serum from patients in fulminant hepatic failure (Ghoda et al., 1988). It was subsequently isolated and purified from rat blood platelets and serum (Nakamura et al., 1987; Zarnegar and Michalopoulos, 1989; Matsumoto and Nakamura, 1991, 1992). HGF was finally cloned and sequenced by Nakamura et al. (1989). HGF was later found to be identical to scatter factor (Naldini et al., 1991), a motility promoting substance secreted by fibroblasts of mouse or human origin, and identified and purified about the same time as HGF (Stoker et al., 1987). HGF is produced by various cells of mesenchymal origin in the liver, pancreas, brain, thyroid, salivary and Brunner's glands, kidney, and lung, but not by the epithelial cells in these organs. Within the liver, HGF is produced principally by the nonparenchymal cells of Ito (fat-storing cells). The high-affinity signaling receptor for HGF is the protein product of the c-met proto-oncogene (see Chapters 2 and 3), which is present in hepatocytes and a variety of other epithelial cells which HGF stimulates to proliferate. Like various other growth factors, HGF also acts negatively, inhibiting growth of several tumors, including HepG 2 cells and even normal

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Liver Regeneration Then and N o w

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hepatocytes when used at high concentrations (Michalopoulos, 1990; Matsumoto and Nakamura, 1991, 1992; DuBois et al., 1994). The serum concentration of HGF rises dramatically within an hour after partial hepatectomy, apparently from extrahepatic sources. About 12 hr later, HGF mRNA increases in the nonparenchymal cells, becoming maximal by 24 hr. The HGF activity also increases during this time, suggesting a paracrine mode of action specifically involving the liver. Although the HGF mRNA also increases in various nonhepatic tissues (e.g., lung) following partial hepatectomy, only the liver cells actively proliferate (DuBois et al., 1994). Similar increases in HGF are found in rat livers following other means of inducing regenerative activity, including liver deficiency induced by damage with CC14, galactosamine, ischemia, or physical injury. The timing of the HGF response is commensurate with the overall type of injury and extent of damage induced (Matsumoto and Nakamura, 1991, 1992). It appears likely from these observations, coupled with the remarkably high levels of HGF in patients with fulminant hepatic failure, as well as a large number of in vitro studies, that HGF has a significant physiological role in liver regeneration, but how it fits into the overall scheme still needs clarification. 4. Acidic Fibroblast Growth Factor

Both aFGF (also termed heparin binding growth factor-I) and HGF bind heparin, and consequently bind to heparin-like sites in the extracellular matrix, notably heparan sulfate proteoglycans, where they may serve as reservoirs from which they can be released as soluble growth factors. Alternatively, they may remain matrix bound and interact with heparan sulfate proteoglycans or specific receptors on the cell surface. In regenerating liver, expression of aFGF precedes the expression of TGFo~, and occurs in both the parenchymal and nonparenchymal cells, persisting for 7 days after the partial hepatectomy. It is secreted by hepatocytes and nonparenchymal cells, the maximal rate approximately coinciding with the peak rate of DNA synthesis. In hepatocyte cultures, the aFGF peptide binds to high affinity receptors and stimulates DNA synthesis with about one-third the potency of EGF (Kan et al., 1989; Michalopoulos, 1990; Matsumoto and Nakamura, 1992). S. Hepatocyte Stimulatory Substance

Hepatocyte stimulatory, substance (HSS) is another hepatocyte mitogen. It was originally observed in 1975 in extracts of weanling and regenerating rat liver, and subsequently in rabbit, mouse, dog, and pig livers (LaBrecque, 1994; LaBrecque et al., 1987). Purified preparations have so far not stimu-

16

Nancy L. R. Bucher

lated growth in any cell type other than hepatoma cells. However, because HSS has not yet become available in sufficient quantity and purity its cell type specificity has not been widely tested (Michalopoulos, 1990; Bucher and Strain, 1992; DuBois et al., 1994). 6. T r a n s f o r m i n g G r o w t h Factor f3

The TGFf3 family of growth factors comprises three isoforms, which are widespread and multifunctional. They can stimulate, or reversibly inhibit proliferation, tending to stimulate growth in mesenchymal cells and inhibiting it in epithelial cells. With regard to hepatocytes, TGFf31 is by far the most widely studied. TGFf31 is secreted in a latent form which is biologically inactive (DuBois et al., 1994), and the proteolytic activation of TGFf31 by plasmin is facilitated by binding of the latent complex of TGF[31 to the M6P/IGF2r (Dennis and Rifkin, 1991). During liver regeneration, expression of all three TGF[3 mRNA isoforms increases, especially TGF[31. The TGF[31 mRNA becomes detectable by 4 hr, increases further during the peak of DNA synthesis, rises steeply to a maximum at about 72 hr and then slowly declines to normal (Fausto and Mead, 1989). The mRNA is found only in the nonparenchymal cells. In contrast, the TGFIB types I, II, and III receptors are rapidly downregulated following a partial hepatectomy reaching a nadir at 24 hr post partial hepatectomy, the time of maximum DNA synthesis (Chari et al., 1995). Furthermore, the kinetics of type II receptor expression suggest it may be primarily responsible for ultimately limiting hepatocyte proliferation. Injection of TGFf31 at the time of partial hepatectomy does not affect the induction or course of DNA synthesis. In contrast, the injection of TGFf3 prior to the start of DNA synthesis substantially reduces the usual peak of DNA synthesis at 24 hr. The inhibition is transient, and continuation of treatment does not prevent the ultimate progression of the regeneration to completion. These results suggest that there is a TGF[31 sensitive restriction point during the late G 1 phase of the cell cycle (see Chapters 8 and 9) (Russell et al., 1988; DuBois et al., 1994). It has also been repeatedly shown that TGF[31 potently but reversibly inhibits DNA synthesis in growth factor stimulated hepatocytes in culture (Fausto, 1990; Michalopoulos, 1990; Bucher, 1991; Bucher and Strain, 1992; Matsumoto and Nakamura, 1992). Immunostaining of regenerating livers for TGF[31 protein is intensely positive in the periportal hepatocytes just before the start of DNA synthesis which also begins in these cells. The TGFf31 localization then progresses in a wave-like fashion toward the pericentral region of the liver lobule just before appearance of hepatocytes undergoing DNA synthesis (Jirtle et al., 1991). Thus, TGFIB appears to have a role in maintaining a ba!anced regenerative response, but a much fuller understanding of the underlying molecu-

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Liver Regeneration Then and N o w

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lar mechanisms is needed before the complexities of TGFf3 function can be interpreted into a coherent description of the regulation of liver regeneration. 7. Other Negative Growth Factors

These have not been extensively studied and are dealt with in recent reviews. They include activin, which is a peptide with homology to TGFI3, hepatocyte proliferation inhibitor (HPI) and platelet derived growth inhibitors cx and 13 (PDGIc~ and PDGI[3) (Fausto, 1990; Michalopoulos, 1990; Bucher, 1991; Bucher and Strain 1992; Matsumoto and Nakamura, 1992).

C. Cytokines 1. Interleukins

Recent studies implicate products of activated nonparenchymal cells in the regenerative process (i.e., cytokines). Some of these substances appear to act during the prereplicative period of hepatocyte regeneration. The relationship of cytokines to the regenerative response is provided by the indirect evidence that partial hepatectomy releases cytokines such as the interleukins (e.g., IL-1 and IL-6) and tumor necrosis factor c~ (TNFc~). Pretreatment of normal mice with lipopolysaccharide (an activator of cytokine production) 24 hr before partial hepatectomy augments DNA synthesis, whereas it is depressed in germ-free euthymic and athymic mice. These observations suggest that cytokines play an important role in stimulating liver regeneration (Cornell, 1990a,b). Moreover, serum IL-6 concentrations increase significantly after partial hepatectomy, and this increase is inhibited by antibodies to TNFa, a factor known to trigger IL-6 release (Akerman et al., 1992). Injection of IL-lc~ or IL-6 in combination with glucagon and ammonium chloride is reported to significantly increase the mitotic index in livers of normal adult rats. In contrast, control animals given only ammonium chloride and glucagon are unaffected (Koga and Ogasawara, 1991). IL-113, and to a lesser extent IL-6, inhibit the proliferation of hepatocytes in culture. Neither cytokine is as effective in this regard as TGF[31 (Michalopoulos, 1990; Bucher, 1991; Matsumoto and Nakamura, 1992). 2. Tumor Necrosis Factor c~

Administration of TNFe~ to normal rats has been reported to stimulate DNA synthesis. At the optimal dose of 25 I~g, DNA synthesis increases by about four-fold over the level in control animals; this is a very low DNA synthetic stimulus compared to partial hepatectomy where the increase is in

18

Nancy L. R. Bucher

the range of 50-fold (Feingold et al., 1988). It was subsequently reported, that the proliferative response is restricted to the nonparenchymal cells, especially the macrophages (Feingold et al., 1991). Meanwhile, intraperitoneal injection of polyclonal antibodies to TNFc~ 1 hr before partial hepatectomy significantly inhibits DNA synthesis in both the parenchymal and nonparenchymal cell populations. The conclusion is that TNFc~ positively modulates liver regeneration (Akerman et al., 1992). Thus, TNFc~ appears to be another of many possible effectors already proposed to function in the process of liver regeneration. D. Interactions

A finely balanced, integrated process such as liver regeneration must require a continuously shifting interplay of many growth effectors to meet the changing demands imposed by a varying array of bodily functions during the activation, progression, and subsidence of a major outburst of proliferative activity in a tissue that is normally in a state of growth arrest. Unraveling of the physiological and molecular biological complexities of this process started more than 100 years ago but still has a long way to go. Instances of hormonal, growth factor, and cytokine interactions have already been mentioned in relation to liver growth. These substances also interact in various ways to influence many metabolic functions of the liver, including amino acid, protein, lipid, carbohydrate, and probably other metabolic processes as well (Andrus et al., 1991). This is relevant, because the metabolic state of the cell can significantly affect its response to various growth effectors. Although hormones are generally available, nearly all of the growth factors and cytokines of interest, with a few very recent exceptions, have been obtainable only in small amounts. As a result, nearly all growth factor studies have been conducted in hepatocyte cultures. Hepatocytes, however, when freshly isolated for culture, are found to already express mRNAs of growth associated immediate-early genes (see Chapter 4). This indicates that cultured cells are primed before the growth factors are added, having been induced to undergo the G O to G 1 transition during the enzymatic preparative procedure (Etienne et al., 1988). Therefore, although in vitro studies may point to differences between effectors that potentiate mitogenesis and those that primarily modulate the process, in most instances their ability to initiate proliferative activity in unprimed (G O arrested) cells has been largely untested. In a recent report, however, EGF, TGF(x, and HGF have been infused directly into the portal vein of normal rats for 24 hr, resulting in remarkably small effects on DNA synthesis. If these growth factors are infused instead into rats previously subjected to a 33% hepatectomy, significant stimula-

1. Liver Regeneration Then and Now

19

tion results. Compared to 33% hepatectomized controls, EGF plus insulin causes DNA synthesis increases of up to four-fold, TGFc~ of up to eight-fold, and HGF of about five-fold. These results suggest that the 33% hepatectomy, which by itself causes little DNA synthesis, primes the normally quiescent hepatocytes, and potentiates the stimulation effects of the growth factors (Webber et al., 1994). This is at variance with an earlier study in which infusion of EGF plus insulin did stimulate hepatic DNA synthesis in normal rats. The discrepancy is probably due to priming induced by the stresses resulting from the less sophisticated infusion technique used in the earlier work (Bucher et al., 1978b). As regards combinations of growth factors, although HGF and TGFc~ augment each other in stimulating DNA synthesis in hepatocyte cultures, the combination is actually less stimulatory than either factor by itself in the in vivo 33 % hepatectomy model (Webber et al., 1994). No model is perfect, but despite the unquestionable value of hepatocyte cultures, it is occasionally worthwhile to emphasize their deficiencies (Bucher et al., 1990), and the enlightening perspectives to be gained from in vivo experiments.

VI. Conclusion After a long slow start, research on liver regeneration is gaining momentum at an accelerating rate that, to one who started long ago, is breathtaking. The framework, based on whole animal studies, is in place. Many of the parts that function within it are recognized and their potential roles are currently under intensive study at the molecular level. Although the complexities that are emerging may appear overwhelming, the technologies are at hand, and the pieces that fit into the puzzle are coming into sharper focus. The final step, assembly of the parts into the integrated whole that encompasses liver regeneration in vivo, is about to begin. Stay tuned!

References Akerman, R, Cote, R, Yang, S. Q., McClain, C., Nelson, S., Bagby, G. J., and Diehl, A. M. (1992). Antibodies to tumor necrosis factor inhibit liver regeneration after partial hepatectomy. Am. J. Physiol. 263, G579-585. Alison, M. R. (1986). Regulation of hepatic growth. Physiol. Rev. 66, 500-541. Andrus, T., Bauer, J., and Gerok, W. (1991). Effects of cytokines on the liver. Hepatology (Baltimore) 13, 364-375. Bach, R, Hamprecht, B., and Lynen, E (1969). Regulation of cholesterol biosynthesis in rat liver: Diurnal changes of activity and influence of bile acids. Arch. Biochem. Biophys. 133, 11-21. Berry, M. N., and Friend, D. S. (1969). High yield preparations of rat liver parenchymal cells. A biochemical and fine structural study. J. Cell Biol. 43, 506-520.

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Bresnick, E. (1971). Regenerating liver: An experimental model for the study of growth. Methods Cancer Res. 6, 347-397. Brues, A. M., Drury, D. R., and Brues, M. C. (1936). A quantitative study of cell growth in regenerating liver. Arch. Pathol. 22, 658-673. Brues, A. M., Tracy, M. M., and Cohn, W. E. (1944). Nucleic acids of rat liver and hepatoma: Their metabolism and turnover in relation to growth. J. Biol. Chem. 155, 619-633. Bucher, N. L. R. (1963). Regeneration of mammalian liver. Int. Rev. Cytol. 15, 245-300. Bucher, N. L. R. (1967a). Experimental aspects of hepatic regeneration. N. Engl. J. Med. 277, 686-696 and 738-746. Bucher, N. L. R. (1982). Thirty years of liver regeneration: A distillate. In "Cold Spring Harbor Conference on Cell Proliferation: Growth of Cells in Hormonally Defined Media" (G. H. Sato and R. Ross, eds.), Vol. 9, pp. 15-26. Cold Spring Harbor Laboratory, Cold Spring Harbor New York. Bucher, N. L. R. (1987). Regulation of liver growth: Historical perspectives and future directions. In "The Isolated Hepatocyte: Use in Toxicology and Xenobiotic Transformations" (E. J. Rauckman and G. M. Padilla, eds.), pp. 1-19. Academic Press, New York. Bucher, N. L. R. (1991). Liver regeneration: An overview. J. Gastroenterol. Hepatol. 6, 615624. Bucher, N. L. R., and Malt, R. A. (1971). "Regeneration of Liver and Kidney," pp. 1-176. Little Brown, Boston. Bucher, N. L. R., and Strain, A. J. (1992). Regulatory mechanisms in hepatic regeneration. In "Wright's Liver and Biliary Disease" (G. H. Millward-Sadler, R. Wright, and M. J. P. Arthur, eds.), 3rd Ed., pp. 258-274. Saunders, London. Bucher, N. L. R., and Swaffield, M. N. (1964). The rate of incorporation of labeled thymidine into the deoxyribonucleic acid of regenerating rat liver in relation to the amount of liver excised. Cancer Res. 24, 509-512. Bucher, N. L. R., and Swaffield, M. N. (1973). Regeneration of liver in rats in the absence of portal splanchnic organs and a portal blood supply. Cancer Res. 33, 3189-3194. Bucher, N. L. R., and Swaffield, M. N. (1975). Regulation of hepatic regeneration in rats by synergistic action of insulin and glucagon. Proc. Natl. Acad. Sci. U.S.A. 72, 11571160. Bucher, N. L. R., Scott, J. E, and Aub, J. C. (1951). Regeneration of the liver in parabiotic rats. Cancer Res. 11,457-465. Bucher, N. L. R., Schrock, T. R., ,and Moolten, E L. (1969). An experimental view of hepatic regeneration. Johns Hopkins Med. J. 125, 150-257. Bucher, N. L. R., McGowan, J. A., and Patel, U. (1978a). Hormonal regulation of liver growth. "ICN/UCLA Symposium: Molecular Cell Biology," Vol. 12, pp. 661-670. Bucher, N. L. R., Patel, U., and Cohen, S. (1978b). Hormonal factors concerned with liver regeneration. "Hepatotrophic Factors" Ciba Foundation Symposium No. 55, pp. 95-107. Elsevier, New York. Bucher, N. L. R., Robinson, G. S., and Farmer, S. R. (1990). Effects of extracellular matrix on hepatocyte growth and gene expression: Implications for hepatic regeneration and the repair of liver injury. Semin. Liver Dis. 10, 11-19. Carpenter, G., and Cohen, S. (1992). Epidermal growth factor. J. Biol. Chem. 265, 7709-7712. Carpenter, G., and Wahl, M. I. (1990). The epidermal growth factor family. In "Handbook of Experimental Pharmacology" (M. B. Sporn and A. B. Roberts, eds.), Vol. 1, pp. 69-171. Springer-Verlag, Berlin. Carter, E. A., Kirkham, S. E., Tompkins, R. G., and Burke, J. E (1989). Inhibition of in vivo DNA synthesis in regenerating rat liver following thermal injury. Biochem. Biophys. Res. Commun. 160, 196-201. Caruana, J. A., Whalen, D. A., Anthony, W. P., Sunby, C. R., and Ciechoski, M. P. (1986).

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Liver Regeneration Then and Now

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Paradoxical effects of glucose feeding on liver regeneration and survival after partial hepatectomy. Endocr. Res. 12, 147-156. Chari, R. S., Price, D. T., Sue, S. R., Meyers, W. C., and Jirtle, R. L. (1995). Down-regulation of transforming growth factor 13type I, II, and III during liver regeneration. Am. J. Surg. 169, 126-132. Cornell, R. P. (1990a). Depressed liver regeneration after partial hepatectomy of germ-flee athymic and lipopolysaccharide-resistant mice. Hepatology (Baltimore) 11, 916-922. Cornell, R. P. (1990b). Acute phase responses after acute liver injury by partial hepatectomy in rats as indicators of cytokine release. Hepatology (Baltimore) 11, 923-931. Dennis, P. A., and Rifkin, D. B. (1991). Cellular activation of latent transforming growth factor 13 requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor. Proc. Natl. Acad. Sci. U.S.A. 88, 580-584. DuBois, R. N., Hunter, E. B., and Russell, W. E. (1994). Molecular aspects of hepatic regeneration. In "Molecular Basis of Medicine" (C. V. Dang and A. M. Feldman, eds.), in press. Edwards, P. A., Muroya, N., and Gould, R. G. (1972). In vivo demonstration of the circadian rhythm of cholesterol biosynthesis in the liver and intestine of the rat. J. Lipid Res. 13,396401. Ekberg, S., Luther, M., Nakamura, T., and Jansson (1992). Growth hormone promotes early initiation of hepatocyte growth factor gene expression on the liver of hypophysectomized rats after partial hepatectomy. J. Endocrinol. 35, 59-67. Etienne, P. L., Baffet, G., Desvergne, B., Boisnard-Rissel, M., Glaise, D., and GuguenGuillouzo, C. (1988). Transient expression of c-los and constant expression of c-myc in freshly isolated and cultured normal adult rat hepatocytes. Oncog. Res. 3, 255-262. Fausto, N. (1990). Hepatic regeneration. In "Hepatology" (D. Zakim and T. D. Boyer, eds.), 2nd Ed., pp. 49-95. Saunders, London. Fausto, N. (1991). Growth factors in liver development, regeneration and carcinogenesis. Prog. Growth Factor Res. 3, 219-234. Fausto, N., and Mead, M. E. (1989). Regulation of liver growth: Protooncogenes and transforming growth factors. Lab. Invest. 60, 4-13. Feingold, K. R., Soued, M., and Grunfeld, C. (1988). Tumor necrosis factor stimulates DNA synthesis in the liver of intact rats. Biochem. Biophys. Res Commun. 153, 576-582. Feingold, K. R., Barker, M., Jones, A. L., and Grunfeld, C. (1991). Localization of tumor necrosis factor-stimulated DNA synthesis in the liver. Hepatology (Baltimore) 13,773-779. Fishback, E C. (1929). A morphologic study of regeneration of the liver after partial removal. AMA Arch. Pathol. 7, 955-977. Fleig, W. E. (1988). Liver-specific growth factors. Scand. J. Gastroenterol. Suppl. 23, 31-36. Gaub, J., and Iversen, J. (1984). Rat liver regeneration after 90% partial hepatectomy. Hepatology (Baltimore) 4, 902-904. Ghahary, A., Minuk, G. Y., Luo, J., Gauthier, T., and Murphy, L. M. (1992). Effects of partial hepatectomy on hepatic insulin-like growth factor binding protein-1 expression. Hepatology (Baltimore) 15, 1125-1131. Ghoda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Miyazaki, H., Hashimoto, S., and Daikuhara, Y. (1988). Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. J. Clin. Invest. 81, 414-419. Grisham, J. W. (1962). A morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver; autoradiography with thymidine-3H. Cancer Res. 22,842-849. Grisham, J. W., Leong, G. E, and Hole, B. V. (1964). Heterotopic partial autotransplantation of rat liver. Technique and demonstration of structure and function of the graft. Cancer Res. 24, 1474-1482. Gumucio, J. J., and Berkowitz, C. M. (1992). Structural organization of the liver and function

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of the hepatic acinus. In "Liver and Biliary Diseases" (N. Kaplowitz, ed.), 3rd Ed., pp. 217. Williams & Wilkins, Baltimore, Maryland. Gumucio, J. J., and Chianale, J. (1988). Liver cell heterogeneity and liver function. In "The Liver: Biology, and Pathobiology" (I. M. Arias, W. B. Jakoby, H. Popper, D. Schachter, and D. A. Shafritz, eds.), 2nd Ed., pp. 931-947. Raven, New York. Harkness, R. D. (1957). Regeneration of liver. Br. Med. Bull. 13, 87-93. Hasegawa, K., and Koga, M. (1977). Induction of liver cell proliferation in intact rats by amines and glucagon. Life Sci. 21, 1723-1728. Hasegawa, K., Sidhartha, K., and Carr, B. I. (1994). Stimulation of hepatocyte DNA synthesis by neurotensin. J. Cell. Physiol. 159, 215-222. Hays, D. M. (1974). Surgical aspects of hepatic regeneration. Surg. Gynecol. Obstet. 139, 609-619. Higashiyama, S., Abraham, J. A., Miller, J., Fiddes, J. C., and Klagsbrun, M. (1991). A heparinbinding growth factor secreted by macrophage-like cells that is related to EGE Science 251, 936-939. Higashiyama, S., Abraham, J. A., and Klagsbrun, M. (1993). Heparin-binding EGF-like growth factor stimulation of smooth muscle cell migration: Dependence on interactions with cell surface heparan sulfate. J. Cell Biol. 122, 933-940. Higgins, G. M., and Anderson, R. M. (1931). Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 12, 186202. Holecek, M., Simek, J., Palicka, V., and Zadak, Z. (1991). Effect of glucose and branched chain amino acid (BCAA) infusion on onset of liver regeneration and plasma amino acid pattern in partially hepatectomized rats. J. Hepatol. 13, 14-20. Hopkins, H. A., Bonney, R. J., Walker, P. R., Yager, J. D., and Potter, V. R. (1973a). Food and light as separate entrainment signals for rat liver enzymes. Adv. Enzyme Regul. 11, 169191. Hopkins, H. A., Campbell, H. A., Barbiroli, B., and Potter, V. R. (1973b). Thymidine kinase and deoxyribonucleic acid metabolism in growing and regenerating livers from rats on controlled feeding schedules. Biochem. J. 136, 955-966. Horikawa, S., Sakata, K., Hatanaka, M., and Tsukada, K. (1986). Expression of c-myc oncogene in rat liver by dietary manipulation. Biochem. Biophys. Res. Commun. 140, 574580. Ingle, D. J., and Baker, B. L. (1957). Histology and regenerative capacity of liver following multiple partial hepatectomies. Proc. Soc. Exp. Biol. Med. 95, 813-815. Ito, N., Kawata, S., Tamura, S., Kiso, S., Tsushima, H., Damm, D., Abraham, J. A., Higashiyama, S., Taniguchi, N., and Matsuzawa, Y. (1994). Heparin-binding EGF-like growth factor is a potent mitogen for rat hepatocytes. Biochem. Biophys. Res. Commun. 198, 25-31. Jirtle, R. L., and Michalopoulos, G. (1982). Effects of partial hepatectomy on transplanted hepatocytes. Cancer Res. 42, 3000-3004. Jirtle, R. L., Cart, B. I., and Scott, C. D. (1991). Modulation of insulin-like growth factor 2/mannose 6-phosphate receptors and transforming growth factor-J31 during liver regeneration. J. Biol. Chem. 266, 22444-22450. Kan, M., Huang, J., Mansson, P.-E., Yasumitsu, H., Carr, B., and McKeehan, W. L. (1989). Heparin-binding growth factor type I (acidic fibroblast growth factor): A potential biphasic autocrine and paracrine regulator of heplatocyte regeneration. Proc. Natl. Acad. Sci. U.S.A. 86, 7432-7436. Klinge, O., and Mathyl, J. (1969). Tagezeitliche mitose-rhythmen in der rattenleber nach modifizierter teilhepatektomierten rattenleber. Klin. Wochenschr. 46, 395-396. Koga, M., and Ogasawara, H. (1991). Induction of hepatocyte mitosis in intact adult rat by interleukin-le~ and interluekin-6. Life Sci. 49, 1263-1270.

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Liver Regeneration Then and Now

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LaBrecque, D. (1994). Liver regeneration: A picture emerges from the puzzle. Am. J. Gastroenterol. 89(Suppl. 8), $86-$96. LaBrecque, D. R., Steele, G., Fogerty, S., Wilson, M., and Barton, J. (1987). Purification and physical-chemical characterization of hepatic stimulator substance. Hepatology (Baltimore) 7, 100-106. Leduc, E. H. (1949). Mitotic activity in the liver of the mouse during inanition followed by refeeding with different levels of protein. Am. J. Anat. 84, 397-430. Leduc, E. H. (1964). Regeneration of the liver. In "The Liver. Morphology, Biochemistry and Physiology" (C. Rouiller, ed.), Vol. 2, pp. 63-89. Academic Press, New York. Lee, J., Greenbaum, L., Haber, B. A., Nagle, D., Lee, V., Niles, V., Mohn, L. L., Bucan, M., and Taub, R. (1994). Structure and localization of the IGFBP-1 gene and its expression during liver regeneration. Hepatology (Baltimore) 19, 656-665. Leffert, H. L., Koch, L. S., Lad, P. J., Shapiro, I. P., Skelly, H., and deHemptinne, B. (1988). Hepatocyte regeneration, replication and differentiation. In "The Liver: Biology and Pathobiology" (I. M. Arias, W. B. Jakoby, H. Popper, D. Schachter, and D. A. Shafritz, eds.), 2nd Ed., pp. 833-850. Raven, New York. Lewan, L., Ynger, T., and Engelbrecht (1977). The biochemistry of the regenerating liver. Int. J. Biochem. 8, 477-487. Mann, E C. (1944). Restoration and pathologic reactions of the liver. J. Mr. Sinai Hosp. (N. Y.) 11, 65-74. Masquilier, D., Foulkes, N. S., Mattei, M.-G., and Sassone-Corsi, P. (1993). Human CREM gene: Evolutionary conservation, chromosomal localization, and inducibility of the transcript. Cell Growth Differ. 4, 931-937. Massagu6, J. (1990). Transforming growth factor cx. J. Biol. Chem. 265, 21393-21396. Matsumoto, K., and Nakamura, T. (1991). Hepatocyte growth factor: Molecular structure and implications for a central role in liver regeneration. J. Gastroenterol. Hepatol. 6, 509519. Matsumoto, K., and Nakamura, T. (1992). Hepatocyte growth factor: Molecular structure, roles in liver regeneration, and other biological functions. Crit. Rev. Oncogen. 3, 27-54. Mead, J. E., Braun, D. A., Martin, D. A., and Fausto, N. (1990). Induction of replicative competence ("priming") in normal liver. Cancer Res. 50, 7023-7030. Michalopoulos, G. K. (1990). Liver regeneration: Molecular mechanisms of growth control. FASEB J. 4, 176-187. Michalopoulos, G. K., Cianciulli, H. D., Novotny, A. R., Kligerman, A. D., Strom, S. C., and Jirtle, R. L. (1982). Liver regeneration studies with rat hepatocytes in primary cultures. Cancer Res. 42, 4673-4682. Mohn, K. L., Laz, T. M., Hsu, J.-C., Melby, A. E., Bravo, R., and Taub, R. (1991a). The immediate-early growth response in regenerating liver and insulin-stimulated H-35 cells: Comparison to serum-stimulated 3T3 cells and identification of 41 novel immediate-early genes. Mol. Cell. Biol. 11, 381-390. Mohn, K. L., Melby, A. E., Teware, D. E., La, T. M., and Taub, R. (1991b). The gene encoding rat insulin like growth factor binding protein-1 is rapidly and highly induced in regenerating liver. Mol. Cell. Biol. 11, 1393-1401. Moolten, E L., and Bucher, N. L. R. (1967). Regeneration of rat liver: Transfer of "humoral" agent by cross circulation. Science 158, 272-274. Moolten, E L., Oakman, N. J., and Bucher, N. L. R. (1970). Accelerated response of hepatic DNA synthesis to partial hepatectomy in rats pre-treated with growth hormone or surgical stress. Cancer Res. 30, 2353-2357. Mueller, C. R., Maire, P. N., and Schibler, U. (1990). DBP, a liver-enriched transcriptional activator, is expressed late in ontogeny and its tissue specificity is determined post transcriptionally. Cell (Cambridge, Mass.) 61,279-291. Mullhaupt, E., Fodor, A. Feren, A., and Jones, A. (1993). The steady-state level of hepatocyte

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epidermal growth factor RNA rapidly increases in the prereplicative phase of liver regeneration. Hepatology (Baltimore) 18, 148A. Nakamura, T., Nawa, K., and Ichihara, A. (1984). Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats. Biochem. Biophys. Res. Commun. 122, 1450-1459. Nakamura, T., Nawa, K., Ichihara, A., Kaise, N., and Nishino, T. (1987). Purification and subunit structure of hepatocyte growth factor from rat platelets. FEBS Lett. 224, 311-316. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K., and Shimizu, S. (1989). Molecular cloning and expression of human hepatocyte growth factor. Nature (London) 342, 440-443. Naldini, L., Weidner, K. M., Vigna, E., Gaudino, G., Bardelli, A., Ponzetto, C., Narsimhan, R. P., Hartmann, G., Zarnegar, R., Michalopoulos, G. K., Birchmeier, W., and Comoglio, P. M. (1991). Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J. 10, 2867-2878. Ni, N., and Yager, J. D. (1994). Co-mitogenic effects of estrogens on DNA synthesis induced by various growth factors in cultured female rat hepatocytes. Hepatology (Baltimore) 19, 183-192. Noguchi, S., Ohba, Y., and Oka, T. (1991). Influence of epidermal growth factor on liver regeneration after partial hepatectomy in mice. J. Endocrinol. 128, 425-431. Ohmori, H., Murakami, A. E, Higashi, K., Hirano, H., Gotoh, S., Kuroiwa, A., Masui, A., Nakamura, T., and Amalric, E (1990). Simultaneous activation of heat shock protein (hsp 70) and nucleolin genes during in vivo and in vitro prereplicative stages of rat hepatocytes. Exp. Cell Res. 189, 227-232. Russell, W. E., and Bucher, N. L. R. (1983). Vasopressin modulates liver regeneration in the Brattleboro rat. Am. J. Physiol. 245, G321-G324. Russell, W. E., McGowan, J. A., and Bucher, N. L. R. (1984a). Partial characterization of a hepatocyte growth factor from rat platelets. J. Cell. Physiol. 119, 183-192. Russell, W. E., McGowan, J. A., and Bucher, N. L. R. (1984b). Biological properties of a hepatocyte growth factor from rat platelets. J. Cell. Physiol. 119, 193-197. Russell, W. E., Coffey, R. J., Ouellette,, A. J., and Moses, H. L. (1988). Type beta transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat. Proc. Natl. Acad. Sci. U.S.A. 85, 5126-5130. Russell, W. E., Dempsey, P. J., Sitaric, S., Peck, A. E, and Coffey, R. G. (1993). Transforming growth factor-e~ (TGF(x) concentrations increase in regenerating rat liver: Evidence for a delayed accumulation of mature TGFe~. Endocrinology (Baltimore) 133, 1731-1738. Schlesinger, M. J. (1990). Heat shock proteins. J. Biol. Chem. 265, 12111-12114. Scott, C. D., Ballesteros, M., and Baxter, R. C. (1990). Increased expression of insulin-like growth factor-II/mannose-6-phosphate receptor in regenerating rat liver. Endocrinology (Baltimore) 127, 2210-2216. Selden, A. C., and Hodgson, H. J. E (1991). Growth factors and the liver. Gut 32, 601-603. Simpson, G. E. C., and Finkh, E. S. (1963). Pattern of regeneration of rat liver after repeated partial hepatectomies. J. Pathol. Bacteriol. 86, 361-370. Sporn, M. B., and Roberts, A. B. (1990). TGFf3: Problems and prospects. Cell Regul. 1, 875882. St. Hilaire, R. J., and Jones, A. L. (1982). Epidermal growth factor: Its biological and metabolic effects with emphasis on the hepatocyte. Hepatology (Baltimore) 2, 601-613. Starzl, T. E., and Terblanche, J. (1979). Hepatotrophic substances. Prog. Liver Dis. 6, 135151. Stoker, M., Gherardi, E., Perryman, M., and Gray, J. (1987). Scatter factor is a fibroblastderived modulator of epithelial cell mobility. Nature (London) 327, 239-242. Strain, A. J., McGowan, J. A., and Bucher, N. L. R. (1982). Stimulation of DNA synthesis in

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primary cultures of adult rat hepatocytes by rat platelet-associated substance(s). In Vitro 18, 106-116. Strom, S. C., Jirtle, R. L., Jones, R. S., Rosenberg, M. R., and Michalopoulos, G. (1982). Isolation, culture and transplantation of human hepatocytes. J. Natl. Cancer Inst. 68, 771778. Tamura, J., Tanaka, J., Fujita, K.-I., Yoshida, M., Kasamatsu, T., Arii, S., and Tobe, T. (1992). Cell kinetics of regenerating liver after 70% hepatectomy in rats. 2-color flow cytometric analysis. HPB Surg. 5, 103-114. Webber, E. M., FiztGerald, M. J., Brown, P. I., Bartlett, M. H., and Fausto, N. (1993). Transforming growth factoroc~ expression during liver regeneration after partial hepatectomy and toxic injury, and potential interactions between transforming growth factor-e~ and hepatocyte growth factor. Hepatology (Baltimore) 18, 1422-1431. Webber, E. M., Godowski, P. J., and Fausto, N. (1994). In vivo response of hepatocytes to growth factors requires an initial priming stimulus. Hepatology (Baltimore) 19, 489-497. Weinbren, K. (1959). Regeneration of the liver. Gastroenterology 37, 657-668. Wilson, J. W., and Leduc, E. H. (1948). The occurrence and formation of binucleate and multinucleate cells and polyploid nuclei in the mouse liver. Am. J. Anat. 82, 353-392. Yager, J. D., Zurlo, J., Sewall, C. H., Lucier, G. W., and He, H. (1994). Growth stimulation followed by growth inhibition in livers of female rats treated with ethinyl estradiol. Carcinogenesis 15, 2117-2124. Zarnegar, R., and Michalopoulos, G. K. (1989). Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor from serum of hepatectomized rats. Biochem. Biophys Res. Commun. 122, 1450-1459.

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2 Hepatocyte Growth Factor (HGF) and Its Receptor (Met) in9 Liver Rege neratlon, " Neoplasia, and Disease George K. Michalopoulos Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania15261

I. Introduction Hepatocyte growth factor (HGF) is a multifunctional cytokine that has pleiotropic effects on several cells and tissues. It was originally identified and isolated based on its capacity to stimulate mitogenesis in cultured hepatocytes (Michalopoulos et al., 1982, 1984; Nakamura et al., 1984). Isolation of HGF was followed shortly by a description of its structure. This was accomplished by deriving the amino acid sequence of the molecule directly (Zarnegar et al., 1989) as well as by cloning and sequencing the HGF gene (Nakamura et al., 1989; Miyazawa et al., 1989). It was subsequently shown that the protein encoded by the proto-oncogene c - m e t was the receptor for HGF (Naldini et al., 1991a; Bottaro et al., 1991). These fundamental discoveries opened the door for several studies that have now thoroughly documented that HGF and its receptor are important in the function of most organs and tissues. Given this realization, it is relevant to consider the question of whether there is any special relationship between HGF and the liver. Are there any hepatic functions that are regulated by HGF and is the mode of action of HGF as a regulator of these functions unique and specific to the liver? This chapter emphasizes the aspects of the relationship Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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between HGF and the liver that play a regulatory role in liver growth and function. There have been several comprehensive reviews that cover the current knowledge of the structural and functional characteristics of HGF and its receptor (see Chapter 3) (Michalopoulos and Zarnegar, 1992; Gherrardi et al., 1993; Matsumoto and Nakamura, 1992; Ponzetto et al., 1994; Weidner et al., 1993). Thus, these topics will only be covered to the extent that they are essential to our understanding of the biological effects of HGE

II. Structural and Functional Aspects of HGF and the HGF Receptor HGF is a protein of 100-kDa molecular weight (Figure 1). It is synthesized as a precursor molecule composed of a single polypeptide chain and activated by proteolytic cleavage (Nakamura et al., 1989). The activation is carried out by urokinase (uPA) (Mars et al., 1993) as well as by HGF activator, a recently identified molecule that has substantial sequence homology to coagulation Factor XII (Miyazawa et al., 1993). Activated HGF is a heterodimer consisting of two chains. The heavy (alpha) chain is composed of a hairpin loop domain followed by four kringle domains. The light (beta) chain has several amino acid sequences that are identical to the consensus sequences for serine proteases (Nakamura et al., 1989). Although the consensus sequences are present, the key amino acids which define the catalytic site for serum proteases are mutated. The final result is a pseudo protease structure that has no documented functional protease activity. The structure of the heterodimeric molecule has been characterized by sitedirected mutagenesis (Lokker et al., 1992), and by studying the properties and function of variant HGF molecules that are naturally produced by alternate splicing of the HGF mRNA (Chan et al., 1991). These studies have have shown that the hairpin loop domain and the second kringle are important for binding HGF to its receptor. HGF is a potent mitogen for many epithelial cells. The list currently includes hepatocytes, keratinocytes, urinary bladder epithelial cells, mammary epithelial cells, bronchial epithelial cells, bile duct cells, and endothelial cells. Given the experience with other growth factors, it is likely that HGF will prove to be a widespread mitogen for all epithelial cells of ectodermal and endodermal origin. In addition to its mitogenic properties, HGF is also a cell motogen. It was independently identified as a scatter factor that increases the motility of several normal and neoplastic cells (Stoker et al., 1987), and the identity between scatter factor iSF) and HGF has now been unequivocally confirmed (Naldini et al., 1991b). Although the effects of HGF on cell motility have been investigated extensively, little is still known

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Figure 1 Diagrammatic structure of hepatocyte growth factor (HGF). The two chains are shown held together by disulfide bonds (S-S). The amino terminal of the alpha chain contains a hairpin loop structure and the four kringle structures (K1, K2, K3, and K4). The hairpin loop and kringle 2 are the most important determinants for the binding of HGF to its receptor. The beta chain has the structure of a pseudo protease. The numbered amino acids in the beta chain are surrounded by consensus sequences characteristic of serine proteases. These amino acids have replaced the three amino acids normally seen within the sequences in true proteases. The Arg494-Va1495 site contains the peptide bond cleaved by HGF activating molecules (urokinase or factor XII homologous HGF activator), thus converting HGF from an inactive (single chain) form to the two chain active heterodimeric form composed of an alpha and beta chain.

a b o u t the signal t r a n s d u c t i o n p a t h w a y s that mediate this aspects of H G F function. The c o m b i n a t i o n of mitogenic and m o t o g e n i c effects p r o b a b l y underlies the recently d e m o n s t r a t e d m o r p h o g e n i c effects of H G F on different cell types. Studies of endothelial cells have s h o w n that H G F functions in angiogenesis (Bussolino et al., 1992; Rosen et al., 1993b). H G F also stimulates kidney t u b u l a r epithelial cells ( M D C K cells) to f o r m tubules in a collagen substrate (Pepper et al., 1992), and stimulates hepatocytes to f o r m plates in type I collagen gels ( M i c h a l o p o u l o s et al., 1993). In m o r e recent studies, H G F has been s h o w n to induce the f o r m a t i o n of ductular structures in

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cultures of hepatocytes maintained in a medium that allows for long-term hepatocyte proliferation (Michalopoulos, 1992). The HGF receptor (HGFr) is the protein encoded for by the protooncogene c - m e t (see Chapter 3) (Naldini et al., 1991b; Bottaro et al., 1991). The Met protein is synthesized in a precursor form that becomes activated by cleavage almost immediately after its synthesis (Zhen et al., 1994). The cleavage results in a heterodimeric molecule composed of both a heavy chain and a light chain attached to one another by disulfide bonds. The heavy (beta) chain contains an extracellular domain, a transmembrane domain, and an intracellular domain, and the light (alpha) chain is attached to the extracellular domain. The mode of action of the HGFr has been thoroughly described in recent papers and reviews by Comoglio and colleagues (see Chapter 3) (Ponzetto et al., 1994). Upon binding to its ligand, HGF, and HGFr dimerizes and cross phosphorylates its tyrosine kinase sites. The activated tyrosine kinase sites then become the docking site for signal transduction proteins that are further phosphorylated at tyrosine residues and migrate to other regions of the cell to transmit the stimulatory signals of the activated HGFr (Ponzetto et al., 1994). Although the HGFr belongs to the overall family of tyrosine kinase receptors, it has unique peculiarities in the structure of its activated region. Phosphorylation of two tyrosine residues results in the formation of a single docking site on which many proteins with src homology (i.e., SH2 and SH3) recognition sites bind (see Chapter 3) (Ponzetto et al., 1994). This contrasts with other members of the tyrosine kinase family which have two docking sites in their active configuration. The receptors encoded by the genes c-sea and c - r o n have substantial sequence homology with c - m e t and a similar structure of the activate tyrosine kinase sites (Gaudino et al., 1994).

III. H G F L o c a l i z a t i o n A. In Liver Several studies have shown that both HGF mRNA and HGF protein are produced by the nonparenchymal cells in the liver. The identity of the HGF producing cells was initially disputed; however, it has now been clearly demonstrated that the liver cells responsible for the production of HGF are the cells of Ito (Noji et al., 1990; Schirmacher et al., 1992). These cells contain lipid droplets that are rich in vitamin A. They also produce other growth factors including acidic fibroblast growth factor (aFGF), transforming growth factor 131 (TGFI31), etc. In addition, these cells are responsible for producing much of the scant connective tissue present around hepatocytes. Ito cells change into myofibroblasts in culture as well as in cirrhosis,

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and when they become myofibroblasts they lose the capacity to store vitamin A (Schirmacher et al., 1992). Since Ito cells are responsible for the production of HGF as well as matrix production in the liver, it is not surprising that HGF is heavily deposited in the hepatic biomatrix. When isolated liver is perfused with high concentrations of NaC1, large amounts of HGF are extracted (Masumoto and Yamamoto, 1991). The localization of growth factors in the biomatrix is a well-documented phenomenon and is partly related to the heparin affinity binding sites that most growth factors possess. Typically, these binding sites are characterized by high capacity and low affinity. In addition to the binding of HGF to high capacity low affinity sites [e.g., heparin sulfated proteoglycans, (HSPG)], HGF also binds to specific matrix sites that have relatively high affinity (Zarnegar et al., 1990). When HGF is injected into the liver there is a discrepancy between the lobular distribution of HGF and the distribution of the high affinity binding receptor, HGFr. The HGFr is uniformly distributed throughout the hepatic lobule (Liu et al., 1994a) whereas, HGF bound to high affinity sites is predominantly localized in the periportal region of the liver lobules; eidermal growth factor (EGF) binding has also been shown to have the same lobular pattern of localization (St. Hilaire et al., 1983). This suggests that the bulk of HGF attached to high affinity sites is not bound to the HGFr. Previous studies have shown that there are two high affinity binding sites for HGF in the liver (Zarnegar et al., 1990). The highest affinity binding site is the HGFr (Met protein). The other lesser, but still relatively high affinity binding site, has not been clearly characterized although recent studies have shown that HGF binds with relatively high affinity to sulfoglycolipids compounds such as galactosylceramide sulfate (SM4), lactosylceramide sulfate (SM3), and gangliotriaosylceramide bis-sulfate (Kobayashi et al., 1994). The precise role that these relatively high affinity nonsignaling receptor binding sites play in the function of HGF is not yet clear. However, the localization of the bulk of the HGF in the periportal region of the liver lobule along with an identical localization for EGF correlates with hepatocyte proliferation starting in the periportal region of the liver lobule following a partial hepatectomy (Rabes et al., 1976). The presence of the two most powerful hepatic mitogens at high concentrations at the lobular location where liver regeneration begins suggests a cause and effect relationship between the presence of these growth factors and hepatocyte recruitment into the cell cycle during liver regeneration. B. In Extra Hepatic Tissues

As previously stated, HGF is produced primarily by mesenchymal cell types. These cells include the Ito cells in the liver and the myofibroblasts in other

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organs. These are cells that often surround blood vessels, have contractile properties, and produce the connective tissue that provides the structural framework of different organs. Immunohistochemical studies, however, show that HGF is also present in most types of epithelial cells (Wolf et al., 1991). The epithelial distribution of HGF, however, closely corresponds to that for the HGFr which is also present in most epithelial cells (Giordano et al., 1989) suggesting that the presence of HGF in epithelial cells reflects receptor uptake rather than cellular production. Both HGF and HGFr are also present in neurons of the brain cortex and hippocampus (Jung et al., 1994; Schirmacher, 1994). In contrast, endothelial cells express the HGFr but contain no detectable HGF (Rosen et al., 1993a). IV. L i v e r

and the Processing of HGF

When radiolabeled HGF is injected into the systemic circulation, it can be traced to several organs (Appasamy et al., 1993; Zioncheck et al., 1994). Approximately 40 to 50% of the injected radioactivity becomes bound in high capacity low affinity sites in the skin, muscle, and bone; most of the remaining HGF is found in the liver. Approximately 30 to 40% of the injected HGF is localized in the liver within 10 min after injection; studies have shown that similar temporal and spatial relationships exist for other growth factors such as EGF and TGF~I. It is not clear at this point, however, whether the uptake of HGF by the liver proceeds through a lysosomal dependent pathway. An unexpected finding was that a portion of the injected HGF becomes secreted intact in the bile (Liu et al., 1994a). Furthermore, approximately 20% of the HGF present in the bile is intact. The percentage of intact HGF increases after treatment with leupeptin, but it is not affected by the administration of chloroquine (Liu and Michalopoulos, 1993). Chloroquine inhibits lysosomal function by raising the intra lysosomal pH. Therefore, the combined evidence suggests that most of the processing of HGF within the hepatocyte proceeds through nonlysosomal pathways. The mechanisms by which intact HGF is secreted into the bile are not clear at this time. In general, liver has a high capacity for the uptake of HGE When increasing amounts of HGF are injected through the portal circulation, a large amount (0.157 + 0.012 ~g/g liver) is taken up by the liver before its capacity to bind HGF becomes saturated (Liu iet al., 1994a). Similar studies have shown that following partial hepatectomy, the capacity of regenerating liver to take up HGF increases more than two-fold. Regenerating liver can still secrete intact HGF in the bile; however, shortly after partial hepatectomy the secreted forms of HGF appear to be processed by alternate pathways (Liu et al., 1994a).

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A. HGF and Liver Regeneration The relationship between HGF and liver regeneration has been at the center of the investigation of the functions of HGF from the beginning of its discovery and it originally drove the process that led to its isolation. Several past studies, repeated by multiple laboratories and investigators, have demonstrated that during liver regeneration there appears in the blood factor(s) that transmit a mitogenic signal to hepatocytes present anywhere in the body (see Chapter 1). Fragments of hepatic tissue engrafted into extra hepatic sites responded with DNA synthesis and growth when the in situ liver is subjected to a partial hepatectomy (Leong et al., 1964). When rat pairs are joined together in parabiosis, a partial hepatectomy of the liver in one member leads to liver regeneration in situ as well as in the intact liver of the unoperated member of the parabiotic pair (Moolten and Bucher, 1967). The maximal regenerative stimulation of the unoperated liver is seen when the liver of other member of the pair is removed entirely (Fisher et al., 1971). Partial hepatectomy also leads to DNA synthesis in isolated hepatocytes engrafted into the adipose tissue (Jirtle and Michalopoulos, 1982). These and other studies provided abundant evidence for the existence of circulating hepatotrophic factors, and led to the studies that eventually concluded with the isolation of HGF and the identification of its structure and function. In the last decade, liver regeneration has been shown to proceed very rapidly, with specific changes in hepatocytes demonstrable immediately after partial hepatectomy. Hyperpolarization of the hepatic plasma membrane occurs within 10 min after partial hepatectomy (de Hemptinne et al., 1985), and an enhanced expression of multiple new mRNAs occurs within the first 30 min after partial hepatectomy (see Chapter 4) (Mohn et al., 1990). The cluster of specific genes expressed immediately after partial hepatectomy is referred to as "immediate early genes" and their nature and control mechanisms are currently being investigated. Intense glycogenolysis also occurs and depletes the liver of glycogen within an hour after partial hepatectomy. The clonogenicity of transplated hepatocytes is dependent on the time between when the recipient animals are partially hepatectomized and when the hepatocytes are injected, with maximum clonogenicity occurring approximately 1 hr after partial hepatectomy (Jirtle and Michalopoulos, 1982). All of these phenomena suggest that the response to a partial hepatectomy is rapid and that it defines a course of events that commits the hepatocytes to proliferate. In view of the above findings, any changes related to HGF following partial hepatectomy should be viewed in terms of whether they are sufficient and capable of explaining the above biological phenomena. As previously mentioned, HGF was originally isolated from both human and rat plasma.

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Thus, it definitely exists as an identifiable growth factor in the peripheral blood. Lindroos et al. (1991) demonstrated that between 1 and 2 hr after partial hepatectomy, the plasma HGF level rapidly rises approximately 15to 20-fold above that found in control animals; a 10-fold increase in the plasma HGF level is already seen within 30 min after the operation. These original studies have now been corroborated by several other groups (Kinoshita et al., 1991; Tomiya et al., 1992b). Similar changes are also seen when the liver is damaged by carbon tetrachloride (CC14). The rise in HGF is immediate and equally as sustained as after partial hepatectomy, but it is elevated for a longer period. Thus, in animals following either a partial hepatectomy or CCI 4 exposure, the rise in plasma HGF occurs rapidly and precedes the peak in hepatocyte DNA synthesis by approximately 20 to 24 hr (Lindross et al., 1991). Furthermore, in terms of a temporal correlation, the rise in HGF occurs during the time frame that is compatible with the induction in immediate early gene expression (see Chapter 4). The fact that the plasma HGF level significantly increases in the peripheral blood also makes HGF compatible with the early findings documenting the rapid emergence of blood borne hepatotrophic factors following a partial hepatectomy (Moolten and Bucher, 1967; Jirtle and Michalopoulos, 1982). The role of HGF as a potential initiator of the hepatic regenerative process was further strengthened by recent studies that showed HGF stimulates the expression of some of the immediate early genes in primary cultures of hepatocytes (Tewari et al., 1992). A characteristic marker of this process is the transcription factor known as liver regeneration factor (LRF), a transcription factor that functions similarly to I kappa B (IKB). Although L R F gene expression is stimulated in cultured hepatocytes by HGF (Weir et al., 1994), other factors such as EGF also enhance the expression of LRF. This suggests that this effect of HGF is not specific, but rather relates to the overall initiation of hepatocyte growth. The number of growth factors that are capable of initiating DNA synthesis in hepatocytes in chemically defined serum-free medium is rather limited. These factors include HGF, EGF, transforming growth factor cx (TGFcx), and aFGF (Michalopoulos, 1990). Of these growth factors, HGF is the only one whose plasma level rises significantly after partial hepatectomy. There is no strong evidence for changes in the plasma EGF concentration after partial hepatectomy, and the TGFcx level rises only during the late stages of regeneration (Tomiya and Fujiwara, 1993); there is no evidence for any change in the plasma level of aFGE It should be pointed out, however, that although the plasma EGF level does not rise following a partial hepatectomy, EGF is continually available to the liver through the portal circulation. EGF is produced by multiple sites in the gastrointestinal tract, including the Brunner's glands of the duodenum (Skov Olsen et al., 1985). Furthermore, there is indirect evidence which suggests that in addition to HGF, EGF may play a

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role in the early stages of the regenerative process. A characteristic finding subsequent to the interaction of most growth factors with their receptors is their downregulation. This has been shown to be the case with the EGF receptor after partial hepatectomy (Earp and O'Keefe, 1981), suggesting that EGF may also participate in triggering the phenomena seen during the earliest stages of the regenerative process. In addition to the rise of HGF in the plasma, norepinephrine, a comitogen for hepatocytes also increases substantially during liver regeneration (Cruise et al., 1987). Circulating norepinephrine is degraded primarily in liver. Thus, the rapid elevation of norepinephrine may reflect the release of catecholamines following the stress of partial hepatectomy as well as a decreased rate of degradation of circulating catecholamines by the reduced mass of hepatic tissue. Norepinephrine has a synergistic interaction with HGF in stimulating hepatocyte DNA synthesis (Lindroos et al., 1991). Therefore, the elevation of HGF plus the synergistic effect of norepinephrine may play an interactive role in stimulating hepatocytes to proliferate. Other growth factors also increase after partial hepatectomy, including TGF]31 (Jirtle and Michalopoulos, 1994). Despite the well-documented rise of HGF after partial hepatectomy, the reasons for this phenomenon are not clearly understood. An obvious hypothesis derives from the fact that the liver is the major site for degradation and processing of HGE Accordingly, a sharp decrease in liver mass would result in a decreased clearance of HGF, leading to a rise in the plasma HGF level. However, when plasma HGF clearance was compared between normal animals and those that had received a partial hepatectomy, it was found that partial hepatectomy only led to a 50% decrease in HGF clearance (Appasamy et al., 1993). This does not account for the observed 15- to 20fold increase in HGF plasma concentration during liver regeneration. It also does not explain the significant rise in the plasma level of TGF~I. Both findings are more compatible with a hypothesis that the rise in HGF occurs because of its release from preexisting storage sites. This hypothesis will be discussed subsequently in this chapter as it relates to the early proteolytic events following partial hepatectomy. Although HGF protein rises sharply in the plasma, HGF mRNA in the liver does not change until approximately 3 to 4 hr after partial hepatectomy (Zarnegar et al., 1991). At that time, the HGF mRNA level in the liver increases and remains elevated for approximately 36 hr after partial hepatectomy. Although the site of production of the HGF mRNA in regenerating liver has not been fully investigated, it is assumed to be the same cellular site as in the normal liver, namely the cells of Ito. The signals that lead nonparenchymal cells to synthesize HGF as well as TGFI31 after partial hepatectomy are not clear. Additionally, HGF mRNA expression is increased in the lungs and kidneys following a partial hepatectorny (Yanagita et al., 1992).

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This led to the hypothesis that a specific substance, termed "injurin" was produced shortly after partial hepatectomy and was responsible for the increase in the level of HGF mRNA in different tissues (Okazaki et al., 1994). Despite extensive studies, however, the existence of this molecule has still not been documented. Interestingly, recent studies have shown that IL-1 (Matsumoto et al., 1992) as well as IL-6 (Moghul et al., 1994) can stimulate production of HGF and its receptor in responding cell lines. IL-6 also increases after partial hepatectomy or during chronic liver failure (Matsunami et al., 1992). Therefore, it is possible that some of the observed systemic changes in the HGF mRNA expression after partial hepatectomy are mediated by IL-1 and/or IL-6. Of interest is that stimulation of HGF production by IL-6 is matched by the existence of several IL-6 responding elements in the regulatory domain of the H G F gene as well as in the regulatory domain of the c - m e t gene coding for the HGFr (Liu et al., 1994c). In fact, IL-6 has been shown to increase the expression of both H G F and c - m e t in cells in which either of these genes are normally expressed. Thus, IL-6 may be a major regulator for reparative growth responses directed by HGF subsequent to acute inflammation in tissues and organs. IL-6 and other interleukins are produced during inflammation by macrophages and cells in liver and other peripheral tissues. IL-6 induction of H G F and c - m e t expression may, in a coordinate fashion, control tissue repair and angiogenesis following tissue damage. The interaction between IL-6 and HGF makes HGF a growth factor of major importance in local tissue repair and raises further questions about the role of IL-6 as one of the early stimulators of the regenerative process. The importance of the increase in HGF mRNA expression, and the presumed production of new HGF following partial hepatectomy, in the sustenance or further enhancement of growth of hepatocytes is not clear. Hepatocytes are already in the G1 phase of the cell cycle by the time HGF mRNA expression reaches its peak; the same is true for the mRNA expression for TGF~ and aFGE It has been hypothesized that these growth factors, produced by hepatocytes or by Ito cells, may further promote the proliferation of hepatocytes. Supportive of this postulate is the finding that sustained growth of hepatocytes, and eventual appearance of neoplasia, is seen in the liver of TGFc~ transgenic mice (Lee et al., 1992). This has not been the case, however, in HGF transgenic mice (Shiota et al., 1994). It is possible that in addition to having effects on hepatocytes themselves, these growth factors may also direct the proliferation of the nonparenchymal cells which occurs 24 hr after the first cycle of hepatocyte DNA synthesis (Grisham, 1962). In addition, HGF has morphogenic effects on hepatocytes (see sections below on HGF and embryogenesis and liver disease). Sustained HGF production by elements in the liver may be important for further directing the late stage morphogenic effects that occur during liver regeneration (i.e.,

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the formation of acinar structures and mature hepatic plates) (Stamatoglou and Hughes, 1994). The transformation of hepatocytes into acinar structures and hepatic plates is an effect induced by HGF in hepatocyte cultures (Michalopoulos, 1992). Thus, the role of HGF in liver regeneration should be seen as an extended one to encompass aspects of hepatic morphogenesis that occur late in the regenerative process and mimic in many regards the changes seen during embryogenesis.

V. HGF and the Early Proteolytic Events Following Partial Hepatectomy If the rise in the plasma concentration of HGF and norepinephrine provides the main stimuli leading to liver regeneration, a derivative prediction is that the injection of HGF in sufficient amounts would initiate hepatocyte proliferation in the livers of normal rats. However, when HGF is administered in large concentrations to normal rats, either through the portal vein or systemically, stimulation of DNA synthesis is seen only in the hepatocytes surrounding the portal triads. In contrast, if HGF is injected into rats following special nutritional manipulations or surgical procedures (30% hepatectomy), it induces a relatively large number of hepatocytes to undergo DNA synthesis (Webber et al., 1994). The precise nature of these experimental "priming" events is not clear, since these manipulations by themselves do not stimulate DNA synthesis. These findings suggest, however, that most hepatocytes, especially those in midzonal and centralobular regions of the liver, are incapable of responding to HGF unless subjected to a prior "priming" modification. In another study (Liu et al., 1994b), it was shown that if minute amounts of collagenase (approximately 1/100 of the amount required for liver dissociation) are injected into the liver prior to the injection of HGF, the proliferative effect of HGF is dramatically enhanced and a large percentage of the quiescent hepatocytes undergo DNA synthesis. Furthermore, the cells recruited into DNA synthesis come from all areas of the lobule, including the hepatocytes present in the midzonal and central lobular regions. The enhanced recruitment of hepatocytes into DNA synthesis by HGF following collagenase treatment is of interest since other studies where hepatocytes isolated from the liver by collagenase treatment show enhanced expression of immediate early genes and behave as though they were already in the G 1 phase of the cell cycle (Kost and Michalopoulos, 1990). Taken together, these findings suggest that proteolytic events, presumably mimicked by collagenase, occur very shortly after partial hepatectomy and render hepatocytes responsive to the influx of HGF. If there is acute proteolysis following partial hepatectomy, it may also be

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responsible for the sharp rise in HGF, as well as other cellular matrix bound factors such as TGFJ31 and hyaluronic acid (HA) (Figure 2). As mentioned above, large amounts of HGF are bound in the liver matrix. These HGF binding sites range from the very low affinity (HSPG) to relatively higher affinity glycosulfopholipids. Proteolytic degradation of these sites may release a large mount of HGF into the circulation that when returned to the liver via the vasculature exerts a mitogenic effect. Currently, the evidence for proteolytic events early after partial hepatectomy is rather small. Enhanced expression of the urokinase receptor (uPAr) in the plasma membrane fraction

Figure 2 Outline of the proposed scheme of the early events triggering liver regeneration. The emergence of the urokinase receptor (uPAr) in the plasma membrane (discussed in the text) leads to activation of urokinase (uPA) which can further activate type IV collagenase and generate plasmin. Abundant literature has shown that plasmin is involved in the activation of matrix metalloproteinases. It is hypothesized that the activation of the matrix metalloproteinases leads to the degradation of the connective tissue matrix around hepatocytes resulting in the observed increases in the plasma levels of the biomatrix bound growth factors HGF and TGFIB. Hyaluronic acid (HA), a well-known matrix component, has also been found to be increased in the plasma soon after partial hepatectomy. This composite of proven facts and hypothesis is most compatible with the finding that collagenase treatment prior to HGF injection into the portal circulation leads to the stimulation of hepatocyte DNA synthesis comparable to that seen in liver regeneration.

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of liver occurs within 5 min after partial hepatectomy (Mars and Michalopoulos, 1994). The enzyme involved in HGF activation, uPA, also converts plasminogen into plasmin. The latter protease is known to activate a variety of tissue bound metalloproteinases (e.g., type IV collagenase, stromelysin, interstitial collagenase, etc.) (Rifkin, 1992; Duffy, 1992; Blasi, 1993; KeskiOja et al., 1993). uPA also directly activates type IV collagenase (Duffy, 1992; Kleiner and Stetler-Stevenson, 1993; Blasi, 1993; Keski-Oja et al., 1992). uPA has a catalytic effect in simple solutions, but its activity is dramatically enhanced after binding to the uPAr. Therefore, the enhanced presence of the uPAr within minutes after partial hepatectomy should increase uPA activity. Given recent evidence about the rapid cascade of events in plasma membrane surface proteolysis (Rifkin, 1992; Duffy, 1992; Blasi, 1993), the elevated presence of the uPAr in hepatic plasma membranes has the potential of leading to a quick cascade of proteolytic events mediated by matrix metalloproteinases activated by uPA. That could further lead to matrix degradation around the hepatocytes, causing the release of matrix bound growth factors such as TGF~31 and HGF (Figure 2). Subsequent to the release of TGF~31 from the matrix, it would be inactivated by cx2-macroglobulin (O'Connor-McCourt and Wakefield, 1987) thereby blocking its mitoinhibitory effects on hepatocytes. In contrast, matrix bound, inactive, single chain HGF in the presence of enhanced uPA activity would be converted to a heterodimeric activated molecule that could stimulate hepatocyte proliferation. This hypothesis would also explain the paradox of the unique effect of plasma HGF on hepatocyte proliferation. Hepatocytes are unique, among all the potential epithelial targets of HGF since they are stimulated into mitogenesis following a partial hepatectomy. HGF in the plasma should theoretically be capable of being mitogenic to all the potential cellular targets. The restricted effect of HGF on hepatocytes could be explained if the biomatrix surrounding the epithelial cells needs to first be degraded so that HGF and other growth factors have a mitogenic effect in tissues. A competition between matrix binding and signaling receptor (i.e., HGFr) binding may also be an issue that relates peculiarly to HGE In contrast to other growth factors, in addition to the very low affinity and high capacity binding sites, there are also relatively high affinity, nonsignaling receptor binding sites for HGF in the biomatrix which may offer a competition between binding of HGF to HGFr and making HGF not available to function in a mitogenic manner. This competition would also be eliminated by early proteolytic events leading to the destruction of such relatively high affinity, nonsignaling HGF matrix binding sites. Although this hypothesis is attractive and may explain some of the peculiar events related to the rise of HGF in the plasma, and the unique targeting

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of the HGF mitogenic effect to the liver, additional investigations are still required. These would include defining the proteolytic targets as well as the enzymes involved in carrying out the actual proteolysis.

VI. H G F Localization A. In Liver Embryogenesis Given the strong mitogenic, motogenic, and morphogenic effects of HGF in several tissues as well as the high concentration of HGF in the placenta, it is likely that HGF plays a major role in embryogenesis. In recent studies involving HGF implanted into the embryo, abnormal release of HGF led to abnormalities in embryonic polarity with the formation of multiple notochords (Stern and Ireland, 1993). Specifically in relationship to the liver, HGF as well as its receptor are intensely expressed during hepatic embryogenesis. Of the multiple tissues in which HGF and the HGFr are expressed, liver is by far the one with the most expression (Sonnenberg et al., 1993; Katyal and Michalopoulos, 1995). Several authors have pointed out that embryogenesis of the liver proceeds through characteristic stages (Stamatoglou and Hughes, 1994). During most of embryogenesis, hepatocytes do not possess the adult canalicular sinusoidal surface orientation. Toward the end of embryogenesis (1 to 2 days prior to birth) and during the first 2 to 3 days of the postnatal period, the apolar hepatocytes become organized into acinar structures. These acinar/ductal structures then eventually become oriented into typical adult looking hepatic plates that are composed of polar hepatocytes with sinusoidal and canalicular surfaces. The high concentrations of HGF and its receptor in the embryonic liver and during the early stages of the postnatal period suggest that HGF may be involved in these morphogenic processes. This postulate is further strengthened by the studies showing that HGF can affect the morphological structures formed by hepatocytes in primary culture. When hepatocytes are cultured in type I collagen gels and exposed to HGF in normal media, hepatic plates form after an intense period of motogenesis and mitogenesis. If the HGF concentration is increased in media that allows for long-term hepatocyte proliferation, acinar structures composed of ductular hepatocytes are formed (Michalopoulos et al., 1993; Bloc and Michalopoulos, 1995). In standard monolayer cultures with the same medium, HGF induces hepatocytes to transform into hepatoblasts. These studies suggest that HGF is involved not only in hepatocyte proliferation in embryonic liver, but also in the overall transformation of an embryonic liver composed of apoiar hepatocytes into an organ of hepatic plates. However, to further investigate the

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role of HGF in liver embryogenesis studies need to be performed with HGF transgenic mice and HGF "knockout" mice. B. In Liver Disease

Acute liver disease (viral hepatitis) or chronic liver disease (chronic active hepatitis, cirrhosis) leads to a rise in the plasma level of HGF (Tsubouchi et al., 1989). This correlates with the severity of the disease, and usually the level of HGF is inversely correlated with the prognosis of the disease; the patients who fare better tend to have lower concentrations of HGF (Tomiya et al., 1992a). Even more striking is the elevation of HGF in patients with fulminant hepatic failure. This syndrome is precipitated by a variety of offending insults (Wiesner, 1991; Riegler and Lake, 1993; Sherlock, 1993). It can be seen in patients infected with hepatitis A, B, or C viruses, patients exposed to toxins (acetaminophen, amanitin poisoning, other chemical poisons, etc.), patients with autoimmune liver disease, etc. Regardless of the etiology, the injury to the liver proceeds with a rapid destruction of a very large number of hepatocytes. If the process is unfettered, it will lead to eventual destruction of all hepatocytes in the liver, resulting in a condition described in standard pathology textbooks as "acute yellow atrophy" of the liver. The liver volume shrinks, liver weight decreases, and hepatic function indicators plummet. This condition occurs in approximately I to 2% of patients with a variety of acute hepatic inflammatory conditions. A percentage of the patients affected with fulminant hepatitis (20 to 30%) spontaneously recover. Histological examination of the liver in patients with this condition is now often performed because many of these patients are treated with orthotopic liver transplantation (see Chapter 13). The resected livers therefore have allowed for histological studies of the processes leading to fulminant hepatitis. Initially, cell proliferation is seen both in hepatocytes and bile ductal epithelial (Wolf and Michalopoulos, 1992). At late stages of the disease, hepatocytes cease to proliferate. Concomitantly, there is proliferation of hepatocytes arranged in acinar structures as well as exuberant proliferation of frank bile duct epithelial cells. These acinar structures composed of hepatocytes are the basis for the term "ductular hepatocytes" which is reserved for hepatocytes participating in the formation of these acinar structures and those having markers of mixed differentiation between bile ducts and hepatocytes (Sirica et al., 1994). In the context of the above description of the role of HGF in embryology, it is perhaps reasonable to speculate that the observed acinar transformation of hepatocytes is recapitulating the embryonic events occurring in the liver. Given the propensity of HGF to induce the same changes in hepatocytes in primary culture, it is reasonable to

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speculate that the very high levels of HGF present in fulminant hepatitis are responsible for the observed transformation of hepatocytes into acinar structures. The effect of HGF in fulminant hepatitis, however, may be even more insidious. In one of its early incarnations, HGF was isolated as "sarcoma cytotoxic factor" (Higasho and Shima, 1993). Sarcoma cytotoxic factor was subsequently found to be identical to HGF, and it has now been shown that high concentrations of HGF have a mitoinhibitory effect on a variety of normal and neoplastic tissues (Shiota et al., 1992). At high concentrations, HGF also loses its mitogenic effect on hepatocytes in primary culture (Michalopoulos, 1992). The high concentrations of HGF present during fulminant hepatitis could be responsible for the eventual cessation in hepatocyte proliferation, the transformation of hepatocytes into acinar structures, and the eventual decline in hepatocyte-supported liver function. This is an avenue worth exploring because it may allow a therapeutic approach for fulminant hepatitis aimed at restricting the very high concentrations of HGF found in this disease state. The very high levels of HGF in the plasma of patients with fulminant hepatic failure appear to render meaningless the pursuit of the notion that this disease can be treated by injecting high concentrations of HGF to stimulate liver regeneration. Hepatocyte proliferation throughout most of the early and middle stages of this disease is even higher than that which occurs following a partial hepatectomy. Therefore, it is doubtful that further stimulation of the process can be achieved by injecting a substance that is already present in high concentrations in the plasma and available to the hepatocytes. It should be pointed out, however, that there have been no detailed studies of the nature of the circulating HGE If the circulating HGF is in the inactive (single chain) form, then activation of HGF may be a problem. This needs to be further studied. C. In Liver Carcinogenesis The role of HGF in liver regeneration, embryogenesis, and liver disease is well documented; however, its role in hepatic carcinogenesis is not as clear. The HGFr is expressed in many hepatocellular carcinomas, in some instances at increased levels and in other instances at decreased levels. Hepatoma cell lines that express the HGFr respond to HGF slightly, intensely or not at all, for reasons which at this time remain obscure. In contrast to TGFcx, which is expressed by the majority of hepatocellular carcinomas, no hepatocellular carcinoma has yet been described that expresses HGE Furthermore, in transgenic mice where HGF-expression was targeted to the liver under the direction of the albumin promoter, there was no observed neoplasms (Shiota et al., 1994). In fact when HGF cDNA is transfected into

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the human hepatoma cell line, HepG2, the cells expressing the HGF cDNA are less tumorigenic than the normal HepG2 cells (Shiota et al., 1992). Growth inhibitory effects of HGF have also been shown in several other tumor cell lines from a wide range of cell origins (Tajima et al., 1991). It should be pointed out, however, that there is no example of a specific neoplasia in which all of the tumors are either entirely negatively or positively affected by H G E Typically, as for most types of neoplasms, some neoplasms are inhibited and others are stimulated. Nevertheless, there is no doubt that HGF and its receptor have a prominent role in neoplasia in general. The motogenic effects of HGF make it likely that it functions in liver metastases. In fact, the very high concentrations of HGF in the liver matrix would make liver an appealing site for neoplasms whose growth is stimulated by HGE Thus, the HGF bound to the liver biomatrix may enhance the metastatic properties of neoplasms which respond positively to HGE These neoplasms may include squamous cell carcinomas, melanomas, breast cancer, uroepithelial neoplasms, and prostatic cancers. In recent studies, we found that when HGF is infused into livers containing a large number of small primary tumors induced by diethylnitrosamine, the proliferation of most of the tumors was reduced by HGF; however, a small number of tumors were found that were totally resistant to this mitoinhibitory effect (Liu and Michalopoulos, 1993). This suggests that HGF may be useful as a component of therapeutic regimens for hepatocellular carcinomas. In view of competitive molecular analogues of HGF naturally produced by mRNA splicing or artificially produced by site directed mutagenesis (Lokker et al., 1992), it may also be possible to affect the growth of HGF-dependent neoplasms by using HGF competitive molecular analogues.

VII. Summary Although HGF is now a recognized multifunctional cytokine with target effects in different organs and tissues, it has some unique aspects in its relationship toward the liver. The paracrine effects of HGF probably regulate local tissue responses in a variety of organs, but the endocrine effects of HGF appear to be limited primarily to the liver. No documented changes in HGF plasma levels are seen in any other conditions comparable to those seen during liver regeneration, fulminant hepatic failure, or chronic liver disease. The very high expression of HGF and its receptor in embryonic liver, totally out of proportion to that seen in the rest of the embryonic organs, however, may obscure the important role that HGF also plays in the development of the central nervous system, another site where HGF and its receptor are highly expressed (Jung et al., 1994). The endocrine effect of

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HGF and its involvement in events related to liver disease make it likely that in contrast to other growth factors, HGF and its molecular competitive analogues may be useful in therapy. Clearly, HGF is involved in the earliest stages of liver regeneration. Undoubtedly, the regenerative process is a very complex one, but the evidence so far indicates that HGF is the best candidate for the initial trigger of hepatocyte proliferation following partial hepatectomy; it is capable of explaining both the phenomena of the blood borne hepatotrophic factors as well as immediate early gene expression. As we learn more about the other complex parameters that are undoubtedly involved in liver growth regulation, the role of HGF is likely to remain an eminent one and will provide substantial justification for the adoption of a hepatic derived name for this otherwise promiscuous cytokine. References Appasamy, R., Tanabe, M., Murase, N., Zarnegar, R., Venkataramanan, R., Van Thiel, D. H., and Michalopoulos, G. K. (1993). Hepatocyte growth factor, blood clearance, organ uptake, and biliary excretion in normal and partially hepatectomized rats. Lab. Invest. 68, 270-276. Blasi, E (1993). Urokinase and urokinase receptor: A paracrine/autocrine system regulating cell migration and invasivenesss. BioEssays 15, 105-111. Bloc, G., and Michalopoulos, G. K. (1995). Unpublished results. Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M. L., Kmiecick, T. E., Vande Woude, G. E, and Aaronson, S. A. (1991). Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene. Science 251,802-804. Bussolino, E, Di Renzo, M. E, Ziche, M., Bocchietto, E., Olivero, M., Naldini, L., Gaudino, G., Tamagnone, L., Coffer, A., and Comoglio, P. M. (1992). Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J. Cell Biol. 119, 629-641. Chan, A. M., Rubin, J. S., Bottaro, D. P., Hirschfield, D. W., Chedid, M., and Aaronson S. A. (1991). Identification of a competitive HGF antagonist encoded by an alternative transcript. Science 254, 1382-1385. Cruise, J. L., Knechtle, S. J., Bollinger, R. R., Kuhn, C., and Michalopoulos, G. K. (1987). Adrenergic effects and liver regeneration. Hepatology (Baltimore) 7, 1189-1194. de Hemptinne, B., Lorge, E, Kestens, P. J., and Lambotte, L. (1985). Hepatocellular hyperpolarizing factors and regeneration after partial hepatectomy in the rat. Acta Gastroenterol. Belg. 48, 424-431. Duffy, M. J. (1992). The role of proteolytic enzymes in cancer invasion and metastasis. Clin. Exp. Metastasis 10, 145-155. Earp, H. S., and O'Keefe, E. J. (1981). Epidermal growth factor receptor number decreases during rat liver regeneration. J. Clin. Invest. 67, 1580-1583. Fisher, B., Szuch, P., Levine, M., and Fisher, E. R. (1971). A portal blood factor as the humor agent in liver regeneration. Science 171,575-577. Gaudino, G., Follenzi, A., Naldini, L., Collesi, C., Santoro, M., Gallo, K. A., Godowski, P. J., and Comoglio, P. M. (1994). RON is a heterodimeric tyrosine kinase receptor activated by the HGF homologue MSP. EMBO J. 13, 3524-3532.

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Hepatocyte Growth Factor (HGF) and Its Receptor (Met)

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Gherardi, E., Sharpe, M., Lane, K., Sirulnik, A., and Stoker, M. (1993). Hepatocyte growth factor/scatter factor (HGF/SF), the c-met receptor and the behaviour of epithelial cells. Symp. Soc. Exp. Biol. 47, 163-181. Giordano, S., Ponzetto, C., Di Renzo, M. E, Cooper, C. S., and Comoglio, P. M. (1989). Tyrosine kinase receptor indistinguishable from the c-met protein. Nature (London) 339, 155-156. Grisham, J. W. (1962). A morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating liver; autoradiography with thymidine-H3. Cancer Res. 22, 842849. Higashio, K., and Shima, N. (1993). Tumor cytotoxic activity of HGF-SE EXS 65, 351-368. Jirtle, R. L., and Michalopoulos, G. K. (1994). unpublished observations. Jirtle, R. L., and Michalopoulos, G. (1982). Effects of partial hepatectomy on transplanted hepatocytes. Cancer Res. 42, 3000-3004. Jung, W., Castren, E., Odenthal, M., Vande Woude, G. E, Ishii, T., Dienes, H. P., and Lindholm, D., and Schirmacher, P. (1994). Expression and functional interaction of hepatocyte growth factor-scatter factor and its receptor c-met in mammalian brain. J. Cell Biol. 126, 485-494. Katyal, S., and Michalopoulos, G. K. (1995). unpublished results. Keski-Oja, J., Lohi, J., Tuuttila, A., Tryggvason, K., and Vartio, T. (1992). Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator. Exp. Cell Res. 202, 471-476. Kinoshita, T., Hirao, S., Matsumoto, K., and Nakamura, T. (1991). Possible endocrine control by hepatocyte growth factor of liver regeneration after partial hepatectomy. Biochem. Biophys. Res. Commun. 177, 330-335. Kleiner, D. E., Jr., and Stetler-Stevenson, W. G. (1993). Structural biochemistry and activation of matrix metalloproteases. Curr. Opin. Cell Biol. 5, 891-897. Kobayashi, T., Honke, K., Miyazaki, T., Matsumoto, K., Nakamura, T., Ishizuka, I., and Makita, A. (1994). Hepatocyte growth factor specifically binds to sulfoglycolipids. J. Biol. Chem. 269, 9817-9821. Kost, D. P., and Michalopoulos, G. K. (1990).Effect of epidermal growth factor on the expression of protooncogenes c-myc and c-Ha-ras in short-term primary hepatocyte culture. J. Cell. Physiol. 144, 122-127. Lee, G. H., Merlino, G., and Fausto, N. (1992). Development of liver tumors in transforming growth factor alpha transgenic mice. Cancer Res. 52, 5162-5170. Leong, G. E, Grisham, J. W., Hole, B. V., and Albright, M. L. (1964). Effect of partial hepatectomy on DNA synthesis and mitosis in heterotopic partial autografts of rat liver. Cancer Res. 24, 1496-1501. Lindroos, P. M., Zarnegar, R., and Michalopoulos, G. K. (1991). Hepatocyte growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration. Hepatology (Baltimore 13, 743-750. Liu, M. L., and Michalopoulos, G. K. (1993). unpublished results. Liu, M. L., Mars, W. M., Zarnegar, R., and Michalopoulos, G. K. (1994a). Uptake and distribution of hepatocyte growth factor in normal and regenerating adult rat liver. Am. J. Pathol. 144, 129-140. Liu, M. L., Mars, W. M., Zarnegar, R., and Michalopoulos, G. K. (1994b). Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver. Hepatology (Baltimore) 19, 1521-1527. Liu, Y., Michalopoulos, G. K., and Zarnegar, R. (1994c). Structural and functional characterization of the mouse hepatocyte growth factor gene promoter. J. Biol. Chem. 269, 41524160.

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Lokker, N. A., Mark, M. R., Luis, E. A., Bennett, G. L., Robbins, K. A., Baker, J. B., and Godowski, P. J. (1992). Structure-function analysis of hepatocyte growth factor: Identification of variants that lack mitogenic activity yet retain high affinity receptor binding. EMBO J. 11, 2503-2510. Mars, W. M., and Michalopoulos, G. K. (1994). unpublished results. Mars, W. M., Zarnegar, R., and Michalopoulos, G. K. (1993). Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA. Am. J. Pathol. 143, 949-958. Masumoto, A., and Yamamoto, N. (1991). Sequestration of a hepatocyte growth factor in extracellular matrix in normal adult rat liver. Biochem. Biophys. Res. Commun. 174, 90-95. Matsumoto, K., and Nakamura, T. (1992). Hepatocyte growth factor: Molecular structure, roles in liver regeneration, and other biological functions. Crit. Rev. Oncog. 3, 27-54. Matsumoto, K., Okazaki, H., and Nakamura, T. (1992). Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts. Biochem. Biophys. Res. Commun. 188, 235-243. Matsunami, H., Kawasaki, S., Ishizone, S., Hashikura, Y., Ikegami, T., Makuuchi, M., Kawarasaki, H., Iwanaka, T., Nose, A., and Takemura, M. (1992). Serial changes of h-HGF and IL-6 in living-related donor liver transplantation with special reference to their relationship to intraoperative portal blood flow. Transplant. Proc. 24, 1971-1972. Michalopoulos, G. (1992). unpublished results. Michalopoulos, G. K. (1990). Liver regeneration: Molecular mechanisms of growth control. FASEBJ 4, 176-187. Michalopoulos, G. K., and Zarnegar, R. (1992). Hepatocyte growth factor (editorial). Hepatology (Baltimore) 15, 149-155. Michalopoulos, G., Cianciulli, H. D., Novotny, A. R., Kligerman, A. D., Strom, S. C., and Jirtle, R. L. (1982). Liver regeneration studies with rat hepatocytes in primary culture. Cancer Res. 42, 4673-4682. Michalopoulos, G., Houck, K. A., Dolan, M. L., and Luetteke, N. C. (1984). Control of hepatocyte replication by two serum factors. Cancer Res. 44, 4414-4419. Michalopoulos, G. K., Bowen, W., Nussler, A. K., Becich, M. J., and Howard, T. A. (1993). Comparative analysis of mitogenic and morphogenic effects of HGF and EGF on rat and human hepatocytes maintained in collagen gels. J. Cell. Physiol. 156, 443-452. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., Arakaki, N., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Gohoda, E., Daikuhara, Y., and Kitamura, N. (1989). Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem. Biophys. Res. Commun. 163, 967-973. Miyazawa, K., Shimomura, T., Kitamura, A., Kondo, J., Morimoto, Y., and Kitamura, N. (1993). Molecular cloning and sequence analysis of the cDNA for a human serine protease responsible for activation of hepatocyte growth factor. Structural similarity of the protease precursor to blood coagulation factor XII. J. Biol. Chem. 268, 10024-10028. Moghul, A., Lin, L., Beedle, A., Kanbour-Shakir, A., DeFrances, M. C., Liu, Y., and Zarnegar, R. (1994). Modulation of c-met proto-oncogene (HGF receptor) mRNA abundance by cytokines and hormones: Evidence for rapid decay of the 8 kb c-met transcript. Oncogene 9, 2045-2052. Mohn, K. L., Laz, T. M., Melby, A. E., and Taub, R. (1990). Immediate-early gene expression differs between regenerating liver, insulin-stimulated H-35 cells, and mitogen-stimulated Balb/c 3T3 cells. Liver-specific induction patterns of gene 33, phosphoenolpyruvate carboxykinase, and the jun, fos, and egr families. J. Biol. Chem. 265, 2914-2921. Moolten, E L., and Bucher, N. L. R. (1967). Regeneration of rat liver: Transfer of humoral agents by cross circulation. Science 158, 272-274. Nakamura, T., Nawa, K., and Ichihara, A. (1984). Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats. Biochem. Biophys. Res. Commun. 122, 1450-1459.

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Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K., and Shimizu, S. (1989). Molecular cloning and expression of human hepatocyte growth factor. Nature (London) 342, 440-443. Naldini, L., Vigna, E., Narsimhan, R. P., Gaudino, G., Zarnegar, R., Michalopoulos, G. K., and Comoglio, P. M. (1991a). Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-met Oncogene 6, 501-504. Naldini, L., Weidner, K. M., Vigna, E., Gaudino, G., Bardelli, A., Ponzetto, C., Narsimhan, R. P., Hartmann, G., Zarnegar, R., Michalopoulos, G. K., Birchmeier, W., and Comoglio, P. M. (1991b). Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J. 10, 2867-2878. Noji, S., Tashiro, K., Koyama, E., Nohno, T., Ohyama, K., Taniguchi, S., and Nakamura, T. (1990). Expression of hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat livers, as revealed by in situ hybridization. Biochem. Biophys. Res. Commun. 173, 42-47. O'Connor-McCourt, M. D., and Wakefield, L. M. (1987). Latent transforming growth factor-J3 in serum. A specific complex with ot2-macroglobulin. J. Biol. Chem. 262, 14090-14099. Okazaki, H., Matsumoto, K., and Nakamura, T. (1994). Partial purification and characterization of "injurin-like" factor which stimulates production of hepatocyte growth factor. Biochim. Biophys. Acta 1220, 291-298. Pepper, M. S., Matsumoto, K., Nakamura, T., Orci, L., and Montesano, R. (1992). Hepatocyte growth factor increases urokinae-type plasminogen activator (u-PA) and u-PA receptor expression in Madin-Darby canine kidney epithelial cells. J. Biol. Chem. 267, 2049320496. Ponzetto, C., Bardelli, A., Zhen, Z., Maina, E, dalla Zonca, P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M. (1994). A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell (Cambridge, Mass.) 77, 261-271. Rabes, H. M., Wirsching, R., Tuczek, H. V., and Iseler, G. (1976). Analysis of cell cycle compartments of hepatocytes after partial hepatecomy. Cell Tissue Kinet. 6, 517-532. Riegler, J. L., and Lake, J. R. (1993). Fulminant hepatic failure. Med. Clin. North Am. 77, 1057-1083. Rifkin, D. B. (1992). Plasminogen activator expression and matrix degradation. Matrix Suppl. 1, 20-22. Rosen, E. M., Grant, D. S., Kleinman, H. K., Goldberg, I. D., Bhargava, M. M., Nickoloff, B. J., Kinsella, J. L., and Polverini, P. (1993a). Scatter factor (Hepatocyte growth factor) is a potent angiogenesis factor in vivo. Symp. Soc. Exp. Biol. 47, 227-234. Rosen, E. M., Zitnik, R. J., Bhargava, M. M., Wines, J., and Goldberg, I. D. (1993b). The interaction of HGF-SF with other cytokines in tumor invasion and angiogenesis. EXS 65, 301-310. St. Hilaire, R. J., Hradek, G. T., and Jones, A. L. (1983). Hepatic sequestration and biliary secretion of epidermal growth factor: Evidence for a high-capacity updtake system. Proc. Natl. Acad. Sci. U.S.A. 80, 3797-3801. Schirmacher, P. (1994). Expression and functional interaction of hepatocyte growth factorscatter and its receptor c-met in mammalian brain. J. Cell Biol. 126, 485-494. Schirmacher, P., Geerts, A., Pietrangelo, A., Dienes, H. P., and Rogler, C. E. (1992). Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells. Hepatology (Baltimore) 15, 5-11. Sherlock, S. (1993). Fulminant hepatic failure. Adv. Intern. Med. 38, 245-267. Shiota, G., Rhoads, D. B., Wang, T. C., Nakamura, T., and Schmidt, E. V. (1992). Hepatocyte growth factor inhibits growth of hepatocetlular carcinoma cells. Proc. Natl. Acad. Sci. U.S.A. 89, 373-377. Shiota, G., Wang, T. C., Nakamura, T., and Schmidt, E. (1994). Hepatocyte growth factor in

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transgenic mice: Effects on hepatocyte growth, liver regeneration and gene expression. Hepatology (Baltimore) 19, 962-972. Sirica, A. E., Gainey, T. W., and Mumaw, V. R. (1994). Ductular hepatocytes: Evidence for a bile ductular cell origin in furan-treated rats. Am. J. Pathol. 145, 375-383. Skov Olsen, P., Poulsen, S. S., and Kirkegaard, P. (1985). Adrenergic effects of secretion of epidermal growth factor from Brunner's glands. Gut 26, 920-927. Sonnenberg, E., Meyer, D., Weidner, K. M., and Birchmeier, C. (1993). Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelial during mouse development. J. Cell Biol. 123,223-235. Stamatoglou, S. C., and Hughes, R. C. (1994). Cell adhesion molecules in liver function and pattern formation. FASEBJ. 8, 420-427. Stern, C. D., and Ireland, G. W. (1993). HGF-SF: A neural inducing molecule in vertebrate embryos? EXS 65,369-380. Stoker, M., Gherardi, E., Perryman, M., and Gray, J. (1987). Scatter factor is a fibroblastderived modulator of epithelial cell mobility. Nature (London) 327, 239-242. Tajima, H., Matsumoto, K., and Nakamura, T. (1991). Hepatocyte growth factor has potent anti-proliferative activity in various tumor cell lines. FEBS Lett. 291, 229-232. Tewari, M., Dobrzanski, P., Mohn, K. L., Cressman, D. E., Hsu, J. C., Bravo, R., and Taub, R. (1992). Rapid induction in regenerating liver of RL/IF-1 (an I kappa B that inhibits NFkappa B, RelB-p50, and c-Rel-p50) and PHF, a novel kappa B site-binding complex. Mol. Cell. Biol. 12, 2898-2908. Tomiya, T., and Fujiwara, K. (1993). Serum levels of transforming growth factor-alpha in patients after partial hepatectomy as determined with an enzyme-linked immunosorbent assay. Hepatology (Baltimore) 18, 304-308. Tomiya, T., Nagoshi, S., and Fujiwara, K. (1992a). Significance of serum human hepatocyte growth factor levels in patients with hepatic failure. Hepatology (Baltimore) 15, 1-4. Tomiya, T., Tani, M., Yamada, S., Hayashi, S., Umeda, N., and Fujiwara, K. (1992b). Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients. Gastroenterology 103, 1621-1624. Tsubouchi, H., Horono, S., Gohda, E., Nakayama, H., Takahashi, K., Sakiyama, O., Miyazaki, H., Sugihara, J., Eiichi, T., Muto, Y., Dakuhara, Y., and Hashimoto, S. (1989). Clinical significance of human hepatocyte growth factor in blood from patients with fulminant hepatic failure. Hepatology (Baltimore) 9, 875-881. Webber, E. M., Godowski, P. J., and Fausto, N. (1994). In vivo response of hepatocytes to growth factors requires an initial priming stimulus. Hepatology (Baltimore) 19, 489-497. Weidner, K. M., Hartmann, G., Sachs, M., and Birchmeier, W. (1993). Properties and functions of scatter factor/hepatocyte growth factor and its receptor c-Met. Am. J. Respir. Cell Mol. Biol. 8, 229-237. Weir, E., Chen, Q., DeFrances, M. C., Bell, A., Taub, R., and Zarnegar, R. (1994). Rapid induction of nRNAs for liver regeneration factor and insulin-like growth factor binding protein-1 in primary cultures of rat hepatocytes by hepatocyte growth factor and epidermal growth factor. Hepatology (Baltimore) 20, 955-960. Wiesner, R. H. (1991). Acute fulminant hepatic failure. Transplant. Proc. 23, 1892-1894. Wolf, H. K., and Michalopoulos, G. K. (1992). Hepatocyte regeneration in acute fulminant and non-fulminant hepatitis: A study of proliferating cell nuclear antigen expression. Hepatology (Baltimore) 15, 707-713. Wolf, H. K., Zarnegar, R., and Michalopoulos, G. K. (1991). Localizatoin of hepatocyte growth factor in human and rat tissues: An immunohistochemical study. Hepatology (Baltimore) 14, 488-494. Yanagita, K., Nagaike, M., Ishibashi, H., Niho, Y., Matsumoto, K., and Nakamura, T. (1992). Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs. Biochem. Biophys. Res. Commun. 182, 802-809.

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Zarnegar, R., Muga, S., Enghild, J., and Michalopoulos, G. (1989). NH2-terminal amino acid sequence of rabbit hepatopoietin A, a heparin-binding polypeptide growth factor for hepatocytes. Biochem. Biophys. Res. Commun. 163, 1370-1376. Zarnegar, R., DeFrances, M. C., Oliver, L., and Michalopoulos, G. (1990). Identification and partial characterization of receptor binding sites for HGF on rat hepatocytes. Biochem. Biophys. Res. Commun. 173, 1179-1185. Zarnegar, R., DeFrances, M. C., Kost, D. P., Lindroos, P., Mich~lopoulos, G. K. (1991). Expression of hepatocyte growth factor mRNA in regenerating rat ~rer after partial hepatectomy. Biochem. Biophys. Res. Commun. 177, 559-565. Zhen, Z., Giordano, S., Longati, P., Medico, E., Campiglio, M., and Comoglio, P. M. (1994). Structural and functional domains critical for constitutive activation of the HGF receptor (Met). Oncogene 9, 1691-1697. Zioncheck, T. E, Richardson, L., DeGuzman, G. G., Modi, N. B., Hansen, S. E., and Godowski, P. J. (1994). The pharmacokinetics, tissue localization, and metabolic processing of recombinant human hepatocyte growth factor after intravenous administration in rats. Endocrinology (Baltimore) 134, 1879-1887.

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3 Structure and Functions of the HGF Receptor (c-Met) Paolo M. Comoglio Elisa Vigna Institute for Cancer Research and Treatment, University of Torino School of Medicine, 10126 Torino, Italy

I. Hepatocyte Growth Factor and Scatter Factor Hepatocyte growth factor (HGF) and scatter factor (SF) are molecules secreted by mesenchymal cells acting in a paracrine way on epithelial cells. The factors were isolated independently because they show different, and apparently unrelated, biological effects. HGF was purified from several sources, such as rat platelets and human and rabbit serum, using a biological assay based on hepatocyte proliferation in vitro (Nakamura et al., 1986; Godha et al., 1988; Zarnegar and Michalopoulos, 1989). SF was isolated from cell culture supernatants, given its ability to induce dissociation of the epithelial monolayers--the so-called "scatter" effect (Stoker et al., 1987; Gherardi et al., 1989; Weidner et al., 1990). Subsequently, biochemical purification and cDNA cloning demonstrated that the two molecules are indistinguishable, and have the same biological activities, the same biochemical and immunological properties, and ultimately the same sequence (Weidner et al., 1991; Naldini et al., 1991c). H G F is a disulfideqinked heterodimer made of a heavy subunit (~) of 60 kDa and a light subunit (13) of 32 to 34 kDa. The two chains are glycosylated and differences in glycosylation generate different forms of the chain. HGF is homologous to serine proteases involved in the blood clotting cascade (e.g., 38% homology with plasminogen). The e~ chain contains a "hairpin" loop and four "kringle" modules. The ~ chain has the same Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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overall structure of the serine proteases, but critical amino acids in the catalytic domain are mutated (Figure 1) (Miyazawa et al., 1989; Nakamura et al., 1989; Weidner, et al., 1990, 1991). No evidence for proteolytic activity of HGF has ever been reported. Interestingly, two independent reports have shown the existence of another protein related to HGF, called "HGFlike" (Han et al., 1991) or macrophage stimulating protein (MSP) (Skeel et al. 1991; Yoshimura et al., 1993), that shares with HGF the overall fourkringle/protease-like structure. HGF is synthesized and secreted as a single chain precursor devoid of biological activity (Naldini et al., 1992; Hartmann et al., 1992; Lokker et al., 1992; Naka et al., 1992; Mizuno et al., 1992). A serine protease in the extracellular environment cleaves the precursor between Arg 494 and Va149s to generate the two-chain mature form. Urokinase-type plasminogen activator (uPA) has been shown to correctly process HGF in vitro (Naldini et al., 1992). A serum-derived serine protease, homologous to coagulation factor XII, can also cleave the HGF precursor (Miyazawa et al., 1993). This enzyme must first, however, be proteolytically activated by thrombin (Shimomura et al., 1993). It is thus possible to postulate two different paths for the activation of HGF, one mediated by uPa, normally present in plasma and tissues, the other activated only during the coagulation process. Recently, a rather broad spectrum of target cells and biological activities have been assigned to HGF. It induces mitogenesis in different epithelial cell

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K1

K2

(66%)

(49%)

K3

K4

I

HGF

(45%)

(50%)

I

D

-S

~ R

V

I

Q

! I

Q

I

MSP

Y

Figure 1 Schematic structure of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP). HPL designates the hairpin loop and K1 to K4 are the four kringle domains of HGF and MSP. The mature oLand 13chains originate from proteolytic cleavage of a single-chain precursor, at a specific site between Arg 494 (R) and Val ags (V). The estimated position of the disulfide bond (S---S) is indicated. The HGF 13 chain is devoid of proteolytic activity due to substitution of critical residues (Q, D, Y) in the catalytic domain; similarly, Q, Q, Y substitutions have occurred in MSP. Numbers in parentheses indicate the overall and the partial homology of the various domains.

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lines (Kan et al., 1991; Rubin et al., 1991) and in primary culture of hepatocytes (Zarnegar and Michalopoulos, 1989), melanocytes (Matsumoto et al., 1991a; Halaban et al., 1992), and keratinocytes (Matsumoto et al., 1991b). HGF also induces growth and wound healing in endothelial cells and behaves as a potent angiogenic factor in vivo (Bussolino et al., 1992; Grant et al., 1993). HGF stimulates invasion of the extracellular matrix by epithelialderived cancer cells (Weidner et al., 1990), and may have a paradoxical growth-inhibitory effect (Higashio et al., 1990; Tajima et al., 1991; Shiota et al., 1992a). A morphogenic effect has also been demonstrated. HGF induces tubule formation by kidney epithelial cells in vitro (Montesano et al., 1991; Santos and Nigam, 1993), and is involved in development of the spinal cord during embryogenesis (Stern et al., 1990). Recently, we showed that HGF also activates and promotes the phenotypic transition between monocytes and macrophages (Galimi et al., 1993), and that it stimulates the growth and differentiation of erythroid hematopoietic precursors (Galimi et al., 1994).

II. HGF Receptor A. Encoding by the c-met Oncogene The expression of the c-met proto-oncogene in the liver, in addition to several other epithelial organs, prompted us to study the possible identity between the Met protein (p190 Met) and the HGF receptor (HGFr). A gastric carcinoma cell line, GTL-16, in which the receptor is overexpressed (Giordano et al., 1989b; Ponzetto et al., 1991), was used as source of the Met protein. Cross-linking experiments showed a specific interaction between the HGF c~ chain and one of the two subunits (IB) of p190 Met. Binding experiments showed a high affinity interaction between the two molecules, with a calculated dissociation constant (KA) of approximately 0.3 nM (NaP dini et al., 1991c; Higuchi and Nakamura, 1991; Zarnegar et al., 1990; Lokker et al., 1992). Another binding site was identified, with low affinityhigh capacity binding properties (K a) = 3 nM) (Naldini et al., 1991c), which corresponds to membrane sulfoglycolipids and/or heparin-sulfate proteoglycans associated to the extracellular matrix (Naldini et al., 1991c; Kobayashi et al., 1994). Formal proof of the identity between the transmembrane tyrosine kinase encoded by the m e t oncogene and the high affinity HGFr was obtained by transfer of the binding activity to insect cells (Spodoptera frugiperda) infected with a recombinant baculovirus carrying human m e t cDNA (Naldini et al., 1991c). Moreover, HGF is capable of stimulating autophosphorylation in tyrosine of p190 Met (Rubin et al., 1991; Bottaro et al., 1991; Naldini et al., 1991b, 1991c).

Paolo M. Comoglio and Elisa Vigna

54

1. Structure a n d Biosynthesis

The c-met proto-oncogene encodes a protein with tyrosine kinase activity (Park et al., 1987). This molecule is a dimer of two subunits, an extracellular a chain (50 kDa) and a transmembrane 13chain (145 kDa) (Giordano et al., 1989a). The 13chain contains an extracellular, a transmembrane, and an intracellular domain endowed with the catalytic function. The a and 13 chains are encoded by the same gene and linked by disulfide bonds. This was proved by transfecting the cloned human M e t cDNA into simian cells; the entire exogenous p]90 Met was expressed and the two-chain structure correctly processed (Giordano et al., 1993). The HGFr (pl90 Met) is synthesized as a single chain precursor of 170 kDa, cleaved and N-glycosylated to generate the mature 190-kDa oLI3heterodimer (Giordano et al., 1989a). The cleavage site (K303RKKR-S308) amino acid numbers are derived from the sequence of the major m e t transcript, (EMBL/GenBank Accession number: X54559, Ponzetto et al., 1991) is a canonical consensus for the endoplasmic reticulum protease furin (Barr, 1991). Experiments of site-directed mutagenesis confirmed that the critical residues for cleavage are the two arginines in position 304 and 307 (Mark et al., 1992). The HGFr is the prototype of a distinct subfamily of heterodimeric tyrosine kinases, including the putative receptors c-Ron and c-Sea (Figure 2) (Ronsin et al., 1993; Huff et al., 1993). These receptors share significant sequence similarities including the extracellular cleavage site between oLand [3 chains and the location of the cysteine residues. In the cytoplasmic domain, the three family members are highly homologous in the kinase region and share an eleven-amino acid motif containing two tyrosines that behaves as a multifunctional docking site for SH2-transducers (Ponzetto et al., 1994). B. Post-Translational Modifications

Two different forms of HGFr devoid of kinase activity were identified using monoclonal antibodies against extracellular epitopes of the Met protein (Prat et al., 1991a). The molecular weight of these truncated forms are 140 and 130 kDa. The first, p140 Met, is composed of the canonical c~ chain linked to an 85 kDa 13 chain lacking most of the cyotplasmic domain. The second, p130 Met, is a soluble protein released from the cell; it differs from the p140 Met by lacking the transmembrane domain. NIH3T3 fibroblasts transfected with the full-length human M e t cDNA express the full-size p190 Met and both p140 Met and P130 Met. This indicates that post-translational events, and not alternative mRNA splicing, are responsible for generation of the truncated receptor forms (Giordano et al., 1993). The generation of truncated receptors was investigated in detail

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55

Figure 2 The HGF receptor family. Schematic representation of the heterodimeric (or-J3) structure of the HGF receptor (c-Met), compared with the structure of the putative receptors encoded by the proto-oncogenes c-ron and c-sea. The cross-hatched boxes represent cysteinerich domains and the solid boxes the tyrosine kinase (TK) domains. The major tyrosine phosphorylation sites (yn) are indicated.

(Crepaldi et al., 1994a). In the endoplasmic reticulum, a fraction of the single-chain p170 Met precursor is cleaved at the cytosolic side, generating a second precursor of 120 kDa. A further proteolytic event, occurring in the trans-Golgi network, converts both precursors into the mature heterodimers, p190 Met and p140 Met, respectively. The soluble p130 Met is generated by proteolytic cleavage at the cell surface (Prat et al., 1991a). Under physiological conditions the p170 Met precursor is inactive. Conditions that cause its accumulation in the endoplasmic reticulum (e.g., overexpression) lead to activation of the tyrosine kinase. Consequently, an accumulation of p120 Met is observed. Overproduction of p120 Met does not occur in cells overexpressing a transfected kinase-dead receptor mutant. Proteolytic cleavage of the cytoplasmic domain of p170 Met may thus represent a safety mechanism aimed at preventing the effect of ligand-independent intracellular activation of the receptor kinase (Crepaldi et al., 1994a).

C. Positive and Negative Regulation The HGFr binds ATP to Lys 111~ which is located in the catalytic pocket of the tyrosine kinase domain. Mutation of this residue leads to a kinase-

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Paolo M. Comoglio and Elisa Vigna

defective receptor (Crepaldi et al., 1994a). Autophosphorylation upregulates the tyrosine kinase activity of the p190 Met receptor (Naldini et al., 1991a). An increase in substrate phosphorylation rate is observed due to a higher Vmax of the phosphotransfer reaction catalyzed by the enzyme. The K m for ATP is not affected while a slight increase in the Km for the peptide substrate is observed. The major autophosphorylation sites of the HGFr have been mapped to y1234 and y1235 (Figure 3) (Ferracini et al., 1991; Longati et al., 1993). This tyrosine doublet is present at homologous locations in the proteins encoded by ron and sea, two putative receptors belonging to the M e t gene family (Ronsin et al., 1993; Huff et al., 1993). The doublet is also conserved and phosphorylated in other tyrosine kinases, including the insulin receptor (Tornqvist et al., 1987, 1988). Mutant receptors have been constructed by substitution of y1234 and/or y1235 with phenylalanine (Longati et al., 1993). These studies showed that single mutations of either y1234 or y1235 generate a receptor with reduced but not abolished kinase activity; the residual activity was sufficient for activation of the kinase by autophosphorylation. However, when both tyrosines were mutated together the re-

Figure 3 Positive and negative regulation of the HGF receptor tyrosine kinase (TK) activity. Inhibition of catalytic activity is observed after Ser 98s phosphorylation, induced either by protein kinase C (PKC) or by calcium-calmodulin kinase III (CAM-K-III). The latter responds to variations of the intracellular Ca + + concentration. Enhancement of catalytic activity follows autophosphorylation of Tyr 1234 and Tyr 1235. The cross-hatched box represents a cysteine rich domain and the solid box a tyrosine kinase (TK) domain.

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57

ceptor lost its kinase activity. These two sites are also the first tyrosines phosphorylated in response to ligand stimulation. Stable transfectants expressing the receptor with single substitutions (either y1234 or y1235) show an impaired response to HGE Phosphorylation on serine and/or threonine residues negatively modulates the tyrosine kinase activity of receptors, such as those for insulin, insulin-like growth factor I (IGF1), and epidermal growth factor (EGF) (Jacobs et al., 1983; Takayama et al., 1988; Davis and Czech, 1984). Stimulation of protein kinase C (PKC) by 12-O-tetradecanoyl-phorbal-13-acetate (TPA) is followed by serine phosphorylation and concomitant decrease in tyrosine kinase activity of the HGFr (Gandino et al., 1990). An independent pathway has been shown to have a similar effect. This negative regulatory pathway is triggered by increased intracellular Ca 2+ concentration, and is mediated by a serine kinase with the biochemical properties of Ca 2+calmodulin kinase III (CAM-K-III)(Gandino et al., 1991). Interestingly, the target residue phosphorylated in both instances is Ser 98s (Figure 3). This residue is located within the juxtamembrane domain of the HGFr, and fits into a canonical consensus sequence for phosphorylation by both PKC and CAM-K-III (Kennelly and Krebs, 1991). The inhibitory effect of either TPA or Ca +2 ionophores on the HGFr kinase activity was abolished if Ser 98s was substituted with an alanine residue by site-directed mutagenesis (Gandino et al., 1994).

D. Signal Transduction Upon ligand binding, tyrosine kinase receptors dimerize and each monomer is transphosphorylated at multiple sites (Ullrich and Schlessinger, 1991). In the HGFr, sites located within the tyrosine kinase domain (i.e., y1234 and y1235) are phosphorylated at high stoichiometry. These are responsible for regulatory functions (see above). Other sites, located outside the kinase domain, are phosphorylated at low stoichiometry and create docking sites for binding to cytoplasmic effectors. These effectors recognize phosphotyrosines via a conserved structural module, known as the Src homology-2 (SH2) domain (Cantley et al., 1991; Koch et al., 1991). Different SH2 domains bind different sequences containing a phosphotyrosine residue. In these sequences, amino acids in position + 1, +2, and + 3 (with respect to phosphotyrosine) are critical in determining the selectivity of interaction with the different SH2 domains (Fantl et al., 1992; Cantley et al., 1991; Rotin et al., 1992; Songyang et al., 1993; Waksman et al., 1992, 1993; Eck et al., 1993). The HGFr docking site for SH2-signal transducers is endowed with unconventional binding properties. Using synthetic phosphopeptides and BIAcore biosensor analysis to measure intermolecular bindings, we showed that

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a phosphotyrosine pair in the sequence y*1349VHVNATY*1356VNV (Y* = phosphorylated tyrosine) located in the receptor C-terminal tail mediates specific association with multiple SH2-containing cytoplasmic effectors (Ponzetto et al., 1994). These include phosphatidylinositol 3-kinase (PI3K), phospholipase C-~/1 (PLC~/), pp60 c-src, the SH2 adapter, and the growth factor receptor binding protein 2/son of sevenless (GRB2/SOS) complex. All the above transducers interact directly with both phosphotyrosine y*1349VHV and y*1356VNV. GRB2, which has a strong requirement for asparagine in the +2 position (Songyang et al., 1993), binds selectively to the motif following y*13s6. Comparison of the HGFr sequence Y V H / N V with a series of binding motifs, optimal for each transducer (Songyang et al. 1993), indicates the Y V H / N V represents a degenerate site permissive for multiple SH2 domains ("SH2-supersite"). All bindings are characterized by fast association and even faster dissociation rates, indicating that the "SH2-supersite" can efficiently and rapidly exchange the bound effectors. The multispecific supersite represents a variation from the common theme of docking sites found in other tyrosine kinase receptors, where a phosphotyrosine embedded within a specific sequence interacts with one effector molecule at a time (Figure 4). In other receptors, the phosphorylation of different specific sites is likely to determine which intracellular transducer will be activated (Pawson and Schlessinger, 1993). For the HGFr, one must postulate a different model; phosphorylation of the single multifunctional site triggers a pleiotropic response involving multiple signal transducers. Modulation of this response may, however, take place by variations in the level of receptor phosphorylation affecting the binding rates of transducers with different affinities. Formal proof for the crucial role of the HGFr multifunctional docking site is the loss of function following mutation of y1349 and y1356. Substitution of both tyrosines with phenylalanine in TPR-MET, the oncogenic counterpart of the receptor (Cooper et al., 1984), abolishes its transforming ability. Single substitution of either tyrosine was less effective for the inhibition, indicating that maximal signaling efficiency of HGFr results from the cooperation between the two tyrosines (Ponzetto et al., 1994). The supersite motif Y-hydrophobic-X-hydrophobic-(X)3-Y-hydrophobic-N-hydrophobic is also conserved in the otherwise divergent C-terminal regions of the Metrelated putative receptors, Sea and Ron (Huff et al., 1993; Ronsin et al., 1993). Thus, the multifunctional docking site (in three subtle variations) represents the main transductional switch for all members of this receptor family. In response to ligand binding in vivo, the tyrosine-phosphorylated HGF receptor activates PI3K (Graziani et al., 1991; Ponzetto et al., 1993), PLC~/, SRC, (Ponzetto et al., 1994), as well as Ras, through stimulation of a guanine nucleotide exchange factor (Graziani et al., 1993). Following HGF

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Figure 4 HGF receptor docking "supersite". Signaling by tyrosine kinase receptors is mediated by selective interactions between individual SH2 domains of cytoplasmic effectors and specific phosphotyrosine residues in the activated receptor. In the HGF receptor (HGFr), phosphorylation of a multifunctional docking site made of the tandemly arranged degenerate sequence YVH/NV (residue y1349 and y1356) mediates high-affinity interactions with multiple (src homology 2 (SH2)-containing signal transducers, including phosphotyrosine phosphatase 2 (PTP2), GTPase activating protein (GAP), Nck (Nishimura et al., 1993), phosphatidylinositol-3-OH kinase (PI3K), phospholipase C-~/1 (PLC~/), Src, and the growth factor receptor binding protein 2/son of sevenless (GRB2/SOS) complex. These transducers bind to individual docking sites on the epidermal growth factor receptor (EGFr) tail or are scattered along the entire cytoplasmic domain of the platelet derived growth factor receptor (PDGFr). The crosshatched boxes represent cysteine rich domains and the solid boxes the tyrosine kinase (TK) domains.

stimulation by association to the receptor, a tyrosine phosphatase is also activated (Villa-Moruzzi et al., 1993). E. Tissue Distribution and Subcellular Localization The H G F r is mainly expressed in tissues of epithelial origin. In h u m a n s , high levels of expression are found in the liver and in epithelial cells of the gastrointestinal tract. Significant levels of expression are also found in the kidney, ovary, and endometrium. Low a m o u n t s of H G F r are detectable in lung epithelium, in the prostate, seminal vesicles, m a m m a r y gland, and in keratinocytes and melanocytes (Di Renzo et al., 1991; Prat et al., 1991b; Natali et al., 1993; M a t s u m o t o et al., 1991a,b; H a l a b a n et al., 1992). In

Paolo M. Comoglio and Elisa Vigna

60

these cells mRNA and protein levels are correlated. In the thyroid follicular epithelium, which expresses low levels of mRNA, no protein is detectable. This suggests regulation of HGFr expression at both the translational and post-translational level (Di Renzo et al., 1991, 1992b). The HGFr is also expressed by oval cells, considered to be the hepatic stem cell compartment (see Chapter 5), and the receptor level increases during liver regeneration after partial hepatectomy (Hu et al., 1993). The HGFr expression in the brain is restricted to a specific cell subset, the microglia, a macrophagederived cell population (Di Renzo et al., 1992a). The HGFr is expressed in some nonepithelial cells, such as endothelial cells (Bussolino et al., 1992), monocytes (Galimi et al., 1993), and hemopoietic precursors (Galimi et al., 1994). The subcellular distribution of HGFrs in polarized epithelial cells has been studied using a variety of techniques. The reported apical distribution of the receptor, observed by immunohistochemistry (Tsarfay et al., 1992), was unexpected as the receptor should have access to HGF that is present in the blood (Nakamura et al., 1986; Zarnegar and Michalopoulos, 1989) and is stored in the pericellular matrix (Matsumoto and Yamamoto, 1991; Naldini et al., 1992). In contrast, studies using domain selective surface biotinylation and immunoprecipitation on cultured polarized kidney epithelial cells showed that HGFrs are selectively exposed at the basolateral surface; the receptors were colocalized with the basolateral marker molecule E-cadherin around the cell-cell contact zone (Crepaldi et al., 1994b). An extensive analysis of human organ frozen sections, using monoclonal antibodies against different epitopes, also showed a consistent localization of the receptors at the basolateral surface and not at the apical surface of epithelia lining the lumen of organs (Crepaldi et al., 1994b).

III. Regulation of

c-met

Expression

The oncogene encoding the HGFr, c-met, is an inducible gene and its expression is controlled at the transcriptional level. The c-met regulatory sequences have been identified and cloned (Gambarotta et al., 1994). The minimal promoter region is included within the first 300 bp upstream to the start site, it lacks TATAA consensus and CCAAT-boxes, and is characterized by GC rich boxes. The promoter contains sequences for binding the transcriptional factors, AP2 and PEA3; these motifs function as PKC responsive elements (Imagawa et al., 1987; Xin et al., 1992). Accordingly, the c-met promoter region responds to the phorbol ester, TPA, in vitro (Gambarotta et al., 1994), and TPA treatment in vivo results in a strong upregulation of the receptor expression, at both the mRNA and protein levels (Boccaccio et al., 1994).

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HGFr levels are regulated in the different phases of growth. In tissue culture, confluent growth-arrested epithelial cells express low amounts of p190 Met. Recruitment into the cell cycle by stimulation with fresh serum, however, results in a significant increase of both mRNA and protein. Interestingly, HGF can also transiently induce expression of its own receptor, in a time- and dose-dependent manner. The appearance of specific c-met mRNA follows c-fos and c-jun induction. It is reduced by the protein synthesis inhibitor, puromycin, but is increased by cycloheximide alone (superinduction) (Boccaccio et al., 1994). Thus, in the response to serum or growth factors, c-met behaves as a delayed-early response gene (see Chapter 4). Using this autoamplification mechanism the ligand seems able to enhance its intracellular signal.

IV. Role of HGF in Tissue Regeneration and Embryogenesis Long before molecular cloning, HGF was known to stimulate hepatocyte growth in physiological as well as pathological conditions. The HGF mRNA markedly increases in nonparenchymal liver cells of rodents treated with carbon tetrachloride or other hepatotoxic compounds within the first 10 hr (Kinoshita et al., 1989). Consequently, the HGF biological activity increases in the plasma, with a peak at 30 hr (Lindroos et al., 1991). Other agents causing liver damage, such as ischemia, mechanical crush (Hamanoue et al., 1992), and fulminant hepatitis (Tsubouchi et al., 1989), cause HGF mRNA increases with similar fast kinetics. Circulating HGF also increases in the plasma of patients with liver cirrhosis (Shimizu et al., 1991). HGF is primarily synthesized in the nonparenchymal liver cells, like Kupffer cells, sinusoidal endothelia, and Ito cells (Kinoshita et al., 1989; Noji et al., 1990; Ramadori et al., 1992); thus, a paracrine mechanism seems to be responsible for hepatocyte growth following liver injuries. If liver regeneration is induced by partial hepatectomy, and not by direct damage of hepatocytes, the peak of liver HGF mRNA expression is observed at 24 hr. In contrast, the plasma level of HGF maximizes within 2 hr. This indicates that hepatocytes respond to exogenous HGF produced and released into the circulation by other distal noninjured organs (see Chapter 2) (Kinoshita et al., 1991). There are thus two possible mechanisms of HGF action: one paracrine and one endocrine. In this respect, one group reported the identification of a protein, named injurin, as the possible inducer of HGF mRNA synthesis in distant organs after partial hepatectomy (Matsumoto et al., 1992). Evidence that HGF acts as a potent hepatotropic factor in vivo was obtained in animals injected intravenously with the factor (Ishiki et al.,

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1992). In these animals, HGF induced hepatocyte DNA synthesis and protected the liver from subsequent injuries. HGF also strongly stimulates DNA synthesis of rabbit kidney tubular epithelial cells in tissue culture (Igawa et al., 1991). Unilateral nephrectomy in experimental animals, or treatment with CC14, markedly increases HGF mRNA in the contralateral kidney. In situ hybridization shows the appearance of HGF mRNA in endothelial cells of the organ (Nagaike et al., 1991). These data suggest that HGF also functions in a paracrine manner as a renotrophic factor after kidney injury. In proliferating hepatocytes or renal tubular cells the specific binding of HGF to its receptor is greatly inhibited. Scatchard analysis showed a decrease in receptor number without changes in affinity, suggesting that HGFr downmodulation occurs during liver and kidney regeneration (Higuchi and Nakamura, 1991; Nagaike et al., 1991). The mitogenic and motogenic activity of HGF, associated with its ability to stimulate the invasion of extracellular matrices and the organization of tubular structures, strongly suggest that the factor may behave as a potent morphogen during embryonal development. Experimental administration of exogenous HGF in chicken embryos affects the development of the spinal cord (Stern et al., 1990). In situ hybridization studies in mouse embryos also demonstrate the nonrandom distribution of HGF and its receptor in developing organs. Epithelial cells express the HGFr, while mesenchymal cells in close vicinity express HGF, suggesting a paracrine relationship (Sonnemberg et al., 1993). Signals of mesenchymal origin are known to govern differentiation and morphogenesis of many epithelial organs. HGF may be one of the long-sought factors mediating these paracrine interactions.

V. R o l e of c - m e t in

Carcinogenesis

The HGFr gene, c-met, was originally identified as an oncogene activated in an osteosarcoma cell line treated with a chemical carcinogen (Cooper et al., 1984). In this cell line, a genomic rearrangement generated a fusion protein containing the aminoterminal sequence of a gene called tpr and the truncated carboxyterminal sequence of c-met, encoding the tyrosine kinase domain (Park et al., 1986). The tpromet rearrangement was also found in human xeroderma pigmentosum (XP) cells after treatment with a chemical carcinogen (Michelin et al., 1993), and at low frequency in naturally occurring human tumors (Soman et al., 1990, 1991). The tpr-met encoded tyrosine kinase is constitutively active and is transforming. Deletion of the tpr moiety yields a protein with reduced transforming potential, indicating a direct role for this region in the mechanism of activation. Two leucine zipper motifs within the tpr region were demonstrated to mediate dimerization of the Tpr-Met receptor; constitutive dimerization allows transphosphoryla-

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tion of the kinase domains and contributes to oncogenic activation (Rodriguez and Park, 1993). Although tpr enhances the transforming activity of the m e t kinase domain, the simple truncation of the extracellular and the transmembrane portion is sufficient to generate an active oncogenic protein. Recent findings show that a short sequence in the juxtamembrane domain is critical for constitutive activation of the truncated met. This sequence, 36 amino acids long, could contain dimerization motifs, as in tpr, or previously unrecognized sequences critical for transformation (Zhen et al., 1994). Transfection of full-length human m e t c-DNA in mouse or rat fibroblasts does not lead to malignant transformation (Giordano et al., 1993; Zhen et al., 1994). In contrast, transfection of human m e t together with human HGF cDNA is transforming; an autocrine loop has been suggested (Rong et al., 1992). In a gastric human carcinoma, the m e t proto-oncogene is amplified and overexpressed and the tyrosine kinase is constitutively activated (Giordano et al., 1989a,b). Because no modification in the sequence has been found in the derived cell line (Ponzetto et al., 1991), constitutive activation of the Met kinase may be initiated by simple overexpression. Overexpression has indeed been found in spontaneously arising human cancers. In a significant fraction of gastrointestinal tract carcinomas, the amount of m e t m R N A and protein are increased (Di Renzo et al., 1991). In thyroid tumors, overexpression of m e t is observed in carcinomas derived from the follicular epithelium and correlates with an aggressive phenotype (Di Renzo et al., 1992b).

Acknowledgments The experimental work reviewed in this paper is the result of the collaborative effort of our coworkers E Di Renzo, G. Gaudino, C. Ponzetto, M. Prat, T. Crepaldi, S. Giordano, A. Graziani, L. Naldini, C. Boccaccio, G. Gambarotta, and P. Longati, who are gratefully acknowledged. The manuscript was written with the excellent assistance of A. Cignetto and E. Wright. Our work is supported by grants from the Associazione Italiana Ricerche Cancro (AIRC) and from the Italian National Research Council (CNR), progetto finalizzato ACRO.

References Barr, P. J. (1991). Mammalian subtilisins: The long-sought dibasic processing endoproteases. Cell (Cambridge, Mass.) 66, 1-3. Boccaccio, C., Gaudino, G., Gambarotta, G., Galimi, E, and Comoglio, P. M. (1994). Hepatocyte growth factor receptor expression is inducible and is part of the delayed-early response to HGE J. Biol. Chem. 269, 12846-12851. Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M. L., Kmiecick, T. E., Vande Woude, G. E,

64

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and Aaronson, S. A. (1991). Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene. Science 251, 802-804. Bussolino, E, Di Renzo, M. E, Ziche, M., Bocchietto, E., Olivero, M., Naldini, L., Gaudino, G., Tamagnone, L., Coffer, A., Marchisio, P. C., and Comoglio, P. M. (1992). Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J. Cell Biol. 119, 629-641. Cantley, L. C., Auger, K., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991). Oncogenes and signal transduction. Cell (Cambridge, Mass.) 64, 281-302. Cooper, C. S., Park, M., Blair, D., Tainsky, M. A., Huebner, K., Croce, C. M., and Vande Woude, G. E (1984). Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature (London) 311, 29-33. Crepaldi, T., Prat, M., Giordano, S., and Comoglio, P. M. (1994a). Generation of a truncated hepatocyte growth factor receptor in the endoplasmic reticulum. J. Biol. Chem. 269, 17501755. Crepaldi, T., Pollack, A. L., Prat, M., Zborek, A., Mostov, K., and Comoglio, P. M. (1994b). Targeting of the SF/HGF receptor to the basolateral domain of polarized epithelial cells. J. Cell Biol. 125, 312-320. Davis, R. J., and Czech, M. P. (1984). Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor. J. Biol. Chem. 259, 8545-8549. Di Renzo, M. E, Narsimhan, R. P., Olivero, M., Bretti, S., Giordano, S., Medico, E., Gaglia, P., Zara, P., and Comoglio, P. M. (1991). Expression of the met/HGF receptor in normal and neoplastic human tissues. Oncogene 6, 1997-2003. Di Renzo, M. E, Bertolotto, A., Olivero, M., Putzolu, P., Crepaldi, T., Schiffer, D., Pagni, C. A., and Comoglio, P. M. (1992a). Selective expression of the met/HGF receptor in human central nervous system microglia. Oncogene 8, 219-222. Di Renzo, M. E, Olivero, M., Ferro, S., Prat, M., Bongarzone, I., Pilotti, S., Belfiore, A., Costantino, A., Vignari, R., Pierotti, M. A., and Comoglio, P. M. (1992b). Overexpression of the c-met/HGF receptor gene in human thyroid carcinomas. Oncogene 7, 2549-2553. Eck, M. J., Shoelson, S. E., and Harrison, S. C. (1993). Recognition of a high affinity phosphotyrosyl peptide by the scr homology 2 domain of p56 lck. Nature (London) 362, 87-91. Fantl, W. J., Escobedo, J. A., Martin, G. A., Turck, C. W., Rosario, M., McCormick, E, and Williams, L. T. (1992). Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell (Cambridge, Mass.) 69, 413-423. Ferracini, R., Longati, P., Naldini, L., Vigna, E., and Comoglio, P. M. (1991). Identification of the major autophosphorylation site of the met/hepatocyte growth factor receptor tyrosine kinase. J. Biol. Chem. 266, 19558-19564. Galimi, E, Brizzi, M. E, and Comoglio, P. M. (1993). The hepatocyte growth factor and its receptor. Stem Cells 11, 22-30. Galimi, E, Bagnara, G. P., Bonsi, L., Cottone, E., Follenzi, A., Simeone, A., and Comoglio, P. M. (1994). Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors. J. Cell Biol. 127, 1743-1754. Gambarotta, G., Pistoi, S., Giordano, S., Comoglio, P. M., and Santoro, C. (1994). Structure and inducible regulation of the human met promoter. J. Biol. Chem. 269, 12852-12857. Gandino, L., Di Renzo, M. E, Giordano, S., Bussolino, E, and Comoglio, P. M. (1990). Protein kinase-C activation inhibits tyrosine phosphorylation of the c-met protein. Oncogene, 5, 721-725. Gandino, L., Munaron, L., Naldini, L., Magni, M., and Comoglio, P. M. (1991). Intracellular calcium regulates the tyrosine kinase receptor encoded by the met oncogene. J. Biol. Chem. 266, 16098-16104. Gandino, L., Longati, P., Medico, E., Prat, M., and Comoglio, P. M. (1994). Phosphorylation

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of serine 985 negatively regulates the hepatocyte growth factor receptor kinase. J. Biol. Chem. 269, 1815-1820. Gherardi, G., Gray, J., Stoker, M., Perryman, M., and Furlong, R. (1989). Purification of scatter factor, a fibroblast-derived basic protein that modulates epithelial interaction and movement. Proc. Natl. Acad. Sci. U.S.A. 86, 5844-5848. Giordano, S., Di Renzo, M. E, Narsimhan, R., Cooper, C. S., Rosa, C., and Comoglio, P. M. (1989a). Biosynthesis of the protein encoded by the c-met proto-oncogene. Oncogene 4, 1383-1388. Giordano, S., Ponzetto, C., Di Renzo, M. E, Cooper, C. S., and Comoglio, P. M. (1989b). Tyrosine kinase receptor indistinguishable from the c-MET protein. Nature (London) 339, 155-156.

Giordano, S., Zhen, Z., Medico, E., Gaudino, G., Galimi, E, and Comoglio, P. M. (1993). Transfer of motogenic and invasive response to scatter factor/hepatocyte growth factor by transfection of human met proto-oncogene. Proc. Natl. Acad. Sci. U.S.A. 90, 649-653. Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Miyazaki, H., Hashimoto, S., and Daikuhara, Y. (1988). Purification and partial characterization of hepatocyte growth factor from plasma of a patient with hepatic failure. J. Clin. Invest. 81, 414-419. Grant, D. S., Kleinman, H. K., Goldberg, I. D., Bhargava, M., Nickoloff, B. J., Kinsella, J. L., Polverini, P. J., and Rosen, E. M. (1993). Scatter factor induces blood vessel formation in vivo. Proc. Natl. Acad. Sci. U.S.A. 90, 1937-1941. Graziani, A., Gramaglia, D., Cantley, L. C., and Comoglio, P. M. (1991). The tyrosine phosphorylated hepatocyte growth factor/scatter factor receptor associates with phosphatidylinositol 3-kinase. J. Biol. Chem. 266, 22087-22090. Graziani, A. Gramaglia, D., Dalla Zonca, P., and Comoglio, P. M. (1993). Hepatocyte growth factor/scatter factor stimulates the ras-guanine nucleotide exchanger. J. Biol. Chem. 268, 9165-9168.

Halaban, R., Rubin, J., Funasaka, Y., Cobb, C., Boulton, T., Faletto, D., Rosen, E., Chan, A., Yoko, K., White, W., Cook, C., and Moellmann, G. (1992). Met and hepatocyte growth factor/scatter factor signal transduction in normal melanocytes and melanoma cells. Oncogene 7, 2195-2206.

Hamanoue, M., Kawaida, K., Takao, S., Shimazu, H., Noji, S., Matsumoto, K., and Nakamura, T. (1992). Rapid and marked induction of hepatocyte growth factor during liver regeneration after ischemic or crush injury. Hepatology (Baltimore) 16, 1485-1492. Han, S., Stuart, L. A., and Friezner Degen, S. J. (1991). Characterization of the DNF15S2 locus on human chromosome 3: Identification of a gene coding for four kringle domains with homology to hepatocyte growth factor. Biochemistry 30, 9768-9780. Hartmann, G., Naldini, L., Weidner, M., Sachs, M., Vigna, E., Comoglio, P., and Birchmeier, W. (1992). A functional domain in the heavy chain of scatter factor/hepatocyte growth factor binds the c-met receptor, induces cell dissociation but not mitogenesis. Proc. Natl. Acad. Sci. U.S.A. 89, 11574-11578. Higashio, K., Shima, N., Goto, M., Itagaki, Y., Nagao, M., Yasuda, H., and Morinaga, T. (1990). Identity of a tumor cytotoxic factor from human fibroblasts and hepatocyte growth factor. Biochem. Biophys. Res. Commun. 170, 397-404. Higuchi, O., and Nakamura, T. (1991). Identification and change in the receptor for hepatocyte growth factor in rat liver after partial hepatectomy or induced hepatitis. Biochem. Biophys. Res. Commun. 176, 599-607. Hu, Z., Evatrs, R. P., Fujio, K., Marsden, E. R., and Thorgeirsson, S. (1993). Expression of hepatocyte growth factor and c-met gene during hepatic differentiation and liver development in the rat. Am. J. Pathol. 142, 1823-1830. Huff, J. I., Jelinek, A. M., Borgman, C. A., Lansing, T. J., and Parsons, J. T. (1993). The proto-

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oncogene c-sea encodes a transmembrane protein-tyrosine kinase related to the met/hepatocyte growth factor/scatter factor receptor. Proc. Natl. Acad. Sci. U.S.A. 90, 6140-6144. Igawa, T., Kanda, S., Kanetake, H., Saitoh, Y., Ichihara, A., Tomita, Y., and Nakamura, T. (1991). Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells. Biochem. Biophys. Res. Commun. 174, 831-838. Imagawa, M., Chiu, R., and Karin, M. (1987). Transcription factor AP-2 mediates induction by two different signal transduction pathways: Protein kinase C and cAMP. Cell (Cambridge, Mass.) 51, 251-260. Ishiki, Y., Ohnishi, H., Muto, Y., Matsumoto, K., and Nakamura, T. (1992). Direct evidence that hepatocyte growth factor is a hepatotrophic factor for liver regeneration and for potent anti-hepatitis action in vitro. Hepatology (Baltimore) 16, 1227-1235. Jacobs, S., Shayoun, N. E., Saltiel, A. R., and Cuatrecasas, P. (1983). Phorbol esters stimulate the phosphorylation of receptors for insulin and somatomedin C. Proc. Natl. Acad. Sci. U.S.A. 80, 6211-6213. Kan, M., Zhang, G. H., Zarnegar, R., Michalopoulos, G., Myoken, Y., Mckeehan, W. L., and Stevens, J. L. (1991). Hepatocyte growth factor/hepatopoietin A stimulates the growth of rat kidney proximal tubule epithelial cells (RPTE), rat nonparenchymal liver cells, human melanoma cells, mouse keratinocytes and stimulates anchorage-independent growth of SV40-transformed RPTE. Biochem. Biophys. Res. Commun. 174, 331-337. Kennelly, P. J., and Krebs, E. G. (1991). Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J. Biol. Chem. 266, 15555-15558. Kinoshita, T., Tashiro, K., and Nakamura, T. (1989). Marked increase of HGF and mRNA in nonparenchymal liver cells of rat treated with hepatotoxin. Biochem. Biophys. Res. Commun. 165, 1229-1234. Kinoshita, T., Hirao, S., Matsumoto, K., and Nakamura, T. (1991 ). Possible endocrine control by hepatocyte growth factor of liver regeneration after partial hepatectomy. Biochem. Biophys. Res. Commun. 177, 330-335. Kobayashi, T., Honke, K., Miyazaki, T., Matsumoto, K., Nakamura, T., Ishizuka, I., and Makita, A. (1994). Hepatocyte growth factor specifically binds to sulfoglycolipids. J. Biol. Chem. 269, 9817-9821. Koch, C. A., Anderson, D., Moran, M. E, Ellis, C., and Pawson, T. (1991). SH2 and SH3 domains: Elements that control interactions of cytoplasmic signaling proteins. Science 252, 668-674. Lindroos, P. M., Zarnegar, R., and Michalopoulos, G. K. (1991). Hepatocyte growth factor (hepatopoietin A) rapidly increases in plasma prior to DNA synthesis and liver regeneration stimulated by partial hepatectomy and C C I 4 administration. Hepatology (Baltimore) 13, 743-750. Lokker, N. A., Mark, M. R., Luis, E. A., Bannet, G. L., Robbins, K. A., Baker, J. B., and Godowski, P. J. (1992). Structure-function analysis of hepatocyte growth factor: Identification of variants that lack mitogenic activity yet retain high affinity receptor binding. EMBO J. 11, 2503-2510. Longati, P., Bardelli, A., Ponzetto, C., Naldini, L., and Comoglio, P. M. (1993). Tyr o s i n e s 1234-1235 a r e critical for activation of the tyrosine kinase encoded by the met protooncogene (HGF receptor). Oncogene 9, 49-57. Mark, M. R., Lokker, N. A., Zioncheck, T. E, Luis, E. A., and Godowski, P. J. (1992). Expression and characterization of hepatocyte growth factor receptor-IgG fusion proteins. J. Biol. Chem. 36, 26166-26171. Matsumoto, A., and Yamamoto, N. (1991). Sequestration of a hepatocyte growth factor in extracellular matrix in normal adult liver. Biochem. Biophys. Res. Commun. 174, 90-95. Matsumoto, K., Tajima, H., and Nakamura, T. (1991a). Hepatocyte growth factor is a potent stimulator of human melanocytes DNA synthesis and growth. Biochem. Biophys. Res. Commun. 176, 45-51.

3.

Structure and Functions of the HGF Receptor (c-Met)

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Matsumoto, K., Hashimoto, K., Yoshikawa, K., and Nakamura, T. (1991b). Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor. Exp. Cell Res. 196, 114-120. Matsumoto, K., Tajima, H., Hamanoue, M., Kohno, S., Kinoshita, T., and Nakamura, T. (1992). Identification and characterization of "injurin", an inducer of the gene expression of hepatocyte growth factor. Proc. Natl. Acad. Sci. U.S.A. 89, 3800-3804. Michelin, S., Daja-Grosjean, L., Sureau, F., Said, S., Sarasin, A., and Suarez, H. G. (1993). Characterization of a c-met proto-oncogene activated in human xeroderma pigmentosum cells after treatment with N-methyl N-nitro N-nitrosoguanidina (MNNG). Oncogene, 8, 1983-1991. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., Arakaki, N., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Gohda, E., Daikuhara, Y., and Kitamura, N. (1989). Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem. Biophys. Res. Commun. 163, 967-973. Miyazawa, K., Shimomura, T., Kitamura, A., Kondo, J., Morimoto, Y., and Kitamura, N. (1993). Molecular cloning and sequence analysis of the c-DNA for a human serine protease responsible for activation of hepatocyte growth factor. J. Biol. Chem. 268, 1002410028. Mizuno, K., Takehara, T., and Nakamura, T. (1992). Proteolytic activation of a single-chain precursor of hepatocyte growth factor by extracellular serine-protease. Biochem. Biophys. Res. Commun. 189, 1631-1638. Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. (1991). Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell (Cambridge, Mass.) 7, 901-908. Nagaike, M., Hirao, S., Tajima, H., Noji, S., Taniguchi, S., Matsumoto, K., and Nakamura, T. (1991). Renotropic functions of hepatocyte growth factor in renal regeneration after unilateral nephrectomy. J. Biol. Chem. 266, 22781-22784. Naka, D., Ishii, T., Yoshiyama, Y., Miyazawa, K., Hara, H., Hishida, T., and Kitamura, N. (1992). Activation of hepatocyte growth factor by proteolytic conversion of a single chain form to a heterodimer. J. Biol. Chem. 267, 20114-20119. Nakamura, T., Teramoto, H., and Ichihara, A. (1986). Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary cultures. Proc. Natl. Acad. Sci. U.S.A. 86, 6489-6493. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K., and Shimizu, S. (1989). Molecular cloning and expression of human hepatocyte growth factor. Nature (London) 342, 440-443. Naldini, L., Vigna, E., Ferracini, R., Longati, P., Gandino, L., Prat, M., and Comoglio, P. M. (1991a). The tyrosine kinase encoded by the met proto-oncogene is activated by autophosphorylation. Mol. Cell. Biol. 11, 1793-1803. Naldini, L., Vigna, E., Narshiman, R. P., Gaudino, G., Zarnegar, R., Michalopoulos, G., and Comoglio, P. M. (1991b). Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded bythe proto-oncogene c-met. Oncogene, 6, 501-504. Naldini, L., Weidner, M., Vigna, E., Gaudino, G., Bardelli, A., Ponzetto, C., Narsimhan, R., Hartmann, G., Zarnegar, R., Michalopoulos, G., Birchmeier, W., and Comoglio, P. M. (1991c). Scatter factor and hepatocyte growth factor are indistinguishable ligands for the met receptor. EMBO J. 10, 2867-2878. Naldini, L., Tamagnone, L., Vigna, E., Sachs, M., Hartmann, G., Birchmeier, W., Daikuhara, Y., Tsubouchi, H., Blasi, E, and Comoglio, P. M. (1992). Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor EMBO J. 11, 4825-4833. Natali, ~P. G., Nicotra, M. R., Di Renzo, M. E, Prat, M., Bigotti, A., Cavaliere, R., and Comoglio, P. M. (1993). Expression of the c-met/HGF receptor in human melanocytic

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neoplasms: Demonstration of the relationship to malignant melanoma tumour. Br. J. Cancer 68, 746-750. Nishimura, R., Li, W., Kashishian, A., Mondino, A., Zhou, M., Cooper, J., and Schlessinger, J. (1993). Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor. Mol. Cell. Biol. 13, 6889-6896. Noji, S., Tashiro, K., Koyama, E., Nohno, T., Osyama, K., Taniguchi, S., and Nakamura, T. (1990). Expression of the hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat liver, as revealed by in situ hybridization. Biochem. Biophys. Res. Commun. 173, 42-47. Park, M., Dean, M., Cooper, C. S., Schmidt, M., O'Brian, S. J., Blair, D. G., and Vande Woude, G. E (1986). Mechanism of met oncogene activation. Cell. (Cambridge, Mass.) 45, 895904. Park, M., Dean, M., Kaul, K., Braun, M. J., Gonda, M. A., and Vande Woude, G. (1987). Sequence of met proto-oncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors. Proc. Natl. Acad. Sci. U.S.A. 84, 6379-6383. Pawson, T., and Schlessinger, J. (1993). SH2 and SH3 domains. Curr. Biol. 3, 434-442. Ponzetto, C., Giordano, S., Peverali, F., Della Valle, G., Abate, M., Vaula, G., and Comoglio, P. M. (1991). c-met is amplified but not mutated in a cell line with an activated MET tyrosine kinase. Oncogene 6, 553-559. Ponzetto, C., Bardelli, A., Maina, F., Longati, P., Panayotou, G., Dhand, R., Waterfield, M. D., and Comoglio, P. M. (1993). A novel recognition motif for phosphatiylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor. Mol. Cell. Biol. 13, 4600-4608. Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., Dalla Zonca, P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M. (1994). A multifunctional docking site mediates signalling and transformation by hepatocyte growth factor/scatter factor (HGF/SF). receptor family. Cell (Cambridge, Mass.) 77, 261-271. Prat, M., Crepaldi, T., Gandino, L., Giordano, S., Longati, P., and Comoglio, P. M. (1991a). C-terminal truncated forms of met, the hepatocyte growth factor receptor Mol. Cell. Biol. 11, 5954-5962. Prat, M. P., Narsimhan, R. P., Crepaldi, T., Nicotra, M. R., Natali, P. G., and Comoglio, P. M. (1991b). The receptor encoded by the human c-met oncogene is expressed in hepatocytes, epithelial cells and solid tumors. Int. J. Cancer 49, 323-328. Ramadori, G., Neubauer, K., Odenthal, M., Nakamura, T., Knittel, T., Schwogler, K. H., and zum Buschenfelde, M. (1992). The gene of hepatocyte growth factor is expressed in fatstoring cells of rat liver and is downregulated during cell growth and by transforming growth factor-beta. Biochem. Biophys. Res. Commun. 183, 739-742. Rodriguez, G. A., and Park, M. (1993). Dimerization mediated through a leucine zipper activates the oncogenic potential of the MET receptor tyrosine kinase. Mol. Cell. Biol. 13, 6711-6722. Rong, S., Bodescot, M., Blair, D., Dunn, J., Nakamura, T., Mizuno, K., Park, M., Chan, A., Aaronson, S., and Vande Woude, G. F. (1992). Tumorigenicity of the met proto-oncogene and the gene for hepatocyte growth factor. Mol. Cell. Biol. 12, 5152-5158. Ronsin, C., Muscatelli, F., Mattei, M. G., and Breathnach, R. (1993). A novel putative receptor tyrosine kinase of the met family. Oncogene 8, 1195-1202. Rotin, D., Margolis, B., Mohammadi, M., Daly, R. J., Daum, G., Li, N., Fischer, E. H., Burgess, W. H., Ullrich, A., and Schlessinger, J. (1992). SH2 domain prevent tyrosine dephosphorylation of the EGF receptor: Identification of tyr 992 as the high affinity binding site for SH2 domains of phospholipase C gamma. EMBO J. 11,559-567. Rubin, J. S., Chan, A. M. L., Bottaro, D. P., Burgess, W. H., Taylor, W. G. Cech, A. C., Hirschfield, D. W., Wong, J., Miki, T., Finch, P. W., and Aaronson, S. A. (1991). A broad-

3.

Structure and Functions of the HGF Receptor (c-Met)

69

spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor. Proc. Natl. Acad. Sci. U.S.A. 88, 415-419. Santos, O. E P., and Nigam, S. K. (1993). HGF-induced tubulogenesis and branching of epithelal cells is modulated by extracellular matrix and TGF-beta. Dev. Biol. 160, 293302. Shimizu, I., Ichihara, A., and Nakamura, T. (1991). Hepatocyte growth factor in ascites from patients with cirrhosis. J. Biochem. (Tokyo) 109, 14-18. Shimomura, T., Kondo, J., Ochiai, M., Naka, D., Miyazawa, K., Morimoto, Y., and Kitamura, N. (1993). Activation of the zymogen of hepatocyte growth factor activator by thrombin. J. Biol. Chem. 268, 22927-22932. Shiota, G., Rhoads, D. B., Wang, T. C., Nakamura, T., and Schmidt, E. V. (1992a). Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells. Proc. Natl. Acad. Sci. U.S.A. 89, 373-377. Skeel, A., Yoshimura, T., Showalter, S. D., Tanaka, A., Appella, E., and Leonard, E. J. (1991). Macrophage stimulating protein: Purification, partial aminoacid sequence, and cellular activity. J. Exp. Med. 173, 1227-1234. Soman, N. R., Wogan, G. N., and Rhim, J. S. (1990). Tpromet oncogenic rearrangement: Detection by polymerase chain reaction amplification of the transcript and expression in human tumor cell lines. Proc. Natl. Acad. Sci. U.S.A. 87, 738-742. Soman, N. R., Correa, P., Ruiz, B. A., and Wogan, G. N. (1991). The tpromet oncogenic rearrangement is present and expressed in human gastric carcinoma and precursor lesions. Proc. Natl. Acad. Sci. U.S.A. 88, 4829-4896. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, E, Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993). SH2 domains recognize specific phosphopeptide sequences. Cell (Cambridge, Mass.) 72, 1-20. Sonnemberg, E., Meyer, D., Weidner, M. K., and Birchmeier, C. (1993). Scatter factor/hepatocyte growth factor and its receptor the c-met tyrosine kinase can mediate a signal exchange between mesenchyme and epithelia during mouse embryogenesis. J. Cell Biol. 123, 223235. Stern, C. D., Ireland, G. W., Herrick, S. E., Gherardi, E. Gray, J., Perryman, M., and Stoker, M. (1990). Epithelial scatter factor and development of the chick embryonic axis. Development (Cambridge, UK) 110, 1271-1284. Stoker, M., Gherardi, E., Perryman, M., and Gray, J. (1987). Scatter factor is a fibroblastderived modulator of epithelial cell mobility. Nature (London) 327, 239-242. Tajima, H., Matsumoto, K , and Nakamura, T. (1991). Hepatocyte growth factor has a potent anti-proliferative activity for various tumor cell lines. FEBS Lett. 291,229-232. Takayama, S., White, M. F., and Kahn, C. R. (1988). Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. J. Biol. Chem. 263, 3440-3447. Tornqvist, H. E., Pierce, M. W., Frackleton, A. R., Nemenoff, R. A., and Avruch, J. (1987). Identification of insulin receptor tyrosine residues autophosphorylated in vitro. J. Biol. Chem. 262, 10212-10219. Tornqvist, H. E., Gunsalus, J. R., Nemenoff, R. A., Frackleton, A. R., Pierce, M. W., and Avruch, J. (1988). Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells. J. Biol. Chem. 263, 350359. Tsarfay, I., Resau, J. H., Rulong, S., Keydar, I., Faletto, D. L., and Vande Woude, G. E (1992). The met proto-oncogene receptor and lumen formation. Science 257, 1258-1261. Tsubouchi, H., Hirono, S., Godha, E., Nakayama, H., Takahashi, K., Sakiyama, O., Miyazaki, H., Sugihara, J., Tomita, E., Muto, Y., Daikuhara, Y., and Hashimoto, S. (1989). Clinical

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significance of human hepatocyte growth factor in blood from patients with fulminant hepatic failure. Hepatology (Baltimore) 9, 875-881, Ullrich, A., and Schlessinger, J. (1991). Signal transduction by receptors with tyrosine kinase activity. Cell (Cambridge, Mass.) 61,203-212. Villa-Moruzzi, E., Lapi, S., Prat, M., Gaudino, G., and Comoglio, R M. (1993). A protein tyrosine phosphatase activity associated with the hepatocyte growth factor/scatter factor receptor. J. Biol. Chem. 268, 18176-18180. Waksman, G., Kominos, D., Robertson, S. R., Pant, N., Baltimore, D., Birge, R. B., Cowburn, D., Hanafusa, H., Mayer, B. J., Overduin, M., Resh, M. D., Rios, C. B., Silverman, L., and Kuriyan, J. (1992). Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides. Nature (London), 358, 646-653. Waksman, G., Shoelson, S., Pant, N., Cowburn, D., and Kuriyan, J. (1993). Binding of a high affinity phosphotyrosil peptide in the src SH2 domain: Crystal structures of the complexed and peptide-free forms. Cell (Cambridge, Mass.) 72, 779-790. Weidner, K. M., Behrens, J., Vandekerckove, J., and Birchmeier, W. (1990). Scatter factor: Molecular characteristics and effect on the invasiveness of epithelial cells. J. Cell Biol. 111, 2097-2108. Weidner, K. M., Arakaki, N., Vandekerchove, J., Weingart, S., Hartmann, G., Rieder, H., Fonatsch, C., Tsubouchi, H., Hishida, T., Daikuhara, Y., and Birchmeier, W. (1991). Evidence for the identity of human scatter factor and hepatocyte growth factor. Proc. Natl. Acad. Sci. U.S.A. 88, 7001-7005. Xin, J. H., Cowie, A., Lachance, R, and Hassel, J. A. (1992). Molecular cloning and characterization of PEA3, a new member of the ETS oncogene family that is differentially expressed in mouse embryonic cells. Gene Dev. 6, 481-496. Yoshimura, T., Yuhki, N., Wang, M.-H., Skeel, A., and Leonard, E. J. (1993). Cloning, sequencing, and expression of human macrophage stimulating protein (MSP, MST1) confirms MSP as a member of the family of the kringle proteins and locates the MSP gene on chromosome 3. J. Biol. Chem. 268, 15461-15468. Zarnegar, R., and Michalopoulos, G. (1989). Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. Cancer Res. 49, 3314-3320. Zarnegar, R., De Frances, M. C., Oliver, L., and Michalopoulos (1990). Identification and partial characterization of receptor binding sites of HGF on rat hepatocytes. Biochem. Bioph. Res. Commun. 173, 1179-1185. Zhen, Z. Giordano, S., Longati, R, Medico, E., Campiglio, M., and Comoglio, R M. (1994). Structural and functional domains critical for constitutive activation of the HGF receptor (MET) Oncogene 9, 1691-1697.

4 Expression and Function of Growth-Induced Genes during Liver Regeneration Rebecca Taub Department of Genetics and Medicine Howard Hughes Medical Institute University of Pennsylvania, School of Medicine Philadelphia, Pennsylvania 19104

I. Liver Regeneration: The Important Questions Although scientists have been studying liver regeneration for many years, until recently there has been little research directed toward understanding its molecular basis. Ultimately, scientists would like to understand the mechanisms that (1) trigger regeneration, (2) allow the liver to concurrently grow and maintain differentiated function, and (3) terminate cell proliferation once the liver has reached the appropriate mass. These questions are fundamentally important to help us understand the mechanisms of cell growth and differentiation. Furthermore, they are directly important in understanding some aspects of clinical liver disease since the liver is required to regenerate following liver transplantation and hepatic damage resulting from hepatitis virus infection and exposure to chemical toxins. In addressing these questions, it is important to first realize some basic facts about cell cycle kinetics in the regenerating liver (see Chapters I and 2) (Fausto and Mead, 1989; Michalopoulos, 1990; Fausto, 1994). Within minutes after a partial hepatectomy, the majority of the cells in the remnant liver undergo a transition from the quiescent or Go state into the G1 phase of the cell cycle (Figure 1). The signals that mediate this transition are not clearly understood, but a number of different growth factors and other signaling Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FigureI Modelof the cell cycle during liver regeneration followingpartial hepatectomyshowing the timing of induction of immediate-early and delayed-early genes.

events have been implicated in this process. Following rapid intracellular signal transduction in hepatic cells, preexisting transcription factors are modified resulting in their activation. These activated transcription factors are then responsible for initiating the transcription of primary or immediateearly response genes within minutes after the partial hepatectomy in a protein synthesis independent manner. As discussed below, immediate-early genes represent diverse functional classes and include transcription factors, growth factors, signal transduction regulators, and other types of proteins (A1mendral et al., 1988; Herschman, 1991; Lau and Nathans, 1985, 1987; Mohn et al., 1991a; Zipfel et al., 1989). Immediate-early genes encode proteins that regulate later phases in G 1 including the induction of the delayed-early response genes. Delayed-early response genes are induced within a few hours of the partial hepatectomy, but their transcription also requires protein synthesis.

II. Immediate-Early Gene Expression in Hepatic Cells To begin to understand the complex interplay between the onset of cell growth and the maintenance of differentiated function during liver regeneration, one must understand the molecular bases of the events that occur during the hepatic cell cycle. A subset of the primary or immediate-early response genes and delayed-early response genes induced in the regenerating liver are likely to be central players in regulating this process. Early studies in regenerating liver showed that immediate-early genes like c-fos and c - m y c that are induced in mitogen-treated cells, are also rapidly induced in regen-

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erating liver (Goyette et al., 1984; Kruijer et al., 1986; Thompson et al., 1986). Initially, our laboratory specifically examined immediate-early gene expression in H35 cells treated with insulin (Taub et al., 1987). H35 cells represent a minimal deviation cell line that becomes quiescent under serumdeprived conditions and rapidly reenters the cell cycle in response to physiologic concentrations of insulin. We demonstrated that c-fos, c-myc, and ~3-actin are induced as immediate-early genes following insulin treatment of serum-deprived H35 cells (Mohn et al., 1990; Taub, et al., 1987). Thus, H35 cells represent an in vitro liver system with some properties of the regenerating liver in which the growth environment could be precisely defined. To further examine the spectrum of immediate-early gene induction in insulin-treated H35 cells and regenerating liver, we performed differential cDNA cloning studies in regenerating liver and insulin-treated H35 cells. We examined more than 1000 differentially expressed clones and found 52 nonoverlapping sequences that were induced in hepatic cells. Of these, 41 were novel genes or genes not previously known to be induced as immediateearly genes (Mohn et al., 1991a). We then extended these studies further by examining a large number of other known and novel immediate-early genes that were isolated by differential cDNA cloning of mitogen-treated fibroblasts. In total we identified more than 70 genes induced as immediateearly genes in regenerating liver and insulin-treated H35 cells. Of these, a small subset was induced as delayed-early genes in regenerating liver and as immediate-early genes in H35 cells. Table 1 presents an updated list of the immediate- and delayed-early genes expressed in the regenerating liver that have been examined in our laboratory. In the past few years, the sequence of a number of these novel genes has been determined in our laboratory or coincidentally in other laboratories. In addition, a number of immediateearly genes induced in regenerating liver has been identified in other labs including plasminogen activator inhibitor (PAI) (Schneiderman et al., 1993; Thornton et al., 1994), metallothionein (Tohyama et al., 1993), and adherens junction proteins (Gliick et al., 1992). Following the initial identification, members of this group of novel growthincluded genes that fell into interesting functional categories were studied in detail. These include novel transcription factors, proteins involved in signal transduction, potential growth factors, liver-specific immediate-early genes, and delayed-early genes (Figure 2). The fact that immediate-early genes represent diverse functional classes is not surprising, because cell growth requires simultaneous activation of multiple different intracellular pathways. The detailed study of any one particular gene may ultimately explain how that protein functions during liver regeneration and other types of cellular growth. In this way, proteins that have central roles in controlling liver growth may be identified.

Table 1 Gene Expression during Liver Regeneration

Pattern Growth regulated

Induction

Gene

Tissue; Hep, NP

Peak exp. (h)

Pattern

Induction

Peak 2 (h)

Gene

Tissue; Hep, NP

ATP synthase B2 microglobulin IGFl ubiquitin

Mult Mult Mult; Hep Mult

0-216 0-216 0-216 0-216

eck ClEBP alpha CL-34 CL-58

Mult Mult; Hep Liver Liver

48-216 60-216 2-216 2-216

c-fos IGFBP-1 Ikpa pip92( CL-14) MKP-1(RL-30) LRF-1 IP-10 CL-6 CL-73 CL-142 G6Pase(RL- 1) PC3(RL-98)

Mult Liver Mult Mult Mult Mult Mult; Hep Mult; Hep Mult; Hep Liver Liver Mult

1 2 1 2 1 2 0.5 3 2 2 0.5 2

Constitutive

IE

beta actin Gene 33 fibronectin junB c-jun JE RL-9 (viral env.protein) albumin PRL-1 (SL-314) RNR-1 (SL-332) egr-1 PEPCK KC c-myc c-ets C/EBPbeta CL-8 p68 RNA helicase(CL-36) CL-97 CL-180 dCBP(CL-183) CL-211 RL-27 RL-53 SL-339 SL-371

Mult Mult Mult Mult Mult Mult Liver; Hep Liver Mult; NP Liver Mult Liver; Hep Mult; Hep Mult Mult; Hep Mult Mult Mult Mult; Hep Liver Mult Mult Mult Mult Mult Mult

L

2 L

1 2 2 6 3 1

Max expression after growth phase

L

L

2 2 I

2 2 2 2 2 nd 1 nd 2 1 nd

Cell-cycle regulated

IE

48 36 48 48 48 48 42 48 48 36 60 48

SnkKinase(B28) Thrombospondinl(B10) Gly96 (M45) elF-2B (S48) HLH462 (S68) MyD 118 (Tr5) Tis7/PC4 (TT40) FK506BP(CL-87) nur77 DE

HRS (CL-4) MHC class 1 tropomyosin alpha FNR beta FNR pre-rnRNABP(CL-20) CL-22 HIBBA18(CL-31) CL-38 CL-39 CL-61 CL-120 FBRNP(CL- 141) CL-182 RL-104

Mult Mult Mult Mult Mult Mult

Mult

nd nd nd nd nd nd nd

nd Mult

nd

Mult Mult; Hep Mult Mult Mult Mult Mult Mult Mult; NP Liver Mult Mult Mult Mult Liver

6 8 24 24 24 24 24 24 24 8 8 24 24 24 24

C

DE S phase

SL-353

Mult

1

48

gGCS(CL-131)

Liver

1

24

Histone H 3

Mult

16-24

Undetectable

EGF EGF receptor IGFl receptor egr-2 alpha-fetoprotein

Pattern of expression, type of induction (IE, immediate-early; DE, delayed-early), tissue expression (Hep, hepatocytes; NP, nonparenchymal; Mult, multiple tissues), and peak expression in hours (Peak exp.) is given for each gene. References for most genes are provided in references of this chapter, and all papers are from Dr. Taub's laboratory except for the following: p68 RNA helicase (Lemaire and Heinlein, 1993); d cBP (Ito et al., 1993); SnkKinase (Simmons et al., 1992);M45, S48, S68, TT5, TT40, original clone numbers from Almendral et al. (1988);HLH462 (Christy et al., 1991);Tis7 (Varnum et al., 1989); FK506BP (Nelson et al., 1991); pre-mRNABP (Adams et al., 1993); HIBBAl8 (Adams et al., 1993); FBRNP (Takiguchi et a/., 1993); MKP-1 (Sun et al., 1993); G6Pase (Shelly et al., 1993); PC3 (Bradbury eta!., 1991);gGCS (Huang et d., 1993); CL-6 is also identical to a recently identified gene RESP-18 (Bloomquist et al., 1994).

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Figure 2 Categoriesof function of immediate-early and delayed-earlygenes.

III. Modification of Preexisting Transcription Factors Immediately Following Partial Hepatectomy Turns on Immediate-Early Genes As immediate-early genes are induced in a protein synthesis independent fashion, their transcription must be activated by transcription factors that are preexisting in hepatic cells. Little is presently known about what preexisting transcription factors are activated by signal transduction pathways in mitogen-stimulated cells. However, in analyzing the c-fos promoter, it was noted that a serum-response element was important in mediating c-los gene

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transcription following mitogen stimulation of 3T3 fibroblasts. Similar elements have been noted in the promoter regions of several other immediateearly genes, and it is now known that serum response factor (SRF) and a cofactor ets-like protein (ELK-l) which bind to the serum response element are activated by phosphorylation following mitogen stimulation (Marais et al., 1993). These transcription factors act in a coordinated fashion to activate c-fos gene transcription. It is likely that members of the SRF family are also activated during liver regeneration, but they have never been studied. Our laboratory also found that members of the Rel transcription factor family that preexist in hepatic cells are activated within minutes post partial hepatectomy. Clues to this activation were provided by the identification of one of the immediate-early genes expressed in the regenerating liver. This gene encodes inhibitor of kappa Bc~ IKB0t, a specific inhibitor of p65/RelA and other Rel family members (Grilli et al., 1993; Tewari et al., 1992). In examining the expression of Rel family members during liver regeneration, we discovered a post hepatectomy factor (PHF) that binds to a nuclear factor-kappa B (NF-KB) site and is induced within minutes after partial hepatectomy in the remnant liver in a protein synthesis independent manner; PHF is not induced by sham surgery. This is one of the most rapid responses that has been measured in the regenerating liver. Since its isolation, increased PHF activity has been used as the first indicator of signal transmission following growth factor addition in isolated hepatocytes and in liver perfusion studies. Further characterization of PHF indicates that it contains NF-KB subunits, but has some differences from standard p65-p50 NF-KB (Haber et al., 1995). Moreover, unlike the NF-KB DNA binding activity that is induced for extended times in many cells following cytokine or mitogen treatment, high molecular weight PHF complexes rapidly disappear 1 hr post partial hepatectomy and only lower molecular weight complexes persist. This occurs through physiologic nuclear proteolysis involving proteasomes. The target genes of PHF during liver regeneration are not known. However, genes such as IKB~ and K C which are immediate-early genes in regenerating liver and are known to be regulated by NF-KB subunits are likely to be induced by PHE Interestingly, NF-KB has been shown to interact with the CAAT enhancer binding protein (C/EBP) and other transcription factors to modulate the activity of certain promoters (Stein and Baldwin, 1993). As both active C/EBP and NF-KB subunits are found in the regenerating liver, a growth-induced factor (i.e., PHF) and a constitutive factor (i.e., C/EBP) could act in a coordinate fashion to regulate target genes in the regenerating liver. Another preexisting transcription factor that is activated by growth factor or interferon treatment of cells is signal transducer and activator of transcription I (SIF/Statl), which binds to the serum inducible element first identified in the c-fos promoter (Ruff-Jamison et al., 1993). This transcrip-

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tion factor family is activated by phosphorylation on a specific tyrosine residue that results in its nuclear translocation and ability to bind to DNA. Interestingly, epidermal growth factor (EGF) treatment of animals induces Stat activity in the liver. In unpublished studies, we have found that partial hepatectomy also induces Stat3 DNA binding within 30 min of partial hepatectomy (Cressman et al., 1995). Thus, it represents another early transcription factor that is activated immediately after the initial growth signal. PHF and Stat3 are activated by the earliest signaling pathways that trigger liver regeneration. Thus, they may be useful as markers to trace back to the triggering mechanism. They also are interesting as potential regulators of immediate-early growth response genes in regenerating liver.

IV. Induction Patterns of 70 Genes Following Partial Hepatectomy Define the Temporal Course of Liver Regeneration The large collection of growth-induced genes provides a useful tool for defining the temporal course of liver regeneration at the level of gene expression. Using dot blot technology, we denatured and immobilized more than 100 different cDNAs corresponding to immediate-early, delayed-early, and liver-specific genes on a nylon membrane (Haber et al., 1993). We then hybridized these filters with labeled remnant liver mRNA corresponding to different times post partial hepatectomy. The signal obtained for each immobilized cDNA correlates with the level of expression for that gene at the given time point. We then were able to use Northern blots to confirm the temporal course of expression of a subset of these genes. The kinetics of gene induction during liver regeneration is shown in Figure 3. Approximately 12 hr after partial hepatectomy, S phase is entered by some hepatocytes and peak DNA synthesis occurs at 24 hr. DNA synthesis peaks significantly later in nonparenchymal cells. The major portion of the mass of the liver is reconstituted within 72 hr and the process stops completely after 7 to 10 days (Grisham, 1962). We found that many of these "early" genes are expressed for extended periods during the hepatic growth response (Haber et al., 1993). Several patterns of expression of immediateearly, delayed-early, and liver-specific genes were defined during the 9-day period post partial hepatectomy. One pattern of induction parallels the major growth period of the liver that ends at 60 to 72 hr post partial hepatectomy. A second pattern has two peaks coincident with the first and second G 1 phases of the two hepatic cell cycles. A third group which in-

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Figure 3 Representation of patterns of regulated gene expression during liver regeneration. The pattern of gene expression is indicated for growth-regulated genes (e.g., f3-actin), cell cycle regulated genes (e.g., IGFBP-1), and genes with maximal expression after the major growth phase (e.g., C/EBPe~). Hours after partial hepatectomy and patterns of DNA synthesis in hepatocytes (H) and nonparenchymal cells (NP) and the regrowth of liver mass are indicated. cludes liver-specific genes such as C/EBPcx shows maximal expression after the growth period. Although the peak in DNA synthesis in nonparenchymal cells occurs 24 hr later than in hepatocytes, most of the genes studies demonstrate similar induction in both cell types. This finding suggests that the G0/G 1 transition occurs simultaneously in all cells in the liver, but that the G1 phase of nonparenchymal cells may be relatively prolonged. These studies define the temporal boundary between proliferation and return to quiescence in the post partial hepatectomized liver. For regeneration to be precisely carried out, multiple hepatic cell types must proliferate in a coordinated fashion, and it is likely that cell-cell interactions and paracrine effects of growth factors are involved. Therefore, it is interesting to compare the immediate-early gene response in the two cell types. We found that the majority of immediate-early genes induced in the regenerating liver are expressed in a cell type independent fashion, but

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approximately one-third show cell type restricted expression and are induced primarily in hepatocytes.

V. Transcription Factors Induced in the Regenerating Liver Members of many different families of transcription factors are induced as immediate-early genes in several different cell types. Transcription factors induced as immediate-early genes in the regenerating liver include members of the large Jun-Fos leucine-zipper family, the Rel family, the nuclear receptor family, the helix-loop-helix family, and the zinc finger family. Immediateearly genes in the regenerating liver and insulin-treated H35 cells include many members of the transcription factor families that are induced differently in mitogen-treated fibroblasts (Mohn et al., 1990). For instance, in mitogen-treated fibroblasts the zinc finger genes, egr-1 and egr-2, are induced, but in H35 cells and regenerating liver, egr-1 is induced but not egr-2. Likewise, in the large Jun-Fos family of transcription factors, c-fos and all of the jun family members are induced in the two liver systems. However, many of the fos family members (e.g., fra-1, fosB, fra-2) that are immediateearly genes in mitogen-treated fibroblasts are not induced in the two liver systems. Instead, liver regeneration factor-1 (LRF-1), another member of this family that was identified in our laboratory, is highly induced in the two experimental liver systems we have used (discussed below) (Hsu et al., 1991, 1992). These findings imply that similar transcription factors responsible for transactivating a specific subset of immediate-early genes may be present in the two liver systems, and that a similar intracellular milieu is responsible for immediate-early gene induction in both liver systems. A. The LRF-1/JunB Story Members of the Jun and Fos families of transcription factors are thought to have a role in activating the transcription of delayed-early genes expressed subsequently in the growth response. In regenerating liver and insulintreated H35 cells, LRF-1, junB, c-jun and c-los among jun/fos/LRF-1 family members are induced post partial hepatectomy. We first identified LRF-1 through differential screening of a regenerating-liver cDNA library (HSU et al., 1991). We found that LRF-1 is a rapidly and highly induced novel gene encoding a 21-kDa leucine-zipper protein. LRF-1 is also highly induced following insulin-treatment of H35 cells, and is induced at a lower level in mitogen-treated fibroblasts. As such, it may be a more important regulator of hepatic than nonhepatic growth.

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LRF-1 has no homology with other leucine-zipper proteins outside the basic leucine-zipper domains. LRF-1 alone can bind DNA, but it preferentially forms heteromeric complexes with c-Jun and JunB and does not interact with c-Fos. In solution, it binds with highest affinity to cAMP response elements but also has affinity for related sites like activator protein-1 (AP-1) elements (i.e., phorbol ester response elements). Early cotransfection studies showed that LRF-1 in combination with c-Jun strongly activates an AP-1 element-containing promoter (Hsu et al., 1991). The induction of the LRF-1 gene in regenerating liver and insulin-treated H35 cells greatly increases the potential variety of heterodimeric combinations of leucine-zipper transcription factors. While LRF-1 mRNA is rapidly induced in the absence of protein synthesis, its peak induction is later than that of c-fos mRNA, suggesting that LRF-1 may regulate responsive genes at a later point in the cell cycle. To further explore the coordinate temporal expression of LRF-1, JunB, c-Jun and c-Fos, we examined the levels of these proteins in hepatic cells following partial hepatectomy or insulin treatment (Hsu et al., 1992). We found that the peak levels of c-Fos and c-Jun occur early in G1, within 1 hr of the stimulus and that the peak levels of JunB and LRF-1 occur later in G 1. Using immunoprecipitations with specific antisera, we found that in insulintreated H35 cells, high levels of c-Fos/c-Jun, c-Fos/JunB, LRF-1/c-Jun, and LRF-1/JunB complexes are present for several hours after the G0/G 1 transition. The relative level of LRF-1/JunB complex increases during G 1. We found dramatic differences in promoter-specific activation by LRF-1 and c-Fos containing complexes. LRF-1 in combination with either Jun protein strongly activates a cyclic AMP response element-containing promoter which c-Fos/Jun does not activate. LRF-1/c-Jun, c-Fos/c-Jun, and c-Fos/JunB activate specific AP-1 and activator of transcription factor (ATF) site-containing promoters. In contrast, LRF-1/JunB potently represses c-Fos and c-Junmediated activation of these promoters (Figure 4). Repression is dependent on a region in LRF-1 that includes amino acids just proximal to the basic domain and the DNA binding domain. We extended these studies to examine the activity of JunB which interacts with LRF-1 to mediate inhibition of AP-1 sites (Hsu et al., 1993). We identified separate regions of JunB required for cAMP element mediated transactivation and AP-1/ATF site-mediated repression. Deletion analysis showed that the region involved in transactivation function is highly conserved among all Jun family members, and corresponds to activator domain (A1) of c-Jun. In contrast, repression is maximal in the presence of both the DNA binding domain and a region proximal to the basic region that is highly divergent among Jun proteins. The regions required for repression in LRF-1 and JunB are positioned similarly just upstream of the DNA binding domain. It is

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Figure 4 Model of the actions of LRF-1/JunB heterodimers in regulation of target genes controlled by AP-1 and cAMP (CRE) promoter elements.

likely that the configuration of the LRF-1/JunB heterodimer varies depending on whether it is interacting with cAMP or AP-1 response elements, thereby determining if the heterodimer will be an activator or repressor. Functional distinctions between Jun proteins during the growth response may be accounted for by promoter-specific activation and repression mediated by regional differences in Jun family proteins. Thus, through complex interactions among LRF-1, JunB, c-Jun, and c-Fos, control of delayed-early gene expression may be established for extended time during the G 1 phase of hepatic growth (Figure 5). As the relative level of LRF-1/JunB complexes increases post partial hepatectomy and following insulin treatment of H35 cells, the c-Fos/Jun-mediated ATF and AP-1 site activation is likely to decrease with simultaneous increased transcriptional activation of the many liver-specific genes whose promoters contain cyclic AMP response element sites. In this way the liver-specific phenotype of the liver or H35 cells can be maintained during the growth response. It is likely that Jun/Fos/LRF-1 interact with other transcription factors such as members of the steroid receptor family and constitutively expressed hepatic factors in regulating target genes during the hepatic growth response. B. RNR-1, a Novel Nuclear Receptor That Acts through the NGFI-B Half-Site

During screening of a subtracted cDNA library of immediate-early genes induced during liver regeneration, we identified a novel member of the

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Figure 5

Model of the regulation of delayed-early genes containing AP-1 and CRE enhancers by LRF-1, JunB, c-Jun, and c-Fos heterodimers during the G 1 phase of liver regeneration and insulin-induced mitogenesis in H35 cells. Various heterodimers are found at the indicated times post partial hepatectomy. Relative activation ("ON," "OFF, .... DECREASED") of delayedearly gene promoters is indicated (adapted from Hsu et al., 1993).

thyroid/steroid receptor superfamily, regenerating liver nuclear receptor-1 (RNR-1) (Scearce et al., 1993). This gene is not expressed in quiescent liver but is rapidly induced following partial hepatectomy and is specific to hepatic growth as it is not induced in other mitogen-treated cells; RNR-1 is also expressed in brain. A full-length cDNA clone of RNR-1 encodes a 66kDa 597-amino acid protein that is highly homologous to nerve growth factor inducible clone B/nuclear receptor clone 77 (r-NGFI-B/m-Nur77) particularly in the DNA binding (94%) and putative ligand binding (59%) domains. Interestingly, m-nur77 is also induced as an immediate-early gene in regenerating liver. RNR-1 specifically binds to the NGFI-B DNA half-site and forms a complex very similar in size to the m-Nur77 complex, suggesting that RNR-1 also may bind as a monomer. Consistent with this finding, the A box region important in mediating half-site binding is 100% conserved between RNR-1 and r-NGFI-B/m-Nur77. Both RNR-1 and r-NGFIB/Nur77 strongly transactivate a reporter driven by a consensus r-NGFIB/m-Nur77 binding site, and their effect together is additive. As both the RNR-1 and r-NGFI-B/m-nur77 genes are induced during liver regeneration, it is possible that RNR-1 acts concomitantly with r-NGFI-B/m-Nur77 in regulating the expression of delayed-early genes during liver regeneration. These factors are also intriguing because they can cooperate with other transcription factor families to modulate transactivation of specific promoters, and they are regulated by specific ligands, many of which have not yet been defined. Such ligands could be, however, important unidentified circulating factors that help regulate regeneration.

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VI. Immediate-Early Genes Involved in Signal Transduction Signal transduction pathways are rapidly activated following mitogen or cytokine addition to cells. The activity of signal transduction molecules is frequently controlled by phosphorylation. For instance, when a growth factor binds to a tyrosine kinase receptor, the tyrosine kinase receptor becomes activated and autophosphorylates itself and may phosphorylate other downstream molecules. Active tyrosine phosphorylated proteins may interact with other downstream targets via Src homology 2 (SH2) domains that interact with the tyrosine phosphate residue. Activation of downstream modulators such as Ras, Raf, mitogen-activated protein (Map) kinase, and phosphoinositol kinase then ensues. Recently, a subset of these pathways has been dissected and is briefly summarized (Figure 6) (Blenis, 1993). Tyrosine kinases and tyrosine phosphorylated substrates can also be regulated by specific tyrosine phosphatases (Brautigan, 1992). In the regenerating liver, we have identified two immediate-early genes, map kinase phosphatase (MKP-1) (Sun et al., 1993), and phosphatase of regenerating liver (PRL-1) (Diamond et al., 1994) that encode distinct tyrosine protein phosphatases.

A. PRL-1, a Member of a Novel Class of Protein-Tyrosine Phosphatases P R L - 1 is a particularly interesting immediate-early gene because it is in-

duced in mitogen-stimulated cells and regenerating liver, but is constitutively expressed in insulin-treated rat H35 hepatoma cells which otherwise show normal regulation of immediate-early genes (Diamond et al., 1994). Sequence analysis revealed that P R L - 1 encodes a novel 20-kDa protein that contains the eight amino acid consensus protein-tyrosine phosphatase (PTPase) active site. PRL-1 is able to dephosphorylate phosphotyrosine substrates, and mutation of the cysteine residue in the active site abolishes this activity. Because P R L - 1 has no homology to other PTPases outside the active site, it is a member of a new class of PTPases. PRL-1 migrates as a 21kDa protein, and is located primarily in the triton-insoluble fraction of the cell nucleus. Stably transfected cells which overexpress PRL-1 demonstrate altered cellular growth and morphology, and a transformed phenotype. Thus far, we have not identified specific intracellular targets for the PRL-1 phosphatase. Tyrosine phosphatases, particularly those of the unique classes such as PRL-1, MKP-1, and the cyclin, cdc25, may have very specific intracellular targets (Figure 6). Several different sites have been identified in

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Figure6 Model of signal transduction pathway and potential sites of interaction of phosphatases (PTPases).

the signal transduction pathway where tyrosine phosphorylation is important. For instance, cdc25 is a regulator of cdc2 that dephosphorylates cdc2 on a specific tyrosine and threonine residue resulting in its activation (Dunphy and Kumagai, 1991). The cyclin, cdc2, and related molecules bind to specific cyclins, and in the active state are important in cell cycle transition between G1/S and G2/M (see Chapter 8). Thus, cdc25 is important in mediating transition through G1/S and G2/M. Likewise, MKP-1 appears to specifically dephosphorylate Map kinase and is important in turning off the initial signal transduction cascade. Because MKP-1 is highly induced in regenerating liver with a peak in expression at 30 min post partial hepatectomy, it is likely that this pathway is important in the regenerating liver. PRL-1 acts at a distinct site in this pathway, or in as yet unidentified pathways. Like MKP-1, it appears that PRL-1 is important in normal cellular growth control during mitogenesis. The specific role of PRL-1 in hepatic growth is implied by the very high level of its expression in liver regeneration and development, and in hepatoma cells. Thus, it is likely that PRL-1 plays a central role in regulating growth during liver regeneration and the development of some hepatic malignancies.

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VII. Immediate-Early Genes That Are Secreted Proteins Several identified immediate-early genes encode secreted proteins. Some of these are known cytokines or growth factors and others are secreted proteins of unknown function. The importance of such proteins in modulating the growth response is that they may be acting as paracrine or autocrine factors in directly stimulating cell growth. Immediate-early genes identified in regenerating liver encode the intercrines, JE, KC, (clone names-PDGF inducible), and interferon inducible protein (IP-10). Their function in liver regeneration is not known but interestingly, expression of both KC and IP-10 is restricted to hepatocytes (Haber et al., 1993). Potentially, they could be important in recruitment of other cells to the regenerating liver or in growth stimulation of neighboring cells. Of the secreted proteins induced in the regenerating liver, insulin-like growth factor binding protein-1 (IGFBP-1) is the most highly induced. IGFBPs are important modulators of the insulin-like growth factors (IGF) that may have both positive and negative effects on the ability of IGFs to stimulate cell growth. One of the most abundant liver-specific immediate-early genes is IGFBP-1 (Mohn et al., 1991b), which is rapidly induced more than 100-fold with peak expression at 1 hr post partial hepatectomy. In a different study, a more modest elevation of IGFBP-1 gene expression was seen after partial hepatectomy in fasted rats (Ghahary et al., 1992). IGFBP-1 is similar to other IGFBPs which have been shown to have important roles in modulating the actions of IGFs (Andress and Birnbaum, 1992; Brinkman et al., 1988; Conover, 1992; Elgin et al., 1987; Hsu and Olefsky, 1992). In particular, IGFBP-1 has been shown to either enhance or inhibit the mitogenic effect of IGFs in certain tissues. We have shown that the 1GFBP-1 gene is transcriptionally activated during liver regeneration, but its transcription is rapidly depressed by insulin treatment of hepatic cells (Mohn et al., 1991b). IGFBP-1 is highly expressed in the liver during fetal development, and is normally tissue restricted with high levels of expression only in the liver and kidney (Mohn et al., 1991b). During liver regeneration, there is a second peak of 1GFBP-1 expression at 36 to 60 hr that corresponds to the second round of cell division, and there appears to be expression in both parenchymal and nonparenchymal cell types (Haber et al., 1993). During liver regeneration, the level of hepatic IGF1 mRNA remains constant, and insulin-like growth factor 1 receptor (IGFlr) gene expression is virtually undetectable (Mohn et al., 1991b). However, because IGFBP-1 increases, the amount of bioactive IGF1 available to tissues may be altered. The mechanism of IGFBP-1 action is postulated to be through modulating the delivery of IGF1 to the IGFlr. In the liver, these receptors are present primarily

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on nonparenchymal cells which therefore may be the targets for IGFBP-1/ IGF1 actions during regeneration. As previously stated, IGFBP-1 may play an important role in both liver growth and metabolism. To begin to examine the regulation of this gene, we cloned and sequenced the entire mouse IGFBP-1 gene (Lee et al., 1994). Its structure is highly similar to the human gene, and in addition to the exonic regions, the two genes are highly conserved within specific regions in the promoter and first intron. Analysis of this conservation allows us to predict important regulatory sites that define the tissue specific and insulinmediated regulation of the gene, as well as to identify potential sites that might be important for the transcriptional induction during liver regeneration. The mouse gene is located on mouse chromosome 11, and is found at the boundary between regions in the mouse genome syntenic to human chromosomes 22 and 7. We have found IGFBP-1 mRNA in both parenchymal and nonparenchymal RNA following partial hepatectomy. By in situ hybridization to IGFBP-1 mRNA in regenerating rat liver tissue, IGFBP-1 transcripts were found to be present in multiple cell types. We find that IGFBP-1 gene induction following partial hepatectomy is paralleled by protein expression, and that the IGFBP-1 protein is found only in hepatocytes post partial hepatectomy. Unlike IGFBP-1 mRNA, serum levels of IGFBP-1 are elevated for a relatively short time with a peak at 2 to 3 hr post partial hepatectomy. Increased levels of IGFBP-1 could be important in modulating IGF1 effects on metabolism and growth during liver regeneration. Studies that modulate the level of IGFBP-1 in the liver during development and regeneration will be an important means of determining its function in the hepatic growth response.

VIII. Liver-Specific Immediate-Early Genes" Relationship to the Maintenance of Hepatocyte Differentiation and Metabolism During liver regeneration the liver must maintain normal glucose homeostasis despite the abrupt loss of two-thirds of its mass. As a result, immediately following partial hepatectomy, insulin levels rapidly fall and glucagon levels rise (Bucher et al., 1975; Shelly et al., 1993). Thus, it is not surprising that some liver-restricted immediate-early genes are important in maintaining metabolic homeostasis. For instance, two immediate-early genes restricted to liver and kidney encode phosphoenol pyruvate carboxykinase (PEPCK) and the recently identified glucose-6-phosphatase ( G6Pase) gene which are important in gluconeogenesis (Haber et al., 1995; Shelly et al., 1993). Likewise, IGFBP-1 has been proposed to have some role in

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metabolic homeostasis. Expression of all three of these genes is elevated in diabetics that lack insulin. A sign for the potential involvement of a particular gene in glucose homeostasis is indicated by distinct differences in its regulation in insulin-treated H35 cells and regenerating liver (Diamond et al., 1993; Mohn et al., 1990, 1991a,b). These differences include suppression of expression of two genes, IGFBP-1 and PEPCK by insulin and their induction in regenerating liver. When insulin levels return to normal a few hours post partial hepatectomy, IGFBP-1, G6Pase, and PEPCK expression also return to normal. Thus, immediate-early genes that are specific to the hepatic growth response may also have some role in the maintenance of hepatic function during liver regeneration. A. Identification of CL-6 Some genes were more highly induced in insulin-treated H35 cells than in the regenerating liver (Diamond et al., 1993). Some of these genes, including hepatic Arg-Ser protein (HRS), are induced as delayed-early genes in the regenerating liver (discussed below). In the differential screening analyses we performed in insulin/cycloheximide treated H35 cells, 12% of over 200 differentially expressed cDNA clones represented HRS clones and 25% represented CL-6 clones (clone name-insulin inducible). These clones were never isolated from differential or subtraction screening of regenerating liver libraries. Both mRNAs showed low level immediate-early induction in serum-treated 3T3 fibroblasts, but were not isolated from 3T3 cell cDNA libraries (Almendral et al., 1988) despite extensive differential screening analyses. Interestingly, CL-6 mRNA shows immediate-early expression in H35 cells, regenerating liver and 3T3 fibroblasts, while HRS shows immediateearly induction in H35 cells and delayed-early (protein synthesis dependent) induction in regenerating liver. CL-6 is much more highly induced in hepatic cells including the regenerating liver than it is in mitogen-treated fibroblasts. Sequence analysis of CL-6, the most abundant insulin-induced gene, resulted in the identification of a highly hydrophobic hepatic protein. CL-6 demonstrates hepatocyte and epithelial organ-specific expression. It is induced in the regenerating liver and fetal liver in the perinatal period (Haber et al., in press). CL-6 migrates as a 43-kDa protein following in vitro translation of synthetic CL-6 mRNA, and on immunoblots of H35 and regenerating liver extracts. Interestingly, although CL-6 mRNA shows strong induction in H35 cells and regenerating liver, the protein level is constitutive in H35 cells, but increases during liver regeneration. Although it is highly hydrophobic, it is not yet clear if CL-6 is a membrane-associated protein. However, because of its high level expression in hepatic tissues, CL-6 is likely to have a role in the tissuespecific aspect of cellular growth, perhaps being involved in the mainte-

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nance of normal liver architecture or metabolism during regeneration and fetal development.

IX. Immediate-Early Genes in H35 Cells That Are Expressed as Delayed-Early Genes in Regenerating Liver Another group of genes shows immediate-early expression in insulin-treated H35 cells, but delayed-early expression in regenerating liver (Table 1) (Diamond et al., 1993). In general, the peak expression in insulin-treated H35 cells occurs a few hours before peak expression in regenerating liver, but later than the induction of many other immediate-early genes (Diamond et al., 1993; Mohn et al., 1990, 1991a). In regenerating liver, the earliest elevation of delayed-early expression occurs at 3 hr post partial hepatectomy, while peak expression occurs between 8 and 24 hr, consistent with late G 1 to mid S phase. Little is known about the delayed-early growth response genes, the kinetics of their induction, or their role as part of the regulatory cascade that results in cell growth (Lemaire and Heinlein, 1993). However, the finding that some cyclins are members of this class establishes the importance of delayed-early genes in cell cycle regulation (Lanahan et al., 1992; Lu et al., 1992). Cyclins are important in cell cycle events (Sherr, 1993). The timing of the induction of the delayed-early genes we have identified indicates that they are first expressed approximately 3 hr post partial hepatectomy in a protein synthesis dependent fashion; however, peak expression frequently occurs several hours later. Therefore, whatever stimuli are responsible for elevating the level of delayed-early gene mRNAs, they are present for extended times during G1 and perhaps continue into the S phase. A. Delayed-Early Genes That Encode RNA Binding Proteins Given the dramatic increase in RNA production during late G1, proteins that control RNA processing are likely to have important roles in cell cycle regulation. Thus far, three delayed-early genes that we have identified fall into this family including HRS, fetal brain ribonucleic protein (FBRNP), and pre messenger R N A binding protein (m-RNABP) (Adams et al., 1993; Diamond et al., 1993; Takiguchi et al., 1993). H R S is of particular interest because it is highly induced by insulin, shows delayed-early induction in regenerating liver, and has two different transcripts [i.e., HRS-Short Form(SF) (1.7 kb) and HRS-Long Form(LF) (3.2 kb)] that demonstrate different temporal patterns of induction. The complete sequence of HRS-SF was determined and an alignment between

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HRS-SF and the protein data bank demonstrated that HRS is a member of the family of regulators of alternative pre-mRNA splicing. It is most similar to Drosophila protein splicing factor, serine arginine p55 (B52/SRp55), and human splicing factor 2/alternative splicing factor (SF2/ASF), and appears to be identical to splicing factor, serine arginine p40 (SRp40) (Ge et al., 1991; Hedley and Maniatis, 1991; Krainer et al., 1991; Li and Bingham, 1991; Mattox et al., 1992; Mayeda et al., 1992; Mayeda and Krainer, 1992; Zahler et al., 1992, 1993a,b). These proteins are structurally similar with two amino RNA binding domains and a carboxyl Arg/Ser domain. These proteins have been shown to function in in vitro splicing assays, and are members of the larger class of Arg/Ser-rich proteins that contain RNA binding regions including the small splicing RNAs U1 and U2, small nuclear ribonucleoproteins (snRNPS) polyadenylation factors, and other splicing factors such as sex lethal (Sxl) and Drosophila developmental splicing factor (Tra-2) which are important in developmental regulation. These Arg/Ser domain proteins target to the speckled subnuclear compartment where mRNA processing occurs. We found a low level of HRS mRNA in most tissues except spleen and thymus in which HRS mRNA is abundant and is composed of both mRNA forms. Interestingly, unlike other tissues, spleen and thymus contain actively proliferating cells. Another interesting feature of HRS expression is the delayed appearance of a higher molecular weight RNA species (HRS-LF). Several splicing regulators have been found to regulate the production of their own spliced forms. For example, in the Drosophila su(w)a system this results in the accumulation of larger differentially processed su(w)a mRNA molecules in which the open reading frame is disrupted by the alternatively spliced exon (Hedley and Maniatis, 1991). Likewise, it seems possible that HRS-SF autoregulates HRS pre-mRNA processing resulting in the production of HRS-LF mRNA which appears later than HRS-SF mRNA following insulin treatment or partial hepatectomy. We identified several longer FIRS cDNA clones derived from HRS-LF mRNA. These cDNAs have an approximately 500 bp "insert" with stop codons in all three reading frames that disrupt the open reading frame of HRS-SF mRNA (Figure 7) (Diamond et al., 1993). This "insert" (E'2) and a smaller cDNA insert (E'I) found in the 5' untranslated region of HRS-SF cDNA may represent alternative exons. In fact, genomic cloning and sequence analyses indicate that E'2 appears to be an alternative exon because it is flanked in the genome by consensus splice acceptor and donor sequences (Figure 7). HRS has all of the features of a RNA binding protein that regulates alternative pre-mRNA splicing. In the absence of such regulators, default splicing pathways are chosen based on recognition of consensus splice site sequences by the general splicing machinery. Regulators like Tra-2, Sxl, SF2/ASF, and B52/SRp55 may function partly by binding to specific sequences in the pre-mRNA to prevent or to enhance splicing at specific

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Figure 7 Modelof hypothetical interaction of hepatic Arg-Serprotein HRS with its own premRNA. The structure of the HRS gene is shown with exons surrounding the E'2 exon alternative exon that is present in HRS-LF mRNA. Splice donor (SD) and acceptor (SA) sites are indicated. Following interaction between HRS and its pre-mRNA, alternative splicing with retention of the E'2 exon could occur resulting in HRS-LF mRNA.

positions. For instance, HRS could autoregulate the production of H R S - L F mRNA by interacting with the pre-mRNA and regulating splice site selection. The interesting question is what are the targets of HRS during hepatic growth. I would propose that one way to regulate cellular events is by activating alternative splicing of existent hepatic mRNAs. For instance, fibronectin mRNA is induced during liver regeneration, and apparently, alternatively spliced forms of fibronectin mRNA are produced (Enrich et al., 1988; Huh and Hynes, 1993). Different forms of fibronectin may be important in defining the hepatic architecture during regeneration. Potentially, fibronectin pre-mRNA could be a target for the HRS splicing factor, resulting in the production of alternatively spliced forms of fibronectin during liver regeneration. The identification of delayed-early genes involved in RNA production is consistent with previous studies that have demonstrated that rRNA, mRNA, and protein levels increase dramatically during the late phase of G1. This is compatible with one of the major functions of G 1 which is to double cell mass. The identification of several RNA processing proteins induced in the regenerating liver that are also insulin-regulated is particularly intriguing because of the involvement of insulin in stimulating mRNA, rRNA, and protein synthesis--major components of insulin-regulated growth (Hutson et al., 1987; Jefferson, 1980; Peavy et al., 1985). Because insulin has been proposed as a major regulator of growth during liver regeneration, and because insulin levels renormalize by the time RNA processing protein genes are expressed, their expression could be tied to insulin regulation of growth during liver regeneration.

X. Conclusions In this chapter, I have tried to demonstrate how the study of immediateearly and delayed-early growth response genes induced in the regenerating

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Figure 8 Summaryof proposed sites of cell cycle regulation by various immediate-early and delayed-early gene products during liver regeneration.

liver can lead to new insights into the central questions regarding liver regeneration. Specifically, analysis of the temporal expression of a large group of these genes defines the time course of liver regeneration and identifies the liver-specific aspects of the growth response. The study of preexisting transcription factors that are activated by partial hepatectomy will provide insights into the triggering mechanisms (Figure 8). Examination of the interplay of growth-induced and constitute transcription factors can provide insight into how the liver maintains its hepatic phenotype during regeneration. Analysis of signal transduction molecules will lead to a greater understanding of the specific regulation of growth processes and the induction of abnormal hepatic growth that occurs in malignancies. Study of liver-specific and secreted proteins induced in the primary growth response provides insight into how the liver maintains its metabolic function and architecture during regeneration. Finally, analysis of certain delayed-early genes allows insight into regulation of later phases of G 1 which may be accomplished not just by transcriptional control but by post-transcriptional regulation of the form of mRNA that are produced. Liver regeneration is a complex process that requires the interplay of many different cellular events. It is only through the analysis of each of these events on a individual basis that we may begin to dissect this complex process, and to form the larger picture of how the liver regenerates.

Acknowledgments I would like to thank the members of my laboratory past and present who were responsible for the data reviewed in this chapter including Kenneth L. Mohn, Thomas M. Laz,Jui-Chou Hsu, Dinesh S. Tewari, Manorama Tewari, Barbara A. Haber, Robert H. Diamond, L. Marie Scearce, Simon E. Chin, Linda E. Greenbaum, Drew E. Cressman, Vashti Miles, Jehyuk Lee, and Leyla Naji. Work in this chapter was in part supported by grants from the Juvenile Diabetes Foundation and NIH (DK44237).

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References Adams, M. D., Soares, M. B., Kerlavage, A. R., Fields, C., and Venter, J. C. (1993). Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library. Nat. Genet. 4, 373-380. Almendral, J. M., Sommer, D., MacDonald-Bravo, H., Burckhardt, J., Perera, J., and Bravo, R. (1988). Complexity of the early genetic response to growth factors in mouse fibroblasts. Mol. Cell. Biol. 8, 2140-2148. Andress, D. L., and Birnbaum, R. S. (1992). Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action. J. Biol. Chem. 267 22467-22472. Blenis, J. (1993). Signal transduction via the Map kinases: Proceed at your own RSK. Proc. Natl. Acad. Sci. U.S.A. 90, 5889-5892. Bloomquist, B. T., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1994). RESP18, a novel endocrine secretory protein transcript, and four other transcripts are regulated in parallel with pro-opiomelanocortin in melanotropes. J. Biol. Chem. 269, 9113-9122. Bradbury, A., Possenti, R., Shooter, E. M., and Tirone, E (1991). Molecular cloning of PC3, a putatively secreted protein whose mRNA is induced by nerve growth factor and depolarization. Proc. Natl. Acad. Sci. U.S.A. 88, 3353-3357. Brautigan, D. L. (1992). Great expectations: protein tyrosine phosphatases in cell regulation. Biochim. Biophys. Acta 1114, 63-77. Brinkman, A., Groffen, C., Kortleve, D. J., Geurts van Kessel, A., and Drop, S. L. S. (1988). Isolation and characterization of cDNA encoding the low molecular weight insulin-like growth factor binding protein (IGFBP-1). EMBO J. 7, 2417-2423. Bucher, N. L. R., and Swaffield, M. N. (1975). Regulation of hepatic regeneration in rats by synergistic action of insulin and glucagon. Proc. Natl. Acad. Sci. U.S.A. 72, 11571160. Christy, B. A., Sanders, L. K., Lau, L. E, Copeland, N. G., Jenkins, N. A., and Nathans, D. (1991). An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene. Proc. Natl. Acad. Sci. U.S.A. 88, 1815-1819. Conover, C. A. (1992). Potentiation of insulin-like growth factor (IGF) action by IGF-binding protein-3: Studies of underlying mechanism. Endocrinology (Baltimore) 130, 31913199. Cressman, D. E., Diamond, R. H., and Taub, R. (1995). Rapid activation of the Stat3 transcription complex in liver regeneration. Hepatology (Baltimore) 21, 1443-1449. Diamond, R. H., Du, K., Lee, V. M., Mohn, K. L., Haber, B. A., Tewari, D. S., and Taub, R. (1993). Novel delayed-early and highly insulin-induced growth response genes. J. Biol. Chem. 268, 15185-15192. Diamond, R. H., Cressman, D. E., Laz, T. M., Abrams, C. S., and Taub, R. (1994). PRL-1 a unique nuclear protein tyrosine phosphatase, affects cell growth. Mol. Cell. Biol. 14, 37523762. Dunphy, W. G., and Kumagai, A. (1991). The cdc25 protein contains an intrinsic phosphatase activity. Cell (Cambridge, Mass.) 67, 189-196. Elgin, R. G., Busby, W. H., and Clemmons, D. R. (1987). An insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF1. Proc. Natl. Acad. Sci. U.S.A. 84, 3254-3258. Enrich, C., Evans, W. H., and Gahmberg, C. G. (1988). Fibronectin isoforms in plasma membrane domains of normal and regenerating rat liver. FEBS Lett. 228, 135-138. Fausto, N. (1994). Liver Regeneration, In "The Liver: Biology and Pathobiology" (I. M. Arias, W. B. Jakoby, H. Popper, D. Schachter, and D. A. Shafritz, eds.), pp. 1059-1084. Raven, New York.

94

Rebecca Taub

Fausto, N., and Mead, M. E. (1989). Regulation of liver growth: Proto-oncogenes and transforming growth factors Lab. Invest. 60, 4-13. Ge, H., Zuo, P., and Manley, J. L. (1991). Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators. Cell (Cambridge, Mass.) 66, 373-382. Ghahary, A., Minuk, G. Y., Luo, J., Gauthier, T., and Murphy, L. J. (1992). Effects of partial hepatectomy on hepatic insulin-like growth factor binding protein-1 expression. Hepatology (Baltimore) 15, 1125-1131. Gliick, U., Fernandez, J. L. R., Pankov, R., and Ben-Ze'ev, A. (1992). Regulation of adherens junction protein expression in growth-activated 3T3 cells and in regenerating liver. Exp. Cell Res. 202, 477-486. Goyette, M., Petropoulos, C., Shanko, P., and Fausto, N. (1984). Regulated transcription of c-Ki-ras and c-myc during compensatory growth of rat liver Mol. Cell. Biol. 4, 1493-1498. Grilli, M., Chiu, J. J.-S., and Lenardo, M. J. (1993). NF-KB and Rel: Participants in a multiform transcriptional regulatory system. Int. Rev. Cytol. 143, 1-62. Grisham, J. W. (1962). A morphologic study of deoxyrobonucleic acid synthesis and cell proliferation in regenerating rat liver: Autoradiography with thymidine-3H. Cancer Res. 22, 842-849. Haber, B. A., Mohn, K. L., Diamond, R. H., and Taub, R. (1993). Induction patterns of 70 genes during nine days after hepatectomy define the temporal course of liver regeneration. J. Clin. Invest. 91, 1319-1326. Haber, B. A., Chin, S., Chuang, E., Buikhuisen, W., Naji, A., and Taub, R. (1995). High levels of glucose-6-phosphtase gene and protein expression reflect an adaptive response in proliferating liver and diabetes. J. Clin. Invest. in press. Haber, B. A., Naji, L., Cressman, D. E., and Taub, R. (1995). Coexpression of liver-specific and growth-induced genes in perinatal and regenerating liver: Attainment and maintenance of the differentiated state during cell growth. Hepatology (Baltimore), in press. Hedley, M. L., and Maniatis, T. (1991). Sex-specific splicing and polyadenylation of dsx premRNA requires a sequence that binds specifically to tra-2 protein in vitro. Cell (Cambridge, Mass.) 68, 365-375. Herschman, H. R. (1991). Primary response genes induced by growth factors and tumor promoters. Annu. Rev. Biochem. 60, 281-319. Hsu, D., and Olefsky, J. M. (1992). Characterization of insulin-like growth factor (IGF) binding proteins and their role in modulating IGF-I action in BHK cells. J. Biol. Chem. 267, 25576-25582. Hsu, J.-C., Laz, T. M., Mohn, K. L., and Taub, R. (1991). LRF-1, a novel leucine-zipper protein rapidly and highly induced in regenerating liver. Proc. Natl. Acad. Sci. U.S.A. 88, 35113515. Hsu, J.-C., Bravo, R., and Taub, R. (1992). Interactions between LRF-1, JunB, c-Jun and c-Fos define a regulatory program in the G 1 phase of liver regeneration. Mol. Cell. Biol. 12, 4654-4665. Hsu, J.-C., Cressman, D. E., and Taub, R. (1993). Promoter-specific trans-activation and inhibition mediated by JunB. Cancer Res. 53, 3789-3794. Huang, C.-S., Anderson, M. E., and Meister, A. (1993). Amino acid sequence and function of the light subunit of rat kidney ~/-glutamylcysteine synthetase. J. Biol. Chem. 268, 2057820583. Huh, G. S., and Hynes, R. O. (1993). Elements regulating an alternatively spliced exon of the rat fibronectin gene. Mol. Cell. Biol. 13, 5301-5314. Hutson, S. M., Stinson-Fisher, C., Shiman, R., and Jefferson, L. S. (1987). Regulation of albumin synthesis by hormones and amino acids in primary cultures of rat hepatocytes. Am. J. Physiol. 252, E291-E298.

4.

Expression and Function of Growth-Induced Genes during Liver Regeneration

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Jefferson, L. S. (1980). Role of insulin in the regulation of protein synthesis. Diabetes 29, 487496. Krainer, A. R., Mayeda, A., Kozak, D., and Binns, G. (1991). Functional expression of cloned human splicing factor SF2: Homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell (Cambridge, Mass.) 66, 383-394. Kruijer, W., Skelly, H., Botteri, E, van der Putten, H., Barber, J., Verma, I., and Leffert, H. (1986). Proto-oncogene expression in regenerating liver is simulated in cultures of primary adult rat hepatocytes. J. Biol. Chem. 261, 7929-7933. Lanahan, A., Williams, J. B., Sanders, L. K., and Nathans, D. (1992). Growth factor-induced delayed early response genes. Mol. Cell. Biol. 12, 3919-3929. Lau, L. E, and Nathans, D. (1985). Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells. EMBO J. 4, 3145-3151. Lau, L. E, and Nathans, D. (1987). Expression of a set of growth-related immediate early genes in BALB/c 3T3 cells: Coordinate regulation with c-fos or c-myc. Proc. Natl. Acad. Sci. U.S.A. 84, 1182-1186. Lee, J., Greenbaum, L., Haber, B. A., Nagle, D., Lee, V., Miles, V., Mohn, K. L., Bucan, M., and Taub, R. (1994). Structure and localization of the IGFBP-1 gene and its expression during liver regeneration. Hepatology (Baltimore) 19, 656-665. Lemaire, L., and Heinlein, U. A. O. (1993). High-level expression in male germ cells of murine P68 RNA helicase mRNA. Life Sci. 52, 917-926. Li, H., and Bingham, P. M. (1991). Arginine/serine-rich domains of the su(wa) and tra RNA processing regulators target proteins to a subnuclear compartment implicated in splicing. Cell (Cambridge, Mass.) 67 335-342. Lu, X. P., Koch, K. S., Lew, D. J., Dulic, V., Pines, J., Reed, S. I., Hunter, T., and Leffert, H. L. (1992). Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration. J. Biol. Chem. 267, 2841-2844. Marais, R., Wynne, J., and Treisman, R. (1993). The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell (Cambridge, Mass.) 73, 381-393. Mattox, W., Ryner, L., and Baker, B. S. (1992). Autoregulation and multifunctionality among trans-acting factors that regulate alternative pre-mRNA processing. J. Biol. Chem. 267, 19023-19026. Mayeda, A., and Krainer, A. R. (1992). Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell (Cambridge, Mass.) 68, 365-375. Mayeda, A., Zahler, A. M., Krainer, A. R., and Roth, M. B. (1992). Two members of a conserved family of nuclear phosphoproteins are involved in pre-mRNA splicing. Proc. Natl. Acad. Sci. U.S.A. 89, 1301-1304. Michalopoulos, G. K. (1990). Liver regeneration: Molecular mechanisms of growth control FASEB J. 4, 176-187. Mohn, K. L., Laz, T. M., Melby, A. E., and Taub, R. (1990). Immediate-early gene expression differs between regeneration liver, insulin-stimulated H-35 cells, and mitogen~ 3T3 cells: Liver specific induction patterns of gene 33, PEPCK, and the jun, fos and egr families. J. Biol. Chem. 265, 21914-21921. Mohn, K. L., Laz, T. M., Hsu, J.-C., Melby, A. E., Bravo, R., and Taub, R. (1991a). The immediate-early growth response in regenerating liver and insulin-stimulated H-35 cells: Comparison to serum-stimulated 3T3 cells and identification of 41 novel immediate-early genes. Mol. Cell. Biol. 11, 381-390. Mohn, K. L., Melby, A. E., Tewari, D. S., Laz, T. M., and Taub, R. (1991b). The gene encoding rat insulin like growth factor-binding protein i is rapidly and highly induced in regenerating liver. Mol. Cell. Biol. 11, 1393-1401.

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Nelson, P. A., Lippke, J. A., Murcko, M. A., Rosborough, S. L., and Peattie, D. A. (1991). cDNA encoding murine FK506-binding protein (FKBP): Nucleotide and deduced amino acid sequences. Gene 109, 255-258. Peavy, D. E., Taylor, J. M., and Jefferson, L. S. (1985). Time course of changes in albumin synthesis and mRNA in diabetic and insulin-treated diabetic rats. Endocrinol. Metal). Clin. North Am. 248,E656-E663. Ruff-Jamison, S., Chen, K., and Cohen, S. (1993). Induction by EGF and interferon-~ of tyrosine phosphorylated DNA binding proteins in mouse liver nuclei. Science 261, 17331736. Scearce, L. M., Laz, R. M., Hazel, T. G., Lau, L. E, and Taub, R. (1993). RNR-1, a nuclear receptor in the NGFI-B/Nur-77 family that is rapidly induced in regenerating liver. J. Biol. Chem. 268, 8855-8861. Schneiderman, J., Sawdey, M., Craig, H., Thinnes, T., Bordin, G., and Loskutoff, D. J. (1993). Type I plasminogen activator inhibitor gene expression following partial hepatectomy. Am. J. Pathol. 143, 753-762. Shelly, L. L., Lei, K.oJ., Pan, C.-J., Sakata, S. E, Ruppert, S., Shutz, G., and Chou, J. Y. (1993). Isolation of the gene for murine glucose-6ophosphatase, the enzyme deficient in glycogen storage disease type 1A. J. Biol. Chem. 268, 21482-21485. Sherr, C. J. (1993). Mammalian G1 cyclins Cell (Cambridge, Mass.) 75, 839-841. Simmons, D. L., Neel, B. G., Stevens, R., Evett, G., and Erikson, R. L. (1992). Identification of an early-growth-response gene encoding a novel putative protein kinase. Mol. Cell. Biol. 12, 4164-4169. Stein, B., and Baldwin, Jr., A. S. (1993). Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-KB. Mol. Cell Biol. 13, 7191-7198. Sun, H., Charles, C. H., Lau, L. E, and Tonks, N. K. (1993). MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates Map kinase in vivo. Cell (Cambridge, Mass.) 75, 487-493. Takiguchi, S., Tokino, T., Imai, T., Tanigami, A., Koyama, K., and Nakamura, Y. (1993). Identification and characterization of a cDNA, which is highly homologous to the ribonucleoprotein gene, from a locus (D10S102) closely linked the MEN2 (multiple endocrine neoplasia type 2). Cytogenet. Cell Genet. 64, 128-130. Taub, R., Roy, A., Dieter, R., and Koontz, J. (1987). Insulin as a growth factor in rat hepatoma cells: Stimulation of protooncogene expression. J. Biol. Chem. 262, 10893-10897. Tewari, M., Dobrzanski, P., Mohn, K. L., Cressman, D. E., Hsu, J.oC., Bravo, R., and Taub, R. (1992). Rapid induction in regenerating liver of RL/IF-1, an IKB that inhibits NH-KB, Relo p50; and PHF, a novel KB site binding complex. Mol. Cell. Biol. 12, 2898-2908. Thompson, N. L., Mead, J. E., Braum, L., Goyette, M., Shank, P. R., and Fausto, N. (1986). Sequential protooncogene expression during rat liver regeneration. Cancer Res. 46, 31113117. Thornton, A. J., Bruzdzinski, C. J., Raper, S. E., and Gelehrter, T. D. (1994). Plasminogen activator inhibitor-1 is an immediate early response gene in regenerating rat liver. Cancer Res. 54, 1337-1343. Tohyama, C., Suzuki, J. S., Hemelraad, J., Nishimura, N., and Nishimura, H. (1993). Induction of metallothionein and its localization in the nucleus of rat hepatocytes after partial hepatectomy. Hepatology (Baltimore) 18, 1193-1201. Varnum, B. C., Lim, R. W., and Herschman, H. R. (1989). Characterization of TIS7, a gene induced in Swiss 3T3 cells by the tumor promoter tetradecanoyl phorbol acetate. Oncogene 4, 1263-1265. Zahler, A. M., Lane, W. S., Stolk, J. A., and Roth, M. B. (1992). SR proteins: A conserved family of pre-mRNA splicing factors. Genes Dev. 6, 837-847.

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Zahler, A. M., Neugebauer, K. M., Stolk, J. A., and Roth, M. B. (1993a). Human SR proteins and isolation of a cDNA encoding SRp75. Mo/. Cell. Biol. 13, 4023-4028. Zahler, A. M., Neugebauer, K. M., Stolk, J. A., and Roth, M. B. (1993b). Alternative premessenger RNA splicing by SR proteins. Science 260, 219-222. Zipfel, P. F., Irving, S. G., Kelly, K., and Siebenlist, U. (1989). Complexity of the primary genetic response to mitogenic activation of human T cells. Mo/. Cell. Biol. 9, 1041-1048.

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5 Stem Cells and Hepat0carc nogenes s Snorri S. Thorgeirsson Laboratory of Experimental Carcinogenesis Division of Cancer Etiology National Cancer Institute National Institutes of Health Bethesda, Maryland 20892

I. Introduction The existence of hepatic stem cells has been, and no doubt will continue to be, a matter of considerable controversy. This controversy is partly fueled by the fact that cell turnover in the liver is very slow and that the two major types of hepatic epithelial cells, hepatocytes and biliary epithelia, are capable of proliferation and can, at least in a healthy liver, meet replacement demands of cellular loss from these two differentiated populations. The best example of the capacity of adult hepatocytes and bile epithelial cells to proliferate is seen following partial hepatectomy in rats and mice in which the compensatory hyperplasia of these cells in the remaining lobes restores the liver mass (see Chapters 1 and 2). The increased use and success of liver transplantation in clinical medicine has shown that these animal models also correctly reflect the capacity of the human liver to regenerate (see Chapter 13) (Van Thiel et al., 1989). The major part of the hepatic stem cell controversy may, however, be due to the failure of recognizing that the adult organism contains many kinds of stem cells. These cells may exist at different stages of differentiation and have very different capacities for generating multilineage progeny.

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The capacity for self maintenance is a fundamental and common trait of all stem cells. A cell population that has an extensive self-maintaining capacity is the only definition that applies to all stem cells (Lajtha, 1979). In this context, the adult liver, which has an extensive capacity for maintaining parenchymal cell number throughout the life span of the organism, can be viewed as a single lineage stem cell system in which the hepatocyte is the stem cell. Recent data from hepatic cell transplantation experiments in a transgenic mouse system (Rhim et al., 1994) have demonstrated the enormous growth potential of adult hepatocytes (12 to 16 doublings per donor cell), further supporting the notion of the liver parenchyma as a single lineage or unipotential stem cell system (see Chapter 11). The endodermal origin of the liver, and the fact that the early fetal hepatocytes or hepatoblasts are progenitor for both adult hepatocytes and bile epithelial cells, suggests that the hepatoblasts are at least bipotential precursors. The question then arises whether either or both of the cell lineages derived from the hepatoblast retain the "bipotential capacity" of the precursor cells. At present there is no substantial evidence to indicate that adult hepatocytes are more than a unipotential stem cell system. The possible exception to this generalization is the concept of ductular metaplasia or transformation of hepatocytes into ductules. This topic has been extensively discussed by Desmet and colleagues (for review see Van Eyken and Desmet, 1992). Studies of chronic cholestatic diseases in humans employing both enzyme histochemistry and cytokeratin immunohistochemistry have provided evidence for gradual transformation of hepatocytes into "bile duct-type" cells (Van Eyken et al., 1989). Evidence showing that these "bile duct-type" cells also exhibit functional characteristics of normal bile epithelium is still lacking. It is therefore questionable but still possible that ductal metaplasia of hepatocytes seen in cholestatic diseases may reflect multipotential or at least bipotential capacity of the hepatocytes. In contrast to the hepatocyte system, there is strong evidence indicating that the bile epithelium harbors a compartment of cells that are capable of differentiating into several lineages including bile epithelia, hepatocytes, intestinal epithelia, and possibly exocrine pancreas (Thorgeirsson, 1993; Fausto, 1990; Sell, 1990; Sigal et al., 1992). Also, as pointed out by Sell (1990), there may exist a periductal system of stem cells capable of differentiating into all the hepatic lineages. These ductular/periductal cells are frequently referred to as the hepatic stem cell system. In light of these observations, the liver system should be viewed as being composed of two stem cell systems: the unipotential (possibly bipotential) hepatocytic and the multipotential nonparenchymal epithelial (ductular) systems (Figure 1). This chapter will review the general cellular biology of the hepatic stem cell compartment, and the possible involvement of these cells in hepatocarcinogenesis.

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Figure I Possiblelineage pathways in the liver.

n. Cellular Biology of the Hepatic Stem Cell Compartment What then is the evidence that a multipotential stem cell compartment exists in the liver? The existence of hepatic stem cells was first postulated by Wilson and Leduc in 1958 based on experiments involving liver regeneration in the mouse after chronic injury induced with a methionine-rich basal diet mixed with an equal amount of bentonite (Wilson and Leduc, 1958). The authors concluded "that prolonged and severe injury to the liver may make direct restoration by division of preexisting parenchymal cells impossible, and that, when this occurs, the new parenchyma is derived from the indifferent cholangiole cells." The major support for the existence of hepatic stem cells has, perhaps not surprisingly in light of the earlier work by Wilson and Leduc, come from extensive studies of hepatic carcinogenesis (Fausto, 1990; Sell, 1990; Sigal et al., 1992; Marceau, 1990). A. Experimental Systems in Vivo The rat has been most extensively used to generate experimental systems in which to study the cell biology of the hepatic stem cell compartment. The three most used model systems are (1) feeding of choline deficient diet with or without 0.1% ethionine (CDE) (Shinozuka et al., 1978); (2) combination of 2-acetylaminofluorene (AAF) treatment and partial hepatectomy (Tatematsu et al., 1985); and (3) administration of hepatotoxic doses of d-galac-

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tosamine (Lemire et al., 1991). Common to all these experimental systems is the extensive destruction of liver parenchyma and/or prevention of regeneration following loss of liver mass as well as compromised function of the remaining hepatocytes. The observation that the regenerative capacity of the hepatocytes has to be compromised before the hepatic stem cell compartment becomes activated and starts to contribute to the regeneration of the liver parenchyma led Grisham (1980) to propose that this be referred to as a facultative stem cell compartment. This is a particularly important concept since, as pointed out above, healthy hepatocytes have extensive capacity for regeneration and should be looked on as an unipotential stem cell system. A common cellular response in rats subjected to the experimental systems listed above is the proliferation of small periportal cells with scant cytoplasm and ovoid nuclei that have been termed oval cells (Farber, 1984, 1990; Evarts, et al., 1987; Marceau, 1990). The existence of similar cells has been reported in human liver (Gerber et al., 1983; Hsia et al., 1992). These oval cells are thought to represent a progeny of the hepatic stem cell compartment, and in some instances to be a precursor for hepatic tumors (Sell, 1990; Sigal et al., 1992; Hixson et al., 1990). The precise anatomical location of the hepatic stem cell compartment in normal liver is still unclear, but present data suggest that the terminal ductule cells connecting the canals of herring with the bile canaliculi and/or a distinct population of periductal cells constitute the hepatic stem cell compartment (Factor et al., 1994; Fausto, 1990; Sell, 1990; Sigal et al., 1992; Wilson and Leduc, 1958). Recent studies in the rat liver have shown that oval cells are capable of differentiating into, in addition to bile epithelium, at least two lineages in vivo including hepatocytes (Evarts et al., 1987; Lemire et al., 1991) and intestinal type epithelium (Tatematsu et al., 1985). Furthermore, isolated oval cells in culture can be induced to differentiate into both hepatocyte-like and biliary types of cells (Hayner et al., 1984; Germain et al., 1985, 1988). These data and other results not reviewed here support the notion that oval cells have lineage options similar to hepatoblasts in the early stages of liver development. As such, the oval cells can be regarded as "bipotential precursors" for the two hepatic parenchymal cell lineages. In addition, they may exhibit a capacity to differentiate into lineages particularly when the hepatic microenvironment is drastically disrupted (Thorgeirsson and Evarts, 1992). B.

ExperimentalSystems in

Vitro

In addition to the in vivo data mentioned above, significant support for the existence of the hepatic stem cell has come from results obtained in studies on hepatic cell cultures. Several research groups have been able to isolate and establish long-term cultures of small, morphologically and functionally

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simple epithelial cells by enzymatic perfusion of fetal and adult rat liver and utilizing culture conditions that exclude differentiated hepatocytes (Grisham et al., 1974; Furukaura et al., 1987; Marceau et al., 1980; Schaeffer, 1980). These rat liver-derived epithelial (RLE) cells share some phenotypic properties with both bile duct epithelial cells and hepatocytes, but are phenotypically much closer to some of the oval cell lines (Hayner et al., 1984; Tsao et al., 1984; Marceau et al., 1986). The notion that oval cells are descendants from hepatic stem cells suggests that the RLE cells are also progeny from such a stem cell compartment because of their phenotypic similarities to oval cells. This suggestion is supported by data obtained from extensive use of RLE cells for in vitro transformation studies with both chemical carcinogens and oncogenes. These studies have demonstrated various transformation properties of RLE cells (Tsao and Grisham, 1987; Garfield et al., 1988; Schaeffer, 1980). Most importantly, when the transformed RLE cells are transplanted into either syngeneic rats or nude mice a spectrum of tumors are encountered that includes highly differentiated hepatocellular carcinomas, hepatoblastomas, cholangiocarcinoma as well as mixed epithelial-mesenchymal tumors (Tsao and Grisham, 1987). This suggests a blastic nature of the RLE cells and demonstrates the potential of the cells to differentiate via both the hepatocytic and biliary lineages. However, no in vitro culture system exists that conclusively demonstrates differentiation of the RLE cells into either hepatocytes or bile epithelial cells. A recent insight into the nature of the RLE cells and the potential role of hepatic stem cells in liver biology was provided by Coleman et al. (1993). They have successfully demonstrated, following intrahepatic transplantation of RLE cells carrying the Escherichia coli 13-galactosidase reporter gene, that the RLE cells integrate into hepatic plates and acquire the size and nuclear structure of mature hepatocytes. In addition, they showed that intrahepatic transplantation of an aneuploid, neoplastically transformed derivative of the RLE cells, which produces aggressively growing tumor when transplanted subcutaneously, does not produce tumors in the liver but rather integrates into the hepatic plates and morphologically differentiates. The results from this study raise several important issues for basic hepatic biology as well as clinical hepatology. Differentiated hepatocytes isolated from enzymatically prepared suspensions of murine liver cells have been shown already to integrate into the hepatic plate following transplantation into liver of congenic animals (Ponder et al., 1991; Gupta et al., 1991). This is, however, the first demonstration that primitive or "stem-like" RLE cells on intrahepatic transplantation also integrate into the hepatic plates and differentiate morphologically into hepatocytes. These results taken together with the large body of data on the blastic nature of the RLE cells strongly support both the existence of a hepatic stem cell compartment and the

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notion that RLE cells are derived from this compartment. Furthermore, the present study has also reinforced the notion that both oval cells and RLE cells represent a progeny from the hepatic stem cell compartment.

III. Neoplastic Development in the Liver Genesis of liver tumors most probably occurs via multiple molecular mechanisms which depend on both the nature of the carcinogen and the lesions induced by it. As previously stated, the liver system should be viewed as being composed of two stem cell systems: the unipotential (possibly bipotential) hepatocytic and the multipotential nonparenchymal epithelial (ductular) systems (Figure 1). Therefore, it seems reasonable to expect that both cell systems could provide progenitor cells for the neoplastic process. There is no doubt that the hepatocyte frequently is the progenitor cell for liver tumors (Farber, 1992). The involvement of the nonparenchymal (ductular) system in the genesis of liver tumors, particularly hepatocellular carcinomas, is still hotly debated. On the one hand, it has been proposed that "the cell of origin of liver cancer is the putative liver stem cell or its progeny, the transitional duct cell" (Sell and Pierce, 1994). Alternatively, Farber (1992) has stated that "rare original mature hepatocytes in zone 1, 2, or 3 of the adult liver appearing after initiation with genotoxic carcinogens have been shown to be the cell of origin for foci or islands of altered hepatocytes and of nodules derived from these foci." The central issue in better understanding the involvement of the nonparenchymal epithelial (ductular) cells in the carcinogenic process is the characterization of the mechanisms that regulate both the proliferation of these cells after carcinogenic as well as noncarcinogenic insults, and the factors governing the lineage commitment processes in this compartment. A. Hepatic Stem Cells and Hepatocarcinogenesis A landmark contribution to the involvement of liver nonparenchymal epithelial cells in hepatocarcinogenesis was provided by Farber (1956)who documented a detailed description of the early histological changes during hepatocarcinogenesis caused by three chemical carcinogens. The carcinogens used by Farber, ethionine, 2-acetylaminofluorene (AAF), and 3'-methyl4-dimethylaminoazobenzene (Me-DAB), in spite of being structurally very different, caused similar histological alterations. The common features included (1) oval cell proliferation which progressively involved most of the liver lobule, beginning in the portal areas, (2) degenerative and hypertrophic changes in hepatocytes adjacent to proliferating oval cells, and (3) nodular regenerative hyperplasia of liver cells. There were, however, important dif-

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ferences in the time course of appearance and fate of the oval cells induced by these three hepatocarcinogens. Whereas, oval cells appeared early following ethionine and AAF administration (7 and 14 days, respectively), their appearance occurred significantly late after Me-DAB treatment (first seen at Day 21). More importantly, the fate of the oval cells in the Me-DAB treated animals were different from those induced by ethionine and AAE In the early stages the oval cells induced by Me-DAB were morphologically indistinguishable from those generated by ethionine and AAE However, at later stages areas of apparent transition between oval cells and hepatocytes were numerous in the Me-DAB treated animals but absent in those receiving ethionine and AAE These observations raise several important issues. First, and most importantly, it is now well established that many different chemical compounds capable of producing liver tumors in rats and mice, induce a similar sequence of histological changes in which oval cell hyperplasia is prominent (Dunsford et al., 1985). Secondly, if the transition from oval cells to hepatocytes can be morphologically observed after Me-DAB treatment, then it is in principle established that oval cells (or at least a subpopulation of oval cells) have the capacity to differentiate into hepatocytes. The fact that administration of ethionine or AAF did not provide the same clear morphological sequence as seen with Me-DAB in which the oval cells merge imperceptibly and were in continuity with the regenerating nodules, suggests that the compounds capable of inducing oval cell proliferation may greatly affect both the rate and extent of oval cell differentiation into hepatocytes. The fact that a large population of oval cells is cycling during the early stages of chemical hepatocarcinogenesis and that these cells can differentiate into hepatocytes strongly suggests that at least a percentage of the hepatocellular carcinomas are derived from oval cell progenitors. Recently there has been accumulating experimental evidence in support of this notion. Hixson and his colleagues (Hixson et al., 1990; Faris et al., 1991) have used a battery of monoclonal antibodies specific for antigens associated with bile duct cells, oval cells and fetal, adult, and neoplastic hepatocytes to analyze the phenotypic relationship between oval cells, foci, nodules, and hepatocellular carcinomas during chemical hepatocarcinogenesis. These investigators found, using the resistant hepatocyte model of Solt and Farber (1976), that oval cells, GGT-positive hepatocellular foci, persistent hepatocyte nodules, and primary hepatocellular carcinomas express both oval cell and hepatocyte antigens. This finding indicates a precursorproduct relationship between oval cells and carcinomas. Similar results were obtained by Dunsford et al. (1989) using different monoclonal antibodies raised against oval cells. These lineage relationships between oval cells and hepatocellular carcinomas also exist in other models of liver carcinogenesis. For example, animals maintained on a CDE diet display markers

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for oval cells and hepatocytes in a significant percentage of nodules and hepatocellular carcinomas (Hixson et al., 1990; Faris et al., 1991). Also, metastatic foci in the lung from the animals harboring these liver tumors show essentially the same phenotype (Hixson et al., 1990). The evidence for oval or ductular cells as progenitors for hepatocellular carcinomas is not restricted to experimental models of chemical hepatocarcinogenesis in rodents. Results from Van Eyken et al. (1988) on the cytokeratin expression in 34 "classical" human hepatocellular carcinomas (HCC) using monospecific anticytokeratin antibodies show that all HCCs were positive for cytokeratins 8 and 18. However, in 17 cases a variable number of the tumor cells were positive for cytokeratin 7 (two cases), cytokeratin 19 (7 cases), or both 7 and 19 (8 cases). The authors also reported that only 3 of 11 well-differentiated tumors display an "unexpected" pattern of immunoreactivity as opposed to 7 of 7 poorly differentiated tumors. This is particularly important in light of the earlier observation by Denk et al. (1982) that cytokeratins continue to be expressed when hepatocytes become neoplastic. These observations are also highly relevant in light of the recent findings of Hsia et al. (1992) and Vandersteenhoven et al. (1990) who demonstrated immunohistochemically the presence of ductular "oval" type cells with characteristics of both bile ducts and hepatocytes in the liver of patients with end stage cirrhosis and/or tumors from hepatitis B infection. It is important at this stage to reemphasize that the relative percentage of primary hepatocellular carcinomas derived from oval cell progenitors may vary over a wide range depending on the carcinogenesis protocol and/or the chemical carcinogen used as well as the extent of oval cell involvement in the early stages of the process. B. Transformation of Liver Derived Epithelial (Oval) Cells The most direct evidence that oval cells and/or RLE cells can progress to hepatocellular carcinomas comes from in vitro transformation of these cells. Spontaneous transformation of RLE and oval cells as well as transformation with chemical carcinogens and dominant oncogenes results in the tumors displaying a wide range of phenotypes including well-differentiated hepatocellular carcinomas, cholangiomas, hepatoblastomas, and poorly differentiated or anaplastic tumors (Tsao and Grisham, 1987; Garfield et al., 1988; Fausto, 1990; Marceau, 1990). In one of the most comprehensive studies on the chemical transformation of the RLE cells by Tsao and Grisham (1987), a wide range of tumors described including carcinomas, sarcomas, mixed epithelia-mesenchymal tumors, and undifferentiated tumors. In addition, several tumors were morphologically indistinguishable from hepatocellular carcinomas. Also, the "mixed epithelial-mesenchymal" tumors reproduced most of the various histologic features of human hepatoblastomas (Table 1).

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Table 1 Light Microscopic Classification of Tumors Produced by ChemicallyTransformed Rat Liver Epithelial Cells Tumor type Carcinomas (epithelial) Epidermoid Adenocarcinoma Hepatocellular Poorly differentiated/anaplastic Sarcomas (mesenchymal) Mixed epithelial-mesenchymaltumors Unclassified

Number of tumors 15 13 4 22 19 30 22

From Tsao and Grisham (1987).

We have recently demonstrated that cytokeratin 14 is expressed in several RLE cell lines (Bisgaard et al., 1994a). Although the partner for cytokeratins 8 and 14 has traditionally been found to be cytokeratins 18 and 5, respectively, it is now well documented that cytokeratins 8 and 14 can be expressed in the complete absence of their traditional partner (Bisgaard and Thorgeirsson, 1991; Bisgaard et al., 1993; Wirth et al., 1992). We have shown that in some RLE cell lines cytokeratins 8 and 14 form heterotypic filaments (Bisgaard et al., 1993). Also, we found that these cell lines express vimentin along with the cytokeratins (Wirth et al., 1992). However, the spontaneous transformation and differentiation of one of our RLE cell lines to a hepatoblast-like phenotype, forming a well-differentiated trabecular hepatocellular carcinoma, results in an abrogation of vimentin protein expression and a change in cytokeratin expression from which cytokeratin 14 was substituted by 18 (Bisgaard et al., 1994b). We have used this RLE transformation system to study the relationship between the expression of cytokeratins 14, 8, as well as 18 and oL-fetoprotein (AFP) during the process of proliferation and differentiation of the RLE cell line to a hepatoblast-like progeny. The steady-state levels of mRNA transcripts for cytokeratin 14 and AFP, as well as for cytokeratins 8 and 18 and vimentin show a significant change in the expression pattern during the process of transformation (Figure 2). Before the cells display morphological signs of transformation, a high steady-state level of cytokeratin 14 transcripts in addition to transcripts for cytokeratin 8 and vimentin is detected (Figure 2). During the process of transformation that occurred within 33 to 35 passages, the steady-state levels of cytokeratin 14 and vimentin abruptly declined, and could not be detected in later passages nor in the clonal transformed B5T cell line. The disappearance of the cytokeratin 14 and vimentin mRNA transcripts closely corresponds with the appearance of a 2.1-kDa transcript for AFP and a 1.4-

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Figure 2 Differential expression of cytokeratins 8, 14, 18, vimentin, and oL-fetoprotein mRNA during spontaneous transformation of RLESF13 cells by prolonged passage in vitro. (From Bisgaard et al., 1994b. With permission).

kDa transcript for cytokeratin 18 (Figure 2). The mRNA transcripts for cytokeratins 8 and 18 as well as those for AFP are present in the transformed clonal B5T cell line (Figure 2). In contrast to the spontaneous transformation, these same RLE cells when transformed by dominant oncogenes yield primitive and anaplastic tumors (Garfield et al., 1988). These data indicate that the tumor phenotypes derived from RLE and/or oval cells may depend on both the mechanism of transformation and the stage of differentiation of the cells when the transformation occurs.

IV. C o n c l u s i o n s The adult organism contains many kinds of stem cells that exist at different stages of differentiation and have very different capacities for generating multilineage progeny. The capacity for self maintenance is a fundamental and common trait of all stem cells. A cell population that has an extensive self-maintaining capacity is the only definition that applies to all stem cells. It is proposed that the liver system be viewed as composed of two stem cell systems: the unipotential hepatocytic and the multipotential nonparenchy-

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mal epithelial (ductular) systems. Although the participation of the nonparenchymal epithelial (ductular) system in the development of liver tumors is still not fully defined, strong evidence now exists indicating that both these systems can and do provide progenitor cells for the neoplastic process in the liver. The central issue in better understanding the involvement of the nonparenchymal epithelial (ductular) cells in the carcinogenic process is the characterization of the mechanisms that regulate both the proliferation of these cells after carcinogenic as well as noncarcinogenic insults and the factors that govern the lineage commitment processes in this stem cell system.

References Bisgaard, H. C., and Thorgeirsson, S. S. (1991). Evidence for a common cell of origin for primitive epithelial cells isolated from rat liver and pancreas. J. Cell. Physiol. 147, 333343. Bisgaard, H. C., Parmelee, D. C., Dunsford, H. A., Sechi, S., and Thorgeirsson, S. S. (1993). Keratiin 14 protein in cultured nonparenchymal rat hepatic epithelial cells: Characterization of keratin 14 and keratin 19 as antigens for the commonly used mouse monoclonal antibody OV-6. Mol. Carcinog. 7, 60-66. Bisgaard, H. C., Nagy, P., Ton, P. T., Hu, Z., and Thorgeirsson, S. S. (1994a). Modulation of keratin 14 and ~-fetoprotein expression during hepatic oval cell proliferation and liver regeneration. J. Cell. Physiol. 159, 475-484. Bisgaard, H. C., Ton, P. T., Nagy, P., and Thorgeirsson, S. S. (1994b). Phenotypic modulation of keratins, vimentin, and cx-fetoprotein in cultured rat liver epithelial cells after chemical, oncogene, and spontaneous transformation. J. Cell. Physiol. 159, 485-494. Coleman, W. B., Wennerberg, A. E., Smith, G. T., and Grisham, J. W. (1993). Regulation of the differentiation of diploid and some aneuploid rat liver epithelial (stem like) cells by the hepatic microenvironment. Am. J. Pathol. 142, 1373-1382. Denk, H., Krepler, R., Lackinger, E., Artlieb, U., and Franke, W. W. (1982). Biochemical and immunocytochemical analysis of the intermediate filament cytoskeleton in human hepatocellular carcinomas and in hepatic neoplastic nodules of mice. Lab. Invest. 46, 584-596. Dunsford, H. A., Maset, R., Salman, J., and Sell, S. (1985). Connection of duct-like structures induced by a chemical hepatocarcinogen to portal bile ducts in the rat liver detected by injection of bile ducts and pigmented barium gelatin medium. Am. J. Pathol. 118,218-224. Dunsford, H. A., Karnasuta, C., Hunt, J. M., and Sell, S. (1989). Different lineages of chemically induced hepatocellular carcinoma in rats defined by monoclonal antibodies. Cancer Res. 49, 4894-4900. Evarts, R. P., Nagy, P., Marsden, E., and Thorgeirsson, S. S. (1987). A precursor relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 8, 1737-1740. Factor, V. M., Radaeva, S. A., and Thorgeirsson, S. S. (1994). Origin and fate of oval cells in Dipin-induced hepatocarcinogenesis in the mouse. Am. J. Pathol. 145,409-422. Farber, E. (1956). Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylaminofluorene, and 3'-methyl-4-dimethylaminoazobenzene. Cancer Res. 16, 142-148. Farber, E. (1984). Cellular biochemistry of the stepwise development of cancer with chemicals. Cancer Res. 44, 5463-5474.

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Farber, E. (1990). Clonal adaptation during carcinogenesis. Biochem. Pbarmacol. 39, 18371846. Farber, E. (1992). On cells of origin of liver cell cancer. In "The Role of Cell Types in Hepatocarcinogenesis" (A. E. Sirica, ed.), pp. 1-28. CRC Press, Boca Raton, Florida. Faris, R. A., Monfils, B. A., Dunsford, H. A., and Hixson, D. (1991). Antigenic relationship between oval cells and a subpopulation of hepatic foci, nodules, and carcinomas induced by the "resistant hepatocyte" model system. Cancer Res. 51, 1308-1317. Fausto, N. (1990). Oval cells and liver carcinogenesis: An analysis of cell lineages in hepatic tumors using oncogene transfection techniques. Prog. Clin. Biol. Res. 331,325-334. Furukaura, K., Schimada, T., England, P., Mochizuki, Y., and Williams, G. M. (1987). Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains. In Vitro Cell. Dev. Biol. 23, 339-348. Garfield, S., Huber, B. E., Nagy, P., Cordingley, M. G., and Thorgeirsson, S. S. (1988). Neoplastic transformation and lineage switching of rat liver epithelial cells by retrovirus-associated oncogenes. Mol. Carcinog. 1, 189-195. Gerber, M. A., Thung, S. N., Shen, S., Stromeyer, E W., and Ishak, K. G. (1983). Phenotypic characterization of hepatic proliferation: Antigenic expression of proliferating epithelial cells in fetal liver, massive hepatic necrosis, and nodular transformation of the liver. Am. J. Pathol. 110, 70-74. Germain, L., Goyette, R., and Marceau, N. (1985). Differential cytokeratin and ot-fetoprotein expression in morphologically distinct epithelial cells emerging at the early stages of rat hepatocarcinogenesis. Cancer Res. 45,673-681. Germain, L., Noel, H., Gourdeau, H., and Marceau, N. (1988). Promotion of growth and differentiation of rat ductular oval cells in primary culture. Cancer Res. 48, 368-378. Grisham, J. W. (1980). Cell types in long-term propagable cultures of rat liver. Ann. N.Y. Acad. Sci. 349, 128-137. Grisham, J. W., Thai, S. B., and Nagel, A. (1974). Cellular derivation of continuously cultured epithelial cells from normal rat liver. In "Gene Expression and Carcinogenesis in Cultured Liver" (L. E. Gerschenson and E. B. Thompson, eds.), pp. 1-23. Academic Press, New York. Gupta, S., Aragona, E., Vemuru, R. P., Bhargava, K. K., Burk, R. D., and Chowdhury, J. R. (1991). Permanent engraftment and function of hepatocytes delivered to the liver: Implications for gene therapy and liver repopulation. Hepatology (Baltimore) 14, 144-149. Hayner, N. T., Braun, L., Yaswen, R, Brooks, M., and Fausto, N. (1984). Isozyme profiles of oval cells, parenchymal cells, and biliary cells isolated by centrifugal elutriation from normal and preneoplastic livers. Cancer Res. 44, 332-338. Hixson, D. C., Faris, R. A., and Thompson, N. L. (1990). An antigenic portrait of the liver during carcinogenesis. Pathobiology 58, 65-77. Hsia, C. C., Evarts, R. P., Nakatsukasa, N., Marsden, E. R., and Thorgeirsson, S. S. (1992). Occurrence of oval-type cells in hepatitis B virus-associated human hepatocarcinogenesis. Hepatology (Baltimore) 16, 1327-1333. Lajtha, L. G. (1979). Stem cell concepts. Nouv. Rev. Fr. Hematol. 21, 59-65. Lemire, J. M., Shiojiri, N., and Fausto, N. (1991). Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine. Am. J. Pathol. 139, 535-552. Marceau, N. (1990). Biology of disease. Cell lineage and differentiation programs in epidermal, urothelial and hepatic tissues and their neoplasms. Lab. Invest. 63, 4-20. Marceau, N., Goyette, R., Deschenes, J., and Valet, J.-P. (1980). Morphological differences between epithelial and fibroblast cells in rat liver cultures and the roles of cell surface fibronectin and cytoskeletal element organization in cell shape. Ann. N.Y. Acad. Sci. 349, 138-152. Marceau, N., Germain, L., Goyette, R., Noel, M., and Gourdeau, H. (1986). Cell of origin of

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distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression. Biochem. Cell Biol. 64, 788-802. Ponder, K. P., Gupta, S., Leland, E, Darlington, G., Finegold, M., DeMayo, J., Ledley, E D., Chowdhury, J. R., and Woo, S. L. C. (1991). Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation. Proc. Natl. Acad. Sci. U.S.A. 88, 1217-1221. Rhim, J. A., Sandgren, E. P., Degen, J. L., Palmiter, R. D., and Brinster, R. L. (1994). Replacement of diseased mouse liver by hepatic cell transplantation. Science 263, 1149-1152. Schaeffer, W. I. (1980). The long term culture of a diploid rat hepatocyte cell strain. Ann. N.Y. Acad. Sci. 349, 165-182. Sell, S. (1990). Is there a liver stem cell? Cancer Res. 50, 3811-3815. Sell, S., and Pierce, G. B. (1994). Biology of disease. Maturation arrest of stem cell differentiation is a common pathway for the cellular origin of teratocarcinomas and epithelial cancers. Lab. Invest. 70, 6-22. Shinozuka, H., Lombardi, B., Sell, S., and Iammarino, R. M. (1978). Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline-deficient diet. Cancer Res. 38, 1092-1098. Sigal, S. H., Brill, S., and Reid, L. M. (1992). The liver as a stem cell and lineage system. Am. J. Physiol. 263, G139-G148. Solt, D. B., and Farber, E. (1976). New principle for the analysis of chemical carcinogenesis. Nature (London) 263, 701-703. Tatematsu, M., Kaku, T., Medline, A., and Farber, E. (1985). Intestinal metaplasia as a common option of oval cells in relation to cholangiofibrosis in the livers of rats exposed to 2-acetylamino-fluorene. Lab. Invest. 52, 354-362. Thorgeirsson, S. S. (1993). Commentary. Hepatic stem cells. Am. J. Pathol. 142, 1331-1333. Thorgeirsson, S. S., and Evarts, R. P. (1992). Growth and differentiation of stem cells in adult liver. In "The Role of Cell Types of Hepatocarcinogenesis" (A. E. Sirica, ed.), pp. 100-120. CRC Press, Boca Raton, Florida. Tsao, M.-S., and Grisham, J. W. (1987). Hepatocarcinomas, cholangiocarcinomas, and hepatoblastomas produced by chemically transformed cultured rat liver epithelial cells. A lightand electron-microscopic analysis. Am. J. Pathol. 127, 168-181. Tsao, M.-S., Smith, J. D., Nelson, K. G., and Grisham, J. W. (1984). A diploid epithelial cell line from normal adult rat liver with phenotypic properties of oval cells. Exp. Cell Res. 154, 38-52. Van Eyken, P., and Desmet, V. J. (1992). Development of intrahepatic bile ducts, ductular metaplasia of hepatocytes, and cytokeratin patterns in various types of human hepatic neoplasms. In "The Role of Cell Types in Hepatocarcinogenesis" (A. E. Sirica, ed.), pp. 227-263. CRC Press, Boca Raton, Florida. Van Eyken, P., Sciot, R., Paterson, A., Callea, E, Kew, M. C., and Desmet, V. J. (1988). Cytokeratin expression in hepatocellular carcinoma: An immunohistochemical study. Hum. Pathol. 19, 562-568. Van Eyken, P., Sciot, R., Callea, E, and Desmet, V. J. (1989). A cytokeratin-immunohistochemical study of focal nodular hyperplasia of the liver: Further evidence that ductular metaplasia of hepatocytes contributes to ductular "proliferation." Liver 9, 372-377. Van Thiel, D. H., Gavaler, J. S., Kam, I., Francavilla, A., Polimeno, L., Schade, P. R., Smith, J., Diven, W., Penkrot, R. J., and Starzl, T. E. (1989). Rapid growth of an intact human liver transplanted into a recipient larger than the donor. Gastroenterology 93, 1414-1419. Vandersteenhoven, A. M., Burchette, J., and Michalopoulos, G. (1990). Characterization of ductular hepatocytes in end-stage cirrhosis. Arch. PathoI. Lab. Med. 114, 403-406. Wilson, J. W., and Leduc, E. H. (1958). Role of cholangioles in restoration of the liver of the mouse after dietary injury. J. Pathol. Bacteriol. 76, 441-449.

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Wirth, P. J., Luo, L.-D., Fujimoto, Y., and Bisgaard, H. C. (1992). Two-dimensional electrophoretic analysis of transformation-sensitive polypeptides during chemically, spontaneously, and oncogene-induced transformation of rat liver epithelial cells. Electrophoresis (Weinheim, Fed. Repub. Ger.) 13, 305-320.

6 Contributions of Hepadnavirus Research to Our Understanding of H ep ato carcin og en esis Charles E. Rogler Leslie E. Rogler Deyun Yang Silvana Breiteneder-Geleef Shih Gong Haiping Wang Marion Bessin Liver Research Center Albert Einstein College of Medicine Bronx, New York 10461

I. General Overview of Hepadnavirus Animal Models and Hepatocarcinogenesis The field of hepadnaviruses and their association with hepatocellular carcinomas has recently been the subject of several extensive reviews (Schirmacher et al., 1993; Rogler and Chisari, 1992; Tennant and Gerin, 1994). Rather than attempt to recapitulate these summaries, we will focus on those aspects of hepadnavirus research which have provided unique approaches to studying hepatocarcinogenesis and some of the insights they have provided into the process. Our discussion will focus on three animal models of hepadnavirus associated hepatocarcinogenesis. These include: (1) overexpression of the hepatitis B virus (HBV)envelope protein (HBsAg) in transgenic mice, (2) persistent infection of woodchucks with woodchuck hepatiLiver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tis virus (WHV), and (3) overexpression of hepatitis B virus X gene (HBx) in transgenic mice. We will also review selected studies on the role of viral DNA integrations in hepatocellular carcinoma (HCC). Hepadnavirus animal models have upheld and reinforced many of the well-established principles of multistage hepatocarcinogenesis which were developed using chemical carcinogenesis animal models (Scherer, 1983; Farber and Sarma, 1987; Sell et al., 1987). The hepadnavirus animal models, as well as previously established models, are now poised to add new insights into the molecular genetics of hepatocarcinogenesis using newly developed tools made available through the murine and human genome projects. Table 1 is a brief list of some of the major cellular and molecular genetic processes which occur during hepatocarcinogenesis in the hepadnavirus associated models. While there are many common features of the overall pathophysiology between hepadnavirus models and chemical carcinogenesis models, the overall picture regarding molecular genetic changes is still unclear. The last section of Table 1 is an attempt to obtain a general consensus of possible changes in proto-oncogene, growth factor, and tumor suppressor gene expression in the hepadnavirus various models. Much of the data for this table was obtained from recent reviews (Tabor, 1994; Strom and Faust, 1990; Pasquinelli et al., 1992). At present, the fields of viral and chemical hepatocarcinogenesis would benefit from a comprehensive overview of all the literature to determine whether any clear general conclusions can be made; however, this is beyond the scope of this review. In spite of the confusion, the existing data suggest that malignant transformation of hepatocytes can be obtained by mutations or deregulation of genes linked to signal transduction pathways which are common to other cancers, and to alterations in tumor suppressor genes which perform critical functions in controlling cell cycle progression. Some of the signal transduction pathways implicated in hepadnavirus associated hepatocarcinogenesis are schematically represented in Figure 1. The interactions between members of these pathways continue to be elucidated, and new members of each interacting pathway are being discovered almost daily. One recent report which illustrates this concept is the discovery that the main function of Ras proto-oncogenes is to recruit the Raf proto-oncogene to the plasma membrane where it functions as a mitogenactivated protein (Map) kinase to activate an entire signal transduction pathway (Leevers et al., 1994). It will be productive for future studies to focus on common pathways which are perturbed in malignant hepatocytes and how these pathways interact with each other to lead to the malignant phenotype. We should not expect one specific link in the pathways to be altered in all cases. When viewed in this light, it is clear that the Ras signal transduction pathways, which are growth factor regulated, are altered at some point in nearly all hepatomas (Figure 1). The tumor suppressor gene

Table 1 Hepadnavirus Associated Hepatocarcinogenesis Virus: HBV Host: Man

1. Long-term persistent infection associated with H C C 2. Limited immune response to persistent infection 3. Persistent cycles of cell death and regeneration 4. Proliferation of oval cells in precancerous stages 5. Development of distinct precancerous lesions a. Altered hepatic foci b. Neoplastic nodules 6. Development of hepatocellular adenoma 7. Development of H C C types: a. Poorly differentiated b. Well differentiated 8. Viral DNA integrations a. Occur in precancerous liver b. Are clonal in HCCs c. Are associated with a commonly activated protooncogene d. Are associated with function genes e. Often contain viral gene 9. Viral X gene is a. Present in genome b. A viral transcription factor c. A cellular gene transcription factor d. Oncogenic

WHV Woodchuck

GSHV Ground squirrel

DHBV Duck

HBsAg Mouse Yes Yes Yes

HBxgene Mouse -

Yes Yes Yes

? Yes Yes

Yes Yes Yes Yes

Yes Yes Yes Yes

Yes Yes Yes ?

Yes Yes > >

Yes Yes Yes

Yes Yes Yes

Yes Yes Yes

? ? ?

Yes Yes

Yes Yes

?

?

?

?

Yes

-

Yes

Yes

Yes Yes No

Yes Yes Yes

?

?

Yes

Yes

?

?

NA NA NA

NA NA NA

Yes Yes

Yes Yes

? ?

? ?

? ?

? ?

Yes Yes Yes Yes

Yes Yes Yes >

Yes Yes Yes

No

Yes > >

Yes

?

Yes

?

> > ?

> >

? ?

(continues )

Table 1-Continued Virus: Host:

10. Frequency of alterations in cellular proto-oncogenes and growth factor expression in HCCs a. Ras point mutations b. Ras overexpression c. Myc overexpression via amplification d. Myc overexpression via insertional mutagenesis of viral DNA e. IGF2 overexpression f. TGFa overexpression 11. Tumor suppressor genes a. Frequent LOH in 17p b. Frequent point mutations in p53 c. Frequent loss of Rb expression

HBV Man

WHV Woodchuck

GSHV Ground squirrel

DHBV Duck

HBsAg Mouse

H B x gene Mouse

Low Med Med Low

?

?

?

Low High

High Low

? ? ?

Low Low Low Low

?

?

?

High High

High ?

?

>

?

?

High Low

? ?

Highllow Highllow Highllow

?

? ? ?

? ? ?

Low Low Low

? ?

?

?

?

? ?

?

LOW:Not believed to be major mechanism. Most studies do not reveal any change by this mechanism; Med: Some studies positive, others negative. Basically, there is some support for mechanism, but its importance is not firmly established. High: Several studies clearly linking this mechanism to hepatocarcinogenesis. HighILow: Results of studies can reveal high or low incidence depending on regions of the world.

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Figure I Schematic diagram of selected signal transduction pathways which lead to transcriptional activation of nuclear genes involved in growth control. In these pathways the following have been identified as oncogenes in various systems: (1) ligands IGF2 and TGFci, (2) receptor tyrosine kinases, (3) SH2-SH3 domain proteins (i.e., SOS), (4) membrane localized protooncogenes, c-Ras and c-Raf, and (5) the nuclear proto-oncogene c-Myc. This illustrates plasticity and redundancy in oncogenic pathways.

p53 which controls the cell cycle, is also clearly implicated in hepatocarcinogenesis (Harris, 1993).

II. Hepatitis B Virus Envelope Protein (HBsAg) Transgenic Mice A. HBsAg (Line 50-4) Transgenic Model of Hepatocarcinogenesis HBsAg expression in transgenic mice is normally non cytopathic; however, when the large envelope protein of HBV is overexpressed in relation to the small envelope protein, the HBsAg particles remain in the endoplasmic reticulum and cause a cytopathic effect (Chisari et al., 1989). In this murine model, chronic hepatocellular injury and inflammation lead to regenerative hyperplasia and eventually to the development of chromosomal abnormalities and HCC (Chisari et al., 1989). This transgenic mouse model thereby reiterates many of the pathophysiological events that occur prior to the

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development of HCC in chronic HBV infection in humans (Schirmacher et al., 1993).

An extensive survey of the structure and expression of a large panel of proto-oncogenes and tumor suppressor genes in the precancerous liver and HCCs of these HBsAg animals did not reveal any commonly activated genes (Pasquinelli et al., 1992). The genes studied included multiple members of the m y c and ras gene families, raf, and others. These findings suggest that mechanisms other than transcriptional activation or mutation of the studied proto-oncogenes must be responsible for malignant transformation in this model (Pasquinelli et al., 1992). Sequence analysis of the frequently mutated regions of the p53 tumor suppressor gene did not reveal any point mutations in p53 in a large set of tumors from HBsAg mice (Pasquinelli et al., 1992). This is consistent with p53 mutations primarily being a late event in hepatocarcinogenesis (Pasquinelli et al., 1994). The authors suggest that the tumors in HBsAg, line 50-4 mice represent an early stage of hepatocellular tumorigenesis. Interestingly, the only growth factor which is commonly found to be overexpressed is insulin-like growth factor 2 (IGF2) (Schirmacher et al., 1992; Pasquinelli et al., 1994). No other changes are observed in the steady-state expression of hepatocyte growth controlling factors including insulin-like growth factor 1 (IGF1), epidermal growth factor (EGF), EGF receptor (EGFr), hepatocyte growth factor (HGF), c-met, transforming growth factor [3 (TGF[3) or transforming growth factor cx (TGFci) in the HCCs for line 50-4 transgenic mice (Pasquinelli et al., 1994). IGF2 is also expressed in HCCs from three other transgenic models suggesting that it may be a common growth factor involved in hepatocarcinogenesis (Schirmacher et al., 1992). This conclusion has been strongly supported by subsequent research from our group and others (see further discussion below).

B. Noncytopathic HBsAg Transgenic Mice: Role of Cytokines in Gene Regulation The cellular immune response to virus encoded antigens is thought to play an important role in viral clearance and the pathogenesis during acute and chronic infection (Moriyama et al., 1990; Ando et al., 1992; Mondelli et al., 1982). Transgenic mice which produce the small HBsAg in the liver do not develop a cytopathic lesion but rather secrete HBsAg into the blood (Chisari et al., 1985; Babinet et al., 1985; Burk et al., 1988). Chisari and colleagues have reconstructed a cellular immune response by adaptive transfer of HBsAg specific cytotoxic T lymphocyte (CTL) clones into the HBsAg transgenic mice (Moriyama et al., 1990, Ando et al., in press). Their studies show that an important component of the disease process upon adaptive transfer is mediated by inflammatory cytokines secreted by the CTLs, especially

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~/-interferon (IFN~/) (Guidotti et al., in press). One of the most interesting aspects of these studies, and several which have followed (Guilhot et al., 1993; Gilles et al., 1992; Guidotti et al., 1994), is that HBV gene expression is downregulated by the cytokines, IFN~/, interleukin-2 (IL-2) and tumor necrosis factor cx (TNFcx) which are secreted from HBV specific T cells and the inflammatory cells they recruit. The regulatory effect of IL-2 and TNFcx on HBV gene expression occurs at the post-transcriptional level and is specific for virus transcripts, suggesting that IL-2 and TNFcx activate genes that selectively destabilize HBV mRNA (Guidotti et al., 1994). Most likely, intrahepatic macrophages in the HBsAg mice release TNFcx in response to activation by IL-2, which is produced by activated class II restricted CD4positive T cells. Therefore, the intrahepatic cellular immune response to HBV may play a previously unsuspected role in the biology of HBV infection by modulating HBV gene expression without destroying infected cells (Guidotti et al., 1994). What relevance do these results have to hepatocarcinogenesis? Their relevance is perhaps strongest in helping us understand possible mechanisms by which gene expression is controlled in precancerous lesions in hepadnavirus carriers. This has relevance to any hepatocarcinogenesis regimen in which an inflammatory response occurs. The early stages of chemical hepatocarcinogenesis regimens include a short period in which there is an inflammatory response to liver damage (Scherer 1983; Farber and Sarma 1987; Sell et al., 1987). In WHV-infected liver, precancerous lesions described as altered hepatic foci (AHF) have the characteristics of polyclonal lesions (Table 2). AHF invariably contain focal accumulations of inflammatory cells within them and the steady-state mRNA level of several cellular genes are coordinately upregulated while others are downregulated in the woodchuck precancerous lesions (Yang and Rogler 1991; Yang et al., 1993) (Figures 2 and 3). Is it possible that cytokines secreted from inflammatory

Table 2 Characteristics of Altered Hepatic Foci (AHF) in Woodchucks 1. Histological analysis reveals: a. Hepatocytes strongly basophilic with clear cell lesions occurring only rarely b. Well-defined cord structure c. Altered nuclear phenotype d. Slight or no compression of surrounding parenchyma e. AHF extend from portal tracts to central veins f. Localized inflammatory cell accumulation in nearly all AHF g. Structural characteristics of polyclonal lesions 2. Nonpermissive or semipermissive for WHV transcription and replication 3. Heterogeneousexpression of gammaglutamyltranspeptidase (GGT)with expression strongest near portal tracts

Figure 2 (A) N-myc, IGF2, and WHV expression in persistently infected woodchuck livers. Autoradiograms of serial sections of two tissue blocks hybridized with WHV, N-myc, or IGF2 antisense riboprobes. Areas of positive hybridization appear as dark regions of the sections. (B) Microscopic analysis of gene expression in serial sections of a single altered hepatic focus (AHF). The AHF analyzed is the one marked by arrows in Figure 2A. In all the serial sections the long solid arrows denote the outer boundaries of the AHF, open arrows denote portal tracts, solid arrowheads denote central veins, and curved solid arrows denote specialized structures. The pattern of portal tracts and central veins provide landmarks to facilitate direct comparison of the serial sections. (1) Structural organization of the AHF and surrounding liver

Figure 3 Expression of genes involved in regulating the biological activity of IGF2 and N-myc in woodchuck tissues. Schematic illustration of representative results from in situ hybridization of antisense riboprobes to Normal, normal uninfected woodchuck liver; N § AHF, persistently infected woodchuck liver containing an altered hepatic loci (circular area within the square); T/N, T, hepatocellular carcinoma, N, peritumor liver. The intensity of hybridization is directly proportional to the darkness of the section. Dark circles within AHF note the presence of "foci within loci" which express higher or lower levels of the gene. (Note results for IGF2, N-myc and IGFBP-1.) IGFBP3 is expressed in Ito cells and therefore its pattern is strippled. When expression was uniform for an entire region, the area is uniformly darkened.

viewed by bright field microscopy (hematoxylin and eosin stained' X40). (2) Variable gamma glutamyl transpetidase (GGT) gene expression within the AHF (bright field microscopy, hematoxylin counter stained, X40). Panels 3 to 6 are dark field micrographs in which silver grains appear as bright spots (X40). (3) N-myc expression. (4) IGF2 expression. Curved arrow denotes the highly elevated expression of IGF2 in the subregion of the AHE (5) WHV expression detected with a WHV antisense riboprobe. Note the low and variable expression, which is strongest in GGT positive regions (compare to Panel 2). (6) Histone H3-2 expression. Note higher frequency of histone III positive cells in the foci compared to the surrounding liver tissue.

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cell lesions in the AHF are responsible for the altered expression of liver specific genes and WHV mRNA described above for HBsAg mice (Guilhot et al., 1993; Gilles et al., 1992; Guidotti et al., 1994). Whether downregulation of a few critical genes required for the maintenance of the hepatocyte differentiated functions would release them from cell cycle controls and start them on a deregulated path toward immortalization and eventually HCCs is an area that needs further investigation. Furthermore, the cytokine effects, once established, may not require a continued cytokine presence. The results from the hepadnavirus models clearly suggest that the roles of various cytokines in the early stages of hepatocarcinogenesis should be more thoroughly investigated.

ni. Woodchuck Hepatitis Virus (WHV) Model of Hepatocarcinogenesis The WHV animal model has recently been the subject of a review (Tennant and Gerin, 1994). Therefore, we will focus on those aspects of the model which directly relate to viral activity in carcinogenesis, and the insights gained into the genetic events associated with precancerous lesions. A. Toxic Oxygen Radicals and WHV Persistent Infection

Point mutations in genes which are responsible for mismatch repair have recently been linked to hereditary nonpolyposis colorectal cancer (Leach et al., 1993). Similarly, point mutations in the MSH1 and MSH2 genes, which are responsible for mismatch repair, lead to microsatellite instability (MIN) within the host genome (Leach et al., 1993). Thus, a direct link has recently been drawn between specific genes involved in a basic genetic mechanism (i.e., mismatch repair), and the genetic instability (i.e., MIN) that predisposes cells to carcinogenic transformation. In this regard, the work of Tennant and his associates is particularly relevant (Liu et al., 1991, 1992, 1993). They have demonstrated that the production of toxic oxygen radicals, specifically nitric oxide (NO), increases in livers persistently infected with WHV. Clearly, the presence of toxic oxygen radicals in hepatocytes will present a risk factor for point mutations and possible development of MIN, especially if the normal detoxification mechanisms in the liver are also compromised. The frequency and the time course of development of MIN in liver of hepadnavirus carriers needs to be determined. B. Insertional Mutagenesis

In addition to predisposing the hepatocyte genome to point mutations, WHV has another mechanism for generating host genomic instability. This

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involves direct integration of its viral DNA genome into the host genome (Table 3). Fortunately for the host, viral integration is an infrequent event, since integration is not required for productive viral infection. However, viral DNA integration does occur and integrations can be cloned from precancerous liver tissue (Rogler and Summers, 1984). During persistent viral infection, open circular viral DNA molecules, containing a single stranded region resembling a stationary replication fork, are recycled into the nucleus at a low rate (Tuttleman et al., 1986a; Summers et al., 1990). These molecules are suicide substrates for the ubiquitous nuclear enzyme, Topoisomerase I (Topo I). This enzyme can cleave and linearize WHV DNA molecules in vitro and can also mediate their integration into the cellular genome (Wang and Rogler, 1991). The integration reaction has been duplicated in vitro using purified Topo I, purified WHV virion DNA, and a cellular target DNA (Wang and Rogler 1991). The in vitro generated WHV integrations were virtually identical to a subset of those which have been cloned from HCCs (Dejean et al., 1984; Yaginuma et al., 1987; Hino et al., 1989; Shih et al., 1987; Nagaya et al., 1987). The Topo I integration mechanism predicts that integrations cloned from HCCs should occur at preferred Topo I cleavage sites and a detailed analysis of cellular and viral sequences at integration sites has revealed that this is true (Schirmacher et al., 1994). The survey showed that 93% of the illegitimate recombination junctions of hepadnavirus DNA and host DNA occur within, or immediately adjacent to, preferred Topo I cleavage sites (Schirmacher et al., 1994). The idea that integrations generate genetic instability comes from the finding that integrations are found at the site of large and small deletions of

Table 3 Information About Hepadnavirus Integrations a 1. Although integration is not required for viral replication, clonally propagated integrations are present in most HCCs from hepadnavirus carriers. 2. Both linear and arranged viral genomes are present in integrations. 3. Integration is not specific for any viral DNA sequence yet highly preferred integration sites exist, especially in the immediate vicinity of the replication origin (DR1). 4. Topoisomerase I, an abundant nuclear enzyme, can mediate illegitimate recombination of hepadnavirus DNA with cellular DNA in vitro. The integrations produced in vitro closely resemble those which occur in vivo. 5. Preferred Topoisomerase I cleavage sites are present at over 90% of the crossover sites in hepadnavirus integrations cloned from HCCs. 6. Integration increases carcinogenic risk by causing micro and macro deletions, inverted and direct duplications, and translocations of host genomic DNA. 7. Activation of c-myc genes by insertion of hepadnavirus DNA is a commnon event in the genesis of HCC in woodchucks. 8. Integrations often contain viral X genes, and/or a truncated PreS2/S, both of which function as transcriptional transactivators; the X gene is also an oncogene. aSee text for reference citations and abbreviations.

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cellular DNA (Rogler et al., 1985; Nakamua et al., 1988), at direct and inverted duplications of cellular DNA (Yaginuma et al., 1985; Mizusawa et al., 1985), and as linker molecules at sites of chromosome translocations (Hino et al., 1986). These findings do not allow us to distinguish whether HBV directly causes genome instability by integrating into the genome or becomes linked at sites in cellular DNA which have already been cleaved and not repaired correctly. Recent studies in our laboratory, using a cell line which replicates hepadnaviruses, have shown that viral DNA integrations can also excise from the cellular genome at a relatively high frequency (Gong and Rogler, unpublished data). Further measurements of the frequency of integration and excision of integrations in stable and unstable genetic backgrounds will help resolve the questions of cause and effect in regard to integrations and genome stability. One previous study has presented evidence for increased in vitro recombination of DNA adjacent to integrated HBV DNA (Hino et al., 1991). The presence of clonal viral DNA integrations, sometimes as the sole form of viral DNA in an HCC, fueled the speculation that they could activate expression of proto-oncogenes involved in hepatocarcinogenesis. Initial cloning experiments of WHV integrations from woodchuck HCCs did not reveal a common integration site (Ogston et al., 1982). However, after many years of diligent searching, the research group headed by Tiollais and Buendia discovered a proto-oncogene in the woodchuck genome which is commonly activated by WHV DNA integration in a large majority of woodchuck HCCs (Fourel et al., 1990; Wei et al., 1992). The chromosome locus identified turned out to be a functional retroposon of the cellular N-myc gene, which they designated N-myc-2 (Fourel et al., 1990). They demonstrated that WHV integration activated transcription of the normally silent N-myc-2 retroposon by an enhancer insertion mechanism. The WHV enhancer has many binding sites for liver specific transcription factors and is a strong enhancer, which when inserted into the 3' untranslated region of N-myc-2, activates transcription from a cryptic promoter that lies within the first exon of N-myc-2, upstream from the translation start site (Wei et al., 1992). Further work by their group has shown that additional integrations, very distant from N-myc-2, but at a common site on the same chromosome, may also activate N-myc expression in those cases where a WHV DNA integration is not found immediately 5' to the N-myc-2 gene, or within the 3' noncoding region of N-myc-2 (Fourel et al., 1993). In those tumors that do not express N-myc, c-myc is overexpressed due to either WHV DNA integration or another mechanism (Hansen et al., 1993; Hsu et al., 1990). Therefore, WHV DNA integration has revealed a cellular proto-oncogene, which may fit the criteria of a "gatekeeper gene" for HCC in woodchucks. The gatekeeper concept states that the gatekeeper gene is a gene whose expression must be altered in order for a tumor to

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develop in a particular organ or cell type. Another example of a gatekeeper gene would be the APC gene in certain hereditary forms of colon carcinoma (Fearon and Vogelstein, 1990).

C. Integration and Human HCC As clear as the function of viral DNA integration appears to be in woodchucks, it is unclear in all the other animal models and in humans (Table 3). After analyzing the flanking sequences of HBV integrations from many tumors from many sources around the world, a common or even frequent integration site has not yet been identified (Nagaya et al., 1987). Chromosomes 17 and 11, both of which contain tumor suppressor genes, and protooncogenes, have been shown to have the highest frequency of HBV integrations. One of the HBV integrations in chromosome 11 was in the vicinity of the Wilms tumor suppressor gene on chromosome 11p13; the HBV integration caused a large deletion in that segment of the chromosome (Rogler et al., 1985). Another HBV integration was identified at a chromosome translocation in which chromosome 17q21-22 was linked to chromosome 18q 11 via HBV DNA (Hino et al., 1986). The breast tumor gene, B r a c l , was recently localized to chromosome 17q21 and ongoing studies are being conducted to determine whether the HBV associated chromosome breakpoints are within the small region that has been recently identified to contain the Bracl gene (Bowcock et al., 1993). Additional HBV integrations have been identified in association with a truncated cyclin A gene (Wang et al., 1990), and the retinoic acid receptor beta gene (deThe et al., 1987), as well as other cellular genes whose functions are not as well established (Etiemble et al., 1989; Ochiya et al., 1986). These data demonstrate that HBV integrations can be utilized to identify new cellular genes which are potentially involved in tumorigenesis. The cloning of a single HBV integration in a cyclin A gene provides a telltale sign that additional alterations in the numerous cell cycle control genes may be important in hepatocarcinogenesis. The HBV integration into the retinoic acid receptor beta gene suggests that altering the expression of genes that control hepatocyte differentiation may also be a step in hepatocarcinogenesis in HBV carriers. D. Analysis of Precancerous Lesions and HCCs: The Case for a Role of IGF2 in Tumor Promotion In order to study the early events of hepatocarcinogenesis in WHV carrier woodchuck liver, we have utilized in situ hybridization and immunocytochemistry to study the expression patterns of genes in AHE Some of the histological characteristics of AHF in woodchuck liver are summarized

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in Table 2. AHF are clearly evident in woodchuck liver as foci of hepatocytes which have a strongly basophilic cytoplasm. The histological characteristics of the AHF strongly suggest that they are polyclonal in nature, although subregions (loci within foci) have characteristics of clonally propagated cells. A schematic summary of the in situ hybridization results to be discussed in this section is presented in Figure 3 and typical data used to support the schematic summary are presented in Figure 2. 1. R a t i o n a l e a n d Genetic Predisposition to H C C

Our original rationale for studying the expression of IGF2 in AHF and HCCs of woodchucks was based on the discovery of HBV integrations in chromosome 11 (Nagaya et al., 1987; Rogler et al., 1985), and the finding of a high frequency of loss of heterozygosity (LOH) on chromosome 11p in human HCCs (Wang and Rogler, 1988). In that study, we discovered that LOH was limited to the 11p15 region in some HCCs but in others it was limited to chromosome 11p13, which is the site of the Wilms tumor suppressor gene (WT1). The WT1 gene at 11p13 is not expressed in adult liver, leading to the hypothesis that a second tumor suppressor might be located at the distal end of chromosome 11 at 11p15 (Wang and Rogler, 1988). Genetic evidence also suggested that genes on chromosome 11p15 were involved in hepatocarcinogenesis because duplications of the distal end of chromosome 11, encompassing 11p15 are characteristic of Beckwith Wiedeman syndrome (Best and Hoeksha, 1981; Waziri et al., 1983). This genetic syndrome is associated with overgrowth and childhood tumors, and particularly with hepatomegaly and heptoblastoma (Koufos et al., 1985). Since hepatoblastoma arises in children with a fairly short latency, this strongly suggests that genes important in hepatocarcinogenesis are located in that region of chromosome 11p. Rapid developments in the recent past have strongly supported this hypothesis and pointed to specific genes in chromosome 11p15 as important candidates for roles as promoters and suppressors of tumorigenesis. Chromosome 11p15 contains a whole series of genes that have been linked to growth and malignancy. The H-ras proto-oncogene at 11p15.5 has been widely implicated in tumorigenesis, and overexpression of ras protooncogenes is a common event in HCCs (Tabor 1994). The other gene cluster of great interest at 11p15 includes the insulin, IGF2, and H 1 9 genes. Insulin is not expressed in normal liver whereas IGF2 is expressed at a low level in normal human liver and woodchuck liver but not in adult mouse liver. Since IGF2 has mitogenic activity in cell cultures (Cohick and Clemmons, 1993) and is expressed in a wide variety of tumors (Fu et al., 1988; Cullen et al., 1992), we focused on the expression of IGF2 in the woodchuck liver and HCCs. The H 1 9 gene produces an abundant developmentally regulated transcript of unknown function in normal embryos (Willison, 1991).

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2. IGF2 Expression in A H F a n d H C C s

Studies of IGF2 expression in woodchuck liver and HCCs strongly suggest that it plays a role in hepatocarcinogenesis. IGF2 is overexpressed in over 90% of AHF in precancerous woodchuck liver (Yang and Rogler, 1991). Interestingly, two levels of IGF2 expression are present in AHF, a moderate level throughout the foci and, in about 5% of loci, we observe localized regions (foci within loci) of highly elevated expression which is equivalent to the high levels most often observed in HCCs (Figure 2). We have suggested that this may be clonal selection of high expressing cells within the AHF because the small regions of very high IGF2 expression in AHF do have higher mitotic indices (Yang and Rogler, 1993). Interestingly, similar findings of localized high level IGF2 expression in precancerous lesions and a higher degree of malignancy in IGF2 producing tumors has recently been reported for SV40 TAg associated pancreatic carcinoma (Chrislofori et al., 1994). 3. IGF2 Functions as a T u m o r P r o m o t e r in Transgenic M i c e

The above studies establish that the pattern of IGF2 expression in precancerous lesions and HCCs is consistent with a causal role in carcinogenesis. In order to determine whether IGF2 was causally involved in tumorigenesis, two new animal models were utilized. We developed IGF2 transgenic mice which overexpress IGF2 primarily in the liver (Rogler et al., 1994). These animals develop HCC plus tumors from organs that do not express the transgene suggesting that the IGF2 in our mice functions by both autocrine and endocrine mechanisms (Rogler et al., 1994). The tumors arise at a slightly higher incidence than in control animals (22% versus 5% in controis), and the tumors arise with a very long latency period (18 to 24 months). These data suggest that although IGF2 is a weak tumor initiator, it can promote the growth of tumors which arise in mice. In order to test this possibility we have taken two approaches. The first approach was to mate the IGF2 transgenic mice with TGFc~ transgenic mice. The TGFc~ mice develop liver tumors with a latency of approximately 13 to 16 months (Jhappan et al., 1990; Takagi et al., 1992) and initial results of such matings have shown that the presence of the IGF2 transgene decreases the latency time for development of liver tumors in the TGF0~ mice by approximately 4 to 6 months (C. E. Rogler, L. E. Rogler, and G. Merlino, unpublished data). Furthermore, at short latency times, a much higher percentage of the tumors have a clearly malignant phenotype. These data suggest that IGF2 expression plays a direct role in tumor progression in the liver and that it can function in cooperation with another liver growth factor. The second approach is to determine whether knocking out the IGF2 gene can prevent HCC. In this approach we crossed TGF0~ transgenic mice

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with mice in which the IGF2 gene had been knocked out (mice kindly provided by Efstradiadis, Robertson and DeChiara, Columbia University). This experiment is nearing completion. However, a parallel experiment using the IGF2 knockout animals has been completed by Hanahan and colleagues at the University of California, San Francisco. Knocking out the IGF2 gene in mice that express the SV40 T A g in pancreatic islets significantly reduces both tumor growth in islets and the degree of tumor malignancy (Chrislofori et al., 1994). Tumors which developed in the SV40 TAg mice are clearly malignant whereas SV40 T Ag plus IGF2 knockout mice develop small benign tumors. The absence of IGF2 is associated with a fivefold increase in apoptosis in the benign tumors suggesting that the presence of IGF2 protects cells in tumors from apoptosis. However, knocking out IGF2 did not block tumor formation per se. Thus, tumor initiation in the SV40 TAg model depends on the action of SV40 TAg plus at least one additional unknown oncogene (Chrislofori et al., 1994). It was proposed that without IGF2 the action of this oncogene may cause cells to undergo apoptosis as opposed to mitogenesis. This hypothesis is based on work from other systems that shows the overexpression of c-myc can induce apoptosis in some immortalized cell lines (Shi et al., 1992). Cells can also be rescued from apoptosis by overexpression of growth factors or other cytokines which block the apoptotic pathway (Rodriguez-Tarduchy et al., 1992; Williams and Smith, 1993). The coordinate expression of IGF2 along with N-myc in AHF of woodchucks (Figure 2) may prevent hepatocytes from entering the apoptosis pathway. In fact, recent studies have shown that IGF2 can block apoptosis in several in vitro systems (Evans, 1994). Bcl-2 which also can prevent apoptosis is not normally expressed in liver and it is not induced in AHF of woodchucks (L. E. Rogler, unpublished data). 4. IGF2 Expression in the Context of Multiple Receptors, Regulatory Proteins, and Genetic Imprinting The biological activity of IGF2 is mediated by its receptors and insulin-like growth factor binding proteins (IGFBPs) (Table 4). In order to understand IGF2 expression in the context of its receptor and binding proteins in woodchuck liver, serial sections of the liver specimens that were used to determine IGF2 and N-myc expression were used to study expression of IGF receptor and IGFBP genes. The general conclusions of in situ hybridization studies are schematically summarized in Figure 3. The most striking results of this study are the coordinate upregulation of IGF2, N-myc, and IGFBP-4 and downregulation of IGFBPs 1 and 2 in AHF and HCCs and the localization of IGFBP-3 expression to Ito cells (represented by a speckled pattern in Figure 3). IGFBP-2 expression is generally downregulated in AHF; however, subregions of AHF sometimes expressed IGFBP-2 at normal levels. These

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Table 4 Major Proteins Which Regulate IGF Biological Activity Ligands

Binding proteins

Receptors

Insulin IGF1 IGF2

IGFBP-1 IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-5 IGFBP-6 IGFBP-?

Insulin receptor IGF1 receptor M6P/IGF2 receptor

regions are immediately adjacent to portal tracts and are represented in Figure 3 as a dark loci within the AHE While some IGFBPs are known to interfere with IGF2 activity, others can enhance its activity in certain cell types. Further work is necessary to determine whether IGFBPs 1 and 2 interfere with IGF2 action in liver in which case downregulation in tissues would enhance the effectiveness of IGF2 or whether they enhance IGF2 activity and their loss reduces the biological activity of IGF2. In the liver, IGF2 signal transduction must be mediated through either the insulin receptor or the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r) since IGF1 receptors are not present (Cohick and Clemmons, 1993). Northern blot analysis of woodchuck liver and HCCs has shown that both insulin and M 6 P / I G F 2 r genes are expressed, as expected (Yang and Rogler, manuscript in preparation) (Figure 3). IGF2 polypeptides in woodchuck HCCs accumulate in the perinuclear region of cells and colocalize with M6P/IGF2r (Yang and Rogler, 1991). Some evidence exists that the M6P/IGF2r may signal through G proteins when bound by IGF2 (Nishimoto et al., 1989; Okamoto et al., 1990); however, they are generally believed to target IGF2 for degradation in lysosomes. Therefore, IGF2 media..ed signal transduction is most likely mediated through its cross-reaction with insulin receptors in the liver (Cohick and Clemmons, 1993).

IV. Hepadnavirus X Gene Encodes an Oncogenic Transcriptional Transactivator A. Background We will present a brief historical overview of major aspects of HBx gene research and then focus more in-depth on the recent transgenic and cell culture models that have clearly demonstrated that the X gene can function

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as an oncogene (Conclusions, Table 5). After the HBV genome was cloned, sequence analysis revealed the presence of an open reading frame whose protein sequence did not match any of the viruses structural proteins (Galibert et al., 1979). Since this gene's function was u n k n o w n , it was given the name, X protein, as opposed to the name of the other viral genes, S (surface antigen), C (nucleocapsid core protein), and P (polymerase). Initial studies suggested that the X gene does not have a highly active promoter that produced a unique X gene message; however, later studies have clearly shown that it does have an active p r o m o t e r (Treinin and Laub, 1987). X gene mutagenesis studies and the lack of an X gene in duck hepatitis B virus (DHBV) initially led to the conclusion that the X gene is not required for viral replication. Recent in v i v o infection studies with m u t a n t W H V viruses lacking the X gene, however, demonstrate that the W H x gene is required for establishment of viral infection in v i v o (Chen et al., 1993). The position of the X gene in the m a m m a l i a n hepadnaviruses ( d o w n s t r e a m from the core and envelope genes and in the same transcriptional orientation), led to speculation that it might have originated as a cellular gene which was picked up by the m a m m a l i a n hepadnaviruses (Miller and Robinson, 1986). H o w ever, to this date a cellular homologue has not been identified. The presence of the complete X open reading frame in hepadnavirus D N A integrations cloned from H C C s while the core (C) and surface antigen (S) genes in the same integrations are rearranged (Nagaya et al., 1987; Ogston et al., 1982),

Table 5 Information about Hepadnavirus X Genea 1. X-ORF encodes a 17-kDa promiscuous transcriptional activator (transactivator). a. Examples of a few enhancers and promoters transactivated include: 1. HBV enhancer + core promoter 2. SV40 enhancer + early promoter 3. LTRs for HSV-TK, HIV~ and RSV 4. c-Myc 5. Many others 2. No cellular homologue yet identified. 3. X proteins of HBV, WHV, and GSHV are active in transactivation assays. 4. HBx acts through multiple cell type-specific transcription factors (AP-1, AP-2, CREB, ATF~ NFKB). 5. Transactivation of HBV and heterologous genes occurs when the X gene is expressed in its native state during productive infection. 6. X gene is required for WHV productive infection. 7. X gene is expressed during acute and persistent infection. 8. X gene is retained in many integrations in HCCs and transactivation activity is maintained. 9. X gene has transforming activity in SV40T Ag immortalized murine hepatocytes. 10. HBx transgenic mice that express high levels of X protein develop HCC after one year. 11. HBx may function through both the PKC and Raf signal transduction pathways. aSee text for reference citations and abbreviations.

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led to additional speculation that the X gene might function in hepatocarcinogenesis. The discovery that the X protein was a transcriptional transactivator added a great deal of focus to the field and provided a potential mechanism by which the X gene might function in viral replication and hepatocarcinogenesis (Twu and Schloemer, 1987; Spandau and Lee, 1988). Antibodies to X protein were detected after acute infection demonstrating that the X protein was expressed in productive infections (Feitelson et al., 1990; Pershing et al., 1986). In vitro replication systems were used to demonstrate that X proteins of the mammalian viruses [HBV, ground squirrel hepatitis virus (GSHV) and WHV] transactivate viral promoters during productive infections (Colgrove et al., 1989). Cloned integrations from HCCs that contain X genes were utilized to demonstrate that integrated X genes are transcribed and have transactivation activity (Yang et al., 1993; Wollersheim et al., 1988; Takada and Koike, 1990b). Many studies of the transactivation mechanism of the X protein demonstrate that it is a promiscuous transactivator that acts through multiple cell type specific transcription factors some of which include AP-1, AP-2, CREB, ATF-2, NFKB, cEBP, and others (Seto et al., 1990; Maguire et al., 1991). B. X Gene Transactivation Mechanism

Researchers in the field of X transactivation agree the X protein is not a DNA binding protein and that its activity is manifest through proteinprotein interactions. The search for proteins that interact with the X protein in vivo is ongoing in many laboratories. This line of research is important because it will give us insights into the molecular mechanism of transactivation and may provide valuable insights into which gene pathways are important to start hepatocytes on the path to malignant transformation. We can say this because the X protein has now been shown to have oncogenic activity in vivo in transgenic mice (Kim et al., 1991; Koike et al., 1994). X protein is not an acute transforming protein but rather appears to be a slow acting oncogene which induces metabolic alterations (e.g., glycogen accumulation) in hepatocytes in a zonal pattern (near central veins) when expressed in the liver under its own native enhancer-promoter (Koike et al., 1994). The mechanism of X protein transactivation is, and has been, the focus of a great deal of research. Work in several laboratories has pointed to two main pathways in which the X protein may function indirectly to regulate transcription of a variety of promoters. These include activation of the protein kinase C (PKC) pathway (Kekule et al., 1992) through mobilization of PKC to the plasma membrane, and activation of a protein kinase cascade pathway through activation of Raf (Cross et al., 1993), which serves a

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serine threonine protein kinase kinase kinase (see signal transduction pathway outlined in Figure 1). Evidence also suggests that the HBx protein might function as a serine threonine protease inhibitor (Takada and Koike, 1990b). Such a function of X might fulfill the broad requirements for its promiscuous action on enhancers with unrelated sequence motifs. Interestingly, the early effects of X gene expression in transgenic mice include alterations in metabolism leading to clear cell foci with glycogen accumulation. This could reflect early actions on gene pathways involved in maintenance of hepatocyte differentiated functions. C. In Vitro Assays for Hepadnavirus Transforming Activity

Rather than extensively discuss the literature on X protein transformation activity, we will focus on a single experimental system dealing with SV40 T antigen immortalized hepatocytes (Paul et al., 1992). SV40 TAg is a powerful oncogene because it binds and functionally inactivates at least two important cellular proteins, Rb and p53 (Levine et al., 1994; Harris and Hollstein, 1993). The tumor suppressor functions of these proteins are due, at least in part, to their ability to control the cell cycle and the fact that their removal or inactivation deregulates the cell cycle. Cells which have experienced DNA damage progress through the cell cycle, when p53 is knocked out, rather than arresting in G 1 (Levine et al., 1994; Harris and Hollstein, 1993). Thus, mutations become fixed in the genome increasing the risk of malignant transformation of that cell. Expression of SV40 TAg in hepatocytes deregulates the cell cycle and the hepatocytes behave as immortalized cells. The immortalized phenotype is stable in culture and the immortalized cells do not exhibit a malignant phenotype (Paul et al., 1992). HBV expression in these cells causes malignant transformation as judged by growth in soft agar and production of HCCs in nude mice (H6hne et al., 1990). Furthermore, exclusive expression of HBx protein in the cells also causes malignant transformation (Seifer et al., 1991). Work on DHBV replication in primary hepatocytes shows that viral replication per se does not lead to malignant transformation (Tuttleman et al., 1986b). Therefore, once cell cycle controls, provided by tumor suppressors, are removed HBV and particularly the X protein have a greater oncogenic potential. The recent report that HBx protein can directly interact with the p53 tumor suppressor protein provides a potential mechanism by which HBx might directly deregulate cell cycle control in hepatocytes (Wang et al., 1994).

D. HBx Transgenic Mice Develop HCC There are now two reports that HBx expression in transgenic mice can lead to a multistep progression toward HCC (Kim et al., 1991; Koike et al.,

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1994). Since the second report provides a more detailed description of the pathophysiology, we will focus our analysis on it. When the HBx gene was expressed under the influence of the natural HBV enhancer and X promoter, transgenic mice with a high level of HBx expression clearly developed HCC beginning around 13 months old. Mice expressing low levels of HBx develop HCC at a low frequency and with a longer latency. These results may explain why HCC has not been observed in some other lines of HBx transgenic mice which express only low levels of the HBx transgenes (Lee et al., 1990). In young HBx transgenic mice, a high level of HBx expression occurs in the centrilobular region (zone 3), and these regions subsequently develop increased vacuolation at which time increased HBx expression in the centrilobular region is evident (Kim et al., 1991; Koike et al., 1994). By the age of 4 months, aneuploid peaks of DNA are present in nuclear preparations of the HBx liver and this corresponds to the development of dysplastic hepatocytes with large nuclei which appear within the altered foci around central veins. At 7 months of age, the regions around the central veins that selectively express higher HBx protein also have an increased DNA synthesis as measured by incorporation of BrdU into newly replicated DNA. The presence of aneuploidy suggests that X protein expression leads to genetic instability in the liver, and that this could be due to alterations in the functional levels of p53 in hepatocytes (Koike et al., 1994). HBx protein expression did not induce an inflammatory response, or elevate glutamic-pyruvic transaminase (SGPT) in blood which is an indicator of ongoing liver damage. Thus cell death, which must occur, most likely occurs via apoptosis which does not elevate blood SGPT levels. Experiments to determine whether p53 is bound to the HBx protein in this transgenic model or whether p53 undergoes genetic alteration (i.e., point mutations or deletions) are of great interest. A change in frequency of apoptotic bodies was not detected in preneoplastic foci of HBx transgenic mice compared to control mice; however, extensive quantitative data on the apoptotic index of foci were not presented in this study. One might predict that if HBx protein really does bind and inactivate p53 in vivo, there would be no need to further mutate p53, and wild type p53 genes would be found in HCCs from these mice. As the HBx mice age and the liver lesions progress to HCC, the expression of HBx protein increases suggesting that a selection for high expressing cells occurs. The mice develop multifocal HCC beginning at 13 months of age. The absence of a distinct period of hepatocyte death and an inflammatory response in the liver, however, separates this model from the HBsAg model of hepatocarcinogenesis. The progressive series of precancerous lesions, adenoma, and HCC which occur in HBx mice are common features in all the other transgenic models. While HBx is capable of initiating metabolic and/or genetic alterations in hepatocytes which lead to HCC, it must func-

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tion in conjunction with other oncogenes/growth factors and/or tumor suppressor genes for malignant transformation to be complete. The activity of HBx as a transactivator and oncogene provides a valuable new tool to begin to dissect the early events in hepatocarcinogenesis in the HBx transgenic mice.

V. C o n c l u s i o n s This article has not attempted to review all the literature on hepadnaviruses and HCC. Instead, we have chosen to focus on a few experimental models that have provided specific insights into hepatocarcinogenesis in hepadnavirus carriers which may be of general significance. The new transgenic models which utilize the HBsAg and the HBx genes should now be exploited to further understand the molecular genetics of HCC. These models are directly relevant to H C C in humans since both the HBsAg and HBx genes are expressed in precancerous liver and HCCs from humans. In addition, these models have at their disposal the vast resources of the human and murine genome projects. In addition, the W H V model provides many opportunities to further analyze molecular genetic changes that occur in a natural host-virus system which is also very relevant to the human disease. So far, work on these models has pointed to the m y c family of protooncogenes and the I G F 2 gene as important genes in the initiation of transformation and tumor growth and malignant progression, respectively. The effects of HBx on the PKC and Raf 1 signal transduction pathways may also lead to the identification of new genes of importance in hepatocarcinogenesis. When the individual genetic components (e.g., Ras, Raf, etc.) of the interactive signal transduction pathways are more fully understood, common mechanisms between HCC and other cancers will undoubtedly become more evident.

References Ando, K., Moriyama, T., Guidotti, L. G., Wirth, S., Schreiber, R. D., Schlicht, H. J., Huang, S., and Chisari, E V. (1992). Mechanisms of class I restricted immunopathology:A transgenic model for fulminant hepatitis. J. Exp. Med. 178, 1541-1554. Ando, K., Guidotti, L. G., Wirth, S., Ishikawa, T., Missale, G., Moriyama, T., Schreiber, R. D., Schlicht, H. J., Huang, S. N., and Chisari, E V. (1994). Class I-restricted cytotoxicT lymphocytes are directly cytopathic for their target cells in vivo. J. Immunol. 152, 3245-3253. Babinet, C., Farza, H., Morello, D., Hadchovel, M., and Pourcel, C. (1985). Specificexpression of hepatitis B surface antigen (HBsAg) in transgenic mice. Science 230, 1160-1163. Best, L. G., and Hoeksha, R. E. (1981). Wiedemann-Beckwith Syndrome:Autosomal dominant inheritance in a family. Am. J. Med. Genet. 9, 291-299. Bowcock, A. M., Anderson, L. A., Friedman, L. S., Black, D. M., Osborne-Lawrence, S.,

6.

Contributions of Hepadnavirus Research

135

Rowell, S. E., Hall, J. M., Solomon, E., and King, M. C. (1993). THRA1 and D17S183 flank an interval of <4 cM for the breast-ovarian cancer gene (BRCA1) on chromosome 17q21. Am. J. Hum. Genet. 52, 718-722. Burk, R. D., DeLoia, J. A., E1 Awady, M. K., and Gearhart, J. D. (1988). Tissue preferential expression of hepatitis B virus (HBV) surface antigen gene in two lines of HBV transgenic mice. J. Virol. 62, 649-654. Chen, H. S., Kareko, S., Girones, R., Anderson, R. W., Hornbuckle, W. E., Tennant, B. C., Cote, P. J., Gerin, J. L., Purcell, R. H., and Miller, R. H. (1993). The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks. J. Virol. 67, 1218-1226. Chisari, E V., Pinkert, C. A., Milich, D. R., Filippi, P., McLachlan, A., Palmiter, R. D., and Brinster, R. L. (1985). A transgenic mouse model of the chronic hepatitis B surface antigen carrier state. Science 230, 1157-1160. Chisari, E V., Klopchin, K., Moriyama, T., Pasquinelli, C., Dunsford, H. A., Sell, S., and Pinkert, C. A. (1989). Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 59, 1145-1156. Chrislofori, G., Nalk, P., and Hanahan, D. (1994). A second signal supplied by insulin like growth factor II in oncogene-induced tumorigenesis. Nature 369, 414-417. Cohick, W. S., and Clemmons, D. R. (1993). The insulin-like growth factors. Ann. Rev. Physiol. 55, 131-153. Colgrove, R., Simon, G., and Ganem, D. (1989). Transcriptional activation of homologous and heterologous genes by the hepatitis B virus gene product in cells permissive for viral replication. J. Virol. 63, 4019-4026. Cross, J. C., Wen, R., and Rutter, W. J. (1993). Transactivation by hepatitis B virus X protein is promiscuous and dependent on mitogen-activated cellular/threonine kinases. Proc. Natl. Acad. Sci. USA 90, 8078-8082. Cullen, K. J., Lippman, M. E., Chow, D., Hill, S., Rosen, N., and Zwiebel, J. A. (1992). Insulinlike growth factor-II overexpression in MCF-7 cells induces phenotypic changes associated with malignant progression. Mol. Endocrinol. 6, 91-100. Dejean, A., Sonigo, P., Wain-Hobson, S., and Tiollais, P. (1984). Specific hepatitis B virus integration in hepatocellular carcinoma DNA through a viral 11-base-pair direct repeat. Proc. Natl. Acad. Sci. USA 81, 5350-5354. deThe, H., Marchio, A., Tiollais, P., and Dejean, A. (1987). A novel steroid thyroid hormone receptor-related gene inappropriately expressed in human hepatocellular carcinoma. Nature 330, 667-670. Etiemble, J., M6r6y, T., Jacquemin, E., Tiollais, P., and Buendia, M. A. (1989). Fused transcripts of c-myc and a new cellular locus, hcr in a primary liver tumor. Oncogene 4, 51-57. Evans, G. (1994). Integration of cell proliferation and programmed cell death (apoptosis) by c-myc. Cold Spring Harbor Meeting on "Molecular Genetics of Cancer". Abstract #261. Farber, E., and Sarma, D. S. R. (1987). Hepatocarcinogenesis: A dynamic cellular perspective. Lab. Invest. 56, 4-22. Fearon, E. R., and Vogelstein, B. (1990). A genetic model colorectal tumorigenesis. Cell 61, 759-767. Feitelson, M. A., Clayton, M. M., and Blumberg, B. (1990). X antigen/antibody markers in hepadnavirus infections. Gastroenterology 98, 1071-1078. Fourel, G., Trepo, C., Bougueleret, L., Henglein, B., Ponzetto, A., Tiol!ais, P., and Buendias, M. (1990). Frequent activation of N-myc genes by hepadnavirus insertion in woodchuck liver tumours. Nature 347, 294-298. Fourel, G., Couturier J., Tiollais, P., and Buendia, M. A. (1993). A new common integration site for WHV DNA in woodchuck liver tumors. Molecular Biology of Hepatitis B Viruses meeting, Georgetown, VA, Abstract #68. Fu, X, Su, X, Lee, C. Y., Hintz, R., Biempica, L., Snyder, R., and Rogler, C. E. (1988). Insulin-

136

Charles E. Rogler et al.

like growth factor II expression and oval cell proliferation associated with hepatocarcinogenesis in woodchuck hepatitis virus carriers. J. Virol. 62, 3422-3430. Galibert, E, Mandart, E., Fitoussi, F., Tiollais, P., and Charnay, P. (1979). Nucleotide sequence of hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature 281,646-650. Gilles, P. N., Fey, G., and Chisari, E V. (1992). Tumor necrosis factor-or negatively regulates hepatitis B virus gene expression in transgenic mice. J. Virol. 66, 3955-3960. Guidotti, L. G., Ando, K., Hobbs, M. V., Ishikawa, T., Runkel, L., Schreiber, R. D., and Chisari, F. V. (1994). Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice. Proc. Natl. Acad. Sci. USA 91, 3764-3768. Guidotti, L. G., Guilhot, S., and Chisari, E V. (1994). Interleukin-2 and cx/13interferon downregulate Hepatitis B virus gene expression in vivo by tumor necrosis factor-dependent and independent pathways. J. Virol. 68, 1265-1270. Guilhot, S., Guidotti, L. G., and Chisari, F. V. (1993). Interleukin-2 downregulates hepatitis B virus gene expression in transgenic mice by a post-transcriptional mechanism. J. Virol. 67, 7444-7449. Harris, C. C. (1993). P53: at the crossroads of molecular carcinogenesis and risk assessment. Science 262, 1980-1981. Harris, C. C., and Hollstein, M. (1993). Clinical implications of the p53 tumor suppressor gene. N. Eng. J. Med. 329, 1318-1327. Hansen, L., Tennant, B. C., Seeger, C., and Ganem, D. (1993). Differential activation of myc gene family members in hepatic carcinogenesis by closely related hepatitis B viruses. Mol. Cell. Biol. 13, 659-667. H6hne, M., Schaefer, S., Seifer, M., Feitelson, M. A., Paul, D., and Gerlich, W. H. (1990). Malignant transformation of immortalized transgenic hepatocytes after transfection with hepatitis B virus. EMBO J. 11, 1137-1145. Hino, O., Shows, T. B., and Rogler, C. E. (1986). Hepatitis B virus integration site in hepatocellular carcinoma at chromosome 17;18 translocation. Proc. Natl. Acad. Sci. USA 83, 8338-8342. Hino, O., Ohtake, K., and Rogler, C. E. (1989). Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration. J. Virol. 63, 2638-2643. Hino, O., Tabata, S., and Hotta, Y. (1991). Evidence for increased in vitro recommendation with insertion of human hepatitis B virus DNA. Proc. Natl. Acad. Sci. USA 88, 9248-9252. Hsu, T., M6r6y, T., Etiemble, J., Louise, A., Trepo, C., Tiollais, P., and Buendia, M. A. (1990). Activation of c-rnyc by woodchuck hepatocellular carcinoma. Gastroent. Jpn. (Suppl. 2) 25, 43-48. Jhappan, C., Stahle, C., Harkins, R. N., Fausto, N., Smith, G. H., and Merlino, G. T. (1990). TGFa overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 61, 1137-1146. Kekule, A. S., Lauer, U., Weiss, L., Luber, B., and Hofschneider, P. H. (1992). Hepatitis B virus transactivator HBx uses a tumor promoter signaling pathway. Nature 361, 742-743. Kim, C. M., Koche, K., Sarto, I., Miyamura, T., and Jay, G. (1991). HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 351,317-319. Koike, K., Moriya, K., Jino, S., Yotsuyanagi, H., Endo, Y., Miyamura, T., and Kurokawa, K. (1994). High level expression of hepatitis B virus, HBx gene and hepatocarcinogenesis in transgenic mice. Hepatology 19, 810-819. Koufos, A., Hansen, M. F., Copeland, N. G., Jenkins, N. A., Lampkin, B. C., and Cavenee, W. K. (1985). Loss of heterozygosity in three embryonal tumors suggests a common pathogenetic mechanism. Nature 316, 330-334. Leach, F. S., Nicolaides, N. C., Popadopoulus, N., Liu, B., and Vogelstein, B. (1993). Mutations of a mut S homolog in hereditary nonpolyposis colorectal cancer. Cell 75, 1215-1225. Lee, T. H., Finegold, M. J., Shen, R. E, DeMayo, J. L., Woo, S. L., and Butel, J. (1990).

6.

Contributions of Hepadnavirus Research

137

Hepatitis B virus transactivator X protein is not tumorigenic in transgenic mice. J. Virol. 64, 5939-5947. Leevers, S. J., Paterson, H. E, and Marshall, C. J. (1994). Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369, 411-414. Levine, A. J., Momand, J., and Finlay, C. A. (1994). The p53 tumor suppressor gene. Nature 351,453-456. Liu, R. H., Baldwin, B., Tennant, B. C., and Hotchkiss, J. H. (1991). Elevated formation of nitrate and N-nitrosodimethylamine in woodchucks (Marmota monax) associated with chronic woodchuck hepatitis virus infection. Cancer Res. 51, 3925-3939. Liu, R. H., Jacob, J. R., Tennant, B. C., and Hotchkiss, J. H. (1992). Nitrite and nitarosamine synthesis by hepatocytes isolated from normal woodchucks (Marmota monax) and woodchucks chronically infected with woodchuck hepatitis virus. Cancer Res. 52, 4139-4143. Liu, R. H., Jacob, J. R., Hotchkiss, J. H., and Tennant, B. C. (1993). Synthesis of nitric oxide and nitrosamine by immortalized woodchuck hepatocytes. Carcinogenesis 14, 1609-1613. Maguire, H. E, Hoeffler, J. P., and Siddiqui, A. (1991). HBV X protein alters the DNA binding specificity of CREB and ATF-2 by protein -protein interactions. Science 252, 842-844. Miller, R. H., and Robinson, W. S. (1986). Common evolutionary origin of hepatitis B virus and retroviruses. Proc. Natl. Acad. Sci. USA 83, 2531-2535. Mizusawa, H., Taira, M., Yaginuma, K., Kobayashi, M., Yoshida, E., and Koike, K. (1985). Inversely repeating integrated hepatitis B virus DNA and cellular flanking sequences in the human hepatoma-derived cell line huSP. Proc. Natl. Acad. Sci. USA 82, 208-212. Mondelli, M., Mieli-Vergani, G., and Alberti, A. (1982). Specificity of T lymphocyte cytotoxicity to autologous hepatocytes in chronic hepatitis B virus infection: Evidence that T cells are directed against HBV core antigen expression on hepatocytes. J. Immunol. 129, 27732778. Moriyama, T., Guilhot, S., Moss, B., Klopchin, K., Pinkert, C., Palmiter, R., Brinster, R., Osami, K., and Chisari, E (1990). Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science 248, 361-364. Nagaya, T., Nakamura, T., Tokino, T., Tsurimoto, T., Imai, M., Mayioni, T., Kamino, K., Yamamura K., and Matsubara, K. (1987). The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinomas. Genes Dev. 1, 773-782. Nakamua, T., Tokino, T., Nagaya, T., and Matsubara, K. (1988). Microdeletion associated with the integration process of hepatitis B virus DNA. Nucleic Acids Res. 16, 4865-4873. Nishimoto, I., Murayama, Y., Katada, T., Michio, U., and Ogata, E. (1989). Possible direct linkage of insulin-like growth factor-II receptor with guanine nucleotide-binding proteins. J. Biol. Chem. 264, 14029-14038. Ochiya, T., Fujiyama, A., Fukushige, S., Hatada, I., and Matsubara, K. (1986). Molecular cloning on an oncogene from a human hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 83, 4993-4997. Ogston, C. W., Jonak, G. J., Rogler, C. E., Astrin, S. M., and Summers, J. (1982). Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks. Cell 29, 385-394. Okamoto, T., Katada, T., Murayama, Y., Ui, M., Ogata, E., and Nishimoto, I. (1990). A simple structure encodes G protein activating function of the IGF2/mannose-6-phosphate receptor. Cell 62, 709-717. Pasquinelli, C., Bhavani, K., and Chisari, E V. (1992). Multiple oncogenes and tumor suppressor genes are structurally and functionally intact during hepatocarcinogenesis in Hepatitis B virus transgenic mice. Cancer Res. 53, 2823-2829. Pasquinelli, C., Bhavani, K., Schirmacher, P., Rogler, C. E., and Chisari, E V. Expression of hepatocyte mitogen and mitogen receptors during hepatocarcinogenesis in hepatitis B virus transgenic mice. J. Virol. (Submitted for publication).

138

Charles E. Rogler et al.

Paul, D., Hohne, M., Pinkert, C., Piasecki, A., Ummelmann, E., and Brinster, R. L. (1992). Immortalized differentiated hepatocyte lines derived from transgenic mice harboring SV40 T-antigen genes. Exp. Cell Res. 175, 354-362. Pershing, D. H., Varmus, H. E., and Ganem, D. (1986). Antibodies to pre-S and X determinants arise during natural infection with ground squirrel hepatitis virus. J. Virol. 60, 177184. Rodriguez-Tarduchy, G., Collins, M. K., Garcia, I., Lopez-Rivas, A. (1992). Insulin-like growth factor I inhibits apoptosis in IL-3 dependent hemopoietc cells. J. Immunol. 149, 535-540. Rogler, C. E., and Summers, J. (1984). Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from a chronically infected liver. J. Virol. 50, 832-837. Rogler, C. E., and Chisari, E V. (1992). Cellular and molecular mechanisms of hepatocarcinogenesis in Seminars. In "Liver Disease" (D. Shafritz, ed), Vol. 12, pp. 265-267, Thieme Medical Publishers, Inc., New York. Rogler, C. E., Sherman, M., Su, C. Y., Shafritz, D. A., Summers, J., Shows, T. B., Henderson, A., and Kerr, M. (1985). Deletion in chromosome 11p associated with a hepatitis B integration site in hepatocellular carcinoma. Science 230, 319-322. Rogler, C. E., Yang, D., Rossetti, L., Donohoe J., Alt, E., Change, C. J., Rosenfeld, R., Neely, K., and Hintz, R. (1994). Altered body composition and increased frequency of diverse malignancies in insulin-like growth factor-II transgenic mice. J. Biol. Chem. 269, 1377913784. Scherer, E. (1983). Neoplastic progression during or in experimental hepatocarcinogenesis. Biochim. Biophys. Acta 738, 219-236. Schirmacher, P., Held, W. A., Yang, D., Chisari, E V., Rustum, Y., and Rogler, C. E. (1992). Reactivation of insulin-like growth factor II during hepatocarcinogenesis in transgenic mice suggests a role in malignant growth. Cancer Res. 52, 2549-2556. Schirmacher, P., Rogler, C. E., and Dienes, H. P. (1993). Current pathogenic and molecular concepts in viral liver carcinogenesis. Virchows Arch. B-Cell Pathol. Incl. Mol. Pathol. 63, 71-89. Schirmacher, P., Wang, H. P., Stahnke, G., Will, H., and Rogler, C. E. (1994). Sequences and structures at hepadnaviral integration-recombination sites implicate topoisomerase I in hepadnaviral DNA rearrangements and integration. J. Hepatol. (in press). Seifer, M., H6hne, M., Schaefer, S., Gerlich, W. H. (1991 ). In vitro tumorigenicity of hepatitis B virus DNA and HBx protein. J. Hepatol. 13, $61-$65. Sell, S., Hunt, J. M., Knoll, B. J., and Dunsford, H. A. (1987). Cellular events during hepatogenesis in rats and the question of premalignancy. Adv. Cancer Res. 48, 37-111. Seto, E., Mitchell, P. J., and Yen, T. S. B. (1990). Transactivation by the hepatitis B virus X protein depends on AP-2 and other transcription factors. Nature 344, 72-74. Shi, Y., Glynn, J. M., Guilbert, L. J., Cotter, T. G., Bissinnette, R. P., and Green, D. R. (1992). Role of c-myc in activation induced apoptotic cell death in T cell hybridomas. Science 257, 212-214. Shih, C., Burke, K., Chou, M.-J., Zeldis, J. B., Yang, C. S., Lee, C. S., Isselbacher, K. J., Wands, J. R., and Goodman, H. M. (1987). Tight clustering of human hepatitis B virus integration sites in hepatomas near a triple-stranded region. J. Virol. 61, 3491-3498. Spandau, D. E, and Lee, C. H. (1988). Trans-activation of viral enhances by the hepatitis B virus X protein. J. Virol. 62, 427-434. Strom, S. C., and Faust, J. B. (1990). Oncogene activation and hepatocarcinogenesis. Pathobiology 58, 153-167. Summers, J., Smith, P. M., and Horwich, A. (1990). Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. J. Virol. 64, 2819-2824. Tabor, E. (1994). Tumor suppressor genes, growth factor genes and oncogenes in hepatitis B virus associated hepatocellular carcinoma. J. Med. Virol. 42, 357-365.

6.

Contributions of Hepadnavirus Research

139

Takada, S., and Koike, K. (1990a). X protein of hepatitis B virus resembles a serine protease inhibitor. Jpn. J. Cancer Res. 81, 1191-1194. Takada, S., and Koike, K. (1990b). Transactivation function of 3r-truncated X gene as an X-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues. Proc. Natl. Acad. Sci. USA 87, 5628-5632. Takada, S., Kido, H., Fukutomi, A., Mori, T., and Koike, K. (1994). Interaction of hepatitis B virus X protein with a serine protease, tryptase TL2 as an inhibitor. Oncogene 9, 341348. Takagi, H., Sharp, R., Hammermeister, C., Goodrow, T., Bradley, M. O., Fausto, N., and Merlino, G. (1992). Molecular and genetic analysis of liver oncogenesis in transforming growth factor ~ transgenic mice. Cancer Res. 52, 5171-5177. Tennant, B. C., and Gerin, J. L. (1994). The woodchuck model of hepatitis B virus infection. In "The Liver: Biology and Pathobiology" (I. M. Arias, J. L. Boyer, N. Fausto, W. B. Jakoby, D. A. Shafritz, and D. A. Schachter, eds.), 3rd ed. Raven Press, New York. Treinin, M., and Laub, O. (1987). Identification of a promoter element located upstream from the hepatitis B virus X gene. Mol. Cell Biol. 7, 545-548. Tuttleman, J., Pourcel, C., and Summers, J. (1986a). Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47, 451-460. Tuttleman, J., Pugh, J., and Summers, J. (1986b). In vitro experimental infection of primary duck hepatocyte cultures with DHBV. J. Virol. 58, 17-25. Twu, J. S., and Schloemer, R. H. (1987). Transcriptional trans-activating function of hepatitis B virus. J. Virol. 61, 3448-3453. Wang, H. P., and Rogler, C. E. (1988). Deletions in chromosome arms 11p and 13q in primary hepatocellular carcinomas. Cytogenet. Cell Genet. 48, 72-78. Wang, H. P., and Rogler, C. E. (1991). Topoisomerase I mediated hepadnavirus DNA integration in vitro. J. Virol. 65, 2381-2392. Wang, J., Chenivesse, X., Henglein, B., and Brechot, C. (1990). Hepatitis B virus-integration in a cyclin A gene in a hepatocellular carcinoma. Nature 348, 555-557. Wang, X. W., Forrester, K., Yeh, H., Feitelson, M. A., Gu, J. R., and Harris, C. C. (1994). Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity and association in transcription factor ERCC3. Proc. Natl. Acad. Sci. USA 91, 2230-2234. Waziri, M., Patil, S. R., Hanson, J. W., and Bartley, J. A. (1983). Abnormality of chromosome 11 in patients with feature of Beckwith Wiedemann Syndrome. J. Pediatr. 102, 873-876. Wei, Y., Fourel, G., Ponzetto, A., Silvestro, M., Tiollias, P., and Buendia, M. A. (1992). Hepadnavirus integration: Mechanisms of activation of the N-myc 2 retropposon in woodchuck HCC. J. Virol. 66, 5265-5276. Willison, K. (1991). Opposite imprinting of mouse Ifg2 and Igf2r genes. Trends Genet. 7, 107109. Williams, G. T., and Smith, C. A. (1993). Molecular regulation of apoptosis: Genetic controls on cell death. Cell 74, 777-779. Wollersheim, M., Debelka, U., and Hofschneider, P. H. (1988). A transactivating function encoded in the hepatitis B virus X gene is conserved in the integrated state. Oncogene 3, 545-554. Yaginuma, K., Kobayashi, M., Yoshida, E., and Koike, K. (1985). Hepatitis B virus integration in hepatocellular carcinoma DNA: Duplication of cellular flanking sequences at the integration site. Proc. Natl. Acad. Sci. USA 82, 4458-4462. Yaginuma, K., Kobayashi, H., Kobayashi, M., Morishima, T., Matsuyama, K., and Koike, K. (1987). Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children. J. Virol. 61, 1807-1813. Yang, D. Y., and Rogler, C. E. (1991). Analysis of insulin-like growth factor II (IGF2) expres-

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sion in neoplastic nodules and hepatocellular carcinomas of woodchucks utilizing in situ hybridization and immunocytochemistry. Carcinogenesis 12, 1893-1901. Yang, D. Y., Alt, E., and Rogler, C. E. (1993). Coordinate expression of N-myc 2 and insulinlike growth factor II in precancerous altered hepatic foci in woodchuck hepatitis virus carriers. Cancer Res. 53, 2020-2027. Zahm, P., Hofschneider, P. H., and Koshy, R. (1988). The HBV X-ORF encodes a transactivator: A potential factor in viral hepatocarcinogenesis. Oncogene 3, 169-177.

7 Apoptosis and Hepatocarcin0genesis Rolf Schulte-Hermann Bettina Grasl-Kraupp Wilfried Bursch 9 Institute fiir rumorblology-CancerResearch A-1090 Vienna, Austria

I. Apoptosis and Other Types of Active Cell Death Cell death in tumors traditionally has been considered a passive, degenerative phenomenon due to toxicity and to insufficient supply of nutrients, oxygen, etc. A number of observations, however, made during the last decades were difficult to reconcile with this view and suggested that under certain conditions physiological mechanisms may be involved in the generation of cell death. These observations include the "cytolytic" effect of glucocorticoid hormones on lymphocytes. The subsequent introduction of glucocorticoids into the therapy of lymphatic leukemia provided one of the early breakthroughs for chemotherapy of malignant disease (Schrek, 1949). Furthermore, human mammary and prostate cancers are hormone dependent, and the removal of trophic hormones by gonadectomy sometimes results in dramatic regression of cancers and complete elimination of metastases (Beatson, 1896; Huggins et al., 1941). In experimentally induced mammary cancers, hormone withdrawal actively triggers cell death as shown by biochemical methods (Lanzerotti and Gullino, 1972; Gullino, 1980). Morphological signs of active cell death are also found in tumors and tumor prestages (Kerr et al., 1972; Bursch et al., 1984, 1991; Wyllie, 1985; Searle et al., 1987; Sarraf and Bowen, 1988; Szende et al., 1989; Trauth et al., 1989), and their incidence is often enhanced by treatment modalities Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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such as irradiation (Kruman et al., 1991; Martin and Cotter, 1991; Sklar et al., 1993; Kerr et al., 1994), cytotoxic drugs (Yoshida et al., 1993; Fisher et al., 1993; Miyashita and Reed, 1993), and hormone antagonists (Szende et al., 1989; Tenniswood et al., 1992; Michna et al., 1992). Active cell death (apoptosis) during hepatocarcinogenesis was first described by Bursch, Schulte-Hermann, and their associates. These authors found that putative preneoplastic cell foci in rat liver showed high rates of apoptosis, which largely counterbalanced the enhanced cell replication in these lesions. Furthermore, tumor promoters were found to reduce this cell death, thereby accelerating the growth of foci and cancer development (Schulte-Hermann et al., 1982; Bursch and Lauer, 1983; Bursch et al., 1984; Schulte-Hermann, 1985; Bursch, 1994). Active cell death (see Table 1 for definition) is a phylogenetically old phenomenon occurring in a wide variety of animals (Wyllie et al., 1980; Bursch et al., 1992b) and plants (Greenberg et al., 1994). Its biological relevance was first recognized by developmental biologists (Nussbaum, 1901; Gliicksmann, 1930; Lockshin and Williams, 1965; Lockshin and Beaulaton, 1974; Schweichel and Merker, 1973). During the embryonal development of mammalian organisms, cell death (at that time called necrosis) was found to be necessary for removing excessive tissues such as interdigital webs, the Muellerian duct in males, and excessive neurons in shaping the final form of the organism. Likewise, during metamorphosis in lower invertebrates and vertebrates massive tissue involution and cell elimination occur as a physiological event. In these situations cell death is governed by Table I Characteristic Features of Active Cell Death Definition

A process of genetically encoded, active self-destruction of a cell. Functions

Counterpart of mitosis in the regulation of cell number in tissues. Elimination of "unwanted" cells (i.e., excessive, damaged or precancerous). During embryogenesis, part of the developmental program (i.e., programmed cell death). Types Apoptosis, Type I: Cell condensation, fragmentation, and heterophagy of fragments. Autophagic, Type II: Increase in lysosomes and autophagic vacuoles. Nonlysosomal disintegration, Type III. Biochemical and Cytological Markers

DNA fragmentation into oligonucleosomal pieces ("DNA ladders"): only in specific cells during apoptosis; not generally valid. In situ end labeling of DNA fragments; not specific for apoptosis. Functional Markers

Inhibition by tissue-specific mitogens and growth factors (i.e., mitogen rescue); occurrence under specific physiological conditions (e.g., withdrawal of tissue-specific mitogen, induction by growth inhibitors).

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the developmental program (i.e., programmed cell death). In adult organisms non toxic types of cell elimination also occur (e.g., involution of the uterus after pregnancy and hormone-dependent organs after removal of the hormone producing organ). Farber and his colleagues recognized the occurrence of an "active" or "suicide" type of cell death that depended on protein synthesis and appeared after treatment with genotoxic (chemotherapeutic) drugs (Liebermann et al., 1970; Farber et al., 1971; Verbin et al., 1972). The recognition of the widespread occurrence and relevance of active cell death, frequently displaying a peculiar morphology, is credited to Kerr and colleagues (Kerr, 1971; Kerr et al., 1972). The characteristic morphology first led to the name "shrinkage necrosis" (Kerr, 1971). Later, they introduced the new term "apoptosis" to describe a category of cell death "which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations" (Kerr et al., 1972). It was understood as an active, inherently programmed phenomenon characterized by a specific morphology which can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological (Table 1). On the other hand, these authors redefined the traditional term "necrosis" as an event usually "not determined by factors intrinsic to the cell itself, but by environmental perturbation, which must be violent and lead to rapid incapacitation of major cell functions and to collapse of internal homeostasis" (Kerr et al., 1972). The definition of apoptosis was based on morphological and functional grounds (Kerr et al., 1972; Wyllie et al., 1980). The morphological characteristics are condensation of the cytoplasm, condensation of chromatin at the nuclear membrane, and fragmentation of the nucleus and the cell, giving rise to apoptotic bodies (AB), which are then phagocytosed by neighboring phagocytes or epithelial cells (Figure 1). Cellular organelles including the cell membrane appear to be well preserved during early stages of apoptosis, so that the cell contents are not liberated, and usually no inflammatory responses are seen around apoptotic cells. Typically, lysosomes do not play a role early in apoptosis although they are involved later in the degradation of phagocytosed apoptotic bodies in the host cells (Kerr, 1971; Kerr et al., 1972; Wyllie et al., 1980; Bursch et al., 1985). Apoptosis is not the only type of active cell death (Table 1). In previous studies a steep increase of lysosomal enzymes was observed in hormonedependent mammary cancer regressing after ovariectomy which does not fit the concept of apoptosis (Lanzerotti and Gullino, 1972; Gullino, 1980). In studies on the developing embryo, Schweichel and Merker (1973-) discovered the occurrence of three morphologically different modes of active cell death: type I: characterized by condensation, fragmentation and heterophagy of cell fragments and is probably identical to apoptosis; type II: cells are destroyed by their own lysosomes, forming autophagic vacuoles (auto-

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DURATION

STAGES intact cell

resence of GF-gl

[ololol 1 [ololo'1

2-3 hours

end of mitogen rescue ca I hour

condensation

few minutes

fragmentation

phagocytosis

1

ca.3 hours

degradation

Figure 1

Stages of apoptosis in rat liver and their approximate duration. See text for details.

phagic cell death); and type III: cells disintegrate into fragments, without involvement of the lysosomal system (Table 1) (Schweichel and Merker, 1973; Clarke, 1990). Nonapoptotic types of active cell death seem to occur, in addition to apoptosis, not only during embryonic development, but also in insects, in certain mammalian organs and tumors (Lockshin and Williams, 1965; Lockshin and Beaulaton, 1974; Zakeri et al., 1993; Schwartz et al., 1993; Bursch et al., 1995), and in developing neuronal tissue (Clarke, 1990). Occurrence of type II cell death may be involved in the regression of mammary tumors which would explain the observed increase in lysosomal

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enzymes after the withdrawal of estrogens (see above) (Lanzerotti and Gullino, 1972). In the human mammary cancer cell line MCF-7 anti estrogens induce active cell death that has been classified as apoptosis (Bardon et al., 1987; W~irri et al., 1993). Recent studies, however, show that the majority of dying MCF-7 cells undergo the type II mode of death rather than apoptosis. The induction of both types of cell death in MCF-7 cells by antiestrogens is prevented by estrogen indicating the active nature of death (Bursch et al., 1995). In the present review, the roles of active cell death will be described during normal liver growth and development and hepatocarcinogenesis. In order to avoid confusion, the term "active cell death" will be used as recently suggested by Tenniswood et al. (1992). Use of "apoptosis" will be restricted to cases where appropriate morphological evidence is available. The term "programmed cell death" which is frequently applied interchangeably with apoptosis will be used only to indicate cell death during embryonal development.

II. Active Cell Death in the Liver A. Detection and Quantification Morphological methods were first used to identify apoptosis. In the liver they are still the only reliable procedures, particularly if quantitative determination of the incidence of apoptosis is required. Classical techniques such as staining with hematoxylin and eosin are useful and can provide unequivocal proof of apoptosis both in tissue sections and in cultured cells when used in conjunction with electron microscopy. Other dyes such as the fluorescent Hoechst 33258 are useful for revealing the morphology of nuclear chromatin. Antibodies to proteins specifically associated with apoptosis may be available in the future. A promising example is the expression of the precursor of transforming growth factor 131 (pre-TGF[31) in apoptotic hepatocytes although its applicability under different situations remains to be studied in more detail (Bursch et al., 1993). Biochemical studies with thymocytes and lymphocytes led to the concept that the characteristic condensation of nuclear chromatin during apoptosis results from the activation of endonuclease(s) and the degradation of chromatin into mono- and oligonucleosomal fragments (Wyllie, 1980; Arends et al., 1990; Cohen and Duke, 1984; Compton and Cidlowski, 1986; Harmon et al., 1990). Gel electrophoresis of these fragments yields the so-called "DNA ladders" which are widely used as biochemical markers of apoptosis. However, DNA ladders are not necessarily specific for apoptosis (Collins et

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al., 1992). Furthermore, their occurrence cannot be demonstrated under

conditions of high rates of apoptosis in rat hepatocytes occurring during the regression of mitogen-induced liver hyperplasia or after TGFI31 treatment of cultured hepatocytes (Oberhammer et al., 1993c). In contrast, Shen et al. (1991) and Ray et al. (1992) have reported the formation of DNA ladders in mouse hepatocytes following administration of toxic chemicals such as acetaminophen and suggested the occurrence of apoptosis. Morphological evidence of apoptosis in mouse liver cells induced by tumor necrosis factor (TNFa) and a transcription inhibitor is also associated with formation of DNA ladders (Leist et al., 1994). The reasons for these apparent discrepancies between these two animal species remain to be determined. In any event, biochemical assays for oligonucleosomal DNA fragmentation patterns (DNA ladders) are questionable for the identification and quantitation of apoptosis in the liver. Recently, methods have been adapted to detect DNA fragmentation in single or double-stranded DNA in situ on histological sections by labeling DNA ends with DNA polymerase or terminal transferase; test kits are commercially available. However, the results should be interpreted with care because DNA degradation into fragments is not specific for apoptosis. A positive response is also found in nuclei of necrotic cells after toxic liver damage (CC14, nitrosamines) and even as a result of tissue autolysis (GraslKraupp, et al., 1995). Thus, in situ DNA fragmentation assays should not be used as a sole indicator of apoptosis. For kinetic studies on cell turnover investigators should be aware of the possible existence of a circadian rhythm of apoptotic activity. As will be described in greater detail below, the feeding/fasting state, and thereby the light/dark rhythm, codetermines the occurrence of apoptosis. In fact, high rates of apoptosis are found at the end of the daily light fasting period, and low rates in the dark feeding period (Schulte-Hermann et al., 1988; GraslKraupp et al., 1994). Thus, in experiments where rodents are sacrificed in the morning according to laboratory routine, apoptotic rates in the liver may be minimal. Identification of nonapoptotic types of active cell death in the liver to our knowledge has not yet been reported. B. Models Normal adult liver shows a very low incidence of cell replication and cell death. In untreated rats there are approximately 2 to 4 apoptoses per 10,000 hepatocytes (Bursch et al., 1985). These low rates may increase considerably under certain conditions. Two principally different pathophysiological states favor the occurrence of active cell death in the liver: (1) involution after adaptive hyperplasia, or atrophy due to starvation, hypophysectomy etc., and (2) certain types of toxic injury.

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1. A d a p t i v e Liver Growth

Liver growth and hyperplasia can be induced by numerous chemical compounds which often show tumor-promoting activity in rodents. These agents include lipophilic xenobiotic agents such as phenobarbital (PB), (x-hexachlorocyclohexane ((x-HCH), cyproterone acetate (CPA), ethinyl estradiol and other steroids (Schulte-Hermann et al., 1980, 1988; Mayol et al., 1992), and peroxisome proliferators such as nafenopin (Reddy and Lalwani, 1983; Levine et al., 1977; Grasl-Kraupp et al., 1993c). The growth response is accompanied by increases in functional proteins such as drug metabolizing enzymes, peroxisomal enzymes and blood clotting proteins; signs of tissue damage are usually absent (Conney et al., 1960). Therefore, it is considered to be an adaptive response of the liver. An inorganic compound, lead nitrate, also induces liver growth in rats (Columbano et al., 1985). In most cases a steep increase in DNA synthesis and mitosis occurs in the first days of treatment resulting in tissue hyperplasia. While cell replication subsequently decreases back to normal, organ enlargement and hyperplasia are maintained at a constantly enhanced level as long as the hepatomitogen is present (Schulte-Hermann, 1974, 1979, 1985). An adaptive type of liver growth is also seen in pathophysiological situations such as overfeeding, pregnancy, and lactation, or during severe serum protein depletion (Schulte-Hermann, 1974, 1979). 2. Involution ol: the Liver "Relative Hyperplasia"

After cessation of treatment with a hepatomitogenic compound, the adaptive changes in liver size and enzyme activity are reversed; the rate of regression will depend on the half-life of the inducing agent. Involution is a well-controlled process which resembles reduction of organ size (atrophy) occurring after functional deprivation of the liver, such as by starvation or hypophysectomy (Table 2). It is remarkable that those organelles that are excessively increased during preceding mitogen treatment apparently are Table 2 Changes during Involution of the Enlarged Liver Decrease in organ size Reduction in cell size Coordinated reduction of liver consituents Selective degradation of excessive enzymes and organelles increased by a preceding treatment or functional overload (e.g., drug-metabolizing enzymes and endoplasmic reticulum after stopping of promoter treatment) Enhanced autophagy Inhibition of DNA replication Increase in active cell death (apoptosis)

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preferentially removed by autophagy (Bolender and Weibel, 1973; Glaumann et al., 1977; St~iubli and Hess, 1975; Moody and Reddy, 1976; Leighton et al., 1975; Levine et al., 1977). Several studies suggest that the involution results from both reduced synthesis de n o v o and from enhanced degradation by lysosomal and autophagic processes. With respect to regression of hyperplasia, variable responses have been obtained. When liver growth was induced by ~-HCH or butylhydroxytoluene, total liver DNA (as an indicator of cell number) shows little decrease in the first 7 weeks following treatment cessation, although the excess liver is completely eliminated after a prolonged period (Schulte-Hermann et al., 1971; Schulte-Hermann and Parzefall, 1981). In contrast, much of the excess of DNA readily disappears when nafenopin, CPA, and lead nitrate are used as the mitogen (Levine et al., 1977; Schulte-Hermann et al., 1980; Bursch et al., 1984, 1986, Columbano et al., 1985; Alison et al., 1987). When regression of liver mass is induced by weaning in lactating rats, by starvation, or by hypophysectomy little or no decrease in total organ DNA is observed (Schulte-Hermann, 1974, 1979; Grasl-Kraupp et al., 1994). Reasons for these apparently discrepant findings may be that: (1) active elimination of cells (see below) probably occurs only when the extent of tissue regression exceeds a certain limit or threshold, and (2) the inducing compound has to be eliminated from the body at a relatively rapid rate (Bursch et al., 1986). The rapid regression of liver hyperplasia seen after CPA withdrawal is explained by the observation of enhanced rates of cell death by apoptosis. Within a few days, 25% of total liver DNA is eliminated by apoptosis. Retreatment with CPA as well as by administration of other hepatomitogens inhibits cell death and prevents the elimination of liver tissue indicating that an active type of cell death is involved ("mitogen rescue") (Table 1) (Bursch et al., 1984, 1986). Likewise, when liver hyperplasia is induced by lead nitrate, cessation of treatment results in a massive increase in apoptosis and elimination of excessive liver DNA within approximately 2 weeks (Columbano et al., 1985). The number of apoptotic cells is also enhanced during the liver regression that occurs after stopping prolonged treatment with nafenopin. However, there are also numerous cells undergoing a type of cell death appearing morphologically different from apoptosis. Since these events are triggered by cessation of nafenopin treatment, an active type of cell death is likely to be involved (possibly type II). More detailed studies on this phenomenon are currently ongoing (B. Grasl-Kraupp, B. RuttkeyNedecky, A. Ellinger, W. Bursch, R. Schulte-Hermann, 1995, unpublished observations). Liver size is also controlled by the type and amount of food consumed. Food restriction or complete starvation results in a decline in liver mass. Since liver DNA (cells) is not eliminated proportionately, the organ may

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contain more cells than needed to meet the functional requirements of the body ("relative hyperplasia"). This state favors the induction of apoptosis and the gradual elimination of cells. Conversely, provision of food suppresses apoptosis, and it was found that each of the major food constituents (protein, fat or carbohydrate), was equally effective (Grasl-Kraupp et al., 1994). The enhanced apoptotic activity in the perivenous region of the liver lobule after portocaval shunt, described by Searle et al. (1987), may partially reflect nutritional deprivation of cells. The states of absolute or relative hyperplasia not only are most susceptible to induction of active cell death, but are also largely refractory to the stimulation of DNA synthesis and cell replication. As mentioned, prolonged mitogen treatment leads to enhanced cell proliferation only in the initial few days; subsequently, cell replication rates return to normal despite continuous presence of the mitogen (Schulte-Hermann and Schmitz, 1980). Likewise, hypophysectomy results in a decrease in liver mass, while the DNA content remains unchanged; this provides another example of relative hyperplasia. In this state the hepatomitogens c~-HCH and PB increased liver mass but not liver DNA. When the relative DNA surplus was surgically removed these mitogens did induce an increase in DNA showing that the relative hyperplasia and not the deficiency in pituitary hormones was responsible for the refractory state of the liver (Schulte-Hermann et al., 1977). The DNA content of the liver (or the number of liver cells) was concluded to be controlled by a feedback system which monitors an excess of DNA (cells) and suppresses cell replication if the content of DNA exceeds the physiological needs. Thus, there appears to be a concerted regulation of cell replication and active cell death: in the hyperplastic state, rates of replication (c~) are inhibited and rates of death (13) are favored; the reverse is true for states of cell deficit (Figure 2). It is of interest that isolated primary hepatocytes can also be kept alive by adding certain hepatomitogens to the culture medium. A potent compound in this respect is nafenopin which can prolong hepatocyte survival by months. Removal of nafenopin results in hepatocyte death with an apoptosis-like morphological pattern (Muakkassah-Kelly et al., 1987; Bayly et al., 1994). 3. Cell Death after Toxic Injury to the Liver

Hepatocytes undergoing an apoptosis-like type of cell death have also been seen after toxic injury to the liver. Apoptotic liver cells appear in the pericentral region 3 to 6 hr after a single dose of thioacetamide to rats; after 12 hr, cell necrosis is also present (Ledda-Columbano et al., 1991). Occasional apoptoses are noted after C C I 4 intoxication in the intermediate zone of the liver lobule at the rim between necrotic and surviving hepatocytes (Bursch et al., 1993; Alison and Sarraf, 1994). In rats after a single high dose of the

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Rolf Schulte-Hermann, Bettina Grasl-Kraupp, and Wilfried Bursch NORMAL LIVER

PRENEOPLASTIC FOCl

Figure 2 Cell kinetics during growth and regression of normal liver and of preneoplastic liver foci. Basal rates of cell birth (cx) and of cell death (13) in normal adult liver are set to one. Changes in these rates as found in putative preneoplastic loci and after mitogen/promoter treatment or withdrawal are given as relative values. These values are approximate data that summarize and illustrate numerous experiments, e~-13indicates the growth rate of the cell population. genotoxic carcinogen, N-nitrosomorpholine, massive necrosis is seen initially. At the same time enhanced apoptosis occurs which persists for some weeks (B. Grasl-Kraupp, A. L6w-Baselli, W. Huber, H. Koudelka, K. Bukowski, R. Schulte-Hermann, 1995, unpublished). A high dose of diethyl-nitrosamine predominantly induces necrosis in rat liver, while an apoptosis-like type of cell death predominates after a low dose (Daoust and Morais, 1986). Likewise, the protein synthesis inhibitor, cycloheximide, induces apoptosis in rat liver (Ledda-Columbano et al., 1992). Thus, whether a cell undergoes necrosis or apoptosis m a y depend on the type and extent of injury. In cases of h u m a n hepatitis, residues of dead cells known as Councilman bodies closely resemble apoptotic cells in rat liver (Schaff et al., 1984; Searle et al., 1987). In various states of rodent and h u m a n liver damage, dying cells show either apoptotic or necrotic morphology or may

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display morphological features of both (Wyllie, 1987; Bursch et al., 1992a, 1993; Alison and Sarraf, 1994, Ogasawara et al., 1993). Due to the lack of reliable histological markers it is frequently difficult or impossible to categorize dying cells as to whether they are undergoing apoptosis, other types of active cell death, or necrosis. 4. Selection o f Cells ]:or Apoptosis

The concept of apoptosis implies that "unwanted" (i.e., damaged or dangerous) cells are selected for apoptosis. Thus, autoaggressive lymphocyte clones are recognized and selectively destroyed in the thymus (Mac Donald and Lees, 1990). There must also be mechanisms by which the organism identifies liver cells that, although still vital, are more seriously damaged than others. The observation of apoptotic cells at the rim of necrotic areas where damage may have been less severe than in the perivenous area supports the existence of this selection mechanism. Furthermore, in a study with the hepatomitogen, CPA, all proliferating hepatocytes are labeled with [3H]thymidine during the growth response; in the subsequent regression period the nonlabeled, "old" hepatocytes preferentially undergo apoptosis (Bursch et al., 1985). Preneoplastic and ~neoplastic liver cells are also removed at a much higher rate than their normal counterparts (see below). The process by which such cells are recognized in the organ and are selectively activated to undergo apoptosis is not known. Recent studies support the concept that intercellular contacts are important in controlling apoptosis. In a colon carcinoma cell line anti-integrin antibodies leads to apoptosis (Bates et al., 1994). Denial of anchorage may induce apoptosis and implicates integrin-mediated signaling in the control of this phenomenon (Ruoslahti and Reed, 1994). Carbohydrate antigens on the cell surfaces recognized by specific lectins may also be involved in the apoptosis of certain cells (Hiraishi et al., 1993; Kim et al., 1993). Further recognition phenomena are required for phagocytosis of apoptotic bodies (Savill et al., 1993). In rat liver regressing after temporary induction of hyperplasia by lead nitrate, asiologlycoprotein receptors of hepatocytes and the galactose-specific receptors of nonparenchymal liver cells appear to be involved in the clearance of apoptotic cells and fragments (Dini et al., 1992, 1993). C. Duration of Apoptosis in the Liver The quantitative role of cell death in the kinetics of growth and involution of tissues cannot be assessed on the basis of histological counts unless the duration of the histologically visible signs of cell death is known. This parameter was determined in the liver by blocking apoptosis with CPA

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("mitogen rescue") and assaying the kinetics of disappearance of histological indicators of cell death. After a short lag phase apoptotic bodies began to disappear with a half-life of about 2 hr; from this result an average duration of 3 hr for the process of apoptosis was calculated. The short duration of apoptosis explains its relatively rare occurrence in histological sections even in states of considerable cell loss. The lag phase between the end of the sensitivity to mitogen rescue (i.e., "point of no return") and chromatin condensation is less than 1 hr. Likewise, the first stages of apoptosis, condensation of chromatin, fragmentation, and phagocytosis by neighboring cells are of very short duration, namely only a few minutes. By far the greatest part of the entire length of the visible portion of the process of apoptosis appears to be due to the intracellular breakdown of apoptotic bodies (Figure 1) (Bursch et al., 1990). Beginning approximately 2 hr before the point of no return, hepatocytes preparing for apoptosis show signs of cytoplasmic condensation; in addition, they are stained with antibodies to pre-TGF~l (see below) (Bursch et al., 1993). The cell loss per hour (13) can be calculated as follows: = (i x f)/d

where i = incidence of ABs, f = a factor to correct for the formation of more than 1 AB from an apoptotic cell, and d = duration (hours) of the visible parts of apoptosis. In regressing liver and in preneoplastic liver foci (see below) we found apoptosis rates of 0.5 % and 2 to 5 % per hour, respectively (Bursch et al., 1990). Such estimates are useful for mathematical modeling of growth kinetics of normal and preneoplastic tissues (Moolgavkar, 1986; Moolgavkar et al., 1990; Luebeck et al., 1995). However, in order to calculate the daily rates of apoptosis, the circadian rhythmicity of apoptosis has to be taken into account.

III. Biochemical and Molecular Aspects of Apoptosis Conceptually, biochemical and molecular events associated with active cell death can be assigned to three different segments of the overall process. One segment comprises the irreversible steps beyond the "point of no return," including cell killing and elimination of the corpse. Possibly, there are a few different death mechanisms available to cells (type I, type II, type III, see above). This irreversible "suicide segment" is preceded by reversible regulatory and preparatory steps. The "preparatory segment" includes activation of early genes, synthesis of crucial proteins, and some cell condensation. The "regulatory segment" comprises the generation of intra- and extracellular "death signals," their recognition and transduction. Events in these latter

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two segments show considerable variability, depending on the cell type, state of differentiation and function, and kind of death signal. They are closely interactive and mutually affect each other. Subsequently, findings pertaining to active cell death in the liver are reviewed. Most of the data available on the biochemical and molecular events during active cell death are not based on studies with hepatocytes; however, these results will be described when they are germane to the discussion. A. Events Associated with Cell Killing 1. Chromatin Condensation a n d D N A Fragmentation

Chromatin condensation at the nuclear membrane is probably the morphological indicator of the irreversible suicide segment of apoptosis. This event was originally associated with the formation of oligonucleosomal fragments (i.e., DNA ladders) in thymocytes and lymphocytes (Wyllie, 1980) (see above). An increase of Ca 2 § which activates endonucleases in isolated liver nuclei has been implicated as a regulatory signal (Jones et al., 1989). Recent evidence suggests that chromatin condensation during apoptosis may involve at least two different, discrete steps of DNA degradation: the first one leads to DNA fragments approximately 300 and then 50 kbp long, probably by cutting off chromatin loops from their anchorage sites at the nuclear scaffold. The enzyme involved appears to be a Mg*-dependent, but not Ca 2 § 2+-dependent, endonuclease possibly identical to topoisomerase II. Inhibitors of this enzyme cause irreversible double-strand breaks of DNA and can lead to cell death exhibiting apoptotic morphology. Formation of these large fragments has been seen during apoptosis in several different cell types (Filipsky et al., 1990; Barry et al., 1990; Walker et al., 1991, 1994; Dive and Hickman, 1991; Brown et al., 1993; Oberhammer et al., 1993b; Sun and Cohen, 1994). In a second step further degradation of chromatin may occur through Ca 2 +/Mg 2+-dependent endonucleases; in some model systems nucleosomal fragments of 180 bp may appear, while in others the formation of DNA fragments of an irregular size seems to predominate (seen as "smears" instead of "ladders" on agarose gels). 2. Transglutaminase

The enzyme tissue-transglutaminase (TGase) was originally associated with late events in apoptosis. It crosslinks lysine and glutamine residues of proteins. This may provide the mechanical strength required for the formation of a cross-linked protein scaffold of apoptotic bodies preventing release of their contents into the extracellular space. TGase appears to be specifically

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expressed in apoptotic cells in the liver as well as in some other tissues (Fesus et al., 1987, 1989; Piacentini et al., 1991; Bursch et al., 1992a). TGase activity in rat liver increases during regression of liver hyperplasia, coinciding with apoptotic activity. More recent studies suggest that TGase may have additional functions in the preparatory and regulatory segments of active cell death. It is required for the activation of latent TGFf31 in endothelial cells (Kojima et al., 1993). Furthermore, TGase has GTP binding activity and participates in the coupling of oL1adrenergic receptors to the phospholipase C signal transduction pathway (Nakaoka et al., 1994). 3. Proteases

Proteases may also play a key role in starting the irreversible part of the cellular suicide program. Studies on cell death induced by cytotoxic T lymphocytes and natural killer cells have revealed remarkable similarities with apoptosis. During direct contact these cells inject into their target cells a pore forming protein (perforin) which is integrated into the membrane, and a group of proteases which are released into the cytoplasm of the target cell. The latter have serine protease activity and are responsible for the appearance of apoptotic changes in the nucleus of the target cell (Shi et al., 1992a; Helgason et al., 1993). Recently, it was shown that the ced3 gene which is required for active cell death in the nematode, Caenorhabditis elegans, is a homologue of the mammalian interleukin converting enzyme (ICE). It is a cysteine protease, and its activation leads to apoptotic cell death in neuronal cells (Gagliardini et al., 1994). The potential role of plasminogen activators and their inhibitors as well as of other proteases during tissue involution has been discussed by Tenniswood et al. (1992). B. Events Associated with Preparation for Active Cell Death Several genes originally identified as oncogenes or tumor suppressor genes seem to participate in the preparation of cells for apoptosis. An example is the c-fos gene, whose expression was found before DNA synthesis as well as prior to apoptosis, at least in the systems studied (Buttyan et al., 1988; Fanidi et al., 1992). Transgenic mice overexpressing c-fos exhibit a strong signal in tissues committed for programmed cell death during development (Smyene et al., 1992). In a recent study the c-Fos protein was studied in normal rat liver as well as in preneoplastic and neoplastic lesions induced by diethylnitrosamine. Although the lesions at all stages contained an average of 50 to 60% of c-Fos immunopositive nuclei compared to <10% in normal liver, no relationship to proliferation or apoptosis was apparent (Alexandre et al., 1994). Continued expression of c-myc in a state of growth arrest due to growth

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factor deprivation in vitro may induce apoptosis (Evan et al., 1992). Furthermore, c-myc antisense oligonucleotides inhibit cell death of T-cell hybridomas suggesting that c-myc expression may be necessary for induction of apoptosis in this model (Shi et al., 1992b). The involvement of c-myc in proliferation or apoptosis is linked to differential cyclin gene expression; cyclin A elevation is associated with both, while expression of cyclins C, D1, and E is associated with protection from apoptosis (Hoang et al., 1994). In the liver of double-transgenic mice overexpressing c-myc and transforming growth factor ~ (TGF0~) apoptotic changes were observed in existing hepatocytes (Murakami et al., 1993). This suggests that also in the liver in vivo an unbalanced overexpression of c-myc may result in apoptosis. Apoptosis triggered by genotoxic damage seems to be mediated by the tumor suppressor gene p53. In a myeloid cell line wildtype p53 induces apoptosis (Yonish-Rouach et al., 1991). Studies in cells of p53 gene knockout mice suggests an involvement of this gene in the induction of apoptosis by genotoxic chemicals and irradiation. Genotoxic damage leads to a rapid appearance of p53 protein. This effect does not seem to involve enhanced transcription but to result from translation of preformed mRNA. In contrast, glucocorticoid-induced apoptosis of thymocytes does not appear to depend on p53 (Clarke et al., 1993; Lowe et al., 1993). p53 is thought to arrest cell replication in G 1 until the repair of genotoxic damage is completed or, if the damage is extensive, to switch on the pathway to cell death. Thus, p53 seems to participate in the intracellular generation of a death signal by genotoxic damage. Although hepatocytes, as well as other cells, may undergo apoptosis after treatment with genotoxic carcinogens (see Section IIB), it is not known whether p53 is enhanced in dying hepatocytes. The apparent association between p53 expression and apoptosis is of particular interest because mutations in the p53 gene at codon 249 frequently occur in human liver cancer in countries with high aflatoxin B 1 exposure (Hsu et al., 1991; Bressac et al., 1991). Recent animal studies suggest that there may be more chemicals inducing p53 mutations in the liver, e.g., the antiestrogen tamoxifen and the cooked food mutagen aminomethylimidazoquinolin (IQ) (Vancutsem et al., 1994; Fujimoto et al., 1994). p53 is also frequently mutated in other human tumors such as of the colon, lung, and skin. It has been speculated that these mutations interfere with the induction of apoptosis in cancer cells, thereby providing a proliferation advantage to the tumor cells. However, the 249 mutation in p53 does not alter TGFf31mediated apoptosis in hepatoma (Hep3B) cells (Ponchel et al., 1994). Other genes have been found which normally serve to inhibit apoptosis, such as the bcl2 gene and its homologue ced9 in the nematode C. elegans. Overexpression of bcl2 inhibits apoptosis in B lymphocytes and some other cells caused by the withdrawal of growth factors. The rescued cells do not proliferate suggesting that Bcl2 protein acts as a survival factor rather than

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as a mitogen. Bcl2 may be involved in the generation of some lymphomas in humans where the gene is translocated from chromosome 18 to chromosome 14 in the region coding for heavy chain immunoglobins. Lymphomas exhibiting this translocation express enhanced levels of the Bcl2 gene product. Transgenic mice overexpressing the bcl2 gene develop B cell hyperplasia and later B cell lymphoma (Cleary and Smith, 1986; Hockenberry et al., 1990; McDonnell and Korsmeyer, 1991; Hengartner et al., 1992; McDonnell, 1993). Bcl2 and ced9 are members of a gene family including the Bax which dimerizes with Bcl2 and antagonizes its effect (Oltvai et al., 1993; Williams and Smith, 1993). This gene family may therefore play an important role in the life/death decision of cells. Recently, it was reported that expression of the bcl2 gene decreases the net cellular generation of reactive oxygen species (Hockenberry et al., 1993; Sarafian and Bredesen, 1994). Indeed, there is evidence that reactive oxygen species may mediate apoptosis in lymphocytes and granulocytes (Sarafian and Bredesen, 1994). These findings are of considerable interest for investigating mechanisms of toxicity and carcinogenesis in the liver where peroxidation can be induced by a variety of toxic factors. One of the first genes found to be associated with active cell death was the clusterin or testosterone repressed prostate message-2 (TRPM2) gene which is strongly expressed in the prostate gland after castration and which can be repressed by testosterone (L~ger et al., 1988; Tenniswood et al., 1992). Clusterin/TRPM2 was expressed in various organs undergoing active cell death including the liver during involution of CPA-induced hyperplasia. However, when clusterin/TRPM2 expression was studied on the single cell level by in situ hybridization on histological liver sections, the label was equally distributed among all hepatocytes. It was concluded that T R P M 2 was not specifically expressed in liver cells actively undergoing apoptosis. Furthermore, clusterin/TRPM2 was also expressed under conditions of massive cell elimination after liver injury with CC14(Bursch et al., 1995). The role of this gene may be to protect cells in the involuting or injured organ from potential damage during the extensive membrane turnover and remodeling (Tenniswood et al., 1992; Koch-Brandt and Morgans, 1995).

C. Signal Factors Induction or inhibition of active cell death are controlled by the network of intercellular signals which maintain homeostasis of cell number in accordance with the needs of the organism. Intercellular signals cooperate with intracellular ones which monitor the extent of genetic damage, state of differentiation, etc.

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Because of the linked but inverse regulation of apoptosis and DNA synthesis (see section IIB) (Figure 2), TGFf31 became of interest. It belongs to a family of peptides with growth inhibitory functions in many cells (Roberts et al., 1988). These peptides include Muellerian inhibiting substance (MIS), inhibins, and activins (Roberts et al., 1988; Ying, 1989; Russo and Russo, 1994). TGFIB1 is known to inhibit hepatocyte DNA synthesis in vitro and in regenerating rat liver in vivo (Carr et al., 1986; Russell et al., 1988). Immunocytochemical studies on rat liver involuting after withdrawal of CPA suggests the presence of pre-TGFf31 specifically in apoptotic hepatocytes (Bursch et al., 1993); mature TGFf31 is also present but at an apparently lower concentration. This may be explained by the short half-life of the mature form of TGFf31. TGFI31 induces apoptosis in cultured hepatocytes and in the liver when injected as a pulse some hours before sacrifice (Oberhammer et al., 1992, 1993a). Interestingly, TGFf31 induces apoptosis much more effectively in liver regressing after withdrawal of CPA than in normal rat liver. Apparently, it acts synergistic on cells already primed for apoptosis by unknown signals which may be generated by mitogen pretreatment or by hyperplasia. Some intact hepatocytes also contain pre-TGF~31 suggesting that cells in preparation for apoptosis synthesize TGFf31 (Bursch et al., 1993). This observation may provide a marker to identify early apoptotic cells at a stage before distinct chromatin condensation. Furthermore, hepatocytes undergoing necrosis in severely injured liver do not stain for pre-TGFI31; pre-TGFf31 may therefore provide a marker to discriminate apoptosis from necrosis (Bursch et al., 1993). Another member of the TGFf3 superfamily of polypeptides is activin. This peptide, like TGFf31, was found to induce apoptosis in intact rat liver as well as in isolated hepatocytes. Activin has only one-tenth the activity of TGFI31, and therefore a 24-hr exposure is required to induce apoptosis (Schwall et al., 1993). These studies support the view that TGFf31 and related peptides are involved in the regulation of the balance between cell proliferation and cell death in the liver. Furthermore, TGF~31 has been implicated in the induction of active cell death in the regressing prostate after castration (Kyprianou and Isaacs, 1989), and in the endometrium (Rotello et al., 1991). TGFf31 also induces apoptosis in cells of preneoplastic liver foci and in some hepatoma cell lines (Lin and Chou, 1992; Fukuda et al., 1993). In FaO hepatoma cells, apoptosis induced by TGFf31 is prevented by nafenopin, a liver tumor promoter (Bayly et al., 1994); other hepatomaderived cells display little response to TGFf31. Malignant cells including human hepatocellular carcinoma (HCC) overexpress the TGFf31 gene (Ito et al., 1991), and an increased level of TGFf31 is found in the plasma of patients with HCC (Shirai et al., 1994). Thus, some tumors that are resistant to TGFp I may inhibit the growth of surrounding normal tissue by their

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enhanced synthesis of TGFI31. The possible role that TGF[31 plays in liver tumor promotion is described in further detail in Chapter 9 (Jirtle et al., 1994). The cell surface antigen Fas can mediate apoptosis as shown with the use of anti-fas antibodies (Itoh et al., 1991). Fas shows sequence identity with the Apo 1 antigen that mediates apoptosis by anti-Apo 1 antibodies in human malignant lymphocytes (Trauth et al., 1989; Oehm et al., 1992). The Fas antigen belongs to the tumor necrosis factor receptor/nerve growth factor receptor family. Fas is involved in the induction of apoptosis by cytotoxic T cells. This effect requires the direct contact between the cytotoxic cell and the target cell, but is independent of the perforin/fragmentin pathway (see Section IIIA) (Ju et al., 1994). Intraperitoneal injection of a monoclonal anti-fas antibody into mice induced extensive cell death within 2 hr, exhibiting morphological signs of apoptosis in the liver. Only few hepatocytes remained intact, and the animals died within 6 hr. Mice carrying mutations in the fas gene (lpr- and lprcg) were protected indicating that fas was directly involved in these lethal effects. Biochemical analysis revealed massive increases in serum enzymes glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT) etc. in the susceptible mice; obviously liver cells were not phagocytosed and the cell contents leaked out. For reasons not quite clear other organs including thymus and heart which express the fas antigen were not affected. These findings may provide a model to study human fulminant hepatitis (Ogasawara et al., 1993). Another factor possibly involved in hepatocyte apoptosis is TNFc~. It is one of the terminal mediators of the cascade of reactions during endotoxin shock. TNFcx alone did not induce cell death in cultured mouse hepatocytes or in mouse liver in vivo, but pretreatment with transcription inhibitors such as actinomycin T, D-galactosamine or cx-amanitin sensitized hepatocytes to TNFc~, leading to massive cell death, apparently of an apoptotic type (Leist et al., 1994).

IV. Active Cell Death in the Stages of Hepatocarcinogenesis A. Cancer Prestages in the Liver Cancer development, in the liver and other organs, usually occurs as a stepwise process via sequential appearance of cellular intermediates with increasing deviation from normal (see Chapters 8-10) (Farber and Cameron, 1980; Pitot and Sirica, 1980; Schulte-Hermann, 1985; Pitot and Drag-

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an, 1994). The first step, initiation, can be induced by genotoxic insults and may result from mutational events in important genes or chromosomes (Dragan et al., 1993). Initiation may also arise spontaneously for unknown reasons as in most human cancers (Cattley et al., 1994; Schulte-Hermann et al., 1983; Kraupp-Grasl et al., 1991; Ward, 1983). Most investigators agree that initiation occurs in single cells which then replicate and gradually form larger clones. Tumor promotion, as the second step of carcinogenesis, accelerates the growth rate of these clones and thereby cancer development. Cells within these clones may undergo further mutational changes and new cell clones with higher proliferative potential may be selected (progression). Multistage carcinogenesis can be studied particularly well in rodent liver. In this organ putative prestages of cancer were discovered 40 years ago (Weiler, 1956), and their biological behavior has been extensively studied. Suitable phenotypic markers allow their identification as single cells or as foci of cells. Adenomas (previously called neoplastic nodules) appear later than foci, are larger, and may show similar phenotypic alterations. It is not clear whether they are obligatory or facultative intermediates on the pathway to cancer. Different types of preneoplastic lesions can be distinguished in rat liver. The first type exhibits eosinophilic or clear cytoplasm, tends to show an enhanced expression of gamma-glutamyltransferase (GGT), glutathione-S-transferases (GST), and other drug metabolizing enzymes and seems to be inducible by PB, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and similarly acting agents. In contrast, the second type (weakly basophilic foci) shows no or very little expression of drug metabolizing enzymes but tends to overexpress peroxisomal enzymes; these loci seem to be promoted by peroxisome proliferators (Grasl-Kraupp et al., 1993a, b, c; Kraupp-Grasl et al., 1991a; Schulte-Hermann et al., 1994; Marsman and Popp, 1994; Sills et al., 1994). Thus, there may be more than one sequence of intermediate cells on the pathway to liver cancer. In addition, oval cells are considered as potential tumor precursors (see Chapter 5). Similar foci have been found in human liver although few systematic studies have been published (Deugnier et al., 1993; Okuda, 1992). Tumor promoters exert two major effects on loci: they accelerate their growth rate, and they enhance the expression of phenotypic (biochemical, morphological) alterations (Schulte-Hermann, 1985; Schulte-Hermann et al., 1990; Grasl-Kraupp et al., 1993c). The extent of phenotypic alteration in individual loci is quite variable. During promotion foci overexpress enzymes that are present at low constitutive levels in normal liver and/or are inducible by hepatomitogenic promoters. This "facilitated expression" may also be responsible for the enhanced proliferative response to liver tumor promoting agents (Schulte-Hermann et al., 1986). Consistent with this hypothesis are recent data suggesting that loci cells, in contrast with normal

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liver cells, exhibit enhanced resistance to mitogenic stimuli (Jirtle and Meyer, 1991). After promoter withdrawal phenotypic expression in foci may decrease again (i.e., remodel).

B. Kinetic Aspects of Cell Proliferation and Death in Cancer Prestages A two-stage version of the stepwise development of cancer is graphically depicted in Figure 3. The whole process can be described and mathematically modeled if a few key determinants are known, namely rates of cell transformation tx (txl, initiation; ~2, progression), and the growth rate cx 13 resulting from birth rate (el) and cell death rate (13). These variables are accessible to experimental estimation. The present discussion will focus on 0~ and 13. The preneoplastic cell populations (c~2, 132) generally exhibit higher growth rates than the tissue of origin (or1, [31) (Moolgavkar and Knudson, 1981; Moolgavkar, 1986), namely: (0[2 = [32) > (or1 - ~1) Traditional thinking assumes that either c~2 is greater than ~1 or that ]32 is smaller than 131. However, the experimental evidence is not fully compatible with this assumption as will be described below. Cells in preneoplastic foci of the liver usually have 5- to 10-fold higher rates of DNA synthesis and mitosis than normal hepatocytes (SchulteHermann et al., 1981; Schulte-Hermann, 1985; Grasl-Kraupp et al., 1994).

@

initiat,on ,, @ orooress,on , .@

@@

@@ promotion

a - ~ = net growth rate

I~/a = probability of extinction

Figure 3 Determinants of carcinogenesis in a two-stage model. N, normal hepatocytes; I, initiated hepatocytes; M, malignant cells, oil, or2,131and [32 indicate birth rates and death rates of normal and initiated hepatocytes, respectively. ~1 and ~2 are probabilities of cell transformation resulting in initiation or progression. Modified from Moolgavkar (1989) with permission.

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Despite their relatively high proliferative activity foci show little if any growth at least during the early stages of carcinogenesis. It was then found that the number of apoptoses in foci was also higher than in normal liver and this counterbalanced to a great degree the increase in cell proliferation (Schulte-Hermann, 1985; Bursch and Lauer, 1983; Bursch et al., 1984; Schulte-Hermann et al., 1990). All types of liver foci exhibit enhanced rates of cell replication and apoptosis as compared to normal liver; among the different foci phenotypes, weakly basophilic ones show the highest rates (Grasl-Kraupp, 1991b). Cells in foci are rescued by tumor promotion indicating the active nature of cell death (see below) (Bursch et al., 1984). Studies on the kinetics of the disappearance of apoptotic bodies in foci after cell rescue show that the duration of apoptosis in foci is similar to that in normal liver (Bursch et al., 1990). This indicates that the enhanced number of apoptoses seen histologically in foci do not result from delays in phagocytosis and digestion of apoptotic bodies. The enhanced occurrence of apoptosis in putative prestages of liver cancer as compared to normal liver was soon confirmed by Columbano et al. (1984, 1986). Garcea et al. (1989a) also reported relatively high rates of apoptosis in foci and even higher ones in neoplastic nodules of rat liver. Marsman et al. (1992) found enhanced rates of apoptosis in weakly basophilic foci. Apparently, apoptosis of preneoplastic cells is generally seen during hepatocarcinogenesis, and may be regarded as one of the defense lines against cancer. C. Active Cell Death and Initiation

Irreversibility of initiation is an important principle of the multistage concept of carcinogenesis and is supported by numerous experimental data. However, if initiated cells occasionally undergo cell death it follows that a certain percentage of initiated cells or cell clones are extinguished. The probability depends on the ratio of birth and death rates of cells in the clone ([3:~) and of the actual clone size (Figure 3). On the basis of mathematical modeling it was predicted that as many as 80% of initiated cells may become extinct (Moolgavkar, 1989; Luebeck et al., 1991). This prediction was experimentally tested by studying single putative initiated cells (detected by antibodies against the placental GST). Such cells are almost absent in untreated young rat liver, but dramatically increase after administration of genotoxic carcinogens. Within a few weeks approximately 80% of these cells disappear again, with morphological evidence of apoptosis (B. GraslKraupp, A. Wagner, A. L6w-Baselli, W. Huber, and R. Schulte-Hermann, unpublished results). A similar observation was made after initiation with diethylnitrosamine (Satoh et al., 1989). These experimental findings support

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the mathematical prediction that in current models of hepatocarcinogenesis the majority of initiated cells may be extinguished (Moolgavkar, 1989; Luebeck et al., 1991). Elimination of initiated cell clones of spontaneous origin was studied in aged rats. A state of "relative hyperplasia" (see Section IIB) of the liver was induced by subjecting the animals to food restriction to 60% of normal for 3 months. This resulted in a decline of DNA synthesis, a tendency toward enhanced apoptosis, and a loss of approximately 20% of liver cells. Qualitatively similar effects occurred in foci but they were much more pronounced, so that foci number and volume declined by 85% (Figure 4). Subsequent treatment with the liver tumor promoter nafenopin induced significantly less tumors than in animals steadily fed ad libitum (GraslKraupp et al., 1994). This indicates that many initiated (promotable) cell

Figure 4 DNA synthesis and apoptosis in normal and preneoplastic hepatocytes during restricted feeding (FR). Nine-month-old male rats received 60% of the food ration of pair-fed control animals for up to 95 days. In (a) total liver DNA is shown as an index of cell number, and foci number as an index of the number of initiated clones per liver. The total foci volume comprised less than 1% of the liver. Liver DNA and foci number in control animals fed ad libitum are set to 100%. In (b) labeling indices (LI) obtained after injection of [3H]-thymidine, and the incidence of apoptotic bodies (AB) per 100 hepatocytes are shown for normal liver and foci. See text and Grasl-Kraupp et al. (1994) for further details.

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clones had actually been extinguished during the fasting period and provides experimental proof for Moolgavkar's prediction. The remarkable selection of foci cells for elimination can be explained by the 5- to 10-fold higher rates of apoptosis in foci than in normal liver; doubling of this rate by food restriction resulted in a 5- to 10-fold greater loss of cells from foci than from normal liver in the same time. Thus, the elevated cell turnover is a critical property of cancer prestages, because shifts in the balance between replication and apoptosis have a much greater impact on cell number in foci than in unaltered tissue (Figure 3). These findings show for the first time that food restriction preferentially enhances apoptosis in preneoplastic lesions. This may provide a new explanation for the partial protection from cancer by low calorie diets in both experimental animals and in humans. Another interesting conclusion relates to the risk assessment of nongenotoxic carcinogens. It is still debated whether these agents, e.g., nafenopin, have initiating potential or not (Grasl-Kraupp et al., 1993c). The study described above (Grasl-Kraupp et al., 1994) suggests that the carcinogenic potency of these agents depends on the presence and number of (spontaneously) initiated cells in the liver. Hypothetically, in a liver carrying no initiated cells nafenopin should produce no tumors. An additional, anti-initiating role of apoptosis can be ascribed to the elimination of cells suffering from genotoxic damage by chemicals or radiation. It was observed that stem cells in intestinal crypts are preferentially eliminated by apoptosis after genotoxic injury due to irradiation (Ijiri and Potten, 1983). Likewise, active cell death occurs in the liver after treatment with genotoxic carcinogens (see Section IIB). In this way apoptosis would eliminate potentially dangerous cells with pro-mutational lesions which may be at risk for initiation. The likely involvement of the tumor suppressor gene p53 in the control of this pathway to cell death has been discussed above (see Section IIIB). D. Active Cell Death and Tumor Promotion

Tumor promoters enhance the growth rate and the size of initiated cell populations. Originally it was expected that this effect would result from selective stimulation of the proliferation of preneoplastic cells and/or from inhibition of proliferation in normal cells (see Chapter 9) (Farber and Cameron, 1980; Solt and Farber, 1976). Detailed experimental studies on proliferation kinetics in foci of rat liver have shown that indeed promoter treatment initially stimulates the proliferative activity in foci much more than in the surrounding tissue. However, this stimulation readily disappears during repeated treatment, and DNA synthesis in foci returns to the level seen in the absence of the promoter (Schulte-Hermann et al., 1981, 1990). Obvi-

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ously, foci still respond to the feedback mechanisms which prevent excessive increases of DNA during treatment with hepatomitogenic promoters as described in Section II. Therefore, the steady growth of foci in the absence of increased proliferation could only be explained by assuming variations in the cell death rates. In fact, PB lowers the high rate of apoptosis normally found in foci, thereby leading to an accumulation of foci cells during prolonged administration. Cessation of PB treatment dramatically increases the incidence of apoptosis in foci, and retreatment decreases it again (Schulte-Hermann et al., 1982, 1990; Bursch and Lauer, 1983; Bursch et al., 1984). CPA, another tumor promoter, also inhibits apoptosis in foci (Bursch et al., 1990). These findings suggest that tumor promoters can act as survival factors for initiated or preneoplastic hepatocytes. A similar mechanism may also apply to TCDD, the most potent liver tumor promoter known. A recent study showed only a slight increase in the rates of DNA synthesis of foci during treatment with TCDD. This suggests the occurrence of a decreased death rate for foci cells during TCDD promotion (Buchmann et al., 1994). The shifts in rates of birth and death of preneoplastic hepatocytes during and after promoter treatment and their consequences for lesion growth are illustrated in Figure 3. The importance of apoptosis during the promotion of preneoplastic lesions in the liver is demonstrated by the mathematical modeling of biological data obtained with PB and a-HCH (Luebeck et al., 1995). This study further suggests an uneven distribution of cell proliferation and apoptosis within foci, both rates being higher close to the surface of foci. The occurrence of apoptosis in foci tends to be associated with weak expression of the altered phenotype. Apoptotic counts are higher in "remodeling" foci than in "persistent" ones, even though the rate of DNA synthesis is not different (Schulte-Hermann et al., 1990). Thus, in remodeling foci cell proliferation and cell death may balance each other, while persistent foci may have an excess in cell proliferation. Accordingly, persistent foci show more rapid growth than remodeling foci and hypothetically may be more prone to progression to cancer (Rotstein et al., 1986). Treatment with PB reinforced the expression of the phenotypic alteration and decreased apoptosis; after stopping promoter treatment both remodeling and apoptosis again increased. Thus, as in normal liver the probability to undergo apoptosis seems to be coupled to the functional state of the cell (altered phenotype = overexpression of a pleiotropic response). However, loss of phenotypic markers is not necessarily associated with enhanced death rates. When rats are switched from PB to clofibrate or nafenopin treatment, GGT and some other markers disappeared in foci, but the rate of apoptosis is not enhanced. This suggests that peroxisome proliferators reduce the expression of the specific phenotype of foci, but did not stimulate the elimination of foci cells (Gerbracht et al., 1990). Reversibility is considered a crucial property of tumor promotion. This

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seems to be the case in the mouse skin model (Boutwell, 1964). Liver foci may at least partially disappear when promoter treatment is stopped. This may simply be due to reversion of the altered phenotype (remodeling), and therefore should not be considered as evidence for reversal of promotion. However, the long-lasting enhancement of apoptosis in foci after promoter withdrawal suggests that tumor promotion may be truly reversible in the liver (Bursch et al., 1984; Schulte-Hermann et al., 1990). Foci, adenomas, and even carcinomas induced by treatment with the tumor promoter, nafenopin, largely disappeared on cessation of treatment (B. Grasl-Kraupp, B. Ruttkay-Nedecky, L. Mfillauer, H. Taper, T. Radaszkiewicz, H. Denk, R. Schulte-Hermann, 1995, in preparation). Although there is increasing evidence supporting the reversibility of tumor promotion in the liver, more detailed studies are still required. An antipromoting effect on liver foci and nodules was seen after treatment with S-adenosyl-methionine (SAM), a methyl group donor enhancing methylation of DNA, which may be involved in suppression of tumor promoting genes. SAM reduced DNA synthesis and enhanced apoptosis and remodeling in foci and nodules. As a result a pronounced regression of nodular lesions occurred. The number of malignant liver tumors developing at 2 years was reduced by SAM treatment for 6 months (Garcea et al., 1989a, b; Pascale et al., 1991, 1992). An antipromoting effect o n rat liver foci was also obtained during 3 months of restricted feeding (see above) (Grasl-Kraupp et al., 1994). E. Active Cell Death and Tumors

Adenomas and carcinomas in rat liver exhibited a dramatically enhanced cell turnover with higher rates of DNA replication and apoptosis than in normal liver. The increase of active cell death from foci to adenomas and carcinomas shows that the enhanced susceptibility of early cancer prestages to undergo apoptosis is not eliminated by selection in the course of progression. Instead, the enhanced rates of both birth and death of cells may be an inherent characteristic of increasing malignancy. The reason for this apparently coupled increase of birth and death rates during malignancy are unknown. From a biological point of view increased birth and death rates, or increased cell turnover, will enhance the chance of the population to acquire additional genetic damage and may thus provide a selection advantage. The relatively high rate of active cell death in tumors is probably not unique to the liver. It has also been seen in other malignant tumors (Bursch et al., 1991). Obviously in a growing tumor, the balance between cell birth and cell death is shifted toward proliferation. In a study where liver adenomas and carcinomas were induced by prolonged treatment with nafenopin cessation of treatment resulted in rapid regression and disappearance of most tumors; DNA synthesis ceased almost completely, while active cell death remained increased and probably caused

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the rapid reduction of tumor mass (B. Grasl-Kraupp, B. Ruttkay-Nedecky, L. Mfillauer, H. Taper, T. Radaszkiewicz, H. Denk, R. Schulte-Hermann, 1995, in preparation). Thus, even carcinoma cells may still depend on the presence of a promoter agent as a "survival factor" (see Chapter 9). The examples presented above and studies with hormone-dependent tumors where massive apoptosis and rapid tumor regression can be induced by hormone withdrawal or antihormone treatment show that the balance between birth and death can be shifted toward cell death. This is of principal interest for tumor therapy since the concept of active cell death as an event under the control of growth regulating signals should provide new strategy for cancer therapy.

V. Conclusions 9

In normal liver both cell replication and cell death by apoptosis are involved in the homeostasis of cell number. Both processes occur in a coordinated, coupled manner. Hepatic growth stimuli tend to increase cell replication and decrease cell death, while during regression of liver size cell replication declines and apoptosis goes up.

9

In the preneoplastic and neoplastic liver, rates of cell replication and of apoptosis are both higher than in the normal tissue. However, the coordination and coupling between both events as noted in normal liver tissue is still maintained. As a result preneoplastic and neoplastic tissue may still exhibit a partial balance between cell replication and cell elimination. Preneoplastic liver cells seem to exhibit an intrinsic defect in growth control rendering them more susceptible to certain signals stimulating growth or regression of liver tissue. Treatment with a tumor promoter will shift the balance favoring cell proliferation; the preferential response of foci results in accumulation of preneoplastic cells and, thereby, in acceleration of tumor development. Conversely, promoter withdrawal or food restriction favors active cell death; this may result in preferential elimination of initiated and preneoplastic cells and, thereby, in extinction of initiated clones and in regression of preneoplastic lesions and even of tumors. Nongenotoxic carcinogens which are liver tumor promoters are mitogens for normal as well as for preneoplastic and neoplastic liver cells. In addition, these agents act as survival factors for preneoplastic cells, and this is a further important property of tumor promoters.

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T h e a n t i - i n i t i a t i n g , a n t i p r o m o t i n g a n d a n t i c a r c i n o g e n i c effects of actively i n d u c e d cell d e a t h is of interest for the d e s i g n of n e w c h e m o p r e v e n t i v e a n d t h e r a p e u t i c strategies a g a i n s t cancer.

References Alexandre, K., Fokan, D., and Galand, P. (1994). Immunohistochemical expression of the c-FOS protein in preneoplastic lesions during hepatocarcinogenesis in rats. Int. J. Oncol. 4, 429-434. Alison, M. R., and Sarraf, C. E. (1994). Liver cell death: Patterns and mechanisms. Gut 35, 577-581. Alison, M. R., Wilkins, M. J. E., Walker, S. M., and Hully, J. R. (1987). Cell population size control in the rat liver: The response of hepatocytes in various proliferative states to a mitogenic stimulus. Epithelia 1, 53-64. Arends, M. J., Morris, R. G., and Wyllie, A. H. (1990). Apoptosis. The role of the endonuclease. Am. J. Pathol. 136, 593-607. Bardon, S., Vignon, E, Montcourrier, P., and Rochefort H. (1987). Steroid receptor-mediated cytotoxicity of an antiestrogen and an antiprogestin in breast cancer cells. Cancer Res. 47, 1441-1448. Barry, M. A., Behnke, C. A., and Eastman, A. (1990). Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Pharmacol. 40, 2353-2362. Bates, R. C., Buret, A., van Helden, D. E, Horton, M. A., and Burns, G. E (1994). Apoptosis induced by inhibition of intercellular contact. J. Cell Biol. 125, 403-415. Bayly, A. C., Roberts, R. A., and Dive C. (1994). Suppression of liver cell apoptosis in vitro by the non-genotoxic hepatocarcinogen and peroxisome proliferator nafenopin. ]. Cell Biol. 125, 197-203. Beatson, G. T. (1896). On the treatment of inoperable cases of carcinoma of the mamma: Suggestion for a new method of treatment, with illustrative cases. Lancet 2, i04-107. Bolender, R. P., and Weibel, E. R. (1973). A morphometric study of the removal of phenobarbitalinduced membranes from hepatocytes after cessation of treatment. J. Cell Biol. 56, 746761. Boutwell, R. K. (1964). Some biological aspects of skin cacinogenesis. In "Progress in Experimental Tumor Research" (E Homburger, ed.), Vol. 4, pp. 207-250. Karger, Basel and New York. Bressac, B., Kew, M., Wands, J., and Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature (London) 350, 429-431. Brown, D. G., Sun, X.-M., and Cohen, G. M. (1993). Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J. Biol. Chem. 268, 3037-3039. Buchmann, A., Stinchcombe, S., K6rner, W., Hagenmaier, H., and Bock, K. W. (1994). Effects of 2,3,7,8-tetrachloro- and 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin on the proliferation of preneoplastic liver cells in the rat. Carcinogenesis 15, 1143-1150. Bursch, W. (1994). Apoptosis and cancer therapy. In "New Approaches in Cancer Pharmacology: Drug Design and Development" (P. Workman, ed.), Vol. 2, pp. 71-85. SpringerVerlag, Berlin. Bursch, W., and Lauer, B. (1983). Influence of liver tumor promoters on cell death by apoptosis in rat liver. Naunyn-Schmiedeberg's Arch. Pharmacol. 322 (Suppl.), Rl19.

168

Roll Schulte-Hermann, Bettina Grasl-Kraupp, and Wilfried Bursch

Bursch, W., Lauer, B., Timmermann-Trosiener, T., Barthel, G., Schuppler, J., and SchulteHermann, R. (1984). Controlled cell death (apoptosis) of normal and putative neoplastic cells in rat liver following withdrawal of tumor promoters. Carcinogenesis 5, 53-58. Bursch, W., Taper, H. S., Lauer B., and Schulte-Hermann, R. (1985). Quantitative histological and histochemical studies on the occurrence and stages of controlled cell death (apoptosis) during regression of rat liver hyperplasia. Virchows Arch. Cell Pathol. 50, 153-166. Bursch, W., Duesterberg, B., and Schulte-Hermann, R. (1986). Growth, regression and cell death in rat liver as related to tissue levels of the hepatomitogen cyproterone acetate. Arch. Toxicol. 59, 221-227. Bursch, W., Paffe, S., Putz, B., Barthel, G., and Schulte-Hermann, R. (1990). Determination of the length of the histological stages of apoptosis in normal liver and in altered hepatic foci of rats. Carcinogenesis 11, 847-853. Bursch, W., Liehr, J. G., Sirbasku, D., Putz, B., Taper H., and Schulte-Hermann, R. (1991). Control of cell death (apoptosis) by diethylstilbestrol in an estrogen dependent kidney tumor. Carcinogenesis 12, 855-860. Bursch, W., Fesus, L., and Schulte-Hermann, R. (1992a). Apoptosis ("programmed" cell death) and its relevance in liver injury and carcinogenesis. In "Tissue Specific Toxicology: Biochemical Mechanisms" (W. Dekant and H. G. Neumann, eds.), pp. 95-115. Academic Press, London. Bursch, W., Oberhammer, E, and Schulte-Hermann, R. (1992b). Cell death by apoptosis and its protective role against disease. Trends Pharmacol. Sci. 13, 245-251. Bursch, W., Oberhammer, E, Jirtle, R. L., Askari, M., Sedivy, R., Grasl-Kraupp, B., and Purchio, A. E (1993). Transforming growth factor-131 as a signal for induction of cell death by apoptosis. Br. J. Cancer 67, 531-536. Bursch, W., Gleeson, T., Kleine, L., and Tenniswood, M. (1995). Expression of clustering (testosterone-repressed prostate message-2) mRNA during growth and regression of rat liver. Arch. Toxicol. 69, 253-258. Bursch, W., Kienzl, H., Ellinger A., and Schulte-Hermann, R. (1995). Cell death in cultured human mammary carcinoma cells (MCF-7) after treatment with the antiestrogens tamoxifen and ICI 164 384. submitted for publication. Buttyan, R., Zakeri, Z., Lockshin, R. A., and Wolgemuth, D. (1988). Cascade induction of c-fos, c-myc, and heat shock 70 k transcripts during regression of the rat vetral prostate gland. Mol. Endocrinol. 2, 650-657. Carr, B. I., Hayashi, I., Branum, E. L., and Moses, H. L. (1986). Inhibition of DNA synthesis in rat hepatocytes by platelet-derived TGF-[31. Cancer Res. 46, 2330-2334. Cattley, R. C., Marsman, D. S., and Popp, J. A. (1991). Age-related susceptibility to the carcinogenic effect of the peroxisome proliferator WY-14,643 in rat liver. Carcinogenesis 12, 469-473. Clarke, P. G. H. (1990). Developmental cell death: Morphological diversity and multiple mechanisms. Anat. Embryol. 181, 195-213. Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. L., and Wyllie, A. H. (1993). Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature (London) 462, 849-852. Cleary, M. L., and Smith, S. D. (1986). Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell (Cambridge, Mass.) 47, 19-28. Cohen, J. J., and Duke, R. C. (1984). Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J. Immunol. 132, 38-42. Collins, R. J., Harmon, B. K., Gobe, G. C., and Kerr, J. E R. (1992). Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int. J. Radiat. Biol. 61, 451-453.

7.

Apoptosis and Hepatocarcinogenesis

169

Columbano, A., Ledda-Columbano, G. M., Rao, P. M., Rajalakshmi, S., and Sarma, D. S. R. (1984). Occurrence of cell death (apoptosis) in preneoplastic and neoplastic liver cells: A sequential study. Am. J. Pathot. 116, 441-446. Columbano, A., Ledda-Columbano, G. M., Coni, P. P., Faa, G., Liguori, C., Santa Cruz, G., and Pani, P. (1985). Occurrence of cell death (apoptosis) during the involution of liver hyperplasia. Lab. Invest. 53, 670. Columbano, A., Ledda-Columbano, G. M., Coni, P., Faa, G., and Pani, P. (1986). Does apoptosis play an opposite role to mitosis in the regulation of animal cell turnover? Cell Biol. Int. Rep. 10, 473-474. Compton, M. M., and Cidlowski, J. A. (1986). Rapid in vivo effects of glucocorticoids on the integrity of rat lymphocyte genomic deoxyribonucleic acid. Endocrinology (Baltimore) 118, 38-45. Conney, A. H., Davison, C., Gastel, R., and Burns, J. J. (1960). Adaptive increases in drug metasbolizing enzymes induced by phenobarbital and other drugs. J. Pharmacol. Exp. Ther. 130, 1-8. Daoust, R., and Morais, R. (1986). Degenerative changes, DNA synthesis and mitotic activity in rat liver following single exposure to diethylnitrosamine. Chem. Biol. Int. 57, 55-64. Deugnier Y. M., Charalambous, P., Le Quilleuc D., Turlin, B., Searle, J., Brissot P., Pokell, L. W., and Halliday, J. W. (1993). Preneoplastic signiciance of hepatic iron-ree loci in genetic hemochromatosis: A study of 185 patients. Hepatology (Baltimore) 18, 13631369. Dini, L., Autuori, F., Lentini, A., Oliverio, S., and Piacentini, M. (1992). The clearance of apoptotic cells in the liver is mediated by the asialoglycoprotein receptor. FEBS Lett. 296, 174-178. Dini, L., Falasca, L., Lentini, A., Mattioli, P., Piacentini, M., Piredda, L., and Autuori, E (1993). Galactose-specific receptor modulation related to the onset of apoptosis in rat liver. Eur. J. Cell Biol. 61, 329-337. Dive, C., and Hickman, J. A. (1991). Drug-target interactions: Only the first step in the commitment to a programmed cell death? Br. J. Cancer 64, 192-196. Dragan, Y. P., Peterson, J., and Pitot, H. C. (1993). Comparison of hepatocyte phenotypes at the glutathione transferase and albumin loci in Sprague-Dawley and Nagase analbuminemic rats and F1 progeny after initiation and promotion. Carcinogenesis 14, 1313-1319. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M., Waters, C. M., Penn, L. Z., and Hancock, D. C. (1992). Induction of apoptosis in fibroblasts by c-myc protein. Cell (Cambridge, Mass.) 69, 119-128. Fanidi, A., Harrington, E. A., and Evan, G. I. (1992). Cooperative interaction between c-myc and bcl-2 proto-oncogenes. Nature (London) 359, 554-556. Farber, E., and Cameron, R. (1980). The sequential analysis of cancer development. Adv. Cancer Res. 31, 125-225. Farber, E., Verbin, R. S., and Lieberman, M. (1971). Cell suicide and cell death, In "A Symposium on Mechanisms of Toxicology" (N. Aldridge, ed.), pp. 163-173. Macmillan, New York. Fesus, L., Thomazy, V., and Falus, A. (1987). Induction and activation of tissue transglutaminase during programmed cells death. FEBS Lett. 224, 104-108. Fesus, L., Thomazy, V., Autuori, F., Ceru, M. P., Tarcsa, E., and Piacentini, M. (1989). Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action. FEBS Lett. 245, 150-154. Filipski, J., Leblanc, J., Youdale, T., Sikorska, M., and Walker, P. R. (1990). Periodicity of DNA folding in higher order chromatin structures. EMBO J. 9, 1319-1327. Fisher, T. C., Milner, A. E., Gregory, C. D., Jackman, A. L., Aherne, G. W., Hartley, J. A., Dive, C., and Hickman, J. A. (1993) Bcl-2 modulation of apoptosis induced by anticancer drugs:

170

Rolf Schulte-Hermann, Bettina Grasl-Kraupp, and Wilfried Bursch

Resistance to thymidylate stress is independent of classical resistance pathways. Cancer Res. 53, 3321-3326. Fujimoto, Y., Hampton, L. L., Snyderwine, E. G., Nagao, M., Sugimura, T., Adamson, R. H., and Thorgeirsson, S. S. (1994). p53 gene mutation in hepatocellular carcinoma induced by 2-amino-3-methylimidazol(4,5-f)quinoline in nonhuman primates. Jpn. J. Cancer Res. 85, 506-509. Fukuda, K., Kojiro, M., and Chiu, J. E (1993). Induction of apoptosis by transforming growth factor-f31 in the rat hepatoma cell line MCA-RH7777: A possible association with tissue transglutaminase expression. Hepatology (Baltimore) 18, 945-953. Gagliardini, V., Fernandez, P. A., Lee, R. K. K., Drexler, H. C. A., Rotello, R. J., Fishman, M. C., and Yuan, J. (1994). Prevention of vertebrate neuronal death by the crmA gene. Science 263, 826-828. Garcea, R., Daino, L., Pascale, R., Simile, M. M., Puddu, M., Frassetto, S., Cozzolino, P., Seddaiu, M. A., Gaspa, L., and Feo, E (1989a). Inhibition of promotion and persistent nodule growth by S-adenosyl-L-methionine in rat liver carcinogenesis: Role of remodeling and apoptosis. Cancer Res. 49, 1850-1856. Garcea, R., Daino, L., Pascale, R., Simile, M. M., Puddu, M. Rugiu, M. E., Seddaiu, M. A., Satta, G., Sequenza, M. J., and Feo, E (1989b). Protooncogene methylation and expression in regenerating liver and preneoplastic liver nodules induced in the rat by diethylnitrosamine: Effect of variations of S-adnosylmethionine: S-adenosylhomocysteine ratio. Carcinogenesis 10, 1183-1192. Gerbracht, U., Bursch, W., Kraus, P., Putz, B., Reinacher, M., Timmermann-Trosiener, I., and Schulte-Hermann, R. (1990). Effects of hypolipidemic drugs on phenotypic expression and cell death (apoptosis) in altered foci of rat liver. Carcinogenesis 11, 617-624. Glaumann, H., Arborgh, B., and Lindeborg, T. (1977). Induction of liver lysosomal enzymes during the autophagic phase following phenobarbital treatment of rat. Virchows Arch. (Zellpathol.) 23, 17-28. Gliicksmann, A. (1930). Uber die Bedeutung von Zellvorg~ingen ffir die Formbildung epithelialer Tumoren. Z. Anat. Entwicklungsgesch. 93, 35-92. Grasl-Kraupp, B., Huber, W., Timmermann-Trosiener, I., and SchulteoHermann, R. (1993a). Peroxisomal enzyme induction uncoupled from enhanced DNA synthesis in putative preneoplastic liver foci of rats treated with a single dose of the peroxisome proliferator nafenopin. Carcinogenesis 14, 2435-2437. Grasl-Kraupp, B., Waldh6r, T., Huber, W., and Schulte-Hermann, R. (1993b). Glutathione S-transferase isoenzyme patterns in different subtypes of enzyme-altered rat liver foci treated with the peroxisome proliferator nafenopin or with phenobarbital. Carcinogenesis 14, 2407-2412. Grasl-Kraupp, B., Huber, W., and Schulte-Hermann, R. (1993c). Are peroxisome proliferators tumour promoters in rat liver? In "Peroxisomes. Biology and Importance in Toxicology and Medicine" (G. Gibson and B. Lake, eds.), pp. 667-693. Taylor & Francis, London. Grasl-Kraupp, B., Bursch, W., Ruttkay-Nedecky, B., Wagner, A., Lauer, B., and SchulteHermann, R. (1994). Food reduction eliminates preneoplastic cells through apoptosis and antagonizes carcinogenesis in rat liver. Proc. Natl. Acad. Sci. U.S.A. 91, 19, 9995-9999. Grasl-Kraupp, B., Ruttkay-Nedecky, R., Koudelka, H., Bukowska, K., Bursch, W., and SchulteHermann, R. (1995). In situ detection of fragmented DNA (TUNEL Assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: A cautionary note. Hepatology 21, 1465-1468. Greenberg, J. T., Guo, A., Klessig, D. E, and Ausubel, E M. (1994). Programmed cell death in plants: A pathogen-triggered response activated coordinately with multiple defense functions. Cell (Cambridge, Mass.) 77, 551-563. Gullino, P. M. (1980). The regression process in hormone-dependent mammary carcinomas. In

7. Apoptosis and Hepatocarcinogenesis

171

"Hormones and Cancer" (S. Iacobelli; R. B. J. King; H. R. Lindner; and M. E. Lippman, eds.), pp. 271-279. Raven, New York. Harmon, B. V., Corder, A. M., Collins, R. J., Gobe, G. C., Allen, J., Allen, D. J., and Kerr, J. E R. (1990). Cell death induced in a murine mastocytoma by 42 to 47~ heating in vitro: Evidence that the form of death changes from apoptosis to necrosis above a critical heat load. Int. J. Radiat. Biol. 58, 845-858. Helgason, C. D., Shi, L., Greenberg, A. H., Shi, Y., Bromley, P., Cotter, T., Green, D. R., and Bleackley, R. C. (1993). DNA fragmentation induced by cytotoxic T lymphocytes can result in target cell death. Exp. Cell Res. 206, 302-310. Hengartner, M. O., Ellis, R. E., and Horvitz, H. R. (1992). Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature (London) 356, 494-499. Hiraishi, K., Suzuki, K., Hakomori, S., and Adachi, M. (1993). Le(y) antigen expression is correlated with apoptosis (programmed cell death). Glycobiology 3, 381-390. Hoang, A. T., Cohen, K. J., Barrett, J. E, Bergstrom, D. A., and Dang, C. V. (1994). Participation of cyclin A in myc-induced apoptosis. Proc. Natl. Acad. Sci. U.S.A. 91, 68756879. Hockenberry, D., Nunez, G., Milliman, C., Schreiber, R. D., and Korsmeyer, S. J. (1990). Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature (London) 348, 334-336. Hockenberry, D. M., Oltvai, Z. N., Yin, X.-M., Milliman, C. L., and Korsmeyer, S. J. (1993). Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell (Cambridge, Mass.) 75, 241-251. Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., and Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature (London) 350, 427-428. Huggins, C., Stevens, R. E., and Hodges, C. V. (1941). Studies on prostatic cancer II. The effects of castration on advanced carcinoma in the prostate gland. Arch. Surg. 43,209-233. Ijiri, K., and Potten, C. S. (1983). Response of intestinal cells of differing topographical and hierarchial status to ten cytotoxic drugs and five sources of radiation. Br. J. Cancer 47, 175185. Ito, N., Kawata, S., Tamura, S., Takaishi, K., Shirai, Y., and Kiso, S. (1991). Elevated levels of transforming growth factor 131 messenger RNA and its polypeptide in human hepatocellular carcinoma. Cancer Res. 51, 4080-4083. Itoh, N., Yonehara, S., Ishi, A., Yonehara, M., Mizushima, S.-I., Sameshima, M., Hase, A., Seto, Y., and Nagata, S. (1991). The polypeptide encoded by the cDNA for human cell surface antigen fas can mediate apoptosis. Cell (Cambridge, Mass.) 66, 233-243. Jirtle, R. L., and Meyer, S. A. (1991). Liver tumor promotion: Effect of phenobarbital on EGF and protein kinase C signal transduction and transforming growth factor 131 expression. Dig. Dis. Sci. 5, 659-668. Jirtle, R. L., Hankins, G. R., Reisenbichler, H., and Boyer, I. J. (1994). Regulation of mannose 6-phosphate/insulin-like growth factor-II receptors and transforming growth factor beta during liver tumor promotion with phenobarbital. Carcinogenesis 15, 1473-1478. Jones, D. P., McConkey, D. J., Nicotera, P., and Orrenius, S. (1989). Calcium-activated DNA fragmentation in rat liver nuclei. J. Biol. Chem. 264, 6398-6403. Ju, S.~ Cui, H., Panka, D. J., Ettinger, R., and Marshak-Rothstein, A. (1994). Participation of target fas protein in apoptosis pathway induced by CD4 § Thl and CD8 § cytotoxic T cells. Proc. Natl. Acad. Sci. U.S.A. 91, 4185-4189. Kerr, J. E R. (1971). Shrinkage necrosis: A distinct mode of cellular death. J. Pathol. 105, 13-20. Kerr, J. E R., Wyllie, A. H., and Currie, A. R. (1972). Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. J. Cancer 26, 239-257.

172

Rolf Schulte-Hermann, Bettina Grasl-Kraupp, and Wilfried Bursch

Kerr, J. E, Winterford, C. M., and Harmon, B. V. (1994). Apoptosis. Its significance in cancer and cancer therapy. Cancer 73, 2013-2026. Kim, M., Rao, M. V., Tweardy, D. J., Prakash, M., Galili, U., and Gorelik, E. (1993). Lectininduced apoptosis of tumour cells. Glycobiology 3, 447-453. Koch-Brandt, C., and Morgans, C. (1995). Clusterin: A role in cell survival in the face of apoptosis? In "Progress in Molecular and Subcellular Biology: Apoptosis" (W. E. G. Mtiller, E Jeanteur, Y. Kuchino, and E Paine, eds.), Vol. 16. Springer-Verlag, Berlin. Kojima, S., Nara, K., and Rifkin, D. B. (1993). Requirement for transglutaminase in the activation of latent transforming growth factor-J3 in bovine endothelial cells. J. Cell Biol. 121,439-448. Kraupp-Grasl, B., Huber, W., Taper, H., and Schulte-Hermann, R. (1991a). Increased susceptibility of aged rats to hepatocarcinogenesis by the peroxisome proliferator nafenopin and the possible involvement of altered liver foci occurring spontaneously. Cancer Res. 51, 666-671. Kraupp-Grasl, B., Huber, W., and Schulte-Hermann, R. (1991b). Cell turnover and expression of GSH-transferase isoenzymes, peroxisomal enzymes, and cytochrom P-452 in weakly basophilic foci, the likely tumor precursors in nafenopin-treated rat livers. Proc. Am. Assoc. Cancer Res. 32, A155. [Abstract] Kruman, I. I., Matylevich, N. E, Beletsky, I. P., Afanasyev, V. N., and Umansky, S. R. (1991). Apoptosis of murine BW 5147 thymoma cells induced by dexamethasone and gammairradiation. J. Cell. Physiol. 148, 267-273. Kyprianou, N., and Isaacs, J. T. (1989). Expression of transforming growth factor in the rat ventral prostate during castration-induced programmed cell death. Mol. Endocrinol. 3, 1515-1522. Lanzerotti, L. H., and Gullino, P. M. (1972). Activity and quantity of lysosomal enzymes during mammary tumor regression. Cancer Res. 32, 2679-2685. Ledda-Columbano, G. M., Coni, E, Curto, M., Giacomini, L., Faa, G., Oliverio, S., Piacentini, M., and Columbano, A. (1991). Induction of two different modes of cell death, apoptosis and necrosis, in rat liver after a single dose of thioacetamide. Am. J. Pathol. 139, 10991109. Ledda-Columbano, G. M., Coni, P., Faa, G., Manenti, G., and Columbano, A. (1992). Rapid induction of apoptosis in rat liver by cycloheximide. Am. J. Pathol. 140, 545-549. L6ger, J., Le Guellec, R., and Tenniswood, P. R. (1988). Treatment with antiandrogens induces an androgen repressed gene in the rat vetral prostate. Prostate 13, 131-142. Leighton, E, Coloma, L., and Koenig, C. (1975). Structure, composition, physical properties, and turnover of proliferated peroxisomes: A study of the trophic effects of Su-13437 on rat liver. J. Cell Biol. 67, 281-309. Leist, M., Gantner, E, Bohlinger, I., Germann, P. G., Tiegs, G., and Wendel, A. (1994). Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-e~ requires transcriptional arrest. J. Immunol. 153, 1778-1788. Levine, W. G., Ord, M. G., and Stocken, L. A. (1977). Some biochemical changes associated with nafenopin-induced liver growth in the rat. Biochem. Pharmacol. 26, 933-942. Liebermann, M. W., Verbin, R. S., Landay M., Liang, H., Farber, E., Lee, T.-N., and Starr, R. (1970). A probable role for protein synthesis in intestinal epithelial cell damage induced in vivo by cytosine arabinoside, nitrogen mustard, or x-irradiation. Cancer Res. 30, 942-951. Lin, J. K., and Chou, C. K. (1992). In vitro apoptosis in the human hepatoma-cell line induced by transforming growth factor [31. Cancer Res. 52, 385-388. Lockshin, R. A., and Beaulaton, J. (1974). Programmed cell death. Cytochemical evidence for lysosomes during the noemal breakdown of the intersegmental muscles. J. Ultrastruct. Res. 46, 43-62. Lockshin, R. A., and Williams, C. M. (1965). Programmed cell death. I. Cytology of degeneration in the intersegmental muscles of the Pernyi silk moth. J. Insect. Physiol. 11, 123-133.

7. Apoptosis and Hepatocarcinogenesis

173

Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A., and Jacks, T. (1993). p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature (London) 362, 847-849. Luebeck, E. G., Moolgavkar, S. H., Buchmann, A., and Schwarz, M. (1991). Effects of polychlorinated biphenyls in rat liver: Quantitative analysis of enzyme-altered foci. Toxicol. Appl. Pharmacol. 111, 469-484. Luebeck, E. G., Grasl-Kraupp, B., Timmermann-Trosiener, I., Bursch, W., Schulte-Hermann, R., and Moolgavkar, S. H. (1995). Growth kinetics of enzyme altered liver foci in rats treated with phenobarbital or oL-hexachlorocyclohexane. Toxicol. Appl. Pharmacol. 130, 304-315. Mac Donald, H. R., and Lees, R. K. (1990). Programmed death of autoreactive thymocytes. Nature (London) 343, 642-644. McDonnell, T. J. (1993). Cell division versus cell death: A functional model of multistep neoplasia. Mol. Carcinog. 8, 209-213. McDonnell, T. J., and Korsmeyer, S. J. (1991). Progression from lymphoid hyperplasia to highgrade malignant lymphoma in mice transgenic for the t(14;18). Nature (London) 349, 254256. Marsman, D. S., and Popp, J. A. (1994). Biological potential of basophilic hepatocellular loci and hepatic adenoma induced by the peroxisome proliferator, Wy-14,643. Carcinogenesis 15, 111-117. Marsman, D. S., Goldsworthy, T. L., and Popp, J. A. (1992). Contrasting hepatocytic peroxisome proliferation, lipofuscin accumulation and cell turnover for the hepatocarcinogens Wy-14,643 and clofibric acid. Carcinogenesis 13, 1011-1017. Martin, S. J., and Cotter, T. G. (1991). Ultraviolet B irradiation of human leukemia HL-60 cells in vitro induces apoptosis. Int. J. Radiat. Biol. 59, 1001-1016. Mayol, X., Neal, G. E., Davies, R., Romero, A., and Domingo, J. (1992). Ethinyl estradiolinduced cell proliferation in rat liver. Involvement of specific populations of hepatocytes. Carcinogenesis 13, 2381-2388. Michna, H., Nishino, Y., Neef, G., McGuire, W. L., and Schneider, M. R. (1992). Progesterone antagonists: Tumor-inhibiting potential and mechanism of action. J. Steroid Biochem. Mol. Biol. 41, 339-348. Miyashita, T., and Reed, J. C. (1993). Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 81, 151-157. Moody, D. E., and Reddy, J. K. (1976). Morphometric analysis of the ultrastructural changes in rat liver induced by the peroxisome proliferator Sail 42-348. J. Cell Biol. 71, 768-780. Moolgavkar, S. H. (1986). Carcinogenesis modeling: From molecular biology to epidemiology. Am. Rev. Publ. Health 7, 151-169. Moolgavkar, S. H. (1989). Multistages models for cancer risk assessment. In "Biologically Based Models for Cancer Risk Assessment" (C. C. Travis, ed.), Vol. 159, pp. 9-20. Plenum, New York. Moolgavkar, S. H., and Knudson, A. G., Jr. (1981). Mutation and cancer: A model for human carcinogenesis. J. Natl. Cancer Inst. 66, 1037-1052. Moolgavkar, S. H., Luebeck, E. G., De Gunst, M., Port, R. E., and Schwarz, M. (1990). Quantitative analysis of enzyme-altered loci in rat hepatocarcinogenesis experiments I: Single agent regimen. Carcinogenesis 11, 1271-1278. Muakkassah-Kelly, S. E, Bieri, F., Waechter, F., Bentley, P., and Staeubli, W. (1987). Long-term maintenance of hepatocytes in primary culture in the presence of DMSO: Further characterization and effect of nafenopin, a peroxisome proliferator. Exp. Cell Res. 171, 37-51. Murakami, H., Sanderson, N. D., Nagy, P., Marino, P. A., Merlino, G., and Thorgeirsson, S. S. (1993). Transgenic mouse model for synergistic effects of nuclear oncogenes and growth factors in tumorigenesis: Interaction of c-myc and transforming growth factor oLin hepatic oncogenesis. Cancer Res. 53, 1719-1723. Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Misono, K., Ira, M.-J., and

174

Rolf Schulte-Hermann, Bettina Grasl-Kraupp, and Wilfried Bursch

Graham, R. M. (1994). Gh: A GTP-binding protein with transglutaminase activity and receptor signaling function. Science 264, 1593-1596. Nussbaum, M. (1901). Zur Riickbildung embryonaler Anlagen. Arch. Mikrosk. Anat. 57, 676-705. Oberhammer, F., Pavelka, M., Sharma, S., Tiefenbacher, R., Purchio, T. A., Bursch, W., and Schulte-Hermann, R. (1992). Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor-J31. Proc. Natl. Acad. Sci. U.S.A. 89, 5408-5412. Oberhammer, E, Bursch, W., Tiefenbacher, R., Fr6schl, G., Pavelka, M., Purchio, T., and Schulte-Hermann, R. (1993a). apoptosis is induced by transforming growth factor-J31 within 5 hours in regressing liver without significant fragmentation of the DNA. Hepatology (Baltimore) 18, 1238-1246. Oberhammer, E, Wilson, J. W., Dive, C., Morris, I. D., Hickman, J. A., Wakeling, A. E., Walker, P. R., and Sikorska, M. (1993b). Apoptotic death in epithelial cells: Cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679-3684. Oberhammer, E, Fritsch, G., Schmied, M., Pavelka, M., Printz, D., Purchio, T., Lassmann, H., and Schulte-Hermann, R. (1993c). Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease. J. Cell Sci. 104, 317-326. Oehm, A., Behrmann, I., Falk, W., Pawlita, M., Maier, G., Klast, C., Li-Weber, M., Richards, S., Dhein, J., Trauth, B. C., Ponstingl, H., and Krammer, P. H. (1992). Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. J. Biol. Chem. 267, 10709-10715. Ogasawara, J., Watanabe-Fukunaga, R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T., and Nagata, S. (1993). Lethal effect of the anti-Fas antibody in mice. Nature (London) 364, 806-809. Okuda, K. (1992). Hepatocellular carcinoma: Recent progress. Hepatology (Baltimore) 15, 948-963. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993). Bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programmed cell death. Cell (Cambridge, Mass.) 74, 609-619. Pascale, R., Simile, M. M., Ruggiu, M. E., Seddaiu, M. A., Satta, G., Sequenza, M. J., Daino, L., Vannini, M. G., Lai, P., and Feo, E (1991 ). Reversal by 5-azacytidine of the S-andenosyl-rmethionine-induced inhibition of the development of putative preneoplastic foci in rat liver carcinogenesis. Cancer Lett. 56, 259-265. Pascale, R. M., Marras, V., Simile, M. M., Daino, L., Pinna, G., Bennati, S., Carta, M., Seddaiu, M. A., Massarelli, G., and Feo, E (1992). Chemoprevention of rat liver carcinogenesis by S-adenosyl-L-methionine: A long-term study. Cancer Res. 52, 4979-4986. Piacentini, M., Autuori, E, Dini, L., Farrace, M. G., Ghibelli, L., Piredda, L., and Fesus, L. (1991). "Tissue" transglutaminase is specifically expressed in neonatal rat liver cells undergoing apoptosis upon epidermal growth factor-stimulation. Cell Tissue Res. 263, 227-235. Pitot, H. C., and Dragan, Y. P. (1994). The multistage nature of chemically induced hepatocarcinogenesis in the rat. Drug Metab. Rev. 26, 209-220. Pitot, H. C., and Sirica, A. E. (1980). The stages of initiation and promotion in hepatocarcinogenesis. Biochim. Biophys. Acta 605, 191-215. Ponchel, F., Puisieux, A., Tabone, E., Michot, P. J., Fr6schl, G., Morel, A. P., Fr~bourg, T., Fontani~re, B., Oberhammer, E, and Ozturk, M. (1994). Hepatocarcinoma-specific mutant p53-249ser induces mitotic activity but has no effect on transforming growth factor ~l-mediated apoptosis. Cancer Res. 54, 264-268. Ray, S. D., Sorge, C. L., Kamendulis, L. M., and Corcoran, G. B. (1992). Ca + +-activated DNA fragmentation and dimethylnitrosamine-induced hepatic necrosis: Effects of Ca ++-

7.

Apoptosis and Hepatocarcinogenesis

175

endonuclease and poly(ADP-ribose) polymerase inhibitors in mice. J. Pharmacol. Exp. Ther. 263, 387-394. Reddy, J. K., and Lalwani, N. D. (1983). Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. Crit. Rev. Toxicol. 12, 1-58. Roberts, A. B., Thompson, N. L., Heine, U., Flanders, C., and Sporn, M. B. (1988). Transforming growth factor-13: Possible roles in carcinogenesis. Br. ]. Cancer 57, 594-600. Rotello, R. J., Liebermann, R. C., Purchio, A., and Gerschenson, L. E. (1991). Coordinated regulation of apoptosis and cell proliferation by transforming growth factor 131 in cultured uterine epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 88, 3412-3415. Rotstein, J., Sarma, D. S. R., and Farber, E. (1986). Sequential alterations in growth control and cell dynamics of rat hepatocytes in early precancerous steps in hepatocarcinogenesis. Cancer Res. 46, 2377-2385. Ruoslahti, E., and Reed, J. C. (1994). Anchorage dependence, integrins and apoptosis. Cell (Cambridge, Mass.) 77, 477-478. Russell, W. E., Coffex, R. J., Jr., Ouellette, A. J., and Moses, H. L. (1988). Type 13transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat. Proc. Natl. Acad. Sci. U.S.A. 85, 5126-5130. Russo, I. H., and Russo, J. (1994). Role of hCG and inhibin in breast cancer (review). Int. J. Oncol. 4, 297-306. Sarafian, T. A., and Bredesen, D. E. (1994). Is apoptosis mediated by reactive oxygen species? Racliat. Res. 21, 1-8. Sarraf, C. E., and Bowen, I. D. (1988). Proportions of mitotic and apoptotic cells in a range of untreated experimental tumors. Cell Tissue Kinet. 21, 45-49. Satoh, K., Hatayama, I., Tateoka, N., Tamai K., Shimizu, T., Tatematsu, M., Ito, N., and Sato, K. (1989). Transient induction of single GST-P positive hepatocytes by DEN. Carcinogenesis 10, 2107-2111. Savill, J., Fadok, V., Henson, P., and Haslett, C. (1993). Phagocyte recognition of cell undergoing apoptosis. Immunol. Today 14, 131-136. Schaff, Z., Lapis, K., and Szendroi, M. (1984). D-galactosamine-induced liver injury in immunosuppressed mice. Acta Morphol. Hung. 32, 67-72. Schrek, B. (1949). Cytotoxic action of hormones of the adrenal cortex according to the method of unstained cell counts. Endocrinology (Baltimore) 45, 317-334. Schulte-Hermann, R. (1974). Induction of liver growth by xenobiotic compounds and other stimuli. Crit. Rev. Toxicol. 3, 97-158. Schulte-Hermann, R. (1979). Reactions of the liver to injury: Adaption. In "Treatise on Liver, Toxic Injury of the Liver" (E. Farber and M. M. Fisher, eds.), pp. 385-444. Dekker, New York. Schulte-Hermann, R. (1985). Tumor promotion in the liver. Arch. Toxicol. 57, 147-215. Schulte-Hermann, R., and Parzefall, W. (1981). Failure to discriminate initiation from promotion of liver tumors in a long-term study with the phenobarbital-type inducer oL-hexachlorocyclohexane and the role of sustained stimulation of hepatic growth and monooxygenases. Cancer Res. 41, 4140-4146. Schulte-Hermann, R., and Schmitz, E. (1980). Feedback inhibition of hepatic DNA synthesis. Cell Tissue Kinet. 13, 371-380. Schulte-Hermann, R., Koransky, W., Leberl, C., and Noack, G. (1971). Hyperplasia and hypertrophy of rat liver induced by o~-hexachlorcyclohexane and butylhydroxytoluene. Retention of the hyperplasia during involution of the enlarged organ. Virchows Arch. (Zellpathol.) 9, 125-134. Schulte-Hermann, R., Landgraf, H., and Koransky, W. (1977). Effect of hypophysectomy on the stimulation of liver growth by ot-hexachlorocyclohexane, phanobarbital, and partial hepatectomy in the rat. Naunyn-Schrniedeberg's Arch. Pharmacol. 298, 137-142.

176

Rolf Schulte-Hermann, Bettina Grasl-Kraupp, and Wilfried Bursch

Schulte-Hermann, R., Hoffmann, V., Parzefall, W., Kallenbach, M., Gerhardt, A., and Schuppier, J. (1980). Adaptive responses of rat liver to the gestagen and anti-androgen cyproterone acetate, other inducers. II, induction of growth. Chem.-Biol. Interact. 31, 287300. Schulte-Hermann, R., Ohde, G., Schuppler, J., and Timmermann-Trosiener, I. (1981). Enhanced proliferation of putative preneoplastic cells following treatment with the tumor promoters phenobarbital, hexachlorocyclohexane, steroid compounds and nafenopin. Cancer Res. 41, 2556-2562. Schulte-Hermann, R., Timmermann-Trosiener, I., and Schuppler, J. (1982). Response of liver foci in rats to hepatic tumor promoters. Toxicol. Pathol. 10, 63-70. Schulte-Hermann, R., Timmermann-Trosiener, I., and Schuppler, J. (1983). Promotion of spontaneous preneoplastic cells in rat liver as a possible explanation of tumor production by nonmutagenic compounds. Cancer Res. 43, 839-844. Schulte-Hermann, R., Timmermann-Trosiener, I., and Schuppler, J. (1986). Facilitated expression of adaptive responses to phenobarbital in putative pre-stages of liver cancer. Carcinogenesis 7, 1651-1655. Schulte-Hermann, R., Ochs, H., Bursch, W., and Parzefall, W. (1988). Quantitative structureactivity studies on effects of sixteen different steroids on growth and monoxygenases of rat liver. Cancer Res. 48, 2462-2468. Schulte-Hermann, R., Timmermann-Trosiener, I., Barthel, G., and Bursch, W. (1990). DNA synthesis, apoptosis and phenotypic expression as determinants of growth of altered foci in rat liver during phenobarbital promotion, Cancer Res. 50, 5127-5135. Schulte-Hermann, R., Bursch, W., Grasl-Kraupp, B., Huber W., and Parzefall W. (1994). Nongenotoxic carcinogenesis in the liver. In "Nongenotoxic Carcinogenesis" (A. Cockburn and L. Smith, eds.), pp. 109-120. Springer-Verlag, Berlin. Schwall, R. H., Robbins, K., Jardieu, P., Chang, L., Lai, C., and Terrell, T. G. (1993). Activin induces cell death in hepatocytes in vivo and in vitro. Hepatology (Baltimore) 18, 347-356. Schwartz, L. M., Smith, S. W., Jones, M. E. E., and Osborne, B. A. (1993). Do all programmed cell deaths occur via apoptosis? Proc. Natl. Acad. Sci. U.S.A. 90, 980-984. Schweichel J.-U., and Merker, H. J. (1973). The morphology of various types of cell death in prenatal tissues. Teratology 7, 253-266. Searle, J., Harmon B. V., Bishop, C. J., and Kerr, J. E R. (1987). The significance of cell death by apoptosis in hepatobiliary disease. J. Gastroentol. Hepatol. 2, 77-96. Shen, W., Kamendulis, L. M., Ray, S. D., and Corcoran, G. B. (1991). Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes correlation of nuclear Ca 2+ accumulation and early DNA fragmentation with cell death. Toxicol. Appl. Pharmacol. 111,242-254. Shi, L., Kraut, R. P., Aebersold, R., and Greenberg, A. H. (1992a). A natural killer cell granule protein that induces DNA fragmentation and apoptosis. J. Exp. Med. 175, 553-566. Shi, Y., Glynn, J. M., Guilbert, L. J., Cotter, T. G., Bissonette, R. P., and Green, D. R. (1992b). Role of c-myc in activation-induced apoptotic cell death in T cell hybridomas. Science 257, 212-214. Shirai, Y., Karwata, S., Tamura, S., Ito, N., Tsushima, H., Takaishi, K., Kiso, S., and Matsuzawa, Y. (1994). Plasma transforming growth factor-J31 in patients with hepatocellular carcinoma. Cancer 73, 2275-2279. Sills, R. C., Goldsworthy, T. L., and Sleight, S. D. (1994). Tumor-promoting effects of 2,3,7,8tetrachlorodibenzo-p-dioxin and phenobarbital in initiated weanling Sprague-Dawley rats: A quantitative, phenotypic, and ras p21 protein study. Toxicol. Pathol. 22, 270-275. Sklar, G. N., Eddy, H. A., Jacobs, S. C., and Kyprianou, N. (1993). Combined anti-tumor effect of suramin plus irradiation in human prostate cancer cells: The role of apoptosis. J. Urol. 150, 1526-1532. Smyene, R. J., Schilling, K., Robertson, L., Curran, T., and Morgan, J. I. (1992). Fos-LacZ

7.

Apoptosis and Hepatocarcinogenesis

177

transgenic mice: Mapping sites if gene induction in the central nervous system. Neuron 8, 13-23. Solt, D., and Farber, E. (1976). New principle for the analysis of chemical carcinogenesis. Nature (London) 263, 701-703. St/iubli, W., and Hess, R. (1975). Liprotein formation in the liver cell (ultrastructural and functional aspects relevant to hypolipidemic action). In "Handbook of Experimental Pharmacology, Hypolipidemic Agents" (D. Krichevsky, ed.), Vol. 41, pp. 229-289. SpringerVerlag, New York. Sun, X.-M., and Cohen, G. M. (1994). Mg 2+-dependent cleavage of DNA into kilobase pair fragments is responsible for the initial degradation of DNA in apoptosis. J. Biol. Chem. 269, 14857-14860. Szende, B., Zalatnai, A., and Schally, A. V. (1989). Programmed cell death (apoptosis) in pancreatic cancers of hamsters after treatment with analogs of both luteinizing hormonereleasing hormone and somatostatin. Proc. Natl. Acad. Sci. U.S.A. 83, 1643-1647. Tenniswood, M. P., Guenette, R. S., Latkins, J., Mooibroek, M., Wong, P., and Welsh, J.-E. (1992). Active cell death in hormone-dependent tissues. Cancer Metastasis Rev. 11, 197220. Trauth, B. C., Klas, C., Peters, A. M. J., Matzku, S., M611er, P., Falk, W., Debatin K., and Krammer, P. H. (1989). Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245, 301-305. Vancutsem, P. M., Lazarus, P., and Williams, G. M. (1994). Frequent and specific mutations of the rat p53 gene in hepatocarcinomas induced by tamoxifen. Cancer Res. 54, 3864-3867. Verbin, R. S., Diluiso, G., Liang, H., and Farber, E. (1972). The effects of cytosine arabinodide upon proliferating epithelial cells. Cancer Res. 12, 1476-1488. Walker, P. R., Smith, C., Youdale, T., Leblanc, J., Whitfield, J. E, and Sikorska, M. (1991). Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in immature thymocytes. Cancer Res. 51, 1078-1085. Walker, P. R., Weaver, V. M., Lach, B., LeBlanc, J., and Sikorska, M. (1994). Endonuclease activities associated with high molecular weight and internucleosomal DNA fragmentation in apoptosis. Exp. Cell Res. 213, 100-106. Ward, J. M. (1983). Increased susceptibility of livers of aged F344/NCr rats to the effects of phenobarbital on the incidence, morphology, and histochemistry of hepatocellular loci and neoplasms. J. Natl. Cancer Inst. 71, 815-823. W~irri, A. M., Huovinen, R. L., Laine, A. M., Martikainen, P. M., and H~irk6nen, P. L. (1993). Apoptosis in toremifene-induced growth inhibition of human breast cancer cells in vivo and in vitro. J. Natl. Cancer Inst. 17, 1412-1418. Weiler, E. (1956). Die )knderung der serologischen Spezifit/it yon Leberzellen der Ratte w~ihrend der Cancerogenese durch p-Dimethylaminoazobenzol. Z. Naturforsch. 11B, 31-37. Williams, G. T., and Smith, C. A. (1993). Molecular regulation of apoptosis: Genetic controls on cell death. Cell (Cambridge, Mass.) 74, 777-779. Wyllie, A. H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature (London) 284, 555-556. Wyllie, A. H. (1985). The biology of cell death in tumours. Anticancer Res. 5, 131-136. Wyllie, A. H. (1987). Apoptosis: Cell death under homeostatic control. Mech. Methods Toxicol. 11, 3-10. Wyllie, A. H., Kerr, J. E R., and Currie, A. R. (1980). Cell death: The significance of apoptosis. Int. Rev. Cytol. 68, 251-300. Ying, S.-Y. (1989). Inhibins, activins and follistatins. J. Steroid Biochem. 33, 705-713. Yonish-Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A., and Oren, M. (1991). Wildtype p53 induces apoptosis of myeloid leukemia cells that is inhibited by interleukin-6. Nature (London) 352, 345-347.

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Rolf Schulte-Hermann, Bettina Grasl-Kraupp, and Wilfried Bursch

Yoshida, A., Ueda, T., Wano, Y., and Nakamura, T. (1993). DNA damage and cell killing by camptothecin and ist derivative in human leukemia HL-60 cells. Jpn. J. Cancer Res. 84, 566-573. Zakeri, Z. E, Quaglino, D., Latham, T., and Lockshin, R. A. (1993). Delayed internucleosomal DNA fragmentation in programmed cell death. FASEB J. 7, 470-478.

8 Liver Tumor Promotion and the Suppression of p53-Dependent Cell Cycle Checkpoint Function Yingchun Zhang Chia Chiao Laura L. Byrd David G. Kaufman William K. Kaufmann Department of Pathology and Laboratory Medicine,and Lineberger ComprehensiveCancerCenter University of North Carolinaat ChapelHill Chapel Hill, North Carolina27599

I. Introduction The natural history of cancer development in a variety of human tissues is characterized by malignant neoplasms that progress from benign precursor lesions by accumulating a series of genetic and epigenetic changes (Boone et al., 1992). The transition of mucociliary columnar epithelium from metaplasic and dysplasic states to carcinoma in situ before the emergence of squamous carcinoma is apparent in both cervical and bronchial carcinogenesis. Similar transitions in neoplastic evolution have been described in in vitro model systems where carcinogen exposure initiates extended-lifespan/ enhanced growth variants (EL/EGVs). Such EL/EGVs continue to proliferate in vitro under conditions in which normal cells growth arrest and either senesce or degenerate. They are also precursors to immortal cell lines with indefinite proliferative lifespans (Newbold, 1985; Barrett et al., 1987; Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Nettesheim and Barrett, 1984). However, when transplanted into a tissue matrix, chemically initiated or oncogene-transformed EL/EGVs and immortal cell lines display dysplastic not malignant growth (Baba et al., 1987; Nettesheim and Barrett, 1984; Sun et al., 1992). Further progression of immortal cell lines to cancer requires inactivation of tumor suppressor genes, activation of proto-oncogenes, or acquisition of autocrine growth stimulation (Barrett et al., 1987; Lee et al., 1991; Newbold, 1985; Nettesheim and Barrett, 1984). In this review, we discuss our recent findings from an in vitro liver carcinogenesis model system that suggest the early stages of hepatocyte transformation are associated with a tumor promoterdependent inactivation of cell cycle growth control pathways.

II. Mechanisms of Cell Cycle Control The regenerating rat liver has proved to be an excellent model system to study the mechanisms of growth control within a natural tissue environment (see Chapters 1 to 4). Following the surgical resection of the median and left lateral lobes, cells in the remaining right lateral and caudate lobes undergo a burst of cell proliferation that restores the mass of lost tissue within a week. This compensatory growth process is tightly controlled with the hepatic cells returning to their original state of replicative quiescence following liver mass restoration. We already know much about the humoral and cellular factors involved in stimulating hepatocytes to enter the cell division cycle after partial hepatectomy (Michalopoulos, 1990). We are now also learning more about the patterns of gene expression that accompany the movement of hepatocytes into and through the cell cycle (see Chapter 4) (Thompson et al., 1986; Fausto, 1991). Studies of cell cycle control in yeast, amphibian, and mammalian cells have identified a large set of gene products that propel cells through the various stages of the cell cycle. These cell cycle regulatory gene products are known as cyclin-dependent kinases (cdks) (Reed, 1992; Sherr, 1994). Cdks are composed of an enzymatic subunit that possesses protein kinase activity and a regulatory component that is essential for kinase activity. This regulatory component usually undergoes a wave of expression through the cell cycle in which protein levels rise and then fall. Proteins in this family are, therefore, known as cyclins. The activities of cdks also rise and fall through the cell cycle under the control of the cyclins. The principal cyclindependent kinase complexes that control the cell cycle in human fibroblasts are shown in Figure 1. Progression through G 1 appears to be the responsibility of two cdks, cyclin D1/cdk4 and cyclin E/cdk2. Both kinase complexes can phosphorylate the retinoblastoma susceptibility gene product, RB, thereby releasing the transcriptional enhancer, E2F, that activates

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Figure I Cyclin-dependent kinase activity during the cell cycle.

downstream genes in the cell cycle such as c-myc, dihydrofolate reductase, D N A polymerase alpha, and cdkl (see Xu et al., 1994 for references). The cyclin D1/cdk4 complex appears early in G 1 while the cyclin E/cdk2 complex reaches a maximal level at or near the G1/S border where RB phosphorylation occurs. Cyclin A/cdk2 activity rises as cells enter the S-phase and continues to increase throughout S and into G 2. Activation of cyclin A/cdk2 depends on the activation of cyclin E/cdk2 (Dulic et al., 1994). Movement from Gz into mitosis is controlled by cyclin B1/cdkl, which when activated can induce all the initial steps in mitosis such as nuclear envelope breakdown, chromatin condensation, microtubule assembly, and alignment of chromosomes on the metaphase plate (King et al., 1994; Norbury et al., 1991). Cyclin B1/cdkl activity rises explosively at the G2/M transition, then falls rapidly as cyclin B1 is degraded after metaphase. Dephosphorylation of RB occurs at the end of mitosis, thereby reestablishing negative control over newly synthesized E2E

III. Cell Cycle Checkpoints, Lifespan Extension, and Genetic Instability Proliferating cells that are being propelled by these cdks respond to DNA damage by delaying progression through the cell cycle (Dulic et al., 1994; Kastan et al., 1992; Kaufmann et al., 1991; Kaufmann and Wilson, 1994). The advantage of delaying cycle phase transitions is that the cell obtains additional time for DNA repair systems to remove potentially toxic, muta-

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genic and clastogenic DNA lesions before they are fixed by DNA replication and mitosis (Kaufmann and Wilson, 1994, Kaufmann and Kaufman, 1993). Cells from patients with the familial cancer syndrome, ataxia telangiectasia (AT), display a reduced ability to express cell cycle delays in response to DNA damage (Dulic et al., 1994; Kastan et al., 1992, Painter and Young, 1980; Zampetti-Bosseler and Scott, 1981 ). Therefore, AT cells are hypersensitive to the lethal effects of certain forms of DNA damage such as those produced by ionizing radiation and chemotherapeutic drugs. AT cells are also genetically unstable and display increased frequencies of spontaneous and carcinogen-induced chromosomal aberrations (Kaufmann and Wilson, 1994, Zampetti-Bosseler and Scott, 1981). The inability to delay cell cycle progression in response to DNA damage is associated with enhanced sensitivity to genotoxic agents, genetic instability, and cancer in AT patients (Cohen and Levy, 1989). DNA damage appears to arrest cell cycle progression within proliferating cells by inhibiting the activities of the cdks that propel the cell cycle. When normal human fibroblasts in the G 1 phase of the cell cycle are irradiated with ionizing radiation, they growth arrest and delay entry into the S-phase (Dulic et al., 1994; E1-Deiry et al., 1994). Recently, we and others have shown that radiation-induced G 1 arrest can be attributed to the induction, via a p53-dependent signal transduction pathway, of an inhibitor of cyclindependent kinases known as p 2 ] W a f l / C i p 1/Sdil (Dulic et al., 1994; E1-Deiry et al., 1994). When induced in G 1 this inhibitor binds to and inhibits cyclin E/cdk2 which mediates the G1/S transition (Reed, 1992; Dulic et al., 1992). G2-delay is another checkpoint response to DNA damage (ZampettiBosseler and Scott, 1981; O'Connor et al., 1992). By delaying entry into mitosis, cells obtain additional time to repair preclastogenic lesions before they are fixed by mitosis. Studies with human lymphocytes have shown that G 2 cells which fail to delay entry into mitosis express the greatest frequencies of chromosomal aberrations (Olivieri and Micheli, 1983). Accordingly, AT cells that express little delay in G 2 display higher frequencies of chromosomal aberrations than do normal cells (Zampetti-Bosseler and Scott, 1981 ). G2-arrest has been associated with alterations in the activity of cyclin B1/cdkl kinase due to the presence of inhibitory phosphates on amino acid residues threonine 14 and tyrosine 15 in the ATP-binding domain of the enzyme (Norbury et al., 1991; O'Connor et al., 1992). Thus, posttranslational modification of this cyclin/kinase complex seems to be involved in controlling the G 2 checkpoint response to DNA damage. Recent observations suggest a connection between inactivation of checkpoint controls and the acquisition of an extended proliferative lifespan and immortality in human fibroblasts (Dulic et al., 1993, 1994; Shay et al., 1993). The biochemistry of senescence arrest at the end of the cellular proliferative lifespan in vitro (Dulic et al., 1993) is quite similar to that of radiation-induced G 1 arrest (Dulic et al., 1994). Under both conditions of

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growth arrest, cells accumulate high levels of cyclin E/cdk2 kinase complexes, which are inactive due to the presence of the cyclin-dependentkinase inhibitor, p21Wan/Cip l/sdil (Noda et al., 1994). Inactivation of p53 function (by viral oncoproteins or by mutation and deletion of p53 alleles) prevents the induction of p21Wafl/Cip 1/sdil, abrogates G 1 arrest, and extends the in vitro proliferative lifespan as an early event in transformation. Evidence suggests that lifespan control in human fibroblasts is linked to the length of the telomeric sequences that cap the ends of chromosomes (Harley et al., 1992; Greider, 1991). Our working hypothesis is that normal senescence arrest of cell proliferation is triggered by the loss of the telomere structure at the end of one or more chromosomes. The chromosome end that lacks a telomere resembles one half of a double-strand break. DNA single- and double-strand breaks activate G 1 arrest that may persist until the breaks are rejoined. Since the half-double-strand break at the end of a chromosome without a telomere has no natural DNA to rejoin, the signal to arrest growth is persistent. Inactivation of p53 permits extension of lifespan presumably by preventing the induction of the growth inhibitory protein, p21Wafl/Cip 1/Sdil. There is also evidence that the attenuation or loss of cell cycle checkpoint response results in genetic instability (Yin et al., 1992; Livingstone et al., 1992). For example, diploid nontransformed human and mouse fibroblasts are unable to amplify the C A D gene to acquire resistance to the DNA metabolic poison N-(phosphonacetyl)-L-aspartate (Tlsty, 1990). However, loss of p53 function and G 1 arrest is permissive for C A D gene amplification in diploid human and mouse fibroblasts (Yin et al., 1992; Livingstone et al., 1992; White et al., 1994). Genetic instability in transformed cells may not be limited to defects in the G 1 checkpoint. Recently, Sanford et al. (1992) showed that human breast epithelial cells acquire a condition of G2 chromosomal hypersensitivity at early stages of transformation, and we have shown that G 2 checkpoint function is attenuated in extended-lifespan and immortal human fibroblasts (Kaufmann et al., 1995; Paules et al., 1995). Because the G 2 checkpoint arrests the progression into mitosis of cells with broken chromatids, its attenuation will provide transformed cells a growth advantage since they can proliferate with levels of chromatid damage that arrest the growth of normal cells. Therefore, defects in both the G 1 and G2 checkpoint functions may contribute to the genetic instability that characterizes tumor progression.

IV. Isolation of EL/EGV Hepatocytes and Promotion of Hepatocarcinogenesis in Vitro Previous studies with rodent fibroblasts in culture have suggested that malignant transformation may require at least two phenotypic alterations:

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immortalization and anchorage independence (Newbold, 1985; Barrett et al., 1987). Thus, it might be possible to isolate immortal rat hepatocytes soon after a carcinogen treatment regimen that initiates hepatocarcinogenesis. Accordingly, rats were treated with a single dose of the methylating agent, methyl(acetoxymethyl)nitrosamine (DMN-OAc), during the peak of hepatocyte DNA synthesis that follows a partial hepatectomy. When these carcinogen-treated animals were fed a diet containing the liver tumor promoter, phenobarbital (PB), hepatocellular adenomas and carcinomas began to emerge after 25 weeks, and by one year, the livers contained an average of 5 neoplasms (Kaufmann et al., 1985). Carcinogen-treated animals that were not fed PB developed fewer liver neoplasms; 11 to 16 % of the number seen in the PB-promoted animals. Thus, the single treatment of rats with DMNOAc initiated a population of hepatocytes, but most did not develop into tumors in the absence of PB treatment. To determine whether any of these initiated rat hepatocytes were immortal and capable of extended growth in vitro, hepatocytes were isolated from chemically initiated rat livers 2 to 60 weeks after carcinogen treatment and cultured in medium containing 10% serum with 2 mM PB. Hepatocytes from noninitiated control livers generally growth arrested and degenerated. Thus, proliferative hepatocyte colonies were rarely seen (frequency of 10-7), and the cultures were ultimately replaced by an outgrowth of fibroblasts and biliary ductular epithelial cells. Hepatocytes that were isolated as early as 2 weeks after initiation of hepatocarcinogenesis with DMN-OAc or benzo(a)pyrene diolepoxide I had a 10- to 300-fold increase in colony forming frequency (Kaufmann et al., 1986, 1988). Optimal conditions for hepatocyte EL/EGV colony formation required the presence of PB in the cell culture medium. It is now thought that these proliferative hepatocyte colonies are biologically equivalent to the EL/EGVs described in other cell transformation systems. If these EL/EGVs represent a stage of neoplastic evolution, they should express onco-developmental markers such as gamma-glutamyltranspeptidase (GGT) which typifies preneoplastic hepatocyte lineages. In fact, rat hepatocyte EL/EGVs do express GGT (Kaufmann et al., 1986; Kitagawa et al., 1980), suggesting that these cells are similar to the hepatocytes in hepatocellular foci that are the precursors to liver adenomas and carcinomas (Kaufmann et al., 1992). Although hepatocyte EL/EGVs are readily isolated from initiated livers and secondary cultures can be performed for several passages, the cells are not immortal. This implies that immortality is not induced during initiation. Rather, the initiation stage of hepatocarcinogenesis generates hepatocytes that display enhanced growth or extended proliferative lifespan in the presence of PB, but that require additional alterations in growth control to become immortal. The frequencies of hepatocyte EL/EGVs in initiated rat livers varies con-

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siderably from one preparation to the next, and it was thought that hepatocyte EL/EGVs could be recovered more consistently by improving the cell culture conditions. As a first step in enhancing the culture conditions, cell feeder layers were employed since hepatocyte EL/EGVs were frequently observed to develop when in contact with contaminating hepatic biliary ductular epithelial cells. Furthermore, other investigators have shown that such epithelial cells support the expression of hepatocyte phenotypic markers in cocultures (Foliot et al., 1985). Therefore, whether epithelial cells would provide a more fertile substrate upon which hepatocyte EL/EGVs might grow was tested (Chiao et al., 1995a). Hepatocytes were isolated from livers 5 or 7 weeks after treating rats with DMN-OAc and seeded into plastic culture dishes or onto confluent monolayers of radiation-sterilized hepatic epithelial cells. While low frequencies of hepatocyte EL/EGVs developed when the isolates were seeded into plastic dishes, exuberant hepatocyte growth occurred on the feeder layers. By 3 months after seeding onto feeder layers, the dishes were confluent with hepatocytes. We then concentrated on subculturing the EL/EGVs to try to establish cell lines. The confluent dishes were treated with collagenase to release the hepatocytes. These were subcultured into plastic dishes in a medium containing PB. Under these conditions normal hepatocytes die and are eliminated from cultures. Proliferative hepatocyte EL/EGVs were identified in the secondary cultures along with fibroblasts that also colonized the feeder layer. Successive subculturing was done on two independently derived lines of hepatocytes (designated 6/15 and 6/27) for more than 36 months; some cultures now have been passaged over 100 times. These lines retain the morphological features of hepatocytes with bile canaliculi and polyploidy. They also express the onco-developmental marker of hepatocyte transformation, glutathione S-transferase-placental form (GST-P), while retaining expression of albumin (Figure 2). When tested at passage 55 (after 2.5 years in culture), growth of both lines of hepatocytes stopped when PB was withdrawn from the culture medium. Thus, propagation of these cell lines in secondary culture requires PB. The immortal 6/15 and 6/27 lines were tested for tumorigenicity by transplantation into the livers of syngeneic rats. At early and intermediate passages (12 to 56), both lines were nontumorigenic. At passage 85 the 6/15 line formed multiple hepatocellular carcinomas when transplanted into the liver, whereas the 6/27 line at a similar passage level continued to be nontumorigenic. Furthermore, the 6/15 line formed carcinomas in the livers of animals that were not fed PB suggesting that some of the hepatocytes had become PB-independent. To test this postulate, we isolated a number of clones of the 6/15 line and evaluated the PB-dependent colony formation. While most of the clones still showed moderate to substantial stimulation of colony formation by PB, the growth of a few clones was unaffected by PB.

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Figure 2 Northern hybridization analysis of albumin and glutathione S-transferase (placental isoform) (GST-P) gene expression in hepatocyte extended-lifespan/enhanced-growthvariants (EL/EGVs). mRNAwas isolated from normal rat hepatocytes, 6/27 hepatocytes, 6/15 hepatocytes, and a rat hepatocellular carcinoma line, RLE-57.

These findings suggest that the tumorigenic 6/15 hepatocytes correspond to the PB-independent cells cloned from the parental population. These studies demonstrate that the liver tumor promoter, PB, greatly enhances the in vitro growth of chemically initiated hepatocyte EL/EGVs. During propagation in culture, two independent hepatocyte lines progressed to immortality. One immortal, nontumorigenic line (6/27) retained strict dependence on PB for growth; clonal expansion was inhibited by more than 95% after withdrawal of PB. The other line (6/15) progressed further to PBindependence and tumorigenicity. These observations suggest that the EL/ EGV hepatocytes lie on a lineage pathway to hepatocellular carcinoma. Thus, this may be a valuable in vitro model system for investigating the molecular mechanisms involved in liver tumor promotion with PB.

V. Immortal Rat Hepatocytes Require PB for Clonal Expansion To investigate the altered growth of these immortal hepatocytes further, we isolated a clone from the 6/27 cell line (6/27C1) and examined its sensitivity to various concentrations of PB (Figure 3). Colony formation by the 6/27C1 hepatocytes displays a strict dependence on PB, and in its absence colony formation efficiency is reduced by 99%. PB at 1 mM provides half-maximal support while 2 to 3 mM is optimal for growth; PB at 4 mM inhibits colony growth. Transforming growth factor beta (TGFI3) has been implicated as a negative regulator of normal hepatocyte growth during promotion of hepa-

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Figure 3 Transforming growth factor [3 (TGF[3) inhibits clonal expansion of PB-dependent 6/27C1 hepatocytes. Hepatocytes were seeded at low cell density in medium containing 2 mM phenobarbital (PB). After overnight incubation for cell attachment, the medium was changed to include PB at various concentrations or to add TGF[3 at various concentrations. After 10 days growth, colonies were fixed, stained, and counted. Panel A shows colony formation in the absence and presence of PB, and with 1 ng/ml of added TGF[3. Panel B shows the PB concentration-response. Panel C shows the TGF[3 concentration-response.

tocarcinogenesis with PB (Jirtle and Meyer, 1991). TGF[3 also inhibits colony formation by the 6/27C1 line in a concentration-dependent manner (Figure 3). Colony-formation efficiency is reduced by 75% when hepatocytes are incubated with 3 ng/ml of TGF[3 in medium containing PB. Thus, the immortal 6/27 hepatocyte cell line retains sensitivity to the growthinhibitory effects of TGF[3, although the sensitivity appears to be somewhat less than that for normal hepatocytes (Jirtle and Meyer, 1991). When PB is withdrawn from the culture medium, growth of the 6/27C1 hepatocyte line is slowed but not completely inhibited. After 4 days of this slowed growth, dead cells that are permeable to trypan-blue dye begin to appear. Twelve days after PB withdrawal, the percentage of dead cells increased by about seven-fold over the low level observed in PB-supplemented medium. In serum-supplemented medium, death of hepatocytes in the absence of PB is by cytoplasmic degeneration and membrane lysis, not apoptosis (Chiao et al., 1995a). Death of hepatocytes after withdrawal of PB resembles the death of SV40-transformed human fibroblasts in crisis (Stein, 1985). At the end of their extended lifespan, during the phase known as crisis, trypan-blue positive fibroblasts are released from dishes even as the viable attached cells continue to synthesize DNA. In contrast, the PB-dependent 6/27C1 hepatocytes die by apoptosis when cell growth is arrested by incubation in low-serum (0.5 %) medium. (Chiao et al., 1995b). When PB-supplemented cells are deprived of serum, cell growth also slows, the fraction of mitotic cells decreases by 75 %, and after 4 days, hepatocytes begin to undergo apoptosis. We confirmed the apoptotic

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mode of cell death by electron microscopic demonstration of nuclear chromatin condensation and cytoplasmic blebbing; chromatin was found to fragment into nucleosomal oligomers. In rat fibroblasts that overexpress c-Myc, serum deprivation also induces apoptosis (Evan et al., 1992). This result and similar results in other systems suggest that the continued expression of c-Myc, as growth-factor-deprived cells attempt to withdraw from the cell cycle, generates conflicting signals that trigger apoptosis. The 6/27C1 hepatocytes also continue to express c-Myc after serum deprivation suggesting that a similar mechanism of conflicting signals may account for the apoptotic death of these rat hepatocytes. These data suggest that PB may condition the growth of the immortal 6/27C1 hepatocytes by interacting with pathways that control cell viability. Based on these observations, we postulated that promoting agents might

Figure 4 Northern hybridization analysis of albumin, glutathione S-transferase (placental isoform) (GSToP),transforming growth factor ot (TGFoL),and p53 gene expression, mRNA was isolated from a Ras-transformed rat hepatic epithelial cell line (R1), freshly isolated rat hepatocytes from quiescent liver, and 6/27 and RLE-57 cell lines. Four different clones of 6/27 were studied (C1, C2, C3, and C5). Cells were harvested for isolation of mRNA when in logarithmic growth phase (L) and after reaching confluence.

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affect tumor suppressor and/or growth factor gene expression. Thus, we determined the expression of p53 and transforming growth factor alpha (TGFcx) genes in 6/27 hepatocytes growing in the presence of PB. Northern hybridization analysis revealed that the expression of p53 mRNA was growth regulated (Figure 4). When two clones of 6/27 hepatocytes (i.e., C2, C3) and the RLE-57 hepatocellular carcinoma line were grown to confluence, the expression of p53 decreased. Expression of TGFc~ mRNA in the 6/27 cell lines was undetectable by Northern analysis. Expression of GST-P was detectable but was not found to be affected by growth to confluence. When 6/27C1 PB-dependent cells in log-phase growth (where the requirement for PB is greatest) were deprived of PB, the expression of TGFcx was suppressed (Figure 5A). In contrast, the signal for p53 mRNA increased substantially when PB was withdrawn (Figures 5A, B). Expression of J3-actin was unaffected by the withdrawal of PB from the medium. In a PBindependent line of 6/27C1, which was selected for growth in the absence of PB, the presence of PB did not affect the level of p53 mRNA (Figure 5B). These results suggest that PB may condition the growth of immortal hepatocytes by inhibiting expression of p53. Inhibition of p53 is expected to inactivate the G1 cell cycle checkpoint control and to allow initiated hepatocytes

Figure 5 PB-responsive p53 and TGFoL gene expression in 6/27 hepatocytes. (A) Reverse transcription-polymerase chain reaction analysis (RT-PCR) of p53, transforming growth factor e~ (TGFcx), and J3-actin mRNAs in PB-dependent hepatocytes grown in the presence or absence of PB. Whole cell lysates were incubated with reverse transcriptase to make cDNA from mRNA. Oligonucleotide primers that hybridize to defined regions of these genes were then added and PCR was used to test for the presence of the gene sequence. (B) Northern hybridization analysis of p53 mRNA. The PB-dependent 6/27C1 line and a PB-independent derivative line (PBI) were incubated for 8 days in medium in the presence or absence of PB, then RNA was extracted and mRNA isolated, mRNAs from freshly isolated normal hepatocytes and from the RLE-57 hepatocellular carcinoma line were also examined. After probing for p53 using a radiolabeled amplimer from panel A, the filter was stripped and probed for [3-actin.

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Figure 6 Radiation-induced G 1 arrest. Log-phase cultures of normal human fibroblasts (A,B), immortal Li-Fraumeni fibroblasts (C,D) and 6/27C1 rat hepatocytes (E,F) were sham-treated (A,C,E) or irradiated with 3 Gy of gamma rays (B,D,F). After 6 hr, 5-bromodeoxyuridine (BrdU) was added to the medium for 2 hr to label DNA in S-phase cells. Nuclei were isolated, incubated with fluoresceinated anti-BrdU antibody, and analyzed by flow cytometry for DNA content and incorporation of BrdU. DNA content is measured on the X axis; BrdU labeling on the Y axis.

g r o w u n d e r c o n d i t i o n s t h a t suppress g r o w t h of n o r m a l hepatocytes. O u r results suggest t h a t PB reduces cell cycle c o n t r o l , at least in the PBd e p e n d e n t 6 / 2 7 C 1 line. To assess cell cycle c h e c k p o i n t f u n c t i o n in the P B - d e p e n d e n t 6 / 2 7 C 1 line of i m m o r t a l h e p a t o c y t e s , G1 c h e c k p o i n t f u n c t i o n w a s assayed using a flow to

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cytometric method, in which a short 2-hr incubation with 5-bromodeoxyuridine (BrdU) was used to label cells in S-phase (Figure 6). Prior irradiation of normal human fibroblasts (F1) with 3 Gy of gamma rays reduced the fraction of cells in the first half of the S-phase (2N-3N DNA content) by 90%. This loss of cells from the first half of S-phase is due to G 1 arrest which stops progression from G 1 into the S-phase. Cells that were in the first half of S at the time of irradiation continued to synthesize DNA and thereby progressed into the second half of the S-phase (3N-4N DNA content). Irradiation of immortal Li-Fraumeni cells (MDAH087) that express only a mutant form of p53 had no effect on the fraction of cells in the first half of the S-phase. These cells had previously been shown to lack G 1 checkpoint function (Dulic et al., 1994). Application of this assay method to the PBdependent 6/27C1 line suggests that these cells also lack a functional G 1 checkpoint. Cell cycle checkpoint responses appear to occur in normal rat hepatocytes both in vivo and in vitro. For example, gamma-irradiation of proliferating hepatocytes during the prereplicative phase of liver regeneration produces a dramatic delay in the initiation of DNA synthesis (G 1 growth arrest) (Kaufmann et al., 1987). A recent study with primary cultures of rat hepatocytes demonstrates that the addition of an oligomer of antisense p53 RNA suppresses expression of p53 and inhibits uv-radiation-induced G 1 arrest (Tsuji and Ogawa, 1994). Finally, mouse hepatic epithelial cells lose uv-induced G1 arrest after transformation with the H-ras oncogene (Kadohama et al., 1994). Therefore, it appears that hepatocytes and other hepatic epithelial cells are proficient in cell cycle checkpoint response to DNA damage, and that elements of checkpoint response are lost or attenuated during carcinogenesis (Hunter and Pines, 1994).

VI. Mechanisms of Promotion of Hepatocarcinogenesis by Phenobarbital Peraino et al. (1971) first showed that PB is a liver tumor promoter in the rat. We showed that inclusion of PB in the diet increased by six- to nine-fold the number of liver tumors that developed following a single treatment with a chemical carcinogen (Kaufmann et al., 1985). Thus, PB promotes the development of liver tumors from initiated cells that would otherwise remain latent. Jirtle et al. (1994) have shown that PB selectively increases the expression of the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r) in normal hepatocytes relative to its expression in initiated cells. Therefore, since the M6P/IGF2r binds latent TGF]3 and facilitates its activation (Dennis and Rifkin, 1991), it-was postulated that PB-exposed initiated hepatocytes gain a growth advantage because of their relative inability to activate TGFI3. Bursch et al. (1984) have shown that PB inhibits

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apoptosis in initiated hepatocytes in vivo. They proposed that this suppression of apoptosis leads to the accelerated growth of the initiated foci (see Chapter 7). With an increased growth rate, foci would progress to malignancy more rapidly, thereby accounting for tumor promotion. These two proposed mechanisms of liver tumor promotion need not, however, be mutually exclusive since PB may mediate both the selective inhibition of growth in normal cells and apoptosis in initiated cells through TGF~-dependent pathways (see Chapters 7 and 9). The PB-dependent immortal rat hepatocytes share a property with c-Myctransformed rat fibroblasts, i.e., induction of apoptosis during incubation in low-serum medium. Recent studies indicate that a similar process occurs in rat fibroblasts that are transformed with adenovirus E1A to inactivate the function of RB and other suppressor proteins (Lowe and Ruley, 1993; Debbas and White, 1993). In these cases, p53 participates in the cell death, as inactivation of p53 in EIA-transformed cells prevents apoptosis. A recent report has extended this result to show that the induction of apoptosis in serum-starved c-Myc-transformed mouse fibroblasts also requires p53 (Hermeking and Eick, 1994). Myc-transformed fibroblasts from p53 - / - "knockout" mouse embryos do not undergo apoptosis when deprived of serum whereas Myc-transformed wildtype fibroblasts (p53 +/+) enter apoptosis when serum-starved. These data implicate p53 in the cellular signaling pathways that induce apoptosis in oncogene-transformed cells that experience growth-arresting signals. TGFI3 is implicated in hepatocarcinogenesis both as an inhibitor of normal hepatocyte proliferation and as an inducer or enhancer of apoptosis (Oberhammer et al., 1992). TGFI3 induces G 1 arrest in mink epithelial cells apparently by increasing the available pool of a cdk inhibitor, p27Kip I (Polyak et al., 1994). p27KipI has also been found to block cdk activity in contact-inhibited cells and in serum-starved fibroblasts, p27Kip 1 is related to p21Waf1/Cip 1/Sdil by conservation of a domain that interacts with cdks (Toyoshima and Hunter, 1994), so it is possible that these two cdk inhibitors may share some functions (Peter and Herskowitz, 1994). Induction of p21Waf1/Cip 1/sdil is associated with G~ arrest in some cells and with apoptosis in others (EI-Deiry, 1994), and the transactivator of p21Wafl/Cip1/Sdil, p53, appears to mediate the induction of apoptosis in serum-starved myctransformed cells. PB may inhibit apoptosis by initiated hepatocytes by disconnecting a signal transduction pathway that links TGFI3 or other growth-arresting signals to the expression of cdk inhibitors that trigger apoptosis or by directly reducing the ability of initiated cells to either activate (Jirtle et al., 1994) or respond to (Reisenbichler et al., 1994) TGFf3 (see Chapter 9). Chemically initiated rat liver foci display enhanced rates of cell proliferation as though carcinogen-induced mutations alter the program of cell

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growth control (Schulte-Hermann et al., 1990). Enhanced rates of cell division occur in foci as growth of the normal surrounding hepatocytes is arrested. It is conceivable that initiated cells experience a conflict between the internal compulsion to divide and external signals to arrest growth, thereby triggering apoptosis. Genetic alterations that could enhance growth include mutations affecting growth factor responsiveness, regulation of c-myc expression, or tumor suppressor gene inactivation. External signals to arrest growth include TGFI3, cell contact, or limiting supply of growth factors. If p53 were to participate in a pathway whereby TGFI3 induces apoptosis in initiated hepatocytes, then the suppression of p53 expression would make the hepatocytes resistant to TGFf3 and inhibit apoptosis. Because PB-induced suppression of p53 expression could also induce a condition of genetic instability by inactivation of the G1 checkpoint, the enhanced clonal expansion of hepatocytes in GST-P positive initiated foci would favor their progression to more advanced stages of transformation, such as GST-P positive, TGFa-positive foci, and neoplasms (Kaufmann et al., 1992). Based on the proposition that PB alters cell cycle checkpoint controls in initiated cells so as to extend lifespan, inhibit apoptosis, and induce genetic instability, one can imagine the development of drugs that would serve as promoter antagonists to reassert a limitation on cellular lifespan, induce apoptosis, and preserve genetic stability in initiated cells. Aspirin has been shown to block promotion of hepatocarcinogenesis by PB (Denda et al., 1989). This supports a view that drug therapy with promoter antagonists may be useful for suppressing the early stages of carcinogenesis. The current clinical trial with tamoxifen appears to represent an analogous approach to block the effects of estrogen on mammary carcinogenesis. Finally, the monoterpene, perillyl alcohol, has been shown recently to significantly reduce the growth of liver tumors by increasing the apoptotic frequency possibly through a TGFJ3-dependent pathway (Mills et al., 1995). These novel agents not only may be effective for treating and preventing cancer, but may also be useful chemical tools for further elucidating the molecular mechanisms of liver tumor promotion. VII. Conclusions In a two-stage model of chemical hepatocarcinogenesis in the rat, the initiation stage is associated with the appearance of increased numbers of hepatocytes that can proliferate and form colonies in vitro under conditions that normal hepatocytes growth arrest, degenerate, and die. Expression of altered growth by such initiated hepatocytes requires the presence of PB in the culture medium. The tumor suppressor gene, p53, participates in a checkpoint that can arrest growth in the G1 phase of the cell cycle. This check-

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point responds to external growth inhibitory signals from factors such as TGF[3, and internal signals generated both by DNA damage and the end of a cell's in vitro proliferative lifespan. This review has presented evidence that suggests that the liver tumor promoter, PB, reduces TGFIB activation and suppresses the expression of p53, thereby enhancing the growth and extending the proliferative lifespan of initiated hepatocytes. Because cell cycle checkpoints also provide more time for DNA repair, suppression of checkpoint function could also induce a condition of chromosomal instability accelerating the progression to malignancy.

Acknowledgments This work was supported by PHS grants CA42765, CA59495 and ES05948.

References Baba, M., Klein-Szanto, A. J. P., Trono, D., Obara, T., Yoakum, G. H., Masui, T., and Harris, C. C. (1987). Preneoplastic and neoplastic growth of xenotransplanted lung-derived human cell lines using deepithelialized rat tracheas. Cancer Res. 47, 573-578. Barrett, J. C., Oshimura, M., and Koi, M. (1987). Role of oncogenes and tumor suppressor genes in a multistep model of carcinogenesis. In "Critical Molecular Determinants of Carcinogenesis" (Becker, E E, and Slaga, T. J., Eds.), Vol. 39, pp. 45-56. Univ. of Texas Press, Austin, TX. Boone, C. W., Kelloff, G. J., and Steele, V. E. (1992). Natural history of intraepithelial neoplasia in humans with implications for cancer chemoprevention strategy. Cancer Res. 52, 1651-1659. Bursch, W., Lauer, B., Timmermann-Trosiener, I., Barthel, G., Schuppler, J., and SchulteHermann, R. (1984). Controlled death (apoptosis) of normal and putative preneoplastic cells in rat liver following withdrawal of tumor promoters. Carcinogenesis 5, 453-458. Chiao, C., Zhang, Y., Kaufman, D. G., and Kaufmann, W. K. (1995a). Derivation of phenobarbitalresponsive immortal rat hepatocytes. Am. J. Path., 146, 1248-1259. Chiao, C., Zhang, Y., Kaufman, D. G., and Kaufmann, W. K. (1995b). Phenobarbital modulates the type of cell death by rat hepatocytes during deprivation of serum in vitro. Hepatology, 22, 297-303. Cohen, M. M., and Levy, H. P. (1989). Chromosomal instability syndromes. Adv. Human Genet. 18, 43-149. Debbas, M., and White, E. (1993). Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev. 7, 546-554. Denda, A., Ura, H., Tsujiuchi, T., Tsutsumi, M., Eimoto, H., Takashima, Y., Kitazawa, S., Kinugasa, T., and Konishi, Y. (1989). Possible involvement of arachidonic acid metabolism in phenobarbital promotion of hepatocarcinogenesis. Carcinogenesis 10, 1929-1935. Dennis, E A., and Rifkin, D. B. (1991 ). Cellular activation of latent transforming growth factor IB requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor. Proc. Natl. Acad. Sci. USA 88, 580-584. Dulic, V., Lees, E., and Reed, S. I. (1992). Association of cyclin E with a periodic G1-S phase protein kinase. Science 257, 1958-1961.

8.

Liver Tumor Promotion

195

Dulic, V., Drullinger, L. E, Lees, E., Reed, S. I., and Stein, G. H. (1993). Altered regulation of G1 cyclins in senescent human diploid fibroblasts-Accumulation of inactive cyclin D 1/cdk2 and cyclin E/cdk2 complexes. Proc. Natl. Acad. Sci. USA 90, 11034-11038. Dulic, V., Kaufmann, W. K., Wilson, S. J., Tlsty, T. D., Lees, E., Harper, J. W., Elledge, S. J., and Reed, S. I. (1994). P53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76, 1013-1023. EI-Deiry, W. S., Harper, J. W., O'Connor, P. M., Velculescu, V. E., Canman, C. E., Jackman, J., Pietenpol, J. A., Burrell, M., Hill, D. E., Wang, Y., Wiman, K. G., Mercer, W. E., Kastan, M. B., Kohn, K. W., Elledge, S. J., Kinzler, K. W., and Vogelstein, B. (1994). Wafl/Cipl is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 54, 1169-1174. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M., Waters, C. M., Penn, L. Z., and Hancock, D. C. (1992). Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119-128. Fausto, N. (1991). Growth factors in liver development, regeneration and carcinogenesis. Prog. Growth Factor Res. 3, 219-234. Foliot, A., Glaise, D., Erlinger, S., and Guguen-Guillouzo, C. (1985). Long-term maintenance of taurocholate uptake by adult rat hepatocytes co-cultured with a liver epithelial cell line. Hepatology 5, 215-219. Greider, C. W. (1991). Telomeres. Curr. Opin. Cell Biol. 3, 444-451. Harley, C. B., Vaziri, H., Counter, C. M., and Allsopp, R. C. (1992). The telomere hypothesis of cellular aging. Exp. Gerontol. 27, 375-382. Hermeking, H., and Eick, D. (1994). Mediation of c-myc-induced apoptosis by p53. Science 265, 2091-2093. Hunter, T., and Pines, J. (1994). Cyclins and cancer II: Cyclin D and CDK inhibitors come of age. Cell 79, 573-582. Jirtle, R. L., and Meyer, S. A. (1991). Liver tumor promotion: Effect of phenobarbital on EGF and protein kinase C signal transduction and transforming growth factor ~31 expression. Dig. Dis. Sci. 36, 659-668. Jirtle, R. L., Hankins, G. R., Reisenbichler, H., and Boyer, I. J. (1994). Regulation of mannose 6-phosphate/insulin-like growth factor-II receptors and transforming growth factor beta during liver tumor promotion with phenobarbital. Carcinogenesis 15, 1473-1478. Kadohama, T., Tsuji, K., and Ogawa, K. (1994). Indistinct cell cycle checkpoint after u.v. damage in H-ras-transformed mouse liver cells despite normal p53 gene expression. Oncogene 9, 2845-2852. Kastan, M. B., Zhan, Q., EI-Deiry, W. S., Carrier, E, Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J., Jr. (1992). A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia telangiectasia. Cell 71, 587-597. Kaufmann, W. K., and Kaufman, D. G. (1993). Cell cycle control, DNA repair and initiation of carcinogenesis. FASEB J. 7, 1188-1191. Kaufmann, W. K., and Wilson, S. R. (1994). G1 arrest during cell cycle dependent clastogenesis in UV-irradiated human fibroblasts. Mutat. Res. 314, 67-76. Kaufmann, W. K., MacKenzie, D., and Kaufman, D. G. (1985). Quantitative relationship between hepatocytic neoplasms and islands of cellular alteration during hepatocarcinogenesis in the male F344 rat. Am. J. Pathol. 119, 171-174. Kaufmann, W. K., Tsao, M-S., and Novicki, D. L. (1986). In vitro colonization ability appears soon after initiation of hepatocarcinogenesis in the rat. Carcinogenesis 7, 669-671. Kaufmann, W. K., MacKenzie, S. A., and Kaufman, D. G. (1987). Factors influencing the initiation by gamma rays of hepatocarcinogenesis in the rat. Teratog. Carcinog. Mutagen. 7, 551-556. Kaufmann, W. K., Ririe, D. G., and Kaufman, D. G. (1988). Phenobarbital-dependent proliferation of putative initiated rat hepatocytes. Carcinogenesis 9, 779-782.

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Yingchun Zhang et al.

Kaufmann, W. K., Boyer, J. C., Estabrooks, L. E., and Wilson, S. R. (1991). Inhibition of replicon initiation in human cells following transient stabilization of topoisomerase-DNA complexes. Mol. Cell. Biol. 11, 3711-3718. Kaufmann, W. K., Zhang, Y. C., and Kaufman, D. G. (1992). Association between expression of transforming growth factor-alpha and conversion of hepatocellular foci to neoplasms. Carcinogenesis 13, 1481-1483. Kaufmann, W. K., Levedakou, E. N., Grady, H. L., Paules, R. S., and Stein, G. H. (1995). Attenuation of G2 checkpoint function precedes human cell immortalization. Cancer Res. 55, 7-11. King, R. W., Jackson, P. K., and Kirschner, M. W. (1994). Mitosis in transition. Cell 79, 563571. Kitagawa, T., Watanabe, R., Kayano, T., and Sugano, H. (1980). In vitro carcinogenesis of hepatocytes obtained from acetylaminofluorene-treated rat liver and promotion of their growth by phenobarbital. Gann 71,747-754. Lee, L. W., Raymond, V. W., Tsao, M. S., Lee, D. C., Earp, H. S., and Grisham, J. W. (1991). Clonal co-segregation of tumorigenicity with overexpression of c-myc and transforming growth factor alpha genes in chemically transformed rat liver epithelial cells. Cancer Res. 51, 5238-5244. Livingstone, L. R., White, A., Sprouse, J., Livanos, E., Jacks, T., and Tlsty, T. D. (1992). Altered cell cycle arrest and gene amplification potential accompany loss of wild type p53. Cell 70, 923-935. Lowe, S. W., and Ruley, H. E. (1993). Stabilization of the p53 tumor suppressor is induced by adenovirus 5 EIA and accompanies apoptosis. Genes Dev. 7, 535-545. Michalopoulos, G. K. (1990). Liver regeneration: Molecular mechanisms of growth control. FASEB J. 4, 176-187. Mills, J. J., Chari, R. S., Boyer, I. J., Gould, M. N., and Jirtle, R. L. (1995). Induction of apoptosis in liver tumors by the monoterpene perillyl alcohol. Cancer Res. 55, 979-983. Nettesheim, P., and Barrett, J. C. (1984). Tracheal epithelial cell transformation: A model system for studies on neoplastic progression. CRC Crit. Rev. Toxicol. 12, 215-240. Newbold, R. E (1985). Multistep malignant transformation of mammalian cells by carcinogens: Induction of immortality as a key event. Carcinog. Compr. Surv. 9, 17-28. Noda, A., Ning, Y., Venable, S. E, Pereira-Smith, O. L., and Smith, J. R. (1994). Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression system. Exp. Cell Res. 211, 90-98. Norbury, C., Blow, J., and Nurse, P. (1991 ). Regulatory phosphorylation of the p34 cac2 protein kinase in vertebrates. EMBO J. 10, 3321-3329. Oberhammer, E A., Pavelka, M., Sharma, S., Tiefenbacher, R., Purchio, A. E, Bursch, W., and Schulte-Hermann, R. (1992). Induction of apoptosis in cultured hepatocytes and in regressing liver by TGF[31. Proc. Natl. Acad. Sci. USA 89, 5408-5412. O'Connor, P. M., Ferris, D. K., White, G. A., Pines, J., Hunter, T., Longo, D. L., and Kohn, K. W. (1992). Relationships between cdc2 kinase, DNA cross-linking and cell cycle perturbations induced by nitrogen mustard. Cell Growth Differ. 3, 43-52. Olivieri, G., and Micheli, A. (1983). Mitotic delay and repair in human lymphocytes. Mutat. Res. 122, 65-72. Painter, R. B., and Young, B. R. (1980). Radiosensitivity in ataxia-telangiectasia: A new explanation. Proc. Natl. Acad. Sci. USA 77, 7315-7317. Paules, R. S., Levedakou, E. N., Wilson, S. J., Innes, C., Rhodes, N., Tlsty, T. D., Galloway, D. A., Tainsky, M., Donehower, L., and Kaufmann, W. K. (1995). Defective G2 checkpoint function in cells from individuals with familial cancer syndromes. Cancer Res. 55, 1763-1773. Peraino, C., Fry, R. J. M., and Staffeldt, E. (1971). Reduction and enhancement by phenobarbital of hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res. 31, 1506-1512.

8.

Liver Tumor Promotion

197

Peter, M., and Herskowitz, I. (1994). Joining the complex: Cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell 79, 181-184. Polyak, K., Kato, J., Solomon, M. J., Sherr, C. J., Massague, J., Roberts, J. M., and Koff, A. (1994). p27Kip1, a cyclin-cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 8, 9-22. Reed, S. I. (1992). Cyclin-dependent protein kinases. Annu. Rev. Cell Biol. 8, 529-561. Reisenbichler, H., Chari, R. S., Boyer, I. J., and Jirtle, R. L. (1994). Transforming growth factor-beta receptors type I, II and III in phenobarbital-promoted rat liver tumors. Carcinogenesis 15, 2763-2767. Sanford, K. K., Price, E M., Rhim, J. S., Stampfer, M. R., and Parshad, R. (1992). Role of DNA repair in malignant neoplastic transformation of human mammary epithelial cells in culture. Carcinogenesis 13, 1137-1141. Schulte-Hermann, R., Timmermann-Trosiener, I., Barthel, G., and Bursch, W. (1990). DNA synthesis, apoptosis, and phenotypic expression as determinants of growth of altered loci in rat liver during phenobarbital promotion. Cancer Res. 50, 5127-5135. Shay, J. W., Wright, W. E., Brasiskyte, D., and Van Der Haegen, B. A. (1993). E6 of human papilloma virus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts. Oncogene 8, 1407-1414. Sherr, C. J. (1994). G1 phase progression: Cycling on cue. Cell 79, 551-555. Stein, G. H. (1985). SV40-transformed human fibroblasts: Evidence for cellular aging in precrisis cells. J. Cell. Physiol. 125, 36-44. Sun, Q., Tsutsumi, K., Kelleher, M. B., Pater, A., and Pater, M. M. (1992). Squamous metaplasia of normal and carcinoma in situ of HPV16-immortalized human endocervical cells. Cancer Res. 52, 4254-4260. Thompson, N. L., Mead, J. E., Braun, L., Goyette, M., Shank, P. R., and Fausto, N. (1986). Sequential protooncogene expression during rat liver regeneration. Cancer Res. 46, 31113117. Tlsty, T. D. (1990). Normal diploid human and rodent cells lack a detectable frequency of gene amplification. Proc. Natl. Acad. Sci. USA 87, 3132-3136. Toyoshima, H., and Hunter, T. (1994). p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity, is related to p21. Cell 78, 67-74. Tsuji, K., and Ogawa, K. (1994). Recovery from ultraviolet-induced growth arrest of primary rat hepatocytes by p53 antisense oligonucleotide treatment. Mol. Carcinog. 9, 167-174. White, A., Livanos, E., and Tlsty, T. D. (1994). Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins. Genes Dev. 8, 666-677. Xu, M., Sheppard, K., Peng, C., Yee, A. S., and Piwnica-Worms, H. (1994). Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA binding activity of E2F-1/DP-1 by phosphorylation. Mol. Cell. Biol. 14, 8420-8431. Yin, Y., Tainsky, M. A., Bischoff, E Z., Strong, L. C., and Wahl, G. M. (1992). Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell 70, 937-948. Zampetti-Bosseler, E, and Scott, D. (1981). Cell death, chromosome damage, and mitotic delay in normal human, ataxia telangiectasia, and retinoblastoma fibroblasts after X-irradiation. Int. J. Rad. Biol. 39, 547-558.

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Mechanisms of Liver Tumor Promotion Jeremy J. Mills

Randy L. Jirtle Department of Radiation Oncology, Duke UniversityMedical Center Durham, North Carolina27710

Ivan J. Boyer MITRE Corporation, Inc. McLean, Virginia22102

I. Introduction Hepatocellular carcinoma (HCC), although relatively uncommon in Western countries, is one of the most common cancers in the Far East and southern Africa. Both hepatitis virus infection (i.e., HBV and HCV) and exposure to aflatoxins have been implicated as etiologic factors for the high incidence of HCC in these geographic areas (see Chapter 6) (Okuda, 1992). Interestingly, approximately 60% of the chemicals determined by the National Toxicology Program to be carcinogenic in rats and mice give rise to liver tumors. Some of these carcinogens, however, are either only weakly genotoxic or have been found to cause no detectable genetic damage. Rather, they appear to function mainly as tumor promoting agents. Substances that may pose a human health risk, and have been shown to act as liver tumor promoters in animal models include contraceptive steroids (Baum et al., 1973; Yager and Yager, 1980), the antiestrogen tamoxifen (Catherino and Jordon, 1993; Williams et al., 1993), benzodiazepine compounds (Diwan et al., 1986), dioxin (Pitot e t a l . , 1987), peroxisome proliferators (Cattley and Popp, 1989), and phenobarbital (PB) (Peraino et al., 1971). Thus, to accurately assess chemical agents for their carcinogenic risk to humans, it Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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is necessary to determine the molecular mechanisms by which liver tumor promoters enhance tumor formation. Carcinogenesis is a complex process that operationally has been divided into three stages (Figure 1) (Dragan and Pitot, 1992), although measurements of age-dependent liver tumor incidence in rats suggest that up to 5 or 6 independent steps may be involved in the malignant conversion of hepatocytes (Okuda, 1992). These three stages of tumor formation have been characterized in many mammalian experimental systems, particularly in the liver (Solt and Farber, 1976; Pitot et al., 1978; Peraino et al., 1981) and skin (Slaga et al., 1982). According to the concept of multistage carcinogenesis, clones of cells arise with increasing autonomy from normal growth regulation at each stage of development, and from these selected populations of cells neoplasms ultimately develop. In the 1940s, it was demonstrated that certain combinations of chemical treatments are synergistic in the formation of skin tumors (Friedwald and Rous, 1944; Berenblum and Shubik, 1947). For example, application of a small amount of a carcinogenic polycyclic hydrocarbon to mouse skin resuited in few tumors; however, when this treatment was followed by continuous exposure to the noncarcinogenic plant extract, croton oil, the mice rapidly developed a large number of skin tumors. In these pioneering studies, the hydrocarbon treatment was described as the initiating event for carcinogenesis while the croton oil acted as a tumor promoter. Since then, evidence has accumulated that this multistage model of carcinogenesis applies to neoplastic formation in virtually all epithelial cells including urinary bladder (Cohen et al., 1979), lung (Witschi et al., 1977), colon (Reddy et al., 1977), liver (Peraino et al., 1971), and breast (Meites, 1972). Since these early skin carcinogenesis studies, many experimental tumor models have been developed for investigating hepatocarcinogenesis (Goldsworthy et al., 1986). In all of these liver tumor models, it is possible to identify three distinct stages of tumor development. Initiation, the first stage in carcinogenesis, involves the formation of cells containing an irreversible

Figure 1 Stages of liver carcinogenesis. Preneoplastic (white circles) and neoplastic (black circles) hepatocytes are shown surrounded by normal liver which increases in size on exposure to phenobarbital (PB).

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genetic change that enables them to expand clonally under the influence of a promoter. This genetic alteration or mutation may occur spontaneously or in response to chemical or physical agents and may result from either genetic and/or epigenetic events. The reversible stage of p r o m o t i o n involves the selective clonal expansion of these initiated cells into foci of altered cells. Subsets of these foci develop further through a process of neoplastic transformation into end stage neoplasms; this final stage has been termed progression (Pitot et al., 1991). During tumor progression, additional genetic alterations occur that involve the activation of oncogenes and/or the inactivation of tumor suppressor genes that ultimately give rise to tumors with autonomous growth and the ability to metastasize (Pitot, 1993; Vogelstein and Kinzler, 1993). These three stages of liver carcinogenesis are described in greater detail below.

II. Stages of Liver Carcinogenesis A. Initiation Initiation is characterized by the irreversible nature of carcinogen-induced genetic damage. For studies of hepatocarcinogenesis, the production of a population of initiated cells can be brought about by a variety of techniques and treatments. The exposure of the liver to a carcinogen by itself is usually not sufficient to produce initiated cells. Most protocols also require increased cell proliferation to "fix" the carcinogen-induced alterations in the cellular genome. Since the adult level of hepatocyte proliferation is relatively low, efficient initiation by a single dose of a genotoxic chemical carcinogen such as diethylnitrosamine (DEN) requires that the animals be treated either directly before a two-thirds partial hepatectomy to induce proliferation (Goldsworthy et al., 1986), or at an early age when the liver is still rapidly growing (Peraino et al., 1981). Alternatively, a large dose of a chemical carcinogen can be administered that leads to liver necrosis followed by compensatory regenerative cell proliferation (Farber, 1991). It has been shown, however, that a large single dose of DEN also leads to increased karyotypic changes and aneuploidy when compared to the chromosomal changes observed when animals are treated with a low dose of DEN soon after birth (Sargent et al., 1989). Recently, it has been suggested that initiation can be a reversible process since initiated cells are removed from the liver by apoptosis under certain experimental conditions such as diet restriction (see Chapter 7) (Grasl-Kraup et al., 1994). However, the elimination of initiated hepatocytes from the liver by the process of apoptosis is not the same as reversing the carcinogen-induced DNA damage caused during the initiating process.

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The study of initiated hepatocytes has been hampered by an inability to detect them at an early stage of development. Recently, the placental isozyme of glutathione-S-transferase (GST-P) has been suggested as a marker for studying initiated hepatocytes at their earliest stage of development (Moore et al., 1987; Cameron, 1989) since the number of GST-P immunohistochemically positive hepatocytes present in the liver correlates positively with DEN dose (Dragan et al., 1993). Although GST-P may be useful in studying the initial stage of carcinogenesis, not all initiated cells are GSTP positive. Furthermore, the genetic lesions responsible for the initiation of hepatocytes have not been elucidated, thus making the relationship between a positive GST-P phenotype and initiation unclear. B. Promotion

The promotion of liver tumors was first demonstrated by Peraino and coworkers (1971). In this study, rats were treated for 2 weeks with the hepatocarcinogen 2-acetylaminofluorene (2-AAF), followed by a diet containing PB. Animals exposed to both the carcinogen and PB had up to ten times the yield of liver tumors compared to rats that were exposed only to 2-AAF. In addition, few of the animals treated with PB alone developed liver tumors. Since the publication of this seminal paper, three important characteristics of liver tumor promotion have been defined. (1) The promotional stage of liver carcinogenesis is reversible (Solt et al., 1980; Teebor and Becker, 1988). Termination of treatment with a promoter leads to a decrease in both the number and size of loci, whereas its readministration causes their rapid reappearance (Hendrich et al., 1986). (2) Animals must be exposed continuously to a tumor promoter for an extended period in order to observe a promotional effect. Exposure to PB (Preat et al., 1987) and orotic acid (Laconi et al., 1993) for approximately 3 months is necessary for maximal tumor promotion. (3) Promoter dose-response curves demonstrate an apparent threshold below which promotional effects are not detectable (Pitot et al., 1980). Prior to developing grossly visible tumors, the livers of carcinogentreated rats contain focal areas of proliferating initiated hepatocytes that can be identified by histochemical staining. These loci are described as altered hepatic loci (AHF) or enzyme altered loci (EAF), and can be detected months before the appearance of adenomas and carcinomas. These hepatic loci have been shown to be clonal in origin (Tsuji et al., 1988) suggesting that they develop in direct response to treatment with a promoting agent and represent the clonal expansion of the initiated cells. In addition, the hepatic foci grow progressively and have increased levels of DNA synthesis compared to the surrounding tissue (Seo et al., 1988). It is from these EAF

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that a subset of nodules develop whose growth is independent of a promoting agent, and hepatic neoplasms ultimately arise (Figure 1). The ability to quantitate initiated hepatocytes has highlighted the fact that although 106 putative initiated GST-P positive cells can be detected in the livers of DEN treated rats, only 104 EAF develop following PB promotion (Pereira, 1983; Kaufmann et al., 1986), and of these, just a small fraction ultimately give rise to HCC. Clearly, even under the influence of this potent promoter, not all of the initiated cells can be clonally expanded into EAE This suggests that the various promotional regimens promote different subpopulations of initiated cells whose genotypic makeup provides a selective growth advantage (Columbano et al., 1982). Indeed, analysis of the phenotypic and histological profiles of liver foci promoted with PB or tamoxifen shows that the majority of PB-promoted foci are positive for GST-P and gamma-glutamyltranspeptidase (GGT), whereas tamoxifenpromoted foci stain positive for the marker glucose 6-phosphate (Pitot et al., 1991). In contrast, the phenotypic markers for EAF following exposure to 2,3,7,8-tetrachlorodibenz-p-dioxin are similar to those for PB (Pitot et al., 1980). Also Yelandi and co-workers (1989) demonstrated that the GGTnegative phenotype of the foci induced by the peroxisome proliferator, ciprofibrate, is not altered when the promotional agent is switched to 2-AAE Furthermore, Gerbracht and colleagues (1990) have shown that upon withdrawal of PB, the administration of cyproterone acetate or hexachlorocyclohexane will maintain the GGT characteristics of the developing foci. These examples of the differences and similarities in the distribution of these phenotypic enzyme markers underscore the fact that although they are useful in the histological demonstration of EAF, they may not be mechanistically involved in the oncogenesis of liver tumors.

C. Progression Progression is the least well-studied and consequently least well-understood stage of liver carcinogenesis. The concept of progression develops from the observation that only a small subset (1 to 5 %) of the clonally expanded EAF persist and develop into true neoplasms (Farber, 1986). In progression, the persistent EAF can undergo a series of irreversible changes that ultimately leads to HCC formation. No markers of the kind used to study promotion have been found for progression although a phenotypically heterogeneous focus, called a focus-in-focus, has been suggested as an intermediate step in the progression of liver tumors in rats (Scherer, 1987) and humans (Matsuno et al., 1990). Aneuploidy, karyotypic instability, and malignant invasion are the most recognized features of progression. Studies at the molecular level have also demonstrated that changes occur

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in the expression of proto-oncogenes and tumor suppressor genes in a variety of tumors (Hsu et al., 1991; Goyette et al., 1992). The cellular expression of the proto-oncogenes c-Ha-ras and c - m y c are not increased in EAF, but are elevated in some HCC (Beer et al., 1986; Embleton and Butler, 1988). Furthermore, alterations in c - m y c and src expression have been demonstrated in HCC following exposure of animals to either 3'-methyl-4dimethylaminobenzene (Cote et al., 1985) or the Solt-Farber protocol (Richmond et al., 1988). The underlying mechanisms for the karyotypic and genetic instability that result in altered oncogene and tumor suppressor gene expression in HCC is, however, just beginning to be unraveled at the molecular level.

III. Cell Cycle R e g u l a t i o n a n d Liver C a r c i n o g e n e s i s The study of cell cycle regulation has become one of fastest developing areas of biological research, and the results of these investigations are particularly germane to understanding the molecular changes that occur during the stages of carcinogenesis described previously (Hunter, 1993; Motokura and Arnold, 1993; Heichman and Roberts, 1994; Hunter and Pines, 1991, 1994; Alexandrow and Moses, 1995) (see Chapter 8). The cell cycle, in a simplified form, can be considered as having four stages, the gap before DNA replication (G1), the stage of DNA synthesis (S), the gap after DNA replication (G2), and the stage of mitosis leading to cell division (M) (Figure 2). In the liver, most hepatocytes are in a quiescent metastable state (Go) with unduplicated DNA. Therefore, depending on the extracellular growth factor stimuli present, cells can either stay in Go, move out of Go into the G~ stage of the cell cycle, or undergo apoptosis (Figure 2). The entry into the cell cycle and passage through the restriction (R) checkpoint late in G1 depends on extracellular growth factor signals; however, after traversing this first R checkpoint, the cell cycle continues according to an internal program independent of extracellular signals (Sager, 1992). The R checkpoints at the G1/S and G2/M interfaces enable the cell to establish that all the prerequisites for the current cell cycle stage have been met before progressing into the next cell cycle stage; i.e., synthesis of DNA polymerases, histones, etc. before DNA replication (G1/S R checkpoint) (Pardee, 1989) and completion of DNA replication, protein synthesis, etc. before mitosis (G2/M R checkpoint) (Hartwell and Weinert, 1989). Thus, the G1/S and G2/M R checkpoints enable the cell to ensure genetic fidelity during cell replication (Bagrodia et al., 1991). Conversely, defects in the cell cycle R checkpoints can lead to genomic instability, an often observed characteristic of cancer cells (Hartwell, 1992).

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Figure 2 Growth factor control of tissue homeostasis. Hepatocytes in vivo are impinged on by both growth inhibitors and growth stimulators. When the effects of these two opposing types

of growth factors balance one another, the hepatocytes remain in Go (i.e., a metastable state). In contrast, an imbalance in growth factor signals can either stimulate hepatocytes to enter the cell cyclethereby increasingthe cell number or induce hepatocytes to undergo programmedcell death (i.e., apoptosis). Once in the cell cycle TGFfl plays an important function in regulating the rate at which hepatocytes transverse the G1/S restriction (R) checkpoint and enter the S-phase. Cyclin-dependentkinases (cdks); epidermal growth factor (EGF); hepatocyte growth factor (HGF); insulin-like growth factor 2 (IGF2); transforming growth factor c~ (TGF~); transforming growth factor 13(TGF~).

A. Cyclins and Cyclin-Dependent Kinases Studies with yeast and early embryonic divisions in Xenopus (Dowdy et al., 1993; Sherr, 1994) have shown that nuclear proteins called cyclins and cyclin-dependent kinases (cdks) are key regulators of the critical steps within the cell cycle. Cdks are present in cycling cells throughout the cell cycle in fairly constant amounts; the presence or absence of associated cyclins, which act as regulatory elements, determines the catalytic activity and substrate specificity of the kinase complexes (reviewed by Sherr, 1994). Recent studies have also determined that inhibitors of cdks, i.e., p27Kip 1, p21Wafl/Cip 1/Sdil, p15 INK4B and p16 INK4A, play a significant role in the regulation of the cell cycle (reviewed by Elledge and Harper, 1994; Alexandrow and Moses, 1995). Specific cyclins are expressed at discrete points in the cell cycle, allowing phosphorylation of substrates by the active cdkcyclin complex. Substrate phosphorylation by the various cdk-cyclin complexes allows for progression through the various stages of the cell cycle

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(Mailer, 1991). Thus, altered expression of cyclins, cdks, or cdk inhibitors could potentially deregulate the cell cycle program leading to an unlimited proliferative potential. This has been seen in HCC with the human cyclin A gene (Wang et al., 1990) and in benign parathyroid tumors (Motokura et al., 1991) and breast and esophageal carcinomas (Jiang et al., 1992; Buckley et al., 1993) with the human cyclin D genes. B. Rb Gene One key substrate operating downstream of the cdk/cyclins during the late G 1 phase of the cell cycle is Rb, a 110-kDa nuclear phosphoprotein, and the product of the retinoblastoma tumor suppressor gene (reviewed by Picksley and Lane, 1994). This gene is inactivated in a variety of tumors (Lee et al., 1987; Buchkovich et al., 1989), and reintroduction of the wild-type form of the Rb gene into Rb negative tumor cells has been shown to reduce their ability to form tumors (Huang et al., 1988). As previously stated, the phosphorylation of Rb is modulated during the cell cycle by cdk-cyclin complexes (Dulic et al., 1989; Kato et al., 1993). In the G Oand early G 1 stages of the cell cycle Rb is in an activated hypophosyphorylated form, but it becomes inactivated hyperphosphorylated in late G 1 and remains so throughout S, G2, and M phases of the cell cycle (DeCaprio et al., 1988; Buchkovich et al., 1989). Rb is again activated by dephosphorylation at the end of M phase by specific phosphatases (Ludlow et al., 1990). The E2F transcription factor preferentially binds to the hypophosphorylated form of Rb preventing it from initiating gene transcription. Since E2F has a reduced affinity for RB when it is hyperphosphorylated, phosphorylation of the Rb protein enables the cells to progress through the cell cycle by releasing the E2F transcription factor (Hiebert et al., 1992; Weintraub et al., 1992). In addition to the E2F transcription factor, viral oncoproteins, including SV40 T antigen and human papilloma virus E7, bind to the hypophosphorylated form of Rb leading to its functional inactivation (DeCaprio et al., 1988; Dyson et al., 1989; Ludlow et al., 1989). Thus, cells containing these viral oncoproteins have lost the ability to regulate cell cycle progression because of Rb inactivation, and this may in part be the mechanism by which these oncoviruses transform mammalian cells. Treatment of mink lung cells with transforming growth factor/31 (TGF[31) retains Rb in its active, hypophosphorylated form, thereby preventing cells from entering the S-phase (Laiho et al., 1990). This inhibition of Rb phosphorylation correlates with decreased DNA synthesis. Furthermore, in cells which express the SV40 large T antigen, TGFf31 fails to decrease DNA synthesis, presumably due to the hypophosphorylated Rb being sequestered by the viral oncoprotein. This implies that an interaction between the TGFI3

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signaling pathway and the hypophosphorylated form of Rb mediates the growth inhibitory effect of TGF]3 on hepatocytes. Recently, TGF[31 was shown to arrest cells at the GI/S R checkpoint by a mechanism that may involve interactions with inhibitors of cdks. In human keratinocytes growth arrested with TGFI3, the mRNA level of cdk inhibitor p15 INK4B is increased 30-fold with a corresponding decrease in cdk6 activity (Hannon and Beach, 1994). Cdk6 associates with the D-cyclins and is responsible in part for the phosphorylation and functional inactivation of Rb potentially leading to the accumulation of the active hypophosphorylated Rb in TGFI3 arrested cells. In addition, p27Kip1, an inhibitor known to complex with multiple cyclin/kinase complexes, inhibits cdk2/E-cyclin complexes in mink lung cells arrested by TGFf3 (Polyak et al., 1994). Although the mechanism by which TGFI3 inhibits cell cycle progression is still not totally clear, based on these findings, it is postulated that treatment with TGF[3 increases p15 INK4B which sequesters cdk6 from the cdk6/D-cyclin complex, releasing p27Kip1 to inhibit ckd2/E-cyclin complexes and therefore, preventing the cells from passing through the G1/S R checkpoint (Peters, 1994). C. p53 Gene

The Rb protein has been described as a tumor suppressor gene due to its ability to abrogate the neoplastic phenotype when transfected back into Rb negative tumor cells (Huang et al., 1988). Another tumor suppressor gene product, p53, may also cause growth arrest at the GI/S phase border (see reviews by Vogelstein and Kinzler, 1992; Picksley and Lane, 1994). As with Rb, the mechanism by which this growth arrest occurs has not been completely delineated; however, data from irradiated cells suggest that p53 normally functions in maintaining genetic stability. For example, following exposure to X-rays, p53 expression in normal cells is increased leading to growth arrest until the DNA damage is repaired. In cells containing mutant p53, growth arrest is incomplete, and the irradiated cells continue to divide and eventually die because of unrepaired DNA damage (Karsten et al., 1991). Indeed, expression of the wild-type p53 in glioblastoma cells inhibits the progression of these cells from G 1 to the S-phase of the cell cycle following growth stimulation (Lin et al., 1992a). As with Rb, p53 is a nuclear phosphoprotein which exists in an hypophosphorylated active form in early G 1 and can be phosphorylated by the same cdk-cyclin complexes that phosphorylate Rb (Bischoff et al., 1990). In addition to arresting cell growth, p53 has been shown to be involved in the induction of apoptosis (YonishRouach et al., 1991; E1-Deiry et al., 1994). Additionally, p53 can bind to DNA at sequence-specific sites, activate or repress transcription of an array

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of genes, stimulate annealing of single stranded DNA, and inhibit replicase activity and DNA replication (see review by Pietenpol and Vogelstein, 1993). Mutations of p53 have been studied intensively and have been observed in a majority of human tumors including those in the liver (Bressac et al., 1991; Hsu et al., 1991). Thus, cells with inactive p53 have a selective growth advantage over the surrounding normal cells. However, a functional p53 protein in the early developmental stages is not vital, as evidenced by the fact that p53 knockout mice are viable and develop normally (Donehower et al., 1992). Nevertheless, the increased tumor incidence observed in these mice demonstrates that p53 has a major role in tumor suppression (Donehower et al., 1992; Pietenpol and Vogelstein, 1993). Clearly, alterations in the function of Rb, p53, cyclins, and cdk proteins and their inhibitors will cause dysfunctional regulation of the normal cellcycle and replication/transcriptional pathways. These alterations in function could occur through genetic means involving direct effects on the chromatin or through epigenetic pathways involving, for example, altered expression of phosphatases and/or kinases. Additionally, a decrease in the extracellular signals from the growth inhibitor, TGF[3, would reduce a cell's ability to both remain quiescent and appropriately regulate its cell cycle once it entered the proliferative state (Figure 2) (Jirtle et al., 1991a). The role of TGF[3 in liver carcinogenesis is described in more detail below.

IV. TGFI3 and Liver Carcinogenesis Tumor promoting agents increase both the number and size of tumors, with the tumor promoter(s) determining which of these effects is more prominent (Dragan and Pitot, 1992). It must be stressed, however, that tumors are not created in a vacuum. Rather, developing liver tumors are surrounded by normal hepatocytes. Therefore, to understand at tissue level the mechanisms by which promoters enhance liver tumor formation, it is important to appreciate the growth effects these agents have not only on the initiated hepatocytes but also on those in the surrounding normal liver. In the liver where homeostatic mechanisms are operating, it is the differential growth effect that liver tumor promoting agents have on the normal versus the initiated hepatocytes that provides the selective pressure which ultimately gives rise to tumor formation (Figure 3). When animals are exposed to PB the liver increases significantly in size (Figures 1 and 3). Part of this increase in liver mass is due to cellular hypertrophy; however, PB also causes a significant degree of hyperplasia, principally in the pericentral region of the liver (Peraino et al., 1975; SchulteHermann et al., 1986; Yager et al., 1986; Jirtle et al., 1991b). This prolifera-

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Figure 3 Liver tumor promotion model in response to PB. Tumors depicted by the open circles do not express the M6P/IGF2r or the TGF[3 Types I, II, and III receptors (Figure 5). Therefore, they have a growth advantage over the surrounding normal tissue because they cannot degrade IGF2 or activate and respond to TGF[3 (Figure 4) as readily as the surrounding normal tissue. Tumors depicted by the gray circles can activate TGF[3, but cannot readily respond to it because their TGF[3 receptors are reduced in number. The tumor shown as a black circle has not lost the ability to upregulate the expression of either the M6P/IGF2r or the TGF[3 receptors in response to PB. Its selective growth is postulated to be regulated through alternative oncogenic pathways. Hepatocyte growth factor (HGF); insulin-like growth factor 2 receptor (IGF2r); phenobarbital (PB); transforming growth factor oL (TGFoL); transforming growth factor 13 (TGF[3); transforming growth factor 13 receptors (TGF[3r).

tive response, which in part may result from an increased plasma concentration of hepatocyte growth factor (HGF) (Lindroos et al., 1992), maximizes approximately 3 days after starting chronic PB treatment, and I to 2 weeks later hepatocyte proliferation returns to normal (Peraino et al., 1975; Yager et al., 1986). Thus, the short-term effect of PB is to transiently increase hepatocyte proliferation. It takes approximately 3 months of continuous PB exposure, however, to maximally promote liver tumor formation (Preat et al., 1987). Therefore, it is necessary to determine the effects that both shortand long-term PB exposure have on the proliferation of normal and initiated hepatocytes to fully characterize the mechanisms of liver tumor promotion. Interestingly, our results (Jirtle et al., 1991b) and those of others (Abanobi et al., 1982; Barbason et al., 1983) demonstrate that the proliferative capacity of hepatocytes is significantly reduced rather than increased by long-term exposure to PB; the liver tumor promoters, orotic acid (Manjeshwar et al., 1993), and ethinyl estradiol (Yager et al., 1994), also have similar long-term mitoinhibitory effects. Thus, although PB initially enhances normal hepatocyte proliferation, the long-term PB exposure required for tumor promotion dramatically decreases the growth potential of normal hepatocytes relative to that of initiated cells (Jirtle and Meyer, 1991; Jirtle et al., 1991b). This reduced capacity of PB-treated hepatocytes to proliferate in response to mitogenic stimuli is correlated with a significant reduction in the EGF receptor number (Eckl et al., 1988; Meyer and Jirtle, 1989), a loss in the ability of

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PKC to be translocated to the membrane (Brockenbrough et al., 1991), and an increase in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r) and TGFIB1 expression in normal but not in initiated hepatocytes (Brockenbrough et al., 1991; Jirtle et al., 1991a, 1994; Jirtle and Meyer, 1991). These latter results emphasize the potential importance of TGFf3 in the oncogenesis of liver tumors. A. TGFf3 and TGFIB Receptors In mammals, TGFI3 consists of a family of three structurally and functionally related isoforms, TGFIB1, 132, and 133 (Figure 4) (see review by Massagu6, 1990). Throughout this chapter, the TGFI3 isoforms are referred to simply as TGFf3 unless a specific isoform was used in the referenced study. TGFI3 inhibits epithelial cell proliferation, stimulates cellular differentiation, and augments connective tissue formation. These biological responses are mediated through TGFIB binding to three distinct receptors, and the TGFf3 Type I (Bassing et al., 1994), Type II (Lin et al., 1992b), and Type III (Lopez-Cassillas et al., 1991; Wang et al., 1991) receptors have now been cloned and sequenced. The type I (Mr:53-kDa) and type II (Mr:80-kDa) serine/threonine kinase receptors form a heteromeric complex on binding TGF~3, and generate the first step in the signal transduction pathway (Figure 5) (Warna et al., 1994). In contrast, the type III receptor (Mr:280-kDa), also called betaglycan, lacks an intracellular signaling domain, and functions primarily in the concentration and presentation of TGFIB to the type I and II

Figure4 Model of the TGFf3latent complex. This model is based on the results of Kankaki et al. (1990). The circles represent putative N-linked glycosylation sites; those that contain M6P residues are depicted by closed circles. TGFf3-LAP is the N-terminal portion of the TGFf3 prepro molecule (Gentry and Nash, 1990). Transforming growth factor 13(TGFf3);transforming growth factor 13latent-associated protein (TGFf3 LAP).

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Figure 5 Model of the M6P/IGF2r and the TGFJ3 Type I, II, and III receptors. The 15 repeat sequences in the extracellular domain of the M6P/IGF2r are indicated by the black boxes. The two M6Ps (O-P) attached to the TGFJ3 latent complex are aligned with the putative binding regions of the M6P/IGF2r (regions a and b) (Westlund et al., 1991; Dahms et al., 1993). IGF2 is also shown bound to this receptor, and region c is its putative binding site (Dahms et al., 1994). Region d denotes that portion of the receptor involved in regulating the intracellular trafficking of lysosomal enzymes (Kornfeld, 1992). The TGFJ3 Type III receptor, also called betaglycan, presents active TGFJ3 to the TGFI3 Type II receptor on which the TGFI3Type I and II receptors form a heterodimeric complex, the Type I receptor is phosphorylated, and an intracel]ular signal is transduced (Wrana et al., 1994). Insulin-like growth factor 2 (IGF2); mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r); transforming growth factor 13 (TGFJ3); transforming growth factor j3 receptors (TGFJ3r).

signal transducing receptors (Massagu6, 1990; Lopez-Casillas et al., 1993; Massagu8 et al., 1993; Segarini, 1993). Although activated TGFJ3 binds to these three receptors, TGFf3 is not secreted in an active form but rather as a latent complex (Figure 4) (Kankaki et al., 1990; MassaguS, 1990). The TGFI3 latent complex cannot bind to the TGFJ3 Type I, II, or III receptors (Wang et at., 1991; Lin et al., 1992b; Bassing et al., 1994; Warna et al., 1994). Instead, TGFIB must first be proteolytically activated (Figures 5 and 6). Because the TGFf3 latent complex contains mannose 6-phosphate (M6P) residues, it binds to the M 6 P / IGF2r (Figure 5) (Kovacina et al., 1989). The binding of the TGFJ3 latent complex to this receptor facilitates the activation of TGFJ3 by the proteolytic enzyme plasmin when in the presence of transglutaminase (Lyons et al., 1990; Dennis and Rifkin, 1991; Kojima et al., 1993). Therefore, the regula-

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Figure 6 Activation model for TGFI3 in liver tumors exposed to PB. TGF[3 latent complex (Figure 4) is produced by the Ito cells. The TGFI3 latent complex binds to the M6P/IGF2r (Figure 5) 1 on the surface of the hepatocytes and the Ito cells where TGF[3 activation by plasmin occurs. Alternatively, the TGFi3 latent complex is internalized into the pre-endosomal compartment 2, and the growth factor is degraded in the lysosomes 3. Once activated, the TGF[3 binds to the TGFI3 Type I and II or Type III receptors (Figure 5) 4 and inhibits the hepatocytes from proliferating while stimulating the Ito cells to produce more latent TGFIB 5; IGF2 also binds to the M6P/IGF2r and is degraded (2 and 3). Mannose 6-phosphate/insulinlike growth factor 2 receptor (M6P/IGF2r); transforming growth factor [3 receptors (TGFf3r).

tion of both the synthesis and the activation of TGF[3 are important in controlling the biological effects of TGF[3 in tissues, and the M6P/IGF2r plays a central role in this activation process (Figures 5 and 6). B. M6P/IGF2 Receptor The structures of the calcium independent M6P receptor and IGF2 receptor are identical (MacDonald et al., 1988; Kornfeld, 1992) (Figure 5). The multifunctional M6P/IGF2r consists of a single polypeptide chain with a large extracellular domain and a cytoplasmic domain consisting of 163 amino acids. In mammals this receptor possesses distinct binding regions for both phosphomannosyl residues and IGF2 (Kornfeld, 1992). Regions a and b of the receptor are necessary for binding the TGFJ3 latent complex (Westlund et al., 1991; Dahms et al., 1993) and region c is required to bind IGF2 (Dahms et al., 1994). Region d denotes that portion of the receptor involved in regulating the intracellular trafficking of lysosomal enzymes (Kornfeld, 1992). The M6P/IGF2r is ubiquitously expressed in tissues and a truncated form of the receptor is also present in the circulation (Kiess et al., 1987). Its

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expression is related to the developmental state in rats with the highest tissue concentration present in fetal and neonatal life; the level dramatically declines between 20 and 40 days of postnatal development (Sklar et al., 1989, 1992). A primary function of the M6P/IGF2r in adults is the sorting of newly synthesized lysosomal enzymes and the endocytosis of extracellular lysosomal enzymes and IGF2 (Dahms et al., 1989). However, secreted growth factors like proliferin (Lee and Nathans, 1988), the proteolytic enzyme cathepsin D (Kornfeld, 1992), and the TGFI3 latent complex (Purchio et al., 1988; Kovacina et al., 1989) also bind to the M6P/IGF2r. Although binding of the TGF~ latent complex to this receptor can lead to its internalization and subsequent degradation in the lysosomes, the extracellular activation of TGF~ is also greatly enhanced (Figures 5 and 6) (Dennis and Rifkin, 1991; Kojima et al., 1993). The M 6 P / I G F 2 r gene is maternally imprinted in mice (i.e., only the maternal copy of this gene is transcribed) and closely linked or identical to the Tme (T-associated maternal effect) locus, a lethal mutation (Barlow et al., 1991). Two regions in the M 6 P / I G F 2 r gene have been implicated as functioning as the imprinting signal (St6ger et al., 1993). Region 1 contains part of the promoter region and the transcription start site and is hypermethylated only on the silent paternal chromosome. Region 2 is contained in an intron 27 kb downstream of the promoter and is methylated only on the expressed maternal chromosome. Methylation of region 2 is inherited from the mother whereas region 1 is methylated following fertilization. Therefore, methylation of region 2 on the expressed maternal allele has been proposed to be the imprint signal for the M6P/IGF2r gene (St6ger et al., 1993). This is particularly interesting since in contrast to other imprinted genes (e.g., IGF2, Snrpn, and H19) (Bartolomei et al., 1991; Left et al., 1992; Liu et al., 1993), where methylation of CpG sites is required to repress gene function, methylation of a CpG site in region 2 of the M 6 P / I G F 2 r gene is required for its expression. Recently, with the use of DNA methyltransferase knockout mice, Li et al. (1993) have clearly shown that DNA methylation is required for controlling the differential expression of the paternal and maternal alleles of imprinted genes. Loss of DNA methylation in the CpG island in region 2 of the M 6 P / I G F 2 r gene resulted in the loss of gene expression from the maternal allele. Thus, loss of M6P/IGF2r gene expression could result from genetic effects or epigenetic effects such as hypomethylation of the maternal allele in region 2 or hypermethylation in region 1. The potential importance of DNA methylation in liver carcinogenesis is discussed in greater detail in Chapter 10. As previously stated, the IGF2 (Liu et al., 1993), H 1 9 (Bartolomei et al., 1991), Snrpn (Left et al., 1992), and M 6 P / I G F 2 r genes (Barlow et al., 1991)

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have been found to be imprinted in mice; however, imprinting of the M 6 P / I G F 2 r gene appears to be a polymorphic trait in humans with only a

small subset of humans potentially being imprinted at this locus (Kalscheuer et al., 1993; Ogawa et al., 1993; Xu et al., 1993). This is the only known

example of differential imprinting of a gene between mice and humans. Because of the potential that the M 6 P / I G F 2 r gene may function as a tumor suppressor gene, the presence of only one functional allele in mice may in part explain why mice are more sensitive to liver tumor formation than humans, at least those humans where M 6 P / I G F 2 r gene imprinting is lost (i.e., both alleles are functional).

C. Experimental Results Consistent with the postulate that the M 6 P / I G F 2 r gene functions as a tumor suppressor gene are our findings that the expression of the M 6 P / I G F 2 r and TGFI31 are significantly lower in PB-promoted rat liver tumors than in the surrounding normal liver (Jirtle et al., 1994). Although initially only a small proportion (i.e., 1 to 5%) of the nodules demonstrate this altered phenotype, the loss of M6P/IGF2r and TGF[31 expression is much more prevalent in HCC. Similarly, TGFI31 is significantly decreased in a subset of early skin lesions, and these tumors have been shown to have a high probability of progressing to carcinomas (Glick et al., 1993; Cui et al., 1994). The M6P/IGF2r and TGFf31 negative phenotype is also seen in liver foci developing in response to the peroxisome proliferator, WY-14,643 (Figures 7A, B). However, in contrast to the DEN-initiated/PB-promoted nodules (Jirtle et al., 1994), virtually all of the Wy-14,643 induced liver tumors have reduced expression of both the M6P/IGF2r and TGFI31 (J. J. Mills, R. L. Jirtle, and R. C. Cattley, unpublished results). This is in contrast to the elevated levels of M6P/IGF2r observed in normal liver of rats exposed to Wy-14,643 (Rumsby et al., 1994). The M6P/IGF2r protein level is not only decreased in rat liver tumors, but has also been found to be reduced in 65% of human HCC (Sue et al., in press). This finding is consistent with our most recent finding that there is loss of heterozygosity (LOH) at M6P/IGF2r locus in 64% of human HCC (De Souza et al., 1995). Since the M6P/IGF2r expression is increased during liver regeneration (Jirtle et al., 1991a), a reduction in its expression in both rat and human liver tumors does not occur simply because of increased cellular proliferation. Rather, these results support strongly our hypothesis that the M6P/IGF2r is mechanistically involved in liver tumor oncogenesis. We have also compared the levels of the TGFIB Type I, II, and III receptors in PB-promoted liver tumors with the receptor levels in the surrounding normal liver tissue (Reisenbichler et al., 1994). PB did not alter the expres-

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Figure 7 (A) Immunohistochemical staining of the liver for transforming growth factor [31 (TGFJ31) and (B) the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r) in Fischer 344 male rats exposed to Wy-14,643 (0.1% in the food) for i year (Research done in collaboration with Dr. R. Cattley, CIIT, Research Triangle Park, NC). Normal tissue (N) and tumor tissue (T) are marked; counterstained with hematoxylin; X160.

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sion of any of the TGFI3 receptors in normal hepatic tissue. In contrast, the steady-state mRNA and membrane protein levels of all three TGFJ3 receptors were significantly reduced in PB-promoted liver tumors when compared to those in the surrounding normal tissue. Similarly, human HCC have a 60% reduction in both the TGFJ3 Type I and II receptor expression relative to the level of these receptors in surrounding normal tissue (Sue et al., in press). Thus, when compared to normal hepatocytes, neoplastic hepatocytes not only possess a reduced ability to activate TGFJ3 (i.e., decreased M 6 P / IGF2r), but also a diminished ability to respond to TGF[3 (i.e., decreased TGF[3 Type I, II, and III receptors). The postulate that TGFf3 is mechanistically involved in liver carcinogenesis is further supported by the recent finding that long-term exposure of cultured rat liver epithelial cells (WB cells) to increasing concentrations of TGF[3 promotes the spontaneous neoplastic transformation of a population of liver cells that express increased levels of proto-oncogenes (Zhang et al., 1994). This in vitro model demonstrates directly that chronic exposure to elevated levels of TGF[3 produces a mitoinhibitory selection environment that promotes the formation of TGF[3 resistant transformed liver cells.

V. Apoptosis and Liver Carcinogenesis Promoting agents can not only increase the size of EAF by enhancing the cell proliferation rate but also by reducing the cell death rate (Schulte-Hermann et al., 1993). The term apoptosis was coined to describe the phenomenon of programmed cell death (Kerr et al., 1972), and it is an important process in both embryogenesis and hematopoiesis (see review by Zakeri and Lockshin, 1994). This form of cell death can be considered as "cellular suicide," and is a genetically encoded process that requires gene transcription and translation (Wyllie et al., 1980; Williams and Smith, 1993). Apoptosis involves a series of morphological stages, including cell condensation and fragmentation, condensation of the nuclear chromatin, and phagocytosis by the surrounding normal epithelial cells. Evidence suggests that apoptosis is not a random event, but rather involves the preferential elimination of diseased, old, preneoplastic, damaged, or excessive cells (Bursch et al., 1992; Levine et al., 1993). The importance of apoptosis in liver tumor promotion has been well documented and discussed in Chapter 7 (Bursch et al., 1993; Marsman and Barrett, 1994). It has been shown that PB-promotion does not significantly alter cellular proliferation in the foci above that observed in unpromoted loci; however, the PB-promoted loci do grow more rapidly (Bursch et al., 1984). These conflicting observations were reconciled by the finding that apoptotic frequency within the developing foci is significantly reduced by

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PB. In addition, TGFJ31 has been shown to induce hepatocytes to undergo apoptosis, both in vitro and in vivo (Bursch et al., 1993; Schulte-Hermann et al., 1993). This suggests that the clonal expansion of TGFJ31 negative loci promoted by PB (Jirtle et al., 1994) and peroxisome proliferators (Figures 7A, B) may directly result from a reduced level of active TGFJ31 leading to a reduction in the level of apoptosis. Recently, we have demonstrated that the monoterpene, perillyl alcohol (POH) greatly inhibits the growth and development of rat liver tumors induced with DEN (Mills et al., 1995). The apoptotic index was increased 10-fold in liver tumors from POH-treated animals when compared to tumors from untreated animals; however, there was no effect of POH treatment on the rate of hepatocyte proliferation. POH was also shown to increase the expression of the M6P/IGF2r and the TGFI3 receptors in tumors when compared to these receptor levels in both the surrounding normal tissue and tumors from untreated animals. This demonstrates that within liver tumors POH-treatment causes an elevation in the levels of receptors involved in both TGFJ3 activation and response. Thus, the cytostatic agent, POH, appears to function mechanistically in a manner opposite to that of the tumor promoter, PB, and could therefore be of use in further elucidating the mechanisms by which tumor promoting agents function.

VI.

Summary

Hepatocellular carcinomas are among the most common malignancies in the world with tumor incidence approaching 150 per 100,000 per year in areas such as China. Although the incidence of liver cancer is lower in the United States, 60% of the chemicals determined by the National Toxicology Program to be carcinogens give rise to liver tumors in rats and mice. Interestingly, a number of these agents appear to function through tumor promoting rather than initiating mechanisms. Consequently, to appropriately assess the risk these xenobiotic agents pose to humans, it is necessary to determine the molecular events by which liver tumor promoters enhance the formation of HCC. In this chapter, we provide evidence that liver tumor promotion by PB and possibly other tumor promoting agents is a process of natural selection for cells resistant to the growth inhibitory environment produced by the tumor promoter. Furthermore, experimental results with PB suggest that relative increases in the expression of the M6P/IGF2r, the TGFJ3 receptors, and TGFJ31 in normal versus initiated hepatocytes establish the growth inhibitory pressure in the liver required for promoting agents to selectively enhance the formation of liver tumors. The concept of liver tumor promotion being a process of natural selection for a resistant phenotype has now given rise to a new risk assessment model based on

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n e g a t i v e selection w h i c h m a y b e t t e r p r e d i c t the c a r c i n o g e n i c risk c h e m i c a l a g e n t s p o s e to h u m a n s ( A n d e r s e n et al., in press).

Acknowledgments This research is supported in part by NIH grant CA25951, MITRE grant #67532, and an ILSI post-doctoral fellowship (J.J.M.).

References Abanobi, S. E., Lombardi, B., and Shinozuka, H. (1982). Stimulation of DNA synthesis and cell proliferation in the liver of rats fed a choline-devoid diet and their suppression by phenobarbital. Cancer Res. 42, 412-415. Andersen, M. E., Mills, J. J., Jirtle, R. L., and Greenlee, W. F. (1995). Negative selection in hepatic tumor promotion in relation to cancer risk assessment. Toxicology in press. Bagrodia, S., Chackalaparampil, I., Kmiecik, T. E., and Shalloway, D. (1991). Altered tyrosine 527 phosphorylation and activation of p60c-src. Nature (London) 349, 172-175. Barbason, H., Rassenfosse, C., and Betz, E. H. (1983). Promotion mechanism of phenobarbital and partial hepatectomy in DENA hepatocarcinogenesis cell kinetics effect. Br. J. Cancer 47, 517-525. Barlow, D. P., St6ger, R., Herrmann, B. G., Saito, K., and Schweifer, N. (1991). The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tree locus. Nature (London) 349, 84-87. Alexandrow, M. G., and Moses, H. L. (1995). Transforming growth factor [3 and cell cycle regulation. Cancer Res. 55, 1452-1457. Bartolomei, M. S., Zemel, S., and Tilghman, S. M. (1991). Parental imprinting of the mouse H19 gene. Nature (London) 351, 153-155. Bassing, C. H., Yingling, J. M., Howe, D. J., Wang, T., He, W. W., Gustafson, M. L., Shah, P., Donah6e, P. K., and Wang, X.-E (1994). A transforming growth factor J3 type I receptor that signals to activate gene expression. Science 263, 87-89. Baum, J. K., Holtz, E, Bookstein, J. J., and Klein, E. W. (1973). Possible association between benign hepatomas and oral contraceptives. Lancet 2, 926-929. Beer, D. G., Schwarz, M., Sawada, N., and Pitot, H. C. (1986). Expression of H-ras and c-myc protooncogenes in isolated GGT gamma-glutamyl transpeptidase-positive rat hepatocytes and in hepatocellular carcinomas induced by diethylnitrosamine. Cancer Res. 46, 24352441. Berenblum, I., and Shubik, P. (1947). The role of croton oil application, associated with a single painting of a carcinogen in tumor induction in the mouse skin. Br. J. Cancer 1,379-383. Bischoff, J. R., Friedman, P. N., Marshak, D. R., Prives, C., and Beach, D. (1990). Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2. Proc. Natl. Acad. Sci. U.S.A. 87, 47664770. Bressac, B., Kew, M., Wands, J., and Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature (London) 350, 429-431. Brockenbrough, J. S., Meyer, S. A., Li, C., and Jirtle, R. L. (1991). A reversible and phorbol ester-specific defect of protein kinase C translocation in hepatocytes isolated from phenobarbital-treated rats. Cancer Res. 51, 130-136. Buchkovich, K., Duffy, L. A., and Harlow, E. (1989). The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell (Cambridge, Mass.) 58, 1097-1105.

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Mechanisms of Liver Tumor Promotion

219

Buckley, M. E, Sweeney, K. E, Hamilton, J. A., Sini, R. L., Manning, D. L., Nicholson, R. I., deFazio, A., Watts, C. K., Musgrove, E. A., and Sutherland, R. L. (1993). Expression and amplification of cyclin genes in human breast cancer. Oncogene 8, 2127-2133. Bursch, W., Lauer, B., Timmermann-Trosierner, I., Barthel, G., Schuppler, J., and SchulteHermann, R. (1984). Controlled death (apoptosis) of normal and putative preneoplastic cells in rat liver following withdrawal of tumor promoters. Carcinogenesis 5, 453-458. Bursch, W., Oberhammer, E, and Schulte-Hermann, R. (1992). Cell death by apoptosis and its protective role against disease. Trends Pharmacol. Sci. 13, 245-251. Bursch, W., Oberhammer, E, Jirtle, R. L., Askari, M., Sedivy, R., Grasl-Kraupp, B., Purchio, A. E, and Schulte-Hermann, R. (1993). Transforming growth factor-131 as a signal for induction of cell death by apoptosis. Br. J. Cancer 67, 531-536. Cameron, R. G. (1989). Identification of the putative first cellular step of chemical hepatocaro cinogenesis. Cancer Lett. 47, 163-167. Catherino, W. H., and Jordon, V. C. (1993). A risk-benefit assessment of tamoxifen therapy. Drug Safety 8, 381-397. Cattley, R. C., and Popp, J. A. (1989). Differences between the promoting activities of the peroxisome proliferator Wy-14,643 and phenobarbital in rat liver. Cancer Res. 49, 32463251. Cohen, S. M., Arai, M., Jocobs, J. B., and Friedell, G. H. (1979). Promoting effect of saccharin and Dt-tryptophan in urinary bladder carcinogenesis. Cancer Res. 39, 1207-1217. Columbano, A., Ledda, G. M., Rao, P. M., Rajalakshmi, S., and Sarma, D. S. R. (1982). Initiation of experimental liver carcinogenesis by chemicals: Are the carcinogenic altered hepatocytes stimulated to grow into foci by different selection procedures identical? In "Chemical Carcinogenesis" (C. Nicolini, ed.), pp. 167-178. Plenum, New York. Cote, G. J., Lastra, B. A., Cook, J. R., Huang, D.-P., and Chiu, J.-E (1985). Oncogene expression in rat hepatoma and during carcinogenesis. Cancer Lett. 26, 121-127. Cui, W., Kemp, C. J., Duffle, E., Balmain, A., and Akhurst, R. J. (1994). Lack of transforming growth factor-131 expression in benign skin tumors of p53 null mice is prognostic for a high risk of malignant conversion. Cancer Res. 54, 5831-5836. Dahms, H. M., Wick, D. A., and Brzycki-Wessell, M. A. (1994). The bovine mannose 6-phosphate/insulin-like growth factor II receptor: Localization of the insulin-like growth factor II binding site to domains 5-11. J. Biol. Chem. 269, 3802-3809. Dahms, N. M., Lobel, P., and Kornfeld, S. (1989). Mannose 6-phosphate receptors and lysosomal enzyme targeting. J. Biol. Chem. 264, 12115-12118. Dahms, N. M., Rose, P. A,, Molkentin, J. D., Zhang, Y., and Brzycki, M. A. (1993). The bovine mannose 6-phosphate/insulin-like growth factor II receptor. The role of arginine residues in mannose 6-phosphate binding. J. Biol. Chem. 268, 5457-5463. DeCaprio, J. A., Ludlow, J. W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica-Worms, H., Huang, C.-M., and Livingston, D. M. (1988). SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell (Cambridge, Mass.) 54, 275-283. Dennis, P. A., and Rifkin, D. B. (1991). Cellular activation of latent transforming growth factor 13 requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor. Proc. Natl. Acad. Sci. U.S.A. 88, 580-584. DeSouza, A. T., Hankins, G. R., Washington, M. K., Fine, R. L., Orton, T. C., and Jirtle, R. L. (1995). Frequent loss of heterozygosity at the mannose 6-phosphate/insulin'like growth factor II receptor locus in human hepatocellular tumors. Oncogene, 10, 1725-1729. Diwan, B. A., Rice, J. M., and Ward; J. M. (1986). Tumor-promoting activity of benzodiazapine tranquilizers, diazepam and oxazepam, in mouse liver. Carcinogenesis 7, 789794. Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Jr., Butel,

220

Jeremy J. Mills and Randy L. Jirtle

J. S., and Bradley, A. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature (London) 356, 215-221. Dowdy, S. E, Winds, P. W., Louie, K., Reed, S. I., Arnold, A., and Weinberg, R. A. (1993). Physical interaction of the retinoblastoma protein with human D cyclins. Cell (Cambridge, Mass.) 73, 499-512. Dragan, Y. P., and Pitot, H. C. (1992). The role of the stages of initiation and promotion in phenotypic diversity during hepatocarcinogenesis in the rat. Carcinogenesis 5, 739-750. Dragan, Y. P., Laufer, C., Koleske, A. J., Drinkwater, N., and Pitot, H. C. (1993). Quantitative comparison of initiation and mutation phenotypes in hepatocytes of the analbuminemic rat. Jpn. J. Cancer Res. 84, 175-183. Dulic, V., Lee, E., and Reed, S. I. (1989). Association of human cyclin E with a periodic G1-S phase protein kinase. Science 243, 934-937. Dyson, N., Howley, P. M., Munger, K., and Harlow, E. (1989). The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma susceptibility gene product. Science 243, 934-937. Eckl, P. M., Meyer, S. A., Whitcombe, W. R., and Jirtle, R. L. (1988). Phenobarbital reduces EGF receptors and the ability of physiological concentrations of calcium to suppress hepatocyte proliferation. Carcinogenesis 9, 479-483. EI-Deiry, W. S., Harper, J. W., O'Connor, P. M., Velculescu, V. E., Canman, C. E., Jackman, J., Pietenpol, J. A., Burrell, M., Hill, D. E., Wang, Y., Wiman, K. G., Mercer, W. E., Kastan, M. B., Kohn, K. W., Elledge, S. J., Kinzler, K. W., and Vogelstein, B. (1994). WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 54, 1169-1174. Elledge, S. J., and Harper, J. W. (1994) Cdk inhibitors: On the threshold of checkpoints and development. Curr. Opin. Cell Biol. 6, 847-852. Embleton, M. J., and Butler, P. C. (1988). Reactivity of monoclonal antibodies to oncoproteins with normal rat liver, carcinogen-induced tumors and premalignant lesions. Br. J. Cancer 57, 48-53. Farber, E. (1986). Some emerging general principles in the pathogenesis of hepatocellular carcinoma. Cancer Surv. 5,695-718. Farber, E. (1991). Hepatocyte proliferation in stepwise development of experimental liver cell cancer. Dig. Dis. Sci. 36, 973-978. Friedwald, W. E, and Rous, P. (1944). The initiating and promoting elements in tumor promotion. J. Exp. Med. 80, 101-125. Gentry, L. E., and Nash, B. W. (1990). The pro domain of pre-pro-transforming growth factor 131 when independently expressed is a functional binding protein for the mature growth factor. Biochemistry 29, 6851-6857. Gerbracht, U., Bursch, W., Kraus, P., Putz, B., Reinacher, M., Timmermann-Trosiener, I., and Schulte-Hermann, R. (1990). Effects of hypolipidemic drugs nafenopin and clofibrate on phenotypic expression and cell death (apoptosis) in altered foci of rat liver. Carcinogenesis 11, 617-624. Glick, A. B., Kulkarni, A. B., Tennenbaum, T., Hennings, H., Flanders, K. C., O'Reilly, M., Sporn, M. B., Karlsson, S., and Yuspa, S. H. (1993). Loss of expression of transforming growth factor 13 in skin and skin tumors is associated with hyperproliferation and a high risk for malignant conversion. Proc. Natl. Acad. Sci. U.S.A. 90, 6076-6080. Goldsworthy, T. L., Hanigan, M. H., and Shinozuka, H. (1986). Models of hepatocarcinogenesis in the rat--contrasts and comparisons. Crit. Rev. Toxicol. 17, 61-89. Goyette, M. C., Cho, K., Fasching, C. L., Levy, D. B., Kinzler, K. W., Paraskeva, C., Vogelstein, B., and Stanbridge, E. J. (1992). Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol. Cell. Biol. 12, 1387-1395. Grasl-Kraup, B., Bursch, W., Ruttkay-Nedecky, N., Wagner, A., Lauer, B., and Schulte-

9.

Mechanisms of Liver Tumor Promotion

221

Hermann, R. (1994). Food reduction eliminates preneoplastic cells through apoptosis and antagonizes carcinogenesis in rat liver. Proc. Natl. Acad. Sci. U.S.A. 91, 9995-9999. Hannon, G. J., and Beach, D. (1994). p15 INKaB is a potential effector of TGF~3-induced cell cycle arrest. Nature (London) 371,257-261. Hartwell, L. H., and Weinert, T. A. (1989). Checkpoints: Controls that ensure the order of cell cycle events. Science 246, 629-634. Hartwell, L. H. (1992). Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell (Cambridge, Mass.) 71, 543-546. Heichman, K. A., and Roberts, J. M. (1994). Rules to replicate by. Cell (Cambridge, Mass.) 79, 547-550. Hendrich, S., Glauert, H. P., and Pitot, H. C. (1986). The phenotypic stability of altered hepatic loci: Effects of withdrawal and subsequent readministration of phenobarbital. Carcinogenesis 7, 2041-2045. Hiebert, S. W., Chellappan, S. P., Horowitz, J. M., and Nevins, J. R. (1992). The interaction of Rb with EZF coincides with an inhibition of the transcriptional activational activity of EZF. Genes Dev. 6, 177-185. Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., and Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature (London) 350, 427-428. Huang, H.-J., Yee, J.-K., Shew, J.-Y., Chen, P.-L., Bookstein, R., Friedman, T., Lee, E. Y., and Lee, W. H. (1988). Suppression of the neoplastic phenotype by replacement of the Rb gene in human cancer cells. Science 242, 1563-1566. Hunter, T., and Pines, J. (1991). Cyclins and cancer. Cell (Cambridge, Mass.) 66, 10711074. Hunter, T., and Pines, J. (1994). Cyclins and cancer II: Cyclin D and CDK inhibitors come of age. Cell (Cambridge, Mass.) 79, 573-582. Hunter, T. (1993). Braking the cycle. Cell (Cambridge, Mass.) 75, 839-841. Jiang, W., Kahn, S. N., Tomita, N., Zhang, Y. J., Lu, S. H., and Weinstein, I. B. (1992). Amplication and expression of the human cyclin D gene in esophageal cancer. Cancer Res. 52, 2980-2983. Jirtle, R. L., and Meyer, S. A. (1991). Liver tumor promotion: Effect of phenobarbital on EGF and protein kinase C signal transduction and transforming growth factor-~l expression. Dig. Dis. Sci. 36, 659-668. Jirtle, R. L., Carr, B. I., and Scott, C. D. (1991a). Modulation of insulin-like growth factor/mannose-6-phosphate receptors and transforming growth factor-J31 during liver regeneration. ]. Biol. Chem. 266, 22444-22450. Jirtle, R. L., Meyer, S. A., and Brockenbrough, J. S. (1991b). Liver tumor promoter phenobarbital: A biphasic modulator of hepatocyte proliferation. In "Chemically Induced Cell Proliferation Implications for Risk Assessment" (B. E. Butterworth, T. J. Slaga, W. Farland, and M. McClain, eds.), pp. 209-216. Wiley-Liss, New York. Jirtle, R. L., Hankins, G. R., Reisenbichler, H., and Boyer, I. J. (1994). Regulation of mannose 6-phosphate/insulin-like growth factorolI receptors and transforming growth factor beta during liver tumor promotion with phenobarbital. Carcinogenesis 15, 1473-1478. Kalscheuer, V. M., Mariman, E. C., Schepens, M. T., Rehder, H., and Ropers, H.-H. (1993). The insulin-like growth factor type-2 receptor gene is imprinted in the mouse but not in humans. Nat. Genet. 5, 74-78. Kankaki, T., Olofsson, A., Moren, A., Wernstedt, C., Hellman, U., Miyazono, K., ClaessonWelsh, L., and Heldin, C.-H. (1990). TGFq31 binding protein: A component of the large latent complex of TGF-~I with multiple repeat sequences. Cell (Cambridge, Mass.) 61, 1051-1061. Karsten, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig, R. W. (1991). Partici-

222

Jeremy J. Mills and Randy L. Jirtle

pation of p53 protein in the cellular response to DNA damage. Cancer Res. 51, 63046311. Kato, J., Matsushime, H., Hiebert, S. W., Ewen, M. E., and Sherr, C. J. (1993). Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev. 7, 331-342. Kaufmann, W. K., Mackenzie, S. A., Rahija, R. J., and Kaufman, D. G. (1986). Quantitative relationship between initiation of hepatocarcinogenesis and induction of altered cell islands. J. Ceil. Biochem. 30, 1-9. Kerr, J. E R., Wyllie, A. H., and Currie, A. R. (1972). Apoptosis: A basic biological phenomenon with wide-ranging implications for tissue kinetics. Br. J. Cancer 26, 239-257. Kiess, W., Greenstein, L. A., White, R. M., Lee, L., Rechler, M. M., and Nissley, S. P. (1987). Type II insulin-like growth factor receptor is present in rat serum. Proc. Natl. Acad. Sci. U.S.A. 84, 7720-7724. Kojima, S., Nara, K., and Rifkin, D. B. (1993). Requirement for transglutaminase in the activation of latent transforming growth factor-~3 in bovine endothelial cells. J. Cell Biol. 121,439-448. Kornfeld, S. (1992). Structure and function of the mannose 6-phosphate/insulin-like growth factor II receptors. Annu. Rev. Biochem. 61,307-330. Kovacina, K. S., Steele-Perkins, G., Purchio, A. E, Lioubin, M., Miyazono, K., Heldin, C.-H., and Roth, R. A. (1989). Interactions of recombinant and platelet transforming growth factor-131 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor. Biochem. Biophys. Res. Commun. 160, 393-403. Laconi, E., Denda, A., Rao, P. M., Rajalakshmi, S., Pani, P., and Sarma, D. S. R. (1993). Studies on liver tumor promotion in the rat by orotic acid: Dose and minimum exposure time required for dietary orotic acid to promote hepatocarcinogenesis. Carcinogenesis 14, 17711775. Laiho, M., DeCaprio, J. A., Ludlow, J. W., Livingston, D. M., and MassaguS, J. (1990). Growth inhibition by TGF-IB linked to suppression of retinoblastoma protein phosphorylation. Cell (Cambridge, Mass.) 62, 175-185. Lee, S. J., and Nathans, D. (1988). Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors. J. Biol. Chem. 263, 3521-3527. Lee, W.-H., Shew, J.-Y., Hong, E D., Sery, T. W., Donoso, L. A., Young, L. J., Bookstein, R., and Lee, E. Y. (1987). The retinoblastoma gene encodes a nuclear phophoprotein associated with DNA binding activity. Nature (London) 329, 642-645. Left, S. E., Brannan, C. I., Reed, M. L., Ozcelik, T., Francke, U., Copeland, N. G., and Jenkins, N. A. (1992). Maternal imprinting of mouse Snrpn gene and conserved linkage homology with the human Prader-Willi syndrome region. Nat. Genet. 4, 259-264. Levine, B., Huang, Q., Isacs, J. T., Reed, J. C., Griffin, D. E., and Hardwick, J. M. (1993). Conversion of lytic to persistent alphavirus infection by bcl-2 cellular oncogene. Nature (London) 361, 739-742. Li, E., Beard, C., and Jaenisch, R. (1993). Role of DNA methylation in genomic imprinting. Nature (London) 366, 362-365. Lin, D., Shields, M. T., Ullrich, S. J., Appella, E., and Mercer, W. E. (1992a). Growth arrest induced by wild-type p53 protein blocks cells prior to or near the restriction point in late G1 phase. Proc. Natl. Acad. Sci. U.S.A. 89, 9210-9214. Lin, H. Y., Wang, X.-E, Hg-Eaton, E., Weinberg, R. A., and Lodish, H. E (1992b). Expression cloning of the TGF-f3 type II receptor, a functional transmembrane serine/threonine kinase. Cell (Cambridge, Mass.) 68, 775-785. Lindroos, P., Tsai, W. H., Zarnegar, R., and Michalopoulos, G. K. (1992). Plasma levels of HGF in rats treated with tumor promoters. Carcinogenesis 13, 139-141. Liu, J.-P., Baker, J., Perkins, A. S., Robertson, E. J., and Efstratiadis, A. (1993). Mice carrying

9.

Mechanisms of Liver Tumor Promotion

223

null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igflr). Cell (Cambridge, Mass.) 75, 59-72. Lopez-Cassillas, E, Cheifetz, S., Doody, J., Andres, J. L., Lane, W. S., and MassaguS, J. (1991). Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-~3 receptor system. Cell (Cambridge, Mass.) 67, 785-795. Lopez-Casillas, E, Wrana, J. L., and MassaguS, J. (1993). Betaglycan presents ligand to the TGFf3 signaling receptor. Cell (Cambridge, Mass.) 73, 1435-1444. Ludlow, J. W., DeCaprio, J. A., Huang, C.-M., Lee, W.-H., Paucha, E., and Livingston, D. M. (1989). SV40 large T antigen binds preferentially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell (Cambridge, Mass.) 56, 57-65. Ludlow, J. W., Shon, J., Pipas, J. M., Livingston, D. M., and DeCaprio, J. A. (1990). The retin~ oblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation binding to and release from SV40 large T-antigen. Cell (Cambridge, Mass.) 60, 387-396. Lyons, R. M., Gentry, L. E., Purchio, A. E, and Moses, H. L. (1990). Mechanism of activation of latent recombinant transforming growth factor ~ 1 by plasmin. J. Cell Biol. 110,1361-1367. MacDonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P., and Ullrich, A. (1988). A single receptor binds both insulin-like growth factor II and mannose-6-phosphate. Science 239, 1134-1137. Mailer, J. L. (1991). Mitotic control. Curr. Opin. Cell Biol. 3, 269-275. Manjeshwar, S., Laconi, E., Rao, P. M., Rajalakshmi, S., and Sarma, D. S. R. (1993). Influence of orotic acid on multistage hepatocarcinogenesis in the rat: Resistance of hepatocytes from nodules to the mitoinhibitory effects of orotic acid. Proc. Soc. Exp. Biol. Med. 202, 25-29. Marsman, D. S., and Barrett, J. C. (1994). Apoptosis and chemical carcinogenesis. Risk Anal. 14, 321-326. MassaguS, J. (1990). The transforming growth factor-f3 family. Annu. Rev. Cell Biol. 6, 597641. MassaguS, J., Cheifetz, S., Boyd, E T., and Andres, J. L. (1993). TGF-~ receptors and TGF-~3 binding proteoglycans: Recent progress in identifying their functional properties. Ann. N.Y. Acad. Sci. 593, 59-72. Matsuno, Y., Hirohashi, S., Furuya, S., Sakamoto, M., Kiyoshi, M., and Shimosato, Y. (1990). Heterogeneity of proliferative activity in nodule-in-nodule lesions of small hepatocellular carcinoma. Jpn. J. Cancer Res. 81, 1137-1140. Meites, J. (1972). Relation of prolactin and estrogen to mammary tumorigenesis in the rat. J. Natl. Cancer Inst. 48, 1217-1224. Meyer, S. A., and Jirtle, R. L. (1989). Phenobarbital decreases hepatocyte EGF receptor expression independent of protein kinase C activation. Biochem. Biophys. Res. Commun. 158, 652-659. Mills, J. J., Chari, R. S., Boyer, I. J., Gould, M. N., and Jirtle, R. L. (1995). Induction of apoptosis in liver tumors by the monoterpene perillyl alcohol. Cancer Res. 55, 979-983. Moore, M. A., Nakagawa, K., Satoh, K., Ishikawa, T., and Sato, K. (1987). Single GST-P positive liver cells-putative initiated hepatocytes. Carcinogenesis 10, 2107-2111. Motokura, T., and Arnold, A. (1993). Cyclins and oncogenesis. Biochim. Biophys. Acta Rev. Cancer 1155, 63-78. Motokura, T., Bloom, T., Kim, H. G., Juppner, H., Ruderman, J. V., Kronenberg, H. M., and Arnold, A. (1991). A BCL-1 linked candidate oncogene which is rearranged in parathyroid tumors encodes a novel cyclin. Nature (London) 334, 503-508. Ogawa, O., McNoe, L. A., Eccles, M. R., Morison, I. M., and Reeve, A. E. (1993). Human insulin-like growth factor type I and type II receptors are not imprinted. Hum. Mol. Genet. 2, 2163-2165. Okuda, K. (1992). Hepatocellular carcinoma: Recent progress. Hepatology (Baltimore) 15, 948-963.

224

Jeremy J. Mills and Randy L. Jirtle

Pardee, A. (1989). G1 events and regulation of cell proliferation. Science 246, 603-608. Peraino, C., Fry, R. J. M., and Staffeldt, E. (1971). Reduction and enhancement by phenobarbital of hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res. 31, 1506-1512. Peraino, C., Fry, R. J. M., Staffeldt, E., and Christopher, J. P. (1975). Comparative enhancing effects of phenobarbital, amobarbital, diphenylhydantoin, and dichlorodiphenyltrichloroethane on 2-acetylaminofluorene-induced hepatic tumorigenesis in the rat. Cancer Res. 35, 2884-2890. Peraino, C., Staffeldt, E. E, and Ludeman, V. A. (1981). Early appearance of histochemically altered hepatocyte foci and liver tumors in female rats treated with carcinogens one day after birth. Carcinogenesis 5,463-465. Pereira, M. A. (1983). Rat liver foci bioassay. J. Am. Coll. Toxicol. 1, 101-117. Peters, G. (1994). Stifled by inhibitions. Nature (London) 371, 204-205. Picksley, S. M., and Lane, D. P. (1994). p53 and Rb: Their cellular roles. Curr. Opin. Cell Biol. 6, 853-858. Pietenpol, J. A., and Vogelstein, B. (1993). Tumour suppressor genes: No room at the p53 inn. Nature (London) 365, 17-18. Pitot, H. C. (1993). The molecular biology of carcinogenesis. Cancer 72(Suppl.), 962-970. Pitot, H. C., Barsness, L., Goldsworthy, T., and Kitagawa, T. (1978). Biochemical characterization of stages of hepatocarcinogenesis after a single dose of diethylnitrosamine. Nature (London) 271,456-458. Pitot, H. C., Goldsworthy, T. L., Campbell, H. A., and Poland, A. (1980). Quantitative evaluation of the promotion by 2,3,7,8-tetrachlorordibenzo-p-dioxin of hepatocarcinogenesis from diethylnitrosamine. Cancer Res. 40, 3616-3620. Pitot, H. C., Goldsworthy, T. L., Moran, S., Kennan, W., Glauert, H. P., Maronpot, R., and Campbell, H. A. (1987). A method to quantitate the relative initiating and promoting potencies of hepatocarcinogenic agents in their dose-response relationships to altered foci. Carcinogenesis 8, 1491-1499. Pitot, H. C., Dragan, Y., Sargent, L., and Xu, Y.-H. (1991). Biochemical markers associated with the stages of promotion and progression during hepatocarcinogenesis in the rat. Environ. Health Perspect. 93, 181-189. Polyak, K., Kato, J. Y., Solomon, M. J., Sherr, C. J., Massagu~, J., and Roberts, J. M. (1994). p27Kip2, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 8, 9-22. Preat, V., Lans, M., de Gerlache, J., Taper, H., and Roberfroid, M. (1987). Influence of the duration and the delay of administration of phenobarbital on its modulating effect on rat hepatocarcinogenesis. Carcinogenesis 8, 333-335. Purchio, A. E, Cooper, J. A., Brunner, A. M., Lioubin, M. N., Gentry, L. E., Kovacina, K. S., Roth, R. A., and Marquardt, H. (1988). Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta-1 precursor. J. Biol. Chem. 263, 14211-14215. Reddy, B. S., Watanabe, J. H., Weisberger, J. H., and Wynder, E. L. (1977). Promoting effects of bile acids in colon carcinogenesis in germ-free and conventional F344 rats. Cancer Res. 37, 3238-3242. Reisenbichler, H., Chari, R. S., Boyer, I. J., and Jirtle, R. L. (1994). Transforming growth factor-beta receptors type I, II and III in phenobarbital-promoted rat liver tumors. Carcinogenesis 15, 2763-2767. Richmond, R. E., Pereira, M. A., Carter, J. H., Carter, H. W., and Long, R. E. (1988). Quantitative and qualitative immunohistochemical detection of myc and src oncogene proteins in normal, nodule and neoplastic rat liver. J. Histochem. Cytochem. 36, 179-184. Rumsby, P. C., Davies, M. J., Price, R. J., and Lake, B. G. (1994). Effect of some peroxisome

9.

Mechanisms of Liver Tumor Promotion

225

proliferators on transforming growth factor-131 gene expression and insulin-like growth factor II/mannose-6-phosphate receptor gene expression in rat liver. Carcinogenesis 15, 419-421. Sager, R. (1992). Tumor suppressor genes in the cell cycle. Curr. Opin. Cell Biol. 4, 155-160. Sargent, L., Xu, Y.-H., Sattler, G. L., Meisner, L., and Pitot, H. C. (1989). Ploidy and karyotype of hepatocytes isolated from enzyme-altered loci in two different protocols of multistage hepatocarcinogenesis in the rat. Carcinogenesis 10, 387-391. Scherer, E. (1987). Relationship among histochemically distinguishable early lesions in multistep-multistage hepatocarcinogenesis. Arch. Toxicol. 10(Suppl.), 81-94. Schulte-Hermann, R., Timmermann-Trosiener, I., and Schuppler, J. (1986). Facilitated expression of adaptive responses to phenobarbital in putative pre-stages of liver cancer. Carcinogenesis 7, 1651-1655. Schulte-Hermann, R., Bursch, W., Kraupp-Grasl, B., Wagner, A., and Jirtle, R. (1993). Cell proliferation and apoptosis in normal liver and preneoplastic loci. Environ. Health Perspect. 101(Suppl. 5), 87-90. Segarini, P. R. (1993). TGF-13 receptors: A complicated system of mulxiple binding proteins. Biochim. Biophys. Acta Rev. Cancer 1155, 269-275. Seo, M. K., Lynch, K. E., and Podolsky, D. K. (1988). Multiplicity of transforming growth factors in human malignant effusions. Cancer Res. 48, 1792-1797. Sherr, C. J. (1994). G1 phase progression: Cycling on cue. Cell (Cambridge, Mass.) 79, 551555. Sklar, M. M., Kiess, W., Thomas, C. L., and Nissley, S. P. (1989). Developmental expression of the tissue insulin-like growth factor II/mannose 6-phosphate receptor in the rat: Measurement by quantitative immunoblotting. J. Biol. Chem. 264, 16733-16738. Sklar, M. M., Thomas, C. L., Municchi, G., Roberts, C. T., Jr., LeRoith, D., Kiess, W., and Nissley, P. (1992). Developmental expression of rat insulin-like growth factor-II/mannose 6-phosphate receptor messenger ribonucleic acid. Endocrinology (Baltimore) 130, 34843491. Slaga, T. J., Fischer, S. M., Weeks, C. E., Klein-Szanto, A. J. P., and Reiners, J. (1982). Studies on the mechanisms involved in multistage carcinogenesis in mouse skin. J. Cell Biol. 18, 99-119. Solt, D., and Farber, E. (1976). New principle for the analysis of chemical carcinogenesis. Nature (London) 263, 701-703. Solt, D. B., Cayama, E., Sarma, D. S. R., and Farber, E. (1980). Persistence of resistant putative preneoplastic hepatocytes induced by N-nitrosodiethylamine or N-methyl-N-nitrosourea. Cancer Res. 40, 1112-1118. St6ger, R., Kubicka, P., Liu, C.-G., Kafri, T., Razin, A., Cedar, H., and Barlow, D. P. (1993). Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell (Cambridge, Mass.) 73, 61-71. Sue, S. R., Chari, R. S., Kong, E-M., Mills, J. J., Fine, R. L., Jirtle, R. L., and Meyers, W. C. (1995). Transforming growth factor beta receptors and mannose 6-phosphate/insulin-like growth factor II receptor expression in human hepatocellular carcinomas. Ann. Surg. in press. Teebor, G. W., and Becker, E E (1988). Regression and resistance of hyperplastic nodules induced by N-2-fluoroacetylamide and there relationship to hepatocarcinogenesis. Cancer Res. 31, 1-3. Tsuji, S., Ogawa, K., Takasaka, H., Sonoda, T., and Mori, M. (1988). Clonal origin of gammaglutamyl transpeptidase positive lesions induced by initiation-promotion in ornithine carbamyltransferase mosaic mice. Jpn. J. Cancer Res. 79, 148-151. Vogelstein, B., and Kinzler, K. W. (1992). p53 function and dysfunction. Cell (Cambridge, Mass.) 70, 523-526.

226

Jeremy J. Mills and Randy L. Jirtle

Vogelstein, B., and Kinzler, K. W. (1993). The multistep nature of cancer. Trends Genet. 4, 138-141. Wang, J., Chenivesse, X., Henglein, B., and Brechot, C. (1990). Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma. Nature (London) 343, 317-322. Wang, X.-E, Lin, H. Y., Ng-Eaton, E., Downward, J., Lodish, H. E, and Weinberg, R. A. (1991). Expression cloning and characterization of the TGF-IB type III receptor. Cell (Cambridge, Mass.) 67, 797-805. Wrana, J. L., Attisano, L., Wieser, R., Ventura, E, and Massagu6, J. (1994). Mechanism of activtion of the TGF-13 receptor. Nature (London) 370, 341-347. Weintraub, S. J., Prater, C. A., and Dean, D. C. (1992). Retinoblastoma protein switches the E2F site from positive to negative element. Nature (London) 358, 259-261. Westlund, B., Dahms, N. M., and Kornfeld, S. (1991). The bovine mannose 6-phosphate/ insulin-like growth factor II receptor. Localization of mannose 6-phosphate binding sites to domains 1-3 and 7-11 of the extracytoplasmic region. J. Biol. Chem. 266, 23233-23239. Williams, G. M., Iatropoulos, M. J., Djordjevic, M. V., and Kaltenberg, O. P. (1993). The triphenylethylene drug tamoxifen is a strong liver carcinogen in the rat. Carcinogenesis 14, 315-317. Williams, G. T., and Smith, C. A. (1993). Molecular regulation of apoptosis: Genetic controls on cell death. Cell (Cambridge, Mass.) 74, 777-779. Witschi, H. D., Williamson, D., and Lock, S. (1977). Enhancement of urethan tumorigenesis in mouse lung by butlylated hydroxytoluene. J. Natl. Cancer Inst. 58, 301-305. Wyllie, A. H., Kerr, J. E R., and Currie, A. R. (1980). Cell death: The significance of apoptosis. Int. Rev. Cytol. 68, 251-300. Xu, Y., Goodyer, C. G., Deal, C., and Polychronakos, C. (1993). Functional polymorphism in the parental imprinting of the human IGF2R gene. Biochem. Biophy. Res. Commun. 197, 747-754. Yager, J. D., Jr., and Yager, R. (1980). Oral contraceptive steroids as promoters of hepatocarcinogenesis in female Sprague-Dawley Rats. Cancer Res. 40, 3680-3685. Yager, J. D., Roebuck, B. D., Paluszcyk, T. L., and Memoli, V. A. (1986). Effects of ethinyl estradiol and tamoxifen on liver DNA turnover and new synthesis and appearance of gamma glutamyl transpeptidase-positive foci in female rats. Carcinogenesis 7, 2007-2014. Yager, J. D., Zurlo, J., Sewall, C. H., Lucier, G. W., and He, H. (1994). Growth stimulation followed by growth inhibition in livers of female rats treated with ethinyl estradiol. Carcinogenesis 15, 2117-2124. Yelandi, A. V., Subbarao, V., Rajan, A., Reddy, J. K., and Rao, M. S. (1989). Gammaglutamyltranspeptidase-negative phenotypic property of preneoplastic and neoplastic liver lesions induced by ciprofibrate does not change following 2-acetylaminofluorene administration. Carcinogenesis 10, 797-799. Yonish-Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A., and Oren, M. (1991). Wildtype p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature (London) 352, 345-347. Zakeri, Z., and Lockshin, R. A. (1994). Physiological cell death during development and its relationship to aging. Ann. N.Y. Acad. Sci. 31,212-229. Zhang, X., Wang, T., Batist, G., and Tsao, M.-S. (1994). Transforming growth factor 131 promotes spontaneous transformation of cultured rat liver epithelial cells. Cancer Res. 54, 6122-6128.

10 Hypomethylation of DNA: An Ep"lgene"tiC Mechanism That Can Facilitate the Aberrant Oncogene Expression Involved in9 Liv e r Carc'nogene" 1 sis Jennifer L. Counts Jay I. Goodman Department of Pharmacology and Toxicology Michigan State University East Lansing, Michigan 48824

I. Introduction Carcinogenesis is a multistage, multistep process consisting of at least three experimentally defined stages: initiation, promotion, and progression (Pitot, 1990; Dragan et al., 1993). In experimental models it is possible to demonstrate that a chemical exerts its carcinogenic effect by acting at a particular stage. For example, many chemicals (but not all) that act as mutagens are effective initiating agents. The initiation stage is defined as an irreversible change to the DNA base sequence due to the interaction of a chemical with DNA. Promoting agents are capable of facilitating the clonal expansion of initiated cells potentially leading to a tumor by increasing cell proliferation (Butterworth et al., 1991) and/or by decreasing apoptosis (SchulteHermann et al., 1990; Bursch et al., 1993). The potential exists for the development of spontaneous mutations (some of which may result in initiation) during periods of cell proliferation e.g., compensatory hyperplasia in response to necrosis (Ames and Gold, 1990) and following treatment with Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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promoting agents. The progression stage of carcinogenesis is characterized by changes in ploidy and autonomous clonal expansion. The terms initiation and promotion refer to modes of action (McClain, 1994). Initiating agents act by a genotoxic mechanism, i.e., either the chemical or one of its metabolites reacts directly with DNA (Williams, 1980). Chemicals that form DNA adducts and have the potential to act as initiating agents have been reviewed in the literature and the reader is referred to several papers for a thorough discussion of that subject (Williams, 1989; Benigni and Andreoli, 1993; Matthews et al., 1993; Sullivan et al., 1993; Chang et al., 1994; Miller, 1994). There is a need to better understand nongenotoxic mechanisms of carcinogenesis. Promoting agents are nongenotoxic, that is, neither the compound nor any of its metabolites directly interacts with DNA (Williams, 1980). We describe here how alterations in DNA methylation, most commonly hypomethylation, can act as an epigenetic, nongenotoxic mechanism to facilitate the aberrant expression of oncogenes that is involved in carcinogenesis. As stated above, nongenotoxic carcinogens do not directly interact with DNA. These compounds may exert their effects in a variety of ways (Williams and Weisburger, 1991; McClain, 1994), for example, by causing cytotoxicity or chronic tissue irritation, enhancing cell proliferation, inducing hormone imbalances, altering immune function, inhibiting normal cellcell communication, or facilitating aberrant gene expression, possibly as a consequence of altering DNA methylation status (Goodman and Counts, 1993; Counts and Goodman, 1994c). DNA methylation plays a role in the regulation of gene activity. Hypomethylation [i.e., decreased 5-methylcytosine (5MeC) content] of a gene is necessary, but not sufficient, for its expression; therefore, a hypomethylated gene can be considered to possess an increased potential for expression compared to a hypermethylated gene (Vorce and Goodman, 1989). Alterations in DNA methylation can enhance the expression of oncogenes and/or interfere with the expression of tumor suppressor genes. This can occur via hypomethylation of DNA (a process that is discussed below) that facilitates increased, aberrant expression of dominant-acting oncogenes and/or by hypermethylation of DNA facilitating aberrant, decreased expression of recessive-acting tumor suppressor genes. Hypomethylation may be a mechanism underlying the role of cell proliferation in carcinogenesis (Goodman and Counts, 1993), and hypomethylation could possibly result from an enzymatic replacement of 5MeC with cytosine that is not linked to DNA replication (Vairapandi and Duker, 1993). The mechanisms believed to be involved in altered DNA methylation will be discussed in this chapter. Additionally, the importance of the presence of the DNA-(cytosine-5)methyltransferase (DNA MTase) gene will be discussed in the context of DNA MTase knockout mice, and emphasis will be placed on the role that

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hypomethylation of D N A may play in tumor promotion (Counts and Goodman, 1994c, 1995). The presence of D N A adducts (e.g., O6-methyl guanine) can interfere with the ability of the maintenance methylase to bind D N A at a hemimethylated site and restore the normal 5MeC content after D N A replication (Weitzman et al., 1994). Thus, we can envision a situation where genotoxic compounds can alter the methylation status of D N A in addition to potential mutagenic effects. With the possible exception of tumor suppressor genes, a mutated gene must be expressed in order to alter the phenotype of a cell. Thus, hypomethylation and mutation can be viewed as complementary with regard to facilitating aberrant gene expression. A schematic illustrating the interplay between factors influencing mutation and hypomethylation of DNA, and how these can lead to aberrant gene expression is provided (Figure 1). Our experimental model is mouse liver tumorigenesis. We focus on the oncogenes that are relevant to this system: Ha-ras and raf. We employ the liver tumor prone B6C3F1 (C57BL/6 female x C 3 H / H e male) mouse and make relevant comparisons with the sensitive C 3 H / H e paternal strain and the resistant C57BL/6 maternal strain (Goodman et al., 1991; Goodman and Counts, 1993; Counts and Goodman, 1995). The degree of sensitivity of the parental strains is heritable and is determined largely by a genetic locus, the hepatocarcinogen sensitivity locus (Hanigan et al., 1988). An elegant study involving C3H/HeN~-~C57BL/6N chimeric mice demonstrated that the different sensitivities of these strains to tumor development exists in the target cells (e.g., a tumor susceptibility gene) and is not milieu (e.g., hormone) dependent (Lee et al., 1991). A unique aspect of our research is that it offers the potential to provide insight regarding molecular

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mechanisms that underlie promotion of carcinogenesis, while at the same time, the results can provide the type of information that is required to take a more rational approach to carcinogen risk assessment. Specifically, it can provide insight regarding dose-response relationships, the existence of thresholds, and species-to-species extrapolation issues (Counts and Goodman, 1994c, 1995). Our rationale for choosing specific oncogenes to study is based on the high involvement of Ha-ras in mouse liver tumors in those strains that are highly susceptible to liver tumorigenesis (e.g., B6C3F1 and C3H/He) (Buchmann et al., 1991). However, approximately one-third to one-half of the liver tumors in B6C3F1 mice do not appear to involve an activated Ha-ras as assessed by mutation (Buchmann et al., 1991; Reynolds et al., 1987) or by the ability of DNA from these tumors to transform NIH-3T3 cells (Reynolds et al., 1987). Thus, gene products other than, or in addition to, that of Ha-ras are likely to be involved in mouse liver tumorigenesis. The rafgene is a reasonable candidate in this regard because it is another oncogene involved in signal transduction to the nucleus.

II. Epigenetics A thorough consideration of inheritance involves making a distinction between the transmission of genes from generation to generation (i.e., inheritance of DNA base sequence) and the mechanisms involved in the transmission of alternative states of gene activity following cell division. Epigenetics is the term used to describe the latter. It may be defined as the study of mechanisms responsible for the temporal and spatial control of gene activity, e.g., changes in gene expression during development, segregation of gene activities such that daughters of an individual cell have different patterns of gene expression, and mechanisms to permit the somatic inheritance of a specific set of active and quiescent genes. DNA methylation is one epigenetic mechanism by which gene activity, including genomic imprinting (Brandeis et al., 1993; Kafri et al., 1993), may be regulated (Holliday, 1990). Changes in the methylation status of a gene provide a mechanism by which its potential for expression can be altered in an epigenetic heritable manner (Holliday, 1990; Jones and Buckley, 1990) and, in light of the enzymatic steps involved in DNA maintenance methylation, it is expected that modifications in DNA methylation would result from threshold-exhibiting events.

III. DNA Methylation DNA methylation (i.e., the 5MeC content of DNA) plays a role in the regulation of gene activity (Razin and Cedar, 1991). Cytosine methylation

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produces a localized pattern of steric, hydrophobic, energetic, conformational, and electrostatic alterations in DNA, primarily in the major grove where most protein interactions with DNA occur (Hausheer et al., 1989); methylation enhances while demethylation reduces stability of the DNA helix (Murchie and Lilly, 1989; Xodo et al., 1994). Differential methylation of DNA is a determinant of higher order chromatin structure (Lewis and Bird, 1991). In addition, the methyl group provides a chemical signal which is recognized by trans-acting factors. Binding or lack of binding of these factors regulate transcription (Holliday, 1990). While it is generally recognized that key cis elements controlling gene expression are located in the 5' flanking region (Jones, 1986), critical domains involved in the regulation of transcription may also be located all along the gene, e.g., toward the middle (Yisraeli et al., 1988; Miinzel et al., 1991; Opdencamp et al., 1992) and at the 3' region (Spandidos and Holmes, 1987; Cohen and Levinson, 1988; Sharrard et al., 1992). Methylation can inhibit transcription in three possible ways, that are not mutually exclusive: (1) methylation may affect transcription by altering chromatin structure (Adams, 1990), (2) 5MeCpG can interfere with transcription directly as methylation in the recognition site might prevent transcription factors from binding to their cognate sequences, or (3) proteins in the nucleus that bind specifically to methylated DNA, e.g., methylated DNA binding protein (MDBP) (Zhang et al., 1990) and methyl-CpG binding proteins (MeCP-1 and MeCP-2) (Boyes and Bird, 1991), may block access of transcription factors. There is evidence in mammalian cells supporting the role for mechanisms 2 and 3 noted above which involve direct or indirect inhibition of transcription factor binding (Meehan et al., 1992). Thus, DNA methylation appears to be a mechanism whereby cells can control the expression of genes with similar promoter regions in the presence of ubiquitous transcription factors (Jones and Buckley, 1990). 5MeC is most often found in DNA at CpG residues and each site is modified on both strands (Razin and Cedar, 1991), i.e., DNA methylation is symmetrical. During periods of cell proliferation the established pattern of DNA methylation is maintained by the action of an S-adenosylmethionine (SAM)-requiring maintenance methylase (Figure 2). 5MeC is not a substrate for nucleoside monophosphate kinase (Vilpo and Vilpo, 1993) and a 5MeC deaminase can metabolize free 5MeC to thymine (Vairapandi and Duker, 1993); therefore, salvage of this modified base and its direct incorporation into DNA is prevented. Immediately following DNA replication the newly synthesized strand is unmodified. However, the maintenance methylase is specific for hemimethylated sites (Gruenbaum et al., 1982) and, therefore, it can restore the DNA to the normal symmetrical pattern of methylation [de n o v o methylation (Szyf, 1991; Hasse et al., 1992) and demethylation without DNA replication (Razin et al., 1986; Vairapandi and Duker, 1993) may also occur]. A CpG site which is initially unmodified

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Figure 2 Schematic representation of maintenance methylation of DNA following DNA replication (modified from Hergersberg, 1991). DNA maintenance methylase is the enzyme responsible for propagating the parental pattern of methylation in daughter cells during DNA replication. Under normal conditions, when DNA replication occurs, the newly synthesized strand is not methylated. Consequently, the new double-stranded DNA contains hemimethylated sites which provide the signal for DNA maintenance methylation that restores the fully methylated pattern. If maintenance methylation does not occur, e.g., due to a decreased level of S-adenosylmethionine (SAM) or a reduced fidelity of DNA maintenance methylase activity, and cell division followed by a second round of DNA replication takes place, then that daughter strand will give rise to double-stranded DNA which has lost a methylated site; generating hypomethylated DNA. This epigenetic change is heritable. Furthermore, it is expected that modifications in DNA methylation would result from threshold-exhibiting events. In addition, de novo methylation (Szyf, 1991; Hasse et al., 1992) and demethylation without DNA replication (Razin et al., 1986; Vairapandi and Duker, 1993) may occur. This paradigm provides a basis for our working hypothesis regarding hypomethylation of DNA as a nongenotoxic, epigenetic mechanism underlying the role of cell proliferation in carcinogenesis (Goodman and Counts, 1993), particularly during the promotion stage (Counts and Goodman, 1994c).

can remain that w a y after replication while a methylated C p G site will be recognized by the methyl group remaining on the parental strand and will be modified on the c o m p l e m e n t a r y strand (Razin and Cedar, 1991). If maintenance methylation does not take place, e.g., due to a decreased level of S A M or a reduced fidelity of D N A maintenance methylase activity, and cell division f o l l o w e d by a second round of D N A replication takes place, then that daughter strand will give rise to double-stranded D N A which has lost a methylated site; generating h y p o m e t h y l a t e d D N A . This epigenetic change is heritable.

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A cDNA encoding DNA MTase of mouse cells, which appears capable of functioning as a maintenance methylase, has been cloned (Bestor et al., 1988). The carboxyl-terminal amino acid sequence of the inferred protein is similar to bacterial type II DNA cytosine methyltransferases. The enzyme was shown to be expressed in a variety of mouse cell types (Bestor et al., 1988). The 5' flanking sequence of DNA MTase does not contain TATA or CAAT boxes (Rouleau et al., 1992). Thus, it shares characteristics of housekeeping or widely expressed genes (Gardiner-Garden and Frommer, 1987). However, it does not contain CpG islands (Rouleau et al., 1992) which are characteristic of these genes (Gardiner-Garden and Frommer, 1987). The 5' flanking sequence of DNA MTase contains activator proteins (Apl and Ap2) and glucocorticoid cis elements, suggesting the possibility for regulation of its expression by cellular signal transduction pathways (Rouleau et al., 1992). The fidelity of DNA MTase is comparable to that of mammalian DNA polymerase (Smith et al., 1992). An example of the key role played by maintenance methylation is provided by the results of a recent study involving targeted mutation of DNA MTase in knockout mice (Li et al., 1992). This resulted in a three-fold reduction in the level of methylation of genomic DNA in embryos causing abnormal development and embryonic lethality. Interestingly, a similar reduction of methylation in embryonic stem cells did not affect their viability or capacity to proliferate (Li et al., 1992). Taken together, these observations illustrate the important role played by DNA methylation in the control of specialized cell functions in a whole animal. Decreases in DNA methylation are frequently observed in tumor tissue and aberrations in methylation might be a key factor in carcinogenesis (Holliday, 1987; Jones and Buckley, 1990). There is a direct relationship between DNA methylation and gene silencing (Holliday and Ho, 1991). Hypomethylation of a gene, i.e., low levels of 5MeC, is necessary but not sufficient for its expression. Therefore, a hypomethylated gene can possess an increased potential for expression as compared to a hypermethylated gene (Vorce and Goodman, 1989). Additional evidence in support of a role for hypomethylation in carcinogenesis comes from studies involving 5-aza-cytidine (AzaC), an analog of cytidine. The deoxynucleotide of AzaC can be incorporated into DNA leading to hypomethylation of newly replicated DNA as a consequence of noncompetitive inhibition of DNA methyltransferase (Glover and LeylandJones, 1987). AzaC can alter gene expression, and thus change differentiation, in rat fetuses and act as a tumor promoter in rat liver (Carr et al., 1984). This observation is supported by a study indicating that exposure of nontumorigenic fibroblasts, expressing ras or m y c oncogenes, to AzaC can lead to malignant transformation (Rimoldi et al., 1991). In addition, both spontaneously transformed rat liver epithelial cells and those transformed

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by AzaC are resistant to the growth inhibitory effects of transforming growth factor IB (TGFI3) (Huggett et al., 1991). This similarity in phenotype between spontaneous and AzaC transformants is consistent with the notion that hypomethylation of DNA is typically involved in the multistep process by which a normal cell is transformed into a malignant cell. Increases in DNA methylation, i.e., hypermethylation, can also play a role in carcinogenesis. Tumor suppressor genes must be active to prevent transformation; hypermethylation can turn off expression of these genes and lead to cancer. Studies involving neural tumors (Makos et al., 1993a) and renal tumors (Makos et al., 1993b) indicate that regional hypermethylation might constitute a molecular change which is associated with genetic instability leading to allelic loss of the chromosome in question and, thus, contribute to progression as a consequence of the loss of a tumor suppressor gene(s). These observations are compatible with an earlier study (Klein et al., 1991) indicating that loss or inactivation of a senescence gene on the human X chromosome by hypermethylation is associated with the acquisition of immortality, and might represent an early event associated with nickel-induced transformation. Increased expression of DNA MTase occurs during colon carcinogenesis and this might play a role in the hypermethylation and genetic instability noted above (EI-Deiry et al., 1991). In addition, spontaneous deamination of 5MeC to thymine can result in C:G to A:T transitions. Thus, 5MeC might act as an endogenous mutagen and deamination-driven point mutations could play a role in carcinogenesis (Rideout et al., 1990; Shen et al., 1994; Zhang and Mathews, 1994). In this context it should be noted that as many as 43% of p53 gene mutations may be due to the presence of 5MeC (Rideout et al., 1990). While it appears that hypermethylation of limited regions of the genome can, indeed, play a role in carcinogenesis, there is a consistent finding that overall hypomethylation of DNA is a general phenomenon involved in carcinogenesis in both rodents and humans. The genomic level of 5MeC is lower in chemical-induced rat liver tumors (Lapeyre and Becker, 1979), and in spontaneous and chemical-induced mouse liver tumors (Lapeyre et al., 1981) as compared to normal liver tissue. In addition, hypomethylation is a relatively early event in carcinogenesis in humans (Feinberg and Vogelstein, 1983; Goelz et al., 1985). Furthermore, most metastatic human neoplasms have significantly lower genomic 5MeC than benign neoplasms or normal tissue, and the percentage of primary malignancies with hypomethylated DNA is intermediate between those of metastases and benign neoplasms (Gama-Sosa et al., 1983). This appears to reflect a role for hypomethylation in tumor progression in addition to promotion. The involvement of regional hypermethylation and a general hypomethylation of the genome are not incompatible. Indeed, hypermethylation leading to loss of tumor suppressor gene activity and hypomethylation facilitating aberrant increases in on-

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cogene expression [although hypomethylation within an intron may decrease expression of the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r) gene, as discussed in Section V] can be viewed as complementary mechanisms involved in multistage carcinogenesis.

IV. W o r k i n g

Hypothesis and E x p e r i m e n t a l

Model

Our experimental interests involve testing the hypothesis (Goodman et al., 1991; Goodman and Counts, 1993; Counts and Goodman, 1995) that hypomethylation of DNA is a nongenotoxic, threshold-exhibiting, mechanism that facilitates aberrant oncogene expression that plays a role in carcinogenesis. In particular, this epigenetic mechanism may play a key role in tumor promotion (Counts and Goodman, 1994c). The phenomenon of de n o v o methylation (Szyf, 1991; Hasse et al., 1992) provides a potential mechanism for reversing hypomethylation. Thus, our hypothesis regarding the role of hypomethylation in tumor promotion is compatible with the notion that reversibility is a fundamental component of the promotion stage of carcinogenesis (Pitot, 1990; Dragan et al., 1993). The B6C3F1 mouse exhibits a high spontaneous incidence of liver tumors (Becker, 1982) and there is a uniquely high involvement of Ha-ras in both chemical-induced and spontaneous B6C3F1 mouse liver tumors. Thus, we chose to study Ha-ras because of its role in B6C3F1 mouse liver tumorigenesis. Activation (i.e., mutation) of Ha-ras is seen in spontaneous and chemical induced liver tumors in the B6C3F1 (C57BL/6 female x C3H/He male) mouse (Wiseman et al., 1986; Reynolds et al., 1987; Stowers et al., 1988; Buchmann et al., 1991), but not in nonliver tumors of the same animal (Candrian et al., 1991). There is also a striking absence of Ha-ras activation in diethylnitrosamine-induced liver tumors of the Fischer-344 rat (Stowers et al., 1988) and the C57BL/6 mouse (Buchmann et al., 1991); it occurs with a relatively low frequency in spontaneous liver tumors of the C3H/He mouse (Enomoto et al., 1993). This last finding, that Ha-ras involvement is seen to a lesser degree in the paternal strain that is also susceptible to spontaneous liver tumorigenesis (Becker, 1982), indicates that liver tumor formation in the B6C3F1 is not simply the sum of what is observed in the parental strains, as the spontaneous liver tumor incidence in the C 5 7 B L / 6 is virtually zero at 18 months (Becker, 1982). The unusually high incidence of liver tumorigenesis in the B6C3F1 mouse t h a t cannot be explained merely by mutation at codon 61 o f Ha-ras (Schwartz et al., 1991) led to our analysis of the methylation status of this oncogene. The results of these studies indicate that Ha-ras in the nascent liver of the liver tumor prone strains (i.e., B6C3F1 and C3H/He) lacks a methylated site that is present in the resistant C 5 7 B L / 6 strain (Vorce and

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Goodman, 1991a). In addition, Ha-ras was shown to be hypomethylated in B6C3F1 mouse liver tumors induced by both a genotoxic (Vorce and Goodman, 1989) and nongenotoxic compounds, as well as in spontaneous liver tumors (Vorce and Goodman, 1991b). These studies demonstrate that hypomethylation of Ha-ras is a common event in mouse liver tumorigenesis and that this could facilitate the aberrant oncogene expression that is seen (Ray et al., 1994; Vorce and Goodman, 1989). In light of the multistep nature of tumorigenesis we did not want to limit our examination of alterations in methylation to Ha-ras; this led us to investigate the raf oncogene as another potential target gene (Ray et al., 1994). Our results indicate that the B6C3F1 mouse inherits a maternal raf allele containing a methylated site that is not present in the paternal allele. At 7 days after partial hepatectomy or after administration of a promoting dose of phenobarbital (PB) for 14 days raf in B6C3F1 mouse liver is hypomethylated. The additional methylated site in the allele inherited from the C57BL/6 is not maintained after partial hepatectomy. However, the methylation status of raf in the liver of the C57BL/6 mouse is not affected by PB treatment. This indicates that the liver tumor prone B6C3F1 mouse has a lower capacity to maintain methylation of raf than the resistant C57BL/6 strain. Additionally, raf is hypomethylated in a nonrandom fashion in both PB-induced and spontaneous B6C3F1 liver tumors. The level of raf mRNA is increased in 7 out of 10 PB-induced tumors but in only 1 out of 5 spontaneous tumors, while Ha-ras mRNA is increased in 9 out of 10 PBinduced tumors and in 4 out of 5 spontaneous tumors (Ray et al., 1994). The results of this investigation (Ray et al., 1994): (a) support the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism involved in tumorigenesis; (b) support the notion that PB promotes liver tumors which develop along a pathway different from that leading to spontaneous tumors; and (c) indicate that differences in DNA methylation between the C57BL/6 and B6C3F1 mice could, in part, account for the unusually high tendency of the latter strain to develop liver tumors.

V. Liver Tumor Promotion: A Role for Hypomethylation of DNA Chronic exposure to PB results in a decrease in the ability of hepatocytes to respond to mitogenic stimuli. This is correlated with a reduction of epidermal growth factor (EGF) receptor number (Eckl et al., 1988), an inhibition of the ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) to cause translocation of protein kinase C (PKC) to the cell membrane (Brockenbrough et al., 1991) and an increase in the level of TGF[3 (Jirtle and Meyer, 1991). In addition, PB treatment enhances the clonability of hepatocytes

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(Jirtle and Michalopoulos, 1986) and stimulates the proliferation of putative initiated hepatocytes under conditions in which normal hepatocytes senesce and die (see Chapters 8 and 9) (Kaufmann et al., 1988). Activation of the EGF receptor results in phosphorylation and increased kinase activity of raf by PKC-dependent and PKC-independent pathways (App et al., 1991; Baccarini et al., 1991). In light of this and the discussion presented above, we propose (Ray et al., 1994) that those hepatocytes exhibiting an increased expression of Ha-ras and raf may be able to overcome the growth inhibitory actions of PB and serve as the progenitors of the PBinduced liver tumors. The development of spontaneous mouse liver tumors which, in contrast to those induced by PB, contain a high frequency of codon 61 mutations in Ha-ras (Fox et al., 1990) involves an increased expression of Ha-ras and not raf (Ray et al., 1994). Furthermore, hypomethylation may be a mechanism that facilitates the crucial aberrant oncogene expression. This proposal is consistent with the observations that resistance to the growth inhibitory action of TGF[3 has been observed (a) following introduction of v-Ha-ras into rat liver epithelial cells (Houck et al., 1989) and the degree of resistance, exhibited by rat intestinal epithelial cells, is related directly to the level of expression of a mutated Ha-ras (Filmus et al., 1992); and (b) in rat liver epithelial cells transformed with v-raf (Huggett et al., 1990), spontaneously, or by treatment with the DNA hypomethylating agent AzaC (Huggett et al., 1991). Several peroxisome proliferators (e.g., nafenopin, methyl-clofenapate, Wy-14,643 and clofibric acid), nongenotoxic compounds that may produce rodent liver tumors following prolonged administration, have been shown to increase the level of TGF[3 following their daily administration for I week (Rumsby et al., 1994). Therefore, we hypothesize that development of resistance to growth inhibition is a general mechanism involved in tumor promotion as opposed to being unique for PB. TGFI3 is secreted from cells as a latent complex containing phosphomannosyl residues which can undergo proteolytic activation following binding to the M6P/IGF2r located on the cell surface (Dennis and Rifkin, 1991; Jirtle et al., 1993). Therefore, the M 6 P / I G F 2 r gene can be viewed as a likely tumor suppressor gene (Jirtle et al., 1994; De Souza et al., 1995). Hypomethylation of the 5' flanking region of this gene is required for its expression and methylation within an intron appears to be an imprinting signal (St6ger et al., 1993). Therefore, it should be noted that failure to maintain methylation of this imprinted intron would be expected to lead to inhibition of M 6 P / I G F 2 r gene transcription, and a decreased capacity of TGF~ to inhibit cell proliferation. This could be one of the specific mechanisms underlying the role of hypomethylation in tumor promotion. In the mouse, only the maternal allele is expressed (Barlow et al., 1991); however, both alleles are usually expressed in the human (Xu et al., 1993; Kalscheuer et al.,

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1993). This could, in part, provide a basis for the observations that rodent cells are generally more susceptible to transformation than human cells (Rhim, 1993), i.e., there is a higher probability of losing tumor suppressor function when one imprinted expressed allele is involved as compared to a situation where both alleles are expressed. Furthermore, differences in methylation between the C57BL/6 and B6C3F1 strains of mice could, in part, explain the high tendency of the latter strain to develop liver tumors (Goodman et al., 1991; Goodman and Counts, 1993; Counts and Goodman, 1995; Ray et al., 1994).

VI. Methyl Deficient Diets An experimental system that can be used to induce rodent liver tumors is that of a methyl deficient (MD) diet (i.e., deficient in choline and methionine and/or folic acid and vitamin B12); both rats and mice will develop hepatocellular carcinomas after administration of a MD diet (Mikol et al., 1983; Ghoshal and Farber, 1984; Newberne et al., 1982). A study by Sawada and co-workers (1990) demonstrated that the MD diet exerts promoting effects in rat liver tumorigenesis. Choline is a required component of the mammalian diet. There are several crucial reactions that require choline or one of its metabolites (Zeisel et al., 1989). Choline is important for neurotransmission, as choline is taken up by a high affinity process in nerve cells and this leads to the formation of the neurotransmitter acetylcholine. Choline contributes the phospholipid phosphatidylcholine to cellular membranes and phosphatidylcholine is important in the transport of lipid out of hepatocytes (Yao and Vance, 1988). In addition, choline is involved in the supply of dietary methyl groups required for methylation of DNA. This occurs via the cofactor, SAM, which is synthesized from choline and methionine. Thus, a MD diet may interfere with many biological processes and is expected, due to the requirement for choline and methionine in the formation of SAM, to lead to hypomethylation of DNA. This system then provides an experimental model that can be utilized to study the effects of hypomethylation of DNA. Another aspect of this experimental system is the possibility for comparisons with a classic rodent liver tumor promoter, PB (Peraino et al., 1973; Weghorst and Klaunig, 1989). Indeed, studies have revealed a synergistic relationship between PB and MD diets with regard to liver tumorigenesis (Shinozuka and Lombardi, 1980). There are currently three hypotheses as to the mechanism of action of MD diet-induced liver tumorigenesis: (1) depletion of choline and methionine leading to decreased levels of SAM and thereby to hypomethylation of DNA which facilitates aberrant oncogene expression, (2) depletion of choline and methionine leading to decreased

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generation of glutathione (a tripeptide important in protection against oxidative damage) which allows for increased levels of reactive oxygen species, and (3) depletion of choline and methionine leading to alterations in PKC and altered expression of growth factor receptors on the plasma membrane (Rogers et al., 1993). The reader is referred to several recent reviews that address the role and likelihood of each of these leading to liver tumor formation in rodents (Rogers et al., 1993; Lombardi and Smith, 1994; Shinozuka et al., 1993; Wainfan and Poirier, 1992; Christman et al., 1993; Ghoshal and Farber, 1993). Our discussion will focus on hypomethylation of DNA as a mechanism involved in liver tumor formation under this dietary deficiency. However, this does not necessarily exclude the possibility of any of the other mechanisms being involved. A discussion of the experimental evidence supporting an important role for hypomethylation of DNA in this system is useful; however, some comparisons are complicated because of the different MD diets utilized. These diets range in degree of methyl deficiency from the relatively moderate choline-devoid, methionine-deficient diet to the more severe lipotrope deficient diet (deficient in all four methyl donors). Hypomethylation of hepatic DNA, assessed by a variety of methods, is seen in all studies in rats (Locker et al., 1986; Wilson et al., 1984; Wainfan et al., 1989) and mice (Shivapurkar et al., 1986), although in the last example the decreases were not statistically significant. These observations led to a more refined analysis of methylation state by examining the methylation status of specific genes using methylation sensitive restriction enzymes. Hypomethylation was evident in Ha-ras (Bhave et al., 1988; Zapisek et al., 1992; Dizik et al., 1991; Wainfan and Poirier, 1992), Ki-ras (Bhave et al., 1988; Zapisek et al., 1992), m y c (Dizik et al., 1991; Wainfan and Poirier, 1992), and fos (Dizik et al., 1991; Zapisek et al., 1992) usually by 1 to 3 weeks. In addition, many of these groups examined level of expression of these genes and observed increases in mRNA after only 1 week on the severely lipotrope deficient diet (Dizik et al., 1991; Wainfan and Poirier, 1992). The significance of this experimental system is readily evident when one considers the potential usefulness of the MD diet in discerning mechanisms underlying tumor promotion when careful comparisons are carried out between strains of mice with well-characterized differences in sensitivities toward liver tumor formation. This approach (being carried out in collaboration with Drs. McClain and Harbison, Hoffmann-La Roche, Inc.) will allow for comparisons of methylation status, availability of SAM, and levels of cell proliferation to be made between the relatively susceptible B6C3F1 and the relatively resistant C57BL/6 mouse strains and to relate any differences to their different propensities toward liver tumorigenesis. The results of our preliminary experiments indicate that hypomethylation of DNA occurs at an

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earlier time and to a larger extent in the B6C3F1 as compared to the C57BL/6 following placement of the animals of a choline-devoid, methionine-deficient diet (Counts and Goodman, 1994b; Counts et al., 1995).

VII. D N A D a m a g e a n d Altered D N A M e t h y l a t i o n The potential for genotoxic carcinogens to influence DNA methylation status has been studied. Wilson et al. (1987a) examined the level of 5MeC present in DNA after treatment of normal human bronchial epithelial cells with a variety of carcinogens (including benzo[a]pyrene, aflatoxin B1, and 7,12-dimethylbenz[a]anthracene), a weak carcinogen (benzo[e]pyrene) and a noncarcinogen (phenanthrene). As a control they used AzaC, a compound known to hypomethylate DNA (see earlier discussion), and decreased levels of 5MeC in DNA compared to controls was observed (Wilson et al., 1987a). Furthermore, all of the carcinogens tested in this study decreased levels of 5MeC compared to controls, while the weak carcinogen and the noncarcinogen did not alter the level of 5MeC (Wilson et al., 1987a). In addition, benzo[a]pyrene was tested at several dose levels and a dose response relationship was exhibited. The presence of O6-methylguanine on the newly synthesized DNA strand at hemimethylated CpG sites may interfere with the ability of the maintenance methylase to incorporate the methyl group onto the cytosine adjacent to the O6-methylguanine during DNA replication (Tan and Li, 1990; Hepburn et al., 1991). Thus, in a proliferating population of cells, alkylation of guanine on the newly synthesized DNA strand, may result in hypomethylation. This heritable epigenetic modification would remain even if the initial DNA damage was eventually repaired. These studies indicate that genotoxic chemicals, commonly thought of as acting by a mechanism involving mutagenesis, may also influence gene expression by altering the methylation status of DNA (Figure 1). Genotoxic compounds act to inhibit methylation either directly, by covalently binding to and inactivating the methylase (Ruchirawat et al., 1984; Wojciechowski and Meehan, 1984) or indirectly, by adduct formation on DNA preventing access of the methylase to the CpG site (Tan and Li, 1990; Hepburn et al., 1991). Additionally, there are reports indicating an increase in methylase activity on removal of the carcinogen (Pfohl-Leszkowicz and Dirheimer, 1986; Boehm et al., 1983). The increased levels of methylase activity after carcinogen removal may reflect increased expression of the methyltransferase protein as it attempts to compensate. Furthermore, 0 6 alkylation of the guanine moiety at a CpG site may not be repaired efficiently when the C is methylated and this could facilitate mutagenesis (Bentivegna and Bresnick, 1994).

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Endogenous oxidative damage (i.e., free radical-induced damage) is becoming an area of increasing interest in studies aimed at discerning mechanisms underlying carcinogenesis. This is due to the potential for oxidative stress to lead to alterations in DNA. For example, 8-hydroxyguanine is a common product of oxidative damage. A study by Weitzman and coworkers (1994) examined the effect of the presence of 8-hydroxyguanine in DNA at 5 P-CCGG-3 p sites in place of guanine. As discussed earlier, 5MeC is most often found in DNA at CpG sites and 5MeC is not directly incorporated into DNA; the parental strand of DNA contains the 5MeC while the daughter strand incorporates cytosine. This generates a hemimethylated site that is recognized by a SAM-requiring maintenance methylase which adds the methyl group to the 5 position of cytosine. It was demonstrated that 8-hydroxyguanine in deoxyoligonucleotides inhibited the ability of the enzyme to methylate DNA (Weitzman et al., 1994). This provides an example of a situation where oxidative damage can generate hypomethylated D N A - - a situation that can facilitate aberrant oncogene expression involved in tumorigenesis. As noted earlier, one hypothesis regarding a mechanism by which an MD diet results in tumorigenesis is the generation of reactive oxygen species. At this time it is useful to point out that the generation of reactive oxygen species may induce hypomethylation of DNA. Several studies have examined the influence of free radical effects in rat liver tumors induced by a choline-devoid, methionine-deficient diet (Rushmore et al,, 1986, 1987) and have shown that lipid peroxidation is an early event. They suggest that oxidative DNA damage plays a role in carcinogenesis. Evidence demonstrating that dietary antioxidants protect against biochemical changes, but not morphological changes seen in methyl deficiency (Jenkins et al., 1993) indicates that factors in addition to oxidative damage may be playing a role in determining the effects of MD diets. In light of the discussion presented above, we would like to point out that to the extent that oxidative DNA damage plays a role in MD diet-induced liver carcinogenesis, hypomethylation of DNA may also be involved.

VIII. DNA Methylation and Chemoprevention While the preceding discussion has focused on the convincing body of literature indicating that hypomethylation of DNA can facilitate aberrant gene expression that is involved in carcinogenesis, it is also important to address the potential for an increased capacity for DNA methylation to protect against carcinogenesis. Ethionine, a chemical carcinogen that interferes with SAM metabolism, is a known rodent liver carcinogen (Farber, 1963). Inter-

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estingly, if animals administered a diet containing ethionine are also given a diet supplemented with choline and/or methionine tumors do not occur (Hoffman, 1984). The protection against liver tumorigenesis afforded by additional choline and methionine in the diet has been demonstrated in both the mouse and rat. B6C3F1 male mice were fed an excess of choline, methionine, or both after administration of a carcinogenic dose of aflatoxin B 1 and the tumor yield was determined at 50 weeks (Newberne et al., 1990). The mice that received aflatoxin B 1 and were fed the excess choline and excess methionine diet had a tumor yield comparable with control animals (Newberne et al., 1990). The groups of animals that received either excess choline or excess methionine alone exhibited some protection from aflatoxin Bl-induced liver tumorigenesis, but this was intermediate between the animals receiving an excess of both methyl donors and those receiving no additional dietary methyl donors. The authors concluded that the protection resulted from increased metabolic inactivation of aflatoxin B1; however, a role for protection against hypomethylation of DNA cannot be discounted. Fullerton and co-workers (1990) noted an inhibition of DEN-initiated and PB-promoted liver carcinoma, but not adenoma, formation in C3H/He mice fed a diet supplemented with both choline and methionine. In addition, in the animals on the choline and methionine supplemented diet, there was a decrease in carcinoma, but not adenoma, formation after PB-treatment alone, and there were also fewer metastases to the lung when tumors did form (Fullerton et al., 1990). A protective effect of the administration of SAM on the development of rat liver tumors has been demonstrated (Pascale et al., 1992; Feo et al., 1991). SAM im injections decrease the size and number of preneoplastic foci in rats that have been initiated with DEN. Indeed this is actual protection, not just a slowing of tumor growth because the tumors do not develop even after cessation of SAM (Pascale et al., 1992). These studies support the hypothesis that hypomethylation of DNA is involved in tumorigenesis as two different methods of restoring methyl groups to the diet can protect against carcinogen-induced liver tumor formation. It also provides a potential source for intervention in some human cancers if similar mechanisms are operative. Another potentially useful preventative measure is caloric restriction, which increases the life span of rodents significantly over rodents fed ad libitum. Cooney (1993) hypothesizes that caloric restriction increases life span in part by allowing for better maintenance methylation of DNA. He supports this hypothesis with the knowledge that less fat intake in the restricted animals will mean less phosphatidylcholine is being used to make VLDL (Cooney, 1993) that is necessary to package fat for transport out of

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the hepatocyte (Yao and Vance, 1988). In this situation a greater proportion of the available methyl group donors may be used for DNA methylation reactions. This hypothesis is supported by a study testing the "permanence" of the effect of caloric restriction in an in vivo to in vitro model (Hass et al., 1993). These authors found that the effects of caloric restriction were passed on to cells taken from an animal and grown in culture; the effects included increased Ha-ras oncogene methylation, decreased Ha-ras expression and mutation, and decreased p53 tumor suppressor gene mutation (Hass et al., 1993). Clearly, dietary restriction, which is known to increase longevity, was able to prevent hypomethylation of DNA that is involved in tumorigenesis. The preventative nature of caloric restriction brings together many of the concepts discussed in this paper and provides support for our hypothesis (Goodman and Counts, 1993; Counts and Goodman, 1994c, 1995; Ray et al., 1994) concerning hypomethylation of DNA being involved in the promotion of tumorigenesis. Interestingly, caloric restriction has recently been shown to also selectively cause the removal of chemical carcinogen initiated hepatocytes by apoptosis thereby reducing liver tumor formation (Grasl-Kraupp et al., 1994).

IX. Differences in D N A Methylation between Rodents and Humans A. Capacity to Maintain DNA Methylation Incomplete DNA methylation may occur during cell proliferation and, when accumulated over many cell cycles, may play a role inaging (Cooney, 1993). In general, the overall level of genomic 5MeC appears to be maintained more effectively in human cells as compared to rodent cells. When normal diploid fibroblasts from mice, hamsters, and humans are grown in culture, the 5MeC content of their DNAs decreases over time, with the rate of loss being highest in mouse cells and lowest in human cells (Wilson and Jones, 1983). This is consistent with a report that 5MeC in human liver does not decline with age (Tawa etal., 1992), and the observation that the rate of loss of 5 M e C from mouse liver DNA is related inversely to lifespan of the strains studied (Wilson et al., 1987b). Additionally, age-linked reactivation (reexpression) of a gene on the silent X-chromosome has been shown to occur in mice (Wareham et al., 1987) but not in humans (Migeon et al., 1988). DNA methylation plays a key role in the inactivation of one X-chromosome in the female (Holliday, 1987), and the silent locus in the human cells could be expressed following treatment with AzaC, an inhibitor of DNA methylation (Migeon et al., 1988). Therefore, the apparent discrep-

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ancy regarding X-chromosome reactivation and aging can be explained on the basis of a higher capacity to maintain methylation in human cells as compared to mouse cells (Holliday, 1989). B. Methylation of the 5' Flanking Region of Ha-ras Ha-ras is a widely expressed gene and the sequence of the promoter region of mouse Ha-ras (Brown et al., 1988; Neades et al., 1991) indicates that it

has some characteristics of housekeeping and other widely expressed genes. Specifically, the promoter region contains CpG islands and lacks a TATA box (Brown et al., 1988). CpG islands are expected to be unmethylated, as has been determined for a variety of housekeeping and tissue specific genes (Gardiner-Garden and Frommer, 1987). In line with this generally established observation, the promoter region of human Ha-ras has been reported to be unmethylated (Borrello et al., 1992). Nevertheless, the high involvement of Ha-ras in mouse liver tumorigenesis and our previous work showing hypomethylation of Ha-ras and raf in these tumors (Vorce and Goodman, 1989, 1991b; Ray et al., 1994) prompted us to determine if the 5' flanking region of Ha-ras in mouse liver is methylated and, if so, whether differences exist between the methylation status of this region in mouse strains that exhibit different propensities for liver tumor formation. As has been discussed, key aspects of the control of gene expression occur in the promoter region (contained within the 5' flanking region) of a gene. This is the area where transcription factors bind and facilitate binding of the RNA polymerase to DNA. Indeed, the 5' flanking region of mouse Ha-ras contains potential binding sites for a variety of transcription factors, including Spl, Apl, Ap2, and ERE (Counts and Goodman, 1994a). There are also several sites similar to the recognition sequence for MDBP (see Figure 1 in Counts and Goodman, 1994a). The studies discussed above focused on the structural portion of the oncogenes, Ha-ras and raf. We employed Southern analysis involving restriction with methylation sensitive enzymes and deoxyoligonucleotide probes to assess the methylation status of this region of Ha-ras in male B6C3F1, C3H/He, and C57BL/6 mouse liver (Counts and Goodman, 1994a). Our study detected the presence of 5MeC in the 5' flanking region of Ha-ras, although many of the CpG sites did appear to be unmethylated, and indicated that the methylation status or base sequence of this region is not the same in the three strains (Counts and Goodman, 1994a). We have recently employed a PCR-based approach to analyze the methylation status of a portion of the 5' flanking region of Ha-ras and this initial investigation confirmed the presence of 5MeC at specific sites (Counts and Goodman, 1994b), indicating that methylation of this region of Ha-ras in mouse liver may play a role in regulating the expression of this oncogene.

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X. Conclusions A. DNA

Methylation and Multistage Carcinogenesis

The compartmentalization of carcinogenesis into three stages (initiation, promotion, and progression) is based on specific experimental paradigms and it provides a very useful conceptualization of the carcinogenic process (Pitot, 1990; Dragan et al., 1993). However, in our view, it was correctly not intended to connote that mutation only occurs during initiation, or that promotion only involves inducing proliferation of and preventing death (apoptosis) of initiated cells (Schulte-Hermann et al., 1990), or that changes in ploidy only occur during progression. Operationally, an initiated cell is one that can be stimulated to proliferate by a promoter and a promoter is capable of stimulating the proliferation of initiated cells. The terms initiation and promotion imply modes of action. In reality, alterations to the genome (e.g., mutation, chromosome loss, change in methylation status) occur at multiple points in the carcinogenic process, as depicted in the Vogelstein model based on colon carcinogenesis (Vogelstein et al., 1988; Marx, 1991). The multiple alterations to the genome, including hypomethylation of DNA, involved in the sequential clonal expansion of cells leading to a frank malignancy is illustrated schematically in Figure 3.

Figure3 Initiation and cell proliferation in multistage carcinogenesis (modifiedfrom Swenberg et al., 1987). The "critical events" referred to involve changes in the genome (e.g., mutation and/or alterations in the methylation status of DNA). Each line through a cell represents a critical event that may play a role in carcinogenesis.

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B. DNA Methylation and Risk Assessment The testing of our hypothesis (Goodman et al., 1991; Goodman and Counts, 1993; Counts and Goodman, 1994c, 1995) that hypomethylation of DNA is an epigenetic, threshold-exhibiting mechanism underlying the role of cell proliferation in the promotion of carcinogenesis has the potential to enhance our understanding of mechanisms of carcinogenesis. At the same time, this research is providing the type of information that is required to take a more rational approach toward risk assessment (Goodman, 1994). Specifically, it is related directly to two practical goals (Counts and Goodman, 1995): (1) a reasoned approach toward species-to-species extrapolation issues (e.g., discerning the molecular basis for the unique susceptibility of the B6C3F1 mouse toward development of liver tumors); and (2) an understanding of dose-response relationships (i.e., this research can provide both a mechanistic and theoretical framework behind the suggestions to employ a safety factor approach toward regulation of nongenotoxic compounds which appear to act as tumor promoters, and the highest dose at which there is no hypomethylation in a potential target organ might be considered as a no adverse effect level). In addition, assessments of DNA methylation can provide insight regarding the interpretation of studies aimed at deciding on high doses that are reasonable to employ in chronic bioassays (Counts and Goodman, 1995). C. DNA Methylation and Chemoprevention Additionally, we have discussed the potential to take information concerning the role of DNA methylation in tumorigenesis and employ this for the prevention of tumorigenesis. Liver tumor development in rodents appears to be inversely related to the level of methyl donors in the diet as diets deficient in methyl donors lead to liver tumors in both rats and mice (Mikol et al., 1983; Ghoshal and Farber, 1984; Newberne et al., 1982). The fact that cells from humans (a longer lived species) grown in culture are better capable of retaining their normal methylation status than cells from rodents (shorter lived species) grown in culture (Wilson and Jones, 1983) may, at first glance, appear to complicate this discussion. However, a possible role for chemoprevention in humans by dietary supplementation with methyl donors (e.g., choline and methionine) exists as hypomethylation of DNA is a consistently seen in human cancer (Feinberg and Vogelstein, 1983; Goelz et al., 1985). XI. Summary In summary, evidence has been presented that suggests hypomethylation of DNA may serve as an epigenetic, nongenotoxic mechanism that facilitates

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the aberrant oncogene expression involved in carcinogenesis, especially the promotion stage. This biologically plausible paradigm may furnish the type of mechanistic information required to provide: (1) a more rational approach to risk assessment (e.g., dose-response relationships that include the existence of thresholds; species-to-species extrapolation that includes a basis for the unique high sensitivity of the B6C3F1 mouse to liver tumorigenesis), and (2) insight into chemopreventative strategies applicable to humans.

Acknowledgments Our research concerning DNA methylation was initiated by support from the International Life Sciences Institute (ILSI) Risk Science Institute. This is currently supported by ILSI, Health and Environmental Sciences Institute, and NIH Grant ES-05299. JLC is the recipient of a Society of Toxicology predoctoral fellowship award.

References Adams, R. L. P. (1990). DNA methylation: The effect of minor bases on DNA-protein interactions. Biochem. J. 265, 309-320. Ames, B. N., and Gold, L. S. (1990). Chemical carcinogenesis: Too many rodent carcinogens. Proc. Natl. Acad. Sci. U.S.A. 87, 7772-7776. App, H., Hazan, R., Zilberstein, A., Ullrich, A., Schlessinger, J., and Rapp, U. (1991). Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor. Mol. Cell. Biol. 11, 913-919. Baccarini, M., Gill, G. N., and Stanley, E. R. (1991). Epidermal growth factor stimulates phosphorylation of RAF-1 independently of receptor autophosphorylation and internalization. J. Biol. Chem. 266, 10941-10945. Barlow, D. P., St6ger, R., Herrmann, B. G., Saito, K., and Schweifer, N. (1991). The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature (London) 349, 84-87. Becker, E E (1982). Morphological classification of mouse liver tumors based on biological characteristics. Cancer Res. 42, 3918-3923. Benigni, R., and Andreoli, C. (1993). Rodent carcinogenicity and toxicity, in vitro mutagenicity, and their physical chemical determinants. Mutat. Res. 297, 281-292. Bentivegna, S. S., and Bresnick, E. (1994). Inhibition of human O6,methylguanine-DNA methyltransferase by 5-methylcytosine. Cancer Res. 54, 327-329. Bestor, T., Laudano, A., Mattaliano, R., and Ingram, V. (1988). Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells: The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases. J. Mol. Biol. 203, 971-983. Bhave, M. R., Wilson, M. J., and Poirier, L. A. (1988). c-H-ras and c-K-ras gene hypomethylation in the livers and hepatomas of rats fed methyl-deficient, amino acid-defined diets. Carcinogenesis 9, 343-348. Boehm, T. L. J., Grunberger, D., and Drahovsky, D. (1983). Aberrant de novo methylation of DNA after treatment of murine cells with N-acetoxy-N-2-acetylaminofluorene. Cancer Res. 43, 6066-6071.

248

Jennifer L. Counts and Jay I. Goodman

Borrello, M. G., Pierotti, M. A., Tamborini, E., Biassoni, D., Rizzetti, M. G., Pilotti, S., and Porta, G. D. (1992). DNA methylation of coding and non-coding regions of the human H-ras gene in normal and tumor tissues. Oncogene 7, 269-275. Boyes, J., and Bird, A. (1991). DNA methylation inhibits transcription indirectly via a methylCpG binding protein. Cell (Cambridge, Mass.) 64, 1123-1134. Brandeis, M., Ariel, M., and Cedar, H. (1993). Dynamics of DNA methylation during development. BioEssays 15, 709-713. Brockenbrough, J. S., Meyer, S. A., Li, C., and Jirtle, R. L. (1991). Reversible and phorbol esterspecific defect of protein kinase C translocation in hepatocytes isolated from phenobarbitaltreated rats. Cancer Res. 51, 130-136. Brown, K., Bailleul, B., Ramsden, M., Fee, F., Krumlauf, R., and Balmain, A. (1988). Isolation and characterization of the 5' flanking region of the mouse c-Harvey-ras gene. Mol. Carcinog. 1, 161-170. Buchmann, A., Bauer-Hofmann, R., Mahr, J., Drinkwater, N. R., Luz, A., and Schwarz, M. (1991). Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: Correlation with susceptibility to hepatocarcinogenesis. Proc. Natl. Acad. Sci. U.S.A. 88, 911-915. Bursch, W., Oberhammer, F., Jirtle, R. L., Askari, M., Sedivy, R., Grasl-Kraupp, B., Purchio, A. F., and Schulte-Hermann, R. (1993). Transforming growth factor~ as a signal for induction of cell death by apoptosis. Br. J. Cancer 67, 531-536. Butterworth, B. E., Slaga, T. J., Farland, W., and McClain, M. (eds.) (1991). In "Chemically Induced Cell Proliferation: Implications for Risk Assessment." Wiley-Liss, New York. Candrian, U., You, M., Goodrow, T., Maranpot, R. R., Reynolds, S. H., and Anderson, M. W. (1991). Activation of protooncogenes in spontaneously occurring non-liver tumors from C57BL/6 • C3H F 1 mice. Cancer Res. 51, 1148-1153. Carr, B. I., Reilly, J. G., Smith, S. S., Winberg, C., and Riggs, A. (1984). The tumorigenicity of 5-azacytidine in the male Fischer rat. Carcinogenesis 5, 1583-1590. Chang, L. W., Hsia, S. M. T., Chan, P.-C., and Hsieh, L.-L. (1994). Macromolecular adducts: Biomarkers for toxicity and carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 34, 41-67. Christman, J. K., Chen, M.-L., Sheikhnejad, G., Dizik, M., Abileah, S., and Wainfan, E. (1993). Methyl deficiency, DNA methylation, and cancer: Studies on the reversibility of the effects of a lipotrope-deficient diet. J. Nutr. Biochem. 4, 672-680. Cohen, J. B., and Levinson, A. D. (1988). A point mutation in the last intron responsible for increased expression and transforming activity of the c-Ha-ras oncogene. Nature (London) 334, 119-124. Cooney, C. A. (1993). Are somatic cells inherently deficient in methylation metabolism? A proposed mechanism for DNA methylation loss, senescence and aging. Growth Dev. Aging 57, 261-273. Counts, J. L., and Goodman, J. I. (1994a). Comparative analysis of the 5' flanking region of Ha-ras in B6C3F1, C3H/He and C57BL/6 mouse liver. Cancer Lett. 75,129-136. Counts, J. L., and Goodman, J. I. (1994b). Site specific methylation in the 5' flanking region of Ha-ras in the liver of the B6C3F1 mouse. Toxicologist 14, 146. Counts, J. L., and Goodman, J. I. (1994c). Hypomethylation of DNA: an epigenetic mechanism involved in tumor promotion. Mol. Carcinog. 11, 185-188. Counts, J. L., and Goodman, J. I. (1995). Hypomethylation of DNA: A possible epigenetic mechanism involved in tumor promotion. In "Growth Factors and Tumor Promotion: Implications for Risk Assessment" (R. M. McClain, T. J. Slaga, R. A. LeBoeuf and H. C. Pitot, eds.), in press. Wiley-Liss, New York. Counts, J. L., McClain, R. M., Harbison, M. L., Downing, J. C., and Goodman, J. I. (1995). Differences in DNA methylation induced by a choline-devoid, methionine-deficient diet or by phenobarbital correlate with liver tumor susceptibility in mice. The Toxicologist 15, 56.

10.

Hypomethylation of DNA

249

Dennis, P. A., and Rifkin, D. B. (1991). Cellular activation of latent transforming growth factor 13 requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor. Proc. Natl. Acad. Sci. U.S.A. 88, 580-584. De Souza, A. T., Hankins, G. R., Washington, M. K., Fine, R. L., Orton, T. C., and Jirtle, R. L. (1995). Frequent loss of heterozygosity on 6q at the mannose 6-phosphate/insulin-like growth factor II locus in human hepatocellular tumors. Oncogene 10, 1725-1729. Dizik, M., Christman, J. K., and Wainfan, E. (1991). Alterations in expression and methylation of specific genes in livers of rats fed a cancer promoting methyl-deficient diet. Carcinogenesis 12, 1307-1312. Dragan, Y. P., Sargent, L., Xu, Y.-D., Xu, Y.-H., and Pitot, H. C. (1993). The initiationpromotion-progression model of rat hepatocarcinogenesis. Proc. Soc. Exp. Biol. Med. 202, 16-24. Eckl, P. M., Meyer, S. A., Whitcombe, W. R., and Jirtle, R. L. (1988). Phenobarbital reduces EGF receptors and the ability of physiological concentrations of calcium to suppress hepatocyte proliferation. Carcinogenesis 9, 479-483. EI-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R.-W. C., Falco, J. P., Hamilton, S. R., and Baylin, S. B. (1991). High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer. Proc. Natl. Acad. Sci. U.S.A. 88, 3470-3474. Enomoto, T., Weghorst, C. M., Ward, J. M., Anderson, L. M., Perantoni, A. O., and Rice, J. M. (1993). Low frequency of H-ras activation in naturally occurring hepatocellular tumors of C3H/HeNCr mice. Carcinogenesis 14, 1939-1944. Farber, E. (1963). Ethionine carcinogenesis. Adv. Cancer Res. 7, 383-474. Feinberg, A. P., and Vogelstein, B. (1983). Hypomethylation of ras oncogenes in primary human cancers. Biochem. Biophys. Res. Commun. 111, 47-54. Feo, E, Pascale, R., Simile, M. M., Ruggiu, M. E., Daino, L., Satta, G., and Seddaiu, M. A. (1991). Chemoprevention of rat liver carcinogenesis by S-adenosylmethionine: Role of remodeling and apoptosis. In "Chemical Carcinogenesis 2" (A. Columbano, E Feo, P. Pascale and P. Pani, eds.), pp. 529-551. Plenum, New York. Filmus, J., Zhao, J., and Buick, R. N. (1992). Overexpression of H-ras oncogene induces resistance to the growth-inhibitory action of transforming growth factor beta-1 (TGF-~I) and alters the number and type of TGF-~I receptors in rat intestinal epithelial cell clones. Oncogene 7, 521-526. Fox, T. R., Schumann, A. M., Watanabe, P. G., Yano, B. L., Maher, V. M., and McCormick, J. J. (1990). Mutational analysis of the H-ras oncogene in spontaneous C57BL/6 x C3H/He mouse liver tumors and tumors induced with genotoxic and nongenotoxic hepatocarcinogens. Cancer Res. 50, 4014-4019. Fullerton, E R., Hoover, K., Mikol, Y. B., Creasia, D. A., and Poirier, L. A. (1990). The inhibition by methionine and choline of liver carcinoma formation in male C3H mice dosed with diethylnitrosamine and fed phenobarbital. Carcinogenesis 11, 1301-1305. Gama-Sosa, M. A., Slagel, V. A., Trewyn, R. W., Oxenhandler, R., Kuo, K. C., Gehrke, C. W., and Ehrlich, M. (1983). The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res. 11, 6883-6894. Gardiner-Garden, M., and Frommer, M. (1987). CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261-282. Ghoshal, A. K., and Farber, E. (1984). The induction of liver cancer by dietary deficiency of choline and methionine without added carcinogens. Carcinogenesis 5, 1367-1370. Ghoshal, A. K., and Farber, E. (1993). Choline deficiency, lipotrope deficiency and the development of liver disease including liver cancer: A new perspective. Lab. Invest. 68, 255-260. Glover, A. B., and Leyland-Jones, B. (1987). Biochemistry of azacytidine: A review. Cancer Treat. Rep. 71, 959-964.

250

Jennifer L. Counts and Jay I. Goodman

Goelz, S. E., Vogelstein, B., Hamilton, S. R., and Feinberg, A. P. (1985). Hypomethylation of DNA from benign and malignant human colon neoplasms. Science 228, 187-190. Goodman, J. I. (1994). A rational approach to risk assessment requires the use of biological information: An analysis of the National Toxicology Program (NTP), final report of the advisory review by the NTP Board of Scientific Counselors. Regul. Toxicol. Pharmacol. 19, 51-59. Goodman, J. I., and Counts, J. L. (1993). Hypomethylation of DNA: a possible nongenotoxic mechanism underlying the role of cell proliferation in carcinogenesis. Environ. Health Perspect. 101(Suppl. 5), 169-172. Goodman, J. I., Ward, J. M., Popp, J. A., Klaunig, J. E., and Fox, T. R. (1991). Mouse liver carcinogenesis: Mechanisms and relevance. Fundam. Appl. Toxicol. 17, 651-665. Grasl-Kraupp, B., Bursch, W., Ruttkay-Nedecky, B., Wagner, A., Lauer, B., and SchulteHermann, R. (1994). Food reduction eliminates preneoplastic cells through apoptosis and antagonizes carcinogenesis in rat liver. Proc. Natl. Acad. Sci. U.S.A. 91, 9995-9999. Gruenbaum, Y., Cedar, H., and Razin, A. (1982). Substrate and sequence specificity of a eukaryotic DNA methylase. Nature (London) 295,260-262. Hanigan, M. H., Kemp, C. J., Ginsler, J. J., and Drinkwater, N. R. (1988). Rapid growth of preneoplastic lesions in hepatocarcinogen-sensitive C3H/HeJ male mice relative to C57BL/6J male mice. Carcinogenesis 9, 885-890. Hass, B. S., Hart, R. W., Lu, M. H., and Lyn-Cook, B. D. (1993). Effects of caloric restriction in animals on cellular function, oncogene expression, and DNA methylation in vitro. Mutat. Res. 295,281-289. Hasse, A., Schultz, W. A., and Sies, H. (1992). De novo methylation of transfected CAT gene plasmid constructs in F9 mouse embryonal carcinoma cell. Biochim. Biophys. Acta 1131, 16-22. Hausheer, E H., Rao, S. N., Gamcsik, M. P., Kollman, P. A., Colvin, O. M., Saxe, J. D., Nelkin, B. D., McLennan, I. J., Barnett, G., and Baylin, S. B. (1989). Computational analysis of structural and energetic consequences of DNA methylation. Carcinogenesis 10, 11311137. Hepburn, P. A., Margison, G. P., and Tisdale, M. J. (1991 ). Enzymatic methylation of cytosine in DNA is prevented by adjacent O-6-methylguanine residues. J. Biol. Chem. 266, 79857987. Hergersberg, M. (1991 ). Biological aspects of cytosine methylation in eukaryotic cells. Experientia 47, 1171-1185. Hoffman, R. M. (1984). Altered methionine metabolism, DNA methylation and oncogene expression in carcinogenesis: A review and synthesis. Biochim. Biophys. Acta 738, 49-87. Holliday, R. (1987). The inheritance of epigenetic defects. Science 238, 163-170. Holliday, R. (1989). X-chromosome reactivation and ageing. Nature (London) 337, 311. Holliday, R. (1990). Mechanisms for the control of gene activity during development. Biol. Rev. 65, 431-471. Holliday, R., and Ho, T. (1991). Gene silencing in mammalian cells by uptake of 5-methyldeoxycytidine-5'-triphosphate. Somatic Cell Mol. Genet. 17, 537-542. Houck, K. A., Michalopoulos, G. K., and Strom, S. C. (1989). Introduction of a Ha-ras oncogene into rat liver epithelial cells and parenchymal hepatocytes confers resistance to the growth inhibitory effects of TGF-f3. Oncogene 4, 19-25. Huggett, A. C., Hampton, L. L., Ford, C. P., Wirth, P. J., and Thorgeirsson, S. S. (1990). Altered responsiveness of rat liver epithelial cells to transforming growth factor ~1 following their transformation with v-raf. Cancer Res. 50, 7468-7475. Huggett, A. C., Ellis, P. A., Ford, C. P., Hampton, L. L., Rimoldi, D., and Thorgeirsson, S. S. (1991). Development of resistance to the growth inhibitory effects of transforming growth factor [31 during the spontaneous transformation of rat liver epithelial cells. Cancer Res. 51, 5929-5936.

10.

Hypomethylation of DNA

251

Jenkins, M. Y., Sheikh, N. M., Mitchell, G. V., Grundel, E., Blakely, S. R., and Carter, C. J. (1993). Dietary carotenoids influenced biochemical but not morphological changes in adult male rats fed a choline-deficient diet. Nutr. Cancer 19, 55-65. Jirtle, R. L., and Meyer, S. A. (1991). Liver tumor promotion: Effect of phenobarbital on EGF and protein kinase C signal transduction and transforming growth factor-]31 expression. Dig. Dis. Sci. 36, 659-668. Jirtle, R. L., and Michalopoulos, G. (1986). Enhancement of the clonability of adult parenchymal hepatocytes with the liver tumor promoter phenobarbital. Carcinogenesis 7, 18131817. Jirtle, R. L., Haag, J. D., Ariazi, E. A., and Gould, M. N. (1993). Increased mannose 6-phosphate/insulin-like growth factor II receptor and transforming growth factor 131 levels during monoterpene-induced regression of mammary tumors. Cancer Res. 53, 3849-3852. Jirtle, R. L., Hankins, G. R., Reisenbichler, H., and Boyer, I. J. (1994). Regulation of mannose 6-phosphate/insulin-like growth factor-II receptors and transforming growth factor beta during liver tumor promotion with phenobarbital. Carcinogenesis 15, 1473-1478. Jones, P. A. (1986). DNA methylation and cancer. Cancer Res. 46, 461-466. Jones, P. A., and Buckley, J. D. (1990). The role of DNA methylation in cancer. Adv. Cancer Res. 54, 1-23. Kafri, T., Gao, X., and Razin, A. (1993). Mechanistic aspects of genome-wide demethylation in the preimplantation mouse embryo. Proc. Natl. Acad. Sci. U.S.A. 90, 10558-10562. Kalscheuer, V. M., Mariman, E. C., Schepens, M. T., Rehder, H., and Ropers, H. H. (1993). The insulin-like growth factor type-2 receptor gene is imprinted in the mouse but not in humans. Nature Genetics 5, 74-78. Kaufmann, W. K., Ririe, D. G., and Kaufman, D. G. (1988). Phenobarbital-dependent proliferation of putative initiated rat hepatocytes. Carcinogenesis 9, 799-782. Klein, C. B., Conway, K., Wang, X. W., Bhamra, R. K., Lin, X., Cohen, M. D., Annab, L., Barrett, J. C., and Costa, M. (1991). Senescence of nickel-transformed cells by an X chromosome: Possible epigenetic control. Science 251, 796-799. Lapeyre, J.-N., and Becker, E E (1979). 5-Methylcytosine content of nuclear DNA during chemical hepatocarcinogenesis and in carcinomas which result. Biochem. Biophys. Res. Commun. 87 698-705. Lapeyre, J.-N., Walker, M. S., and Becker, E E (1981). DNA methylation and methylase levels in normal and malignant mouse hepatic tissue. Carcinogenesis 2, 873-878. Lee, G. H., Nomura, K., Kanda, H., Kusakabe, M., Yoshiki, A., Sakakura, T., and Kitagawa, T. (1991). Strain specific sensitivity to diethylnitrosamine-induced carcinogenesis is maintained in hepatocytes of C3H/HeN~-)C57BL/6N chimeric mice. Cancer Res. 51, 32573260. Lewis, J., and Bird, A. (1991). DNA methylation and chromatin structure. FEBS Lett. 285, 155-159. Li, E., Bestor, T. H., and Jaenisch, R. (1992). Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell (Cambridge, Mass.) 69, 915-926. Locker, J., Reddy, T. V., and Lombardi, B. (1986). DNA methylation and hepatocarcinogenesis in rats fed a choline-devoid diet. Carcinogenesis 7, 1309-1312. Lombardi, B., and Smith, M. L. (1994). Tumorigenesis, protooncogene activation, and other gene abnormalities in methyl deficiency. J. Nutr. Biochem. 5, 2-9. McClain, R. M. (1994). Mechanistic considerations in the regulation and classification of chemical carcinogens. In "Nutritional Toxicology" (E N. Kotsonis, M. Mackey, and J. Hjelle, eds.), pp. 273-304. Raven, New York. Makos, M., Nelkin, B. D., Chazin, V. R., Cavenee, W. K., Brodeur, G. M., and Baylin, S. B. (1993a). DNA hypermethylation is associated with 17p allelic loss in neural tumors. Cancer Res. 53, 2715-2718. Makos, M., Nelkin, B. D., Reiter, R. E., Gnarra, J. R., Brooks, J., Isaacs, W., Linehan, M., and

252

Jennifer L. Counts and Jay I. Goodman

Baylin, S. B. (1993b). Regional hypermethylation at D17S5 precedes 17p structural changes in the progression of renal tumors. Cancer Res. 53, 2719-2722. Marx, J. (1991). Possible new colon cancer gene found. Science 251, 1317. Matthews, E. J., Spalding, J. W., and Tennant, R. W. (1993). Transformation of BALB/c-3T3 cells: v. Transformation responses of 168 chemicals compared with mutagenicity in salmonella and carcinogenicity in rodent bioassays. Environ. Health Perspect. 101, (Suppl. 2), 347-482. Meehan, R., Lewis, J., Cross, S., Nan, X., Jeppesen, P., and Bird, A. (1992). Transcriptional repression by methylation of CpG. J. Cell Sci. 16 (Suppl.), 9-14. Migeon, B. R., Axelman, J., and Beggs, A. H. (1988). Effect of ageing on reactivation of the human X-linked HPRT locus. Nature (London) 335, 93-96. Mikol, Y. B., Hoover, K. L., Creasia, D., and Poirier, L. A. (1983). Hepatocarcinogenesis in rats fed methyl-deficient, amino acid-defined diets. Carcinogenesis 4, 1619-1629. Miller, J. A. (1994). Recent studies on the metabolic activation of chemical carcinogens. Cancer Res. 54, (Suppl.), 1879s-1881s. Mfinzel, P. A., Pfohl-Leszkowicz, A., R6hrdanz, E., Keith, G., Dirheimer, G., and Bock, K. W. (1991). Site-specific hypomethylation of comyc protooncogene in liver nodules and inhibition of DNA methylation by Nonitrosomorpholine. Biochem. Pharmacol. 42, 365-371. Murchie, A. I. H., and Lilley, D. M. J. (1989). Base methylation and local DNA helix stability: Effect of the kinetics of cruciform extrusion. J. Mol. Biol. 205,593-602. Neades, R., Betz, N. A., Sheng, X., and Pelling, J. C. (1991). Transient expression of the cloned mouse c-Ha-ras 5' upstream region in transfected primary SENCAR mouse keratinocytes demonstrates its power as a promoter element. Mol. Carcinog. 4, 369-375. Newberne, P. M., deCamargo, J. L. V., and Clark, A. J. (1982). Choline deficiency, partial hepatectomy, and liver tumors in rats and mice. Toxicol. Pathol. 10, 95-106. Newberne, P. M., Suphiphat, V., Locniskar, M., and deCamargo, J. L. V. (1990). Inhibition of hepatocarcinogenesis in mice by dietary methyl donors methionine and choline. Nutr. Cancer 14, 175-181. Opdencamp, K., Rivi~re, M., Moln~, M., Szpirer, J., and Szpirer, C. (1992). Methylation of an oL-foetoprotein gene intragenic site modulates gene activity. Nucliec Acids Res. 20, 171178. Pascale, R. M., Marras, V., Simile, M. M., Diano, L., Pinna, G., Bennati, S., Carta, M., Seddaiu, M. A., Massarelli, G., and Feo, E (1992). Chemoprevention of rat liver carcinogenesis by S-adenosyl-L-methionine: A long-term study. Cancer Res. 52, 4979-4986. Peraino, C., Fry, R. J. M., Staffeldt, E., and Kisieleski, W. E. (1973). Effects of varying the exposure to phenobarbital on its enhancement of 2-acetylaminofluorene-induced hepatic tumorigenesis in the rat. Cancer Res. 33, 2701-2705. Pfohl-Leszkowicz, A., and Dirheimer, G. (1986). Changes in de novo DNA (cytosine-5-)methyltransferase activity in oncogenically susceptible rat target tissues induced by N-methyl-N-nitrosourea. Cancer Res. 46, 1110-1113. Pitot, H. C. (1990). Altered hepatic foci: Their role in murine hepatocarcinogenesis. Annu. Rev. Pharmacol. Toxicol. 30, 465-500. Ray, J. S., Harbison, M. L., McClain, R. M., and Goodman, J. I. (1994). Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse liver tumors. Mol. Carcinog. 9, 155-166. Razin, A., and Cedar, H. (1991). DNA methylation and gene expression. Microbiol. Rev. 55, 451-458. Razin, A., Szyf, M., Kafri, T., Roll, M., Giloh, H., Scarpa, S., Carotti, D., and Cantoni, G. L. (1986). Replacement of 5-methylcytosine by cytosine: A possible mechanism for transient DNA demethylation during differentiation. Proc. Natl. Acad. Sci. U.S.A. 83, 2827-2831. Reynolds, S. H., Stowers, S. J., Patterson, R. M., Maronpot, R. R., Aaronson, S. A., Anderson,

10.

Hypomethylation of DNA

253

M. W. (1987). Activated oncogenes in B6C3F1 mouse liver tumors: Implications for risk assessment. Science 237, 1309-1316. Rhim, J. S. (1993). Neoplastic transformation of human cells in vitro. Crit. Rev. Oncogen. 4, 313-335. Rideout, W. M., Coetzee, G. A., Olumi, A. E, and Jones, P. A. (1990). 5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science 249, 1288-1290. Rimoldi, D., Srikantan, V., Wilson, V. L., Bassin, R. H., and Samid, D. (1991). Increased sensitivity of nontumorigenic fibroblasts expressing ras or myc oncogenes to malignant transformation induced by 5-aza-2'-deoxycytidine. Cancer Res. 51, 324-330. Rogers, A. E., Zeisel, S. H., and Groopman, J. (1993). Diet and carcinogenesis. Carcinogenesis 14, 2205-2217. Rouleau, J., Tanigawa, G., and Szyf, M. (1992). The mouse DNA methyltransferase 5'-region--a unique housekeeping gene promoter. J. Biol. Chem. 267, 7368-7377. Ruchirawat, M., Becker, E E, and Lapeyre, J.-N. (1984). Mechanism of rat liver DNA methyltransferase interaction with anti-benzo[a]pyrene diol epoxide modified DNA templates. Biochemistry 23, 5426-5432. Rumsby, P. C., Davies, M. J., Price, R. J., and Lake, B. G. (1994). Effect of some peroxisome proliferators on transforming growth factor-f31 gene expression and insulin-like growth factor II/mannose-6-phosphate receptor gene expression in rat liver. Carcinogenesis 15, 419-421. Rushmore, T. H., Farber, E., Ghoshal, A. K., Parodi, S., Pala, M., and Taningher, M. (1986). A choline-devoid diet, carcinogenic in the rat, induces DNA damage and repair. Carcinogenesis 7, 1677-1680. Rushmore, T. H., Ghazarian, D. M., Subrahmanyan, V., Farber, E., and Ghoshal, A. K. (1987). Probable free radical effects on rat liver nuclei during early hepatocarcinogenesis with a choline-devoid low methionine diet. Cancer Res. 47, 6731-6740. Sawada, N., Poirier, L., Moran, S., Xu, Y. H., and Pitot, H. C. (1990). The effect of choline and methionine deficiencies on the number and volume percentage of altered hepatic foci in the presence or absence of diethylnitrosamine initiation in rat liver. Carcinogenesis 11, 273281. Schulte-Hermann, R., Timmermann-Trosiener, I., Barthel, G., and Bursch, W. (1990). DNA synthesis, apoptosis, and phenotypic expression as determinants of growth of altered foci in rat liver during phenobarbital promotion. Cancer Res. 50, 5127-5135. Schwartz, M., Buchmann, A., Bauer-Hofmann, R., and Mahr, J. (1991). Molecular genetic analysis of preneoplastic and neoplastic liver lesions in the mouse. Prog. Histochem. Cytochem. 23, 107-114. Sharrard, R. M., Royds, J. A., Rogers, S., and Shorthouse, A. J. (1992). Patterns of methylation of the c-myc gene in human colorectal cancer progression. Br. J. Cancer 65, 667-672. Shen, J.-C., Rideout, W. M., and Jones, P. A. (1994). The rate of hydrolytic deamination of 5-methylcytosine in double-stranded DNA. Nucleic Acids Res. 22, 972-976. Shinozuka, H., and Lombardi, B. (1980). Synergistic effect of a choline-devoid diet and phenobarbital in promoting the emergence of foci of ~/-glutamyltranspeptidase-positive hepatocytes in the liver of carcinogen-treated rats. Cancer Res. 40, 3846-3849. Shinozuka, H., Masuhara, M., Kubo, Y., and Katyal, S. L. (1993). Growth factor and receptor modulations in rat liver by choline-methionine deficiency. J. Nutr. Biochem. 4, 610-617. Shivapurkar, N., Wilson, M. J., Hoover, K. L., Mikol, Y. B., Creasia, D., and Poirier, L. A. (1986). Hepatic DNA methylation and liver tumor formation in male C3H mice fed methionine- and choline-deficient diets. J. Natl. Cancer Inst. 77, 213-217. Smith, S. S., Kaplan, B. E., Sowers, L. C., and Newman, E. M. (1992). Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation. Proc. Natl. Acad. Sci. U.S.A. 89, 4744-4748.

254

Jennifer L. Counts and Jay I. Goodman

Spandidos, D. A., and Holmes, L. (1987). Transcriptional enhancer activity in the variable tandem repeat DNA sequence downstream of the human Ha-ras 1 gene. FEBS Lett. 218, 41-46. St6ger, R., Kubicka, P., Liu, C.-G., Kafri, T., Razin, A., Cedar, H., and Barlow, D. P. (1993). Maternal-specific methylation of the imprinted mouse IGF2 locus identifies the expressed locus as carrying the imprinting signal. Cell (Cambridge, Mass.) 73, 61-71. Stowers, S. J., Wiseman, R. W., Ward, J. M., Miller, E. C., Miller, J. A., Anderson, M. W., and Eva, A. (1988). Detection of activated proto-oncogenes in N-nitrosodiethylamine-induced liver tumors: A comparison between B6C3F 1 mice and Fischer 344 rats. Carcinogenesis 9, 271-276. Sullivan, N., Gatehouse, D., and Tweats, D. (1993). Mutation, cancer and transgenic models: Relevance to the toxicology industry. Mutagenesis 8, 167-174. Swenberg, J. A., Richardson, F. C., Boucheron, J. A., Deal, E H., Belinsky, S. A., Charbonneau, M., and Short, B. G. (1987). High- to low-dose extrapolation: Critical determinants involved in the dose response of carcinogenic substances. Environ. Health Perspect. 76, 57-63. Szyf, M. (1991). DNA methylation patterns: An additional level of information? Biochem. Cell Biol. 69, 764-767. Tan, N.-W., and Li, B. E L. (1990). Interaction of oligonucleotides containing 6-0methylguanine with human DNA (cytosine-5)-methyltransferase. Biochemistry 29, 92349240. Tawa, R., Ueno, S., Yamamoto, K., Yamamoto, Y., Sagisaka, K., Katakura, R., Kayama, T., Yoshimoto, T., Sakurai, H., and Ono, T. (1992). Methylated cytosine level in human liver DNA does not decline in aging process. Mech. Ageing Dev. 62, 255-261. Vairapandi, M., and Duker, N. J. (1993). Enzymic removal of 5-methylcytosine from DNA by a human DNA-glycosylase. Nucleic Acids Res. 21, 5323-5327. Vilpo, J. A., and Vilpo, L. M. (1993). Nucleoside monophosphate kinase may be the key enzyme preventing salvage of DNA 5-methylcytosine. Mutat. Res. 286, 217-220. Vogelstein, B., Fearon, E. R., Hamilton, S. R., Kern, S. E., Preisinger, A. C., Leppert, M., Nakamura, Y., White, R., Smits, A. M. M., and Bos, J. L. (1988). Genetic alterations during colorectal-tumor development. N. Eng. J. Med. 319, 525-532. Vorce, R. L., and Goodman, J. I. (1989). Altered methylation of ras oncogenes in benzidineinduced B6C3F1 mouse liver tumors. Toxicol. Appl. Pharmacol. 100, 398-410. Vorce, R. L., and Goodman, J. I. (1991a). Differential DNaseI hypersensitivity of ras oncogenes in B6C3F1, C3H/He and C57BL/6 mouse liver. J. Toxicol. Environ. Health 34, 385-395. Vorce, R. L., and Goodman, J. I. (1991b). Hypomethylation of ras oncogenes in chemically induced and spontaneous B6C3F1 mouse liver tumors. J. Toxicol. Environ. Health 34, 367-384. Wainfan, E., and Poirier, L. A. (1992). Methyl groups in carcinogenesis: Effects on DNA methylation and gene expression. Cancer Res. 52(Suppl.), 2071s-2077s. Wainfan, E., Dizik, M., Stender, M., and Christman, J. K. (1989). Rapid appearance of hypomethylated DNA in livers of rats fed cancer-promoting, methyl-deficient diets. Cancer Res. 49, 4094-4097. Wareham, K. A., Lyon, M. E, Glenister, P. H., and Williams, E. D. (1987). Age related reactivation of an X-linked gene. Nature (London) 327, 725-727. Weghorst, C. M., and Klaunig, J. E. (1989). Phenobarbital promotion in diethylnitrosamineinitiated infant B6C3F1 mice: Influence of gender. Carcinogenesis 10, 609-612. Weitzman, S. A., Turk, P. W., Milkowski, D. H., and Kozlowski, K. (1994). Free radical adducts induce alterations in DNA cytosine methylation. Proc. Natl. Acad. Sci. U.S.A. 91, 1261-1264.

10.

Hypomethylation of D N A

255

Williams, G. M. (1980). Classification of genotoxic and epigenetic hepatocarcinogens using liver culture assays. Ann. N.Y. Acad. Sci. 349, 273-282. Williams, G. M. (1989). Methods for evaluating chemical genotoxicity. Annu. Rev. Pharmacol. Toxicol. 29, 189-211. Williams, G. M., and Weisburger, J. H. (1991). Chemical carcinogens. In "Casarett and Doull's Toxicology: The Basic Science of Poisons" (M. A. Amdur, J. Doull, and C. D. Klaassen, eds.), pp. 127-200. Pergamon, New York. Wilson, V. L., and Jones, P. A. (1983). DNA methylation decreases in aging but not in immortal cells. Science 220, 1055-1057. Wilson, M. J., Shivapurkar, N., and Poirier, L. A. (1984). Hypomethylation of hepatic nuclear DNA in rats fed with a carcinogenic methyl-deficient diet. Biochem. J. 218, 987-990. Wilson, V. L., Smith, R. A., Longoria, J., Liotta, M. A., Harper, C. M., and Harris, C. C. (1987a). Chemical carcinogen-induced decreases in genomic 5-methyldeoxycytidine content of normal human bronchial epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 84, 32983301. Wilson, V. L., Smith, R. A., Ma, S., and Cutler, R. G. (1987b). Genomic 5-methyldeoxycytidine decreases with age. J. Biol. Chem. 262, 9948-9951. Wiseman, R. W., Stowers, S. J., Miller, E. C., Anderson, M. W., and Miller, J. A. (1986). Activating mutations of the c-Ha-ras proto-oncogene in chemically induced hepatomas of the male B6C3F 1 mouse. Proc. Natl. Acad. Sci. U.S.A. 83, 5825-5829. Wojciechowski, M. E, and Meehan, T. (1984). Inhibition of DNA methyltransferases in vitro by benzo[a]pyrene diol epoxide-modified substrates. J. Biol. Chem. 259, 9711-9716. Xodo, L. E., Alunnifabbroni, M., and Manzini, G. (1994). Effect of 5-methylcytosine on the structure and stability of DNA--formation of triple-stranded concatenamers by overlapping oligonucleotides. J. Biomol. Struct. Dyn. 11, 703-720. Xu, Y., Goodyer, C. G., Deal, C., and Polychronakos, C. (1983). Functional polymorphism in the parental imprinting of the human IGF2R gene. Biochem. Biophys. Res. Commun. 197, 747-754. Yao, Z., and Vance, D. E. (1988). The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes. J. Biol. Chem. 263, 29983004. Yisraeli, J., Frank, D., Razin, A., and Cedar, H. (1988). Effect of in vitro DNA methylation on J3-globin gene expression. Proc. Natl. Acad. Sci. U.S.A. 85, 4638-4642. Zapisek, W. E, Cronin, G. M., Lyn-Cook, B. D., and Poirier, L. A. (1992). The onset of oncogene hypomethylation in the livers of rats fed methyl-deficient, amino acid-defined diets. Carcinogenesis 13, 1869-1872. Zeisel, S. H., Zola, T., daCosta, K.-A., and Pomfret, E. A. (1989). Effect of choline deficiency on S-adenosylmethionine and methionine concentrations in rat liver. Biochem. J. 259, 725729. Zhang, X., and Mathews, C. K. (1994). Effect of DNA cytosine methylation upon deaminationinduced mutagenesis in a natural target sequence in duplex DNA. J. Biol. Chem. 269, 7066-7069. Zhang, X.-Y., Supakar, P. C., Wu, K., Ehrlich, K. C., and Ehrlich, M. (1990). An MDBP site in the first intron of the human c-myc gene. Cancer Res. 50, 6865-6869.

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11 Transgenic Models of Hepatic Growth Regulation and Hep ato carcinogenesis Eric R Sandgren Department of PathobiologicalSciences School of VeterinaryMedicine University of Wisconsin-Madison Madison, Wisconsin53706

I. Transgene-Based Strategies for Studying Liver Growth, Development, and Cancer To the hepatologist, use of in v i v o m o d e l s to study cellular growth regulation has become second nature. This is true mainly because of the ability of two-thirds partial hepatectomy to provide synchronized populations of dividing cells, and, in fact, much of our understanding of liver growth and regeneration is based on studies employing this protocol in the rat (reviewed in Fausto and Webber, 1993; Fausto and Webber, 1994). Therefore, following development of technologies to modify the mammalian genome through production of transgenic or embryonic stem (ES) cell-derived mice, it was not surprising that investigators would apply these techniques to study the liver. This chapter will review pertinent studies employing genetically modified animals that address liver growth regulation and neoplasia. The mechanics of production of transgenic and ES cell-derived mice will be described only briefly, as these have been reviewed elsewhere (Gordon, 1993; Ramirez-Solis et al., 1993; Stewart, 1993). General reviews of transgenic models of liver gene expression and disease also have been recently published (Sell and Knoll, 1992; Macri and Gordon, 1993, Merlino, 1994). The most common method of producing genetically altered animals is to introduce DNA into fertilized mouse eggs. This process gives rise to transLiver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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genic mice that have incorporated the foreign DNA into their genome (Brinster et al., 1985). To accomplish this, fertilized one-cell mouse eggs in egg culture medium are fixed in place on the stage of a microscope and pierced by a sharp-tipped microinjection needle containing a DNA solution. The solution is expelled into one of the two egg pronuclei, and surviving eggs are surgically transferred into the oviducts of pseudopregnant foster mothers. Offspring resulting from this procedure are screened to identify those in which the foreign DNA (transgene) has become integrated into the genome; these mice should carry a copy of the transgene in every cell of the body. Generally, multiple (up to several hundred) copies of the transgene in a head-to-tail linear array integrate into a single, presumably random, chromosomal location. Transgene-positive mice are termed founder animals, and their transgene-bearing offspring constitute lineages, all members of which share the same chromosomal transgene integration site and generally display a similar pattern of transgene expression. Different chromosomal insertion sites often mediate different patterns or levels of expression of the same transgene, so it is necessary to analyze lineages separately. For the same reason, it also is necessary to reproduce key findings in more than one lineage as a way to ensure that a phenotype is due to transgene expression, not disruption of an endogenous gene. A further consideration in interpreting the results of experiments employing transgenic animals must be the genetic background into which the transgene is introduced, which can affect dramatically the transgene-associated phenotypes. With this methodology, experimental design is critically dependent on the nature of the transgene DNA. Transgene constructs generally contain two functional elements: the DNA sequence encoding the protein whose activity is being studied, and the DNA regulatory elements (enhancer/promoters) that target gene expression specifically to one or more cell types. (Specific enhancer/promoters that have been used to target transgene expression to liver are discussed in Section VI.A of this chapter.) Thus, chimeric genes contain a novel combination of regulatory and coding elements, and can target gene expression to novel tissues. In contrast, the recently developed ES cell-derived or gene-targeted mice provide a way to "knock out" or otherwise modify endogenous genes (Ramirez-Solis et al., 1993; Stewart, 1993). Embryonic stem cells are derived from the inner cell mass of the blastocyst and are totipotent, which means that they can, under appropriate conditions, give rise to every cell type of the body. When they are reinjected into blastocysts, they often become incorporated into the recipient inner cell mass and thereafter develop into a part of the resulting fetus. Chimeric or "tetraparental" mice derived from injected blastocysts will generate some offspring of the ES cell genotype, provided that ES cells colonized the recipient germline. The strength of this system lies in its ability to modify and select cultured ES

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cells. For example, a gene targeting construct with homology to endogenous DNA can be introduced into ES cells. This construct will undergo homologous recombination in a small fraction of cells, resulting in transfer to an endogenous gene of a defined deletion or mutation carried in the targeting vector. These infrequent gene-targeted cells can be isolated by one of several available selections protocols, expanded, and injected into blastocysts, ultimately giving rise to mice carrying the desired gene alteration. Subsequent interbreeding can yield homozygous mutant mice. Gene-targeted mice demonstrate the effects of altering the expression of endogenous genes, and therefore provide models that complement transgenic mice in assessing the role of gene expression during animal development, growth, and neoplasia. To date, transgenic mice have provided the principal models of genetically altered animals employed in studies of liver growth regulation and carcinogenesis (Merlino, 1994). Transgenic studies can be divided into several categories based on the character of the coding sequence employed. Reflecting this, the following sections of this chapter will describe hepatocarcinogenesis induced by oncogenes, growth factors, and hepatotoxic gene products, and then transgene-based analyses of multistage carcinogenesis and hepatic growth regulation. The closing section discusses approaches to the study of liver growth control employing genetically modified animals that are likely to become important over the next several years.

II. Oncogenic Transgenes and Hepatic Neoplasia A. Viral

Oncogenes

1. Simian Virus 40 T Antigens

By the mid-1980s, evidence had accumulated linking expression of viral and altered endogenous genes ("oncogenes") with tumor initiation and progression. To test directly the oncogenicity of these genes, several groups developed transgenic mice in which oncogene expression was targeted to specific cell types. The first transgene shown to produce liver tumors in vivo encoded the simian virus 40 T or transforming antigens (SV40 TAg) (Messing et al., 1985). Since that report, transgenic mouse models employing a variety of targeting strategies have confirmed TAg's hepatocarcinogenicity (Table 1). TAg induces multiple biochemical changes in target cells, which in liver inexorably produce a sequence of morphological alterations reminiscent of those observed during chemical carcinogenesis: the development of (1) altered hepatic foci of basophilic, clear cell, or eosinophilic type, (2) hepatic adenomas, and (3) solid or trabecular hepatocellular carcinomas (Cullen et al., 1993). Each of these lesions is focal, and TAg-induced tumors have been shown to be clonal (Sandgren et al., 1989; Dubois et al., 1991) but

Table I Viral Oncogene-Induced Hepatic Neoplasia in Transgenic Animals Oncogene a

Gene regulatory elementb, ,

SV40-TAg

MT; MUP; AL; AAT; ATIII; SAP; L-PK; CRP

SV40-Tag

Gastrin

HBx HBx

AAT HBx

H I V tat

HIV-LTR (no/little hepatic expression)

H I V tat

HIV-LTR plus BKVearly (hepatic expression)

STP-C

H2, MT

Sequence of hepatic lesions `/ 1. Diffuse hepatocellular hyperplasia, dysplasia by 1 to 3 months of age. 2. Multiple altered hepatic foci, principally basophilic or clear cell types; more severe dysplasia of remaining liver; by 2 to 6 months of age. 3. Multiple basophilic or clear cell hyperplastic nodules or adenomas; trabecular hepatocellular carcinomas; occasional cholangiomas or cholangiocarcinomas; by 3 to 10 months of age. 4. Incidence 100%. 1. Hepatocellular and biliary dysplasia and neoplasia by 3 to 8 months of age. Incidence up to 100%. 1. No significant lesions. 1. Pericentral dysplasias by 2 months of age. 2. Altered hepatocellular loci by 8 to 12 months of age. 3. Eosinophilic, basophilic, or clear cell adenomas; trabecular hepatocellular carcinomas; beginning at 10 months of age. Incidence 60 to 85%. 1. Diffuse hepatocellular dysplasia (onset not determined; all mice examined at over 12 months of age). 2. Basophilic or eosinophilic hepatic adenomas; solid or trabecular hepatocellular carcinomas; beginning at 12 months of age. Incidence of all lesions 42%; incidence of carcinomas 27%. 1. Diffuse hepatocellular dysplasia by 6 to 7 months of age. Incidence 85%. 2. One hepatocellular carcinoma at 15 months of age. 1. Bile duct hyperplasia by 2 weeks of age. 2. Cholangiofibromas by 1 to 3 months of age. Incidence of all lesions 65-100%.

a Abbreviations: SV40-TAg, simian virus 40 T antigens; HBx, hepatitis B virus x gene; HIV tat, human immunodeficiency virus tat gene; STP-C, saimiri transformation associated protein subgroup C gene. bAbbreviations: MT, metallothionein-I; MUP, major urinary protein; AL, albumin; AAT, alpha-l-antitrypsin; ATIII, antithrombin III; SAP, serum amyloid protein; L-PK, liver type pyruvate kinase; CRP, C reactive protein; HBx, hepatitis B virus x gene; HIV-LTR, human immunodeficiency virus long terminal repeat; BKV, BK virus; H2, histocompatibility 2. cReferences: MT-TAg, Messing et al., 1985, Dyer and Messing, 1989; MUP-TAg, Held et al., 1989, Schirmacher et al., 1991, Held et al., 1994; AL-TAg, Sandgren et al., 1989, Hino et al., 1991, Cullen et al., 1993; AAT-TAg, Sepulveda et al., 1989, Butel et al., 1990; ATIIITAg, Dubois et al., 1991; SAP-TAg, Araki et al., 1991; L-PK-TAg, Cartier et al., 1992; CRP-TAg, Ruther et al., 1993; gastrin-TAg, Montag et al., 1993; AAT-HBx, Lee et al., 1990b; HBx-HBx, Kim et al., 1991, Koike et al., 1994; HIV-LTR-Tat, Vogel et al., 1991; BKV/HIV-LTR-tat, Corallini et al., 1993; H2-STP-C and MT-STP-C, Murphy et al., 1994. aThe precise onet and rate of progression of the principal hepatic lesions in each group of transgenic mice will vary for different constructs and for different lineages with the same construct. Information summarized in this column provides typical lesions and the sequence and range of ages at which they appear. Lesions developing in other organs are not described.

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their presence in transgenic mouse liver is generally preceded by the appearance of diffuse, progressively worsening hepatocellular dysplasia and hyperplasia. In some lineages dysplasia was not a prominent early finding (Araki et al., 1991), but in this case a correspondingly focal rather than diffuse pattern of TAg expression was present in liver, preserving the association between TAg expression and dysplastic lesions. (Nonuniform transgene expression, though not expected with promoters that target all cells of a particular type, is a frequent complication in transgenic studies, and is reproducible among offspring of affected lineages.) Thus, dysplasia and hyperplasia likely constitute direct effects of TAg on hepatocytes, and these effects may represent a state of hepatocellular preneoplasia. However, because subsequent lesions are focal, TAg does not appear sufficient to produce further neoplastic changes; additional, stochastic alterations in target cells must be required for induction of altered foci, adenomas, and carcinomas. This observation is consistent with a multistage mechanism of tumor progression in TAg transgenic mice. The sequence of changes described above was observed whether transgene expression was constitutive or inducible (Cartier et al., 1992; Ruther et al., 1993). Indeed, there is remarkable similarity in the character of TAg-induced liver tumor progression when diverse targeting schemes are employed, with an average latency from onset of TAg expression to development of liver tumors of 3 to 5 months, although the pattern of extrahepatic lesions observed could vary significantly (in each case reflecting the pattern of extrahepatic transgene expression). The variability in the timing of lesions noted in Table 1 reflects the variable onset and level of expression in different lineages of transgenic mice. Interestingly, continuous high-level expression of TAg may not be required for subsequent tumor progression. Alpha-l-antitrypsin-TAg (AATTAg) transgenic mice exhibited a decline of TAg expression at and beyond 3 weeks of age yet efficiently developed multiple tumors within several months (Sepulveda et al., 1989), and halting LPS-mediated induction of the C reactive protein-TAg (CRP-TAg) transgene after 30 versus 90 days of treatment delayed only slightly the appearance of tumors (although a postLPS decline in transgene expression was not documented in the latter study; Ruther et al., 1993). Thus, tumor progression may become a transgeneindependent event once the early stages of tumorigenesis are completed. Not all liver tumors that developed in these models were hepatocytic. A notable finding associated with expression of four transgene constructs listed in Table 1 was the development of biliary epithelial lesions. Cholangiofibromas and occasionally cholangiocarcinomas were observed in multiple gastrin-TAg lineages (Montag et al., 1993). Biliary epithelial tumors also appeared in albumin-TAg (AL-TAg) (Sandgren et al., 1989; Cullen et al., 1993), major urinary protein-TAg (MUP-TAg) (Held et al., 1989), and

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CRP-TAg mice (Ruther et al., 1993), although at a lower frequency than hepatocellular carcinomas and in only a subset of lineages. Because MUP and the inducible CRP are likely to be expressed only in postnatal mouse liver, the appearance of biliary lesions is unlikely to reflect TAg expression in a fetal bipotential precursor cell in liver. Rather, occurrence of lesions in both the hepatocellular and biliary epithelial lineages suggests either (1) that a transgene-expressing bipotential cell remains present in postnatal mouse liver, or (2) that the respective promoters are expressed at a low level and/or in a subset of adult biliary epithelial cells. Regardless of mechanism, this finding indicates that both biliary epithelial cells and hepatocytes are susceptible to neoplastic transformation by TAg in vivo. Given the historical preponderance of rat models of hepatocarcinogenesis, it is comforting to report that hepatic lesions in the recently described AL-TAg transgenic rat (Hully et al., 1994) generally reproduce those described above for transgenic mice. In this model, the earliest lesions tended to be focal, but so did the pattern of TAg expression. Subsequent lesions included adenomas and carcinomas, as in mice.

2. Hepatitis B Virus x-Antigen Identifying pathogenic mechanisms of hepatitis 13 virus (HBV) infection has been the principal aim of several transgenic mouse studies, and these have taken one of two approaches. The first, introduction of the whole virus into transgenic animals, has not produced preneoplastic lesions or cancer (Farza et al., 1988; Burk et al., 1988; Araki et al., 1989). Targeting expression to liver of specific HBV genes has been a second approach, and this also has taken two forms. Hepatocyte-directed expression of HBV surface antigen is hepatotoxic and results in hepatic inflammation and necrosis, nodular regeneration, and carcinogenesis; these mice will be described further in Section IV.A of this chapter. Expression of the HBV x-protein (HBx), a transactivator of viral and cellular genes, also has proved capable of transforming mouse liver, and this is discussed below. The first model of liver-targeted HBx expression, employing an AATHBx transgene, was characterized by relatively low-level x gene expression that declined further in 2-month-old and older mice (Table 1; Lee, T.-H., et al., 1990). These mice and their livers remained essentially normal. In contrast, mice bearing a portion of the HBV genome that included the viral enhancer and HBx coding elements displayed higher-level hepatic HBx expression at all ages (Table 1; Kim et al., 1991; Koff, 1992; Koike et al., 1994). These mice developed focal, pericentral dysplasias with increased HBx protein and elevated DNA synthesis that progressed to hepatocellular adenomas and carcinomas beginning at one year of age (Koike et al., 1994). The focal lesions appeared early, by 2 months of age, yet further mor-

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phological evidence of progression was not apparent for an additional 10 months. Two lineages displayed lesions of this type, although in one the tumors were evident only in mice homozygous for the transgene, which expressed more HBx than heterozygous littermates. Most importantly, lesions developed in the absence of hepatic inflammation or marked hepatocellular necrosis. These findings suggest that hepatic expression of HBx can have a causative role in hepatocarcinogenesis. However, like SV40 TAg, HBx alone was not sufficient to transform this target cell population. The creators of these mice argued that HBx induces focal proliferative lesions within which cells become susceptible to secondary transformation events (Koike et al., 1994), a theme echoed throughout this chapter. 3. Other Viral Antigens

Two groups of mice bearing transgenes encoding the human immunodeficiency virus (HIV) tat transactivating protein displayed diffuse hepatocellular dysplasias that progressed to carcinoma with variable frequency (Table 1; Vogel et al., 1991; Corallini et al., 1993). Interestingly, mice bearing the HIV-long terminal repeat tat gene (HIV-LTR-tat) construct displayed little or no hepatic transgene expression but did have high level expression in other organs, most notably skin. This observation raised the possibility that hepatic lesions were induced by tat-mediated perturbations of extrahepatic growth signaling pathways originating in other tissues (Vogel et al., 1991). Given the range of lesions and other functional changes that appear in each group of HIV-tat transgenic mice, the precise mechanisms involved in this effect are not yet apparent. However, results of this type illustrate the need for in vivo studies to provide evidence of pathogenetic associations that would not exist in vitro. The most recent report of viral-induced hepatic neoplasia involved expression of the herpes virus saimiri transformation associated protein of subgroup C (STP-C) (Murphy et al., 1994). STP-C is a transforming protein produced by a herpes virus that causes lymphoma in certain primate hosts. Surprisingly, widespread STP-C targeting with the histocompatibility H2 gene enhancer/promoter caused rapid (but still focal) neoplastic transformation of biliary epithelium as well as salivary, pancreatic, and thymic epithelia; T cells were not affected (Table 1; Murphy et al., 1994). Metallothionein-I (MT)-driven STP-C expression reproduced this phenomenon in biliary epithelium. These observations provide a fascinating example of the effect of separating expression of a pathogenic viral gene from its typical hosts and the natural route of viral infection. Thus, the range of tissues that STP-C can transform is dramatically extended, and these mice provide a means to examine mechanisms of herpes virus-mediated tumorigenesis in novel tissue contexts. However, this also points to an important caveat in

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the design and interpretation of transgenic animal experiments: engineered expression of a transforming gene may establish an artificial model of disease with little direct relevance to the gene's typical pathogenic role. B. Cellular Oncogenes 1. H - R a s

The genetic change most commonly identified in rodent liver tumors is mutational activation of the Harvey-ras (H-ras) gene (Barrett and Wiseman, 1992; Sell and Knoll, 1992). The Ras p21 protein is an integral component of growth stimulatory signal transduction pathways, and carcinogenic mutations cause Ras to become orders of magnitude more active (reviewed in Boguski and McCormick, 1993). To examine the hepatocarcinogenic role of mutation of this gene in hepatocarcinogenesis, a mutant H-ras coding sequence (encoding a glycine to valine substitution at amino acid 12) was ligated to the albumin enhancer/promoter and introduced into mouse eggs. The result was striking. Three-fourths of the AL-ras transgenic founder mice were born with hyperplastic livers up to four times the normal weight and composed of morphologically immature hepatocytes (Table 2; Sandgren et al., 1989). The uniform appearance of hyperplastic neonatal AL-ras liver cells and the inability of those cells to grow when transplanted into syngeneic hosts (Sandgren, unpublished observation) suggested that overexpression of mutant H-Ras caused excessive activation of a potent fetal growth stimulatory signal transduction pathway in all targeted cells, inducing a diffuse growth disorder rather than neoplasia. Consistent with this interpretation was a finding of elevated 1,2-diacylglycerol, a product of Ras activity, in hyperplastic newborn transgenic liver (Wilkison et al., 1989). The surviving one-fourth of founder mice appeared to express lower levels of the transgene in liver (Sandgren et al., 1989). However, each developed multiple and lethal lung adenomas and adenocarcinomas (Sandgren et al., 1989; Maronpot et al., 1991). Occasionally, they also displayed solitary hepatic neoplasms, indicating that adult liver was subject to transformation by mutant H-Ras, although lung appeared to be even more susceptible. These findings underscore another limitation to current transgene targeting schemes. Most spontaneous tumors develop in the context of adult tissues, and it is most appropriate to model these conditions by altering the pattern of gene expression in adults. However, transgene targeting is accomplished by using existing gene regulatory elements, which seldom initiate expression in the adult organ. Because of the dramatic and lethal effect of mutant H-ras in fetal liver, experiments with this oncogene need to be reproduced using a different targeting strategy (perhaps employing inducible promoters such as CRP, liver type pyruvate kinase (L-PK) or phosphoenolpyruvate carboxy-

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Table 2 Cellular Oncogene-Induced Hepatic Neoplasia in Transgenic Animals

Oncogene a

Gene regulatory elementb,r

(mutant)

AL

H-ras (normal)

AL

c-myc

AL, AAT, L-PK

c-myc

WHV

H~

Sequence of hepatic lesions`/ 1. Fetal heptocellular hyperplasia; liver enlarged 2 to 4 fold at birth; mice die within 1 week. Incidence 75% among founder mice. 2. Survivors (with lower hepatic transgene expression) develop hepatocellular adenocarcinomas (incidence 20 to 50%) and lung adenomas and adenocarcinomas. 3. Incidence 100% by 1 to 12 months of age. 1. Hepatocellular adenomas and carcinomas beginning at 10 months of age. Incidence 80%. 1. Centrilobular hepatocytomegally at 2 months of age. 2. Hepatocellular adenomas and adenocarcinomas beginning at 10 months of age. Incidence 50 to 75%. 1. Hepatocellular dysplasia by 2 months of age. 2. Hyperplastic nodules, adenomas, and hepatocellular carcinomas by 6 to 12 months of age. Incidence 90 to 100%.

aAbbreviations: H-ras, Harvey ras. bAbbreviations: AL, albumin; AAT, alpha-l-antitrypsin; L-PK, liver type pyruvate kinase; WHV, woodchuck hepatitis virus. cReferences: AL-H-ras mutant, Sandgren et al., 1989; AL-H-ras normal, Sandgren et al., unpublished; AL-c-myc, Sandgren et al., 1989; AAT-c-myc, Dalemans et al., 1990; L-PKc-myc, Cartier et al., 1992; WHV-c-myc, Etiemble et al., 1994. aThe precise onset and rate of progression of the principal hepatic lesions in each group of transgenic mice will vary for different constructs and for different lineages with the same construct. Information summarized in this column provides typical lesions and the sequence and range of ages at which they appear.

kinase (PEPCK); see Table 7 to better define the h e p a t o c a r c i n o g e n i c role of m u t a n t H - R a s in the adult. In contrast, AL-directed o v e r e x p r e s s i o n of the n o n m u t a t e d f o r m of H - R a s had no effect on fetal liver (Sandgren, P a l m i t e r a n d Brinster, unpublished observations). However, in three lines e x a m i n e d , m o s t mice over one year of age d e v e l o p e d multiple h e p a t o c e l l u l a r a d e n o m a s and carcinom a s (Table 2). The m e c h a n i s m of this effect was n o t e x a m i n e d , but transgenic mice bearing the w h o l e h u m a n c - H - r a s gene ( p r o m o t e r and coding elements) also developed t u m o r s in multiple o r g a n s by 18 m o n t h s of age (Saitoh et al., 1990). O v e r 7 0 % of the t u m o r s in those mice displayed activating point m u t a t i o n s in the transgene, suggesting t h a t engineered provision of a d d i t i o n a l c - H - r a s gene " t a r g e t s " increased the incidence of H - r a s

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gene activation and thereby of H-Ras-induced tumors. A similar mechanism may have been active in AL-c-H-ras transgenic mouse liver. This raises the possibility that AL-c-H-ras transgenic mice may provide highly sensitive hosts for bioassays designed to examine hepatic effects of chemical mutagens. However, treatment of mice bearing the whole c-H-ras gene with carbon tetrachloride, while accelerating hepatic tumor formation, did not produce tumors with activating point mutations in the transgene, implying that overexpression of unmutated Ras may stimulate hepatocarcinogenesis (Tsunematsu et al., 1994).

2. c-Myc The influence of c-Myc expression on rodent hepatic neoplasia has been less clearly defined than has mutational activation of H-Ras. Thus, several transgenic mouse models were established to identify the effect of constitutive c-Myc overexpression in liver. AL-directed expression of the c-myc gene in transgenic mice had no effect on fetal liver, but rather induced benign hypertrophy of pericentral hepatocytes in young adults (Table 2; Sandgren et al., 1989). Eventually, transgenic mice over 15 months of age began to develop focal hepatocellular adenomas or carcinomas. Similar findings were reported in mice bearing L-PK-myc (Cartier et al., 1992) or AAT-myc (Dalemans et al., 1990) transgenes. However, when fed a carbohydrate-rich diet, which induces expression from the L-PK promoter, L-PK-myc transgenic mice displayed elevated levels of c-Myc expression and accelerated tumorigenesis, suggesting that hepatic tumor pathogenesis may be influenced by the level of c-Myc produced. A notable recent study described the oncogenic effect of a fusion gene that combined the woodchuck hepatitis virus (WHV) enhancer region with the woodchuck c-myc promoter and coding region (Etiemble et al., 1994). This DNA was isolated as an intact unit from a spontaneous woodchuck liver tumor and thus reflected a naturally occurring insertion event during the course of WHV infection. The resulting transgene was particularly potent in mouse liver compared to AL- or AAT-myc transgenes. Dysplastic lesions appeared during the first 2 months of age in two WHV-myc lineages (Table 2). These were followed by small hyperplastic foci or adenomas by 4 months of age and finally progression to hepatocellular carcinoma between 6 and 12 months of age in most transgenic mice. These findings demonstrate the potency of novel viral/c-myc fusion genes that by virtue of their presence in tumors must have been selected for oncogenicity (even if this is not a widespread mechanism of viral oncogenicity). In this model, there was no production of the viral X transactivator protein or large surface protein (corresponding to HBx and HBsAg of hepatitis B virus, respectively; see

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Sections II.A and IV.A). Nevertheless, small surface viral proteins were produced, and their contribution to tumor progression remained undefined. C. Oncogene-Transformed Immortalized Cell Lines Every cell in the body of a transgenic mouse carries transgene DNA. Thus, primary cell cultures derived from tissues that express an oncogenic transgene may display immortalized or transformed characteristics due to continued transgene expression. This has been demonstrated for hepatocytes isolated from several different lineages of transgenic mice, most bearing SV40-TAg constructs. Paul et al. (1988) developed cultures of differentiated, immortalized, nonmalignant hepatocytes from day 19 fetal liver isolated from MT-TAg transgenic mice, and have used these cells to examine regulation of c-fos and c-myc protooncogenes in vitro (Hirsch-Ernst et al., 1993). Pavirani and colleagues reported synthesis of active human factor IX (hFIX) by cells derived from tumors that developed in mice carrying AAThFIX, AAT-c-myc, and AAT-TAg transgenes (Jallet et al., 1990). They used a similar approach to generate cell lines producing human AAT, and further compared the differentiation status of cell lines derived from c-Myc- versus TAg-expressing transgenic liver tumors (Dalemans et al., 1990; Perraud et al., 1991). Once established, cell lines carrying either oncogene displayed similar growth patterns and morphology. Finally, antithrombin III-TAg (ATIII-TAg) mouse livers have been used to produce well-differentiated mouse hepatocytic cell lines (Antoine et al., 1992), and these have been used to study the effects of transcription factors on expression of liver specific genes (Levrat et al., 1993). These cell lines, plus several described in Section III.A of this chapter that express an MT-transforming growth factor alpha (TGF0~) transgene, provide suitable in vitro hosts for a range of studies best addressed using cultured cells, but that may be difficult or impossible to examine in primary cultures of normal hepatocytes, which under most conditions rapidly lose their differentiated phenotype when removed from the organ. D. Conclusions Transgenic mouse technology provides a direct means to assess the influence on tissue growth and neoplasia of specific forms of altered gene expression. In the context of liver, the studies described above have produced several general conclusions: (1) oncogenes can have a causative role in hepatocarcinogenesis; (2) oncogenes are not equivalent in transforming potency; each distinct phenotype represents an interaction between the oncogene's biochemical activity and the tissue's repertoire of signal transducing or other transformation-associated target molecules; and (3) oncogenes are not suffi-

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cient to neoplastically transform adult hepatocytes since relatively few tumors develop from millions of transgene-expressing cells. Hepatocarcinogenesis is clearly a multistep phenomenon that includes but is not limited to expression of single oncogenic genes. The following sections consider transgenic mouse studies o~ additional ~actors that may influence tumor progression in liver.

III. Growth Factor Transgenes and Hepatic Neoplasia A. Transforming Growth Factor Alpha TGF~ is a 50 amino acid peptide ligand for the epidermal growth factor receptor (EGFr) (reviewed in Lee et al., 1993). It is produced by proteolytic cleavage of a larger transmembrane precursor, which itself is capable of binding to and initiating tyrosine kinase activity from EGFr on neighboring cells. TGFcx is a potent hepatic mitogen, and its expression is increased during liver regeneration (Fausto and Webber, 1993, 1994). Furthermore, increased TGFcx production is associated with hepatic transformation (Lee et al., 1993). As a means to identify the significance of TGF~ expression during hepatic regeneration and neoplasia, two groups introduced MTregulated, TGFcx-encoding transgenes into mice (Sandgren et al., 1990; Jhappan et al., 1990). MT is widely expressed in the body, most notably in the endodermally derived liver, pancreas, and gastrointestinal tract. Expression from the MT promoter also is highly inducible by heavy metals, including zinc. Thus, by providing zinc sulfate in the drinking water, maximal levels of MT-driven transgene expression can be attained. The most striking phenotype observed in young MT-TGFc~ transgenic mice was diffuse epithelial hyperplasia in most organs that expressed the transgene (Sandgren et al., 1990). In contrast, both fat and muscle mass were decreased in these animals, underscoring the importance of tissue context when evaluating growth factor effects in vivo (Luetteke et al., 1993a). Liver weight was increased up to two-fold, due to a generalized increase in cell number (Table 3). This process was not progressive, however; liver mass reached a new and elevated "set point" (determined as a percent of total body weight), then stabilized at this new equilibrium. Liver hyperplasia was accompanied by a persistent increase in the basal rate of hepatocyte replication relative to nontransgenic liver (Webber et al., 1994). Eventually, livers developed focal hepatocellular dysplasia, adenomas, and carcinomas (Lee et al., 1992; Takagi et al., 1992; Sandgren et al., 1993); chronic zinc induction of transgene expression in MT-TGF~ mice produced an incidence of hepatic tumors up to 100% (Table 3). Despite the wide-

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Table 3 Growth Factor-Induced Hepatic Neoplasia in Transgenic Mice Growth factor a

Gene regulatory elementb, r

TGF~

MT

IGF1

MT

IGF2

MUP

HGF

AL

Sequence of hepatic lesions a 1. Hepatocellular hyperplasia and increased mitotic index by 0.5 months of age. 2. Focal centrilobular hepatocellular pleomorphism by 1 to 2 months of age. 3. Focal hepatocellular dysplasia by 6 months of age. 4. Hepatocellular adenomas, solid or trabecular hepatocellular carcinomas by 8 to 12 months of age. 5. Incidence 50 to 100%. 1. Hepatocellular hyperplasia and centrilobular hypertrophy; no tumors. 1. Hepatocellular carcinomas by 18 months of age. Incidence less than 10%. 1. Mild, focal hepatocellular pleomorphism and increased mitotic index; no hepatic tumors at 18 months of age.

aAbbreviations: TGFcx, transforming growth factor alpha; IGF, insulin-like growth factor; HGF, hepatocyte growth factor. bAbbreviations: MT, metallothionein-I; MUP, major urinary protein; AL, albumin. cReferences: MT-TGFa, Sandgren et al., 1990, 1993, Jhappan et al., 1990, Lee et al., 1992, Takagi et al., 1992; MT-IGF1, Mathews, et al., 1988, Quaife et al., 1989; MUP-IGF2, Rogler et al., 1994; AL-HGF, Shiota, et al., 1992, 1994. dThe precise onset and rate of progression of the principal hepatic lesions in each group of transgenic mice will vary for different constructs and for different lineages with the same construct. Information summarized in this column provides typical lesions and the sequence and range of ages at which they appear. Lesions developing in other organs are not described.

spread hyperplasia, only hepatic, mammary, and coagulation gland epithelia developed tumors (Sandgren et al., 1990; Jhappan et al., 1990). (Coagulation gland is a rodent derivative of the ventral prostate.) Thus, in the context of liver, TGFcx was oncogenic, and at least as potent a transforming agent as c-Myc, consistent with a role for this growth factor in spontaneous tumor formation in liver. However, although TGFcx induced diffuse hepatic hyperplasia in young mice, transgenic mouse hepatocytes remained well differentiated at early stages of TGFc~ stimulation and the process of growth was still regulated, unlike the uncontrolled growth and altered hepatocyte differentiation induced by mutant H-Ras. Insight into the mechanisms of TGFc~-induced transformation will be discussed further in Section V.A. Hepatocytes derived from MT-TGFe~ transgenic mice have been used to establish apparently immortalized hepatocytic cell lineages with a differentiated but nontransformed phenotype (Wu et al., 1994). This finding is consistent with TGFc~'s postulated role as an hepatocyte growth factor, but it

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nevertheless is striking that autocrine production of a single growth factor apparently can overcome the difficulties (rapid loss of differentiated characteristics or replicative capability) associated with primary cultures of nontransgenic hepatocytes. It will be important to examine how relatively "normal" these cells remain, by determining further the stability of their phenotype in culture and whether they can participate in normal hepatic regeneration following reintroduction into the liver. B. Insulin-Like Growth Factors

In contrast to the effects of TGF(x, other growth factors examined for hepatic transforming activity in transgenic mice have proved to be only weakly oncogenic when tested alone. The insulin-like growth factors (IGF) 1 and 2 can initiate signal transduction through a family of IGF1 receptors as well as the insulin receptor (reviewed in Schofield, 1992). IGF1 is a principal mediator of growth hormone effects in adult organisms, whereas IGF2 is viewed mainly as a fetal growth factor. However, expression of the latter often is reactivated in liver neoplasms, suggesting that IGF2, like TGFcx, may have a role in hepatocarcinogenesis. Despite these characteristics, neither IGF is highly oncogenic in liver. Elevated expression of IGF1 was associated with increased weight of multiple organs, including liver. MT-IGF1 mice contained livers that were 1.4-fold larger than those of nontransgenic littermates (Mathews et al., 1988). (When expressed as a percent of total body weight, livers show only a 1.1-fold increase.) This change was due primarily to increased cell number. Livers also displayed pericentral hepatocellular hypertrophy (Quaife et al., 1989), yet liver tumors have not been observed in these mice (Table 3). (It remains possible that, by examining additional MT-IGF1 mice older than 1 year of age, a small increase in the incidence of hepatic neoplasms would become apparent.) Thus, hypercellularity and focal dysplasia in liver are not necessarily associated with subsequent neoplasia. In contrast, IGF2 expression in MUP-IGF2 transgenic mouse liver was not reported to increase liver weight (Rogler et al., 1994). Instead, these mice displayed a slight reduction in lean body mass but a major reduction in fat mass. The microscopic appearance of liver in these mice was not described, but a small fraction (<10%) of MUP-IGF2 transgenic mice examined at over 18 months of age developed hepatocellular carcinomas (Table 3; nontransgenic mouse background incidence in this experiment was <2%). Although these findings are consistent with a role for IGF2 in hepatocarcinogenesis, that role is unlikely to include enhanced tumor initiation, given the very small increase in incidence of hepatic neoplasms observed in these mice.

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C. Hepatocyte Growth Factor Hepatocyte growth factor (HGF) is perhaps the most potent hepatic mitogen and, like TGFct, is almost certainly involved in hepatic regeneration (see Chapters 1 to 3) (reviewed in Zarnegar et al., 1992, 1994). For these reasons, AL-HGF transgenic mice were created as a means to assess the in vivo transforming potential of this growth factor (Shiota et al., 1992, 1994). Unexpectedly, and in contrast to TGFcx, HGF was not oncogenic in liver, although hepatic regeneration following two-thirds partial hepatectomy in transgenic mice proceeded more rapidly than in nontransgenic control mice (Table 3; Shiota et al., 1994). In fact, in vitro evidence suggested that HGF may even inhibit growth of hepatocellular carcinoma cells (Shiota et al., 1992). These findings underscore the unpredictability of the effects exhibited by growth factors when their expression is altered in vivo, and further emphasize the need for caution when applying conclusions based on in vitro experiments to the intact animal. However, transgene expression in these animals was low; it remains possible that high-level transgene expression may result in a more distinctive phenotype. D. Conclusions As for viral and cellular oncogenes, transgenic mouse studies permit evaluation of growth factor oncogenicity in vivo. The studies undertaken to date emphasize the diversity of the hepatic response to different growth factors. This is particularly striking when considering the contrasting effects of two potent hepatic mitogens, TGF~ and HGF; the former induces tumors with high incidence while the latter does not. Thus, each growth factor appears to produce a unique range of effects. Nevertheless, studies of unregulated growth factor production in transgenic mice provide only partial information regarding growth factor carcinogenicity, because altered growth factor gene expression in tumors is often superimposed on other genetic aberrations. A crucial next step is examination of the effects of combined oncogene/growth factor expression, and studies of this type are described in Section V.

IV. Hepatotoxic Transgenes and Liver Neoplasia A. Hepatitis B Virus Surface Antigen Certain hepatocarcinogenic gene products, including the HBsAg, appear to act through induction of hepatonecrosis. As noted in Section II.A, several approaches have been taken to study HBV pathogenesis in transgenic mice.

Table 4 Hepatotoxic Transgene-Induced Hepatic Neoplasia in Transgenic Mice

Toxic factor a

Gene regulatory elementb, c

HBsAg

AL

uPA

AL

AAT (mutant)

AAT

mdr-2

Gene knockout

IFN~/

SAP

K14

TTR

Sequence of hepatic lesions a 1. Moderately severe hepatitis and hepatic necrosis from 4 months of age to death. 2. Regenerative hepatocellular nodules, occasionally dysplastic, by 6 months of age. 3. Hepatocellular adenomas by 8 months of age. 4. Hepatocellular carcinomas by 12 to 20 months of age. 5. Incidence 100%. 1. Total hepatocellular regeneration by clones of cells that have deleted transgene DNA, completed by 2 to 3 months of age. No subsequent inflammation. 2. Multiple eosinophilic or clear cell altered hepatocellular foci by 6 months of age. 3. Hepatocellular hyperplastic nodules and adenomas by 10 months of age. 4. Hepatocellular carcinomas by 12 to 20 months of age. 5. Incidence 100%. 1. Transient hepatitis, hepatic necrosis, and regeneration, completed by 1.5 to 3 months of age. 2. Focal hepatocellular dysplasia by 1.5 months of age. 3. Altered hepatocellular foci by 3 months of age. 4. Hepatocellular adenomas and carcinomas by 12 to 24 months of age. Incidence 100%. 1. Diffuse hepatocyte pleomorphism and necrosis, elevated mitotic rate; extensive bile duct proliferation and portal inflammation by 2 to 3 weeks of age. Incidence 100%. 2. Multiple hepatic nodules beginning by 5 months of age. Incidence: 3 of 4 mice examined. 1. Multifocal hepatocellular necrosis and inflammation by 1 month of age. Incidence 100%. 2. Slight hepatic fibrosis by 6 months of age. 3. Ductal proliferation by 8 to 9 months of age. 4. Death by 1 year of age from bacteremia. Incidence 80%. 1. Multifocal hepatocellular degenerative changes and inflammation after 4 months of age. Incidence up to 75%. 2. Hepatocellular hyperplastic nodules in 3 of 60 mice examined. (Age not specified.)

aAbbreviations: HBsAg, hepatitis B virus surface antigen; uPA, urokinase-type plasminogen activator; AAT (mutant), the Z type mutant alpha-l-antitrypsin; mdr-2, multiple drug resistance-2; IFN~/, interferon gamma; K14, cytokeratin 14. bAbbreviations: AL, albumin; AAT, alpha-l-antitrypsin; SAP, serum amyloid P; TTR, transthyretin. cReferences: AL-HBsAg, Chisari et aL, 1985, 1986, 1987, 1989, Dunsford et al., 1990; ALuPA, Heckel et al., 1990, Sandgren et al., 1991, 1992; AAT-AAT (mutant), Dycaico et al., 1988, Geller et al., 1994; mdr-2 knockout, Smit et al., 1993; SAP-IFN3,, Toyonaga et al., 1994; TTR-K14, Alberts et al., 1995. dThe precise onset and rate of progression of the principal hepatic lesions in each group of transgenic mice will vary for different lineages with the same construct. Information summarized in this column provides typical lesions and the sequence and range of ages at which they appear.

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One involved targeting expression of the HBV large envelope polypeptide using the AL enhancer/promoter (Chisari et al., 1985). Overproduction of HBV large envelope polypeptide in hepatocytes led to the accumulation in smooth endoplasmic reticulum of nonsecretable, filamentous HBsAg particles (Chisari et al., 1986, 1987). In young adults, this condition eventually was cytotoxic, and produces focal hepatic necrosis and inflammation, nodular hepatic regeneration, and finally development of hyperplastic loci, adenomas, and hepatocellular carcinomas (Table 4; Chisari et al., 1989; Dunsford et al., 1990; Toshkov et al., 1994). Incidence approached 100% in the most severely affected lineage. The carcinogenic mechanism in this model did not involve overexpression of potentially oncogenic genes that are members of growth regulatory signal transduction pathways. Instead, the mechanism appeared to involve transgene-induced hepatocellular injury and subsequent chronic hepatic regeneration (Chisari et al., 1989; Dunsford et al., 1990). The proliferative state of regenerative hepatocytes, particularly in the presence of inflammation and associated free radical generation, favored both the generation and fixation of potentially oncogenic mutations in the susceptible cell population (Hagen et al., 1994). The chronicity of these conditions eventually could lead to the appearance of clones of cells displaying unregulated growth. Given the similarity of lesions and the course of the disease in AL-HBsAg transgenic mice and humans with chronic HBV infection, this model has provided important information regarding possible mechanisms of hepatocarcinogenesis in the human population.

B. Urokinase-Type Plasminogen Activator A second example of the association between transgene-induced hepatotoxicity and hepatocarcinogenesis was provided by mice carrying an AL enhancer/promoter urokinase-type plasminogen activator fusion transgene (AL-uPA) (Heckel et al., 1990; Sandgren et al., 1991, 1992). Urokinase proteolytically activates plasminogen to plasmin, which is itself a protease with broad specificity. Plasmin's major physiological effect is degradation of fibrin clots. The endogenous urokinase gene is not expressed in hepatocytes. However, targeted expression of the AL-uPA transgene created a condition in which both plasminogen and its activator, uPA, were transported into the same intracellular compartment, the rough endoplasmic reticulum. The effect of this manipulation shared features with that of hepatocyte-targeted HBsAg: development of intracellular lesions, in this case vacuolation of rough endoplasmic reticulum, and eventually cell necrosis (Heckel et al., 1990). Resolution of this condition, however, was remarkably different for this model. Hepatocyte clones derived from progenitor cells that had physically

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deleted transgene DNA, and therefore no longer were subject to the toxic effects of transgene expression, eventually repopulated the entire liver by 2 to 3 months of age (Sandgren et al., 1991). At this stage, the microscopic appearance of liver became normal, and no transgene expression was detectable. However, beginning several months later, a stereotypical sequence of changes was observed in liver of all A L - u P A transgenic mice: development of altered hepatocellular foci, hyperplastic foci, adenomas, and hepatocellular carcinomas (Table 4; Sandgren et al., 1992). The early clonal hepatocellular regeneration was not the sole determinant of subsequent neoplasia, although tumors appeared to be derived only from cells that had lost both the transgene and surrounding chromosomal DNA during early stages of the regenerative response (Sandgren et al., 1992). Thus, loss of linked tumor suppressor genes, altered balance of gene dosage, or destabilization of the tumor precursor cell's genome could each contribute to tumor progression in this model. The central feature of this model is the possible association between loss of genomic DNA and subsequent transformation. This was not observed in the AL-HBsAg model, nor was inflammation a prominent feature of the A L - u P A model.

C. Alpha-l-Antitrypsin A third example of toxin-induced hepatocarcinogenesis involves hepatocyte-targeted expression of the mutant Z allele of A A T . AAT is an important serum proteinase inhibitor; deficiency of this enzyme is associated with emphysema secondary to proteinase-(elastase-) mediated destruction of alveolar tissue (Crystal, 1990). The mutant Z allele of A A T produces an altered protein that is improperly transported from the endoplasmic reticulum and therefore accumulates in rough endoplasmic reticulum, causing hepatonecrosis and hepatitis. When introduced into transgenic mice, the human Z A A T gene (with its own enhancer/promoter region) produces a similar effect (Dycaico et al., 1988; Carlson et al., 1989; Sifers et al., 1989). Specifically, mutant protein accumulates in rough endoplasmic reticulum, causing necrosis, hepatitis, and liver regeneration in young mice (Table 4). This has now been shown to be followed, in at least one transgenic mouse lineage, by dysplasia, altered hepatocellular foci, and, after an 8- to 10month delay, adenomas and hepatocellular carcinomas (Table 4; Geller et al., 1994; Gordon, 1994). (Development of hepatic neoplasia also is a component of the AAT-deficiency disease in humans.) The sequence and morphological character of lesions are similar to those observed in AL-HBsAg transgenic mice (and, more generally, in oncogene-bearing transgenic mice and in rodents administered hepatocarcinogenic chemicals), prompting speculation by the authors that progression of liver tumors induced by

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275

diverse causes follows a common pathway determined by the hepatocyte itself (Geller et al., 1994). An independently generated lineage of mutant AAT-expressing mice, though displaying similar early lesions (inflammation, necrosis), were reported to have remained free of hepatic tumors for at least 20 months (Carlson et al., 1989). This is difficult to reconcile with the notion of a universal pathway leading from hepatic injury to carcinoma, yet differences in mouse genotype and/or diet, for example, could contribute to variable susceptibility to neoplastic transformation. D. Conclusions The studies described in this and preceding sections underscore the diverse nature of genetic and cellular lesions that can lead to a stereotypical pattern of tumor development; hepatic injuries associated with viral or endogenous mutant gene expression are as capable of initiating hepatocarcinogenesis as targeted expression of certain oncogenes. Identification of this strong association between hepatic injury and cancer suggests that neoplasia may be observed in other model systems involving chronic liver injury. For example, mice deficient for the multidrug resistance-2 (mdr-2) gene produced phospholipid-deficient bile; this caused hepatocellular dysplasia, necrosis, and, in 3 of 4 mice examined at 5 months of age, development of hepatic nodules (Table 4; Smit et al., 1993). Mice expressing in the liver a serum amyloid P component-interferon ~/transgene displayed hepatonecrosis and hepatitis as young adults (Table 4; Toyonaga et al., 1994). Death associated with bacteria occurred in 80% of these mice by 1 year of age. Finally, transthyretin-cytokeratin 14 (TTR-K14) transgenic mice developed abnormal keratin filament networks in the hepatocyte cytoplasm and subsequent hepatic degeneration and inflammation (Albers et al., 1995). It will be interesting to learn whether older mice bearing any of these transgenes show evidence of hepatic neoplasia, which would support the suggested association between injury and cancer in this organ. A further strength of the transgenic approach is that it provides the ability to break down the study of complex molecular lesions into component parts, and to determine the effects of each separately. In this manner, pathogenesis of HBV virus was studied by independently targeting expression of the HBx transactivator and the HBsAg to hepatocytes. These studies identified a carcinogenic effect of both agents, though each almost certainly employed different mechanisms. Thus, hepatocarcinogenesis associated with expression of intact HBV is likely to have at least two molecular components, and the ability of these components to act together can be tested directly by crossbreeding the respective lineages to produce bitransgenic mice carrying both transgenes.

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V. Transgenes and Multistage Carcinogenesis A. Cooperating Events in Transgene-Induced

Hepatocarcinogenesis A key feature of transgene-induced liver tumor development is the focal nature of tumors. Despite the targeting of transgene expression to millions of cells, only single to at most several hundred tumors are evident as end stage lesions. Thus, each tumor progenitor cell must accumulate additional cellular alterations before completing malignant transformation. Transgenic models have been used to seek out the identity of these changes in several ways. The most straightforward approach is to screen tumors for mutation or altered expression of candidate genes previously suggested to have a role in hepatocarcinogenesis. For example, 10 of 25 TAg-induced hepatic tumors were shown to contain activating mutations in codon 61 of the endogenous H-ras gene (Lee et al., 1990). In contrast, MT-TGFcx liver tumors appeared to maintain normal K- and H-ras alleles (Takagi et al., 1992). Pasquinelli et al. (1992) used an expanded approach to examine the molecular pathogenesis of HBsAg tumorigenesis. They could detect no mutations in, or altered expression of, 19 genes, including H-, K-, and N-ras, C- and N - m y c , and the RB-1 and p53 tumor suppressor genes. In contrast, in this and two additional transgenic mouse models, MUP-TAg and ATIII-TAg (Table 1), reactivation of IGF2 expression was observed frequently at a late stage of progression, principally in tumors (although in ATIII-TAg mice, some early lesions were reported to show this change) (Cariani et al., 1991; Schirmacher et al., 1992). Similarly, up to 75% of tumors developing in MT-TGFc~ transgenic mice had reactivated IGF2 expression, and 37% overexpressed c-Myc (Takagi et al., 1992). Thus, a strong association has been established between transgene-induced liver tumor progression and overexpression of IGF2, which normally is not expressed in mice more than several weeks old. However, this change apparently occurs during late stages of tumor progression; this fact, and the observation that large numbers of candidate genes remain unaffected (at least in the AL-HBsAg model), underscore our limited ability to detect pertinent oncogenic changes that collaborate with transgene expression during early stages of hepatocarcinogenesis in these models.

B. Coexpression of Multiple Transgenes A second approach to examining multistage carcinogenesis in transgenic mouse liver is to test directly the ability of two genetic changes to cooperate during tumor formation by creating bitransgenic mice that carry two different transgenes. This is most commonly accomplished by interbreeding sepa-

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Table 5 Transgene Cooperativity in Hepatic Neoplasia Transgenesa,b AL-TAg/AL-ras

AL-TAg/AL-myc

AL-ras/AL-myc

AL-TAg/MT-TGFcx

AL-myc/ MT-TGFoL

Sequence of hepatic lesionsc 1. Hyperplasia of ductal cells, liver enlargement, and death by 10 days of age. 2. Incidence 100%. 1. Hepatocellular hyperplasia and dysplasia by I to 2 weeks of age. 2. Altered hepatocellular foci by 2 to 4 weeks of age. 3. Hepatocellular and biliary adenomas and carcinomas by 4 to 6 weeks of age. 4. Incidence 100%. 1. Hepatocellular adenomas and adenocarcinomas by 2 to 3 months of age. 2. Incidence up to 30%. Remainder die from lung tumor development. 1. Hepatocellular hyperplasia and dysplasia by 2 to 4 weeks of age. 2. Altered hepatocellular foci by 4 to 8 weeks of age. 3. Hepatocellular and biliary adenomas and carcinomas by 8 to 10 weeks of age. 4. Incidence 100%. 1. Hepatocellular dysplasia by 3 to 6 weeks of age. 2. Clear cell or basophilic altered hepatic foci by 4 months of age. Basophilic foci greatly overexpress the MT-TGFe~ transgene. 3. Hepatocellular hyperplastic foci, adenomas, and carcinomas by 4 to 9 months of age. Tumors greatly overexpress the MTTGFc~ transgene. 4. Incidence 100%.

aAbbreviations: AL, albumin; MT, metallothionein-I; TAg, simian virus 40 T antigens; TGFe~, transforming growth factor alpha. 6References: AL-TAg/AL-ras, AL-TAg/AL-myc, and AL-ras/AL-myc, Sandgren et al., 1989; AL-TAg/MT-TGFoL, Sandgren et al., 1993; AL-myc/MT-TGFc~, Sandgren et al. 1993; Murakami et al., 1993. cInformation summarized in this column provides typical lesions and the sequence and range of ages at which they appear.

rate transgenic m o u s e lineages, and assessing t u m o r p r o g r e s s i o n in mice carrying both transgenes. Sandgren et al. (1989) r e p o r t e d t h a t pairwise expression of TAg, c-Myc, or m u t a n t H - R a s accelerated the d e v e l o p m e n t of hepatic lesions, in s o m e cases d r a m a t i c a l l y (Table 5). C o e x p r e s s i o n of TAg a n d m u t a n t H - R a s induced diffuse hyperplastic changes in p o s t n a t a l m o u s e liver, and tissue h a r v e s t e d f r o m these mice was not t r a n s p l a n t a b l e , thus not fully t r a n s f o r m e d (Sandgren et al., 1989; E. P. Sandgren, unpublished). H o w e v e r , c o e x p r e s s i o n of TAg and c-Myc or m u t a n t H - R a s a n d c-Myc increased both t u m o r incidence and rate of p r o g r e s s i o n , yet b o t h c o m b i n a tions were insufficient to t r a n s f o r m all h e p a t o c y t e s in one step (Sandgren et

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Eric P. Sandgren

al., 1989). This approach also was used to assess the ability of TGFc~ overexpression to influence the progression of tumors induced by AL-TAg (Sandgren et al., 1993) or A L - m y c (Sandgren et al., 1993; Murakami et al., 1993) transgenes (Table 5). Again, TGFcx acted synergistically with TAg and c-myc to increase the incidence and accelerate the rate of tumor progression of liver tumors in bitransgenic mice. Interestingly, tumor progression in one c-myc/TGFcx bitransgenic mouse model always was accompanied by "superinduction" of TGFc~ transgene expression relative to surrounding nontumorous liver (by 5- to 20-fold), and often was accompanied by increased c-myc transgene expression (Sandgren et al., 1993). A class of basophilic altered cell foci present in these mice also displayed TGFc~ superinduction, suggesting that these foci represented preneoplasia. Similar tumor-specific transgene superinduction was reported in a different line of MT-TGFcx monotransgenic mice (Takagi et al., 1992). Thus, a key feature of tumor progression associated with deregulated TGFc~ expression appears to be a selection for more highly expressing cells. These reports confirm the oncogenic activity of TGFcx in the context of liver, and emphasize the significance of elevated TGFc~ expression in tumors of human liver.

C. Transgenes and Chemical Carcinogens A third approach to the study of multistage carcinogenesis using transgenic models is the administration of genotoxic and nongenotoxic hepatocarcinogenic chemicals to mice expressing potentially oncogenic transgenes in liver. The outcome provides information regarding how chemical lesions complement expression of a precisely defined transgene, thus helping to identify the stage or stages of tumor development affected by transgene expression. The first described chemical/transgene combination involved administration of the genotoxic chemicals diethylnitrosamine (DEN) or dimethylaminobenzene (DAB) to 7-day-old HBV-HBsAg transgenic mice (Table 6; Dragani et al., 1989). Mice in this lineage express HBsAg and HBx proteins, but do not display hepatic lesions. Transgenic male mice receiving either chemical displayed a 1.6-fold increase in tumor (adenoma and carcinoma) incidence 29 weeks later relative to similarly treated nontransgenic mice. Transgenic mouse tumors were also larger and tended to represent more advanced stages of progression (Dragani et al., 1989). Similarly, ALHBsAg transgenic mice, in this case females, also displayed an enhanced response to treatment with chemical carcinogens (Table 6; Sell et al., 1991). Using protocols that did not induce tumors in nontransgenic littermates, both aflatoxin and DEN-induced tumors (adenomas or carcinomas) in ALHBsAg transgenic mice by 15 months of age. Thus, liver specific HBsAg expression acted cooperatively with chemical carcinogens to enhance the tumorigenic response. In contrast, chronic feeding to these female mice of

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Table 6 Transgenes and Chemical Carcinogens in Hepatic Neoplasia Transgene a,b

Carcinogen c

Tumor incidence (transgenic/nontransgenic)'t

HBV-HBsAg

DEN DAB none aflatoxin DEN PB none DEN DMN PB DEN low DMN low DMN high

0.52/0.32 0.50/0.32 0/0 0.58/0 1.00/0 0/0 0.13/0 1.00/0.44 0.90/0.30 0.78/0 0.86/0.95 e 0.38/0.90 1.00/0.88

AL-HBsAg

MT-TGFol

MT-MGMT

aAbbreviations: HBV, hepatitis B virus; HBsAg, hepatitis B virus surface antigen; AL, albumin; MT, metallothionein-I; TGFoL, transforming growth factor alpha; MGMT, O6-methylguanine-DNA methyltransferase. bReferences: HBV-HBsAg, Dragani et al., 1989; AL-HBsAg, Sell et al. 1991, Sell, personal communication; MT-TGFot, Takagi et al., 1993; MT-MGMT, Nakatsuru et al., 1993. cAbbreviations: DEN, diethylnitrosamine; DAB, dimethylaminobenzene; DMN, dimethylnitrosamine; PB, phenobarbital. aData are summarized as the fraction of transgenic mice developing tumors (adenomas or carcinomas) compared to the fraction of nontransgenic mice developing tumors. See original references for details of experimental design. eTumor multiplicity was decreased in this group of DEN-treated transgenic mice relative to nontransgenic controls.

phenobarbital (PB), a nongenotoxic tumor promoting agent, did not induce tumors (Sell et al., 1991). Also in this model, although high levels of dietary cadmium did not increase male transgenic mouse tumor incidence, these mice displayed increased tumor malignancy, as assessed by morphological criteria (Sell and Ilic, 1994). A similar study of chemical effects in MT-TGFot transgenic mice suggested specific mechanisms by which TGFcx might influence tumor development in the liver. In this study, MT-TGFot mice were administered DEN, dimethylnitrosamine (DMN), or PB. In males, each regimen increased hepatic tumor incidence at 24 to 32 weeks post-treatment (Table 6; Takagi et al., 1993). Tumor size increased more rapidly in treated transgenic versus nontransgenic mice; correspondingly, hepatic loci and adenomas within transgenic mice displayed an increased index of proliferation (Tamano et

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al., 1994). Finally, the rate of tumor progression, as determined by morphological criteria, was also accelerated. Thus, TGFcx expression complemented both genotoxic tumor initiators and the classical tumor promoter, PB. This led to the conclusion that TGFe~ can act at several stages along the tumorigenic pathway, both being subject to and capable of providing tumor promoting activity (Takagi et al., 1993). A recent twist on this subject is the creation of mice that should be more resistant to the effects of chemical carcinogens. These mice carry transgenes targeting hepatocytic expression of the bacterial 06-methylguanine-DNA methyltransferase (MGMT) gene (Lim et al., 1990; Nakatsuru et al., 1993). This gene catalyzes removal of a methyl group from O6-methylguanine adducts, thus repairing this form of potentially mutagenic DNA damage. Nitrosamines such as DEN or DMN can produce this lesion, raising the possibility that elevated hepatic MGMT could reduce the incidence and/or multiplicity of nitrosamine-induced hepatic neoplasia. In fact, protection was observed following treatment of M T - M G M T transgenic mice with either chemical (Table 6; Nakatsuru et al., 1993). Tumor multiplicity but not incidence was reduced in DEN-treated transgenic males. Protection was not afforded against the high dose of DMN (5 mg/kg) in male mice, indicating a dose-dependence of the response, as predicted if protection provided by endogenous and transgene-produced MGMT would reach a threshold or upper limit once the enzyme became saturated. This avenue of experimentation provides not only supportive evidence for specific biochemical mechanisms of tumor initiation associated with particular chemicals, but also suggests means to enhance tumor resistance or explain tumor susceptibility.

D. Transgene-Based Mutagenesis Assay Systems In contrast to the studies described above, which sought to evaluate the interaction between tumor-enhancing or protective transgene products and carcinogenic chemicals, the studies described in this section have been designed to detect and quantitate mutagenic events in vivo (reviewed in Gossen and Vijg, 1993; Provost et al., 1993; Mirsalis et al., 1994). These procedures rely on the introduction into the mouse genome of bacteriophage shuttle vectors containing the bacterial lacZ or lacI gene. LacZ encodes betagalactosidase, which catalyzes cleavage of a colorless substrate molecule into a blue product. The lacI gene product encodes a repressor of lacZ. In simplest form, the assays proceed in the following manner. Transgenic mice are exposed to a chemical mutagen and then sacrificed at selected times thereafter. Potential target tissues are harvested, transgene vectors are recovered by in vitro packaging of total genomic DNA, and the resulting phage are used to infect appropriate strains of Escherichia coli. Alternatively, vectors can be recovered directly from restriction enzyme digested genomic

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DNA, and used to transform E. coli. The frequency of mutations in the lacZ target sequence is determined by comparing the number of colorless colonies (which must carry a mutated lacZ gene) to the total number of colonies. Analysis of mutations in lacI is handled similarly; in this case, E. coli recipients carry lacZ but not a functional lacI gene, so that colonies with a mutated transgene-derived lacI will be blue (permissive for lacZ gene expression) and colonies with intact lacI will be colorless. In any experiment, a large number of controls must be established, including determination of the background rate of mutation. These mice have been employed to analyze chemical mutagenicity in liver. For example, Mirsalis et al. (1993) demonstrated that the hepatocarcinogen DMN produced an elevated mutation frequency in the lacI transgene, but that the nonhepatocarcinogenic methylmethane sulfanate (MMS) did not. Both DMN and MMS are genotoxic and produce DNA adducts in liver, but only the former induces hepatocyte proliferation (which is necessary to transform adducts into mutations). Shephard et al. (1993) demonstrated increased lacI mutagenicity following 28 day feeding of 2-acetylaminofluorene (AAF) at a dosage much lower than typically employed in acute mutagenicity studies, although still somewhat higher than a carcinogenic dose employed in the long-term carcinogenicity bioassay. These studies illustrate the ability of transgene-based mutagenesis assays to predict carcinogenicity, and demonstrate progress toward development of short-term, low dose experimental protocols that more accurately reproduce the nature of human exposure and should complement the long-term rodent bioassay in risk assessment. In addition, these mice can be mated with mice bearing hepatotoxic transgenes, such as ALHBsAg, and bitransgenic offspring used to assess the mutagenic effect of hepatonecrosis and inflammation. E. Conclusions In vitro studies of growth regulatory signal transduction pathways have proved exceptionally productive in identifying individual elements that comprise signaling pathways and documenting the nature of their interactions. Transgenic animals, in turn, provide the means to explore the interactions between signaling pathways and the tissue, organ, and organismal contexts in which they exist. The study of altered TGFcx expression in vivo serves as a paradigm for the latter type of analysis. When overexpressed in liver, TGFcx was moderately oncogenic, and the extent of this effect appeared to be proportional to the magnitude of overexpression (Takagi et al., 1992; Sandgren et al., 1993). When its expression was combined with that of other growth regulatory molecules, c-Myc or SV40 TAg, TGFcx acted synergistically with those agents to accelerate tumor onset and rate of growth, and to increase tumor malignancy (Sandgren et al., 1993; Mu-

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rakami et al., 1993). Finally, the hepatocarcinogenic potency of chemical initiating and promoting agents was greatly enhanced by TGFcx overexpression (Takagi et al., 1993). Thus, a picture emerges of TGFcx as an agent capable of influencing several of the classically defined stages of liver tumor development. It stimulates the rate of tumor growth and increases the fraction of tumors classified as carcinomas, thus acting as a promoting and/or progression factor. However, TGFcx-associated tumorigenesis was also complemented by PB, a promoting agent, indicating the presence of initiated cells in TGFo~ transgenic mouse liver. (In contrast, although expression of the AL-HBsAg transgene enhanced DEN-induced hepatocarcinogenesis, under the same conditions there was no synergism between HBsAg and PB; Sell et al., 1991.) These diverse observations provide insight into the mechanisms by which TGFcx contributes to hepatic neoplasia, and in a way that only could have been provided by in vivo models of deregulated TGF0~ expression. The studies described in this section also underscore the multifactorial nature of hepatocarcinogenesis. Even combined expression of oncogenes or oncogene/growth factor pairs in adult liver was insufficient to fully transform all expressing cells. However, by superimposing expression of multiple transgenes on a genetic background rendered deficient (by gene knockout technology) in one or more tumor suppressor genes, we ultimately should be able to use genetically modified animals to define precisely the elements that constitute sufficiency for hepatocarcinogenesis.

VI. Transgenes and Hepatic Growth Regulation A. Liver Gene Expression Regulation of liver growth can be examined from several perspectives, ranging from organismal (What signals initiate the stimulus for hepatic regeneration following loss of liver mass? What signals halt liver regrowth once the appropriate mass is attained?) to cellular (What roles are played by different liver cell populations in liver growth? Do hepatic stem cells exist in adult liver?) to molecular (What signal transduction pathways are active in liver growth? What factors regulate the pattern of gene expression during liver development?). The latter question has been addressed, at least indirectly, by multiple studies in transgenic animals that seek to identify DNA regulatory elements associated with genes expressed in the liver. In most cases, transgenes have consisted of the intact genes (coding sequence plus variable amounts of upstream and downstream DNA) or upstream DNA fused to a reporter gene. Selected studies in which transgene expression has been targeted to hepatocytes are summarized in Table 7. I will not attempt a

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Table 7. Gene Regulatory Elements Used to Target Hepatic Expression in Transgenic Mice a Gene regulatory element (species)

Characteristics

Albumin (AL) (mouse) High-level expression in liver; variable, low-level expression in gut, kidney, lung. a-fetoprotein (AFP) High-level expression in fetal liver, (mouse) gut, yolk sac; expression often reactivated in hepatic neoplasms. a-l-Antitrypsin (human)

~-l-Acid glycoprotein (rat)

~-2u-Globulin (rat)

Angiotensinogen (mouse) Amylase (mouse)

Antithrombin III (ATIII) (human) C-reactive protein (CRP) (human) Cholesterol ester transfer protein (CETP) (human) Cholesterol 7~hydroxylase (rat)

High-level expression in liver; lowlevel expression in pancreas, gastrointestinal tract, kidney, brain, chondrocytes, adrenal gland, and testes. Expression in liver inducible by dexamethasone, LPS, IL-1, and IL-6; low-level expression in spleen, heart, and submaxillary gland. Onset of expression in liver at puberty in males; expression in female liver inducible by ovariectomy and testosterone administration; expression in preputial gland; homologous to MUP gene family in mouse. Expression in liver inducible by estrogen, glucocorticoids, and LPS; expression in brain, salivary gland, and testis. Expression in liver; low-level expression in brown and white fat, skeletal muscle, and testes; lacked expected expression in parotid gland. Expression specific for liver. Expression in liver highly inducible by LPS. Expression in liver inducible by high fat, high cholesterol diet; expression in spleen, small intestine, kidney, and fat; variable expression in heart and brain. When combined with albumin enhancer, expression in liver; hepat-

Primary reference(s) Pinkert et al., 1987

Krumlauf et al., 1985; Hammer et al., 1987; Camper and Tilghman, 1989; Vacher and Tilghman, 1990; Vacher et al., 1992 Kelsey et al., 1987; Sifers et al., 1987; Carlson et al., 1988

Dewey et al., 1990

Da Costa Soares et al., 1987

Clouston et al., 1989

Jones et al., 1989

Dubois et al., 1991 Toniatti et al., 1990; Murphy et al., 1995 Jiang et al., 1992

Ramirez et al., 1994

( continues )

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Table 7 (continued) Gene regulatory element (species)

Characteristics

ic expression induced by cholesterol and colestipol, reduced by cholic acid; very lowlevel expression in heart. Cypla~ ( P 4 5 0 ) Expression in liver highly inducible (mouse) by 3-methylcholanthrene; expression in spleen, colon, kidney, and lung. Cyp2B-2 (P450) (rat) Expression in liver highly inducible by phenobarbital; no detectable expression in other tissues for construct with longest 5' region. Erythropoietin (human) Expression in liver inducible by anemia or cobalt; variable expression in kidney. Fumarylacetoacetate Expression in liver and kidney; rehydrolase (FAH) verses lesions in mice homo(mouse) zygous for lethal albino deletions. Glutathione transExpression analyzed in transgenic ferase P (GST-P) rats. Expression in liver highly (rat) induced within focal lesions and tumors following treatment with DEN plus AAF (Solt-Farber protocol); variable expression in lung and kidney. Liver-enriched activaTransgene copy number-dependent tor protein ( L A P ) expression in liver, spleen, lung, (rat) brain, heart, and kidney. Liver fatty acid bindExpression in liver and gut; pattern ing protein of gastrointestinal expression in(L-FABP) (rat) fluenced by multiple suppressor and activator elements. Liver expression inducible by peroxisome proliferators. L-type pyruvate kinase Expression in liver highly inducible (LPK) (rat) by high carbohydrate diet, inhibited by glucagon; low-level expression in kidney and small intestine. Metallothionein (MT) Expression in liver inducible by (mouse) heavy metals (cadmium, zinc); metal-inducible expression in kidney; pancreas, gastrointestinal tract; variable expression in most organs.

Primary reference(s)

Jones et al., 1991

Ramsden et al., 1993

Semenza et al., 1990; Semenza et al., 1991 Kelsey et al., 1993

Morimura et al., 1993

Talbot et al., 1994

Sweetser et al., 1988; Simon et al., 1993

Tremp et al., 1989; Boquet et al., 1992; Cuif et al., 1993

Brinster et al., 1981; Palmiter et al., 1993

( continues )

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Table 7 (continued) Gene regulatory element (species) Major urinary protein (MUP) (mouse)

Ornithine decarboxylase (ODC)(human) Ornithine transcarbamylase (OTC) (rat, mouse) Peroxisomal

hydratase/ dehydrogenase (rat) Phenylalanine hydroxylase (human) Phosphoenolpyruvate carboxykinase (PEPCK) (rat)

Characteristics Onset of expression in liver at puberty in males; expression inducible in females by ovariectomy and testosterone administration; low-level expression in sebaceous glands and preputial glands; variable expression in mammary gland and kidney. Expressed in multiple tissues; expression in liver inducible by partial hepatectomy. Expression in liver and small intestine; corrects OTC deficiency in spf-ash mutant mice. Expression in liver highly inducible by peroxisome proliferators.

Primary reference(s) Held et al., 1989

Halmekyto et al., 1993 Murakami et al., 1989; Jones et al., 1990; Shimada et al., 1991 Alvares et al., 1994

Expression in liver; low-level exWang et al., 1992, 1994 pression in kidney. Expression in liver inducible by low McGrane et al., 1988, 1990; carbohydrate, high protein diet; Yamada et al., 1990; expression in kidney, intestine, Short et al., 1992; Eisensublingual gland, and white and berger et al., 1992; Patel brown fat; expression in all sites et al., 1994 influence by multiple regulatory elements. Expression specific for liver. Tan, 1991

Retinol binding protein (RBP)(human) Serum amyloid P com- Expression specific for liver. ponent (SAP) (human) Transferrin (mouse, Expression in liver and brain human) (oligodendrocytes).

Transthyretin (TTR) Expression in liver and choroid (mouse) (human) plexus. Tryptophan oxygenase Onset of expression in liver be(TO) (rat) tween 11 days of age and adulthood. Tyrosine aminotransExpression specific for liver. ferase (TAT) (rat)

Zhao et al., 1992 Idzerda et al., 1989; Adrian et al., 1990; Theisen et al., 1993 Yan et al., 1990; Nagata et al., 1995 Kaltschmidt et al., 1994 Beermann et al., 1993

aSee also Macri and Gordon, 1993; Merlino, 1994 detailed discussion of these reports, as this subject f o r m s the basis of a recent review (Macri a n d G o r d o n , 1993). Nevertheless, several n o t a b l e features emerge f r o m the reports. First, a large n u m b e r of gene r e g u l a t o r y

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elements have been examined, indicating the widespread interest in liver gene expression. Second, for several genes, including ~-fetoprotein (AFP), PEPCK, and liver fatty acid binding protein (L-FABP), extensive mapping of gene regulatory elements has been accomplished that would have been extremely difficult in vitro, because of the lack of appropriate cell lines. Third, some enhancer/promoters are inducible, providing the potential for gene targeting strategies whereby transgene expression can be upregulated when desired. This is true for metallothionein gene regulatory elements, which are induced by heavy metals such as zinc, and this has been exploited most notably in studies examining the effects of hepatic TGFoL expression. Finally, from a practical point of view, the range of characteristics of cloned regulatory elements with respect to level of expression, inducibility, and tissue specificity permits design of multiple targeting strategies for transgene-based analyses of liver function, growth, or disease. B. Fetal and Neonatal Liver Development

Several studies employing genetically engineered mice have addressed liver growth, focusing on molecular pathways that influence the rate and fidelity of DNA synthesis. As noted earlier, mutant H-Ras induced uniform fetal liver hyperplasia (Sandgren et al., 1989), thus indicating the ability of H-Ras to modulate a potent growth stimulatory signal transduction pathway that must be active during liver growth. TGF(x also induced liver hyperplasia, but in older animals (Sandgren et al., 1990; Jhappan et al., 1990). However, in this case, the process remained tightly regulated: once two-fold increase in liver weight was achieved, further growth stopped. Additional studies identified growth-related effects of several genes by employing gene targeting in ES cells. Mice lacking the c-jun gene died at mid to late gestation and displayed multiple defects including hepatic epithelial hypoplasia and dysplasia (Hilberg et al., 1993). Chimeric mice composed of normal cells and c-Jun-deficient ES cells were phenotypically normal; however, no ES-derived cells were present in liver despite the presence of roughly equivalent numbers of normal and ES-derived cells in other organs. This finding indicated the requirement for intrinsic (cell-autonomous) expression of c-Jun in hepatoblasts during early stages of liver development. Alterations of postnatal liver were also observed in knockout mice lacking the mdr-2 gene, the DNA excision repair cross complementing gene-1 (ERCC-1), or the HGF gene. The mdr-2-deficient mice developed hepatocellular dysplasia and necrosis secondary to a toxic effect of phospholipiddeficient bile (Smit et al., 1993). This report illustrates an example in which the likely function of a gene, in this case phospholipid transport, was first identified in genetically modified mice. ERCC-l-deficient mice displayed accelerated hepatic polyploidization associated with aneuploidy, and most

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died at or before 4 weeks of age with signs of liver disease (McWhir et al., 1993). This finding suggests that during the course of normal development the liver either is highly susceptible to or experiences a higher rate of DNA damage (perhaps mediated by oxidative events) than other organs. Inefficient repair of resulting damage leads to hepatic dysfunction and death (McWhir et al., 1993). The HGF null mice died at mid to late gestation. The lack of HGF affected the development of the embryonic liver, which was reduced in size and demonstrated extensive loss of parenchymal cells (Schmidt et al., 1995). Finally, two groups knocked out the metallothionein I and II genes in mice (Michalska and Choo, 1993; Masters et al., 1994). Unmanipulated MT-null mice developed normally, but both groups displayed increased hepatic cadmium sensitivity, underscoring the protective effects of MT proteins against heavy metal-induced toxicity. Interestingly, one study employing transgenic mice actually demonstrated reversal of a neonatal lethal phenotype. Certain deletions on mouse chromosome 7 that flank the albino locus are lethal in the recessive state, and are associated with severe lesions in liver. Mice homozygous for the neonatal lethal albino deletions but also carrying a fumarylacetoacetate hydroxylase (FAH) transgene did not die at birth and were phenotypically normal (Kelsey et al., 1993). FAH catalyzes the final step in tyrosine catabolism. This finding indicated that the F A H gene was the critical locus deleted in the lethal albino genotype, and demonstrated that a tyrosine metabolism defect caused hepatic lesions and death.

C. Liver Regeneration Regeneration of liver following partial hepatectomy or other forms of liver injury also has been examined in transgenic mice. Despite many years of intensive study, major questions remain regarding the identity of molecular events that initiate and stop regeneration (see Chapters 1 to 4) (Fausto and Webber, 1993, 1994). Because growth factors play key roles, hepatic regeneration in mice overexpressing relevant growth factors has been studied to determine how altering the basal level of growth factor expression influences the regeneration process. Surprisingly, overexpression of either of the two most potent hepatic mitogens, TGF~ and HGF, had relatively minor effects on regeneration, at most shortening the time required to fully replace lost mass (Webber et al., 1994; Shiota et al., 1994). It is possible that normal physiological levels of these factors are sufficient to produce a maximal regenerative response, so that adding more has little effect. Gene-targeted mice lacking an intact TGFc~ gene displayed normal liver growth (and, in fact, normal growth of most tissues; Luetteke et al., 1993b; Mann et al., 1993). For the reason noted above, examination of hepatic regeneration in these mice may provide more information about the regenerative influence of TGFe~.

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The remarkable regenerative capacity of individual hepatocytes has been demonstrated by studies involving AL-urokinase-type plasminogen activator (AL-uPA) transgenic mice. In this model, clones of cells derived from progenitor cells that deleted transgene DNA and no longer produced the hepatotoxic gene product repopulated the entire hepatic parenchyma (Sandgren et al., 1991; see Section IV. B.). As few as one clone could replace up to 95% of the liver, in effect regenerating the entire organ. The progenitor cells, however, were derived from fetal or early postnatal mouse liver. To identify the regenerative capacity of adult liver cells, genetically marked cells were harvested by perfusion from adult mouse liver and injected into spleens of young AL-uPA transgenic mice (Rhim et al., 1994). These cells traveled to liver and began to divide like endogenous transgene-deficient cells in AL-uPA mouse liver. When examined 8 weeks post-transplant, recipient liver was composed of up to 70% donor-derived cells (Rhim et al., 1994). Donor-derived parenchyma took the form of hundreds to thousands of confluent nodules containing several thousand cells each. These likely represent clonal progeny of single transplanted cells, and suggest that adult liver cells can undergo multiple rounds of cellular division as previously suggested by Jirtle and Michalopoulos (1982). Donor nodule cells retained the ability to divide following two-thirds partial hepatectomy, indicating that they remained mitotically competent. By using this approach while restricting the number of donor cells introduced, so that only a few reach the liver, an even greater mitotic potential of individual adult liver cells than the 12 doublings presently observed may be demonstrable. These mice may also be used to examine the biology and pathology of xenogeneic liver cells. Rhim and co-workers (Rhim et al., 1995) were able to replace up to 100% of immunocompromised AL-uPA mouse hepatic parenchyma by rat hepatocytes. Transplants of human liver cells into immunocompromised AL-uPA mice could produce a mouse with a humanized liver, as accomplished for human lymphoid tissue in severe combined immune deficient (SCID) mice (reviewed in McCune et al., 1991). D. Conclusions

Complex mechanisms underlie liver development, growth, and regeneration, with regulatory influences active at the molecular, cellular, tissue, and organismal levels. To fully address this complexity, at least some studies must be conducted that experimentally examine these phenomena in living organisms. The reports described above using genetically altered mice have begun to address this need. Coupled with new approaches introduced in the following section, future in vivo experimentation should define with greater precision the genetic basis of liver growth.

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VII. Assessment and Future Directions Establishment of methods for altering the mammalian genome has revolutionized our ability to explore the genetic bases of development, growth, and disease. The experimental models described in this chapter warrant continued study to identify detailed mechanisms linking altered gene expression with disorders of hepatic growth. Furthermore, similar models targeting expression of additional growth modulating genes to liver must be created and examined both alone and in mice carrying two or more transgenes. In this way, we can create a catalog of genetic phenotypes in liver, linking expression patterns of individual genes with specific alterations of tissue architecture and growth. The combination of transgene- and EScell-based technologies to examine the interplay between excessive and deficient gene expression should prove particularly important in this respect. A major hurdle that must be overcome is the current lack of fully inducible, liver-specific enhancer/promoters that display no basal expression in the uninduced state and that have biologically inert inducing agents (LPS, for example, is potently bioactive). Similarly, it has been difficult to abolish expression of target genes in a tissue-specific manner; gene knockouts affect all cells of the body. However, these limitations are being addressed (Furth et al., 1994; Gu et al., 1994), opening new possibilities for even more precise manipulation of the genetics of liver growth. Future studies also must address the cellular basis of liver growth and regeneration. Targeting of growth modulatory substances to nonparenchymal cells can be used to examine cellular interactions in liver. Furthermore, strategies currently exist for ablating selected cell types by cell-specific targeting of toxin gene expression (for example, diphtheria toxin or herpes simplex virus thymidine kinase; reviewed in Evans, 1989); appropriate experimental design can provide insight into the effects on hepatic regeneration of loss of specific liver cell subpopulations. Metabolic aspects of liver growth and neoplasia can be studied by modulating the complement or activities of enzymes present in the hepatocyte (Goodridge, 1990). Finally, clinically important issues should be addressed. TAg-bearing transgenic mice have been the subject of several therapeutic trials designed to delay development of hepatic tumors (Allemand et al., 1993; Macri and Gordon, 1994; Kimura et al., 1994). Mice expressing a creatine kinase-encoding transgene displayed elevated hepatic creatine kinase activity, and administration of phosphocreatine rendered the livers of these mice more resistant to hypoxia (an important consideration for transplanted tissues; Koretsky et al., 1990; Miller et al., 1993). Transplantation of human liver cells into immunodeficient AL-uPA mice should provide a model that can be manipulated in a controlled manner to examine the effects of toxic or infectious

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a g e n t s o n h u m a n liver cells in v i v o . Similar studies s h o u l d b e c o m e m o r e c o m m o n as r e d e s i g n of the m a m m a l i a n g e n o m e c o n t i n u e s to increase o u r u n d e r s t a n d i n g of genetic m e c h a n i s m s of disease a n d to p r o v i d e a n i m a l s e n g i n e e r e d to m o r e precisely m o d e l diseases of h u m a n liver.

References Adrian, G. S., Bowman, B. H., Herbert, D. C., Weaker, E J., Adrian, E. K., Robinson, L. K., Walter, C. A., Eddy, C. A., Riehl, R., Pauerstein, C. J., and Yang, E (1990). Human transferrin. J. Biol. Chem. 265, 13344-13350. Albers, K. M., Davis, F. E., Perrone, T. N., Lee, E. Y., Liu, Y., and Vore, M. (1995). Expression of an epidermal keratin protein in liver of transgenic mice causes structural and functional abnormalities. J. Cell Biol. 128, 157-169. Allemand, I., Christ, M., Pannecoucke, X., Molina, T., Luu, B., and Briand, P. (1993). Effect of oxysterol derivatives on the time course development of hepatocarcinoma in transgenic mice. Anticancer Res. 13, 1097-1102. Alvares, K., Fan, C., Dadras, S. S., Yeldandi, A. V., Rachubinski, R. A., Capone, J. P., Subramani, S., Iannaccone, P. M., Rao, M. S., and Reddy, J. K. (1994). An upstream region of the enoyl-coenzyme A hydratase/3-hydroxyacyl-coenzyme A dehydrogenase gene directs luciferase expression in liver in response to peroxisome proliferators in transgenic mice. Cancer Res. 54, 2303-2306. Antoine, B., Levrat, E, Vallet, V., Berbar, T., Cartier, N., Dubois, N., Briand, P., and Kahn, A. (1992). Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice. Exp. Cell Res. 200, 175-185. Araki, K., Miyazaki, J., Hino, O., Tomita, N., Chisaka, O., Matsubara, K., and Yamamura, K. (1989). Expression and replication of hepatitis B virus genome in transgenic mice. Proc. Natl. Acad. Sci. USA 86, 207-211. Araki, K., Hino, O., Miyazaki, J., and Yamamura, K. (1991). Development of two types of hepatocellular carcinoma in transgenic mice carrying the SV40 large T-antigen gene. Carcinogenesis 12, 2059-2062. Barrett, J. C., and Wiseman, R. W. (1992). Molecular carcinogenesis in humans and ~odents. Prog. Clin. Biol. Res. 376, 1-30. Beermann, E, Hummler, E., Schmid, E., and Schiitz, G. (1993). Perinatal activation of a tyrosine animotransferase fusion gene does not occur in albino lethal mice. Mechanisms Dev. 42, 59-65. Boguski, M. S., and McCormick, E (1993). Proteins regulating ras and its relatives. Nature 366, 643-654. Boquet, D., Vaulont, S., Tremp, G., Ripoche, M.-A., Daegelen, D., Jami, J., Kahn, A., and Raymondjean, M. (1992). DNase-I hypersensitivity analysis of the L-type pyruvate kinase gene in rats and transgenic mice. Eur. J. Biochem. 207, 13-21. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Senear, A. W., Warren, R., and Palmiter, R. D. (1981). Somatic expression of a herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27, 223-231. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., and Palmiter, R. D. (1985). Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82, 4438-4442. Burk, R. D., DeLoia, J. A., El Awady, M. K., and Gearhart, J. D. (1988). Tissue preferential

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Transgenic Models of Hepatic Growth Regulation and Hepatocarcinogenesis

291

expression of the hepatitis B virus (HBV) surface antigen gene in two lines of HBV transgenic mice. J. Virol. 62, 649-654. Butel, J. S., Sepulveda, A. R., Finegold, M. J., and Woo, S. L. C. (1990). SV40 large T antigen directed by regulatory elements of the human c~-l-antitrypsin gene. Intervirology 31, 85100. Camper, S. A., and Tilghman, S. M. (1989). Postnatal repression of the ot-fetoprotein gene is enhancer independent. Genes Dev. 3, 537-546. Cariani, E., Dubois, N., Lasserre, C., Briand, P., and Brechot, C. (1991). Insulin-like growth factor II (IGF2) mRNA expression during hepatocarcinogenesis in transgenic mice. J. Hepatol. 13, 220-226. Carlson, J. A., Rogers, B. B., Sifers, R. N., Hawkins, H. K., Finegold, M. J., and Woo, S. L. C. (1988). Multiple tissues express Cxl-antitrypsin in transgenic mice and man. J. C/in. Invest. 82, 26-36. Carlson, J. A., Rogers, B. B., Sifers, R. N., Finegold, M. J., Clift, S. M., DeMayo, E J., Bullock, D. W., and Woo, S. L. C. (1989). Accumulation of PiZ Otl-antitrypsin causes liver damage in transgenic mice. J. Clin. Invest. 83, 1183-1190. Cartier, N., Miquerol, L., Tulliez, M., Lepetit, N., Levrat, F., Grimber, G., Briand, P., and Kahn, A. (1992). Diet-dependent carcinogenesis of pancreatic islets and liver in transgenic mice expressing oncogenes under the control of the L-type pyruvate kinase gene promoter. Oncogene 7, 1413-1422. Chisari, F. V., Pinkert, C. A., Milich, D. R., Filippi, P., McLachlan, A., Palmiter, R. D., and Brinster, R. L. (1985). A transgenic mouse model of the chronic hepatitis B surface antigen carrier state. Science 230, 1157-1160. Chisari, E V., Filippi, P., McLachlan, A., Milich, D. R., Riggs, M., Lee, S., Palmiter, R. D., Pinkert, C. A., and Brinster, R. L. (1986). Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. J. Virol. 60, 880-887. Chisari, F. V., Filippi, P., Buras, J., McLachlan, A., Popper, H., Pinkert, C. A., Palmiter, R. D., and Brinster, R. L. (1987). Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice. Proc. Natl. Acad. Sci. USA 84, 69096913. Chisari, F. V., Klopchin, K., Moriyama, T., Pasquinelli, C., Dunsford, H. A., Sell, S., Pinkert, C. A., Brinster, R. L., and Palmiter, R. D. (1989). Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 59, 1145-1156. Cloustin, W. M., Lyons, I. G., and Richards, R. I. (1989). Tissue-specific and hormonal regulation of angiotensinogen minigenes in transgenic mice. EMBO J. 8, 3337-3343. Corallini, A., Altavilla, G., Pozzi, L., Bignozzi, E, Negrini, M., Rimessi, P., Gualandi, E, and Barbanti-Brodano, G. (1993). Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes. Cancer Res. 53, 55695575. Crystal, R. G. (1990). cx-l-antitrypsin deficiency, emphysema, and liver disease: Genetic basis and strategies for therapy. J. C/in. Invest. 85, 1343-1352. Cuif, M.-H., Porteu, A., Kahn, A., and Vaulont, S. (1993). Exploration of a liver-specific, glucose/insulin-responsive promoter in transgenic mice. J. Biol. Chem. 268, 13769-13772. Cullen, J. M., Sandgren, E. P., Brinster, R. L., and Maronpot, R. R. (1993). Histologic characterization of hepatic carcinogenesis in transgenic mice expressing SV40 T-antigens. Vet. Pathol. 30, 111-118. DaCosta Soares, V., Gubits, R. M., Feigelson, P., and Costantini, F. (1987). Tissue-specific and hormonally regulated expression of a rat e~2u globulin gene in transgenic mice. Mol. Cell. Biol. 7, 3749-3758.

292

Eric P. Sandgren

Dalemans, W., Perraud, E, Le Meur, M., Gerlinger, P., Courtney, M., and Pavirani, A. (1990). Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice. Biologicals 18, 191-198. Dewey, M. J., Rheaume, C., Berger, E G., and Baumann, H. (1990). Inducible and tissuespecific expression of rat or-l-acid glycoprotein in transgenic mice. J. Immunol. 144, 43924398. Dragani, T. A., Manenti, G., Farza, H., Della Porta, G., Tiollais, P., and Pourcel, C. (1989). Transgenic mice containing hepatitis B virus sequences are more susceptible to carcinogeninduced hepatocarcinogenesis. Carcinogenesis 11, 953-956. Dubois, N., Bennoun, M., Allemand, I., Molina, T., Grimber, G., Daudet-Monsac, M., Abelanet, R., and Briand, P. (1991). Time-course development of differentiated hepatocarcinoma and lung metastasis in transgenic mice. J. Hepatol. 13, 227-239. Dunsford, H. A., Sell, S., and Chisari, E V. (1990). Hepatocarcinogenesis due to chronic liver cell injury in hepatitis B virus transgenic mice. Cancer Res. 50, 3400-3407. Dycaico, M. J., Grant, S. G. N., Felts, K., Nichols, W. S., Geller, S. A., Hager, J. H., Pollard, A. J., Kohler, S. W., Short, H. P., Jirik, E R., Hanahan, D., and Sorge, J. A. (1988). Neonatal hepatitis induced by Otl-antitrypsin: A transgenic mouse model. Science 242, 1409-1412. Dyer, K. R., and Messing, A. (1989). Metal-inducible pathology in the liver, pancreas, and kidney of transgenic mice expressing SV40 early region genes. Am. J. Pathol. 135, 401410. Eisenberger, C. L., Nechushtan, H., Cohen, H., Shani, M., and Reshef, L. (1992). Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice. Mol. Cell. Biol. 12, 1396-1403. Etiemble, J., Degott, C., Renard, C. A., Fourel, G., Shamoon, B., Vitcitski-Trepo, L., Hsu, T. Y., Tiollais, P., Babinet, C., and Buendia, M. A. (1994). Liver-specific expression and high oncogenic efficiency of a c-myc transgene activated by woodchuck hepatitis virus insertion. Oncogene 9, 727-737. Evans, G. A. (1989). Dissecting mouse development with toxigenics. Genes Dev. 3,259-263. Farza, H., Hadchouel, M., Scotto, J. Tiollais, P., Babinet, C., and Pourcel, C. (1988). Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome. J. Virol. 62, 4144-4152. Fausto, N., and Webber, E. M. (1993). Control of liver growth. Crit. Rev. Eukaryotic Gene Exp. 3, 117-135. Fausto, N., and Webber, E. M. (1994). Liver regeneration. In "The Liver: Biology and Pathobiology" (I. M. Arias, J. L. Boyer, N., Fausto, W. B. Jakoby, D. Schachter, and D. A. Shafritz, eds.), 3rd ed., pp. 1059-1084. Raven Press, New York. Furth, E A., St. Onge, L., Boger, H., Gruss, E, Gossen, M., Kistner, A., Bujard, H., and Hennighausen, L. (1994). Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl. Acad. Sci. USA 91, 9302-9306. Geller, S. A., Nichols, W. S., Kim, S., Tolmachoff, T., Lee, S., Dycaico, M. J., Felts, K., and Sorge, J. A. (1994). Hepatocarcinogenesis is the sequel to hepatitis in Z#2 ot~-antitrypsin transgenic mice: Histopathological and DNA ploidy studies. Hepatology 19, 389-397. Goodridge, A. G. (1990). The new metabolism: Molecular genetics in the analysis of metabolic regulation. FASEB J. 4, 3099-3110. Gordon, J. W. (1993). Production of transgenic mice. Method Enzymol. 225, 747-771. Gordon, J. W. (1994). Transgenic mouse models of hepatocellular carcinoma. Hepatology 19, 538-539. Gossen, J., and Vijg, J. (1993). Transgenic mice as model systems for studying gene mutations in vivo. Trends in Genet. 9, 27-31. Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., and Rajewsky, K. (1994). Deletion of a DNA polymerase 13gene segment in T cells using cell-type-specific gene targeting. Science 265, 103-106.

11.

Transgenic Models of Hepatic Growth Regulation and Hepatocarcinogenesis

293

Hagen, T. M., Huang, S., Curnutte, J., Fowler, P., Martinez, V., Wehr, C. M., Ames, B. N., and Chisari, E V. (1994). Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 91, 12808-12812. Halmekyt6, M., Hyttinen, J.-M., Sinervirta, R., Lepp/inen, P., J/inne, J., and Alhonen, L. (1993). Regulation of the expression of human ornithine decarboxylase gene and ornithine decarboxylase promoter-driven reporter gene in transgenic mice. Biochem. J. 292, 927932. Hammer, R. E., Krumlauf, R., Camper, S. A., Brinster, R. L., and Tilghman, S. M. (1987). Diversity of ~t-fetoprotein gene expression in mice is generated by a combination of separate enhancer elements. Science 235, 53-58. Heckel, J. L., Sandgren, E. P., Degen, J. L., Palmiter, R. D., and Brinster, R. L. (1990). Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator. Cell 62, 447456. Held, W. A., MuUins, J. J., Kuhn, N. J., Gallagher, J. E, Gu, G. D., and Gross, K. W. (1989). T antigen expression and tumorigenesis in transgenic mice containing a mouse major urinary protein/SV40 T antigen hybrid gene. EMBO J. 8, 183-191. Held, W. A., Pazik, J., O'Brien, J. G., Kerns, K., Gobey, M., Meis, R., Kenney, L., and Rustum, Y. (1994). Genetic analysis of liver tumorigenesis in SV40 T antigen transgenic mice implies a role for imprinted genes. Cancer Res. 54, 6489-6495. Hilberg, E, Aguzzi, A., Howells, N., and Wagner, E. E (1993). c-Jun is essential for normal mouse development and hepatogenesis. Nature 365, 179-181. Hino, O., Kitagawa, T., Nomura, K., Ohtake, K., Cui, L., Furuta, Y., and Aizawa, S. (1991). Hepatocarcinogenesis in transgenic mice carrying albumin-promoted SV40 T antigen gene. Jpn. J. Cancer Res. 82, 1226-1233. Hirsch-Ernst, K. I., Paul, D., Kahl, G. E, and H6hne, M. W. (1993). Expression of c-los and c-myc protooncogenes in an immortalized hepatocyte line harbouring SV40 T antigen and hGH as transgenes. Transgenic Res. 2, 101-108. Hully, J. R., Su, Y., Lohse, J. K., Griep, A. E., Sattler, C. A., Haas, M. J., Dragan, Y., Peterson, J., Neveu, M., and Pitot, H. C. (1994). Transgenic hepatocarcinogenesis in the rat. Am. J. Pathol. 145, 384-397. Idzerda, R. L., Behringer, R. R., Theisen, M., Huggenvik, J. I., McKnight, G. S., and Brinster, R. L. (1989). Expression from the transferrin gene promoter in transgenic mice. Mol. Cell. Biol. 9, 5154-5162. Jallat, S., Perraud, E, Dalemans, W., Balland, A., Dieterle, A., Faure, T., Meulien, P., and Pavirani, A. (1990). Characterization of recombinant human Factor IX expresed in transgenic mice and in derived trans-immortalized hepatic cell lines. EMBO J. 9, 3295-3301. Jhappan, C., Stahle, C., Harkins, R. N., Fausto, N., Smith, G. H., and Merlino, G. T. (1990). TGFot overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 61, 1137-1146. Jiang, X. C., Agellon, L. B., Walsh, A., Breslow, J. L., and Tall, A. (1992). Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. J. Clin. Invest. 90, 1290-1295. Jirtle, R. L., and Michalopoulos, G. (1982). Effects of partial hepatectomy on transplanted hepatocytes. Cancer Res. 42, 3000-3004. Jones, J. M., Keller, S. A., Samuelson, L. C., Osborn, L., Rosenberg, M. P., and Meisler, M. H. (1989). A salivary amylase transgene is efficiently expressed in liver but not in parotid gland of transgenic mice. Nucleic Acids Res. 17, 6613-6623. Jones, S. N., Grompe, M., Munir, M. I., Veres, G., Craigen, W. J., and Caskey, C. T. (1990). Ectopic correction of ornithine transcarbamylase deficiency in sparse fur mice. J. Biol. Chem. 265, 14684-14690. Jones, S. N., Jones, P. G., Ibarguen, H., Caskey, C. T., and Craigen, W. J. (1991). Induction of

294

Eric P. Sandgren

the Cypla-1 dioxin-responsive enhancer in transgenic mice. Nucleic Acids Res. 19, 65476551. Kaltschmidt, C., Muller, M., Brem, G., and Renkawitz, R. (1994). DNase I hypersensative sites far upstream of the rat tryptophan oxygenase gene direct developmentally regulated transcription in livers of transgenic mice. Mechanisms Dev. 45, 203-210. Kelsey, G. D., Povey, S., Bygrave, A. E., and Lovell-Badge, R. H. (1987). Species- and tissue' specific expression of human Otl-antitrypsin in transgenic mice. Genes Dev. 1, 161-171. Kelsey, G., Ruppert, S., Beermann, E, Grund, C., Tanguay, R. M., and Schiitz, G. (1993). Rescue of mice homozygous for lethal albino deletions: Implications for an animal model for the human liver disease tyrosinemia type 1. Genes Dev. 7, 2285-2297. Kim. C.-M., Koike, K., Saito, I., Miyamura, T., and Jay, G. (1991). HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 351,317-320. Kimura, O., Yamaguchi, Y., Gunning, K. B., Teeter, L. D., Husain, E, and Kuo, M. T. (1994). Retroviral delivery of DNA into the livers of transgenic mice bearing premalignant and malignant hepatocellular carcinomas. Human Gene Therapy 5, 845-852. Koff, R. S. (1992). Hepatocellular carcinoma in transgenic mice: A consequence of continued expression of the HBx gene? Gastroenterology 102, 1081-1082. Koike, K., Moriya, K., Iino, S., Yotsuyanagi, H., Endo, Y., Miyamura, T., and Kurokawa, K. (1994). High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice. Hepatology 19, 810-819. Koretsky, A. P., Brosnan, M. J., Chen, L., Chen, J., and Van Dyke, T. A. (1990). NMR detection of creatine kinase expressed in liver of transgenic mice: Determination of free ADP levels. Proc. Natl. Acad. Sci. USA 87, 3112-3116. Krumlauf, R., Hammer, R. E., Tilghman, S. M., and Brinster, R. L. (1985). Developmental regulation of ~-fetoprotein genes in transgenic mice. Mol. Cell. Biol. 5, 1639-1648. Lee, G.-H., Li, H., Ohtake, K., Nomura, K., Hino, O., Furuta, Y., Aizawa, S., and Kitagawa, T. (1990). Detection of activated c-H-ras oncogene in hepatocellular carcinomas developing in transgenic mice harboring albumin promoter-regulated simian virus 40 gene. Carcinogenesis 11, 1145-1148. Lee, T.-H., Finegold, M. J., Shen, R.-E, DeMayo, J. L., Woo, S. L. C., and Butel, J. S. (1990). Hepatitis B virus transactivator protein is not tumorigenic in transgenic mice. J. Virol. 64, 5939-5947. Lee, G.-H., Merlino, G., and Fausto, N. (1992). Development of liver tumors in transforming growth factor ~ transgenic mice. Cancer Res. 52, 5162-5170. Lee, D. C., Luetteke, N. C., and Petch, L. A. (1993). Transforming growth factor-alpha and its role in neoplastic transformation. In "Oncogenes and tumor suppressor genes in human malignancies" (C. C. Benz and E. T. Liu, eds.), pp. 233-254. Kluwer Academic Publishers, Boston. Levrat, E, Vallet, V., Berbar, T., Miquerol, L., Kahn, A., and Antoine, B. (1993). Influence of the content in transcription factors on the phenotype of mouse hepatocyte-like cell lines (mhAT). Exp. Cell Res. 209, 307-316. Lim, I. K., Dumenco, L. L., Yun, J., Donovan, C., Warman, B., Gorodetzkaya, N. Wagner, T. E., Clapp, D. W., Hanson, R. W., and Gerson, S. L. (1990). High level, regulated expression of the chimeric p-enolpyruvate carboxykinase (GTP)-bacterial O6-alkylguanine DNA alkyltransferase (ada) gene in transgenic mice. Cancer Res. 50, 1701-1708. Luetteke, N. C., Lee, D. C., Palmiter, R. D., Brinster, R. L., and Sandgren, E. P. (1993a). Regulation of fat and muscle development by transforming growth factor ~ in transgenic mice and in cultured cells. Cell Growth Differentiation 4, 203-213. Luetteke, N. C., Qui, T. H., Peiffer, R. L., Oliver, P., Smithies, O., and Lee, D. C. (1993b). TGF~ deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice. Cell 73, 263-278.

11.

Transgenic Models of Hepatic Growth Regulation and Hepatocarcinogenesis

295

Macri, P., and Gordon, J. W. (1993). Transgenic animals as tools for investigating hepatocyte gene regulation and liver disease. Prog. Liver Dis. 11, 1-25. Macri, P., and Gordon, J. W. (1994). Delayed morbidity and mortality of albumin/SV40 T-antigen transgenic mice after insertion of an ot-fetoprotein/herpes virus thymidine kinase transgene and treatment with ganciclovir. Hum. Gene Ther. 5, 175-182. Mann, C. B., Fowler, K. J., Babriel, A., Nice, E. C., Williams, R. L., and Dunn, A. R. (1993). Mice with a null mutation of the TGFot gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation. Cell 73, 249-261. Maronpot, R. R., Palmiter, R. D., Brinster, R. L., and Sandgren, E. E (1991). Pulmonary carcinogenesis in transgenic mice. Exp. Lung Res. 17, 305-320. Masters, B. A., Kelly, E. J., Quaife, C. J., Brinster, R. L., and Palmiter, R. D. (1994). Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. USA 91, 584-588. Mathews, L. S., Hammer, R. E., Behringer, R. R., D'Ercole, A. J., Bell, G. I., Brinster, R. L., and Palmiter, R. D. (1988). Growth enhancement of transgenic mice expressing human insulinlike growth factor I. Endocrinol. 123, 2827-2833. McCune, J., Kaneshima, H., Krowka, J., Namikawa, R., Outzen, H., Peault, B., Rabin, L., Shih, C.-C., Yee, E., Lieberman, M., Weissman, I., and Shultz, L. (1991). The SCID-hu mouse: A small animal model for HIV infection and pathogenesis. Ann. Rev. Immunol. 9, 399-429. McGrane, M. M., de Vente, J., Yun, J., Bloom, J., Park, E., Wynshaw-Boris, A., Wagner, T., Rottman, E M., and Hanson, R. W. (1988). Tissue-specific expression and dietary regulation of a chimeric phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic mice. J. Biol. Chem. 263, 11443-11451. McWhir, J., Selfridge, J., Harrison, D. J., Squires, S., and Melton, D. W. (1993). Mice with DNA repair gene (ERCC-1) deficiency have elevated levels of p53, liver nuclear abnormalities and die before weaning. Nat. Genet. 5,217-223. Merlino, G. (1994). Transgenic mice: Designing genes for molecular models. In "The Liver: Biology and Pathobiology" (I. M. Arias, J. L. Boyer, N. Fausto, W. B. Jakoby, D. Schachter, and D. A. Shafritz, eds.), 3rd ed., pp. 1579-1589. Raven Press, New York. Messing, A., Chen, H. Y., Palmiter, R. D., and Brinster, R. L. (1985). Peripheral neuropathies, hepatocellular carcinomas, and islet cell adenomas in transgenic mice. Nature 316, 461463. Michalska, A. E., and Choo, K. H. A. (1993). Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. USA 90, 8088-8092. Miller, K., Halow, J., and Koretsky, A. P. (1993). Phosphocreatine protects transgenic mouse liver expressing creatine kinase from hypoxia and ischemia. Am. J. Physiol. 265, C15441551. Mirsalis, J. C., Provost, G. S., Matthews, C. D., Hamner, R. T., Schindler, J. E., O'Loughlin, K. G., MacGregor, J. T., and Short, J. M. (1993). Induction of hepatic mutations in lacI transgenic mice. Mutagenesis 8, 265-271. Mirsalis, J. C., Monforte, J. A., and Winegar, R. A. (1994). Transgenic animal models for measuring mutations in vivo. Crit. Rev. Toxicol. 24, 255-280. Montag, A. G., Oka, T., Baek, K. H., Choi, C. S., Jay, G., and Agarwal, K. (1993). Tumors in hepatobiliary tract and pancreatic islet tissues of transgenic mice harboring gastrin simian virus 40 large tumor antigen fusion gene. Proc. Natl. Acad. Sci. USA 90, 6696-6700. Morimura, S., Suzuki, T., Hochi, S.-I., Yuki, A., Nomura, K., Kitagawa, T., Nagatsu, I., Imagawa, M., and Muramatsu, M. (1993). Trans-activation of glutathione transferase P gene during chemical hepatocarcinogenesis of the rat. Proc. Natl. Acad. Sci. USA 90, 20652068.

296

Eric P. Sandgren

Murakami, T., Takiguchi, M., Inomoto, T., Yamamura, K.-I., and Mori, M. (1989). Tissueand developmental stage-specific expression of the rat ornithine carbamoyltransferase gene in transgenic mice. Dev. Genet. 10, 393-401. Murakami, H., Sanderson, N. D., Nagy, P., Marino, P. A., Merlino, G., and Thorgeirsson, S. S. (1993). Transgenic mouse model for synergistic effects of nuclear oncogenes and growth factors in tumorigenesis: Interaction of c-myc and transforming growth factor et in hepatic oncogenesis. Cancer Res. 53, 1719-1723. Murphy, C., Kretschmer, C., Biesinger, B., Beckers, J., Jung, J., Desrosiers, R. C., MtillerHermelink, H. K., Fleckenstein, B. W., and Riither, U. (1994). Epithelial tumors induced by a Herpesvirus oncogene in transgenic mice. Oncogene 9, 221-226. Murphy, C., Beckers, J., and Riither, U. (1995). Regulation of the human C-reactive protein gene in transgenic mice. J. Biol. Chem. 270, 704-708. Nagata, Y., Tashiro, E, Yi, S., Murakami, T., Maeda, S., Takahashi, K., Shimada, K., Okamura, H., and Yamamura, K.-I. (1995). A 6-kb upstream region of the human transthyretin gene can direct developmental, tissue-specific, and quantitatively normal expression in transgenic mouse. ]. Biochem. 117, 169-175. Nakatsuru, Y., Matsukuma, S., Nemoto, N., Sugano, H., Sekiguchi, M., and Ishikawa, T. (1993). O6-methylguanine-DNA methyltransferase protects against nitrosamine-induced hepatocarcinogenesis. Proc. Natl. Acad. Sci. USA 90, 6468-6472. Palmiter, R. D., Sandgren, E. E, Koeller, D. M., and Brinster, R. L. (1993). Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice. Mol. Cell. Biol. 13, 5266-5275. Pasquinelli, C., Bhavani, K., and Chisari, E V. (1992). Multiple oncogenes and tumor suppressor genes are structurally and functionally intact during hepatocarcinogenesis in hepatitis B virus transgenic mice. Cancer Res. 52, 2823-2829. Patel, Y. M., Yun, J. S., Liu, J., McGrane, M. M., and Hanson, R. W. (1994). An analysis of regulatory elements in the phosphoenolpyruvate carboxykinase (GTP) gene which are responsible for its tissue-specific expression and metabolic control in transgenic mice. J. Biol. Chem. 269, 5619-5628. Paul, D., Hohne, M., Pinkert, C., Piasecki, A., Ummelmann, E., and Brinster, R. L. (1988). Immortalized differentiated hepatocyte lines derived from transgenic mice harboring SV40 T-antigen genes. Exp. Cell Res. 175, 354-362. Perraud, F., Dalemans, W., Gendrault, J.-L., Dreyer, D., Ali-Hadji, D., Faure, T., and Pavirani, A. (1991). Characterization of trans~ hepatic cell lines established from transgenic mice. Exp. Cell Res. 195, 59-65. Pinkert, C. A., Ornitz, D. M., Brinster, R. L., and Palmiter, R. D. (1987). An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice. Genes Dev. 1,268-276. Provost, G. S., Kretz, E L., Hamner, R. T., Matthews, C. D., Rogers, B. J., Lundberg, K. S., Dycaico, M. J., and Short, J. M. (1993). Transgenic systems for in vivo mutation analysis. Mutation Res. 288, 133-149. Quaife, C. J., Mathews, L. S., Pinkert, C. A., Hammer, R. E., Brinster, R. L., and Palmiter, R. D. (1989). Histopathology associated with elevated levels of growth hormone and insulin-like growth factor I in transgenic mice. Endocrinol. 124, 40-48. Ramirez, M. I., Karaoglu, D., Haro, D., Barillas, C., Bashirzadeh, R., and Gil, G. (1994). Cholesterol and bile acids regulate cholesterol 7et-hydroxylase expression at the transcriptional level in culture and in transgenic mice. Mol. Cell. Biol. 14, 2809-2821. Ramirez-Solis, R., Davis, A. C., and Bradley, A. (1993). Gene targeting in embryonic stem cells. Meth. Enzymol. 225, 855-878. Ramsden, R., Sommer, K. M., and Omiecinski, C. J. (1993). Phenobarbital induction and tissue-specific expression of the rat CYP2B2 gene in transgenic mice. ]. Biol. Chem. 268, 21722-21726.

11.

Transgenic Models of Hepatic Growth Regulation and Hepatocarcinogenesis

297

Rhim, J. A., Sandgren, E. P., Degen, J. L., Palmiter, R. D., and Brinster, R. L. (1994). Replacement of diseased mouse liver by hepatic cell transplantation. Science 263, 1149-1152. Rhim, J. A., Sandgren, E. P., Palmiter, R. D., and Brinster, R. L. (1995). Complete reconstitution of mouse liver with xenogeneic hepatocytes. Proc. Natl. Acad. Sci. USA, in press. Rogler, C. E., Yang, D., Rossetti, L., Donohoe, J., Alt, E., Chang, C. J., Rosenfeld, R., Neely, K., and Hintz, R. (1994). Altered body composition and increased frequency of diverse malignancies in insulin-like growth factoroII transgenic mice. J. Biol. Chem. 269, 1377913784. Rfither, U., Woodroofe, C., Fattori, E., and Ciliberto, G. (1993). Inducible formation of liver tumors in transgenic mice. Oncogene 8, 87-93. Saitoh, A., Kimura, M., Takahashi, R., Yokoyama, M., Nomura, T., Izawa, M., Sekiya, T., Nishimura, S., and Katsuki, M. (1990). Most tumors in transgenic mice with human c-Haras gene contained somatically activated transgenes. Oncogene 5, 1195-1200. Sandgren, E. P., Quaife, C. J., Pinkert, C. A., Palmiter, R. D., and Brinster, R. L. (1989). Oncogene-induced liver neoplasia in transgenic mice. Oncogene 4, 715-724. Sandgren, E. P., Luetteke, N. C., Palmiter, R. D., Brinster, R. L., and Lee, D. C. (1990). Overexpression of TGFoL in transgenic mice: Induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61, 1121-1135. Sandgren, E. P., Palmiter, R. D., Heckel, J. L., Daugherty, C. C., Brinster, R. L., and Degen, J. L. (1991). Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene. Cell 66, 245-256. Sandgren, E. P., Palmiter, R. D., Heckel, J. L., Brinster, R. L., and Degen, J. L. (1992). DNA rearrangement causes hepatocarcinogenesis in albumin-plasminogen activator transgenic mice. Proc. Natl. Acad. Sci. USA 89, 11523-11527. Sandgren, E. P., Luetteke, N. C., Qiu, T. H., Palmiter, R. D., Brinster, R. L., and Lee, D. C. (1993). Transforming growth factor alpha dramatically enhances oncogene-induced carcinogenesis in transgenic mouse pancreas and liver. Mol. Cell. Biol. 13, 320-330. Schirmacher, P., Held, W. A., Yang, D., Biempica, L., and Rogler, C. E. (1991). Selective amplification of periportal transitional cells preceeds formation of hepatocellular carcinoma in SV40 large Tag transgenic mice. Am. J. Pathol. 139, 231-241. Schirmacher, P., Held, W. A., Yang, D., Chisari, E V., Rustum, Y., and Rogler, C. E. (1992). Reactivation of insulin-like growth factor II during hepatocarcinogenesis in transgenic mice suggests a role in malignant growth. Cancer Res. 52, 2549-2556. Schmidt, C., Bladt, E, Goedecke, S., Brinkmann, V., Zschiesche, W., Sharpe, M., Gherardi, E., Birchmeier, C. (1995). Scatter factor/hepatocyte growth factor is essential for liver development. Nature 373, 699-702. Schofield, P. N. (1992). "The Insulin-like Growth Factors: Structure and Biological Function." Oxford Univ. Press, Oxford. Sell, S., Hunt, J. M., Dunsford, H. A., and Chisari, E V. (1991). Synergy between hepatitis B virus expression and chemical hepatocarcinogenesis in transgenic mice. Cancer Res. 51, 1278-1285. Sell, S., and Knoll, B. (1992). Transgenic mouse models of hepatocarcinogenesis. In "The Role of Cell Types in Hepatocarcinogenesis" (A. E. Sirica, ed.), pp. 299-321. CRC Press, Boca Raton. Sell, S., and Ilic, Z. (1994). Dietary cadmium may enhance the progression of hepatocellular tumors in hepatitis B transgenic mice. Carcinogenesis 15, 2057-2060. Semenza, G. L., Dureza, R. C., Traystman, M. D., Gearhart, J. D., and Antonarakis, S. E. (1990). Human erythropoietin gene expression in transgenic mice: Multiple transcription initiation sites and cis-acting regulatory elements. Mol. Cell. Biol. 10, 930-938. Semenza, G. L., Nejfelt, M. K., Chi, S. M., and Antonarakis, S. E. (1991). Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene. Proc. Natl. Acad. Sci. USA 88, 5680-5684.

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Eric P. Sandgren

Sepulveda, A. R., Finegold, M. J., Smith, B., Slagle, B. L., DeMayo, J. L., Shen, R.-E, Woo, S. L. C., and Butel, J. S. (1989). Development of a transgenic mouse system for the analysis of stages in liver carcinogenesis using tissue-specific expression of SV40 large T-antigen controlled by regulatory elements of the human ~-l-antitrypsin gene. Cancer Res. 49, 6108-6117. Shephard, S. E., Sengstag, C., Lutz, W. K., and Schlatter, C. (1993). Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene. Mutation Res. 302, 91-96. Shimada, T., Noda, T., Tashiro, M., Murakami, T., Takiguchi, M., Mori, M., Yamamura, K.-I., and Saheki, T. (1991). Correction of ornithine transcarbamylase (OTC) deficiency in spfash mice by introduction of rat OTC gene. FEBS Let. 279, 198-200. Shiota, G., Rhoads, D. B., Wang, T. C., Nakamura, T., and Schmidt, E. V. (1992). Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells. Proc. Natl. Acad. Sci. USA 89, 373-377. Shiota, G., Wang, T. C., Nakamura, T., and Schmidt, E. V. (1994). Hepatocyte growth factor in transgenic mice: Effects on hepatocyte growth, liver regeneration and gene expression. Hepatology 19, 962-972. Short, M. K., Clouthier, D. E., Schaefer, I. M., Hammer, R. E., Magnuson, M. A., and Beale, E. G. (1992). Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice. Mol. Cell. Biol. 12, 1007-1020. Sifers, R. N., Carlson, J. A., Clift, S. M., DeMayo, E J., Bullock, D. W., and Woo, S. L. C. (1987). Tissue specific expression of the human cxl-antitrypsin gene in transgenic mice. Nucleic Acids Res. 15, 1459-1475. Sifers, R. N., Rogers, B. B., Hawkins, H. K., Finegold, M. J., and Woo, S. L. C. (1989). Elevated synthesis of human cx~-antitrypsin hinders secretion of murine cxl-antitrypsin from hepatocytes of transgenic mice. J. Biol. Chem. 264, 15696-15700. Simon, T. C., Roth, K. A., and Gordon, J. I. (1993). Use of transgenic mice to map cis-acting elements in the liver fatty acid-binding protein gene (Fabpl) that regulate its cell lineagespecific, differentiation-dependent, and spatial patterns of expression in the gut epithelium and in the liver acinus. J. Biol. Chem. 268, 18345-18358. Smit, J. J. M., Schinkel, A. H., Oude Elferink, R. P. J., Groen, A. K., Wagenaar, E., van Deemter, L., Mol, C. A. A. M., Ottenhoff, R., van der Lugt, N. M. T., van Roon, M. A., van der Valk, M. A., Offerhaus, G. J. A., Berns, A. J. M., and Borst, P. (1993). Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 75, 451-462. Stewart, C. L. (1993). Production of chimeras between embryonic stem cells and embryos. Methods Enzymol. 225,823-855. Sweetser, D. A., Birkenmeier, E. H., Hoppe, P. C., McKeel, D. W., and Gordon, J. I. (1988). Mechanisms underlying generation of gradients in gene expression within the intestine: An analysis using transgenic mice containing fatty acid binding protein-human growth hormone fusion genes. Genes Dev. 2, 1318-1332. Takagi, H., Sharp, R., Hammermeister, C., Goodrow, T., Bradley, M. O., Fausto, N., and Merlino, G. (1992). Molecular and genetic analysis of liver oncogenesis in transforming growth factor ot transgenic mice. Cancer Res. 52, 5171-5177. Takagi, H., Sharp, R., Takayama, H., Anver, M. R., Ward, J. M., and Merlino, G. (1993). Collaboration between growth factors and diverse chemical carcinogens in hepatocarcinogenesis of transforming growth factor cx transgenic mice. Cancer Res. 53, 4329-4336. Talbot, D., Descombes, P., and Schibler, U. (1994). The 5' flanking region of the rat LAP (C/EBPI3) gene can direct high-level, position-independent, copy number-dependent expression in multiple tissues in transgenic mice. Nucleic Acids Res. 22, 756-766. Tamano, S., Merlino, G. T., and Ward, J. M. (1994). Rapid development of hepatic tumors in

11.

Transgenic Models of Hepatic Growth Regulation and Hepatocarcinogenesis

299

transforming growth factor oLtransgenic mice associated with increased cell proliferation in precancerous hepatocellular lesions initiated by N-nitrosodiethylamine and promoted by phenobarbital. Carcinogenesis 15, 1791-1798. Tan, S.-S. (1991). Liver-specific and position-effect expression of a retinol-binding protein-lacZ fusion (RBP-lacZ) in transgenic mice. Dev. Biol. 146, 24-37. Theisen, M., Behringer, R. R., Cadd, G. C., Brinster, R. L., and McKnight, G. S. (1993). A C/EBP-binding site in the transferrin promoter is essential for expression in the liver but not the brain of transgenic mice. Mol. Cell. Biol. 13, 7666-7676. Toniatti, C., Arcone, R., Majello, B., Ganter, U., Arpaia, G., and Ciliberto, G. (1990). Regulation of the human C-reative protein gene, a major marker of inflammation and cancer. Mol. Biol. Med. 7, 199-212. Toshkov, I., Chisari, E V., and Bannasch, P. (1994). Hepatic preneoplasia in hepatitis B virus transgenic mice. Hepatology 20, 1162-1172. Toyonaga, T., Hino, O., Sugai, S., Wakasugi, S., Abe, K., Shichiri, M., and Yamamura, K.-I. (1994). Chronic active hepatitis in transgenic mice expressing interferon-~/in the liver. Proc. Natl. Acad. Sci. USA 91, 614-618. Tremp, G. L., Boquet, D., Ripoche, M.-A., Cognet, M., Lone, Y.-C., Jami, J., Kahn, A., and Daegelen, D. (1989). Expression of the rat L-type pyruvate kinase gene from its dual erythroid- and liver-specific promoter in transgenic mice. J. Biol. Chem. 264, 1990419910. Tsunematsu, S., Saito, H., Kagawa, T., Morizane, T., Hata, J.-I., Nakamura, T., Ishii, H., Tsuchiya, M., Nomura, T., and Katsuki, M. (1994). Hepatic tumors induced by carbon tetrachloride in transgenic mice carrying a human c-H-ras proto-oncogene without mutations. Int. J. Cancer 59, 554-559. Vacher, J., Camper, S. A., Krumlauf, R., Compton, R. S., and Tilghman, S. M. (1992). Raf regulates the postnatal repression of the mouse oL-fetoprotein gene at the posttranscriptional level. Mol. Cell. Biol. 12, 856-864. Vacher, J., and Tilghman, S. M. (1990). Dominant negative regulation of the mouse o~-fetoprotein gene in adult liver. Science 250, 1732-1735. Vogel, J., Hinrichs, S. H., Napolitano, L. A., Ngo, L., and Jay, G. (1991). Liver cancer in transgenic mice carrying the human immunodeficiency virus tat gene. Cancer Res. 51, 6686-6690. Wang, Y., DeMayo, J. L., Hahn, T. M., Finegold, M. J., Konecki, D. S., Lichter-Konecki, U., and Woo, S. L. C. (1992). Tissue- and development-specific expression of the human phenylalanine hydroxylase/chloramphenicol acetyltransferase fusion gene in transgenic mice. J. Biol. Chem. 267, 15105-15110. Wang, Y., Hahn, T. M., Tsai, S. Y., and Woo, S. L. C. (1994). Functional characterization of a unique liver gene promoter. J. Biol. Chem. 269, 9137-9146. Webber, E. M., Wu, J. C., Wang, L., Merlino, G., and Fausto, N. (1994). Overexpression of transforming growth factor-oL causes liver enlargement and increases hepatocyte proliferation in transgenic mice. Am. J. Pathol. 145, 398-408. Wilkison, W. O., Sandgren, E. P., Palmiter, R. D., Brinster, R. L., and Bell, R. M. (1989). Elevation of 1,2-diacylglycerol in ras-transformed neonatal liver and pancreas of transgenic mice. Oncogene 4, 625-628. Wu, J. C., Merlino, G., and Fausto, N. (1994). Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor oL. Proc. Natl. Acad. Sci. USA 91, 674-678. Yamada, K., Noguchi, T., Miyazaki, J.-I., Matsuda, T., Takenaka, M., Yamamura, K.-I., and Tanaka, T. (1990). Tissue-specific expression of rat pyruvate kinase L/chloramphenicol acetyltransferase fusion gene in transgenic mice and its regulation by diet and insulin. Biochem. Biophys. Res. Comm. 171, 243-249. Yan, C., Costa, R. H., Darnell, J. E., Jr., Chen, J., and Van Dyke, T. A. (1990). Distinct positive

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and negative elements control the limited hepatocyte and choroid plexus expression of transthyretin in transgenic mice. E M B O J. 9, 869-878. Zarnegar, R., DeFrancis, M. C., and Michalopoulos, G. K. (1994). Hepatocyte growth factor: Its role in hepatic growth and pathobiology. In "The Liver: Biology and Pathobiology" (I. M. Arias, J. L. Boyer, N. Fausto, W. B. Jakoby, D. Schachter, and D. A. Shafritz, eds.), 3rd ed., pp. 1047-1057. Raven Press, New York. Zhao, X., Araki, K., Miyazaki, J.-I., and Yamamura, K.-I. (1992). Developmental and liverspecific expression directed by the serum amyloid P component promoter in transgenic mice. J. Biochem. 111, 736-378.

12 Genetic Susceptibility to Liver Cancer Norman R. Drinkwater McArdle Laboratory for CancerResearch University of WisconsinMedicalSchool Madison, Wisconsin53076

Gang-Hong Lee Department of Pathology Asahikawa Medical College Asahikawa 078, Japan

I. Introduction The genetic background of the host has a profound impact on risk for cancer development in both humans and experimental animals. Recent studies have led to the identification and molecular characterization of the genes responsible for several familial cancers in people, including retinoblastoma, Wilm's tumor, and familial adenomatous polyposis (Friend et al., 1986; Rose et al., 1990; Kinzler et al., 1991). These rare genetic diseases are inherited with very high penetrance, allowing the identification of the relevant genes through formal genetic studies in affected families. However, human genetic studies have been less effective in identifying "risk-modifier" genes that may increase by 5- to 50-fold an individual's susceptibility to the development of more common malignancies. The lower effective penetrance of such genes and the large variation in environmental factors that influence cancer development in humans increase the difficulty of genetic analysis. The elucidation of the identity of the B R C A 1 gene, which predisposes affected women to breast and ovarian cancer, represents a tour de force in human cancer genetics. Following the inference from family studies of the existence of this gene in 1988 (Newma n et al., 1988), its location in the

Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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genome was determined from linkage studies (Hall et al., 1990; Narod et al., 1991) and the gene was isolated by molecular cloning (Miki et al., 1994; Futreal et al., 1994) during the space of 6 years. Animal models provide a powerful tool for analysis of the effects of risk modifier genes because the effects of these genes can be studied in wellcontrolled experiments. The existence of such genes can be inferred from the large variation (up to 100-fold) among inbred strains of mice in their sensitivities to tumor induction at numerous tissue sites (Drinkwater and Bennett, 1991). Genetic studies also provide a useful approach to mechanistic studies of carcinogenesis. For example, the role of a specific gene product during carcinogenesis can be inferred from studies comparing tumor development in mice carrying a mutant allele with that in wild type animals. Our knowledge of the genetic basis for liver cancer risk in humans is somewhat limited, as summarized below. Accordingly, this chapter will focus mainly on the genetic regulation of liver cancer development in mice. Understanding the mechanism of action of specific genes that control hepatocarcinogenesis in rodents will provide insight into the underlying biology of carcinogenesis and paradigms for understanding the action of riskmodifier genes in humans.

II. Genetics of Human Liver Cancer Primary liver cancer results in more than 250,000 deaths each year. This disease shows striking geographic variation, with incidence rates ranging from 4 per 100,000 in the United States to 150 per 100,000 in regions of Asia and sub-Saharan Africa (DiBesceglie et al., 1988). Chronic infection by hepatitis B virus (HBV) has been shown to be the primary risk factor for liver cancer through both case-control and prospective epidemiological studies (Beasley et al., 1981; Beasley, 1988). Recently, the hepatitis C virus was found to be a risk factor for hepatocellular carcinoma (Tsukuma et al., 1993). Exposure to the carcinogen, aflatoxin B1, may act synergistically with HBV infection to further increase the risk for liver cancer (Qian et al., 1994). Given these strong environmental influences on human hepatocarcinogenesis, it has been difficult to discern a genetic component of liver cancer risk. However, both family studies and clinical studies of several genetic diseases demonstrate that genetic factors may play a role in human liver cancer.

A. Genetic Epidemiology Several case reports and some formal studies have suggested that liver cancer may cluster in families. A recent case-control study (Fernandez et al.,

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1994) indicated a three fold increased risk for hepatocellular carcinoma among individuals with a family history of the disease. However, it is difficult to exclude from these studies the familial clustering of environmental risk factors, such as HBV infection, in favor of a genetic basis for increased risk. Two studies provide evidence that the risk of liver cancer development among HBV-infected individuals may be determined by specific susceptibility genes. Shen and co-workers (1991) studied 490 families identified through a case-control study of liver cancer in eastern China. Extended families of both cases and control patients were analyzed for both HBV status and liver cancer. As expected, HBV infection was associated with an approximately 26-fold increased risk for liver cancer within this population. A significantly increased risk was also observed among the parents and siblings of the patients with liver cancer, even when the HBV status was taken into account. The best genetic model for this familial clustering postulated that a single recessive allele, with a population frequency of 0.25, contributed to cancer risk. Among HBV infected men, lifetime risks for liver cancer development of 0.84 and 0.09 were predicted for the genetically susceptible and resistant populations, respectively. A similar familial clustering was observed in a study of the Alaskan Native population, in which the incidences of both HBV infection and primary liver cancer are higher than among the U.S. general population (Alberts et al., 1991; Buetow, 1992). HBV-positive individuals were identified through screening of the population and were followed for signs of liver cancer. Pedigree information was obtained for the first and second degree relatives of each patient developing liver cancer. Approximately one-third of the 47 cases identified in this study were found to cluster in five families. The median age at diagnosis in these families was younger than that for the remaining cases (22 versus 54 years). Buetow (1992) proposed that a single dominant allele could account for these results. HBV-infected carriers of the gene were predicted to have a lifetime risk in excess of 90% of developing liver cancer, in comparison to a 9% risk for those individuals infected with HBV and resistant in genotype. B. Genetic Diseases Associated with an Increased Risk for Liver Cancer

Several human genetic diseases are associated with the frequent development of hepatocellular adenomas or carcinomas. In most cases, the development of hepatocellular tumors is preceded by chronic liver damage or cirrhosis. The most frequent of these diseases, hereditary hemochromatosis (HFE), is characterized by increased intestinal absorption of iron and progressive iron overload. The disease shows autosomal recessive inheritance

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and has been linked to markers on chromosome 6p21 (Gasparini et al., 1993). Population based screening of prospective blood donors in Utah found that the frequency of HFE homozygotes was approximately 0.5%, indicating a mutant allele frequency of about 1 in 15 in the population (Edwards et al., 1988). If affected individuals are left untreated (by phlebotomy), iron accumulation damages a variety of organ systems, resulting in diabetes, cardiomyopathy, or hepatic cirrhosis (Niederau et al., 1985). Associated with the development of cirrhosis (Deugnier et al., 1993), HFE patients are at a 200-fold increased risk for hepatocellular carcinomas relative to the population as a whole (Niederau et al., 1985). Hereditary deficiency of Cxl-antitrypsin (AAT), the major inhibitor of neutrophil elastase, is an autosomal recessive disease that occurs with a frequency of approximately 1 in 2000 births in the United States (Crystal, 1989). This locus maps to chromosome 14q3 .~Cox et al., 1982) and a variety of mutant alleles have been described. The most frequent allele (PI Z) associated with disease often results in the development of emphysema in homozygotes by age 40 (reviewed in Crystal, 1989). Approximately 10% of ZZ homozygotes develop cirrhosis as a consequence of the accumulation of AAT in the hepatic endoplasmic reticulum (Perlmutter et al., 1985); these individuals are at a greatly increased risk for the development of hepatocellular carcinoma (Eriksson et al., 1986). Wu et al. (1994) have shown that overexpression of the Z mutant protein in fibroblasts from ZZ homozygotes resulted in slower intracellular degradation of the protein when the cells were isolated from individuals with liver disease when compared with cells from individuals without liver disease. Thus, the increased risk for liver cancer may result from the interaction of the AAT mutation and other polymorphic genes. Type I tyrosinemia results from a defect in the fumarylacetoacetase gene, which maps to chromosome 15q23-q25 (Phaneuf et al., 1991). This autosomal recessive disease is rare in the general population (1 per 10,000 births) but is five fold more frequent in a particular region of Quebec (De Braekeleer and Larochelle, 1990). Severe kidney and liver damage occurs in affected individuals either acutely or chronically, depending on the mutant allele. Cirrhosis in these patients precedes the frequent development of hepatocellular carcinoma early in adolescence (Dehner et al., 1989). Recessive mutations in the glucose-6-phosphatase gene result in Type I glycogen storage disease, leading to severe hypoglycemia, hepatomegaly, and short stature (Nordlie et al., 1993). Pathogenesis of liver cancer in these patients differs from that in the preceding three diseases in that chronic liver damage and cirrhosis are not generally observed. By age 20, most patients develop hepatic adenomas, with subsequent development of hepatocellular carcinoma in a minority of individuals (Bianchi, 1993).

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III. Genetics of Experimental Liver Cancer Liver tumor induction in the mouse and rat closely follows the paradigm of multistage carcinogenesis first established in mouse skin (Drinkwater, 1990). Initiation of carcinogenesis is thought to result from the induction of a mutation in a critical cellular gene. The promotion stage of carcinogenesis is complex, representing a composite of events involving alteration in gene expression, the proliferation and clonal expansion of the initiated cell, and accumulation of additional genetic alterations (see Chapters 6 to 10). Genetic approaches to understanding the nature and regulation of the events associated with these two stages of carcinogenesis include comparative studies of inbred animal strains, linkage analysis to identify cancer susceptibility genes, and analysis of carcinogenesis in animals that carry specific mutations.

A. Variation among Inbred Strains The first inbred strains of mice ~were developed early in the century with the aim of understanding the genetic basis for the development of cancer (Morse, 1981). Studies of both spontaneous and chemically induced tumors in mice and rats have demonstrated that there is considerable variation among inbred strains in their susceptibilities to carcinogenesis (Drinkwater and Bennett, 1991). For a variety of tissue sites, including the lung, liver, mammary gland, and skin, the range of susceptibilities among inbred strains is as great as 100-fold. These strain differences provide direct evidence that the genetic background of the host plays a critical role in determining cancer susceptibility. The cancer susceptibility genes that determine the differences in susceptibility between inbred strains can be identified through a "reverse genetic" approach. The inheritance of susceptibility to tumor induction is studied in crosses between sensitive and resistant animals and the chromosomal location of the gene is identified by linkage analysis. Once the location of the gene is known, detailed genetic and physical maps of the region can be constructed. From this information, candidate genes in the appropriate regions can be identified and evaluated for their abilities to confer the phenotype of interest. This general approach has been used successfully to isolate the genes responsible for several human diseases (Monaco et al., 1986; Rommens et al., 1989). Although the first application of these methods represented extraordinary efforts, recent developments will facilitate greatly the use of a reverse genetic approach to isolate murine cancer susceptibility genes. A critical requirement for approaches based on gene mapping is the

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availability of a large number of informative genetic polymorphisms, whose location in the genome is known. Within the last 2 years, a dense genetic map of the mouse genome based on simple sequence length polymorphisms (SSLP) has emerged as a powerful tool for genetic mapping (Dietrich et al., 1992). These markers are based on repeated dinucleotide sequences, such as (CA)n , that are scattered r a n d o m l y t h r o u g h o u t the genomes of mice and other vertebrates. This set of markers has distinct advantages compared to more conventional restriction fragment length polymorphisms or isozyme markers, including the fact that (a) they are highly polymorphic among standard inbred mouse strains; (b) they can be analyzed readily by use of the polymerase chain reaction; and (c) the c h r o m o s o m a l location of more than 5000 SSLP markers are known, providing a high density linkage map for the mouse (Copeland et al., 1993; Dietrich et al., 1994). 1. S p o n t a n e o u s a n d P e r i n a t a l H e p a t o c a r c i n o g e n e s i s

Inbred mouse strains vary widely in their susceptibilities to both spontaneous and chemically induced hepatocarcinogenesis. The incidences of spontaneous liver tumors and t u m o r yields for male mice treated as preweanlings with N,N-diethylnitrosamine (DEN) are compared in Table 1. The range of variation among strains exceeds 20-fold for spontaneous liver tumors and is nearly 100-fold for DEN-induced hepatocarcinogenesis. In general, there is a good correlation between these two measures of suscep-

Table I Strain Variation in Susceptibility to Spontaneous and Chemically

Induced Hepatocarcinogenesis in Male Mice Spontaneous liver tumorsa

DEN-induced liver tumorsb

Strain

Number of mice

Incidence (%)

Number of m i c e

Mean tumor multiplicity (sd)c

C3H/He DBA/2J C57BR/cdJ BALB/c C57BL/6J SWR

61 67 48 637 609 269

51 1.5 25 3.9 3.6 1.1

22 23 32 23 23 24

78 (30) 55 (20) 37 (23) 9 (6) 1.4 (1.6) 0.8 (1.3)

aData on lifetime spontaneous incidences of liver tumors taken from Smith and Walford (1978), Frith and Wiley (1982), Storer (1966), Rabstein et al. (1973), and Smith et aI. (1973). 6Data from unpublished experiments by M. Bennett, T. Poole, M. Winkler, and N. Drinkwater. Male mice were treated at 12 days of age with diethylnitrosamine (0.1 mmol/g body weight) and sacrificed at 32 weeks of age. cValues in the table are mean tumor multiplicity (standard deviation).

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tibility and the sensitivities of the strains to other carcinogen treatment protocols (Drinkwater, 1989). The genetic basis for the high susceptibility of three strains, C3H, DBA/2, and C57BR, have been studied in detail, as described below. a. C 3 H Mice. The high susceptibilities of the C3H and closely related CBA mouse to liver cancer development were first noted more than 40 years ago (Andervont, 1950). The genetics and biology of hepatocarcinogenesis in the susceptible C3H mouse have been studied extensively by several laboratories. We have studied the genetic basis for the high susceptibility of C3H/HeJ male mice relative to C57BL/6J male mice by segregation analysis and by comparison of recombinant inbred (RI) strains derived from these two inbred strains (Drinkwater and Ginsler, 1986). The simplest genetic model that could account for the results of these experiments postulated at least two independent loci, one of which accounted for a majority of the 50-fold difference in susceptibility between the two inbred parental strains. The major locus, designated as Hcs ( H e p a t o c a r c i n o g e n sensitivity), is autosomal and the C3H/HeJ and C57BL/6J alleles are semidominant, such that the heterozygous B6C3F 1 mouse is intermediate in sensitivity to the two parental strains. Comparison of the strain distribution patterns for chemically induced and spontaneous hepatocarcinogenesis in male BXH RI mice (G. H. Lee and N. Drinkwater, submitted for publication) demonstrated a nearly perfect concordance between these two phenotypes, indicating that the same locus influences both traits. The high susceptibility of the C3H male mouse is largely mediated through effects on the growth rate of preneoplastic hepatic lesions. Preneoplastic hepatic lesions in carcinogen-treated mice can be identified histochemically as basophilic or glucose-6-phosphatase-deficient hepatic foci. Several studies have demonstrated that these altered foci grew more rapidly in susceptible C3H or B6C3F 1 male mice than in resistant C57BL/6, A, BALB/c, or (C57BL/6xBALB/c)F 1 male mice (Dragani et al., 1987; Dragani et al., 1991a; Hanigan et al., 1988; Kakizoe et al., 1989; Pugh and Goldfarb, 1992). Studies of spontaneous (Condamine et al., 1971) and chemically induced (Lee et al., 1991a) hepatocarcinogenesis in chimeric mice, derived by fusion of C3H and C57BL/6 embryos, demonstrated that the susceptibility phenotype was cell autonomous, i.e., it was expressed at the level of the target hepatocyte. In studies by Lee et al. (1991a), in which C3H-C57BL/6 chimeric mice were treated as preweanlings with DEN, the preneoplastic hepatic lesions of C3H origin were five fold larger than the lesions in the same livers derived from C57BL/6 cells. The same gene(s) may also play an important role in the growth regulation of normal hepatocytes. The response of C3H/HeJ mice to the growth stimulus of partial hepatectomy was significantly greater than that for C57BL/6 mice as determined by

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the level of DNA synthesis and the expression of the E2F1 and dhfr genes at the G1/S boundary of the first cell cycle after hepatectomy (Bennett et al., 1995). The C3H susceptibility genes may preferentially influence the growth of hepatic lesions containing initiating mutations in the H-ras I locus. A high proportion of liver tumors induced in C3H or B6C3F 1 mice carried mutant H-rasl genes, while such mutations were infrequent in tumors from C57BL/6, BALB/c, or (C57BL/6 x BALB/c)F 1 mice (Buchmann et al., 1991; Dragani et al., 1991b). Two groups have attempted to identify liver tumor susceptibility genes carried by the C3H mouse through linkage analysis. Gariboldi et al. (1993) treated preweanling male and female mice from the F2 generation of a cross between C3H/He and A/J mice with ethyl carbamate and analyzed the animals for the volume fraction of the liver occupied by tumors and for 83 genetic markers. Their results indicated that three chromosomal regions, near the markers D 7 N d s l (chromosome 7), D8Mit14 (chromosome 8), and Odc8 (chromosome 12), together accounted for approximately 40% of the more rapid growth of the liver tumors in animals inheriting alleles from the C3H parent. These susceptibility genes were designated Hcsl, -2, and-3. It is difficult, however, to evaluate the strength of these linkage assignments from the data provided in the paper. Recently, the same group (Manenti et al., 1994) performed a linkage analysis in an interspecific backcross, (C3H x Mus spretus)F 1 x C57BL/6. In this experiment, 106 male and female mice were treated perinatally with ethyl carbamate and analyzed for tumor multiplicity, tumor volume, and 222 genetic markers. A region of distal chromosome 2 (near D2Mit25) demonstrated significant linkage to liver tumor susceptibility and the authors designated this sensitivity locus Hcs4. The LOD score, a measure of the statistical significance of a linkage assignment, was 3.7 (i.e., the region is 1037 = 5011 times more likely to carry a susceptibility gene than elsewhere in the genome), well above the significance threshold of 3.3 (Lander and Botstein, 1989). Two additional susceptibility loci, Hcs5 and -6, were suggested to occur on chromosomes 5 (near D5Mit6) and 19 (near D19Mit27). However, the LOD scores for these assignments were 2.3 and 2.8, respectively. The Hcs locus was originally defined in crosses between sensitive C3H/HeJ and resistant C57BL/6J mice. We have analyzed 58 B6C3F 1 x C57BL/6 backcross and 57 B6C3F 2 intercross male mice for their susceptibilities to perinatal liver tumor induction by DEN and for 94 genetic markers (Bennett et al., 1993). Inheritance of C3H alleles for markers on chromosome 1, near the locus D I M i t 1 4 , was correlated with increased tumor yield, with a LOD score of approximately 5.9 (significance level p < 0.002). In the F2 cross, mice homozygous for the C3H alleles of these markers had a six fold higher yield of tumors than animals homozygous for the C57BL/6 alleles. No other region of the genome was significantly associated with increased tumor multiplicity.

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Based on these three studies, the genetic basis for the high susceptibility of the C3H mouse to hepatocarcinogenesis may be more complex than originally appreciated. The specific genes identified by linkage analysis depended on the resistant strain used in the segregation analysis, the sex of the animals, and the susceptibility phenotype that was assayed. Development of congenic strains for each of the proposed susceptibility loci, in which the C3H susceptibility allele is carried on a standard, resistant genetic background (e.g., C57BL/6), will allow the detailed characterization of the independent effects of each of these loci on hepatocarcinogenesis. b. D B A / 2 Mice. The DBA/2 mouse is an exception to the general correlation between susceptibility to spontaneous liver tumor development and sensitivity to the induction of tumors by perinatal treatment with chemical carcinogens (Table 1). While male mice of this strain have a low spontaneous incidence of liver tumors (Smith et al., 1973), they are 70% as sensitive as C3H/HeJ mice to DEN-induced hepatocarcinogenesis and 20-fold more sensitive than C57BL/6 mice. In contrast, carcinogen treatment of DBA/2 mice at older ages, when postnatal proliferation of hepatocytes has ceased, results in a low yield of tumors, similar to that observed for the C57BL/6 mouse (Diwan et al., 1986). The high susceptibility of DBA/2 mice to liver tumor induction by treatment of preweanling animals with carcinogen is related to the influence of this genetic background on the growth of preneoplastic lesions. Comparative studies of the development of glucose-6-phosphatase-deficient hepatic loci in male mice treated at 12 days of age with DEN revealed that the apparent growth rate of the hepatic foci in DBA/2 mice was similar to that observed for C3H mice, and significantly greater than the growth rate of foci in C57BL/6 mice (Bennett et al., 1992). However, the numbers of preneoplastic lesions observed in DBA/2 and C57BL/6 mice were similar and were significantly lower than that for C3H/HeJ mice. The genetic basis for the high susceptibility of DBA/2 mice to hepatocarcinogenesis is complex (Lee et al., 1995). Segregation of susceptibility to tumor induction was studied in 71 backcross (D2B6F 1 x C57BL/6) and 46 intercross (D2B6F2) mice. Analysis of these animals for 100 genetic markers resulted in the identification of two chromosomal regions, on chromosomes 4 (near D4Mit31) and 10 (near D10Mit15), for which inheritance of DBA/2 alleles resulted in the dominant suppression of susceptibility. The LOD scores for these two linkage assignments (5.3 and 4.3, respectively) were highly significant and the two resistance loci were designated Hcrl (Hepatocarcinogen resistance) and Hcr2, respectively. These results present a paradox, in that no genes for which the DBA/2 alleles conferred susceptibility could be mapped, in spite of the 20-fold greater sensitivity of this strain relative to C57BL/6 mice and the fact that more than 95 % of the mouse genome was tested for linkage. The best model

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to explain these results postulates that the DBA/2 mouse carries multiple sensitivity genes, each with a relatively small individual effect, in addition to the two resistance loci. Support for this model was obtained by analyzing a series of 23 recombinant inbred (RI) strains derived from DBA/2 and C57BL/6 mice (Lee et al., 1995). Consistent with a polygenic model for cancer susceptibility, three of the strains showed sensitivity phenotypes that were more extreme than either parental strain while the remaining strains demonstrated variable intermediate phenotypes.

c. CS7BR/cdJ Mice. Hepatocarcinogenesis in mice is strongly affected by sex hormones. Male mice are significantly more susceptible to liver tumor induction because of the contrasting effects of male and female sex hormones on hepatocarcinogenesis; androgens promote and ovarian hormones inhibit liver tumor development through effects on the growth rate of preneoplastic lesions (Vesselinovitch et al., 1980, 1982; Hanigan et al., 1988; Kemp et al., 1989). Female mice are generally quite resistant to hepatocarcinogenesis because of the inhibiting effects of ovarian hormones on liver tumor induction. Although C3H females are three times more susceptible than C57BL/6 females, they are only 3% as sensitive as C3H males. The range of sensitivities in females, albeit narrow, follows a similar pattern to that seen in the males with the exception of the C57BR/cdJ mouse in which the females are highly sensitive while the males are only intermediately susceptible (Kemp and Drinkwater, 1989; Poole et al., 1993). C57BR females are 15-30 times more sensitive than females of any other strain. The high susceptibility of the females of this strain results from a failure of the ovarian hormones to inhibit hepatocarcinogenesis. Ovariectomy of C3H or C57BL/6 female mice 6 weeks after treatment with DEN at 12 days of age, resulted in an eightfold increase in the yield of tumors relative to intact females, but only a 25 % increase in tumor yield for C57BR/cdJ mice (Poole et al., 1993). In intact female mice, preneoplastic hepatic lesions demonstrated a significantly more rapid growth rate than those in C57BL/6 females (Poole et al., 1993). To determine the genetic basis for the hepatocarcinogen sensitivity of the BR female mouse, we analyzed segregating crosses between C57BR and C57BL/6 mice (Poole et al., 1994). Male and female mice from a backcross (B6BRF 1 • C57BL/6; 114 animals) and intercross (B6BRF2; 135 animals) were analyzed independently for their susceptibility to tumor induction and 70 genetic markers. Inheritance of C57BR alleles at two chromosomal regions, chromosomes 17 (near locus D17Nds2, LOD score = 15.8) and 1 (near D i M i t l O , LOD score = 6.2) was correlated with high liver tumor multiplicity in both sexes. We have designated the chromosome 17 and the chromosome 1 gene Hcfl and Hcf2, respectively, based on their effects on "hepatocarcinogenesis in females." In female mice, these two genes have a

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synergistic effect on tumor development. Mice that carry C57BR alleles at both loci are 12-fold more sensitive than mice with only C57BL/6 alleles present; together Hcfl and Hcf2 account for nearly all of the difference in sensitivity between the female mice of the two strains. The same two loci act additively to determine the moderate susceptibility of C57BR male mice. 2. Genetic Control of Tumor Promotion A wide variety of agents, including microsomal enzyme inducers, hypolipidemic agents, and steroid hormones, act as promoters of hepatocarcinogenesis in rats or mice (Moore and Kitagawa, 1986). Differences between inbred mouse strains in their responses to hepatic tumor promotion were first reported by Diwan and co-workers (1986). Chronic treatment with phenobarbital (PB), a strong promoter of hepatocarcinogenesis in the rat (Peraino et al., 1971), resulted in significant increases in the number of preneoplastic and neoplastic hepatic lesions in DEN-treated C3H/He and DBA/2, but not C57BL/6 mice. They also observed that serum levels of PB were higher in DBA/2 mice than in the other two strains, consistent with differences between the strains in the activity of the cytochrome P450 that metabolizes this drug (Nabeshima and Ho, 1981). In the absence of treatment with initiating agents, long-term feeding of PB induces liver tumors in C3H mice (Peraino et al., 1973; Ward et al., 1988) but is not carcinogenic to C57BL/6 mice (Becker, 1982). Phenobarbital also promotes the development of hepatoblastomas by a novel genetic mechanism. Treatment of D2B6F1 mice with a single injection of DEN followed by long-term treatment with PB results in the induction of a high incidence of hepatoblastomas by 47 weeks of age (Diwan et al., 1989). However, this type of tumor was rarely observed in similarly treated B6D2F1, C57BL/6, or DBA/2 mice. The difference between the two reciprocal F 1 hybrids in susceptibility may indicate a role for genome imprinting (Sapienza, 1990) in the development of these tumors. Lee and co-workers (1989a) compared C3H/He, BALB/c, and C57BL/6 mice for their sensitivities to promotion of hepatocarcinogenesis by PB, clofibrate, and ethynyl estradiol following DEN initiation. Consistent with earlier studies (Diwan et al., 1986), C3H mice were significantly more susceptible to the promoting effects of PB than were C57BL/6 mice. BALB/c mice were also found to be responsive to this tumor promoter. Preneoplastic hepatic lesions in PB-treated animals from the two sensitive strains were significantly larger than those observed in C57BL/6 mice. Only the C3H mice responded to promotion by clofibrate. Ethynyl estradiol, a promoter of hepatocarcinogenesis in rats (Cameron et al., 1982), inhibited liver tumor induction by DEN in all three strains. Strain variation in sensitivity to promotion by halogenated aromatic hy-

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drocarbons is largely controlled by the Ah locus (Polandet al., 1984), which encodes a specific receptor. Smith and co-workers (1990) have demonstrated a synergistic effect on liver tumor induction between treatment of C57BL/10 mice with iron-dextran and chronic feeding of polychlorinated or polybrominated biphenyls. Similarly treated DBA/2 mice, which carried a nonresponsive Ah allele, were resistant to hepatocarcinogenesis by this regimen. Subsequent studies revealed that genetic variation in sensitivity to iron-induced uroporphyria may also have contributed to the higher susceptibility of the C57BL/10 mice to hepatocarcinogenesis (Smith and Francis, 1993). 3. Transformation of Hepatocytes In Vitro

The ability to establish immortalized cell lines from murine hepatocytes occurs in a strain dependent manner. Long-term culture of primary hepatocytes from untreated, young adult C3H/He mice frequently resulted in the outgrowth of cell clones with the capacity to grow indefinitely in culture (Lee et al., 1989b). Addition of PB into the culture medium enhanced the frequency of immortalization several fold. Cultures of hepatocytes derived from C57BL/6 mice rarely gave rise to immortalized clones, in the presence or absence of PB. Cell lines established from the C3H hepatocytes were nontumorigenic in nude mice and did not contain transforming genes that could be detected by transfection of cellular DNA into NIH3T3 cells (Lee et al., 1991b). However, transformation of the cell lines by transfection with a mutationally activated H-rasl gene gave rise to tumorigenic lines that formed moderately differentiated hepatocellular carcinomas when injected into nude mice (Lee et al., 1991b). Yoshie and co-workers (1994) compared the frequencies of colony formation in cultures of primary hepatocytes from C3H, C57BL/6, and C3B6F 1 mice and observed that cells from the F 1 hybrid animal were as susceptible to in vitro transformation as cells from C3H mice, indicating that this phenotype is dominant. Recent studies have provided clues to the molecular basis for the high susceptibility of C3H hepatocytes to in vitro transformation. Karyotypic analysis of cell lines derived from C3H hepatocytes revealed that all of the lines contained monosomy or partial deletion of chromosome 4 (Nishimori et al., 1994). Furthermore, analysis of 20 hepatocyte cell lines from C3B6F 1 mice demonstrated that 95 % of the lines exhibited loss of heterozygosity for a region of chromosome 4 and that, in most cases, the allele from the C57BL/6 parent was lost. These results led the authors to propose that this region of chromosome 4 contained a suppressor gene, designated Lci (liver cell immortalization), for which the C57BL/6 allele was more active than the C3H allele (Nishimori et al., 1994). Detailed characterization of the F 1 hybrid cell lines for loss of heterozygosity allowed the assignment of the Lci

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locus to a 2-cm interval near the D 4 M i t 1 2 3 locus (G. H. Lee, K. Ogawa, H. Nishimori, and N. Drinkwater, submitted for publication). The homologous region of the human genome, chromosome l p, is often deleted or rearranged in hepatocellular carcinomas (Simon et al., 1991; Yeh et al., 1994). It is also of interest to note that this putative suppressor gene maps to the same region containing the H c r l locus, identified as a gene conferring resistance to liver tumor induction in DBA/2 mice (Lee et al., 1995).

B. Specific Mutations Affecting Hepatocarcinogenesis Studies of hepatocarcinogenesis in mouse or rat strains carrying specific mutations provide a direct approach to testing hypotheses regarding the role of those gene products in the development of liver cancer. Mutant alleles of particular genes have been shown to modulate the metabolism of chemical carcinogens, responsiveness to tumor promoters, and the growth or progression of preneoplastic lesions. The A locus (agouti, mouse chromosome 2) influences the microenvironment of hair follicles and thus regulates coat color (Silvers, 1979). This locus also has pleitropic effects on a variety of tissues and two alleles, Ay and Avy, increase susceptibility to spontaneous and chemically induced tumors of the skin, mammary gland, bladder, and liver in heterozygous animals (Heston and Vlahakis, 1961; Wolff et al., 1986). When carried on a susceptible C3H background (see Section III.A.l.a), these two mutant alleles result in a twoto four fold increase in both spontaneous and PB-induced liver tumors (Heston and Vlahakis, 1961, 1968; Wolff, 1970; Wolff et al., 1986). The progression of adenomas to carcinomas was enhanced in C57BL/6 mice carrying the Avy allele relative to wild type animals (Becker, 1986). Although the mechanism by which this locus affects hepatocarcinogenesis is unknown, it may be related to the alterations in growth or energy metabolism exhibited by mice carrying the Ay or Avy alleles (Wolff et al., 1986). The LEC rat spontaneously develops hepatitis by 4 months of age and a high incidence of hepatocellular carcinomas is observed among animals that survive past 12 months of age (Masuda et al., 1988). A single recessive locus was found to be responsible for the hereditary hepatitis (Yoshida et al., 1987). The hepatitis observed in LEC rats cosegregated with an abnormally high accumulation of copper in the liver (Li et al., 1991; Sone et al., 1992), features similar to the pathogenesis of Wilson disease in humans. Subsequent studies demonstrated that the LEC rat carries a deletion mutation in the A t p 7 b gene, which encodes a copper-transporting ATPase and is the same gene responsible for Wilson disease (Wu et al., 1994). Homozygous b m / b m (chromosome 19) mutant mice suffer from a disproportionate dwarfing and a reduction in the development of the long bones as a consequence of reduced levels of sulfated glycosaminoglycans

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(Sugahara and Schwartz, 1979). The primary defect in these mice is in the enzyme, adenylsulfate kinase, that converts adenosine-5'-phosphosulfate to 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (Sugahara and Schwartz, 1979). PAPS is also the cofactor for sulfotransferases involved in xenobiotic metabolism. The decreased tissue levels of PAPS in mutant mice can result in a reduction in the synthesis of electrophilic sulfuric acid esters for several proximate carcinogens. The Millers and their co-workers (Boberg et al., 1983; Lai et al., 1985; Delclos et al., 1986) have shown that treatment of bm/bm mutant mice with l'-hydroxysafrole, 4-aminoazobenzene, or Nhydroxy-2-acetylaminofluorene resulted in levels of hepatic DNA adducts and yields of liver tumors that were 10 to 20% of those observed in bm/+ or + / + littermates. As noted above (Section III.A.l.c), male mice are significantly more susceptible to liver tumor induction than female mice as a result of the contrasting effects on hepatocarcinogenesis of promotion by testosterone and inhibition by estrogen. The promoting effects of testosterone are regulated by the Tfm locus (X-chromosome). Male mice hemizygous for the Tfm mutation do not express androgen receptor mRNA or functional receptor protein at detectable levels (Attardi and Ohno, 1974; Gaspar et al., 1990; Charest et al., 1991). Mutant male mice (Tfm/Y) are resistant to liver tumor induction relative to wild type males even when the animals were treated chronically with supraphysiological doses of testosterone (Kemp et al., 1989). However, analysis of tumors induced in Tfm/+ female mice for androgen receptor activity demonstrated that testosterone promotes tumor development by an indirect mechanism. Equal numbers of tumors that were deficient or expressed androgen receptors were induced in these mosaic animals when they were treated with DEN, ovariectomized, and received repetitive treatments with testosterone (Kemp et al., 1989). These results indicate that the required receptor-hormone interaction does not take place in the target preneoplastic hepatocyte and implicates a second messenger produced in either the liver or another tissue as the direct stimulus for promotion by androgens.

IV. Conclusion Recent advances in our ability to dissect genetically complex interactions have led to the identification of several loci that play a role in the development of liver cancer. The next difficult step, proceeding from positional information to molecular identification, is essential if we are to understand the mechanisms by which these genes control liver cancer risk. Ultimately, the challenge will be to relate the function of these genes to the development of liver cancer in humans.

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Acknowledgments We are grateful to Sue Schadewald, Rey Carabeo, and Dr. Therese Poole for their helpful comments on the manuscript.

References Alberts, S. R., Lanier, A. R, McMahon, B. J., Harpster, A., Bulkow, L. R., Heyward, W. L., and Murray, C. (1991). Clustering of hepatocellular carcinoma in Alaska native families. Genet. Epidemiol. 8, 127-139. Andervont, H. B. (1950). Studies on the occurrence of spontaneous hepatomas in mice of strains C3H and CBA. J. Natl. Cancer Inst. 11, 581-592. Attardi, B., and Ohno, S. (1974). Cytosol androgen receptor from kidney of normal and testicular feminized (Tfm) mice. Cell 2, 205-212. Beasley, R. R (1988). Hepatitis B virus: The major etiology of hepatocellular carcinoma. Cancer 61, 1942-1956. Beasley, R. R, Hwang, L. Y., Lin, C. C., and Chien, C. S. (1981). Hepatocellular carcinoma: A prospective study of 22,707 men in Taiwan. Lancet 2, 1129-1133. Becker, E E (1982). Morphological classification of mouse liver tumors based on biological characteristics. Cancer Res. 42, 3918-3923. Becker, E E (1986). Progression of tumor histiotype during mouse hepatocarcinogenesis associated with the viable yellow (Avy) gene. Cancer Res. 46, 2241-2244 (1986). Bennett, L. M., Winkler, M. L., and Drinkwater, N. R. (1992). Analysis of the genetic basis for the high susceptibility of DBA/2 mice to liver tumor induction by N,N-diethylnitrosoamine. Proc. Am. Assoc. Cancer Res. 33, 164. Bennett, L. M., Winkler, M. L., and Drinkwater, N. R. (1993). A gene that determines the high susceptibility of the C3H/HeJ strain of mouse to liver tumor induction is located on chromosome one. Proc. Am. Assoc. Cancer Res. 34, 144. Bennett, L. M., Farnham, P. J., and Drinkwater, N. R. (1995). Strain-dependent differences in DNA synthesis and gene expression in the regenerating livers of C57BL/6J and C3H/HeJ mice. Mol. Carcinog., in press. Bianchi, L. (1993). Glycogen storage disease I and hepatocellular tumours. Eur. J. Pediatr. 152 (Suppl. 1), $63-$70, 1993. Boberg, E. W., Miller, E. C., Miller, J. A., Poland, A., and Liem, A. (1983). Strong evidence from studies with brachymorphic mice and pentachlorophenol that l'-sulfooxysafrole is the major ultimate electrophilic and carcinogenic metabolite of l'-hydroxysafrole in mouse liver. Cancer Res. 43, 5163-5173. Buchmann, A. R., Bauer-Hoffmann, Mahr, J., Drinkwater, N. R., Luz, A., and Schwartz, M. (1991). Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: Correlation with susceptibility to hepatocarcinogenesis. Proc. Natl. Acad. Sci. USA 88, 911-915. Buetow, K. H. (1992). Genetic studies of human primary hepatocellular carcinoma. Prog. Clin. Biol. Res. 376, 155-172. Cameron, R. G., Imaida, K., Tsuda, H., and Ito, N. (1982). Promotive effects of steroids and bile acids on hepatocarcinogenesis initiated by diethylnitrosamine. Cancer Res. 42, 24262428. Charest, N. J., Zhou, Z-X., Lubahn, D. B., Olsen, K. L., Wilson, E. M., and French, E S. (1991). A frameshift mutation destabilizes androgen receptor messenger RNA in the Tfm mouse. Mol. Endrocrinol. 5, 573-581.

316

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Condamine, H., Custer, R. P., and Mintz, B. (1971). Pure-strain and genetically mosaic liver tumors histochemically identified with the (beta)-glucoronidase marker in allophenic mice. Proc. Natl. Acad. Sci. USA 68, 2032-2036. Copeland, N. G., Gilbert, D. J. Jenkins, N. A., Nadeau, J. H., Eppig, J. T., Maltais, L. J., Miller, J. C., Dietrich, W. E, Steen, R. G., Lincoln, S. E., Weaver, A., Joyce, D. C., Merchant, M., Wessel, M., Katz, H., Stein, L. D., Reeve, M. P., Daly, M. J., Dredge, R. D., Marquis, A., Goodman, N., and Lander, E. S. (1993). Genome maps IV. Science 262, 57-66. Cox, D. W., Markovic, V. D., and Teshima, I. E. (1982). Genes for immunoglobulin heavy chains and for alpha-l-antitrypsin are localized to specific regions of chromosome 14q. Nature 297, 428-430. Crystal, R. G. (1989). The alpha-l-antitrypsin gene and its deficiency states. Trends Genet. 5, 411-417. De Braekeleer, M., and Larochelle, J. (1990). Genetic epidemiology of hereditary tyrosinemia in Quebec and in Saguenay-Lac-St-Jean. Am. J. Hum. Genet. 47, 302-307. Dehner, L. P., Snover, D. C., Sharp, H. L., Ascher, N., Nakhleh, R., and Day, D. L. (1989). Hereditary tyrosinemia type I (chronic form), pathologic findings in the liver. Hum. Pathol. 20, 149-158. Delclos, K. B., Miller, E. C., Miller, J. A., and Liem, A. (1986). Sulfuric acid esters as major ultimate electrophilic and carcinogenic metabolites of 4-aminoazobenzene and its N-methyl derivatives in infant male C57BL/6J x C3H/HeJ F 1 (B6C3F1) mice. Carcinogenesis 7, 277287. Deugnier, Y. M., Guyader, D., Crantock, L., Lopez, J.-M., Turlin, B., Yaouanq, J., Jouanolle, H., Campion, J.-P., Launois, B., Halliday, J. W., Powell, L. W., and Brissot, P. (1993). Primary liver cancer in genetic hemochromatosis: A clinical, pathological, and pathogenetic study of 54 cases. Gastroenterology 104, 228-234. DiBisceglie, A. M., Rustgi, V. K., Hooffnagle, J. H., Dusheiko, G. M., and Lotze, M. T. (1988). Hepatocellular carcinoma. Ann. Intern. Med. 108, 390-401. Dietrich, W., Katz, H., Lincoln, S. E., Shin, H-S., Friedman, J., Dracopoli, N. C., and Lander, E. S. (1992). A genetic map of the mouse suitable for typing intraspecific crosses. Genetics 131, 423-447. Dietrich, W. E, Miller, J. C., Steen, R. G., Merchant, M., Damron, D., Nahf, R., Gross, A., Joyce, D. C., Wessel, M., Dredge, R. D., Marquis, A., Stein, L. D., Goodman, N., Page, D. C., and Lander, E. S. (1994). A genetic map of the mouse with 4,006 simple sequence length polymorphisms. Nature Genet. 7, 220-225. Diwan, B. A., Rice, J. M., Ohshima, M., and Ward, J. M. (1986). Interstrain differences in susceptibility to liver carcinogenesis initiated by N-nitrosodiethylamine and its promotion by phenobarbital in C57BL/6NCr, C3H/HeNCrMTV- and DBA/2NCr mice. Carcinogenesis 7, 215-220. Diwan, B. A., Ward, J. M., and Rice, J. M. (1989). Promotion of malignant 'embryonal' liver tumors by phenobarbital: Increased incidence and shortened latency of hepatoblastomas in (DBA/2 x C57BL/6) F1 mice initiated with N-nitrodiethylamine. Carcinogenesis 10, 1345-1348. Dragani, T. A., Manenti, G., and Della Porta, G. (1987). Genetic susceptibility to murine hepatocarcinogenesis is associated with high growth rate of NDEA-initiated hepatoctyes. J. Cancer Res. Clin. Oncol. 113, 223-229. Dragani, T. A., Manenti, G., and DellaPorta, G. (1991a). Quantitative analysis of genetic susceptibility to liver and lung carcinogenesis in mice. Cancer Res. 51, 6299-6303. Dragani, T. A., Manenti, G., Colombo, B. M., Falvella, E S., Gariboldi, M., Pierotti, M. A., and Della Porta, G. (1991b). Incidence of mutations at codon 61 of the Ha-ras gene in liver tumors of mice genetically susceptible and resistant to hepatocarcinogenesis. Oncogene 6, 333-338. Drinkwater, N. R. (1989). Genetic control of hepatocarcinogenesis in inbred mice. In "Genes

12.

Genetic Susceptibility to Liver Cancer

317

and Signal Transduction in Multistage Carcinogenesis" (Colburn, N., Ed.), pp. 3-17. Dekker, New York. Drinkwater, N. R. (1990). Experimental models and biological mechanisms for tumor promotion. Cancer Cells 2, 8-14. Drinkwater, N. R., and Ginsler, J. J. (1986). Genetic control of hepatocarcinogenesis in C57BL/6J and C3H/HeJ inbred mice. Carcinogenesis 7, 1701-1707. Drinkwater, N. R., and Bennett, L. M. (1991). Genetic control of carcinogenesis in experimental animals. In "Modification of Tumor Development in Rodents" (Ito, N., Ed.), pp. 1-20. Karger A.G., Basel. Edwards, C. Q., Griffen, L. M., Goldgar, D., Drummond, C., Skolnick, M. H., and Kushner, J. P. (1988). Prevalence of hemochromatosis among 11,065 presumably healthy blood donors. N. Engl. J. Med. 318, 1355-1362. Eriksson, S., Carlson, J., and Velez, R. (1986). Risk of cirrhosis and primary liver cancer in alpha-l-antitrypsin deficiency. N. Engl. J. Med. 314, 736-739. Fernandez, E., La Vecchia, C., D'Avanzo, B., Negri, E., and Franceschi, S. (1994). Family history and the risk of liver, gallbladder, and pancreatic cancer. Cancer Epidemiol. Biomarkers Prev. 3, 209-212. Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, J. M., Albert, D. M., and Dryja, T. P. (1986). A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323, 643-646. Frith, C. H., and Wiley, L. (1982). Spontaneous hepatocellular neoplasms and hepatic hemangiosarcomas in several strains of mice. Lab. Anita. Sci. 32, 157-162. Futreal, P. A., Liu, Q., Shattuck-Eidens, D., Cochran, C., Harshman, K., Tavtigian, S., Bennett, L. M., Haugen-Strano, A., Swensen, J., Miki, Y., Eddington, K., McClure, M., Frye, C., Weaver-Feldhaus, J., Ding, W., Gholami, Z., Soderkvist, P., Terry, L., Jhanwar, S., Berchuck, A., Iglehart, J. D., Marks, J., Ballinger, D. G., Barrett, J. C., Skolnick, M. H., Kamb, A., and Wiseman, R. (1994). BRCA1 mutation in primary and ovarian carcinomas. Science 266, 120-122. Gariboldi, M., Manenti, G., Canzian, E, Falvella, E S., Pierotti, M. A., Della Porta, G., Binelli, G., and Dragani, T. A. (1993). Chromosome mapping of murine susceptibility loci to liver carcinogenesis. Cancer Res. 53,209-211. Gaspar, M. L., Meo, T., and Tosi, M. (1990). Structure and size distribution of the androgen receptor mRNA in wild-type and Tfm/Y mutant mice. Mol. Endocrinol. 4, 1600-1610. Gasparini, P., Borgato, L., Piperno, A., Girelli, D., Olivieri, O., Gottardi, E., Roetto, A., Dianzani, I., Fargion, S., Schinaia, G., Cappellini, G., Pignatti, P., Fiorelli, G., DeSandre, G., and Camaschella, C. (1993). Linkage analysis of 6p21 polymorphic markers and the hereditary hemochromatosis: Localization of the gene centromeric to HLA-E Hum. Mol. Genet. 2, 571-576. Hall, J. M., Lee, M. K., Newman, B., Morrow, J. E., Anderson, L. A., Huey, B., and King, M.C. (1990). Linkage of early-onset familial breast cancer to chromosome 17q21. Science 250, 1684-1689. Hanigan, M. H., Kemp, C. J., Ginsler, J. J., and Drinkwater, N. R. (1988). Rapid growth of preneoplastic lesions in hepatocarcinogen-sensitive C3H/HeJ male mice relative to C57BL/6J male mice. Carcinogenesis 9, 885-890. Heston, W. E., and Vlahakis, G. (1961). Influence of the Ay gene on mammary gland tumors, hepatomas, and normal growth in mice. J. Natl. Cancer Inst. 26, 969-983. Heston, W. E., and Vlahakis, G. (1968). C3H-AvymA high hepatoma and high mammary tumor strain of mice. J. Natl. Cancer Inst. 40, 1161-1166. Kakizoe, S., Goldfarb, S., and Pugh, T. D. (1989). Focal impairment of growth in hepatocellular neoplasms of C57BL/6 mice: A possible explanation for the strain's resistance to hepatocarcinogenesis. Cancer Res. 49, 3985-3989. Kemp, C. J., and Drinkwater, N. R. (1989). Genetic variation in liver tumor susceptibility,

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Norman R. Drinkwater and Gang-Hong Lee

plasma testosterone levels, and androgen receptor binding in sex inbred strains of mice. Cancer Res. 49, 5044-5047. Kemp, C. J., Leary, C. N., and Drinkwater, N. R. (1989). Promotion of murine hepatocarcinogenesis by testosterone is androgen receptor-dependent but not cell autonomous. Proc. Natl. Acad. Sci. USA 86, 7505-7509. Kinzler, K. W., Nilbert, M. C., Su, L., Vogelstein, B., Bryan, T. M., Levy, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M. S., Altschul, S. F., Horii, A., Ando, H., Miyoshi, Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991). Identification of FAP locus genes from chromosome 5q21. Science 253, 661-664. Lai, C.-C., Miller, J. A., Miller, E. C., and Liem, A. (1985). N-Sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-2-acetylaminofluorene in the livers of infant male C57BL/6J 3< C3H/HeJ F 1 (B6C3F1) mice. Carcinogenesis 6, 1037-1045. Lander, E. S., and Botstein, D. (1989). Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121, 185-199. Lee, G.-H., Nomura, K., and Kitagawa, T. (1989a). Comparative study of diethylnitrosoamineinitiated two-stage hepatocarcinogenesis in C3H, C57BL and BALB mice promoted by various hepatopromoters. Carcinogenesis 10, 2227-2230. Lee, G.-H., Sawada, N., Mochizuki, Y., Nomura, K., and Kitagawa, T. (1989b). Immortal epithelial cells of normal C3H mouse liver in culture: Possible precursor populations for spontaneous hepatocellular carcinoma. Cancer Res. 49, 403-409. Lee, G.-H., Nomura, K., Kanda, H., Kusakabe, M., Yoshiki, A., Sakakura, T., and Kitagawa, T. (1991a). Strain specific sensitivity to diethylnitrosamine-induced carcinogenesis is maintained in hepatocytes of C3H/HeN-C57BL/6N chimeric mice. Cancer Res. 51, 3257-3260. Lee, G.-H., Sakai, R., Nagao, M., and Kitagawa, T. (1991b). Role of activated C-H-ras oncogene in the induction and progression of immortal epithelial cell lines derived from normal C3H mice. Int. J. Cancer 47, 60-65. Lee, G.-H., Bennett, L. M., Carabeo, R. A., and Drinkwater, N. R. (1995). Identification of hepatocarcinogen-resistance genes in DBA/2 mice. Genetics 139, 387-395. Li, Y., Togashi, Y., Sato, S., Emoto, T., Kang, J., Takeichi, N., Kobayashi, H., Kojima, Y., Uno, Y., and Uchino, J. (1991 ). Spontaneous hepatic copper accumulation in Long-Evans Cinnamon rats with hereditary hepatitis: A model of Wilson's disease. J. Clin. Invest. 87, 18581861. Manenti, G., Binelli, G., Gariboldi, M., Canzian, E, DeGregorio, L., Falvella, E S., Dargani, T. A., and Pierotti, M. A. (1994). Multiple loci affect genetic predisposition to hepatocarcinogenesis in mice. Genomics 23, 118-124. Masuda, R., Yoshida, M. C., Sasaki, M., Dempo, K., and Mori, M. (1988). High susceptibility to hepatocellular carcinoma development in LEC rats with hereditary hepatitis. Jpn. J. Cancer Res. 79, 828-835. Miki, Y., and 44 others. (1994). A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266, 66-71. Monaco, A. P., Neve, R. L., Colletti-Feener, C., Bertelson, C. J., Kurnit, D. M., and Kunkel, L. M. (1986). Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature 323, 646-650. Moore, M. A., and Kitagawa, T. (1986). Hepatocarcinogenesis in the rat: The effect of promoters and carcinogens in vivo and in vitro. Int. Rev. Cytol. 101,125-173. Morse, H. C. (1981). The laboratory mousenA historical perspective. In "The Mouse in Biomedical Research" (Foster, H. L., Small, D. J., and Fox, J. G., Eds.), Vol. 1, pp. 1-16. Academic Press, New York. Nabeshima, T., and Ho, I. K. (1981). Pharmacological responses to pentobarbital in different strains of mice. J. Pharmacol. Exp. Ther. 216, 198-204.

12.

Genetic Susceptibility to Liver Cancer

319

Narod, S. A., Feunteun, J., Lynch, H. T., Watson, P., Conway, T., Lynch, J., and Lenoir, G. M. (1991). Familial breast-ovarian cancer locus on chromosome 17q12-q23. Lancet 338, 82-83. Newman, B., Austin, M. A., Lee, M., and King, M.-C. (1988). Inheritance of human breast cancer: Evidence for autosomal dominant transmission in high-risk families. Proc. Natl. Acad. Sci. USA 85, 3044-3048. Niederau, C., Fischer, R., Sonnenberg, A., Stremmel, W., Trampisch, H. J., and Stohmeyer, G. (1985). Survival and causes of death in cirrhotic and in noncirrhotic patients with primary hemochromatosis. N. Engl. J. Med. 313, 1256-1262. Nishimori, H., Ogawa, K., and Tateno, H. (1994). Frequent deletion in chromosome 4 and duplication of chromosome 15 in liver epithelial cells derived from long-term culture of C3H mouse hepatocytes. Int. J. Cancer 59, 108-113. Nordlie, R. C., Sukalski, K. A., and Johnson, W. T. (1993). Human microsomal glucose-6phosphatase system. Eur. J. Pediatr. 152, $2-$6. Peraino, C., Fry, R. J. M., and Staffeldt, E. (1971). Reduction and enhancement by phenobarbital of hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res. 31, 1506-1512. Peraino, C., Fry, R. J. M., and Staffeldt, E. (1973). Enhancement of spontaneous hepatic tumorigenesis in C3H mice by dietary phenobarbital. J. Natl. Cancer Inst. 51, 1349-1350. Perlmutter, D. H., Kay, R. M., Cole, E S., Rossing, T. H., Van Thiel, D. H., and Colten, H. R. (1985). The cellular defect in alpha 1-proteinase inhibitor (alpha 1-PI) deficiency is expressed in human monocytes and in Xenopus oocytes injected with human liver mRNA. Proc. Natl. Acad. Sci. USA 82, 6918-6921. Phaneuf, D., Labelle, Y., Berube, D., Arden, K., Cavenee, W., Gagne, R., and Tanguay, R. M. (1991). Cloning and expression of the cDNA encoding human fumarylacetoacetate hydrolase, the enzyme deficient in hereditary tyrosinemia: Assignment of the gene to chromosome 15. Am. J. Hum. Genet. 48, 525-535. Poland, A., Knutson, J. C., and Glover, E. (1984). Histologic changes produced by 2,3,7,8tetrachlorodibenzo-p-dioxin in the skin of mice carrying mutations that affect the integument. J. Invest. Dermatol. 83, 454-459. Poole, T. M., Winkler, M. L., Kaehler, D. A., and Drinkwater, N. R. (1993). C57BR/cdJ mice are genetically insensitive to inhibition of hepatocarcinogenesis by ovarian hormones. Proc. Am. Assoc. Cancer Res. 34, 255. Poole, T. M., Winkler, M. L., Kaehler, D. A., and Drinkwater, N. R. (1994). Two loci account for the sensitivity of male C57BR/cdJ mice to heptocarcinogenesis. Proc. Am. Assoc. Cancer Res. 35, 122. Pugh, T. D., and Goldfarb, S. (1992). Growth kinetics of microscopic hepatocellular neoplasms in carcinogen-resistant and carcinogen-sensitive strains of mice. Cancer Res. 52, 280-284. Qian, G. S., Ross, R. K., Yu, M. C., Yuan, J. M., Gao, Y. T., Henderson, B. E., Wogan, G. N., and Groopman, J. D. (1994). A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People's Republic of China. Cancer Epidemiol. Biomarkers Prey. 3, 3-10. Rabstein, L. S., Peters, R. L., and Spahn, G. J. (1973). Spontaneous tumors and pathologic lesions 'in SWR/J mice. J. Natl. Cancer Inst. 50, 751-758. Rommens, J. M., Iannuzzi, M. C., Kere, B.-S., Drumm, M. L., Melmer, G., Dean, M., Rozmahel, R., Cole, J. L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J. R., Tsui, L.-C., and Collins, E S. (1989). Identification of the cystic fibrosis gene: Chromosome walking and jumping. Science 245, 1059-1065. Rose, E. A., Glaser, T., Jones, C., Smith, C. L., Lewis, W. H., Call, K. M., Minden, M., Champagne, E., Bonetta, L., Yeger, H., and Housman, D. E. (1990). Complete physical map of the WAGR region of 11p13 localizes a candidate Wilm's tumor gene. Cell 60, 495508.

320

Norman R. Drinkwater and Gang-Hong Lee

Sapienza, C. (1990). Genome imprinting, cellular mosaicism, and carcinogenesis. Mol. Carcinog. 3, 118-121. Shen, E-M., Lee, M. K., Gong, H.-M., Cai, X.-Q., and King, M.-C. (1991). Complex segregation analysis of primary hepatocellular carcinoma in Chinese families: Interaction of inherited susceptibility and hepatitis B viral infection. Am. J. Hum. Genet. 49, 88-93. Silvers, W. K. (1979). The agouti and extension series of alleles, umbrous and sable. In "The Coat Colors of Mice" (Silvers, W. K., Ed.), pp. 6-23. Springer-Verlag, New York. Simon, D., Knowles, B. B., and Weith, A. (1991). Abnormalities of chromosome 1 and loss of heterozygosity in primary hepatomas. Oncogene 6, 765-770. Smith, A. G., and Francis, J. E. (1993). Genetic variation in iron-induced uroporphyria in mice. Biochem. J. 291, 29-35. Smith, A. G., Francis, J. E., and Carthew, P. (1990). Iron as a synergist for hepatocellular carcinoma induced by polychlorinated biphenyls in Ah-responsive C57BL/10ScSn mice. Carcinogenesis 11, 437-444. Smith, G. S., and Walford, R. L. (1978). Influence of H-2 and H-1 histocompatibility systems upon life span and spontaneous cancer incidences in congenic mice. Birth Defects Orig. Artic. Ser. 14, 281-312. Smith, G. S., Walford, R. L., and Mickey, M. R. (1973). Lifespan and incidence of cancer and other diseases in selected long-lived inbred mice and their F1 hybrids. J. Natl. Cancer Inst. 50, 1195-1213. Sone, H., Maeda, M., Gotoh, M., Wakabayashi, K., Ono, T., Yoshida, M. C., Takeichi, N., Mori, M., Hirohashi, S., Sugimura, T., and Nagao, M. (1992). Genetic linkage between copper accumulation and hepatitis/hepatoma development in LEC rats. Mol. Carcinog. 5, 199-204. Storer, J. B. (1966). Longevity and gross pathology at death in 22 inbred mouse strains. J. Gerontol. 21,404-409. Sugahara, K., and Schwartz, N. B. (1979). Defect in 3'-phosphoadenosine 5'-phosphosulfate formation in brachymorphic mice. Proc. Natl. Acad. Sci. USA 76, 6615-6618. Tsukuma, H., Hiyama, T., Tanaka, S., Nakao, M., Yabuuchi, T., Kitamura, T., Nakanishi, K., Fujimoto, I., Inoue, A., Yamazaki, H., and Kawashima, T. (1993). Risk factors for hepatocellular carcinoma among patients with chronic liver disease. N. Engl. J. Med. 328, 17971801. Vesselinovitch, S. D., Itze, L., Mihailovich, N., and Rao, K. V. N. (1980). Modifying role of partial hepatectomy and gonadectomy in ethylnitrosourea~ hepatocarcinogenesis. Cancer Res. 40, 1538-1542. Vesselinovitch, S. D., Mihailovich, N., Rao, K. V. N., and Goldfarb, S. (1982). Relevance of basophilic foci to promoting effect of sex hormones on hepatocarcinogenesis. In "Carcinogenesis" (Hecker, E., Fusenig, N., Kunz, W., Marks, E, and Thielmann, H., Eds.), Vol. 7, pp. 127-131. Raven Press, New York. Ward, J. M., Lynch, P., and Riggs, C. (1988). Rapid development of hepatocellular neoplasms in aging male C3H/HeNVr mice given phenobarbital. Cancer Lett. 39, 9-18. Wolff, G. L. (1970). Differential growth of hepatoma-susceptible liver induced by gene x genome interaction. Cancer Res. 30, 1722-1725. Wolff, G. L., Roberts, D. W., and Galbraith, D. B. (1986). Prenatal determination of obesity, tumor susceptibility, and coat color pattern in viable yellow (Avy/a) mice. J. Hered. 77, 151-158. Wu, J., Forbes, J. R., Chen, H. S., and Cox, D. W. (1994). The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene. Nature Genet. 7, 541-545. Wu, Y., Whitman, I., Molmenti, E., Moore, K., Hippenmeyer, P., and Perlmutter, D. H. (1994). A lag in intracellular degradation of mutant alpha-l-antitrypsin correlates with the liver

12.

Genetic Susceptibility to Liver Cancer

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disease phenotype in homozygous PiZZ alpha-l-antitrypsin deficiency. Proc. Natl. Acad. Sci. USA 91, 9104-9018.

Yeh, S-H., Chen, P. J., Chen, H. L., Lai, M. Y., Wang, C. C., and Chen, D. S. (1994). Frequent genetic alterations at the distal region of chromosome lp in human hepatocellular carcinomas. Cancer Res. 54, 4188-4192. Yoshida, M. C., Masuda, R., Sasaki, M., Takeichi, N., Kobayashi, H., Dempo, K., and Mori, M. (1987). New mutation causing hereditary hepatitis in the laboratory rat. J. Hered. 78, 361-365. Yoshie, M., Nishimori, H., Kadohawa, T., and Ogawa, K. (1994). Comparison of colonyforming ability primary hepatocyte cultivated from various mouse strains and their F1 hybrids. Trends Soc. Pathot. Jpn. 88, 157.

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13 Surgical Treatment of Hepatic Tumors and Its Molecular Basis Ravi S. Chari Department of Surgery Duke UniversityMedical Center Durham, North Carolina27710

R. Daniel Beauchamp Department of Surgery Vanderbilt University Nashville, Tennessee37240

I. Introduction For many years the liver has been a mysterious organ from a surgical standpoint. Even at the turn of the century, it was regarded as a noli m e tangere (do not touch me) organ, as its complex anatomy, overwhelming number of functions, and abundant blood supply were poorly understood. With the improved understanding of liver physiology and pathophysiology that has occurred over the last 20 years, surgeons have witnessed the acquisition of new surgical techniques, the establishment of far less restrictive indications for these surgical procedures, and the realization of acceptable morbidity and mortality rates. Currently, there are many surgical options available for the management of hepatic tumors. Furthermore, the increasing sophistication of radiological and other diagnostic techniques has given surgeons a number of alternatives in the diagnosis and treatment of patients with hepatobiliary disorders. This chapter reviews the basic features and techniques of hepatic surgery related to hepatic tumors and the current molecular basis on which these Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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practices are based. It is both important and intriguing to note that many of the practices in hepatic surgery are based on years of accumulated clinical experience rather than basic science observation. Therefore, most of the molecular properties of the liver that allow for modern hepatobiliary surgery to be successfully performed have yet to be defined.

II. Diagnosis of Surgical Liver Tumors The operative management of hepatic tumors begins with their recognition and proper preoperative diagnosis. Disorders of the liver, and the hepatobiliary system may give rise to a wide variety of signs and symptoms. Most of these have long been recognized: the Babylonians (ca. 2000 Bc) observed hepatomegaly, and also knew of a special relationship between the liver and mental function, and conjectured that the liver was the seat of the soul (Meyers and Jones, 1990). Skin changes, ascites, bleeding disorders, and the inanition of liver disease have also been appreciated for centuries as manifestations of liver disease (Hardy, 1990). Once there is clinical evidence of liver disease, further diagnostic investigation should be initiated. Today the widespread application of refined techniques of abdominal imaging has contributed greatly to the increased discovery of tumors of the liver that previously went undiagnosed, or were detected only when associated with dramatic clinical presentation (Jenkins et al., 1994). The increasing sophistication of diagnostic techniques has given surgeons a number of possibilities with respect to defining the underlying disorder. Table 1 (Chari and Meyers, 1993) summarizes the diagnostic tests, their applications and complications. The exact indications and use of each test are beyond the scope of this chapter; however, several tests are often used in conjunction to delineate operable and inoperable liver pathology.

A. Evaluation of Asymptomatic Liver Mass For most suspected liver masses ultrasound is a reasonable first choice for diagnosis since it differentiates between cystic and solid lesions. A cystic lesion by ultrasound is more suggestive of benign disease, and these include parasitic, pyogenic, or congenital lesions. A spherical cystic mass with welldefined margins, and no intraluminal septations or debris in an otherwise asymptomatic patient is almost certainly a developmental cyst. Multiple cystic lesions in an afebrile patient, with no abnormalities of the liver function tests, most probably represents polycystic liver disease, especially if concomitant cysts are found in the kidneys. If the patient has lived in a region endemic for echinococcal disease, and ultrasound shows evidence of multiple daughter cysts within a larger cyst, then echinococcal cyst is the

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Table I Diagnostic Modalities in Liver Disease Test Ultrasound

PTC

ERCP

Tech-99m DISIDA

CT

Arteriography

MRI

Major indication Screening test Noninvasive, inexpensive, safe Detect cystic lesions 80-90% accuracy Detects lesions 1-2 cm Intraoperative/intraductal ultrasound aid in biopsy Definition of hepatobiliary tree Therapeutic role: stents may be placed

Definition of hepatobiliary tree Performance of needle biopsy Performance of papillotomy Stone extraction Sometimes more sensitive than CT: FNH, cirrhosis, hepatoma Acute cholecystitis Hepatobiliary tree function Bile leak Biliary tree injury Liver imaging modality of choice CT AP most sensitive detection rate Cannot always determine resectability Can aid in biopsy Defines hepatic artery anatomy Diagnostic in hemangiomas Assess vascularity of tumor Possible embolization of arteries in hemangioma, tumors More sensitive than plain CT Multiplanar scanning Safe

Liver Biopsy

Pathological diagnosis possible

Laparoscopy

Assess resectability of tumors Biopsy under direct vision

Drawbacks/complications Gas in bowel interferes Operator dependent Difficult to interpret images

Biliary leakage Sepsis Hemorrhage Pneumothorax Pancreatitis Cholangitis Hemorrhage Replaced by CT Hepatic cell injury interferes with resolution

Inferior to ultrasound for demonstration of hepatobiliary anatomy, stones Risks of arteriography

Increased cost Longer time to scan Insensitive for metastatic disease Mortality of <0.2% Morbidity of 5-8% Needle tract seeding Misdiagnosis Sampling error Inexperience of users Cannot assess deep parenchymal lesions

PTC, percutaneous transhepatic cholangiogram; ERCP, endoscopic retrograde cholangiopancreatography; Tech-99m, technetium-99m; DISIDA, diisopropyl immuno diacetic acid; CT, computerized tomography; MRI, magnetic resonance imaging; FNH, focal nodular hyperplasia.

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leading diagnosis. Serological testing for this entity would clarify the issue, but aspiration of the cyst should be avoided, as a lethal anaphylactic response is possible. A cystic lesion with debris, or multiple loculations, in a patient with fever suggests pyogenic or amebic abscessmaspiration of "anchovy paste" liquid, or clinical improvement on metronidazol will support a diagnosis of the latter. If the ultrasound reveals a solid, or cystic mass with a solid component, a computerized tomography (CT) should be performedmthe first step should be to exclude a hemangioma. If there is still doubt after CT scan, angiography, or isotope scanning should be undertaken. The development of spiral CT angiography may supplant conventional angiography in the evaluation of liver masses. If the mass is not a hemangioma, then it probably represents a tumormprimary or metastatic. In general, magnetic resonance imaging (MRI) confers little benefit over CT scanning for identification of liver masses, but can occasionally demonstrate characteristics of hemangioma on T2-weighted imaging. In addition, MRI is helpful in displaying the relationship of liver masses and the hepatic vasculature, especially the hepatic veins. The most common solid primary lesions include hepatocellular carcinoma (HCC), hepatic adenoma, and focal nodular hyperplasia (FNH). The diagnosis of FNH may be made by biopsy, but if there is any doubt, the lesion should be removed. In reality, adenomas should all be resected, as there is a 10% chance of malignancy, and a risk of spontaneous hemorrhage. With respect to metastatic lesions, the workup should involve identification of the primary (if unknown), and preoperative evaluation for possible resection (i.e., if the primary tumor is of colorectal or carcinoid origin with no evidence of extrahepatic tumor).

III. Indications for Surgical Treatment of Liver Tumors The surgeon divides liver disease into two basic categories: operable and inoperable. Bridging the gap between these two extremes is transplantable liver disease. Table 2 outlines this new classification of liver disease (Chari and Meyers, 1993). When surgically treated diseases of the liver are considered, one thinks most commonly of liver masses. The following is a brief discussion of the more commonly encountered hepatic tumors.

A. Cystic Liver Disease There are three types of liver cysts: developmental, inflammatory, and neoplastic. Developmental cysts can be divided into parenchymal or biliary; other adjectives to describe biliary cysts are communicating and choledoch-

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Table 2 Surgical Classification of Liver Disease 1. Resectable liver disease Cystic disease Neoplastic disease Liver abscessmunusual types Hydatid disease Large hemangiomas Infarctions Carolli's disease Pseudotumors Hepatic stone disease 2. Disorders involving other surgical options Polycystic disease Unresectable tumors Liver abscess Portal hypertension Hemobilia Variceal bleeding Infections and infestations of the liver Ascites Ruptured hepatocellular carcinoma 3. Transplantable liver disease Chronic advanced liver disease Unresectable hepatic malignancy Fulminant hepatic failure Inborn errors of metabolism Budd-Chiari 4. Nonsurgical liver disease Disease inappropriate for surgery Patient inappropriate for surgery Overriding factors

al. Inflammatory cysts refers mainly to liver abscesses, and by definition are N O T true cysts as they lack an epithelial l i n i n g n t h e s e are distinct from infected liver cysts. Traumatic pseudocysts of the liver are very rare. Most hepatic parenchymal cysts probably develop from aberrant bile ducts. The ductal epithelium secretes water and electrolytes. Thus, a cyst is formed as fluid accumulates in this aberrant noncommunicating ductal tissue. Although cysts may appear solitary, they are usually multiple, and multilocular; the term "solitary" is useful nonetheless in the clinical setting to describe a dominant, single lesion predominantly in one portion of the liver, and to distinguish this entity from polycystic disease. Solitary liver cysts vary in size from a few millimeters to massive lesions involving the upper abdomen. Most are small and peripheral, but any part of the liver may be involved. The right lobe is more common than t h e left, and anterior and inferior

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location are slightly more common than posterior or superior. In the operating room, liver cysts are smooth, have a bluish hue, are ballotable from outside the liver capsule, and are covered by a thin layer of hepatic parenchyma. Unlike parasitic cysts, these are usually low pressure cysts, unless hemorrhage has occurred. The specific gravity is between 1.007, and 1.024, and unless superinfected, the gram stain is negative. Autopsy series report a 0.14 to 0.30% incidence, with a male to female ratio of 1:3. B. Neoplasms of the Liver Neoplasms of the liver continue to be a significant worldwide problem. Improved diagnostic modalities and increasing correlation between the clinical course and pathological features have resulted in progress in the identification, management, and prognosis of these tumors. 1. Benign Tumors

The nonbiliary tumors of the liver may be classified as: (1) hepatocellular in origin, (2) vascular in origin, and (3) other nonvascular tumors (Table 2). Characteristic features of most benign tumors can be ascertained by sequential radiological investigation, but malignancy may be difficult to exclude, and resection is often the optimal method to provide histological diagnosis and definitive treatment. The primary reasons to pursue operative management of benign tumors of the liver are: (1) inability to exclude malignancy, (2) risk of rupture or hemorrhage, and (3) incapacitating or debilitating symptoms. These specific operative indications will vary among individuals as well as by the nature of the tumor. a. Hepatic A d e n o m a : Hepatic adenoma, or benign hepatocellular neoplasms, can arise in otherwise normal livers where they appear as a focal abnormality or mass--well circumscribed, from 2 cm to more than 30 cm in diameter. Although uncommon, the incidence of hepatic adenoma has increased with usage of oral contraceptives--90% of patients with adenomas have a history of birth control pill usage (Baum et al., 1973). The annual incidence among oral contraceptive users appears to be 3 to 4 per 100,000 among those who have used it for more than 2 years. Pregnancy is reported to stimulate the growth of adenomas and the development of complications. The incidence of hepatic adenomas appears to depend on the duration of oral contraceptive use, dosage of drug, and the age over 30. Several case reports document regression of liver cell tumors after the discontinuation of birth control pills (Edmonson et al., 1977; Chistopherson and Mays, 1977). Development of hepatocellular carcinoma at the site of a regressing adenoma also has been reported (Kerlin et al., 1983; Neuberger et al., 1980). Hepatic adenomas also are associated with anabolic steroid use, diabetes,

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glycogen storage diseases (may have multiple adenomas), and iron overload. Pathologically, these tumors are usually solitary, round, and not encapsulated; a pseudocapsule can be formed by compression of surrounding hepatic parenchyma. Liver adenomatosis is defined as the presence of liver adenomas in otherwise normal hepatic parenchyma in patients without glycogen storage disease. Intraoperatively, adenomas often bulge from the surface of the liver; when the tumor is incised, the cut surface will be yellow or tan, and often has areas of hemorrhage and necrosis. The microscopic diagnosis of adenomas is based on finding uniform masses of benign-appearing hepatocytes, without ducts or portal triads. Adenomas are composed of broad sheets of hepatocytes, often loaded with glycogen. About 10% of surgically excised hepatic adenomas harbor loci of hepatocellular carcinoma (Gyoffy et al., 1989). Clinically, more than one-half of the patients with hepatic adenomas have abdominal pain. Some experience chronic or episodic mild upper abdominal pain; others may have repeated acute attacks of severe pain caused by hemorrhage into the tumor or adjacent liver. One-fourth to one-third of patients have a pain syndrome caused by rupture and hemoperitoneum, which frequently leads to shock (Flowers et al., 1990). The remainder of hepatic adenomas are found incidentally at laparotomy, or during radiological evaluation of another problem. The most common imaging studies performed are ultrasound and CT. While all of the imaging modalities may suggest a diagnosis, none has exact diagnostic features (Welch et al., 1985; Clouse, 1989). Ultrasound can demonstrate adenomas between 1 to 2 c m m characteristically, they can be hyperechoic, hypoechoic, or isoechoic. CT generally yields little in terms of additional diagnostic information. Both of these tests lack specificity. The liver scan (i.e., sulfur colloid Tech-99m) will show an adenoma as a cold spot, as there are no functioning biliary elements. Arteriography will show hypervascularity of the tumor, with regions of hypovascularity owing to areas of necrosis or hemorrhage; this test will also exclude a diagnosis of hemangioma. The best treatment option for suspected adenomas is operative, primarily because the diagnosis of hepatocellular carcinoma cannot be excluded with certainty in the preoperative evaluation. Secondly, as noted above, 10% of adenomas will harbor loci of hepatocellular carcinoma. The risk of spontaneous lethal hemorrhage is also not trivial. Oral contraceptive use should cease immediately with the discovery of a suspected hepatic neoplasm. For incidental adenomas found at laparotomy for other procedures, resection should be performed if the surgeon is skilled in hepatic resection and if other contraindications are absent. b. Focal N o d u l a r H y p e r p l a s i a : FNH is an unusual hepatic lesion, which like benign hepatic adenoma, is most frequently seen in women (Jenkins et al., 1994). The documented incidence of FNH is increasing,

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which is a reflection of the increased number of imaging procedures performed on the abdomen (Kerlin et al., 1983; Brady and Coit, 1990). Ninety percent of the lesions occur in women in the second and third decades. Unlike benign hepatic adenoma, however, no convincing evidence links oral contraceptive use to the development of FNH. Oral contraceptive use may foster growth of FNH, and a tendency for established FNH to bleed, but few cases of FNH have been reported to regress after cessation of the use of oral contraceptives. There is no evidence to link these lesions with primary liver cancer. Unlike adenomas, they seldom bleed. FNH can occur in any portion of the liver and in 12 to 13 % of the cases there are multiple lesions. FNH probably represents a hyperplastic response of the hepatic parenchyma to a preexisting arterial malformation rather than a true neoplasm. FNH consists of one or more grossly visible localized nodule(s) in an otherwise normal liver. The majority of tumors are less than 5 cm, with less than 5% greater than 10 cm in diameter. Grossly, FNH appears as a well-circumscribed lesion with a central scar having stellate radiationsmsometimes giving this tumor a nodular and umbilicated visage. It is usually lighter than the surrounding tissue. Microscopically, FNH consists of many normal hepatic cells mixed with bile ducts and ductules, divided by fibrous bands, or septa, into nodules (Jenkins et al., 1994). The fibrous septa contain numerous bile ducts, and a moderate, predominantly lymphocytic infiltration. There is usually no evidence of malignancy. Clinical symptoms are reported in only 10% of these patients; these usually consist of mild, chronic, intermittent abdominal pain. Hemorrhage as a cause for acute presentation is rare. As with adenomas, most imaging techniques cannot reliably establish the diagnosis of FNH. A central scar is considered a characteristic feature of FNH and may be seen by CT, ultrasound, MRI or angiography (Welch et al., 1985; Butch et al., 1986). The central scar is not completely diagnostic, however, as it can be falsely read in other lesions, including hemangioma, lymphoma, and fibrolamellar hepatoma. Arteriography is highly sensitive, but lacks specificity. The appropriate management of this tumor is again surgical if malignancy or adenoma remain as a possible diagnosis. The fibrolamellar variant of HCC has occasionally been mistaken for FNH, lending support to the principle of resection if the pathological diagnosis cannot otherwise be established unequivocally (Saul et al., 1987). FNH can be followed, however, if the diagnosis is secure. c. H e m a n g i o m a s : Cavernous hemangiomas are found in about 2 to 7% of all autopsied livers, making this the most common benign liver tumor, and is most frequently seen in patients in the 3rd to 5th decades of life (Ochsner and Halpert, 1958; Ishak and Robin, 1975). This lesion occurs

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in all age groups, and like benign adenoma, and FNH, it is more common in women. The pathology is similar to that of hemangiomas in other parts of the body. In the liver, there is usually one dominant mass, although they may be numerous and virtually replace the whole liver. The size is extremely variable, with the largest reported at more than 30 cm in diameter; most are less than 4 cm in diameter. Most hepatic hemangiomas produce no symptoms. Less than half of the tumors smaller than 4 cm in diameter are associated with the symptoms of feeling full or upper abdominal pain, believed to result from stretching of Glisson's capsule (Adams et al., 1970). Ten percent of patients with a clinically detected mass are febrile. Occasionally, these may rupture as a result of trauma. However, spontaneous rupture is rare, with only 29 cases reported in the literature to date (Yamamoto et al., 1991). Special note is made of some hepatic hemangiomas in children and infants where the course may be more aggressive than in adults. Rarely do children develop high output cardiac failure or thrombocytopenia from platelet trapping. As opposed to the previously mentioned benign tumors, radiological studies play an integral role in the definitive diagnosis of hemangiomas; occasionally, laparotomy is necessary to make the diagnosis. The ultrasound appearance of hemangioma is characteristic, but not pathognomonic: a well-defined, solitary, hyperechoic, homogeneous mass, with well-defined margins and posterior acoustic enhancements (Bruneton et al., 1983). Hemangiomas may occur in any lobe, and are usually subcapsular in locationmmost frequently in the posterior right lobe. As an independent study, ultrasound rarely is diagnostic for hemangioma, thus the diagnosis is made in conjunction with CT, MRI, or radionuclide imaging. Most hemangiomas have a benign course: the risk of spontaneous rupture, malignant conversion, or growth is negligible. Thus if a hemangioma is diagnosed by imaging studies with "classic" appearance, most should remain undisturbed. Indications for surgical intervention are: (1) intraperitoneal, or intrahepatic rupture of the lesion is suspected, (2) persistent and bothersome symptomatology such as fever or pain, (3) lesion is large enough to be considered at high risk for traumatic rupture (e.g., a palpable mass in an athlete), and (4) uncertain diagnosis. In the case of multiple lesions, resection should be limited to the one at risk or responsible for the problems. Morbidity and mortality rates for hepatic hemangioma resection remain low, and patients enjoy full recovery with normal life expectancy. 2. Primary Malignant Tumors a. Hepatocellular Carcinoma: HCC is the most prevalent malignant disease in the world killing up to 1.25 million people annually. Accordingly, it is also the most frequent malignant liver disease seen. Hepatocellu-

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lar carcinoma constitutes 90 to 95 % of primary liver cancers. In the western world, hepatoblastoma is the most common primary hepatic malignancy of young children. Within the United States, there is an increased death rate in the western, southern, and central states due to HCC. Asians in the United States have about eight times the risk of developing HCC as do Caucasians. HCC is 4 to 9 times more frequent in males, except in the group of patients with preexisting liver disease, where there is unity in incidence. Epidemiological and laboratory studies have firmly established a strong and specific association between hepatitis B virus (HBV) and HCC (see Chapter 6). The relative risk of developing HCC among HBV carriers is 9.7. Although HBV is the major predisposing factor for HCC worldwide, alcoholic cirrhosis appears to be the major predisposing factor for the development of HCC in the United States. There is also an increased incidence of HCC following hepatitis C infection (Farmer et al., 1994). The oral birth control pill has been implicated, but has not been proven to be a risk factor. There appears, however, to be a slight increase in the development of HCC, as well as an increased risk for benign adenomas which are associated with HCC in oral contraceptive users. HCC has also developed in males taking androgens or other anabolic steroids. Histological patterns of HCC have various classifications. The tumors have been described as trabecular ("sinusoidal"), pseudoglandular ("acinar"), solid ("compact"), scirrhous, clear-cell ("replacing"), giant-cell pseudocapsular and sarcomatous. One distinct pathological type that does seem to behave differently is the fibrolamellar variant of HCC. This variant usually occurs in noncirrhotic livers and is characterized microscopically by parallel bundles of collagen separating clusters of acidophilic hepatocytes. Two misconceptions about HCC are that it is rapid growing and that it is universally fatal. HCC is relatively slow growing as compared with other neoplasms such as colon cancer and bronchogenic carcinoma, and many cases result in cure if resected early. The tumor most commonly spreads locally through the vascular system, primarily through the portal vein branches, but also via the hepatic veins, and arteries and lymphatics. Occasionally, the tumor can grow into the hepatic vein and spread intraluminally into the inferior vena cava, and right atrium. Metastases are reported more frequently in the absence of cirrhosis, although this observation is debated. The lymph nodes most frequently involved are those of the portal hilum, retroperitoneum, mediastinum, and periaortic regions. The most common extrahepatic organ affected is the lung. Other common sites of involvement are the adrenal gland, bone marrow, and spleen. The most common symptoms of HCC are weakness, malaise, anorexia, upper abdominal pain, and weight loss. Jaundice is found infrequently

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(24%), and an abdominal mass is the chief complaint in 14% of patients; obstructive jaundice is the initial problem in 1 to 10% of patients. Physical findings in HCC depend primarily on the stage at the time of presentation. The most common finding is hepatomegaly, which occurs 50 to 98% of the time. The mean overall survival of patients with untreated hepatocellular carcinoma is generally reported to be between 3 to 4 months after symptoms appear. Chemotherapy rarely impacts on this surviorship, or length of survival. Arterial embolization without resection has reportedly increased the length of survival from time of diagnosis to 9.5 months. On the other hand, the average survival in patients who have undergone resection is 3 years. Liver resection is the only therapy that substantially prolongs survival. Therefore, exploratory surgery is recommended for all patients if there are no obvious contraindications. The decision to operate on a suspected tumor should be based on the likelihood of anatomic resectability, hepatic functional reserve, and comorbid diseases which may preclude a major operative procedure. The operative mortality has decreased from close to 20% before 1950 to less than 5% currently with the advent of newer surgical techniques, better anesthesia, and improved intraoperative and postoperative monitoring. Hepatic resection is performed on half of the patients undergoing laparotomy. Wedge resection is as effective as radical procedures if adequate margins can be achieved. Repeat hepatectomy has been advocated for patients with recurrent HCC (Shimada et al., 1994; Suenaga et al., 1994). Liver transplantation has been performed in otherwise unresectable cancers, with a tumor recurrence rates of 60% and 5-year survival between 20 and 40% (Makowka et al., 1989; Yokoyama et al., 1990); patients with fibrolamellar cancer generally have a better survival after transplantation. Current indications for transplantation in patients with minimal to moderate underlying hepatic dysfunction with HCC include those with endstage liver disease with no other comordities, no evidence of extrahepatic disease, patent portal vein, and a unilobar HCC less than 5 cm in diameter (Farmer et al., 1994). The role of transplantation in hepatic malignancy is discussed in detail later in this chapter. Alternatives to surgical resection include hepatic artery embolization, or intraoperative ligation, intra-arterial and systemic chemotherapy, targeted radiotherapy and chemotherapy, direct tumor injection, cryoablation, or a combination of these methods (Farmer et al., 1994; Steele, 1994). As discussed previously, the fibrolamellar variant of HCC has, within the past decade, emerged as a distinct clinical and pathological form. This tumor occurs in young patients with noncirrhotic livers and is associated with a more favorable prognosis than HCC. The distinguishing histological features are sheets of well-differentiated hepatocytes located between

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Table 3 Hepatocellular Carcinoma Compared to Fibrolamellar Carcinoma

Male to female Tumor Resectability Mean survival Cirrhosis Increased cx-fetoprotein Hepatitis B positive

Hepatocellular carcinoma 4 : 1-8:1 Multiple nodules Large, invasive tumors Less than 50% 3-4 months 77% 83 % 65%

Fibrolamellar 1 : 1-1:2 Well localized at time of diagnosis 50-75% 32-68 months 4% 7% 6%

lamellae of collagen and fibroblasts. This neoplasm accounts for 1 to 2% of all hepatocellular carcinomas, and 4 0 % of these are seen in patients less than 35 years old. A female sex predilection has been reported. Two-thirds of these tumors occur in the left lobe of the liver. Table 3 (Chari and Meyers, 1993) compares H C C and fibrolamellar carcinoma. b. Cholangiocarcinoma: Another type of primary liver cancer is cholangiocarcinoma, which appears to arise in a peripheral portion in hepatic parenchyma. The intrahepatic form of bile duct cancer is rare, but has some characteristic clinical features. The average age of patients is in the sixtiesmabout a decade later than HCC. The male-female ratio is 1.7:1. Chronic liver disease is rarely present, however, jaundice is more frequently encountered in this tumor than in HCC, but is not nearly as common as in extrahepatic bile duct carcinoma. The average survival time is 6.5 months. AFP is occasionally elevated. The pattern of metastasis is similar to that of HCC. Treatment is resection whenever possible (Bismuth et al., 1992; Schoenthaler et al., 1994; Sugiura et al., 1994). c. M a l i g n a n t M e s e n c h y m a l Tumors: Primary malignant mesenchymal tumors make up less than 2% of primary malignant hepatic tumors. Most of the tumors are sarcomas, and the prognosis varies with cell type and resectability. Most sarcomas are fast growing, and often fatal before surgical resection can be performed, but exceptions occur. 3. Metastatic Tumors

Metastatic cancer comprises the largest group of malignant tumors of the liver in the United States. The most frequent tumor to metastasize to the liver is bronchogenic carcinoma. Overall, approximately 40% of patients with a solid tumor will develop liver metastases, but exact totals and percentages of patients who develop metastasis are unavailable. The diagnosis

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of liver metastases, regardless of primary is associated with a dismal prognosis with most patients not surviving more than 3 years (Hughes et al., 1989; Wagner et al., 1984). From a surgical standpoint, the metastases of most concern are those from colorectal primaries. The mean survival time can be measured in months in colorectal carcinoma, and less in other visceral tumors metastatic to the liver. The factors responsible for the increased propensity of tumors to metastasize to the liver are poorly understood. It seems that the liver's natural functions as a filtering organ make it susceptible to deposition of embolic tumor cells. It is known that tumors which drain into the portal system contribute seven times as many tumors to the livers as compared to tumors arising outside of the portal drainage system. Growth factors present in the liver at high concentrations may also make the liver a fertile environment for metastatic tumor growth (see Chapter 2). Understanding that there is a great tendency for tumors to metastasize to the liver, many attempts have been made to define the subset of patients that are amenable to surgical resection with cure as the goal. Numerous reports throughout the last two decades have expounded on the role of resection in metastatic colorectal carcinoma, but for metastases from other primary sites, there is a lack of sufficient data to draw meaningful conclusions about the effectiveness of medical and surgical treatment. Thus, except for resectable metastases from colorectal primaries, resection of metastatic liver tumors should be considered experimental and should be done only in larger centers under protocol. The most important step in the surgical management of liver metastases is the detection of these tumors. The most common symptoms of hepatic metastatic disease are pain, ascites, jaundice, palpable mass, weight loss, inanition, fever, and vague abdominal complaints. Unfortunately, patients presenting with these complaints are usually manifesting advanced disease. Thus, symptomatology can N O T be relied on as an indicator of metastatic spread; rather, one must detect the disease before it becomes clinically overt. Close follow-up, with regularly scheduled laboratory tests such as alkaline phosphatase and carcino-embryonic antigen (CEA) should be performed to detect metastases. Elevations should be followed-up with imaging studies. Routine imaging after colorectal primary is controversial because of expense and lack of proven benefit; however, routine ultrasonography in experienced hands has the potential to detect lesions before elevation of biochemical markers occurs. The 5-year survival of patients with hepatic metastases undergoing resection is 25 to 30%. Factors found in retrospective studies that do not impact on the 5-year survival are listed in Table 4, and contraindications to resection are listed in Table 5. Some authors advocate using preoperative portal embolization to increase the number of patients eligible for operative resec-

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Table 4 Factors Not Influencing 5-Year Survival after Resection of Hepatic Metastases of Colorectal Cancer Age Sex Latency of primary Stage of primary at initial surgery Bilobar liver involvement Number of hepatic metastases (<3) Type of hepatic resection

tion (Kawasaki et al., 1994). At the time of surgery, a meticulous exploration should be performed to uncover extrahepatic disease or previously undetected liver involvement. Intraoperative ultrasound and nodal biopsy should be performed routinely. When synchronous liver lesions are found at the time of operation for colon resection, the hepatic lesion may be removed at the same setting, or at a second procedure. The decision to resect is based on the adequacy of the colon preparation, the magnitude of the principal procedure, the extent of the hepatic resection that would be involved, the general status of the patient, and adequacy with which extrahepatic disease has been excluded. Generally, it is recommended to delay simultaneous resection when the resection is extensive. The features of liver tumors are summarized in Table 6 (Chari and Meyers, 1993). The diagnostic steps and appropriate treatments described above for hepatic tumors are depicted schematically in Figure 1.

IV. Surgical A n a t o m y Historical concepts of hepatobiliary anatomy evolved from initial misinterpretations of ligamentous reflections of the peritoneum (i.e., the falciform ligament and its division of the "right" and "left" lobes of the liver) into the

Table 5 Factors Adversely Influencing 5-Year Survival after Resection of Hepatic Metastases Noncolorectal primary Evidence of extrahepatic metastases Positive margins at the time of resection

Table 6 Liver Tumor Features

Disorder

Epidemiology

Solitary cyst

0.14-0.30% F:M = 3 : l pt < 40 years

Adenoma

3-4 per 100,000 F>M 3rd to 5th decades Linked to oral contraceptive ? incidence F>M Not linked to OCP 2-7% F>M 3rd to 5th decades 1-7 per 100,000 M : F = 4:l to 8 : l Associated with cirrhosislHBV

Focal nodular hyperplasia Hemangioma

Hepatocellular carcinoma

Metastatic cancer 30% Synchronous (colorectal) lesions

Signs/symptom Fullness in abdomen Nausea Vomiting Hepatomegaly Abdominal pain in 50% 10% rupture Palpable mass in 30% Only 10% with symptoms Abdominal pain Painldiscomfort Hepatomegaly RUQ mass Weakness Malaise Upper abdominal pain Mass in RUQ Hepatomegaly Asymptomatic Symptomaticnonsurgical

Diagnostic modality of choice Ultrasound

Findings

Indication for operation

Operative management

Thin walls Smooth-contoured anechoic mass Variable size No definitive findings Single mass Hypovascular on arteriogram

Pain Rupturelbleeding Infection Diagnosis Rule out malignancy Risk of malignant transformation

No definitive findings Tumor often <5cm and solitary IV time-sequenced Vascular “puddling” CT arteriogram on arteriogram

Symptomatic Rule out malignancy Risk of rupture Symptomatic

Arterial ligation, or ernbolization

CT-IV contrast

No definitive finding

Resection for cure No metastasis No cirrhosis Pt in good health

Resect if possible Arterial ligation Tumor debridement

CT-portography

Hypovascular lesions

Colorectal primary No extrahepatic metastasis

Resect for margins >1 cm

UltrasoundlCT

UltrasoundlCT

Resection Roux-en-Y jejunal loop if fluid is bilious Resection If adenoma is found during laparotomy, resection Resection

Figure 1. Schematic for diagnosis and management of hepatic tumor. Most commonly, the diagnosis of an hepatic mass is made incidentally during abdominal ultrasonography. The other important initiation point is an abnormal laboratory test in follow-up for primary colorectal cancer. The algorithm outlines the diagnostic steps and the appropriate treatments. Please refer to text for details.

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modern understanding of the hepatic lobar. The lobar anatomy, as defined by the distribution of the major branches of the portal vein, seldom conforms to the topographical right and left lobes. Cantlie first reported in 1898 that the division of the right and left lobes of the liver was not the falciform ligament, but rather a line (Cantlie's line) passing through the fossa of the gall bladder and to the left of the inferior vena cava; this plane is also called the lobar fissure. Further investigation in the 1950s (Chari and Meyers, 1993) revealed information suggesting that each true lobe could be further subdivided into two segments: an anterior and posterior on the right, and the medial and lateral on the left (Figure 2A). Furthermore, these researchers also established congruity of the arterial, portal venous, and biliary duct branches with the four segments. Couinaud further divided each of the four segments into two, resulting in a total of eight subsegments: four on the right, three on the left, and one corresponding to the caudate lobemsegment I (Figure 2B). Segments II to IV constitute the anatomical left lobe of the liver, while segments V to VIII comprise the right. The caudate lobe is its own segment in the French nomenclature as described by Couinaud. Anatomically, the branches of the portal vein in the caudate are two in number, one arising from each the right and left portal branches. Thus, the caudate is actually part of both the true left and right lobes. The portal vein is formed by a confluence of the splenic and superior mesenteric veins, and provides threefourths of the total blood supply to the liver. The hepatic artery most commonly derives its origin from the celiac axis. Draining blood from the liver into the inferior vena cava are three major hepatic veins and multiple smaller veins along the posterior surface of the liver. The biliary drainage of the liver begins at the hepatocyte level, and these tributaries unite to form progressively larger channels.

A. Surgical Resection and Transplantation The techniques of resection used today have been developed within this century, and more specifically within the last 4 decades. Two kinds of resections can be performed: anatomical and nonanatomical. The anatomical resections include right and left lobectomies, right and left trisegmentectomies, left-lateral segmentectomy, and a subsegmentectomy. The leftlateral segmentectomy includes segments II and III; the right trisegmentectomy includes the right lobe and segment IV, while a left trisegmentectomy involves the resection of the left lobe and the anterior segments (segments V and VIII) of the right lobe (Figure 3). Bisegmentectomy of VI and V is performed in patients with cancer of the gallbladder or with a Klatskin tumor.

The indications for the procedure have become broadened with the advent of newer technologies, better antibiotics, and more stringent intra-

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Figure 2. (A) The American system divides the liver into two lobes determined by a plane through the gallbladder fossa and inferior vena cava. (B) The French segmental system divides the liver into eight segments determined by hepatic veins and portal anatomy. The caudate lobar represents segment 1. From Sugarbaker (1990)with permission.

operative and postoperative monitoring; in fact, the indications are still evolving! Fewer than 5 0 % of malignant primaries are amenable to surgical resection (fibrolamellar variant not withstanding), and the cure rates are reported at 10 to 4 0 % . The extreme r e s e c t i o n - - t r a n s p l a n t a t i o n m d o e s not improve these rates. Hepatic resection for colorectal primaries, with no evidence of extrahepatic metastases is associated with a 5-year survival between 20 and 34%. Resection of noncolonic primaries is generally not

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Figure 3.

In experienced hands, liver resection can be carried out along the lines of the French segmental system. This figure shows six major hepatic resections involving en bloc removal of multiple hepatic segments. (A) Right hepatic lobectomy, (B) right trisegmentectomy, (C) left hepatic lobectomy, (D) left trisegmentectomy, (E) middle hepatectomy for gallbladder or Klatskin tumor, and (F) transverse hepatectomy. Another classic resection not depicted is the left lateral segmentectomy, corresponding to segments 2 and 3. From Sugarbaker (1990) with permission.

recommended. Indications for surgical resection in benign, or suspected benign cysts or tumors, include: (1) presence of symptoms, (2) enlargement of the neoplasm, (3) uncertainty of the diagnosis (i.e., cannot reliably exclude malignancy), (4) a significant risk of malignant transformation, and (5) risk of complication (i.e., rupture or hemorrhage). Contraindications to liver resection in malignant disease are unresectability of the tumor (determined either preoperatively or intraoperatively), cirrhosis, and inoperability (i.e., patients with severe ascites, marked hyperbilirubinemia, severe coagulation defect all suggestive of compromise liver function). When there is doubt about the synthetic function of the liver, biopsy of the nontumorous liver before resection may allow more insight into the "resectability" of the tumor than a biopsy of the tumor itself. Resectability of an isolated liver lesion is determined by its proximity to the portal vein, hepatic arteries, hepatic veins, and vena cava. Invasion of the common duct is a relative contraindication. Current techniques allow the surgeon to perform resection that until two decades ago was thought to be impossible. Scoring systems have been established which can predict the safe limit of partial hepatectomy, and thereby, eliminate deaths related to excessive resection for patients with normal or injured livers (Yamanaka et al., 1994). Modern operative mortality is less than 1% in resection for benign pathology and less than 5 % for malignant

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tumor resection; primary HCC has a higher operative mortality compared to resection of metastatic disease. The highest mortality is reported in decreasing order, for left trisegmentectomies, right and left lobectomy, and left-lateral segmentectomy, and finally nonanatomical wedge resection. In the face of cirrhosis, the operative mortality can be increased as much as sevenfold. Results of liver transplantation for metastatic disease are generally poor, with only a few long-term survivors (Penn, 1991; Pichlmayr, 1988). In HCC, transplantation is widely accepted in patients with centrally located tumors, tumors unresectable by conventional techniques but confined to the liver, multifocal tumors in more than one lobe, and patients with limited hepatic reserve (Farmer et al., 1994). Transplantation in patients with colorectal, breast, and other nonendocrine breast cancers has resulted in short disease-free intervals, and in only a few patients was palliation achieved (Penn, 1991; Pichlmayr, 1988; Margreiter, 1986). Given the critical shortage of donor organs, liver transplantation for metastatic disease should be reserved for patients with symptomatic neuroendocrine tumors in whom long-term survival is possible (Makowka et al., 1994; Alsina et al., 1990; Bussutil et al., 1994).

V. Hepatic Regeneration after Resection and Transplantation" Current Clinical Concepts Clinically, the regenerative capacity of the liver is well known and is typically triggered by partial hepatectomy (Higgins and Anderson, 1931; Schaffner, 1991). Heterotopic liver transplantation (HLT) has also been reported to induce hepatic regeneration in the hepatic remnant (Moritz et al., 1993) as well as the transplanted liver (Lee et al., 1968; Chandler et al., 1971; Fisher et al., 1971). Mitosis has also been reported in iso- and allotransplantation models of small-for-size whole or reduced orthotopic liver transplantation (OLT) (Rossi et al., 1987; Ishikura et al., 1987). In middle-aged patients, the liver will regenerate to a volume of 25 +_ 1.2 mL/kg (Van Thiel et al., 1985). Extensive work in rat models suggests that after partial hepatectomy the transplanted liver proliferates to reestablish an adequate liver mass:body weight ratio (Conn, 1989). The exact time course for the regenerative phase in humans is lacking. Van Thiel et al. (1987) have previously Figure 4. (A) Computerized tomogram of the liver, in a patient with an abscess of the right lobe of the liver (arrows). The right lobe was resected surgically. (B) Six weeks after surgery, there is almost complete regeneration of the liver. (Courtesy of Dr. William C. Meyers, Duke University Medical Center)

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presented clinical data on two patients undergoing OLT from donors on an average 10 kg smaller than the recipients, which resulted in livers 29 and 59% smaller than expected had the donor liver and recipient been ideally matched. Serial CT scans after transplantation revealed an average increase of 70 ml/day of liver volume, until achievement of a liver volume consistent with that expected for the recipients size, age, or sex was achieved. In the nontransplant setting, this enlargement to estimated mass, but not shape, has occurred in 2 to 3 weeks (Lin and Chen, 1965; Zoli et al., 1986). Interestingly, while plasma levels of amino acids, glucagon, insulin, and standard liver injury tests have been monitored, none have correlated with changes in graft size (Van Thiel et al., 1987). Most of these are generally felt to be poor indices of hepatocyte proliferation (Conn, 1989; Van Thiel et al., 1991), and currently there is still no specific clinical marker for hepatocyte proliferation. Schaffner (1991) reported a consensus timetable of test results during regeneration, in which bilirubin returns to normal at 3 weeks, and albumin after 5 weeks (Van Thiel et al., 1991). In a recent study by Teramoto et al. (1990), DNA synthesis in rat hepatocytes was examined during liver allograft rejection. Four rat recipient/donor combinations were investigated (isogeneic, allogeneic nonrejector, allogeneic rejecter, and allogeneic intermediate rejecter). The four combinations of reduced OLT produced different degrees of rejection, and DNA synthesizing hepatocytes were most abundant where tissue damage was most severe. Therefore, immunological injury actively stimulated liver regeneration, probably through growth promoting agents released during rejection. Potential growth promoters include prostaglandins (MacManus and Braceland, 1976), platelet derived growth factor (PDGF) (Hiyama et al., 1981), epidermal growth factor (EGF), hepatocyte growth factor (HGF) (Nakamura et al., 1986), and epinephrine (Takai et al., 1988) (see Chapters 1 and 2). Ongoing low-grade ischemia/thrombosis, or infection in the remaining liver tissue could all produce similar tissue injury; thereby releasing similar agents. Furthermore, other mediators released during this kind of injury, such as oxygen radicals, cytokines, and tumor necrosis factor (Jones and Summerfield, 1988; Bhatnager et al., 1981; Kark et al., 1988) might contribute to microcirculatory disturbances and further disrupt the regenerative response. In contrast with regeneration seen after partial hepatectomy, hepatocyte growth and regeneration in these types of injury would occur only in areas of necrosis irrespective of lobular gradients (Rappaport, 1976), most probably resulting in nodular regeneration (Callea et al., 1991). The effect of cyclosporin A on regeneration has been extensively researched (Kahn et al., 1990; Coughlin et al., 1987). Evidence points to cyclosporin A augmenting the liver regenerative response. Cyclosporin A appears to affect basic control mechanisms independent of its effect on the immune system (Kim et al., 1990; Henderson et al., 1989). Evidence also

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suggests that cyclosporin may have hepatotoxic effects (Kikuchi et al., 1993) in addition to its hepatotrophic effects. The balance of these two effects depends on the dose administered (Kim et al., 1990). Finally, Kawasaki has recently suggested that the regenerative capacity of the hepatocytes is determined by factors other than the hepatocytes themselves (Kawasaki et al., 1992). These data result from studies of reduced-size liver grafts from related donors in which the transplanted livers regenerated faster than the residual liver in the donor. The factors responsible could be the rejection reaction, influence of cyclosporin, or increased liver blood flow in transplanted livers (Henderson et al., 1989). In all cases, the livers regenerated to approximately a graft volume/standard liver volume ratio of 1. In some cases of small-for-size livers, during the initial stage of regeneration, attenuation of liver function might be critical (Conn, 1989). These observations substantiate previous findings that regeneration of a transplanted liver is determined by an intrinsic liver:host body volume ratio, independent of functional liver mass (Kam et al., 1987). The regenerative capacity of the liver has another important clinical correlate. In patients with fulminant hepatic failure, recent work involving the development of auxiliary livers in the form of ex v i v o xeno-perfused organs (Chari et al., 1994), or bioartificial hybrid systems (Nyberg et al., 1992; Cattral and Levy, 1994) are being employed in several centers. The regenerative potential of liver may allow the auxiliary hepatic support to provide enough while the native liver regenerates hepatocyte mass to replace that lost as a result of resection, ischemia, cirrhosis, or infection. Administration of hepatotrophic factors may also play a role in the augmentation of this response. In the future, it is evident that molecular knowledge of hepatocyte regenerative and repair processes will play an increasing role in the management of hepatobiliary disorders, both from a operative and nonoperative standpoint. Although many of the current practices of hepatic surgery for tumors are based on years of accumulated experience, the future of hepatobiliary surgery rests on the incorporation of basic science concepts of normal and hepatic tumor physiology into current surgical practice.

References Adams, Y. G., Hunos, A. G., and Fortnes, J. G. (1970). Giant hemangiomas of the liver. Ann. Surg. 172, 239-245. Alsina, A. E., Bartus, S., and Hull, D. (1990). Liver transplantation of metastatic neuroendocrine tumor. J. Clin. Gastroenterol. 12, 533-537. Baum, J. K., Holtz, E, Bookstein, J. J., and Kelin, E. W. (1973). Possible association between benign hepatic hepatomas, spontaneous rupture and oral contraceptives. Lancet 2, 926929.

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Bhatnager, R., Schirmir, R., Ernst, M., and Decker, K. (1981). Superoxide release by zymogenstimulated rat Kupffer cells in vitro. Eur. J. Biochem. 119, 171-175. Bismuth, H., Nakache, R., and Diamond, T. (1992). Management strategies on resection for hilar cholangiocarcinoma. Ann. Surg. 215, 31-38. Brady, M. S., and Coit, D. G. (1990). Focal nodular hyperplasia of the liver. Surg. Gynecol. Obstet. 171, 377-381. Bruneton, J. N., Drouilland, J., and Feneart, D. (1983). Ultrasonography of hepatic cavernous hemagiomas. Br. J. Radiol. 56, 791-795. Bussutil, R. W., Shaked, A., Millis, J. M., Jurim, O., Colquhoun, S. D., Shackleton, C. R., Neusse, B. J., Csets, M., Goldstein, L. I., and McDiarmid, S. V. (1994). One thousand liver transplants: The lessons learned. Ann. Surg. 219, 490-499. Butch, R. J., Stark, D. D., and Malt, R. A. (1986). MR imaging of focal nodular hyperplasia. J. Comput. Assist. Tomog. 10, 874-881. Callea, E, Brisigotti, M., Fabbretti, G., Sciot, R., Van Eyken, P., and Favret, M. (1991). Cirrhosis of the liver: A regenerative process. Dig. Dis. Sci. 36, 1287-1293. Cattral, M. S., and Levy, G. A. (1994). Artificial liverwpipe dream or reality? N. Eng. J. Med. 331,268-269. Chandler, J. G., Lee, S., Krubel, R., Rosen, H., Nakaji, H. T., and Orloff, M. J. (1971). The inter-liver competition and portal blood in regeneration of auxiliary liver transplants. Surg. Forum 22, 341-343. Chari, R. S., and Meyers, W. C. (1993). Liver surgery. In "Current Practice of Surgery" (B. A. Levine, E. M. Copeland, R. J. Howard, H. J. Sugerman, and A. L. Warshaw, eds.), pp. 152. Churchill Livingstone. New York. Chari, R. S., Collins, B. H., Macgee, J. C., DiMaio, J. M., Kirk, A. D., Harland, R. C., McCann, R. L., Platt, J. L., and Meyers, W. C. (1994). Treatment of hepatic failure with exvivo pig-liver perfusion, followed by liver transplantation. N. Eng. J. Med. 331,234-237. Chistopherson, W. M., and Mays, E. T. (1977). Liver tumors and contraceptive steroids: Experience with the first 100 registry patients. J. Natl. Cancer Inst. 58, 161-171. Clouse, M. E. (1989). Current diagnostic imaging modalities of the liver. Surg. Clin. North Am. 69, 193-234. Conn, H. O. (1989). Do transplanted human livers regenerate? Hepatology (Baltimore) 9, 789-793. Coughlin, J. P., Austen, W. G., Donohoe, P. K., and Russell, W. E. (1987). Liver regeneration during immunosuppresion. J. Pediatr. Surg. 22, 566-570. Edmonson, H. A., Reynolds, T. B., Hendersond, B., and Benton, B. (1977). Regression of liver cell adenomas with oral contraceptives. Ann. Intern. Med. 86, 180-182. Farmer, D. G., Rosove, M. H., Shaked, A., and Busutil, R. W. (1994). Current treatment modalities for hepatocellular carcinoma. Ann. Surg. 219, 236-247. Fisher, B., Szuch, P., and Fisher, E. R. (1971). Evaluation of a humoral factor in liver regeneration utilizing liver transplants. Cancer Res. 31,322-331. Flowers, B. E, McBurney, R. P., and Vera, S. R. (1990). Ruptured hepatic adenoma. A spectrum of presentation and treatment. Am. J. Surg. 56, 380-383. Gyoffy, E. J., Bredfeldt, J. E., and Black, W. C. (1989). Transformation of hepatic cell adenoma to hepatocellular cancinoma due to oral contraceptive use. Ann. Intern. Med. 110, 489490. Hardy, K. J. (1990). Liver surgery: The past 2000 years. Aust. N. Z. J. Surg. 60, 811-817. Henderson, J. M., Millikan, W. J., Hooks, M., Noe, B., Kutner, M. H., and Warren, W. D. (1989). Increased galactose clearance after live transplantation: A measure of increased blood flow though denervated liver? Hepatology (Baltimore) 10, 288-291. Higgins, G. M., and Anderson, R. M. (1931). Experimental pathology of the liver: I. Restoration of the liver of the white rat following partial surgical resection. Arch. Pathol. 12, 186192.

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Hiyama, Y., Mahmud, I., Karima-tari, E, Fujita, S., Fukui, N., and Miura, Y. (1981). Platelet derived growth factor and thromboxane are necessary for liver regeneration. Cell. Mol. Biol. 27, 593-597. Hughes, K., Scheele, J., and Sugarbaker, P. H. (1989). Surgery for colorectal cancer metastatic to the liver. Surg. Clin. North Am. 69, 339-359. Ishak, K. J., and Robin, L. (1975). Benign tumors of the liver. Med. Clin. North Am. 59, 9951000. Ishikura, H., Tsuchimoto, S., Misonou, J., Natori, T., and Aizawa, M. (1987). Leukocyte subsets infiltrating into fully allogeneic, long-surviving rat liver allografts. Transplantation 43, 709-714. Jenkins, R. L., Johnson, L. B., and Lewis, W. D. (1994). Surgical approach to benign liver tumors. Semin. Liver Dis. 14, 178-189. Jones, E. A., and Summerfield, J. A. (1988). Kupffer cells. In "The Liver: Biology and Pathobiology" (M. Arias, W. B. Jakoby, H. Popper, D. Schacter, and D. A. Shafritz, eds.), pp. 683-704. Raven, New York. Kahn, D., Makowka, L., Lai, H., Eagon, P. K., Dindzan, V., Starzl, T. E., and Van Thiel, D. H. (1990). Cyclosporin augments hepatic regenerative response in rats. Dig. Dis. Sci. 35,392398. Kam, I., Lynch, S., Svanas, G., Todo, S., Polimeno, L., Francavilla, A., Takaya, S., Ericzon, B. G., Starzl, T. E., and Van Thiel, D. H. (1987). Evidence that host size determines liver size: Studies in dogs receiving orthotopic liver transplants. Hepatology (Baltimore) 7, 362366. Kark, U., Peters, T., and Decker, K. (1988). The release of tumor necrosis factor from endotoxinstimulated rat Kupffer cells is regulated by prostaglandin E2 and dexamethasone. J. Hepatol. 7, 352-361. Kawasaki, S., Makuuchi, M., Ishizone, I., Matsunami, H., Terada, M., and Kawarazaki, H. (1992). Liver regeneration in recipients and donors after transplantation. Lancet 339, 580581. Kawasaki, S., Makuuchi, M., Kakazu, T., Miyagawa, S., Takayama, T., Kosuge, T., Sugihara, K., and Moriya, Y. (1994). Resection of multiple metastatic liver tumors after portal embolization. Surgery 115, 674-677. Kerlin, P., Davis, G. L., McGill, D. B., Weiland, L. H., Adson, M. A., Sheedy II, P. E (1983). Hepatic adenoma and focal nodular hyperplasia: Clinical, pathological and radiological features. Gastroenterology 84, 994'1002. Kikuchi, N., Yamaguchi, Y., Mori, K., Takata, N., Goto, M., Makino, Y., Hamaguchi, H., Hisama, N., and Ogawa, M. (1993). Effect of cyclosporin on liver regeneration after orthotopic reduced-size heaptic transplantation in the rat. Dig. Dis. Sci. 38, 1492-1499. Kim, Y. I., Nakashima, K., Kawano, K., and Kobayashi, M. (1990). Evidence that cyclosporin is hepatotoxic and hepatotrophic in 70% hepatectomized rats and mice. Eur. Surg. Res. 22, 231-237. Lee, S., Edington, T. S., and Orloff, M. J. (1968). The role of the afferent blood supply in regeneration of liver isografts in rats. Surg. Forum 22, 360-362. Lin, T., and Chen, C. (1965). Metabolic function and regeneration of cirrhotic and noncirrhotic livers after hepatic lobectomy in man. Ann. Surg. 162, 959-971. MacManus, J. P., and Braceland, B. M. (1976). A connection between the production of prostaglandins during liver regeneration and the DNA synthetic repsonse. Prostaglandins 11, 609-620. Makowka, L., Svanas, G., and Esquivel, C. O. (1989). Effect of cyclosporin on hepatic regeneration. Surg. Forum 37, 352-354. Makowka, L,, Tzakis, A. G., and Massaferro, V. (1994). Transplantation of the liver for metastatic endocrine tumors of the intestine and pancreas. Surg. Gynecol. Obstet. 168, 107-111.

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Margreiter, R. (1986). Indications for liver transplantation for primary and secondary liver tumors. Transplant. Proc. 18(Suppl. 3), 74-77. Meyers, W. C., and Jones, R. S. (1990). Development of liver and biliary surgery. In "Textbook of Liver And Biliary Surgery" (W. C. Meyers and R. S. Jones, eds.), pp. 1-13. Lippincott, Philadelphia. Moritz, M. J., Jarrell, B. E., Munoz, S. J., and Maddrey, W. C. (1993). Regeneration of the native liver after heterotopic liver transplantation for fulminant hepatic failure. Transplantation 50, 952-954. Nakamura, T., Teramoto, H., and Ichihara, A. (1986). Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary culture. Proc. Natl. Acad. Sci. U.S.A. 83, 6489-6493. Neuberger, J., Portmen, B., Nunnerly, H. B., Laws, J. W., Davis, M., and Williams, R. (1980). Oral contraceptives associated with liver tumors: Occurrence of malignancy and difficulties in diagnosis. Lancet 9, 273-276. Nyberg, S. L., Shatford, R. A., Hu, W. S., Payne, W. D., and Cerra, E B. (1992). Hepatocyte culture systems for artificial liver support: Implications for critical care medicine (bioartificial liver support). Crit. Care Med. 20, 1157-1168. Ochsner, J. L., and Halpert, B. (1958). Cavernous hemangiomas of the liver. Surgery 43, 577582. Penn, I. (1991). Hepatic transplantation for primary or metastatic cancer of the liver. Surgery 110, 726-735. Pichlmayr, R. (1988). Is there a place for grafting in malignancy? Transplant Proc. 20, 478482. Rappaport, A. M. (1976). The microcirculatory acinar concept of normal and pathalogical hepatic structure. Beitr. Pathol. 157, 215-243. Rossi, G., de Carlis, L., Doglia, M., Fassati, L. R., Tarenzi, L., and Galmarini, D. (1987). Orthotopic liver transplantation of partially hepatectomized liver in the pig. Transplantation 43, 362-365. Saul, H. C., Tietlebaum, D. S., Gansler, T. S., Varello, M., Burke, D. R., Atkinson, B. E, and Rosato, E. E (1987). The fibrolammellar variant of hepatocellular carcinoma. Cancer 60, 3049-3055. Schaffner, E (1991). Structural and functional aspects of regeneration of the human liver. Dig. Dis. Sci. 36, 1282-1286. Schoenthaler, R., Phillips, T. L., Castro, J., Efrid, J. T., Better, A., and Way, L. W. (1994). Carcinoma of the extrahepatic bile ducts: The University of California at San Francisco experience. Ann. Surg. 219, 267-274. Shimada, M., Matsumata, T., Taketomi, A., Yamamoto, K., Itasaka, H., and Sugimachi, K. (1994). Repeat hepatectomy for recurrent hepatocellular carcinoma. Surgery 115, 703706. Steele, G. (1994). Cryoablation in hepatic surgery. Semin. Liver Dis. 14, 120-125. Suenaga, M., Sugiura, H., Kokuba, Y., Uehara, S., and Kurumiya, T. (1994). Repeated hepatic resection for recurrent hepatocellular carcinoma in eighteen cases. Surgery 115, 452-457. Sugarbaker, P. H. (1990). En block resection of hepatic segments 4B, 5 and 6 by transverse hepatectomy. Surg. Gynecol. Obstet. 170, 250-252. Sugiura, Y., Nakamura, S., Iida, S., Hosoda, Y., Ikeuchi, S., Mori, S., Sugioka, A., and Tsuzuki, T. (1994). Extensive resection of the bile ducts combined with liver resection for cancer of the main hepatic duct junction: A cooperative study of the Keio Bile Duct Cancer Study Group. Surgery 115,445-451. Takai, S., Nakamura, T., Komi, N., and Ichihara, A. (1988). Mechanism of stimulation of DNA synthesis induced by epinephrine in primary culture of adult rat hepatocytes. J. Biochem. (Tokyo) 103, 848-852.

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Teramoto, K., Shimizu, K., Tsukada, K., and Kamada, N. (1990). DNA synthesis in hepatocytes during liver allograft rejection in rats. Transplantation 50, 199-201. Van Thiel, D. H., Hayler, H. G., Schade, R. R., Skolmick, M. L., Pollitt-Heyl, A., Rosenblum, E., Gavaler, J. S., and Penkrot, R. J. (1985). In vivo hepatic volume determination using sonography and computed tomography validation and a comparison of the two techniques. Gastroenterology 88, 1812-1817. Van Thiel, D. H., Gavaler, J. S., Kam, I., Francavilla, A., Polomeno, L., Schade, R. R., Smith, J., Diven, W., Penkrot, R. J., and Starzl, T. E. (1987). Rapid growth of an intact human liver transplanted into a recipient larger than the donor. Gastroenterology 93, 1414-1419. Van Thiel, D. H., Stauber, R., Gavaler, J. S., and Francavilla, A. (1991). Hepatic regeneration: Effects of age, sex hormone status, prolatin and cyclosporin. Dig. Dis. Sci. 36, 1309-1912. Wagner, J. S., Adson, M. A., and Van Heerdon, J. A. (1984). The natural history of hepatic metastases from colorectal cancer. Ann. Surg. 199, 502-510. Welch, T. J., Sheedy, P. F., and Johnson, C. M. (1985). Focal nodular hyperplasia and hepatic adenomas: Comparison of angiography, CT, US and scintigraphy. Radiology 156, 593595. Yamamoto, T., Karaward, Y., and Yano, T. (1991). Spontaneous rupture of hemangioma of the liver: Treatment with transcather hepatic arterial embolization. Am. J. Gastroenterol. 86, 1645-1649. Yamanaka, N., Okamoto, E., Oriyama, T., Fujimoto, J., Furakawa, K., Kawamura, E., Tanaka, T., and Tomodo, E (1994). A prediction scoring system to select the surgical treatment of liver cancer. Ann. Surg. 219, 342-346. Yokoyama, I., Todo, S., and Iwatsuki, S. (1990). Liver transplantation in treatment of primary liver cancer. Hepatogastroenterology 37, 188-193. Zoli, M., Marchesini, G., Melli, A., Vita, G., Marra, D., Marrano, D., and Pisi, E. (1986). Evaluation of liver volume and liver function following hepatic resection in man. Liver 6, 286-291.

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14 Gene Therapy for the Treatment of Inherited and Acquired Diseases of the Liver Brian E. Huber Wellcome Research Laboratories Research TrianglePark, North Carolina27709

I. Human Gene Therapy---A Definition A broad, encompassing definition of human gene therapy is the in v i v o or e x v i v o transfer of defined genetic material to specific target cells of a patient, thereby altering the genotype and, in most situations, altering the phenotype of those target cells for the ultimate purpose of preventing or altering a particular disease state. As this definition states, the underlying premise is that these therapeutic genetic procedures are designed to ultimately prevent, treat, or alter an overt or covert pathological condition. In most situations, the ultimate therapeutic goal of gene therapy procedures is to alter the phenotype of a specific target cell population. However, there may be particular medical situations, such as in latent disease states, where the therapeutic goal will only be to alter the genotype of a particular target cell population before the occurrence of any overt pathological condition. In certain virally associated diseases, the causative viruses may not produce any overt pathological condition for many years. In these "latent" disease states, a therapeutic goal would be to target those potentially pathogenic cells to disrupt the integrity and stability of the integrated but transcriptionally silent viral genomic sequences. In other situations, important clinical questions can be addressed by genetically "tagging" or marking particular cell types, such as transplanted hepatocytes, by stably altering their genotype. Genetic marking will permanently tag those

Liver Regeneration and Carcinogenesis Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cells for their lifetime as well as all progeny cells which arise from them. Genetic marking will make it possible to address important questions involving transplantation efficiency, distribution, half-life, and reproductive potential of transplanted hepatocytes. The ability to accurately address these issues may prove to be critically important in the clinical development of hepatocellular transplantation. Gene therapy can be divided into two categories: cellular therapy and gene therapy. The distinction between these two types of therapy is that the former involves removal of particular target cells (e.g., hepatocytes) from a patient or an appropriate donor, ex vivo gene transfer and genetic alteration, and the reintroduction of the genetically altered cells back into the patient. The latter, gene therapy, involves the direct in vivo genetic alteration of a patient's target cells. However, it is quite common and acceptable to describe both categories as gene therapy without distinguishing between ex vivo and in vi vo gene transfer. Illustrations of ex vivo and in vivo liverdirected gene therapy will follow in this chapter. Debates concerning the ethics of human somatic cell gene therapy have occurred during the last decade. It is now generally accepted that human somatic cell gene therapy is ethically acceptable within strict scientific, medical, and regulatory guidelines.

II. Strategies for Liver-Directed Gene Therapy One can envision two distinctly different strategies for liver-directed gene therapy; namely, (1) gene replacement (repair) or excision therapy, and (2) gene addition therapy.

A. Gene Replacement (Repair) or Excision Therapy A first hypothetical strategy for liver-directed gene therapy is gene replacement (repair) or excision therapy (Figure 1A). In this scenario, one can envision a mutated hepatic gene(s) producing an aberrant hepatic protein that ultimately results in or contributes to a disease state. The pathogenicity of the hepatic gene may be qualitative or quantitative in nature resulting in either an altered hepatic protein or a hepatic protein which is inappropriately expressed, respectively. In the gene replacement or excision strategy of liver-directed gene therapy, one could theoretically deliver to the liver a defined sequence of a normal gene. Then, through a homologous recombination event, the mutated sequence in the pathogenic gene could be replaced with the normal sequence of the unmutated gene (Figure 1A). Hence, the mutated hepatic gene (acquired or inherited mutations) is repaired in situ.

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This site-specific sequence replacement or correction is intellectually very attractive for a variety of reasons. It would be theoretically possible to correct any identified point mutation in a gene, such as the mutations found in the low density lipoprotein (LDL) receptor gene creating the disease state of familial hypercholesterolemia (Turley and Dietschy, 1988). Another important theoretical application of this gene therapy strategy is the ability to stop expression of specific genes. Through a homologous recombination event, one could envision being able to engineer stop codons or "nonsense" sequences into the internal domains of specific genes in situ. With this technical ability, it would be possible to stop the expression of specific genes, such as the gene encoding hepatitis B surface antigen. A final theoretical application of this gene therapy strategy is the potential ability to excise a sequence of genetic material from a target cell's genome. With this technical ability, one could theoretically excise target sequences from hepatocytes, such as a stably integrated viral DNA sequence. For the above reasons, the gene replacement (repair) or excision strategy is an intellectually attractive one. However, this strategy requires extremely efficient and specific homologous recombination events to occur in the liver cell population in situ. Although homologous recombination has been demonstrated to take place in mammalian cells (for reviews see Capecchi, 1989; Bollag et al., 1989), homologous recombination is such a rare event, it makes this approach for liver-directed gene therapy currently impracticable with present technology.

B. Gene Addition Therapy Since present day technology does not allow for efficient homologous recombination in the liver genome in situ, a second, more feasible strategy for liver-directed gene therapy has been developed. This second, more practical strategy is gene addition therapy (Figure 1B). For this strategy, we again can envision a mutated hepatic gene producing a quantitatively or qualitatively altered hepatic protein which ultimately results in or contributes to a disease state. For gene addition therapy, a complete copy of a normal gene is delivered to the hepatic target cells. Depending on the gene delivery system used (see below), this complete copy of a normal gene can either be randomly integrated into the genome of the liver cell or it may remain extrachromosomal. If the delivered genetic material is integrated into the genome, it is not site specific. An important application of the gene addition strategy is the theoretical ability to replace important genetic information that has been functionally lost through mutational events. Another important application of gene addition strategy is the ability to create entirely new and unique phenotypes in hepatocytes by genetically modifying them to synthesize and secrete pro-

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teins t h a t h e p a t o c y t e s do n o t n a t u r a l l y synthesize. Finally, this t e c h n i q u e will allow the "genetic t a g g i n g " of h e p a t o c y t e s used in h e p a t o c e l l u l a r transp l a n t a t i o n p r o c e d u r e s . Since these genetic tags are n o t lost or diluted over time a n d are p r o p a g a t e d to all p r o g e n y cells, i m p o r t a n t clinical q u e s t i o n s r e g a r d i n g the efficacy of allogenic or a u t o l o g o u s h e p a t o c y t e t r a n s p l a n t can be addressed.

C. Gene Addition Therapy and the Hepatocyte Liver-directed gene t h e r a p y via the gene a d d i t i o n s t r a t e g y shares s o m e similar issues p e r t i n e n t to all gene t h e r a p y a p p r o a c h e s . Some issues are u n i q u e , however, to the liver milieu a n d p r e s e n t b o t h obstacles a n d o p p o r t u n i t i e s . Some of these issues are s u m m a r i z e d below: 9 9 9

adult h e p a t o c y t e s in vivo do n o t divide to a n y great extent; adult h e p a t o c y t e s in vivo can be driven into the cell cycle for c o m p e n s a t o r y liver g r o w t h by surgical or chemical h e p a t e c t o m y ; adult h e p a t o c y t e s can be r e m o v e d by surgical resection and, s u b s e q u e n t to collagenase p e r f u s i o n , can be placed into cell culture. In culture, h e p a t o c y t e s have real but limited cellular

Figure 1. Strategies of gene therapy. A normal eukaryotic gene is characterized by a noncoding, 5' transcriptional regulatory sequence (TRS; single line) linked to the gene's sequence which is transcribed into messenger RNA (TS; triple line). Portions of the transcribed sequence may be translated into amino acids while other portions of the messenger RNA remain untranslated. The transcribed sequence (exons) may also be interspersed with untranscribed sequences called introns. Inherited or acquired mutations (open symbols) may arise in the transcriptional regulatory sequence of genes causing altered expression and/or regulation of that particular gene (and possible other genes as well). These are termed quantitative alterations in gene function resulting from altered expression. Alternatively, inherited or acquired mutations may arise in the coding domain of a gene generating a gene product which has different biochemical properties compared to the unmutated, normal gene. These altered biochemical properties may result in a completely nonfunctional protein or a protein which functions aberantly. These alterations are collectively called qualitative alterations in gene function resulting from the production of an altered gene product. (A) Gene replacement (repair) or excision therapy. In this strategy, a portion of the normal gene is delivered to the cells which harbor theemutant gene. Through a homologous recombination event in situ, the portion of the mutant gene is replaced with the normal sequence, thereby generating a repaired and normal gene and hence, a normally regulated gene product. (B) Gene addition therapy. In this strategy a complete copy of a normal gene is delivered to the cell. Depending on the delivery system, this complete copy can remain extrachromosomal or become randomly integrated into the cell's genome. In this case, in addition to the mutated gene, the cell also contains a normal gene. In many situations both the mutant allele and the newly delivered gene will be expressed.

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replication potential but can remain viable and differentiated for extended periods; 9 adult hepatocytes may be used for autologous or allogenic transplant; 9 intact liver is amenable for orthotopic transplant; and 9 in unstressed conditions, adults can lose significant liver mass and still maintain homeostasis. D. Gene Addition T h e r a p y u E x Vivo versus In Vivo Liver-Directed Gene Therapy As described above, technology is presently available to implement a gene addition type strategy for liver-directed gene therapy. To reduce this to clinical practice, two different approaches can be taken: an ex vivo gene transfer approach or an in vivo gene transfer approach. For the ex vivo approach, a portion of the liver is surgically removed from the patient, enzymatically isolated hepatocytes are placed into culture, genetically manipulated, and then returned to the patients liver or potentially a heterotopic site (Figure 2). For in vivo gene transfer, the genetic alteration is performed in situ. 1. E x Vivo

Compared to other complex solid organs, the liver is an organ that has proved to be amenable to surgical manipulation, resection, and transplantation since it undergoes a regenerative process after surgical resection or tissue injury (Francavilla et al., 1990). a. H e p a t o c e l l u l a r Culture: Substantial advances have been made in identifying conditions in which nonhuman primate and human hepatocytes can be successfully placed into cell culture and maintained while still retaining hepatic morphology and differentiated characteristics (Ismail et al., 1991; Li et al., 1992; Lanford et al., 1989; Gibson-D'Ambrosio et al., 1990). It is anticipated that the technical ability to maintain differentiated human hepatocytes in culture will become routine. As growth- and differentiation-associated factors become better understood, it may eventually be possible to continually propagate human hepatocytes in a growthassociated media then switch them to a differentiation-associated media for transplantation. Please refer to Chapters 1 to 4 in this book for more information regarding growth control and growth-associated factors for human hepatocytes. b. H e p a t i c Stem Cells: Another potential advancement in the ex vivo approach for liver-directed gene therapy is the specific selection of

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Figure 2. Ex vivo and in vivo liver-directed gene therapy. Illustrated are ex vivo and in vivo liver-directed gene therapy. For the ex vivo approach, hepatocytes are isolated after surgical resection. Gene transfer is done ex vivo and these genetically modified hepatocytes are transplanted back into the patient. These hepatocytes can be autologous or potentially from another donor (allogenic). For in vivo gene transfer, the genetic manipulation is done in situ. This can be accomplished by delivering the gene transfer vector to the liver directly via regional administration (i.e., portal vein or hepatic artery) or by engineering a vector with hepatocyte targeting potential (hepatotrophic) for systemic delivery.

hepatic stem cells for genetic m a n i p u l a t i o n and transplant. It was previously a s s u m e d that the hepatic c o m p a r t m e n t did not contain a stem cell p o p u l a tion. This was based on historical data indicating that after a chemical or surgical partial hepatectomy, m o s t m a t u r e hepatocytes divide to regain the original liver mass. This regenerative capacity does not d e p e n d on the existence of a stem cell p o p u l a t i o n . Indeed, it has been definitively illustrated that adult hepatocytes located near the portal spaces have a significant

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proliferative capacity and further, that all hepatocytes in the lobule have some proliferative potential (Zajicek et al., 1985; Reid et al., 1992). There is increasing evidence to suggest, however, that the liver may also contain a stem cell and/or progenitor cell compartment (for reviews see Fausto, 1990; Thorgeirsson and Evarts, 1992; Fausto et al., 1992, 1993; Hixson et al., 1992; Thorgeirsson et al., 1993; and Chapter 5). This cellular compartment has been called the oval cell compartment. It appears that the oval cell compartment is heterogeneous in make-up, consisting of cell populations at various stages of differentiation in either the hepatocyte or the bile duct lineages (Fausto et al., 1993). However, there may be a small population of oval cells that are not yet committed to either lineage, and in specific situations, may serve as progenitors for both hepatocyte and bile ductal lineages. This oval cell compartment may also give rise to intestinal-type epithelia and pancreatic tissues (Thorgeirsson et al., 1993; Evarts et al., 1987; Tatematsu et al., 1985; Kimbrough et al., 1972; Rao et al., 1986). Hence, these uncommitted oval cells may be considered a hepatic stem cell. It is unclear what cellular signals and mechanisms activate this facultative stem cell compartment and dictate the cell lineage that will emerge. Nevertheless, a better understanding of this facultative stem cell system and the molecular mechanisms of its activation and lineage commitment may have a significant impact on liver-directed gene therapy. c. H e p a t o c e l l u l a r E x p a n s i o n : As indicated above, much progress has been made in the harvesting, cultivating, cyropreservation, and transplantation of nonhuman primate and human hepatocytes. It is becoming more routine to successfully isolate human hepatocytes via sequential perfusions of EDTA and collagenase, and to be able to maintain these differentiated cells in a hormonally defined media for several weeks (for reviews see Ismail et al., 1991; Li et al., 1992; Lanford et al., 1989; Ledley et al., 1991). Nonhuman primate hepatocytes and human hepatocytes also have been demonstrated to undergo limited proliferation in vitro (for reviews see Ismail et al., 1991; Li et al., 1992; Lanford etal., 1989; Ledley et al., 1991). It appears that the most important growth control factors for human hepatocyte proliferation are transforming growth factor 0~ (TGFo~), hepatocyte growth factor (HGF), acidic fibroblast growth factor (aFGF), insulin, dexamethasone, and transforming growth factor 13 (TGFI3) (acting as a potential negative growth control signal following compensatory hyperplasia) (for reviews see Ismail et al., 1991; Li et al., 1992; Lanford et al., 1989; Ledley et al., 1991; Michalopoulos and Zarnegar, 1992; Michalopoulos, 1990; Fausto, 1991). Unlike rodent hepatocytes, arginine-vasopressin fails to stimulate human hepatocytes (Ismail et al., 1991). Despite this information, it is clear that we presently have only a rudimentary understanding of the molecular events associated with the control of human hepatocellular growth.

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It is very likely that continued investigations in this area will lead to the ability to significantly expand human hepatocytes in culture, similar to cells in the hematopoietic lineage. This, of course, will have significant positive implications for ex vivo liver-directed gene therapy. One potential possibility may be the use of punch biopsy material to provide the cell seed (and hepatic stem cells) for ex vivo expansion. This would eliminate the necessity for major invasive surgical resection of the patients liver (see below). d. G e n e Transfer: Once the human hepatocytes are established in culture, gene transfer is performed. This can be accomplished with a number of different gene delivery vector systems, which will be described in Section III. An important consideration at this point is that certain gene delivery vectors will only be applicable for ex vivo gene transfer. Human hepatocytes in situ do not significantly divide under normal physiological conditions. However, as stated above, human hepatocytes in culture can go through a limited number of cell divisions. This property permits the use in an ex vivo setting of certain gene delivery vectors (i.e., retroviral vectors) which require cell proliferation for efficient gene insertion and expression. e. A u t o l o g o u s versus A l l o g e n e i c H e p a t o c y t e s : This may become the most intensely investigated area of research and controversial aspect of ex vivo liver-directed gene therapy. Clearly, the use of autologous hepatocytes will eliminate a significant concern regarding rejection of the genetically modified transplanted hepatocytes (see Section II.D and Figure 2). However, isolation and cultivation of autologous hepatocytes will entail major invasive surgical resection of the liver that will inherently be associated with a certain percentage of morbidity and mortality. In addition, these surgical procedures and primary autologous hepatocyte cultivation are inordinately expressive, time consuming, and not 100% effective. Finally, in many situations the medical condition of the patient undergoing gene therapy is inherently compromised. Therefore, it seems likely that the use of allogenic hepatocytes will receive significant attention, especially if a series of crossmatched, HLA compatible, serially passaged human hepatocytes (or human hepatic stem cells) are available (Baumgartner et al., 1983). It is interesting to note that cross-matched, allogenic human hepatocytes for hepatocellular transplant may have less rejection potential compared to allogenic orthotopic liver transplantation for the following reasons:

Cellular elements, such as blood cells, endothelial cells, Kupffer cells, and tissue matrix components, are eliminated in hepatocellular transplantation procedures. These nonhepatocyte cellular components may be more immunogenic than hepatocytes themselves (Lafferty et al., 1983);

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The antigenicity, and hence rejection potential, of hepatocytes may be decreased by the procedures of ex vivo harvest and cultivation (Bowen et al., 1980; Naji et al., 1981). [. Transplant: Once the autologous or allogenic hepatocytes (or hepatic stem cells) are genetically modified, these cells will be transplanted back into the host. After genetic modification and before transplant, these genetically modified cells may be expanded (see above), and/or selected based on the incorporation and expression of a dominate selectable marker. Transplant may be back into the hepatic compartment or a heterotopic site. For transplant into the hepatic compartment, portal vein infusion or intrasplenic injection seem logical approaches. Experimental data suggest that intrasplenic injection may be the administration route of choice. Portal vein infusions have been noted to be associated with thrombosis, hepatic infarction, and limited by the amount of cells that can be transplanted (Ponder et al., 1991). In contrast, intrasplenic injections of genetically modified autologous hepatocytes have shown that (Ponder et al., 1991):

9 hepatocytes transplanted via an intrasplenic injection seed into the hepatic compartment; 9 once in the hepatic compartment, the transplanted, genetically modified hepatocytes retain a normal hepatocyte morphology and reside in a normal histological location; 9 very little, if any, of the transplanted hepatocytes are retained in the spleen; 9 a larger mass of cells can be transplanted via the intrasplenic route compared to the portal vein infusion route. Additional medical procedures may assist in the efficacy of hepatic engraftment of the transplanted hepatocytes. Animal models have demonstrated that partial hepatectomy (Jirtle and Michalopoulos, 1982), administration of angiogenesis factors (Thompson et al., 1988), and other growth factors (Michalopoulos and Zarnegar, 1992; Michalopoulos, 1990; Fausto, 1991) aid in the engraftment and viability of the transplanted hepatocytes. An alternative approach is to transplant the genetically modified hepatocytes to a heterotopic site. Experimental animal data demonstrate the utility of fat pads, fascia, lens of the eye, and peritoneal cavity (Jirtle and Michalopoulos, 1982; Jirtle et al., 1980; Demtriou et al., 1986). Heterotopic transplant can also be added in the use of biologic and/or organic supports (as examples see Demtriou et al., 1986; Anderson et al., 1989; Vacanti et al., 1988; Moscioni et al., 1989).

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2. In V i v o

The alternative to ex vivo liver-directed gene therapy is in vivo liver-directed gene therapy. In this approach the gene delivery vector is delivered to the hepatic compartment via regional infusion into the hepatic artery or portal vein. If the gene delivery system is by nature hepatotrophic or has engineered hepatic targeting capability, then the vector may be able to be delivered via the systemic circulation.

III. Gene Transfer Techniques for Liver-Directed Gene Therapy Central to gene addition therapy is the in vivo or ex vivo transfer of genetic material into particular liver target cells, for the purpose of transiently or stably altering the genotype, and, in most cases, the phenotype of that liver target cell population. There are many established techniques for transferring genetic material into mammalian cells for the subsequent purpose of having that genetic material transcribed and translated (Table 1). Some of these gene transfer techniques have no present application to liver-directed gene therapy. Some of these techniques have limited applications while others have very specific applications to liver-directed gene therapy. The techniques which have received the most attention to date are retroviral and adenoviral delivery. It is beyond the scope of this chapter to describe in detail all gene transfer techniques outlined in Table 1. Only abbreviated comments will be made.

Table 1 Established Techniques for the Transfer of Genetic Material into Mammalian Cells Methods Chemical transfer methods Electroporation Microinjection Scrape loading Macroinjection Ballistic barrage Receptor-mediated delivery Liposomal transfer Viral delivery DNA viruses RNA viruses

Utility in gene therapy protocols

Very little utility

Limited utility in defined therapeutic situations Specific utility in defined therapeutic situations

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A. Chemical Transfer Methods Methods which rely on the physicochemical interaction between DNA and different chemical carrier complexes have been extensively utilized to facilitate the transfer of DNA through intact biological membranes of living mammalian cells. Agents such as calcium phosphate, DEAE-dextran, poly-1ornithine, and strontium phosphate have been used to form DNA-carrier precipitation complexes which mediate an interaction with biological membranes to facilitate the internalization of the precipitated DNA. Chemicalmediated transfer systems can be relatively efficient for transient expression (10 to 50% efficiencies), but relatively inefficient for genomic integration and stable expression of the delivered DNA (0.0001 to 1% efficiencies). If genomic integration does occur, multiple copies of the transferred DNA are usually integrated in head-to-head or head-to-tail concatemers since the DNA is taken up by the cell as a precipitate. In addition to concatemerization, it has been reported that from 1% to more than 10% of the transfected DNA can acquire other rearrangements, deletions, or point mutations by the chemical transfection procedure (for reviews see Kucherlapati and Skoultchi, 1984; Anderson, 1984). As such, it is difficult to achieve with chemical transfer methods the stable integration of one or a few copies of DNA which retain the integrity and symmetry of the starting DNA molecule. In addition, chemical transfer methods are extremely toxic to a wide range of cell types. Nevertheless, chemical transfer methods have been extensively utilized in experimental in vitro DNA transfection procedures with much success. There has been at least one report describing the use of calcium phosphate precipitated DNA for in vivo liver-directed gene transfer (Benvenisty and Reshef, 1986). Precipitated DNA was injected intraperitoneally into newborn rats and gene expression was subsequently observed in the liver and spleen. Despite this very interesting report, it is unlikely that chemical transfer techniques will be significantly utilized in human gene therapy protocols due to the lack of efficacy for stable gene transfer, the high propensity for gene rearrangements, and the toxicity exhibited in most cell types.

B. Electroporation Electroporation has been used to transfer macromolecules (DNA, RNA, and proteins) as well as small molecules (cations, dyes, nucleotides, and drugs) into prokaryotic, eukaryotic, and plant cells (for review see Shigekawa and Dower, 1988). This technique developed from the observation that a high electrical discharge through a chamber containing a cell suspension caused cell fusion. Shortly afterward, it was demonstrated that plasmid

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DNA could be induced to be taken up and expressed in cells via electroporation. Electroporation is based on the principle that a brief high voltage electricaldischarge between suspended cells causes transient holes or pores to form in cell membranes. These transient pores make a cell permeable to exogenous agents, such as DNA and other macromolecules, which can diffuse into a cell through these pores before they reseal and repair. Electroporation-mediated gene transfer can be fairly efficient for transient gene expression (similar or slightly better than chemical transfer methods), but again relatively inefficient for stable genomic integration and expression of the delivered DNA (i.e., 0.0001 to 1% efficiencies). Electroporated DNA that does become stably integrated into the target cell's genome has a greater propensity to integrate as a single copy gene and less of a propensity to form concatemers compared to chemical delivery systems. It has also been suggested that electroporation may be applicable to a greater number of different cell types compared to chemical transfer methods. Indeed, electroporation has been extensively utilized in many research experiments requiring in vitro gene transfer. Electroporation has not received wide attention for its use in human gene therapy procedures due to the inefficiency of stable gene transfer, and methodological constraints using this transfer technique. In addition, electroporation may not be easily adapted for in vivo gene transfer. However, with the increasing ability to isolate and selectively culture desirable cell populations, such as hepatocytes and liver stem cells (see above), electroporation may find a role in ex vivo liver-directed gene therapy protocols.

C. Microinjection into the Nucleus Microinjection is a gene transfer technique involving the physical transfer of genetic material into the nucleus of particular target cells (for review see DePamphilis et al., 1988). This technique utilizes microinjection pipettes with tips of less than 1 ~m outside diameter to physically transfer genetic material into the cell nucleus. Microinjection has been extensively used for injection into the pronuclei of fertilized one-cell zygotes to produce transgenic animals. Microinjection into the cell nucleus has the primary advantage that stable expression efficiencies of 10 to 30% can be achieved (DePamphilis et al., 1988). Despite having very high efficiency of gene transfer, the microinjection technique has little present utility in human gene therapy procedures due to the very low potency of gene transfer (i.e., only a small number of cells can be microinjected for a given period), and many important human target cell types are not amenable to the technical procedures required for this technique.

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D. Scrape Loading Scrape loading is another technique used to transfer small, intermediate, and macromolecules into cells. Molecules such as antibodies, antisense oligonucleotides, RNA templates, and double-stranded DNA have been transferred into cells using this technique. Scrape loading involves the physical detachment of adherent cells by scraping, which disrupts the integrity of the cell membrane. Before resealing, the now "permeablized" cells can take up extracellular molecules into their cytoplasm. No direct comparisons have been made between chemical or electroporation transfer methods and scrape loading, but it is assumed that scrape loading is less efficient for both transient and stable gene transfer. The sole advantage of this technique for gene transfer is its technical ease. There appears to be little utility for the scrape loading technique of gene transfer in human gene therapy procedures. Delivery systems which have limited utility for liver-directed gene therapy are macroinjection, ballistic barrage, and receptor-mediated delivery.

E. Macroinjection Macroinjection is a gene transfer technique involving the direct tissue injection of RNA templates or double-stranded DNA. A very small percentage of the injected genetic material is internalized by the cells and thus remains in an unintegrated, extrachromosomal form. This technique has been shown to result in the transient expression of the delivered genetic material. This particular technique may have a very defined but limited role in certain human gene therapy protocols requiring transient gene expression (Wolff et al., 1990).

E Ballistic Barrage Submicron-sized tungsten or gold particles spontaneously adsorb DNA. These DNA-coated heavy particles can be accelerated to high velocity to literally bombard a tissue for the purpose of introducing genetic material into it. Expression of the DNA which is bound to microparticle beads has been demonstrated in skin, muscle, liver, intestine, and mammary glands. The utility of this gene transfer system is still in the early stages of development and the applicability to liver-directed gene therapy has yet to be demonstrated.

G. Receptor-Mediated Gene Delivery One gene delivery approach which may have very specific applications for liver-directed gene therapy is receptor-mediated gene delivery. Many ci~ll

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surface receptors are found on many different cell types throughout the body. There are, however, certain receptors which are cell-type specific. One "liver-specific" receptor is the asiaolglycoprotein receptor (ASGPr) (for reviews see Ashwell and Harford, 1982; Stockert and Morell, 1983). This receptor has been used to specifically and selectively internalize and express DNA. The ligands for this receptor are asialoglycoproteins, such as asialoorsomucoid or asialo-fetuin. These ligands can be covalently linked to a positively charged moiety, such as poly-L-lysine. When mixed at proper rations with DNA, a ionic complex is formed between the negatively charged nucleic acid and the positively charged poly'L-lysine. The ligand portion of this complex binds to the liver-specific ASGPr and is subsequently internalized into the hepatocyte. This approach has been used to target and express DNA into hepatocytes in vitro and in vivo (Wu and Wu, 1987, 1988; Wu et al., 1989, 1991; Wilson et al., 1992b). One specific application of this approach was the in vivo delivery of a functional low density lipoprotein (LDL) receptor gene to the Watanabe heritable hyperlipidemic rabbit (WHHL) (Wilson et al., 1992b). The gene-protein complex was rapidly cleared from the circulation and specifically taken up by the liver subsequent to peripheral administration. Most importantly, there was also the appearance of LDL receptor RNA in the liver with a concomitant decrease in total serum cholesterol. However, this effect only lasted 6 days. Similar type results have been obtained for the delivery and expression of the albumin gene in Nagase analbuminemic rats (Wu et al., 1991) and the gene for methylmalonyl CoA mutase gene in mice (Stankovics et al., 1992). Another variation on this approach has been the use of synthetic ligands for the ASGPr rather than the big and bulky asialo-proteins. Since the receptor recognizes the terminal sugar complexes on these bulky serum proteins, trigalactosylated bisacridine analogues have been utilized (Haenser and Szoka 1993). Although intellectually very attractive in concept, the approach of hepatocyte receptor-mediated uptake and expression of systemically delivered gene/ligand complexes has significant practical limitations. First, the liverspecific ASGPr is actually not liver specific. It is expressed and functional in late-stage sperm (Monroe and Huber, 1994a,b), as well as many other tissues, including human tissues (Huber and Monroe, unpublished data). Since gene delivery to germ cells is ethically problematic and presently unacceptable from a regulatory standpoint, this may dramatically limit the human application of this approach. It remains to be determined if the blood/testis barrier will functionally circumvent this problem (Monroe and Huber, 1994a,b). A second limitation of this approach is the fact that subsequent to receptor binding and internalization, lysosomal degradation of the endosome-internalized gene/ligand complex degrades most of the internalized complex, hence limiting efficiency and persistence of expression

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(Cotten et al., 1990). One approach to circumvent lysosomal degradation is the coadministration of chemical lysosomatropic agents, but this has met with mixed results (Cotten et al., 1990). An alternative strategy to the use of chemical lysosomatropic agents is the coadministration of adenoviral components. Adenoviruses disrupt endocytic vesicles as part of their normal infection life cycle. This property was shown to significantly increase gene expression subsequent to gene/ligand complex administration (Curiel et al., 1994; Cristiano et al., 1993). The third limitation of this technique is the fact that expression is not persistent, which may require repeated administration of the complexes. However, the complexes have been shown to be antigenic (Ledley, 1993), which may limit their repeated administration and correspondingly limit their clinical utility. H. Liposomal Gene Delivery Another gene delivery system which may find limited clinical utility for liverdirected gene therapy is the use of liposomal transfer. The introduction of cationic liposome formulations has made liposomal-mediated gene transfer both practical, and in certain situations, efficient (Gao and Huang, 1993; Singhal and Huang, 1994). Cationic liposomes form a complex with negatively charged DNA. These complexes easily bind and are internalized by cells, probably by adsorption-mediated endocytosis (Zhou and Huang, 1994). A number of reports have demonstrated that liposomal vesicles can deliver to and express foreign genes in the liver compartment (Kato et al., 1992; Leibiger et al., 1990, 1991; Kaneda et al., 1989). However, much of the work involving liposomal transfer has been disappointing due to limited efficacy and persistence of gene expression. Liposomal-gene transfer may find the greatest utility for direct intralesional delivery, such as to the vascular endothelium to prevent restenosis after angioplasty. A specific limitation which may present itself for liposomal-mediated liver-directed gene therapy is the fact that liposomes are efficiently cleared by the reticuloendothelial (RES) system, which includes the Kupffer cells of the liver. Recent data suggest that modification of the chemical make-up of the liposome may help bypass RES degradation thereby decreasing Kupffer cell degradation (Singhal and Huang, 1994). Another interesting potential application of liposomal-mediated gene transfer is the further development of "targeted" liposomes. If this property is achieved, then systemically delivered genes may be targeted to specific cell types, including hepatocytes, through these new vectors. I. Retroviruses Retroviruses, a term designated to RNA viruses, have been extensively studied and applied to gene therapy in experimental and clinical settings. Retro-

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viruses have been extensively studied for decades based on their ability to cause tumors in animals with an extraordinary short latency period, their unusual mechanism of progressing through a D N A intermediate in their life cycle (see Figure 3 for retroviral life cycle), their propensity to propagate vertically once stably integrated into an appropriate host genome and finally, their ability to transmit genetic information horizontally. It is this last characteristic that has made them a very attractive vehicle for shuttle vectors in gene therapy experiments. Retroviral features which have fostered their use as a gene transfer vector include: 9 9 9 9

retroviruses potentially can be extremely efficient and expressing genetic information; retroviruses carry dispensable genetic information structural genes; in most situations, retroviral integration has little viability of the infected target cell; retroviruses can display a very broad host, tissue, range for infection.

at transferring in their effect on the and cell type

The Moloney murine leukemia virus has been the most extensively utilized retroviral vector to date. These vectors have been extensively modified so that they remain infectious but are replication defective. Hence, they can

Figure3. Retrovirallife cycle. Generic life cycle of a typical RNA virus using reverse transcriptase to convert an RNA genome into a DNA form which becomes randomly integrated into the host cell's genome.

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infect target cells and shuttle into the genome of those target cells therapeutic genes of choice. However, they are engineered to be replication defective being incapable of producing viral gene products and other viruses (for review see Jolly, 1994). These retroviral vectors have been shown to efficiently infect human hepatocytes in culture (Grossman et al., 1992a; Adams et al., 1992; Kaleko et al., 1991). Two critical features regarding retroviral vectors are: (1) integration and subsequent expression of the retroviral genome requires DNA synthesis to be occurring in the target cell at the time of infection, and (2) retroviruses become stably integrated into the target cells genome, theoretically enabling long-term, persistent expression of the therapeutic gene. These features, of course, have significant implications for liver-directed gene therapy. Since human hepatocytes in primary culture can go through a limited number of cell divisions, retroviruses have been successfully used in this ex vivo setting (Grossman et al., 1992; Adams et al., 1992). Due to the natural quiescent property of hepatocytes in situ, in vivo retroviral delivery to intact, normal liver is problematic. However, compensatory liver growth can be induced by surgical or chemical partial hepatectomy. In this regenerative milieu, retroviruses have been shown to be effective at transferring and expressing genes subsequent to direct liver injection or portal vein infusion (Kaleko et al., 1991; Ferry et al., 1991; Kay et al., 1992). To be practical in a clinical setting, methods other than subtotal hepatectomy will have to be developed to generate limited liver cell division. This again points to the importance of achieving a greater understanding of the molecular mechanisms involved in normal and pathogenic liver growth and differentiation.

J. Adenoviruses Similar to retroviruses, infectious but replication defective adenoviruses can be created to act as genetic shuttle vectors for liver-directed gene therapy. However, adenoviral vectors have three distinctly different characteristics compared to retroviral vectors: 9 adenoviruses do not require cell replication in the target cell enabling their use in normal hepatocytes in vivo; 9 adenoviruses do not stably integrate into the genome of the target cell, thus generating only transient expression for several weeks of the therapeutic gene; 9 some strains of adenoviruses appear to be significantly hepatotropic. The utility of in vivo administration of adenoviral vectors for liver-directed gene therapy has been demonstrated (Stratford-Perricaudet et al., 1990; Jaffe et al., 1992).

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IV. Clinical Applications of Gene Therapy Directed to the Hepatic Compartment The liver is an organ which presents very specific opportunities and challenges for the clinical application of gene therapy. The liver-associated clinical targets can be divided into four categories: 9 9 9 9

metabolic and plasma protein disorders oncology viral diseases hepatocellular transplantation

A. Metabolic and Plasma Protein Disorders The liver is an essential organ for many diverse life supporting functions, including the production site of essential serum proteins and the site for critical steps in intermediary metabolism to maintain homeostasis. Since many heritable disorders result from altered or disrupted hepatic gene expression, it is obvious that liver-directed gene therapy may have diverse clinical applications. There are two important issues, however, concerning liver-directed gene therapy which must be contemplated when considering a gene therapy approach for the correction of metabolic or plasma protein disorders: the generation of neoantigens, and the use of surrogate cells.

1. Generation o]: Neoantigens Many, if not all, of the metabolic or plasma protein disorders involve the loss of gene function by mutation. These mutation events result in either the complete absence of expression of a key protein or the expression of a qualitatively altered, nonfunctional protein. One potential concern about gene addition therapy for the type of disorder caused by loss of gene function is that the new protein product of the therapeutic gene may be recognized by the recipient as a neoantigen. This of course may lead to an immune response to the genetically corrected cells (Figure 4). Ascertaining the molecular etiology of the metabolic disorder will of course prove to be very important. Certain disorders have been shown to result from either mutations which completely stop expression of the essential metabolic protein or from missense mutations generating a qualitatively nonfunctional protein. Familial hypercholesterolemia (FH) is a good example of this (see below). FH is characterized by an inherited deficiency in functional low density lipoprotein (LDL) receptors. This deficiency may be caused by mutations which totally ablate LDL receptor gene expression or missense mutations (i.e., Try66Gly, exon 3) which cause the expression of a nonfunctional

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GENETIC METABOLIC DISORDERS

GENETIC THERAPY

POTENTIAL IMMUNOLOGICAL RESPONSE

HC

protein

\

NEOANTIGEN?

HC

pr

protein

Figure 4. Potential of neoantigen creation. Genetic metabolic disorders and plasma protein disorders are usually the result of mutations in important genes. These mutations may result in either the specific loss of expression of the important protein (A) or the translation of a qualitatively altered, nonfunctional protein (B). Genetic therapy may restore expression of the essential protein, thereby correcting the pathogenic state. In doing so, the new protein will be displayed by the major histocompatibility complex and may be recognized by the immune system as a neoantigen.

receptor (Leitersdorf et al., 1990; Turley and Dietschy, 1988). It is possible that there may be less immunological response if there is only one base difference between the therapeutic protein and the nonfunctional protein compared to the case where there is a complete absence of protein. Precedence for this has been demonstrated in the clinical protocols of Wilson and colleagues (see below, and Grossman et al., 1993a; Wilson et al., 1992a). In discussing the possibility of generating neoantigens by genetic therapy, it is important to put this in perspective with current therapy. Many of these disorders are routinely treated by continually supplying, exogenously, the essential liver enzyme or plasma protein. This, of course, has the same inherent problem with the possibility of producing an antigenic response. This is especially true if the protein or enzyme source is nonhuman. In addition, there are additional problems of contaminated products resulting in the contraction of other disease states, such as AIDS or viral hepatitis. It is important to point out, however, that there may be dynamic differences in the type of immune response when one compares providing the therapeutic

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protein exogenously, or via genetic therapy where it is produced inside a cell via gene transcription and translation. Finally, certain metabolic disorders have been treated by hepatic transplant (Starzl et al., 1984; Bilheimer et al., 1984). When one considers the inherent problems associated with hepatic transplant, not the least of which are organ rejection, continually immunosuppression, and organ procurement, it seems likely that genetic therapy has intrinsically less likelihood of immunological failure compared to hepatic transplant.

2. Surrogate Cells As mentioned, the liver compartment is the production site of some essential serum proteins as well as enzymes critical for intermediary metabolism to maintain homeostasis. For the gene addition strategy for human gene therapy, it may be able to replace the defective gene in cells other than the hepatocyte. This may be an obvious technical advantage in some situations. However, in many situations, surrogate cells for the hepatocyte are not practical. For many of the hepatocyte gene products (enzymes and serum proteins) to be functional, hepatocyte specific cofactors and post-translational modifications are essential. Surrogate cell populations would not provide these essential hepatocyte-specific properties to make functional proteins. Hence, for certain heritable inborn errors of metabolism, surrogate cells in the body would not be appropriate since they lack hepatocytespecific elements. An example of this is phenylalanine hydroxlase (PAH) deficiency, creating the disease state of phenylketonuria (PKU). PAH converts the essential amino acid phenylalanine to tyrosine. Without functional PAH activity there is both a build-up of phenylpyruvic acid and a deficiency of tyrosine. Although many cells in the body can be genetically engineered through gene addition therapy to produce PAH, it appears that this enzyme may only be functional in the hepatocyte since hepatocytes are the only cells which can synthesize and reduce the bioterin cofactor required for the phenylalanine hydroxylation reaction (Figure 5). There are many other examples that, based on essential hepatocyte-specific cofactors and/or post-transitional modifications, will limit the target cell population of gene therapy directly to the hepatocyte.

3. Applications of Liver-Directed Gene Therapy a. Familial Hypercholesterolemia (FH): FH is caused by an inherited deficiency of functional low density lipoprotein (LDL) receptors. These receptors are found on most cells but it appears that hepatic LDL receptors are critically important in the quantitative and qualitative regulation of

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PHENYLALANINE

o~~tarate NADPH + H+ + Diiydrobiopterin Tetrahydrobiopterin

l"

Phenylalanine "~'~e. 4-monooxygenase "~,., (PAH)

O2

NADP+ + Dihydrobiopterin + H20

Phenylpyruvio acid

Tyrosine

Figure 5.

Phenylalanine metabolism. The enzyme phenylalanine 4-monooxygenase or phenylalanine hydroxylase is used to convert phenylalanine to tyrosine. A deficiency of this enzyme increases the transamination of phenylalanine which increases the levels of phenylpyruvic acid.

cholesterol levels in the body (Leitersdorf et al., 1990). In the homozygous condition, without functional LDL receptors, cholesterol levels are not properly regulated, resulting in whole body cholesterol burden becoming pathogenically high. These high levels of cholesterol result in progressively severe atherosclerotic pathology and premature death. This genetic disorder can manifest itself in either two predominate ways. There can be either a missense mutation in the LDL receptor gene which produces a nonfunctional receptor or mutations which totally eliminate LDL receptor gene expression (Leitersdorf et al., 1990; Turley and Dietschy, 1988). FH is refractory to convention medical intervention. However, orthotopic liver transplantation has been demonstrated to correct the hypercholesterolemic condition, lowering both total cholesterol levels and LDL concentrations in the blood (Starzl et al., 1984; Bilheimer et al., 1984; Hoeg et al., 1987). These transplantation data indicate that correction of LDL receptors in the hepatic compartment may be sufficient to reverse the course of this inherited disease. However, it is also clear that medical intervention strategy other than orthotopic transplantation must be developed to treat this genetic disorder. This genetic disorder is a logical disease candidate for the clinical evaluation of gene therapy directed at the hepatic compartment since orthotopic liver transplantation is not without severe medical and practical limitations. Furthermore, there are no conventional treatment modalities for FH and the disease is lethal. To develop the gene therapy methodology to treat FH, two essential components were in place to evaluate the preclinical efficacy and safety of this approach. The cDNA for the human LDL receptor gene had been

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cloned and engineered into an appropriate retroviral vector (Grossman et al., 1992b). In the clinical study described below, this cDNA for human LDL receptor gene was constitutively expressed from a J3-actin promoter functional linked to a CMV enhancer sequence. Secondly, a very appropriate animal model existed which closely mimicked the human disease state. This model was a strain of rabbit called the WHHL rabbit which was genetically deficient functional LDL receptors. The original paradigm proposed for liver-directed gene therapy to treat FH was an ex vivo gene transfer approach. This approach involved the surgical resection of a portion of the liver which was then perfuse with collagenase to generate a single cell suspension of hepatocytes which were cultivated ex vivo. These hepatocytes were then infected with a retrovirus containing the human LDL receptor gene. Subsequent to infection, these hepatocytes were infused into the liver via the portal vein. The safety and efficacy of this autologous hepatocellular transplantation approach was demonstrated in the WHHL rabbit as well as in dogs and baboons (Chowdhury et al., 1991; Grossman et al., 1992b, 1993a). Due to these preclinical safety and efficacy studies, the RAC and FDA approved an ex vivo gene therapy protocol to treat FH. This clinical effort, directed by Dr. Jim Wilson, was initiated on June 5, 1992 when a 28-year-old female was first treated (the outline of this protocol is described in detail by Grossman et al., 1993a; Wilson et al., 1992a). Briefly, the left lateral segment of the liver was removed (15% of total mass) and perfused with collagenase to generate approximately 3 billion autologous hepatocytes which were plated into 800 10 cm 2 plates. These hepatocytes were then infected with a replication defective recombinant retrovirus described above. Based on one determination, it was estimated that a functional LDL receptor phenotype was generated in 20% of the hepatocytes. These hepatocytes were then infused slowly over 30 min into the portal vein via the inferior mesenteric vein in three separate aliquots. The detailed results of this initial clinical study are found in Grossman et al. (1993a). Briefly, the patient tolerated the procedure remarkably well and based on needle biopsy specimens, there were no apparent gross histopathology. In situ hybridization analysis of these biopsy samples indicated that LDL receptor expressing hepatocytes were still present at least 4 months after the therapy. b. ~ - l - a n t i t r y p s i n Deficiency: oL-1antitrypsin is a major circulating serum protein which has broad activity at inhibiting proteases. These proteases include trypsin, chymotrypsin, certain clotting factors, and elastase. Elastase in the lower respiratory tract is a particularly important substrate for this enzyme, oL-1 antitrypsin is normally produced predominately in the liver. Deficiencies of this enzyme usually result in the clinical symptoms of panacinar emphysema and cirrhosis of the liver.

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Prenatal genetic screening can identify individuals at risk for this disease. Currently, enzyme replacement therapy is being evaluated. However, like most enzyme replacement approaches, infusions of replacement enzyme would mean chronic (possibly weekly) therapy since the half-life of the enzyme is measured in days. An alternative approach is replacement of the defective gene through gene therapy to potentially have a more permanent correction. In this case, surrogate cells may be more practical than directing the gene therapy to the hepatic compartment. There may be no reason why hepatocytes would make a better oL-l-antitrypsin cell factory compared to other more accessible cells, such as lymphocytes. Since expression in these surrogate cells would also decrease with time, it would be less burdensome to infuse in T cells twice yearly than attempting liver-directed gene therapy at the same interval. c. Clotting Factor Deficiencies: Hemophilia is usually related to deficiencies in either clotting factor VIII (hemophilia A) or clotting factor IX (hemophilia B); these clotting factors are normally produced in the liver. Current therapy is based on continual replacement of the missing factors. Again, an alternative approach is to replace the defective gene(s) through gene therapy to potentially have a more permanent correction. Similar to o~-l-antitrypsin, surrogate cells may be the most appropriate target cells for replacement of these serum clotting factors. Ex vivo modification of skin fibroblasts, followed by skin grafting has demonstrated the principle of this approach. In fact, a human clinical trial was initiated in 1992 in China for factor IX replacement. In this trial, autologous skin fibroblasts obtained by biopsy, were infected with a replication defective retroviral vector containing the human factor IX gene. These modified fibroblasts were then grafted onto the backs of the patients. Initial results indicated that there was an increase in circulating factor IX levels following grafting. Challenges for further development of this approach include achieving both long-term expression and robust expression to correct this disorder.

B. Primary and Metastatic Liver Cancer Tremendous advances have been made in our understanding of the molecular events which contribute to the progressive development of cancer. For many types of cancers, including primary liver cancer, quantitative and qualitative alterations in oncogenes and tumor suppressor genes have been identified (see Chapters 6 to 12). Gene therapy approaches are also being developed to clinically address either primary or metastatic cancer in the hepatic compartment (for review see Huber, 1994). Like other liver-directed gene therapy approaches, these are based on a gene addition type strategy. The loss of tumor suppressor gene function has been demonstrated to be an important aspect of primary and metastatic tumors in the liver. One

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obvious approach would be to attempt to replace genes encoding tumor suppressing genes. Tumor suppressor gene replacement may have a unique theoretical advantage of being inherently nontoxic (for reviews see Finlay et al., 1989; Baker et al., 1990); however, the feasibility of tumor suppressor gene replacement may be limited by the following: 9

it may require very efficient gene transfer into most, if not all, tumor cells in a solid tumor mass; 9 it may require long-term expression unless the tumor cells go through an apoptotic process; 9 it may require direct knowledge of the causative genetic lesions in a tumor; 9 it may require that a tumor does not have redundant lesions that could substitute for that particular tumor suppressor gene. For these reasons, it appears that tumor suppressor gene replacement for primary or metastatic tumors in the liver faces many hurdles which present day technology may not be able to sufficiently overcome. An alternative approach is to deliver to primary or metastatic tumors in the liver genes that directly or indirectly generate toxins in the tumor (for review see Huber, 1994; Huber et al., 1991, 1993, 1994; Huber and Lazo, 1994). Genes that encode a toxin will be directly toxic to the tumor cell. Genes that encode nonmammalian enzymes can produce toxicity when a nontoxic prodrug is administered. This nontoxic prodrug is converted to a toxic metabolite at the tumor site by expression of the nonmammalian enzyme in the tumor. These approaches may have neighboring cell effects that will not require the delivery of the therapeutic gene to every tumor cell and not require continued long-term expression (Huber et al., 1991, 1993, 1994). Another approach is to deliver genes to the tumor cells that encode immunostimulatory proteins. These proteins may directly or indirectly stimulate the immune system to recognize the tumor as foreign and reject it. Most importantly, if the immunological cascade is initiated, then the possibility exists that the immune system may also recognize genetically unmodified tumor cells as well (for reviews see Huber and Lazo, 1994). A final approach is the use of genes encoding drug resistance genes, such as the multidrug resistance gene (MDR) or an altered dihydrofolate reductase gene, to transfer into normal cells (i.e., bone marrow stem cells) to attempt to make traditional cancer chemotherapy more efficacious (for reviews see Huber and Lazo, 1994). C. Viral Diseases of the Liver

Gene therapy treatment of viruses which directly or indirectly cause liver damage, such as hepatitis B virus and hepatitis C virus has yet to be exploit-

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ed. This is due, in part, to the inability to propagate and establish productive infections systems in vitro. In the future it may be possible to deliver genes to the liver which encode proteins that: 9 9 9 9

make the hepatocyte noninfectable; make the hepatocyte incompetent to sustain viral replication; make the hepatocyte resistant to the toxic effects of the virus; and make the hepatocyte present to the immune system viral antigens which can enhance the immunological response to the virus.

D. Hepatocellular Transplantation For patients in endstage liver disease resulting from environmental influences (hepatitis B virus, drug-induced toxicity), inborn errors of metabolism, or congenital malformations of the liver and biliary tract, orthotopic liver transplantation (OLT) may have previously represented the last and only therapeutic option (see Chapter 13) (Starzl et al., 1989a,b,c; Whitngton and Balistreri, 1991). The overall success rate of OLT has dramatically increased in the last decade due to the following: 9

introduction of effective immunosuppressive agents, such as cyclosporin and immuran, to inhibit transplant rejection; 9 greater awareness in the general population regarding organ donation; 9 improved methods for preserving and transporting donated organs (Benichou et al., 1977; Sumimoto et al., 1989); 9 improved surgical techniques (Bismuth and Houssin, 1984; Broelsch et al., 1990; Otte et al., 1990). However, the general and widespread application of this procedure is limited because there is: 9 a high incidence of morbidity and mortality even in the best circumstances; 9 difficulty in donor liver procurement creating a constant shortage of transplantable organs; 9 a tremendous cost in terms of real dollars and burden on our health care system (Whitington and Balistreri, 1991). As an alternative approach, hepatocellular transplantation (HCT) has been suggested. In this approach, allogenic hepatocytes from a donor liver are prepared and infused into the patient. Site of infusion and engraftment could be the diseased liver to provide the essential milieu for engraftment but it could conceivably be alternative sites in the body as well. Indeed, it has been well established that primary hepatocytes can engraft and function

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for limited periods in sites other than the liver compartment (Jirtle and Michalopoulos, 1982; Jirtle et al., 1980; Demtriou et al., 1986). Transplanted hepatocytes would then provide the essential hepatic functions to maintain homeostasis. This type of approach may be useful in the following medical situations: 9 providing short-term hepatic function as a "stop gap" until liver procurement for OLT; 9 providing a "bridge to recovery" allowing patients with fulminate hepatic failure to recover; 9 if the transplanted hepatocytes stably engraft and remain functional, then conceivably this therapeutic approach could be an alternative therapy to OLT. HCT has been demonstrated to reverse hepatic insufficiency in animal models. Techniques optimized for rodents or primates have shown that hepatocytes (autologous or allogenic) can be harvested, maintained ex v i v o in a cell culture environment, perhaps expanded for a limited number of cell divisions, and transplanted into a recipient animal while still maintaining expression of liver-specific functions. Hepatocyte marking with genetic tags may also allow critical questions to be addressed which will help in the clinical development of hepatocellular transplant.

V. Conclusions In conclusion, technology is presently available to initiate the first clinical evaluation of gene addition type gene therapy protocols directed at inherited or acquired diseases of the liver. Gene therapy in the liver compartment presents some unique issues and challenges. The nature of these challenges are related to the fact that adult liver is normally a nondividing organ; the liver has a remarkable ability for regenerative and compensatory growth; and autologous hepatocyte transplantation is feasible. A direct consequence regarding these liver-specific issues will be the development and refinement of the types of technology needed for the widespread application of hepatic gene therapy. It is anticipated that gene transfer vectors which can efficiently transduce nondividing hepatocytes and maintain long-term expression will eventually be developed. Gene delivery vectors that efficiently target hepatocytes may make systemic delivery a practical reality. Isolation and e x v i v o propagation of hepatic stem cells may also impact the clinical application of hepatic gene therapy. The first clinical applications of hepatic gene therapy have been important milestones for the "clinical proof of principle" for this new area of medicine. It is with much anticipation that we look to the future for the

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technological advances that will make hepatic gene therapy a practical reality for many debilitation acquired and inherited diseases of the liver.

References Adams, R. M., Soriano, H. E., Wang, M., Darlington, G., Steffen, D., and Ledley, E D. (1992). Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors. Proc. Natl. Acad. Sci. USA 89, 8981-8985. Anderson, W. E (1984). Prospects of human gene therapy. Science 226, 401-409. Anderson, K. D., Thompson, J. A., DiPietro, J. M., Montgomery, K. T., Reid, L. M., and Anderson, W. E (1989). Gene expression in implanted rat hepatocytes following retroviralmediated gene transfer. Somat. Cell Mol. Genet. 15,215-227. Ashwell, G., and Harford, J. (1982). Carbohydrate-specific receptors of the liver. Annu. Rev. Biochem. 51,531-540. Baker, S. J., Markowitz, S., Fearon, E. R., Willson, J. K., and Volgelstein, B., (1990). Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 249, 912-915. Baumgartner, D., LaPlante-O'Neil, P. M., Sutherland, D. E., and Najarian, J. S. (1983). Effects of intrasplenic injection of hepatocytes, hepatocyte fragments and hepatocyte culture supernatants of o-galactosamine-induced liver failure in rats. Eur. Surg. Res. 15, 129-135. Benichou, J., Halgrimson, C. G., Weil, R. III, Koep, L. J., and Starzl, T. E. (1977). Canine and human liver preservation for 6 to 18 hours by gold infusion. Transplantation 24, 407-411. Benvenisty, N., and Reshef, L. (1986). Direct introduction of genes into rats and expression of the genes. Proc. Natl. Acad. Sci. USA 83, 9551-9555. Bilheimer, D. W., Goldstein, J. L., Grundy, S. M., Starzl, T. E., and Brown, M. (1984). Liver transplantation to provide low density lipoprotein receptors and lower plasma cholesterol in a child with homozygous familial hypercholesterolemia. N. Engl. J. Med. 311, 16581664. Bismuth, H., and Houssin, D. (1984). Reduced-size orthotopic liver graft in hepatic transplantation in children. Surgery 95, 367-370. Bollag, R. J., Waldman, A. S., and Liskay, R. M. (1989). Homologous recombination in mammalian cells. Annu. Rev. Genet. 23, 199-225. Bowen, K. M., Andrus, L., and Lafferty, K. J. (1980). Successful allotransplantation of mouse pancreatic islets to nonimmunosuppressed recipients. Diabetes 29, 98-104. Broelsch, C. E., Edmond, J. C., Whitington, P. E, Thistlethwaite, J. R., Baker, A. L., and Lichtor, J. L. (1990). Application of reduced-size liver transplants as split grafts, auxiliary orthotopic grafts and living related segmental transplants. Ann. Surg. 212, 368-375. Capecchi, M. R. (1989). Altering the genome by homologous recombination. Science 244, 1288-1292. Chowdhury, J. R., Grossman, M., Gupta, S., Chowdhury, N. R., Baker, J. R., Jr., and Wilson, J. M. (1991). Long-term improvement of hypercholesterolemia after ex vivo gene therapy in the LDLR deficient rabbits. Science 254, 1802-1805. Cotten, M., Laengle-Rouault, E, Kirlappos, H., Wangner, E., Mechtler, K., Zenke, M., Beug, H., and Birnstiel, M. L. (1990). Transferrin-polycation-mediated introduction of DNA into human leukemic cells: Stimulation by agents that affect the survival of transpected DNA or modulate transferrin receptor levels. Proc. Natl. Acad. Sci. U.S.A. 87, 4033-4037. Cristiano, R. J., Smith, L. C., and Woo, S. L. (1993). Hepatic gene therapy, adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90, 2122-2126. Curiel, D. T., Agarwal, S., Wagner, E., and Cotten, M. (1991). Adenovirus enhancement of

14.

Gene Therapy for the Treatment of Inherited and Acquired Diseases of the Liver

379

transferrin-polylysine-mediated gene delivery. Proc. Natl. Acad. Sci. U.S.A. 88, 88508854. Demetriou, A. A., Whitinig, J., Levenson, S. M., Chowdhury, N. R., Schechner, R., Michalski, S., Feldman, D., and Chowdhury, J. R. (1986). New method of hepatocyte transplantation and extra corporeal liver support. Ann. Surg. 204, 259-271. DePamphilis, M. L., Herman, S. A., Martinez-Salas E., Chalifour, L. E., Wirak, D. O., Cupo, D. Y., and Miranda, M. (1988). Microinjecting DNA into mouse ova to study DNA replication and gene expression and to produce transgenic animals. Bio technology 6, 662680. Evarts, R. P., Nagy, P., Marsden, E., and Thorgeirsson, S. S. (1987). A precursor-product relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 8, 17371740. Fausto, N. (1990). Hepatocyte differentiation and liver progenitor cells. Curr. Opin. Cell Biol. 2, 1036-1042. Fausto, N. (1991). Protooncogenes and growth factors associated with normal and abnormal liver growth. Dig. Dis. Sci. 36, 653-658. Fausto, N., Lemire, J. M., and Shiojiri, N. (1992). Oval cells in liver carciniogenesis. Cell lineages in hepatic development and the identification of stem cells in normal liver. In "The Role of Cell Types in Hepatocarcinogenesis" (A. E. Sirica, ed.), pp. 89-108. CRC Press, Boca Raton, FL. Fausto, N., Lemire, J. M., and Shiojiri, N. (1993). Cell lineages in hepatic development and the identification of progenitor cells in normal and injured liver. Proc. Soc. Exp. Biol. Med. 204, 237-241. Ferry, N., Duplessis, O., Houssin, D., Danos, O., and Heard, J. M. (1991). Retroviral mediated gene transfer into hepatocytes in vivo. Proc, Natl. Acad. Sci. U.S.A. 88, 8377-8381. Finlay, C. A., Hinds, P. W., and Levine, A. J. (1989). The p53 proto-oncogene can act as a suppressor of transformation. Cell 57, 1083-1093. Francavilla, A., Panella, C., Polimeno, L., Giangaspero, A., Mazzaferro, V., Pan, C. E., Van Thiel, D. H., and Starzl, T. E. (1990). Hormonal and enzymatic parameters of hepatic regeneration in patients undergoing major liver resections. Hepatology 12, 1134-1138. Gao, X., and Huang, L. (1993). Cationic liposomes and polymers for gene transfer. J. Liposome Res. 3, 399-410. Gibson-D'Ambrosio, R. E., Crowe, D, L., Shuler, C. E., and D'Ambrosio, S. M. (1993). The establishment and continuous subculturing of normal human adult hepatocytes, and expression of differentiated liver functions. Cell Biol. Toxicol. 9, 385-403. Grossman, M., Raper, S. E., and Wilson, J. M. (1991). Towards liver-directed gene therapy: Retrovirus-mediated gene transfer into human hepatocytes. Somat. Cell Mol. Genet. 17, 601-607. Grossman, M., Raper, S. E., and Wilson, J. M. (1992). Transplantation of genetically modified autologous hepatocytes in non-human primates: Feasibility and short-term toxicity. Hum. Gene Ther. 3, 501-510. Grossman, M., Wilson, J. M., and Raper, S. E. (1993). A novel approach for introducing hepatocytes into the portal circulation. J. Lab. Clin. Med. 121, 472-478. Grossman, M., Raper, S. E., Kozarsky, K., Stein, E. A., Engelhardt, J. F., Muller, D., Lupien, P. J., and Wilson, J. M. (1994). Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolaemia. Nat. Genet. 6, 335-341. Haensler, J., and Szoka, F. C, Jr. (1993). Synthesis and characterization of a trigalactosylated bisacridine compound to target DNA to hepatocytes. Bioconjug. Chem. 4, 85-93. Hixson, D. C., Faris, R. A., Yang, L., and Novikoff, P. (1992). Antigenic cues to liver development, renewal, and carcinogenesis: An integrated model. In "The Role of Cell Types in Hepatocarcinogenesis" (A. E. Sirica, ed.), pp. 151-182. CRC Press, Boca Raton, FL.

380

Brian E. Huber

Hoeg, J. M., Starzl, T. E., and Brewer, H. B., Jr. (1987). Liver transplantation for the treatment of cardiovascular disease. Comparison with medication and plasma exchange in homozygous familial hypercholesterolemia. Am. J. Cardiol. 59, 705-707. Huber, B. E. (1994). Gene therapy strategies for treating neoplastic disease. In "Gene Therapy for Neoplastic Diseases" (B. E. Huber and J. S. Lazo, eds.) Ann. N e w York Acad. Sci. 716, 6-11. Huber, B. E., Richards, C. A., and Krenitsky, T. A. (1991). Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma. An innovative approach for cancer therapy. Proc. Natl. Acad. Sci U.S.A. 88, 8039-8043. Huber, B. E., Austin, E. A., Good, S. S., Knick, V. C., Tibbels, S., and Richards, C. A. (1993). In Vivo antitumor activity of 5-flurocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer Res. 53, 4619-4626. Huber, B. E., Austin, E. A., Richards, C. A., Davis, S. T., and Good, S. S. (1994). Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene. Significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc. Natl. Acad. Sci. U.S.A. 91, 8302-8306. Huber, B. E., and Lazo, J. S. (1994). Gene Therapy for Neoplastic Diseases. Ann. New York Acad. Sci., Vol. 716, New York. Ismail, T., Howl, J., Wheatley, M., McMaster, P., Neuberger, J. M., and Strain A. J. (1991). Growth of normal human hepatocytes in primary culture. Effect of hormones and growth factors on DNA synthesis. Hepatology 14, 1076-1082. Jaffe, H. A., Danel, C., Longenecker, G., Metzger, M., Setoguchi, Y., Rosenfield, M. A., Gant, T. W., Thorgeirsson, S. S., Stratford-Perricaudet, and Perricaudet, M. (1992). Adenovirusmediated in vivo gene transfer and expression in normal rat liver. Nature Genet. 1, 372378. Jirtle, R. L., and Michalopoulos, G. (1982). Effects of partial hepatectomy on transplanted hepatocytes. Cancer Res. 42, 3000-3004. Jirtle, R. L., Biles, C., and Michalopoulos, G. (1980). Morphological and histochemical analysis of hepatocytes transplanted into syngeneic hosts. Am. J. Pathol. 101, 115-126. Jolly, D. (1994). Viral vector systems for gene therapy. Cancer Gene Ther. 1, 51-64. Kaleko, M., Garcia, J. V., and Miller, A. D. (1991). Persistent gene expression after retroviral gene transfer into liver cells in vivo. Hum. Gene Ther. 2, 27-32. Kaneda, Y., Iwai, K., and Uchida, T. (1989). Introduction and expression of the human insulin gene in adult rat liver. J. Biol. Chem. 264, 12126-12129. Kato, K., Nakanishi, M., Kaneda, Y., Uchida, T., and Okada, Y. (1991). Expression of hepatitis B virus surface antigene in adult rat liver. Co-introduction of DNA and nuclear protein by a simplified liposome method. J. Biol. Chem. 266, 3361-3364. Kay, M. A., Li, O., Liu, T. J., Leland, E, Toman, C., Finegold, M., and Woo, S. L. (1992). Hepatic gene therapy. Persistent expression of human alpha-1 antitrypsin in mice after direct gene delivery in vivo. Hum. Gene Ther. 3, 641-647. Kimbrough, R. D., Linder, R. E., and Gaines, T. B. (1972). Morphological changes in liver of rats fed polychlorinated biphenyls: Light microscopy and ultrastructure. Arch. Environ. Health 25, 354-364. Kucherlapati, R., and Skoultchi, A. I. (1984). Introduction of purified genes into mammalian cells. Crit. Rev. Biochem. 16, 349-388. Lafferty, K. J., Prowse, S. J., Simeonovic, C. J., and Warren, H. S. (1983). Immunobiology of tissue transplantation: A return to the passenger leukocyte concept. Ann. Rev. Immunol. 1, 143-173. Lanford, R. E., Carey, K. D., Estlack, L. E., Smith, G. C., and Hay, R. V. (1989). Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-flee medium in vitro. Cell. Dev. Biol. 25, 174-182.

14.

Gene Therapy for the Treatment of Inherited and Acquired Diseases of the Liver

381

Ledley, E D. (1994). Pharmacokinetic issues in the use of genes as medicines. In "Gene Therapeutics: Methods and Applications of Direct Gene Transfer" (J. A. Wollf, ed.). Birkhauser, New York. Ledley, F. D., Woo, S. L., Ferry, G. D., Whisennand, H. H., Brandt, M. L., Darlington, G. J., Demmler, G. J., Finegold, M. J., Pokorny, W. J., Rosenblatt, H., Schwartz, P., Anderson, W. E, and Moen, R. C. (1991). Hepatocellular transplantation in acute hepatic failure and targeting genetic markers to hepatic cells. Hum. Gene Ther. 2, 331-358. Leibiger, I., Leibiger, B., Sarrach, D., Walther, R., and Zuhlke, H. (1990). Genetic manipulation of rat hepatocytes in vivo. Implications for a therapy model of type-1 diabetes. Biomed. Biochim. Acta 49, 1193-1200. Leibiger, B., Leibiger, I., Sarrach, D., and Zuhlke, H. (1991). Expression of exogenous DNA in rat liver cells after liposome-mediated transfection in vivo. Biochem. Biophys. Res. Commun. 174, 1223-1231. Leitersdorf, E., Tobin, E. J., Davignon, J., and Hobbs, H. H. (1990). Common low-density lipoprotein receptor mutations in the French canadian population. ]. Clin. Invest. 85, 1014-1023. Li, A. P., Colburn, S. M., and Beck, D. J. (1992). A simplified method for the culturing of primary adult rat and human hepatocytes as multicellular spheroids. In Vitro Cell. Dev. Biol. 28A, 673-677. Michalopoulos, G. K. (1990). Liver regeneration. Molecular mechanisms of growth control. FASEB J. 4, 176-187. Michalopoulos, G. K., and Zarnegar, R. (1992). Hepatocyte growth factor. Hepatology 15, 149-155. Monroe, R. S., and Huber, B. E. (1994a). Characterization of the hepatic asialoglycoprotein receptor in rat late-stage spermatids and epididymal sperm. Gene 148, 261-268. Monroe, R. S., and Huber, B. E. (1994b). The major form of the murine asialoglycoprotein receptor, cDNA sequence and expression in liver, testis and epididymis. Gene 148, 237244. Moscioni, A. D., Roy-Chowdhury, J., Barbour, R., Brown, L. L., Roy-Chowdhury, N., Competiello, L. S., Lahiri, P., and Demetriou, A. A. (1989). Human liver cell transplantation: Prolonged function in athymic-athymic-analbuminemic hybrid rats. Gastroenterology 96, 1546-1551. Naji, A., Silver, W. K., and Barker, C. F. (1981). Influence of organ culture on the survival of major histocompatible complex-compatible and incompatible parathyroid allografts in rats. Transplantation 32, 296-298. Otte, J. B., deVille-deGoyet, J., Stokal, E., Albberti, D., Mouslin, D., deHemptinne, B., Veyckemas, F., van Obbergh, L., Carlier, M., and Clapuyt, P. (1990). Size reduction of the donor liver is a safe way to alleviate the shortage of size-matched organs in pediatric liver transplantation. Ann. Surg. 211, 146-157. Ponder, K. P., Gupta, S., Leland, E, Darlington, G., Finegold, M., Demayo, J., Ledley, E D., Chowdhury, J. R., and Woo, S. L. (1991). Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation. Proc. Natl. Acad. Sci. U.S.A. 88, 1217-1221. Rao, M. S., Bendayan, R. D., Kimbrough, R. D., and Reddy, J. K. (1986). Characterization of pancreatic-type tissue in the liver of rats induced by polychlorinated biphenyls. J. Histochem. Cytochem. 34, 197-201. Reid, L., Fiorino, A., Sigal, S., Brill, S., and Hoist, P. (1992). Extracellular matrix gradients in the space of Disse: Relevance to liver biology. Hepatology 15, 1198-1203. Shigekawa, K., and Dower, W. J. (1988). Electroporation of eukaryotes and prokaryotes: A general approach to the introduction of macromolecules into cells. Biotechnology 6, 742751.

382

Brian E. Huber

Singhal, A., and Huang, L. (1994). Gene transfer in mammalian cells using liposomes as carries. In "Gene Therapeutics Methods and Applications of Direct Transfer" (J. A. Wolff, ed.). Birkhauser, New York. Sratford-Perricaudet, L. D., Levrero, M., Chasse, J. E, Perricaudet, M., and Briand, P. (1990). Evaluation of the transfer and expression in mice of an enzyme-encoding gene using a human adenoviral vector. Hum. Gene Ther. 1, 241-256. Stankovics, J., Andrews, E., Wu, G., and Ledley, E D. (1992). Overexpression of human methylmalonyl CoA mutase (MCM) in mouse liver after in vivo gene delivery using asialoglycoprotein complexes. Am. J. Hum. Genet. 51, A177. Starzl, T. E., Bilheimer, D. W., Bahnson, H. T., Shaw, B. W., Jr., Hardesty, R. L., Griffith, B. P., Iwatsuki, S., Sitelli, B. J., Gartner, J. C., Jr., Malatack, J. J., et al. (1984). Heart-liver transplantation in a patient with familial hypercholesterolemia. Lancet 1(8391), 13821383. Starzl, T. E., Demetris, A. J., and Van-Thiel, D. (1989a). Liver transplantation (1). N. Engl. J. Med. 321, 1014-1022. Starzl, T. E., Demetris, A. J., and Van-Thiel, D. N. (1989b). Liver transplantation (2). N. Engl. J. Med. 321, 1092-1099. Starzl, T. E., Todo, S., Tzakis, A..G, Gordon, R. D., Makowka, L., Stieber, A., Podesta, L., Yanaga, K., Concepcion, W., and Iwatsuki, S. (1989c). Transplant Proc. 21, 2197-2200. Stockert, R. J., and Morell, A. G. (1983). Mepatic binding protein: The galactose-specific receptor of mammalian hepatocytes. Hepatology 3, 750-757. Sumimoto, R., Jamieson, N. V., Wake, K., and Kamada, N. (1989). 24-hour rat liver preservation using UW solution and some simplified variants. Transplantation 48, 1-5. Tatematsu, M., Kaka, T., Medline, A., and Farber, E. (1985). Intestinal metaplasia as a common option of oval cells in relation to cholangiofibrosis in livers of rats exposed to 2-acetylaminofluorene. Lab. Invest. 52, 354-362. Thompson, J. A., Anderson, K. D., and Dipietro, J. M., Zwiebel, J. A., Zametta, M., Anderson, W. E, and Maciag, T. (1988). Site-directed neovessel formation in vivo. Science 241, 1349. Thorgeirsson, S. S., and Evarts, R. P. (1992). Growth and differentiation of stem cells in adult rat liver. In "The Role of Cell Types in Hepatocarcinogenesis" (A. E. Sirica, ed.), pp. 102120. CRC Press, Boca Raton, FL. Thorgeirsson, S. S., Evarts, R. P., Bisgaard, H. C., Fujio, K., and Hu Z. (1993). Hepatic stem cell compartment: Activation and lineage commitment. Proc. Soc. Exp. Biol. Med. 204, 253-260. Turley, S. D., and Dietschy, J. M. (1988). The metabolism and excretion of cholesterol by the liver. In "Liver Biology and Pathology" (I. M. Arias, W. B. Jakoby, H. Popper, D. Schachter, and D. A. Shafritz, eds.), pp. 617-641. Raven Press, New York. Vacanti, J. P., Morse, M. A., Saltzman, W. M., Domb, A. J., Perez-Atayde, A., and Langer, R. (1988). Selective cell transplantation using bioabsorbable artificial polymers as matrices. J. Pediatr. Surg. 23, 3-9. Whitington, P. E, and Balisteri, W. E (1991). Liver transplantation in pediatrics: Indications, contraindication, and pretransplant management. J. Pediatr. 118, 169-177. Wilson, J. M., Grossman, M., Raper, S. E., Baker, J. R., Jr., Newton, R. S., and Thoene, J. G. (1992a). Ex vivo gene therapy of familial hypercholesterolemia. Hum. Gene Ther. 3, 179222. Wilson, J. M., Grosman, M., Wu, C. H., Chowdhury, N. R., Wu, G. Y., and Chowdhury, J. R. (1992b). Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits. J. Biol. Chem. 276, 963-967.

14.

Gene Therapy for the Treatment of Inherited and Acquired Diseases of the Liver

383

Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A., and Feigner, P. L. (1990). Direct gene transfer into mouse muscle in vivo. Science 247, 1465-1468. Wu, G. Y., and Wu, C. H. (1987). Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. 262, 4429-4432. Wu, G. Y., and Wu, C. H. (1988). Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263, 14621-14624. Wu, C., Wilson, J. M., and Wu, G. Y. (1989). Targeting genes: Delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J. Biol. Chem. 262, 16985-16987. Wu, G. Y., Wilson, J. M., Shalaby, E, Grossman, M., Shafritz, D. A., and Wu, C. H. (1991). Receptor-mediated gene delivery in vivo. J. Biol. Chem. 266, 14338-14342. Zajicek, G., Oren, R., and Weinreb, M. J. (1985). The streaming liver. Liver 5, 293-300. Zhou, X., and Huang, L. (1994). DNA transfection mediated by cationin liposomes containing lipopolysine: Characterization and mechanism of action. Biochim. Biophys. Acta 1189(2), 195-203.

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Index Page citations followed by "f" or "t" refer to figure or table, respectively.

A AAF, see 2-Acetylaminofluorene 2-Acetylaminofluorene, and hepatocarcinogenesis, 104-105 Activator protein- 1, promoter, regulation of target genes controlled by, 80-82,

82[, 83/ Activin apoptosis and, 157 in liver regeneration, 17 Acute yellow atrophy, see Hepatitis, fulminant Adenoma, hepatic, 328-329 active cell death and, 165-166 clinical features, 329, 337t epidemiology, 328, 337t pathology, 329 surgical treatment, 329, 337t S-Adenosylmethionine antipromoting effect on liver foci and nodules, 165 DNA maintenance methylase activity and, 231, 232f protective effect against liver tumorigenesis, 242 rodent liver tumorigenesis and, 238-239 Adenoviruses, as gene transfer vectors, 368 aFGF, see Fibroblast growth factor, acidic Aflatoxin hepatocellular carcinoma and, 199, 302 transgenes and, in hepatic neoplasia, 278-280, 279t Alpha- 1-antitrypsin deficiency liver cancer risk with, 304 liver-directed gene therapy in, 373374

mutant gene regulatory element, 272t hepatic neoplasia and sequence of hepatic lesions, 272t, 274-275 in transgenic animals, 272t, 274275 Alpha-fetoprotein gene, regulatory elements, 286 in RLE cell lines, during transformation in vitro, 107, 108f Androgens, in liver regeneration, 11-12 Angiotensin II, in liver regeneration, 9 Antigens, see specific antigen AP-1, see Activator protein-1 Apoptosis, 204, 2 0 5 f biochemistry, 152-158 cell loss per hour, calculation, 152 cell selection for, 151 chromatin condensation in, 153 clearance of apoptotic cells and fragments after, 151, 152 death signals, 152-153 definition, 142t, 143, 216 DNA fragmentation assays and, 146 DNA fragmentation in, 153 DNA ladders and, 145-146, 153 with genotoxic damage, and p53 gene, 155

in hepatocarcinogenesis, 142, 216-217 in liver detection, 145-146 duration, 151-152 models, 146-151 quantification, 145-146 mitogen rescue and, 148, 151-152 molecular aspects, 152-158

385

386 Apoptosis (continued) morphologic characteristics, 143, 144f, 216 and necrosis, discrimination, pre-TGFJ31 as marker, 157 point of no return, 152 in preneoplastic liver foci, 161-164 preparatory segment, 152 genes participating in, 154-156 proteases in, 154 regulation, by intercellular contacts, 151 regulatory segment, 152 stages, 143, 144f, 216 suicide segment, 152, 153 transforming growth factor 131 and, 157, 217 transglutaminase in, 153-154 in tumor cells, 1 4 1 - 145 blocked by IGF2, 128 Arteriography, in liver disease, 325t Ataxia telangiectasia, cell cycle kinetics in, 182 5-Azacytidine, gene expression and, 233234

B Ballistic barrage, gene transfer by, 364 Beckwith Wiedeman syndrome, 126 Benzodiazepine compounds, as tumor promoters, 199 Biliary tumors, in viral oncogene transgenic mice, 261-262

C CAAT enhancer binding protein, 77 Caenorhabditis elegans

active cell death in, 154 genes ced3, 154 ced9, 155 Calcitonin, in liver regeneration, 10-11 Calcium, in liver regeneration, 10-11 Caloric restriction DNA maintenance methylation and, 242-243 rodent lifespan and, 242 cAMP response element, promoter, regulation of target genes controlled by, 8082, 82f, 83f Carbon tetrachloride, induction of apoptosis in liver, 149

Index

Carcinogenesis cell proliferation in, 245, 245f c-met in, 62-63 critical events in, 245f hepatic, see also Tumor promotion active cell death in, 158-166 apoptosis and, 142, 216-217 cell cycle regulation and, 181-183, 204-208, 205f hepadnavirus research and, 113-140 hepatic stem cells and, 104-106 HGF in, 42-43 human environmental factors, 302 genetics, 302-304 initiation, 159, 200-202, 200f active cell death and, 161-163 prestages, 158-160 cell proliferation and death in, kinetics, 160f, 160-161 progression, 159, 201,200f, 203204 promotion, 201-203, 200f sex hormones and, in mice, 310-311 stages, 200-204, 200f transforming growth factor [3 and, 208-216 transgene cooperativity in, 276-278, 277t transgene-induced, cooperating events in, 276 transgenic models, principles and rationale for, 257-259 two-stage model, 160f, 160-161 initiating agents, 227-228; see also Tumor promoters initiation, 245, 245f, 305 definition, 227 mechanism genotoxic, 228-229 nongenotoxic, 227-228 multistage, 200, 200f, 305 DNA methylation and, 245, 245f in SV40-Tag transgenic mice, 261 in SV40-Tag transgenic rat, 262 transgene cooperativity in, 276-278, 277t natural history of, 179-180 progression, 203, 228 promotion, 202, 305 stages, 201-204, 227

387

Index

Carcinogens chemical, 199, 227 transgenes and, in hepatic neoplasia, 278-280, 279t genotoxic, 199 as mutagens, 227 non-genotoxic, and spontaneously initiated cells, 163, 235 as tumor promoters, 199 Carcinoma, see Hepatocellular carcinoma; Liver cancer; Liver tumors, malignant cdks, see Cyclin-dependent kinases C/EBP, see CAAT enhancer binding protein Cell cycle checkpoints, 181-183 genetic instability and, 183, 204 kinetics, in liver regeneration, 4, 18, 7172, 72f lifespan extension, 182-183 regulation hepatic carcinogenesis and, 204-208, 205f

mechanisms, 180-181 restriction (R) checkpoint, 204, 2 0 5 f stages, 204 Cell death, see also Apoptosis active characteristics, 142, 142t functions, 142-143 in hepatocarcinogenesis, 158-166 in hormone-dependent tumors, 141, 144-145 and initiation of tumorigenesis, 161163 in liver, 145-152 after adaptive hyperplasia, 146-149, 147t detection, 145-146 models, 146-151 quantification, 145-146 after toxic injury, 146, 149-151 in mammary cancer, 141, 144-145 mechanisms, 152-153 morphologic characteristics, 143 phylogenetic distribution, 142-143 in prostate cancer, 141 signal factors in, 156-158 treatments aimed at, 141-142 tumor promotion and, 163-165 tumors and, 165-166

in tumors and tumor prestages, 141145 types, 142-145, 142t programmed definition, 145 in embryonal development, 142, 145 Cholangiocarcinoma epidemiology, 334 pathology, 334 surgical treatment, 334 in viral oncogene transgenic mice, 261262 Cholangiofibroma, in viral oncogene transgenic mice, 261-262 Choline depletion, liver tumorigenesis and, 238239 dietary, protective effect against liver tumorigenesis, 242 Chromatin condensation, in apoptosis, 153 Chromosome 11p genes, in hepatocarcinogenesis, 126 loss of heterozygosity, in HCC, 126 CL-6, identification, 88-89 Clofibrate, as tumor promoter, in inbred mice, genetic factors affecting, 311 Clotting factor deficiencies, liver-directed gene therapy in, 374 Clusterin, gene, 156 c - m e t , see Hepatocyte growth factor, receptor; Proto-oncogene, c - m e t Collagenase, pretreatment, and mitogenic effects of hepatocyte growth factor, 37 Computed tomography, in liver disease, 325t, 326 Contraceptive steroids, as tumor promoters, 199 Councilman bodies, 150 CPA, see Cyproterone acetate CRE, see cAMP response element Cross-circulation experiments, in rats, 5-6 Cyclin-dependent kinases activity, 180-181,181f inhibitor p21Wafl/Cip1/sdil, 182-183 in regulation of cell cycle, 180-183, 205-207 in regulation of cell cycle, 205-206 Cyclins cdc25, in signal transduction pathway, 84-85, 85f

388

Index

Cyclins (continued) definition, 180 in regenerating liver, 89 in regulation of cell cycle, 205-206 Cycloheximide, induction of apoptosis in liver, 150 Cyclosporin A, liver regeneration and, 344-345 Cyproterone acetate adaptive liver growth in response to, 147, 151 involution after cessation of treatment, 148 effects on apoptosis, 164 Cytokeratins in hepatocellular carcinoma, 106 in RLE cell lines, during transformation in vitro, 107-108, 108f Cytokines in gene regulation, in noncytopathic HBsAg transgenic mouse model, 118-122 and growth factors, comparison, 12 in liver regeneration, 7, 8t, 17-18 interactions with growth factors and hormones, 18-19

D Delayed-early response genes, 72 containing AP-1 and CRE enhancer, regulation by Jun/Fos/LRF-1, 80-82,

82/, 83f functional categories, 73, 76f in liver regeneration, 73, 75t in regenerating liver expressed as immediate-early genes in H35 cells, 89-91 RNA binding proteins encoded by, 89 DHBV, see Duck hepatitis B virus Diethylnitrosamine, transgenes and, in hepatic neoplasia, 278-280, 279t Dihydroxycholecalciferol, in liver regeneration, 10-11 Diisopropyl iminodiacetic acid, in liver disease, 325t Dimethylaminobenzene, transgenes and, in hepatic neoplasia, 278-280, 279t Dimethylnitrosamine, transgenes and, in hepatic neoplasia, 278-280, 279t

DNA damage and altered DNA methylation, 240241 cell cycle progression and, 181-182 checkpoint responses to, 182, 207 fragmentation, in apoptosis, 153 assays, 146 hypermethylation, in carcinogenesis, 234-235 hypomethylation in carcinogenesis, 230-235 in liver tumor promotion, 236-238 with methyl deficient diet, 239-240 oncogene expression and, 228-229, 229f experimental model, 235-236 working hypothesis, 235-236 in regulation of gene activity, 228, 230-231 methylation, 230-235 altered, DNA damage and, 240-241 caloric restriction and, 242-243 chemoprevention and, 241-243, 246 in epigenetics, 230 gene silencing and, 213, 233 in liver carcinogenesis, 213, 227-255 maintenance, 231-233, 232f in rodents and humans, comparison, 243-244 multistage carcinogenesis and, 245, 245f oncogene expression and, 228-229, 229f in regulation of gene activity, 228, 230-231 risk assessment and, 246 in rodents and humans, comparison, 243-244 transcription and, 231 synthesis, in regenerating rat liver, 4-6 viral, integration into host genome and human HCC, 125 and WHV-associated HCC in woodchuck, 122-125, 123t DNA-(cytosine-5)-methyltransferase gene, in DNA knockout mice, 213, 233, 228-229 DNA ladders, and apoptosis, 145-146, 153 DNA maintenance methylase, 231,232f

389

Index

immortal, 185 isolation, 184 and promotion of hepatocaro cinogenesis in vitro, 185-186

Drosophila

developmental splicing factor, 90 protein splicing factor, serine arginine p55, 9O Duck hepatitis B virus, 130 and hepatocarcinogenesis, 115t-116t

E Electroporation, gene transfer by, 362-363 EL/EGVs, see Extended-lifespan/enhanced growth variants Elk-l, see ets-like protein Embryogenesis, hepatocyte growth factor in, 40-41 Endoscopic retrograde cholangiopancreatography, in liver disease, 325t Epidermal growth factor heparin-binding, 12 in liver regeneration, 12, 13, 18-19 Epidermal growth factor receptor, number, phenobarbital's effect on, 209-210, 236-237 Epigenetics, definition, 230 Epithelial cells, rat liver-derived culture, 102-103 differentiation into hepatocytes, 103104 integration into hepatic plates, 103-104 intrahepatic transplantation experiments, 103-104 transformation in vitro, tumors produced by, 106-108, 107t transformation properties, 102 Estrogens hepatocarcinogenesis and, in mice, 310311,314 in liver regeneration, 11-12 Ethinyl estradiol adaptive liver growth in response to, 147 as tumor promoter, in inbred mice, genetic factors affecting, 311 Ethionine carcinogenicity, 241-242 and hepatocarcinogenesis, 104-105 ets-like protein, in transcriptional activation, 77 Extended-lifespan/enhanced growth variants growth and proliferation, 179-180 hepatocyte culture, 184-185

F Familial hypercholesterolemia, liverdirected gene therapy in, 371-373 Fas antigen, in apoptosis, 158 FBRNP, see Fetal brain ribonucleic protein Fetal brain ribonucleic protein, in regenerating liver, 89 FGF, see Fibroblast growth factor Fibroblast growth factor, acidic, in liver regeneration, 12, 15, 34 Focal nocular hyperplasia clinical features, 330, 337t diagnosis, 330, 337t epidemiology, 329-330, 337t pathology, 330 surgical treatment, 330, 337t Food restriction DNA synthesis and apoptosis during, in normal and preneoplastic hepatocytes, 162f, 162-163 liver involution in response to, 148-149

G Gatekeeper gene, cellular proto-oncogene as, for HCC in woodchucks, 124125 Genes, see also Delayed-early response genes in active cell death, 154-156 bcl2, effects on apoptosis, 155-156 BRCA1, cancer susceptibility and, 301302 clusterin/TRPM2, 156 gatekeeper, cellular proto-oncogene as, for HCC in woodchucks, 124125 growth-induced, expression and function in liver regeneration, 71-97 patterns, 78-80, 79f H19, 126 imprinting, 213 IGF2, 126 expression in WHV-infected woodchuck liver, 120f-121f, 126128 imprinting, 213

390

Genes (continued) imprinted, 213-214 insulin, 126 expression, in WHV-infected woodchuck liver, 121f, 129 insulin-inducible, 87-89 in liver regeneration, see also Immediateearly genes; Proto-oncogenes histone H4, 10 MGMT, chemical carcinogens and, in hepatic neoplasia, in transgenic animals, 279t, 280 M6P/IGF2r, expression, in WHVinfected woodchuck liver, 121f, 129 multidrug resistance-2 (mdr-2), deficiency, hepatic neoplasia and, in gene knockout mouse model, 272t, 275 mutations, studied in vivo, in transgenic mice, 280-281 Rb, see Retinoblastoma tumor suppressor gene risk modifier, 301-302 Snrpn, imprinting, 213 tpr-met rearrangement, in oncogenesis, 62-63 Gene therapy human cellular type, 352 definition, 351-352 gene therapy type, 352 liver-directed in otl-antitrypsin deficiency, 373-374 clinical applications, 369-377 in clotting factor deficiencies, 374 ex vivo transfer approach, 356-361, 357f autologous versus allogeneic hepatocytes for, 359-360 gene transfer procedure, 359 hepatic stem cells for, 356-358 hepatocellular culture for, 356 hepatocellular expansion, 358-359 transplant, 360 in familial hypercholesterolemia, 371373 gene addition strategy, 353-355, 354f-355f ex vivo transfer approach, 356-360, 357/ hepatocyte and, 355-356 in vivo transfer approach, 357f

Index

gene replacement (repair) or excision strategy, 352-353, 3 5 4 f - 3 5 5 f gene transfer for adenovirus gene transfer vectors for, 368 ballistic barrage method, 364 chemical methods, 362 electroporation method, 362-363 liposomal gene delivery method, 366 macroinjection method, 364 microinjection method, 363 receptor-mediated gene delivery method, 364-366 retroviral gene transfer vectors for, 366-368 scrape loading method, 364 gene transfer techniques, 361-368, 362t in vivo transfer approach, 361 for liver tumors, 374-375 in metabolic and plasma protein disorders, 369-374 neoantigen generation in, 369-371, 370f strategies for, 352-361 surrogate cells in, 371 for viral liver disease, 375-376 Genetic susceptibility, to liver cancer, 301321 Genotoxic damage, apoptosis with anti-initiating effects, 163 and p53 gene, 155 Glucagon, in liver regeneration, 7-9 Glucocorticoids, in liver regeneration, 11 Glucose feeding, effects on liver regeneration after partial hepatectomy, 6-7 Glucose-6-phosphatase, in gluconeogenesis, 87-88 Glutathione S-transferase-placental form gene expression, in hepatocyte extendedlifespan/enhanced growth variants, 185, 186f as marker for initiated hepatocytes, 202 Glycogen storage disease, type I, liver cancer risk with, 304 G6Pase, see Glucose-6-phosphatase Growth factors in liver regeneration, 7, 8t, 12-17 interactions with cytokines and hormones, 18-19

391

Index

regulation of tissue homeostasis, 204, 205f

Growth hormone, in liver regeneration, 10

H Halogenetic aromatic hydrocarbons, tumor-promoting effects, in inbred mice, strain variation in, 311-312 Harvey-ras gene, see Oncogenes, H-ras HB-EGF, see Epidermal growth factor, heparin-binding Heat shock protein, hsp 70, activation, in liver regeneration, 6 Hemangioma clinical features, 331,337t diagnosis, 331, 337t epidemiology, 330-331,337t surgical treatment, 331, 337t Hemophilia, liver-directed gene therapy in, 374 Hepadnaviruses DNA integration into host genome, 122125, 123t and hepatocarcinogenesis, 113-140 animal models, 113-117 DHBV in duck, 115t-116t GSHV in ground squirrel, 115t116t HBsAg transgenic mouse, 113, 115t-116t, 117-118 woodchuck hepatitis virus infection, 113-114, 115t-116t, 122-129 cellular and molecular genetic processes in, 114, 115t-116t in humans, 115t-116t signal transduction in, 114, 117f X gene characteristics, 129-130, 130t and hepatocellular carcinoma in HBx transgenic mice, 132-134 historical perspective on, 129-130 overexpression in transgenic mice, 114, 115t-116t, 129-134 transactivation mechanism, 131-132 transforming activity, assays in vitro, 132 in viral replication and hepatocarcinogenesis, 131 Hepatectomy liver regeneration after, 342-345, 343f in malignant disease, 339-342

partial early proteolytic events after, HGF and, 37-40 gene induction patterns after, and temporal course of liver regeneration, 78-80, 79f HGF levels after, 34-36, 39-40 immediate-early gene expression after, preexisting transcription factors in, 76-78 liver regrowth after, 4 glucose feeding and, 6-7 stress and, 6-7 studies, historical perspective on, 12 in rat, historical perspective on, 1 techniques, 339-341, 341f Hepatic Arg-Ser protein HRS-Long form, 89 HRS-Short form, 89-90 interaction with its pre-mRNA, 90, 91f in regenerating liver, 88, 89-91 in RNA production, 90-91 Hepatic stem cell system cellular biology, 101-104 studies in vitro, 102-104 in vivo, 101-102 definition, 100 Hepatitis fulminant HGF in, 41-42 histopathology, 41-42 HGF in, 41-42 virus, see also Hepatitis B virus; Hepatitis C virus hepatocellular carcinoma and, 199 Hepatitis B virus envelope protein, overexpression in transgenic mice as model of hepatocarcinogenesis, 113, 115t-116t, 117-118, 271-273, 272t noncytopathic, 118-122 hepatic neoplasia and, 302 , genetic epidemiology, 303 susceptibility genes and, 303 Hepatitis B virus surface antigen gene regulatory element, 272t hepatic neoplasia and sequence of hepatic lesions, 272t, 273

392 Hepatitis B virus surface antigen (continued) in transgenic animals, 271-273, 272t, 275 Hepatitis B virus X gene, see also Hepadnaviruses, X gene gene regulatory elements, 260t hepatic neoplasia and sequence of hepatic lesions with, 260t, 262-263 in transgenic animals, 262-263, 275 Hepatitis C virus, hepatic neoplasia and, 302 Hepatobiliary system, surgical anatomy, 336-342 Hepatoblasts, bipotential capacity, 100 Hepatocarcinogenesis, see Carcinogenesis, hepatic HepatoceUular carcinoma clinical features, 332-333, 334t, 337t ductular cells as progenitors for, 105106 epidemiology, 199, 331-332, 337t fibrolamellar variant, 332-334, 334t geographic distribution, 199 in HBx transgenic mice, 132-134 hepadnavirus-associated, and viral DNA integration into host genome in humans, 125 in woodchuck, 122-125, 123t HGFr expression, 42-43 histology, 332 liver transplantation for, 333 management, 333 mortality, 331-332 oval cells as progenitors for, 105-106 spread, 332 surgical treatment, 333, 337t TGFa expression, 42-43 TG Ff31 gene in, 157-158 Hepatocellular transplantation, clinical applications, 376-377 Hepatocyte growth factor, 51-53 activation, 28, 52 in bile, intact, 32 binding sites, in liver, 31, 38 biological activity, 52-53 in central nervous system development, 43 in disease, 41-42

Index

and early proteolytic events after partial hepatectomy, 37-40 effects on hepatocytes, after partial hepatectomy, 38f, 38-40 in embryogenesis, 40-41, 61-62 endocrine effect, 43-44 epithelial distribution, 31-32 functions, 27-30 hepatic neoplasia in transgenic animals, 269t, 271 isolation, 27, 51 in liver regeneration, 7, 12, 14-15, 1819, 27-49 localization, 30-32 in extrahepatic tissues, 31-32 in liver, 30-31 mitogenic effects, 28-29, 52-53, 61-62 and collagenase pretreatment, 37 mitoinhibitory effect, 42, 43 morphogenic effects, 29-30, 36-37, 53, 62 motogenic effects, 28-29, 61-62 in neoplasia, 42-43 plasma levels, after partial hepatectomy, 34-36, 39-40, 61 properties, 27 radiolabeled, distribution in body, 32 receptor, 14, 27, 53-60 biosynthesis, 54 encoding by c-met oncogene, 53-54 family of proteins, 54, 55f functional characteristics, 30 gene, 27, 30 post-translational modifications, 5455 SH2-supersite, 58, 59f signal transduction, 57-59, 59f Src homology-2 (SH2) domain, 5758 structure, 30, 54 subcellular localization, 59-60 tissue distribution, 59-60 tyrosine kinase activity, positive and negative regulation, 55-57, 56f structure, 27-30, 29f, 51-52, 52f synthesis, 52 target cells, 52-53 in tissue regeneration, 61-62 uptake, in liver, 32 Hepatocyte proliferation inhibitor, in liver regeneration, 17

393

Index

Hepatocytes ductular, 41-42 extended-lifespan/enhanced growth variants culture, 184-185 immortal, 185 isolation, 184 and promotion of hepatocarcinogenesis in vitro, 185-186 from inbred mice, transformation in vitro, strain variation in, 312-313 oncogene-transformed, immortalized, 267 priming, 5-7 and response to HGF, 37 in vitro versus in vivo, 18

rat, 2-3 binucleate, 3 immortal, clonal expansion, phenobarbital requirement, 186-191, 187f mitotic activity, diurnal periodicity, 3 ploidy, 3 regenerating diurnal periodicity, 3 DNA synthesis, 4-5 as stem cells, 100 TGFoL-transformed, immortalized, 269270 transformation into bile duct-type cells, 100 Hepatocyte stimulatory substance, in liver regeneration, 15-16 Hepatomitogens adaptive liver growth in response to, 147 involution after, 147-149, 147t effects on isolated hepatocytes, 149 Hereditary hemochromatosis, liver cancer risk with, 303-304 oL-Hexachlorocyclohexane, adaptive liver growth in response to, 147, 149 involution after cessation of treatment, 148 HFE, see Hereditary hemochromatosis HGF, see Hepatocyte growth factor Hormones, in liver regeneration, 7-12, 8t interactions with growth factors and cytokines, 18-19 HPI, see Hepatocyte proliferation inhibitor HRS, see Hepatic Arg-Ser protein HSS, see Hepatocyte stimulatory substance

Human immunodeficiency virus tat gene gene regulatory elements, 260t hepatic neoplasia and sequence of hepatic lesions with, 260t in transgenic animals, 263 Human splicing factor 2/alternative splicing factor, 90 Hypophysectomy, liver involution in response to, 149

I IGF, see Insulin-like growth factor IGFBP-1, see Insulin-like growth factor binding protein-1 IL, see Interleukins Immediate-early genes expression in hepatic cells, 72-73 after partial hepatectomy, by preexisting transcription factors, 76-78 functional categories, 73, 76f in H35 cells, expressed as delayed-early genes in regenerating liver, 89-91 in liver growth and metabolism, IGFBP-1, 86-87 in liver regeneration, 6, 73, 74t-75t f3-actin, 73 c-los, 6, 61, 72-73, 80-82 c-jun, 6, 61, 80-82 c-myb, 10 c-myc, 6, 10, 72-73

liver-specific, 87-89 CL-6, 88-89 G6Pase, 87-88 PEPCK, 87-88 in metabolic homeostasis, IGFBP-1, 8788 secreted proteins encoded by, 86-87 in signal transduction, 84-85 PRL-1, 84-85, 85f Immortalized cell lines, oncogenetransformed, 267 Injurin, 36, 61 Insertional mutagenesis, in woodchuck hepatitis virus infection, 122-125, 123t Insulin, in liver regeneration, 7-9, 19, 91 Insulin-like growth factor hepatic neoplasia in transgenic animals, 269t, 270

394

Insulin-like growth factor ( c o n t i n u e d ) IGF1, in liver regeneration, 10, 86-87 IGF2 biological activity, regulation, 128129, 129t expression in transgene-induced liver tumor, 276 in WHV-associated altered hepatic foci and HCC, 120f, 121f, 126-127, 128-129 in WHV-infected woodchuck liver, 128-129 in liver regeneration, 10 as tumor promoter in transgenic mice, 127-128 receptor, expression, in WHV-infected woodchuck liver, 121f, 128-129 Insulin-like growth factor binding proteins expression, in WHV-infected woodchuck liver, 121f, 128-129 IGFBP-1 biologic activity, 86 in liver growth and metabolism, 8687 in metabolic homeostasis, 87-88 Interferon % regulatory effect on HBV gene expression, 119 Interferon ~ transgene, hepatic neoplasia and, in mouse model, 272t, 275 Interferon inducible protein, 86 Interleukin converting enzyme, 154 Interleukins IL-1, and HGF expression, 36 IL-let, in liver regeneration, 17 IL-113, in liver regeneration, 17 IL-2, regulatory effect on HBV gene expression, 119 IL-6 and HGF expression, 36 in liver regeneration, 17, 36 IP-10, see Interferon inducible protein Ito cells, hepatocyte growth factor production, 30-31

L Lactation, adaptive liver growth in response to, 147 Laparoscopy, in liver disease, 325t Lead nitrate, adaptive liver growth in response to, 147

Index

involution after cessation of treatment, 148 Liver active cell death in, see Apoptosis; Cell death, active acute yellow atrophy, see Hepatitis, fulminant adaptive growth (hyperplasia), 146, 147 involution after, 147-149, 147t bioartificial, 345 biopsy indications for, 325t, 326 limitations, 325t cell lineage pathways in, 100, 101f cell replication and cell death in, see also Apoptosis; Cell death normal, 146 e x v i v o xeno-perfused, 345 fetal development, transgenic models, 286-287 growth regulation, transgenic models, 282-288 involution, after relative hyperplasia, 147-149, 147t lobar anatomy, 337-339, 340f mass, asymtomatic, evaluation, 324326 neonatal development, transgenic models, 286-287 parenchyma, as single lineage or unipotential stem cell system, 100 surgical anatomy, 336-342 surgical resection, 339-342, 341f transplantation liver regeneration after, 342-345 in malignant disease, 333, 342 tumor promotion in, see Tumor promotion Liver cancer, see also Carcinogenesis, hepatic; Hepatocellular carcinoma; Liver tumors; Tumor promotion active cell death and, 165-166 experimental genetics, 305-314 mutations affecting, 313-314 genetic susceptibility to, 301-321 human epidemiology, 199, 302 genetic diseases associated with, 303304 genetic epidemiology, 302-303

395

Index

genetics, 302-304 mortality, 302 Liver disease cystic clinical features, 337t diagnosis, 324-326, 337t pathogenesis, 326-327 pathophysiology, 327-328 surgical treatment, 328, 337t types, 326-327 diagnosis, 324-326, 325t surgical classification, 326, 327t viral, liver-directed gene therapy for, 375-376 Liver failure, fulminant, HGF in, 41-42 Liver regeneration as compensatory hyperplasia, 4 effectors, 7-19, 8t historical perspective on, 1-25 metabolic workload and, 4-5 regulation, 5-7 after resection, 342-345, 343f review articles on, 2 signals, 7-19, 8t stimuli, 5-7 temporal course, 78-80 transgenic models, 287-288 whole animal studies, 3-4 Liver regeneration factor, expression, 34 Liver regeneration factor-i, in regenerating liver, 80-82, 82f Liver tumors benign, 328-331,337t diagnosis, 338f malignant, primary, 331-334, 337t malignant mesenchymal clinical features, 334 surgical treatment, 334 management, 338f metastatic, 334-336, 337t clinical features, 337t diagnosis, 337t epidemiology, 334, 337t liver-directed gene therapy in, 374375 pathophysiology, 335 surgical treatment, 335-336, 337t survival with, 335-336, 336t primary, liver-directed gene therapy in, 374-375 surgical, diagnosis, 324-326, 325t

surgical treatment, 323-349 indications for, 326-336 LRF, s e e Liver regeneration factor LRF-1, s e e Liver regeneration factor-1

M Macroinjection, gene transfer by, 364 Macrophage stimulating protein, structure,

52, s2f Magnetic resonance imaging, in liver disease, 325t, 326 Malignant transformation, phenotypic alterations in, 183-184 and tumor promoters, 159-160 Mannose 6-phosphate/insulin-like growth factor 2 receptor gene imprinting, 213-214 as tumor suppressor gene, 213-214,

21sf,

237

liver tumor promotion and, 210, 212214, 237, 237 structure, 21 l f, 212-213 Map kinase phosphatase, in signal transduction pathway, 84-85, 85f Me-DAB, s e e 3'-Methyl-4-dimethylaminoazobenzene Messenger RNA binding protein, in regenerating liver, 89 Methionine depletion, liver tumorigenesis and, 238239 dietary, protective effect against liver tumorigenesis, 242 Methylated DNA binding protein, 231 Methyl-CpG binding proteins, 231 Methyl deficient diet, ~rodent liver tumorigenesis and, 238-240 mechanisms, 238-240, 241 3'-Methyl-4-dimethylaminoazobenzene, ' and hepatocarcinogenesis, 104-105 O6-Methylguanine-DNA methyltransferase gene, chemical carcinogens and, in hepatic neoplasia, 279t, 280 Met protein, s e e Hepatocyte growth factor, receptor Microinjection, gene transfer by, 363 Microsatellite instability in carcinogenesis, 122 in hepadnavirus carriers, 122 MKP-1, s e e Map kinase phosphatase

396 Moloney murine leukemia virus, as gene transfer vector, 367-368 Mouse BALB/c, tumor promotion in, genetic control, 311-312 B6C3F1, liver tumorigenesis, 229, 235 DNA methylation and, 235-236, 239-240 oncogenes in, 229-230, 235 bm/bm mutant, 3'-phosphoadenosine-5'phosphosulfate defect in, 313-314 C57BL/6 liver tumor resistance, DNA methylation and, 229, 235-236, 239240 tumor promotion in, genetic control, 311-312 C57BR/cdJ Hcfl and Hcf2 (hepatocarcinogenesis in females) loci, 310-311 liver tumor susceptibility, 310-311 sex hormones and, 310-311 C3H Hcs (Hepatocarcinogen sensitivity) locus, 307-308 Lci (liver cell immortalization) gene, 312-313 liver tumor susceptibility, 307-309 tumor promotion in, genetic control, 311-312 C3H/He, liver tumorigenesis in, 229230, 235 DBA/2 Hcr l and Hcr2 (Hepatocarcinogen resistance) loci, 309-310 liver tumor susceptibility, 309-310 embryonic stem cell-derived, 257 methodology for, 258 principles and rationale for, 258-259 for studying liver growth, development, and neoplasia, 257, 259, 286-287 gene mapping in, 305-306 gene-targeted, see also Mouse, embryonic stem cell-derived for studying liver growth, development, and neoplasia, 258-259, 286-287 assessment, 289-290 research needs, 289-290

Index

hepatocarcinogenesis in mutations affecting, 313-314 sex hormones and, 310-311,314 inbred Ah locus, tumor susceptibility and, 311-312 cancer susceptibility, variation among, 305-313 hepatocarcinogenesis perinatal, 306-311,307t spontaneous, 306-311,307t hepatocytes from, transformation in vitro, strain variation in, 312313 tumor promotion in, genetic control, 311-312 transgenic gene mutations in, studied in vivo, 280-281 HBx, hepatocellular carcinoma in, 132-134, 260t, 262-263 IGF2 knockout experiments, 127-128 IGF2 overexpression experiments, 127, 269t, 270 liver development in fetal, 286-287 neonatal, 286-287 liver regeneration in, 287-288 production, 257-259 for studying liver growth, development, and neoplasia, 258-259, 286-287 assessment, 289-290 research needs, 289-290 targeted gene expression, in liver, 282286, 283t-285t tumor promotion in, genetic control, in inbred strains, 311-312 m-RNABP, see Messenger RNA binding protein MSP, see Macrophage stimulating protein Mutagenesis, assays in vivo, transgenebased systems, 280-281

N Nafenopin adaptive liver growth in response to, 147 involution after cessation of treatment, 147-148

397

Index

carcinogenicity, and spontaneously initiated cells, 163 effects on isolated hepatocytes, 149 Necrosis definition, 143 toxin-induced, in liver, 150-151 Neurotensin, in liver regeneration, 9 NF-KB, see Nuclear factor-kappa B N-Nitrosomorpholine induction of apoptosis in liver, 150 induction of necrosis in liver, 150 Norepinephrine, in liver regeneration, 9, 35 Nuclear factor-kappa B, 77

O Oncogenes, see also Proto-oncogenes cellular, hepatic neoplasia and, in transgenic animals, 264-267, 265t C-lOS

in liver regeneration, 6, 61, 72-73, 80-82 in preparation for active cell death, 154 c-myc

hepatic neoplasia in transgenic animals, 265t, 266267 in liver regeneration, 6, 10, 72-73 in preparation for active cell death, 154-155 C-myc, expression, in transgene-induced liver tumor, 276 5' flanking region of Ha-ras, methylation, 244 Ha-ras, in mouse liver tumorigenesis, 229-230, 235-237 HBx, hepatic neoplasia and, in transgenic animals, 260t, 262-263 hepatic neoplasia and, in transgenic animals, 267-268 HIV tat, hepatic neoplasia and, in transgenic animals, 132-134, 260t, 263 H-ras

expression, in transgene-induced liver tumor, 276 hepatic neoplasia in transgenic animals, 264-266, 265t mutant, effects in fetal liver, 264-265 K-ras, expression, in transgene~ liver tumor, 276

in mouse liver tumorigenesis, 229-230 DNA hypomethylation and, 229-230, 237 N-myc, expression, in transgene-induced liver tumor, 276 N-ras, expression, in transgene-induced liver tumor, 276 raf, in mouse liver tumorigenesis, 229230, 236, 237 STP-C, hepatic neoplasia and, in transgenic animals, 260t, 263-264 SV40-Tag, hepatic neoplasia and, in transgenic animals, 259-262, 260t viral, hepatic neoplasia and, in transgenic animals, 259-264, 260t Oval cells as bipotential precursors, 102 hyperplasia, in hepatocarcinogenesis, 104-106 transformation in vitro, tumors produced by, 106-108 as tumor precursors, 159 Overfeeding, adaptive liver growth in response to, 147 Oxygen reactive species (toxic oxygen radicals) and hepadnavirus infection, 122 rodent liver tumorigenesis and, 239, 241

P Parathyroid hormone, in liver regeneration, 10-11 PDGI, see Platelet-derived growth inhibitor PEPCK, see Phosphoenol pyruvate carboxy-kinase Percutaneous transhepatic cholangiography, in liver disease, 325t Perillyl alcohol, growth inhibitory effects on liver tumors, 217 Peroxisome proliferators, as tumor promoters, 199, 237 PG, see Prostaglandins Phenobarbital adaptive liver growth in response to, 147, 149, 200f, 208, 209f apoptosis and, 164, 216 carcinogenic risk with, 199 in clonal expansion of immortal rat hepatocytes, 186-191, 187f hepatocyte extended-lifespan/enhanced

398

Index

Phenobarbital (continued) growth variants and, 185-186, 236-237 transgenes and, in hepatic neoplasia, 278-280, 279t as tumor promoter, 199, 208-210, 209/ in inbred mice, genetic factors affecting, 311 mechanisms of action, 191-193 pathomechanism, 236 Phenylalanine metabolism, 371,372f PHF, see Post hepatectomy factor Phosphatase of regenerating liver, in signal transduction, 84-85, 85f Phosphoenol pyruvate carboxykinase, in gluconeogenesis, 87-88 Platelet-derived growth inhibitor oL, in liver regeneration, 17 Platelet-derived growth inhibitor 13, in liver regeneration, 17 Point mutations and hepadnavirus infection, 122 in hereditary nonpolyposis colorectal cancer, 122 Post hepatectomy factor, 7 7 - 7 8 Pregnancy, adaptive liver growth in response to, 147 Preneoplastic liver foci apoptosis in, 161-164 cell proliferation and death in kinetics, 160f, 160-161 and tumor promotion, 163-165 growth and regression, cell kinetics in,

~sof persistent, cell proliferation and death in, 164 phenotypic alterations, and tumor promoters, 159-160 in rat, 159 types, 159 remodeling, cell proliferation and death in, 164 PRL-1, see Phosphatase of regenerating liver Prolactin, in liver regeneration, 11 Prostaglandins, in liver regeneration, 11 Proteases, in apoptosis, 154 Protein, serum, depletion, adaptive liver growth in response to, 147

Protein deprivation, induction of hepatocyte priming, 6 Protein kinase C pathway, effects of X gene on, 131 translocation, in phenobarbital-treated hepatocytes, 236 Proto-oncogenes, see also Oncogenes cellular as gatekeeper gene, for HCC in woodchucks, 124-125 in hepadnavirus-associated hepatocarcinogenesis, 114, 116t c-met

in carcinogenesis, 62-63 expression, regulation, 60-61 receptor encoded by, 14, 27, 30, 5354 c-ron, receptor encoded by, 30, 54, 55f c-sea, receptor encoded by, 30, 54, 55f N - m y c , expression in WHV-infected woodchuck liver, 120f-121f, 128 raf, signal transduction pathway, effects of HBx on, 131-132 ras

in hepatocellular carcinoma, 126 signal transduction pathways, in hepatomas, 114

R Rat cross-circulation experiments in, 5-6 hepatectomy in, historical perspective on, 1 hepatic stem cell system, studies in vivo, 101-102 hepatocarcinogenesis in, mutations affecting, 313-314 inbred strains, cancer susceptibility, variation among, 305-313 LEC A t t p 7 b gene mutation, 313 hepatitis in, 313 liver, 1 multistage carcinogenesis in, 159 normal adult, 2-3 preneoplastic lesions in, 159 regeneration, 3-5 Rb gene, see Retinoblastoma tumor suppressor gene

399

Index

Regenerating liver nuclear receptor-1 expression, 83 in liver regeneration, 82-83 structure, 83 tissue distribution, 83 Rel transcription factors, activation after partial hepatectomy, 7 7 Retinoblastoma susceptibility gene product (RB), phosphorylation, 180, 181f, 206-207 Retinoblastoma tumor suppressor gene in cell cycle regulation, 206-207 protein Rb, SV40 T Ag interaction with, 132 Retroviruses as gene transfer vectors, 366-368 life cycle, 367, 3 6 7 f Reverse genetics, of cancer susceptibility genes, in inbred rodents, 304 RLE cells, see Epithelial cells, rat liverderived RNR-1, see Regenerating liver nuclear receptor-1

S Saimiri transformation associated protein subgroup C gene hepatic neoplasia in transgenic animals, 263-264 SAM, see S-Adenosylmethionine Sarcoma cytotoxic factor, 42 Scatter factor, 51-53; see also Hepatocyte growth factor isolation, 51 Scrape loading, gene transfer by, 364 Serum response factor, transcriptional activation by, 7 7 Sex hormones, hepatocarcinogenesis and, in mice, 310-311, 314 Sexual dimorphism, in liver function, 11 SF, see Scatter factor SF2/ASF, see Human splicing factor 2/alternative splicing factor SIF/Stat 1, see Signal transducer and activator of transcription 1 Signal transducer and activator of transcription 1, 7 7 - 7 8 Signal transduction in hepadnavirus-associated hepatocarcinogenesis, 114, 117f

hepatocyte growth factor receptor in,

s7-sg, sgf immediate-early genes in, 84-85 pathway, 84, 85f phosphatase interaction in, 84-85, 85f tyrosine phosphorylation in, 84-85, 85/ Simian virus 40 T antigens hepatic neoplasia in transgenic animals, 259-262, 2 6 0 t interaction with p53, 132 with Rb, 132 Simple sequence length polymorphisms, in genetic mapping of mouse, 304 SRF, see Serum response factor Starvation, liver involution in response to, 148-149 Stem cells, see also Hepatic stem cell system definition, 100 ductular/periductal, 100, 101f; see also Hepatic stem cell system hepatic controversial aspects, 99-100 and hepatocarcinogenesis, 104-106 hepatocytes as, 100 Steroids, adaptive liver growth in response to, 147 Stress, effects on liver regeneration after partial hepatectomy, 6-7 Surgical anatomy, of hepatobiliary system, 336-342 Surgical treatment, of hepatic tumors, 323349

T Tamoxifen, as tumor promoter, 199 TCDD, see 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin Technetium-99m, in liver disease, 325t Testosterone, hepatocarcinogenesis and, in mice, 310-311,314 Testosterone repressed prostate message gene, 156 2,3,7, 8-Tetrachlorodibenzo-p-dioxin, tumor-promoting effects, 164 TGF, see Transforming growth factor Thioacetamide, induction of apoptosis in liver, 149

400

Thyroid hormones, in liver regeneration, 11 TNFoL, see Tumor necrosis factor ot Tra-2, see Drosophila, developmental splicing factor Transcription factors, see also Immediateearly genes; specific factor Fos, in regenerating liver, 80-82 induced in regenerating liver, 80-83 Jun, in regenerating liver, 80-82 Transformation, malignant, phenotypic alterations in, 183-184 and tumor promoters, 159-160 Transforming growth factor gene regulatory element, 269t hepatic neoplasia and sequence of hepatic lesions, 268-269, 269t in transgenic animals, 268-270, 269t, 281-282 in liver regeneration, 12, 13-14, 18-19 Transforming growth factor 13 activation, in liver tumors, 211-212, 211/, 212f effects on clonal expansion of phenobarbital-dependent hepatocytes, 187, 187/ isoforms, 210 latent complex, 210/, 237 receptor binding, 211-212, 211 f liver carcinogenesis and, 208-216 in liver regeneration, 7, 16-17 in liver tumor promotion activation, 211-212, 211/, 212f experimental results, 214-216 in phenobarbital-treated hepatocytes, 236-237 receptors, 210-212, 21 If in phenobarbital-treated hepatocytes, 209/, 210 Transforming growth factor 131 apoptosis and, 157, 217 effect on Rb phosphorylation, 206-207 hepatocarcinogenesis and, 157-158, 214 Transgenes AL-HBsAg, chemical carcinogens and, in hepatic neoplasia, 278-280, 279t AL-myc/MT-TGFcx, multistage carcinogenesis and, 276-278, 277t

Index AL-ras/AL-myc, multistage carcinogen-

esis and, 276-278, 277t AL-TAg/AL-myc, multistage carcinogen-

esis and, 276-278, 277t AL-TAg/AL-ras, multistage carcinogen-

esis and, 276-278, 277t AL-TAg/MT-TGFot, multistage carcinogenesis and, 276-278, 277t gene regulatory elements, 258 growth factor, hepatic neoplasia and, 268-271 HBV-HBsAg, chemical carcinogens and, in hepatic neoplasia, 278-280, 279t hepatic expression, targeted, 282-286 characteristics, 283t-285t gene regulatory elements used, 283t285t, 285-286 in hepatic growth regulation, 282-288 in hepatic neoplasia, chemical carcinogens and, 278-280, 279t hepatotoxic, hepatic neoplasia and, 271275 MT-MGMT, chemical carcinogens and, in hepatic neoplasia, 278-280, 279t MT-TGFoL, chemical carcinogens and, in hepatic neoplasia, 278-280, 279t multiple, coexpression, 276-278, 277t multistage carcinogenesis and, 276-282 transgene cooperativity in, 276-278, 277t oncogenic, hepatic neoplasia and, 259268 protein-encoding sequence, 258 Transgenic models, 257-300, see also Mouse, transgenic assessment, 289-290 with growth factors, 268-271 of hepatocarcinogenesis, principles and rationale for, 257-259 with hepatotoxic transgenes, 271-275 of liver growth and development, principles and rationale for, 257-259 multistage carcinogenesis in, 276-282 coexpression of multiple transgenes in, 276-278, 277t cooperating events in, 278 transgene-based mutagenesis assay systems, 280-281 transgenes and chemical carcinogens in, 278-280, 279t

401

Index

research needs, 289-290 with viral oncogenes, 259-265, 260t Transglutaminase, in apoptosis, 153154 Tumorigenesis, see Carcinogenesis Tumor necrosis factor in apoptosis, 158 in liver regeneration, 17-18 regulatory effect on HBV gene expression, 119 Tumor promoters, liver effects on preneoplastic loci, 159-160 mechanism of action, 199-200 Tumor promotion active cell death and, 163-165 genetic control, in rodents, 311-312 liver, 159, 208-209, 209f and suppression of p53-dependent cell cycle checkpoint function, 179197 mechanisms, 199-226 molecular mechanisms, 229-230 reversibility, 165 Tumor suppressor genes in hepadnavirus-associated hepatocarcinogenesis, 114, 116t M 6 P / I G F 2 r as, 213-214, 214f, 237 p53

in cell cycle regulation, 179-197, 207-208 expression, in transgene-induced liver tumor, 276 in hepadnavirus-associated hepatocarcinogenesis, 114-117, 116t mutation, caloric restriction's protective effect against, 243 in preparation for active cell death, 155 product, see Tumor suppressor protein p53 Rb, see Retinoblastoma tumor suppressor gene RB-1, expression, in transgene-induced liver tumor, 276 Tumor suppressor protein p53 in cell cycle regulation, 179-197, 207208 HBx protein interaction with, 132 and hepatocellular carcinoma in HBx transgenic mice, 133 SV40 TAg interaction with, 132

Tyrosinemia, type I, liver cancer risk with, 304

U Ultrasound, in liver disease, 324-326, 325t uPA, see Urokinase-type plasminogen activator Urokinase-type plasminogen activator hepatic neoplasia in transgenic animals, 272t, 273274 in HGF activation, 38f, 38-39, 52 receptor, expression, after hepatectomy, 38f, 38-39

V Vasopressin, in liver regeneration, 9 Vimentin, in RLE cell lines, during transformation in vitro, 107, 108f

W WHV, see Woodchuck hepatitis virus Wilms tumor suppressor gene, 126 Woodchuck hepatitis virus altered hepatic foci due to, 119-122, 119t, 120f-121f gene expression in, 125-129 histologic characteristics, 119t, 125126 IGF2 expression in, 120f, 121f, 126129 - c - m y c fusion gene, hepatic neoplasia and, in transgenic animals, 265t, 266-267 DNA integration into host genome, 122125, 123t HCC due to, IGF2 expression in, 120f, 126-127 infection model of hepatocarcinogenesis, 113114, 115t-116t, 122-129 toxic oxygen radicals and, 122

X X-chromosome reactivation, aging and, in rodents and humans, 243-244 Xenobiotic agents, adaptive liver growth in response to, 147

402 X gene, see Hepadnaviruses, X gene X protein, as oncogenic transcriptional transactivator, 131 deregulation of cell cycle control in hepatocytes, 132

Index

interaction with p53 tumor suppressor protein, 132 promiscuous action on enhancers with unrelated sequence motifs, 131132