Advances in Cancer Research Volume 86

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Advances in

CANCER RESEARCH Volume 86

This Page Intentionally Left Blank

Advances in

CANCER RESEARCH Volume 86

Edited by

George F. Vande Woude Van Andel Research Institute Grand Rapids, Michigan

George Klein Microbiology and Tumor Biology Center Karolinska Institute Stockholm, Sweden

Amsterdam Boston London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

∞ This book is printed on acid-free paper. C 2002, Elsevier Science (USA). Copyright

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. The appearance of the code at the bottom of the ﬁrst page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of speciﬁc clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2002 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-230X/2002 $35.00 Explicit permission from Academic Press is not required to reproduce a maximum of two ﬁgures or tables from an Academic Press chapter in another scientiﬁc or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given.

Academic Press An imprint of Elsevier Science. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com

Academic Press 84 Theobalds Road, London WC1X 8RR, UK http://www.academicpress.com International Standard Book Number: 0-12-006686-6 PRINTED IN THE UNITED STATES OF AMERICA 02 03 04 05 06 07 MM 9 8 7 6 5 4

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Contents

Contributors to Volume 86 vii

Coordinate Regulation of Translation by the PI 3-Kinase and mTOR Pathways Kathleen A. Martin and John Blenis I. II. III. IV. V.

Overview of Translational Regulatory Pathways 2 PI 3-K and mTOR Effectors which Regulate Translation 7 Regulation of Translational Effectors 10 Coordinated Translational Control 20 Conclusions 29 References 30

Histone Acetyltransferases and Deacetylases in the Control of Cell Proliferation and Differentiation Heike Lehrmann, Linda Louise Pritchard, and Annick Harel-Bellan I. II. III. IV. V. VI. VII. VIII. IX.

Introduction 42 Acetylation of Histones 43 Histone Acetyltransferases 44 Histone Deacetylases and Cell Cycle Regulation 48 Muscle Differentiation 51 Hematopoiesis 53 Huntington’s Disease 55 Histone Acetylation in Combination with Other Chromatin Modiﬁcations 56 Conclusion 57 References 58

Molecular Pathogenesis of Human Hepatocellular Carcinoma Michael A. Kern, Kai Breuhahn, and Peter Schirmacher I. Introduction 67 II. Morphology of Human Hepatocarcinogenesis 69

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Contents

Molecular Etiology 72 Host Carcinogenic Events 77 Functional Consequences 88 Therapeutic Implications 90 References 92

The Cell-Mediated Immune Response to Human Papillomavirus-Induced Cervical Cancer: Implications for Immunotherapy Gretchen L. Eiben, Markwin P. Velders, and W. Martin Kast I. II. III. IV. V.

Introduction 113 Human Papillomaviruses 114 Cellular Immunity to HPV 116 Immunotherapy against HPV-Induced Carcinomas 123 Conclusion 136 References 137

The T-Cell Response in Patients with Cancer Chiara Castelli and Markus J. Maeurer I. Deﬁnition of Immune Effector Functions 149 II. Deﬁning the Bait for Antigen-Speciﬁc T Cells 152 III. The Role of the Coreceptor CD8 in Mediating Antitumor Restricted T-Cell Responses 158 IV. Tools to Measure ex Vivo T-Cell Avidity 160 V. Biomarkers or True Surrogate Markers? 163 VI. T-Cell Crossreactivity 172 VII. Questions of Speciﬁcity and Alternate T-Cell Effector Functions 173 References 176

The Life and Death of a B Cell ` and Peter H. Krammer Thierry Defrance, Montserrat Casamayor-Palleja, I. II. III. IV. V. VI.

Introduction 196 The Maintenance of B Cell Tolerance 197 The Regulation of B Cell Homeostasis 201 Control of the Speciﬁcity and Afﬁnity of the Ab Response 206 Regulation of Survival in the Memory B Cell Compartment 212 Conclusive Remarks 216 References 218

Contributors

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

John Blenis, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 (1) Kai Breuhahn, Institute of Pathology, University of Cologne, D-50931 Cologne, Germany (67) ` INSERM U404, “Immunity and VaccinaMontserrat Casamayor-Palleja, tion,” 69365, Lyon, Cedex 07, France (195) Chiara Castelli, Unit of Immunotherapy of Human Tumors, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milano, Italy (149) Thierry Defrance, INSERM U404, “Immunity and Vaccination,” 69365, Lyon, Cedex 07, France (195) Gretchen L. Eiben, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois 60153 (113) Annick Harel-Bellan, CNRS UPR 9079, Institut Andr´e Lwoff, 94800 Villejuif, France (41) W. Martin Kast, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois 60153 (113) Michael A. Kern, Institute of Pathology, University of Cologne, D-50931 Cologne, Germany (67) Peter H. Krammer, Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany (195) Heike Lehrmann, CNRS UPR 9079, Institut Andr´e Lwoff, 94800 Villejuif, France (41) Markus J. Maeurer, Department of Medical Microbiology, University of Mainz, 55101 Mainz, Germany (149) Kathleen A. Martin, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 (1)∗ Linda Louise Pritchard, CNRS UPR 9079, Institut Andr´e Lwoff, 94800 Villejuif, France (41) ∗ Present address: Departments of Surgery and of Pharmacology and Toxicology, Dartmouth Medical School, Lebanon, New Hampshire 03756

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Contributors

Peter Schirmacher, Institute of Pathology, University of Cologne, D-50931 Cologne, Germany (67) Markwin P. Velders, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois 60153 (113)

Coordinate Regulation of Translation by the PI 3-Kinase and mTOR Pathways Kathleen A. Martin and John Blenis Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

I. Overview of Translational Regulatory Pathways A. The PI 3-Kinase Pathway B. The mTOR Pathway II. PI 3-K and mTOR Effectors which Regulate Translation A. 5’ TOP mRNA and S6K1 B. Capped mRNA and eIF-4E C. Other PI 3-K- and mTOR-Regulated Translation Initiation Factors III. Regulation of Translational Effectors A. 4E-BP1 Regulation B. Regulation of S6K1 C. S6K2 IV. Coordinated Translational Control A. Coordination of mRNA Splicing and Translation B. Coordinated Growth and Proliferation in Liver Regeneration C. PI 3-K, mTOR, Translation, and Cell Size D. Coordination of the PI 3-K and mTOR Pathways E. PI 3-K and mTOR Pathways in Cancer V. Conclusions References

Control of translation initiation is an important means by which cells tightly regulate the critical processes of growth and proliferation. Multiple effector proteins contribute to translation initiation of specially modiﬁed mRNAs that modulate these processes. Coordinated regulation of these translational effectors by multiple signaling pathways allows the integration of information regarding mitogenic signals, energy levels, and nutrient sufﬁciency. The mTOR protein, in particular, serves as a sensor of all of these signals and is thought to thus serve as a crucial checkpoint control protein. Signals from the mTOR pathway converge with mitogenic inputs from the phosphoinositide (PI) 3-kinase pathway on translational effector proteins to coordinately control cellular growth, size, and cell proliferation. The translational effectors regulated by the PI 3-kinase and mTOR pathways and their roles in regulation of cellular growth will be the primary focus of this review. C 2002, Elsevier Science (USA).

Advances in CANCER RESEARCH 0065-230X/02 $35.00

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Copyright 2002, Elsevier Science (USA). All rights reserved.

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Martin and Blenis

I. OVERVIEW OF TRANSLATIONAL REGULATORY PATHWAYS A. The PI 3-Kinase Pathway Inputs from a wide range of extracellular signals converge on the phosphoinositide 3-kinase (PI 3-K) family enzymes, which, in turn, regulate a host of critical cellular processes, including proliferation, survival, motility, vesicle trafﬁcking, transcription, and protein synthesis (Fig. 1) (Rameh and Cantley, 1999). The role of this pathway in cell survival, in large part through regulation of the effector kinase Akt/PKB, is widely appreciated (Downward, 1998). The importance of this pathway in maintaining the balance between proliferation, survival, and apoptosis is further underscored by the fact that several effectors are protooncogenes, and the functional antagonist of this pathway, PTEN, is a tumor suppressor gene frequently found to be mutated in human tumors (Cantley and Neel, 1999). Notably, the PI 3-K pathway coordinates the separable but related processes of cellular proliferation (an increase in cell number) and growth (an increase in cell mass) in large part through regulation of protein synthesis. In this review, we focus on the role of PI 3-K targets in regulation of protein synthesis and cell growth. Class I PI 3-Ks, including adapter (p85 family) and catalytic (p110 family) subunits, lie at the apex of an important growth factor stimulated signaling pathway that governs many aspects of cell behavior. While PI 3-kinase enzymes catalyze both lipid and protein phosphorylation, the lipid MITOGENS

PI 3-Kinase

phospholipid second messengers

PTEN

effector proteins

survival

proliferation

growth

motility

Fig. 1 The PI 3-kinase pathway regulates multiple cellular functions in response to mitogenic stimulation. Lipid second messengers generated by PI 3-K bind to and regulate multiple effector proteins, which in turn modulate numerous critical processes in mammalian cells.

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kinase-dependent functions are more thoroughly understood (Bondeva et al., 1998). PI 3-Ks phosphorylate phosphatidylinositols at the 3 -OH position to generate lipid second messengers that target a diverse array of downstream effector proteins, allowing coordinated modulation of multiple cellular processes. The major growth factor-induced PI 3-K-derived lipid messengers are phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5P3) (Rameh and Cantley, 1999). Binding of these lipids to effector proteins, especially those containing the plekstrin homology (PH) domain motif, may induce conformational changes and/or membrane targeting which alters their activities and access to other regulatory proteins or substrates. The PTEN lipid phosphatase dephosphorylates PI 3-K-generated phospholipid second messengers, and thus coordinately inhibits the activation of the numerous downstream effectors of PI 3-K (Cantley and Neel, 1999). The pharmacological inhibitors of PI 3-K, wortmannin and LY294002, have been important tools for dissecting the roles of this pathway and its effectors in vivo.

B. The mTOR Pathway The lipid-sensitive PI 3-K effectors that regulate translation will be discussed in detail in this review. Many of these mediators are also regulated by mTOR signaling, another pathway critical for translational control (Gingras et al., 2001b). The integration of signals from the PI 3-K and mTOR pathways is an important emerging theme in the coordinated regulation of cell growth and proliferation. A current model suggests that translational control is regulated by distinct, parallel signaling pathways that converge on common effectors: mTOR senses nutrient sufﬁciency, energy levels, and perhaps some mitogenic signals via phosphatidic acid (Fang et al., 2001) or PI 3-K/Akt (Fig. 2) (Nave, 1999; Sekulic et al., 2000), while growth factoror mitogen-induced translation is mediated largely by the PI 3-K pathway, with additional contributions from PKCs and MAPKs. Consistent with a role in a nutrient checkpoint, inhibition of the mTOR pathway can override PI 3-K and other growth factor-derived signals to multiple translational effectors. Thus, no discussion of PI 3-K signaling would be complete without an understanding of the inputs contributed by the mTOR pathway (Fig. 3). mTOR is the mammalian target of rapamycin (Sabers et al., 1995), also known as FRAP (FKBP and rapamycin-associated protein) (Brown et al., 1994), RAFT (rapamycin and FKBP12 target) (Sabatini et al., 1994), or RAPT (Chiu et al., 1994). This protein is a serine/threonine protein kinase that autophosphorylates and regulates exogenous substrates in translation pathways, including S6Ks and 4E-BPs (to be discussed in detail in following sections) (Brown et al., 1995; Brunn et al., 1997). mTOR signaling is

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NUTRIENTS (amino acids)

ENERGY (ATP)

MITOGENS (phosphatidic acid)

amino acid withdrawal

mTOR

TRANSLATION INITIATION

Fig. 2 Convergence of multiple upstream inputs on mTOR. Because mTOR integrates signals from multiple stimuli, and because its inhibition suppresses the activity of translation initiation effectors despite the presence of other mitogenic stimuli, mTOR can serve as an effective sensor in checkpoint control of protein synthesis.

inhibited by a complex formed between the lipophilic macrolide antibiotic rapamycin (also known as Sirolimus) and the ubiquitous cellular protein FKBP12 (FK506-binding protein 12 kDa) (Peterson et al., 2000). Rapamycin inhibits multiple important functions of mammalian cells, including protein synthesis, cell proliferation (Pyronnet and Sonenberg, 2001), growth factors PI3K

ERK

nutrients Rapamycin mTOR S6K

4E-BP

S6 eIF-4F structured-mRNA

40S 60S

5' TOP mRNA

Fig. 3 Coordinate regulation of translational effectors by growth factor and nutrient pathways. S6 kinases and 4E-BPs are regulated by multiple phosphorylations. Growth factor signals are transduced to these effectors by the PI 3-K, ERK, and mTOR pathways, and nutrient/energy sufﬁciency signals are mediated by the mTOR pathway. Both growth factor and nutrient/energy signals are required for full activation of these translational effector proteins.

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muscle hypertrophy (Rommel et al., 2001; Bodine et al., 2001), and cell growth/cell size (Fingar, et al., 2002). While mTOR and FKBP12 are ubiquitously expressed, the effects of rapamycin are more pronounced in certain cell types. Lymphocyte proliferation is highly sensitive to rapamycin, which likely accounts for the drug’s utility as an immunosuppressant that reduces organ transplant rejection in clinical trials (Podbielski and Schoenberg, 2001). mTOR contains a C-terminal domain with homology to the PI 3-K kinase domain. It does not appear to be a functional lipid kinase, but is a member of a growing family of proteins containing this homologous region known as PIKKs, or phosphoinositide kinase-related kinases (Hoekstra, 1997). Many members of the PIKK family are thought to serve checkpoint functions, including ATM, ATR, and DNA-PK, which act as sensors for DNA damage and repair (Hoekstra, 1997). A connection between DNA damage and mTORdependent signaling has been observed (Tee and Proud, 2000), suggesting that mTOR may also serve as a sensor of DNA damage. mTOR appears to function in a nutritional checkpoint that senses amino acid availability. Phosphorylation of critical translational effectors of mTOR, including S6K1 and 4E-BP1, is sensitive to the availability of leucine and other branched chain amino acids, and this effect is rapamycin-sensitive (Hara et al., 1998). Conversely, phosphorylation is inhibited by amino acid deprivation. Amino acid signaling and rapamycin may employ similar mTOR regulatory mechanisms, as a rapamycin-resistant mTOR mutant relieves the amino acid dependence of S6K1 (Iiboshi et al., 1999). The mechanism by which leucine and other amino acids signal to the mTOR pathway is not yet known but has been postulated to involve tRNA charging (Iiboshi et al., 1999), or leucine-stimulated increased mitochondrial metabolism via oxidative decarboxylation and activation of glutamate dehydrogenase (Xu et al., 2001; see Lynch, 2001, for review). While amino acid-sensitive mTOR effectors are also subject to PI 3-K regulation, a role for PI 3-K in amino acid signaling is unlikely, since PI 3-K and Akt activities are unaffected by amino acid stimulation or deprivation (Hara et al., 1998). As protein synthesis is the most energetically expensive function performed by the cell (Schmidt, 1999), integrated control of this process by a nutrientand energy-sensing checkpoint would be optimal. It has recently been suggested that mTOR may indeed serve as a sensor of energy levels (Dennis et al., 2001). Dennis et al. have shown that reduction of cellular ATP levels using the glycolytic inhibitor 2-deoxyglucose prevented insulin-induced activation of mTOR-regulated translational effectors. While all kinases require ATP, the apparent Km for ATP for mTOR was estimated to be greater than 1 mM, as opposed to 10–20 μM for most other known kinases (Dennis et al., 2001). This requirement for high, but physiological, concentrations of ATP may reﬂect the ability of mTOR to physically sense cellular energy

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levels, as well as explain the technical challenges that have faced researchers studying mTOR enzymatic activity in vitro. Nutrient regulation of the yeast mTOR homologs, TOR1 and TOR2, has been well documented (Rohde et al., 2001). The yeast TOR proteins have provided many important insights into the roles and regulation of this pathway in mammalian cells. While some mTOR functions may be mediated through phosphorylation of targets, yeast models have suggested that TOR 1/2 may have more sweeping effects on multiple downstream cellular targets through regulation of a phosphatase. In S. cerevisiae, TOR proteins stimulate the association of the phosphatases Sit4 (PP6 homolog) and Pph21/22 (PP2A homolog) with the regulatory protein Tap42. This association is inhibited by nutrient insufﬁciency or rapamycin, and different mutations in Tap42 can inhibit translation or confer rapamycin resistance, demonstrating the importance of this protein in TOR-mediated translational control (Di Como and Arndt, 1996; Jiang and Broach, 1999). α4, the murine homolog of Tap42, associates with the catalytic subunits of human phosphatases PP2A, PP6, or PP4 (Murata et al., 1997; Chen et al., 1998). α4 binding alters PP2A substrate speciﬁcity (Murata et al., 1997), and rapamycin inhibits the association of α4 and PP2A (Murata et al., 1997), suggesting that an analogous pathway may exist in mammals. Inhibition of S6K1 and 4E-BP1 by rapamycin may be mediated via phosphatase activity, and PP2A has been shown to associate with S6K1, but not with a rapamycin-resistant S6K1 mutant lacking the N- and C-terminal regulatory domains (Fig. 4) (Peterson et al., 1999). mTOR α4

Rapamycin or Amino acid withdrawal α4

PP2A-C PP2A-A

PP2A-C

altered substrate specificity

PP2A-B

S6K1

S6K1

(inactive)

(active)

Fig. 4 Hypothetical model for mTOR regulation of phosphatase activity and translational effectors in mammalian cells. Evidence from the TOR analogs in yeast, and from mammalian cell experiments, suggests a model by which mTOR may modulate the activity and/or substrate speciﬁcity of PP2A-family serine/threonine phosphatases by regulating binding of adapter subunits such as α4.

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Activation of a phosphatase, as opposed to inhibition of a kinase, is an attractive model to explain the rapid rapamycin-induced dephosphorylation of at least 12 different sites on 4E-BP1 and S6K1 with dissimilar motifs (Peterson et al., 1999). In this way, a nutrient-sensing checkpoint protein could coordinately inhibit multiple diverse translational effectors when the essential amino acid leucine is lacking, despite the continued presence of positive growth factor signals (i.e., from the active PI 3-K pathway). Recent work has suggested a novel mechanism by which serum mitogens may also signal through mTOR to translational effectors. Serum-induced phospholipase D (PLD) activity generates the second messenger phosphatidic acid (PA), which binds to the FRB domain of mTOR (the same domain that binds the FKBP12/rapamycin complex). This PA-mediated signaling stimulates phosphorylation and activation of S6K1 and 4E-BP1 in a rapamycinand wortmannin-sensitive manner. Interestingly, PA does not alter the kinase activity of mTOR. The authors suggest a model in which S6K1 and 4E-BP1 integrate nutrient signals through mTOR, as well as mitogenic signals via PI 3-K and PA/mTOR (Fang et al., 2001). The roles and regulation of translational effector targets of both the mTOR and PI 3-K pathways will be presented in detail below.

II. PI 3-K AND mTOR EFFECTORS WHICH REGULATE TRANSLATION Two downstream targets of PI 3-K and mTOR signaling, S6K1 and 4EBP1/eIF-4E, are major regulators of protein synthesis (Fig. 3). These effectors will be introduced here, followed by a discussion of the intermediates that transduce these signals to these targets.

A. 5’ TOP mRNA and S6K1 Translation initiation rates for different mRNAs can vary dramatically depending on the degree and type of secondary structure present in the 5 untranslated region of the message. One modiﬁcation, the 5 terminal oligopyrimidine tract (5 TOP), a stretch of 4–14 pyrimidine bases at the extreme 5 end of the message, marks a particular subset of mRNAs for inefﬁcient translation initiation under basal conditions (Meyuhas, 2000). Insulin or other growth factor stimulation rapidly induces translation of these messages in a rapamycin-sensitive manner (Jefferies et al., 1994). These 5 TOP mRNAs typically encode components of the protein synthetic machinery itself, including ribosomal proteins and translation elongation factors. Induction

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of 5 TOP mRNA translation upregulates ribosome biogenesis and overall translational capacity (Meyuhas, 2000). Translation initiation of 5 TOP mRNAs is thought to be mediated by the 40S ribosomal protein S6. Growth factor-stimulated phosphorylation of S6 at multiple C-terminal residues correlates with translation initiation of these 5 TOP mRNAs (Jefferies et al., 1997). S6 is phosphorylated by the 70-kDa S6 kinase 1 (p70 S6K1) (Kozma et al., 1990), a ubiquitously expressed mitogen- and amino acid-sensitive protein kinase. S6K1 activation and subsequent S6 phosphorylation is a conserved mitogenic response, as all mitogenic stimuli, including growth factors, protooncogene products, phorbol esters, and cytokines induce S6K1 activity (Dufner and Thomas, 1999). Enhanced ribosome biogenesis and translational capacity is a conserved response to growth signals. Recent data have questioned whether S6K1 regulates 5 TOP-mediated mRNA translation through S6 phosphorylation, suggesting that this process is instead dependent on PI 3-K and mTOR signaling, but not on S6K1 or the ribosomal S6 protein (Tang et al., 2001). Although many aspects of this study contradict the published work of others (Jefferies et al., 1997; Lee-Fruman et al., 1999), this and other studies suggest the likely possibility that other as yet unidentiﬁed targets for the S6 kinases exist that are important in mediating their physiological function (Shima et al., 1998; Montagne et al., 1999). Notably, mice lacking S6K1 exhibit a small-animal phenotype despite normal phosphorylation of S6 and 5 TOP translation (likely due to redundant function by S6K2) (Shima et al., 1998), thus other S6K1 targets may contribute to regulation of animal size. Whether or not ribosomal S6 phosphorylation modulates 5 TOP mRNA translation, it is likely an essential component of a proliferation checkpoint mechanism as revealed by conditional S6 deletion studies (Volarevic et al., 2000) (see Section IV.B).

B. Capped mRNA and eIF-4E A methyl cap structure (m7GpppN) is found at the 5 end of all mRNAs transcribed in the nucleus. Interaction of this cap with translation initiation factor eIF-4E (eukaryotic initiation factor-4E) is an important step in loading these messages on the ribosome (Sonenberg and Gingras, 1998). The capbinding subunit eIF-4E is present in rate limiting quantities relative to other components of the translational apparatus (Rau et al., 1996), and thus serves as a key regulatory translation factor. Notably, eIF-4E provides an additional degree of translational control toward mRNAs containing a high degree of secondary structure in the 5 UTR (Rousseau et al., 1996). These stable secondary structures include hairpin loops and upstream AUGs, modiﬁcations which mark mRNAs encoding proteins important for cell growth and cell

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Fig. 5 Regulation of eIF-4F formation by 4E-BP1. eIF-4E is sequestered by hypophosphorylated 4E-BP1. Phosphorylation of 4E-BP1 by growth factor- and nutrient-sensing signaling pathways allows release of eIF-4E, which subsequently associates with the scaffolding protein eIF-4G and helicase eIF-4A to form the eIF-4F complex.

cycle progression, including growth factors, receptors, cyclins, and signaling proteins (Sonenberg and Gingras, 1998). Efﬁcient translation initiation of these highly structured mRNAs requires unwinding by a helicase, eIF-4A. eIF-4E links these messages to the helicase by binding both the mRNA cap and a scaffolding protein, eIF-4G, which also binds eIF-4A. This complex of eIF-4E, eIF-4G, and eIF-4A is referred to collectively as eIF-4F, and is important for recruiting the 40S ribosome to the message (Fig. 5) (Sonenberg and Gingras, 1998). Like 5 TOP mRNAs, eIF-4E-dependent growth regulatory messages are poorly translated in quiescent cells, and translation of these structured mRNAs is similarly upregulated following growth factor stimulation. In quiescent cells, eIF-4E is sequestered by the 4E-BP (eIF-4E-binding protein) family repressor proteins (Pause et al., 1994) (4E-BP1 is also known as PHAS-I; phosphorylated, heat- and acid-stable-Insulin responsive (Lin et al., 1994)). In their hypophosphorylated state, 4E-BPs bind eIF-4E in a manner mutually exclusive with eIF-4G, a scaffolding subunit of the eIF-4F initiation complex. This complex binds capped mRNAs (Haghighat, 1995; Marcotrigiano et al., 1997). 4EB-P1 binding to eIF-4E inhibits formation of eIF-4F complexes. This inhibition is relieved when 4E-BPs are phosphorylated in response to growth factors and dissociate from eIF-4E (Sonenberg and Gingras, 1998). In addition to regulation by 4E-BPs, phosphorylation of eIF-4E itself by the ERK effectors Mnk1/2 is thought to promote translation initiation (Waskiewicz et al., 1997; Scheper et al., 2001). Phosphorylation of eIF-4E alone, however, is not sufﬁcient for eIF-4F assembly (Herbert et al., 2000). 4E-BP1, the best characterized 4E-BP, is regulated by signals from

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the PI-3K, ERK, and mTOR pathways, and these inputs will be discussed in detail below.

C. Other PI 3-K- and mTOR-Regulated Translation Initiation Factors Initiation factor eIF-4G also likely integrates PI 3-K and mTOR signals. It undergoes serum- or insulin-stimulated phosphorylation on at least three sites that are sensitive to wortmannin and rapamycin, but the upstream kinases are not yet known (Raught et al., 2000). The effects on eIF-4G function are also unknown, but it has been postulated that such phosphorylations may alter its structure, which may modulate its scaffold function during eIF-4F assembly. Another translational regulator responsive to both PI 3-K and mTOR signals is eIF-4B. This RNA binding protein may function as a link between ribosomal and messenger RNAs, and stimulates eIF-4A helicase activity (Rozen et al., 1990). eIF-4B is phosphorylated in response to growth factors in a rapamycin- and wortmannin-sensitive manner, and is a substrate for S6K1 in vitro (Morley and Traugh, 1993). The effect of phosphorylation is not known, but it is likely that regulation of eIF-4B may have the most profound effect on translation of mRNAs with highly structured 5 UTRs. A translational effector that appears to be regulated by the PI 3-K but not by the mTOR or MAPK pathways is eIF-2B. This guanine nucleotide exchange factor is a component of eIF2, which catalyzes binding of the initiator Met-tRNA to the ribosome, an important step in initiation (Price and Proud, 1994). The eIF-2Bε subunit is inhibited when phosphorylated by GSK3 (Welsh et al., 1998). Insulin relieves this suppression through an Aktinduced inhibition of GSK3 (Cross et al., 1995; Takata et al., 1999). Thus, the PI 3-K pathway regulates translation through multiple effectors of the initiation apparatus, including S6K1, 4E-BP1, eIF-4G, eIF-4B, and eIF-2B.

III. REGULATION OF TRANSLATIONAL EFFECTORS A. 4E-BP1 Regulation 4E-BPs are the major factors regulating eIF-4E activity, and thus, subsequent formation of the eIF-4F initiation complex. Hypophosphorylated 4E-BP1 binds eIF-4E with high afﬁnity (Pause et al., 1994; Lin et al., 1994). The 4E-BP1–eIF-4E interaction is disrupted following sequential 4E-BP1

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Fig. 6 Regulation of 4E-BP1 phosphorylation. Sequential phosphorylation of 4E-BP1 is mediated by nutrient- and growth factor-sensing signaling pathways. Phosphorylation of the priming sites Thr37 and Thr46 precedes phosphorylation of Thr70 and Ser65, to allow release of eIF-4E.

phosphorylation at multiple sites. Thr37 and Thr46 are basally phosphorylated and are substrates for phosphorylation by mTOR in vitro (Fig. 6) (Gingras et al., 1999; Mothe-Satney et al., 2000a). 4E-BP1 undergoes ordered, sequential phosphorylation, with Thr37 and Thr46 serving as prerequisite priming sites for serum-induced phosphorylation at Thr70 and Ser65 (Gingras et al., 1999, 2001a; Mothe-Satney et al., 2000b). Studies with phosphospeciﬁc antibodies suggest that Ser65 may be the ﬁnal site to be phosphorylated (Mothe-Satney et al., 2000a; Gingras et al., 2001a). This site is also likely critical for disruption of the eIF-4E interaction, as the eIF-4E binding site in 4E-BP1 is ﬂanked by Thr46 and Ser65 (Haghighat, 1995), and phosphorylation of Ser65 prevents this association in vitro. Due to the multiple phosphorylations, 4E-BP1 from stimulated cells migrates as a triplet of bands in SDS-polyacrylamide gels. Only the most slowly migrating band reacts with phospho-Ser65, and this species fails to copurify with eIF-4E (Mothe-Satney et al., 2000a). Like S6K1, Ser65 and Thr70 are regulated by both PI 3-K and mTOR pathways, as they are sensitive to wortmannin and rapamycin (Gingras et al., 1998; Mothe-Satney et al., 2000a). S6K1 is not an in vivo 4E-BP1 kinase, but S6K1 and 4E-BP1 are thought to function in parallel pathways which bifurcate downstream of mTOR. S6K1 and 4E-BP1 may share a common rapamycin-sensitive activator, since overexpression of S6K1 can inhibit 4E-BP1 phosphorylation (von Manteuffel et al., 1997). One report states that Ser65 and Thr70 are also phosphorylated by mTOR in vitro, but only when

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mTOR is incubated with an “activating” antibody, mTAb1 (Mothe-Satney et al., 2000a). Alternately, dissociation of eIF-4E from 4E-BP1 may involve 4E-BP1 phosphorylation by an mTOR-associated kinase (Heesom and Denton, 1999). Another model to explain 4E-BP1 inactivation is that these sites are phosphorylated by PI 3-K-regulated kinases, and dephosphorylated by PP2A-type phosphatases, which are activated following mTOR inhibition (Peterson et al., 1999). Akt appears to be an important regulator of 4E-BP1 in vivo, as expression of a constitutively active mutant induces 4E-BP1 phosphorylation, while a dominant negative is inhibitory (Gingras et al., 1998; Dufner et al., 1999; Takata et al., 1999). Akt may regulate a 4E-BP1 kinase, as Akt itself does not directly phosphorylate 4E-BP1 in vitro (Gingras et al., 1998). It has recently been shown that the mTOR-regulated nPKCδ (Parekh et al., 1999) can phosphorylate 4E-BP1 in a rapamycin-sensitive manner, although the target site has not yet been identiﬁed (Kumar et al., 2000a). Through an inhibitory association with mTOR, the c-abl tyrosine kinase is a negative regulator of 4E-BP1 function in response to DNA damage (Kumar et al., 2000b). Finally, 4E-BP1 phosphorylation may occur by a phorbol ester-stimulated, PI 3-K independent mechanism which may be mediated by the MEK/ERK cascade (Herbert et al., 2000, 2002). Basal MEK activity is also required for insulin-stimulated 4E-BP1 phosphorylation and eIF-4F assembly cascade (Herbert et al., 2000). ERK2 can phosphorylate 4E-BP1 in vitro (Fadden et al., 1997) and in vivo in vascular smooth muscle cells (Rao et al., 1999). Other 4E-BP family members, including 4E-BP2 and 4E-BP3 (Poulin et al., 1998) have been identiﬁed. These are related to 4E-BP1, with the greatest sequence conservation in the eIF-4E-binding motif (Gingras et al., 2001b). These proteins seem to serve a similar function and are likewise regulated by phosphorylation. 4E-BP1 and −2, but not 4E-BP3, contain a four-amino acid motif (Arg-Ala-Ile-Pro “RAIP”) in the N-terminus that is required for efﬁcient phosphorylation (Tee and Proud, 2002). Early studies with 4E-BP2 suggest that slight differences in regulation may lead to different kinetics of eIF-4E dissociation (Gingras et al., 2001b; Grolleau, 1999). Thus, the function of these isoforms may allow for tissue-speciﬁc differences in growth factor stimulated translational responses (Grolleau, 1999).

B. Regulation of S6K1 The PI 3-K and mTOR pathways are major signaling pathways regulating S6K1 activity (Chung et al., 1992, 1994). The S6K1 (and 4E-BP1) requirement for inputs from both the PI 3-K and mTOR pathways, as well as by other mitogen activated signaling pathways, suggests a mechanism by which

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cells can integrate nutrient capacity and growth factor stimulated protein synthesis. S6K1 exists as two isoforms. The 70-kDa αII isoform is largely cytosolic in localization. An 85-kDa αI isoform is identical to p70 with the exception of an additional 23 amino acids at the N-terminus encoding a nuclear localization signal (Coffer and Woodgett, 1991; Reinhard et al., 1994). The function of nuclear S6K1 is unclear, but S6K1 can phosphorylate the transcription factor cremτ , suggesting a possible role in transcriptional control (de Groot et al., 1994). The two S6K1 isoforms appear to be regulated similarly in all systems examined, except for slightly delayed kinetics of p85 activation relative to p70 in response to pressure overload in cardiomyocytes (Laser et al., 1998). S6K1 kinase activity is regulated by at least nine growth factor-induced phosphorylation events. The ﬁrst of these, targeting multiple sites in the C-terminus, is thought to relieve autoinhibition by intramolecular interactions (Cheatham et al., 1995; Weng et al., 1995). The kinase domain at the core of the molecule is ﬂanked by N- and C-terminal regulatory domains (Fig. 7). A model of S6K1 activation based on structure/function mutagenesis studies suggests that acidic amino acids in the N-terminus interact with basic residues in the C-terminus to stabilize an autoinhibitory inactive conformation. In this inactive state, a pseudosubstrate region in the C-terminus with high homology to ribosomal S6 occludes the kinase

Fig. 7 Structure of S6 kinases. Shown is a schematic of the primary structures of the shorter isoforms of S6K1 (αII) and S6K2 (βII). Mitogen-stimulated phosphorylation sites are indicated by amino acid number. The activation loop (T229/228 TFCGT), linker region (S371/370 SPDD) and hydrophobic motif (T389/388 FLGFTY) sites are perfectly conserved between S6K1 and S6K2, but there is some divergence in the proline-directed C-terminal sites. S6K2 exhibits regions of divergence from S6K1 in the N- and C-terminal regulatory domains, including the presence of a C-terminal polyproline-rich domain and nuclear localization signal.

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domain (Cheatham et al., 1995; Weng et al., 1995). An early step in S6K1 activation is mitogen-induced phosphorylation of C-terminal regulatory sites (Ser404, Ser411, Ser418, Thr421, Ser424), which disrupt this interaction, perhaps mediated by ERK or p38 MAP kinases (Weng et al., 1998; A. Romanelli, unpublished results). Phospho-speciﬁc antibodies reveal that these sites are also sensitive to inhibition by rapamycin or wortmannin (Weng et al., 1998). Phosphorylation of sites in an internal regulatory domain and the kinase domain follow, including Ser371, Thr389, and Thr229. Ser371 phosphorylation is essential for S6K1 activity, and is mediated by an as yet undetermined mechanism but is insensitive to rapamycin and wortmannin (Moser et al., 1997; A. Romanelli, unpublished observations). Thr229, located in the catalytic activation loop, is essential for kinase activity and sensitive to wortmannin and rapamycin (Weng et al., 1998). This site is phosphorylated by phosphoinositide-dependent kinase 1 (PDK1), a constitutively active kinase whose subcellular localization and access to substrates is regulated by PI 3-K-derived phospholipids (Williams et al., 2000; Alessi et al., 1998; Pullen et al., 1998). The importance of Thr229 is highlighted by the fact that an inhibitor of PDK1 signaling, n-alpha-tosyl-lphenylalanyl chloromethyl ketone (TPCK), is a potent inhibitor of S6K1 activity (Grammer and Blenis, 1996; Ballif et al., 2001). Further, mutation of this site (T229A) abolishes S6K1 activity and subsequent Thr389 phosphorylation (Weng et al., 1998). Phosphorylation of Thr389 in the regulatory domain appears to be central to S6K1 activation, as it is exquisitely sensitive to inhibition by rapamycin, and thought to be the ﬁnal and rate-limiting step in S6K1 activation (Weng et al., 1998; A. Romanelli, unpublished observations). While the importance of this amino acid in S6K1 function is universally accepted, the mechanism underlying Thr389 regulation is currently controversial. This site has been reported to be directly phosphorylated by mTOR in vitro (Burnett et al., 1998a). However, Thr389 is still phosphorylated in a mitogen-sensitive manner in a truncation mutant of S6K1 that is rapamycin resistant, indicating that mitogens can stimulate Thr389 phosphorylation independently of mTOR (S. Schalm, unpublished observations). The NIMA family kinases NEK6/7 have been shown to phosphorylate Thr389 in vivo and in vitro (Belham et al., 2001). Activity of these kinases, however, is only weakly stimulated by insulin and partially sensitive to wortmannin, while Thr389 phosphorylation is entirely wortmannin sensitive, suggesting that other mechanisms may contribute to Thr389 regulation. Regulation of the critical Thr389 site is also dependent on PDK1, as IGF-I fails to induce Thr389 phosphorylation in PDK1 null cells (Williams et al., 2000), and TPCK inhibits phosphorylation of both Thr229 and Thr389 (Ballif et al., 2001). It is also possible that S6K1 autophosphorylation

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contributes to the phosphorylation state of Thr389, as we have observed that optimal and prolonged Thr389 phosphorylation does not occur in a kinase inactive S6K1 (K100R) point mutant (A. Romanelli, unpublished observations). This mechanism is consistent with the requirement for PDK1 phosphorylation of the catalytic activation loop site (Thr229). Finally, the many mitogen-stimulated phosphorylation sites in S6K1, including Thr389, are rapidly dephosphorylated after rapamycin treatment. As discussed earlier, an mTOR-regulated phosphatase is a possible mechanism which could rapidly inhibit many sites regulated by diverse upstream kinases (Peterson et al., 1999). A highly conserved sequence in the N-terminus of S6K1 and S6K2 (amino acids 5 to 9 of S6K1) has recently been revealed to be a critical TOR signaling (TOS) motif that is essential for mTOR-dependent signaling. The TOS domain mediates mTOR regulation of two distinct regions of S6K1: this motif is required for phosphorylation of Thr389, as well as for release of a negative regulatory mechanism mediated by the S6K1 C-terminus. The importance of the TOS motif in mTOR signaling is underscored by its identiﬁcation in other mTOR-regulated proteins. This motif is conserved throughout evolution in the C-termini of 4E-BPs. Mutation of the TOS motif in 4E-BP1 similarly blocks its mitogen-dependent phosphorylation (Schalm and Blenis, 2002). It has been long known that conventional PKCs (cPKCs) can also contribute to S6K1 activation (Blenis and Erikson, 1986), but the precise molecular mechanism is still not well documented. PDGF receptor mutants deﬁcient in PLCγ binding and subsequent cPKC activation are impaired in signaling to S6K1, while mutants lacking the PI 3-K binding site suggest that the PI 3-K pathway is the major contributor to S6K1 activation (Chung et al., 1994). Separable PI 3-K and cPKC inputs were also documented in B cells (Monfar et al., 1995). Similarly, S6K1 activation by B cell antigen receptor cross-linking is dependent on both PI 3-K and cPKC-dependent pathways and involves the tyrosine kinase Syk (Li et al., 1999).

1. SUBCELLULAR LOCALIZATION AND REGULATION OF S6Ks S6K1 is activated by multiple PI 3-K effectors, including Cdc42, Rac (Chou and Blenis, 1996), PDK1 (Alessi et al., 1998; Pullen et al., 1998), and atypical PKCζ (Romanelli et al., 1999) and PKCλ (Fig. 8) (Akimoto et al., 1998). While PDK1 directly phosphorylates S6K1 (Alessi et al., 1998; Pullen et al., 1998), the mechanism of activation by these other effectors is an ongoing subject of investigation. The low-molecular-weight G proteins Cdc42 and Rac activate S6K1 independent of the p38 or JNK pathways (Chou and Blenis, 1996). These G proteins, however, can bind to S6K1 in vivo in a

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PI3Kp110

Cdc42/Rac

PKCζ

mTOR

PI3K p85

PDK1

Akt

PP2A?

S6K1

Fig. 8 Regulation of S6K1 by PI 3-K and mTOR. The PI 3-K effectors Cdc42, Rac, PKCζ , PDK1, and Akt have all been implicated in regulation of S6K1. Only PDK1 is known to directly phosphorylate S6K1 (at Thr229). Evidence suggests that mTOR may directly phosphorylate S6K1 at Thr389, and/or may regulate all S6K1 phosphorylation sites through regulation of a PP2A-type phosphatase. Multiple PI 3-K effectors exist in a complex with S6K1, and the PI 3-K p85 adapter subunit may mediate association of mTOR and S6K1.

rapamycin- and wortmannin-insensitive manner. This association is required for Cdc42 activation of S6K1. Isoprenylation of the G protein is also required (Chou and Blenis, 1996). These data suggest that interaction of these G proteins may activate S6K1 by inducing a conformational change in the kinase, and/or by targeting the kinase to a membrane, bringing it in proximity with other membrane-localized activators including PDK1, aPKCs, and Akt. We have also reported that S6K1 coimmunoprecipitates with PDK1 or PKCζ , and that PDK1 and PKCζ can associate with each other (Romanelli et al., 1999). Constitutively active mutants of Akt support a membrane targeting model, as only those which localize to the plasma membrane are capable of activating S6K1 (Dufner et al., 1999). These combined data suggest that S6K1 may participate in a complex of PI 3-K-regulated effector proteins which facilitates signaling in this pathway. The existence of such scaffolding mechanisms has been suggested for other pathways, including MAPK pathways (Kholodenko et al., 2000; Garrington and Johnson, 1999; Burack and Shaw, 2000). It is not known whether a common central scaffolding molecule exists in this PI 3-K model, but other evidence suggests that these interactions may take place at the plasma membrane and/or cytoskeleton. Finally, coprecipitation of S6K1, mTOR, and the p85 subunit of PI 3-K suggest that a ternary (or larger) complex integrates S6K1 activation by the PI 3-K and mTOR pathways (Gonzalez-Garcia et al., 2002) (see Section IV.D for further detail).

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The S6K1 activators Cdc42 and Rac are also important regulators of the cytoskeleton that mediate many structural changes contributing to cell motility (Erickson and Cerione, 2001). Several studies suggest that S6K1 might also play a role in motility, as it has been shown to colocalize with stress ﬁbers, and that thrombin-induced elongation and organization of stress ﬁbers is rapamycin sensitive in ﬁbroblasts (Crouch, 1997). Furthermore, S6K1 colocalizes with actin arc structures at the leading edge of migrating cells (Berven and Crouch, 2000). Interestingly, several factors known to regulate and associate with S6K1, the atypical PKCs ζ or λ (Romanelli et al., 1999; Akimoto et al., 1998) and Cdc42 (Chou and Blenis, 1996), have recently been reported to associate with each other in a GTP-dependent manner (Coghlan et al., 2000). This study demonstrated that activated Cdc42 induced stress ﬁber loss, and that this process required active aPKCs. A role for S6K1 in this process was not addressed, but these data further document the clustering of similarly regulated signaling proteins at cytoskeletal structures. Two-hybrid and biochemical analyses identiﬁed the F-actin binding protein neurabin as a binding partner for S6K1 in neural cells (Burnett et al., 1998b). A PDZ domain in this neural-speciﬁc cytoskeletal-associated protein binds to the extreme C-terminus of S6K1 in a serum- and rapamycinindependent manner. The mRNAs for neurabin and S6K1 colocalize in various brain structures, including the hippocampus and cerebellum, and it has been suggested that these proteins colocalize at nerve terminals. Coexpression of S6K1 and neurabin in nonneural tissues leads to a modest induction of S6K1 activity (Burnett et al., 1998b), supporting the model that “scaffold”-mediated targeting of S6K1 to cellular locales enriched in regulatory molecules may facilitate S6K1 activation. Subcellular localization is important for regulation of the mTOR pathway, as well as for PI 3-K effectors, and perhaps especially so for common effectors of both pathways such as S6Ks. The ubiquitously expressed protein gephyrin serves to cluster glycine receptors at postsynaptic nerve terminals, and has been identiﬁed as a binding partner for mTOR (Sabatini et al., 1999). mTOR interaction with gephyrin mediates its subcellular localization and is essential for its ability to regulate S6K1 and 4E-BP1. Furthermore, an intriguing study has suggested that nuclear shuttling of mTOR may be necessary for mitogen-stimulated activation of translational effectors, as inhibition of nuclear export using leptomycin B inhibited phosphorylation of both S6K1 and 4E-BP1 (Kim and Chen, 2000). Such nuclear/cytoplasmic shuttling of mTOR suggests a possible regulatory mechanism for primarily nuclear isoforms such as p85-S6K1 or the S6K2 proteins. It is likely that future studies will further clarify the mechanisms by which components of signaling pathways are brought together to function efﬁciently and speciﬁcally.

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C. S6K2 For many years it was thought that S6K1 was the only in vivo S6 kinase. However, several groups recently identiﬁed a homolog closely related to S6K1, now called S6K2 (also called p70β, SRK) (Shima et al., 1998; Gout et al., 1998; Lee-Fruman et al., 1999; Koh et al., 1999). In addition to isolation based on database searches for novel proteins with homology to S6K1 (Shima et al., 1998; Gout et al., 1998; Lee-Fruman et al., 1999; Koh et al., 1999), S6K2 was identiﬁed when normal S6 phosphorylation and 5 TOP mRNA translation was discovered in cells derived from mice lacking both p70 and p85 isoforms of S6K1 (Shima et al., 1998). As the mRNA for S6K2 was found to be upregulated in these mice, it is likely that S6K2 may supply the S6 phosphorylation function in vivo in the absence of S6K1. Using a rapamycin-resistant S6K2 mutant, we ﬁnd that S6K2 is indeed an in vivo S6 kinase, as S6 phosphorylation persists in rapamycin-treated cells stably overexpressing this mutant, when S6K1 and other mTOR-effectors are maximally inhibited (K. Martin, unpublished observations). Whether additional in vivo S6 kinases might also exist remains to be determined. Mice lacking S6K1 through homozygous deletion demonstrate a smallanimal phenotype despite normal S6 phosphorylation and 5 TOP mRNA translation (Shima et al., 1998), suggesting that S6 regulation is not the critical determinant of animal size, but that S6K1 may mediate other important nonredundant functions that contribute to size regulation. Because, unlike S6K1, both S6K2 isoforms encode a common C-terminal nuclear localization signal (Koh et al., 1999), it is likely that S6K2 mediates unique nuclear functions. Several structural features and differential regulation further suggest that S6K2 mediates distinct functions. While S6K2 is highly homologous to S6K1 overall, there are regions of divergence in both the amino- and carboxyl-terminal regulatory regions (Fig. 7). In addition to the unique Cterminal NLS, S6K2 contains a C-terminal polyproline-rich region absent in S6K1. The role of the polyproline domain is not yet known, but deletion of this domain reportedly does not affect wortmannin or rapamycin sensitivity (Gout et al., 1998). Chimeras swapping the region of the polyproline domain between S6K1 and S6K2 failed to reveal an obvious function for this domain (S. Schalm, unpublished observations). S6K2 is activated by the same stimuli that activate S6K1, and is potently inhibited by wortmannin and rapamycin (Shima et al., 1998; Gout et al., 1998; Lee-Fruman et al., 1999; Koh et al., 1999), suggesting that it also functions as an effector of the PI 3-K and mTOR pathways. Regulation of S6K2 is largely similar to S6K1, with some intriguing differences, likely arising due to sequence variations and/or differential subcellular localization. Despite the primarily nuclear expression of S6K2, it is regulated

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by the PI 3-K effectors Cdc42, Rac, PDK1, PKCζ , (Martin et al., 2001a), and Akt (Koh et al., 1999). One difference in S6K regulation was that atypical PKCζ was a more potent activator of S6K2. Interestingly, point mutation which destroys the S6K2 nuclear localization signal modestly potentiates its activation by PI 3-K effectors (Martin et al., 2001a). This regulation by cytosolic effectors suggests that S6K2 may exit the nucleus during the course of activation. Like S6K1, S6K2 exists as two alternately spliced isoforms, which differ by an additional N-terminal 13 amino acids present in S6K2β1 but lacking in S6K2β2 (Gout et al., 1998). Although the C-terminal nuclear localization signal is common to both S6K2 isoforms (Koh et al., 1999), it has been suggested that the unique basic sequence at the N-terminus of S6K2β1 contributes to its nuclear localization as well, as overexpressed S6K2β1 is found primarily in the nucleus, while overexpressed S6K2β2 can be found in both the nucleus and the cytosol (Minami et al., 2001). It has been suggested that S6K2 is less sensitive to rapamycin or wortmannin than is S6K1 (Gout et al., 1998; Minami et al., 2001). It is likely, however, that this reﬂects a difference in inactivation, as opposed to initial activation. Minami et al. observed that S6K2 overexpressed in cells continually cultured in 10% serum was less sensitive to inhibition by addition of rapamycin or wortmannin to the medium. We and others, however, have found that activation of S6K2 in serum-starved cells by the addition of insulin or serum is nearly completely inhibited by rapamycin or wortmannin (Lee-Fruman et al., 1999; Koh et al., 1999). The most notable functional difference between S6K1 and S6K2 is the role of the C-terminal autoinhibitory domain. Deletion of this regulatory region, which lies just C-terminal to the kinase domain, has a modest inhibitory effect on S6K1, yet has a potent stimulatory effect on S6K2 that is dramatically enhanced by coexpression of PI 3-K effectors, such as Cdc42 or PDK1 (Martin et al., 2001b). These data suggest that this domain participates in potent repression of S6K2 kinase activity. Furthermore, this repression of S6K2 may be relieved by inputs from the MEK/ERK1/2 pathway, as fulllength S6K2 is highly sensitive to inhibition by the MEK inhibitor U0126, while S6K1 is far less sensitive to MEK inhibition (Martin et al., 2001b). The S6K2 U0126 sensitivity was evident for EGF, a potent ERK agonist, but not for insulin, a poor ERK agonist in the HEK293 cells used. Activation of S6K2 by G protein-coupled receptor agonists in cardiomyocytes was also found to be highly MEK dependent (Wang et al., 2001). The MEK pathway has been implicated in regulation of the C-terminal phosphorylation sites in both S6K1 and S6K2 (Lenormand et al., 1996; Scott and Lawrence, 1997; Mukhopadhyay et al., 1992; Herbert et al., 2000; A. Romanelli, unpublished results), but may play a more critical role in the initial steps of S6K2

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activation. It is also possible that the presence of the polyproline-rich domain in proximity to the C-terminal regulatory phosphorylation sites may confer differential S6K2 regulation. While S6K1 contains a motif at its C-terminus which may allow its regulation by PDZ domain proteins such as neurabin (Burnett et al., 1998b), S6K2 lacks such a motif. The absence of a C-terminal PDZ-binding sequence may account for some differences in S6K2 regulation by this domain (Martin et al., 2001b). However, both S6K1 and S6K2 are regulated by isoprenylated Cdc42 and Rac (Chou and Blenis, 1996; Martin et al., 2001a), suggesting that membrane association may be important in regulation of both kinases.

IV. COORDINATED TRANSLATIONAL CONTROL A. Coordination of mRNA Splicing and Translation Recent work on regulation of mRNA splicing by the low-molecular-weight G protein Cdc42 suggests that the PI 3-K and mTOR pathways may coordinate the related processes of mRNA splicing and translation, in part, through S6K1 (Wilson et al., 2000). Cdc42 has been shown to be an effector of the PI 3-K pathway, and an activator of both S6K1 and S6K2 (Chou and Blenis, 1996; Martin et al., 2001a). Activated Cdc42 stimulates binding of the capbinding complex (CBC) to nuclear capped mRNAs (Wilson et al., 1999) and stimulates pre-mRNA splicing independent of its PAK, ACK, or WASP effector functions (Wilson et al., 2000). Notably, splicing stimulated by an activated Cdc42 mutant is sensitive to rapamycin, but not to wortmannin. Signaling from Cdc42 to S6K1 has been proposed to mediate this effect, as S6K1 was found to phosphorylate the CBC subunit CBP80 in vitro at growth factor-stimulated rapamycin-sensitive sites. It is likely that PI 3-K lies upstream in this pathway, but that the downstream constitutively activated Cdc42 mutant employed is resistant to the effects of wortmannin, as overexpression of p70 S6K1 and constitutively active PI 3-K enhances splicing in the presence of endogenous Cdc42. The S6K1 phosphorylation sites ﬂank a nuclear localization signal, but ablation or acidic substitution of the sites does not alter nuclear localization of CBC. S6K1 phosphorylates CBP80 in vitro, but it has not yet been determined whether the cytosolic p70 or nuclear p85 S6K1, or even the nuclear S6K2 isoforms, may mediate this event in vivo. One model suggests that the nuclear CBC escorts the capped mRNA complex from the nucleus to the cytosol, where it binds to the eIF-4E capbinding translation initiation factor (Wilson et al., 2000; Visa et al., 1996). Thus, Cdc42/S6K inputs may coordinate mRNA splicing with PI 3-K- and mTOR-regulated translation initiation. Cdc42 has been implicated in cell

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transformation (Lin et al., 1997). Interestingly, an activated Cdc42 double mutant deﬁcient in splicing regulation is also deﬁcient in transformation, suggesting that this new S6K-mediated function of Cdc42 may contribute to the transformed phenotype (Wilson et al., 2000).

B. Coordinated Growth and Proliferation in Liver Regeneration p85 alpha PI 3-K knockout mice reveal that these enzymes play an essential role in the liver (Fruman et al., 2000). Liver regeneration following partial hepatectomy provides a model system for assessing the intertwined processes of cell growth and proliferation. Partial hepatectomy increases circulating levels of hepatocyte growth factors, and markedly induces the activities of PI 3-K, Akt, and S6K1 (Michalopoulos and DeFrances, 1997; Hong et al., 2000; Jiang et al., 2001), and expression of a novel liver-speciﬁc PI 3-K, termed PI 3-KIIγ (Ono et al., 1998). Mice with diminished levels (CRE/lox conditional knockout) of ribosomal S6 protein in liver are impaired in recovery from partial hepatectomy (Volarevic et al., 2000). Hepatocytes from these livers exhibit abnormal ribosome proﬁles and progress through early G1 phase, as indicated by normal induction of cyclin D. The cells do not progress to S phase, as cyclins E and A mRNA and protein are lacking. The authors propose that this arrest may be due to activation of a checkpoint which senses abnormal ribosome biogenesis. This study raises interesting questions regarding the coordination of protein synthesis and proliferation. Interpretation of these studies is complicated, however, by the long half-life of the S6 protein. Because S6 protein was present at even 5 days after CRE induction, it should be noted that the data were observed in the presence of reduced levels, but not complete lack, of S6. A complete knockout of S6 protein, if viable, may have a more immediate and potent effect on liver regeneration following both starvation and hepatectomy. Another interesting ﬁnding from the partial hepatectomy model is that 4E-BP1 is not required for rapamycin-sensitive liver regeneration. Partial hepatectomy induces S6K1 activation and 4E-BP1 phosphorylation and subsequent reduction in eIF-4E binding and repression (Jiang et al., 2001). Normal animals treated with rapamycin suffer impaired liver regeneration (Francavilla et al., 1992; Jiang et al., 2001). It was found that under these conditions, S6K1 phosphorylation and activity is markedly inhibited, while 4E-BP1 phosphorylation persists, and eIF-4E cap-binding activity consequently increases in hepatocytes from rapamycin-treated rats after partial hepatectomy (Jiang et al., 2001). These data suggest not only an mTORindependent phosphorylation of 4E-BP1, but also that S6K1 may be an essential target in rapamycin-sensitive regeneration in vivo. The role of the

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rapamycin-sensitive related kinase S6K2 in hepatocyte models has not yet been addressed, but this kinase may be another candidate for regulation of protein synthesis and proliferation in regenerating liver. These ﬁndings differ from reports of rapamycin-sensitive 4E-BP1 phosphorylation in vitro, and suggest that the in vivo environment following hepatectomy is not mirrored in tissue culture experiments. The Erk pathway has been implicated in 4E-BP phosphorylation in other cell types (Fadden et al., 1997; Rao et al., 1999), but Erk1/2, p38, or Jnk activation was not detected following hepatectomy (Jiang et al., 2001). Other rapamycin-insensitive effector kinases in the PI 3-K or PKC pathways known to regulate 4E-BPs in other systems may be responsible for the rapamycin-insensitive phosphorylation following hepatectomy. In light of these studies suggesting the importance of S6K and S6 in liver regeneration, it will be of interest to determine the effect of partial hepatectomy in S6K1 knockout mice. Embryonic ﬁbroblasts from these mice are reported to have normal S6 phosphorylation, perhaps due to compensation by S6K2 (Shima et al., 1998). Whether S6 phosphorylation is entirely normal in adult hepatocytes in vivo, however, has not yet been established. This may shed light on the relative roles of S6K1 versus S6K2 in the dynamic liver model. Comparison of individual and combined knockouts of S6K1 and S6K2 would be the best tool to address this question.

C. PI 3-K, mTOR, Translation, and Cell Size The critical role of PI 3-K in cell growth and proliferation is underscored by the conservation of this pathway from C. elegans to humans. This conservation has allowed for elegant genetic studies in multiple organisms, which have revealed the importance of the PI 3-K pathway and speciﬁc effectors in control of cell size. Mutations of the Drosophila insulin receptor (INR) (Chen et al., 1996), IRS homolog (chico) (Bohni et al., 1999), PI 3-K (dp110) (Leevers et al., 1996), Akt (dAkt) (Verdu et al., 1999), TOR/FRAP (dTOR) (Zhang et al., 2000; Oldham et al., 2000), or S6K1 (dS6K) (Montagne et al., 1999) give rise to a small cell phenotype. Similarly, overexpression of a d4EBP mutant having high afﬁnity for deIF4E reduces Drosophila cell size (Miron et al., 2001). In the dS6K mutant ﬂies, cell number is preserved, but each individual cell is smaller in size than wild-type cells, producing normally proportioned animals with a 50% reduction in body size (Montagne et al., 1999). These data suggest that dS6K regulates cell size and, subsequently, organ and body size, but does not inﬂuence patterning, cell-fate, or spatial decisions. S6-dependent control of ribosome biogenesis and, translational capacity is an attractive hypothesis to explain these phenotypes, but it is more likely that other targets of S6K1 are involved in cell size control: Homozygous deletion of S6K1 in mice results in a small animal phenotype

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despite the presence of normal S6 phosphorylation and 5 TOP mRNA regulation, presumably mediated by the intact function of the homolog S6K2 (Shima et al., 1998). These mice have pancreatic β-cells of reduced size, rendering them hypoinsulinemic (Pende et al., 2000). Notably, the dS6K mutant phenotype is far more severe in ﬂies, where it includes female sterility, developmental delay, and early death (Montagne et al., 1999). The S6K1-/- mice are viable, fertile, and only 20% smaller than wild type (Shima et al., 1998). This may reﬂect the role played by S6K2 in mammalian cells (Shima et al., 1998), while there is thought to be only one S6K species in Drosophila. The PI 3-K pathway has also been implicated in cell size regulation in a cardiac hypertrophy model, where the variable of cell proliferation is eliminated due to the terminally differentiated status of cardiomyocytes (Shioi et al., 2000). Cardiac-speciﬁc expression of activated or dominant negative PI 3-K resulted in transgenic mice with larger or smaller hearts, respectively, with corresponding regulation of Akt and S6K1. Similarly, overexpression of active PTEN inhibited, and catalytically inactive PTEN promoted, cardiomyocyte hypertrophy (Schwartzbauer and Robbins, 2001). Genetic studies demonstrate that upstream PI 3-K components may play more diverse roles in regulation of both growth and proliferation. While chico and dS6K mutant ﬂies are viable, deletions of INR, PI 3-K, or dAkt result in embryonic lethality (Chen et al., 1996; Leevers et al., 1996; Verdu et al., 1999). Mutations in INR, chico, PI 3-K, and dAkt affect cell number as well as cell size (Leevers, 1999). dPTEN has been shown to act as a tumor suppressor in ﬂies, reducing cell number and cell size, by antagonizing the PI 3-K pathway (Goberdhan et al., 1999). Other evidence, however, points to a role for S6K1 in proliferation as well as translational control and cell size. A rapamycin-resistant S6K1 mutant rescued rapamycin-sensitive inhibition of E2F transcriptional responses in lymphocytes (Brennan et al., 1999). Others suggest that S6K1 is not essential for proliferation after observation of rapamycin-sensitive S6K1 activity in cells whose proliferation is rapamycin resistant (Hosoi et al., 1998; Louro et al., 1999; Slavik et al., 2001). We propose that there may be multiple downstream targets of the mTOR pathway that may confer rapamycin resistance to proliferation. A lesion in the pathway downstream of S6K1 does not necessarily exclude a role for S6K1 in proliferation. While genetic data from Drosophila and in vivo mouse models have begun to identify signaling molecules that function to regulate cell growth and cell size, the biochemical signaling pathways that cooperate to control cell growth in mammalian cells are less well understood. Treatment of asynchronously cycling cultured mammalian cells with rapamycin or LY294002 reduces cell size as well as cell cycle progression and proliferation, implicating a role for mTOR and PI3-K in control of mammalian cell size (Fingar et al., 2002). It is the inhibition of mTOR that mediates

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rapamycin’s effect on cell size, as expression of a rapamycinresistant mutant of mTOR rescues the rapamycin-induced small cell size phenotype. Similarly, treatment of differentiated C2C12 myotubes or isolated rat skeletal muscle with rapamycin blocks muscle hypertrophy (Bodine et al., 2001; Rommel et al., 2001). Consistent with a role for mTOR and PI3-K in control of cell size, overexpression of either S6K1 or eIF4E increases cell size, while coexpression of both proteins additively increases cell size, demonstrating that the mTOR to S6K1 and mTOR to 4EBP/eIF4E pathways function in parallel downstream of mTOR to coordinately regulate cell size (Fingar et al., 2002). Similarly, overexpression of S6K1 or Akt1 in differentiated C2C12 myotubes induces muscle cell hypertrophy (Rommel et al., 2001). Consistent with the reduction in cell size observed upon overexpression of a d4EBP mutant with high afﬁnity for deIF4E in Drosophila (Miron et al., 2001), overexpression of a phosphorylation site mutant of 4EBP1 (Thr37/46Ala) in cultured mammalian cells also reduces cell size (Fingar et al., 2002). Further evidence supporting a role for mTOR-mediated signaling in control of mammalian cell size is that an S6K1 phosphorylation site acidic substitution mutant (E389D3E) that exhibits partial rapamycin resistance, or overexpression of eIF4E, partially rescues the reduced cell size phenotype induced by rapamycin (Fingar et al., 2002). Lastly, while cell cycle progression and cell growth are normally tightly coordinated during cellular proliferation (cells tend to remain fairly constant in size through multiple cell division cycles), the two processes can be experimentally dissociated in mammalian cells (Fingar et al., 2002), conﬁrming what Lee Hartwell and colleagues ﬁrst observed in budding yeast 25 years ago (Johnston et al., 1977). When cell cycle progression is blocked upon expression of cell cycle inhibitory proteins (such as the cdk inhibitors p16 or p21, or dominant-negative cdk2), cells continue to grow to increased size, indicating that cell cycle progression and cell growth are separable and distinct processes. Importantly, this growth to increased cell size is blocked by rapamycin or LY294002. The mechanisms that allow tight coordination of cell cycle progression and cell growth are poorly understood (see Section IV.D). Data from numerous experimental systems all point to an important role for signaling molecules that regulate protein translation as important regulators of cell growth and cell size.

D. Coordination of the PI 3-K and mTOR Pathways Two recent genetic studies of mutations in the Drosophila TOR (dTOR) protein elegantly assessed the relationship between the PI 3-K and TOR pathways (Oldham et al., 2000; Zhang et al., 2000). Both studies conclude that the dTOR pathway serves a nutrient checkpoint function that converges

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on growth factor-regulated translational effectors of the PI 3-K pathway. Disruption of dTOR function prevents ﬂies from developing past the larval stage (Zhang et al., 2000). The cellular phenotypes perfectly mimic the effects of amino acid starvation (Oldham et al., 2000). As with mutations in the PI 3-K pathway, including dS6K (Montagne et al., 1999), loss of dTOR function results in a cell autonomous cell size defect, resulting in reductions in organ and body size (Zhang et al., 2000; Oldham et al., 2000). In dTOR mutants, proliferation rates are also reduced, characterized by an increased number of cells in G1 phase, and fewer in S and G2 (Zhang et al., 2000). Cyclin E overexpression rescued the G1 arrest, and cyclin E protein levels were greatly reduced in dTOR mutant ﬂies, suggesting that this cyclin is a critical downstream target of TOR regulation (Zhang et al., 2000). The dTOR mutants provide important observations on the role and regulation of dS6K. Oldham et al. observed diminished S6 phosphorylation, but an upregulation of dS6K protein levels in dTOR mutants or following amino acid deprivation, suggesting dTOR-mediated feedback regulation of dS6K. Interestingly, mutations in the PI 3-K pathway do not affect dS6K levels (Oldham et al., 2000). The role of dS6K as an essential downstream target of dTOR was emphasized by Zhang et al. (2000), who demonstrated that overexpression of dS6K could rescue development of ﬂies with diminished dTOR function, allowing them to develop to adulthood. The dS6Krescued ﬂies were fertile and developed normally, but were slightly reduced in size, suggesting that while dS6K is a major in vivo effector of the TOR pathway, other dTOR-mediated functions may be necessary. Consistent with this hypothesis, constitutively activated dS6K could not rescue the most severe dTOR mutants to adulthood, but did allow progression to the pupal stage (Zhang et al., 2000). Oldham et al. also report a failure of dS6K to rescue severe dTOR mutants. Thus, a low level of dTOR activity is necessary to supply functions of dTOR effectors in addition to dS6K. One such function may be eIF-4E activation, as eIF-4E mutants exhibit a growth arrest phenotype (Zhang et al., 2000). However, eIF-4E overexpression alone was not sufﬁcient to rescue the dTOR mutant phenotype (Zhang et al., 2000). The common effectors of both PI 3-K and mTOR have lead some to hypothesize that these proteins may function in a linear signaling pathway. The dTOR studies in Drosophila, along with other evidence, suggest that this is not the case. dTOR mutations complement mutations in PTEN, suggesting that dTOR affects downstream components in the PI 3-K pathway (Oldham et al., 2000; Zhang et al., 2000). Mutations in dTOR are more severe than those in PI 3-K, however, as dTOR mutants arrest at an earlier larval stage than those of the Drosophila PI 3-K subunits Dp110 or Dp60 (Weinkove et al., 1999; Zhang et al., 2000). In contrast to dTOR mutants, PI 3-K

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mutants fail to upregulate dS6K, and exhibit dissimilar larval phenotypes (Oldham et al., 2000). Thus, the Drosophila genetic data are inconsistent with a role for dTOR as a downstream effector of PI 3-K. Studies in mammalian cells also support the model that PI 3-K and mTOR regulate independent but parallel pathways that converge on common effectors. While the autokinase activity of mTOR is indeed wortmannin sensitive, this inhibition requires a concentration 100-fold greater than the dose that effectively inhibits PI 3-K activity (Brunn et al., 1996). Other studies have suggested that the PI 3-K effector Akt phosphorylates mTOR in a putative negative regulatory domain (Ser2448) (Nave et al., 1999; Sekulic et al., 2000). Mutation of this site, however, does not inhibit mTOR activation of S6K1 (Nave, 1999; Sekulic et al., 2000), and, notably, this site is not conserved in dTOR, despite high sequence conservation between dTOR and mTOR in other regions, including the kinase and HEAT domains (Zhang et al., 2000). In addition, growth factor activation of the PI 3-K pathway and Akt induces little to no change in mTOR kinase activity (Scott et al., 1998; Sekulic et al., 2000). Notably, S6K1 is activated by constitutively active Akt mutants only when these mutants are targeted to the plasma membrane (Dufner et al., 1999), and dominant negative Akt inhibits S6K1 (Takehashi, et al., 2002). This Akt regulation of S6K1 may be explained by its inhibition of TSC2 (Manning, unpublished observations, see Section IV.E.). S6K1 itself suggests separable mTOR and PI 3-K pathways, as an S6K1 truncation mutant is rapamycin resistant, yet retains sensitivity to wortmannin (Cheatham et al., 1995; Weng et al., 1995). Genetic studies, however, suggest that the PI3K and mTOR pathways may also function linearly, as rapamycin analogs inhibit malignancies induced by lesions in PI3K/PTEN signaling (Podsypanina et al., 2001; Neshat et al., 2001; Aoki et al., 2001) (see Section IV.E). The mTOR gene has been mutated, but not deleted, in mice. This study reveals mTOR is essential for embryonic development (Hentges et al., 2001). Mutation of mTOR or treatment of embryos with rapamycin results in a “ﬂat top” embryonic lethal phenotype, characterized by lack of the telencephalon. Interestingly, cells from the mutant mouse exhibited a defect in proliferation, but not in cell size. It should be noted, however, that S6K1 and 4E-BP1 phosphorylation and activities were reduced, but not absent, and may have been sufﬁcient to maintain cell size regulation. This study suggests, however, that mTOR is an important mediator of mitogenic signaling in the developing mouse. The issue of the relationship between PI 3-K and mTOR remains controversial, as experimental evidence is difﬁcult to interpret for several reasons. Much of the controversy revolves around the ability of the PI 3-K pathway, and speciﬁcally, Akt, to regulate mTOR kinase activity. Measurement

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of mTOR kinase activity is technically challenging and may account for disparate results from different groups. A larger issue, however, is the misleading assumption that mTOR kinase activity always mediates mTOR function. For example, PA activation of mTOR substrates is dependent upon PA binding to mTOR, but stimulation or inhibition of PLD/PA signaling does not alter mTOR kinase activity (Fang et al., 2001). There is evidence for direct phosphorylation of S6K1 and 4E-BP1 by mTOR in vitro, but there is also compelling evidence that mTOR-mediated phosphatase regulation may be more important in regulating translational effectors. Equally plausible is involvement of both mechanisms. Finally, whether or not PI 3-K can signal to mTOR is difﬁcult to address by pharmacologic means, because rapamycin completely overrides all other signals that regulate mTOR effectors. Much of the data regarding PI 3-K and mTOR can be explained by two possible models. One model suggests that PI 3-K and mTOR function in distinct and parallel signaling pathways, converging upon common downstream effectors. Inputs from both pathways are required, as inhibition of either pathway is sufﬁcient to override incoming signals from the other. This model is consistent with a checkpoint function for mTOR. Another model would suggest that in addition to functioning independently in parallel, PI 3-K-derived signals may also contribute to activation of mTOR, creating a linear pathway from PI 3-K to mTOR. Because of the technical concerns described, it is difﬁcult to discriminate between these views. Finally, a physical basis by which parallel PI 3-K and mTOR inputs converge upon activation of S6K1 has been proposed in a recent study, which suggests that the p85 adapter subunit of PI 3-K nucleates a complex between S6K1 and mTOR (Gonzalez-Garcia et al., 2002). A p85 truncation mutant (p65PI3K) which lacks the C-terminal SH2 domain was shown to retain the ability to activate the p110 PI 3-K catalytic subunit and Akt. However, this mutant failed to promote serum-stimulated activation of S6K1. While p85PI3K forms a complex with S6K1 and mTOR, p65PI3K does so inefﬁciently. Interestingly, a rapamycin-resistant S6K1 truncation mutant can be activated by either p65PI3K or p85PI3K, suggesting that the crucial function of the PI 3-K regulatory subunit is recruitment of mTOR. This study suggests a model by which PI 3-K is necessary for (1) phosphorylation of S6K1 at Thr229 and Thr389 by PI 3-K-regulated kinases, and (2) recruitment of mTOR to an S6K1-containing complex, which confers protection from mTOR-regulated phosphatases (Peterson et al., 1999). This model suggests a physical basis by which the PI 3-K and mTOR pathways integrate growth factor- and nutrient-derived signals at the level of the translational effector S6K1. It will be interesting to determine whether this complex formation is mediated by the TOS domain of S6K1, whether this regulation applies to activation of primarily nuclear S6 kinases (p85 S6K1 and S6K2 isoforms), and whether

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PI 3-K and mTOR signals similarly converge in a physical complex on 4E-BP factors.

E. PI 3-K and mTOR Pathways in Cancer The importance of these signaling pathways in regulating cell growth and proliferation is underscored by the presence of mutations in multiple components of these pathways in human cancers. Gain of function mutations in PI 3-Kp110, Akt, mTOR, and eIF-4E, and loss of PTEN function lesions have been detected in multiple human cancers (Vogt, 2001). Germline mutation of PTEN has been implicated as the lesion in Cowden’s disease, a human genetic disorder characterized by multiple hamartomas and susceptibility to multiple benign and malignant tumors (Marsh et al., 1999). Elevated S6K activity has been observed in uterine tumors from mice heterozygous for PTEN, and treatment with the rapamycin ester CCI 779, an analog designed for intravenous delivery, reduced tumor size and proliferation (Podsypanina et al., 2001). The growth and proliferation of tumor cell lines lacking PTEN were found to be more sensitive to CCI 779 than PTEN+/+ cells (Neshat et al., 2001). These PTEN null cells also exhibited increased S6K1 activity and 4E-BP1 phosphorylation. Interestingly, oncogenic transformation of chick embryo ﬁbroblasts by PI 3-K or Akt gain of function could be inhibited by rapamycin (Aoki et al., 2001). This study found that mTOR was essential for PI 3-K-induced oncogenesis, but not for transformation induced by oncogenes that function in other signaling pathways. These studies suggest that rapamycin may be an effective anticancer agent, particularly for tumors arising due to lesions in the PI 3-K pathway. The tuberous sclerosis complex (TSC) genes have been recently identiﬁed as tumor suppressors that negatively regulate both cell growth and proliferation. These genes are responsible for a human inherited disorder characterized by development of benign hamartomatous tumors in multiple organs, and predisposition to malignant tumor formation (Jones et al., 1997). Loss of function of TSC1 or 2 results in benign tumors characterized by large cells. Genetic analyses in Drosophila suggest that the TSC1/2 complex lies on a parallel pathway that inhibits insulin signaling downstream of Akt (Gao and Pan, 2001). Interestingly, genetic epistasis experiments place dS6K downstream of TSC1/2 and demonstrate that dS6K and TSC1/2 serve antagonistic functions (Fig. 9) (Potter et al., 2001; Tapon et al., 2001). This also appears to be true in mammalian cells, as S6K1 activity is upregulated in mouse cells lacking TSC1 (Kwiatkowski et al., 2002) and in human cells lacking TSC2 (Goncharova et al., 2002). Akt phosphorylation and inhibition of the TSC2 gene product tuberin suggests a mechanism by which Akt contributes to S6K1 activation (Manning, unpublished observations), and

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Fig. 9 The tuberous sclerosis complex (TSC) opposes insulin signaling. The TSC1/2 genes have been identiﬁed as negative regulators of S6K function and cell growth. The PI 3-K effectors Akt, PDK1, and PKCζ contribute to S6K activation. Akt relieves TSC inhibition by phosphorylation and inhibition of the TSC2 gene product tuberin. It is not yet known whether tuberin inhibits S6K1 directly or via mTOR.

may explain S6K1 inhibition by dominant negative Akt (Takehashi et al., 2002). These examples from the cancer literature further highlight the interdependence of the PI 3-K and mTOR pathways, and the contribution of their effectors to growth and proliferation.

V. CONCLUSIONS The PI 3-K pathway mediates many essential cellular functions, including survival, proliferation, and cell growth/cell size. We have described PI 3-K effectors which transduce signals to regulate translation initiation, ribosome biogenesis, and translational capacity. These include the S6 kinases, 4E-BPs and eIF-4E, eIF-4G, and eIF-4B. These PI 3-K effectors also integrate signals from other growth factor-stimulated pathways, including conventional PKCs and MAPKs. Importantly, these effectors integrate the incoming growth factor signals with mitogenic inputs from the mTOR pathway, a crucial nutrient- and energy-sensing checkpoint pathway, ensuring adequate amino acid resources to meet the demand for new protein synthesis. Common subcellular localization of effectors and regulatory molecules may facilitate such integrated signal transduction. The inﬂuence of growth factorand nutrient-sensitive pathways on growth and proliferation is a continuing subject of investigation that will likely reveal important new insights into normal physiology and pathological conditions including hypertrophy, diabetes, and cancer.

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ACKNOWLEDGMENTS The authors thank Diane Fingar, Angela Romanelli, Stefanie Schalm, Celeste Richardson, and Andrew Tee for their contributions to this manuscript.

REFERENCES Akimoto, K., Nakaya, M., Yamanaka, T., Tanaka, J., Matsuda, S., Weng, Q. P., Avruch, J., and Ohno, S. (1998). Atypical protein kinase Clambda binds and regulates p70 S6 kinase. Biochem. J. 335, 417–24. Alessi, D. R., Kozlowski, M. T., Weng, Q. P., Morrice, N., and Avruch, J. (1998). 3Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr. Biol. 8, 69–81. Aoki, M., Blazek, E., and Vogt, P. K. (2001). A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt. Proc. Natl. Acad. Sci. USA 98, 136– 141. Ballif, B. A., Shimamura, A., Pae, E., and Blenis, J. (2001). Disruption of 3-phosphoinositidedependent kinase 1 (PDK1) signaling by the anti-tumorigenic and anti-proliferative agent n-alpha-tosyl-l-phenylalanyl chloromethyl ketone. J. Biol. Chem. 276, 12,466–12,475. Belham, C., Comb, M. J., and Avruch, J. (2001). Identiﬁcation of the NIMA family kinases NEK6/7 as regulators of the p70 ribosomal S6 kinase. Curr. Biol. 11, 1155–1167. Berven, L. A., and Crouch, M. F. (2000). Cellular function of p70S6K: A role in regulating cell motility. Immunol. Cell Biol. 78, 447–451. Blenis, J., and Erikson, R. L. (1986). Stimulation of ribosomal protein S6 kinase activity by pp60v-src or by serum: Dissociation from phorbol ester-stimulated activity. Proc. Natl. Acad. Sci. USA 83, 1733–1737. Bodine, S. C., Stitt, T. N., Gonzalez, M., Kline, W. O., Stover, G. L., Bauerlein, R., Zlotchenko, E., Scrimgeour, A., Lawrence, J. C., Glass, D. J., and Yancopoulos, G. D. (2001). Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat. Cell. Biol. 3, 1014–1019. Bohni, R., Riesgo-Escovar, J., Oldham, S., Brogiolo, W., Stocker, H., Andruss, B. F., Beckingham, K., and Hafen, E. (1999). Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1–4. Cell 97, 865–875. Bondeva, T., Pirola, L., Bulgarelli-Leva, G., Rubio, I., Wetzker, R., and Wymann, M. P. (1998). Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK. Science 282, 293–296. Brennan, P., Babbage, J. W., Thomas, G., and Cantrell, D. (1999). p70(s6k) integrates phosphatidylinositol 3-kinase and rapamycin-regulated signals for E2F regulation in T lymphocytes. Mol. Cell. Biol. 19, 4729–4738. Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. (1994). A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369, 756–758. Brown, E. J., Beal, P. A., Keith, C. T., Chen, J., Shin, T. B., and Schreiber, S. L. (1995). Control of p70 S6 kinase by kinase activity of FRAP in vivo. Nature 377, 441–446. Brunn, G. J., Hudson, C. C., Sekulic, A., Williams, J. M., Hosoi, H., Houghton, P. J., Lawrence, J. C., Jr., and Abraham, R. T. (1997). Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science 277, 99–101.

Coordinate Regulation of Translation

31

Brunn, G. J., Williams, J., Sabers, C., Wiederrecht, G., Lawrence, J. C., and Abraham, R. T. (1996). Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 15, 5256– 5267. Burack, W. R., and Shaw, A. S. (2000). Signal transduction: Hanging on a scaffold. Curr. Opin. Cell Biol. 12, 211–216. Burnett, P. E., Barrow, R. K., Cohen, N. A., Snyder, S. H., and Sabatini, D. M. (1998a). RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl. Acad. Sci. USA 95, 1432–1437. Burnett, P. E., Blackshaw, S., Lai, M. M., Qureshi, I. A., Burnett, A. F., Sabatini, D. M., and Snyder, S. H. (1998b). Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton. Proc. Natl. Acad. Sci. USA 95, 8351–8356. Cantley, L. C., and Neel, B. G. (1999). New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl. Acad. Sci. USA 96, 4240–4245. Cheatham, L., Monfar, M., Chou, M. M., and Blenis, J. (1995). Structural and functional analysis of p70 S6 kinase. Proc. Natl. Acad. Sci. USA 92, 11,696–11,700. Chen, C., Jack, J., and Garofalo, R. S. (1996). The Drosophila insulin receptor is required for normal growth. Endocrinology 137, 846–856. Chen, J., Peterson, R. T., and Schreiber, S. L. (1998). Alpha 4 associates with protein phosphatases 2A, 4, and 6. Biochem. Biophys. Res. Commun. 247, 827–832. Chiu, M. I., Katz, H., and Berlin, V. (1994). RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex. Proc. Natl. Acad. Sci. USA 91, 12,574– 12,578. Chou, M. M., and Blenis, J. (1996). The 70kD S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1. Cell 85, 573–583. Chung, J., Grammer, T., Lemon, K., Kazlauskas, A., and Blenis, J. (1994). PDGF- and insulindependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature 370, 71–75. Chung, J., Kuo, C. J., Crabtree, G. R., and Blenis, J. (1992). Rapamycin-FKBP speciﬁcally blocks growth-dependent activation of and signalling by the 70kD S6 protein kinases. Cell 69, 1227–1236. Coffer, P. J., and Woodgett, J. R. (1991). Molecular cloning and characterization of a novel putative protein serine kinase related to the cAMP-dependent and protein kinase C families. Eur. J. Biochem. 201, 475–481. Coghlan, M. P., Chou, M. M., and Carpenter, C. L. (2000). Atypical protein kinases Clambda and -zeta associate with the GTP-binding protein Cdc42 and mediate stress ﬁber loss. Mol. Cell. Biol. 20, 2880–2889. Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785–789. Crouch, M. F. (1997). Regulation of thrombin-induced sress ﬁbre formation in Swiss 3T3 cells by the 70-kDa S6 kinase. Biochem. Biophys. Res. Commun. 233, 193–199. de Groot, R. P., Ballou, L. M., and Sasssone-Corsi, P. (1994). Positive Regulation of the cAMPresponsive activator CREM by the p70 S6 kinase: An alternative route to mitogen-induced gene expression. Cell 79, 81–91. Dennis, P. B., Jaeschke, A., Saitoh, M., Fowler, B., Kozma, S. C., and Thomas, G. (2001). Mammalian TOR: A homeostatic ATP sensor. Science 294, 1102–1105. Di Como, C. J., and Arndt, K. T. (1996). Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Dev. 10, 1904–1916. Downward, J. (1998). Mechanisms and consequences of activation of protein kinase B/Akt. Curr. Opin. Cell Biol. 10, 262–267.

32

Martin and Blenis

Dufner, A., Andjelkovic, M., Burgering, B. M., Hemmings, B. A., and Thomas, G. (1999). Protein kinase B localization and activation differentially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation. Mol. Cell. Biol. 19, 4525–4534. Dufner, A., and Thomas, G. (1999). Ribosomal S6 kinase signaling and the control of translation. Exp. Cell Res. 253, 100–109. Erickson, J. W., and Cerione, R. A. (2001). Multiple roles for Cdc42 in cell regulation. Curr. Opin. Cell Biol. 13, 153–157. Fadden, P., Haystead, T. A., and Lawrence, J. C. J. (1997). Identiﬁcation of phosphorylation sites in the translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes. J. Biol. Chem. 272, 10,240–10,247. Fang, Y., Vilella-Bach, M., Bachmann, R., Flanigan, A., and Chen, J. (2001). Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science 294, 1942–1945. Fingar, D. C., Salama, S., Tsou, C., Harlow, E., and Blenis, J. (2002). Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev. 16, 1472–1487. Francavilla, A., Starzl, T. E., Scotti, C., Carrieri, G., Azzarone, A., Zeng, Q. H., Porter, K. A., and Schreiber, S. L. (1992). Inhibition of liver, kidney, and intestine regeneration by rapamycin. Transplantation 53, 496–498. Fruman, D. A., Mauvais-Jarvis, F., Yballe, C. M., Bronson, R. T., Kahn, C. R., and Cantely, L. C. (2000). Hypoglycemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha. Nat. Genet. 26, 379–382. Gao, X., and Pan, D. (2001). TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev. 15, 1383–1392. Garrington, T. P., and Johnson, G. L. (1999). Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11, 211–218. Gingras, A. C., Gygi, S. P., Raught, B., Polakiewicz, R. D., Abraham, R. T., Hoekstra, M. F., Aebersold, R., and Sonenberg, N. (1999). Regulation of 4E-BP1 phosphorylation: A novel two-step mechanism. Genes Dev. 13, 1422–1437. Gingras, A. C., Kennedy, S. G., O’Leary, M. A., Sonenberg, N., and Hay, N. (1998). 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. Genes Dev. 12, 502–513. Gingras, A. C., Raught, B., Gygi, S. P., Niedzwiecka, A., Miron, M., Burley, S. K., Polakiewicz, R. D., Wyslouch-Cieszynska, A., Aebersold, R., and Sonenberg, N. (2001a). Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev. 15, 2852–2864. Gingras, A. C., Raught, B., and Sonenberg, N. (2001b). Regulation of translation initiation by FRAP/mTOR. Genes Dev. 15, 807–826. Goberdhan, D., Paricio, N., Goodman, E. C., Mlodzik, M., and Wilson, C. (1999). Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling pathway. Genes Dev. 13, 3244–3258. Goncharova, E. A., Goncharov, D. A., Eszterhas, A., Hunter, D. S., Glassberg, M. K., Yeung, R. S., Walker, C. L., Noonan, D., Kwiatkowski, D. J., Chou, M. M., Panettieri, Jr., R. A., and Krymskaya, V. P. (2002). Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation: A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM). J. Biol. Chem. e-published June 3. Gonzalez-Garcia, A., Garrido, E., Hernandez, C., Alvarez, B., Jimenez, C., Cantrell, D. A., Pullen, N., and Carrera, A. C. (2002). A new role for the p85-phosphatidylinositol 3-kinase regulatory subunit linking FRAP to p70 S6 kinase activation. J. Biol. Chem. 277, 1500– 1508. Gout, I., Minami, T., Hara, K., Tsujishita, Y., Filonenko, V., Waterﬁrld, M. D., and Yonezawa, K. (1998). Molecular cloning and characterization of a novel S6 kinase, p70 S6 kinase beta containing a proline-rich region. J. Biol. Chem. 273, 30,061–30,064.

Coordinate Regulation of Translation

33

Grammer, T. C., and Blenis, J. (1996). The serine protease inhibitors, tosylphenylalanine chloromethyl ketone and tosyllysine chloromethyl ketone, potently inhibit pp70S6k activation. J. Biol. Chem. 23,650–23,652. Grolleau, A., Sonenberg, N., Wietzerbin, J., and Beretta, L. (1999). Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation. J. Immunol. 162, 3491–3497. Haghighat, A., Mader, S., Pause, A., and Sonenberg, N. (1995). Repression of cap-dependent translation by 4E-binding protein 1: Competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J. 14, 5701–5709. Hara, K., Yonezawa, K., Weng, Q. P., Kozlowski, M. T., Belham, C., and Avruch, J. (1998). Amino acid sufﬁciency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J. Biol. Chem. 273, 14,484–14,494. Heesom, K. J., and Denton, R. M. (1999). Dissociation of the eukaryotic initiation factor4E/4E-BP1 complex involves phosphorylation of 4E-BP1 by an mTOR-associated kinase. FEBS Lett. 457, 489–493. Hentges, K. E., Sirry, B., Gingeras, A. C., Sarbassov, D., Sonenberg, N., Sabatini, D., and Peterson, A. S. (2001). FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse. Proc. Natl. Acad. Sci. USA 98, 13,796– 13,801. Herbert, T. P., Kilhams, G. R., Batty, I. H., and Proud, C. G. (2000). Distinct signalling pathways mediate insulin and phorbol ester-stimulated eukaryotic initiation factor 4F assembly and protein synthesis in HEK 293 cells. J. Biol. Chem. 275, 11,249–11,256. Herbert, T. P., Tee, A. R., and Proud, C. G. (2002). The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites. J. Biol. Chem. 277, 11,591–11,596. Hoekstra, M. F. (1997). Responses to DNA damage and regulation of cell cycle checkpoints by the ATM protein kinase family. Curr. Opin. Genet. Dev. 7, 170–175. Hong, F., Nguyen, V. A., Shen, X., Kunos, G., and Gao, B. (2000). Rapid activation of protein kinase B/Akt has a key role in antipoptotic signaling during liver regeneration. Biochem. Biophys. Res. Commun. 279, 947–979. Hosoi, H., Dilling, M. B., Liu, L. N., Danks, M. K., Shikata, T., Sekulic, A., Abraham, R. T., Lawrence, J. C. J., and Houghton, P. J. (1998). Studies on the mechanism of resistance to rapamycin in human cancer cells. Mol. Pharmacol. 54, 815–824. Iiboshi, Y., Papst, P. J., Kawasome, H., Hosoi, H., Abraham, R. T., Houghton, P. J., and Terada, N. (1999). Amino acid-dependent control of p70(s6k). Involvement of tRNA aminoacylation in the regulation. J. Biol. Chem. 274, 1092–1099. Jefferies, H. B., Fumagalli, S., Dennis, P. B., Reinhard, C., Pearson, R. B., and Thomas, G. (1997). Rapamycin suppresses 5 TOP mRNA translation through inhibition of p70s6k. EMBO J. 16, 3693–3704. Jefferies, H. B. J., Reinhard, C., Kozma, S. C., and Thomas, G. (1994). Rapamycin selectively represses translation of the “polypryimidine tract” nRNA family. Proc. Natl. Acad. Sci. USA 91, 4441–4445. Jiang, Y., and Broach, J. R. (1999). Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J. 18, 2782–2792. Jiang, Y.-P., Ballou, L. M., and Lin, R. Z. (2001). Rapamycin-insensitive regulation of 4E-BP1 in regenerating rat liver. J. Biol. Chem. 276, 10,943–10,951. Johnston, G. C., Pringle, J. R., and Hartwell, L. H. (1977). Coordination of growth with cell division in the yeast Saccharomyces cerevisiae. Exp. Cell. Res. 105, 79–98. Jones, A. C., Daniells, C. E., Snell, R. G., Tachataki, M., Idziaszczyk, S. A., Krawczak, M., Sampson, J. R., and Cheadle, J. P. (1997). Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis. Hum. Mol. Genet. 6, 2155–2161.

34

Martin and Blenis

Kholodenko, B. N., Hoek, J. B., and Westerhoff, H. V. (2000). Why cytoplasmic signalling proteins should be recruited to cell membranes. Trends Cell Biol. 10, 173–178. Kim, J. E., and Chen, J. (2000). Cytoplasmic-nuclear shuttling of FKBP12-rapamycin-associated protein is involved in rapamycin-sensitive signaling and translation initiation. Proc. Natl. Acad. Sci. USA 97, 14,340–14,345. Koh, H., Jee, K., Lee, B., Kim, J., Kim, D., Yun, Y. H., Kim, J. W., Choi, H. S., and Chung, J. (1999). Cloning and characterization of a nuclear S6 kinase, S6 kinase-related kinase (SRK); a novel nuclear target of Akt. Oncogene 18, 5115–5119. Kozma, S. C., Ferrari, S., Bassand, P., Siegmann, M., Totty, N., and Thomas, G. (1990). Cloning of the mitogen-activated S6 kinase from the rat liver reveals an enzyme of the second messanger subfamily. Proc. Natl. Acad. Sci. USA 87, 7365–7369. Kumar, V., Pandey, P., Sabatini, D., Kumar, M., Majumder, P. K., Bharti, A., Carmichael, G., Kufe, D., and Kharbanda, S. (2000a). Functional interaction betwen RAFT1/FRAP/mTOR and protein kinase Cδ in the regulation of cap-dependent initiation of translation. EMBO J. 19, 1087–1097. Kumar, V., Sabatini, D., Pandey, P., Gingras, A. C., Majumder, P. K., Kumar, M., Yuan, Z. M., Carmichael, G., Weichselbaum, R., Sonenberg, N., Kufe, D., and Kharbanda, S. (2000b). Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and capdependent initiation of translation by the c-Abl protein-tyrosine kinase. J. Biol. Chem. 275, 10,779–10,787. Kwiatkowski, D. J., Zhang, H., Bandura, J. L., Heiberger, K. M., Glogauer, M., el-Hashemite, N., and Onda, H. (2002). A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6K kinase activity in Tsc1 null cells. Hum. Mol. Genet. 11, 525–534. Laser, M., Kasi, V. S., Hamawaki, M., Cooper, G. T., Kerr, C. M., and Kuppuswamy, D. (1998). Differential activation of p70 and p85 S6 kinase isoforms during cardiac hypertrophy in the adult mammal. J. Biol. Chem. 273, 24,610–24,619. Lee-Fruman, K. K., Kuo, C. J., Lippincott, J., Terada, N., and Blenis, J. (1999). Characterization of S6K2, a novel kinase homologous to S6K1. Oncogene 18, 5108–5114. Leevers, S. J. (1999). All creatures great and small. Science 285, 2082–2083. Leevers, S. J., Weinkove, D., MacDougall, L. K., Hafen, E., and Waterﬁeld, M. D. (1996). The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J. 15, 6584– 6594. Lenormand, P., McMahon, M., and Pouyssegur, J. (1996). Oncogenic Raf-1 activates p70 S6 kinase via a mitogen-activated protein kinase-independent pathway. J. Biol. Chem. 271, 15,762–15,768. Li, H. L., Davis, W., and Pure, E. (1999). Suboptimal cross-linking of antigen receptor induces Syk-dependent activation of p70S6 kinase through protein kinase C and phosphoinositol 3-kinase. J. Biol. Chem. 274, 9812–9820. Lin, R., Bagrodia, S., Cerione, R., and Manor, D. (1997). A novel Cdc42Hs mutant induces cellular transformation. Curr. Biol. 7, 794–797. Lin, T., Kong, X., Haystead, T. A. J., Pause, A., Belsham, G., Sonenberg, N., and Lawrence, J. C. J. (1994). PHAS-1 as a link between mitogen-activated protein kinase and translation initiation. Science 266, 653–656. Louro, I. D., McKie-Bell, P., Gosnell, H., Brindley, B. C., Bucy, R. P., and Ruppert, J. M. (1999). The zinc ﬁnger protein GLI induces cellular sensitivity to the mTOR inhibitor rapamycin. Cell. Growth Differ. 10, 503–516. Lynch, C. J. (2001). Role of leucine in the regulation of mTOR by amino acids: Revelations from structure-activity studies. J. Nutr. 131, 861S–865S. Marcotrigiano, J., Gingras, A. C., Sonenberg, N., and Burley, S. K. (1997). Cocrystal structure of the messenger RNA 5 cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell 89, 951–961.

Coordinate Regulation of Translation

35

Marsh, D. J., Kum, J. B., Lunetta, K. L., Bennett, M. J., Gorlin, R. J., Ahmed, S. F., Bodurtha, J., Crowe, C., Curtis, M. A., Dasouki, M., Dunn, T., Feit, H., Geraghty, M. T., Graham, J., Hodgson, S. V., Hunter, A., Korf, B. R., Manchester, D., Miesfeldt, S., Murday, V. A., Nathanson, K. L., Parisi, M., Pober, B., Romano, C., Eng, C. et al. (1999). PTEN mutation spectrum and genotype-phenotype correlations in Bannayan–Riley–Ruvalcaba syndrome suggest a single entity with Cowden syndrome. Hum. Mol. Genet. 8, 1461–1472. Martin, K. A., Schalm, S. S., Richardson, C., Romanelli, A., Keon, K. L., and Blenis, J. (2001a). Regulation of S6K2 by effectors of the PI3-kinase pathway. J. Biol. Chem. 276, 7884–7891. Martin, K. A., Schalm, S. S., Romanelli, A., Keon, K. L., and Blenis, J. (2001b). Ribosomal S6 kinase 2 inhibition by a potent C-terminal repressor domain is relieved by mitogenactivated protein-extracellular signal-regulated kinase kinase-regulated phosphorylation. J. Biol. Chem. 276, 7892–7898. Meyuhas, O. (2000). Synthesis of the translational apparatus is regulated at the translational level. Eur. J. Biochem. 267, 6321–6330. Michalopoulos, G. K., and DeFrances, M. C. (1997). Liver regeneration. Science 276, 60–66. Minami, T., Hara, K., Oshiro, N., Ueoku, S., Yoshino, K., Tokunaga, C., Shirai, Y., Saito, N., Gout, I., and Yonezawa, K. (2001). Distinct regulatory mechanism for p70 S6 kinase beta from that for p70 S6 kinase alpha. Genes Cells 6, 1003–1015. Miron, M., Verdu, J., Lachance, P. E., Birnbaum, M. J., Lasko, P. F., and Sonenberg, N. (2001). The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat. Cell Biol. 3, 596–601. Monfar, M., Lemon, K. P., Grammer, T. C., Cheatham, L., Chung, J., Vlahos, C. J., and Blenis, J. (1995). Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP. Mol. Cell Biol. 15, 326–337. Montagne, J., Stewart, M. J., Stocker, H., Hafen, E., Kozma, S. C., and Thomas, G. (1999). Drosophila S6 kinase: A regulator of cell size. Science 285, 2126–2129. Morley, S. J., and Traugh, J. A. (1993). Stimulation of translation in 3T3-L1 cells in response to insulin and phorbol ester is directly correlated with increased phosphate labelling of initiation factor (eIF-) 4F and ribosomal protein S6. Biochimie 75, 985–989. Moser, B. A., Dennis, P. B., Pullen, N., Pearson, R. B., Williamson, N. A., Wettenhall, R. E., Kozma, S. C., and Thomas, G. (1997). Dual requirement for a newly identiﬁed phosphorylation site in p70s6k. Mol. Cell. Biol. 17, 5648–5655. Mothe-Satney, I., Brunn, G. J., McMahon, L. P., Capaldo, C. T., Abraham, R. T., and Lawrence, J. C. J. (2000a). Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-speciﬁc antibodies. J. Biol. Chem. 275, 33,836–33,843. Mothe-Satney, I., Yang, D., Fadden, P., Haystead, T. A., and Lawrence, J. C. J. (2000b). Multiple mechanisms control phosphorylation of PHAS-I in ﬁve (S/T)P sites that govern translational repression. Mol. Cell. Biol. 20, 3558–3567. Mukhopadhyay, N. K., Price, D. J., Kyriakis, J. M., Pelech, S., Sanghera, J., and Avruch, J. (1992). An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase. J. Biol. Chem. 267, 3325–3335. Murata, K., Wu, J., and Brautigan, D. L. (1997). B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proc. Natl. Acad. Sci. USA 94, 10,624–10,629. Nave, B., Ouwens, M., Withers, D. J., Alessi, D. R., and Shepherd, P. R. (1999). Mammalian target of rapamycin is a direct target for protein kinase B: Identiﬁcation of a convergence point for opposing effects of insulin and amino-acid deﬁciency on protein translation. Biochem. J. 344, 427–431. Neshat, M. S., Mellinghoff, I. K., Tran, C., Stiles, B., Thomas, G., Petersen, R., Frost, P., Gibbons, J. J., Wu, H., and Sawyers, C. L. (2001). Enhanced sensitivity of PTEN-deﬁcient tumors to inhibition of FRAP/mTOR. Proc. Natl. Acad. Sci. USA 98, 10,314–10,319.

36

Martin and Blenis

Oldham, S., Montagne, J., Radimerski, T., Thomas, G., and Hafen, E. (2000). Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14, 2689–2694. Ono, F., Nakagawa, T., Saito, S., Owada, Y., Sakagami, H., Goto, K., Suzuki, M., Matsuno, S., and Kondo, H. (1998). A novel class II phosphoinositide 3-kinase predominantly expressed in the liver and its enhanced expression during liver regneration. J. Biol. Chem. 273, 7731– 7736. Parekh, D., Ziegler, W., Yonezawa, K., Hara, K., and Parker, P. J. (1999). Mammalian TOR controls one of two kinase pathways acting upon nPKCdelta and nPKCepsilon. J. Biol. Chem. 274, 34,758–34,764. Pause, A., Belsham, G. J., Gingras, A., Donze, O., Lin, T., Lawrence, J. C. J., and Sonenberg, N. (1994). Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5 -cap function. Nature 371, 762–767. Pende, M., Kozma, S. C., Jaquet, M., Oorschot, V., Burcelin, R., Le Marchand-Brustel, Y., Klumperman, J., Thorens, B., and Thomas, G. (2000). Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1-deﬁcient mice. Nature 408, 994–997. Peterson, R. T., Comb, M. J., and Schreiber, S. L. (2000). FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions. J. Biol. Chem. 275, 7416–7423. Peterson, R. T., Desai, B. N., Hardwick, J. S., and Schreiber, S. L. (1999). Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin associated protein. Proc. Natl. Acad. Sci. USA 96, 4438–4442. Podbielski, J., and Schoenberg, L. (2001). Use of sirolimus in kidney transplantation. Prog. Transplant 11, 29–32. Podsypanina, K., Lee, R. T., Politis, C., Hennessy, I., Crane, A., Puc, J., Neshat, M., Wang, H., Yang, L., Gibbons, J., Frost, P., Dreisbach, V., Blenis, J., Gaciong, Z., Fisher, P., Sawyers, C., Hedrick-Ellenson, L., and Parsons, R. (2001). An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/− mice. Proc. Natl. Acad. Sci. USA 98, 10,320–10,325. Potter, C. J., Huang, H., and Xu, T. (2001). Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 105, 357– 368. Poulin, F., Olsen, H., Chevalier, S., and Sonenberg, N. (1998). 4E-BP3, a new member of the eukaryotic initiation factor 4E-binding protein family. J. Biol. Chem. 273, 14,002– 14007. Price, N., and Proud, C. (1994). The guanine nucleotide-exchange factor, eIF-2B. Biochimie 76, 748–760. Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S. C., Hemmings, B. A., and Thomas, G. (1998). Phosphorylation and activation of p70s6k by PDK1. Science 279, 707– 710. Pyronnet, S., and Sonenberg, N. (2001). Cell-cycle-dependent translational control. Curr. Opin. Genet. Dev. 11, 13–18. Rameh, L. E., and Cantley, L. C. (1999). The role of phosphoinositide 3-kinase lipid products in cell function. J. Biol. Chem. 274, 8347–8350. Rao, G. N., Madamanchi, N. R., Lele, M., Gadiparthi, L., Gingras, A. C., Eling, T. E., and Sonenberg, N. (1999). A potential role for extracellular signal-regulated kinases in prostaglandin F2alpha-induced protein synthesis in smooth muscle cells. J. Biol. Chem. 274, 12,925–12,932. Rau, M., Ohlmann, T., Morley, S. J., and Pain, V. M. (1996). A reevaluation of the cap-binding protein, eIF4E, as a rate-limiting factor for initiation of translation in reticulocyte lysate. J. Biol. Chem. 271, 8983–8990.

Coordinate Regulation of Translation

37

Raught, B., Gingras, A. C., Gygi, S. P., Imataka, H., Morino, S., Gradi, A., Aebersold, R., and Sonenberg, N. (2000). Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI. EMBO J. 19, 434–444. Reinhard, C., Fernandez, A., Lamb, N. J. C., and Thomas, G. (1994). Nuclear localization of p85s6k: functional requirement for entry into S phase. EMBO J. 13, 1557–1565. Rohde, J., Heitman, J., and Cardenas, M. E. (2001). The TOR kinases link nutrient sensing to cell growth. J. Biol. Chem. 276, 9583–9586. Romanelli, A., Martin, K. A., Toker, A., and Blenis, J. (1999). p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex. Mol. Cell. Biol. 19, 2921–2928. Rommel, C., Bodine, S. C., Clarke, B. A., Rossman, R., Nunez, L., Stitt, T. N., Yancopoulos, G. D., and Glass, D. J. (2001). Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat. Cell Biol. 3, 1009–1013. Rousseau, D., Kaspar, R., Rosenwald, I., Gehrke, L., and Sonenberg, N. (1996). Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E. Proc. Natl. Acad. Sci. USA 93, 1065–1070. Rozen, F., Edery, I., Meerovitch, K., Dever, T. E., Merrick, W. C., and Sonenberg, N. (1990). Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol. Cell. Biol. 10, 1134–1144. Sabatini, D. M., Barrow, R. K., Blackshaw, S., Burnett, P. E., Lai, M. M., Field, M. E., Bahr, B. A., Kirsch, J., Betz, H., and Snyder, S. H. (1999). Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling. Science 284, 1161–1164. Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S. H. (1994). RAFT1: A mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78, 35–43. Sabers, C. J., Martin, M. M., Brunn, G. J., Williams, J. M., Dumont, F. J., Wiederrecht, G., and Abraham, R. T. (1995). Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J. Biol. Chem. 270, 815–822. Schalm, S. S., and Blenis, J. (2002). Identiﬁcation of conserved motif required for mTOR signaling. Curr. Biol. 12, 632–639. Scheper, G. C., Morrice, N. A., Kleijn, M., and Proud, C. G. (2001). The mitogen-activated protein kinase signal-integrating kinase Mnk2 is a eukaryotic initiation factor 4E kinase with high levels of basal activity in mammalian cells. Mol. Cell. Biol. 21, 743–754. Schmidt, E. V. (1999). The role of c-myc in cellular growth control. Oncogene 18, 2988– 2996. Schwartzbauer, G., and Robbins, J. (2001). The tumor suppressor gene PTEN can regulate cardiac hypertrophy and survival. J. Biol. Chem. 276, 35,786–35,793. Scott, P. H., Brunn, G. J., Kohn, A. D., Roth, R. A., and Lawrence, J. C., Jr. (1998). Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc. Natl. Acad. Sci. USA 95, 7772– 7777. Scott, P. H., and Lawrence, J. C., Jr. (1997). Insulin activates a PD 098059-sensitive kinase that is involved in the regulation of p70S6K and PHAS-I. FEBS Lett. 409, 171–176. Sekulic, A., Hudson, C. C., Homme, J. L., Yin, P., Otterness, D. M., Karnitz, L. M., and Abraham, R. T. (2000). A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res. 60, 3504–3513. Shima, H., Pende, M., Chen, Y., Fumagalli, S., Thomas, G., and Kozma, S. C. (1998). Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase. EMBO J. 17, 6649–6659.

38

Martin and Blenis

Shioi, T., Kang, P. M., Douglas, P. S., Hampe, J., Yballe, C. M., Lawitts, J., Cantley, L. C., and Izumo, S. (2000). The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J. 19, 2537–2548. Slavik, J. M., Lim, D. G., Burakoff, S. J., and Haﬂer, D. A. (2001). Uncoupling p70(s6) kinase activation and proliferation: Rapamycin-resistant proliferation of human CD8(+) T lymphocytes. J. Immunol. 166, 3201–3209. Sonenberg, N., and Gingras, A. C. (1998). The mRNA 5 cap-binding protein eIF4E and control of cell growth. Curr. Opin. Cell Biol. 10, 268–275. Takata, M., Ogawa, W., Kitamura, T., Hino, Y., Kuroda, S., Kotani, K., Klip, A., Gingras, A. C., Sonenberg, N., and Kasuga, M. (1999). Requirement for Akt (protein kinase B) in insulin-induced activation of glycogen synthase and phosphorylation of 4E-BP1 (PHAS-1). J. Biol. Chem. 274, 20,611–20,618. Takehashi, A., Kureishi, Y., Yang, J., Luo, Z., Guo, K., Mukhopadhyay, D., Ivashchenko, Y., Branellec, B., and Walsh, K. (2002). Myogenic Akt signaling regulates blood vessel recruitment during myoﬁber growth. Mol. Cell. Biol. in press. Tang, H., Hornstein, E., Stolovich, M., Levy, G., Livingstone, M., Templeton, D., Avruch, J., and Meyuhas, O. (2001). Amino acid-induced translation of TOP mRNAs is fully dependent on phosphatidylinositol 3-kinase-mediated signaling, is partially inhibited by rapamycin, and is independent of S6K1 and rpS6 phosphorylation. Mol. Cell. Biochem. 21, 8671–8683. Tapon, N., Ito, N., Dickson, B. J., Treisman, J. E., and Hariharan, I. K. (2001). The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell 105, 345–355. Tee, A., and Proud, C. (2002). Caspase cleavage of initiation factor 4E-binding protein 1 yields a dominant inhibitor of cap-dependent translation and reveals a novel regulatory motif. Mol. Cell. Biol. 22, 1674–1683. Tee, A. R., and Proud, C. G. (2000). DNA-damaging agents cause inactivation of translational regulators linked to mTOR signalling. Oncogene 19, 3021–3031. Verdu, J., Buratovich, M. A., Wilder, E. L., and Birnbaum, M. J. (1999). Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB. Nat. Cell Biol. 1, 500–506. Visa, N., Izaurralde, E., Ferreira, J., Daneholt, B., and Mattaj, I. (1996). A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export. J. Cell. Biol. 133, 5–14. Vogt, P. K. (2001). PI 3-kinase, mTOR, protein synthesis and cancer. Trends Mol. Med. 7, 482–484. Volarevic, S., Steward, M. J., Ledermann, B., Zilberman, F., Terracciano, L., Montini, E., Grompe, M., Kozma, S. C., and Thomas, G. (2000). Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6. Science 288, 2045–2047. von Manteuffel, S. R., Dennis, P. B., Pullen, N., Gingras, A. C., Sonenberg, N., and Thomas, G. (1997). The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k. Mol. Cell. Biol. 17, 5426–5436. Wang, L., Gout, I., and Proud, C. G. (2001). Cross-talk between the ERK and p70 S6 kinase (S6K) signaling pathways. MEK-dependent activation of S6K2 in cardiomyocytes. J. Biol. Chem. 276, 32,670–32,677. Waskiewicz, A. J., Flynn, A., Proud, C. G., and Cooper, J. A. (1997). Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J. 16, 1909–1920. Weinkove, D., Neufeld, T. P., Twardzik, T., Waterﬁeld, M. D., and Leevers, S. J. (1999). Regulation of imaginal disc cell size, cell number and organ size by Drosophila class I(A) phosphoinositide 3-kinase and its adaptor. Curr. Biol. 9, 1019–1029. Welsh, G., Miller, C. M., Loughlin, A. J., Price, N. T., and Proud, C. G. (1998). Regulation of eukaryotic initiation factor eIF2B: Glycogen synthase kinase-3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin. FEBS Lett. 421, 125–130.

Coordinate Regulation of Translation

39

Weng, Q., Andrabi, K., Kozlowski, M. T., Grove, J. R., and Avruch, J. (1995). Multiple independent inputs are required for activation of the p70 S6 kinase. Mol. Cell. Biol. 15, 2333–2340. Weng, Q. P., Kozlowski, M., Belham, C., Zhang, A., Comb, M. J., and Avruch, J. (1998). Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-speciﬁc anti-phosphopeptide antibodies. J. Biol. Chem. 273, 16,621–16,629. Williams, M. R., Arthur, J. S., Balendran, A., van der Kaay, J., Poli, V., Cohen, P., and Alessi, D. R. (2000). The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases deﬁned in embryonic stem cells. Curr. Biol. 10, 439–448. Wilson, K., Fortes, P., Singh, U. S., Ohno, M., Mattaj, I. W., and Cerione, R. A. (1999). The nuclear cap-binding complex is a novel target of growth factor receptor-coupled signal transduction. J. Biol. Chem. 274, 4166–4173. Wilson, K., Wu, W. J., and Cerione, R. A. (2000). Cdc42 stimulates RNA splicing via the S6 kinase and a novel S6 kinase target, the nuclear cap-binding complex. J. Biol. Chem. 275, 37,307–37,310. Xu, G., Kwon, G., Cruz, W. S., Marshall, C. A., and McDaniel, M. L. (2001). Metabolic regulation by leucine of translation initiation through the mTOR-signaling pathway by pancreatic beta-cells. Diabetes 50, 353–360. Zhang, H., Stallock, J. P., Ng, J. C., Reinhard, C., and Neufeld, T. P. (2000). Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 14, 2712–2724.

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Histone Acetyltransferases and Deacetylases in the Control of Cell Proliferation and Differentiation Heike Lehrmann, Linda Louise Pritchard, and Annick Harel-Bellan CNRS UPR 9079, Institut Andr´e Lwoff, 94800 Villejuif, France

I. Introduction II. Acetylation of Histones III. Histone Acetyltransferases A. GNAT-Family (Gcn5-Related N-acetyltransferases) B. MYST-Family C. CBP/p300 Family IV. Histone Deacetylases and Cell Cycle Regulation A. Mad /Max and HDACs B. Rb and HDACs C. HDACs and Cancer V. Muscle Differentiation A. Interaction of MyoD with CBP/p300 and PCAF B. MyoD and HDAC1 C. MEF2 and Deacetylases VI. Hematopoiesis A. GATA-1 B. EKLF C. CBP/p300 and Hematopoietic Disorders VII. Huntington’s Disease VIII. Histone Acetylation in Combination with Other Chromatin Modiﬁcations IX. Conclusion References

Histone acetylation and deacetylation are chromatin-modifying processes that have fundamental importance for transcriptional regulation. Transcriptionally active chromatin regions show a high degree of histone acetylation, whereas deacetylation events are generally linked to transcriptional silencing. Many of the acetylating and deacetylating enzymes were originally identiﬁed as transcriptional coactivators or repressors. Their histone-modifying enzymatic activity was discovered more recently, opening up a whole new area of research. Histone acetyltransferases such as CREB-binding protein

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Copyright 2002, Elsevier Science (USA). All rights reserved.

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(CBP) and PCAF are involved in processes as diverse as promoting cell cycle progression and regulating differentiation. A controlled balance between histone acetylation and deacetylation seems to be essential for normal cell growth. Both histone acetyltransferases and deacetylases are involved in the development of diseases, including neurodegenerative disorders and cancer. Treatments that target these enzymes are already under clinical investigation. C 2002, Elsevier Science (USA).

I. INTRODUCTION Packaging of DNA is based on interactions between the negatively charged DNA and positively charged histones. The fundamental unit of this packaging system is the nucleosome, consisting of 146 base pairs of DNA helix wrapped around one histone octamer. Although this packaging system was originally believed to serve mainly scaffolding functions (e.g., condensation of DNA), this view has changed dramatically in recent years, and a complex interplay between histone modiﬁcations, chromatin structure, and transcriptional regulation has emerged. Two main chromatin-modifying principles have been identiﬁed: ATP-dependent helicases and histone-modifying enzymes. The former can induce changes in nucleosomal structure by ATPdependent processes. Based on homology to yeast and Drosophila ATPases, these chromatin remodeling complexes can be divided into three subclasses (SWI/SNF, ISWI, Mi-2) (Kingston and Narlikar, 1999). Chromatin-remodeling complexes can interact with and recruit the second class of chromatinmodifying complexes, leading to covalent modiﬁcations of histones. Of special interest are enzymes that modify the N-terminal histone tails that protrude from the histone octamer, and serve functions in histone–DNA and/or histone–histone interactions. An intensively studied modiﬁcation is acetylation of the epsilon-amino group of lysine residues in histone tails. Acetylation of histones is effected by histone acetyltransferases (HATs) and counteracted by histone deacetylases (HDACs). Acetylation and deacetylation of histone tails are rapidly changing events that mediate a link between cellular signaling pathways, on the one hand, and activation or repression of transcription due to changes in the chromatin structure, on the other hand. Additional modiﬁcations of histone tails include methylation, phosphorylation, ubiquitination, and ADP-ribosylation. Although these modiﬁcations are less well studied, recent evidence suggests a complex regulation system involving different types of histone modiﬁcations. The importance of acetylation and deacetylation events in cell cycle regulation and differentiation is the subject of this review.

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II. ACETYLATION OF HISTONES Histone acetylation is generally associated with transcriptionally active genomic regions (Allfrey, 1977). The most relevant acetylation sites map to the tail regions of histones H3 and H4, although H2A and H2B are also acetylated in vivo. Modiﬁed lysine residues of these regions include lysines 9, 14, 18, and 23 of H3 and lysines 5, 8, 12, and 16 of H4 (Strahl and Allis, 2000). Acetylation of lysines neutralizes the charge of the lysine amino group. This is thought to weaken the interaction between histone tails and DNA or adjacent histones. Weakening of these interactions can lead to a more open chromatin structure and can facilitate the access of transcription factors to their cognate DNA binding site. In addition, several transcription factors have themselves been shown to be substrates for HATs. In some cases, acetylation of transcription factors is suspected to increase their DNA binding activity through an uncharacterized mechanism and to result in more stable protein–DNA complexes. Alternatively, acetylated transcription factors can recruit additional proteins or protein complexes that are essential for the initiation of promoter activation. HATs target either single lysine residues or combinations of them. The speciﬁcity of HATs for one or the other lysine residue is determined not only by the enzyme itself but also by interacting proteins. Recombinant yeast Gcn5 preferentially acetylates lysine 14 of H3 and lysine 8 of H4 (Kuo et al., 1996). Gcn5 in cells, however, is part of the multiprotein complexes SAGA and ADA. In this context, Gcn5 not only efﬁciently acetylates histones within assembled nucleosomes but also targets histone H2B as well as most lysine residues of the H3 tail (Grant et al., 1999). Certain patterns of lysine acetylation in histone tails have already been linked to physiological functions. Acetylation of lysines 5 and 12 of histone H4 is characteristic for newly synthesized histones and seems to be necessary for nuclear transport and deposition of histones on replicated DNA (Sobel et al., 1995). Acetylation of lysine 16 of H4 is linked to dosage compensation in Drosophila (Bone et al., 1994; Turner et al., 1992), whereas acetylation of H4 lysine 12 is found in heterochromatic regions of the genome (Turner et al., 1992). The involvement of histone acetylation in transcriptional activation has been demonstrated in yeast: mutation of acetylatable lysine residues in the H4 tail resulted in loss of gene expression from the GAL1 and PHO5 promoters (Durrin et al., 1991). Characterization of the enzymes involved in the acetylation process has led to intensiﬁed research in the ﬁeld, so that a large body of information on histone acetylation is now available. Many of these enzymes were ﬁrst identiﬁed as transcriptional coactivators, again emphasizing the importance

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of acetylation for transcriptional regulation. Nuclear HATs are classed in several families; three of them will be discussed here.

III. HISTONE ACETYLTRANSFERASES A. GNAT-Family (Gcn5-Related N-acetyltransferases) Gcn5, the founding member of this family, was the ﬁrst HAT to be identiﬁed (Brownell et al., 1996). Using an in-gel HAT assay, Brownell and colleagues isolated a protein from Tetrahymena macronuclear extracts that exhibited histone acetyltransferase activity. Characterization of this protein revealed homology to a known transcriptional regulator, the yeast Gcn5 protein. Mutation of yeast Gcn5 demonstrated a direct correlation between its HAT activity and transcriptional control (Wang et al., 1998). Mammalian cells express two homologues of yGcn5, hGcn5 and PCAF. Both yGcn5 and hPCAF have been shown to be members of multisubunit complexes. Gcn5 can be found in the yeast ADA and SAGA complexes. The SAGA complex shows a subunit composition similar to the mammalian PCAF complex (Schiltz and Nakatani, 2000). In addition to proteins of the Ada and Spt families, both complexes contain TBP associated factors (TAFs). Deletion of speciﬁc TAFs renders the whole complex inactive for nucleosomal acetylation, emphasizing the importance of regulated protein–protein interactions for proper HAT activity (Grant et al., 1998). Homozygous deletion of Gcn5 in mice leads to embryonic lethality, whereas PCAF null mice show no aberrant phenotype. The GNAT family contains additional members that are less well characterized than Gcn5 and PCAF. Elp3, for example, has been identiﬁed as part of the yeast elongator complex and associates with RNA polymerase II transcription complexes (Wittschieben et al., 1999). Characteristic for the GNAT family is the presence of four conserved protein domains. Of special interest is the acetyl-coenzyme A binding site, which is conserved between distinct HAT families, and the presence of a C-terminal bromodomain (Fig. 1). No involvement of GNAT family members in diseases has been reported.

B. MYST-Family This family is named for its founding members MOZ, Ybf2/Sas3, Sas2, and Tip60. Sas2 and Sas3 (Sas for something about silencing) are yeast HATs that have been identiﬁed as members of multiprotein complexes, similar to HATs of the GNAT family. Sas2 is part of the SAS-I complex that also

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Fig. 1 Schematic presentation of human HATs with conserved protein motifs and binding sites for associated proteins. HAT: histone acetyltransferase enzymatic domain; Bromo: bromodomain; Zn: zinc ﬁnger region; Poly-Q: polyglutamine region.

contains Sas4 and Sas5 proteins. Recent ﬁndings demonstrate an association of Sas2 with chromatin assembly factors. Sas2 associates with Cac1, a component of the yeast chromatin assembly factor-1 complex (CAF-I) and with the nucleosome assembly factor Asf1. Association with the chromatin assembly factors targets SAS-I to newly replicated DNA. Deletions encompassing either components of the SAS-I complex or Cac1 or Asf1 all have similar effects on derepression of silenced telomeric heterochromatin. Requirement of lysine 16 of histone H4 for Sas2 function suggests an involvement of histone acetylation in the biologic activity of Sas2, although HAT activity of Sas2 remains to be demonstrated (Meijsing and Ehrenhofer-Murray, 2001; Osada et al., 2001). Sas3 is part of the yeast H3 speciﬁc NuA3 complex (John et al., 2000). While deletion of Sas3 shows almost no detectable phenotype in yeast, combined deletion of Sas3 and Gcn5 (both of which target H3 for acetylation) results in extensive loss of H3 acetylation and G2/M-phase arrest. These ﬁndings lead to the conclusion that acetylation of histone H3 is essential for yeast viability and that this need is governed by several complexes with at least partially overlapping activities (Howe et al., 2001). In contrast, H4 in yeast is only acetylated by the NuA4 complex, which contains Esa1p (a MYST family member, Esa for essential Sas family acetyltransferase) as the catalytic HAT (Allard et al., 1999). Deletion of Esa1p is lethal, demonstrating also the requirement of H4 acetylation for yeast viability (Clarke et al., 1999; Smith et al., 1998). Esa1p is homologous to the Drosophila MOF (males absent on the first) protein. MOF is required

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for dosage compensation in Drosophila. Members of the MSL complex (see below) target MOF to the male X chromosome, where acetylation of H4 lysine 16 leads to increased transcription. Deletion of members of the MSL complex leads to male-speciﬁc lethality (MSL) (Akhtar and Becker, 2000; Smith et al., 2000). Tip60 (Tat-interacting protein) represents the catalytic subunit of the human TIP60 complex (Ikura et al., 2000). Tip60 has been identiﬁed as an HIV-Tat interacting protein; and Tat inhibits Tip60 HAT activity (Creaven et al., 1999; Kamine et al., 1996). The predicted involvement of Tip60 in DNA repair could have implications for cancer development in the event of loss or mutation of members of the Tip60 complex.

C. CBP/p300 Family CBP (CREB-binding protein) and p300 are considered to be functional homologues. CBP was originally characterized as a transcriptional coactivator. Both proteins contain N- and C-terminal regions that are able to activate transcription when artiﬁcially tethered to promoters, even though these regions are devoid of HAT activity. These regions contain zinc ﬁnger domains that are characterized interaction sites for a variety of proteins. Viral proteins, transcription factors, and other HATs have been shown to bind to CBP/p300 through these regions (Fig. 1). Some of the interactions seem to be mutually exclusive (e.g., interaction of p300 with either E1A or PCAF). Due to limiting cellular CBP/p300 levels, competition for CBP/p300 is thought to be a means for regulating promoter activity and to account for the changes observed during viral infections or differentiation processes (Kamei et al., 1996). CBP and p300 are also characterized HATs, with the catalytic domain residing in the central region of the protein (Bannister and Kouzarides, 1996; Ogryzko et al., 1996). Therefore, CBP/p300 exhibit their transcriptional coactivator function through multiple facets: through offering a surface for the binding of various proteins, CBP/p300 contribute to the formation of a multiprotein activation complex and ensure the integration of different signaling pathways. In addition, the enzymatic activity conferred by the HAT domain of these proteins can inﬂuence promoter activity directly by acetylating histones in the corresponding promoter region. Besides opening of the chromatin structure due to reduced interaction between DNA and acetylated histone tails, acetylation of histones can also inﬂuence the histone composition of nucleosomes. p300 has been shown to interact with NAP-1, a histone chaperone involved in nucleosome remodeling (Ito et al., 2000). p300 acetylates the H2A/H2B dimer, which facilitates the transfer of H2A/H2B from the nucleosome to NAP-1. It is not clear whether acetylation reduces the interaction between histones in the octamer, or if it increases the afﬁnity of H2A/H2B for NAP-1. Nucleosomes depleted

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of H2A/H2B histones may be responsible for the altered chromatin structure of actively transcribed regions and facilitate the access for the transcription machinery (Baer and Rhodes, 1983). p300 and CBP are implicated in a variety of cellular processes. A wide range of promoters can be activated by overexpression of either of these proteins. Pathways as different as cell cycle progression, apoptosis, and differentiation seem to require CBP and/or p300. Both proteins are also targets for viral transforming proteins, which highlights their importance in the regulation of cellular processes. E1A, for example, has been shown to interact with p300 (Eckner et al., 1994). The binding site of E1A on CBP/p300 overlaps the interaction site for PCAF (Yang et al., 1996). This interaction is essential, since deletion of the N-terminal p300-binding region of E1A eliminates the transforming potential of E1A (Harlow et al., 1986). CBP/p300, and to a lesser extent PCAF, also acetylate nonhistone substrates. Among them are many transcription factors (e.g., E2F, MyoD, HNF-4) (MartinezBalbas et al., 2000; Polesskaya et al., 2000; Sartorelli et al., 1999; Soutoglou et al., 2000), but also structural proteins such as alpha-tubulin (MacRae, 1997) and the cytoplasmic-nuclear shuttle protein importin alpha (Bannister et al., 2000).

1. INTERACTION WITH p53 Of special interest here is the interaction of CBP and p300 with p53. p53 is a classical tumor suppressor. Its function as “guardian of the genome” ensures proper transmission of the genomic information from cell division to cell division. Aberrations such as DNA damage due to UV irradiation or deregulated gene expression after viral infection lead to activation of p53 and concomitant cell cycle arrest or apoptosis. Activation of p53 is regulated at the posttranslational level by multiple mechanisms, including phosphorylation and association with the ubiquitin ligase MDM2. Gu and Roeder (1997) recently demonstrated that this regulation cascade also includes acetylation of p53 by CBP/p300 and PCAF. In vivo, acetylation of p53 increases upon DNA damage and activates the transcriptional activity of p53. As for many other acetylated transcription factors, initial studies suggested that an increase in DNA binding due to acetylation of p53 might be the molecular explanation for the observed increase in transcriptional activity. However, it is not clear whether these data, which are based on electrophoretic mobility shift assays in vitro, reﬂect the in vivo situation. Acetylation of p53 might rather inﬂuence the recruitment of speciﬁc factors to a given promoter region or alter the subnuclear localization of p53 (i.e., association with PML and localization to nuclear bodies). In contrast, p300 also participates in the degradation of p53. p53 degradation involves a ternary complex of p300, MDM2, and p53. Interestingly, TAFII250, another HAT, shows

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ubiquitin-conjugating activity (Wassarman and Sauer, 2001). The potential for competition between acetylation and ubiquitination on certain lysine residues is especially intriguing. Furthermore, the participation of p300 in both activation and degradation of p53 puts it in a critical position for cell cycle regulation and reﬂects its dual role in promoting cell cycle progression and acting as a tumor suppressor gene.

2. CBP/p300 AND CELL CYCLE REGULATION A direct involvement of CBP/p300 in the regulation of S-phase entry has been demonstrated by Ait-Si-Ali et al. (1998). Serum stimulation of arrested cells leads to phosphorylation of CBP/p300 and an increase in its intrinsic HAT activity; E1A binding to CBP seems to mimic the effect of phosphorylation although under different conditions, maybe depending on the dose of E1A, it can have inhibitory effects. Both phosphorylation and HAT activity of CBP/p300 peak at the G1/S boundary. A candidate enzyme for phosphorylating CBP is the cell cycle regulated cyclinE/cdk2 complex. Cyclin E can interact directly with p300 (Perkins et al., 1997). Additionally, p300 regulates the expression of cyclin D1, and its HAT activity is required for this effect (Albanese et al., 1999). p300 also coactivates the transcription factor E2F, which is critically involved in the regulation of S-phase speciﬁc genes (Martinez-Balbas et al., 2000; Trouche et al., 1996). Inhibition of CBP/p300 by antibodies results in reduced numbers of cells in S phase, strongly suggesting the necessity of CBP/p300 in cell cycle progression (Ait-Si-Ali et al., 2000). An involvement of CBP and p300 in the regulation of proliferation was also predicted from knockout mice, based on the observation that CBP or p300 null cells show reduced proliferation (Yao et al., 1998).

IV. HISTONE DEACETYLASES AND CELL CYCLE REGULATION Histone deacetylases can be grouped into three classes based on their homology to yeast enzymes. The ﬁrst class contains HDACs 1–3 and 8 and shows homology to the yeast enzyme RPD3. HDACs 4–7, 9, and 10 are grouped in the second class with similarity to the yeast HDA1 enzyme, while the third class of HDACs displays NAD-dependent deacetylation and homology to the yeast enzyme Sir2. Extensive studies in yeast have demonstrated an involvement of histone deacetylases in transcriptional control. The transcriptional repressor Tup1 recruits HDA1 to the promoter regions of ENA1 and STE6 genes. Recruitment of HDA1 leads to a speciﬁc deacetylation of H3 and H2B that is limited to the promoter’s vicinity (Wu et al., 2001).

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The transcription factor UME6, on the other hand, represses target promoters by recruitment of RPD3, which deacetylates all four core histones of nucleosomes in the entire promoter region. The speciﬁcity of mammalian HDACs is less well documented. However, association with many transcriptional repressors has been reported (Cress and Seto, 2000).

A. Mad/Max and HDACs The Mad/Max and Myc/Max complexes are thought to be involved in cell cycle control. In particular, Mad/Max is induced upon cell terminal differentiation (Ayer and Eisenman, 1993). Mad inhibits cell cycle progression through association with Max and binding to E-box containing promoters (McArthur et al., 1998). Promoters that are repressed by Mad/Max include E2F and cdc25 promoters. The repressive function of Mad/Max requires the association with a repressor complex that contains mSin3, N-CoR, and HDAC (Alland et al., 1997; Ayer et al., 1995; Hassig et al., 1997; Heinzel et al., 1997; Laherty et al., 1997; Nagy et al., 1997; Schreiber-Agus et al., 1995).

B. Rb and HDACs An intensively studied interaction is the association of HDAC with the retinoblastoma protein (RB). RB is frequently mutated in human tumors and plays a key role in regulating cell cycle progression from G1 to S phase. The main mechanism of this regulatory function depends on the association of RB with the transcription factor E2F. E2F regulates the expression of many genes involved in progression from G1 to S phase. Association of E2F with RB inhibits transcriptional activation by E2F via several mechanisms. First of all, RB interacts with the transcriptional activation domain of E2F and masks its activity. Active repression of promoters additionally requires an association with HDAC. This interaction depends on the pocket region of RB and leads to deacetylation of histones in the promoter region of repressed genes (Ferreira et al., 2001). Recent ﬁndings suggest a concomitant association of RB with the DNA methyltransferase DNMT1 (Robertson et al., 2000) as well as with the histone methyltransferase SUV39 and methylation of histones (Nielsen et al., 2001). However, methylation is currently thought to be an irreversible mechanism, and methylation of promoters regulated during the cell cycle would require periodic methylation and demethylation. If this occurs, it would extend the properties of histone methylation from an epigenetic marker to a ﬂexible label comparable with acetylation and

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phosphorylation. Characterization of an enzyme involved in the demethylation process has not yet been reported. In addition to regulation of G1/S progression, RB is also involved in regulating additional stages of the cell cycle. M-phase entry of cells requires the expression of cyclin A and cdc2. RB controls the expression of these genes through association with Brg1 or Brm, components of the histone remodeling complex SWI/SNF. Progressive phosphorylation of RB during the G1 phase of the cell cycle disrupts its association with Brg1 and leads to expression of cyclinA and cdc2 and, as a result, to progression into M phase (Zhang et al., 2000). Although these examples show an involvement of HDAC proteins in the control of cell cycle progression, the cellular regulation system seems to be complex. Administration of HDAC inhibitors like TSA leads to cell cycle arrest in G1 and G2. TSA treatment induces histone hyperacetylation and activates expression of the cdk inhibitor p21/WAF/Cip1 (Nakano et al., 1997; Sowa et al., 1997). In addition, Rb accumulates in its hypophosphorylated form. Although cyclin D1 levels increase following TSA treatment, no associated kinase activity is detectable. This is potentially due to eleveated levels of the cyclin-dependent inhibitor p27. However, since even p21- and p27-deﬁcient cells respond to TSA treatment by growth arrest, additional factors must be regulated by TSA, and the molecular mechanism of growth arrest triggered by TSA remains elusive (Wharton et al., 2000).

C. HDACs and Cancer A direct involvement of HDACs in human cancers is well documented in the case of promyelocytic leukemia (PML)– and PLZF–RAR fusion proteins (Melnick and Licht, 1999; Minucci and Pelicci, 1999). In certain types of acute promyelocytic leukemia, genomic translocations result in fusion of PML or PLZF to the retinoic acid receptor (RAR), thus preventing hematopoietic differentiation. Both fusion proteins are known to associate with transcriptional repressor complexes (NcoR/SMRT) that contain HDAC activity. Wild-type RAR is also found to associate with these repressor complexes. However, whereas physiological doses of retinoic acid (RA) release the transcriptional repression mediated by wtRAR, the PML– and PLZF–RAR repressive complexes are resistant to these doses of RA. Increased doses of RA release the repressor complex from PML–RAR fusion proteins, leading to progression in the differentiation process, whereas PLZF-RAR is resistant. Both PML– and PLZF–RAR fusion proteins form oligomers that result in increased concentrations of repressor complex at the promoter region of target genes, explaining the need for higher doses of RA to overcome the augmented repressor activity (Lin and Evans, 2000; Minucci et al., 2000);

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however, for PLZF–RAR fusion proteins repressor complexes also associate with the PLZF part of the protein that does not respond to RA administration, and release from transcriptional repression can only be achieved by additional administration of HDAC inhibitors (Melnick and Licht, 1999). Treatment of PLZF–RAR patients with a combination of HDAC inhibitors and pharmacological doses of RA has been proven efﬁcient in inducing myeloid differentiation (Warrell et al., 1998) but often results in the emergence of resistant clones. For a detailed description of HDAC inhibitors in clinical trials readers are referred to Wang et al. (2001).

V. MUSCLE DIFFERENTIATION A. Interaction of MyoD with CBP/p300 and PCAF Muscle differentiation depends on two families of transcription factors: myogenic basic helix-loop-helix proteins (bHLH) and myocyte enhancer factors-2 (MEF2s). Overexpression of myogenic bHLHs (i.e., MyoD, Myf-5, Myogenin, MRF-4) is sufﬁcient to induce the muscle-speciﬁc differentiation program. Expression of MyoD and Myf-5 results in chromatin remodeling at muscle-speciﬁc promoters (Gerber et al., 1997). MyoD has been shown to interact with p300; and p300 coactivates MyoD-dependent transcription of muscle-speciﬁc promoters (Eckner et al., 1996; Sartorelli et al., 1997; Yuan et al., 1996). The N-terminal KIX domain and the C-terminal CH3 region of p300 are characterized binding sites for MyoD (Riou et al., 2000; Yuan et al., 1996). The CH3 region of p300 is involved in the interaction with several cellular and also viral proteins that regulate processes as diverse as proliferation (E2F-1), differentiation (GATA-1), apoptosis (p53), and transformation (E1A) (Goodman and Smolik, 2000). Competition for this binding region is likely to be involved in cellular decision processes. MyoD and PCAF seem to be able to bind to the CH3 region of p300 simultaneously, whereas the adenoviral protein E1A, which also interacts with the CH3 region of p300, inhibits binding of MyoD and PCAF to p300. Injection of antibodies against PCAF and p300 demonstrated that both HATs are necessary for the muscle differentiation process (Eckner et al., 1996; Puri et al., 1997). Although initial studies with deletion mutants of p300 and PCAF suggested that only the enzymatic HAT activity of PCAF was required for muscle differentiation (Puri et al., 1997), this model has been revised recently. Administration of p300-speciﬁc HAT inhibitors demonstrated the necessity of the p300 HAT domain for the muscle differentiation process. The requirement for PCAF vs. p300 HAT activity might be regulated in a timely ordered manner. p300, for

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example, shows speciﬁcity for induction of late muscle genes, concomitant with an increase in its intrinsic HAT activity (Polesskaya et al., 2001a). Interestingly, MyoD itself can serve as a substrate for p300 and PCAF (Polesskaya et al., 2000; Sartorelli et al., 1999). The importance of MyoD acetylation in the differentiation process is not completely understood. Sartorelli et al. have proposed a structural change in the conformation of MyoD and increased DNA binding of MyoD following acetylation by PCAF (Sartorelli et al., 1999). Polesskaya et al., however, demonstrated an increased afﬁnity of acetylated MyoD for CBP (Polesskaya et al., 2001b). This interaction requires the bromodomain of CBP/p300 and is likely to involve a recognition process similar to that observed for acetylated histone tails and the bromodomains of Gcn5 and TAFII250. Tighter binding of CBP/p300 to muscle-speciﬁc promoters might lead to increased histone acetylation in the promoter region and enhanced transcription of muscle-speciﬁc genes.

B. MyoD and HDAC1 In undifferentiated myoblasts, MyoD is found in association with HDAC1, which represses the transcriptional activation potential of MyoD. Upon differentiation, HDAC1 dissociates from MyoD; and MyoD associates with PCAF (Mal et al., 2001). The interactions of HDAC1 and PCAF with MyoD might therefore be mutually exclusive. The mechanism responsible for release of HDAC1 from MyoD is not completely understood. Puri et al. (2001) have suggested a link to the Rb pathway: during the differentiation process, Rb becomes progressively hypophosphorylated and associates with HDAC1. A mutant HDAC1 protein, deﬁcient for Rb-binding, was unable to relieve repression of transcription from muscle-speciﬁc promoters. They postulate that redistribution of HDAC1 into Rb-containing complexes during the muscle differentiation process could be one of the means that regulates MyoD/HDAC repressor complexes (Fig. 2).

C. MEF2 and Deacetylases The class II HDACs 4 and 5 interact directly with MEF2 (Miska et al., 1999), and overexpression of HDACs 4 and 5 inhibits muscle differentiation (Lu et al., 2000a). Activation of MEF-2 dependent promoters requires dissociation of the MEF-2/HDAC II complex. Phosphorylation of class II HDACs by CaMK triggers the release from MEF-2 (Lu et al., 2000b). In addition, phosphorylation of HDACs 4 and 5 induces cytoplasmic localization of these proteins, via interaction with proteins of the 14-3-3 family (Grozinger and Schreiber, 2000; McKinsey et al., 2000; Wang et al., 2000).

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Fig. 2 Activation of MyoD regulated promoters during muscle differentiation. In proliferating myoblast, MyoD and MEF-2 are associated with HDAC proteins. Induction of differentiation leads to release of HDAC proteins and the formation of transcriptional activator complexes that contain CBP and PCAF. HD: HDAC; P: phosphorylation; Ac: acetylation.

VI. HEMATOPOIESIS A. GATA-1 GATA-1 is a zinc ﬁnger-containing transcription factor that is required for the erythroid and megakaryocytic differentiation processes (Blobel, 2000). GATA-1 interacts with CBP in vivo and in vitro; and GATA-1-dependent transcription is augmented by overexpression of CBP/p300 (Blobel et al., 1998; Boyes et al., 1998). The GATA-1 binding site on CBP has been mapped to the CH3 region of CBP (Blobel et al., 1998). Overexpression of the adenovirus protein E1A, which also interacts with the CH3 region of CBP, blocks the erythroid differentiation process and inhibits GATA-1-dependent transcription. CBP and p300 acetylate GATA-1 in vitro (Boyes et al., 1998; Hung et al., 1999). The importance of this acetylation event in vivo, however, is controversial. Whereas Boyes et al. showed increased DNA binding by acetylated GATA-1, the second study failed to detect this effect.

B. EKLF ¨ The erythroid Kruppel-like factor EKLF is essential for the regulation of beta-globin expression (Miller and Bieker, 1993). In overexpression experiments, EKLF can interact with CBP, p300, and PCAF. However, only CBP and p300 seem to acetylate EKLF in vitro, and only CBP and p300 enhance transcriptional activation by EKLF in vivo (Zhang and Bieker, 1998).

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Acetylation of EKLF does not affect its DNA-binding afﬁnity. Mutation of one of the target lysine residues of EKLF resulted in reduced transactivation potential and nonresponsiveness to coactivation by CBP or p300. Zhang et al. (2001) demonstrated that acetylation of EKLF results in higher afﬁnity for the SWI/SNF complex. The transcription factor EKLF therefore seems to integrate different chromatin modifying complexes at speciﬁc target promoters. Transcriptional activation in this case is most likely a result of histone acetylation combined with remodeling of the nucleosomal structure of the promoter region.

C. CBP/p300 and Hematopoietic Disorders 1. HEMATOPOIETIC DISORDERS IN CBP/p300 KNOCKOUT MICE Although CBP and p300 show many overlapping functions, divergence has been observed for certain differentiation processes. Common features of CBP and p300 knockout mice are growth retardation and developmental defects such as lack of neural tube closure and embryonic lethality (Tanaka et al., 1997; Yao et al., 1998). p300 homozygous animals show characteristic defects in heart development, whereas heterozygous CBP knockout mice display a high incidence of hematological malignancies. In addition, some of the defects observed in CBP knockout mice are reminiscent of alterations seen in the Rubinstein–Taybi syndrome (RTS). This human disorder is characterized by cranial and digital malformations, mental retardation, and hematopoietic abnormalities. Patients show mutation of one CBP allele (Petrij et al., 1995) and are predisposed to certain types of cancer, speciﬁcally, childhood tumors of neural crest origin. Tumor development is usually accompanied by loss of the second CBP allele (Miller and Rubinstein, 1995).

2. GENOMIC TRANSLOCATIONS AND LEUKEMIAS Genomic translocations of CBP and p300 are also associated with certain subtypes of leukemia. As discussed below, fusion of CBP to MOZ results in the development of acute myeloid leukemia (AML). Another genomic translocation fuses CBP to the mixed lineage leukemia gene MLL/ALL-1, a homologue of the Drosophila Trithorax protein. MLL is found in several translocations in childhood leukemias and in chemotherapy-induced secondary leukemias (Dimartino and Cleary, 1999). All of the MLL fusion proteins contain the N-terminal region of MLL and the C-terminal part of the translocation partner, for example, CBP (Sobulo et al., 1997). In many cases,

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MLL retains its DNA binding domain, transcriptional repression domain, and the SET domain, a region conserved in methyltransferases. Fusion to CBP adds the C-terminal part of this protein, including the bromodomain and HAT region. Although the molecular mechanism that leads to tumorigenesis is not understood, transcriptional deregulation due to aberrant chromatin remodeling is a likely hypothesis. Mutations of p300 have also been found in certain tumors (Gayther et al., 2000; Muraoka et al., 1996). Although the incidence of CBP/p300 mutations in tumors is low, their occurrence suggests a possible role for both proteins in tumor suppression.

3. MYST-FAMILY MEMBERS AND LEUKEMIAS As mentioned above, another member of the MYST family is MOZ (monocytic leukemia zinc ﬁnger protein), which is a human homologue of the yeast Sas3 protein (Reifsnyder et al., 1996). Although an actual HAT activity has not been demonstrated for MOZ, such a catalytic activity is strongly suggested due to homology to other MYST family members. MOZ is essential for proper differentiation of hematopoietic cells. Genomic translocations of MOZ and fusion of the gene to that encoding other HATs (CBP, p300, TIF2) is observed in some leukemias (Borrow et al., 1996; Carapeti et al., 1998; Kitabayashi et al., 2001). All of these fusion proteins contain the N-terminal region of MOZ, including its potential HAT domain, linked to the C-terminal regions of CBP, p300, or TIF2, containing a second HAT domain (Fig. 1). In addition, TIF2 has been shown to associate with CBP/p300, potentially generating a protein complex with three functional HAT domains. The increased number of HAT domains and/or the lost regulatory regions can lead to aberrant promoter activation and development of leukemias. Interestingly, like MOZ, many of the other MYST family proteins contain zinc ﬁnger domains and chromodomains.

VII. HUNTINGTON’S DISEASE Huntington’s disease is a neurodegenerative disorder that is characterized by movement disorders and progressive dementia (Gusella et al., 1996). The only identiﬁed mutation implicated in this disease is the expansion of a polyglutamine stretch in the N-terminal region of huntingtin (Htt). Expression of polyglutamine-containing N-terminal fragments of Htt is sufﬁcient to trigger the disease phenotype in mice (Mangiarini et al., 1996). Histologically, the disease is accompanied by the appearance of intranuclear and cytoplasmic inclusions that contain Htt and ubiquitin (Davies et al., 1997; DiFiglia et al., 1997). Recent reports demonstrate that the polyglutamine form of Htt

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associates with a glutamine repeat sequence of CBP and sequesters it in nuclear and cytoplasmic inclusions both in transgenic mice and in the brains of Huntington patients (Nucifora et al., 2001; Steffan et al., 2000). In addition, it has been observed that mutant Htt reduces the transcriptional coactivating function of CBP (Nucifora et al., 2001; Steffan et al., 2000). Overexpression of CBP, on the other hand, rescues some of the disease-related cellular phenotypes (McCampbell et al., 2000; Nucifora et al., 2001). Furthermore, Steffan et al. have shown that mutant Htt interacts directly with the HAT domains of CBP and PCAF and represses the HAT activities of CBP, p300, and PCAF in vitro (Steffan et al., 2001). Expression of the mutated Htt protein in cell culture leads to a reduction in the acetylation levels of H3 and H4, and this effect is reversed by the administration of histone deacetylase inhibitors (Steffan et al., 2001). It should be mentioned that Htt also associates with transcriptional repressor complexes such as mSin3A and N-CoR that can recruit histone deacetylase activity (Boutell et al., 1999; Steffan et al., 2000). Although the molecular mechanism leading to the development of Huntington’s disease is not completely understood, the use of HDAC inhibitors shows encouraging effects. This is especially important since, to date, no effective treatment exists for this lethal disease.

VIII. HISTONE ACETYLATION IN COMBINATION WITH OTHER CHROMATIN MODIFICATIONS Although it is not the subject of this review, it should be kept in mind that other histone modiﬁcations exert profound inﬂuences on transcriptional regulation. Interplay between different chromatin-modifying systems has been observed in several diseases. The combination of DNA methylation and histone deacetylation in the silencing of promoters is especially intriguing. The genetic loci of MLH1, TIMP3, and CDKN2A (p16) are hypermethylated in the colorectal carcinoma cell line RKO. CDKN2B is hypermethylated in the leukemic cell line KG1a. A combined treatment by demethylating agents and histone deacetylase inhibitors has been proven most efﬁcient for activation of transcription from these loci (Cameron et al., 1999). Similar experience was reported from studies of the fragile X-syndrome. The genomic locus for this disease (FMR-1) is mutated and inappropriately silenced. Loss of FMR-1 expression is manifested by mental retardation. Recent studies have revealed that silencing of FMR-1 is linked to cytosine methylation of the DNA and deacetylation of histones H3 and H4 in the 5 region of the gene. Although treatment with the HDAC inhibitor TSA restores acetylation of histone H4, this does not result in activation of gene expression. Administration of methyltransferase inhibitors, however,

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resulted in both absence of methylation and acetylation of the genomic region, with activation of gene expression (Coffee et al., 1999).

IX. CONCLUSION Although histones were long considered as simple packaging units for DNA, it is now clear that modiﬁcations of histones and nucleosome architecture actively participate in transcriptional regulation and thus in many pathways involved in cell fate control. Recent studies demonstrate close interactions between different modiﬁcation systems. Sequential recruitment of SWI/SNF and HAT complexes has been demonstrated for the HO locus in yeast (Cosma et al., 1999). Cell cycle-dependent recruitment of chromatin remodeling complexes has been reported in several systems. Whereas Gcn5 alone can activate certain promoters during interphase, transcriptional activation of promoters during mitosis also requires the presence of SWI/SNF complexes (Krebs et al., 1999). Multiple modiﬁcations on histone tails suggest the existence of a histone “code” (Strahl and Allis, 2000) that is deciphered by speciﬁc protein modules (e.g., chromo- and bromodomains). Additionally, Thomson and colleagues have postulated a dynamic model for histone modiﬁcations, with speciﬁc modiﬁcations actively changing as transcription proceeds (Thomson et al., 2001). Acetylation of histones can occur on a small number of nucleosomes in the promoter region of a gene or extend over several hundreds of base pairs of DNA, and the interrelationship between the acetylation and methylation status on speciﬁc histones is currently under active investigation. The association of distinct site-speciﬁc histone H3 methylation patterns with heterochromatic vs. euchromatic compartments, and the fact that boundary elements indeed prevent the spread of silencing into neighboring regions of the chromosome have now been directly demonstrated in a ﬁssion yeast model (Noma et al., 2001). Deletion of these boundary elements can lead to silencing of normally active genes in adjacent regions, and it might be anticipated that, in some human diseases, chromosomal translocations could result in inappropriate silencing of normally active genes (or vice versa) through disruption of boundary elements. Downregulation of cell cycle regulators and tumor suppressor genes and loss of differentiation-speciﬁc gene expression are frequently associated with tumor development. Analysis of the molecular defects that lead to inappropriate promoter activation or silencing will be necessary in order to design speciﬁc tools for treatments. In this respect it will be important to identify the enzymes and complexes that are crucially involved in histone modiﬁcation and promoter regulation. The recent success in unraveling HAT and HDAC

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complexes and the growing information on histone and DNA methyltransferases are encouraging. Use of HDAC inhibitors and demethylating agents in the treatment of cancer has already proven successful. Since both treatments may cause severe side effects, the design of disease- and cell type-speciﬁc drugs is likely to prove necessary. The development of speciﬁc inhibitors for certain HATs or HDACs is in progress. However, the documented involvement of these enzymes in multiple complexes makes it necessary to target the inhibitors speciﬁcally to one or the other complex or promoter region. Misfunctioning enzymes (i.e., fusion proteins that arise from genomic translocations) could be targeted for speciﬁc degradation or inactivation through complexing with dominant negative proteins.

ACKNOWLEDGMENTS Work from the authors is supported by the European Community, 5th Framework Programme (QLG1-CT-1999-00866 and QLGA-CT-2000-51259) and by La Ligue Nationale Contre le Cancer.

REFERENCES Ait-Si-Ali, S., Polesskaya, A., Filleur, S., Ferreira, R., Duquet, A., Robin, P., Vervish, A., Trouche, D., Cabon, F., and Harel-Bellan, A. (2000). CBP/p300 histone acetyl-transferase activity is important for the G1/S transition. Oncogene 19, 2430–2437. Ait-Si-Ali, S., Ramirez, S., Barre, F. X., Dkhissi, F., Magnaghi-Jaulin, L., Girault, J. A., Robin, P., Knibiehler, M., Pritchard, L. L., Ducommun, B., et al. (1998). Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A. Nature 396, 184–186. Akhtar, A., and Becker, P. B. (2000). Activation of transcription through histone H4 acetylation by MOF, an acetyltransferase essential for dosage compensation in Drosophila. Mol. Cell 5, 367–375. Albanese, C., D’Amico, M., Reutens, A. T., Fu, M., Watanabe, G., Lee, R. J., Kitsis, R. N., Henglein, B., Avantaggiati, M., Somasundaram, K., et al. (1999). Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis. J. Biol. Chem. 274, 34,186–34,195. Alland, L., Muhle, R., Hou, H., Jr., Potes, J., Chin, L., Schreiber-Agus, N., and DePinho, R. A. (1997). Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature 387, 49–55. Allard, S., Utley, R. T., Savard, J., Clarke, A., Grant, P., Brandl, C. J., Pillus, L., Workman, J. L., and Cote, J. (1999). NuA4, an essential transcription adaptor/histone H4 acetyltransferase complex containing Esa1p and the ATM-related cofactor Tra1p. EMBO J. 18, 5108–5119. Allfrey, V. (1977). Post-synthetic modiﬁcations of histone structure: A mechanism for the control of chromosome structure by the modiﬁcation of histone-DNA interactions. In “Chromatin

Histone Acetylation and Cell Fate Control

59

and Chromosome Structure” (H. J. Li and R. Echhadt, Eds.). Academic Press, San Diego, pp. 167–191. Ayer, D. E., and Eisenman, R. N. (1993). A switch from Myc:Max to Mad:Max heterocomplexes accompanies monocyte/macrophage differentiation. Genes Dev. 7, 2110–2119. Ayer, D. E., Lawrence, Q. A., and Eisenman, R. N. (1995). Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3. Cell 80, 767–776. Baer, B. W., and Rhodes, D. (1983). Eukaryotic RNA polymerase II binds to nucleosome cores from transcribed genes. Nature 301, 482–488. Bannister, A. J., and Kouzarides, T. (1996). The CBP co-activator is a histone acetyltransferase. Nature 384, 641–643. Bannister, A. J., Miska, E. A., Gorlich, D., and Kouzarides, T. (2000). Acetylation of importinalpha nuclear import factors by CBP/p300. Curr. Biol. 10, 467–470. Blobel, G. A. (2000). CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blood 95, 745–755. Blobel, G. A., Nakajima, T., Eckner, R., Montminy, M., and Orkin, S. H. (1998). CREBbinding protein cooperates with transcription factor GATA-1 and is required for erythroid differentiation. Proc. Natl. Acad. Sci. USA 95, 2061–2066. Bone, J. R., Lavender, J., Richman, R., Palmer, M. J., Turner, B. M., and Kuroda, M. I. (1994). Acetylated histone H4 on the male X chromosome is associated with dosage compensation in Drosophila. Genes Dev. 8, 96–104. Borrow, J., Stanton, V. P., Jr., Andresen, J. M., Becher, R., Behm, F. G., Chaganti, R. S., Civin, C. I., Disteche, C., Dube, I., Frischauf, A. M., et al. (1996). The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein. Nat. Genet. 14, 33–41. Boutell, J. M., Thomas, P., Neal, J. W., Weston, V. J., Duce, J., Harper, P. S., and Jones, A. L. (1999). Aberrant interactions of transcriptional repressor proteins with the Huntington’s disease gene product, huntingtin. Hum. Mol. Genet. 8, 1647–1655. Boyes, J., Byﬁeld, P., Nakatani, Y., and Ogryzko, V. (1998). Regulation of activity of the transcription factor GATA-1 by acetylation. Nature 396, 594–598. Brownell, J. E., Zhou, J., Ranalli, T., Kobayashi, R., Edmondson, D. G., Roth, S. Y., and Allis, C. D. (1996). Tetrahymena histone acetyltransferase A: A homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell 84, 843–851. Cameron, E. E., Bachman, K. E., Myohanen, S., Herman, J. G., and Baylin, S. B. (1999). Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21, 103–107. Carapeti, M., Aguiar, R. C., Goldman, J. M., and Cross, N. C. (1998). A novel fusion between MOZ and the nuclear receptor coactivator TIF2 in acute myeloid leukemia. Blood 91, 3127– 3133. Clarke, A. S., Lowell, J. E., Jacobson, S. J., and Pillus, L. (1999). Esa1p is an essential histone acetyltransferase required for cell cycle progression. Mol. Cell. Biol. 19, 2515–2526. Coffee, B., Zhang, F., Warren, S. T., and Reines, D. (1999). Acetylated histones are associated with FMR1 in normal but not fragile X-syndrome cells. Nat. Genet. 22, 98–101. Cosma, M. P., Tanaka, T., and Nasmyth, K. (1999). Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter. Cell 97, 299–311. Creaven, M., Hans, F., Mutskov, V., Col, E., Caron, C., Dimitrov, S., and Khochbin, S. (1999). Control of the histone-acetyltransferase activity of Tip60 by the HIV-1 transactivator protein, Tat. Biochemistry 38, 8826–8830. Cress, W. D., and Seto, E. (2000). Histone deacetylases, transcriptional control, and cancer. J. Cell Physiol. 184, 1–16.

60

Lehrmann et al.

Davies, S. W., Turmaine, M., Cozens, B. A., DiFiglia, M., Sharp, A. H., Ross, C. A., Scherzinger, E., Wanker, E. E., Mangiarini, L., and Bates, G. P. (1997). Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90, 537–548. DiFiglia, M., Sapp, E., Chase, K. O., Davies, S. W., Bates, G. P., Vonsattel, J. P., and Aronin, N. (1997). Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277, 1990–1993. Dimartino, J. F., and Cleary, M. L. (1999). Mll rearrangements in haematological malignancies: Lessons from clinical and biological studies. Br. J. Haematol. 106, 614–626. Durrin, L. K., Mann, R. K., Kayne, P. S., and Grunstein, M. (1991). Yeast histone H4 N-terminal sequence is required for promoter activation in vivo. Cell 65, 1023–1031. Eckner, R., Ewen, M. E., Newsome, D., Gerdes, M., DeCaprio, J. A., Lawrence, J. B., and Livingston, D. M. (1994). Molecular cloning and functional analysis of the adenovirus E1A-associated 300-kD protein (p300) reveals a protein with properties of a transcriptional adaptor. Genes Dev. 8, 869–884. Eckner, R., Yao, T. P., Oldread, E., and Livingston, D. M. (1996). Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation. Genes Dev. 10, 2478–2490. Ferreira, R., Naguibneva, I., Pritchard, L. L., Ait-Si-Ali, S., and Harel-Bellan, A. (2001). The Rb/chromatin connection and epigenetic control: Opinion. Oncogene 20, 3128–3133. Gayther, S. A., Batley, S. J., Linger, L., Bannister, A., Thorpe, K., Chin, S. F., Daigo, Y., Russell, P., Wilson, A., Sowter, H. M., et al. (2000). Mutations truncating the EP300 acetylase in human cancers. Nat. Genet. 24, 300–303. Gerber, A. N., Klesert, T. R., Bergstrom, D. A., and Tapscott, S. J. (1997). Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: A mechanism for lineage determination in myogenesis. Genes Dev. 11, 436–450. Goodman, R. H., and Smolik, S. (2000). CBP/p300 in cell growth, transformation, and development. Genes Dev. 14, 1553–1577. Grant, P. A., Eberharter, A., John, S., Cook, R. G., Turner, B. M., and Workman, J. L. (1999). Expanded lysine acetylation speciﬁcity of Gcn5 in native complexes. J. Biol. Chem. 274, 5895–5900. Grant, P. A., Schieltz, D., Pray-Grant, M. G., Steger, D. J., Reese, J. C., Yates, J. R. 3rd, and Workman, J. L. (1998). A subset of TAF(II)s are integral components of the SAGA complex required for nucleosome acetylation and transcriptional stimulation. Cell 94, 45–53. Grozinger, C. M., and Schreiber, S. L. (2000). Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14-3-3-dependent cellular localization. Proc. Natl. Acad. Sci. USA 97, 7835–7840. Gu, W., and Roeder, R. G. (1997). Activation of p53 sequence-speciﬁc DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595–606. Gusella, J. F., McNeil, S., Persichetti, F., Srinidhi, J., Novelletto, A., Bird, E., Faber, P., Vonsattel, J. P., Myers, R. H., and MacDonald, M. E. (1996). Huntington’s disease. Cold Spring Harb. Symp. Quant. Biol. 61, 615–626. Harlow, E., Whyte, P., Franza, B. R., Jr., and Schley, C. (1986). Association of adenovirus early-region 1A proteins with cellular polypeptides. Mol. Cell. Biol. 6, 1579–1589. Hassig, C. A., Fleischer, T. C., Billin, A. N., Schreiber, S. L., and Ayer, D. E. (1997). Histone deacetylase activity is required for full transcriptional repression by mSin3A. Cell 89, 341–347. Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J., Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., et al. (1997). A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387, 43–48.

Histone Acetylation and Cell Fate Control

61

Howe, L., Auston, D., Grant, P., John, S., Cook, R. G., Workman, J. L., and Pillus, L. (2001). Histone H3 speciﬁc acetyltransferases are essential for cell cycle progression. Genes Dev. 15, 3144–3154. Hung, H. L., Lau, J., Kim, A. Y., Weiss, M. J., and Blobel, G. A. (1999). CREB-Binding protein acetylates hematopoietic transcription factor GATA-1 at functionally important sites. Mol. Cell. Biol. 19, 3496–3505. Ikura, T., Ogryzko, V. V., Grigoriev, M., Groisman, R., Wang, J., Horikoshi, M., Scully, R., Qin, J., and Nakatani, Y. (2000). Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell 102, 463–473. Ito, T., Ikehara, T., Nakagawa, T., Kraus, W. L., and Muramatsu, M. (2000). p300-mediated acetylation facilitates the transfer of histone H2A-H2B dimers from nucleosomes to a histone chaperone. Genes Dev. 14, 1899–1907. John, S., Howe, L., Tafrov, S. T., Grant, P. A., Sternglanz, R., and Workman, J. L. (2000). The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)FACT complex. Genes Dev. 14, 1196–1208. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996). A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85, 403–414. Kamine, J., Elangovan, B., Subramanian, T., Coleman, D., and Chinnadurai, G. (1996). Identiﬁcation of a cellular protein that speciﬁcally interacts with the essential cysteine region of the HIV-1 Tat transactivator. Virology 216, 357–366. Kingston, R. E., and Narlikar, G. J. (1999). ATP-dependent remodeling and acetylation as regulators of chromatin ﬂuidity. Genes Dev. 13, 2339–2352. Kitabayashi, I., Aikawa, Y., Yokoyama, A., Hosoda, F., Nagai, M., Kakazu, N., Abe, T., and Ohki, M. (2001). Fusion of MOZ and p300 histone acetyltransferases in acute monocytic leukemia with a t(8;22)(p11;q13) chromosome translocation. Leukemia 15, 89–94. Krebs, J. E., Kuo, M. H., Allis, C. D., and Peterson, C. L. (1999). Cell cycle-regulated histone acetylation required for expression of the yeast HO gene. Genes Dev. 13, 1412–1421. Kuo, M. H., Brownell, J. E., Sobel, R. E., Ranalli, T. A., Cook, R. G., Edmondson, D. G., Roth, S. Y., and Allis, C. D. (1996). Transcription-linked acetylation by Gcn5p of histones H3 and H4 at speciﬁc lysines. Nature 383, 269–272. Laherty, C. D., Yang, W. M., Sun, J. M., Davie, J. R., Seto, E., and Eisenman, R. N. (1997). Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression. Cell 89, 349–356. Lin, R. J., and Evans, R. M. (2000). Acquisition of oncogenic potential by RAR chimeras in acute promyelocytic leukemia through formation of homodimers. Mol. Cell 5, 821–830. Lu, J., McKinsey, T. A., Nicol, R. L., and Olson, E. N. (2000b). Signal-dependent activation of the MEF2 transcription factor by dissociation from histone deacetylases. Proc. Natl. Acad. Sci. USA 97, 4070–4075. Lu, J., McKinsey, T. A., Zhang, C. L., and Olson, E. N. (2000a). Regulation of skeletal myogenesis by association of the MEF2 transcription factor with class II histone deacetylases. Mol. Cell 6, 233–244. MacRae, T. H. (1997). Tubulin post-translational modiﬁcations—enzymes and their mechanisms of action. Eur. J. Biochem. 244, 265–278. Mal, A., Sturniolo, M., Schiltz, R. L., Ghosh, M. K., and Harter, M. L. (2001). A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: Inhibition of the myogenic program. EMBO J. 20, 1739–1753. Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S. W., and Bates, G. P. (1996). Exon 1 of

62

Lehrmann et al.

the HD gene with an expanded CAG repeat is sufﬁcient to cause a progressive neurological phenotype in transgenic mice. Cell 87, 493–506. Martinez-Balbas, M. A., Bauer, U. M., Nielsen, S. J., Brehm, A., and Kouzarides, T. (2000). Regulation of E2F1 activity by acetylation. EMBO J. 19, 662–671. McArthur, G. A., Laherty, C. D., Queva, C., Hurlin, P. J., Loo, L., James, L., Grandori, C., Gallant, P., Shiio, Y., Hokanson, W. C., et al. (1998). The Mad protein family links transcriptional repression to cell differentiation. Cold Spring Harb. Symp. Quant. Biol. 63, 423–433. McCampbell, A., Taylor, J. P., Taye, A. A., Robitschek, J., Li, M., Walcott, J., Merry, D., Chai, Y., Paulson, H., Sobue, G., and Fischbeck, K. H. (2000). CREB-binding protein sequestration by expanded polyglutamine. Hum. Mol. Genet. 9, 2197–2202. McKinsey, T. A., Zhang, C. L., Lu, J., and Olson, E. N. (2000). Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation. Nature 408, 106–111. Meijsing, S. H., and Ehrenhofer-Murray, A. E. (2001). The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae. Genes Dev. 15, 3169–3182. Melnick, A., and Licht, J. D. (1999). Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 93, 3167–3215. Miller, I. J., and Bieker, J. J. (1993). A novel, erythroid cell-speciﬁc murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Mol. Cell. Biol. 13, 2776–2786. Miller, R. W., and Rubinstein, J. H. (1995). Tumors in Rubinstein–Taybi syndrome. Am. J. Med. Genet. 56, 112–115. Minucci, S., Maccarana, M., Cioce, M., De Luca, P., Gelmetti, V., Segalla, S., Di Croce, L., Giavara, S., Matteucci, C., Gobbi, A., et al. (2000). Oligomerization of RAR and AML1 transcription factors as a novel mechanism of oncogenic activation. Mol. Cell 5, 811–820. Minucci, S., and Pelicci, P. G. (1999). Retinoid receptors in health and disease: Co-regulators and the chromatin connection. Semin. Cell Dev. Biol. 10, 215–225. Miska, E. A., Karlsson, C., Langley, E., Nielsen, S. J., Pines, J., and Kouzarides, T. (1999). HDAC4 deacetylase associates with and represses the MEF2 transcription factor. EMBO J. 18, 5099–5107. Muraoka, M., Konishi, M., Kikuchi-Yanoshita, R., Tanaka, K., Shitara, N., Chong, J. M., Iwama, T., and Miyaki, M. (1996). p300 gene alterations in colorectal and gastric carcinomas. Oncogene 12, 1565–1569. Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S. L., and Evans, R. M. (1997). Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89, 373–380. Nakano, K., Mizuno, T., Sowa, Y., Orita, T., Yoshino, T., Okuyama, Y., Fujita, T., OhtaniFujita, N., Matsukawa, Y., Tokino, T., et al. (1997). Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line. J. Biol. Chem. 272, 22199–22206. Nielsen, S. J., Schneider, R., Bauer, U. M., Bannister, A. J., Morrison, A., O’Carroll, D., Firestein, R., Cleary, M., Jenuwein, T., Herrera, R. E., and Kouzarides, T. (2001). Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561–565. Noma, K., Allis, C. D., and Grewal, S. I. (2001). Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries. Science 293, 1150–1155. Nucifora, F. C., Jr., Sasaki, M., Peters, M. F., Huang, H., Cooper, J. K., Yamada, M., Takahashi, H., Tsuji, S., Troncoso, J., Dawson, V. L., et al. (2001). Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity. Science 291, 2423–2428. Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996). The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953–959.

Histone Acetylation and Cell Fate Control

63

Osada, S., Sutton, A., Muster, N., Brown, C. E., Yates, J. R. 3rd, Sternglanz, R., and Workman, J. L. (2001). The yeast SAS (something about silencing) protein complex contains a MYSTtype putative acetyltransferase and functions with chromatin assembly factor ASF1. Genes Dev. 15, 3155–3168. Perkins, N. D., Felzien, L. K., Betts, J. C., Leung, K., Beach, D. H., and Nabel, G. J. (1997). Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator. Science 275, 523–527. Petrij, F., Giles, R. H., Dauwerse, H. G., Saris, J. J., Hennekam, R. C., Masuno, M., Tommerup, N., van Ommen, G. J., Goodman, R. H., Peters, D. J., et al. (1995). Rubinstein– Taybi syndrome caused by mutations in the transcriptional co-activator CBP. Nature 376, 348–351. Polesskaya, A., Duquet, A., Naguibneva, I., Weise, C., Vervisch, A., Bengal, E., Hucho, F., Robin, P., and Harel-Bellan, A. (2000). CREB-binding protein/p300 activates MyoD by acetylation. J. Biol. Chem. 275, 34,359–34,364. Polesskaya, A., Naguibneva, I., Duquet, A., Bengal, E., Robin, P., and Harel-Bellan, A. (2001b). Interaction between acetylated MyoD and the bromodomain of CBP and/or p300. Mol. Cell. Biol. 21, 5312–5320. Polesskaya, A., Naguibneva, I., Fritsch, L., Duquet, A., Ait-Si-Ali, S., Robin, P., Vervisch, A., Pritchard, L. L., Cole, P., and Harel-Bellan, A. (2001a). CBP/p300 and muscle differentiation: no HAT, no muscle. EMBO J. 20, 6816–6825. Puri, P. L., Iezzi, S., Stiegler, P., Chen, T. T., Schiltz, R. L., Muscat, G. E., Giordano, A., Kedes L., Wang, J. Y., and Sartorelli, V. (2001). Class I histone deacetylases sequentially interact with MyoD and pRb during skeletal myogenesis. Mol. Cell 8, 885–897. Puri, P. L., Sartorelli, V., Yang, X. J., Hamamori, Y., Ogryzko, V. V., Howard, B. H., Kedes, L., Wang, J. Y., Graessmann, A., Nakatani, Y., and Levrero, M. (1997). Differential roles of p300 and PCAF acetyltransferases in muscle differentiation. Mol. Cell 1, 35–45. Reifsnyder, C., Lowell, J., Clarke, A., and Pillus, L. (1996). Yeast SAS silencing genes and human genes associated with AML and HIV-1 Tat interactions are homologous with acetyltransferases. Nat. Genet. 14, 42–49. Riou, P., Bex, F., and Gazzolo, L. (2000). The human T cell leukemia/lymphotropic virus type 1 Tax protein represses MyoD-dependent transcription by inhibiting MyoD-binding to the KIX domain of p300. A potential mechanism for Tax-mediated repression of the transcriptional activity of basic helix-loop-helix factors. J. Biol. Chem. 275, 10,551–10,560. Robertson, K. D., Ait-Si-Ali, S., Yokochi, T., Wade, P. A., Jones, P. L., and Wolffe, A. P. (2000). DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat. Genet. 25, 338–342. Sartorelli, V., Huang, J., Hamamori, Y., and Kedes, L. (1997). Molecular mechanisms of myogenic coactivation by p300: Direct interaction with the activation domain of MyoD and with the MADS box of MEF2C. Mol. Cell. Biol. 17, 1010–1026. Sartorelli, V., Puri, P. L., Hamamori, Y., Ogryzko, V., Chung, G., Nakatani, Y., Wang, J. Y., and Kedes, L. (1999). Acetylation of MyoD directed by PCAF is necessary for the execution of the muscle program. Mol. Cell 4, 725–734. Schiltz, R. L., and Nakatani, Y. (2000). The PCAF acetylase complex as a potential tumor suppressor. Biochim Biophys Acta 1470, M37–M53. Schreiber-Agus, N., Chin, L., Chen, K., Torres, R., Rao, G., Guida, P., Skoultchi, A. I., and DePinho, R. A. (1995). An amino-terminal domain of Mxi1 mediates anti-Myc oncogenic activity and interacts with a homolog of the yeast transcriptional repressor SIN3. Cell 80, 777–786. Smith, E. R., Eisen, A., Gu, W., Sattah, M., Pannuti, A., Zhou, J., Cook, R. G., Lucchesi, J. C., and Allis, C. D. (1998). ESA1 is a histone acetyltransferase that is essential for growth in yeast. Proc. Natl. Acad. Sci. USA 95, 3561–3565.

64

Lehrmann et al.

Smith, E. R., Pannuti, A., Gu, W., Steurnagel, A., Cook, R. G., Allis, C. D., and Lucchesi, J. C. (2000). The drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modiﬁcation linked to dosage compensation. Mol. Cell. Biol. 20, 312–318. Sobel, R. E., Cook, R. G., Perry, C. A., Annunziato, A. T., and Allis, C. D. (1995). Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4. Proc. Natl. Acad. Sci. USA 92, 1237–1241. Sobulo, O. M., Borrow, J., Tomek, R., Reshmi, S., Harden, A., Schlegelberger, B., Housman, D., Doggett, N. A., Rowley, J. D., and Zeleznik-Le, N. J. (1997). MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3). Proc. Natl. Acad. Sci. USA 94, 8732–8737. Soutoglou, E., Katrakili, N., and Talianidis, I. (2000). Acetylation regulates transcription factor activity at multiple levels. Mol. Cell 5, 745–751. Sowa, Y., Orita, T., Minamikawa, S., Nakano, K., Mizuno, T., Nomura, H., and Sakai, T. (1997). Histone deacetylase inhibitor activates the WAF1/Cip1 gene promoter through the Sp1 sites. Biochem. Biophys. Res. Commun. 241, 142–150. Steffan, J. S., Bodai, L., Pallos, J., Poelman, M., McCampbell, A., Apostol, B. L., Kazantsev, A., Schmidt, E., Zhu, Y. Z., Greenwald, M., et al. (2001). Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature 413, 739–743. Steffan, J. S., Kazantsev, A., Spasic-Boskovic, O., Greenwald, M., Zhu, Y. Z., Gohler, H., Wanker, E. E., Bates, G. P., Housman, D. E., and Thompson, L. M. (2000). The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc. Natl. Acad. Sci. USA 97, 6763–6768. Strahl, B. D., and Allis, C. D. (2000). The language of covalent histone modiﬁcations. Nature 403, 41–45. Tanaka, Y., Naruse, I., Maekawa, T., Masuya, H., Shiroishi, T., and Ishii, S. (1997). Abnormal skeletal patterning in embryos lacking a single Cbp allele: A partial similarity with Rubinstein–Taybi syndrome. Proc. Natl. Acad. Sci. USA 94, 10,215–10,220. Thomson, S., Clayton, A. L., and Mahadevan, L. C. (2001). Independent dynamic regulation of histone phosphorylation and acetylation during immediate-early gene induction. Mol. Cell 8, 1231–1241. Trouche, D., Cook, A., and Kouzarides, T. (1996). The CBP co-activator stimulates E2F1/DP1 activity. Nucleic Acids Res. 24, 4139–4145. Turner, B. M., Birley, A. J., and Lavender, J. (1992). Histone H4 isoforms acetylated at speciﬁc lysine residues deﬁne individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 69, 375–384. Wang, A. H., Kruhlak, M. J., Wu, J., Bertos, N. R., Vezmar, M., Posner, B. I., Bazett-Jones, D. P., and Yang, X. J. (2000). Regulation of histone deacetylase 4 by binding of 14-3-3 proteins. Mol. Cell. Biol. 20, 6904–6912. Wang, C., Fu, M., Mani, S., Wadler, S., Senderowicz, A. M., and Pestell, R. G. (2001). Histone acetylation and the cell-cycle in cancer. Front. Biosci. 6, D610–D629. Wang, L., Liu, L., and Berger, S. L. (1998). Critical residues for histone acetylation by Gcn5, functioning in Ada and SAGA complexes, are also required for transcriptional function in vivo. Genes Dev. 12, 640–653. Warrell, R. P., Jr., He, L. Z., Richon, V., Calleja, E., and Pandolﬁ, P. P. (1998). Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J. Natl. Cancer Inst. 90, 1621–1625. Wassarman, D. A., and Sauer, F. (2001). TAF(II)250: A transcription toolbox. J. Cell. Sci. 114, 2895–2902. Wharton, W., Savell, J., Cress, W. D., Seto, E., and Pledger, W. J. (2000). Inhibition of mitogenesis in Balb/c-3T3 cells by trichostatin A. Multiple alterations in the induction and activation of cyclin-cyclin-dependent kinase complexes. J. Biol. Chem. 275, 33,981–33,987.

Histone Acetylation and Cell Fate Control

65

Wittschieben, B. O., Otero, G., de Bizemont, T., Fellows, J., Erdjument-Bromage, H., Ohba, R., Li, Y., Allis, C. D., Tempst, P., and Svejstrup, J. Q. (1999). A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol. Cell 4, 123–128. Wu, J., Suka, N., Carlson, M., and Grunstein, M. (2001). TUP1 utilizes histone H3/H2B-speciﬁc HDA1 deacetylase to repress gene activity in yeast. Mol. Cell 7, 117–126. Yang, X. J., Ogryzko, V. V., Nishikawa, J., Howard, B. H., and Nakatani, Y. (1996). A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382, 319–324. Yao, T. P., Oh, S. P., Fuchs, M., Zhou, N. D., Ch’ng, L. E., Newsome, D., Bronson, R. T., Li, E., Livingston, D. M., and Eckner, R. (1998). Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell 93, 361–372. Yuan, W., Condorelli, G., Caruso, M., Felsani, A., and Giordano, A. (1996). Human p300 protein is a coactivator for the transcription factor MyoD. J. Biol. Chem. 271, 9009–9013. Zhang, H. S., Gavin, M., Dahiya, A., Postigo, A. A., Ma, D., Luo, R. X., Harbour, J. W., and Dean, D. C. (2000). Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF. Cell 101, 79–89. Zhang, W., and Bieker, J. J. (1998). Acetylation and modulation of erythroid Kruppel-like factor (EKLF) activity by interaction with histone acetyltransferases. Proc. Natl. Acad. Sci. USA 95, 9855–9860. Zhang, W., Kadam, S., Emerson, B. M., and Bieker, J. J. (2001). Site-speciﬁc acetylation by p300 or CREB binding protein regulates erythroid Kruppel-like factor transcriptional activity via its interaction with the SWI-SNF complex. Mol. Cell. Biol. 21, 2413–2422.

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Molecular Pathogenesis of Human Hepatocellular Carcinoma Michael A. Kern, Kai Breuhahn, and Peter Schirmacher∗ Institute of Pathology, University of Cologne, D-50931 Cologne, Germany

I. Introduction II. Morphology of Human Hepatocarcinogenesis III. Molecular Etiology A. General (Nonspeciﬁc) Mechanisms B. Hepatitis B Virus C. Hepatitis C Virus D. Other Causes IV. Host Carcinogenic Events A. Genomic Alterations B. Alterations in Speciﬁc Tumor-Relevant Host Genes C. Oncogenic Molecular Cross-Talk V. Functional Consequences A. Cell Growth B. Neoangiogenesis C. Invasion and Metastasis VI. Therapeutic Implications References

I. INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most frequent human cancers. Depending on the type of statistic, it is the ﬁfth to seventh most frequent malignancy worldwide (Bosch, 1997). It is most predominant in subsaharan Africa and far eastern Asia, but its presence is not limited to developing countries; it is also high in some industrialized countries, such as Japan. In Europe there is a strong north-to-south gradient, with relatively high incidence in Spain, Italy, and Greece. The incidence appears to be rising, even in countries with low HCC-frequencies. In more than 80% of the cases, a ∗ Address correspondence to Peter Schirmacher, M.D., Institute of Pathology, University of Cologne, Joseph-Stelzmann-Str. 9, D-50931 Cologne, Germany; Tel: +49-221-478-5257; Fax: +49-221-478-6360; E-mail: [email protected]

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Table I Etiology of Human HCC

High frequency Moderate frequency Low frequency

High risk

Low risk

Chronic HBV infection Chronic HCV infection (stage 4) Haemochromatosis (stage 3) [Aﬂatoxin B1] Glycogen-storage disease (type1) Hereditary tyrosinemia

Chronic alcohol abuse “Healed” HBV infection

Wilson’s disease α1–AT-deﬁciency Chemicals (vinyl chloride, Thorotrast)

well-deﬁned etiology causes chronic liver disease and leads to the development of HCC (Table I). In the western world, where infections with hepatitis viruses are usually obtained during adulthood, 70–80% of the HCCs are accompanied by complete cirrhosis in the nontumorous liver. In areas with endemic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, the time point of infection is frequently around birth in a state of immunological tolerance and therefore the cirrhosis rate in these HCC patients is frequently below 50%. Overall, the geographic distribution of HCCs matches well the prevalence of chronic hepatitis virus infections (Szmuness, 1978). Clinical diagnosis of HCC is difﬁcult, especially in early stages (<2 cm) that are well accessible to curative treatment. No reliable serum markers exist, especially since α-fetoprotein concentrations are nondiagnostic in most early HCCs (Ebara, 1997). Also radiologic diagnosis of HCC is cumbersome on the background of cirrhosis. Small HCCs frequently escape detection even in pretransplantation settings (Mion et al., 1996), and in clinical experience, the size and extension (angioinvasion, intrahepatic spread) is frequently underestimated during preoperative workup. Even pathohistological evaluation is challenging, since early HCCs are highly differentiated in most cases and pose signiﬁcant problems in biopsy diagnosis (Kondo, 1997). The therapeutic options for HCC are sobering, even when compared with other solid malignant tumors. Only partial hepatic resection or liver transplantation offer potential cure, but only a minority of cases is amenable to surgical treatment for various reasons (cirrhosis, intrahepatic tumor spread) (Bathe et al., 1997; Rolles, 1997). Even among surgically treated patients, 5-year survival rates are low, and thus enthusiasm for liver transplantation in HCC patients has faded. There is no curative systemic therapy and response rates to chemotherapy (Okada, 1997), radiation (Osuga et al., 1997), or immunotherapy (Shouval, 1997) are very low (usually below 10%, often including partial remission). Thus even palliative systemic treatment is not recommended currently. Local palliation with ethanol injections or

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thermoablation may offer some symptomatic relief and minor beneﬁts in terms of survival (Livraghi, 1997). Overall, there is a great need for efﬁcient therapies as well as early and sensitive diagnostic markers of HCC based on an improved understanding of the molecular mechanisms of human hepatocarcinogenesis. There are few tumors that offer better perspectives for experimental evaluation. Liver as an organ and hepatocellular cell lines are the canonical human systems in biochemistry and molecular biology and many cell culture studies, and an impressive number of different animal models provide a solid basis for speciﬁc analyses of the tumorigenic mechanisms in vivo. This review focuses on the compilation of molecular and functional changes identiﬁed in human HCCs and their interrelation and impact on tumor formation and therapy. It does not cover the complex interaction of the tumor with the nonneoplastic environment including the host immune response. It furthermore passes over a large body of experimental data obtained in animal models, such as transgenic mouse lines, chemical rodent hepatocarcinogenesis and models of virus-induced liver cancer. Since the research in human molecular hepatocarcinogenesis has almost exploded, we apologize to those colleagues whose work may not be adequately represented for reasons of comprehensibility.

II. MORPHOLOGY OF HUMAN HEPATOCARCINOGENESIS Stepwise morphological hepatocarcinogenesis has been deﬁned and studied extensively in various rodent models for decades (Schirmacher et al., 1991; Pitot, 1994; Sandgren, 1995; Farber, 1996; Grisham, 1997; Bannasch ¨ and Schroder, 2002), but in contrast to other tumorigenic processes, premalignant hepatocellular lesions have evaded detection and deﬁnition for a long time. Due to increasing experience with resected and explanted liver specimens and improved diagnostic procedures, these premalignant liver lesions are well known now, and a uniﬁed histological concept of nodular hepatic lesions including premalignant lesions (International Working Party, 1995) has been largely adopted worldwide. It is well accepted that human hepatocarcinogenesis represents a stepwise process (Fig. 1), including different premalignant stages, correlating well with the fact that typically many years, even decades, are required from the onset of a chronic liver disease until the existence of an HCC. Human hepatocarcinogenesis usually develops on the histological background of a chronic liver disease (e.g., chronic hepatitis, chronic alcoholic liver disease, hemochromatosis, stage 3; compare Table I). Dysplastic foci

Fig. 1 Schematic representation of human morphological hepatocarcinogenesis.

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(DFs) and dysplastic nodules (DN, syn. adenomatous hyperplasia) are regarded as premalignant precursors preceding the development of HCC (International Working Party, 1995; Hirohashi et al., 2000). Furthermore, several authors have speciﬁcally distinguished “early” HCCs, smaller than 2 cm in diameter, from other fully developed HCCs (Kondo, 1997). At this early stage, the tumor is usually highly differentiated, slowly growing, noninvasive, and often not even encapsulated. It is usually resectable and exhibits a much better prognosis. Although these different lesions are reasonably well deﬁned, human hepatocarcinogenesis represents rather a continuum, and a clear cut classiﬁcation of a given lesion may be impossible in certain cases (Fig. 1). The earliest known premalignant lesions are DFs. These are subacinar (<0.1 cm diameter), clonal appearing, coherent hepatocellular proliferations, which often show signs of expansive growth (concentric expansion, sometimes with mild compression of adjacent cells). There has been some debate as to whether large cell dysplasia (groups of large atypical cells) (Anthony, 1976) or small cell dysplasia (Watanabe et al., 1983) are the relevant premalignant lesions. Meanwhile, the overwhelming cytological and molecular evidence points toward the latter (Zhao and Zimmermann, 1998; Marchio et al., 2001), whereas large cell dysplasia is likely to represent a regression phenomenon not directly involved in tumorigenesis. When DFs grow and exceed 0.1 cm in diameter they are termed DNs. These hepatocellular lesions show a clonal growth pattern and are visible to the naked eye. By histology they exhibit some of the features present in HCCs (increased nuclear– cytoplasmic ratio, cytoplasmic basophilia, mild trabecular disarray), but none of the deﬁnite signs of malignant transformation (see below) (International Working Party, 1995). DNs may accompany HCCs in resection specimens, but sometimes are found in noncancerous explantation or autopsy specimens. Occasionally, formations of a DN are still preserved at the margin of a HCC. Clonality of DNs is well supported by molecular data (Aihara et al., 1996) and in selected cases, a precursor–follower relationship between DN and HCC has been proven by the analysis of clonal HBV-integrations (Tsuda et al., 1988). Open questions remain as to whether premalignant liver lesions are obligatory precancerous or whether they may partly undergo regression or remodeling and which lesions are at higher or lower risk for malignant transformation (Terada et al., 1993). The point of no return has to be deﬁned better, most likely with the aid of molecular markers. In a minority of cases, this outlined stepwise HCC-development may be bypassed. Few HCCs, especially in older patients, develop in otherwise normal livers. Furthermore hepatocellular adenoma, a lesion which is promoted by gonadal and anabolic steroids and found predominantly in younger females, has a very low but recognizable rate of malignant transformation.

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Deﬁnite morphological signs of malignancy in HCC are vascular or interstitial invasion, gross trabecular disarray, and intra- or extrahepatic metastasis, but complete malignant transformation at the cellular level may precede these overt histological signs. The biology and prognosis of hepatocellular carcinoma signiﬁcantly correlates with tumor stage and grade, but currently there is no evidence that tumor behavior is predictable from the macroscopic growth pattern, microscopic subtype, or any of the available immunohistological markers. Only ﬁbrolamellar HCC, which is a rare, stroma-rich, oncocytic variant occurring predominantly in younger patients, seems to have a better prognosis.

III. MOLECULAR ETIOLOGY A. General (Nonspeciﬁc) Mechanisms Most HCCs are caused by chronic liver diseases that exhibit some etiologyspeciﬁc protumorigenic interactions between the causative agent and liver cells (Fig. 2). In addition, mechanisms exist that induce and promote tumor growth independent of the underlying etiology and that are based on the continuous presence of chronic liver damage (Chisari et al., 1989). Chronic viral hepatitis as well as chronic alcoholic liver disease and later stages of hemochromatosis lead to persistently increased hepatocellular death

inactivation of tumor suppressor genes (e.g.,, p53, IGF-IIR, E-cadherin, RB-1)

activation of protooncogenes (e.g.,, β-catenin, MET, cyclins)

Fig. 2 Mechanisms of virus-induced human hepatocarcinogenesis.

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rates. In consequence, steady-state levels of growth-promoting cytokines are increased and lead to elevated hepatocellular proliferation (regeneration). A higher proliferation rate statistically increases the total number of replication errors and mutations, including those in tumor-relevant genes. Additionally, chronic liver disease may also promote tumor progression. Frequently, tumor cells have developed antiapoptotic mechanisms that eliminate physiological growth restriction or have lost permissiveness for viral infection (e.g., in HBV infection) (Raimondo et al., 1988) or iron storage (in hemochromatosis) (Deugnier et al., 1993), but may still carry intact growth signaling pathways. Under these conditions, they will recruit increased paracrine growth factor concentrations without succumbing to elevated cell death rates as with the surrounding nontumorous cells. This state of growth factor dependence is still preserved in many fully transformed HCC cell lines, although these tumor cells usually secrete extreme concentrations of growth factors, like insulin-like growth factor (IGF)-II or transforming growth factor (TGF)α, leading to autocrine stimulation. Still, it has to be determined how and at what stage of hepatocarcinogenesis premalignant liver cells or tumor cells switch from paracrine to autocrine stimulation and by which mechanisms they lose permissiveness to the pathogenic agents (e.g., virus or iron).

B. Hepatitis B Virus HBV, the prototype member of the class of hepadnaviridae, is a small, enveloped virus with a compact, partially double-stranded DNA genome of roughly 3 kbp. In about 10% of the cases, HBV infection develops into a chronic hepatitis. There is overwhelming epidemiological evidence connecting chronic hepatitis B with the development of HCC (Beasley et al., 1981). For example, patients with chronic HBV infection carry a more than 100-fold increased risk for developing a HCC compared to uninfected persons. This dramatic difference cannot be explained sufﬁciently by the nonspeciﬁc mechanisms described above, especially since inﬂammatory activity has frequently subsided in livers carrying premalignant lesions or HCCs (Raimondo et al., 1988). Intense research over more than two decades has characterized two major HBV-speciﬁc mechanisms that contribute to HCC development. One mechanism resides on the mode of HBV replication, especially its inherent potential of chromosomal integration; the other mechanism is based on the expression of transactivating factors derived from the viral genome. Hepadnaviruses exhibit a unique replication pathway that turns the retroviral life cycle upside down (for reviews, see Ganem and Varmus, 1987; Nassal, 2000). The viral genome is a partially double-stranded circular DNA that is completed once it enters the hepatocyte nucleus and gives rise to an RNA pregenome (intermediate). This RNA pregenome is packaged and

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represents the matrix for ﬁrst-strand DNA synthesis, which itself is the template for incomplete second-strand synthesis. In contrast to retroviruses, competent HBV replication is purely episomal. Nevertheless, chromosomal integrations of HBV DNA do occur and in vitro experiments using fetal hepatocytes have generated chromosomal integrations within several hours (Ochiya et al., 1989; Okubo et al., 1990). HBV integrations seem to occur randomly in terms of chromosomes or genomic loci involved and do not support competent viral replication. They may be colinear or highly rearranged, but in the overwhelming majority of integrations at least one end is located in the cohesive overlap of the viral genome that is ﬂanked by the 5 ends of the viral (+) and (−) strands (Nagaya et al., 1987; Shih et al., 1987). HBV integrations are generated by nonhomologous recombination events, potentially facilitated by small stretches of sequence homology or speciﬁc local characteristics of the chromosomal integration site (Schirmacher et al., 1995). The HBV genome does not code for an integrase, but cellular topoisomerase I is able to generate in vitro recombinations of hepadnaviral DNA with chromosomal DNA that closely resemble the type and structure of in vivo integrations, and is therefore a candidate enzyme supporting the integration process (Wang and Rogler, 1991). How do HBV integrations contribute to hepatocarcinogenesis? HBV integrations have a general destabilizing effect for the host genome (Hino et al., 1991; Luber et al., 1996), although the responsible mechanisms are unknown. Furthermore, it has been known for more than two decades that HBV integrations may become clonally propagated in HCCs (Brechot et al., 1980; Chakraborty et al., 1980). None of these integrations are replication competent, and further research has been hampered by the fact that tumor cells often carry many different integrations, of which many may be irrelevant (Nagaya et al., 1987). But some cases have unequivocally been demonstrated where HBV integrations have activated a tumor-relevant gene [epidermal growth factor receptor (EGFR)-homolog, cyclin A, retinoic receptor γ , mevalonate kinase] by promoter/enhancer insertion (Dejean et al., 1986; Wang et al., 1990; Graef et al., 1994). Furthermore, HBV integrations have been found in tumor suppressor loci or close by (Pasquinelli et al., 1988; Buetow et al., 1989; Slagle et al., 1991), suggesting that inactivating mutations may have contributed to the loss of tumor suppressor gene function. In addition, the HBV genome encodes two different transactivating factors, the HBxAg and the preS/SAg, which can both be expressed from episomal viral DNA (e.g., in chronic hepatitis) as well as from integrated viral sequences or viral–host fusion sequences (mainly in HCCs) and thus may lead to constitutive protein expression derived from the integrated state despite the lack of competent HBV replication (Wollersheim et al., 1988; Zahm et al., 1988; Caselmann et al., 1990; Kekule et al., 1990; Takada and Koike, 1990;

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¨ Schluter, 1994; Hildt et al., 1995). Both polypeptides encode transcriptional transactivating factors that not only act during viral replication and maturation but also modify the expression of a broad range of host genes (Wu et al., 2001). Both transactivators do not bind DNA directly, but a bewildering complexity of changes in gene expression, intracellular signaling, and cellular functional responses have been reported. For example, HBx has been described to disrupt adherens junctions in a src-dependent manner and induce a migratory phenotype (Lara-Pezzi et al., 2001a,b), inhibit nucleotide excision repair (Becker et al., 1998; Jia et al., 1999), or inhibit apoptosis via interference with p53 (Huo et al., 2001), PI-3K/Akt/Bad (Lee et al., 2001b), caspase-3 (Gottlob et al., 1998), or activation of growth factors (Lee et al., 1998). Other potentially protumorigenic interactions with Ras, c-fos, c-src, JNK, Stat, NFκB, p21/WAF-1, and CREB/ATF have also been reported (Colgrove et al., 1989; Kekule et al., 1990, 1993; Mahe et al., 1991; Avantaggiati et al., 1993; Park et al., 2000a; Bouchard et al., 2001). This has led to the suggestion that HBx acts in a manner comparable to phorbol esters as a tumor promoter (Cross et al., 1993; Kekule et al., 1993). Recent data suggest that HBx effects on viral replication and host gene transcription are connected with increased cytosolic calcium concentrations and stimulation of the tyrosine kinase Pyk2 and subsequently src (Bouchard et al., 2001). Whether this pathway sufﬁciently explains the broad range of activity of HBx remains to be determined. Furthermore, it will have to be tested whether structural mutations of HBx, which are frequent in HCCs (Poussin et al., 1999), may also generate polypeptides with different functions.

C. Hepatitis C Virus Hepatitis C Virus (HCV) is an enveloped Flavivirus with a single-stranded RNA genome of about 9 kb (Major and Feinstone, 1997; Lohmann et al., 2000). HCV infections are a dramatic worldwide problem. Meanwhile, some countries show prevalence rates of up to 20% and numbers are rising. The inﬂammatory activity and thus the course of chronic HCV infection is frequently mild, but the chronicity rate (∼80%) is much higher compared to HBV and approximately 30% of patients with chronic HCV infection experience cirrhosis. Although the HCC risk seems to be somewhat lower, HCV has replaced HBV as the major etiology of HCC in many developed countries (Deufﬁc et al., 1999; Kubicka et al., 2000). The HCV RNA genome not only gives rise to viral replication but also serves translation, generating a precursor polypeptide from a single large open reading frame. This polypeptide undergoes (autocatalytic) cleavage generating the different structural and nonstructural (NS) viral proteins. In contrast to HBV, HCV

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replication does not involve DNA intermediates and does not generate detectable chromosomal integrations. Studies on the oncogenic potential of HCV have focused mainly on two genes, the core gene and the nonstructural protein NS5A (Wands, 2000). NS5A is thought to be a transcriptional activator (Kato et al., 1997), which may act indirectly via the transcription factor SRCAP (Ghosh et al., 2000b). It may promote cell growth also by targeting p53– and cdk–cyclin complexes (Arima et al., 2001; Majumder et al., 2001) or protecting against tumor necrosis factor (TNF)α-mediated apoptosis (Ghosh et al., 2000a). HCV core induces liver carcinogenesis in transgenic mice (Moriya et al., 1998), but experimental evidence for the protumorigenic role of HCV-core in human cells is not conclusive at all. It has been described to act on p53 and p21/WAF-1 expression (Ray et al., 1997, 1998b) and to functionally modulate MAPK (Tsuchihara et al., 1999), p53 (Lu et al., 1999; Otsuka et al., 2000), TNFα signaling (Ray et al., 1998a; Zhu et al., 1998, 2001), 14-3-3 protein (Aoki et al., 2000), and MYC (Terradillos et al., 1997; Su et al., 2001a), but the data are highly contradictory. It is even unclear whether HCV-core promotes cell death or protects against it (Ruggieri et al., 1997; Ray et al., 1998a; Zhu et al., 1998, 2001; Dumoulin et al., 1999; Machida et al., 2001), and whether it signiﬁcantly contributes to transformation in vitro or not (Ray et al., 1996, 2000; Chang et al., 1998). Furthermore, it is unknown whether core polypeptides remain solely cytoplasmic or enter the nucleus. While there is no indication for nuclear localization of full-length HCV-core (Barba et al., 1997), C-terminal truncation of the core polypeptide may unmask N-terminal nuclear localization signals and induce nuclear translocation (Chang et al., 1994; Suzuki et al., 1995). Truncated core polypeptides have been observed in HCCs (Ruster et al., 1996). Currently the major problem of these studies resides in the fact that most of the data have been obtained in different heterologous cell lines using different artiﬁcial constructs. They may therefore reﬂect cell type-speciﬁc differences or experimental artifacts. Thus, future studies regarding the oncogenic potential of HCV await expression systems (Huang et al., 2001) and models that resemble more closely the in vivo situation.

D. Other Causes Aﬂatoxin B1 is produced by the fungus Aspergillus ﬂavus and represents one of the major food-contaminating toxins, and is a severe hygienic problem in tropical third-world countries. Aﬂatoxin B1 is an experimental hepatocarcinogen and there is epidemiological evidence connecting high aﬂatoxin B1 exposure with an increased HCC risk (Wogan, 1992). Its tumorigenic

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mechanisms in the liver has been linked to somatic G-T transversions in codon 249 of the p53 gene that were found to a signiﬁcant percentage exclusively in HCCs from countries with high aﬂatoxin exposure (Aguilar et al., 1993, 1994; Ozturk, 1999). These mutations represent a paradigm for molecular epidemiology in carcinogenesis and a direct mechanistic link between the responsible human carcinogen and a known protumorigenic mechanism. Nevertheless, it is currently unclear why only codon 249 of the p53 gene is preferred and whether other tumor-relevant genes are also affected (Chao et al., 1999). Patients with genetic hemochromatosis at stage 3 (cirrhosis) carry a 20–25% risk for developing an HCC (Niederau et al., 1985). In contrast, other genetic diseases, like Wilson’s disease, that equally lead to cirrhosis do not show a comparable frequency of HCCs. Therefore, it has been assumed that speciﬁcally high intracellular iron concentrations are oncogenic. Indeed, increased intracellular iron levels appear to generate higher concentrations of free oxidative radicals that result in oxidative stress. The mutagenic potential of oxidative radicals has been clearly demonstrated in experimental systems and the increased carcinogenic potential is likely to be a consequence of either direct oxidative DNA damage or the activation of reducing and oxy-stress protective signaling pathways (Toyokuni, 1996). Thus, after some lag period, elimination of hepatocellular iron reduces the tumor risk in hemochromatosis patients.

IV. HOST CARCINOGENIC EVENTS A. Genomic Alterations Currently there is only little evidence supporting germline predisposition for HCC development. None of the known inherited tumor syndromes includes HCC. There have been, however, a few reports of HCCs in patients with familial polyposis coli (FAP) (Zeze et al., 1983; Gruner et al., 1998) or some germline mutations of the p16 gene in HCC patients (Chaubert et al., 1997). Studies of genomic polymorphisms that may lead to an increased risk for HCC development have not gained much success so far. UGF-1A7 polymorphisms have been reported to correlate with HCC risk (Vogel et al., 2001), but other analyses, involving the cytochrome P450 2E1, glutathioneS-transferase M1/T1, and microsomal epoxide hydrolase loci have failed to detect signiﬁcant correlations (Lee et al., 1997; Yu et al., 1999a; Wong et al., 2000c). A contribution of the codon 72 polymorphism in the p53 gene in cooperation with other risk factors, like viral hepatitis, has recently been suggested (Yu et al., 1999b).

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Studies of the overall somatic genomic alterations in HCCs are based on either comparative genomic hybridization (CGH) or loss of heterozygosity (LOH) analyses. CGH has been performed in numerous studies, analyzing more than 600 HCCs (Kusano et al., 1999; Qin et al., 1999; Sakakura et al., 1999; Wong et al., 1999, 2000b; Guan et al., 2000; Marchio et al., 1997, 2000; Tornillo et al., 2000; Balsara et al., 2001; Collonge-Rame et al., 2001; Niketeghad et al., 2001; Rao et al., 2001; Shiraishi et al., 2001; Wang et al., 2001a; Wilkens et al., 2001). The different studies are largely consistent and comparable and generate a clear picture of the frequent large-scale genomic alterations in HCCs: the most frequent gains involve 1q(24-25; 45–80%) and 8q(21-24; 30–80%), while losses affect predominantely 17p(13; 35–65%), 4q(12-22; 30–50%), 16q(21-23; 30–50%), and 13q (13-14; 20–40%). Additionally, a few high-level ampliﬁcations were assigned to 11q13. Interestingly, losses at 4q, 17p, and 16q predominate in HBV-induced HCCs, suggesting etiology-speciﬁc alterations of the host genome and thus gene expression. To most of these chromosomal locations, speciﬁc tumor-relevant genes have been assigned, like p53 (17p), RB-1 (13q), E-cadherin (16q), COX-2/cPLA2 (1q), and c-myc (8q). However, the long-time suspected tumor suppressor at 4q (Pasquinelli et al., 1988; Buetow et al., 1989), which appears to be preferentially HBV related, has not been identiﬁed so far. Interestingly, clonally propagated macroscopic genomic imbalances, predominantly at 1q, have also been found in DNs although at lower frequencies (Zondervan et al., 2000), further supporting their premalignant nature and suggesting that genomic macroimbalances precede malignant transformation. In contrast, losses at 8p appear to be associated with HCC progression (Qin et al., 1999). The disadvantage of these studies is their low resolution (∼1 Mb) and the fact that so far only genomic imbalances, but not balanced chromosomal mutations, have been analyzed. Further information can be expected from multicolor ﬂuorescent in situ hybridization (FISH) analyses, which may also unravel balanced mutations, and from high-density chip-CGHs. A different manner of genome-wide analyses are LOH-studies. Their restriction is that only genomic losses can be reliably detected and that the different studies are hardly comparable, for example due to densities and types of markers utilized. Consequently, the results are highly variable. Comprehensive allelotype studies in HCCs (Nagai et al., 1997; Piao et al., 1998; Okabe et al., 2000; Laurent-Puig et al., 2001) basically agree on highfrequency LOHs at 1p, 4q, 6q, 8p, 13q, and 17p. Respective mapping approaches have narrowed down these regions to 1p36 (Yeh et al., 1994; Fang et al., 2000, 2001), 4q35 (Bando et al., 1999), 6q23 (Koyama et al., 2000), and 8p21-22 (Pineau et al., 1999). Furthermore, losses have been described at the RB-1 locus at a frequency of 20–50% and at the p53 locus (17p13) in 10–60% of the HCCs (Ozturk, 1999).

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B. Alterations in Speciﬁc Tumor-Relevant Host Genes The activity of host oncogenic proteins has also been studied in animal models, especially in chemically induced rodent liver carcinogenesis and also in the woodchuck hepadnaviral model. But these data do not match the human situation well. Ras protein activation, especially due to codon 12 and 13 mutations, which is frequent in rodent chemical carcinogenesis (Funato et al., 1987; Sinha et al., 1988), does not play a signiﬁcant role in human HCCs (Tada et al., 1990; Chao et al., 1999). Also the frequent activation of Nmyc or the Nmyc2 pseudogene in woodchucks due to WHV integration (Fourel et al., 1990, 1994) does not have its human counterpart, not even in HBV-induced HCCs. Beside characteristic aﬂatoxin B1- and vinyl chloride (VC)-typical mutations, there is also strong evidence for virus-speciﬁc molecular alterations of host genes in human HCCs. For example, speciﬁc genomic imbalances occur at different frequencies in HBV- versus HCV-induced HCCs and activation of β-catenin occurs more frequently in HCV-induced HCCs. Novel data about this important topic as well as many new molecular markers and targets have to be expected from ongoing large-scale expression arrays (Okabe et al., 2001; Xu et al., 2001). Further studies will also have to reﬂect that not all oncogenic events are well represented in expression screening, since they may depend, for example, on point mutations (ras genes, p53), protein stabilization (β-catenin), or distinct topographic and subcellular localization (e.g., E-cadherin, β-catenin).

1. p53 More than 50% of human primary tumors contain loss of function mutations in the p53 tumor suppressor gene, which codes for a central regulatory protein in cell cycle arrest, DNA repair, and induction of apoptosis after genotoxic stress (Levine, 1997; Sheikh and Fornace, 2000). Inactivation of the transcriptional regulatory function of p53 by point mutations, deletion, or interacting viral proteins enables cancer cells to proliferate and escape apoptotic stimuli. Moreover, gain of function mutations in the p53 gene may lead to tumor promotion by increased mitosis, cell survival, and angiogenesis (Sigal and Rotter, 2000; Cadwell and Zambetti, 2001). In human hepatocarcinogenesis, viral proteins with transforming activity (e.g., HBxAg) bind wild-type p53 early during tumor formation (Thomas et al., 1996; Feitelson, 1998). Furthermore, aﬂatoxin B1-speciﬁc G-T mutations in codon 249 represent about 1/3 of the p53 mutations recognized in HCCs so far and are speciﬁc for geographic areas with intense food contamination with A. ﬂavus (Ozturk, 1999). Other inactivating p53 mutations typically arise during progression in about 20–40% of HCCs (Hsu et al.,

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1991; Nishida et al., 1993), suggesting that this type of mutational p53 inactivation is a rather late event in multistage hepatocarcinogenesis. Inactivating p53 gene mutations lead to a fault protein with a prolonged half-life, resulting in strong nuclear accumulation of p53 (Hsia et al., 2000). Mutational (Wang et al., 2000) and physical (Giannoudis and Herrington, 2000) loss of wild-type function of p53 correlates with changes in p53-target gene expression (e.g., mdm-2, p21, Cyclin G, bax, IGF-BP3) (Buckbinder et al., 1995; Levine, 1997) resulting in hepatocellular growth, decreased apoptosis, genomic instability, reduced DNA repair, and further accumulation of gene mutations. During HCC-development, mutated p53 has also been described to supply an oncogenic gain of function by leading to transcriptional activation of genes involved in mitosis and cell survival (Lee et al., 2000). Elevated levels of mutated nuclear p53 protein correlate with larger tumor size, poorer differentiation (Hayashi et al., 1995), higher proliferation (Itoh et al., 2000), and increased invasiveness of HCCs (Qiu et al., 1998) and a poor prognosis in HCC patients (Heinze et al., 1999). In fact, increased levels of negative regulators of p53 (MDM2) were also observed in HCCs with wild-type p53, leading to the conclusion that mutations in the p53 gene are not the only way of p53 inactivation (Qiu et al., 1998). MDM2 overexpression signiﬁcantly correlates with small HCC size, but also with poorer prognosis of patients (Endo et al., 2000). p73, which is structurally and functionally related to p53, was not found to be mutated in HCCs. Nevertheless, p73 overexpression correlates with p53 inactivation and a poorer prognosis, suggesting a tumor promoter function (Tannapfel et al., 1999; Herath et al., 2000; Sayan et al., 2001).

2. CELL CYCLE REGULATORY PROTEINS Key regulators of the cell cycle such as cyclins, cyclin dependent kinases (cdks), and cdk inhibitors, are frequently mutated or aberrantly expressed in HCCs. During late G1 phase, cyclin D1 promotes the hyperphosphorylation of the retinoblastoma protein (RB-1) in conjunction with cdk4/6, resulting in E2F release followed by transcriptional activation of S-phase relevant gene products. Cyclin D1 ampliﬁcation and overexpression occurs in about 10–20% of the HCCs, usually in the presence of intact RB-1, and results in relaxed cell cycle control and correlates with poorer differentiation, microvascular invasion, and large tumor size (Zhang et al., 1993; Nishida et al., 1994; Choi et al., 2001). Overexpression of cyclin D1, however, has occasionally been described in dysplastic nodules and in nonneoplastic hepatocytes (Joo et al., 2001). Cyclin E, which is responsible for guiding cells through the late G1–S transition, is also strongly

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expressed in most HCCs (Kohzato et al., 2001), as is cyclin A. Nevertheless, only increased cyclin E but not cyclin A concentrations correlate with enhanced RB-1 phosphorylation and increased mitotic indices (Ohashi et al., 2001), although in one HCC case, a singular HBV integration led to constitutively increased cyclin A levels (Wang et al., 1990). However, increased cyclin A concentrations have been linked to a worse prognosis in HCC patients (Chao et al., 1998). Reactive antibodies against cyclin B1 and a cyclin B1/glutathione-S-transferase fusion protein were found in 15% of HCC patients, suggesting aberrant stimulation of the immune system (Covini et al., 1997). During cell cycle, cdk4 activity is linked to cyclin presence, since cyclin D forms active complexes with cdk4 to release RB-1 bound transcription factors, promoting the initiation of replication. Dysregulation of RB-1 function, induced cdk4, and enhanced p15/p16 activity have been described frequently in HCCs (Murakami et al., 1991; Hui et al., 2000; Wong et al., 2000a; Azechi et al., 2001) and may lead to aberrant expression of target genes (e.g., c-myc and cyclin A) resulting in enhanced cell proliferation. The loss of RB-1 gene expression correlated with LOH of the RB-1 gene locus in nearly all HCC cases, while tumor-speciﬁc mutations were seldom shown (Zhang et al., 1994). Interestingly, gankyrin, which binds RB-1 as well as the 26S proteasome, was activated in all HCCs investigated in one study. Gankyrin accelerates RB-1 degradation and is able to transform NIH3T3 cells (Higashitsuji et al., 2000). Reduced RB-1 activity may also contribute to hepatocarcinogenesis, since absence as well as overexpression of RB-1 levels were both associated with poorly differentiated tumors and metastasis (Hui et al., 1999). So far, functional inactivation of other RB family members (p107 and p130) in hepatic tumors has not been observed (Paggi and Giordano, 2001). The cyclin D/cdk4/cdk6 inhibitor p16 (INK4A) is also a major cell cycle protagonist involved in HCC development. Overall, genetic or epigenetic inactivation of p16, including promoter hypermethylation, point mutations, and deletions, occurs in 50–70% of the HCCs leading to the loss of p16-dependent cell cycle arrest (Kim et al., 1998a; Liew et al., 1999; Matsuda et al., 1999; Esteller et al., 2001; Kondoh et al., 2001). Furthermore, absence of p16 is associated with poorer differentiation of HCCs (Hui et al., 2000). The rate of p16 losses is doubled in metastatic lesions compared with primary HCCs, suggesting that p16 mutations accumulate during HCC progression. Combined with other inhibitors (p21 and p27), nearly 90% of all HCCs exhibit at least one mutation in a cdk inhibitor (Hui et al., 1999). Thus, different alterations at all levels of the cell cycle control suggest that constitutive dysregulation of cell cycle control is obligatory during HCC development.

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3. E-CADHERIN/wnt/β-CATENIN Genetic (Kondoh et al., 2001) as well as epigenetic (Esteller et al., 2001) alterations are frequently responsible for the loss of E-cadherin gene (CDH1) function, an intercellular adhesion molecule, in HCCs (Matsumura et al., 2001). Loss of E-cadherin expression in HCCs correlates with intrahepatic metastasis (Osada et al., 1996), large tumor size, dedifferentiation (Kozyraki et al., 1996), and has been frequently found in the macrotrabecular subtype (Ihara et al., 1996). Furthermore, the cytoplasmic part of E-cadherin interacts with several proteins including β-catenin. Thus, loss of E-cadherin expression is seen as a prerequisite for reduced membranous β-catenin and its potential to translocate into the nucleus. Besides its role as an interaction partner of E-cadherin, β-catenin is a crucial protagonist of the wnt signal transduction pathway. Wnt (wingless) glycoproteins recognize the fzr receptors (frizzled), which leads to diminished GSK-3β-mediated β-catenin degradation (reviewed in Novak and Dedhar, 1999). Nuclear translocation and accumulation of β-catenin together with the transcription factor TCF/LEF leads to the transcriptional activation of TCF/LEF target genes relevant for mitosis and tumor development (e.g., cyclin D1, c-myc and TCF) (Behrens et al., 1996; Tetsu and McCormick, 1999). In HCCs, the β-catenin degradation complex is a frequent target of inactivating mutations, leading to nuclear accumulation of β-catenin in about 40% of all cases. Stabilizing mutations and deletions in the GSK-3β phosphorylation domain of β-catenin, which is essential for reduced protein ubiquitination, are present in 25–30% of HCCs (de La Coste et al., 1998; Miyoshi et al., 1998) and most frequently affect HCV-induced HCCs (Huang et al., 1999; Hsu et al., 2000). In addition, mutational inactivation of other proteins involved in β-catenin degradation may also be responsible for constitutively elevated wnt/β-catenin signal transduction in liver tumorigenesis. For example, inactivating mutations in the axin-1 gene are present in about 5% of HCCs (Satoh et al., 2000). In contrast, mutational APC gene inactivation, typical for colon cancer, is extremely rare in HCCs (Chen et al., 1998; Huang et al., 1999; Su et al., 2001b). However, methylation-dependent silencing of the APC promoter region may be more common (Esteller et al., 2001). Recently published data establish a direct link between p53-dependent cell cycle control and wnt/β-catenin signal transduction (Liu et al., 2001; Matsuzawa and Reed, 2001; for review, see Polakis, 2001). p53-induced Siah-1 participates in a multiprotein complex (including APC, ebi, Skp-1, and SIP) (Matsuzawa and Reed, 2001), resulting in GSK-3β-independent β-catenin degradation (Liu et al., 2001). However, new data have revealed also a p53-dependent β-catenin degradation based on GSK-3β activity (Sadot et al., 2001). On the other hand, β-catenin may induce p53 accumulation via induction of ARF, an alternative reading frame product of INK4 (p16),

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and inhibition of MDM-2 mediated p53-degradation (Damalas et al., 2001). Thus, inactivation of the p53 tumor suppressor gene may uncover the oncogenic potential of activated β-catenin. Consequently, accumulation of active β-catenin may exert selective pressure on the inactivation of the p53 tumor suppressor pathway, thus leading to an increased frequency of p53 mutations. Whether this kind of forced inactivation is also relevant in HCCs, where p53 mutations are suspected to be a rather late event, remains to be explored.

4. PLEIOTROPIC GROWTH FACTORS As in other solid malignant tumors, autocrine stimulation derived from diffusible growth factors is a multifunctional and central protumorigenic principle. It contributes to proliferation, antiapoptosis, neoangiogenesis, and invasive behavior. In HCCs, overexpression and functional relevance of three different growth factors, IGF-II, TGFα, and vascular endothelial growth factor (VEGF), are well documented. IGF-II is a potent fetal growth factor that becomes downregulated shortly after birth. Its regulation is extremely complex involving multiple mechanisms at all levels of gene and protein expression and interaction. Transcription is governed by four different tissue-speciﬁc and developmentally regulated promoters that are subjected to promoter-speciﬁc (P2-P4) genomic imprinting (Ohlsson et al., 1993; Vu and Hoffman, 1994). The many different transcripts are in part alternatively spliced, differentially 3 processed, and even endonucleolytically cleaved (Nielsen, 1992). Most transcripts give rise to the preproIGF-II polypeptide, but translational utilization of the different transcripts is regulated by the cell status (Nielsen et al., 1995). The preproIGF-II polypeptide is differentially processed leading to IGF-II polypeptides of different sizes and activities (Duguay, 1999). Even when secreted, IGF-II interacts with a multiplicity of different serum proteins [IGF-binding proteins (IGF-BPs) and IGF-BP-like proteins] (Murphy, 1998; Rechler and Clemmons, 1998). Furthermore, IGF-II displays high-afﬁnity interactions with three different membrane-bound receptors: the IGF-IIR, which directs IGF-II to lysosomal degradation (Wang et al., 1994; Braulke, 1999), the IGF-IR (D’Ambrosio et al., 1996; Esposito et al., 1997), and the alternatively spliced insulin receptor (IR) isoform A (Frasca et al., 1999; Sciacca et al., 1999), the last two representing tyrosine kinases. All these receptors appear to be expressed in HCCs, and the interactions with these receptors are complex as they compete with other ligands, like insulin and IGF-I. IGF-II is overexpressed in many different animal models of hepatocarcinogenesis (Fu et al., 1988; Ueno et al., 1988; Cariani et al., 1991; Schirmacher et al., 1991, 1992; Yang et al., 1993; Pasquinelli et al., 1994; Casola et al., 1995; Liu et al., 1997; Harris et al., 1998), HCC cell lines, at least 40%

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of human HCCs, and possibly even in some premalignant lesions (Cariani et al., 1988; D’Errico et al., 1994; Kulik et al., 1997; Li et al., 1997; Sohda et al., 1997; Aihara et al., 1998; Ng et al., 1998). Its overexpression results predominantely from transcriptional activation, although the responsible mechanisms are heterogeneous, including loss of promoter speciﬁc (P2-P4) genomic imprinting, dysbalance of promoter activity, transcriptional transactivation, and also higher order chromosomal mechanisms (Sohda et al., 1996; Li et al., 1997; Uchida et al., 1997; Vernucci et al., 2000). Tumorderived IGF-II interacts with several other oncogenic pathways: p53 carrying the aﬂatoxin-induced codon 249-mutation transactivates the IGF-II promoter P4 (Lee et al., 2000). Regulation of IGF-BP3 expression by wildtype but not mutated p53 (Buckbinder et al., 1995) suggests that regulation of IGF-II bioavailability may be an effector mechanism of p53. Furthermore, IGF-II transcription is increased as a consequence of the potentially oncogenic viral proteins HBx (by PKC, MAPK, and SP-1) (Feitelson and Duan, 1997; Lee et al., 1998; Kang-Park et al., 2001) and HCV core (by Egr-1 and Sp-1) (Lee et al., 2001a) and due to tumor hypoxia (by Egr-1) (Kim et al., 1998b; Bae et al., 1999). IGF-II, in turn, activates VEGF transcription, which is also increased in hypoxic tumor areas (Suzuki et al., 1996). TGFα is a short secreted polypeptide with sequence homology to epidermal growth factor (EGF) and is derived from a precursor polypeptide by proteolytic cleavage. TGFα may, however, also be present as a longer, transmembranous form. TGFα stimulates hepatocellular DNA synthesis in an autocrine and paracrine manner and competes with EGF for binding to the EGFR tyrosine kinase. Convincing experimental evidence for the protumorigenic role of TGFα in liver carcinogenesis has been derived from transgenic mice that consistently develop HCCs (Jhappan et al., 1990; Lee et al., 1992; Takagi et al., 1992). It is overexpressed in many human HCCs and HCC cell lines (Yeh et al., 1987; Derynck et al., 1987; Hsia et al., 1992; Morimitsu et al., 1995), and elevated urine concentrations of TGFα have been found in 65% of HCC patients (Yeh et al., 1987). TGFα seems to act during the early stages of hepatocarcinogenesis and has been described to correlate with tumor differentiation and proliferation (Kira et al., 1997; Nalesnik et al., 1998). Whether intracellular TGFα overexpression observed in preneoplastic liver conditions, especially in chronic hepatitis B, represents an early predisposing condition for malignant transformation or an inactive intracellular retention due to occlusion of the endoplasmatic reticulum by HBV surface antigens, remains to be determined (Hsia et al., 1992, 1994; Schirmacher et al., 1996; Ono et al., 1998). VEGF is activated in the majority of HCCs preferentially in hypoxic areas of the tumor and may contribute to early neoangiogenesis in a paracrine manner (Suzuki et al., 1996; Park et al., 2000b). VEGF expression is also present in DNs, increases during progression of hepatocarcinogenesis

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preferentially under hypoxic conditions (Park et al., 2000b; von Marschall et al., 2001), and may exert its signaling via protein kinase C (Yoshiji et al., 1999). It is activated by HIF/p300CBP complexes in HCC cells (Arany et al., 1996), and HIF-1 expression, like IGF-II expression, is strongly activated in hypoxic tumor areas (Maxwell et al., 1997). The role of hepatocyte growth factor (HGF) in liver carcinogenesis is still not clear. It is the most potent growth factor for hepatocytes but transgenic mouse studies have suggested even antitumorigenic activity (Santoni-Rugiu et al., 1996). In consequence, the quality of the HGF effects may be concentration dependent (Tavian et al., 2000). HGF is not expressed by HCC cells (Selden et al., 1994; Noguchi et al., 1996), but a paracrine circuit by stimulation of HGF expression in tumor-associated myoﬁbroblasts by HCC cells and resulting stimulation of tumor cell invasiveness via myoﬁbroblastderived HGF have been described (Neaud et al., 1997; Guirouilh et al., 2000, 2001).

5. MITOINHIBITORY PATHWAYS TGFβ signaling is one of the central mitoinhibitory pathways for epithelial cells, induces growth inhibition and apoptosis in hepatocytes, and is responsible for downregulation of physiological growth response, for example, in organ regeneration. Experimental evidence demonstrates that functional inactivation of TGFβ signaling promotes hepatocarcinogenesis (Kanzler et al., 2001). Intracellular transmission of TGFβ signals is constitutively disrupted in many different cancer types, although upregulation of TGFβ has also been observed during HCC progression (Ito et al., 1991; Matsuzaki et al., 2000). It has been suggested that once tumor cells are insensitive to growth inhibitory signals from TGFβ, they may beneﬁt from its promotion of the angiogenic, invasive, and metastatic phenotype. This may explain the apparently paradoxical ﬁnding that increased tissue expression and serum concentrations of TGFβ can be found in HCC patients (Shirai et al., 1992; Unsal et al., 1994). In HCCs, intracellular TGFβ signaling appears to be constitutively inactivated only in a minority of tumors so far, but comprehensive analyses of intracellular TGFβ signaling molecules, especially of transcription cofactors, in HCCs are lacking. Potentially inactivating mutations of the Smad 2 and 4 signal transducers are rare and may be present in about 10% of HCCs (Kawate et al., 1999; Yakicier et al., 1999), while TGFβRII and Smad 6 and 7 are apparently not affected (Kawate et al., 1999, 2001). IRS-1, an intracellular signaling molecule downstream of IGFIR is frequently activated in human HCCs and appears to protect against TGFβ-induced apoptosis (Tanaka and Wands, 1996) providing an example of a functional link between mitogenic stimulation and an antiapoptotic pathway.

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Another growth suppressive pathway frequently inactivated in HCCs involves the IGF-IIR [cation-independent mannose 6-phosphate (M6P) receptor]. IGF-IIR binds to M6P-containing proteins, facilitates activation of latent TGFβ by plasmin cleavage (Dennis and Rifkin, 1991), and directs IGF-II to lysosomal degradation (Wang et al., 1994; Braulke, 1999). Consequently, deletion of the IGF-IIR leads to increased tissue concentrations of IGF-II (Nolan and Lawlor, 1999). Loss of heterozygosity in the IGF-IIR locus has been described in about 60% of the HCCs and also in some DNs. Inactivating mutations of the second allele have been detected in about 25% of the cases (De Souza et al., 1995). Consequently, tumor suppressive activity has been ascribed to the IGF-IIR (De Souza et al., 1995; Yamada et al., 1997), but the results have remained controversial (Wada et al., 1999). It is currently unclear whether this proposed activity is via increased IGF-II availability, lack of TGFβ activation, or a different mechanism. The signiﬁcance and intracellular interactions of two recently identiﬁed potential tumor suppressor genes, DLC-1 (Yuan et al., 1998; Ng et al., 2000) and HCCS1 (Zhao et al., 2001), which are both growth suppressive and frequently inactivated in HCC cells, remains to be clariﬁed further.

6. OTHER PROTUMORIGENIC MOLECULAR CHANGES Although tyrosine kinases represent an important subclass among transforming oncogenes, evidence for their constitutive alteration in HCCs is rather sparse. MET (HGF-receptor) overexpression occurs in about 30–50% of the HCCs (Boix et al., 1994; Suzuki et al., 1994; Tavian et al., 2000) and correlates with a worse prognosis and an increased incidence of intrahepatic satellite metastases (Ueki et al., 1997; D’Errico et al., 1996). However, the mechanism of activation in HCCs (mutation, transcriptional activation, posttranslational modiﬁcation) has not been clariﬁed thoroughly. Missense mutations in the c-met gene have been found in some childhood HCCs (Park et al., 1999; Trusolino et al., 2001). It will be interesting to determine whether in HCCs MET activation by cell attachment (Wang et al., 2001b) represents a function of a direct MET–α6β4–integrin interaction, as recently described for a gastric carcinoma cell line (Park et al., 1999; Trusolino et al., 2001). IGF-IR and EGFR are both generally expressed in HCCs, but there is no indication for their constitutive overexpression (Kiss et al., 1997). Furthermore no activation of c-erbB2 (HER-2/neu) is detectable in HCCs (Prange and Schirmacher, 2001). Little evidence has accumulated for activated cytokine receptor signaling, but recently, constitutive induction of JAK/STAT signaling in 65% of HCCs was described to be due to the methylation-dependent inactivation of the suppressor of cytokine signaling (SOCS-1) (Yoshikawa et al., 2001). Ras gene activation by codon 12/13 mutations, which is frequent in rodent chemical hepatocarcinogenesis (Funato et al., 1987; Sinha et al., 1988), does

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not play a signiﬁcant role in human HCCs (Tada et al., 1990; Chao et al., 1999), with the exception of signature codon 12/13 mutations in the K-ras-2 gene (Weihrauch et al., 2001) and a few codon 61 mutations of the Ha-ras gene (Boivin-Angele et al., 2000) in the rare HCC cases due to VC exposure. As in many other carcinomas, telomerase activity is detectable in 70–80% of HCCs (Tahara et al., 1995) and closely correlates with the expression of hTERT, the catalytic telomerase subunit (Nakayama et al., 1998). A telomerase suppressor gene may be located at 10p15 and inactivated frequently in HCCs (Nishimoto et al., 2001). Telomerase activation may be a relatively early event in human hepatocarcinogenesis, since it has been detected in premalignant liver lesions (Takaishi et al., 2000; Youssef et al., 2001). On the other hand, telomerase activity in HCCs has been positively correlated with tumor cell dedifferentiation and recurrence after resection (Suda et al., 1998; Kawakami et al., 2000). Several types of human carcinomas, especially colon carcinomas, overexpress cyclooxygenase 2 (COX-2), an enzyme that converts arachidonic acid into prostaglandins. In extrahepatic tumors, COX-2 has been shown to contribute to proliferation, antiapoptosis, activation of procarcinogens, and potentially neoangiogenesis and invasiveness (Fosslien, 2000a,b; Dempke et al., 2001). The antiapoptotic mechanism of COX-2 activity is still unsolved. Our own data demonstrate that initiator (caspase-9) as well as downstream executioner caspases (caspase-3 and caspase-6) are rapidly activated by intramolecular cleavage after COX-2 inhibition in HCC cells (Kern et al., manuscript submitted), but the responsible upstream initiating events have not yet been identiﬁed. HCCs overexpress COX-2 in 50–97% of the cases, and expression was more intense in well-differentiated HCCs (Koga et al., 1999; Bae et al., 2001). The mode of COX-2 overexpression is still unknown, but interestingly 1q25.2-q25.3, the chromosomal location of the COX-2 gene, is overrepresented in approximately 60% of HCCs (Marchio et al., 1997; Niketeghad et al., 2001). In addition, cytosolic phospholipase A2 (cPLA2), which is responsible for arachidonic acid synthesis from membranous phospholipids, is also located at 1q25 and is coregulated with COX-2 (Tay et al., 1995; Kudo and Murakami, 1999). Furthermore, IGF-II, which is frequently overexpressed in HCCs, may upregulate COX-2 expression through activation of the IGF-I receptor, as demonstrated in Caco-2 human colon carcinoma cells (Di Popolo et al., 2000).

C. Oncogenic Molecular Cross-Talk Most of the outlined molecular events do not just add up, but form a molecular network, in which the different factors are functionally interrelated and further increase the probability of novel oncogenic hits. This is exempliﬁed by the interconnection of the E-cadherin, β-catenin, p53, and IGF-II

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pathways. β-catenin is activated in about 40% of human HCCs and expression analyses suggest that loss of E-cadherin expression is a necessary prerequisite for its nuclear translocation. But studies in nonhepatic tumor systems have shown that mutated β-catenin, on the other hand exerts a strong selective pressure on the development of inactivating E-cadherin mutations. The transcriptional effector mechanisms of activated β-catenin (c-myc, MMP-7, cyclin D1) via TCF/LEF-4 are well known, but their presence and relevance in human hepatocarcinogenesis are still not evident. In contrast, recent data suggest a correlation of nuclear β-catenin and nuclear (mutated?) p53. Via a second β-catenin degradation pathway involving the p53-regulated Siah-1 gene, especially oncogenic β-catenin mutations involving the GSK-3β binding site may exert selective pressure on functional inactivation of p53. Once p53 function is lost, it not only increases genomic instability but may also activate IGF-II, either by direct transcriptional transactivation or indirectly by downregulating IGF-BP3 expression. IGF-II in turn appears to induce VEGF and COX-2 expression and kinase signaling pathways via IGF-IR (including antiapoptotic inhibition of TGFβ-signaling) or IR (isoform A) activation.

V. FUNCTIONAL CONSEQUENCES A. Cell Growth The tightly controlled homeostasis of cell proliferation and apoptosis is disrupted during hepatocarcinogenesis. Numerous mechanisms by which proliferation is stimulated have been identiﬁed in HCCs: overexpression of secreted growth factors, like IGF-II and TGFα (in 40–90%), constitutive activation of growth factor receptors, like MET (in 30–50%), and deregulation of cell cycle control genes, like RB-1, cyclins, cdks, and cdk-inhibitors (in more than 90%). Furthermore inactivation of mitoinhibitory pathways, as demonstrated for TGFβ-signaling and IGF-IIR, may exhibit similar effects. There are several mechanisms that in HCC cells are used to evade programmed cell death: loss of Fas receptor expression, (de novo) expression of FasL by the tumor cells, and activation of antiapoptotic growth factors ¨ (Kiso et al., 1994; Moller et al., 1994; Hahne et al., 1996; Higaki et al., 1996; Strand et al., 1996). HCCs partially or completely lose the expression of Fas and induce lymphocyte apoptosis by expressing FasL in the tumor cells (Strand et al., 1996). Data regarding the antiapoptotic effects of IGF-II in hepatocarcinogenesis are equally convincing. In woodchuck hepatocarcinogenesis, IGF-II blocks N-myc-induced apoptosis and in a mouse model, loss of IGF-II expression strongly enhances tumor cell apoptosis (Ueda and Ganem, 1996; Yang et al., 1996). Using antisense oligonucleotides or neutralizing antibodies against IGF-II, the growth of HCC cells was strongly

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inhibited (Lin et al., 1997; Scharf et al., 1998). Neutralizing antibodies against IGF-IR also affect rat tumors and at least some human HCC cells in the same way (Zhang et al., 1997). IGF-II treatment of HCC cell lines that already overexpress IGF-II has no functional effects, indicating maximal autocrine stimulation, which is further supported by the progressive accumulation of unbound, active IGF-II in the cell culture supernatant. In addition, in vitro neutralization of IGF-II increases liver tumor cell apoptosis, when performed together with chemotherapeutic treatment (Lund et al., manuscript submitted).

B. Neoangiogenesis Neovascularization is essential for tumor cell nutrition and efﬁcient tumor growth, and is negatively correlated with the survival of HCC patients (Tanigawa et al., 1997). It has been calculated that tumor growth exceeding the size of 0.2–0.3 cm diameter depends on neoangiogenesis. Since malignant transformation of premalignant liver lesions occurs at a size between 0.4 and 2 cm diameter, neoangiogenesis is an early, rather premalignant event, which is also supported by microvascularization analyses in DNs (Roncalli et al., 1999; Park et al., 2000b). The angiogenic phenotype depends on the strict balance of positive (e.g., IGF-II, FGF, VEGF, angiogenin) and negative (e.g., thombospondin, endostatin) factors. Both IGF-II and VEGF are frequently overexpressed in HCCs, exhibit direct angiogenic activity, and are likely to cooperate in the stimulation of tumor neoangiogenesis (Nguyen et al., 1994). Both factors provide a direct functional link between hypoxia and neoangiogenesis, since transcription of IGF-II and VEGF is activated in areas of tumor hypoxia, and hypoxia furthermore stabilizes VEGF transcripts (von Marschall et al., 2001). Furthermore, IGF-II increases VEGF transcription in human HCC cells (Kim et al., 1998b). Additionally, several negative angiogenic factors are regulated by the wildtype p53 tumor suppressor. Thus, inactivation of p53, which occurs in at least 1/3 of HCCs, may further promote neoangiogenesis (Mise et al., 1996; Kim et al., 1998b; Bae et al., 1998; Yoshiji et al., 1998; Shimoda et al., 1999; Gorrin-Rivas et al., 2000). In human HCCs, the collagen XVIII/endostatin precursor is frequently downregulated and negatively correlates with microvessel density of the tumor (Musso et al., 2001).

C. Invasion and Metastasis Invasion and metastasis of HCC may be facilitated by proteins that stimulate malignant cell attachment to host cellular elements, tumor cell-dependent

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barrier proteolysis, tumor cell migration, and colony formation in the target organ (Liotta et al., 1986, 1991). Any gene product that inhibits one of these steps by interfering with tumor cell motility and adhesiveness, proteolysis, neoangiogenesis, and modulation of immune recognition may diminish tumor invasion and metastasis. Three lines of events have been correlated with invasion and metastasis of human HCCs so far: (1) activation of paracrine or autocrine growth factor signaling, (2) reduced expression of metastasis suppressor genes as upstream modulators, and (3) activation of matrix metalloproteinases (MMPs) or downregulation of their inhibitors (TIMPs) as downstream execution events. Activation of autocrine growth factors with angiogenic properties in HCCs has been demonstrated for IGF-II and VEGF as described above. Activation of paracrine growth factor signaling has been shown for HGF-MET interaction: HCC cells do not express HGF but MET and induce HGF secretion in HCC-associated nonmalignant stromal myoﬁbroblasts (Guirouilh et al., 2000, 2001). Myoﬁbroblast-derived HGF then stimulates invasiveness of MET-expressing HCC cells (Neaud et al., 1997). Reduced expression of established metastasis suppressor genes has been described for nm23-H1 (Iizuka et al., 1995; Lin et al., 1995). High frequency overexpression of MMPs has been demonstrated for gelatinase A (Yamamoto et al., 1997), gelatinase B (Ashida et al., 1996), and MMP-7 (Ozaki et al., 2000). Furthermore, a higher ratio of MMP-9/TIMP-1 mRNA expression in HCCs with capsular invasion suggests that the MMP-9/TIMP-1 balance inﬂuences HCC invasion and metastasis (Arii et al., 1996). How these different events are activated and functionally related in HCCs is largely unknown, but activation of both MMP-7 and gelatinase B by the ets-1 oncogene in HCCs has been reported (Ozaki et al., 2000). The functional relevance and interactions of other differentially expressed genes in HCCs that have been linked with an altered invasive or metastatic phenotype, like the KAI1 genes (Guo et al., 1998), the RECK genes (Furumoto et al., 2001), and the HCCA2 genes (Wang et al., 2001), still have to be determined.

VI. THERAPEUTIC IMPLICATIONS The ﬁrst line of novel molecular antitumorigenic strategies in human hepatocarcinogenesis is primary prevention. With the aid of molecular biology screening techniques, all major etiologies of HCC can be diagnosed early and reliably (Dries et al., 1999; Pawlotsky, 1999; Weston and Martin, 2001). HBV infection is efﬁciently prevented by a recombinant vaccine. Broad nationwide HBV vaccination programs have been inaugurated in several countries and will reduce respective HCC frequencies although with a delay

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of up to several decades. HCV infection can now be diagnosed reliably in serum and tissue, but no immunization is currently available to prevent infection. Treatment of both forms of chronic viral hepatitis has signiﬁcantly improved and will further beneﬁt from therapeutics based on detailed knowledge of the viral life cycle, like novel nucleoside analogs or protease inhibitors. Early molecular diagnosis and thus treatment of genetic hemochromatosis has beneﬁted much from the identiﬁcation of the C282Y mutation in the HFE gene, which is responsible for more than 90% of the hemochro¨ matosis cases in the western world (Feder et al., 1996; Hohler et al., 2000). Currently, secondary prevention of HCC is not feasible, but it has come into sight, since COX-2 is frequently overexpressed in HCCs. Thus, similar to FAP, HCC development may be delayed by constant application of established COX-2 inhibitors. In addition, COX-2 inhibition may offer also some prospect for systemic palliation in fully developed HCCs. Interference with growth factor signaling pathways by small molecules is an attractive therapeutic scheme with potential application in HCCs. It may result in pleiotropic antitumorous effects by reducing proliferation and increasing tumor cell apoptosis, but also affecting neoangiogenesis and invasion. Inhibition of growth factor-mediated antiapoptosis is likely to amplify chemotherapy response and thus may offer potential in systemic combination therapy, as demonstrated for IGF-II signaling. Tyrosine kinase inhibition has already gained clinical success in chronic myeloid leukemia and gastrointestinal stromal tumors. IGF-IR and MET represent respective valid targets in human HCCs, of which IGF-IR inhibition is already approaching clinical application. Furthermore, downstream signaling, like a constitutively active JAK/STAT pathway, may also be targeted with small molecule inhibitors (Yoshikawa et al., 2001). Efﬁcient modes of liver tumor cell delivery have been established, namely, genetically modiﬁed cytolytic HSV vectors (Pawlik et al., 2000) and adenoviral infection. The latter allows targeting of tumor suppressor pathways or sensitization of the tumor cells to cytoreductive therapy (Kanai et al., 1997). A prime target is p53, which is altered in more than 30% of HCCs. These HCCs may be targeted either for reconstitution of wild-type p53 function or for selectively activating adenoviral cytolysis in tumor cells with defective p53. In addition, antiangiogenesis is promising in HCCs. PKC inhibitors may interfere with VEGF-mediated neoangiogenesis, and neovascular endothelial cells may be inhibited with anti-CD34 antibodies and supplementation of angiogenesis inhibitors, like endostatin. All therapeutic approaches have to respect that in the majority of HCC patients the surrounding liver is severely damaged, often showing complete cirrhosis. This imposes restrictions on therapeutic intervention and is often life-limiting in a manner comparable to the HCC itself.

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ACKNOWLEDGMENTS Research by the authors has been supported by the Deutsche Forschungsgemeinschaft, the Dr. ¨ Krebsforschung, the Deutsche Krebshilfe, the Bundesministerium Mildred-Scheel-Stiftung fur ¨ Bildung und Forschung (ZMMK), and Koln ¨ Fortune. fur

REFERENCES Aguilar, F., Harris, C. C., Sun, T., Hollstein, M., and Cerutti, P. (1994). Geographic variation of p53 mutational proﬁle in nonmalignant human liver. Science 264, 1317–1319. Aguilar, F., Hussain, S. P., and Cerutti, P. (1993). Aﬂatoxin B1 induces the transversion of G → T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc. Natl. Acad. Sci. USA 90, 8586–8590. Aihara, T., Noguchi, S., Miyoshi, Y., Nakano, H., Sasaki, Y., Nakamura, Y., Monden, M., and Imaoka, S. (1998). Allelic imbalance of insulin-like growth factor II gene expression in cancerous and precancerous lesions of the liver. Hepatology 28, 86–89. Aihara, T., Noguchi, S., Sasaki, Y., Nakano, H., Monden, M., and Imaoka, S. (1996). Clonal analysis of precancerous lesion of hepatocellular carcinoma. Gastroenterology 111, 455– 461. Anthony, P. P. (1976). Precursor lesions for liver cancer in humans. Cancer Res. 36, 2579–2583. Aoki, H., Hayashi, J., Moriyama, M., Arakawa, Y., and Hino, O. (2000). Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1. J. Virol. 74, 1736–1741. Arany, Z., Huang, L. E., Eckner, R., Bhattacharya, S., Jiang, C., Goldberg, M. A., Bunn, H. F., and Livingston, D. M. (1996). An essential role for p300/CBP in the cellular response to hypoxia. Proc. Natl. Acad. Sci. USA 93, 12969–12973. Arii, S., Mise, M., Harada, T., Furutani, M., Ishigami, S., Niwano, M., Mizumoto, M., Fukumoto, M., and Imamura, M. (1996). Overexpression of matrix metalloproteinase 9 gene in hepatocellular carcinoma with invasive potential. Hepatology 24, 316–322. Arima, N., Kao, C. Y., Licht, T., Padmanabhan, R., and Sasaguri, Y. (2001). Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A. J. Biol. Chem. 276, 12675– 12684. Ashida, K., Nakatsukasa, H., Higashi, T., Ohguchi, S., Hino, N., Nouso, K., Urabe, Y., Yoshida, K., Kinugasa, N., and Tsuji, T. (1996). Cellular distribution of 92-kd type IV collagenase/gelatinase B in human hepatocellular carcinoma. Am. J. Pathol. 149, 1803–1811. Avantaggiati, M. L., Natoli, G., Balsano, C., Chirillo, P., Artini, M., De Marzio, E., Collepardo, D., and Levrero, M. (1993). The hepatitis B virus (HBV) pX transactivates the c-fos promoter through multiple cis-acting elements. Oncogene 8, 1567–1574. Azechi, H., Nishida, N., Fukuda, Y., Nishimura, T., Minata, M., Katsuma, H., Kuno, M., Ito, T., Komeda, T., Kita, R., et al. (2001). Disruption of the p16/cyclin D1/retinoblastoma protein pathway in the majority of human hepatocellular carcinomas. Oncology 60, 346–354. Bae, M. H., Lee, M. J., Bae, S. K., Lee, O. H., Lee, Y. M., Park, B. C., and Kim, K. W. (1998). Insulin-like growth factor II (IGF-II) secreted from HepG2 human hepatocellular carcinoma cells shows angiogenic activity. Cancer Lett. 128, 41–46. Bae, S. H., Jung, E. S., Park, Y. M., Kim, B. S., Kim, B. K., Kim, D. G., and Ryu, W. S. (2001). Expression of cyclooxygenase-2 (COX-2) in hepatocellular carcinoma and growth inhibition of hepatoma cell lines by a COX-2 inhibitor, NS-398. Clin. Cancer Res. 7, 1410– 1418.

Human Hepatocellular Carcinoma Pathogenesis

93

Bae, S. K., Bae, M. H., Ahn, M. Y., Son, M. J., Lee, Y. M., Bae, M. K., Lee, O. H., Park, B. C., and Kim, K. W. (1999). Egr-1 mediates transcriptional activation of IGF-II gene in response to hypoxia. Cancer Res. 59, 5989–5994. Balsara, B. R., Pei, J., De Rienzo, A., Simon, D., Tosolini, A., Lu, Y. Y., Shen, F. M., Fan, X., Lin, W. Y., Buetow, K. H., et al. (2001). Human hepatocellular carcinoma is characterized by a highly consistent pattern of genomic imbalances, including frequent loss of 16q23.1-24.1. Genes Chromosomes Cancer 30, 245–253. Bando, K., Nagai, H., Matsumoto, S., Koyama, M., Kawamura, N., Onda, M., and Emi, M. (1999). Identiﬁcation of a 1-cM region of common deletion on 4q35 associated with progression of hepatocellular carcinoma. Genes Chromosomes Cancer 25, 284–289. ¨ Bannasch, P., and Schroder, C. H. (2002). Tumours and tumour-like lesions of the liver and biliary tract: pathogenesis of primary liver tumours. In “Pathology of the Liver” (R. N. M. MacSween, A. D. Burt, B. C. Portmann, K. G. Ishak, P. J. Scheuer, and P. P. Anthony, Eds.), pp. 777–825. Churchill Livingstone, New York. Barba, G., Harper, F., Harada, T., Kohara, M., Goulinet, S., Matsuura, Y., Eder, G., Schaff, Z., Chapman, M. J., Miyamura, T., and Brechot, C. (1997). Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proc. Natl. Acad. Sci. USA 94, 1200–1205. Bathe, O. F., Scudamore, C. H., Caron, N. R., and Buczkowski, A. (1997). Resection of hepatocellular carcinoma. In “Liver Cancer” (K. Okuda and E. Tabor, Eds.), pp. 511–530. Churchill Livingstone, New York. Beasley, R. P., Hwang, L. Y., Lin, C. C., and Chien, C. S. (1981). Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan. Lancet 2, 1129–1133. Becker, S. A., Lee, T. H., Butel, J. S., and Slagle, B. L. (1998). Hepatitis B virus X protein interferes with cellular DNA repair. J. Virol. 72, 266–272. Behrens, J., von Kries, J. P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R., and Birchmeier, W. (1996). Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382, 638–642. Boivin-Angele, S., Lefrancois, L., Froment, O., Spiethoff, A., Bogdanffy, M. S., Wegener, K., Wesch, H., Barbin, A., Bancel, B., Trepo, C., et al. (2000). Ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-speciﬁc but vary with cell type and species. Int. J. Cancer 85, 223–227. Boix, L., Rosa, J. L., Ventura, F., Castells, A., Bruix, J., Rodes, J., and Bartrons, R. (1994). c-met mRNA overexpression in human hepatocellular carcinoma. Hepatology 19, 88–91. Bosch, F. X. (1997). Global epidemiology of hepatocellular carcinoma. In “Liver Cancer” (K. Okuda and E. Tabor, Eds.), pp. 13–28. Churchill Livingstone, New York. Bouchard, M. J., Wang, L. H., and Schneider, R. J. (2001). Calcium signaling by HBx protein in hepatitis B virus DNA replication. Science 294, 2376–2378. Braulke, T. (1999). Type-2 IGF receptor: a multi-ligand binding protein. Horm. Metab. Res. 31, 242–246. Brechot, C., Pourcel, C., Louise, A., Rain, B., and Tiollais, P. (1980). Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma. Nature 286, 533–535. Buckbinder, L., Talbott, R., Velasco-Miguel, S., Takenaka, I., Faha, B., Seizinger, B. R., and Kley, N. (1995). Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature 377, 646–649. Buetow, K. H., Murray, J. C., Israel, J. L., London, W. T., Smith, M., Kew, M., Blanquet, V., Brechot, C., Redeker, A., and Govindarajah, S. (1989). Loss of heterozygosity suggests tumor suppressor gene responsible for primary hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 86, 8852–8856.

94

Kern et al.

Cadwell, C., and Zambetti, G. P. (2001). The effects of wild-type p53 tumor suppressor activity and mutant p53 gain-of-function on cell growth. Gene 277, 15–30. Cariani, E., Dubois, N., Lasserre, C., Briand, P., and Brechot, C. (1991). Insulin-like growth factor II (IGF-II) mRNA expression during hepatocarcinogenesis in transgenic mice. J. Hepatol. 13, 220–226. Cariani, E., Lasserre, C., Seurin, D., Hamelin, B., Kemeny, F., Franco, D., Czech, M. P., Ullrich, A., and Brechot, C. (1988). Differential expression of insulin-like growth factor II mRNA in human primary liver cancers, benign liver tumors, and liver cirrhosis. Cancer Res. 48, 6844–6849. Caselmann, W. H., Meyer, M., Kekule, A. S., Lauer, U., Hofschneider, P. H., and Koshy, R. (1990). A trans-activator function is generated by integration of hepatitis B virus preS/S sequences in human hepatocellular carcinoma DNA. Proc. Natl. Acad. Sci. USA 87, 2970– 2974. Casola, S., Ungaro, P., Pedone, P. V., Lazzaro, D., Fattori, E., Ciliberto, G., Zarrilli, R., Bruni, C. B., and Riccio, A. (1995). Loss of heterozygosity of imprinted genes in SV40 t/T antigen-induced hepatocellular carcinomas. Oncogene 11, 711–721. Chakraborty, P. R., Ruiz-Opazo, N., Shouval, D., and Shafritz, D. A. (1980). Identiﬁcation of integrated hepatitis B virus DNA and expression of viral RNA in an HBsAg-producing human hepatocellular carcinoma cell line. Nature 286, 531–533. Chang, J., Yang, S. H., Cho, Y. G., Hwang, S. B., Hahn, Y. S., and Sung, Y. C. (1998). Hepatitis C virus core from two different genotypes has an oncogenic potential but is not sufﬁcient for transforming primary rat embryo ﬁbroblasts in cooperation with the H-ras oncogene. J. Virol. 72, 3060–3065. Chang, S. C., Yen, J. H., Kang, H. Y., Jang, M. H., and Chang, M. F. (1994). Nuclear localization signals in the core protein of hepatitis C virus. Biochem. Biophys. Res. Commun. 205, 1284– 1290. Chao, H. K., Tsai, T. F., Lin, C. S., and Su, T. S. (1999). Evidence that mutational activation of the ras genes may not be involved in aﬂatoxin B(1)-induced human hepatocarcinogenesis, based on sequence analysis of the ras and p53 genes. Mol. Carcinog. 26, 69–73. Chao, Y., Shih, Y. L., Chiu, J. H., Chau, G. Y., Lui, W. Y., Yang, W. K., Lee, S. D., and Huang, T. S. (1998). Overexpression of cyclin A but not Skp 2 correlates with the tumor relapse of human hepatocellular carcinoma. Cancer Res. 58, 985–990. Chaubert, P., Gayer, R., Zimmermann, A., Fontolliet, C., Stamm, B., Bosman, F., and Shaw, P. (1997). Germ-line mutations of the p16INK4(MTS1) gene occur in a subset of patients with hepatocellular carcinoma. Hepatology 25, 1376–1381. Chen, T. C., Hsieh, L. L., Ng, K. F., Jeng, L. B., and Chen, M. F. (1998). Absence of APC gene mutation in the mutation cluster region in hepatocellular carcinoma. Cancer Lett. 134, 23–28. 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. Choi, Y. L., Park, S. H., Jang, J. J., and Park, C. K. (2001). Expression of the G1-S modulators in hepatitis B virus-related hepatocellular carcinoma and dysplastic nodule: Association of cyclin D1 and p53 proteins with the progression of hepatocellular carcinoma. J. Korean Med. Sci. 16, 424–432. Colgrove, R., Simon, G., and Ganem, D. (1989). Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication. J. Virol. 63, 4019–4026. Collonge-Rame, M. A., Bresson-Hadni, S., Koch, S., Carbillet, J. P., Blagosklonova, O., Mantion, G., Miguet, J. P., Heyd, B., and Bresson, J. L. (2001). Pattern of chromosomal imbalances in non-B virus related hepatocellular carcinoma detected by comparative genomic hybridization. Cancer Genet. Cytogenet. 127, 49–52.

Human Hepatocellular Carcinoma Pathogenesis

95

Covini, G., Chan, E. K., Nishioka, M., Morshed, S. A., Reed, S. I., and Tan, E. M. (1997). Immune response to cyclin B1 in hepatocellular carcinoma. Hepatology 25, 75–80. Cross, J. C., Wen, P., and Rutter, W. J. (1993). Transactivation by hepatitis B virus X protein is promiscuous and dependent on mitogen-activated cellular serine/threonine kinases. Proc. Natl. Acad. Sci. USA 90, 8078–8082. Damalas, A., Kahan, S., Shtutman, M., Ben-Ze’ev, A., and Oren, M. (2001). Deregulated beta-catenin induces a p53- and ARF-dependent growth arrest and cooperates with Ras in transformation. EMBO J. 20, 4912–4922. D’Ambrosio, C., Ferber, A., Resnicoff, M., and Baserga, R. (1996). A soluble insulin-like growth factor I receptor that induces apoptosis of tumor cells in vivo and inhibits tumorigenesis. Cancer Res. 56, 4013–4020. Dejean, A., Bougueleret, L., Grzeschik, K. H., and Tiollais, P. (1986). Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma. Nature 322, 70–72. de La Coste, A., Romagnolo, B., Billuart, P., Renard, C. A., Buendia, M. A., Soubrane, O., Fabre, M., Chelly, J., Beldjord, C., Kahn, A., and Perret, C. (1998). Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc. Natl. Acad. Sci. USA 95, 8847–8851. Dempke, W., Rie, C., Grothey, A., and Schmoll, H. J. (2001). Cyclooxygenase-2: A novel target for cancer chemotherapy? J. Cancer Res. Clin. Oncol. 127, 411–417. Dennis, P. A., and Rifkin, D. B. (1991). Cellular activation of latent transforming growth factor beta requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor. Proc. Natl. Acad. Sci. USA 88, 580–584. D’Errico, A., Fiorentino, M., Ponzetto, A., Daikuhara, Y., Tsubouchi, H., Brechot, C., Scoazec, J. Y., and Grigioni, W. F. (1996). Liver hepatocyte growth factor does not always correlate with hepatocellular proliferation in human liver lesions: Its speciﬁc receptor c-met does. Hepatology 24, 60–64. D’Errico, A., Grigioni, W. F., Fiorentino, M., Baccarini, P., Lamas, E., De Mitri, S., Gozzetti, G., Mancini, A. M., and Brechot, C. (1994). Expression of insulin-like growth factor II (IGF-II) in human hepatocellular carcinomas: An immunohistochemical study. Pathol. Int. 44, 131–137. Derynck, R., Goeddel, D. V., Ullrich, A., Gutterman, J. U., Williams, R. D., Bringman, T. S., and Berger, W. H. (1987). Synthesis of messenger RNAs for transforming growth factors alpha and beta and the epidermal growth factor receptor by human tumors. Cancer Res. 47, 707–712. De Souza, A. T., Hankins, G. R., Washington, M. K., Orton, T. C., and Jirtle, R. L. (1995). M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozygosity. Nat. Genet. 11, 447–449. Deufﬁc, S., Poynard, T., and Valleron, A. J. (1999). Correlation between hepatitis C virus prevalence and hepatocellular carcinoma mortality in Europe. J. Viral. Hepat. 6, 411– 413. Deugnier, Y. M., Charalambous, P., Le Quilleuc, D., Turlin, B., Searle, J., Brissot, P., Powell, L. W., and Halliday, J. W. (1993). Preneoplastic signiﬁcance of hepatic iron-free foci in genetic hemochromatosis: A study of 185 patients. Hepatology 18, 1363–1369. Di Popolo, A., Memoli, A., Apicella, A., Tuccillo, C., di Palma, A., Ricchi, P., Acquaviva, A. M., and Zarrilli, R. (2000). IGF-II/IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE2 synthesis in Caco-2 human colon carcinoma cells. Oncogene 19, 5517– 5524. ¨ Dries, V., von Both, I., Muller, M., Gerken, G., Schirmacher, P., Odenthal, M., ¨ Bartenschlager, R., Drebber, U., Meyer zum Buschenfelde, K. H., and Dienes, H. P. (1999). Detection of hepatitis C virus in parafﬁn-embedded liver biopsies of patients negative for viral RNA in serum. Hepatology 29, 223–229.

96

Kern et al.

Duguay, S. J. (1999). Post-translational processing of insulin-like growth factors. Horm. Metab. Res. 31, 43–49. Dumoulin, F. L., van dem Bussche, A., Sohne, J., Sauerbruch, T., and Spengler, U. (1999). Hepatitis C virus core protein does not inhibit apoptosis in human hepatoma cells. Eur. J. Clin. Invest. 29, 940–946. Ebara, M. (1997). MRI diagnosis of hepatocellular carcinoma. In: “Liver Cancer” (K. Okuda and E. Tabor, Eds.), pp. 361–370. Churchill Livingstone, New York. Endo, K., Ueda, T., Ohta, T., and Terada, T. (2000). Protein expression of MDM2 and its clinicopathological relationships in human hepatocellular carcinoma. Liver 20, 209–215. Esposito, D. L., Blakesley, V. A., Koval, A. P., Scrimgeour, A. G., and LeRoith, D. (1997). Tyrosine residues in the C-terminal domain of the insulin-like growth factor-I receptor mediate mitogenic and tumorigenic signals. Endocrinology 138, 2979–2988. Esteller, M., Corn, P. G., Baylin, S. B., and Herman, J. G. (2001). A gene hypermethylation proﬁle of human cancer. Cancer Res. 61, 3225–3229. Fang, W., Piao, Z., Buyse, I. M., Simon, D., Sheu, J. C., Perucho, M., and Huang, S. (2001). Preferential loss of a polymorphic RIZ allele in human hepatocellular carcinoma. Br. J. Cancer 84, 743–747. Fang, W., Piao, Z., Simon, D., Sheu, J. C., and Huang, S. (2000). Mapping of a minimal deleted region in human hepatocellular carcinoma to 1p36.13-p36.23 and mutational analysis of the RIZ (PRDM2) gene localized to the region. Genes Chromosomes Cancer 28, 269–275. Farber, E. (1996). The step-by-step development of epithelial cancer: From phenotype to genotype. Adv. Cancer Res. 70, 21–48. Feder, J. N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D. A., Basava, A., Dormishian, F., Domingo, R., Jr., Ellis, M. C., Fullan, A., et al. (1996). A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat. Genet. 13, 399–408. Feitelson, M. A. (1998). Hepatitis B x antigen and p53 in the development of hepatocellular carcinoma. J. Hepatobiliary Pancreat. Surg. 5, 367–374. Feitelson, M. A., and Duan, L. X. (1997). Hepatitis B virus X antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma. Am. J. Pathol. 150, 1141–1157. Fosslien, E. (2000a). Biochemistry of cyclooxygenase (COX)-2 inhibitors and molecular pathology of COX-2 in neoplasia. Crit. Rev. Clin. Lab. Sci. 37, 431–502. Fosslien, E. (2000b). Molecular pathology of cyclooxygenase-2 in neoplasia. Ann. Clin. Lab. Sci. 30, 3–21. Fourel, G., Couturier, J., Wei, Y., Apiou, F., Tiollais, P., and Buendia, M. A. (1994). Evidence for long-range oncogene activation by hepadnavirus insertion. EMBO J. 13, 2526–2534. Fourel, G., Trepo, C., Bougueleret, L., Henglein, B., Ponzetto, A., Tiollais, P., and Buendia, M. A. (1990). Frequent activation of N-myc genes by hepadnavirus insertion in woodchuck liver tumours. Nature 347, 294–298. Frasca, F., Pandini, G., Scalia, P., Sciacca, L., Mineo, R., Costantino, A., Goldﬁne, I. D., Belﬁore, A., and Vigneri, R. (1999). Insulin receptor isoform A, a newly recognized, high-afﬁnity insulin-like growth factor II receptor in fetal and cancer cells. Mol. Cell. Biol. 19, 3278– 3288. Fu, X. X., Su, C. Y., Lee, Y., Hintz, R., Biempica, L., Snyder, R., and Rogler, C. E. (1988). Insulinlike growth factor II expression and oval cell proliferation associated with hepatocarcinogenesis in woodchuck hepatitis virus carriers. J. Virol. 62, 3422–3430. Funato, T., Yokota, J., Sakamoto, H., Kameya, T., Fukushima, S., Ito, N., Terada, M., and Sugimura, T. (1987). Activation of N-ras gene in a rat hepatocellular carcinoma induced by dibutylnitrosamine and butylated hydroxytoluene. Jpn. J. Cancer Res. 78, 689–694. Furumoto, K., Arii, S., Mori, A., Furuyama, H., Gorrin Rivas, M. J., Nakao, T., Isobe, N., Murata, T., Takahashi, C., Noda, M., and Imamura, M. (2001). RECK gene expression

Human Hepatocellular Carcinoma Pathogenesis

97

in hepatocellular carcinoma: correlation with invasion-related clinicopathological factors and its clinical signiﬁcance. Reverse-inducing—cysteine-rich protein with Kazal motifs. Hepatology 33, 189–195. Ganem, D., and Varmus, H. E. (1987). The molecular biology of the hepatitis B viruses. Annu. Rev. Biochem. 56, 651–693. Ghosh, A. K., Majumder, M., Steele, R., Meyer, K., Ray, R., and Ray, R. B. (2000a). Hepatitis C virus NS5A protein protects against TNF-alpha mediated apoptotic cell death. Virus Res. 67, 173–178. Ghosh, A. K., Majumder, M., Steele, R., Yaciuk, P., Chrivia, J., Ray, R., and Ray, R. B. (2000b). Hepatitis C virus NS5A protein modulates transcription through a novel cellular transcription factor SRCAP. J. Biol. Chem. 275, 7184–7188. Giannoudis, A., and Herrington, C. S. (2000). Differential expression of p53 and p21 in low grade cervical squamous intraepithelial lesions infected with low, intermediate, and high risk human papillomaviruses. Cancer 89, 1300–1307. Gorrin-Rivas, M. J., Arii, S., Mori, A., Takeda, Y., Mizumoto, M., Furutani, M., and Imamura, M. (2000). Implications of human macrophage metalloelastase and vascular endothelial growth factor gene expression in angiogenesis of hepatocellular carcinoma. Ann. Surg. 231, 67–73. Gottlob, K., Fulco, M., Levrero, M., and Graessmann, A. (1998). The hepatitis B virus HBx protein inhibits caspase 3 activity. J. Biol. Chem. 273, 33347–33353. Graef, E., Caselmann, W. H., Wells, J., and Koshy, R. (1994). Insertional activation of mevalonate kinase by hepatitis B virus DNA in a human hepatoma cell line. Oncogene 9, 81–87. Grisham, J. W. (1997). Interspecies comparison of liver carcinogenesis: Implications for cancer risk assessment. Carcinogenesis 18, 59–81. Gruner, B. A., DeNapoli, T. S., Andrews, W., Tomlinson, G., Bowman, L., and Weitman, S. D. (1998). Hepatocellular carcinoma in children associated with Gardner syndrome or familial adenomatous polyposis. J. Pediatr. Hematol. Oncol. 20, 274–278. Guan, X. Y., Fang, Y., Sham, J. S., Kwong, D. L., Zhang, Y., Liang, Q., Li, H., Zhou, H., and Trent, J. M. (2000). Recurrent chromosome alterations in hepatocellular carcinoma detected by comparative genomic hybridization. Genes Chromosomes Cancer 29, 110–116. Guirouilh, J., Castroviejo, M., Balabaud, C., Desmouliere, A., and Rosenbaum, J. (2000). Hepatocarcinoma cells stimulate hepatocyte growth factor secretion in human liver myoﬁbroblasts. Int. J. Oncol. 17, 777–781. Guirouilh, J., Le Bail, B., Boussarie, L., Balabaud, C., Bioulac-Sage, P., Desmouliere, A., Schuppan, D., and Rosenbaum, J. (2001). Expression of hepatocyte growth factor in human hepatocellular carcinoma. J. Hepatol. 34, 78–83. Guo, X. Z., Friess, H., Di Mola, F. F., Heinicke, J. M., Abou-Shady, M., Graber, H. U., ¨ Zimmermann, A., Korc, M., and Buchler, M. W. (1998). KAI1, a new metastasis suppressor gene, is reduced in metastatic hepatocellular carcinoma. Hepatology 28, 1481–1488. Hahne, M., Rimoldi, D., Schroter, M., Romero, P., Schreier, M., French, L. E., Schneider, P., Bornand, T., Fontana, A., Lienard, D., et al. (1996). Melanoma cell expression of Fas(Apo1/CD95) ligand: Implications for tumor immune escape. Science 274, 1363–1366. Harris, T. M., Rogler, L. E., and Rogler, C. E. (1998). Reactivation of the maternally imprinted IGF2 allele in TGFalpha induced hepatocellular carcinoma in mice. Oncogene 16, 203– 209. Hayashi, H., Sugio, K., Matsumata, T., Adachi, E., Takenaka, K., and Sugimachi, K. (1995). The clinical signiﬁcance of p53 gene mutation in hepatocellular carcinomas from Japan. Hepatology 22, 1702–1707. Heinze, T., Jonas, S., Karsten, A., and Neuhaus, P. (1999). Determination of the oncogenes p53 and C-erb B2 in the tumour cytosols of advanced hepatocellular carcinoma (HCC) and correlation to survival time. Anticancer Res. 19, 2501–2503.

98

Kern et al.

Herath, N. I., Kew, M. C., Whitehall, V. L., Walsh, M. D., Jass, J. R., Khanna, K. K., Young, J., Powell, L. W., Leggett, B. A., and Macdonald, G. A. (2000). p73 is up-regulated in a subset of hepatocellular carcinomas. Hepatology 31, 601–605. Higaki, K., Yano, H., and Kojiro, M. (1996). Fas antigen expression and its relationship with apoptosis in human hepatocellular carcinoma and noncancerous tissues. Am. J. Pathol. 149, 429–437. Higashitsuji, H., Itoh, K., Nagao, T., Dawson, S., Nonoguchi, K., Kido, T., Mayer, R. J., Arii, S., and Fujita, J. (2000). Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas. Nat. Med. 6, 96–99. Hildt, E., Urban, S., and Hofschneider, P. H. (1995). Characterization of essential domains for the functionality of the MHBst transcriptional activator and identiﬁcation of a minimal MHBst activator. Oncogene 11, 2055–2066. Hino, O., Tabata, S., and Hotta, Y. (1991). Evidence for increased in vitro recombination with insertion of human hepatitis B virus DNA. Proc. Natl. Acad. Sci. USA 88, 9248–9252. Hirohashi, S., Ishak, K. G., Kojiro, M., Wanless, I. R., Theise, N. D., Tsukuma, H., Blum, H. E., Deugnier, Y., Laurent Puig, P., Fischer, H. P., and Sakamoto, M. (2000). Hepatocellular carcinoma. In: “Pathology & Genetics. Tumors of the Digestive System” (S. T. Hamilton and L. A. Aaltonen, Eds.), pp. 159–180. WHO, Geneva. ¨ ¨ Hohler, T., Leininger, S., Kohler, H. H., Schirmacher, P., and Galle, P. R. (2000). Heterozygosity for the hemochromatosis gene in liver diseases—prevalence and effects on liver histology. Liver 20, 482–486. Hsia, C. C., Axiotis, C. A., Di Bisceglie, A. M., and Tabor, E. (1992). Transforming growth factor-alpha in human hepatocellular carcinoma and coexpression with hepatitis B surface antigen in adjacent liver. Cancer 70, 1049–1056. Hsia, C. C., Nakashima, Y., Thorgeirsson, S. S., Harris, C. C., Minemura, M., Momosaki, S., Wang, N. J., and Tabor, E. (2000). Correlation of immunohistochemical staining and mutations of p53 in human hepatocellular carcinoma. Oncol. Rep. 7, 353–356. Hsia, C. C., Thorgeirsson, S. S., and Tabor, E. (1994). Expression of hepatitis B surface and core antigens and transforming growth factor-alpha in “oval cells” of the liver in patients with hepatocellular carcinoma. J. Med. Virol. 43, 216–221. Hsu, H. C., Jeng, Y. M., Mao, T. L., Chu, J. S., Lai, P. L., and Peng, S. Y. (2000). Beta-catenin mutations are associated with a subset of low-stage hepatocellular carcinoma negative for hepatitis B virus and with favorable prognosis. Am. J. Pathol. 157, 763–770. 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 350, 427–428. Huang, H., Fujii, H., Sankila, A., Mahler-Araujo, B. M., Matsuda, M., Cathomas, G., and Ohgaki, H. (1999). Beta-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection. Am. J. Pathol. 155, 1795–1801. Huang, Y., Uchiyama, Y., Fujimura, T., Kanamori, H., Doi, T., Takamizawa, A., Hamakubo, T., and Kodama, T. (2001). A human hepatoma cell line expressing hepatitis C virus nonstructural proteins tightly regulated by tetracycline. Biochem. Biophys. Res. Commun. 281, 732–740. Hui, A. M., Li, X., Makuuchi, M., Takayama, T., and Kubota, K. (1999). Over-expression and lack of retinoblastoma protein are associated with tumor progression and metastasis in hepatocellular carcinoma. Int. J. Cancer 84, 604–608. Hui, A. M., Shi, Y. Z., Li, X., Takayama, T., and Makuuchi, M. (2000). Loss of p16(INK4) protein, alone and together with loss of retinoblastoma protein, correlate with hepatocellular carcinoma progression. Cancer Lett. 154, 93–99. Huo, T. I., Wang, X. W., Forgues, M., Wu, C. G., Spillare, E. A., Giannini, C., Brechot, C., and Harris, C. C. (2001). Hepatitis B virus X mutants derived from human hepatocellular carcinoma retain the ability to abrogate p53-induced apoptosis. Oncogene 20, 3620–3628.

Human Hepatocellular Carcinoma Pathogenesis

99

Ihara, A., Koizumi, H., Hashizume, R., and Uchikoshi, T. (1996). Expression of epithelial cadherin and alpha- and beta-catenins in nontumoral livers and hepatocellular carcinomas. Hepatology 23, 1441–1447. Iizuka, N., Oka, M., Noma, T., Nakazawa, A., Hirose, K., and Suzuki, T. (1995). NM23-H1 and NM23-H2 messenger RNA abundance in human hepatocellular carcinoma. Cancer Res. 55, 652–657. International Working Party. (1995). Terminology of nodular hepatocellular lesions. Hepatology 22, 983–93. Ito, N., Kawata, S., Tamura, S., Takaishi, K., Shirai, Y., Kiso, S., Yabuuchi, I., Matsuda, Y., Nishioka, M., and Tarui, S. (1991). Elevated levels of transforming growth factor beta messenger RNA and its polypeptide in human hepatocellular carcinoma. Cancer Res. 51, 4080– 4083. Itoh, T., Shiro, T., Seki, T., Nakagawa, T., Wakabayashi, M., Inoue, K., and Okamura, A. (2000). Relationship between p53 overexpression and the proliferative activity in hepatocellular carcinoma. Int. J. Mol. Med. 6, 137–142. Jhappan, C., Stahle, C., Harkins, R. N., Fausto, N., Smith, G. H., and Merlino, G. T. (1990). TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 61, 1137–1146. Jia, L., Wang, X. W., and Harris, C. C. (1999). Hepatitis B virus X protein inhibits nucleotide excision repair. Int. J. Cancer 80, 875–879. Joo, M., Kang, Y. K., Kim, M. R., Lee, H. K., and Jang, J. J. (2001). Cyclin D1 overexpression in hepatocellular carcinoma. Liver 21, 89–95. Kanai, F., Lan, K. H., Shiratori, Y., Tanaka, T., Ohashi, M., Okudaira, T., Yoshida, Y., Wakimoto, H., Hamada, H., Nakabayashi, H., et al. (1997). In vivo gene therapy for alphafetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene. Cancer Res. 57, 461–465. Kang-Park, S., Lee, J. H., Shin, J. H., and Lee, Y. I. (2001). Activation of the IGF-II gene by HBV-X protein requires PKC and p44/p42 map kinase signalings. Biochem. Biophys. Res. Commun. 283, 303–307. Kanzler, S., Meyer, E., Lohse, A. W., Schirmacher, P., Henninger, J., Galle, P. R., and Blessing, M. (2001). Hepatocellular expression of a dominant-negative mutant TGF-beta type II receptor accelerates chemically induced hepatocarcinogenesis. Oncogene 20, 5015–5024. Kato, N., Lan, K. H., Ono-Nita, S. K., Shiratori, Y., and Omata, M. (1997). Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator. J. Virol. 71, 8856–8859. Kawakami, Y., Kitamoto, M., Nakanishi, T., Yasui, W., Tahara, E., Nakayama, J., Ishikawa, F., Tahara, H., Ide, T., and Kajiyama, G. (2000). Immuno-histochemical detection of human telomerase reverse transcriptase in human liver tissues. Oncogene 19, 3888–3893. Kawate, S., Ohwada, S., Hamada, K., Koyama, T., Takenoshita, S., Morishita, Y., and Hagiwara, K. (2001). Mutational analysis of the Smad6 and Smad7 genes in hepatocellular carcinoma. Int. J. Mol. Med. 8, 49–52. Kawate, S., Takenoshita, S., Ohwada, S., Mogi, A., Fukusato, T., Makita, F., Kuwano, H., and Morishita, Y. (1999). Mutation analysis of transforming growth factor beta type II receptor, Smad2, and Smad4 in hepatocellular carcinoma. Int. J. Oncol. 14, 127–131. Kekule, A. S., Lauer, U., Meyer, M., Caselmann, W. H., Hofschneider, P. H., and Koshy, R. (1990). The preS2/S region of integrated hepatitis B virus DNA encodes a transcriptional transactivator. Nature 343, 457–461. Kekule, A. S., Lauer, U., Weiss, L., Luber, B., and Hofschneider, P. H. (1993). Hepatitis B virus transactivator HBx uses a tumour promoter signalling pathway. Nature 361, 742–745. Kim, J. R., Kim, S. Y., Kim, M. J., and Kim, J. H. (1998a). Alterations of CDKN2 (MTS1/p16INK4A) gene in parafﬁn-embedded tumor tissues of human stomach, lung, cervix and liver cancers. Exp. Mol. Med. 30, 109–114.

100

Kern et al.

Kim, K. W., Bae, S. K., Lee, O. H., Bae, M. H., Lee, M. J., and Park, B. C. (1998b). Insulin-like growth factor II induced by hypoxia may contribute to angiogenesis of human hepatocellular carcinoma. Cancer Res. 58, 348–351. Kira, S., Nakanishi, T., Suemori, S., Kitamoto, M., Watanabe, Y., and Kajiyama, G. (1997). Expression of transforming growth factor alpha and epidermal growth factor receptor in human hepatocellular carcinoma. Liver 17, 177–182. Kiso, S., Kawata, S., Tamura, S., Ito, N., Takaishi, K., Shirai, Y., Tsushima, H., and Matsuzawa, Y. (1994). Alteration in growth regulation of hepatocytes in primary culture obtained from cirrhotic rat: Poor response to transforming growth factor-beta 1 and interferons. Hepatology 20, 1303–1308. Kiss, A., Wang, N. J., Xie, J. P., and Thorgeirsson, S. S. (1997). Analysis of transforming growth factor (TGF)-alpha/epidermal growth factor receptor, hepatocyte growth Factor/c-met, TGFbeta receptor type II, and p53 expression in human hepatocellular carcinomas. Clin. Cancer Res. 3, 1059–1066. Koga, H., Sakisaka, S., Ohishi, M., Kawaguchi, T., Taniguchi, E., Sasatomi, K., Harada, M., Kusaba, T., Tanaka, M., Kimura, R., et al. (1999). Expression of cyclooxygenase-2 in human hepatocellular carcinoma: Relevance to tumor dedifferentiation. Hepatology 29, 688–696. Kohzato, N., Dong, Y., Sui, L., Masaki, T., Nagahata, S., Nishioka, M., Konishi, R., and Tokuda, M. (2001). Overexpression of cyclin E and cyclin-dependent kinase 2 is correlated with development of hepatocellular carcinomas. Hepatol. Res. 21, 27–39. Kondo, Y. (1997). Pathology of early hepatocellular carcinoma and preneoplastic lesions in the liver. In: “Liver Cancer” (K. Okud and E. Tabor, Eds.), pp. 135–154. Churchill Livingstone, New York. Kondoh, N., Wakatsuki, T., Hada, A., Shuda, M., Tanaka, K., Arai, M., and Yamamoto, M. (2001). Genetic and epigenetic events in human hepatocarcinogenesis. Int. J. Oncol. 18, 1271–1278. Koyama, M., Nagai, H., Bando, K., Matsumoto, S., Tajiri, T., Onda, M., Ito, M., Moriyama, Y., and Emi, M. (2000). New target region of allelic loss in hepatocellular carcinomas within a 1-cM interval on chromosome 6q23. J. Hepatol. 33, 85–90. Kozyraki, R., Scoazec, J. Y., Flejou, J. F., D’Errico, A., Bedossa, P., Terris, B., Fiorentino, M., Bringuier, A. F., Grigioni, W. F., and Feldmann, G. (1996). Expression of cadherins and alpha-catenin in primary epithelial tumors of the liver. Gastroenterology 110, 1137–1149. Kubicka, S., Rudolph, K. L., Hanke, M., Tietze, M. K., Tillmann, H. L., Trautwein, C., and Manns, M. (2000). Hepatocellular carcinoma in Germany: A retrospective epidemiological study from a low-endemic area. Liver 20, 312–318. Kudo, I., and Murakami, M. (1999). Diverse functional coupling of prostanoid biosynthetic enzymes in various cell types. Adv. Exp. Med. Biol. 469, 29–35. Kulik, G., Klippel, A., and Weber, M. J. (1997). Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol. Cell. Biol. 17, 1595– 1606. Kusano, N., Shiraishi, K., Kubo, K., Oga, A., Okita, K., and Sasaki, K. (1999). Genetic aberrations detected by comparative genomic hybridization in hepatocellular carcinomas: Their relationship to clinicopathological features. Hepatology 29, 1858–1862. Lara-Pezzi, E., Roche, S., Andrisani, O. M., Sanchez-Madrid, F., and Lopez-Cabrera, M. (2001a). The hepatitis B virus HBx protein induces adherens junction disruption in a srcdependent manner. Oncogene 20, 3323–3331. Lara-Pezzi, E., Serrador, J. M., Montoya, M. C., Zamora, D., Yanez-Mo, M., Carretero, M., Furthmayr, H., Sanchez-Madrid, F., and Lopez-Cabrera, M. (2001b). The hepatitis B virus X protein (HBx) induces a migratory phenotype in a CD44-dependent manner: Possible role of HBx in invasion and metastasis. Hepatology 33, 1270–1281.

Human Hepatocellular Carcinoma Pathogenesis

101

Laurent-Puig, P., Legoix, P., Bluteau, O., Belghiti, J., Franco, D., Binot, F., Monges, G., Thomas, G., Bioulac-Sage, P., and Zucman-Rossi, J. (2001). Genetic alterations associated with hepatocellular carcinomas deﬁne distinct pathways of hepatocarcinogenesis. Gastroenterology 120, 1763–1773. 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. Lee, H. S., Yoon, J. H., Kamimura, S., Iwata, K., Watanabe, H., and Kim, C. Y. (1997). Lack of association of cytochrome P450 2E1 genetic polymorphisms with the risk of human hepatocellular carcinoma. Int. J. Cancer 71, 737–740. Lee, S., Park, U., and Lee, Y. I. (2001a). Hepatitis C virus core protein transactivates insulinlike growth factor II gene transcription through acting concurrently on Egr1 and Sp1 sites. Virology 283, 167–177. Lee, Y. I., Kang-Park, S., and Do, S. I. (2001b). The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade. J. Biol. Chem. 276, 16969– 16977. Lee, Y. I., Lee, S., Das, G. C., Park, U. S., and Park, S. M. (2000). Activation of the insulin-like growth factor II transcription by aﬂatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes: Implications for a gain-of-function during the formation of hepatocellular carcinoma. Oncogene 19, 3717–3726. Lee, Y. I., Lee, S., Lee, Y., Bong, Y. S., Hyun, S. W., Yoo, Y. D., Kim, S. J., Kim, Y. W., and Poo, H. R. (1998). The human hepatitis B virus transactivator X gene product regulates Sp1 mediated transcription of an insulin-like growth factor II promoter 4. Oncogene 16, 2367–2380. Levine, A. J. (1997). p53, the cellular gatekeeper for growth and divison. Cell 88, 323–331. ¨ Li, X., Nong, Z., Ekstrom, C., Larsson, E., Nordlinder, H., Hofmann, W. J., Trautwein, C., ¨ Odenthal, M., Dienes, H. P., Ekstrom, T. J., and Schirmacher, P. (1997). Disrupted IGF2 promoter control by silencing of promoter P1 in human hepatocellular carcinoma. Cancer Res. 57, 2048–2054. Liew, C. T., Li, H. M., Lo, K. W., Leow, C. K., Chan, J. Y., Hin, L. Y., Lau, W. Y., Lai, P. B., Lim, B. K., Huang, J., et al. (1999). High frequency of p16INK4A gene alterations in hepatocellular carcinoma. Oncogene 18, 789–795. Lin, K. H., Lin, Y. W., Lee, H. F., Liu, W. L., Chen, S. T., Chang, K. S., and Cheng, S. Y. (1995). Increased invasive activity of human hepatocellular carcinoma cells is associated with an overexpression of thyroid hormone beta 1 nuclear receptor and low expression of the anti-metastatic nm23 gene. Cancer Lett. 98, 89–95. Lin, S. B., Hsieh, S. H., Hsu, H. L., Lai, M. Y., Kan, L. S., and Au, L. C. (1997). Antisense oligodeoxynucleotides of IGF-II selectively inhibit growth of human hepatoma cells overproducing IGF-II. J. Biochem. (Tokyo) 122, 717–722. Liotta, L. A., Rao, C. N., and Wewer, U. M. (1986). Biochemical interactions of tumor cells with the basement membrane. Annu. Rev. Biochem. 55, 1037–1057. Liotta, L. A., Steeg, P. S., and Stetler-Stevenson, W. G. (1991). Cancer metastasis and angiogenesis: An imbalance of positive and negative regulation. Cell 64, 327–336. Liu, J., Stevens, J., Rote, C. A., Yost, H. J., Hu, Y., Neufeld, K. L., White, R. L., and Matsunami, N. (2001). Siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein. Mol. Cell 7, 927–936. Liu, P., Terradillos, O., Renard, C. A., Feldmann, G., Buendia, M. A., and Bernuau, D. (1997). Hepatocarcinogenesis in woodchuck hepatitis virus/c-myc mice: Sustained cell proliferation and biphasic activation of insulin-like growth factor II. Hepatology 25, 874–883. Livraghi, T. (1997). Ethanol injection for the treatment of hepatocellular carcinoma. In: “Liver Cancer” (K. Okuda and E. Tabor, Eds.), pp. 497–510. Churchill Livingstone, New York.

102

Kern et al.

¨ Lohmann, V., Korner, F., Koch, J. O., and Bartenschlager, R. (2000). Subgenomic hepatitis C virus replicons: A novel system to study replication and pathogenesis in cell culture. In: “Chronic Hepatitis” (H. P. Dienes, P. Schirmacher, C. Brechot, and K. Okuda, Eds.), pp. 34– 44. Kluwer Academic Publishers, Boston. Lu, W., Lo, S. Y., Chen, M., Wu, K., Fung, Y. K., and Ou, J. H. (1999). Activation of p53 tumor suppressor by hepatitis C virus core protein. Virology 264, 134–141. ¨ ¨ Luber, B., Arnold, N., Sturzl, M., Hohne, M., Schirmacher, P., Lauer, U., Wienberg, J., Hofschneider, P. H., and Kekule, A. S. (1996). Hepatoma-derived integrated HBV DNA causes multi-stage transformation in vitro. Oncogene 12, 1597–1608. Machida, K., Tsukiyama-Kohara, K., Seike, E., Tone, S., Shibasaki, F., Shimizu, M., Takahashi, H., Hayashi, Y., Funata, N., Taya, C., et al. (2001). Inhibition of cytochrome c release in Fasmediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins. J. Biol. Chem. 276, 12140–12146. Mahe, Y., Mukaida, N., Kuno, K., Akiyama, M., Ikeda, N., Matsushima, K., and Murakami, S. (1991). Hepatitis B virus X protein transactivates human interleukin-8 gene through acting on nuclear factor kB and CCAAT/enhancer-binding protein- like cis-elements. J. Biol. Chem. 266, 13759–13763. Major, M. E., and Feinstone, S. M. (1997). The molecular virology of hepatitis C. Hepatology 25, 1527–1538. Majumder, M., Ghosh, A. K., Steele, R., Ray, R., and Ray, R. B. (2001). Hepatitis C virus NS5A physically associates with p53 and regulates p21/waf1 gene expression in a p53-dependent manner. J. Virol. 75, 1401–1407. Marchio, A., Meddeb, M., Pineau, P., Danglot, G., Tiollais, P., Bernheim, A., and Dejean, A. (1997). Recurrent chromosomal abnormalities in hepatocellular carcinoma detected by comparative genomic hybridization. Genes Chromosomes Cancer 18, 59–65. Marchio, A., Pineau, P., Meddeb, M., Terris, B., Tiollais, P., Bernheim, A., and Dejean, A. (2000). Distinct chromosomal abnormality pattern in primary liver cancer of non-B, non-C patients. Oncogene 19, 3733–3738. Marchio, A., Terris, B., Meddeb, M., Pineau, P., Duverger, A., Tiollais, P., Bernheim, A., and Dejean, A. (2001). Chromosomal abnormalities in liver cell dysplasia detected by comparative genomic hybridisation. Mol. Pathol. 54, 270–274. Matsuda, Y., Ichida, T., Matsuzawa, J., Sugimura, K., and Asakura, H. (1999). p16(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology 116, 394–400. Matsumura, T., Makino, R., and Mitamura, K. (2001). Frequent down-regulation of E-cadherin by genetic and epigenetic changes in the malignant progression of hepatocellular carcinomas. Clin. Cancer Res. 7, 594–599. Matsuzaki, K., Date, M., Furukawa, F., Tahashi, Y., Matsushita, M., Sakitani, K., Yamashiki, N., Seki, T., Saito, H., Nishizawa, M., et al. (2000). Autocrine stimulatory mechanism by transforming growth factor beta in human hepatocellular carcinoma. Cancer Res. 60, 1394–1402. Matsuzawa, S. I., and Reed, J. C. (2001). Siah-1, SIP, and Ebi collaborate in a novel pathway for beta-catenin degradation linked to p53 responses. Mol. Cell 7, 915–926. Maxwell, P. H., Dachs, G. U., Gleadle, J. M., Nicholls, L. G., Harris, A. L., Stratford, I. J., Hankinson, O., Pugh, C. W., and Ratcliffe, P. J. (1997). Hypoxia-inducible factor-1 modulates gene expression in solid tumors and inﬂuences both angiogenesis and tumor growth. Proc. Natl. Acad. Sci. USA 94, 8104–8109. Mion, F., Grozel, L., Boillot, O., Paliard, P., and Berger, F. (1996). Adult cirrhotic liver explants: Precancerous lesions and undetected small hepatocellular carcinomas. Gastroenterology 111, 1587–1592.

Human Hepatocellular Carcinoma Pathogenesis

103

Mise, M., Arii, S., Higashituji, H., Furutani, M., Niwano, M., Harada, T., Ishigami, S., Toda, Y., Nakayama, H., Fukumoto, M., et al. (1996). Clinical signiﬁcance of vascular endothelial growth factor and basic ﬁbroblast growth factor gene expression in liver tumor. Hepatology 23, 455–464. Miyoshi, Y., Iwao, K., Nagasawa, Y., Aihara, T., Sasaki, Y., Imaoka, S., Murata, M., Shimano, T., and Nakamura, Y. (1998). Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3. Cancer Res. 58, 2524–2527. ¨ Moller, P., Koretz, K., Leithauser, F., Bruderlein, S., Henne, C., Quentmeier, A., and Krammer, P. H. (1994). Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium. Int. J. Cancer 57, 371–377. Morimitsu, Y., Hsia, C. C., Kojiro, M., and Tabor, E. (1995). Nodules of less-differentiated tumor within or adjacent to hepatocellular carcinoma: Relative expression of transforming growth factor-alpha and its receptor in the different areas of tumor. Hum. Pathol. 26, 1126– 1132. Moriya, K., Fujie, H., Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K., Matsuura, Y., Kimura, S., Miyamura, T., and Koike, K. (1998). The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat. Med. 4, 1065–1067. Murakami, Y., Hayashi, K., Hirohashi, S., and Sekiya, T. (1991). Aberrations of the tumor suppressor p53 and retinoblastoma genes in human hepatocellular carcinomas. Cancer Res. 51, 5520–5525. Murphy, L. J. (1998). Insulin-like growth factor-binding proteins: Functional diversity or redundancy. J. Mol. Endocrinol. 21, 97–107. Musso, O., Rehn, M., Theret, N., Turlin, B., Bioulac-Sage, P., Lotrian, D., Campion, J. P., Pihlajaniemi, T., and Clement, B. (2001). Tumor progression is associated with a signiﬁcant decrease in the expression of the endostatin precursor collagen XVIII in human hepatocellular carcinomas. Cancer Res. 61, 45–49. Nagai, H., Pineau, P., Tiollais, P., Buendia, M. A., and Dejean, A. (1997). Comprehensive allelotyping of human hepatocellular carcinoma. Oncogene 14, 2927–2933. Nagaya, T., Nakamura, T., Tokino, T., Tsurimoto, T., Imai, M., Mayumi, T., Kamino, K., Yamamura, K., and Matsubara, K. (1987). The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma. Genes Dev. 1, 773–782. Nakayama, J., Tahara, H., Tahara, E., Saito, M., Ito, K., Nakamura, H., Nakanishi, T., Ide, T., and Ishikawa, F. (1998). Telomerase activation by hTRT in human normal ﬁbroblasts and hepatocellular carcinomas. Nat. Genet. 18, 65–68. Nalesnik, M. A., Lee, R. G., and Carr, B. I. (1998). Transforming growth factor alpha (TGFalpha) in hepatocellular carcinomas and adjacent hepatic parenchyma. Hum. Pathol. 29, 228–234. Nassal, M. (2000). Mechanisms of hepatitis B virus replication. In “Chronic Hepatitis” (H. P. Dienes, P. Schirmacher, C. Brechot, and K. Okuda, Eds.), pp. 3–22. Kluwer Academic Publishers, Boston. Neaud, V., Faouzi, S., Guirouilh, J., Le Bail, B., Balabaud, C., Bioulac-Sage, P., and Rosenbaum, J. (1997). Human hepatic myoﬁbroblasts increase invasiveness of hepatocellular carcinoma cells: Evidence for a role of hepatocyte growth factor. Hepatology 26, 1458–1466. Ng, I. O., Lee, J. M., Srivastava, G., and Ng, M. (1998). Expression of insulin-like growth factor II mRNA in hepatocellular carcinoma. J. Gastroenterol. Hepatol. 13, 152–157. Ng, I. O., Liang, Z. D., Cao, L., and Lee, T. K. (2000). DLC-1 is deleted in primary hepatocellular carcinoma and exerts inhibitory effects on the proliferation of hepatoma cell lines with deleted DLC-1. Cancer Res. 60, 6581–6584. Nguyen, M., Shing, Y., and Folkman, J. (1994). Quantitation of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane. Microvasc. Res. 47, 31–40.

104

Kern et al.

Niederau, C., Fischer, R., Sonnenberg, A., Stremmel, W., Trampisch, H. J., and Strohmeyer, G. (1985). Survival and causes of death in cirrhotic and in noncirrhotic patients with primary hemochromatosis. N. Engl. J. Med. 313, 1256–1262. Nielsen, F. C. (1992). The molecular and cellular biology of insulin-like growth factor II. Prog. Growth Factor Res. 4, 257–290. Nielsen, F. C., Ostergaard, L., Nielsen, J., and Christiansen, J. (1995). Growth-dependent translation of IGF-II mRNA by a rapamycin-sensitive pathway. Nature 377, 358–362. Niketeghad, F., Decker, H. J., Caselmann, W. H., Lund, P., Geissler, F., Dienes, H. P., and Schirmacher, P. (2001). Frequent genomic imbalances suggest commonly altered tumour genes in human hepatocarcinogenesis. Br. J. Cancer 85, 697–704. Nishida, N., Fukuda, Y., Kokuryu, H., Toguchida, J., Yandell, D. W., Ikenega, M., Imura, H., and Ishizaki, K. (1993). Role and mutational heterogeneity of the p53 gene in hepatocellular carcinoma. Cancer Res. 53, 368–372. Nishida, N., Fukuda, Y., Komeda, T., Kita, R., Sando, T., Furukawa, M., Amenomori, M., Shibagaki, I., Nakao, K., Ikenaga, M., et al. (1994). Ampliﬁcation and overexpression of the cyclin D1 gene in aggressive human hepatocellular carcinoma. Cancer Res. 54, 3107– 3110. Nishimoto, A., Miura, N., Horikawa, I., Kugoh, H., Murakami, Y., Hirohashi, S., Kawasaki, H., Gazdar, A. F., Shay, J. W., Barrett, J. C., and Oshimura, M. (2001). Functional evidence for a telomerase repressor gene on human chromosome 10p15.1. Oncogene 20, 828–835. Noguchi, O., Enomoto, N., Ikeda, T., Kobayashi, F., Marumo, F., and Sato, C. (1996). Gene expressions of c-met and hepatocyte growth factor in chronic liver disease and hepatocellular carcinoma. J. Hepatol. 24, 286–292. Nolan, C. M., and Lawlor, M. A. (1999). Variable accumulation of insulin-like growth factor II in mouse tissues deﬁcient in insulin-like growth factor II receptor. Int. J. Biochem. Cell Biol. 31, 1421–1433. Novak, A., and Dedhar, S. (1999). Signaling through beta-catenin and Lef/Tcf. Cell. Mol. Life Sci. 56, 523–537. Ochiya, T., Tsurimoto, T., Ueda, K., Okubo, K., Shiozawa, M., and Matsubara, K. (1989). An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes. Proc. Natl. Acad. Sci. USA 86, 1875–1879. Ohashi, R., Gao, C., Miyazaki, M., Hamazaki, K., Tsuji, T., Inoue, Y., Uemura, T., Hirai, R., Shimizu, N., and Namba, M. (2001). Enhanced expression of cyclin E and cyclin A in human hepatocellular carcinomas. Anticancer Res. 21, 657–662. Ohlsson, R., Nystrom, A., Pfeifer-Ohlsson, S., Tohonen, V., Hedborg, F., Schoﬁeld, P., Flam, F., ¨ T. J. (1993). IGF2 is parentally imprinted during human embryogenesis and in and Ekstrom, the Beckwith-Wiedemann syndrome. Nat. Genet. 4, 94–97. Okabe, H., Ikai, I., Matsuo, K., Satoh, S., Momoi, H., Kamikawa, T., Katsura, N., Nishitai, R., Takeyama, O., Fukumoto, M., and Yamaoka, Y. (2000). Comprehensive allelotype study of hepatocellular carcinoma: Potential differences in pathways to hepatocellular carcinoma between hepatitis B virus-positive and -negative tumors. Hepatology 31, 1073– 1079. Okabe, H., Satoh, S., Kato, T., Kitahara, O., Yanagawa, R., Yamaoka, Y., Tsunoda, T., Furukawa, Y., and Nakamura, Y. (2001). Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: Identiﬁcation of genes involved in viral carcinogenesis and tumor progression. Cancer Res. 61, 2129–2137. Okada, S. (1997). Chemotherapy for hepatocellular carcinoma. In: “Liver Cancer” (K. Okuda and E. Tabor, Eds.), pp. 441–448. Churchill Livingstone, New York. Okubo, K., Nakamura, T., Tokino, T., and Matsubara, K. (1990). Different type of hepatitis B virus (HBV) DNA integrants that may reﬂect the integration process. Gastroenterol. Jpn. 25 (Suppl 2), 23–30.

Human Hepatocellular Carcinoma Pathogenesis

105

Ono, M., Morisawa, K., Nie, J., Ota, K., Taniguchi, T., Saibara, T., and Onishi, S. (1998). Transactivation of transforming growth factor alpha gene by hepatitis B virus preS1. Cancer Res. 58, 1813–1816. Osada, T., Sakamoto, M., Ino, Y., Iwamatsu, A., Matsuno, Y., Muto, T., and Hirohashi, S. (1996). E-cadherin is involved in the intrahepatic metastasis of hepatocellular carcinoma. Hepatology 24, 1460–1467. Osuga, T., Matsuzaki, Y., Chiba, T., and Tsujii, H. (1997). Radiotherapy. In: “Liver Cancer” (K. Okuda and E. Tabor, Eds.), pp. 481–496. Churchill Livingstone, New York. Otsuka, M., Kato, N., Lan, K., Yoshida, H., Kato, J., Goto, T., Shiratori, Y., and Omata, M. (2000). Hepatitis C virus core protein enhances p53 function through augmentation of DNA binding afﬁnity and transcriptional ability. J. Biol. Chem. 275, 34,122–34,130. Ozaki, I., Mizuta, T., Zhao, G., Yotsumoto, H., Hara, T., Kajihara, S., Hisatomi, A., Sakai, T., and Yamamoto, K. (2000). Involvement of the Ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma. Cancer Res. 60, 6519–6525. Ozturk, M. (1999). Genetic aspects of hepatocellular carcinogenesis. Semin. Liver. Dis. 19, 235–242. Paggi, M. G., and Giordano, A. (2001). Who is the boss in the retinoblastoma family? The point of view of Rb2/p130, the little brother. Cancer Res. 61, 4651–4654. Park, U. S., Park, S. K., Lee, Y. I., and Park, J. G. (2000a). Hepatitis B virus-X protein upregulates the expression of p21waf1/cip1 and prolongs G1→S transition via a p53-independent pathway in human hepatoma cells. Oncogene 19, 3384–3394. Park, W. S., Dong, S. M., Kim, S. Y., Na, E. Y., Shin, M. S., Pi, J. H., Kim, B. J., Bae, J. H., Hong, Y. K., Lee, K. S., et al. (1999). Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas. Cancer Res. 59, 307–310. Park, Y. N., Kim, Y. B., Yang, K. M., and Park, C. (2000b). Increased expression of vascular endothelial growth factor and angiogenesis in the early stage of multistep hepatocarcinogenesis. Arch. Pathol. Lab. Med. 124, 1061–1065. Pasquinelli, C., Bhavani, K., Schirmacher, P., Rogler, C. E., and Chisari, F. V. (1994). Expression of hepatocyte mitogens and mitogen receptors during hepatocarcinogenesis in hepatitis B virus transgenic mice. Transgenics 1, 451–457. Pasquinelli, C., Garreau, F., Bougueleret, L., Cariani, E., Grzeschik, K. H., Thiers, V., Croissant, O., Hadchouel, M., Tiollais, P., and Brechot, C. (1988). Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors. J. Virol. 62, 629–632. Pawlik, T. M., Nakamura, H., Yoon, S. S., Mullen, J. T., Chandrasekhar, S., Chiocca, E. A., and Tanabe, K. K. (2000). Oncolysis of diffuse hepatocellular carcinoma by intravascular administration of a replication-competent, genetically engineered herpesvirus. Cancer Res. 60, 2790–2795. Pawlotsky, J. M. (1999). Diagnostic tests for hepatitis C. J. Hepatol. 31, 71–79. Piao, Z., Park, C., Park, J. H., and Kim, H. (1998). Allelotype analysis of hepatocellular carcinoma. Int. J. Cancer 75, 29–33. Pineau, P., Nagai, H., Prigent, S., Wei, Y., Gyapay, G., Weissenbach, J., Tiollais, P., Buendia, M. A., and Dejean, A. (1999). Identiﬁcation of three distinct regions of allelic deletions on the short arm of chromosome 8 in hepatocellular carcinoma. Oncogene 18, 3127–3134. Pitot, H. C. (1994). Hepatic neoplasia: Chemical induction. In: “The liver. Biology and pathobiology,” 3rd ed. (I. M. Arias, W. B. Jakoby, H. Popper, D. Schachter, and D. A. Shaftritz, Eds.), pp. 1467–1495. Raven, New York. Polakis, P. (2001). More than one way to skin a catenin. Cell 105, 563–566. Poussin, K., Dienes, H., Sirma, H., Urban, S., Beaugrand, M., Franco, D., Schirmacher, P., Brechot, C., and Paterlini Brechot, P. (1999). Expression of mutated hepatitis B virus X genes in human hepatocellular carcinomas. Int. J. Cancer 80, 497–505.

106

Kern et al.

Prange, W., and Schirmacher, P. (2001). Absence of therapeutically relevant c-erbB-2 expression in human hepatocellular carcinomas. Oncol. Rep. 8, 727–730. Qin, L. X., Tang, Z. Y., Sham, J. S., Ma, Z. C., Ye, S. L., Zhou, X. D., Wu, Z. Q., Trent, J. M., and Guan, X. Y. (1999). The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma. Cancer Res. 59, 5662–5665. Qiu, S. J., Ye, S. L., Wu, Z. Q., Tang, Z. Y., and Liu, Y. K. (1998). The expression of the mdm2 gene may be related to the aberration of the p53 gene in human hepatocellular carcinoma. J. Cancer Res. Clin. Oncol. 124, 253–258. Raimondo, G., Burk, R. D., Lieberman, H. M., Muschel, J., Hadziyannis, S. J., Will, H., Kew, M. C., Dusheiko, G. M., and Shafritz, D. A. (1988). Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma. Virology 166, 103– 112. Rao, U. N., Gollin, S. M., Beaves, S., Cieply, K., Nalesnik, M., and Michalopoulos, G. K. (2001). Comparative genomic hybridization of hepatocellular carcinoma: Correlation with ﬂuorescence in situ hybridization in parafﬁn-embedded tissue. Mol. Diagn. 6, 27–37. Ray, R. B., Lagging, L. M., Meyer, K., and Ray, R. (1996). Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo ﬁbroblasts to tumorigenic phenotype. J. Virol. 70, 4438–4443. Ray, R. B., Meyer, K., and Ray, R. (2000). Hepatitis C virus core protein promotes immortalization of primary human hepatocytes. Virology 271, 197–204. Ray, R. B., Meyer, K., Steele, R., Shrivastava, A., Aggarwal, B. B., and Ray, R. (1998a). Inhibition of tumor necrosis factor (TNF-alpha)-mediated apoptosis by hepatitis C virus core protein. J. Biol. Chem. 273, 2256–2259. Ray, R. B., Steele, R., Meyer, K., and Ray, R. (1997). Transcriptional repression of p53 promoter by hepatitis C virus core protein. J. Biol. Chem. 272, 10983–10986. Ray, R. B., Steele, R., Meyer, K., and Ray, R. (1998b). Hepatitis C virus core protein represses p21WAF1/Cip1/Sid1 promoter activity. Gene 208, 331–336. Rechler, M. M., and Clemmons, D. R. (1998). Regulatory actions of insulin-like growth factor binding proteins. Trends Endocrinol. Metabol. 9, 176–183. Rolles, K. (1997). Transplantation for liver cancer. In: “Liver Cancer” (K. Okuda, and E. Tabor, Eds.), pp. 531–536. Churchill Livingstone, New York. Roncalli, M., Roz, E., Coggi, G., Di Rocco, M. G., Bossi, P., Minola, E., Gambacorta, M., and Borzio, M. (1999). The vascular proﬁle of regenerative and dysplastic nodules of the cirrhotic liver: Implications for diagnosis and classiﬁcation. Hepatology 30, 1174– 1178. Ruggieri, A., Harada, T., Matsuura, Y., and Miyamura, T. (1997). Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein. Virology 229, 68–76. Ruster, B., Zeuzem, S., and Roth, W. K. (1996). Hepatitis C virus sequences encoding truncated core proteins detected in a hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 219, 911–915. Sadot, E., Geiger, B., Oren, M., and Ben-Ze’ev, A. (2001). Down-regulation of beta-catenin by activated p53. Mol. Cell. Biol. 21, 6768–6781. Sakakura, C., Hagiwara, A., Taniguchi, H., Yamaguchi, T., Yamagishi, H., Takahashi, T., Koyama, K., Nakamura, Y., Abe, T., and Inazawa, J. (1999). Chromosomal aberrations in human hepatocellular carcinomas associated with hepatitis C virus infection detected by comparative genomic hybridization. Br. J. Cancer 80, 2034–2039. Sandgren, E. P. (1995). Transgenic models of hepatic growth regulation and hepatocarcinogenesis. In “Liver regeneration and carcinogenesis” (R. L. Jirtle, Ed.), pp. 257–300. Academic Press, San Diego. Santoni-Rugiu, E., Preisegger, K. H., Kiss, A., Audolfsson, T., Shiota, G., Schmidt, E. V., and Thorgeirsson, S. S. (1996). Inhibition of neoplastic development in the liver by

Human Hepatocellular Carcinoma Pathogenesis

107

hepatocyte growth factor in a transgenic mouse model. Proc. Natl. Acad. Sci. USA 93, 9577– 9582. Satoh, S., Daigo, Y., Furukawa, Y., Kato, T., Miwa, N., Nishiwaki, T., Kawasoe, T., Ishiguro, H., Fujita, M., Tokino, T., et al. (2000). AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat. Genet. 24, 245–250. Sayan, A. E., Sayan, B. S., Findikli, N., and Ozturk, M. (2001). Acquired expression of transcriptionally active p73 in hepatocellular carcinoma cells. Oncogene 20, 5111–5117. Scharf, J. G., Schmidt-Sandte, W., Pahernik, S. A., Ramadori, G., Braulke, T., and Hartmann, H. (1998). Characterization of the insulin-like growth factor axis in a human hepatoma cell line (PLC). Carcinogenesis 19, 2121–2128. Schirmacher, P., Held, W. A., Yang, D., Biempica, L., and Rogler, C. E. (1991). Selective ampliﬁcation of periportal transitional cells precedes formation of hepatocellular carcinoma in SV40 large tag transgenic mice. Am. J. Pathol. 139, 231–241. Schirmacher, P., Held, W. A., Yang, D., Chisari, F. 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., Schauss, D., and Dienes, H. P. (1996). Intracellular accumulation of incompletely processed transforming growth factor-alpha polypeptides in ground glass hepatocytes of chronic hepatitis B virus infection. J. Hepatol. 24, 547–554. Schirmacher, P., Wang, H., Stahnke, G., Will, H., and Rogler, C. E. (1995). Sequences and structures at hepadnaviral integration: Recombination sites implicate topoisomerase I in hepadnaviral DNA rearrangements and integration. J. Hepatol. 22(Supp 1), 21–33. ¨ Schluter, V., Meyer, M., Hofschneider, P. H., Koshy, R., and Caselmann, W. H. (1994). Integrated hepatitis B virus X and 3 truncated preS/S sequences derived from human hepatomas encode functionally active transactivators. Oncogene 9, 3335–3344. Sciacca, L., Costantino, A., Pandini, G., Mineo, R., Frasca, F., Scalia, P., Sbraccia, P., Goldﬁne, I. D., Vigneri, R., and Belﬁore, A. (1999). Insulin receptor activation by IGF-II in breast cancers: Evidence for a new autocrine/paracrine mechanism. Oncogene 18, 2471–2479. Selden, C., Farnaud, S., Ding, S. F., Habib, N., Foster, C., and Hodgson, H. J. (1994). Expression of hepatocyte growth factor mRNA, and c-met mRNA (hepatocyte growth factor receptor) in human liver tumours. J. Hepatol. 21, 227–234. Sheikh, M. S., and Fornace, A. J., Jr. (2000). Role of p53 family members in apoptosis. J. Cell Physiol. 182, 171–181. 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. Shimoda, K., Mori, M., Shibuta, K., Banner, B. F., and Barnard, G. F. (1999). Vascular endothelial growth factor/vascular permeability factor mRNA expression in patients with chronic hepatitis C and hepatocellular carcinoma. Int. J. Oncol. 14, 353–359. Shirai, Y., Kawata, S., Ito, N., Tamura, S., Takaishi, K., Kiso, S., Tsushima, H., and Matsuzawa, Y. (1992). Elevated levels of plasma transforming growth factor-beta in patients with hepatocellular carcinoma. Jpn. J. Cancer Res. 83, 676–679. Shiraishi, K., Okita, K., Kusano, N., Harada, T., Kondoh, S., Okita, S., Ryozawa, S., Ohmura, R., Noguchi, T., Iida, Y., et al. (2001). A comparison of DNA copy number changes detected by comparative genomic hybridization in malignancies of the liver, biliary tract and pancreas. Oncology 60, 151–161. Shouval, D. (1997). Immunotherapy for hepatocellular carcinoma. In: “Liver Cancer” (K. Okuda and E. Tabor, Eds.), pp. 471–480. Churchill Livingstone, New York. Sigal, A., and Rotter, V. (2000). Oncogenic mutation of the p53 tumor suppressor: The demons of the guardian of the genome. Cancer Res. 60, 6788–6793.

108

Kern et al.

Sinha, S., Webber, C., Marshall, C. J., Knowles, M. A., Proctor, A., Barrass, N. C., and Neal, G. E. (1988). Activation of ras oncogene in aﬂatoxin-induced rat liver carcinogenesis. Proc. Natl. Acad. Sci. USA 85, 3673–3677. Slagle, B. L., Zhou, Y. Z., and Butel, J. S. (1991). Hepatitis B virus integration event in human chromosome 17p near the p53 gene identiﬁes the region of the chromosome commonly deleted in virus-positive hepatocellular carcinomas. Cancer Res. 51, 49–54. Sohda, T., Oka, Y., Iwata, K., Gunn, J., Kamimura, S., Shijo, H., Okumura, M., and Yun, K. (1997). Co-localisation of insulin-like growth factor II and the proliferation marker MIB1 in hepatocellular carcinoma cells. J. Clin. Pathol. 50, 135–137. Sohda, T., Yun, K., Iwata, K., Soejima, H., and Okumura, M. (1996). Increased expression of insulin-like growth factor 2 in hepatocellular carcinoma is primarily regulated at the transcriptional level. Lab. Invest. 75, 307–311. ¨ Strand, S., Hofmann, W. J., Hug, H., Muller, M., Otto, G., Strand, D., Mariani, S. M., Stremmel, W., Krammer, P. H., and Galle, P. R. (1996). Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells—a mechanism of immune evasion? Nat. Med. 2, 1361–1366. Su, F., Theodosis, C. N., and Schneider, R. J. (2001a). Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx protein. J. Virol. 75, 215–225. Su, L. K., Abdalla, E. K., Law, C. H., Kohlmann, W., Rashid, A., Vauthey, J. N. (2001b). Biallelic inactivation of the APC gene is associated with hepatocellular carcinoma in familial adenomatous polyposis coli. Cancer 92, 332–339. Suda, T., Isokawa, O., Aoyagi, Y., Nomoto, M., Tsukada, K., Shimizu, T., Suzuki, Y., Naito, A., Igarashi, H., Yanagi, M., et al. (1998). Quantitation of telomerase activity in hepatocellular carcinoma: A possible aid for a prediction of recurrent diseases in the remnant liver. Hepatology 27, 402–406. Suzuki, K., Hayashi, N., Miyamoto, Y., Yamamoto, M., Ohkawa, K., Ito, Y., Sasaki, Y., Yamaguchi, Y., Nakase, H., Noda, K., et al. (1996). Expression of vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma. Cancer Res. 56, 3004–3009. Suzuki, K., Hayashi, N., Yamada, Y., Yoshihara, H., Miyamoto, Y., Ito, Y., Ito, T., Katayama, K., Sasaki, Y., Ito, A., et al. (1994). Expression of the c-met protooncogene in human hepatocellular carcinoma. Hepatology 20, 1231–1236. Suzuki, R., Matsuura, Y., Suzuki, T., Ando, A., Chiba, J., Harada, S., Saito, I., and Miyamura, T. (1995). Nuclear localization of the truncated hepatitis C virus core protein with its hydrophobic C terminus deleted. J. Gen. Virol. 76, 53–61. Szmuness, W. (1978). Hepatocellular carcinoma and the hepatitis B virus: Evidence for a causal association. Prog. Med. Virol. 24, 40–69. Tada, M., Omata, M., and Ohto, M. (1990). Analysis of ras gene mutations in human hepatic malignant tumors by polymerase chain reaction and direct sequencing. Cancer Res. 50, 1121–1124. Tahara, H., Nakanishi, T., Kitamoto, M., Nakashio, R., Shay, J. W., Tahara, E., Kajiyama, G., and Ide, T. (1995). Telomerase activity in human liver tissues: Comparison between chronic liver disease and hepatocellular carcinomas. Cancer Res. 55, 2734–2736. Takada, S., and Koike, K. (1990). Trans-activation function of a 3 truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues. Proc. Natl. Acad. Sci. USA 87, 5628–5632. 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 alpha transgenic mice. Cancer Res. 52, 5171–5177. Takaishi, H., Kitamoto, M., Takahashi, S., Aikata, H., Kawakami, Y., Nakanishi, T., Nakamura, Y., Shimamoto, F., Kajiyama, G., and Ide, T. (2000). Precancerous hepatic nodules had

Human Hepatocellular Carcinoma Pathogenesis

109

signiﬁcant levels of telomerase activity determined by sensitive quantitation using a hybridization protection assay. Cancer 88, 312–317. Tanaka, S., and Wands, J. R. (1996). Insulin receptor substrate 1 overexpression in human hepatocellular carcinoma cells prevents transforming growth factor beta1-induced apoptosis. Cancer Res. 56, 3391–3394. Tanigawa, N., Lu, C., Mitsui, T., and Miura, S. (1997). Quantitation of sinusoid-like vessels in hepatocellular carcinoma: Its clinical and prognostic signiﬁcance. Hepatology 26, 1216– 1223. Tannapfel, A., Wasner, M., Krause, K., Geissler, F., Katalinic, A., Hauss, J., Mossner, J., Engeland, K., and Wittekind, C. (1999). Expression of p73 and its relation to histopathology and prognosis in hepatocellular carcinoma. J. Natl. Cancer Inst. 91, 1154–1158. Tavian, D., De Petro, G., Benetti, A., Portolani, N., Giulini, S. M., and Barlati, S. (2000). u-PA and c-MET mRNA expression is co-ordinately enhanced while hepatocyte growth factor mRNA is down-regulated in human hepatocellular carcinoma. Int. J. Cancer 87, 644–649. Tay, A., Simon, J. S., Squire, J., Hamel, K., Jacob, H. J., and Skorecki, K. (1995). Cytosolic phospholipase A2 gene in human and rat: Chromosomal localization and polymorphic markers. Genomics 26, 138–141. Terada, T., Terasaki, S., and Nakanuma, Y. (1993). A clinicopathologic study of adenomatous hyperplasia of the liver in 209 consecutive cirrhotic livers examined by autopsy. Cancer 72, 1551–1556. Terradillos, O., Billet, O., Renard, C. A., Levy, R., Molina, T., Briand, P., and Buendia, M. A. (1997). The hepatitis B virus X gene potentiates c-myc-induced liver oncogenesis in transgenic mice. Oncogene 14, 395–404. Tetsu, O., and McCormick, F. (1999). Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398, 422–426. Thomas, M., Matlashewski, G., Pim, D., and Banks, L. (1996). Induction of apoptosis by p53 is independent of its oligomeric state and can be abolished by HPV-18 E6 through ubiquitin mediated degradation. Oncogene 13, 265–273. Tornillo, L., Carafa, V., Richter, J., Sauter, G., Moch, H., Minola, E., Gambacorta, M., Bianchi, L., Vecchione, R., and Terracciano, L. M. (2000). Marked genetic similarities between hepatitis B virus-positive and hepatitis C virus-positive hepatocellular carcinomas. J. Pathol. 192, 307–312. Toyokuni, S. (1996). Iron-induced carcinogenesis: The role of redox regulation. Free Radic. Biol. Med. 20, 553–566. Trusolino, L., Bertotti, A., and Comoglio, P. M. (2001). A signaling adapter function for alpha6beta4 integrin in the control of HGF-dependent invasive growth. Cell 107, 643–654. Tsuchihara, K., Hijikata, M., Fukuda, K., Kuroki, T., Yamamoto, N., and Shimotohno, K. (1999). Hepatitis C virus core protein regulates cell growth and signal transduction pathway transmitting growth stimuli. Virology 258, 100–107. Tsuda, H., Hirohashi, S., Shimosato, Y., Terada, M., and Hasegawa, H. (1988). Clonal origin of atypical adenomatous hyperplasia of the liver and clonal identity with hepatocellular carcinoma. Gastroenterology 95, 1664–1666. Uchida, K., Kondo, M., Takeda, S., Osada, H., Takahashi, T., and Nakao, A. (1997). Altered transcriptional regulation of the insulin-like growth factor 2 gene in human hepatocellular carcinoma. Mol. Carcinog. 18, 193–198. Ueda, K., and Ganem, D. (1996). Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II. J. Virol. 70, 1375–1383. Ueki, T., Fujimoto, J., Suzuki, T., Yamamoto, H., and Okamoto, E. (1997). Expression of hepatocyte growth factor and its receptor, the c-met proto-oncogene, in hepatocellular carcinoma. Hepatology 25, 619–623.

110

Kern et al.

Ueno, T., Takahashi, K., Matsuguchi, T., Ikejiri, K., Endo, H., and Yamamoto, M. (1988). Reactivation of rat insulin-like growth factor II gene during hepatocarcinogenesis. Carcinogenesis 9, 1779–1783. Unsal, H., Yakicier, C., Marcais, C., Kew, M., Volkmann, M., Zentgraf, H., Isselbacher, K. J., and Ozturk, M. (1994). Genetic heterogeneity of hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 91, 822–826. Vernucci, M., Cerrato, F., Besnard, N., Casola, S., Pedone, P. V., Bruni, C. B., and Riccio, A. (2000). The H19 endodermal enhancer is required for Igf2 activation and tumor formation in experimental liver carcinogenesis. Oncogene 19, 6376–6385. Vogel, A., Kneip, S., Barut, A., Ehmer, U., Tukey, R. H., Manns, M. P., and Strassburg, C. P. (2001). Genetic link of hepatocellular carcinoma with polymorphisms of the UDPglucuronosyltransferase UGT1A7 gene. Gastroenterology 121, 1136–1144. von Marschall, Z., Cramer, T., Hocker, M., Finkenzeller, G., Wiedenmann, B., and Rosewicz, S. (2001). Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma. Gut 48, 87–96. Vu, T. H., and Hoffman, A. R. (1994). Promoter-speciﬁc imprinting of the human insulin-like growth factor-II gene. Nature 371, 714–717. Wada, I., Kanada, H., Nomura, K., Kato, Y., Machinami, R., and Kitagawa, T. (1999). Failure to detect genetic alteration of the mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) gene in hepatocellular carcinomas in Japan. Hepatology 29, 1718–1721. Wands, J. R. (2000). Hepatitis C virus-induced hepatocarcinogenesis. In: “Chronic Hepatitis” (H. P. Dienes, P. Schirmacher, C. Brechot, and K. Okuda, Eds.), pp. 214–26. Kluwer Academic Publishers, Boston. Wang, G., Huang, C. H., Zhao, Y., Cai, L., Wang, Y., Xiu, S. J., Jiang, Z. W., Yang, S., Zhao, T., Huang, W., and Gu, J. R. (2000). Genetic aberration in primary hepatocellular carcinoma: Correlation between p53 gene mutation and loss-of-heterozygosity on chromosome 16q21q23 and 9p21-p23. Cell Res. 10, 311–323. Wang, G., Zhao, Y., Liu, X., Wang, L., Wu, C., Zhang, W., Liu, W., Zhang, P., Cong, W., Zhu, Y., et al. (2001a). Allelic loss and gain, but not genomic instability, as the major somatic mutation in primary hepatocellular carcinoma. Genes Chromosomes Cancer 31, 221–227. Wang, H. P., and Rogler, C. E. (1991). Topoisomerase I-mediated integration of hepadnavirus DNA 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 343, 555–557. Wang, R., Ferrell, L. D., Faouzi, S., Maher, J. J., and Bishop, J. M. (2001b). Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice. J. Cell Biol. 153, 1023–1034. Wang, Z. Q., Fung, M. R., Barlow, D. P., and Wagner, E. F. (1994). Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene. Nature 372, 464–467. Watanabe, S., Okita, K., Harada, T., Kodama, T., Numa, Y., Takemoto, T., and Takahashi, T. (1983). Morphologic studies of the liver cell dysplasia. Cancer 51, 2197–2205. Weihrauch, M., Benicke, M., Lehnert, G., Wittekind, C., Wrbitzky, R., and Tannapfel, A. (2001). Frequent k-ras-2 mutations and p16(INK4A)methylation in hepatocellular carcinomas in workers exposed to vinyl chloride. Br. J. Cancer 84, 982–989. Weston, S. R., and Martin, P. (2001). Serological and molecular testing in viral hepatitis: An update. Can. J. Gastroenterol. 15, 177–184. Wilkens, L., Bredt, M., Flemming, P., Becker, T., Klempnauer, J., and Kreipe, H. H. (2001). Differentiation of liver cell adenomas from well-differentiated hepatocellular carcinomas by comparative genomic hybridization. J. Pathol. 193, 476–482. Wogan, G. N. (1992). Aﬂatoxins as risk factors for hepatocellular carcinoma in humans. Cancer Res. 52, 2114s–2118s.

Human Hepatocellular Carcinoma Pathogenesis

111

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, 545–552. Wong, I. H., Lo, Y. M., Yeo, W., Lau, W. Y., and Johnson, P. J. (2000a). Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients. Clin. Cancer Res. 6, 3516–3521. Wong, N., Lai, P., Lee, S. W., Fan, S., Pang, E., Liew, C. T., Sheng, Z., Lau, J. W., and Johnson, P. J. (1999). Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: Relationship to disease stage, tumor size, and cirrhosis. Am. J. Pathol. 154, 37–43. Wong, N., Lai, P., E. Pang, Fung, L. F., Sheng, Z., Wong, V., Wang, W., Hayashi, Y., Perlman, E., Yuna, S., et al. (2000b). Genomic aberrations in human hepatocellular carcinomas of differing etiologies. Clin. Cancer Res. 6, 4000–4009. Wong, N. A., Rae, F., Bathgate, A., Smith, C. A., and Harrison, D. J. (2000c). Polymorphisms of the gene for microsomal epoxide hydrolase and susceptibility to alcoholic liver disease and hepatocellular carcinoma in a Caucasian population. Toxicol. Lett. 115, 17–22. Wu, C. G., Salvay, D. M., Forgues, M., Valerie, K., Farnsworth, J., Markin, R. S., and Wang, X. W., (2001). Distinctive gene expression proﬁles associated with Hepatitis B virus x protein. Oncogene 20, 3674–3682. Xu, L., Hui, L., Wang, S., Gong, J., Jin, Y., Wang, Y., Ji, Y., Wu, X., Han, Z., and Hu, G. (2001). Expression proﬁling suggested a regulatory role of liver-enriched transcription factors in human hepatocellular carcinoma. Cancer Res. 61, 3176–3181. Yakicier, M. C., Irmak, M. B., Romano, A., Kew, M., and Ozturk, M. (1999). Smad2 and Smad4 gene mutations in hepatocellular carcinoma. Oncogene 18, 4879–4883. Yamada, T., De Souza, A. T., Finkelstein, S., and Jirtle, R. L. (1997). Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis. Proc. Natl. Acad. Sci. USA 94, 10351–10355. Yamamoto, H., Itoh, F., Adachi, Y., Sakamoto, H., Adachi, M., Hinoda, Y., and Imai, K. (1997). Relation of enhanced secretion of active matrix metalloproteinases with tumor spread in human hepatocellular carcinoma. Gastroenterology 112, 1290–1296. Yang, D., Alt, E., and Rogler, C. E. (1993). Coordinate expression of N-myc 2 and insulin-like growth factor II in precancerous altered hepatic foci in woodchuck hepatitis virus carriers. Cancer Res. 53, 2020–2027. Yang, D., Faris, R., Hixson, D., Afﬁgne, S., and Rogler, C. E. (1996). Insulin-like growth factor II blocks apoptosis of N-myc2-expressing woodchuck liver epithelial cells. J. Virol. 70, 6260–6268. 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 1p in human hepatocellular carcinomas. Cancer Res. 54, 4188–4192. Yeh, Y. C., Tsai, J. F., Chuang, L. Y., Yeh, H. W., Tsai, J. H., Florine, D. L., and Tam, J. P. (1987). Elevation of transforming growth factor alpha and its relationship to the epidermal growth factor and alpha-fetoprotein levels in patients with hepatocellular carcinoma. Cancer Res. 47, 896–901. Yoshiji, H., Kuriyama, S., Ways, D. K., Yoshii, J., Miyamoto, Y., Kawata, M., Ikenaka, Y., Tsujinoue, H., Nakatani, T., Shibuya, M., and Fukui, H. (1999). Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis. Cancer Res. 59, 4413–4418. Yoshiji, H., Kuriyama, S., Yoshii, J., Yamazaki, M., Kikukawa, M., Tsujinoue, H., Nakatani, T., and Fukui, H. (1998). Vascular endothelial growth factor tightly regulates in vivo development of murine hepatocellular carcinoma cells. Hepatology 28, 1489– 1496.

112

Kern et al.

Yoshikawa, H., Matsubara, K., Qian, G. S., Jackson, P., Groopman, J. D., Manning, J. E., Harris, C. C., and Herman, J. G. (2001). SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growthsuppression activity. Nat. Genet. 28, 29–35. Youssef, N., Paradis, V., Ferlicot, S., and Bedossa, P. (2001). In situ detection of telomerase enzymatic activity in human hepatocellular carcinogenesis. J. Pathol. 194, 459–465. Yu, M. W., Chiu, Y. H., Chiang, Y. C., Chen, C. H., Lee, T. H., Santella, R. M., Chern, H. D., Liaw, Y. F., and Chen, C. J. (1999a). Plasma carotenoids, glutathione S-transferase M1 and T1 genetic polymorphisms, and risk of hepatocellular carcinoma: Independent and interactive effects. Am. J. Epidemiol. 149, 621–629. Yu, M. W., Yang, S. Y., Chiu, Y. H., Chiang, Y. C., Liaw, Y. F., and Chen, C. J. (1999b). A p53 genetic polymorphism as a modulator of hepatocellular carcinoma risk in relation to chronic liver disease, familial tendency, and cigarette smoking in hepatitis B carriers. Hepatology 29, 697–702. Yuan, B. Z., Miller, M. J., Keck, C. L., Zimonjic, D. B., Thorgeirsson, S. S., and Popescu, N. C. (1998). Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP. Cancer Res. 58, 2196–2199. 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. Zeze, F., Ohsato, K., Mitani, H., Ohkuma, R., and Koide, O. (1983). Hepatocellular carcinoma associated with familial polyposis of the colon. Report of case. Dis. Colon Rectum 26, 465– 468. Zhang, N., Siegel, K., Odenthal, M., Becker, R., Oesch, F., Dienes, H. P., Schirmacher, P., and Steinberg, P. (1997). The role of insulin-like growth factor II in the malignant transformation of rat liver oval cells. Hepatology 25, 900–905. Zhang, X., Xu, H. J., Murakami, Y., Sachse, R., Yashima, K., Hirohashi, S., Hu, S. X., Benedict, W. F., and Sekiya, T. (1994). Deletions of chromosome 13q, mutations in Retinoblastoma 1, and retinoblastoma protein state in human hepatocellular carcinoma. Cancer Res. 54, 4177– 4182. Zhang, Y. J., Jiang, W., Chen, C. J., Lee, C. S., Kahn, S. M., Santella, R. M., and Weinstein, I. B. (1993). Ampliﬁcation and overexpression of cyclin D1 in human hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 196, 1010–1016. Zhao, M., and Zimmermann, A. (1998). Liver cell dysplasia: Reactivities for c-met protein, Rb protein, E-cadherin and transforming growth factor-beta 1 in comparison with hepatocellular carcinoma. Histol. Histopathol. 13, 657–670. Zhao, X., Li, J., He, Y., Lan, F., Fu, L., Guo, J., Zhao, R., Ye, Y., He, M., Chong, W., et al. (2001). A novel growth suppressor gene on chromosome 17p13.3 with a high frequency of mutation in human hepatocellular carcinoma. Cancer Res. 61, 7383–7387. Zhu, N., Khoshnan, A., Schneider, R., Matsumoto, M., Dennert, G., Ware, C., and Lai, M. M. (1998). Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis. J. Virol. 72, 3691–3697. Zhu, N., Ware, C. F., and Lai, M. M. (2001). Hepatitis C virus core protein enhances FADDmediated apoptosis and suppresses TRADD signaling of tumor necrosis factor receptor. Virology 283, 178–187. Zondervan, P. E., Wink, J., Alers, J. C., JN, I. J., Schalm, S. W., de Man, R. A., and van Dekken, H. (2000). Molecular cytogenetic evaluation of virus-associated and non-viral hepatocellular carcinoma: Analysis of 26 carcinomas and 12 concurrent dysplasias. J. Pathol. 192, 207– 215.

The Cell-Mediated Immune Response to Human Papillomavirus-Induced Cervical Cancer: Implications for Immunotherapy Gretchen L. Eiben, Markwin P. Velders, and W. Martin Kast* Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood Illinois 60153

I. Introduction II. Human Papillomaviruses A. Epidemiology of HPV Infection B. Molecular Genetics of HPV C. Immunology to Viral Assault III. Cellular Immunity to HPV A. Immune Activation B. Immune Evasion of HPV-Induced Cervical Cancer C. The Importance of the CD4 Helper Response D. HLA Polymorphisms and HPV-Associated Carcinogenesis IV. Immunotherapy against HPV-Induced Carcinomas A. Rationale for Immunotherapy B. The Significance of Murine Models C. Immunotherapeutic Approaches V. Conclusion References

I. INTRODUCTION From molecular, clinical and epidemiological studies it has become evident that human papillomaviruses (HPV) are linked with the development of several human malignancies, including carcinomas of the upper aerodigestive tract and the anogenital tract, as well as conjunctival carcinomas (Breitburd ∗ To whom correspondence should be addressed at the present address: Cardinal Bernardin Cancer Center, Loyola University, 2160 S. First Avenue, Maywood, IL 60153.

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and Coursaget, 1999). This review will concentrate on the role of cellular immunity and prospects for immunotherapy in HPV-related cervical malignancies. Cervical cancer remains the third most common cancer among women worldwide, with approximately 400,000 new cases per year (Parkin et al., 1999). HPV DNA has been demonstrated in more than 99.7% of tumor biopsy specimens, with high risk HPV16 and HPV18 sequences being the most prevalent (Munoz, 1997; Walboomers et al., 1997). Cell-mediated immunity is likely to play an important role in protecting against tumor progression, as shown by the increased frequency of HPV-associated tumors in individuals treated with immunosuppressive drugs or suffering from AIDS (Sun et al., 1997; Frisch et al., 2000). In addition, malignant lesions are characterized by an inﬁltration of CD8+ T cells (Evans et al., 1997). Therefore, an effective vaccine that would mount a cellular immune response against HPV-related proteins might contribute to the elimination or prevention of HPV-expressing lesions, including cervical carcinomas.

II. HUMAN PAPILLOMAVIRUSES A. Epidemiology of HPV Infection Genital HPV infection is one of the most prevalent sexually transmitted diseases, with over 1 million new cases per year in the United States alone (Stone, 1995). Fortunately, HPV-induced lesions regress spontaneously in the majority of individuals, suggesting that our natural immunity is capable of clearing HPV-infected cells. HPV types infecting the genital tract have been classiﬁed as either high risk or low risk depending on the outcome and prognosis of the lesion they cause. Low risk HPV types -6, -11, -42, -43, and -44 are frequently associated with low-grade cervical intraepithelial neoplasia (CIN) and genital condyloma (Stone, 1995). While the high risk types -16, -18, -31, -33, and -45 are associated with premalignant displasia and cancer (Schiffman et al., 1993; Remmink et al., 1995). Speciﬁcally HPV-16 and -18 infections represent the major causal factors for cervical cancer (Walboomers et al., 1999). It appears that infection with HPV predisposes one to cervical cancer in a multistage tumorigenic process. Whereas the HPV genome is episomal in early infection, it is commonly integrated into the host genome in late preinvasive and invasive cervical cancer. Early detection and treatment have successfully reduced the incidence and mortality from cervical cancer in most Western countries. Despite this success, cervical cancer is still one of the most common cancers in women worldwide. This is especially true in developing countries, where Papanicolaou (PAP) smear screening, an effective preventive measure against cervical cancer, is insufﬁciently implemented. With growing evidence for HPV as a central etiologic factor in

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cervical cancer, development of a vaccine against this virus has emerged as an important objective.

B. Molecular Genetics of HPV HPV are double stranded DNA viruses of approximately 8 kb that infect basal and suprabasal layers of stratiﬁed epithelium. The early genes, which include E1, E2, E4, E5, E6, and E7, code for proteins involved in viral DNA replication, transcriptional control, and cellular transformation. Late genes encode the major viral capsid protein, L1, and a minor capsid protein, L2. The E1 and E2 genes code for important regulatory proteins of HPV. E1 facilitates the binding of E2 in the promoter region to repress transcription. Integration of the viral DNA in the genome of the host cell, which is an essential step in HPV16- or HPV18-induced development of cervical carcinoma, generally disrupts the E2 segment and results in the loss of E1- or E2-mediated transcriptional control. As a consequence, the transformed cells overexpress the E6 and E7 proteins, initiating the malignant transformation process (Pei, 1996). It has been demonstrated that E6 and E7 expression is required for the immortalization of primary cells as well as for maintenance of the transformed state (Seedorf et al., 1987; Von Knebel Doeberitz et al., 1991). E6 and E7 expression delay keratinocyte differentiation and stimulate cell cycle progression, allowing the virus to utilize host DNA polymerases to replicate its genome. Speciﬁcally, the E6 protein has been shown to bind to p53 and target its degradation by the proteasome (Werness et al., 1990). p53 is a central transcription activator that regulates responses to stress and DNA damage. Loss of p53 leads to genetic instability and rapid malignant progression. The other critical oncoprotein, E7, protein binds to the retinoblastoma protein (pRb) (Dyson et al., 1989). This interaction occurs primarily with the hypophoshorylated form of pRb causing the release of active E2F transcription factors, which in turn stimulate expression of genes involved in cell cycle progression and DNA synthesis. The E6 and E7 viral genes are expressed at low levels in proliferating basal cells, but are highly expressed in HPV-associated genital cancers (Crish et al., 2000; Durst et al., 1992). As a consequence, the E6 and E7 proteins can serve as major targets for the cell-mediated immune response and are, therefore, attractive targets for speciﬁc immunotherapies.

C. Immunology to Viral Assault To fully comprehend the potential of the immune system to reject virusinduced tumors and to understand why the natural immune response sometimes fails to do so, complete knowledge of T cell activation is crucial.

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Cellular immune responses, especially antigen-speciﬁc T lymphocytes, are most likely the critical defense mechanism against HPV-infected cells. The immunological recognition of viral antigens by T cells is restricted by the polymorphic human leukocyte antigen (HLA) class I and class II of the major histocompatibility complex (MHC). HLA class I antigens (HLA-A, -B, or -C) are expressed on virtually all nucleated cells and present intracellularly processed peptides to CD8+ T cells. HLA class II antigens (HLA-DR, -DQ, or –DP) are present on some cells of the immune system and present antigen to CD4+ T helper cells. The extensive polymorphisms of HLA molecules are concentrated within the peptide binding groove and the different HLA haplotypes provide a broad repertoire of peptide binding capacity. Viral immunity is initiated by T cell receptor engagement of speciﬁc peptides, derived from the virus or tumor, presented by the cell-surface MHC class I molecule. An efﬁcient CD8+ T cell response may therefore be characterized as the generation and clonal expansion of effector cytotoxic T lymphocytes (CTLs) that are capable of recognizing and eliminating the source of speciﬁc antigen. Through their cytolytic activity and cytokine production, CD8+ T cells are key components of the cellular immune response against infections and tumors. The differentiation of na¨ıve CD8 T cells into CTLs occurs in response to two signals: antigenic peptide on MHC class I and a costimulatory signal. Only professional antigen-presenting cells (APCs), such as dendritic cells (DCs), express the required costimulatory molecules to provide the second signal. Numerous studies have also demonstrated a requirement for CD4+ T cell help during the generation of primary CD8+ T cell responses in vivo (Matloubian et al., 1994; von Herrath et al., 1996; Stohlman et al., 1998). A major pathway of T help (Th) for CTL priming was shown to be mediated through CD40–CD40 ligand interactions (Schoenberger et al., 1998). The recognition of a Th epitope on a DC allows for their simultaneous activation through the interaction of the CD40 ligand on the Th cell with CD40 on the DC, thereby activating the DC to express additional costimulatory molecules. By their cytokine secretion and activation of DCs, Th cells enhance the differentiation of CD8+ T cells into CTLs that can eradicate virus-infected cells or tumor cells.

III. CELLULAR IMMUNITY TO HPV A. Immune Activation More than 70% of HPV-positive lesions resolve spontaneously, suggesting that a natural immune response against HPV is capable of clearing most infections (Hilders et al., 1993; Ho et al., 1998). Futhermore, while there

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are approximately 300 million cases of HPV cervical infection per year, only 400,000 of these proceed on to invasive cervical cancer, less than 0.13% (Centers for Disease Control and Prevention, 1999). The small number of infected individuals eventually developing cancer of the cervix and the long latency period between primary infection and cancer emergence suggest that host immune factors are involved in the prevention of malignant progression. In this context, the local immune state within the transformation zone of the cervix, where the majority of intraepithelial and invasive neoplasms develop, might be expected to play a key role in the host defense against HPV infection and associated precancerous lesions. There are two main arms to the immune response that may play a role in the host’s natural clearance of HPV infection: the innate and the adaptive immunity. Innate immunity consists of a rapidly induced, nonspeciﬁc response, which does not result in memory. The innate immune system is localized at epithelial borders, where viral infections take place, and is delivered via cytokines and cellular effectors. Activation of the innate immune system then induces a milieu of effector cytokines, which are capable of activating the adaptive immune response. There is considerable evidence to suggest that the effective activation of both the innate and adaptive immune systems may be crucial for the eradication of HPV infection. The presence of inﬁltrating macrophages and natural killer (NK) cells in regressing genital warts suggests that the innate immune system is most likely the ﬁrst line of defense against HPV infection (Coleman et al., 1994). The regression of HPV lesions is controlled by cytokines, most importantly interleukin-2 (IL-2) and interferon-γ (IFN-γ ), which boost the adaptive immune response by activating CTLs (Stellato et al., 1997). IL-2, which is secreted primarily by Th cells, is the principle cytokine required for proliferation and differentiation of activated precursor CTLs into effector CTLs. IFN-γ has also been shown to repress HPV16 gene expression in HPV16 immortalized cell lines and inhibit cell growth (Woodworth et al., 1992). Another key factor of immunosurveillance against HPV may be supplied by interferons released from keratinocytes. Interferons are proteins that interfere with viral replication in eukaryotic cells and form an early cytokine barrier against viral disease, including HPV infection, by inhibiting the differentiation of HPV-infected cells (Rockley and Tyring, 1995). Keratinocytes also release tumor necrosis factor-α (TNF-α) upon infection with HPV (Arany et al., 1993). This cytokine exerts an inhibitory effect on HPV replication as well, and attracts T cells via the upregulation of ICAM-1 and HLA-DR on keratinocytes, which encourages the presentation of antigens to inﬁltrating CD4+ T cells and can result in the resolution of infection (Coleman et al., 1994; Al-Saleh et al., 1998). Furthermore, TNF-α enhances the migration of Langerhans cells (LCs) into the regional lymph nodes where they present antigen to CD8+ T cells (Cumberbatch and Kimber, 1992).

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TNF-α is also able to recruit NK cells to the tumor, providing a valuable mechanism to eliminate tumor cells (Glas et al., 2000; Kashii et al., 1999). Taken together, it is evident that a combination of innate and adaptive immune responses to HPV is capable of clearing most infections and can protect an individual from the development of tumors. It can then be postulated that in the case of cervical malignancy, the host immune response to HPV has failed. Therefore, the enhancement of the immune response against HPV may contribute to virus recognition and tumor regression.

B. Immune Evasion of HPV-Induced Cervical Cancer Numerous studies have focused on the mechanisms whereby HPV can modify the immune response to interfere with antigen presentation in order to hide from immune recognition and establish a persistent infection. Histology from invasive carcinoma as compared to regressing lesions sheds light on the immune escape mechanisms of HPV. Cervical neoplasias are reported to exhibit a downregulation of HLA class I antigens and upregulation of class II molecules (Cromme et al., 1993; Glew et al., 1992; Keating et al., 1995; Coleman et al., 1994; Al-Saleh et al., 1998). HLA class I antigens are typically expressed on the surface of tumor cells and are essential for presenting epitopes of the rejection antigens to T cells. This HLA class I downregulation would undoubtedly hamper tumor presentation of antigen to APCs, leading to a decrease in immunological recognition. Furthermore, loss of class I molecules on the surface of tumor cells will reduce their potential as targets for CTLs. In contrast, the signiﬁcance of the upregulation of class II molecules on tumor cells, namely HLA-DR, is unclear. As opposed to malignant tissue, normal cervical tissue shows no detectable level of HLA-DR on keratinocytes. It has been postulated that expression of HLA-DR on keratinocytes after HPV infection may contribute to the host’s immunity by presenting antigen to CD4+ T cells (Coleman et al., 1994; Konya and Dillner, 2001). However, this assumption is contradicted by the observation that an increase in HLA-DR expression on cervical lesions is positively correlated with the grade of the lesion (Cromme et al., 1993; Glew et al., 1992; Al-Saleh et al., 1998). This would suggest that HLA-DR expression has a suppressive effect on the immune function. One such mechanism may be the activation of CD4+ CD25+ suppressive T cells. Upon antigen-speciﬁc stimulation, these regulatory CD25+ CD4+ T cells have been shown to contribute to tumor growth by inhibiting the activation of normally responsive T cells (Thornton and Shevach, 1998; Onizuka et al., 1999). Thus, the increased expression of class II molecules may lead to an increased activation of these suppressive T cells, which in turn could inhibit both CTLs and potentially cytotoxic CD4+ T cells.

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Another immune evasion mechanism that has been observed in cervical malignancies is the loss of LCs. A decline of LCs in cervical lesions is exhibited by a marked decrease in CD1a expression in correlation with the severity of the lesion. (Morelli et al., 1993; Al-Saleh et al., 1998). This suggests that interference with local APCs of the cervix may protect the infected tissue from the attention of the host adaptive immunity. In conclusion, the loss of LCs and class I molecules as well as the upregulation of class II molecules on keratinocytes suggests that there may be several cellular mechanism working in concert to disrupt both antigen presentation and T cell activation against HPV-infected cells during cervical carcinogenesis. Additionally, E7 and other HPV early gene products might act indirectly by affecting different cytokines, which may inhibit the innate immune response and the activation of adaptive immunity. One such critical immunomodulatory cytokine is IL-2. As opposed to the high levels of IL-2 observed in regressing papillomas, high squamous intraepithelial lesions (SIL) exhibit a dramatic decrease in IL-2 expression (Al-Saleh et al., 1998). Because IL-2 production is primarily mediated by lymphocytes, such a loss indicates a decrease in the activation of lymphocytes in cervical cancer. In addition, the production of IL-10 was demonstrated in keratinocytes of cervical carcinomas, which may provide another mechanism to weaken the T cell-mediated immune surveillance (Kim et al., 1995; El-Sherif et al., 2001; Sheu et al., 2001). IL-10 downregulates costimulatory molecules on DC and inhibits their migration to regional lymph nodes, thereby preventing tumor antigen presentation to CD8+ CTLs. Studies have shown that IL-10-treated human DCs can induce anergy in tumor-speciﬁc CTLs, resulting in a failure to lyse tumor cells (Steinbrink et al., 1999). Another cytokine capable of inhibiting CTL generation and diminishing the production of immunostimulatory cytokines, such as TNF-α and IFN-γ , is transforming growth factor-β (TGF-β). Recently, the role of TGF-β in cervical cancer was investigated by examining cytokine proﬁles from 10 different human cervical cancer lines, all of which displayed an increase in TGF-β expression (Hazelbag et al., 2001). TGF-β has been directly implicated in the escape of tumor recognition in immunocompetent hosts (Torre-Amione et al., 1990; Chang et al., 1993). Furthermore, TGF-β is reported to be secreted by CD4+ CD25+ suppressive T cells and is necessary for the immune suppressive function of these cells (Read et al., 2000). TGF-β also enhances IL-10 production by macrophages (Kitani et al., 2000). All of these data directly imply that an excess of TGF-β secreted in precancerous lesions may prevent the development of an efﬁcient cell-mediated response to HPV. Therefore, depleting TGF-β may be an effective strategy to boost cellular immunity. This approach was recently implemented by examining the antibody-mediated depletion of TGF-β in HPV16+ tumor-bearing mice (Gunn et al., 2001). It was found that the addition of anti-TGF-β treatment to HPV vaccination dramatically

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increased the regression of HPV established tumors (Gunn et al., 2001). As mentioned before, TGF-β can inhibit the expression of TNF-α and IFN-γ ; accordingly, the same cervical cancer cytokine proﬁles found a sharp decline in TNF-α and IFN-γ expression in the cervical cancer lines (Hazelberg et al., 2001; El-Sherif et al., 2001). The reduced transcription of these cytokines in the higher grades of HPV-associated precancerous lesions may contribute to a local environment which favors progression of these lesions by downregulating the synthesis of MHC class I expression (Maudsley, 1991). Studies have shown that the tumor suppressor function of the immune system is critically dependent on the actions of IFN-γ molecules, which are directed at regulating tumor cell immunogenicity (Boursnell et al., 1996). Furthermore, IFN-γ expression is required for the formation and the catalytic activity of the immunoproteasome (Morel et al., 2000). In the absence of the immunoproteasome, antigen processing takes place via the standard proteasome (Macagno et al., 1999). It has been shown that processing of some antigenic tumor peptides by the standard proteasome rather than the immunoproteasome has resulted in differentially processed epitopes and therefore the lack of T cell recognition (Boes et al., 1994; Morel et al., 2000). This implies that in the absence of IFN-γ , tumor cells could switch their proteasome from the immunoproteasome to the standard type and thereby escape an immune response directed against epitopes not properly processed by that proteasome. Whether there is a difference in the processing of HPV epitopes processed by the standard and the immunoproteasome remains to be determined. Taken together, all of these data indicate that HPV-infected cells, via the production of TGF-β, upregulate IL-10 while downregulating TNF-α and IFN-γ . This can result in the reduced level of antigen presentation and decreased recognition by cytotoxic T cells. Such immune deﬁciency might result in prolonged HPV infection and thus increase the chance of malignancy. Another immune escape mechanism observed during the progression toward cervical cancer is derived from the T cell itself. Expression of the ζ chain of the T cell receptor (TCR) is required for T cell activation and the reduced levels or the absence of ζ chain expression is indicative of defects in T cell activation (Chan et al., 1992). Reduced expression of the ζ chain in T cells has been identiﬁed in large percentages of cancer patients for many different tumor types (Whiteside, 1999). Data have indicated that the tumor microenvironment has negative effects on the inﬁltrating T cells (De Gruijl et al., 1999). A decrease in ζ chain expression has also been found after exposure to hydrogen peroxide produced by activated granulocytes and macrophages that was prevented by addition of hydrogen peroxide scavengers (Kono et al., 1996b; Schmielau and Finn, 2001). The downregulation of ζ chains in T cells is not limited to TILs but can also be measured in peripheral blood lymphocytes (PBLs) and NK cells of tumor-bearing patients

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(Guadagno et al., 1993). Furthermore, studies on the ζ expression in PBLs of patients with CIN or cervical cancer have indicated that the ζ chain expression may correlate with disease progression (Kono et al., 1996a). These data indicate that reduced levels of ζ chain expression may contribute to the host inability to mount an effective immune response against HPV-infected cells and furthermore may be a prognostic marker for survival.

C. The Importance of the CD4 Helper Response A positive role for HPV-speciﬁc Th immunity was suggested by the predominance of CD4+ T cells in regressing genital warts as well as by the detection of delayed-type hypersensitivity responses to HPV16 E7 in the majority of subjects with spontaneous regressing CIN lesions (Coleman et al., 1994; Hopﬂ et al., 2000). It is now well accepted that the induction of a strong CD4+ T-helper response is advantageous for the activation and perpetuation of a strong cell-mediated immune response. Upon recognition of antigenic peptides presented by APCs in MHC class II molecules, CD4+ helper T cells release cytokines that regulate the immune response to the antigen. Cytokines are also important immunological mediators of cell-mediated defenses against tumors. CD4 T helper cells are divided into two subsets on the basis of the immunomodulatory cytokines they produce and their effector functions. Th1 cytokines (IL-2, IFN-γ , TNF-α) are proinﬂammatory or immunostimulatory cytokines that boost the cellular immune response by promoting the outgrowth of CTLs. Th2 cytokines (IL-4, IL-5, IL-10, IL-13) are assumed to have the opposite effect, that of impairing the cellmediated immune response. A shift toward cytokines produced by Th2 cells has been associated with a less effective immune response in a number of cancer types (Nakagomi et al., 1995; Huang et al., 1995). It has been demonstrated that na¨ıve CD8+ T cells are able to differentiate into a T cytotoxic 1 (Tc1) subset that produces IL-2 and IFN-γ , or a Tc2 subset that produces IL-4, IL-5, and IL-10 (Carter and Dutton, 1996; Seder et al., 1992; Sad et al., 1995; Mosmann et al., 1997). Although both subsets are cytolytic in humans, Th1/Tc1 cells seem to enhance cell-mediated immunity. In contrast, Th2/Tc2 cells predominate during parasite inﬁltrations and are less protective than Tc1 cells against viral infection. It has been shown that cervical cancer cells can directly drive the tumor-encountered T cells toward the Th2/Tc2 polarity through an IL-10 and TGF-β mediated pathway (Sheu et al., 2001). Thus, a type 2 cytokine release from Th2 cells drives the production of Tc2 cells and further hampers the immune response. This inappropriate response to viral infection may lead to the persistence of HPV, preventing infected cells from being eliminated by cell-mediated immune responses.

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Several T helper epitopes have been identiﬁed in HPV16 L1, E6, and E7 proteins by their ability to induce T cell proliferative responses peripheral blood mononuclear cells from PBMCs (Strang et al., 1990; Van der Burg et al., 2001). T cell proliferative responses against HPV16 E7 proteins or peptides were found in healthy individuals and more frequently in patients with CIN or carcinomas (Kadish et al., 1994). Immunization protocols that generate strong effector CTL responses in the absence of CD4+ T cells illustrate the signiﬁcance of this helper population, through their inability to induce long-term CTL memory (Clarke, 2000). Indirect evidence that CD4+ T cells may indeed be important in mediating or augmenting antitumor immune responses stems from the observation that regression of genital warts is associated with increased numbers of CD4+ and that women with invasive cancer are at a higher risk of developing progressive disease if they exhibit impaired or decreased CD4+ T cells (Gemignani et al., 1995). Recently, it has been observed that tumor-inﬁltrating lymphocytes (TILs) from a cervical cancer patient can recognize the autologous tumor in an MHC class II restricted fashion (Hohn et al., 1999). Thus, MHC class II molecules expressed on cervical cancer cells may serve as restricting molecules to present HPV derived epitopes to CD4+ T cells. MHC class II epitopes may then be implemented into vaccination strategies to augment T cell responses against tumor cells. However, a major obstacle for the development of optimal cancer vaccines is the lack of effective methods for identifying MHC class II restricted tumor antigens that can stimulate CD4+ T cells (Wang, 2001). Identiﬁcation of such antigens would provide new opportunities for developing effective cancer vaccines and improve our understanding of the mechanisms by which CD4+ T cells regulate the host immune system.

D. HLA Polymorphisms and HPV-Associated Carcinogenesis Given the pivotal role of HLA molecules in the recognition of foreign tumors by CTLs, several studies have been performed to examine the association of speciﬁc HLA alleles with HPV infection status and development of cervical cancer. Since individual HLA molecules can present different viral peptides, the various combinations of inherited HLA alleles should result in different immune responsiveness to HPV-encoded antigens among individuals. Several groups have reported positive or negative associations of particular HLA class II alleles with CIN or invasive cervical carcinomas (Duggan-Keen et al., 1996; Glew et al., 1992, 1993; Wank and Thomssen, 1991; David et al., 1992; Helland et al., 1994; Vandenvelde et al., 1993; Apple et al., 1994). As a consequence, carriers of these alleles may be at

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a higher or lower risk of developing cervical cancer. Unfortunately, results from different groups evaluating HLA types and HPV-induced disease have been inconsistent. This is in part due to the heterogeneity in HLA frequencies among different populations and to differences in the prevalence of HPV-type speciﬁc infections. The most frequently reported positive association was the presence of the HLA class II, DQ3 antigen or DQB1∗ 03 allele (Wank and Thomssen, 1991; Odunsi et al., 1995; Wang, 2001). Associations with persistent HPV16 infection or CIN III were also described for DRB1∗ 07, DRB1∗ 1501/DQB1∗ 0602, and DRB1∗ 07/DQB1∗ 0201 haplotypes (Apple et al., 1995). In contrast, it has been observed that DRB1∗ 1301 protects against HPV infection and cervical disease (Sastre-Garau et al., 1996; Bontkes et al., 1998). These ﬁndings support the hypothesis that multiple-risk alleles are required to confer an increased risk that is detectable at the population level, possibly through inadequate presentation of viral antigens to the immune system. In contrast, the presence of a single protective allele may be sufﬁcient to confer protection. Recent studies have revealed no HLA class I associations with HPV16 infection but have demonstrated an HLA-A2, HLA-B44, and HLA-B7 correlation with disease progression (Bontkes et al., 1998; Montoya et al., 1998; Duggan-Keen et al., 1996). HLA-A2 and B44 are the predominant haplotypes in patients with late-stage carcinomas compared with patients with early-stage carcinomas and are among the alleles most commonly downregulated in carcinoma cells (Keating et al., 1995). Furthermore, downregulation of HLA-B7 on cervical cancer cells is associated with a worse survival compared to normal expression of this antigen (Duggan-Keen et al., 1996). All of this suggests that certain individuals may or may not be more prone to a cellular response against HPV-infected cells. However, no correlation between disease progression and the presence or absence of cellular immunity has been established. Therefore, studies are required to determine whether certain HLA types may express more or less immunodominant epitopes for CTL recognition that may contribute to the progression of premalignant lesions of the cervix.

IV. IMMUNOTHERAPY AGAINST HPV-INDUCED CARCINOMAS A. Rationale for Immunotherapy The importance of cell-mediated immunity is directly implied by the increased incidence of HPV lesions and carcinomas in individuals with impaired cellular immune function, including HIV and renal transplant patients

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(Petry et al., 1994; Halpert et al., 1986). On the basis of the largest population-based dataset available for the study of cancer in immunosuppressed individuals, HIV-infected individuals demonstrated a considerably increased risk for HPV-associated cervical cancer (Frisch et al., 2000). The importance of cellular immunity is also supported by immunocompetent patients with cervical lesions exhibiting natural CTL responses against HPV16 and -18 that are absent in healthy donors (Evans et al., 1997). In addition, HPV16 E6 and E7 speciﬁc CD4+ T helper cells have been identiﬁed in patients with abnormal cytology that were not detectable in individuals without viral infection (De Gruijl et al., 1996, 1998; Luxton et al., 1996; Kadish et al., 1997). Recent studies have been aimed at substantiating the role of cellular immunity to high-risk HPV antigens in humans with cervical neoplasias. The signiﬁcance of MHC class I restricted HPV-speciﬁc CD8+ CTLs in HLA-A∗ 0201-positive individuals has been examined in tumor inﬁltrating lymphocytes (Evans et al. 1997). In humans, HLA-A2 has become an important restriction element for CTL-based immunotherapy because it is the most common HLA class I haplotype in the Caucasian population. Peptides that bind to ﬁve common haplotypes of HLA-A were identiﬁed in HPV16 E6 and E7 oncoproteins (Kast et al., 1996). Several CTL epitopes were characterized among HLA-A∗ 0201 binding peptides by their capacity to induce CTL responses in vitro from PBMCs of healthy HLA-A∗ 0201 donors (Ressing et al., 1995), suggesting that these peptides represent naturally processed human CTL epitopes of HPV16. Using individual epitopes based on HLA-A∗ 201 binding HPV16 E7 peptides, memory CTLs could also be detected in patients with cervical carcinomas or CIN but not in controls (Evans et al., 1997; Ressing et al., 1996). It has been shown that CTLs from cancer patients lysed HLA-A∗ 201+, HPV16+ cervical carcinoma-derived Caski cells but not HLA-A∗ 201+ MS751 cells harboring HPV45 (Ressing et al., 1996; Geisbill et al., 1997). In patients infected with HPV16, cell mediated immunity to the E7 protein has been demonstrated (De Gruijl et al., 1996; Kadish et al., 1994). Furthermore, women who are capable of mounting a cellmediated immune response to HPV16 E6 and E7 proteins in vitro are likely to undergo regression of CIN and resolution of HPV infection, whereas patients who fail to exhibit these responses are likely to have persistent disease (Kadish et al., 1997). In conclusion, the presence of inﬁltrating lymphocytes and HPV-speciﬁc T cells found in spontaneously regressing papillomas strongly imply that the immune response does in fact control HPV infection and associated disease (Coleman et al., 1994; Evans et al., 1997; Ghosh and Moore, 1992). This along with evidence that cellular immunity may be inhibited during HPV infection provides the rationale for immunotherapy to boost a CTL response against HPV-associated cancer.

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B. The Significance of Murine Models The various studies on T cell recognition of HPV antigens in mice may have only limited relevance to human disease. Nevertheless, such work has served to encourage the search for human T cell recognition of HPV-derived antigens. Furthermore, immune responses to vaccination in mice are expected to reﬂect natural human responses and have helped to design reliable tests and deﬁne endpoints to assess immunization in humans. Mouse cells can be transformed by high-risk genital HPV E6 and E7 oncogenes and develop into tumors. This has provided multiple experimental HPV models to decipher the role of T helper and CTL responses in the defense against HPV-expressing tumor cells and the identiﬁcation of the E6 and E7 T cell epitopes and their efﬁciency as vaccines to prevent tumor growth. Several lines of experimental evidence in mice reinforce the notion that the induction of cellular immunity is critical for the elimination of HPV-infected cells. In the ﬁeld of immunology, the use of HLA class I transgenic mice can potentially overcome some of the main limitations of human studies. Some HLA class I molecules have been modiﬁed such that the human A2 α3 domain has been replaced with the murine MHC class I α3 domain (Faulkner et al., 1998). The α1 and α2 domains form the peptide binding groove, while the α3 domain interacts with the CD8 molecule; thus this transgenic MHC class I molecule can present human peptides and elicit a murine CTL response. Identiﬁcation of human CTL epitopes and successful peptide vaccination were performed using mice expressing the transgenic HLA-A2 allele (Kast et al., 1996; Ressing et al., 1999). The potential usefulness of HLA class I transgenic mice as animal models of the human immune system depends on the ability of the murine TCR repertoire to recognize the same antigenic determinants as those recognized by human T cells. In this regard, demonstration of HLA-A2 restricted responses against HPV16 E6 and E7 in HLA-A2 transgenic mice has been particularly important (Ressing et al., 1995). These studies indicate that the same epitopes of HPV16 E7 (11-20, 86-93) and E6 (29-38) are recognized by both HLA-A2 mice and humans (Ressing et al., 1995). Recently, an HLA-A2+ HPV16+ tumor model was developed for the study of antitumor vaccination strategies in HLA-A2 transgenic mice (Eiben et al., in press). This tumor model has real potential both for investigating immune responses to human epitopes and for vaccine development and testing. Furthermore, work with other HLA transgenic mice such as HLA-A3, HLA-A24, and HLA-A11 (Barra et al., 1993; Alexander et al., 1997) may provide additional HPV epitope to vaccinate against and yield results that may be directly translatable to human studies.

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C. Immunotherapeutic Approaches Cancers that do not naturally result in an immune response induction can still be susceptible to T cell-mediated eradication when activated CTLs are generated by vaccination. The therapeutic approach to patients with preinvasive and invasive cervical cancer is to develop vaccine strategies to induce speciﬁc CD8+ CTL responses to the relevant class I speciﬁc epitopes of HPV E6 and E7 proteins presented by cervical tumors. Thus, the goal of therapeutic immunization is to stimulate the adaptive immune response to eliminate infected or transformed cells. The aim of prophylactic vaccines is the generation of neutralizing antibodies against the HPV L1 and L2 capsid proteins. Neutralizing antibodies may be an effective way of preventing viral infection and spread, but cell-mediated immune responses are required for the ultimate resolution of infection or disease. This is strengthened by the observation that humoral immunodeﬁciency, characterized by a failure to produce antibodies, does not increase susceptibility to the development of HPV lesions (Benton et al., 1999). Furthermore, antibodies to HPV16 E6 or E7 oncoproteins are rarely detected in patients with premalignant cervical lesions and are found in only 50% of patients with late-stage cervical cancers (Stern, 1996; Viscidi et al., 1993). These data suggest that it is unlikely that antibodies alone are capable of viral clearance of HPV or sufﬁcient to protect an individual from the development of HPV-induced tumors. Furthermore, the intracellular locations of E6 and E7 strongly suggest that a cellular immune response is more efﬁcacious than a humoral response in clearing HPV lesions. Additionally, numerous studies have demonstrated the critical role of cellular immunity mediated by T lymphocytes in the control of HPV-induced malignancies in humans. Although a natural cellular immunity against HPV clearly exists, it may be insufﬁcient or induced too late to eradicate cervical tumor cells. It is clear that in a signiﬁcant proportion of patients with persistent CIN or carcinoma, HPV16 T cell responses do not resolve the lesions, reﬂecting an immune escape mechanism at the effector or target level (Rudolf et al., 1999b). To prevent escape of virally infected cells, it will be important to induce activated immune responses against the viral proteins until full clearance of either infection or the cancer cells is achieved. Furthermore, the time of immunotherapeutic delivery is critical. To minimize the risk for immune escape, anticancer therapy should be administered in the early stages of cervical disease, because progression is accompanied by genetic instability and tumor heterogeneity (Fleuren et al., 1995). The most promising vaccines tested in both murine and human models include viral and bacterial vectors, peptides, recombinant fusion proteins, chimeric virus-like particles, and plasmid DNA, which will be discussed in the following sections.

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1. VACCINIA DELIVERY Vaccines based on proteins present the advantages of being well characterized and presenting both CD4 and CD8 epitopes to the immune system, allowing for the simultaneous induction of a T helper and CTL response. Vaccination with entire proteins permits all possible epitopes of E6 and E7 to be processed and presented for CTL recognition. Thus, the limitations of peptide-based vaccines, such as knowledge of a patient’s HLA type in order to choose the appropriate peptides, can be overcome and may therefore permit a more potent immune response. Their main disadvantage is that puriﬁed proteins elicit relatively poor immune responses on their own (Germain and Hendrix, 1991). This may be related to the protein processing route. Extracellular proteins are mainly targeted to the MHC class II processing route, which may skew the T cell response toward a predominant helper phenotype. To enhance the effect of protein vaccination, studies have focused upon the delivery of DNA coding for the proteins via viral vectors, such as vaccinia. This results in an intracellular protein processing through MHC class I, which predominantly results in a CTL response. Vaccinia has a lytic life cycle, minimizing the risk of viral integration and oncogenic transformation. Several groups have described the induction of HPV-speciﬁc CTL responses in mice upon immunization with recombinant vaccinia virus expressing HPV E6 or E7 (Boursnell et al., 1996; Lin et al., 1996). Vaccination with this vaccinia virus expressing HPV16 and HPV18 E6 and E7 proteins was also shown to induce HPV-speciﬁc CTLs in patients with late-stage cervical cancer (Borysiewicz et al., 1996). In this ﬁrst phase I/II trial, immunization with vaccinia virus was shown to be safe and to have no side effects, which was critical for ongoing trials. The major disadvantage of using E6 or E7 oncoproteins is their potential for integration in the host genome and subsequent transformation of the host cell. This problem might be overcome by using mutated forms of E6 and E7 in the region required for transformation, but these mutations may compromise the immunogenicity of the proteins (Gerard et al., 2001). The possibility of ensuring a safe antitumor CTL response has been studied using recombinant vaccinia viruses encoding modiﬁed forms of HPV16 and 18 E6 and E7 proteins (Boursnell et al., 1996; Wu et al., 1995). In one such model the E7 ORF was mutated to eliminate its Rb protein binding site thus reducing its oncogenic potential (Boursnell et al., 1996). Recent Phase I trials with this vaccine strategy have been shown to induce HPV-speciﬁc CTL responses in patients with preinvasive and invasive cancer. Responses were seen in one of three patients with advanced cervical cancer, 3 of 12 CIN III patients, and in 4 of 29 patients with early invasive cancer (Adams et al., 2001). Ongoing trials will optimize and assess the potential of such immunotherapy. In another model, immunogenicity of the HPV16 E7 protein has been markedly

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improved by fusing it with two parts of the lysosome-associated membrane protein 1 (LAMP-1). This resulted in localization of the E7/LAMP-1 fusion protein into the endosomal and lysosomal compartments and enhancement of presentation of the E7 antigen by MHC class II molecules (Wu et al., 1995). To measure the effect of LAMP-1 targeting of antigen in vivo, the vaccinia virus system was used for delivery. The increased stimulation of CD4+ T cells in this recombinant vaccinia strategy was shown to protect mice against a subsequent HPV tumor challenge, and was signiﬁcantly better at curing small established tumors than was the wild-type E7 vaccine (Lin et al., 1996). Such studies may prove useful in developing therapeutic vaccines for use in patients with HPV-induced disease. Potential drawbacks from the use of viral vector systems include the induction of neutralizing antibodies against viral capsid proteins in preimmune patients or after subsequent vaccination as well as the risk of integration of recombinant genes into the host cell genome. The use of an alphavirus delivery vector may eliminate such drawbacks.

2. ALPHAVIRUSES Another approach to deliver HPV proteins is to use the Semliki Forest virus (SFV) or the Venezuelan equine encephalitis (VEE) virus system. SFV and VEE belong to the genus Alphavirus of the family Togaviridae. Alphaviruses consist of a nucleocapsid with one copy of a single-stranded RNA molecule surrounded by an envelope containing spike proteins. Alphaviruses replicate in the cell cytosol and are lytic after 72 hr, so there is no risk of integration of the E6 and E7 oncogenes into the cellular genome. VEE and SFV have been engineered as nonpropagating replicon vectors that contain a foreign gene in place of the viral structural protein genes. To generate viral particles, the structural protein genes need to be provided in trans. This split helper package system minimizes the likelihood of the in vivo generation of replication competent recombinant viruses. Studies have shown that SFV-E6E7 particles can induce a CTL response against HPV-transformed cells and protected 40% of the mice from tumor challenge (Daemen et al., 2000). An encouraging study has recently reported that VEE-HPV16 E7 vaccination induced immunogenic CTLs capable of 100% tumor protection and elimination of 67% of established HPV tumors in mice (Velders et al., 2001a). By infecting DCs, VEE replicon vectors target expression to lymphoid tissue, a preferred site for induction of immunity (MacDonald and Johnston, 2000). Delivery of VEE to DCs may be of vital importance for HPV proteins with low immunogenicity. Futhermore, there is no widespread existing immunity to VEE in humans, allowing for repeated immunizations and bringing this model closer to clinical evaluation.

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3. VIRUS-LIKE PARTICLES Another possible protein delivery system that has been widely studied is the use of virus-like particles (VLPs). Expression of the L1 major capsid protein of HPV16 in eukaryotic cells results in the formation of empty VLPs (Schiller and Roden, 1995). Chimeric virus-like particles (CVLP) consist of the carboxy-terminally truncated L1 protein fused to the E7 protein. The chimeric protein is able to build viral capsids that retain the ability to penetrate cells similar to the natural virus (DaSilva et al., 2001a). CVLPs are found in the cytosol and endoplasmic reticulum suggesting that they enter the MHC class I processing pathway and should be able to induce a cytotoxic immune response (Schafer et al., 1999). This response was shown to be associated with the production of Th1 type cytokines and delivery of the HPV antigens to the HLA class I processing pathway for priming of CTLs (Dupuy et al., 1997; Peng et al., 1998). CVLPs containing E7 have been synthesized and used to vaccinate against E7 in a murine model system (Greenstone et al., 1998). After vaccination, mice were protected from tumor challenge. Recently, in vitro pulsed CVLPs were shown to induce an E7 speciﬁc CTL in humans by an in vitro vaccination with PBLs (Kaufmann et al., 2001; Rudolf et al., 2001a). These CTLs were cytolytic to autologous B cell pulsed with CVLP or peptides as well as HPV16 positive cervical cancer cells. Furthermore, no difference in CTL response was observed when CVLPs were ﬁrst incubated with the potent antigen-presenting DCs, suggesting the immunogenicity of CVLPs (Kaufmann et al., 2001). This observation reﬂects the potential of a CVLP vaccine administered directly to the patient without the need to isolate, mature, or infect DCs for antigen presentation. Based on the observation of a low prevalence of L1 antibodies in HPV-infected women, it seems probable that this CVLP will induce an immune response in HPV16-positive patients with CIN lesions or cervical cancer. In addition, CVLPs may have the unique advantage of inducing both a therapeutic CTL response and a prophylactic L1 response to prevent further spread of the virus. Mice vaccinated with VLPs consisting of L1 capsid proteins alone were protected against a subsequent tumor challenge with an HPV16induced tumor cell line expressing HPV16 L1 proteins (De Bruijn et al., 1998a). Furthermore, it has been shown that VLPs are capable of stimulating an MHC restricted CTL response against L1 and L2 proteins of HPV16 (De Bruijn et al., 1998a; Rudolf et al., 1999a). The L1 protein is expressed in terminally differentiated epithelial cells, which make up only a small portion of the infected cells within a lesion (Taichman and LaPorta, 1987). However, these cells are the producers of new virus particles, and eradication of these cells would prevent further spreading of the viral infection (Rudolf et al., 1999a), suggesting and another advantage to CVLP vaccination, strategies. In addition to proteins, VLPs can also deliver DNA (Combita et al., 2001).

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A major disadvantage of vaccinating with CVLPs is the possible development of a B cell response against the VLP leading to the generation of virus-neutralizing antibodies. VLP vaccines given to mice after the ﬁrst vaccination are hindered by antibodies generated on the ﬁrst exposure to the VLP (Da Silva et al., 2001b). The use of other papillomavirus VLPs as antigen delivery vehicles (bovine, cottontail rabbit) may be able to overcome any preexisting humoral immunity generated during vaccination, since neutralizing antibodies are species speciﬁc and not cross reactive (Rose et al., 1994; Kirnbauer et al., 1996).

4. BACTERIAL VECTOR-BASED VACCINES Attenuated bacterial strains are effective vehicles for delivering heterologous antigens to the mucosal and systemic immune systems, where in addition to humoral immunity, they can induce speciﬁc CTLs and provide antitumor immunity. It has been reported that a mucosal route of immunization may be necessary in order to provide long-term immunity and CTL-mediated protection from infections of the genital tract (Gallichan and Rosenthal, 1998). Intranasal vaccination has been shown to induce speciﬁc CTLs in the vaginal draining lymph nodes, suggesting that this immunization route allows induction of a mucosal immunity that might be able to inhibit tumor growth in the genital mucosal (Dupuy et al., 1999). Since HPV infects the genital mucosa, this route of vaccination may enhance the local CTL response which may be efﬁcacious for clinical application. Salmonella bacteria invade the host by crossing the gut mucosa and colonize the gut-associated lymphoid tissue, as well as the spleen and liver. Deletion of various genes can render Salmonella bacteria avirulent while preserving its invasiveness and immunogenicity (Benyacoub et al., 1999). Therefore, Salmonella bacterial vectors seem to be attractive vaccine candidates to replicate and express genes of interest. Nevertheless, studies with Salmonella carrying E6 and E7 proteins of HPV16 have failed to induce E7 speciﬁc CTLs (Street et al., 1999) or have elicited a humoral but not a cell mediated response (Krul et al., 1996; Londono et al., 1996). It has been shown that intranasal immunization with the PhoPc S. typhimurium strain expressing HPV16 L1 VLPs induced HPV16 neutralizing antibodies in mice (Nardelli-Haeﬂiger et al., 1997). Furthermore, mucosal immunization with this strain expressing HPV16 L1 VLPs also provides growth inhibition of HPV-expressing tumor cells, both therapeutically and prophylactically (Revaz et al., 2001). Thus, recombinant Salmonella engineered to express chimeric E7 VLPs may be even more effective by inducing both VLP antibodies and E7 speciﬁc CTLs. Unfortunately, the attenuated PhoPc bacteria strain which contains a point mutation in the phoQ gene can revert to wild type at high frequencies (Miller and Mekalanos, 1990). Evaluations of

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different Salmonella strains harboring attenuation that may be more suitable for human vaccines have not been as effective (Benyacoub et al., 1999). In addition, Salmonella has also been shown to have an inherent antitumor activity (Revaz et al., 2001), possibly mediated by the innate immunity induced upon bacterial infection, which may account for some of its therapeutic effects. Vaccination with the recombinant Listeria monocytogenes provides another model for the induction of a mucosal cellular immune response. Upon infection with Listeria, both Th1 helper and CD8+ CTL speciﬁc immune responses are induced, making it a suitable vehicle for vaccine delivery. Like Salmonella, Listeria is a foodborne pathogen and can, therefore, induce mucosal immune responses at the site of infection as well as at other mucosal areas (Mata et al., 2001). Listeria is an intracellular pathogen that secretes the protein listeriolysin O (LLO), which can perforate the phagosomal membrane and allow access to the cytoplasm of APCs (Gedde et al., 2000). By fusing HPV16 E7 to LLO, researchers have taken advantage of its unique ability to target the cellular immune system (Gunn et al., 2001). This LLO–E7 fusion protein induces E7 speciﬁc CTLs and the regression of 75% of HPV16 E7 established tumors. The ability to transform Listeria to express and secrete a foreign antigen and retain sufﬁcient virulence to induce an immune response is an important step toward using attenuated mutants as host strains for recombinant vectors. These data support the idea that Listeria can be used for immunotherapy in humans.

5. PEPTIDE VACCINES There has been considerable progress over the past several years in identifying peptides derived from human tumor antigens recognized by CD8+ T cells (Kast, 2000). Because peptides are relatively easy to prepare for clinical use, peptide based cancer vaccines are being widely tested, with peptides alone, with an adjuvant, or presented by DCs. Experience to date indicates that peptide vaccines can elicit speciﬁc CD8+ T cell responses and that such responses are occasionally associated with tumor regression. Studies have shown that vaccination of mice with an HPV16 E7 peptide with a high binding afﬁnity for the H-2Db class I molecule completely prevented tumor growth upon subsequent challenge with HPV16-positive tumor cells (Feltkamp et al., 1993; Campo et al., 1993). Moreover, adoptive transfer of a CTL clone obtained via peptide vaccination with the E7 peptide led to the eradication of established HPV16 tumors in T cell-deﬁcient mice (Feltkamp et al., 1995). Identiﬁcation of human HLA-A∗ 0201 restricted CTL epitopes of HPV16 and -18 has allowed for successful peptide based vaccination in transgenic mice expressing an HLA-A2 allele (Ressing et al., 1995; Kast et al., 1994; Rudolf et al., 2001b). Futhermore, speciﬁc A2 CTLs have been

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raised after in vitro vaccination of PBLs from normal human donors with A2 peptides (Ressing et al., 1995). Memory CTL responses against some of these HLA-restricted peptides from E6 and E7 are also detectable in some patients with CIN and cervical carcinomas but not in normal subjects (reviewed in DaSilva et al., 2001a). All of this has prompted the basis for clinical trials of a peptide vaccine against HVP. Initially discouraging results were obtained from a phase I-II clinical trial involving E7 peptide vaccination in patients with recurrent or residual cervical carcinoma. (van Driel et al., 1999; Ressing et al., 2000). Of the 19 patients, 15 showed progressive disease, 2 remained stable, and 2 showed tumor regression only after additional chemotherapy (van Driel et al., 1999). Furthermore, no increase in lymphocyte inﬁltration was observed after vaccination. The advanced state of the disease and degree of immunosuppression in these patients strongly emphasized the need for clinical trials at earlier stages of the disease. In one such encouraging study, 18 women with high-grade CIN who were positive for HPV16 and were HLA-A2 positive were treated with escalating doses of a E7 CTL peptide (Muderspach et al., 2000). Three of the 18 patients cleared their dysplasia after vaccination, but partial regression of CIN lesions was observed in an additional 6 patients. Most signiﬁcantly, E7 speciﬁc CTLs and cytokine release increased in 10 of 16 patients tested, suggesting that a cell-mediated response was activated after vaccination. This study is the ﬁrst to demonstrate that patients with high-grade cervical dysplasia can be vaccinated against HPV16, overcome host immunosuppression, and mount a detectable immune response in the peripheral blood (Muderspach et al., 2000). It is possible that peptide vaccine intervention at even earlier stages of disease may further improve the clinical efﬁcacy of such studies. Besides the limitation to predetermined target HLA alleles, another major drawback associated with peptide based vaccination includes the risk of inducing T cell tolerance rather than activation. Due to the induction of speciﬁc T cell tolerance, vaccination with a tumor-speciﬁc peptide has been shown to result in an enhanced tumor outgrowth (Toes et al., 1996). The balance between whether a peptide can induce T cell activation or tolerization is determined by peptide concentration over time and peptide persistence (den Boer et al., 2001; Weijzen et al., 2001). Therefore, knowledge of the kinetics of a peptide are critical for the safety and efﬁcacy of peptide-based vaccines. There is also the possibility of the tumor escaping immune recognition by losing expression of its surface antigen and allowing the tumor to hide from a single peptide vaccination (Gajewski et al., 2001). Therefore, vaccine strategies that include more than one CTL peptide against different tumor antigens may be the most effective at eliminating tumor cells.

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6. DENDRITIC CELLS An alternative method to generate powerful anticancer immune responses may be to generate large numbers of autologous antigen loaded DCs for vaccination. Therapeutic immune intervention appears to require optimal presentation of the tumor antigen to the immune system and in this respect the administration of APCs as carriers appears to be effective. DCs are professional APCs and are capable of presenting antigen to and priming na¨ıve T cells in vivo and in vitro. E7 peptide pulsed DCs have been shown to sensitize na¨ıve mice against an HPV tumor and to induce a peptide-speciﬁc tumor protective CTL response in vivo (Ossevoort et al., 1995). Primed DCs have also been shown to produce a signiﬁcant antitumor effect against established HPV tumors in mice and in patients with metastatic melanoma (Mayordomo et al., 1995; Nestle et al., 1998). It has been observed that when immature DCs prepulsed with the HPV16 E7 H2-Db CTL peptide were injected into mice bearing HPV16+ tumors, tumor regression and long-term immunity was observed in 80% of the animals (Mayordomo et al., 1995). Monocyte derived human DCs have also been shown to induce class I speciﬁc responses to targets expressing HPV antigens in vitro (Adams et al., 2001). The potential of immunotherapy in humans with DCs was also demonstrated in a patient with HPV18 advanced cervical carcinoma who received adoptive transfer of T cells stimulated with HPV18 E7 pulsed DCs (Santin et al., 2000). Characterization of peripheral blood after vaccination revealed an increase in E7 speciﬁc CTLs against autologous tumor cells and an increased expression of Th1 immunostimulatory cytokines. These results strongly illustrate the potential of DCs in inducing tumor-speciﬁc CTLs for the treatment of patients with invasive cervical cancer. In addition, the adoptive transfer of ex vivo HPV16 or 18 positive autologous tumor lysate pulsed DCs is currently being tested as a means of expanding HPV-speciﬁc CTL responses in advanced cervical cancer patients (Adams et al., 2001). Antitumor responses have been observed in humans with the use of both peptide primed mature and immature DCs (Nestle et al., 1998). Maturation of the DC may offer the advantage of optimum migratory capacity to the lymph nodes and a stable state that is not susceptible to the cancer associated inﬂuences such as IL-10. However, immature DCs are more efﬁcient in antigen uptake, which can then lead to their subsequent maturation and migration. Consequently, a trial is currently being tested with an initial group of cervical cancer patients treated with immature DCs that will be followed by a group treated with mature DCs (Adams et al., 2001). The superior antigen presentation capabilities of DCs have been exploited in other vaccination strategies as well. DCs have been utilized for the delivery of E6 and E7 proteins (De Bruijn et al., 1998b; Murakami et al., 1999), E6 and E7

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RNA (Thornburg et al., 2000), and have been directly infected with VLPs (Kaufmann et al., 2001) and viral vectors (Dietz and Vukpavlovic, 1998) to present the proteins within. The manufacture of a DC vaccine, however, is complex and the optimum dose, timing, and coordination of antigen loading and vaccination are essential and may present limitations for clinical application.

7. PLASMID DNA IMMUNIZATIONS A fairly new approach in vaccine research is the development of antigen encoding plasmid DNA to produce immunizing proteins in the vaccinated host. DNA vaccines are particularly attractive because they can raise both CTL and antibody responses. This is due to the ability of proteins that are synthesized within the cells to access pathways for presentation by both MHC class I and class II. Popular methods of DNA vaccine delivery are intramuscular and intradermal saline injections of DNA and, most successfully, gene gun bombardment of skin with DNA-coated gold beads. Gene gun immunizations with plasmid DNA expressing HPV16 E7 have resulted in complete protection of mice against a normally lethal challenge of HPV-expressing tumors (Tuting et al., 1999). In DNA immunizations, the use of unmodiﬁed oncogenes is unacceptable because of the potential risk of oncogenic transformation of the host cells. Fortunately, plasmid DNA is easily modiﬁable, allowing for many combinations of genes or DNA encoding epitopes to be engineered together and thereby eliminating this risk of transformation (Da Silva et al., 2001a). One such strategy involves the shufﬂing of HPV E7 in such a way that the resulting protein still contains all CTL epitopes but has lost its transforming capabilities (Osen et al., 2001). Intramuscular vaccination with this shufﬂed DNA was shown to induce E7 H2-Db restricted CTLs and protect mice from subsequent E7 tumor challenge. Furthermore, no transforming activity of the E7 shufﬂed DNA was observed upon transfection of NIH3T3 cells (Osen et al., 2001). Another strategy to develop a safe DNA vaccine with low or no transforming activity is to increase the instability of the E7 protein. Besides eliminating its transforming activity, it has been shown that an unstable protein has a greater potential to generate CTL responses (Tobery and Siliciano, 1997; Townsend et al., 1988). Therefore, an E7 DNA vaccine has been produced encoding two mutations in the zinc binding motifs, which are critical for protein stability (Shi et al., 1999). Encouragingly, this mutated vaccine was shown to induce a signiﬁcantly stronger E7 speciﬁc CTL response and better tumor protection than a wild-type E7 DNA vaccine expressing a stable E7 protein (Shi et al., 1999). The potential of E7 DNA vaccines has been further enhanced by fusing DNA encoding E7 CTL epitopes to the Mycobacterium heat shock protein 70 gene

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(hsp70) (Liu et al., 2000). Due to their unusual immunogenicity, hsp fusions have been shown to enhance the induction of speciﬁc CTLs sufﬁcient to mediate rejection of tumors expressing the fusion partner (Suzue et al., 1997; Suzue and Young, 1996). Delivering this chimeric E7 gene via an adenoassociated virus (AAV) gene vector has been shown to clear E7 established tumors in mice (Liu et al., 2000). Although AAV can elicit a humoral immune response which results in neutralizing activity after a second administration, repeated vaccination was shown not to be necessary given that the originally transduced cells can escape a CTL response and persist in the long term (Liu et al., 2000). Another successful approach has eliminated the need for delivery through viral vectors. By fusing E7 DNA to the Mycobacterium bovis hsp65 gene, it was shown that a single adjuvant free immunization protects mice from HPV16+ tumor challenge and also protects the mice against rechallenge with higher doses of tumor (Chu et al., 2000). Similarly, immunization of tumor-bearing mice with hsp E7 alone leads to tumor regression and long-term survival. Based on these encouraging data, clinical trials are currently underway to test the efﬁcacy of hspE7 to target cervical and anal dysplasias associated with HPV infection (Chu et al., 2000). In contrast to other approaches, this hspE7 immunotherapy eliminates the need to select HLA restrictive epitopes and avoids the use of viral vectors and potentially oncogenic HPV DNA sequences. Another promising strategy in DNA vaccination is to develop a polyepitope string DNA vaccine, encoding several CTL epitopes from varying HLA types, rather than entire proteins, thus eliminating any possible risk of oncogenic transformation. Early reports of epitope string DNA constructs indicated inconsistent efﬁcacy (Whitton et al., 1993; Rodriguez et al., 1998; Corr et al., 1997); however, recent studies have attempted, with much success, to enhance the epitope vaccines strategy. One such polyepitope string DNA vaccine designed for human clinical use has been constructed which contains four HLA-A2 CTL epitopes, a T helper epitope, a B cell epitope, and an HLA-A24 epitope which is also a murine Db epitope (Velders et al., 2001b). The presence of deﬁned amino acid spacers between the epitopes appeared necessary for correct cleavage of peptides by the proteosome and increased the vaccination efﬁcacy in a murine tumor model. It was also shown that the addition of the ubiquitin molecule to the epitope DNA vaccine targeted the protein to the protein degradation pathway and increased CTL precursor frequencies and a Db speciﬁc CTL response (Velders et al., 2001b). Moreover, this report showed protection from and complete eradication of well established tumors in gene gun vaccinated mice. In addition, after immunizing with this construct in HLA-A2 transgenic mice, A2-speciﬁc CTL responses have been obtained against three different HLA-A2 restricted E6/E7 epitopes, suggesting the relevance for the induction of an immune

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response in humans (Eiben et al., manuscript in press). The delivery of DNA via the gene gun has proven to be crucial in this model. It has been proposed that the gene gun delivers DNA on gold particles directly into skin LCs, inducing their migration to the draining lymph nodes to induce CTL responses (Condon et al., 1996). This vaccination strategy is particularly attractive because one DNA polyepitope vaccine can be used to immunize people with many different HLA backgrounds. Unlike virus-based delivery methods, delivery of DNA will allow vaccination repeats as often as necessary without the establishment of preexisting immunity. Furthermore, DNA vaccination is inexpensive, more stable than RNA vaccinations and relatively simple to prepare and deliver.

V. CONCLUSION Evidence has clearly shown that cell-mediated immunity (i.e., T cells), are crucial for the eradication of HPV-infected cells. Furthermore, the failure to induce or maintain a T cell response leads to persistent infection and the development of malignancy. There are several escape mechanisms whereby the activity of T cells is compromised upon HPV infection. Suppressive Th2 cytokines released within the tumor environment have been shown to inhibit CTL activation and antigen presentation. The downregulation of class I molecules on the surface of tumor cells prevents both antigen presentation and recognition of target antigens by effector CTLs, while the upregulation of class II molecules may activate CD4+ CD25+ suppressor T cells to inhibit CTLs. Additionally, there are other mechanisms, such as the downregulation of the TCR ζ chain, which can inhibit T cell activation and contribute to immune escape. The goal of therapeutic immunization against HPV-induced carcinomas is to induce cellular components of the immune system to recognize and attack cells infected with HPV, including malignant tissue. Modulation of the immune response to HPV-infected cells is possible through many different vaccination strategies. Thus far, clinical trials have not been very successful in achieving signiﬁcant tumor regression, most likely because the diseases were too advanced to treat with immunological intervention. However, their ability to stimulate HPV-speciﬁc immune responses in patients is very promising. Further studies that are directed toward patients with early cervical lesions should result in stronger cell-mediated immune responses and improved clinical outcomes. Numerous vaccination strategies against HPV-induced tumors in animal models have shown that both prophylactic and therapeutic vaccines yield HPV-speciﬁc cellular immune responses. Determining which approaches are most applicable to the clinic proves to be a major challenge in the

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development of cancer vaccines. DNA or RNA vaccination encoding the whole HPV E6 and E7 proteins allows for the processing and presentation of all HPV epitopes, including B cell, T helper, and CTL epitopes for every possible HLA haplotype. However, due to the possibility of oncogenic protein transformation when delivered as DNA, an extensive multiepitope approach may be more preferable for DNA vaccination against high-risk HPV. Given the central role of CD4+ T cells in initiating and maintaining an immune response, it will be beneﬁcial to incorporate both MHC class I and MHC class II epitopes into these vaccines. The utilization of an alphavirus vector to deliver HPV E6/E7 will allow for the generation of a universal vaccine without the before-mentioned difﬁculties. This RNA-based system eliminates any risk for transformation, and according to our data, offers the best therapeutic candidate for clinical evaluation. Additionally, the data obtained with bacterial-based delivery systems is promising, especially for the ability to induce mucosal immunity, and ranks among the best clinical candidates as well. Taken together, major steps have been made in the development of therapeutic vaccines against cervical cancer. Their ability to stimulate the cellmediated immune response against HPV may substantially decrease the worldwide morbidity from this disease in the near future.

ACKNOWLEDGMENTS Part of this review is based on studies supported by NIH grants RO1 CA74397 and PO1 CA 74182 (to W.M.K.). G.L.E. is supported by NIH Training Grant T32 AI07508. M.P.V. is a fellow of the Cancer Research Institute. We thank Caroline Le Poole for critically reading this manuscript.

REFERENCES Adams, M., Borysiewicz, L., Fiander, A., Man, S., Jasani, B., Navabi, H., Lipetz, C., Evans, A. S., and Mason, M. (2001). Clinical studies of human papilloma vaccines in pre-invasive and invasive cancer. Vaccine 19, 2549–2556. Alexander, J., Oseroff, C., Sidney, J., Wentworth, P., Keogh, E., Hermanson, G., Chisari, F. V., Kubo, R. T., Grey, H. M., and Sette, A. (1997). Derivation of HLA-A11/Kb transgenic mice. Functional CTL repertoire and recognition of human A11-restricted CTL epitopes. J. Immunol. 159, 4753–4761. Al Saleh, W., Giannini, S. L., Jacobs, N., Moutschen, M., Doyen, J., Boniver, J., and Delvenne, P. (1998). Correlation of T-helper secretory differentiation and types of antigen-presenting cells in squamous intraepithelial lesions of the uterine cervix. J. Pathol. 184, 283–290. Apple, R. J., Becker, T. M., Wheeler, C. M., and Erlich, H. A. (1995). Comparison of human leukocyte antigen DR-DQ disease associations found with cervical dysplasia and invasive cervical carcinoma. J. Natl. Cancer Inst. 87, 427–436.

138

Eiben et al.

Apple, R. J., Erlich, H. A., Klitz, W., Manos, M. M., Becker, T. M., and Wheeler, C. M. (1994). HLA DR-DQ associations with cervical carcinoma show papillomavirus-type speciﬁcity. Nat. Genet. 6, 157–162. Arany, I., Rady, P., and Tyring, S. K. (1993). Alterations in cytokine/antioncogene expression in skin lesions caused by “low-risk” types of human papillomaviruses. Viral Immunol. 6, 255–265. Barra, C., Gourneir, H., Garcia, Z., Marche, P. N., Jouvin-Marche, E., Briand, P., Fillipi, P., and Lemonnier, F. A. (1993). Abrogation of H-2-restricted CTL responses and efﬁcient recognition of HLA-A3 molecules in DBA/2 HLA/A24 responder mice. J. Immunol. 150, 3681–3689. Benton, C., Shyahidullah, H., and Hunter, J. A. M. (1999). Human papillomavirus in the immunocompromised. Papillomavirus Rep. 3, 23–26. Benyacoub, J., Hopkins, S., Potts, A., Kelly, S., Kraehenbuhl, J. P., Curtiss, R., III, De Grandi, P., and Nardelli-Haeﬂiger, D. (1999). The nature of the attenuation of Salmonella typhimurium strains expressing human papillomavirus type 16 virus-like particles determines the systemic and mucosal antibody responses in nasally immunized mice. Infect. Immun. 67, 3674–3679. Boes, B., Hengel, H., Ruppert, T., Multhaup, G., Koszinowski, U. H., and Kloetzel, P. M. (1994). Interferon gamma stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes. J. Exp. Med. 179, 901–909. Bontkes, H. J., van Duin, M., De Gruijl, T. D., Duggan-Keen, M. F., Walboomers, J. M., Stukart, M. J., Verheijen, R. H., Helmerhorst, T. J., Meijer, C. J., Scheper, R. J., Stevens, F. R., Dyer, P. A., Sinnott, P., and Stern, P. L. (1998). HPV 16 infection and progression of cervical intra-epithelial neoplasia: Analysis of HLA polymorphism and HPV 16 E6 sequence variants. Int. J. Cancer. 78, 166–171. Borysiewicz, L. K., Fiander, A., Nimako, M., Man, S., Wilkinson, G. W., Westmoreland, D., Evans, A. S., Adams, M., Stacey, S. N., Boursnell, M. E., Rutherford, E., Hickling, J. K., and Inglis, S. C. (1996). A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet 347, 1523–1527. Boursnell, M. E. G., Rutherford, E., Hickling, J. K., Rollinson, E. A., Munro, A. J., Rolley, N., McLean, C. S., Borysiewicz, L. K., Vousden, K., and Inglis, S. C. (1996). Construction and characterisation of a recombinant vaccinia virus expressing human papillomavirus proteins for immunotherapy of cervical cancer. Vaccine 14, 1485–1494. Breitburd, F., and Coursaget, P. (1999). Human papillomavirus vaccines. Semin. Cancer Biol. 9, 431–444. Campo, M. S., Grindley, G. J., O’Neil, B. W., Chandrachud, L. M., McGarvie, G. M., and Jarret, W. F. (1993). Prophylactic and therapeutic vaccination against a mucosal papillomavirus. J. Gen. Virol. 74 (Pt 6), 945–953. Carter, L. L., and Dutton, R. W. (1996). Type 1 and type 2: A fundamental dichotomy for all T-cell subsets. Curr. Opin. Immunol. 8, 336–342. Centers for Disease Control and Prevention (1999). Division of STD Prevention. Prevention of Genital HPV Infection and Sequelae: Report of an External Consultants’ Meeting. Department of Health and Human Services, Atlanta. Chan, A. C., Irving, B. A., and Weiss, A. (1992). New insights into T-cell antigen receptor structure and signal transduction. Curr. Opin. Immunol. 4, 246–251. Chang, H. L., Gillett, N., Figari, I., Lopez, A. R., Palladino, M. A., and Derynck, R. (1993). Increased transforming growth factor beta expression inhibits cell proliferation in vitro, yet increases tumorigenicity and tumor growth of Meth A sarcoma cells. Cancer Res. 53, 4391–4398. Chu, N. R., Wu, H. B., Wu, T., Boux, L. J., Siegel, M. I., and Mizzen, L. A. (2000). Immunotherapy of a human papillomavirus (HPV) type 16 E7-expressing tumour by administration of fusion protein comprising Mycobacterium bovis bacille Calmette–Guerin (BCG) hsp65 and HPV 16 E7. Clin. Exp. Immunol. 121, 216–225.

HPV-Induced Cervical Cancer

139

Clarke, S. R. (2000). The critical role of CD40/CD40L in the CD4-dependent generation of CD8+ T cell immunity. J. Leukoc. Biol. 67, 607–614. Coleman, N., Birley, H. D., Renton, A. M., Hanna, N. F., Ryait, B. K., Byrne, M., TaylorRobinson, D., and Stanley, M. A. (1994). Immunological events in regressing genital warts. Am. J. Clin. Pathol. 102, 768–774. Combita, A. L., Touze, A., Bousarghin, L., Sizaret, P. Y., Munoz, N., and Coursaget, P. (2001). Gene transfer using human papillomavirus pseudovirions varies according to virus genotype and requires cell surface heparan sulfate. FEMS Microbiol. Lett. 204, 183–188. Condon, C., Watkins, S. C., Celluzzi, C. M., Thompson, K., and Falo, L. D. (1996). DNA-based immunization by in vivo transfection of dendritic cells. Nat. Med. 2, 1122–1128. Corr, M., Tighe, H., Lee, D., Dudler, J., Trieu, M., Brinson, D., and Carson, D. A. (1997). Costimulation provided by DNA immunization enhances antitumor immunity. J Immunol. 159, 4999–5004. Crish, J. F., Bone, F., Balasubramanian, S., Zaim, T. M., Wagner, T., Yun, J., Rorke, E. A., and Eckert, R. L. (2000). Suprabasal expression of the human papillomavirus type 16 oncoproteins in mouse epidermis alters expression of cell cycle regulatory proteins. Carcinogenesis 21, 1031–1037. Cromme, F. V., Meijer, C. J., Snijders, P. J., Uyterlinde, A., Kenemans, P., Helmerhorst, T., Stern, P. L., van den Brule, A. J., and Walboomers, J. M. (1993). Analysis of MHC class I and II expression in relation to presence of HPV genotypes in premalignant and malignant cervical lesions. Br. J. Cancer 67, 1372–1380. Cumberbatch, M., and Kimber, I. (1992). Dermal tumour necrosis factor-alpha induces dendritic cell migration to draining lymph nodes, and possibly provides one stimulus for Langerhans’ cell migration. Immunology 75, 257–263. Daemen, T., Pries, F., Bungener, L., Kraak, M., Regts, J., and Wilschut, J. (2000). Genetic immunization against cervical carcinoma: Induction of cytotoxic T lymphocyte activity with a recombinant alphavirus vector expressing human papillomavirus type 16 E6 and E7. Gene Ther. 7, 1859–1866. Da Silva, D. M., Eiben, G. L., Fausch, S. C., Wakabayashi, M. T., Rudolf, M. P., Velders, M. P., and Kast, W. M. (2001a). Cervical cancer vaccines: Emerging concepts and developments. J. Cell Physiol. 186, 169–182. Da Silva, D. M., Pastrana, D. V., Schiller, J. T., and Kast, W. M. (2001b). Effect of preexisting neutralizing antibodies on the anti-tumor immune response induced by chimeric human papillomavirus virus-like particle vaccines. Virology 290, 350–360. David, A. L., Taylor, G. M., Gokhale, D., Aplin, J. D., Seif, M. W., and Tindall, V. R. (1992). HLA-DQB1∗ 03 and cervical intraepithelial neoplasia type III. Lancet 340, 52. De Bruijn, M. L., Greenstone, H. L., Vermeulen, H., Melief, C. J. M., Lowy, D. R., Schiller, J. T., and Kast, W. M. (1998a). L1-speciﬁc protection from tumor challenge elicited by HPV16 virus-like particles. Virology 250, 371–376. De Bruijn, M. L., Schuurhuis, D. H., Vierboom, M. P., Vermeulen, H., de Cock, K. A., Ooms, M. E., Ressing, M. E., Toebes, M., Franken, K. L., Drijfhout, J. W., Ottenhoff, T. H., Offringa, R., and Melief, C. J. (1998b). Immunization with human papillomavirus type 16 (HPV 16) oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I-restricted protection to HPV 16-induced tumor cells. Cancer Res. 58, 724–731. De Gruijl, T. D., Bontkes, H. J., Peccatori, F., Gallee, M. P., Helmerhorst, T. J., Verheijen, R. H., Aarbiou, J., Mulder, W. M., Walboomers, J. M., Meijer, C. J., van, d., V, and Scheper, R. J. (1999). Expression of CD3-zeta on T-cells in primary cervical carcinoma and in metastasispositive and -negative pelvic lymph nodes. Br. J. Cancer 79, 1127–1132. De Gruijl, T. D., Bontkes, H. J., Stukart, M. J., Walboomers, J. M., Remmink, A. J., Verheijen, R. H., Helmerhorst, T. J., Meijer, C. J., and Scheper, R. J. (1996). T cell proliferative responses against human papillomavirus type 16 E7 oncoprotein are most prominent in cervical

140

Eiben et al.

intraepithelial neoplasia patients with a persistent viral infection. J. Gen. Virol. 77 (Pt 9), 2183–2191. De Gruijl, T. D., Bontkes, H. J., Walboomers, J. M., Stukart, M. J., Doekhie, F. S., Remmink, A. J., Helmerhorst, T. J., Verheijen, R. H., Duggan-Keen, M. F., Stern, P. L., Meijer, C. J., and Scheper, R. J. (1998). Differential T helper cell responses to human papillomavirus type 16 E7 related to viral clearance or persistence in patients with cervical neoplasia: a longitudinal study. Cancer Res. 58, 1700–1706. Den Boer, A. T., Diehl, L., van Mierlo, G. J. D., van der Voort, E. I. H., Fransen, M. F., Krimpenfort, P., Melief, C. J. M., Offringa, R., and Toes, R. E. M. (2001). Longevity of antigen presentation and activation status of APC are decisive factors in the balance between CTL immunity versus tolerance. J. Immunol. 167, 2522–2528. Dietz, A. B., and Vukpavlovic, S. (1998). High efﬁciency adenovirus-mediated gene transfer to human dendritic cells. Blood 91, 392–398. Duggan-Keen, M. F., Keating, P. J., Stevens, F. R. A., Sinnott, P., Snijders, P. J. F., Walboomers, J. M. M., Davidson, S., Hunter, R. D., Dyer, P. A., and Stern, P. L. (1996). Immunogenetic factors in HPV-associated cervical cancer—Inﬂuence on disease progression. Eur. J. Immunogenet. 23, 275–284. Dupuy, C., Buzoni-Gatel, D., Touze, A., Bout, D., and Coursaget, P. (1999). Nasal immunization of mice with human papillomavirus type 16 (HPV-16) virus-like particles or with the HPV-16 L1 gene elicits speciﬁc cytotoxic T lymphocytes in vaginal draining lymph nodes. J. Virol. 73, 9063–9071. Dupuy, C., Buzoni-Gatel, D., Touze, A., Le Cann, P., Bout, D., and Coursaget, P. (1997). Cell mediated immunity induced in mice by HPV 16 L1 virus-like particles. Microbiol. Pathogen. 22, 219–225. Durst, M., Glitz, D., Schneider, A., and Zur, H. H. (1992). Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: Analysis by in situ hybridization. Virology 189, 132–140. Dyson, N., Howley, P. M., Munger, K., and Harlow, E. (1989). The human papillomavirus16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243, 934– 940. El Sherif, A. M., Seth, R., Tighe, P. J., and Jenkins, D. (2001). Quantitative analysis of IL-10 and IFN-gamma mRNA levels in normal cervix and human papillomavirus type 16 associated cervical precancer. J. Pathol. 195, 179–185. Evans, E. M., Man, S., Evans, A. S., and Borysiewicz, L. K. (1997). Inﬁltration of cervical cancer tissue with human papillomavirus-speciﬁc cytotoxic T-lymphocytes. Cancer Res. 57, 2943–2950. Faulkner, L., Borysiewicz, L. K., and Man, S. (1998). The use of human leucocyte antigen class I transgenic mice to investigate human immune function. J. Immunol. Methods 221, 1–16. Feltkamp, M. C., Vreugdenhil, G. R., Vierboom, M. P., Ras, E., van der Burg, S. H., ter Schegget, J., Melief, C. J. M., and Kast, W. M. (1995). Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16-induced tumors. Eur. J. Immunol. 25, 2638–2642. Feltkamp, M. C. W., Smits, H. L., Vierboom, M. P. M., Minnaar, R. P., de Jongh, B. M., Drijfhout, J. W., ter Schegget, J., Melief, C. J. M., and Kast, W. M. (1993). Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur. J. Immunol. 23, 2242–2249. Fleuren, G. J., Gorter, A., Kuppen, P. J. K., Litvinov, S. V., and Warnaar, S. O. (1995). Tumor heterogeneity and immunotherapy of cancer. Immunol. Rev. 145, 91–122. Frisch, M., Biggar, R. J., and Goedert, J. J. (2000). Human papillomavirus-associated cancers in patients with human immunodeﬁciency virus infection and acquired immunodeﬁciency syndrome. J. Natl. Cancer Inst. 92, 1500–1510.

HPV-Induced Cervical Cancer

141

Gajewski, T. F., Fallarino, F., Ashikari, A., and Sherman, A. (2001). Immunization of HLA-A2+ melanoma patients with MAGE-3 or MelanA peptide-pulsed autologous peripheral blood mononuclear cells plus recombinant human interleukin 12. Clin. Cancer Res. 7, 895s–901s. Gallichan, W. S., and Rosenthal, K. L. (1998). Long-term immunity and protection against herpes simplex virus type 2 in the murine female genital tract after mucosal but not systemic immunization. J. Infect. Dis. 177, 1155–1161. Gedde, M. M., Higgins, D. E., Tilney, L. G., and Portnoy, D. A. (2000). Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes. Infect. Immun. 68, 999–1003. Geisbill, J., Osmers, U., and Durst, M. (1997). Detection and characterization of human papillomavirus type 45 DNA in the cervical carcinoma cell line MS751. J. Gen. Virol. 78 (Pt 3), 655–658. Gemignani, M., Maiman, M., Fruchter, R. G., Arrastia, C. D., Gibbon, D., and Ellison, T. (1995). CD4 lymphocytes in women with invasive and preinvasive cervical neoplasia. Gynecol. Oncol. 59, 364–369. Gerard, C. M., Baudson, N., Kraemer, K., Bruck, C., Garcon, N., Paterson, Y., Pan, Z. K., and Pardoll, D. (2001). Therapeutic potential of protein and adjuvant vaccinations on tumour growth. Vaccine 19, 2583–2589. Germain, R. N., and Hendrix, L. R. (1991). MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding. Nature 353, 134–139. Ghosh, A. K., and Moore, M. (1992). Tumour-inﬁltrating lymphocytes in cervical carcinoma. Eur. J. Cancer 28A, 1910–1916. Glas, R., Franksson, L., Une, C., Eloranta, M. L., Ohlen, C., Orn, A., and Karre, K. (2000). Recruitment and activation of natural killer (NK) cells in vivo determined by the target cell phenotype. An adaptive component of NK cell-mediated responses. J. Exp. Med. 191, 129–138. Glew, S. S., Connor, M. E., Snijders, P. J., Stanbridge, C. M., Buckley, C. H., Walboomers, J. M., Meijer, C. J., and Stern, P. L. (1993). HLA expression in pre-invasive cervical neoplasia in relation to human papilloma virus infection. Eur. J. Cancer 29A, 1963–1970. Glew, S. S., Duggan-Keen, M., Cabrera, T., and Stern, P. L. (1992). HLA class II antigen expression in human papillomavirus-associated cervical cancer. Cancer Res. 52, 4009–4016. Greenstone, H. L., Nieland, J. D., de Visser, K. E., De Bruijn, M. L., Kirnbauer, R., Roden, R. B. S., Lowy, D. R., Kast, W. M., and Schiller, J. T. (1998). Chimeric papillomavirus viruslike particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model. Proc. Natl. Acad. Sci. USA 95, 1800–1805. Guadagno, T. M., Ohtsubo, M., Roberts, J. M., and Assoian, R. K. (1993). A link between cyclin A expression and adhesion-dependent cell cycle progression. Science 262, 1572–1575. [erratum: Science (1994, Jan. 28); 263(5146):455]. Gunn, G. R., Zubair, A., Peters, C., Pan, Z. K., Wu, T. C., and Paterson, Y. (2001). Two listeria monocytogenes vaccine vectors that express different molecular forms of human papilloma virus-16 (HPV-16) E7 induce qualitatively different T cell immunity that correlates with their ability to induce regression of established tumors immortalized by HPV-16. J. Immunol. 167, 6471–6479. Halpert, R., Fruchter, R. G., Sedlis, A., Butt, K., Boyce, J. G., and Sillman, F. H. (1986). Human papillomavirus and lower genital neoplasia in renal transplant patients. Obstet. Gynecol. 68, 251–258. Hazelbag, S., Fleuren, G. J., Baelde, J. J., Schuuring, E., Kenter, G. G., and Gorter, A. (2001). Cytokine proﬁle of cervical cancer cells. Gynecol. Oncol. 83, 235–243. Helland, A., Borresen, A. L., Kristensen, G., and Ronningen, K. S. (1994). DQA1 and DQB1 genes in patients with squamous cell carcinoma of the cervix: Relationship to human papillomavirus infection and prognosis. Cancer Epidemiol. Biomarkers Prev. 3, 479–486.

142

Eiben et al.

Hilders, C. G. J. M., Houbiers, J. G. A., Van Ravenswaay Claasen, H. H., Veldhuizen, R. W., and Fleuren, G. J. (1993). Association between HLA-expression and inﬁltration of immune cells in cervical carcinoma. Lab. Invest. 69, 651–659. Ho, G. Y., Bierman, R., Beardsley, L., Chang, C. J., and Burk, R. D. (1998). Natural history of cervicovaginal papillomavirus infection in young women. N. Engl. J. Med. 338, 423–428. Hohn, H., Pilch, H., Gunzel, S., Neukirch, C., Hilmes, C., Kaufman, A., Seliger, B., and Maeurer, M. J. (1999). CD4+ tumor-inﬁltrating lymphocytes in cervical cancer recognize HLA-DR-restricted peptides provided by human papillomavirus-E7. J. Immunol. 163, 5715– 5722. Hopﬂ, R., Heim, K., Christensen, N., Zumbach, K., Wieland, U., Volgger, B., Widschwendter, A., Haimbuchner, S., Muller-Holzner, E., Pawlita, M., Pﬁster, H., and Fritsch, P. (2000). Spontaneous regression of CIN and delayed-type hypersensitivity to HPV-16 oncoprotein E7. Lancet 356, 1985–1986. Huang, M., Wang, J., Lee, P., Sharma, S., Mao, J. T., Meissner, H., Uyemura, K., Modlin, R., Wollman, J., and Dubinett, S. M. (1995). Human non-small cell lung cancer cells express a type 2 cytokine pattern. Cancer Res. 55, 3847–3853. Kadish, A. S., Ho, G. Y., Burk, R. D., Wang, Y., Romney, S. L., Ledwidge, R., and Angeletti, R. H. (1997). Lymphoproliferative responses to human papillomavirus (HPV) type 16 proteins E6 and E7: outcome of HPV infection and associated neoplasia. J. Natl. Cancer Inst. 89, 1285–1293. Kadish, A. S., Romney, S. L., Ledwidge, R., Tindle, R., Fernando, G. J., Zee, S. Y., Van Ranst, M. A., and Burk, R. D. (1994). Cell-mediated immune responses to E7 peptides of human papillomavirus (HPV) type 16 are dependent on the HPV type infecting the cervix whereas serological reactivity is not type-speciﬁc. J. Gen. Virol. 75 (Pt 9), 2277–2284. Kashii, Y., Giorda, R., Herberman, R. B., Whiteside, T. L., and Vujanovic, N. L. (1999). Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells. J. Immunol. 163, 5358–5366. Kast, W. M. (2000). In “Peptide Based Cancer Vaccines,” Georgetown, Texas, USA. Kast, W. M., Brandt, R. M. P., Sidney, J., Drijfhout, J. W., Kubo, R. T., Grey, H. M., Melief, C. J. M., and Sette, A. (1994). Role of HLA-A motifs in identiﬁcation of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins. J. Immunol. 152, 3904– 3912. Kast, W. M., Feltkamp, M. C. W., Ressing, M. E., Vierboom, M. P. M., Brandt, R. M. P., and Melief, C. J. M. (1996). Cellular immunity against human papillomavirus associated cervical cancer. Semin. Virol. 7, 117–123. Kaufmann, A. M., Nieland, J., Schinz, M., Nonn, M., Gabelsberger, J., Meissner, H., Muller, R. T., Jochmus, I., Gissman, L., Schneider, A., and Durst, M. (2001). HPV16 L1E7 chimeric virus-like particles induce speciﬁc HLA-restricted T cells in humans after in vitro vaccination. Int. J. Cancer. 92, 285–293. Keating, P. J., Cromme, F. V., Duggan-Keen, M., Snijders, P. J., Walboomers, J. M., Hunter, R. D., Dyer, P. A., and Stern, P. L. (1995). Frequency of down-regulation of individual HLA-A and -B alleles in cervical carcinomas in relation to TAP-1 expression. Br. J. Cancer 72, 405–411. Kim, J., Modlin, R. L., Moy, R. L., Dubinett, S. M., McHugh, T., Nickoloff, B. J., and Uyemura, K. (1995). IL-10 production in cutaneous basal and squamous cell carcinomas. A mechanism for evading the local T cell immune response. J. Immunol. 155, 2240–2247. Kirnbauer, R., Chandrachud, L. M., O’Neil, B. W., Wagner, E. R., Grindlay, G. J., Armstrong, A., McGarvie, G. M., Schiller, J. T., Lowy, D. R., and Campo, M. S. (1996). Virus-like particles of bovine papillomavirus type 4 in prophylactic and therapeutic immunization. Virology 219, 37–44. Kitani, A., Fuss, I. J., Nakamura, K., Schwartz, O. M., Usui, T., and Strober, W. (2000). Treatment of experimental (trinitrobenzene sulfonic acid) colitis by intranasal administration

HPV-Induced Cervical Cancer

143

of transforming growth factor (TGF)-beta1 plasmid: TGF-beta1-mediated suppression of T helper cell type 1 response occurs by interleukin (IL)-10 induction and IL-12 receptor beta2 chain downregulation. J. Exp. Med. 192, 41–52. Kono, K., Ressing, M. E., Brandt, R. M. P., Melief, C. J. M., Potkul, R. K., Andersson, B., Petersson, M., Kast, W. M., and Kiessling, R. (1996a). Decreased expression of signaltransducing zeta chain in peripheral T cells and natural killer cells in patients with cervical cancer. Clin. Cancer Res. 2, 1825–1828. Kono, K., Salazar-Onfray, F., Peterson, M., Hansson, J., Masucci, G., Wasserman, K., Nakazawa, T., Anderson, P., and Kiessling, R. (1996b). Hydrogen peroxide secreted by tumor-derived macrophages down-modulates signal-transducing zeta molecules and inhibits tumor-speciﬁc T cell- and natural killer cell-mediated cytotoxicity. Eur. J. Immunol. 26, 1308–1313. Konya, J., and Dillner, J. (2001). Immunity to oncogenic human papillomaviruses. Adv. Cancer Res. 82, 205–235. Krul, M. R., Tijhaar, E. J., Kleijne, J. A., Van Loon, A. M., Nievers, M. G., Schipper, H., Geerse, L., Van der, K. M., Steerenberg, P. A., Mooi, F. R., and Den Otter, W. (1996). Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains. Cancer Immunol. Immunother. 43, 44–48. Lin, K. Y., Guarnieri, F. G., Staveley-O’Carroll, K. F., Levitsky, H. I., August, J. T., Pardoll, D. M., and Wu, T. C. (1996). Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 56, 21–26. Liu, D. W., Tsao, Y. P., Hsieh, C. H., Hsieh, J. T., Kung, J. T., Chiang, C. L., Huang, S. J., and Chen, S. L. (2000). Induction of CD8 T cells by vaccination with recombinant adenovirus expressing human papillomavirus type 16 E5 gene reduces tumor growth. J. Virol. 74, 9083–9089. Londono, L. P., Chatﬁeld, S., Tindle, R. W., Herd, K., Gao, X. M., Frazer, I., and Dougan, G. (1996). Immunisation of mice using Salmonella typhimurium expressing human papillomavirus type 16 E7 epitopes inserted into hepatitis B virus core antigen. Vaccine 14, 545–552. Luxton, J. C., Rowe, A. J., Cridland, J. C., Coletart, T., Wilson, P., and Shepherd, P. S. (1996). Proliferative T cell responses to the human papillomavirus type 16 E7 protein in women with cervical dysplasia and cervical carcinoma and in healthy individuals. J. Gen. Virol. 77, 1585–1593. Macagno, A., Gilliet, M., Sallusto, F., Lanzavecchia, A., Nestle, F. O., and Groettrup, M. (1999). Dendritic cell up-regulate immunoproteasomes and the proteasome regulator PA28 during maturation. Eur. J. Immunol. 29, 4037–4042. MacDonald, G. H., and Johnston, R. E. (2000). Role of dendritic cell targeting in Venezuelan equine encephalitis virus pathogenesis. J. Virol. 74, 914–922. Mata, M., Yao, Z. J., Zubair, A., Syres, K., and Paterson, Y. (2001). Evaluation of a recombinant Listeria monocytogenes expressing an HIV protein that protects mice against viral challenge. Vaccine 19, 1435–1445. Matloubian, M., Concepcion, R. J., and Ahmed, R. (1994). CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J. Virol. 68, 8056–8063. Maudsley, D. J. (1991). Role of oncogenes in the regulation of MHC antigen expression. Biochem. Soc. Trans. 19, 291–296. Mayordomo, J. I., Zorina, T., Storkus, W. J., Zitvogel, L., Celluzzi, C., Falo, L. D., Melief, C. J. M., Ildstad, S. T., Kast, W. M., Deleo, A. B., and Lotze, M. T. (1995). Bone marrowderived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat. Med. 1, 1297–1302. Miller, S. I., and Mekalanos, J. J. (1990). Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages. J. Bacteriol. 172, 2485–2490.

144

Eiben et al.

Montoya, L., Saiz, I., Rey, G., Vela, F., and Clerici-Larradet, N. (1998). Cervical carcinoma: human papillomavirus infection and HLA-associated risk factors in the Spanish population. Eur. J. Immunogenet. 25, 329–337. Morel, S., Levy, F., Burlet-Schiltz, O., Brasseur, F., Probst-Kepper, M., Peitrequin, A. L., Monsarrat, B., van velthoven, R., Cerottini, J.-C., Boon, T., Gairin, J. E., and Van den Eynde, B. (2000). Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells. Immunity. 12, 107–117. Morelli, A. E., Sananes, C., Di Paola, G., Paredes, A., and Fainboim, L. (1993). Relationship between types of human papillomavirus and Langerhans’ cells in cervical condyloma and intraepithelial neoplasia. Am. J. Clin. Pathol. 99, 200–206. Mosmann, T. R., Li, L., and Sad, S. (1997). Functions of CD8 T-cell subsets secreting different cytokine patterns. Semin. Immunol. 9, 87–92. Muderspach, L., Wilczyneki, S., Roman, L., Bade, L., Felix, J., Small, L. A., Kast, W. M., Fascia, G., Marty, V., and Weber, J. (2000). A phase I trial of a HPV peptide vaccine for women with high grade cervical and vulvar intraepithelial neoplasia who are HPV 16 positive. Clin. Cancer Res. 6, 3406–3416. Munoz, N. (1997). In “New Developments in Cervical Cancer Screening and Prevention” (E. Franco and J. Monsonego, Eds.). Blackwell Science, Oxford, pp. 3–13. Murakami, M., Gurski, K. J., Marincola, F. M., Ackland, J., and Steller, M. A. (1999). Induction of speciﬁc CD8+ T-lymphocyte responses using a human papillomavirus-16 E6/E7 fusion protein and autologous dendritic cells. Cancer Res. 59, 1184–87. Nakagomi, H., Pisa, P., Pisa, E. K., Yamamoto, Y., Halapi, E., Backlin, K., Juhlin, C., and Kiessling, R. (1995). Lack of interleukin-2 (IL-2) expression and selective expression of IL-10 mRNA in human renal cell carcinoma. Int. J. Cancer 63, 366–371. Nardelli-Haeﬂiger, D., Roden, R. B., Benyacoub, J., Sahli, R., Kraehenbuhl, J. P., Schiller, J. T., Lachat, P., Potts, A., and De Grandi, P. (1997). Human papillomavirus type 16 viruslike particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice. Infect. Immun. 65, 3328–3336. Nestle, F. O., Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer, R., Burg, G., and Schadendorf, D. (1998). Vaccination of melanoma patients with peptide- or tumor lysatepulsed dendritic cells. Nat. Med. 4, 328–332. Odunsi, K., Terry, G., Ho, L., Bell, J., Cuzick, J., and Ganesan, T. S. (1995). Association between HLA DQB1∗ 03 and cervical intra-epithelial neoplasia. Mol. Med. 1, 161–171. Onizuka, S., Tawara, I., Shimizu, J., Sakaguchi, S., Fujita, T., and Nakayama, E. (1999). Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res. 59, 3128–3133. Osen, W., Peiler, T., Ohlschlager, P., Caldeira, S., Faath, S., Michel, N., Muller, M., Tommasino, M., Jochmus, I., and Gissmann, L. (2001). A DNA vaccine based on a shufﬂed E7 oncogene of the human papillomavirus type 16 (HPV 16) induces E7-speciﬁc cytotoxic T cells but lacks transforming activity. Vaccine 19, 4276–4286. Ossevoort, M. A., Feltkamp, M. C., van Veen, K. J., Melief, C. J. M., and Kast, W. M. (1995). Dendritic cells as carriers for a cytotoxic T-lymphocyte epitope-based peptide vaccine in protection against a human papillomavirus type 16-induced tumor. J. Immunother. 18, 86–94. Parkin, D. M., Pisani, P., and Ferlay, J. (1999). Estimates of the worldwide incidence of 25 major cancers in 1990. Int. J. Cancer 80, 827–841. Pei, X. F. (1996). The human papillomavirus E6/E7 genes induce discordant changes in the expression of cell growth regulatory proteins. Carcinogenesis 17, 1395–1401. Peng, S., Frazer, I. H., Fernando, G. J., and Zhou, J. (1998). Papillomavirus virus-like particles can deliver deﬁned CTL epitopes to the MHC class I pathway. Virology 240, 147–157. Petry, K. U., Schefﬂe, D., Bode, U., Gabrysiak, T., Kochel, H., Kupsch, E., Glaubitz, M., Niesert, S., Kuhnle, H., and Schedel, I. (1994). Cellular immunodeﬁciency enhances the progression of human papillomavirus-associated cervical lesions. Int. J. Cancer 54, 836–840.

HPV-Induced Cervical Cancer

145

Read, S., Malmstrom, V., and Powrie, F. (2000). Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inﬂammation. J. Exp. Med. 192, 295–302. Remmink, A. J., Walboomers, J. M., Helmerhorst, T. J., Voorhorst, F. J., Rozendaal, L., Risse, E. K., Meijer, C. J., and Kenemans, P. (1995). The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease: Natural history up to 36 months. Int. J. Cancer 61, 306–311. Ressing, M. E., de Jong, J. H., Brandt, R. M., Drijfhout, J. W., Benckhuijsen, W. E., Schreuder, G. M., Offringa, R., Kast, W. M., and Melief, C. J. (1999). Differential binding of viral peptides to HLA-A2 alleles. Implications for human papillomavirus type 16 E7 peptidebased vaccination against cervical carcinoma. Eur. J. Immunol. 29, 1292–1303. Ressing, M. E., Sette, A., Brandt, R. M. P., Ruppert, J., Wentworth, P. A., Hartman, M., Oseroff, C., Grey, H. M., Melief, C. J. M., and Kast, W. M. (1995). Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identiﬁed through in vivo and in vitro immunogenicity studies of HLA-A∗ 0201-binding peptides. J. Immunol. 154, 5934–5943. Ressing, M. E., van Driel, W. J., Brandt, R. M. P., Kenter, G. G., de Jong, J. H., Bauknecht, T., Fleuren, G. J., Hoogerhout P., Offringa, R., Sette, A., Celis, E., Grey, H., Trimbos, B. J., Kast, W. M., and Melief, C. J. M. (2000). Detection of T helper responses, but not of human papillomavirus-speciﬁc cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma. J. Immunother. 23, 255–266. Ressing, M. E., van Driel, W. J., Celis, E., Sette, A., Brandt, R. M. P., Hartman, M., Anholts, J. D. H., Schreuder, G. M. T., Bram ter Harmsel, W., Fleuren, G. J., Trimbos, B. J., Kast, W. M., and Melief, C. J. M. (1996). Occasional memory cytotoxic T-cell responses of patients with human papillomavirus type 16-positive cervical lesions against a human leukocyte antigen-A ∗ 0201-restricted E7-encoded epitope. Cancer Res. 56, 582–588. Revaz, V., Benyacoub, J., Kast, W. M., Schiller, J. T., De Grandi, P., and Nardelli-Haeﬂiger, D. (2001). Mucosal vaccination with a recombinant salmonella typhimurium expressing human papillomavirus type 16 (HPV16) L1 virus-like particles (VLPs) or HPV16 VLPs puriﬁed from insect cells inhibits the growth of HPV16-expressing tumor cells in mice. Virology 279, 354–360. Rockley, P. F., and Tyring, S. K. (1995). Interferons alpha, beta and gamma therapy of anogenital human papillomavirus infections. Pharmacol. Ther. 65, 265–287. Rodriguez, F., An, L. L., Harkins, S., Zhang, J., Yokoyama, M., Widera, G., Fuller, J. T., Kincaid, C., Campbell, I., and Whitton, J. L. (1998). DNA immunization with minigenes: Low frequency of memory cytotoxic T lymphocytes and inefﬁcient antiviral protection are rectiﬁed by ubiquitination. J. Virol. 72, 5174–5181. Rose, R. C., Reichmann, R. C., and Bonnez, W. (1994). Human papillomavirus (HPV) type 11 recombinant virus-like particles induce the formation of neutralizing antibodies and detect HPV-speciﬁc antibodies in human sera. J. Gen. Virol. 75 (Pt 8), 2075–2079. Rudolf, M. P., Fausch, S. C., Da Silva, D. M., and Kast, W. M. (2001a). Human dendritic cells are activated by chimeric human papillomavirus type-16 virus-like particles and induce epitope-speciﬁc human T cell responses in vitro. J. Immunol. 166, 5917–5924. Rudolf, M. P., Man, S., Melief, C. J., Sette, A., and Kast, W. M. (2001b). Human T-cell responses to HLA-A-restricted high binding afﬁnity peptides of human papillomavirus type 18 proteins E6 and E7. Clin. Cancer Res. 7, 788s–795s. Rudolf, M. P., Nieland, J. D., Da Silva, D. M., Velders, M. P., Muller, M., Greenstone, H. L., Schiller, J. T., and Kast, W. M. (1999a). Induction of HPV16 capsid protein-speciﬁc human T cell responses by virus-like particles. Biol. Chem. 380, 335–340. Rudolf, M. P., Small, L. A., Velders, M. P., Da Silva, D. M., Weijzen, S., and Kast, W. M. (1999b). Crucial role of vaccine delivery system and immune suppressed state in cervical cancer patients undergoing treatment with therapeutic vaccines. In “Cancer Vaccines: Strategies,

146

Eiben et al.

Prospects and Realities of Immune Intervention” (P. L. Stern, M. Carroll, and P. C. L. Beverly, Eds.). Cambridge University Press, Cambridge. Sad, S., Marcotte, R., and Mosmann, T. R. (1995). Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines. Immunity 2, 271–279. Santin, A. D., Hermonat, P. L., Ravaggi, A., Bellone, S., Pecorelli, S., Roman, J. J., Parham, G. P., and Cannon, M. J. (2000). Interleukin-10 increases Th1 cytokine production and cytotoxic potential in human papillomavirus-speciﬁc CD8+ cytotoxic T lymphocytes. J. Virol. 74, 4729–4737. Sastre-Garau, X., Loste, M. N., Vincent-Salomon, A., Favre, M., Mouret, E., de la, R. A., Durand, J. C., Tartour, E., Lepage, V., and Charron, D. (1996). Decreased frequency of HLA-DRB1 13 alleles in Frenchwomen with HPV-positive carcinoma of the cervix. Int. J. Cancer 69, 159–164. Schafer, K., Muller, M., Faath, S., Henn, A., Osen, W., Zentgraf, H, Benner, A., Gissman, L., and Jochmus, I. (1999). Immune response to human papillomavirus 16 L1E7 chimeric viruslike particles: Induction of cytotoxic T cells and speciﬁc tumor protection. Int. J. Cancer 81, 881–888. Schiffman, M. H., Bauer, H. M., Hoover, R. N., Glass, A. G., Cadell, D. M., Rush, B. B., Scott, D. R., Sherman, M. E., Kurman, R. J., Wacholder, S. et al. (1993). Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J. Natl. Cancer Inst. 85, 958–964. Schiller, J. T., and Roden, R. B. S. (1995). Papillomavirus-like particles. Papillomavirus Rep. 6, 121–128. Schmielau, J., and Finn, O. J. (2001). Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients. Cancer Res. 61, 4756–4760. Schoenberger, S. P., Toes, R. E. M., Vandervoort, E. I. H., Offringa, R., and Melief, C. J. M. (1998). T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393, 480–483. Seder, R. A., Boulay, J. L., Finkelman, F., Barbier, S., Ben Sasson, S. Z., Le Gros, G., and Paul, W. E. (1992). CD8+ T cells can be primed in vitro to produce IL-4. J. Immunol. 148, 1652–1656. Seedorf, K., Oltersdorf, T., Krammer, G., and Rowekamp, W. (1987). Identiﬁcation of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells. EMBO J. 6, 139–144. Sheu, B.-C., Lin, R.-H., Lien, H.-C., Ho, H.-N., Hsu, S.-M., and Huang, S.-C. (2001). Predominant Th2/Tc2 polarity of tumor-inﬁltrating lymphocytes in human cervical cancer. J. Virol. 167, 2972–2978. Shi, W., Bu, P., Liu, J., Polack, A., Fisher, S., and Qiao, L. (1999). Human papillomavirus type 16 E7 DNA vaccine: Mutation in the open reading frame of E7 enhances speciﬁc cytotoxic T-lymphocyte induction and antitumor activity. J. Virol. 73, 7877–7881. Steinbrink, K., Jonuleit, H., Muller, G., Schuler, G., Knop, J., and Enk, A. H. (1999). Interleukin10-treated human dendritic cells induce a melanoma-antigen-speciﬁc anergy in CD8(+) T cells resulting in a failure to lyse tumor cells. Blood 93, 1634–1642. Stellato, G., Nieminem, P., Aho, M., Lehtinen, T., Lehtinen, M., and Paavonen, J. (1997). Type 1 cytokine response and treatment outcome of genital HPV lesions. Genitourin. Med. 73, 387–390. Stern, P. L. (1996). Immunity to human papillomavirus-associated cervical neoplasia. Adv. Cancer Res. 69, 175–211. Stohlman, S. A., Bergmann, C. C., Lin, M. T., Cua, D. J., and Hinton, D. R. (1998). CTL effector function within the central nervous system requires CD4+ T cells. J. Immunol. 160, 2896–2904.

HPV-Induced Cervical Cancer

147

Stone, K. M. (1995). Human papillomavirus infection and genital warts: Update on epidemiology and treatment. Clin. Infect. Dis. 20 (Suppl 1), S91–S97. Strang, G., Hickling, J. K., McIndoe, G. A., Howland, K., Wilkinson, D, Ikeda, H., and Rothbard, J. B. (1990). Human T cell responses to human papillomavirus type 16 L1 and E6 synthetic peptides: Identiﬁcation of T cell determinants, HLA-DR restriction and virus type speciﬁcity. J. Gen. Virol. 71, 423–431. Street, M., Herd, K., Londono, P., Doan, T., Dougan, G., Kast, W. M., and Tindle, R. W. (1999). Differences in the effectiveness of delivery of B- and CTL-epitopes incorporated into the hepatitis B core antigen (HBcAg) c/e1-region. Arch. Virol. 144, 1323–1343. Sun, W., Kuhn, L., Ellerbrock, T., Chiasson, M., Bush, T., and Wright, T. (1997). Human papillomavirus infection in women infected with the human immunodeﬁciency virus. N. Engl. J. Med. 337, 1343–1349. Suzue, K., and Young, R. A. (1996). Adjuvant-free hsp70 fusion protein system elicits humoral and cellular immune responses to HIV-1 p24. J. Immunol. 156, 873–879. Suzue, K., Zhou, X., Eisen, H. N., and Young, R. A. (1997). Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway. Proc. Natl. Acad. Sci USA 94, 13,146–13,151. Taichman, L. B., and LaPorta, R. F. (1987). In “The Papovaviridiae,” Volume 2. Plenum Press, New York, pp. 109–139. Thornburg, C., Boczkowski, D., Gilboa, E., and Nair, S. K. (2000). Induction of cytotoxic T lymphocytes with dendritic cells transfected with human papillomavirus E6 and E7 RNA: Implications for cervical cancer immunotherapy. J. Immunother. 23, 412–418. Thornton, A. M., and Shevach, E. M. (1998). CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188, 287–296. Tobery, T. W., and Siliciano, R. F. (1997). Targeting of HIV-1 antigens for rapid intracellular degradation enhances cytotoxic T lymphocyte (CTL) recognition and the induction of de novo CTL responses in vivo after immunization. J. Exp. Med. 185, 909–920. Toes, R. E., Offringa, R., Blom, R. J., Melief, C. J., and Kast, W. M. (1996). Peptide vaccination can lead to enhanced tumor growth through speciﬁc T-cell tolerance induction. Proc. Natl. Acad. Sci. USA 93, 7855–7860. Torre-Amione, G., Beauchamp, R. D., Koeppen, H., Park, B. H., Schreiber, H., Moses, H. L., and Rowley, D. A. (1990). A highly immunogenic tumor transfected with a murine transforming growth factor type beta1 cDNA escapes immune surveillance. Proc. Natl. Acad. Sci. USA 87, 1486–1490. Townsend, A., Bastin, J., Gould, K., Brownlee, G., Andrew, M., Coupar, B., Boyle, D., Chan, S., and Smith, G. (1988). Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen. J. Exp. Med. 168, 1211–1224. Tuting, T., Gambotto, A., DeLeo, A., Lotze, M. T., Robbins, P. D., and Storkus, W. J. (1999). Induction of tumor antigen-speciﬁc immunity using plasmid DNA immunization in mice. Cancer Gene Ther. 6, 73–80. van der Burg, S. H., Ressing, M. E., Kwappenberg, K. M., de Jong, A., Straathof, K., de Jong, J., Geluk, A., van Meijgaarden, K. E., Franken, K. L., Ottenhoff, T. H., Fleuren, G. J., Kenter, G., Melief, C. J., and Offringa, R. (2001). Natural T-helper immunity against human papillomavirus type 16 (HPV16) E7-derived peptide epitopes in patients with HPV16positive cervical lesions: Identiﬁcation of 3 human leukocyte antigen class II-restricted epitopes. Int. J. Cancer 91, 612–618. Vandenvelde, C., De Foor, M., and Van Beers, D. (1993). Precision about the association between cervical carcinoma and HLA-DQB1∗ 03 alleles. Lancet 342, 553. van Driel, W. J., Ressing, M. E., Kenter, G. G., Brandt, R. M., Krul, E. J., van Rossum, A. B., Schuuring, E., Offringa, R., Bauknecht, T., Tamm-Hermelink, A., van Dam, P. A.,

148

Eiben et al.

Fleuren, G. J., Kast, W. M., Melief, C. J., and Trimbos, J. B. (1999). Vaccination with HPV16 peptides of patients with advanced cervical carcinoma: Clinical evaluation of a phase I-II trial. Eur. J. Cancer. 35, 946–952. Velders, M. P., McElhiney, S., Cassetti, M. C., Eiben, G. L., Higgins, T., Kovacs, G. R., Elmishad, A. G., Kast, W. M., and Smith, L. R. (2001a). Eradication of established tumors by vaccination with Venezuelan equine encephalitis virus replicon particles delivering human papillomavirus 16 E7 RNA. Cancer Res. 61, 7861–7867. Velders, M. P., Weijzen, S., Eiben, G. L., Elmishad, A. G., Kloetzel, P. M., Higgins, T., Ciccarelli, R. B., Evans, M., Man, S., Smith, L., and Kast, W. M. (2001b). Deﬁned ﬂanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine. J. Immunol. 166, 5366–5373. Viscidi, R. P., Sun, Y., Tsuzaki, B., Bosch, F. X., Munoz, N., and Shah, K. V. (1993). Serologic response in human papillomavirus-associated invasive cervical cancer. Int. J. Cancer 55, 780–784. von Herrath, M. G., Yokoyama, M., Dockter, J., Oldstone, M. B., and Whitton, J. L. (1996). CD4-deﬁcient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge. J. Virol. 70, 1072–1079. Von Knebel Doeberitz, M., Bauknecht, T., Bartsch, D., and zur Hausen, H. (1991). Inﬂuence of chromosomal integration on glucocorticoid regulated transcription of growth-stimulating papillomavirus E6 E7 genes in cervical carcinoma cell. Proc. Natl. Acad. Sci. USA 88, 1411– 1415. Walboomers, J., Jacobs, M., van Oostveen, J. W., van der Brule, A. J. C., Snijders, P., and Meijer, C. (1997). In “Human Papillomavirus Infections in Dermatovenereology” (G. Gross and G. von Krough, Eds.). pp. 341–364. CRC Press, New York. Walboomers, J. M., Jacobs, M. V., Manos, M. M., Bosch, F. X., Kummer, J. A., Shah, K. V., Snijders, P. J., Peto, J., Meijer, C. J., and Munoz, N. (1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189, 12–29. Wang, R.-F. (2001). The role of MHC class II-restricted tumor antigens and CD4+ T cells in antitumor immunity. Trends Immunol. 22, 269–276. Wank, R., and Thomssen, C. (1991). High risk of squamous cell carcinoma of the cervix for women with HLA-DQw3. Nature 352, 723–725. Weijzen, S., Meredith, S. C., Velders, M. P., Elmishad, A. G., Schreiber, H., and Kast, W. M. (2001). Pharmacokinetic difference between a T cell-tolerizing and a T cell-activating peptide. J. Immunol. 166, 7151–7157. Werness, B. A., Levine, A. J., and Howley, P. M. (1990). Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76–79. Whiteside, T. L. (1999). Signaling defects in T lymphocytes of patients with malignancy. Cancer Immunol. Immunother. 48, 346–352. Whitton, J. L., Sheng, N., Oldstone, M. B., and McKee, T. A. (1993). A “string-of-beads” vaccine, comprising linked minigenes, confers protection from lethal-dose virus challenge. J. Virol. 67, 348–352. Woodworth, C. D., Lichti, U., Simpson, S., Evans, C. H., and DiPaolo, J. A. (1992). Leukoregulin and gamma-interferon inhibit human papillomavirus type 16 gene transcription in human papillomavirus-immortalized human cervical cells. Cancer Res. 52, 456–463. Wu, T. C., Guarnieri, F. G., Staveley-O’Carroll, K. F., Viscidi, R. P., Levitsky, H. I., Hedrick, L., Cho, K. R., August, J. T., and Pardoll, D. M. (1995). Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens. Proc. Natl. Acad. Sci. USA 92, 11,671–11,675.

The T-Cell Response in Patients with Cancer Chiara Castelli1 and Markus J. Maeurer2,∗ 1 Unit of Immunotherapy of Human Tumors, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milano, Italy 2Department of Medical Microbiology, University of Mainz, 55101 Mainz, Germany

I. Deﬁnition of Immune Effector Functions II. Deﬁning the Bait for Antigen-Speciﬁc T Cells A. MHC–Peptide Complexes: Targets for T-Cell-Mediated Recognition of Tumor Cells B. Tumor-Derived Peptides Recognized by T Cells: Classiﬁcation of Tumor Antigens and Strategies for Deﬁning Novel, More Immunogenic Epitopes III. The Role of the Coreceptor CD8 in Mediating Antitumor Restricted T-Cell Responses IV. Tools to Measure ex Vivo T-Cell Avidity V. Biomarkers or True Surrogate Markers? VI. T-Cell Crossreactivity VII. Questions of Speciﬁcity and Alternate T-Cell Effector Functions References

I. DEFINITION OF IMMUNE EFFECTOR FUNCTIONS Several methods to measure human T-cell responses have been examined, including ELISPOT analysis, intracellular cytokine staining of immune cells after antigenic stimulation, limiting dilution analysis, conventional cloning, and molecular deﬁnition of the T-cell response either in the peripheral circulation or in situ in patients with cancer (for reference see Bercovici et al., 2000). These technologies provided only a partial picture of an antigenspeciﬁc immune response. An important step forward in the ex vivo analysis of cellular immune responses would be the combination of the enumeration of tumor-reactive T cells either in the periphery or in situ, with the analysis of immune effector functions (e.g., cytokine release). The ultimate goal of immunomonitoring in patients with cancer is the association of these ∗ Corresponding author: M. J. Maeurer, Dept. of Medical Microbiology, University of Mainz, Hochhaus Augustusplatz, 55101 Mainz, Germany. Tel. +49.(0)6131-3933645. Fax. +49.(0)6131-3935580. E-mail: [email protected]

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biomarkers with the clinical outcome (prognostic markers) or with response to therapy. Thus, biomarkers may turn into real surrogate markers. They would be endowed with a predictive value for disease prognosis or response to therapy. Given the multifaceted immunological aspects of human cancers (Schultze and Vonderheide, 2001), it is unlikely that such a “holy grail” of immunomonitoring exists. However, major histocompatibility complex (MHC)-restricted peptides have been utilized in patients with immunogenic tumors, and the immune response directed to the immunizing agents has been examined in this population. In addition, the development of the MHC tetramer technology allowed a novel approach for the ex vivo visualization of antigen-speciﬁc and MHC-restricted T cells (Altman et al., 1996; McMichael and O’Callaghan, 1998). This technology is based on the observation that multimeric MHC/peptide complexes exhibit higher avidity for the T-cell receptor (TCR) as compared to monomeric molecules (Boniface et al., 1998; Cochran et al., 2000). Such tetramer reagents are now being widely used to detect antigen-speciﬁc T cells without the need for in vitro manipulation. Some antigen-speciﬁc CD8+ T-cell responses could not be readily detected in ex vivo analysis in peripheral blood lymphocytes (PBL) from patients with cancer, but they could be detected after a short in vitro stimulation with peptides. This information provides useful data pertaining to the capacity of T-cell precursors to proliferate upon activation with the nominal peptide; however, such in vitro manipulation may skew the T-cell activation phenotype as well as T-cell differentiation and T-cell homing markers (Jager et al., 2000d; Lee et al., 1999; Valmori et al., 1999b). Not only MHC class I and class II (for review, see Novak et al., 2001a,b; Reichstetter et al., 2000), but also human nonclassical MHC molecules, including HLA-E (Braud et al., 1998), HLA-G (Allan et al., 1999), and CD1d (Metelitsa et al., 2001), have been utilized to deﬁne antigen-speciﬁc T-cell responses. One of the practical questions in the context of immune monitoring is whether a good surrogate marker for antitumor directed cellular immune responses exists in the peripheral circulation which is easily accessible for diagnostic purposes. It is most likely that there is no simple answer to this rather complex question. With the advent of tetramer complexes, we are now in a position to address the question of how many antigen-speciﬁc T cells are present in any anatomic compartment (not in situ, i.e., in the tumor lesion). What is the “bait” implemented to detect antigen-speciﬁc T cells? Tumorassociated antigens. This deﬁnition is cautious, since the nature of true tumorrejection antigens has not yet been deﬁned in humans. This situation is different in infectious diseases. Most studies addressing surrogate markers for protection rely on the determination of serum antibodies and, more recently, on the determination of antigen-speciﬁc T-cell responses. Thus, the nature of protective cellular immune responses is better deﬁned in viral or bacterial

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infections in man (Koralnik et al., 2002; McCloskey et al., 2001) as compared to tumor-associated antigens (Belyakov et al., 2001; Coles et al., 2002; Hel et al., 2001; Mothe et al., 2002; Stevceva et al., 2002). Thus, if one is so inclined to rephrase the question pertaining to effective anticancer immune responses, one could ask if a certain phenotype of immune effector T cells is associated with clinical responsiveness to therapy (Lauvau et al., 2001) or, more generally, to overall enhanced survival. More recent data obtained from patients with an immunogenic tumor (i.e., melanoma), suggest that T-cell effector populations can be deﬁned with certain markers that are indicative of the T cells’ past experience and homing characteristics. These lessons may by helpful for monitoring T-cell responses in patients with cancer of a different histology. We emphasize in this review the cellular immune response in patients with cancer. Recent studies have suggested that humoral immunity and T-cellmediated immunity is closely linked (Jager et al., 2000d; Valmori et al., 2000b) and it may very well be that the appreciation of humoral immune responses directed against relevant tumor target antigens may experience a renaissance. In addition, most of the data concerning antitumor immune responses have been generated using MHC class I tetramer reagents. However, CD4+ T-cell responses are also instrumental in anticancer immunity (Halder et al., 1997; Hohn et al., 1999a, 2000; Housseau et al., 2001; Marzo et al., 2000; Pardoll and Topalian, 1998; Pieper et al., 1999; Topalian, 1994; Topalian et al., 1994, 1996; Wang et al., 1999b), and MHC class II tetramer complexes have been utilized to characterize CD4+ T-cell responses (Novak et al., 2001a). It is possible that only the entire scenario of an anticancer directed immune response, including CD4+, CD8+ αβ+ T cells, γ δ, or natural killer (NK) cells, as well as humoral immune responses may aid in deﬁning biologically meaningful surrogate markers. In addition, the differentiation of the immediate effector and the memory T-cell response is also instrumental in determining useful surrogate markers, for either clinical prognosis or response to therapy. Could this be translated into a clinical setting? At least two approaches could be possible. First, one needs to postulate potential surrogate markers and test them in clinical studies. Second, of the patients treated with immunotherapeutic approaches, some have responded and others have failed to respond. It is challenging to evaluate the arsenal of available biomarkers in these patients and to test for markers associated with clinical response, or nonresponse to therapy. At least some markers appear to be associated with T cells recognizing the nominal target antigens expressed on tumor cells. We predict that with improved molecular and functional deﬁnitions of anticancer immune responses, particularly in situ, better immune markers will be available in the foreseeable future.

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II. DEFINING THE BAIT FOR ANTIGEN-SPECIFIC T CELLS A. MHC–Peptide Complexes: Targets for T-Cell-Mediated Recognition of Tumor Cells Identiﬁcation of T cell deﬁned tumor antigens ultimately implies the identiﬁcation of MHC–peptide complexes able to speciﬁcally activate the TCR expressed by CD4 or CD8 T cells. Short peptides of 8–10 or 15–18 amino acids are derived from the intracellular processing of tumor-associated proteins. These peptides, bound to MHC class I and class II molecules at the cell surface, represent the minimal structure required to initiate, regulate, and sustain a speciﬁc immune response (Cresswell and Lanzavecchia, 2001). More recent data, obtained from murine studies, suggest that the initiation of a T-cell response impacts the quality of a cellular immune response and hence the cellular immunity measured in clinical trials. Primary T-cell responses appear to be polyclonal in nature and secondary T-cell responses exhibit a more restricted TCR repertoire, usually with increased T-cell avidity (Busch and Pamer, 1999; McHeyzer-Williams and Davis, 1995). Highafﬁnity T cells may be associated with better immune effector functions (reviewed in Lanzavecchia, 2002). It is possible that high-afﬁnity T cells may compete for antigens displayed on the same antigen-presenting cell (APC), presumably by extracting MHC–peptide complexes from APCs by highafﬁnity T cells, thereby preventing activation of low-afﬁnity T cells during a secondary immune response (Kedl et al., 2002). This hypothesis may impact the formulation and delivery of peptides used for immunization. Amino acid sequencing of naturally processed peptides eluted from a given MHC allele has revealed that allelic variants of each MHC molecule efﬁciently bind only a subset of peptides sharing conserved amino acid residues in particular ﬁxed positions (Falk et al., 1991; Godkin et al., 1997). The position and the chemical nature of the amino acids directly affect the ability of a peptide to effectively bind to the corresponding MHC allele: the peptide binding motifs (Bjorkman, 1997; van Bleek and Nathenson, 1991). The peptide-binding groove of each MHC allele contains a variable number of pockets differing in their chemical nature and ability to allocate distinctive side chains of the antigenic peptides. A network of multiple hydrogen bonds fastens the peptide backbone to the main groove of the MHC molecules, while the side chains of the anchor residues establish speciﬁc chemical links with the MHC positioned inside the binding pockets. Besides having a direct contact with the MHC molecules, some peptide residues are more available to establish a direct contact with the TCR. For MHC class I presented peptides, the residues directly involved in TCR triggering reside in the center

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of the peptide sequence, while amino-terminal and carboxy-terminal amino acids anchor the immunogenic peptide to the MHC molecule (Bjorkman et al., 1987; Brown et al., 1988). The immunogenicity of a given peptide, that is, the capacity to stimulate a T-cell-mediated response in vitro and in vivo, is therefore directly affected by both its ability to bind the presenting MHC allele and by its afﬁnity for the clonotypic TCR expressed by T cells.

B. Tumor-Derived Peptides Recognized by T Cells: Classiﬁcation of Tumor Antigens and Strategies for Deﬁning Novel, More Immunogenic Epitopes Several different approaches have been successfully applied to the identiﬁcation of tumor-associated antigens recognized by tumor-speciﬁc T cells expanded in vitro from tumor-inﬁltrating lymphocytes (TIL) or from PBL (Castelli et al., 2000). According to their expression pattern, tumor-associated, T cell deﬁned epitopes can be grouped in different categories (Table I). Tumor-associated antigens include peptides derived from normal proteins expressed by tumor cells but not by normal, transformed, tissues. To this category belong peptides derived from cancer-testis antigens, not expressed in any normal tissue with the exception of testis, and peptides derived from differentiation antigens whose expression is lineage related and detectable in the normal counterpart of the neoplastic tissue. In addition to peptides derived from shared tumor antigens, T-cell epitopes can also be generated from mutated proteins. In the latter case, the immunogenic determinant generally includes the mutated amino acid which directly confers immunogenicity through the exposure of an altered non-self-epitope (Chiari et al., 1999; Coulie et al., 1995; Robbins et al., 1996). An exception to this rule has been recently reported for a peptide recognized by CD4, MHC class II restricted Table I Classiﬁcation of Tumor Antigens Recognized by T Cells Antigen Cancer/testis Differentiation antigens Tumor-restricted: —Shared

—Unique

Pattern of expression Shared among tumors of different histology, expressed by testis, placenta Expressed in normal and neoplastic tissues but lineage related Expressed in neoplastic tissues only, shared among lineage-related tumors or shared also among tumors of different histology Exclusively expressed in a single tumor

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T cells. Although derived from a mutated protein, the immunogenic epitope included the amino acid from the wild-type part of the protein. However, the mutation was associated with a different intracellular distribution of the index protein, enabling this molecule to gain access to the MHC class II processing pathway and resulting in the generation of a novel epitope recognized by CD4+ T-cells (Wang et al., 1999b). T-cell epitopes derived from mutated proteins represent individual antigens uniquely expressed by the tumor bearing that particular mutation. However, there is emerging evidence suggesting that the strong immunogenic nature of this type of antigen, for which a high frequency of speciﬁc circulating cytotoxic T lymphocytes (CTL) has been detected in patients with cancer (Baurain et al., 2000; Echchakir et al., 2001), is associated with a better prognosis. Patients displaying a strong tumor-speciﬁc reactivity directed against unique antigens enjoyed an unexpected favorable clinical outcome. Thus, these epitopes may determine the efﬁcacy of T-cell-mediated responses controlling tumor growth in vivo. If such mutated target epitopes exist in patients with cancer, it may be desirable to implement these antigens for therapy and for an individually tailored monitoring. Again, the feasibility of this approach appears to be limited due to technical and ﬁnancial constraints, since this approach would represent an individually tailored immunotherapy and immunomonitoring. The majority of tumor-associated peptides recognized by tumor-speciﬁc T cells, generated from TIL or from a patient’s peripheral blood mononuclear cells (PBMC), represent peptides recognized by CD8+ CTL in association with MHC class I molecules. Only a few epitopes recognized by CD4+ T cells have been deﬁned (Chiari et al., 2000; Pieper et al., 1999; Topalian et al., 1996; Wang et al., 1999b,c). This relatively low abundance of MHC class II tumor-associated epitopes reﬂects the technical difﬁculties in applying the genetic approach (De Plaen et al., 1997), a strategy which has been very successful in deﬁning CD8 recognized epitopes. In general, a large number of CD8+ T-cell-deﬁned epitopes have been identiﬁed from patients with immunogenic tumors (i.e., melanoma). However, patients with tumors of a different histology are also enrolled in clinical trials, but the knowledge of antigenic determinants capable of stimulating a T-cellmediated response is still limited. The majority of T-cell epitopes presented by solid tumors that show a signiﬁcant incidence in the Caucasian population (e.g., colorectal cancer) (Brossart et al., 1998; Kawashima et al., 1999; Ras et al., 1997), were predominantly identiﬁed using the reverse immunology strategy which has also been successfully applied to the deﬁnition of HLA class II-restricted epitopes (Chaux et al., 1999; Jager et al., 2000b; Kobayashi et al., 2000; Manici et al., 1999; Schultz et al., 2000; Zarour et al., 2000a,b; Zeng et al., 2000). Exploiting the available information concerning the molecular structure of the MHC–peptide complexes, reverse immunology

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uses algorithms, based on peptide binding motifs, to predict potentially novel CTL epitopes in tumor-associated proteins (Brusic et al., 1998a,b; Hammer et al., 1994; Honeyman et al., 1998; Lu and Celis, 2000; Parker et al., 1994). The peptide prediction is then followed by in vitro sensitization of PBMC to prove the immunogenicity of the putative epitope. This strategy allows a systematic search for new T-cell-deﬁned epitopes from tumor-associated antigens without the requirement for TIL or patient-derived T cells. However, this reverse approach has been beset with difﬁculties since T cells generated in vitro using the candidate peptides were often unable to recognize tumor cells, that is, naturally processed and presented peptides (Disis et al., 1994; Gedde-Dahl et al., 1994; Nijman et al., 1994; Van Elsas et al., 1995; Zaks and Rosenberg, 1998): The identiﬁed target epitopes were often not generated by the specialized processing machinery in tumor cells (Valmori et al., 1999a) or the immunogenic epitope was the result of a posttranscriptinal modiﬁcation which could not be predicted from the primary structure of the target protein (Chen et al., 1996; Kittlesen et al., 1998; Meadows et al., 1997; Skipper et al., 1996; Yague et al., 2000; Zarling et al., 2000). One of the limiting factors for peptide prediction appears to be the correct determination of its COOH terminus (Beekman et al., 2000). In fact, the COOH end of a naturally processed peptide results from the speciﬁc and specialized trimming operated by the proteosome, a multicatalytic protease responsible for generating peptides with the proper carboxyl terminus for binding to MHC class I molecules (Yewdell and Bennink, 2001). An additional point of weakness, the in vitro raised CTL generated by the reverse immunology approach, may express TCRs whose afﬁnity for the nominal MHC–peptide complexes is too low to induce a functional triggering when tumor cells, often expressing a suboptimal amount of antigen, are used as targets (Labarriere et al., 1997). Realizing these limitations, the reverse immunology approach has recently been supplemented with additional tools facilitating the ability to predict naturally processed peptides. This strategy has been used for the identiﬁcation of novel T-cell epitopes inside the tumor-associated antigen PRAME. Proteosome-mediated digestion of polypeptides encompassing the candidate epitopes predicted by conventional MHC binding motif algorithms was included in the prediction procedure. By this modiﬁcation, only 20% of the total predicted peptides exhibiting a high afﬁnity/stability for the corresponding HLA molecules was found to be efﬁciently generated by the proteosome as deﬁned by in vitro digestion (Kessler et al., 2001). These ﬁndings indicate the utility of this approach in predicting epitopes with a better chance to be naturally processed and recognized by functionally active T cells. An alternative approach for selecting naturally processed peptide relies on the combination of epitope prediction and the biochemical analysis of naturally processed tumor-eluted peptides (Schirle et al., 2000). The predicted

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synthetic peptide is used as a marker in a high-performance liquid chromatography (HPLC) puriﬁcation to identify the elution fraction which potentially contains the natural analogue. Natural peptides displaying the same retention time in an HPLC run are then subjected to mass spectrometry to conﬁrm the presence of peptides with the same molecular mass and amino acid sequence as compared to the predicted peptide. Using this T-cell independent methodology, tumor-associated proteins could be screened for naturally processed peptides presented by different MHC alleles and the selected candidate further analyzed for in vitro immunogenicity. Although several T-cell-deﬁned epitopes are now available for clinical application in cancer vaccines, they are still of limited usage for tumors other than melanoma. In addition, only very few epitopes have been deﬁned for an extended number of HLA alleles represented in the Caucasian population. Moreover, the majority of T-cell epitopes derived form tumor-associated antigens belong to the category of differentiation antigens for which immune tolerance may be active, thereby limiting their immunogenicity. The search for new T-cell epitopes is therefore still a primary goal. The reverse immunology approach, implemented as described above, will derive new impetus by the integration with the new molecular techniques (e.g., microarray and differential library screening). Furthermore, it will undergo a major expansion, since the entire human genome sequences represent an unlimited source for novel proteins, potential targets for an immunological evaluation of an anticancer-directed immune response. Other than identifying new epitopes, improvement of cancer vaccine approaches could also be achieved, modifying the already available T-cell epitopes to augment their immunogenicity. Different approaches have been applied to a limited number of tumor-associated peptides, strategies that have resulted in intriguing and challenging clinical responses. The immunogenicity of the tumor-associated peptides (excluding MART1/Melan-A) is relatively low. This scarce immunogenic potential can be explained by several factors involving the self-nature of the tumor peptides (Nossal, 2001). Mechanisms, including low frequency of T-cell precursors, inappropriate antigen presentation by specialized APCs, limited HLA peptide binding or TCR afﬁnity for the HLA/peptide complex (Marincola et al., 2000), can be directly involved in limiting the in vivo immunogenicity of tumor-derived peptides. Thus, alternate approaches have been designed in order to increase the ability of a given peptide to induce a more effective T-cell response. These strategies always involved the modiﬁcation of the primary structure of the natural peptide to generate a peptide analogue characterized by (i) a better bioavailability (chemical modiﬁcation of the C- or N-peptide terminus), (ii) an increase in HLA-binding capacity (modiﬁcation of residues in the main MHC anchor positions), and (iii) a heteroclitic activity (modiﬁcation of residues supposed to affect the TCR interaction) (Table II).

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Table II Different Strategies for Designing Altered Peptide Ligands with Increased Immunogenicity Mechanism involved in increasing the T-cell stimulation activity

Chemical modiﬁcations

Examples of tumor-associated antigens

References

Melan-A/Mart-1 (Brinckerhoff et al., 1999) Terminal modiﬁcation Mage-1 NY-Eso-1 (Chen et al., 2000) inserting pegylation or N-acetylation, modiﬁcation of cysteine residues Melan-A/Mart-1 (Valmori et al., 1998) Increase in HLA-binding Substitution of amino Gp100 Ep-cam (Parkhurst et al., 1996) capacity acid residues in (Trojan et al., 2001) anchor position Melan-A/Mart-1 CEA (Rivoltini et al., 1999) Heteroclitic activity Substitution of amino Mage-2, Mage-3, (Salazar et al., 2000) acid residues not (Tangri et al., 2001) inﬂuencing the HLA-peptide binding but likely the TCR afﬁnity for the MHC– peptide complex

Better bioavailability

Modiﬁcations leading to a better HLA binding have been successfully applied to the Melan-A/MART-1 (26-35)2L and gp100 (209-217)2M epitopes. The more hydrophilic amino acid residue leucine replaces the original alanine, and the methionine substitutes for the natural threonine in the second position of MART-1/Melan-A and gp100, respectively (Parkhurst et al., 1996; Valmori et al., 1998). MART-1/Melan-A- and Mage-1-derived epitopes have also been modiﬁed by terminal pegylation or N-acetylation to inhibit proteolytic digestion, thereby enhancing the in vivo peptide stability. Peptide stability has also been increased for a NY-Eso-1 peptide derivative by modifying its cysteine residues. The introduced modiﬁcation prevents peptide dimerization, thereby incrementing in vivo availability (Brinckerhoff et al., 1999; Chen et al., 2000). However, a different approach is the modiﬁcation of peptide residues which do not interact with the HLA molecule, but presumably directly affect TCR contacts. Such analogue peptides, called heteroclitic analogues, have shown the ability to fully trigger the speciﬁc clonotypic TCR inducing a quantitatively and qualitatively enhanced T-cell response associated with cytokine production. For example, interleukin-2 plays a crucial role in maintaining and expanding the cellular immune response. These modiﬁed peptides have shown the ability to break or overcome tolerance by recovering T cells from anergy or inducing a skewed T cell repertoire in vivo (Hernandez et al., 2000; Hoffmann et al., 2002; Wang et al., 1999a). A modiﬁed peptide has been designed for a CEA-HLA-A2 binding peptide by introducing an asparagine

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in position 6, a modiﬁcation leading to a 1000-fold increase in cytokine release as compared with the wild-type peptide (Salazar et al., 2000). This target peptide, admixed as a component in a dendritic cell-based vaccine, induced the expansion of CD8+ T cells recognizing both the altered and the wild-type peptide. Moreover, the increase in peptide-speciﬁc T cells correlated with clinical response to therapy. The heteroclitic peptide was able to generate in vivo a potent T-cell response efﬁciently controlling tumor growth (Fong et al., 2001). Recently, the identiﬁcation of heteroclitic analogues of ﬁve different T-cell epitopes of both cancer and viral origin have also indicated some generally applicable rules for identiﬁcation of heteroclitic variants of HLA-A2restricted T-cell epitopes. Modiﬁcations of peptides at odd-numbered positions were more frequently associated with heteroclitic functions (Tangri et al., 2001). However, the introduced amino acid substitutions should maintain chemical similarities with the original native residues, indicating that subtle alterations in peptide structure seen by the TCR may indeed account for heteroclicity. However, heteroclitic peptide variants have also been described in which the HLA-A2 binding peptides were modiﬁed in positions 1 and 2 (Rivoltini et al., 1999; Salazar et al., 2000). It appears well accepted that the action of heteroclitic analogue peptides is mediated by an enhancement in the TCR binding afﬁnity for the peptide– MHC complex as compared to the wild-type peptide (Slansky et al., 2000). Data pertaining to the cellular mechanisms associated with different TCR triggering are scant. Data from murine studies suggest that heteroclicity is associated with a different pattern of Zap70 phosphorylation. However, the roles of many other costimulatory and coupling molecules such as LAT remain to be fully investigated (Fong et al., 2001; Rivoltini et al., 1999; Salazar et al., 2000; Slansky et al., 2000; Tangri et al., 2001). Discovery of the signal transduction events associated with the heteroclitic activity of analogue peptides is of crucial importance. And the peptides are novel targets for a speciﬁc intervention aimed at boosting a more efﬁcient immune response directed against natural tumor-derived epitopes. These insights aid to deﬁne better target structures for anticancer-directed immune responses for vaccination as well as for immunomonitoring.

III. THE ROLE OF THE CORECEPTOR CD8 IN MEDIATING ANTITUMOR RESTRICTED T-CELL RESPONSES In contrast to antibodies which are able to mature and recombine their antigen receptor (i.e., immunoglobulin), T cells cannot mature. However, recent data suggest that the density of CD8 molecules on the T-cell surface is

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able to modulate T-cell afﬁnity. The exact role of CD8 in the TCR/MHC peptide interaction is still under debate: it is possible that CD8 strengthens the association between the TCR and the MHC/peptide complex, CD8 may also aid in phosphorylation of signaling molecules involved in T-cell activation. This leads to the apposition of CD8:Lck and the TCR/CD3:ZAP70 complex (Delon et al., 1998) and increases the T-cell afﬁnity for the MHC/peptide complex (Luescher et al., 1995). Disparate results regarding the role of CD8 in mediating stronger TCR/MHC peptide interaction may derive from either the nature of the antigen: self-antigens, for example, Melan-A/MART-1, gp100, or tyrosinase, may require a TCR with different afﬁnity as compared to mutated self-antigens present in the autologous tumor. Several studies have also suggested that CD8, like CD4 (Boniface et al., 1998; Crawford et al., 1998; Hamad et al., 1998), does not facilitate MHC/peptide binding, but contributes to TCR signaling after engagement of the TCR with its nominal ligand. Other studies clearly demonstrated that CD8 represents an active part of the initial contact of the TCR/MHC peptide complex (Daniels and Jameson, 2000; Luescher et al., 1995). There is also a considerable amount of controversy as to what component of the CD8 heterodimer is involved in TCR/MHC peptide engagement. In general, T cells without CD8 need more time for TCR engagement in order to elicit T-cell effector functions (Renard et al., 1996). Most TCR αβ+ T cells, as well as peripheral γ δ T cells (predominantly TCR delta2+ T cells), express the CD8αβ heterodimer. In contrast, intestinal T cells including γ δ+ T cells (predominantly TCR delta1+ T-cells) and NK cells express the homodimeric CD8αα molecule. The pivotal role of CD8, particularly the CD8αα homodimer, in modulating T-cell responses has recently been underlined in a murine model of intraepithelial T lymphocyte (iIEL) interaction with TL, an MHC class I-related molecule expressed on intestinal epithelial cells. T cells are very sensitive to the structural composition of MHC/peptide complexes on APCs. TL expression on APCs results in enhanced cytokine production but decreased proliferation and cytotoxicity in iIEL. This was presumably due to the fact that CD8 did not bind to the MHC molecule on the APC recognized by the TCR, but to the TL antigen on the same cells. Thus, CD8αα potentiate TCR-mediated functions by TCR-independent clustering and p56lck phosphorylation (Bonneville and Lang, 2002; Leishman et al., 2001). This observation supports the notion that not only the number, but also the spatial arrangement of MHC molecules presenting peptide antigens to CD8+ αβ+ T cells (Maeurer et al., 2001) or to CD4+ T cells impacts on T-cell activation and function (Kropshofer et al., 2002). Previous studies have shown that the heterodimeric CD8αβ form appears to represent a more potent CD8 coreceptor as compared to the homodimeric CD8αα form. The contribution of CD8β for a better binding to the MHC/peptide complex has recently been examined in greater detail. The cytoplasmic part of CD8β mediates the engagement of CD8 in lipid rafts

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which leads to p56lck association, and the cytoplasmic portion of CD8β facilitates the constitutive association with the TCR/CD3 complex. Since the CD8αβ heterodimer is inserted in lipid rafts of the T-cell membrane, it promotes association with the TCR/CD3 complex and induces Lck activation and in turn CD3 phosphorylation (Arcaro et al., 2001). The notion that TCRs as well as CD8 coreceptors are associated with lipid rafts impacts the practical applications if tetramer complexes are used to enumerate peptide-speciﬁc T cells. Disparate results may in part derive from the fact that T cells have been exposed to tetramer complexes at different temperatures, for example, at 4◦ C, room temperature, or 37◦ C, the latter condition enabling the cell membrane to react physiologically. Obvious differences in regard to tetramer binding at various temperatures has also been utilized to differentiate between high and low avidity T cells. It has been suggested that both low- and high-afﬁnity T cells bind to tetramer complexes at 4◦ C. On the other hand, high-afﬁnity T cells have been suggested to preferentially bind tetramers at 37◦ C due to the different on/off rate of the clonotypic TCR (Reichstetter et al., 2000; Whelan et al., 1999). A different approach to estimate the TCR afﬁnity is the amount of MHC/peptide binding; that is, low-afﬁnity T cells have been suggested to bind lower numbers of MHC/peptide complexes (Busch and Pamer, 1999; Crawford et al., 1998; Daniels and Jameson, 2000; Nugent et al., 2000; Yee et al., 1999).

IV. TOOLS TO MEASURE EX VIVO T-CELL AVIDITY Mutations within the alpha 3 domain of the human HLA-A2 molecules show that MHC class I-restricted T-cell activation is not only dependent on the proper interaction with the CD8 coreceptor, but also that MHC/peptideTCR/CD8 engagement enhances T-cell sensitivity to the nominal peptide ligand reﬂected by complete phosphorylation of the T-cell receptor zeta chain (Purbhoo et al., 2001). The rationale for investigating CD8-mediated effects on T-cell recognition in patients with cancer is that high-avidity T cells may be beneﬁcial for the host in mediating and maintaining an effective antitumor immune response. This may be of clinical importance since high-afﬁnity T cells may be able to detect a few MHC class I/peptide molecules on tumor cells (Bullock et al., 2001). The detection of tumor antigens deﬁned by highor low-avidity T cells in patients with cancer is a critical issue. The patient’s immune system has been exposed for a considerably extended period, presumably years, to transformed cells before the tumor is clinically detectable. From a conceptual standpoint, it may be advisable to focus, to sharpen the antitumor-directed cellular immune response. However, unlike the B-cell receptor (BCR), the TCR is not able to undergo afﬁnity maturation. Recent

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pivotal data suggest that T cells are able to respond with enhanced sensitivity to antigen during an immune response and imply that T cells may undergo afﬁnity maturation by modulation of the CD8 coreceptor or CD8associated molecules without alterations in the TCR, that is, the intrinsic afﬁnity. This notion has also been consolidated by the observation that binding of the coreceptor CD8 to MHC-I molecules is modulated by the T-cell glycosylation state, which is associated with T-cell maturation (Daniels et al., 2001). Both BCR and TCR create the antigen-speciﬁc receptors by somatic recombination. In contrast to T cells, B cells are able to alter the antigen receptor through somatic recombination. T cells create the repertoire in the thymus, and once the na¨ıve progenitor T cells leave the thymus, the TCR cannot be altered and does not show the capacity to accumulate point mutations, like B cells. However, two- to four-fold increase in TCR afﬁnity in animal models during secondary immune responses have been described (Busch and Pamer, 1999; Savage et al., 1999). The exact mechanisms conferring the maturation have not yet been deﬁned. Considering the afﬁnity of the TCR/MHC-peptide interaction per se, the afﬁnity of TCR appears to be low, in the range of 104–107 M, in contrast to BCR which exhibit afﬁnities for nominal antigens in the range of 107–1012 M (Eisen et al., 1996; Valitutti and Lanzavecchia, 1997). According to the serial TCR triggering model (Valitutti and Lanzavecchia, 1997), T cells with very high TCR afﬁnity may even become dysfunctional due to prolonged TCR/MHC-peptide binding and consecutive on/off rates which do not lead to successful TCR downstream events and ultimately T-cell effector functions, for example, proliferation, cytokine release, or cytotoxicity (Salzmann and Bachmann, 1998; Savage et al., 1999; Sykulev et al., 1995; Valitutti and Lanzavecchia, 1997). Recent studies have addressed the questions of whether T cells with a genetically ﬁxed TCR are able to adjust the TCR avidity by modulating CD8 expression. These experiments have shown that MHC-restricted and antigen-speciﬁc T cells are able to respond with enhanced sensitivity to antigens without the instrument of TCR maturation. This has been coined “functional avidity” by Slifka and Whitton (2001). CD8+ T-cell responsiveness to the nominal target antigen increased up to 50-fold in vivo, using a murine model of infection with the choriomeningitis virus (LCMV). In this infection model, T cells were ex vivo stimulated with various amounts of peptides and interferon-γ (IFNγ ) production was measured intracellularly. T-cell avidity increased daily after infections, and antigen-speciﬁc T cells needed less peptide to induce immune effector functions [IFNγ , tumor necrosis factor-α (TNFα), and cytotoxicity] in antigen-speciﬁc T cells. In addition, blocking of the CD8 molecule on effector T cells, or transgenic TCR expression in recipient cell lines without the coreceptor CD8 (Holler et al., 2001), showed dramatic effects on T-cell responses (i.e., reduction of IFNγ production),

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in early infection. In contrast, T cells analyzed later after infection were insensitive to CD8 blockade, suggesting that the overall avidity of the T-cell population increased. Formally three possibilities may account for this observation: (i) TCR afﬁnity, (ii) better signaling, and (iii) differential expression of T-cell costimulatory molecules. Careful studies have shown that the apparent TCR afﬁnity stayed identical over time during the infection in regard to the clonotypic TCR even in polyclonal T-cell populations. The same was true for the expression of adhesion/costimulatory molecules including LFA-1, CD2, CD8, CD28, CD43, and CD49d. The substantial difference in high/low avidity T cells was associated with intracellular Lck expression. Thus, the increase of functional avidity, at least in a murine model, is due to alterations in the T-cell signaling machinery associated with downstream events induced upon TCR triggering. Preassembly of molecules associated with signal transduction may be more important for enhancing T-cell functions as compared to increased afﬁnity of the clonotypic TCR. These observations are also supported by recent data addressing the assembly of the TCR with the CD8 coreceptor in lipid rafts (see above). Thus, better cellular immune responses to viral infections (as a model system) is due to long-term CD8+ T-cell memory and functional high-avidity TCRs, which are able to control the virus in a magnitude of 100-fold up to 1000-fold better as compared to low-avidity T cells (Alexander-Miller et al., 1996). In addition, prime-boost immunization (Estcourt et al., 2002) is able to generate high frequency and high-avidity CD8+ T cell populations in animal models with SIV, inﬂuenza, or Plasmodium berghei infections (Degano et al., 1999; Robinson et al., 1999; Schneider et al., 1998). This has at least two implications. Methods which allow one to expand and maintain such highly effective and high-avidity T cells (Riberdy et al., 2001) in patients with cancer may aid in expanding antitumor-directed T cells and induce strong antitumor-directed immune responses. Several ways to achieve this goal may be available, including different tumor-associated antigens +/− adjuvants, different routes of immunization, prime-boost strategies (reviewed in Kawakami and Rosenberg, 1997; Pardoll, 1998) or, alternatively, the nature of the APC (Salio et al., 2001). But not only novel vaccine strategies may beneﬁt from these observations. The detection of high-avidity T cells in patients with cancer may aid in delineating better surrogate markers for patients undergoing vaccination with deﬁned target antigens. More recent data obtained in patients with cancer suggest that there are indeed antigenspeciﬁc T-cell populations which differ in respect to the avidity of the nominal target antigen (e.g., the melanoma associated antigen MART-1/Melan-A or gp100). Three lines of evidence suggest differences in TCR avidity in patients with cancer. First is the dependence of human antimelanoma-directed T-cell clones on the coreceptor CD8 in regard to binding to soluble MHC/peptide

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complexes (Denkberg et al., 2001; Maeurer et al., 2002). Second is the differential binding characteristics of PBL from patients with melanoma to wild-type HLA-A2 molecules loaded with the Melan-A/MART-1 peptide as compared to mutant HLA-A2 molecules which reduced the engagement with the CD8 coreceptor (Bodinier et al., 2000; Jager et al., 2002a,b). And third, differential staining intensities of tetramer high/low-positive T cells in patients with cancer indicates that tetramer-high+ T cells are associated with enhanced immune effector functions (Dutoit et al., 2001). The latter point is particularly important in regard to monitoring clinical trials or for providing biologically meaningful surrogate markers in the context of cancer vaccines (Oelke et al., 2000). Most of the data addressing the issue of T-cell avidity suggest that high-avidity T cells are associated with better tumor recognition and display a tetramer high phenotype (Yee et al., 1999). It is noteworthy that tetramer-ﬂuorescence intensity appears to represent a good marker of TCR afﬁnity, but not of the overall avidity of an antigen-speciﬁc T-cell response (Palermo et al., 2001). Thus, maturation of T-cell responses directed against tumor cells may be achieved by addressing differences in (i) tetramer-staining intensity (afﬁnity) and (ii), dependence on the coreceptor CD8 (T-cell avidity), potentially by evaluating Lck expression. Until now, only a few studies have addressed the questions of whether there is avidity maturation in antitumor responses in patients with cancer either during natural progression, or in patients undergoing peptide vaccination. T cells which recognize melanoma-associated differentiation antigens in patients with primary tumor lesions appear to be rare in PBL or exhibit low avidity (Nanda and Sercarz, 1995; Staveley-O’Carroll et al., 1998). However, the frequency of tumor antigen-speciﬁc T cells in patients with cancer, or in healthy individuals, is at least associated with two variables: ﬁrst, the nature of the tumor antigen and second the T-cell population capable of reacting to the nominal T-cell epitopes. For instance, Melan-A/MART-1-reactive T cells are not only present in patients with cancer, but also in healthy adults (Pittet et al., 1999). Of note, this Melan-A/MART-1-reactive T-cell subset resided in the CD8+ CD45RA+ CD45RO- T-cell population. This phenotype is consistent with either a na¨ıve or a terminally differentiated phenotype deﬁned by CD8+ CD45RA+ CD27-CD28-expression (Hamann et al., 1997).

V. BIOMARKERS OR TRUE SURROGATE MARKERS? The enumeration of MHC-restricted and antigen-speciﬁc T cells in patients with cancer allows us to address the question of whether the number of antigen-experienced T cells are associated with the disease, or alternatively, with antigen-speciﬁc vaccination. However, the ultimate goal would

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be that either the number of antigen-speciﬁc T cells or the quality of T cells (i.e., deﬁned by cytokine release, or by homing markers) be associated with a good or bad prognosis in the course of the natural disease or predict response to vaccination. Only a few studies have addressed this central question up to now. A recent study evaluated the frequency of CD8+ T-cell responses in patients diagnosed with primary cutaneous melanoma directed against the melanoma-associated differentiation antigens Melan-A/MART-1, gp100, tyrosinase and the cancer-testis antigen MAGE-3 in the context of the HLA-A2 antigen. In some patients, up to 1 in 220 T cells appeared to be speciﬁc for one of these tumor-associated antigens (Palermo et al., 2001). The clinical follow-up of these patients will reveal whether such antigenspeciﬁc T cells are expanded during the course of the disease or acquire a different phenotype. A different study in which the frequency of CD8+ T cells directed against Melan-A/MART-1 has been evaluated in patients with metastatic melanoma lesions has indicated that, at least in some patients with metastatic lesions, the number of Melan-A/MART-1 speciﬁc T cells appear to be elevated (Anichini et al., 1999). These cells exhibited an antigen-experienced CD45RO+ phenotype, and only a small fraction stained positive for CD45RA, which implies that antigen-speciﬁc T cells reside either in the na¨ıve or in the terminally differentiated T-cell effector population (Champagne et al., 2001; Hamann et al., 1996). However, the presence of antigen-speciﬁc CD8+ T cells did not correlate with tumor regression, nor with improved immune surveillance in patients with metastatic lesions, but with better in vitro responsiveness after restimulation with the nominal target peptide (Anichini et al., 1999). A similar situation was found in patients undergoing antigen-speciﬁc vaccination with an analogue peptide derived from the melanoma-associated differentiation antigen gp100 (aa 209–217 with a methionine substitution at position 210) (Lee et al., 1999). Peptide-speciﬁc CD8+ T cells were increased after vaccination, and T cells obtained from peripheral blood could be readily restimulated with the index peptide utilized for immunization. These data suggested that vaccination with deﬁned antigens may be associated with an measurable increase of antigen-speciﬁc T cells in PBL after vaccination without any correlation of regression (or progression) of the disease. It is noteworthy that the T-cell precursor frequency, as measured with soluble MHC/peptide tetramer complexes, was paradoxically not elevated in patients who received in addition to the tumor vaccine recombinant IL-2, a situation linked with tumor regression. This could formally imply at least two situations. First, the antigen-speciﬁc T cells may not be detectable since they may have undergone apoptotic death. Second, these T cells may be present in the patient(s), but not in the peripheral circulation at the time of PBL harvest. This is an important notion that may be underestimated. T cells in the peripheral circulation constitute approximately 2% of the entire T-cell

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pool and may represent the entire T-cell pool if there is indeed an equilibrium of all the T-cell populations in the body. However, this may not be the case if T cells are present in lymph nodes, or are attracted to sites of inﬂammation. A tumor lesion represents an inﬂammatory lesion and it may very well be that antigen-speciﬁc T cells reside in either the tumor lesion itself or in draining lymph nodes (Bousso et al., 1999; Maini et al., 1999; Westermann and Pabst, 1990). Thus, the discrepancy between enhanced cellular immune responses in peripheral blood, as deﬁned by tetramer analysis (or alternate methods to deﬁne the number of antigen-speciﬁc T cells), may indicate either that antigen-speciﬁc T cells are trapped in the tumor lesion or that they may be in the periphery and not able to access the tumor site. A careful examination of the local and systemic cellular immune response has revealed that CD8+ T-cell responses in a patient vaccinated with a gp100 derivative (gp100 209–217 M) exhibited a predominant CTL clone in PBL recognizing the immunizing peptide. In contrast, T cells obtained from TIL showed reactivity directed against naturally processed and presented tumor peptides, but not against the index peptide implemented for vaccination (Lee et al., 1998). Thus, a functional discrepancy may exist between the easily accessible PBL and the tumor lesion, as deﬁned by tetramer reagents (Andersen et al., 2001; Gray et al., 2000; Haanen et al., 2000; Schmitz et al., 2001; Skinner et al., 2000). The implication that vaccination efforts should also be monitored in the target tissue may be clinically feasible in skin metastases, or in patients with carcinoma of the cervix, but computer tomography (CT) guided ﬁne needle aspirates may not be easily obtained in vaccine trials. A different site for T cells conferring tumor regression may be vitiligo lesions from patients with melanoma who are enrolled in immunotherapy trials. The CD8+ T-cell response directed against some of the melanomaassociated antigens is an autoimmune response in nature and is directed against the nonmutated self-melanocyte differentiation antigens Melan-A/ MART-1, gp100, or tyrosinase. The development of vitiligo appears to be associated with response to therapy and a better clinical prognosis. Of note, Melan-A/MART-1 speciﬁc CD8+ T cells in PBL from patients with melanoma exhibiting vitiligo express high levels of the skin homing receptor, the cutaneous lymphocyte-associated antigen (Ogg et al., 1998). This homing receptor was not expressed by Melan-A/MART-1-reactive T cells in PBL from healthy individuals, suggesting that this T-cell population may indeed gain access to skin and be associated with the development of an autoimmune reaction (vitiligo). The same T-cell population may confer tumor regression. Indeed, a patient with stage IV melanoma who responded favorably to peptide vaccination with Melan-A/MART-1/gp100 and tyrosinase peptides showed a predominant Melan-A/MART-1 speciﬁc T-cell clone in the peripheral circulation deﬁned by a monoclonal TCR VB16 (Jager et al., 2000c).

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Remarkably, the same monoclonal TCR was present in skin lesions with vitiligo, presumably associated with the destruction of melanocytes. Thus, at least in patients with clinically accessible lesions, antigen-speciﬁc T cells associated with tumor regression may be present in situ, but not necessarily in PBL. Are there other validated data which link the detection of T cells with clinical regression? Are these T cells antigen speciﬁc and do they express certain markers which could be used to deﬁne T-cell subsets associated with clinical response to therapy? Several reports have indicated the presence of antigenreactive T cells in ex vivo analyses with tumor regression. A female patient presented in 1981 with melanoma, progressed, presented with metastatic disease, and underwent immunization with peptides derived from gp100, Melan-A/MART-1, and tyrosinase (Khong and Rosenberg, 2002). This patient experienced a dramatic response to immunotherapy. What is the repertoire of epitopes recognized by TIL in this patient? First of all, TIL associated with regressing tumor lesions did not recognize the immunizing peptides, but rather epitopes provided by the tyrosinase related proteins (TRP)-2 and the cancer-testis antigen NY-ESO-1. These epitopes were previously described and identiﬁed using T-cell lymphocytes from in vitro stimulation. In addition, TIL recognized a naturally processed and presented peptide which was identiﬁed by screening a cDNA library from an HLA-matched melanoma cell line. This epitope turned out be a previously unknown TRP2 mRNA isoform created by alternative splicing designated as TRP-2-6b. Immunomonitoring of PBL from this patient revealed high reactivity directed against the NY-ESO-1, TRP-2, and TRP-2-6b antigens prior to immunization. In contrast, immune reactivity directed against the immunizing peptides was less pronounced. The above-described case study pinpoints the problem in identifying biologically meaningful markers for predicting the successful outcome of vaccination. First, no dominant T-cell response associated with the immunizing peptides was identiﬁed in PBL or TIL. Most likely, the immune response associated with regression was directed against a different set of target antigens, and one of these antigens turned out to be a private antigen created by alternative splicing. This could imply that we may not receive the proper answer to the question of what are the most successful surrogate markers in cancer vaccine trials, simply because we did not pose the right question. In patients with melanoma, at least four categories of tumor-associated antigens have been identiﬁed: the cancer/testis antigens (e.g., MAGE-family and NY-ESO-1) (Chen et al., 1997; Rosenberg, 1999; Takahashi et al., 1995); the melanoma differentiation antigens (e.g., Melan-A/MART-1, gp100, tyrosinase, TRP-1, and TRP-2) (Brichard et al., 1993; Coulie et al., 1994; Kawakami et al., 1994a,b; Wang et al., 1995, 1996, 1998); antigens which are widely expressed in nontransformed tissues, but overexpressed in cancer cells (e.g., p15 or Prame) (Ikeda et al., 1997; Robbins et al., 1995); and

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private, unique antigens which result from mutations identiﬁed in individual patients (e.g., beta-catenin, CDK4, or MUM-1) (Coulie et al., 1995; Robbins et al., 1996; Wolfel et al., 1995). If the latter group represents true tumor rejection antigens associated with clinical regression of the disease, the quest for identiﬁcation of useful cellular surrogate markers would presumably not be successful. The identiﬁcation of unique antigens in individual patients is too cost- and labor-intensive (Shipp et al., 2002; Van’t Veer and De Jong, 2002). Again, it has not yet been clearly demonstrated that the cellular immune response targeting unique tumor association antigens is deﬁnitely associated with clinical regression. Several points are also to be considered. Most of the data accumulated up to now have been collected from patients with immunogenic tumors, predominantly patients with melanoma or renal cell cancer. Despite the extended list of tumor-associated antigens in these patients, only a fraction of peptide epitopes has been identiﬁed, since a single target antigen may be able to provide several epitopes in the context of up to six individual MCH class I, in the case of CD8-, or MHC class II for CD4 T-cell responses. Thus, a clear picture pertaining to the identiﬁcation of good surrogate markers may ultimately emerge in the case of vaccination with deﬁned target antigens. This picture is deﬁnitively less clear if tumor lysates are used in vaccine trials in patients with melanoma (Nestle et al., 1998). Thus, the problem of deﬁning good surrogate markers not only starts with the strategic questions where (i.e., in which anatomic compartment) and when (i.e, at what intervals after vaccination) to screen for tumor-directed immune responses, but also with the choice of the index antigen. Most of the studies up to now have failed to indicate a good correlation with the immunizing agent and the detection of antigen-directed immune responses; for example, in patients undergoing vaccination with a deﬁned tumor antigen (MAGE-3), peptide speciﬁc CD8+ T cells could be observed. However, it was difﬁcult to link these HLA-A1-restricted and MAGE-3-speciﬁc T-cell responses to clinically relevant tumor reduction, since the frequency of the CTL response was considerably low (Coulie et al., 2001). A remarkable feature of this CD8+ T-cell response was the monoclonality. Thus, a different marker for a potentially successful cellular immune response could be the focus of the cellular immune response. At least in two reports, clinically relevant tumor regression was associated with monoclonal or oligoclonal T-cell responses directed against tumor-associated antigens (Coulie et al., 2001; Jager et al., 2002a). Yet in a different study, successful immunization was achieved with an altered peptide provided from the carcinoembryonic antigen CEA (CEA605–613, with aspartate substituting for asparagine at position 610) with Flt3 ligand expanded DC in patients with colon or non-small cell lung cancer (Fong et al., 2001). Clinical regression was associated with expansion of tetramer-positive cells

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in the peripheral circulation. The phenotype of tetramer-positive cells was CD8+CD45RA+CD27-CCR7-, which is indicative of terminally differentiated CD8+ effector T cells (Fong et al., 2001). A similar picture was found in patients with melanoma who clinically responded to vaccination with peptides from melanoma differentiation antigens (Jager et al., 2002a). By combining tetramer technology, molecular and functional T-cell assays (e.g., TCR CDR3 analysis and cytokine release assays), T cells responding to the wild-type Melan-A/MART-1 peptide (AAGIGILTV) show substantial differences compared to those responding to the Melan-A/MART-1 analogue peptide (ELAGIGILTV) (Fig. 1A; see color insert). AAGIGILTV-reactive T cells were found in different T-cell subsets (e.g., in true-memory CD45RO+, CD45RA-CCR7+ T cells), whereas memory-effector phenotype CD45RO- CD45RA+CCR7- CD8+ T cells almost exclusively reacted with the Melan-A/MART-1 analogue peptide ELA GIGILTV in PBL of a patient who experienced complete tumor regression. The mirror image of the above observation has been identiﬁed in a different study. The CD45RA+/RO-/CCR7+ (na¨ıve) phenotype in Melan-A/MART-1 tetramer-positive T cells was associated with no detectable T-cell response to exposure of antigenic peptides in vitro. In contrast, CD45RO+RA-/CCR7+ (activated/memory) T cells readily responded to the nominal antigen (Dunbar et al., 2000). Thus, the lineage differentiation of human CD8+ αβ+ T cells aids in segregating CD8+ into four categories based on maturation. Segregation into CD45RA+/RO-, CCR7+ (precursor, na¨ıve T cells), CD45RA-/ RO+, CCR7+ (activated, memory T cells), CD45RA-/RO+, CCR7- (memory, preterminally differentiated T cells), and CD45RA+/RO-, CCR7(mature, terminally differentiated effector T cells) (Champagne et al., 2001) T-cell subsets in patients with cancer may aid in deﬁning the most potent effector T-cell population (Fig. 1B). For instance, HIV-speciﬁc CD8+ T cells appear to reside in the preterminally differentiated CD45RA-/CCR7- T-cell population and only a minor proportion resides in the terminally differentiated effector T-cell population (CD45RA+/CCR7-). In contrast to HIV, most of the cytomegalovirus (CMV)-speciﬁc T-cell response (approximately 50%) is presented in the terminally differentiated T-cell population characterized by CD45RA+CCR7- marker expression in the same individual. It may be advantageous for a reencounter with the nominal antigen if effector T cells are present in the population with the highest functional activity. However, it may be equally important that antigen-speciﬁc T cells are present in the precursor pool which enables to replenishment of the T-cell compartment if terminally differentiated T cells apoptose upon antigen (re)exposure. Similarly, the presence of antigen-speciﬁc T-cell responses within the memory T-cell pool may also be clinically beneﬁcial in order to generate an effective recall T-cell response. The same situation may be true in patients with cancer. Detection of antigen-speciﬁc T-cell responses should be associated with T-cell marker

Fig. 1 Enumeration of antigen-speciﬁc T cells using tetramer complexes. (B) Antigen-speciﬁc T cells reside in the CD45RA+/− T-cell subsets. PBL were gated on CD3+ CD8+ T cells and then segregated into CD45RA+ and CD45RA− T cells. Note that tetramer (ELAGIGILTV)reactive T cells reside in each subset and that the CD45RA+ T-cell pool represents either na¨ıve (CCR7+ CD27+ CD28+) or terminally differentiated effector T cells (CCR7− CD27− CD28−) (Champagne et al., 2001; Hamann et al., 1997).

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analysis which provides information pertaining to T-cell maturation and homing characteristics (Marrack et al., 2000). This information may also add important value to tetramer-positive staining cells. Based on T-cell marker analysis, alterations in the composition of T-cell subsets in the peripheral circulation may indicate whether or not the PBL sample drawn for immunomonitoring is representative of the entire T-cell compartment in the body (Hazenberg et al., 2000; Roos et al., 2000), since the TCR repertoire in healthy individuals appears to be stable over time (Hohn et al., 2002) as compared to PBL obtained from patients with cancer (Fig. 2; see color insert). In addition, phenotypic marker analysis of CD8+ T cells which reﬂect the migratory properties of immune effector cells may be helpful in determining T-cell subsets capable of entering the tumor lesion (Weninger et al., 2001). Thus, the combined analysis of antigen speciﬁcity and T-cell phenotype (D’Souza et al., 1998; Moss et al., 1995) may help to characterize the most effective T-cell populations responding to peptide vaccination and mediating clinically measurable tumor repression in future clinical trials. Of note, there are differences in T-cell responses directed against wild-type or analogue peptides. Tetramer+ T cells resided in the T-cell effector population deﬁned by the CD45RA+CCR7- phenotype, and this CD8+ T-cell response did not react to the naturally processed and presented epitope displayed on the tumor, but reacted to analogue peptides from Melan-A/MART-1 (Jager et al., 2002a) or CEA (Fong et al., 2001). Indeed, in vitro studies have suggested that peptide analogues (e.g., derived from gp100) (Rosenberg et al., 1998) and NY-ESO-1 (Jager et al., 2000a), may induce more effective immune responses as compared to the natural epitope (see above). Of clinical interest, the T-cell response directed against the Melan-A/MART-1 epitope ELAGIG ILTV appeared to be more focused and comprised a different T-cell population as compared to the TCR repertoire binding to the peptide AAGIGILTV presented on tumor cells (Jager et al., 2002a). If the available T-cell repertoire consists of low-afﬁnity TCR, or if the TCR repertoire is only capable of targeting partial agonistic peptides, an effective antitumor response may not be achieved. Modiﬁed peptide epitopes capable of stimulating T cells which have not yet been activated by either the nominal tumor-associated antigen (i.e., Melan-A/MART-1) or closely related epitopes provided by viral or bacterial products may represent excellent candidates for improved vaccines. These agonistic peptides ; should be able to elicit IFNγ as well as IL-2 secretion in responding T cells (Hohn et al., 1999b; Kuroda et al., 1998; Pannetier et al., 1995). This ﬁnding may have an important impact on the selection of patients and the design of immunogens in future vaccination studies. Earlier data suggested that tetramer-positive T cells in patients with melanoma directed against Melan-A/MART-1, gp100, or tyrosinase are anergic (Lee et al., 1999). Indeed, differential cytokine production in Melan-A/MART-1 tetramer-sorted T cells has been observed in other studies as well (Jager et al., 2002a). Noteworthy is that the segregation of IL-2 versus IFNγ /GMCSF

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production in Melan-A/MART-1-reactive T cells was speciﬁc for the tumorassociated antigen Melan-A/MART-1; CMV-reactive T cells from the same patient were capable of secretion of IL-2, IFNγ , and GM-CSF. This observation may be related to the nature of the antigen. Melan-A/MART-1 has been reported to act as a partial agonist leading exclusively to IL-2, but not to IFNγ , secretion in responding T cells. However, it may also indicate a general failure of the CD8+ T-cell response to productively react to the antigenic peptides. This has also been observed in patients with HIV: HIV-speciﬁc T cells lack perforin expression concomitant with sustained CD27 and loss of CD28 expression (Appay et al., 2000). The characteristics of T-cell responses associated with potential clinical efﬁcacy are compiled in Table III. Table III Immune Markers Potentially Associated with Better Clinical Prognosis Marker/Analysis T-cell effector phenotype CD8+ CD45RA+ CCR7-, CD27-, CD28-, or activated/memory phenotype

Focused, monoclonal, or oligoclonal TCR repertoire reacting to the immunizing peptide or monoclonal TCRs in situ Increased numbers of peptide-reactive T cells using tetramer complexes

In situ detection/deﬁnition of immune responses

High avidity T cells show superior antitumor reactivity and may be associated with a better clinical prognosis

Comments Association with more sensitive target cell recognition, enhanced immune effector functions, allows access to different tissues due to differential homing receptor expression associated with T-cell homeostasis A focused TCR repertoire may be dependent on the nature of the immunogen and on the exposure time to the nominal T-cell epitope after initiation of a T-cell response Only a limited number of studies showed a clear association between the number of antigen-speciﬁc T cells and clinical response Effective anticancer responses may be targeting individual unique antigens; in situ immunity may not be present in PBL TCR/MHC-peptide afﬁnity may be measured by T cell/tetramer interaction and T-cell avidity by evaluating the necessity for CD8 coreceptor help and/or TCR signaling proteins, e.g., Lck. Activated or memory T cells exhibit better sensitivity to low-dose antigen

References (Faint et al., 2001) (Dunbar et al., 2000) (Ogg et al., 1998) (Pittet et al., 1999) (Jager et al., 2002a) (Fong et al., 2001)

(Jager et al., 2002a) (Coulie et al., 2001) (Jager et al., 2000c) (Hohn et al., 2000)

(Fong et al., 2001)

(Khong and Rosenberg, 2002) (Lee et al., 1998)

(Yee et al., 1999) (Dutoit et al., 2001) (Palermo et al., 2001) (Purbhoo et al., 2001) (Denkberg et al., 2001) (Maeurer et al., 2002) (Slifka and Whitton, 2001) (Daniels et al., 2001)

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VI. T-CELL CROSSREACTIVITY So far, we have addressed the question of whether the detection of antigenspeciﬁc T cells is associated with regression of tumor cells in patients. Particularly in patients with melanoma, T cells have been described which crossreact with peptides derived from viral or bacterial origins (Loftus et al., 1996, 1998). This may represent a fortuitous ﬁnding. This crossreactivity may in part be responsible for the polyclonal TCR repertoire binding to the wild-type AAGIGILTV epitope, as compared to the analogue peptide ELAGIGILTV. However, T-cell crossreactivity appears to represent the inherent nature of T-cell recognition (Mason, 1998). Indeed, a polyclonal TCR repertoire has been identiﬁed in patients with melanoma, and is directed against the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV (Jager et al., 2002a; Valmori et al., 2000a). In contrast, the TCR repertoire in PBL directed against the MelanA/MART-1 peptide analogue ELAGIGILTV was different, broadened over time upon vaccination with the wild-type peptide. This suggests that analogue peptides can be used to stimulate crossreactive T cells capable of recognizing tumor cells. These T cells may not have yet been activated by peptides presented on autologous tumor cells (Jager et al., 2002a). This bears several implications. First, previous encounters with crossreactive antigens may shape the TCR repertoire needed to ﬁght off cancer cells. Such preexposure may have beneﬁts: The already preexisiting memory T-cell pool may be directed against target cells. In contrast, a phenomenon called T-cell exhaustion may indicate that preexposure of T cells results in apoptosis of antigen-speciﬁc T cells. In general, the coexistance of different infectious diseases in humans as well as in animals suggests that such T-cell crossreactivity impacts also the outcome of diseases. For instance, simultaneous infection of patients with the hepatitis G virus and HIV appear to slow down the clinical progression of AIDS. Simultaneous scrub typhus infection and HIV appears also beneﬁcial for the host. In contrast, simultaneous infection of HIV and hepatitis C can be deleterious (Tillmann et al., 2001; Watt et al., 2000; Xiang et al., 2001). Indeed, a recent animal study showed that such crossreactivity impacts clinically the outcome of infectious disease. Memory CD8+ T cells expanded after infection with lymphocytic choriomeningitis virus (LCMV) were selectively activated upon infection with the unrelated vaccina virus. Presence of LCMV-speciﬁc T cells was associated with enhanced vaccinia virus clearance, decreased mortality, and lung pathology (Chen et al., 2001). Thus, if multiple, closely related (at least as seen from a T-cell perspective) antigens are encountered serially, memory or memory-like T cells may occur. For instance, Melan-A/MART-1-speciﬁc T-cell reactivity can be detected in healthy individuals and in melanoma patients (Loftus et al., 1996,

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1998; Mohagheghpour et al., 1998); it can also be induced by viral or bacterial proteins (Loftus et al., 1996, 1998) or by naturally presented melanoma antigens (Castelli et al., 1995; Coulie et al., 1994; Kawakami et al., 1994c).

VII. QUESTIONS OF SPECIFICITY AND ALTERNATE T-CELL EFFECTOR FUNCTIONS Most of the T cells associated with tumor regression have been identiﬁed as TCR αβ+ T cells. However, the nature of an effective antitumor directed immune response may also be mediated by γ δ+ T cells, NK T cells, or NK cells (reviewed in Ferrarini et al., 2002). The speciﬁcity of some of these T-cell subpopulations has been elucidated; others remain to be identiﬁed. Two major γ δ T-cell subsets have been described: delta 1+ T cells which reside primarily in epithelial tissues and provide a ﬁrst-line defense against infections or malignancies (Fisch et al., 2000; Hayday, 2000; Kaufmann and Kabelitz, 1991; Triebel and Hercend, 1989). Human TCR delta 1+ T cells have been reported to speciﬁcally recognize tumor cells of epithelial origin including renal cell cancer (Choudhary et al., 1995), lung cancer (Zocchi et al., 1990), and colorectal cancer (Maeurer et al., 1996) and appear to be expanded in PBL from patients with Epstein–Barr virus or HIV infection, in pulmonary lesions from patients with sarcoidosis, in synovial ﬂuid from patients with RA, in patients with multiple sclerosis, or in intestinal lesions from individuals with celiac disease (Boullier et al., 1995; Bucht et al., 1992; Halstensen et al., 1989; Hinz et al., 1994; Hvas et al., 1993; Jacobs and Haynes, 1992; Orsini et al., 1994; Shimonkevitz et al., 1993; Trejdosiewicz et al., 1991; Uyemura et al., 1992). More recent data suggest that some TCR delta 1+ T cells recognize the human MHC class I-related antigens MICA and MICB (Groh et al., 1998, 1999). This observation is in line with the notion that this T-cell subset is able to recognize a common antigen on epithelial tumor cells. The MICA and MICB antigens are stress induced under the heat-shock responsive promoters of epithelial cells including intestinal cells and lung and kidney epithelium. Of note, tumors derived from these tissues are inﬁltrated with TCR delta1+ T cells (Groh et al., 1999). Not only the clonotypic TCR, but also additional NK cell receptor molecules (e.g., NKG2D, a ligand for MICA and MICB), may be instrumental for target cell engagement and tumor cell recognition (Hayday, 2000; Poccia et al., 1997). In contrast to TCR delta1+ T cells, delta2+ T cells constitute the majority of circulating γ δ T cells. This T-cell subset plays a role in the defense of infection with intracellular bacteria and hematological tumor cells. TCR delta 2+ T cells kill lymphoma and myeloma cells in vitro (Fisch et al., 2000;

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Kaufmann and Kabelitz, 1991; Kunzmann et al., 2000) and appear to be expanded in PBL from disease-free survivors of acute leukemia after bone marrow transplantation (Haas et al., 1993). Of note, the observed crossreactivity of TCR delta2+ T cells to infectious agents and tumor cells may in part be related to the nature of the target antigens. The TCR delta2+ T-cell subsets recognize nonpeptide antigens, for example, phosphorylated thymidine-related cellular products which are involved in the salvage pathway in nucleic acid synthesis and are overexpressed in stressed target cells (Constant et al., 1994). A similar situation may be true for the recognition of prenyl-pyrophosphates (Tanaka et al., 1995). In addition, synthetic bisphononates are able to expand TCR delta2+ T cells which show a strong cytolytic activity directed against myeloma cells (Fisch et al., 2000). Thus, TCR delta1+ as well as delta2+ T cells may be expanded either at the site of the tumor lesions or in the peripheral blood from patients with cancer. Enhanced frequency of this T-cell subset may be indicative of a better prognosis; however, future experimental data implementing speciﬁc target antigens will provide a better correlation between the presence and functional activity of γ δ+ T cells. For instance, soluble MICA or MICB may be implemented to probe for TCR delta1+ T cells capable of recognizing transformed epithelial cells. Noteworthy is that the clonotypic TCR may be able to confer antigen speciﬁcity. In addition, the NK cell receptor (NKG2D) also serves as a ligand for MICA and MICB and it can be expressed on γ δ T cells. More recent data suggest that invasion of tumor lesions with γ δ T cells is indeed a sign of better prognosis, or that γ δ+ T cells negatively regulate cutaneous malignancies (Girardi et al., 2001; Pardoll, 2001; Thomas et al., 2001), potentially due to cytokine release elaborated in situ by γ δ T cells. Resident as well as circulating γ δ T cells secrete chemokines, including RANTES, IL-8, or MIP, which serve as attractors for immune cells (Biswas et al., 2000; Boismenu et al., 1996; Cipriani et al., 2000). A different T-cell subpopulation of NK T cells expresses the invariant TCR VA14 (mouse) or VA24 (human) reactive to the lipoglycan alphagalactosylceramide presented by CD1d (Bendelac et al., 1997; Gumperz et al., 2000; Joyce et al., 1998; Schoﬁeld et al., 1999). Human TCR VA24+ T cells often express an NK-associated C-type lectin NKR-P1A antigen (CD161). This T-cell subset is of major interest in the context of orchestrating an immune response, since it is endowed with producing large amounts of IL-4, IL-10, IL-13, IFNγ , TNFα, and GM-CSF upon activation (Exley et al., 1997, 1998; Spada et al., 1998; Yang et al., 2000). Recent studies in mice have shown that these cells can be tracked using CD1d tetramer complexes loaded with alpha-galactosylceramide (Matsuda et al., 2000). This proof of prinicple implies that this T-cell subset can also be monitored in human PBL. Indeed, human CD1d/alpha-galactosylceramide tetramer complexes were used to isolate NK T cells from PBL (Metelitsa et al., 2001). Exclusively CD1d-positive tumor cells were recognized including the constitutive

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CD1d+ hematopoetic tumor cell lines Jurkat and U937 (Kawano et al., 1999; Metelitsa et al., 2001). In addition, TCR-stimulated NK T cells secreted IL-2, a growth factor critical for NK activation. Thus, NK T cells may act directly be recognizing CD1d+ target cells and indirectly by providing growth factors necessary for the survival and expansion of NK or αβ+ T cells. However, tumor-speciﬁc T-cell memory may also be facilitated by NK-mediated tumor rejection (Kelly et al., 2002). Murine studies have shown that the ligand alpha-galactosylceraminde (derived from the marine sponge Agelas mauritanius) is capable of expanding this T-cell subset in mice associated with potent antitumor immune responses (Morita et al., 1995; Nakagawa et al., 1998, 2000; Toura et al., 1999). In addition, recent experiments have shown that such TCR VA24/VB11 + NKT effector cells are also able to confer cytoxicity against a variety of human tumor cell lines (Kawano et al., 1999; Nicol et al., 2000; Takahashi et al., 2000). Thus, in situ staining of regressing/progressing tumor lesion for NK T cells or the implementation of human CD1d tetramer reagents may aid in better deﬁning this T-cell subset in the context of biologically meaningful surrogate markers associated with a protective antitumor immune response (Moodycliffe et al., 2000; Smyth and Godfrey, 2000; Terabe et al., 2000). CD1d is expressed by cells of the monocyte lineage, by some B-cell intestinal epithelial cells, skin keratinocytes, and activated T cells (Blumberg et al., 1991; Bonish et al., 2000; Salamone et al., 2001; Spada et al., 2000). Malignancies derived from these cells/tissues may present targets for NK T cells or may be able to expand this T-cell subset in situ. This may have clinical potential, since targets deﬁned by either γ δ or NK T cells are independent of the TAP protein whose expression is often lost during tumor progression. Inﬂammation in situ, elaborated by NK T cells, or by any cell type, may determine the quality and quantitiy of an antitumor directed immune response (Frazer et al., 2001). This notion is supported by examining alternate immune effector cells with ill-deﬁned speciﬁcities, particularly in the intestine. These immune cells may play a crucial role in initiating a protective immune response in patients with intestinal malignancies. Their distribution and behavior in human tumors, however, has not yet been evaluated. For instance, about 10–20 intraepithelial lymphocytes (IEL) are present in situ per 100 enterocytes (Ferguson, 1977). This resident T-cell subset is highly cytolytic and bears immunoregulatory capacities (reviewed in Hayday et al., 2001) and functions which may determine the ultimate outcome of the battle between immune cells and transformed epithelial cells. To conclude, a combination of immune marker analysis listed in Table III may aid in deﬁning the most useful surrogate marker for immunomonitoring in patients with cancer. An individually tailored gauging of the immune response may—at least in some patients—represent the most reliable marker for detecting anticancer directed immune reactivity. Carefully designed clinical studies are needed to deﬁne true surrogate markers indicative of better

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prognosis or response to therapy. Potentially negative or positive associations with suppressor T cells, deﬁned by expression of CD25 (Gavin et al., 2002), awaits further clariﬁcation in patients with cancer. However, recent advances in genetic approaches and cell biology hold great promise both for therapy of malignancies and for the capability to deﬁne bona ﬁde surrogate markers for an effective anticancer directed immune response.

ACKNOWLEDGMENTS This work was supported by Grant QLK3-CT-1999-00064 from the European Community to C. C. and Grants SFB 432 A9, SFB 490 C4, and in part by a core grant from the Deutsche Krebshilfe to M. M. We are indebted to Ms. Monika Wiedmann for the extraordinary secretarial assistance.

REFERENCES Alexander-Miller, M. A., Leggatt, G. R., Sarin, A., and Berzofsky, J. A. (1996). Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL. J. Exp. Med. 184, 485–492. Allan, D. S., Colonna, M., Lanier, L. L., Churakova, T. D., Abrams, J. S., Ellis, S. A., McMichael, A. J., and Braud, V. M. (1999). Tetrameric complexes of human histocompatibility leukocyte antigen (HLA)-G bind to peripheral blood myelomonocytic cells. J. Exp. Med. 189, 1149– 1156. Altman, J. D., Moss, P. A., Goulder, P. J., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., McMichael, A. J., and Davis, M. M. (1996). Phenotypic analysis of antigen-speciﬁc T lymphocytes. Science 274, 94–96. Andersen, M. H., Pedersen, L. O., Capeller, B., Brocker, E. B., Becker, J. C., and thor Straten, P. (2001). Spontaneous cytotoxic T-cell responses against survivin-derived MHC class I-restricted T-cell epitopes in situ as well as ex vivo in cancer patients. Cancer Res. 61, 5964–5968. Anichini, A., Molla, A., Mortarini, R., Tragni, G., Bersani, I., Di Nicola, M., Gianni, A. M., Pilotti, S., Dunbar, R., Cerundolo, V., and Parmiani, G. (1999). An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-deﬁned, melanocyte-speciﬁc antigen in metastatic melanoma patients impacts on generation of peptide-speciﬁc CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions. J. Exp. Med. 190, 651–667. Appay, V., Nixon, D. F., Donahoe, S. M., Gillespie, G. M., Dong, T., King, A., Ogg, G. S., Spiegel, H. M., Conlon, C., Spina, C. A., Havlir, D. V., Richman, D. D., Waters, A., Easterbrook, P., McMichael, A. J., and Rowland-Jones, S. L. (2000). HIV-speciﬁc CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. J. Exp. Med. 192, 63–75. Arcaro, A., Gregoire, C., Bakker, T. R., Baldi, L., Jordan, M., Gofﬁn, L., Boucheron, N., Wurm, F., van der Merwe, P. A., Malissen, B., and Luescher, I. F. (2001). CD8beta endows CD8 with efﬁcient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes. J. Exp. Med. 194, 1485–1495.

T-Cell Response in Cancer Patients

177

Baurain, J. F., Colau, D., van Baren, N., Landry, C., Martelange, V., Vikkula, M., Boon, T., and Coulie, P. G. (2000). High frequency of autologous anti-melanoma CTL directed against an antigen generated by a point mutation in a new helicase gene. J. Immunol. 164, 6057–6066. Beekman, N. J., van Veelen, P. A., van Hall, T., Neisig, A., Sijts, A., Camps, M., Kloetzel, P. M., Neefjes, J. J., Melief, C. J., and Ossendorp, F. (2000). Abrogation of CTL epitope processing by single amino acid substitution ﬂanking the C-terminal proteasome cleavage site. J. Immunol. 164, 1898–1905. Belyakov, I. M., Wang, J., Koka, R., Ahlers, J. D., Snyder, J. T., Tse, R., Cox, J., Gibbs, J. S., Margulies, D. H., and Berzofsky, J. A. (2001). Activating CTL precursors to reveal CTL function without skewing the repertoire by in vitro expansion. Eur. J. Immunol. 31, 3557– 3566. Bendelac, A., Rivera, M. N., Park, S. H., and Roark, J. H. (1997). Mouse CD1-speciﬁc NK1 T cells: Development, speciﬁcity, and function. Annu. Rev. Immunol. 15, 535–562. Bercovici, N., Duffour, M. T., Agrawal, S., Salcedo, M., and Abastado, J. P. (2000). New methods for assessing T-cell responses. Clin. Diagn. Lab. Immunol. 7, 859–864. Biswas, P., Rovere, P., Filippi, C. D., Heltai, S., Smith, C., Dagna, L., Poli, G., Manfredi, A. A., and Ferrarini, M. (2000). Engagement of CD30 shapes the secretion of cytokines by human gamma delta T cells. Eur. J. Immunol. 30, 2172–2180. Bjorkman, P. J. (1997). MHC restriction in three dimensions: a view of T cell receptor/ligand interactions. Cell 89, 167–170. Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L., and Wiley, D. C. (1987). The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329, 512–518. Blumberg, R. S., Terhorst, C., Bleicher, P., McDermott, F. V., Allan, C. H., Landau, S. B., Trier, J. S., and Balk, S. P. (1991). Expression of a nonpolymorphic MHC class I-like molecule, CD1D, by human intestinal epithelial cells. J. Immunol. 147, 2518–2524. Bodinier, M., Peyrat, M. A., Tournay, C., Davodeau, F., Romagne, F., Bonneville, M., and Lang, F. (2000). Efﬁcient detection and immunomagnetic sorting of speciﬁc T cells using multimers of MHC class I and peptide with reduced CD8 binding. Nat. Med. 6, 707–710. Boismenu, R., Feng, L., Xia, Y. Y., Chang, J. C., and Havran, W. L. (1996). Chemokine expression by intraepithelial gamma delta T cells. Implications for the recruitment of inﬂammatory cells to damaged epithelia. J. Immunol. 157, 985–992. Boniface, J. J., Rabinowitz, J. D., Wulﬁng, C., Hampl, J., Reich, Z., Altman, J. D., Kantor, R. M., Beeson, C., McConnell, H. M., and Davis, M. M. (1998). Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected]. Immunity 9, 459–466. Bonish, B., Jullien, D., Dutronc, Y., Huang, B. B., Modlin, R., Spada, F. M., Porcelli, S. A., and Nickoloff, B. J. (2000). Overexpression of CD1d by keratinocytes in psoriasis and CD1ddependent IFN-gamma production by NK-T cells. J. Immunol. 165, 4076–4085. Bonneville, M., and Lang, F. (2002). CD8: From coreceptor to comodulator. Nat. Immunol. 3, 12–14. Boullier, S., Cochet, M., Poccia, F., and Gougeon, M. L. (1995). CDR3-independent gamma delta V delta 1+ T cell expansion in the peripheral blood of HIV-infected persons. J. Immunol. 154, 1418–1431. Bousso, P., Levraud, J. P., Kourilsky, P., and Abastado, J. P. (1999). The composition of a primary T cell response is largely determined by the timing of recruitment of individual T cell clones. J. Exp. Med. 189, 1591–1600. Braud, V. M., Allan, D. S., O’Callaghan, C. A., Soderstrom, K., D’Andrea, A., Ogg, G. S., Lazetic, S., Young, N. T., Bell, J. I., Phillips, J. H., Lanier, L. L., and McMichael, A. J. (1998). HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391, 795–799.

178

Castelli and Maeurer

Brichard, V., Van Pel, A., Wolfel, T., Wolfel, C., De Plaen, E., Lethe, B., Coulie, P., and Boon, T. (1993). The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J. Exp. Med. 178, 489–495. Brinckerhoff, L. H., Kalashnikov, V. V., Thompson, L. W., Yamshchikov, G. V., Pierce, R. A., Galavotti, H. S., Engelhard, V. H., and Slingluff, C. L., Jr. (1999). Terminal modiﬁcations inhibit proteolytic degradation of an immunogenic MART-1(27-35) peptide: Implications for peptide vaccines. Int. J. Cancer 83, 326-334. Brossart, P., Stuhler, G., Flad, T., Stevanovic, S., Rammensee, H. G., Kanz, L., and Brugger, W. (1998). Her-2/neu-derived peptides are tumor-associated antigens expressed by human renal cell and colon carcinoma lines and are recognized by in vitro induced speciﬁc cytotoxic T lymphocytes. Cancer Res. 58, 732–736. Brown, J. H., Jardetzky, T., Saper, M. A., Samraoui, B., Bjorkman, P. J., and Wiley, D. C. (1988). A hypothetical model of the foreign antigen binding site of class II histocompatibility molecules. Nature 332, 845–850. Brusic, V., Rudy, G., and Harrison, L. C. (1998a). MHCPEP, a database of MHC-binding peptides: Update 1997. Nucleic Acids Res. 26, 368–371. Brusic, V., Rudy, G., Honeyman, G., Hammer, J., and Harrison, L. (1998b). Prediction of MHC class II-binding peptides using an evolutionary algorithm and artiﬁcial neural network. Bioinformatics 14, 121–130. Bucht, A., Soderstrom, K., Hultman, T., Uhlen, M., Nilsson, E., Kiessling, R., and Gronberg, A. (1992). T cell receptor diversity and activation markers in the V delta 1 subset of rheumatoid synovial ﬂuid and peripheral blood T lymphocytes. Eur. J. Immunol. 22, 567–574. Bullock, T. N., Mullins, D. W., Colella, T. A., and Engelhard, V. H. (2001). Manipulation of avidity to improve effectiveness of adoptively transferred CD8(+) T cells for melanoma immunotherapy in human MHC class I-transgenic mice. J. Immunol. 167, 5824– 5831. Busch, D. H., and Pamer, E. G. (1999). T cell afﬁnity maturation by selective expansion during infection. J. Exp. Med. 189, 701–710. Castelli, C., Rivoltini, L., Andreola, G., Carrabba, M., Renkvist, N., and Parmiani, G. (2000). T-cell recognition of melanoma-associated antigens. J. Cell Physiol. 182, 323–331. Castelli, C., Storkus, W. J., Maeurer, M. J., Martin, D. M., Huang, E. C., Pramanik, B. N., Nagabhushan, T. L., Parmiani, G., and Lotze, M. T. (1995). Mass spectrometric identiﬁcation of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes. J. Exp. Med. 181, 363–368. Champagne, P., Ogg, G. S., King, A. S., Knabenhans, C., Ellefsen, K., Nobile, M., Appay, V., Rizzardi, G. P., Fleury, S., Lipp, M., Forster, R., Rowland-Jones, S., Sekaly, R. P., McMichael, A. J., and Pantaleo, G. (2001). Skewed maturation of memory HIV-speciﬁc CD8 T lymphocytes. Nature 410, 106–111. Chaux, P., Vantomme, V., Stroobant, V., Thielemans, K., Corthals, J., Luiten, R., Eggermont, A. M., Boon, T., and van der Bruggen, P. (1999). Identiﬁcation of MAGE-3 epitopes presented by HLA-DR molecules to CD4(+) T lymphocytes. J. Exp. Med. 189, 767–778. Chen, H. D., Fraire, A. E., Joris, I., Brehm, M. A., Welsh, R. M., and Selin, L. K. (2001). Memory CD8+ T cells in heterologous antiviral immunity and immunopathology in the lung. Nat. Immunol. 2, 1067–1076. Chen, J. L., Dunbar, P. R., Gileadi, U., Jager, E., Gnjatic, S., Nagata, Y., Stockert, E., Panicali, D. L., Chen, Y. T., Knuth, A., Old, L. J., and Cerundolo, V. (2000). Identiﬁcation of NY-ESO-1 peptide analogues capable of improved stimulation of tumor-reactive CTL. J. Immunol. 165, 948–955. Chen, W., Ede, N. J., Jackson, D. C., McCluskey, J., and Purcell, A. W. (1996). CTL recognition of an altered peptide associated with asparagine bond rearrangement. Implications for immunity and vaccine design. J. Immunol. 157, 1000–1005.

T-Cell Response in Cancer Patients

179

Chen, Y. T., Scanlan, M. J., Sahin, U., Tureci, O., Gure, A. O., Tsang, S., Williamson, B., Stockert, E., Pfreundschuh, M., and Old, L. J. (1997). A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc. Natl. Acad. Sci. USA 94, 1914–1918. Chiari, R., Foury, F., De Plaen, E., Baurain, J. F., Thonnard, J., and Coulie, P. G. (1999). Two antigens recognized by autologous cytolytic T lymphocytes on a melanoma result from a single point mutation in an essential housekeeping gene. Cancer Res. 59, 5785–5792. Chiari, R., Hames, G., Stroobant, V., Texier, C., Maillere, B., Boon, T., and Coulie, P. G. (2000). Identiﬁcation of a tumor-speciﬁc shared antigen derived from an Eph receptor and presented to CD4 T cells on HLA class II molecules. Cancer Res. 60, 4855–4863. Choudhary, A., Davodeau, F., Moreau, A., Peyrat, M. A., Bonneville, M., and Jotereau, F. (1995). Selective lysis of autologous tumor cells by recurrent gamma delta tumor-inﬁltrating lymphocytes from renal carcinoma. J. Immunol. 154, 3932–3940. Cipriani, B., Borsellino, G., Poccia, F., Placido, R., Tramonti, D., Bach, S., Battistini, L., and Brosnan, C. F. (2000). Activation of C-C beta-chemokines in human peripheral blood gammadelta T cells by isopentenyl pyrophosphate and regulation by cytokines. Blood 95, 39–47. Cochran, J. R., Cameron, T. O., and Stern, L. J. (2000). The relationship of MHC-peptide binding and T cell activation probed using chemically deﬁned MHC class II oligomers. Immunity 12, 241–250. Coles, R. M., Mueller, S. N., Heath, W. R., Carbone, F. R., and Brooks, A. G. (2002). Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1. J. Immunol. 168, 834–838. Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M., and Fournie, J. J. (1994). Stimulation of human gamma delta T cells by nonpeptidic mycobacterial ligands. Science 264, 267–270. Coulie, P. G., Brichard, V., Van Pel, A., Wolfel, T., Schneider, J., Traversari, C., Mattei, S., De Plaen, E., Lurquin, C., Szikora, J. P., et al. (1994). A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J. Exp. Med. 180, 35–42. Coulie, P. G., Karanikas, V., Colau, D., Lurquin, C., Landry, C., Marchand, M., Dorval, T., Brichard, V., and Boon, T. (2001). A monoclonal cytolytic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-speciﬁc antigenic peptide encoded by gene MAGE-3. Proc. Natl. Acad. Sci. USA 98, 10,290–10,295. Coulie, P. G., Lehmann, F., Lethe, B., Herman, J., Lurquin, C., Andrawiss, M., and Boon, T. (1995). A mutated intron sequence code for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc. Natl. Acad. Sci. USA 92, 7976–7980. Crawford, F., Kozono, H., White, J., Marrack, P., and Kappler, J. (1998). Detection of antigenspeciﬁc T cells with multivalent soluble class II MHC covalent peptide complexes. Immunity 8, 675–682. Cresswell, P., and Lanzavecchia, A. (2001). Antigen processing and recognition. Curr. Opin. Immunol. 13, 11–12. Daniels, M. A., Devine, L., Miller, J. D., Moser, J. M., Lukacher, A. E., Altman, J. D., Kavathas, P., Hogquist, K. A., and Jameson, S. C. (2001). CD8 binding to MHC class I molecules is inﬂuenced by T cell maturation and glycosylation. Immunity 15, 1051–1061. Daniels, M. A., and Jameson, S. C. (2000). Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers. J. Exp. Med. 191, 335–346. Degano, P., Schneider, J., Hannan, C. M., Gilbert, S. C., and Hill, A. V. (1999). Gene gun intradermal DNA immunization followed by boosting with modiﬁed vaccinia virus Ankara: Enhanced CD8+ T cell immunogenicity and protective efﬁcacy in the inﬂuenza and malaria models. Vaccine 18, 623–632.

180

Castelli and Maeurer

Delon, J., Gregoire, C., Malissen, B., Darche, S., Lemaitre, F., Kourilsky, P., Abastado, J. P., and Trautmann, A. (1998). CD8 expression allows T cell signaling by monomeric peptide-MHC complexes. Immunity 9, 467–473. Denkberg, G., Cohen, C. J., and Reiter, Y. (2001). Critical role for CD8 in binding of MHC tetramers to TCR: CD8 antibodies block speciﬁc binding of human tumor-speciﬁc MHCpeptide tetramers to TCR. J. Immunol. 167, 270–276. De Plaen, E., Lurquin, C., Lethe, B., van der Bruggen, P., Brichard, V., Renauld, J. C., Coulie, P., Van Pel, A., and Boon, T. (1997). Identiﬁcation of genes coding for tumor antigens recognized by cytolytic T lymphocytes. Methods 12, 125–142. Disis, M. L., Smith, J. W., Murphy, A. E., Chen, W., and Cheever, M. A. (1994). In vitro generation of human cytolytic T-cells speciﬁc for peptides derived from the HER-2/neu protooncogene protein. Cancer Res. 54, 1071–1076. D’Souza, S., Rimoldi, D., Lienard, D., Lejeune, F., Cerottini, J. C., and Romero, P. (1998). Circulating Melan-A/Mart-1 speciﬁc cytolytic T lymphocyte precursors in HLA-A2+ melanoma patients have a memory phenotype. Int. J. Cancer 78, 699–706. Dunbar, P. R., Smith, C. L., Chao, D., Salio, M., Shepherd, D., Mirza, F., Lipp, M., Lanzavecchia, A., Sallusto, F., Evans, A., Russell-Jones, R., Harris, A. L., and Cerundolo, V. (2000). A shift in the phenotype of melan-A-speciﬁc CTL identiﬁes melanoma patients with an active tumorspeciﬁc immune response. J. Immunol. 165, 6644–6652. Dutoit, V., Rubio-Godoy, V., Dietrich, P. Y., Quiqueres, A. L., Schnuriger, V., Rimoldi, D., Lienard, D., Speiser, D., Guillaume, P., Batard, P., Cerottini, J. C., Romero, P., and Valmori, D. (2001). Heterogeneous T-cell response to MAGE-A10(254-262): High avidity-speciﬁc cytolytic T lymphocytes show superior antitumor activity. Cancer Res. 61, 5850–5856. Echchakir, H., Mami-Chouaib, F., Vergnon, I., Baurain, J. F., Karanikas, V., Chouaib, S., and Coulie, P. G. (2001). A point mutation in the alpha-actinin-4 gene generates an antigenic peptide recognized by autologous cytolytic T lymphocytes on a human lung carcinoma. Cancer Res. 61, 4078–4083. Eisen, H. N., Sykulev, Y., and Tsomides, T. J. (1996). Antigen-speciﬁc T-cell receptors and their reactions with complexes formed by peptides with major histocompatibility complex proteins. Adv. Protein Chem. 49, 1–56. Estcourt, M. J., Ramsay, A. J., Brooks, A., Thomson, S. A., Medveckzy, C. J., and Ramshaw, I. A. (2002). Prime-boost immunization generates a high frequency, high-avidity CD8(+) cytotoxic T lymphocyte population. Int. Immunol. 14, 31–37. Exley, M., Garcia, J., Balk, S. P., and Porcelli, S. (1997). Requirements for CD1d recognition by human invariant Valpha24+ CD4-CD8- T cells. J. Exp. Med. 186, 109–120. Exley, M., Porcelli, S., Furman, M., Garcia, J., and Balk, S. (1998). CD161 (NKR-P1A) costimulation of CD1d-dependent activation of human T cells expressing invariant V alpha 24 J alpha Q T cell receptor alpha chains. J. Exp. Med. 188, 867–876. Faint, J. M., Annels, N. E., Curnow, S. J., Shields, P., Pilling, D., Hislop, A. D., Wu, L., Akbar, A. N., Buckley, C. D., Moss, P. A., Adams, H., Rickinson, A. B., and Salmon, M. (2001). Memory T cells constitute a subset of the human CD8+CD45RA+ pool with distinct phenotypic and migratory characteristics. J. Immunol. 167, 212–220. Falk, K., Rotzschke, O., Stevanovic, S., Jung, G., and Rammensee, H. G. (1991). Allele-speciﬁc motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351, 290–296. Ferguson, A. (1977). Intraepithelial lymphocytes of the small intestine. Gut 18, 921–937. Ferrarini, M., Ferrero, E., Dagna, L., Poggi, A., and Zocchi, M. R. (2002). Human gammadelta T cells: A nonredundant system in the immune-surveillance against cancer. Trends Immunol. 23, 14–18. Fisch, P., Moris, A., Rammensee, H. G., and Handgretinger, R. (2000). Inhibitory MHC class I receptors on gammadelta T cells in tumour immunity and autoimmunity. Immunol. Today 21, 187–191.

T-Cell Response in Cancer Patients

181

Fong, L., Hou, Y., Rivas, A., Benike, C., Yuen, A., Fisher, G. A., Davis, M., and Engleman, E. G. (2001). Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy. Proc. Natl. Acad. Sci. USA 98, 8809–8814. Frazer, I. H., De Kluyver, R., Leggatt, G. R., Guo, H. Y., Dunn, L., White, O., Harris, C., Liem, A., and Lambert, P. (2001). Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinﬂammatory signals at the time of ﬁrst antigen exposure. J. Immunol. 167, 6180–6187. Gavin, M. A., Clarke, S. R., Negrou, E., Gallegos, A., and Rudensky, A. (2002). Homeostasis and anergy of CD4+CD25+ suppressor T cells in vivo. Nat. Immunol. 3, 33–41. Gedde-Dahl, T., 3rd, Spurkland, A., Fossum, B., Wittinghofer, A., Thorsby, E., and Gaudernack, G. (1994). T cell epitopes encompassing the mutational hot spot position 61 of p21 ras. Promiscuity in ras peptide binding to HLA. Eur. J. Immunol. 24, 410–414. Girardi, M., Oppenheim, D. E., Steele, C. R., Lewis, J. M., Glusac, E., Filler, R., Hobby, P., Sutton, B., Tigelaar, R. E., and Hayday, A. C. (2001). Regulation of cutaneous malignancy by gammadelta T cells. Science 294, 605–609. Godkin, A., Friede, T., Davenport, M., Stevanovic, S., Willis, A., Jewell, D., Hill, A., and Rammensee, H. G. (1997). Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2). Int. Immunol. 9, 905–911. Gorochov, G., Neumann, A. U., Kereveur, A., Parizot, C., Li, T., Katlama, C., Karmochkine, M., Raguin, G., Autran, B., and Debre, P. (1998). Perturbation of CD4+ and CD8+ T-cell repertoires during progression to AIDS and regulation of the CD4+ repertoire during antiviral therapy. Nat. Med. 4, 215–221. Gray, C. M., Lawrence, J., Ranheim, E. A., Vierra, M., Zupancic, M., Winters, M., Altman, J., Montoya, J., Zolopa, A., Schapiro, J., Haase, A. T., and Merigan, T. C. (2000). Highly active antiretroviral therapy results in HIV type 1 suppression in lymph nodes, increased pools of naive T cells, decreased pools of activated T cells, and diminished frequencies of peripheral activated HIV type 1-speciﬁc CD8+ T cells. AIDS Res. Hum. Retroviruses 16, 1357– 1369. Groh, V., Rhinehart, R., Secrist, H., Bauer, S., Grabstein, K. H., and Spies, T. (1999). Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB. Proc. Natl. Acad. Sci. USA 96, 6879–6884. Groh, V., Steinle, A., Bauer, S., and Spies, T. (1998). Recognition of stress-induced MHC molecules by intestinal epithelial gammadelta T cells. Science 279, 1737–1740. Gumperz, J. E., Roy, C., Makowska, A., Lum, D., Sugita, M., Podrebarac, T., Koezuka, Y., Porcelli, S. A., Cardell, S., Brenner, M. B., and Behar, S. M. (2000). Murine CD1d-restricted T cell recognition of cellular lipids. Immunity 12, 211–221. Haanen, J. B., van Oijen, M. G., Tirion, F., Oomen, L. C., Kruisbeek, A. M., Vyth-Dreese, F. A., and Schumacher, T. N. (2000). In situ detection of virus- and tumor-speciﬁc T-cell immunity. Nat. Med. 6, 1056–1060. Haas, W., Pereira, P., and Tonegawa, S. (1993). Gamma/delta cells. Annu. Rev. Immunol. 11, 637–685. Halder, T., Pawelec, G., Kirkin, A. F., Zeuthen, J., Meyer, H. E., Kun, L., and Kalbacher, H. (1997). Isolation of novel HLA-DR restricted potential tumor-associated antigens from the melanoma cell line FM3. Cancer Res. 57, 3238–3244. Halstensen, T. S., Scott, H., and Brandtzaeg, P. (1989). Intraepithelial T cells of the TcR gamma/delta+ CD8- and V delta 1/J delta 1+ phenotypes are increased in coeliac disease. Scand. J. Immunol. 30, 665–672. Hamad, A. R., O’Herrin, S. M., Lebowitz, M. S., Srikrishnan, A., Bieler, J., Schneck, J., and Pardoll, D. (1998). Potent T cell activation with dimeric peptide-major histocompatibility complex class II ligand: The role of CD4 coreceptor. J. Exp. Med. 188, 1633– 1640.

182

Castelli and Maeurer

Hamann, D., Baars, P. A., Rep, M. H., Hooibrink, B., Kerkhof-Garde, S. R., Klein, M. R., and van Lier, R. A. (1997). Phenotypic and functional separation of memory and effector human CD8+ T cells. J. Exp. Med. 186, 1407–1418. Hamann, J., Vogel, B., van Schijndel, G. M., and van Lier, R. A. (1996). The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF). J. Exp. Med. 184, 1185–1189. Hammer, J., Bono, E., Gallazzi, F., Belunis, C., Nagy, Z., and Sinigaglia, F. (1994). Precise prediction of major histocompatibility complex class II-peptide interaction based on peptide side chain scanning. J. Exp. Med. 180, 2353–2358. Hayday, A., Theodoridis, E., Ramsburg, E., and Shires, J. (2001). Intraepithelial lymphocytes: Exploring the Third Way in immunology. Nat. Immunol. 2, 997–1003. Hayday, A. C. (2000). [gamma][delta] cells: A right time and a right place for a conserved third way of protection. Annu. Rev. Immunol. 18, 975–1026. Hazenberg, M. D., Otto, S. A., Cohen Stuart, J. W., Verschuren, M. C., Borleffs, J. C., Boucher, C. A., Coutinho, R. A., Lange, J. M., Rinke de Wit, T. F., Tsegaye, A., van Dongen, J. J., Hamann, D., de Boer, R. J., and Miedema, F. (2000). Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection. Nat. Med. 6, 1036–1042. Hel, Z., Nacsa, J., Kelsall, B., Tsai, W. P., Letvin, N., Parks, R. W., Tryniszewska, E., Picker, L., Lewis, M. G., Edghill-Smith, Y., Moniuszko, M., Pal, R., Stevceva, L., Altman, J. D., Allen, T. M., Watkins, D., Torres, J. V., Berzofsky, J. A., Belyakov, I. M., Strober, W., and Franchini, G. (2001). Impairment of Gag-speciﬁc CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeﬁciency virus mac251- and simian-human immunodeﬁciency virus KU2-infected macaques. J. Virol. 75, 11,483–11,495. Hernandez, J., Lee, P. P., Davis, M. M., and Sherman, L. A. (2000). The use of HLA A2.1/p53 peptide tetramers to visualize the impact of self tolerance on the TCR repertoire. J. Immunol. 164, 596–602. Hinz, T., Wesch, D., Friese, K., Reckziegel, A., Arden, B., and Kabelitz, D. (1994). T cell receptor gamma delta repertoire in HIV-1-infected individuals. Eur. J. Immunol. 24, 3044–3049. Hoffmann, T. K., Loftus, D. J., Nakano, K., Maeurer, M. J., Chikamatsu, K., Appella, E., Whiteside, T. L., and DeLeo, A. B. (2002). The ability of variant peptides to reverse the nonresponsiveness of T lymphocytes to the wild-type sequence p53(264-272) epitope. J. Immunol. 168, 1338–1347. Hohn, H., Neukirch, C., Freitag, K., Necker, A., Hitzler, W., Seliger, B., and Maeurer, M. (2002). Longitudinal analysis of the T-cell receptor (TCR) VA and VB repertoire in CD8+ T-cells from individuals immunized with recombinant Hepatitis B surface antigen. Clin. Exp. Immunol. 129 (2). Hohn, H., Pilch, H., Gunzel, S., Neukirch, C., Freitag, K., Necker, A., and Maeurer, M. J. (2000). Human papillomavirus type 33 E7 peptides presented by HLA-DR∗ 0402 to tumorinﬁltrating T cells in cervical cancer. J. Virol. 74, 6632–6636. Hohn, H., Pilch, H., Gunzel, S., Neukirch, C., Hilmes, C., Kaufmann, A., Seliger, B., and Maeurer, M. J. (1999a). CD4+ tumor-inﬁltrating lymphocytes in cervical cancer recognize HLA-DR- restricted peptides provided by human papillomavirus-E7. J. Immunol. 163, 5715– 5722. Hohn, H., Reichert, T., Neukirch, C., Pilch, H., and Maeurer, M. J. (1999b). Monoclonal TCR mRNA transcripts are preferentially detected in the TCR variable alpha chain in CD8(+) T-lymphocytes: Implications for immunomonitoring. Int. J. Mol. Med. 3, 139–144. Holler, P. D., Lim, A. R., Cho, B. K., Rund, L. A., and Kranz, D. M. (2001). CD8(−) T cell transfectants that express a high afﬁnity T cell receptor exhibit enhanced peptide-dependent activation. J. Exp. Med. 194, 1043–1052. Honeyman, M. C., Brusic, V., Stone, N. L., and Harrison, L. C. (1998). Neural network-based prediction of candidate T-cell epitopes. Nat. Biotechnol. 16, 966–969.

T-Cell Response in Cancer Patients

183

Housseau, F., Moorthy, A., Langer, D. A., Robbins, P. F., Gonzales, M. I., and Topalian, S. L. (2001). N-linked carbohydrates in tyrosinase are required for its recognition by human MHC class II-restricted CD4(+) T cells. Eur. J. Immunol. 31, 2690–2701. Hvas, J., Oksenberg, J. R., Fernando, R., Steinman, L., and Bernard, C. C. (1993). Gamma delta T cell receptor repertoire in brain lesions of patients with multiple sclerosis. J. Neuroimmunol. 46, 225–234. Ikeda, H., Lethe, B., Lehmann, F., van Baren, N., Baurain, J. F., de Smet, C., Chambost, H., Vitale, M., Moretta, A., Boon, T., and Coulie, P. G. (1997). Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity 6, 199–208. Jacobs, M. R., and Haynes, B. F. (1992). Increase in TCR gamma delta T lymphocytes in synovia from rheumatoid arthritis patients with active synovitis. J. Clin. Immunol. 12, 130–138. Jager, E., Gnjatic, S., Nagata, Y., Stockert, E., Jager, D., Karbach, J., Neumann, A., Rieckenberg, J., Chen, Y. T., Ritter, G., Hoffman, E., Arand, M., Old, L. J., and Knuth, A. (2000a). Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers. Proc. Natl. Acad. Sci. USA 97, 12,198–12,203. ¨ ¨ Jager, E., Hohn, H., Necker, A., Forster, R., Karbach, J., Freitag, K., Neukirch, C., Castelli, C., Salter, R. D., Knuth, A., and Maeurer, M. J. (2002a). Peptide-speciﬁc CD8+ T-cell evolution in vivo: Response to peptide vaccination with Melan-A/MART-1. Int. J. Cancer. 98, 376–388. Jager, E., Jager, D., Karbach, J., Chen, Y. T., Ritter, G., Nagata, Y., Gnjatic, S., Stockert, E., Arand, M., Old, L. J., and Knuth, A. (2000b). Identiﬁcation of NY-ESO-1 epitopes presented by human histocompatibility antigen (HLA)-DRB4∗ 0101-0103 and recognized by CD4(+) T lymphocytes of patients with NY-ESO-1-expressing melanoma. J. Exp. Med. 191, 625– 630. Jager, E., Maeurer, M., Hohn, H., Karbach, J., Jager, D., Zidianakis, Z., Bakhshandeh-Bath, A., Orth, J., Neukirch, C., Necker, A., Reichert, T. E., and Knuth, A. (2000c). Clonal expansion of Melan A-speciﬁc cytotoxic T lymphocytes in a melanoma patient responding to continued immunization with melanoma-associated peptides. Int. J. Cancer. 86, 538–547. Jager, E., Nagata, Y., Gnjatic, S., Wada, H., Stockert, E., Karbach, J., Dunbar, P. R., Lee, S. Y., Jungbluth, A., Jager, D., Arand, M., Ritter, G., Cerundolo, V., Dupont, B., Chen, Y. T., Old, L. J., and Knuth, A. (2000d). Monitoring CD8 T cell responses to NY-ESO-1: Correlation of humoral and cellular immune responses. Proc. Natl. Acad. Sc.i USA 97, 4760–4765. ¨ Jager, E., Salter, R., Castelli, C., Hohn, H., Freitag, K., Karbach, J., Neukirch, C., Necker, A., Knuth, A., and Maeurer, M. (2002b). Impact of antigen presentation on TCR modulation and cytokine release. Implications for detection and sorting of antigen-speciﬁc CD8+ T-cells using HLA-A2 wild type or HLA-A2 mutant tetrameric complexes. J. Immunol. 168, 2766– 2772. Joyce, S., Woods, A. S., Yewdell, J. W., Bennink, J. R., De Silva, A. D., Boesteanu, A., Balk, S. P., Cotter, R. J., and Brutkiewicz, R. R. (1998). Natural ligand of mouse CD1d1: Cellular glycosylphosphatidylinositol. Science 279, 1541–1544. Kaufmann, S. H., and Kabelitz, D. (1991). Gamma/delta T lymphocytes and heat shock proteins. Curr. Top. Microbiol. Immunol. 167, 191–207. Kawakami, Y., Eliyahu, S., Delgado, C. H., Robbins, P. F., Rivoltini, L., Topalian, S. L., Miki, T., and Rosenberg, S. A. (1994a). Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells inﬁltrating into tumor. Proc. Natl. Acad. Sci. USA 91, 3515–3519. Kawakami, Y., Eliyahu, S., Delgado, C. H., Robbins, P. F., Sakaguchi, K., Appella, E., Yannelli, J. R., Adema, G. J., Miki, T., and Rosenberg, S. A. (1994b). Identiﬁcation of a human melanoma antigen recognized by tumor-inﬁltrating lymphocytes associated with in vivo tumor rejection. Proc. Natl. Acad. Sci. USA 91, 6458–6462.

184

Castelli and Maeurer

Kawakami, Y., Eliyahu, S., Sakaguchi, K., Robbins, P. F., Rivoltini, L., Yannelli, J. R., Appella, E., and Rosenberg, S. A. (1994c). Identiﬁcation of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor inﬁltrating lymphocytes. J. Exp. Med. 180, 347–352. Kawakami, Y., and Rosenberg, S. A. (1997). Human tumor antigens recognized by T-cells. Immunol. Res. 16, 313–339. Kawano, T., Nakayama, T., Kamada, N., Kaneko, Y., Harada, M., Ogura, N., Akutsu, Y., Motohashi, S., Iizasa, T., Endo, H., Fujisawa, T., Shinkai, H., and Taniguchi, M. (1999). Antitumor cytotoxicity mediated by ligand-activated human V alpha24 NKT cells. Cancer Res. 59, 5102–5105. Kawashima, I., Tsai, V., Southwood, S., Takesako, K., Sette, A., and Celis, E. (1999). Identiﬁcation of HLA-A3-restricted cytotoxic T lymphocyte epitopes from carcinoembryonic antigen and HER-2/neu by primary in vitro immunization with peptide-pulsed dendritic cells. Cancer Res. 59, 431–435. Kedl, R. M., Schaefer, B. C., Kappler, J. W., and Marrack, P. (2002). T cells down-modulate peptide-MHC complexes on APCs in vivo. Nat. Immunol. 3, 27–32. Kelly, J. M., Darcy, P. K., Markby, J. L., Godfrey, D. I., Takeda, K., Yagita, H., and Smyth, M. J. (2002). Induction of tumor-speciﬁc T cell memory by NK cell-mediated tumor rejection. Nat. Immunol. 3, 83–90. Kessler, J. H., Beekman, N. J., Bres-Vloemans, S. A., Verdijk, P., van Veelen, P. A., KloostermanJoosten, A. M., Vissers, D. C., ten Bosch, G. J., Kester, M. G., Sijts, A., Wouter Drijfhout, J., Ossendorp, F., Offringa, R., and Melief, C. J. (2001). Efﬁcient identiﬁcation of novel HLAA(∗ )0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis. J. Exp. Med. 193, 73–88. Khong, H. T., and Rosenberg, S. A. (2002). Pre-existing immunity to tyrosinase-related protein (TRP)-2, a new TRP-2 isoform, and the NY-ESO-1 melanoma antigen in a patient with a dramatic response to immunotherapy. J. Immunol. 168, 951–956. Kittlesen, D. J., Thompson, L. W., Gulden, P. H., Skipper, J. C., Colella, T. A., Shabanowitz, J., Hunt, D. F., Engelhard, V. H., Slingluff, C. L., Jr., and Shabanowitz, J. A. (1998). Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: Implications for tumor vaccine development. J. Immunol. 160, 2099–2106. Kobayashi, H., Wood, M., Song, Y., Appella, E., and Celis, E. (2000). Deﬁning promiscuous MHC class II helper T-cell epitopes for the HER2/neu tumor antigen. Cancer Res. 60, 5228– 5236. Koralnik, I. J., Du Pasquier, R. A., Kuroda, M. J., Schmitz, J. E., Dang, X., Zheng, Y., Lifton, M., and Letvin, N. L. (2002). Association of prolonged survival in HLA-A2+ progressive multifocal leukoencephalopathy patients with a CTL response speciﬁc for a commonly recognized JC virus epitope. J. Immunol. 168, 499–504. Kropshofer, H., Spindeldreher, S., Rohn, T. A., Platania, N., Grygar, C., Daniel, N., Wolpl, A., Langen, H., Horejsi, V., and Vogt, A. B. (2002). Tetraspan microdomains distinct from lipid rafts enrich select peptide-MHC class II complexes. Nat. Immunol. 3, 61–68. Kunzmann, V., Bauer, E., Feurle, J., Weissinger, F., Tony, H. P., and Wilhelm, M. (2000). Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 96, 384–392. Kuroda, M. J., Schmitz, J. E., Barouch, D. H., Craiu, A., Allen, T. M., Sette, A., Watkins, D. I., Forman, M. A., and Letvin, N. L. (1998). Analysis of Gag-speciﬁc cytotoxic T lymphocytes in simian immunodeﬁciency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex. J. Exp. Med. 187, 1373–1381. Labarriere, N., Diez, E., Pandolﬁno, M. C., Viret, C., Guilloux, Y., Le Guiner, S., Fonteneau, J. F., Dreno, B., and Jotereau, F. (1997). Optimal T cell activation by melanoma cells depends on a minimal level of antigen transcription. J. Immunol. 158, 1238–1245.

T-Cell Response in Cancer Patients

185

Lanzavecchia, A. (2002). Lack of fair play in the T cell response. Nat. Immunol. 3, 9–10. Lauvau, G., Vijh, S., Kong, P., Horng, T., Kerksiek, K., Serbina, N., Tuma, R. A., and Pamer, E. G. (2001). Priming of memory but not effector CD8 T cells by a killed bacterial vaccine. Science 294, 1735–1739. Lee, K. H., Panelli, M. C., Kim, C. J., Riker, A. I., Bettinotti, M. P., Roden, M. M., Fetsch, P., Abati, A., Rosenberg, S. A., and Marincola, F. M. (1998). Functional dissociation between local and systemic immune response during anti-melanoma peptide vaccination. J. Immunol. 161, 4183–4194. Lee, K. H., Wang, E., Nielsen, M. B., Wunderlich, J., Migueles, S., Connors, M., Steinberg, S. M., Rosenberg, S. A., and Marincola, F. M. (1999). Increased vaccine-speciﬁc T cell frequency after peptide-based vaccination correlates with increased susceptibility to in vitro stimulation but does not lead to tumor regression. J. Immunol. 163, 6292– 6300. Leishman, A. J., Naidenko, O. V., Attinger, A., Koning, F., Lena, C. J., Xiong, Y., Chang, H. C., Reinherz, E., Kronenberg, M., and Cheroutre, H. (2001). T cell responses modulated through interaction between CD8alphaalpha an the nonclassical MHC class I molecule, TL. Science 294, 1936–1939. Loftus, D. J., Castelli, C., Clay, T. M., Squarcina, P., Marincola, F. M., Nishimura, M. I., Parmiani, G., Appella, E., and Rivoltini, L. (1996). Identiﬁcation of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1(27–35). J. Exp. Med. 184, 647–657. Loftus, D. J., Squarcina, P., Nielsen, M. B., Geisler, C., Castelli, C., Odum, N., Appella, E., Parmiani, G., and Rivoltini, L. (1998). Peptides derived from self-proteins as partial agonists and antagonists of human CD8+ T-cell clones reactive to melanoma/melanocyte epitope MART1(27–35). Cancer Res. 58, 2433–2439. Lu, J., and Celis, E. (2000). Use of two predictive algorithms of the world wide web for the identiﬁcation of tumor-reactive T-cell epitopes. Cancer Res. 60, 5223–5227. Luescher, I. F., Vivier, E., Layer, A., Mahiou, J., Godeau, F., Malissen, B., and Romero, P. (1995). CD8 modulation of T-cell antigen receptor–ligand interactions on living cytotoxic T lymphocytes. Nature 373, 353–356. Maeurer, M., Hohn, H., Castelli, C., Salter, R., Necker, A., Reichert, T., Knuth, A., and Jager, E. (2001). Antigen recognition by T cells: A strong sense of structure. Trends Immunol. 22, 599–601. Maeurer, M., Necker, A., Salter, R., Castelli, C., Hoehn, H., Karbach, J., Freitag, K., Neukirch, C., Knuth, A., and Jager, E. (2002). Improved detection of melanoma antigenspeciﬁc T-cells expressing low or high levels of CD8 by HLA-A2 tetramers presenting a Melan-A/MART-1 peptide analogue. Int. J. Cancer 97, 64–71. Maeurer, M. J., Martin, D., Walter, W., Liu, K., Zitvogel, L., Halusczcak, K., Rabinowich, H., Duquesnoy, R., Storkus, W., and Lotze, M. T. (1996). Human intestinal Vdelta1+ lymphocytes recognize tumor cells of epithelial origin. J. Exp. Med. 183, 1681–1696. Maini, M. K., Casorati, G., Dellabona, P., Wack, A., and Beverley, P. C. (1999). T-cell clonality in immune responses. Immunol. Today 20, 262–266. Manici, S., Sturniolo, T., Imro, M. A., Hammer, J., Sinigaglia, F., Noppen, C., Spagnoli, G., Mazzi, B., Bellone, M., Dellabona, P., and Protti, M. P. (1999). Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11. J. Exp. Med. 189, 871–876. Marincola, F. M., Jaffee, E. M., Hicklin, D. J., and Ferrone, S. (2000). Escape of human solid tumors from T-cell recognition: Molecular mechanisms and functional signiﬁcance. Adv. Immunol. 74, 181–273. Marrack, P., Bender, J., Hildeman, D., Jordan, M., Mitchell, T., Murakami, M., Sakamoto, A., Schaefer, B. C., Swanson, B., and Kappler, J. (2000). Homeostasis of alpha beta TCR+ T cells. Nat. Immunol. 1, 107–111.

186

Castelli and Maeurer

Marzo, A. L., Kinnear, B. F., Lake, R. A., Frelinger, J. J., Collins, E. J., Robinson, B. W., and Scott, B. (2000). Tumor-speciﬁc CD4+ T cells have a major “post-licensing” role in CTL mediated anti-tumor immunity. J. Immunol. 165, 6047–6055. Mason, D. (1998). A very high level of crossreactivity is an essential feature of the T-cell receptor. Immunol. Today 19, 395–404. Matsuda, J. L., Naidenko, O. V., Gapin, L., Nakayama, T., Taniguchi, M., Wang, C. R., Koezuka, Y., and Kronenberg, M. (2000). Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J. Exp. Med. 192, 741–754. McCloskey, T. W., Haridas, V., Pahwa, R., and Pahwa, S. (2001). Human immunodeﬁciency virus gag and pol-speciﬁc CD8 T cells in perinatal HIV infection. Cytometry 46, 265–270. McHeyzer-Williams, M. G., and Davis, M. M. (1995). Antigen-speciﬁc development of primary and memory T cells in vivo. Science 268, 106–111. McMichael, A. J., and O’Callaghan, C. A. (1998). A new look at T cells. J. Exp. Med. 187, 1367–1371. Meadows, L., Wang, W., den Haan, J. M., Blokland, E., Reinhardus, C., Drijfhout, J. W., Shabanowitz, J., Pierce, R., Agulnik, A. I., Bishop, C. E., Hunt, D. F., Goulmy, E., and Engelhard, V. H. (1997). The HLA-A∗ 0201-restricted H-Y antigen contains a posttranslationally modiﬁed cysteine that signiﬁcantly affects T cell recognition. Immunity 6, 273– 281. Metelitsa, L. S., Naidenko, O. V., Kant, A., Wu, H. W., Loza, M. J., Perussia, B., Kronenberg, M., and Seeger, R. C. (2001). Human NKT cells mediate antitumor cytotoxicity directly by rcognizing target cell CD1d with bound ligand or indirectly by producing IL-2 to activate NK cells. J. Immunol. 167, 3114–3122. Mohagheghpour, N., Gammon, D., Kawamura, L. M., van Vollenhoven, A., Benike, C. J., and Engleman, E. G. (1998). CTL response to Mycobacterium tuberculosis: Identiﬁcation of an immunogenic epitope in the 19-kDa lipoprotein. J. Immunol. 161, 2400–2406. Moodycliffe, A. M., Nghiem, D., Clydesdale, G., and Ullrich, S. E. (2000). Immune suppression and skin cancer development: Regulation by NKT cells. Nat. Immunol. 1, 521–525. Morita, M., Motoki, K., Akimoto, K., Natori, T., Sakai, T., Sawa, E., Yamaji, K., Koezuka, Y., Kobayashi, E., and Fukushima, H. (1995). Structure-activity relationship of alphagalactosylceramides against B16-bearing mice. J. Med. Chem. 38, 2176–2187. Moss, P. A., Rowland-Jones, S. L., Frodsham, P. M., McAdam, S., Giangrande, P., McMichael, A. J., and Bell, J. I. (1995). Persistent high frequency of human immunodeﬁciency virusspeciﬁc cytotoxic T cells in peripheral blood of infected donors. Proc. Natl. Acad. Sci. USA 92, 5773–5777. Mothe, B. R., Horton, H., Carter, D. K., Allen, T. M., Liebl, M. E., Skinner, P., Vogel, T. U., Fuenger, S., Vielhuber, K., Rehrauer, W., Wilson, N., Franchini, G., Altman, J. D., Haase, A., Picker, L. J., Allison, D. B., and Watkins, D. I. (2002). Dominance of CD8 responses speciﬁc for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection. J. Virol. 76, 875–884. Nakagawa, R., Motoki, K., Ueno, H., Iijima, R., Nakamura, H., Kobayashi, E., Shimosaka, A., and Koezuka, Y. (1998). Treatment of hepatic metastasis of the colon26 adenocarcinoma with an alpha-galactosylceramide, KRN7000. Cancer Res. 58, 1202–1207. Nakagawa, R., Serizawa, I., Motoki, K., Sato, M., Ueno, H., Iijima, R., Nakamura, H., Shimosaka, A., and Koezuka, Y. (2000). Antitumor activity of alpha-galactosylceramide, KRN7000, in mice with the melanoma B16 hepatic metastasis and immunohistological study of tumor inﬁltrating cells. Oncol. Res. 12, 51–58. Nanda, N. K., and Sercarz, E. E. (1995). Induction of anti-self-immunity to cure cancer. Cell 82, 13–17. Nestle, F. O., Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer, R., Burg, G., and Schadendorf, D. (1998). Vaccination of melanoma patients with peptide- or tumor lysatepulsed dendritic cells. Nat. Med. 4, 328–332.

T-Cell Response in Cancer Patients

187

Nicol, A., Nieda, M., Koezuka, Y., Porcelli, S., Suzuki, K., Tadokoro, K., Durrant, S., and Juji, T. (2000). Human invariant valpha24+ natural killer T cells activated by alpha- galactosylceramide (KRN7000) have cytotoxic anti-tumour activity through mechanisms distinct from T cells and natural killer cells. Immunology 99, 229–234. Nijman, H. W., Van der Burg, S. H., Vierboom, M. P., Houbiers, J. G., Kast, W. M., and Melief, C. J. (1994). p53, a potential target for tumor-directed T cells. Immunol. Lett. 40, 171–178. Nossal, G. J. (2001). A purgative mastery. Nature 412, 685–686. Novak, E. J., Liu, A. W., Gebe, J. A., Falk, B. A., Nepom, G. T., Koelle, D. M., and Kwok, W. W. (2001a). Tetramer-guided epitope mapping: rapid identiﬁcation and characterization of immunodominant CD+ T cell epitopes from complex antigens. J. Immunol. 166, 6665–6670. Novak, E. J., Masewicz, S. A., Liu, A. W., Lernmark, A., Kwok, W. W., and Nepom, G. T. (2001b). Activated human epitope-speciﬁc T cells identiﬁed by class II tetramers reside within a CD4high, proliferating subset. Int. Immunol. 13, 799–806. Nugent, C. T., Morgan, D. J., Biggs, J. A., Ko, A., Pilip, I. M., Pamer, E. G., and Sherman, L. A. (2000). Characterization of CD8+ T lymphocytes that persist after peripheral tolerance to a self antigen expressed in the pancreas. J. Immunol. 164, 191–200. Oelke, M., Kurokawa, T., Hentrich, I., Behringer, D., Cerundolo, V., Lindemann, A., and Mackensen, A. (2000). Functional characterization of CD8(+) antigen-speciﬁc cytotoxic T lymphocytes after enrichment based on cytokine secretion: Comparison with the MHCtetramer technology. Scand. J. Immunol. 52, 544–549. Ogg, G. S., Rod Dunbar, P., Romero, P., Chen, J. L., and Cerundolo, V. (1998). High frequency of skin-homing melanocyte-speciﬁc cytotoxic T lymphocytes in autoimmune vitiligo. J. Exp. Med. 188, 1203–1208. Orsini, D. L., van Gils, M., Kooy, Y. M., Struyk, L., Klein, G., van den Elsen, P., and Koning, F. (1994). Functional and molecular characterization of B cell-responsive V delta 1+ gamma delta T cells. Eur. J. Immunol. 24, 3199–3204. Palermo, B., Campanelli, R., Mantovani, S., Lantelme, E., Manganoni, A. M., Carella, G., Da Prada, G., della Cuna, G. R., Romagne, F., Gauthier, L., Necker, A., and Giachino, C. (2001). Diverse expansion potential and heterogeneous avidity in tumor-associated antigen-speciﬁc T lymphocytes from primary melanoma patients. Eur. J. Immunol. 31, 412–420. Pannetier, C., Even, J., and Kourilsky, P. (1995). T-cell repertoire diversity and clonal expansions in normal and clinical samples. Immunol. Today 16, 176–181. Pardoll, D. M. (1998). Cancer vaccines. Nat. Med. 4, 525–531. Pardoll, D. M. (2001). Immunology. Stress, NK receptors, and immune surveillance. Science 294, 534–536. Pardoll, D. M., and Topalian, S. L. (1998). The role of CD4+ T cell responses in antitumor immunity. Curr. Opin. Immunol. 10, 588–594. Parker, K. C., Bednarek, M. A., and Coligan, J. E. (1994). Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 152, 163–175. Parkhurst, M. R., Salgaller, M. L., Southwood, S., Robbins, P. F., Sette, A., Rosenberg, S. A., and Kawakami, Y. (1996). Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modiﬁed at HLA-A∗ 0201-binding residues. J. Immunol. 157, 2539–2548. Pieper, R., Christian, R. E., Gonzales, M. I., Nishimura, M. I., Gupta, G., Settlage, R. E., Shabanowitz, J., Rosenberg, S. A., Hunt, D. F., and Topalian, S. L. (1999). Biochemical identiﬁcation of a mutated human melanoma antigen recognized by CD4(+) T cells. J. Exp. Med. 189, 757–766. Pilch, H., Hohn, H., Freitag, K., Neukirch, C., Necker, A., Haddad, P., Tanner, B., Knapstein, P., and Maeurer, M. J. (2002a). Improved assessment of the TCR VB-repertoire in clinical specimens: Combination of TCR-CDR3 spectratyping with ﬂow cytometry based TCR VB-frequency analysis. Clin. Diagn. Lab. Immunol. 9, 257–266.

188

Castelli and Maeurer

Pilch, H., Hohn, H., Neukirch, C., Freitag, K., Knapstein, P., Tanner, B., and Maeurer, M. J. (2002b). Evidence for antigen-driven T-cell selection in patients with cervical cancer deﬁned by T-cell receptor analysis and recognition of autologous tumor. Clin. Diagn. Lab. Immunol. 9, 267–278. Pittet, M. J., Valmori, D., Dunbar, P. R., Speiser, D. E., Lienard, D., Lejeune, F., Fleischhauer, K., Cerundolo, V., Cerottini, J. C., and Romero, P. (1999). High frequencies of naive Melan-A/MART-1-speciﬁc CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J. Exp. Med. 190, 705–715. Poccia, F., Cipriani, B., Vendetti, S., Colizzi, V., Poquet, Y., Battistini, L., Lopez-Botet, M., Fournie, J. J., and Gougeon, M. L. (1997). CD94/NKG2 inhibitory receptor complex modulates both anti-viral and anti-tumoral responses of polyclonal phosphoantigen-reactive V gamma 9V delta 2 T lymphocytes. J. Immunol. 159, 6009–6017. Purbhoo, M. A., Boulter, J. M., Price, D. A., Vuidepot, A. L., Hourigan, C. S., Dunbar, P. R., Olson, K., Dawson, S. J., Phillips, R. E., Jakobsen, B. K., Bell, J. I., and Sewell, A. K. (2001). The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain. J. Biol. Chem. 276, 32,786–32,792. Ras, E., van der Burg, S. H., Zegveld, S. T., Brandt, R. M., Kuppen, P. J., Offringa, R., Warnarr, S. O., van de Velde, C. J., and Melief, C. J. (1997). Identiﬁcation of potential HLA-A ∗ 0201 restricted CTL epitopes derived from the epithelial cell adhesion molecule (Ep-CAM) and the carcinoembryonic antigen (CEA). Hum. Immunol. 53, 81–89. Reichstetter, S., Ettinger, R. A., Liu, A. W., Gebe, J. A., Nepom, G. T., and Kwok, W. W. (2000). Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR afﬁnities in the human antigen-speciﬁc T cell response. J. Immunol. 165, 6994–6998. Renard, V., Romero, P., Vivier, E., Malissen, B., and Luescher, I. F. (1996). CD8 beta increases CD8 coreceptor function and participation in TCR-ligand binding. J. Exp. Med. 184, 2439– 2444. Riberdy, J. M., Zirkel, A., Surman, S., Hurwitz, J. L., and Doherty, P. C. (2001). Cutting edge: Culture with high doses of viral peptide induces previously unprimed CD8(+) T cells to produce cytokine. J. Immunol. 167, 2437–2440. Rivoltini, L., Squarcina, P., Loftus, D. J., Castelli, C., Tarsini, P., Mazzocchi, A., Rini, F., Viggiano, V., Belli, F., and Parmiani, G. (1999). A superagonist variant of peptide MART1/Melan A27–35 elicits anti-melanoma CD8+ T cells with enhanced functional characteristics: Implication for more effective immunotherapy. Cancer Res. 59, 301–306. Robbins, P. F., El-Gamil, M., Li, Y. F., Kawakami, Y., Loftus, D., Appella, E., and Rosenberg, S. A. (1996). A mutated beta-catenin gene encodes a melanoma-speciﬁc antigen recognized by tumor inﬁltrating lymphocytes. J. Exp. Med. 183, 1185–1192. Robbins, P. F., el-Gamil, M., Li, Y. F., Topalian, S. L., Rivoltini, L., Sakaguchi, K., Appella, E., Kawakami, Y., and Rosenberg, S. A. (1995). Cloning of a new gene encoding an antigen recognized by melanoma-speciﬁc HLA-A24-restricted tumor-inﬁltrating lymphocytes. J. Immunol. 154, 5944–5950. Robinson, H. L., Monteﬁori, D. C., Johnson, R. P., Manson, K. H., Kalish, M. L., Lifson, J. D., Rizvi, T. A., Lu, S., Hu, S. L., Mazzara, G. P., Panicali, D. L., Herndon, J. G., Glickman, R., Candido, M. A., Lydy, S. L., Wyand, M. S., and McClure, H. M. (1999). Neutralizing antibody-independent containment of immunodeﬁciency virus challenges by DNA priming and recombinant pox virus booster immunizations. Nat. Med. 5, 526–534. Roos, M. T., van Lier, R. A., Hamann, D., Knol, G. J., Verhoofstad, I., van Baarle, D., Miedema, F., and Schellekens, P. T. (2000). Changes in the composition of circulating CD8+ T cell subsets during acute epstein-barr and human immunodeﬁciency virus infections in humans. J. Infect. Dis. 182, 451–458. Rosenberg, S. A. (1999). A new era for cancer immunotherapy based on the genes that encode cancer antigens. Immunity 10, 281–287.

T-Cell Response in Cancer Patients

189

Rosenberg, S. A., Yang, J. C., Schwartzentruber, D. J., Hwu, P., Marincola, F. M., Topalian, S. L., Restifo, N. P., Dudley, M. E., Schwarz, S. L., Spiess, P. J., Wunderlich, J. R., Parkhurst, M. R., Kawakami, Y., Seipp, C. A., Einhorn, J. H., and White, D. E. (1998). Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat. Med. 4, 321–327. Salamone, M. C., Rabinovich, G. A., Mendiguren, A. K., Salamone, G. V., and Fainboim, L. (2001). Activation-induced expression of CD1d antigen on mature T cells. J. Leukoc. Biol. 69, 207–214. Salazar, E., Zaremba, S., Arlen, P. M., Tsang, K. Y., and Schlom, J. (2000). Agonist peptide from a cytotoxic T-lymphocyte epitope of human carcinoembryonic antigen stimulates production of tc1-type cytokines and increases tyrosine phosphorylation more efﬁciently than cognate peptide. Int. J. Cancer 85, 829–838. Salio, M., Shepherd, D., Dunbar, P. R., Palmowski, M., Murphy, K., Wu, L., and Cerundolo, V. (2001). Mature dendritic cells prime functionally superior melan-A-speciﬁc CD8+ lymphocytes as compared with nonprofessional APC. J. Immunol. 167, 1188–1197. Salzmann, M., and Bachmann, M. F. (1998). Estimation of maximal afﬁnities between T-cell receptors and MHC/peptide complexes. Mol. Immunol. 35, 65–71. Savage, P. A., Boniface, J. J., and Davis, M. M. (1999). A kinetic basis for T cell receptor repertoire selection during an immune response. Immunity 10, 485–492. Schirle, M., Keilholz, W., Weber, B., Gouttefangeas, C., Dumrese, T., Becker, H. D., Stevanovic, S., and Rammensee, H. G. (2000). Identiﬁcation of tumor-associated MHC class I ligands by a novel T cell-independent approach. Eur. J. Immunol. 30, 2216–2225. Schmitz, J. E., Veazey, R. S., Kuroda, M. J., Levy, D. B., Seth, A., Mansﬁeld, K. G., Nickerson, C. E., Lifton, M. A., Alvarez, X., Lackner, A. A., and Letvin, N. L. (2001). Simian immunodeﬁciency virus (SIV)-speciﬁc cytotoxic T lymphocytes in gastrointestinal tissues of chronically SIV-infected rhesus monkeys. Blood 98, 3757–3761. Schneider, J., Gilbert, S. C., Blanchard, T. J., Hanke, T., Robson, K. J., Hannan, C. M., Becker, M., Sinden, R., Smith, G. L., and Hill, A. V. (1998). Enhanced immunogenicity for CD8+ T cell induction and complete protective efﬁcacy of malaria DNA vaccination by boosting with modiﬁed vaccinia virus Ankara. Nat. Med. 4, 397–402. Schoﬁeld, L., McConville, M. J., Hansen, D., Campbell, A. S., Fraser-Reid, B., Grusby, M. J., and Tachado, S. D. (1999). CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells. Science 283, 225–229. Schultz, E. S., Lethe, B., Cambiaso, C. L., Van Snick, J., Chaux, P., Corthals, J., Heirman, C., Thielemans, K., Boon, T., and van der Bruggen, P. (2000). A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes. Cancer Res. 60, 6272–6275. Schultze, J. L., and Vonderheide, R. H. (2001). From cancer genomics to cancer immunotherapy: Toward second-generation tumor antigens. Trends Immunol. 22, 516–523. Shimonkevitz, R., Colburn, C., Burnham, J. A., Murray, R. S., and Kotzin, B. L. (1993). Clonal expansions of activated gamma/delta T cells in recent-onset multiple sclerosis. Proc. Natl. Acad. Sci. USA 90, 923–927. Shipp, M. A., Ross, K. N., Tamayo, P., Weng, A. P., Kutok, J. L., Aguiar, R. C., Gaasenbeek, M., Angelo, M., Reich, M., Pinkus, G. S., Ray, T. S., Koval, M. A., Last, K. W., Norton, A., Lister, T. A., Mesirov, J., Neuberg, D. S., Lander, E. S., Aster, J. C., and Golub, T. R. (2002). Diffuse large B-cell lymphoma outcome prediction by gene-expression proﬁling and supervised machine learning. Nat. Med. 8, 68–74. Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., and Haase, A. T. (2000). Cutting edge: In situ tetramer staining of antigen-speciﬁc T cells in tissues. J. Immunol. 165, 613–617. Skipper, J. C., Hendrickson, R. C., Gulden, P. H., Brichard, V., Van Pel, A., Chen, Y., Shabanowitz, J., Wolfel, T., Slingluff, C. L., Jr., Boon, T., Hunt, D. F., and Engelhard, V. H. (1996). An HLA-A2-restricted tyrosinase antigen on melanoma cells results from

190

Castelli and Maeurer

posttranslational modiﬁcation and suggests a novel pathway for processing of membrane proteins. J. Exp. Med. 183, 527–534. Slansky, J. E., Rattis, F. M., Boyd, L. F., Fahmy, T., Jaffee, E. M., Schneck, J. P., Margulies, D. H., and Pardoll, D. M. (2000). Enhanced antigen-speciﬁc antitumor immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex. Immunity 13, 529–538. Slifka, M. K., and Whitton, J. L. (2001). Functional avidity maturation of CD8(+) T cells without selection of higher afﬁnity TCR. Nat. Immunol. 2, 711–717. Smyth, M. J., and Godfrey, D. I. (2000). NKT cells and tumor immunity—a double-edged sword. Nat. Immunol. 1, 459–460. Spada, F. M., Borriello, F., Sugita, M., Watts, G. F., Koezuka, Y., and Porcelli, S. A. (2000). Low expression level but potent antigen presenting function of CD1d on monocyte lineage cells. Eur. J. Immunol. 30, 3468–3477. Spada, F. M., Koezuka, Y., and Porcelli, S. A. (1998). CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells. J. Exp. Med. 188, 1529–1534. Staveley-O’Carroll, K., Sotomayor, E., Montgomery, J., Borrello, I., Hwang, L., Fein, S., Pardoll, D., and Levitsky, H. (1998). Induction of antigen-speciﬁc T cell anergy: An early event in the course of tumor progression. Proc. Natl. Acad. Sci. USA 95, 1178–1183. Stevceva, L., Kelsall, B., Nacsa, J., Moniuszko, M., Hel, Z., Tryniszewska, E., and Franchini, G. (2002). Cervicovaginal lamina propria lymphocytes: Phenotypic characterization and their importance in cytotoxic T-lymphocyte responses to simian immunodeﬁciency virus SIVmac251. J. Virol. 76, 9–18. Sykulev, Y., Cohen, R. J., and Eisen, H. N. (1995). The law of mass action governs antigenstimulated cytolytic activity of CD8+ cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA 92, 11,990–11,992. Takahashi, K., Shichijo, S., Noguchi, M., Hirohata, M., and Itoh, K. (1995). Identiﬁcation of MAGE-1 and MAGE-4 proteins in spermatogonia and primary spermatocytes of testis. Cancer Res. 55, 3478–3482. Takahashi, T., Nieda, M., Koezuka, Y., Nicol, A., Porcelli, S. A., Ishikawa, Y., Tadokoro, K., Hirai, H., and Juji, T. (2000). Analysis of human V alpha 24+ CD4+ NKT cells activated by alpha-glycosylceramide-pulsed monocyte-derived dendritic cells. J. Immunol. 164, 4458– 4464. Tanaka, Y., Morita, C. T., Nieves, E., Brenner, M. B., and Bloom, B. R. (1995). Natural and synthetic non-peptide antigens recognized by human gamma delta T cells. Nature 375, 155–158. Tangri, S., Ishioka, G. Y., Huang, X., Sidney, J., Southwood, S., Fikes, J., and Sette, A. (2001). Structural features of peptide analogs of human histocompatibility leukocyte antigen class I epitopes that are more potent and immunogenic than wild-type peptide. J. Exp. Med. 194, 833–846. Terabe, M., Matsui, S., Noben-Trauth, N., Chen, H., Watson, C., Donaldson, D. D., Carbone, D. P., Paul, W. E., and Berzofsky, J. A. (2000). NKT cell-mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nat. Immunol. 1, 515– 520. Thomas, M. L., Badwe, R. A., Deshpande, R. K., Samant, U. C., and Chiplunkar, S. V. (2001). Role of adhesion molecules in recruitment of Vdelta1 T cells from the peripheral blood to the tumor tissue of esophageal cancer patients. Cancer Immunol. Immunother. 50, 218– 225. Tillmann, H. L., Heiken, H., Knapik-Botor, A., Heringlake, S., Ockenga, J., Wilber, J. C., Goergen, B., Detmer, J., McMorrow, M., Stoll, M., Schmidt, R. E., and Manns, M. P. (2001). Infection with GB virus C and reduced mortality among HIV-infected patients. N. Engl. J. Med. 345, 715–724. Topalian, S. L. (1994). MHC class II restricted tumor antigens and the role of CD4+ T cells in cancer immunotherapy. Curr. Opin. Immunol. 6, 741–745.

T-Cell Response in Cancer Patients

191

Topalian, S. L., Gonzales, M. I., Parkhurst, M., Li, Y. F., Southwood, S., Sette, A., Rosenberg, S. A., and Robbins, P. F. (1996). Melanoma-speciﬁc CD4+ T cells recognize nonmutated HLA-DR-restricted tyrosinase epitopes. J. Exp. Med. 183, 1965–1971. Topalian, S. L., Rivoltini, L., Mancini, M., Markus, N. R., Robbins, P. F., Kawakami, Y., and Rosenberg, S. A. (1994). Human CD4+ T cells speciﬁcally recognize a shared melanomaassociated antigen encoded by the tyrosinase gene. Proc. Natl. Acad. Sci. USA 91, 9461– 9465. Toura, I., Kawano, T., Akutsu, Y., Nakayama, T., Ochiai, T., and Taniguchi, M. (1999). Cutting edge: Inhibition of experimental tumor metastasis by dendritic cells pulsed with alphagalactosylceramide. J. Immunol. 163, 2387–2391. Trejdosiewicz, L. K., Calabrese, A., Smart, C. J., Oakes, D. J., Howdle, P. D., Crabtree, J. E., Losowsky, M. S., Lancaster, F., and Boylston, A. W. (1991). Gamma delta T cell receptorpositive cells of the human gastrointestinal mucosa: occurrence and V region gene expression in Helicobacter pylori-associated gastritis, coeliac disease and inﬂammatory bowel disease. Clin. Exp. Immunol. 84, 440–444. Triebel, F., and Hercend, T. (1989). Subpopulations of human peripheral T gamma delta lymphocytes. Immunol. Today 10, 186–188. Trojan, A., Witzens, M., Schultze, J. L., Vonderheide, R. H., Harig, S., Krackhardt, A. M., Stahel, R. A., and Gribben, J. G. (2001). Generation of cytotoxic T lymphocytes against native and altered peptides of human leukocyte antigen-A∗ 0201 restricted epitopes from the human epithelial cell adhesion molecule. Cancer Res. 61, 4761–4765. Uyemura, K., Ho, C. T., Ohmen, J. D., Rea, T. H., and Modlin, R. L. (1992). Selective expansion of V delta 1 + T cells from leprosy skin lesions. J. Invest. Dermatol. 99, 848–852. Valitutti, S., and Lanzavecchia, A. (1997). Serial triggering of TCRs: A basis for the sensitivity and speciﬁcity of antigen recognition. Immunol. Today 18, 299–304. Valmori, D., Dutoit, V., Lienard, D., Lejeune, F., Speiser, D., Rimoldi, D., Cerundolo, V., Dietrich, P. Y., Cerottini, J. C., and Romero, P. (2000a). Tetramer-guided analysis of TCR beta-chain usage reveals a large repertoire of melan-A-speciﬁc CD8+ T cells in melanoma patients. J. Immunol. 165, 533–538. Valmori, D., Dutoit, V., Lienard, D., Rimoldi, D., Pittet, M. J., Champagne, P., Ellefsen, K., Sahin, U., Speiser, D., Lejeune, F., Cerottini, J. C., and Romero, P. (2000b). Naturally occurring human lymphocyte antigen-A2 restricted CD8+ T-cell response to the cancer testis antigen NY-ESO-1 in melanoma patients. Cancer Res. 60, 4499–4506. Valmori, D., Fonteneau, J. F., Lizana, C. M., Gervois, N., Lienard, D., Rimoldi, D., Jongeneel, V., Jotereau, F., Cerottini, J. C., and Romero, P. (1998). Enhanced generation of speciﬁc tumorreactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues. J. Immunol. 160, 1750–1758. Valmori, D., Gileadi, U., Servis, C., Dunbar, P. R., Cerottini, J. C., Romero, P., Cerundolo, V., and Levy, F. (1999a). Modulation of proteasomal activity required for the generation of a cytotoxic T lymphocyte-deﬁned peptide derived from the tumor antigen MAGE-3. J. Exp. Med. 189, 895–906. Valmori, D., Pittet, M. J., Vonarbourg, C., Rimoldi, D., Lienard, D., Speiser, D., Dunbar, R., Cerundolo, V., Cerottini, J. C., and Romero, P. (1999b). Analysis of the cytolytic T lymphocyte response of melanoma patients to the naturally HLA-A∗ 0201-associated tyrosinase peptide 368-376. Cancer Res. 59, 4050–4055. van Bleek, G. M., and Nathenson, S. G. (1991). The structure of the antigen-binding groove of major histocompatibility complex class I molecules determines speciﬁc selection of selfpeptides. Proc. Natl. Acad. Sci. USA 88, 11,032–11,036. Van Elsas, A., Nijman, H. W., Van der Minne, C. E., Mourer, J. S., Kast, W. M., Melief, C. J., and Schrier, P. I. (1995). Induction and characterization of cytotoxic T-lymphocytes recognizing a mutated p21ras peptide presented by HLA-A∗ 0201. Int. J. Cancer 61, 389–396.

192

Castelli and Maeurer

Van’t Veer, L. J., and De Jong, D. (2002). The microarray way to tailored cancer treatment. Nat. Med. 8, 13–14. Wang, R., Wang-Zhu, Y., Gabaglia, C. R., Kimachi, K., and Grey, H. M. (1999a). The stimulation of low-afﬁnity, nontolerized clones by heteroclitic antigen analogues causes the breaking of tolerance established to an immunodominant T cell epitope. J. Exp. Med. 190, 983– 994. Wang, R. F., Appella, E., Kawakami, Y., Kang, X., and Rosenberg, S. A. (1996). Identiﬁcation of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes. J. Exp. Med. 184, 2207–2216. Wang, R. F., Johnston, S. L., Southwood, S., Sette, A., and Rosenberg, S. A. (1998). Recognition of an antigenic peptide derived from tyrosinase-related protein-2 by CTL in the context of HLA-A31 and -A33. J. Immunol. 160, 890–897. Wang, R. F., Robbins, P. F., Kawakami, Y., Kang, X. Q., and Rosenberg, S. A. (1995). Identiﬁcation of a gene encoding a melanoma tumor antigen recognized by HLA-A31-restricted tumor-inﬁltrating lymphocytes. J. Exp. Med. 181, 799–804. Wang, R. F., Wang, X., Atwood, A. C., Topalian, S. L., and Rosenberg, S. A. (1999b). Cloning genes encoding MHC class II-restricted antigens: Mutated CDC27 as tumor antigen. Science 284, 1351–1354. Wang, R. F., Wang, X., and Rosenberg, S. A. (1999c). Identiﬁcation of a novel major histocompatibility complex class II-restricted tumor antigen resulting from a chromosomal rearrangement recognized by CD4(+) T cells. J. Exp. Med. 189, 1659–1668. Watt, G., Kantipong, P., de Souza, M., Chanbancherd, P., Jongsakul, K., Ruangweerayud, R., Loomis-Price, L. D., Polonis, V., Myint, K. S., Birx, D. L., Brown, A. E., and Krishna, S. (2000). HIV-1 suppression during acute scrub-typhus infection. Lancet 356, 475–479. Weninger, W., Crowley, M. A., Manjunath, N., and von Andrian, U. H. (2001). Migratory properties of naive, effector, and memory CD8(+) T cells. J. Exp. Med. 194, 953–966. Westermann, J., and Pabst, R. (1990). Lymphocyte subsets in the blood: A diagnostic window on the lymphoid system? Immunol. Today 11, 406–410. Whelan, J. A., Dunbar, P. R., Price, D. A., Purbhoo, M. A., Lechner, F., Ogg, G. S., Grifﬁths, G., Phillips, R. E., Cerundolo, V., and Sewell, A. K. (1999). Speciﬁcity of CTL interactions with peptide-MHC class I tetrameric complexes is temperature dependent. J. Immunol. 163, 4342–4348. Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C., Klehmann-Hieb, E., De Plaen, E., Hankeln, T., Meyer zum Buschenfelde, K. H., and Beach, D. (1995). A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 1281– 1284. Xiang, J., Wunschmann, S., Diekema, D. J., Klinzman, D., Patrick, K. D., George, S. L., and Stapleton, J. T. (2001). Effect of coinfection with GB virus C on survival among patients with HIV infection. N. Engl. J. Med. 345, 707–714. Yague, J., Vazquez, J., and Lopez de Castro, J. A. (2000). A post-translational modiﬁcation of nuclear proteins, N(G), N(G)-dimethyl-Arg, found in a natural HLA class I peptide ligand. Protein Sci. 9, 2210–2217. Yang, O. O., Racke, F. K., Nguyen, P. T., Gausling, R., Severino, M. E., Horton, H. F., Byrne, M. C., Strominger, J. L., and Wilson, S. B. (2000). CD1d on myeloid dendritic cells stimulates cytokine secretion from and cytolytic activity of V alpha 24J alpha Q T cells: A feedback mechanism for immune regulation. J. Immunol. 165, 3756–3762. Yee, C., Savage, P. A., Lee, P. P., Davis, M. M., and Greenberg, P. D. (1999). Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers. J. Immunol. 162, 2227–2234. Yewdell, J. W., and Bennink, J. R. (2001). Cut and trim: Generating MHC class I peptide ligands. Curr. Opin. Immunol. 13, 13–18.

T-Cell Response in Cancer Patients

193

Zaks, T. Z., and Rosenberg, S. A. (1998). Immunization with a peptide epitope (p369-377) from HER-2/neu leads to peptide-speciﬁc cytotoxic T lymphocytes that fail to recognize HER-2/neu+ tumors. Cancer Res. 58, 4902–4908. Zarling, A. L., Ficarro, S. B., White, F. M., Shabanowitz, J., Hunt, D. F., and Engelhard, V. H. (2000). Phosphorylated peptides are naturally processed and presented by major histocompatibility complex class I molecules in vivo. J. Exp. Med. 192, 1755–1762. Zarour, H. M., Kirkwood, J. M., Kierstead, L. S., Herr, W., Brusic, V., Slingluff, C. L., Jr., Sidney, J., Sette, A., and Storkus, W. J. (2000a). Melan-A/MART-1(51-73) represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4(+) T cells. Proc. Natl. Acad. Sci. USA 97, 400–405. Zarour, H. M., Storkus, W. J., Brusic, V., Williams, E., and Kirkwood, J. M. (2000b). NY-ESO-1 encodes DRB1∗ 0401-restricted epitopes recognized by melanoma-reactive CD4+ T cells. Cancer Res. 60, 4946–4952. Zeng, G., Touloukian, C. E., Wang, X., Restifo, N. P., Rosenberg, S. A., and Wang, R. F. (2000). Identiﬁcation of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules. J. Immunol. 165, 1153–1159. Zocchi, M. R., Ferrarini, M., and Rugarli, C. (1990). Selective lysis of the autologous tumor by delta TCS1+ gamma/delta+ tumor-inﬁltrating lymphocytes from human lung carcinomas. Eur. J. Immunol. 20, 2685–2689.

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The Life and Death of a B Cell Thierry Defrance,1,* Montserrat Casamayor-Palleja, ` 1 and Peter H. Krammer 2 1

INSERM U404, “Immunity and Vaccination,” 69365, Lyon, Cedex 07, France,2Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany

I. Introduction II. The Maintenance of B Cell Tolerance A. B Cell Lymphopoiesis B. Mechanisms of Negative Selection C. Biochemistry of BCR-Induced Apoptotic Signals III. The Regulation of B Cell Homeostasis A. In Primary Lymphoid Organs B. In the Periphery IV. Control of the Speciﬁcity and Afﬁnity of the Ab Response A. At the Onset of the Immune Response B. During Affinity-Maturation of the Ab Response V. Regulation of Survival in the Memory B Cell Compartment A. The Memory B Cell Pool B. The Plasma Cell Pool C. The Molecular Regulators of PC Survival VI. Conclusive Remarks References

Regulation of apoptosis in the B cell lineage has implications for homeostasis, quality control of the antibody response, and tolerance. In this chapter we examine the different checkpoints that control life and death decisions of B cells during the antigenindependent and antigen-dependent phases of their development. We discuss the cell death mechanism involved in elimination of unwanted B cells at different stages of their development as well as the signals that trigger or repress the apoptotic process. At the steady state, before or after development of an immune response, B cell apoptosis ensures that the antigen receptor (BCR) on newly produced B cells is functional and does not recognize self-antigens with high avidity. It also ensures that the size of the peripheral B cell compartment remains constant in spite of the continuous input of B cells from the bone marrow. All these processes are controlled by the mitochondrial death pathway and are thus perturbed by overexpression of the antiapoptotic members of the bcl-2 gene family. By contrast, the death receptor pathway plays a prominent role during ∗ To whom correspondence should be addressed at INSERM U404, “Immunity and Vaccination,” 21 Avenue Tony Garnier, 69365, Lyon, Cedex 07, France.

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Copyright 2002, Elsevier Science (USA). All rights reserved.

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the antigen-dependent phase of B cell development. Three sets of membrane molecules stand as crucial regulators of B cell survival. First, the BCR which plays a central but ambiguous role. On the one hand, it triggers death of B cells that recognize self-antigens or have been exposed to repeated antigenic stimulations. On the other hand, it promotes survival of the peripheral mature B cell pool and protects activated B cells from CD95induced killing. Second, the death receptor Fas/CD95 which is instrumental in censoring B cells activated in a bystander fashion at the initiation of the response to T-dependent antigens. It also drives elimination of low-afﬁnity and self-reactive B cell clones that arise through the process of somatic mutations during the germinal center reaction. As such, it contributes to the afﬁnity maturation of the antibody response. Finally, three membrane receptors (TACI, BCMA, and BAFF-R) which bind a newly discovered member of the tumor necrosis factor family named BAFF. BAFF acts speciﬁcally on peripheral B cells but its cellular targets seem to be restricted to two splenic B cell populations: (i) transitional immature B cells and (ii) marginal zone B cells, known to be responsible for the response to thymus-independent type 2 antigens. This suggests its possible implication in positive selection of peripheral B cells and in the antibacterial B cell responses. C

2002, Elsevier Science (USA).

I. INTRODUCTION Apoptosis intervenes at different stages of natural B cell development. It fulﬁlls three major functions, each of which will be covered in a distinct section of the present review. First, it is one of the molecular mechanisms that preserves B cell tolerance during both the antigen (Ag)-independent (lymphopoiesis) and Ag-dependent (immunopoiesis) phases of B cell development. Second, it preserves homeostasis of the B cell compartment and avoids dysregulated expansion of B cells that might lead to neoplasia. Finally, it contributes to improving the antibody (Ab) response by eliminating irrelevant B cell clones that might have received misdirected T cell help and by counterselecting low-afﬁnity mutant B cell clones that arise during Ag-driven diversiﬁcation of the B cell repertoire in the germinal center (GC). The last section is devoted to the regulation of survival in memory B cells and plasma cells (PC) which constitute the two arms of B cell memory. This chapter is justiﬁed by the emerging concept that persistance of B cell memory could rely upon the maintenance of a pool of long-lived cells located in a deﬁned microenvironment of the lymphoid tissue. The biochemical mechanisms of apoptosis have been extensively documented in other reviews and will not be described here in detail. We rather overview the key control points that determine life and death decisions of B cells during their development and in the course of the immune response. Emphasis will be put on the extrinsic and intrinsic molecular signals which induce or repress cell death at these different developmental stages.

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II. THE MAINTENANCE OF B CELL TOLERANCE A. B Cell Lymphopoiesis Essentially, B cell lymphopoiesis takes place in the liver in the embryo and in the bone marrow (BM) in adults. This process allows for the somatic diversiﬁcation of the B cell repertoire and production of immunocompetent B cells while preserving self-tolerance. At the molecular level, the diversity of the immunoglobulin (Ig) repertoire is generated by the ordered rearrangement of the gene segments of the Ig heavy (V, D, J) and light (V, J) chain loci (Tonegawa, 1983). Rearrangement of the heavy (H) chain locus precedes rearrangement of the light (L) chain locus. Early B lymphocyte development is traditionally divided into three major stages. Commitment of the pluripotent progenitors to the B cell lineage starts at the pro-B cell stage when the ﬁrst D-J rearrangement occurs (Fig. 1; see color insert). Rearrangement of the μ H chain is completed by the V-DJ recombination at the pre-B cell stage. If the rearrangement of the IgH locus is successful, a complete μ chain is exported to the membrane and associates with the pseudo-light chain (L) and the transmembrane molecules Igα and Igβ to form an Ig-like signaling complex. Rearrangement of the L chain begins at this stage, ﬁrst at the κ locus and then at the λ locus. A successful rearrangement of either one or the other of the L chain isotypes will yield an immature B lymphocyte characterized by the surface expression of a complete IgM molecule (see Melchers et al., 2000 for review). The transition from immature (IgM only) to mature (IgM and IgD) B cells takes place in the spleen. Studies of cell population dynamics have led to the conclusion that the precursor B cell population undergoes a dramatic cell loss in the course of early B cell development (Osmond et al., 1994). There are two phases of enhanced apoptosis during B cell lymphopoiesis (Lu and Osmond, 1997). The ﬁrst one takes place after completion of the VDJ rearrangement at the pro-B/pre-B transition stage. It reﬂects the elimination of B cell precursors with faulty rearrangement of the H μ chain (or deﬁcient pairing with the L). The second phase occurs at the immature B cell stage and allows for the elimination of both B cells which failed to successfully rearrange the L chain and B cells which express reactivity against self-Ag. One of the primary functions of the B cell receptor (BCR) on pre-B and immature B cells is to drive selection of the B cell repertoire. First, it ensures that only those B cells which have productively rearranged their H and L chain, and for which the H/L chain pairing is correct, escape apoptosis. This process is referred to as positive selection and takes place at both the pre-B and the immature B cell stages. Second, it ensures that immature B cells whose BCR recognizes auto-Ag present in the microenvironment with a sufﬁciently high

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avidity are deleted. This latter process is referred to as negative selection. It constitutes one of the molecular mechanisms which preserve B cell tolerance (see Rolink et al., 2001 for review).

B. Mechanisms of Negative Selection 1. THE MITOCHONDRIAL PATHWAY Negative selection begins in primary lymphoid organs (fetal liver, BM) and is pursued in the spleen. Continuation of the selection process in the periphery permits maintenance of B cell tolerance toward self-Ag that would not be expressed within the microenvironment of the BM. The actual concept is that the strength of the interaction between BCR and self-Ag dictates the outcome of the selection process at the immature B cell stage. High-avidity interactions will cause apoptosis (Norvell et al., 1995) or will drive immature B cells to undergo a secondary VJ rearrangement (receptor editing) at the L chain locus (Nemazee and Buerki, 1989). Low-avidity interactions will lead immature B cells to take the B1 cell development pathway (Lam and Rajewsky, 1999) or to become anergic (Goodnow et al., 1995). Immature B cells with little BCR occupancy are kept alive and migrate to the periphery where they undergo a second selection step in the spleen before giving rise to mature B lymphocytes. The ﬁrst indication that the mitochondrial death pathway contributes to negative selection of the B cell repertoire during lymphopoiesis came from the pattern of expression of bcl-2 and bcl-xL during B cell development. These two prominent antiapoptotic members of the bcl-2 family known to block all apoptogenic activities of the mitochondria are both repressed at the immature B cell stage during which deletion of self-reactive B cells occurs (Grillot et al., 1996; Merino et al., 1994). The capacity of bcl-2 and bcl-xL to impair negative selection was further examined in clonal deletion models in which mice expressing a transgenic BCR directed against a natural or artiﬁcial self-Ag (such as the Hen Egg Lysozyme/HEL) are exposed to this self-Ag during early development. However, these studies led to contradictory ﬁndings. In double transgenic mice that express both HEL and a BCR speciﬁcally recognizing HEL, enforced bcl-2 expression could rescue self-reactive B cells from apoptosis but failed to allow them to differentiate further into mature B cells. (Hartley et al., 1993). By contrast, a bcl-2 transgene did not impair clonal deletion of self-reactive B cells in the BM of mice transgenic for an antierythrocyte Ab (Nisitani et al., 1993). Overexpression of bcl-xL generated more consistent results. In the HEL/anti-HEL model, transgenic bcl-xL expression not only prevented the loss of self-reactive B cells but also released B cells from their maturation block (Fang et al., 1998). Moreover, the bcl-xL

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transgene was consistently found to efﬁciently protect the WEHI-231 cell line from BCR-induced apoptosis (Merino et al., 1995). Altogether, these results are compatible with the notion that clonal deletion of self-reactive B cells occurs through a bcl-xL-inhibitable mitochondrial pathway.

2. THE DEATH RECEPTOR PATHWAY So far, little is known about the pattern of expression of death receptors (DR) on developing B cell precursors. In normal human BM, expression of CD95 (previously known as Fas or APO-1) seems to be restricted to a fraction of immature B cells (Nilsson et al., 2000). Analysis of mouse BM B cell precursors conﬁrmed that CD95 is marginally expressed on pro- and pre-B cells (Li et al., 1996). Experiments conducted on a leukemic mouse pre-B cell line showed that these cells acquire CD95 as they differentiate into immature B cells in vitro (Onel et al., 1995). By breeding the HEL/anti-HEL-BCR double transgenic mice to the congenic lpr/lpr strain, the group of Goodnow demonstrated that deﬁcient expression of CD95 leaves deletion of self-reactive B cells unperturbed (Rathmell and Goodnow, 1994). This observation rules out CD95 as the key player of negative selection. Until now, there has been no information available on the possible expression of the other ﬁve members of the DR subfamily (TNF-R1, TRAIL-R1 and 2, DR3, and DR6) during early B cell development. However, several groups have invalidated the function of the Fas-Associated Death Domain-containing protein (FADD) to examine the possible contribution of other DR to lymphocyte development. FADD has been chosen because it is one of the most receptor-proximal transduction elements common to all DR (Krammer, 2000). Because disruption of the FADD gene in the mouse is lethal perinatally (Yeh et al., 1999), alternative approaches have been used to address this question. One of them consisted of creating chimeric mice in which the FADD disruption was targeted to lymphocytes using a Rag-deﬁcient blastocyst complementation system, that is, injection of FADD − /− embryonic stem cells into Rag1 − /− blastocysts (Zhang et al., 1998). As expected, T cells from such mice were found to be insensitive to CD95-mediated killing. However, these studies surprisingly revealed that FADD − /− T cells also exhibit a severely defective proliferative capacity and that FADD − /− mice completely lack mature B cells in the periphery. Altogether, these ﬁndings support the notion that DR or other effector molecules using FADD as a downstream signaling element exert a stimulatory activity during early T and B lymphocyte development. This hypothesis is further substantiated by recent studies conducted by the group of Strasser showing that thymocyte negative selection is potentiated by expression of a transgene encoding a dominant negative form of FADD (FADD-DN) (Newton et al., 1998). These data suggest that during T cell ontogeny, DR do not trigger clonal deletion but rather antagonize this process. It is not known

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yet whether DR exert a similar function during negative selection of B cells. In any case, the protection afforded by certain antiapoptotic members of the bcl-2 family against deletion of self-reactive immature B cells does not deﬁnitely exclude the implication of DR in the mechanism of central B cell tolerance. As we shall discuss in the following sections, there is evidence that CD95, and possibly other DR, can be connected to a bcl-2-inhibitable pathway of apoptosis.

C. Biochemistry of BCR-Induced Apoptotic Signals A vast literature has been devoted to the molecular mechanism of BCRinduced apoptosis (reviewed by Carey et al., 2000; Healy and Goodnow, 1998), but an extensive description of this process lies outside the scope of the present review. Therefore, in the following section we shall only present the most salient features of the death pathway connected to the BCR. Most studies have relied on either one or the other of the following in vitro models: (1) immature B cell lines such as the murine B lymphoma cell line WEHI-231 and (2) mature neoplastic B cells exempliﬁed by the group I Burkitt lymphoma cell lines. However, the bulk of experimental data indicate that the global features of BCR-induced apoptosis are conserved between immature and mature B cells. Therefore, the biochemical pathways of BCR-induced death that we describe below also apply to AICD of mature B lymphocytes, developed in Section III.B.2. In accordance with the observation that central B cell tolerance can be abolished by overexpression of antiapoptotic members of the bcl-2 family, all studies which dissected the biochemistry of the BCR death pathway concur in identifying the mitochondria as the central executors of BCR-induced apoptosis. In particular, it has been shown that (i) blockade of the apoptosome function by pharmacological agents prevents BCR-induced death, and (ii) alterations of the mitochondrial functions precedes activation of downstream caspases (Berard et al., 1999b; Bouchon et al., 2000). There is general consensus that execution of the apoptotic program downstream of the mitochondria requires caspase activation and that caspases-2, -3, and -9 are involved (Berard et al., 1999b; Bouchon et al., 2000; Chen et al., 1999). On the contrary, the mechanisms whereby the apoptotic signal is transduced from the BCR to the mitochondria are still unclear. The recent study of Besnault et al. (2001) suggests that the BCR can initiate two distinct death pathways upstream of the mitochondria depending on the valency of the surrogate Ag. When the Ag receptor is heavily cross-linked (anti-Ig antibodies plus a cross-linker), transduction of the death signal from the BCR to the mitochondria relies on the sequential activation of caspase-8 and the subsequent cleavage of Bid. However, initiation of caspase-8 cleavage in this model does not require caspase-8 association with FADD since

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it is unaffected by the overexpression of an FADD-DN lacking the death effector domain involved in caspase-8 recruitment (Besnault et al., 2001). When the degree of cross-linking of the BCR is minimal (soluble anti-Ig Abs), connection between the BCR and the mitochondria is strictly caspase independent (Berard et al., 1999b; Bouchon et al., 2000) but requires an inducible noncaspase protein mediator. This latter assumption is based on the observation that an actinomycin D or cycloheximide treatment prevent loss of membrane potential of the mitochondria and the subsequent death of Burkitt’s lymphoma cell lines treated with soluble anti-Ig Abs (Bouchon et al., 2000). In any case, these two BCR death pathways converge at the mitochondrial level and do not involve the DR. This has been convincingly demonstrated by the observation that overexpression of a dominant negative form of FADD fails to prevent apoptosis of Burkitt’s lymphoma cell lines exposed to either divalent (Lens et al., 1998) or multivalent forms of surrogate Ag (Besnault et al., 2001). A model summarizing the biochemical death pathways connected to the BCR is shown in Fig. 2 (see color insert).

III. THE REGULATION OF B CELL HOMEOSTASIS A. In Primary Lymphoid Organs Survival of developing B lymphocytes is dependent on the correct rearrangement of the gene segments of the H and L chain loci and efﬁcient pairing of the H and L chain products. As we mentioned above, only B cells that express a functional BCR are positively selected during early B cell development. The driving force of the positive selection process is the Ag receptor itself, that is, the μ chain associated with the products of the λ5 and V-pre B genes at the pre-B cell stage or the complete IgM molecule at the immature B cell stage. It is not completely clear how B cells sense the presence of a functional BCR. Existence of a putative BCR ligand securing the survival of B cells which have successfully rearranged their V genes has not been convincingly demonstrated so far. Some lines of evidence suggest that positive selection might function in a ligand-independent fashion. This hypothesis is substantiated in particular by the recent work of the group of D. Rawlings (Guo et al., 2000) showing that a substantial fraction of the preBCR in normal B cell progenitors is constitutively associated with lipid rafts, that is, juxtaposed to certain cytoplasmic signaling elements of the BCR such as Lyn. The pre-BCR or other molecules which might segregate and associate with the pre-BCR in lipid rafts (such as Gpi-anchored molecules) might be sufﬁcient to promote survival of pre-B cells with a productive μ chain rearrangement.

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Once again, the phenotype of mice expressing a bcl-2 or bcl-xL transgene argue for the implication of the mitochondrial death pathway in elimination of functionless B cells. For example, expression of a B cell-restricted bcl-xL transgene dramatically perturbs positive selection as reﬂected by the expansion of pro-B cells carrying nonproductive Ig heavy chain gene rearrangements (Grillot et al., 1996). How the apoptotic process is initiated in functionless B cells is presently unknown. A possible role of DR should be examined since integrity of the DR signaling pathway has been reported to be compulsory for elimination of functionless T cells in the thymus (Newton et al., 2000). Finally, the importance of stromal cells and stromal cell-derived factors such as interleukin-7 (IL-7) for survival of B cell precursors should not be underestimated. This assertion is supported by the observation that B cell development is arrested at the pro-B cell stage in IL-7 deﬁcient mice (von Freeden-Jeffry et al., 1995).

B. In the Periphery 1. BEFORE INDUCTION OF THE IMMUNE RESPONSE a. Role of the BCR In healthy adult mice the number of B cells in the periphery is tightly controlled and remains constant in spite of continuous input of newly formed B cells from the BM and of selective expansion of certain B cell clones consecutive to antigenic stimulation. It has been estimated that the daily production of newly formed B cells would be sufﬁcient to completely renew the peripheral pool in 5 or 6 days (Osmond, 1986). Therefore, this excess input of freshly produced B lymphocytes has to be compensated for by the demise of B cells of the peripheral B cell pool. It is now accepted that the size of the peripheral B cell pool is regulated through interclonal competition. The studies of the group of Freitas in particular (Freitas and Rocha, 2000) have established that both newly produced B cells and resident peripheral B lymphocytes are competing with one another for a limited supply of resources presumably available in deﬁned ecological niches. The precise nature of these resources remains elusive to date, but access to these resources is dependent on the membrane expression of a functional Ag receptor. This concept is supported by an elegant study from the group of Rajewsky showing that ablation of the BCR (using an inducible Cre/Lox-P system) results in rapid death of peripheral B lymphocytes (Lam et al., 1997). Speciﬁcity of the BCR is also an important parameter of the interclonal competition among the peripheral B cell pool. This notion stems from observations made in chimeric animals resulting from the reconstitution of lethally irradiated recipients with BM from both wild-type and BCR-transgenic animals. These

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experiments have shown that competition between monoclonal and polyclonal B cell populations results in counterselection of B cells which express a transgenic BCR (Freitas et al., 1995). These data raise the question as to whether BCR requires ligand binding to deliver survival signals to peripheral B cells. This issue has been addressed by reconstituting recipient mice with mixtures of BM from two transgenic animals expressing either a complete or a truncated form (lacking the V region) of the same human IgM molecule (Rosado and Freitas, 1998). The observation that deletion of the V region of the BCR dramatically reduces the competitive ability and life-span of transgenic B cells is in favor of the notion that ligand recognition through the BCR is required for persistence of the peripheral B cell pool.

b. Role of BAFF and APRIL and Their Receptors The importance of BCR ligands to prevent decay of B cells in the periphery does not rule out that other factors may also contribute to the survival of B cells in secondary lymphoid tissues. A ﬂurry of papers has recently documented the prominent role played by two novel members of the TNF ligand family in the survival of mature B lymphocytes in the periphery. These two molecules, known as APRIL (A Proliferation-Inducing Ligand) and BAFF (B-Cell-Activating Factor, also designated BlyS, THANK, TALL, zTNF4), respectively, were cloned by homology with other members of the TNF family (Hahne et al., 1998; Moore et al., 1999; Schneider et al., 1999). Both factors are constitutively expressed by monocytes, activated T cells, and possibly dendritic cells. APRIL and BAFF both bind with a relatively high afﬁnity to two distinct receptors identiﬁed as TACI (Transmembrane Activator and CAML Interactor) and BCMA (B Cell Maturation Antigen) (Gross et al., 2000; Marsters et al., 2000; Wu et al., 2000). While the expression of BCMA is highly restricted to mature B cells, TACI was found to be expressed also on some T cells. In addition, a third receptor for BAFF, termed BAFF-R (Thompson et al., 2001), has recently been identiﬁed. This receptor is likely to be a crucial mediator of the effect of BAFF on B cell responses since partial disruption of the BAFF-R gene in the mouse results in a phenotype similar to that of BAFF-knockout mice (Thompson et al., 2001). The profound loss of peripheral mature B cells in BAFF-deﬁcient mice (Gross et al., 2001; Schiemann et al., 2001) indicates that, although APRIL and BAFF share two receptors, there is little functional redundancy between BAFF and APRIL on B cells. The putative functions of BAFF receptors are summarized in Fig. 3 (see color insert). Two complementary lines of evidence indicate that the activity of BAFF is restricted to the mature B cell compartment and more speciﬁcally to cells belonging to the B2 lineage. First, transgenic overexpression of BAFF results in a dramatic enlargement of the peripheral B cell pool but affects neither the

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pool of BM B cell precursors nor the peritoneal B1 cell population (Mackay et al., 1999). Second, BAFF-deﬁcient mice have profoundly reduced numbers of mature B cells in the spleen, but their populations of B cell precursors in the BM and B1 cells in the peritoneum are normal (Gross et al., 2001; Schiemann et al., 2001). The issue whether BAFF exerts its antiapoptotic function at a precise stage of the B cell maturation pathway or whether it has a broader spectrum of cellular targets within the mature B cell compartment is not deﬁnitely resolved. On the one hand, studies of mice which express a soluble decoy form of one of the BAFF receptors (TACI) (Gross et al., 2001) and of BAFFdeﬁcient mice (Schiemann et al., 2001) speak in favor of the notion that BAFF has a narrow window of action on peripheral B cells. In both models, B cell development is arrested at the transitional T1 stage in the spleen, suggesting that BAFF may be involved either in the transition of immature splenic B cells from the T1 to the T2 stage or in the survival of T2 cells. The latter hypothesis is further supported by the observation that BAFF selectively promotes survival of T2 B cells in vitro (Batten et al., 2000). On the other hand, there is experimental evidence that BAFF could at least promote survival of another B cell subset since BAFF-transgenic mice are also characterized by an enlargement of the marginal zone (MZ) B cell population (Mackay et al., 1999). In agreement with this ﬁnding, the Ab response to type 2 T-independent Ag, known to be conﬁned to a B cell subset residing in the MZ, is profoundly defective in TACI null mice (von Bulow et al., 2001; Yan et al., 2001). Further studies will be required to determine whether BAFF can also deliver survival signals at other stages of the B cell maturation pathway such as PC or memory B cells. Finally, it is noteworthy that conditional ablation of sIgs and inactivation of the BAFF gene have a strikingly similar outcome: they both induce a dramatic loss of the peripheral B cell pool. This evokes a possible crosstalk between BAFF receptors and BCR.

c. Biochemistry of Peripheral B Cell Survival Signals Enforced expression of bcl-2 can partially prevent elimination of peripheral B cells which have lost expression of their Ag receptor in the model of inducible BCR ablation of Lam and colleagues (Lam et al., 1997). In many respects the phenotype of BAFF-transgenic mice (Gross et al., 2000; Mackay et al., 1999) is similar to that of mice in which a bcl-2 transgene is targeted to the B cell compartment (Strasser et al., 1991). Both types of mice exhibit a large excess of peripheral B cells, increased numbers of PC, and hypergammaglobulinemia associated with the production of autoantibodies. Furthermore, splenic B cells in BAFF-transgenic mice display elevated levels of bcl-2 (Mackay et al., 1999). Taken together, these observations suggest that, at the steady state, homeostasis of the peripheral resting B cell pool is at least partly controlled by bcl-2 family members. By inference, elimination

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of B cells produced in excess should proceed primarily through activation of the mitochondrial apoptotic pathway.

2. AT THE PEAK OF THE IMMUNE RESPONSE The notion that Ag itself can regulate the size of an effector lymphoid population is accepted for T lymphocytes but is still controversial for B cells. Ag-induced apoptosis, more commonly designated AICD (ActivationInduced Cell Death), is generally regarded as a feedback regulatory mechanism preventing overexpansion of T cells exposed to prolonged or repeated antigenic stimulation (Krammer, 2000). AICD involves induction through the TCR of two ligands of the TNF family: CD95 ligand (now assigned as CD178) and TNFα, which act at the early and late stage of AICD, respectively (Alderson et al., 1995; Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995; Zheng et al., 1995). For B cells, the prevailing concept has long been that the ability of the BCR to transduce an apoptotic signal was restricted to the immature B cell stage where it is instrumental for driving deletion of self-reactive B cells. Nevertheless, it had been documented since the early 1990s that engagement of the BCR can promote apoptosis of mature B cell neoplasias such as Burkitt’s lymphomas (Gregory et al., 1991). There is now evidence that primary mature human B cells that have been previously stimulated by engagement of CD40 or the BCR are also sensitive to BCR-induced apoptosis (Berard et al., 1999a; Galibert et al., 1996). The susceptibility of mature B cells to BCR-induced killing is positively correlated with the cycling status and proliferative capacities of the B cells (Berard et al., 1999b). These observations suggest that the outcome of BCR signaling on mature B lymphocytes is apoptosis if the signal delivered by Ag is supraoptimal, or provided to B cells already activated and cycling. In this respect, BCR-induced apoptosis of mature B cells is highly reminiscent of AICD in T cells. By regulatory pressure, B cell AICD may prevent overexpansion of the activated B cell pool at the peak of the immune response. As discussed in Section II.C, two distinct BCR death pathways can be engaged depending on the degree of cross-linking by the Ag. This raises the possibility that type 2 T-independent Ag which cross-link the BCR in a multivalent fashion because of their repetitive structure and conventional T-dependent Ag elicit a different death program. Whether both types of apoptotic cells are handled similarly by the immune system and what message these two modes of B cell death potentially deliver to the organism is totally unknown. Several cosignals can oppose BCR-induced death. These include (i) activated T cells (Billian et al., 1997), (ii) membrane-bound molecules such as CD40 ligand (Parry et al., 1994) or CD5 (Nomura et al., 1996), and (iii) soluble mediators such as type I interferons (Su and David, 1999) or IL-4 (Billian et al., 1997; Galibert et al., 1996; Parry et al., 1994). One might assume that B cell AICD

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is repressed as long as activated B cells have access to resources (cytokines, growth factors, T cell help) available in deﬁned ecological niches or from a limited number of accessory cells. As soon as the size of the activated B cell population exceeds the capacity of these niches or outnumbers accessory cells, competition occurs and resources become limiting. Cells unable to access costimulatory signals and exposed to the sole inﬂuence of Ag may thus be eliminated by AICD.

IV. CONTROL OF THE SPECIFICITY AND AFFINITY OF THE AB RESPONSE A. At the Onset of the Immune Response At the onset of the immune response, B cell apoptosis fulﬁlls two main functions. The ﬁrst one is preservation of peripheral B cell tolerance by elimination of self-reactive B cells that have escaped deletion during early development. The second one is to prevent bystander activation of lowafﬁnity or irrelevant B cell clones in the context of T-dependent Ab responses. The ﬁrst encounter between T and B cells takes place at the border between B cell follicles and the T cell area. It is secondary to the activation of T cells through recognition of the antigenic peptides presented by dendritic cells in the context of MHC (Garside et al., 1998). Primed T cells express the CD40 ligand (Noelle et al., 1992), now assigned as CD154, and have the potential to stimulate relevant Ag-speciﬁc B cells but also useless or potentially harmful bystander B cells. Engagement of the TCR/CD3 complex on T cells by APCs presenting antigenic peptides also results in induction of CD178 (Suda et al., 1995). It is now established that T cell-bound CD178 is instrumental in eliminating inappropriate B cells that might have received T cell help in a noncognate fashion. Ag-speciﬁc B cells are protected from CD95-induced death during T/B cell interaction by signals delivered through the BCR. This assumption stems from the observation that engagement of the BCR either by anti-Ig Abs or by nominal Ag protects activated virgin B cells from CD95-mediated killing both in humans (Lagresle et al., 1996) and in mice (Rathmell et al., 1996; Rothstein et al., 1995). In sum, these data indicate that only those B cells efﬁciently stimulated by the BCR survive the interaction with primed T cells and differentiate further. Because of the desensitization of their Ag receptor (Cooke et al., 1994), the level of BCR stimulation in tolerized self-reactive B, however, is too low to protect them from CD95-induced killing. These cells will be deleted upon encounter with primed T cells. The same mechanism causes apoptosis of B cells expressing an irrelevant BCR if they happen to interact with primed T cells.

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Interestingly, the group of Rothstein has shown that hypercross-linking of sIgs can promote CD95 resistance in tolerized self-reactive B cells. Such cells are normally refractory to the protection delivered by more physiological doses and forms of Ag (Foote et al., 1998). This reinforces the notion that the degree of protection brought about by ligation of the BCR is related to the strength of the signal it transduces. This raises the possibility that the BCR signal could fall below the threshold required for CD95 protection if the afﬁnity of the BCR for Ag is too low. Consequently, certain low-afﬁnity B cell clones could remain sensitive to CD95-mediated killing in spite of their reactivity with the immunizing Ag. Therefore, the CD95/CD178 system might also contribute to the selection of B cells with the best-ﬁtting Ag receptors before the GC reaction is initiated. The observation that the number of GC in lpr mice immunized with NP-CGG is 3- to 4-fold higher than in immunized wild-type mice (Takahashi et al., 2001) is compatible with the hypothesis that CD95 intervenes in the interclonal competition between GC founders.

1. THE CD95 SIGNALING PATHWAY IN B CELLS CD95 may be coupled to two distinct apoptotic pathways. Two cell types (designated as type I and type II) that each preferentially use one or the other of these CD95 pathways have been identiﬁed (Scafﬁdi et al., 1998). In type I cells, such as activated primary T cells, cleavage of caspase-8 occurs at the level of the Death-Inducing Signaling Complex (DISC) within minutes of CD95 ligation before disruption of the mitochondrial transmembrane potential. It is followed by the rapid activation of caspase-3 and other downstream caspases. In type II cells, such as hepatocytes (Lacronique et al., 1996; Rodriguez et al., 1996), activation of caspase-8 at the level of the DISC is strongly reduced and a mitochondrial ampliﬁcation loop is required for full activation of downstream caspases. This loop is initiated by truncation of Bid and subsequent activation of the mitochondria. Although the mitochondrial functions are pertubed in both types of cells, only type II cells can be protected from CD95-induced killing by overexpression of the antiapoptotic members of the bcl-2 family (see Krammer, 2000, for a review). B cells from bcl-xL transgenic mice are resistant to CD95-induced apoptosis (Schneider et al., 1997). Nonetheless, apoptosis initiated by ligation of CD95 on activated primary human B lymphocytes presents more homologies with the type I than with the type II pathway. In particular, activation of caspase-8 occurs at the level of the DISC and is independent of the activity of the mitochondrial initiator complex of apoptosis (apoptosome) (Hennino et al., 2000). However, the CD95 death pathway in activated primary human B cells does not fulﬁll all criteria of deﬁnition of the type I pathway either. While activation of caspase-8 in bona ﬁde type I cells takes place within

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minutes of CD95 ligation, it is not detectable before hours of CD95 ligation in activated primary B cells. The delayed activation of caspase-8 in activated B cells in response to the CD95 signal could be related to the fact that the DR antagonist cFLIP is expressed in these cells (Hennino et al., 2000). This suggests that CD95-mediated killing of bystander B cells might not immediately follow their encounter of CD178-expressing T cells. This temporary suspension of the apoptotic program might offer B cells that have been signaled through CD95 an ultimate opportunity to be rescued by trophic factors or Ag or to revise their BCR through receptor editing.

2. MOLECULAR BASIS OF THE CD95 RESISTANCE PROMOTED BY BCR LIGATION The mechanisms whereby engagement of the BCR promotes CD95 resistance in B cells has been studied by several groups, but a general consensus has not yet been reached. The available data support three different possibilities. The ﬁrst one has been proposed by the group of Rothstein. Using a differential display strategy, they have identiﬁed a novel antiapoptotic gene induced by BCR ligation and designated as Fas (CD95) Apoptosis-Inhibitory Molecule/FAIM (Schneider et al., 1999). The molecular partners of FAIM and the mechanism whereby it interferes with the CD95 signaling pathways remain to be deﬁned. It has been proposed that BCR-induced CD95 resistance could be multifactorial and might rely both on the de novo induction of new gene products such as FAIM and on upregulation of antiapoptotic members of the bcl-2 family such as bcl-xL (Foote et al., 1996; Schneider et al., 1997). The second one has been proposed by Catlett and Bishop (1999). Their study has led to the conclusion that the BCR-induced CD95 resistance results from a receptor-proximal blockade of the CD95 signaling pathway. These authors show that engagement of sIgs prevents recruitment of FADD to the oligomerized death domains of CD95 and subsequent formation of the DISC. The biochemical events which preclude association of FADD with CD95 are unknown. The third possibility stems from a study conducted by the group of Lenardo (Wang et al., 2000). These authors have suggested that cFLIP could contribute to BCR-induced CD95 resistance since BCR crosslinking was found to upregulate FLIP both in primary B cells and in B cell lines.

B. During Affinity-Maturation of the Ab Response In the late phase of the primary immune response, afﬁnity maturation of the Ab response allows for the production of Abs with higher Ag-binding afﬁnities than those produced in the early phase of the response. Completion

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of this process is contingent upon the development of GC inasmuch as most mutations generated by gene targeting which result in the absence of GC formation also abolish the production of high-afﬁnity Abs in response to immunization (Kosco-Vilbois et al., 1997). Afﬁnity maturation of the Ab response is the result of two processes that take place in distinct compartments of the GC (Przylepa et al., 1998). The ﬁrst process is the diversiﬁcation of the Ab repertoire of Ag-activated B lymphocytes in the dark zone of the GC. It operates by random introduction of point mutations in the Ig variable region genes. Mutant B cells can display higher or reduced afﬁnity for the immunizing Ag or will express new speciﬁcities which can either be inocuous or harmful if they lead to reactivity against self-Ag. The second process that takes place in the light zone of the GC is a selection step which allows rescue of B cell clones with high Ag-binding capacity while those with low afﬁnity or undesired speciﬁcity are eliminated. Positive selection of somatic mutants in the light zone relies on their capacity to efﬁciently bind, take up, and present Ag to helper T lymphocytes. The group of MacLennan ﬁrst demonstrated in 1989 that apoptosis is the driving force that underlies the Ag-driven selection of B cells in the GC (Liu et al., 1989). This survey also showed that both the BCR and CD40 contribute to positive selection by preventing B cell apoptosis. The concept of positive selection that ensued and still prevails today is that high-afﬁnity B cell clones are selected because they sequentially receive two signals which concur to block execution of the apoptotic program. The ﬁrst survival signal is provided by the Ag itself trapped as immune complexes on the follicular dendritic cell (FDC) network. Only those B cells whose BCR shows a sufﬁciently high afﬁnity (i.e., capable of displacing the low-afﬁnity IgM Abs forming the immune complexes), gain access to Ag and receive a survival signal. The second survival signal is provided through CD40 when B cells present the processed antigenic peptides in a classical MHC-restricted cognate fashion to helper T cells in the light zone of the GC.

1. APOPTOTIC PATHWAYS IN GC B CELLS The importance of the mitochondrial apoptotic pathway during the GC reaction has been highlighted by a series of papers showing that expression of a bcl-2 or bcl-xL transgene targeted to the B cell compartment impairs the afﬁnity maturation process. Smith et al. showed that enforced bcl-2 expression in mouse B cells increases the size of the GC (Smith et al., 1994) and allows a population of low-afﬁnity B cells, normally counterselected, to enter the memory B cell pool (Smith et al., 2000). Similarly, Takahashi et al. (1999) have shown that targeting of a bcl-xL transgene to the B cell compartment reduces apoptosis in the GC and causes abnormal selection of B cells with low-afﬁnity Ag-binding capacity. Furthermore, the study of Smith et al.

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(1995) showing that the CD95 deﬁciency in lpr mice affected neither the elimination of low-afﬁnity somatic mutants in the GC nor the kinetics and rate of production of memory B cells and high-afﬁnity Ab-secreting cells gave ground to the idea that CD95 is not involved in the GC reaction. Altogether, these ﬁndings support the concept that the death pathway in GC B cells involves the mitochondria but not the DR. However, this view of the regulation of GC B cell death has been challenged by a series of recent observations. First of all, we have shown that spontaneous apoptosis of human GC B cells in vitro is associated with spontaneous activation of caspase-8 and concomitant downregulation of cFLIP expression (Hennino et al., 2001). The biochemical analysis of the CD95 DISC in GC B cells revealed that CD95 is constitutively connected to FADD, pro-caspase-8, and the long form of cFLIP in these cells. Thus, GC B cells are unique in the sense that they contain a preformed CD95 signaling membrane complex. We have proposed that the association of cFLIPL with the CD95 DISC is instrumental in keeping the CD95 death pathway silent until GC B cells are put to the test in the light zone of the GC. This hypothesis is substantiated by two lines of evidence. First, our results and those of van Eijk et al. (2001) indicate that cFLIP rapidly dissociates from the CD95 DISC as GC B cells activate the apoptosis program in vitro. Second, the decay of cFLIP expression in GC B cells is prevented if B cells remain in contact with FDC (van Eijk et al., 2001) or if they are activated by CD154 or a surrogate Ag (Hennino et al., 2001). Based on these in vitro ﬁndings, we proposed a model that postulates that CD95 drives elimination of somatic mutants which display an irrelevant or low-afﬁnity BCR (Fig. 4; see color insert). Association of cFLIPL with the CD95 DISC prevents GC B cells from undergoing apoptosis while they expand and somatically mutate their BCR. During the selection process in the light zone, expression of cFLIPL is maintained only in B cells that receive survival signals through the BCR and CD40 sequentially. The CD95 DISC is converted into its active form by the loss of cFLIPL if B cells fail to receive one or the other of these rescue signals. The model we propose for the regulation of GC B cell apoptosis has received support from two recent in vivo studies in which the pattern of somatic mutations was followed in wild-type and lpr mice immunized with the NP hapten coupled to chicken gamma globulin. Takahashi and colleagues (Takahashi et al., 2001) demonstrate that the afﬁnity maturation process induced by this Ag is perturbed in lpr mice and differs from that in wild-type mice by two major characteristics. First, appearance of the canonical mutations that confer high-afﬁnity NP binding is delayed in lpr mice. Second, the frequency of these mutations is lower in lpr mice at the early stages of the primary response. Using a similar experimental approach, Hoch and colleagues (Hoch et al., 2000) report that in lpr mice, B cells which constitute the GC at

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the late phase of the primary response have lost the ability to recognize NP but have acquired reactivity against self-Ag. These two studies indicate that the impact of CD95 on the afﬁnity maturation process is subtle. In the absence of CD95, elimination of irrelevant or autoreactive B cells is not totally abrogated but is less efﬁcient than in wild-type animals. In other words, the negative selection process in lpr mice is leaky. This might explain why the role of CD95 in this process has escaped notice in the early survey of Smith et al. (1995). One possible explanation for the mild impact of the CD95 deﬁciency on the GC reaction could be that the function played by CD95 is redundant and can be taken over by other members of the DR family in CD95-deﬁcient mice. Selective invalidation of the FADD function in mature B lymphocytes would be a way to address this question. At face value, the above-mentioned data supporting the involvement of DR in the GC reaction are difﬁcult to reconcile with the fact that targeting of a bcl-2 or bcl-xL transgene to the B cell compartment impairs the afﬁnity maturation process. One possible explanation for these apparently contradictory ﬁndings would be that long-lasting survival of high-afﬁnity B cell mutants requires the sequential blockade of the caspase and of the mitochondrial pathways. In Fig. 5 (see color insert) we present a hypothetical model in which it is postulated that Ag provides the primary rescue signal by preventing the decay of cFLIP. By blocking execution of the CD95 death pathway, cFLIP will allow B cells that have received a signal through the BCR to proceed to the second step (i.e., Ag-presentation to T cells). The second signal, provided by CD154, will secure long-term survival of B cells by reinducing expression of antagonists of the mitochondrial death pathway such as bcl-2. For certain low-afﬁnity B cell clones, the signal received through the BCR may be sufﬁcient to maintain cFLIP expression but suboptimal for the development of an efﬁcient Ag-presenting ability (defective expression of the CD80/CD86, for example). A bcl-2 transgene would preserve such cells and allow their recruitment into the memory B cell compartment. The putative source of CD178 in the GC is another puzzling question. The fact that GC B cells express a preformed CD95 DISC naturally raises the question of what causes the oligomerization of CD95 in these cells. Autocrine production of CD178 by B cells themselves is unlikely since human tonsillar GC B cells lack expression of the CD178 transcript (Hennino et al., 2001). The paracrine production of CD178 by non-B cells in the GC is possible since the CD178 transcript and protein have been detected in T cells (Kondo et al., 1997) and FDC (Verbeke et al., 1999), respectively. The hypothesis of an autonomous, ligand-independent, oligomerization of CD95 on GC B cells cannot be excluded either. Spontaneous aggregation of DR is not unprecedented and it has already been documented for TNF-R1 and CD95 (Boldin et al., 1995). Cytoplasmic regulators equivalent to the SODD

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molecule described to prevent spontaneous association of TNF-R1 (Jiang et al., 1999) could operate to maintain CD95 in a monomeric form outside of the GC.

2. ROLE OF FOLLICULAR DENDRITIC CELLS IN GC B CELL SURVIVAL FDC are crucial for the control of B cell survival in the GC because they present the immune complexes. However, their antiapoptotic function extends beyond the mere presentation of Ag. They could also protect B cells from apoptosis at the early phase of the GC reaction while they are proliferating and mutating their Ig V region genes. The published literature is compatible with the notion that FDC interfere with at least two distinct levels of the apoptosis process in GC B cells. First, they block induction of the CD95-induced death signal by preventing the decay of cFLIP, as we previously alluded to (van Eijk et al., 2001). Second, they block a distal point of the apoptotic cascade by switching off a cathepsin-dependent nuclease activity in the GC B cell nuclei (Lindhout et al., 1995). Several signals are likely to contribute to the antiapoptotic function of FDC. Silencing of the GC endonuclease has been reported to be mediated by the release of cystatin A, a natural cathepsin inhibitor possibly made by FDC (van Eijk and de Groot, 1999). Convincing data also point toward an important role of the adhesion molecules ICAM-1 and VCAM-1 on the FDC and their countereceptors LFA-1 and VLA-4, respectively, on B lymphocytes (Koopman et al., 1994; Lindhout and de Groot, 1995). Adhesive interactions between FDC and B cells are probably not directly responsible for the rescue of high-afﬁnity somatic mutants but they might contribute to strengthening and amplifying the primary survival signal delivered by Ag held on FDC. The nature of the FDC-derived signal which might permit the maintenance of cFLIP expression in GC B cells is still unknown. A summarized overview of the apoptosis checkpoints in the mature B cell compartment is presented in Fig. 6 (see color insert).

V. REGULATION OF SURVIVAL IN THE MEMORY B CELL COMPARTMENT A. The Memory B Cell Pool The key observation supporting the concept that the persistance of B cell memory requires contact with either the immunizing or cross-reacting Ag is that memory B cells could only survive in naive hosts if they had been

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cotransfered with Ag (Gray and Skarvall, 1988). The assumption that Ag participates in regulating the lifespan of memory B cells implies that there are sites in the body in which B cells can still gain access to Ag long after resolution of the infection. The GC appears to be a plausible candidate for preservation of a stock of Ag, as suggested by the study of Bachmann and colleagues (Bachmann et al., 1996) conducted in mice infected with vesicular stomatitis virus (VSV). These authors have shown that GC containing viral proteins associated with FDC can persist up to 100 days after infection and that VSV-speciﬁc B lymphocytes can still be detected in these late GC long after extinction of the immune response and resolution of the infection. Nevertheless, the long-term persistence of a nonreplicating Ag still awaits formal demonstration. The notion that contact with Ag is required for survival of memory B cells has recently been challenged by the elegant experiments conducted by the group of Rajewsky (Maruyama et al., 2000) in which an inducible Cre-LoxP system was used to change the Ag speciﬁcity of the BCR on an established pool of memory B cells. In these experiments, a signiﬁcant proportion of memory B cells, generated by immunization with the hapten NP, were induced to irreversibly lose their NP-speciﬁc BCR and to acquire a novel BCR recognizing a structurally unrelated Ag that the mice had never encountered. This study demonstrated that memory B cells which have swapped their initial BCR for a second one which cannot recognize the immunizing Ag are maintained for long periods of time. It provides an almost deﬁnitive argument in favor of the lack of Ag-dependency for memory B cell persistence. Moreover, the earlier study of Schittek and Rajewsky (1990) had come up with the conclusion that the memory B cell compartment is mostly composed of nondividing cells. Taken together, these ﬁndings support the concept that B cell memory is maintained by long-lived quiescent cells. Two strong lines of evidence indicate that integrity of the mitochondrial functions is required for long-term survival of B cells that compose the established memory B cell pool. First of all, expression of a bcl-2 transgene targeted to B cells dramatically extends the half-life of memory B cells after transfer into naive recipients (Nunez et al., 1991). Second, the size of the memory B cell pool in bcl-2-transgenic animals is greatly increased (Smith et al., 1994). It is not known how the mitochondrial death pathway is kept silent in memory B cells, but indirect evidence points toward the possible inﬂuence of a specialized microenvironment. It is noteworthy that resident memory B cells are located in deﬁned microanatomical areas such as the MZ in the spleen or the subepithelial area in tonsils. In light of the reported increased numbers of MZ B cells in BAFF-transgenic mice (Mackay et al., 1999), it will be interesting to determine the impact of this factor on the life-span of memory B lymphocytes. Generations of mice in which the genes for BAFF or its receptors could be conditionally deleted will help to address this issue.

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B. The Plasma Cell Pool PC produced during the early phase of the primary response differentiate in the so-called extrafollicular foci in the outer periarteriolar lymphocytic sheath (PALS) of the splenic white pulp (MacLennan, 1994). These PC produce low-afﬁnity Abs because they do not accumulate somatic mutations and disappear due to apoptosis within 14 days after immunization (Smith et al., 1996). The low-afﬁnity Abs are progressively replaced by higher afﬁnity somatically mutated Abs produced by the differentiated progeny of GC B cells. It is documented that the PC produced at the early phase of the response in the extrafollicular foci of the spleen have a half-life of only a few days (Ho et al., 1986). Although the longevity of somatically mutated PC which arise at later stages of the immune response is higher, their estimated life-span in the spleen does not exceed 6 or 7 days (Slifka et al., 1998). Until recently, the fact that certain vaccines or natural infections could result in the presence of protective circulating Abs for several years was interpreted as the consequence of continued restimulation and terminal differentiation of memory B cells into Ab-secreting cells. Two sets of data have recently challenged this dogma. Slifka and colleagues have exploited the model of acute infection of mice with the lymphocytic choriomeningitis virus (LCMV) to study PC longevity (Slifka et al., 1998). The conclusions of their study can brieﬂy be summarized as follows. First, the seric anti-LCMV Ab titers are maintained throughout life despite the absence of detectable persisting viral Ag in lymphoid tissues as assessed by reverse transcriptase-polymerase chain reaction. Second, selective depletion of the memory B cell pool by in vivo irradiation does not reduce LCMV Ab titers. Third, LCMV-speciﬁc PC are localized in the spleen within the 15 days that follow infection but then relocate in the BM where they persist and continue secreting Abs for more than 1 year. Fourth, adoptively transferred BM PC continue to secrete Abs in the hosts for long periods of time, in the absence of Ag. Similarly, BrdU labeling experiments conducted by Manz and colleagues (Manz et al., 1997) in mice immunized with OVA, demonstrated that the numbers of PC remained constant in the BM up until 100 days following immunization, in the absence of cell divisions. Taken together, these ﬁndings have important implications for our understanding of B cell memory and how it is maintained. They suggest, ﬁrst of all, that there are two types of long-lived memory B cells. The ﬁrst one is a resting small non-Ab-secreting lymphocyte which resides in specialized microanatomical compartments of secondary lymphoid tissues such as the MZ of the spleen. The other one is a long-lived PC located in the BM. The primary function of these long-lived BM PC would be to maintain elevated levels of protective Abs which will constitute the ﬁrst defense barrier in case of pathogen reinfection. The long-lived PC pool of the BM is not replenished through differentiation of memory B cells and does not require

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the immunizing Ag for its persistence (see Slifka and Ahmed, 1998, for a review). As such, these long-lived PC can be envisaged as a component of innate immunity which is acquired de novo through the adaptative immune response. The mechanisms which regulate PC longevity in the BM are completely unknown but several lines of evidence indicate that PC survival is dependent on integrity of the mitochondrial apoptosis pathway and can be maintained through enhanced expression of certain antiapoptotic members of the bcl-2 family. For example, enforced expression of bcl-2 but not of bcl-xL in B cells results in enhanced longevity of PC (Smith et al., 2000; Takahashi et al., 1999). This is conﬁrmed by the observation that a fraction of BM PC shows strong Bcl-2 expression (Pettersson et al., 1992). Expression of Bcl-2 is probably not sufﬁcient to prevent apoptosis of PC since isolated tonsillar PC undergo rapid apoptosis in vitro despite constitutive expression of Bcl-2 (Merville et al., 1996). Another member of the bcl-2 family, the ¨ A1/Bﬂ-1 gene, could be more speciﬁcally involved in PC survival. Knodel et al. (1999) have reported that the immature mouse B cell line WEHI 231 adopts the phenotypic and functional characteristics of PC upon transfection of the transcription factor Blimp-1. They showed that the differentiation into PC was accompanied by a dramatic loss of cell viability which could be compensated by overexpression of A1.

C. The Molecular Regulators of PC Survival The biology of PC is poorly understood. Most information stems from their malignant counterparts, the myelomas. Several factors have been reported to promote survival of myeloma cells such as IL-6 (Xu et al., 1998; Catlett-Falcone et al., 1999), interferon α (Ferlin-Bezombes et al., 1998), insulin-like growth factor 1 (Ferlin et al., 2000), and IL-15 (Hjorth-Hansen et al., 1999). Although IL-6 behaves as a survival factor for plasmablasts (Jego et al., 1999, 2001; Kawano et al., 1995) and malignant PC (CatlettFalcone et al., 1999; Kawano et al., 1988; Xu et al., 1998), it cannot prevent spontaneous apoptosis of fully mature tonsillar PC in vitro (Merville et al., 1996). So far, the only efﬁcient way to block apoptosis of PC in vitro is to coculture them with ﬁbroblasts originating from BM (Kawano et al., 1995; Merville et al., 1996) or from the rheumatoid synovium (Dechanet et al., 1995). Although the nature of the signals provided by BM stromal cells is presently unknown, it is tempting to speculate that they are involved in the long-term survival of PC that have migrated to the BM. Stromal cells may not be the only cell type susceptible to prevent apoptosis of PC since plasmablasts have been found to be physically associated with a subset of dendritic cells in the spleen (Garcia De Vinuesa et al., 1999). Contribution

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of DR to the regulation of PC survival is unknown. The few data available regarding the expression of DR and CD95 in particular on normal PC are still controversial.

VI. CONCLUSIVE REMARKS The mechanisms underlying B cell death and survival at different stages of their development are far from being completely elucidated. However, from the data available so far (summarized in Table I) a picture emerges which shows that B cell death is primarily controlled by the mitochondria at the steady state, while DR and the caspase pathway largely intervene in the course of an immune response. Until now, only CD95 has been seriously considered as a possible regulator of B cell functions but it is probable that future studies will reveal that other members of the DR family are involved in B cell death regulation. It is already known that other DR are either constitutively expressed (DR3) or can be induced (TRAIL-R1 and 2) on B cells upon activation, although their biological function is presently completely unknown. These receptors may not necessarily modulate B cell responses through induction of apoptosis. The most unexpected ﬁnding concerning DR which have emerged during these past 5 years has been the demonstration of their dual capacity to transduce either apoptosis or activation signals depending on the context. It has been clearly demonstrated that the stimulating function of DR contribute to T cell development in the thymus, and there are now strong lines of evidence suggesting that DR, or at least receptors using FADD as a downstream signaling molecule, positively regulate early B cell development as well. It is also important to bear in mind that multiple cellular organelles can contribute to elicit an apoptotic signal (Ferri and Kroemer, 2001). Moreover, the target of action of bcl-2 family members is not restricted to the mitochondria inasmuch as these molecules can also localize in the endoplasmic reticulum, for example. Finally, it is most likely that multiple connections exist between the pathways triggered by the mitochondria, the endoplasmic reticulum, and the DR. Therefore, overexpression or disruption of bcl-2-related genes will affect the functional integrity of more than one organelle and could indirectly perturb multiple pathways. Consequently, the dichotomy between the mitochondrial and the caspase pathways we have made in the present review is probably a reductionist view. However, it was imposed by the fact that the ﬁeld of apoptosis is expanding so rapidly that the mechanisms regulating B cell death have not yet been reexamined in the light of the latest apoptosis concepts.

Table I Regulation of B Cell Survival during Lymphopoiesis and Immunopoiesis: A Tentative Summary At the steady state

Tolerance

Homeostasis

During the immune response Maintenance of memory cells

Tolerance

Homeostasis

Afﬁnity of the Ab response

Death pathway

Mitochondrial

Mitochondrial

Mitochondrial

Caspases

Mitochondrial

Caspases/ mitochondrial

Death-inducing signals

BCR

Unknown

Unknown

CD95

BCR

CD95

Death-repressing signals

Unknown

BCR BAFF/APRIL

Unknown

BCR, IL-4

T cells, IL-4, CD40, type I IFNs

BCR, CD40

Survival mediators involved

Bcl-2 members

Bcl-2 members

Bcl-2 members

cFLIP, FAIM

Bcl-2 members

cFLIPL, Bcl-2 members

Death receptor dependency

No

No

Unknown

Yes

No

Yes

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A major future challenge of investigations in the B cell ﬁeld will be to understand how the survival of memory B cells is regulated and how the nonlymphoid microenvironment contributes to repress their apoptosis. It is clear that the longevity of the two cellular components of B cell memory (resting memory B cells and long-lived PC) is also dependent on their ability to locate and reside in appropriate microanatomical niches of the lymphoid tissues. Therefore regulation of B cell trafﬁcking probably weighs indirectly on their survival. The resident pools of memory B cells and PC are located in the splenic marginal zone and in the BM, respectively. Until now, the precise nature of the resources or cellular interactions which are put at their disposal remains elusive, but an important step has been made by the recent discovery of two B cell-speciﬁc survival factors named BAFF and APRIL. Among the several questions still pending is whether the latter factors can also promote survival of memory B cells, how they repress cell death on a biochemical basis, and whether their signaling pathway can counteract the death signals initiated by the BCR or DR. Determining the sites of production of BAFF and APRIL within secondary lymphoid tissues as well as the developmental regulation of their receptors on B cells is also an important issue. Better knowledge of the extrinsic and intrinsic B cell death regulators that intervene during B cell development will help in understanding the molecular defects underlying immunodeﬁciencies and neoplasia and in designing therapeutical approaches that correct them.

ACKNOWLEDGMENT We thank Dr. Christophe Arpin (INSERM U503) for constructive comments and discussions.

REFERENCES Alderson, M. R., Tough, T. W., Davis-Smith, T., Braddy, S., Falk, B., Schooley, K. A., Goodwin, R. G., Smith, C. A., Ramsdell, F., and Lynch, D. H. (1995). Fas ligand mediates activationinduced cell death in human T lymphocytes. J. Exp. Med. 181, 71–77. Bachmann, M. F., Odermatt, B., Hengartner, H., and Zinkernagel, R. M. (1996). Induction of long-lived germinal centers associated with persisting antigen after viral infection. J. Exp. Med. 183, 2259–2269. Batten, M., Groom, J., Cachero, T. G., Qian, F., Schneider, P., Tschopp, J., Browning, J. L., and Mackay, F. (2000). BAFF mediates survival of peripheral immature B lymphocytes. J. Exp. Med. 192, 1453–1466. Berard, M., Casamayor-Palleja, M., Billian, G., Bella, C., Mondiere, P., and Defrance, T. (1999a). Activation sensitizes human memory B cells to B-cell receptor-induced apoptosis. Immunology 98, 47–54.

Defrance et al.

219

Berard, M., Mondiere, P., Casamayor-Palleja, M., Hennino, A., Bella, C., and Defrance, T. (1999b). Mitochondria connects the antigen receptor to effector caspases during B cell receptor-induced apoptosis in normal human B cells. J. Immunol. 163, 4655–4662. Besnault, L., Schrantz, N., Auffredou, M. T., Leca, G., Bourgeade, M. F., and Vazquez, A. (2001). B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein. J. Immunol. 167, 733–740. Billian, G., Mondiere, P., Berard, M., Bella, C., and Defrance, T. (1997). Antigen receptorinduced apoptosis of human germinal center B cells is targeted to a centrocytic subset. Eur. J. Immunol. 27, 405–414. Boldin, M. P., Mett, I. L., Varfolomeev, E. E., Chumakov, I., Shemer-Avni, Y., Camonis, J. H., and Wallach, D. (1995). Self-association of the “death domains” of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF and Fas/APO1 effects. J. Biol. Chem. 270, 387–391. Bouchon, A., Krammer, P. H., and Walczak, H. (2000). Critical role for mitochondria in B cell receptor-mediated apoptosis. Eur. J. Immunol. 30, 69–77. Brunner, T., Mogil, R. J., LaFace, D., Yoo, N. J., Mahboubi, A., Echeverri, F., Martin, S. J., Force, W. R., Lynch, D. H., Ware, C. F., et al. (1995). Cell-autonomous Fas (CD95)/Fasligand interaction mediates activation- induced apoptosis in T-cell hybridomas. Nature 373, 441–444. Carey, G. B., Donjerkovic, D., Mueller, C. M., Liu, S., Hinshaw, J. A., Tonnetti, L., Davidson, W., and Scott, D. W. (2000). B-cell receptor and Fas-mediated signals for life and death. Immunol. Rev. 176, 105–115. Catlett, I. M., and Bishop, G. A. (1999). Cutting edge: A novel mechanism for rescue of B cells from CD95/Fas-mediated apoptosis. J. Immunol. 163, 2378–2381. Catlett-Falcone, R., Landowski, T. H., Oshiro, M. M., Turkson, J., Levitzki, A., Savino, R., Ciliberto, G., Moscinski, L., Fernandez-Luna, J. L., Nunez, G., et al. (1999). Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity 10, 105–115. Chen, W., Wang, H. G., Srinivasula, S. M., Alnemri, E. S., and Cooper, N. R. (1999). B cell apoptosis triggered by antigen receptor ligation proceeds via a novel caspase-dependent pathway. J. Immunol. 163, 2483–2491. Cooke, M. P., Heath, A. W., Shokat, K. M., Zeng, Y., Finkelman, F. D., Linsley, P. S., Howard, M., and Goodnow, C. C. (1994). Immunoglobulin signal transduction guides the speciﬁcity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells. J. Exp. Med. 179, 425–438. Dechanet, J., Merville, P., Durand, I., Banchereau, J., and Miossec, P. (1995). The ability of synoviocytes to support terminal differentiation of activated B cells may explain plasma cell accumulation in rheumatoid synovium. J. Clin. Invest. 95, 456–463. Dhein, J., Walczak, H., Baumler, C., Debatin, K. M., and Krammer, P. H. (1995). Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature 373, 438–441. Fang, W., Weintraub, B. C., Dunlap, B., Garside, P., Pape, K. A., Jenkins, M. K., Goodnow, C. C., Mueller, D. L., and Behrens, T. W. (1998). Self-reactive B lymphocytes overexpressing Bcl-xL escape negative selection and are tolerized by clonal anergy and receptor editing. Immunity 9, 35–45. Ferlin, M., Noraz, N., Hertogh, C., Brochier, J., Taylor, N., and Klein, B. (2000). Insulin-like growth factor induces the survival and proliferation of myeloma cells through an interleukin6-independent transduction pathway. Br. J. Haematol. 111, 626–634. Ferlin-Bezombes, M., Jourdan, M., Liautard, J., Brochier, J., Rossi, J. F., and Klein, B. (1998). IFN-alpha is a survival factor for human myeloma cells and reduces dexamethasone-induced apoptosis. J. Immunol. 161, 2692–2699.

220

The Life and Death of a B Cell

Ferri, K. F., and Kroemer, G. (2001). Organelle-speciﬁc initiation of cell death pathways. Nat. Cell Biol. 3, E255–263. Foote, L. C., Marshak-Rothstein, A., and Rothstein, T. L. (1998). Tolerant B lymphocytes acquire resistance to Fas-mediated apoptosis after treatment with interleukin 4 but not after treatment with speciﬁc antigen unless a surface immunoglobulin threshold is exceeded. J. Exp. Med. 187, 847–853. Foote, L. C., Schneider, T. J., Fischer, G. M., Wang, J. K., Rasmussen, B., Campbell, K. A., Lynch, D. H., Ju, S. T., Marshak-Rothstein, A., and Rothstein, T. L. (1996). Intracellular signaling for inducible antigen receptor-mediated Fas resistance in B cells. J. Immunol. 157, 1878–1885. Freitas, A. A., and Rocha, B. (2000). Population biology of lymphocytes: The ﬂight for survival. Annu. Rev. Immunol. 18, 83–111. Freitas, A. A., Rosado, M. M., Viale, A. C., and Grandien, A. (1995). The role of cellular competition in B cell survival and selection of B cell repertoires. Eur. J. Immunol. 25, 1729– 1738. Galibert, L., Burdin, N., Barthelemy, C., Meffre, G., Durand, I., Garcia, E., Garrone, P., Rousset, F., Banchereau, J., and Liu, Y. J. (1996). Negative selection of human germinal center B cells by prolonged BCR cross-linking. J. Exp. Med. 183, 2075–2085. Garcia De Vinuesa, C., Gulbranson-Judge, A., Khan, M., O’Leary, P., Cascalho, M., Wabl, M., Klaus, G. G., Owen, M. J., and MacLennan, I. C. (1999). Dendritic cells associated with plasmablast survival. Eur. J. Immunol. 29, 3712–3721. Garside, P., Ingulli, E., Merica, R. R., Johnson, J. G., Noelle, R. J., and Jenkins, M. K. (1998). Visualization of speciﬁc B and T lymphocyte interactions in the lymph node. Science 281, 96–99. Goodnow, C. C., Cyster, J. G., Hartley, S. B., Bell, S. E., Cooke, M. P., Healy, J. I., Akkaraju, S., Rathmell, J. C., Pogue, S. L., and Shokat, K. P. (1995). Self-tolerance checkpoints in B lymphocyte development. Adv. Immunol. 59, 279–368. Gray, D., and Skarvall, H. (1988). B-cell memory is short-lived in the absence of antigen. Nature 336, 70–73. Gregory, C. D., Dive, C., Henderson, S., Smith, C. A., Williams, G. T., Gordon, J., and Rickinson, A. B. (1991). Activation of Epstein–Barr virus latent genes protects human B cells from death by apoptosis. Nature 349, 612–614. Grillot, D. A., Merino, R., Pena, J. C., Fanslow, W. C., Finkelman, F. D., Thompson, C. B., and Nunez, G. (1996). bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice. J. Exp. Med. 183, 381–391. Gross, J. A., Dillon, S. R., Mudri, S., Johnston, J., Littau, A., Roque, R., Rixon, M., Schou, O., Foley, K. P., Haugen, H., et al. (2001). TACI-Ig neutralizes molecules critical for B cell development and autoimmune disease: Impaired B cell maturation in mice lacking BLyS. Immunity 15, 289–302. Gross, J. A., Johnston, J., Mudri, S., Enselman, R., Dillon, S. R., Madden, K., Xu, W., ParrishNovak, J., Foster, D., Lofton-Day, C., et al. (2000). TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature 404, 995–999. Guo, B., Kato, R. M., Garcia-Lloret, M., Wahl, M. I., and Rawlings, D. J. (2000). Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex. Immunity 13, 243–253. Hahne, M., Kataoka, T., Schroter, M., Hofmann, K., Irmler, M., Bodmer, J. L., Schneider, P., Bornand, T., Holler, N., French, L. E., et al. (1998). APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth. J. Exp. Med. 188, 1185–1190. Hartley, S. B., Cooke, M. P., Fulcher, D. A., Harris, A. W., Cory, S., Basten, A., and Goodnow, C. C. (1993). Elimination of self-reactive B lymphocytes proceeds in two stages: Arrested development and cell death. Cell 72, 325–335.

Defrance et al.

221

Healy, J. I., and Goodnow, C. C. (1998). Positive versus negative signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 16, 645–670. Hennino, A., Berard, M., Casamayor-Palleja, M., Krammer, P. H., and Defrance, T. (2000). Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells. J. Immunol. 165, 3023–3030. Hennino, A., Berard, M., Krammer, P. H., and Defrance, T. (2001). FLICE-inhibitory protein is a key regulator of germinal center B cell apoptosis. J. Exp. Med. 193, 447–458. Hjorth-Hansen, H., Waage, A., and Borset, M. (1999). Interleukin-15 blocks apoptosis and induces proliferation of the human myeloma cell line OH-2 and freshly isolated myeloma cells. Br. J. Haematol. 106, 28–34. Ho, F., Lortan, J. E., MacLennan, I. C., and Khan, M. (1986). Distinct short-lived and long-lived antibody-producing cell populations. Eur. J. Immunol. 16, 1297–1301. Hoch, S., Boyd, M., Malone, B., Gonye, G., and Schwaber, J. (2000). Fas-mediated apoptosis eliminates B cells that acquire self-reactivity during the germinal center response to NP. Cell. Immunol. 203, 103–110. Jego, G., Bataille, R., and Pellat-Deceunynck, C. (2001). Interleukin-6 is a growth factor for nonmalignant human plasmablasts. Blood 97, 1817–1822. Jego, G., Robillard, N., Puthier, D., Amiot, M., Accard, F., Pineau, D., Harousseau, J. L., Bataille, R., and Pellat-Deceunynck, C. (1999). Reactive plasmacytoses are expansions of plasmablasts retaining the capacity to differentiate into plasma cells. Blood 94, 701– 712. Jiang, Y., Woronicz, J. D., Liu, W., and Goeddel, D. V. (1999). Prevention of constitutive TNF receptor 1 signaling by silencer of death domains. Science 283, 543–546. Ju, S. T., Panka, D. J., Cui, H., Ettinger, R., el-Khatib, M., Sherr, D. H., Stanger, B. Z., and Marshak-Rothstein, A. (1995). Fas(CD95)/FasL interactions required for programmed cell death after T- cell activation. Nature 373, 444–448. Kawano, M., Hirano, T., Matsuda, T., Taga, T., Horii, Y., Iwato, K., Asaoku, H., Tang, B., Tanabe, O., Tanaka, H., et al. (1988). Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 332, 83–85. Kawano, M. M., Mihara, K., Huang, N., Tsujimoto, T., and Kuramoto, A. (1995). Differentiation of early plasma cells on bone marrow stromal cells requires interleukin-6 for escaping from apoptosis. Blood 85, 487–494. ¨ Knodel, M., Kuss, A. W., Lindemann, D., Berberich, I., and Schimpl, A. (1999). Reversal of Blimp-1-mediated apoptosis by A1, a member of the Bcl-2 family. Eur. J. Immunol. 29, 2988–2998. Kondo, E., Yoshino, T., Nishiuchi, R., Sakuma, I., Nishizaki, K., Kayagaki, N., Yagita, H., and Akagi, T. (1997). Expression of Fas ligand mRNA in germinal centres of the human tonsil. J. Pathol. 183, 75–79. Koopman, G., Keehnen, R. M., Lindhout, E., Newman, W., Shimizu, Y., van Seventer, G. A., de Groot, C., and Pals, S. T. (1994). Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells. J. Immunol. 152, 3760–3767. Kosco-Vilbois, M. H., Zentgraf, H., Gerdes, J., and Bonnefoy, J. Y. (1997). To “B” or not to “B” a germinal center? Immunol. Today 18, 225–230. Krammer, P. H. (2000). CD95’s deadly mission in the immune system. Nature 407, 789–795. Lacronique, V., Mignon, A., Fabre, M., Viollet, B., Rouquet, N., Molina, T., Porteu, A., Henrion, A., Bouscary, D., Varlet, P., et al. (1996). Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice. Nat. Med. 2, 80–86. Lagresle, C., Mondiere, P., Bella, C., Krammer, P. H., and Defrance, T. (1996). Concurrent engagement of CD40 and the antigen receptor protects naive and memory human B cells from APO-1/Fas-mediated apoptosis. J. Exp. Med. 183, 1377–1388.

222

The Life and Death of a B Cell

Lam, K. P., Kuhn, R., and Rajewsky, K. (1997). In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90, 1073–1083. Lam, K. P., and Rajewsky, K. (1999). B cell antigen receptor speciﬁcity and surface density together determine B-1 versus B-2 cell development. J. Exp. Med. 190, 471–477. Lens, S. M., den Drijver, B. F., Potgens, A. J., Tesselaar, K., van Oers, M. H., and van Lier, R. A. (1998). Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells. J. Immunol. 160, 6083–6092. Li, T., Ramirez, K., and Palacios, R. (1996). Distinct patterns of Fas cell surface expression during development of T- or B-lymphocyte lineages in normal, scid, and mutant mice lacking or overexpressing p53, bcl-2, or rag-2 genes. Cell Growth Differ. 7, 107–114. Lindhout, E., and de Groot, C. (1995). Follicular dendritic cells and apoptosis: Life and death in the germinal centre. Histochem. J. 27, 167–183. Lindhout, E., Lakeman, A., and de Groot, C. (1995). Follicular dendritic cells inhibit apoptosis in human B lymphocytes by a rapid and irreversible blockade of preexisting endonuclease. J. Exp. Med. 181, 1985–1995. Liu, Y. J., Joshua, D. E., Williams, G. T., Smith, C. A., Gordon, J., and MacLennan, I. C. (1989). Mechanism of antigen-driven selection in germinal centres. Nature 342, 929–931. Lu, L., and Osmond, D. G. (1997). Apoptosis during B lymphopoiesis in mouse bone marrow. J. Immunol. 158, 5136–5145. Mackay, F., Woodcock, S. A., Lawton, P., Ambrose, C., Baetscher, M., Schneider, P., Tschopp, J., and Browning, J. L. (1999). Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J. Exp. Med. 190, 1697–1710. MacLennan, I. C. (1994). Germinal centers. Annu. Rev. Immunol. 12, 117–139. Manz, R. A., Thiel, A., and Radbruch, A. (1997). Lifetime of plasma cells in the bone marrow. Nature 388, 133–134. Marsters, S. A., Yan, M., Pitti, R. M., Haas, P. E., Dixit, V. M., and Ashkenazi, A. (2000). Interaction of the TNF homologues BLyS and APRIL with the TNF receptor homologues BCMA and TACI. Curr. Biol. 10, 785–788. Maruyama, M., Lam, K. P., and Rajewsky, K. (2000). Memory B-cell persistence is independent of persisting immunizing antigen. Nature 407, 636–642. Melchers, F., ten Boekel, E., Seidl, T., Kong, X. C., Yamagami, T., Onishi, K., Shimizu, T., Rolink, A. G., and Andersson, J. (2000). Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells. Immunol. Rev. 175, 33–46. Merino, R., Ding, L., Veis, D. J., Korsmeyer, S. J., and Nunez, G. (1994). Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes. EMBO J. 13, 683–691. Merino, R., Grillot, D. A., Simonian, P. L., Muthukkumar, S., Fanslow, W. C., Bondada, S., and Nunez, G. (1995). Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40 in WEHI-231 cells. Dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction. J. Immunol. 155, 3830–3838. Merville, P., Dechanet, J., Desmouliere, A., Durand, I., de Bouteiller, O., Garrone, P., Banchereau, J., and Liu, Y. J. (1996). Bcl-2+ tonsillar plasma cells are rescued from apoptosis by bone marrow ﬁbroblasts. J. Exp. Med. 183, 227–236. Moore, P. A., Belvedere, O., Orr, A., Pieri, K., LaFleur, D. W., Feng, P., Soppet, D., Charters, M., Gentz, R., Parmelee, D., et al. (1999). BLyS: Member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285, 260–263. Nemazee, D., and Buerki, K. (1989). Clonal deletion of autoreactive B lymphocytes in bone marrow chimeras. Proc. Natl. Acad. Sci. USA 86, 8039–8043. Newton, K., Harris, A. W., Bath, M. L., Smith, K. G., and Strasser, A. (1998). A dominant interfering mutant of FADD/MORT1 enhances deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes. EMBO J. 17, 706–718.

Defrance et al.

223

Newton, K., Harris, A. W., and Strasser, A. (2000). FADD/MORT1 regulates the pre-TCR checkpoint and can function as a tumour suppressor. EMBO J. 19, 931–941. Nilsson, N., Ingvarsson, S., and Borrebaeck, C. A. (2000). Immature B cells in bone marrow express Fas/FasL. Scand. J. Immunol. 51, 279–284. Nisitani, S., Tsubata, T., Murakami, M., Okamoto, M., and Honjo, T. (1993). The bcl-2 gene product inhibits clonal deletion of self-reactive B lymphocytes in the periphery but not in the bone marrow. J. Exp. Med. 178, 1247–1254. Noelle, R. J., Roy, M., Shepherd, D. M., Stamenkovic, I., Ledbetter, J. A., and Aruffo, A. (1992). A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc. Natl. Acad. Sci. USA 89, 6550–6554. Nomura, T., Han, H., Howard, M. C., Yagita, H., Yakura, H., Honjo, T., and Tsubata, T. (1996). Antigen receptor-mediated B cell death is blocked by signaling via CD72 or treatment with dextran sulfate and is defective in autoimmunity-prone mice. Int. Immunol. 8, 867–875. Norvell, A., Mandik, L., and Monroe, J. G. (1995). Engagement of the antigen-receptor on immature murine B lymphocytes results in death by apoptosis. J. Immunol. 154, 4404– 4413. Nunez, G., Hockenbery, D., McDonnell, T. J., Sorensen, C. M., and Korsmeyer, S. J. (1991). Bcl-2 maintains B cell memory. Nature 353, 71–73. Onel, K. B., Tucek-Szabo, C. L., Ashany, D., Lacy, E., Nikolic-Zugic, J., and Elkon, K. B. (1995). Expression and function of the murine CD95/FasR/APO-1 receptor in relation to B cell ontogeny. Eur. J. Immunol. 25, 2940–2947. Osmond, D. G. (1986). Population dynamics of bone marrow B lymphocytes. Immunol. Rev. 93, 103–124. Osmond, D. G., Rico-Vargas, S., Valenzona, H., Fauteux, L., Liu, L., Janani, R., Lu, L., and Jacobsen, K. (1994). Apoptosis and macrophage-mediated cell deletion in the regulation of B lymphopoiesis in mouse bone marrow. Immunol. Rev. 142, 209–230. Parry, S. L., Hasbold, J., Holman, M., and Klaus, G. G. (1994). Hypercross-linking surface IgM or IgD receptors on mature B cells induces apoptosis that is reversed by costimulation with IL-4 and anti-CD40. J. Immunol. 152, 2821–2829. Pettersson, M., Jernberg-Wiklund, H., Larsson, L. G., Sundstrom, C., Givol, I., Tsujimoto, Y., and Nilsson, K. (1992). Expression of the bcl-2 gene in human multiple myeloma cell lines and normal plasma cells. Blood 79, 495–502. Przylepa, J., Himes, C., and Kelsoe, G. (1998). Lymphocyte development and selection in germinal centers. Curr. Top. Microbiol. Immunol. 229, 85–104. Rathmell, J. C., and Goodnow, C. C. (1994). Effects of the lpr mutation on elimination and inactivation of self-reactive B cells. J. Immunol. 153, 2831–2842. Rathmell, J. C., Townsend, S. E., Xu, J. C., Flavell, R. A., and Goodnow, C. C. (1996). Expansion or elimination of B cells in vivo: Dual roles for CD40- and Fas (CD95)-ligands modulated by the B cell antigen receptor. Cell 87, 319–329. Rodriguez, I., Matsuura, K., Khatib, K., Reed, J. C., Nagata, S., and Vassalli, P. (1996). A bcl-2 transgene expressed in hepatocytes protects mice from fulminant liver destruction but not from rapid death induced by anti-Fas antibody injection. J. Exp. Med. 183, 1031–1036. Rolink, A. G., Schaniel, C., Andersson, J., and Melchers, F. (2001). Selection events operating at various stages in B cell development. Curr. Opin. Immunol. 13, 202–207. Rosado, M. M., and Freitas, A. A. (1998). The role of the B cell receptor V region in peripheral B cell survival. Eur. J. Immunol. 28, 2685–2693. Rothstein, T. L., Wang, J. K., Panka, D. J., Foote, L. C., Wang, Z., Stanger, B., Cui, H., Ju, S. T., and Marshak-Rothstein, A. (1995). Protection against Fas-dependent Th1-mediated apoptosis by antigen receptor engagement in B cells. Nature 374, 163–165. Scafﬁdi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K. J., Debatin, K. M., Krammer, P. H., and Peter, M. E. (1998). Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17, 1675–1687.

224

The Life and Death of a B Cell

Schiemann, B., Gommerman, J. L., Vora, K., Cachero, T. G., Shulga-Morskaya, S., Dobles, M., Frew, E., and Scott, M. L. (2001). An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293, 2111–2114. Schittek, B., and Rajewsky, K. (1990). Maintenance of B-cell memory by long-lived cells generated from proliferating precursors. Nature 346, 749–751. Schneider, P., MacKay, F., Steiner, V., Hofmann, K., Bodmer, J. L., Holler, N., Ambrose, C., Lawton, P., Bixler, S., Acha-Orbea, H., et al. (1999). BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J. Exp. Med. 189, 1747–1756. Schneider, T. J., Grillot, D., Foote, L. C., Nunez, G. E., and Rothstein, T. L. (1997). Bcl-x protects primary B cells against Fas-mediated apoptosis. J. Immunol. 159, 4834–4839. Slifka, M. K., and Ahmed, R. (1998). Long-lived plasma cells: A mechanism for maintaining persistent antibody production. Curr. Opin. Immunol. 10, 252–258. Slifka, M. K., Antia, R., Whitmire, J. K., and Ahmed, R. (1998). Humoral immunity due to long-lived plasma cells. Immunity 8, 363–372. Smith, K. G., Hewitson, T. D., Nossal, G. J., and Tarlinton, D. M. (1996). The phenotype and fate of the antibody-forming cells of the splenic foci. Eur. J. Immunol. 26, 444–448. Smith, K. G., Light, A., O’Reilly, L. A., Ang, S. M., Strasser, A., and Tarlinton, D. (2000). bcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells. J. Exp. Med. 191, 475–484. Smith, K. G., Nossal, G. J., and Tarlinton, D. M. (1995). FAS is highly expressed in the germinal center but is not required for regulation of the B-cell response to antigen. Proc. Natl. Acad. Sci. USA 92, 11628–11632. Smith, K. G., Weiss, U., Rajewsky, K., Nossal, G. J., and Tarlinton, D. M. (1994). Bcl-2 increases memory B cell recruitment but does not perturb selection in germinal centers. Immunity 1, 803–813. Strasser, A., Whittingham, S., Vaux, D. L., Bath, M. L., Adams, J. M., Cory, S., and Harris, A. W. (1991). Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA 88, 8661–8665. Su, L., and David, M. (1999). Inhibition of B cell receptor-mediated apoptosis by IFN. J. Immunol. 162, 6317–6321. Suda, T., Okazaki, T., Naito, Y., Yokota, T., Arai, N., Ozaki, S., Nakao, K., and Nagata, S. (1995). Expression of the Fas ligand in cells of T cell lineage. J. Immunol. 154, 3806–3813. Takahashi, Y., Cerasoli, D. M., Dal Porto, J. M., Shimoda, M., Freund, R., Fang, W., Telander, D. G., Malvey, E. N., Mueller, D. L., Behrens, T. W., and Kelsoe, G. (1999). Relaxed negative selection in germinal centers and impaired afﬁnity maturation in bcl-xL transgenic mice. J. Exp. Med. 190, 399–410. Takahashi, Y., Ohta, H., and Takemori, T. (2001). Fas is required for clonal selection in germinal centers and the subsequent establishment of the memory B cell repertoire. Immunity 14, 181– 192. Thompson, J. S., Bixler, S. A., Qian, F., Vora, K., Scott, M. L., Cachero, T. G., Hession, C., Schneider, P., Sizing, I. D., Mullen, C., et al. (2001). BAFF-R, a newly identiﬁed TNF receptor that speciﬁcally interacts with BAFF. Science 293, 2108–2111. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302, 575–581. van Eijk, M., and de Groot, C. (1999). Germinal center B cell apoptosis requires both caspase and cathepsin activity. J. Immunol. 163, 2478–2482. van Eijk, M., Medema, J. P., and de Groot, C. (2001). Cutting edge: Cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein protects germinal center B cells from apoptosis during germinal center reactions. J. Immunol. 166, 6473–6476. Verbeke, C. S., Wenthe, U., and Zentgraf, H. (1999). Fas ligand expression in the germinal centre. J. Pathol. 189, 155–160.

Defrance et al.

225

von Bulow, G. U., van Deursen, J. M., and Bram, R. J. (2001). Regulation of the T-independent humoral response by TACI. Immunity 14, 573–582. von Freeden-Jeffry, U., Vieira, P., Lucian, L. A., McNeil, T., Burdach, S. E., and Murray, R. (1995). Lymphopenia in interleukin (IL)-7 gene-deleted mice identiﬁes IL-7 as a nonredundant cytokine. J. Exp. Med. 181, 1519–1526. Wang, J., Lobito, A. A., Shen, F., Hornung, F., Winoto, A., and Lenardo, M. J. (2000). Inhibition of Fas-mediated apoptosis by the B cell antigen receptor through c-FLIP. Eur. J. Immunol. 30, 155–163. Wu, Y., Bressette, D., Carrell, J. A., Kaufman, T., Feng, P., Taylor, K., Gan, Y., Cho, Y. H., Garcia, A. D., Gollatz, E., et al. (2000). Tumor necrosis factor (TNF) receptor superfamily member TACI is a high afﬁnity receptor for TNF family members APRIL and BLyS. J. Biol. Chem. 275, 35478–35485. Xu, F. H., Sharma, S., Gardner, A., Tu, Y., Raitano, A., Sawyers, C., and Lichtenstein, A. (1998). Interleukin-6-induced inhibition of multiple myeloma cell apoptosis: Support for the hypothesis that protection is mediated via inhibition of the JNK/SAPK pathway. Blood 92, 241–251. Yan, M., Wang, H., Chan, B., Roose-Girma, M., Erickson, S., Baker, T., Tumas, D., Grewal, I. S., and Dixit, V. M. (2001). Activation and accumulation of B cells in TACI-deﬁcient mice. Nat. Immunol. 2, 638–643. Yeh, W. C., Hakem, R., Woo, M., and Mak, T. W. (1999). Gene targeting in the analysis of mammalian apoptosis and TNF receptor superfamily signaling. Immunol. Rev. 169, 283– 302. Zhang, J., Cado, D., Chen, A., Kabra, N. H., and Winoto, A. (1998). Fas-mediated apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD/Mort1. Nature 392, 296–300. Zheng, L., Fisher, G., Miller, R. E., Peschon, J., Lynch, D. H., and Lenardo, M. J. (1995). Induction of apoptosis in mature T cells by tumour necrosis factor. Nature 377, 348–351.

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Index

A Ab response, see Antibody response Acetylation, histones, 43–44, 56–57 Afﬁnity maturation, Ab response, 208–212 Aﬂatoxin B1, human hepatocarcinogenesis, 76–77 Alphaviruses, HPV-induced carcinoma immunotherapy, 128 Antibody response afﬁnity maturation, 208–212 immune response onset, 206–208 Apoptosis BCR-induced signals, 200–201 GC B cells, 209–212 APRIL, B cell homeostasis, periphery, 203–204

B Bacterial vector-based vaccines, 130–131 BAFF, B cell homeostasis, periphery, 203–204 B cells Ab response, immune response onset, 206–208 GC apoptotic pathways, 209–212 survival, follicular dendritic cells, 212 homeostasis regulation immune response peak, 205–206 periphery immune response induction, 202–205 primary lymphoid organs, 201–202 lymphopoiesis, 197–198 negative selection mechanisms death receptor pathway, 199–200 mitochondrial pathway, 198–199 overview, 196 survival regulation memory B cell pool, 212–213

plasma cell pool, 214–215 plasma cell regulators, 215–216 BCR B cell homeostasis, periphery, 202–203 B cell homeostasis regulation, 201 induced apoptotic signals, 200–201 ligation, CD95 resistance, 208 memory B cell compartment, 213 Biomarkers, T-cell responses in cancer patients, 163–171

C E-Cadherin, hepatocellular carcinoma, 82–83 Cancer patients, T-cell responses alternate effector functions, 173–176 antitumor restricted, CD8 role, 158–160 biomarkers vs. surrogate markers, 163–171 CD8 role, 158–160 crossreactivity, 172–173 immune effector functions deﬁnition, 149–151 MHC–peptide complexes, 152–153 T-cell avidity measurement, 160–163 tumor-derived peptides, 153–158 Carcinogenesis, HPV-associated, HLA polymorphisms, 122–123 Carcinomas, see Hepatocellular carcinoma; Human papillomavirus-induced carcinomas β-Catenin, hepatocellular carcinoma, 82–83 CBP, see CREB-binding protein CD4 helper response, HPV, 121–122 CD8 antitumor restricted T-cell responses, 158–160 T-cell avidity measurement, 160–163 T-cell responses in cancer patients, 164–165, 167–168, 170

227

228 CD95 B cells, 207–208 BCR ligation, 208 cdks, see Cyclin dependent kinases Cell cycle CBP/p300 regulation, 48 histone deacetylase regulation cancer, 50–51 Mad/Max, 49 overview, 48–49 retinoblastoma protein, 49–50 Cell cycle regulatory proteins, 80–81 Cell growth, hepatocellular carcinoma, 88–89 Cell size, PI 3-K, mTOR, translation, 22–24 Cell survival, B cells GC, follicular dendritic cells, 212 homeostasis, periphery, 204–205 memory B cell pool, 212–213 plasma cell pool, 214–215 plasma cell regulators, 215–216 Cellular immunity, HPV activation, 116–118 carcinogenesis, 122–123 CD4 helper response, 121–122 cervical cancer, 118–121 Cervical cancer, HPV-induced, immune invasion, 118–121 Chimeric virus-like particles, 129–130 Chromatin, histone acetylation, 56–57 COX-2, see Cyclooxygenase 2 CREB-binding protein cell cycle regulation, 48 characteristics, 46–47 hematopoietic disorders, 54–55 MyoD interaction, 51–52 p53 interaction, 47–48 CTLs, see Cytotoxic T lymphocytes CVLP, see Chimeric virus-like particles Cyclin dependent kinases, hepatocellular carcinoma, 80–81 Cyclins, hepatocellular carcinoma, 80–81 Cyclooxygenase 2, hepatocellular carcinoma, 87, 91 Cytotoxic T lymphocytes HPV-induced carcinoma alphaviruses, 128 bacterial vector-based vaccines, 130–131 dendritic cells, 133 overview, 124

Index

peptide vaccines, 131–132 plasmid DNA immunizations, 134–136 vaccinia delivery, 127 virus-like particles, 129 HPV-induced cervical cancer, 118–119 tumor-derived peptides, 154–155

D DCs, see Dendritic cells Death receptor, B cell negative selection, 199–200 Dendritic cells follicular, GC B cell survival, 212 HPV-induced carcinoma immunotherapy, 133–134 DF, see Dysplastic foci DN, see Dysplastic nodules DNA packaging, overview, 42 Dysplastic foci, human hepatocarcinogenesis, 69, 71 Dysplastic nodules, human hepatocarcinogenesis, 71

E 4E-BP1, see Eukaryotic initiation factor-4E-binding protein 1 eIF, see Eukaryotic initiation factors ¨ EKLF, see Erythroid Kruppel-like factor Endothelial growth factor, hepatocellular carcinoma, 84–85 Epidemiology, HPV infection, 114–115 ¨ Erythroid Kruppel-like factor, hematopoiesis, 53–54 Eukaryotic initiation factor-4E-binding protein, eIF-4E, 9–10 Eukaryotic initiation factor-4E-binding protein 1 eIF-4E regulation, 10–12 liver regeneration, 21–22 Eukaryotic initiation factors eIF-2B, PI 3-K and mTOR regulation, 10 eIF-4B, PI 3-K and mTOR regulation, 10 eIF-4G, PI 3-K and mTOR regulation, 10 eIF-4E, capped mRNA, 8–10 eIF-4E, 4E-BP1 regulation, 10–12

F FKBP and rapamycin-associated protein, see Mammalian target of rapamycin

Index

FMR-1, histone acetylation, 56 Follicular dendritic cells, GC B cell survival, 212 FRAP, see Mammalian target of rapamycin

G GATA-1, hematopoiesis, 53 Gcn5-related N-acetyltransferases, 44 GC reaction, B cells apoptotic pathways, 209–212 survival, follicular dendritic cells, 212 Genomic translocations, leukemias, 54–55 GNAT-family, see Gcn5-related N-acetyltransferases

H HATs, see Histone acetyltransferases HBV, see Hepatitis B virus HCC, see Hepatocellular carcinoma HCV, see Hepatitis C virus HDACs, see Histone deacetylases Hematopoiesis EKLF, 53–54 GATA-1, 53 Hematopoietic disorders, CBP/p300, 54–55 Hemochromatosis, human hepatocarcinogenesis, 77 Hepatitis B virus hepatocellular carcinoma, 90 human hepatocarcinogenesis, 73–75 Hepatitis C virus, human hepatocarcinogenesis, 75–76 Hepatocarcinogenesis, human, morphology, 69–72 Hepatocellular carcinoma aﬂatoxin B1, 76–77 E-cadherin/wnt/β-catenin, 82–83 cell cycle regulatory proteins, 80–81 cell growth, 88–89 characteristics, 67–68 clinical diagnosis, 68–69 general mechanisms, 72–73 genomic alterations, 77–78 hemochromatosis, 77 hepatitis B virus, 73–75 hepatitis C virus, 75–76 invasion and metastasis, 89–90 mitoinhibitory pathways, 85–86

229 oncogenic molecular cross-talk, 87–88 p53 alterations, 79–80 pleiotropic growth factors, 83–85 protumorigenic changes, 86–87 therapeutic implications, 90–91 Hepatocyte growth factor, hepatocellular carcinoma, 85 HGF, see Hepatocyte growth factor Histone acetyltransferases CBP/p300 and cell cycle regulation, 48 CBP/p300 family characteristics, 46–47 CBP/p300–p53 interaction, 47–48 characteristics, 43–44 GNAT-family, 44 MYST-family, 44–46 Histone deacetylases cell cycle regulation cancer, 50–51 Mad/Max, 49 overview, 48–49 retinoblastoma protein, 49–50 MEF2 interaction, 52 MyoD association, 52 Histones, acetylation, 43–44, 56–57 HLA, see Human leukocyte antigen HPV, see Human papillomavirus Human cancers histone deacetylases, 50–51 PI 3-K and mTOR pathways, 28–29 Human hepatocarcinogenesis, morphology, 69–72 Human leukocyte antigen HPV-induced carcinoma immunotherapy, 132 HPV-induced carcinoma murine models, 125 HPV-induced cervical cancer, 118 immunology to HPV assault, 116 polymorphisms, 122–123 tumor-derived peptides, 154–158 Human papillomavirus cellular immunity activation, 116–118 carcinogenesis, 122–123 CD4 helper response, 121–122 cervical cancer, 118–121 immunology to assault, 115–116 infection epidemiology, 114–115 molecular genetics, 115 overview, 113–114

230 Human papillomavirus-induced carcinomas, immunotherapy alphaviruses, 128 approaches, 126 bacterial vector-based vaccines, 130–131 dendritic cells, 133–134 murine models, 125 peptide vaccines, 131–132 plasmid DNA immunizations, 134–136 rationale, 123–124 vaccinia delivery, 127–128 virus-like particles, 129–130 Huntington’s disease, characteristics, 55–56

I

IFN-γ , see Interferon γ IGF-II, see Insulin-like growth factor II IGF-IIR, see Insulin-like growth factor IIR IL-2, see Interleukin-2 Immune effector, functions, deﬁnition, 149–151 Immune invasion, HPV-induced cervical cancer, 118–121 Immune response Ab response, immune response onset, 206–208 B cell homeostasis in periphery BAFF and APRIL role, 203–204 BCR role, 202–203 peak, 205–206 survival signals, 204–205 HPV, 115–116 Immunizations, HPV-induced carcinoma immunotherapy, 134–136 Immunotherapy, HPV-induced carcinomas alphaviruses, 128 approaches, 126 bacterial vector-based vaccines, 130–131 dendritic cells, 133–134 murine models, 125 peptide vaccines, 131–132 plasmid DNA immunizations, 134–136 rationale, 123–124 vaccinia delivery, 127–128 virus-like particles, 129–130 Insulin-like growth factor II, hepatocellular carcinoma, 83–84 Insulin-like growth factor IIR, hepatocellular carcinoma, 86

Index Interferon γ , HPV-induced cervical cancer, 120 Interleukin-2, HPV-induced cervical cancer, 119 Interleukin-6, plasma cell survival, 215 Invasion, hepatocellular carcinoma, 89–90

K Knockout mouse, CBP/p300, hematopoietic disorders, 54

L LAMP-1, see Lysosome-associated membrane protein 1 Leukemias genomic translocations, 54–55 MYST-family members, 55 Liver regeneration, coordinated growth and proliferation, 21–22 Lymphoid organs, B cell homeostasis regulation, 201–202 Lymphopoiesis, B cells, 197–198 Lysosome-associated membrane protein 1, 128

M Mad/Max, histone deacetylases, 49 Major histocompatibility complex immune effector function, 150–151 immunology to HPV assault, 116 peptide complexes, tumor cell recognition, 152–153 Mammalian target of rapamycin alternate names, 3–5 cell size, 22–24 4E-BP1 regulation, 11–12 energy levels, 5–6 mRNA splicing and translation, 20–21 mTOR pathway coordination, 24–28 nutrient regulation, 6–7 pathways in cancer, 28–29 PI 3-K signal integration, 3 PI 3-K C-terminal domain, 5 S6K1 regulation overview, 12–15 S6K1 regulation subcellular localization, 17 serum mitogens, 7 translation initiation factors, 10

231

Index

MEF2, histone deacetylase interaction, 52 Melan-A/MART-1, T-cell responses in cancer patients, 164, 166, 170–171 Memory B cell compartment, survival regulation, 212–213 Messenger RNA capped, eIF-4E, 8–10 splicing and translation, 20–21 5 TOP, 7–8 MET, hepatocellular carcinoma, 86 Metastasis, hepatocellular carcinoma, 89–90 MHC, see Major histocompatibility complex Mitochondria, B cell negative selection, 198–199 Mitoinhibitory pathways, hepatocellular carcinoma, 85–86 mTOR pathway, see Mammalian target of rapamycin Murine models, HPV-induced carcinomas, 125 Muscle differentiation, MyoD interactions, 51–52 MyoD CBP/p300 and PCAF interactions, 51–52 histone deacetylase association, 52 MYST-family characteristics, 44–46 leukemias, 55

N Negative selection, B cells death receptor receptor, 199–200 mitochondrial pathway, 198–199 Nutrient regulation, mTOR homologs, 6–7

P p53 alterations in HCC, 79–80 CBP/p300 interaction, 47–48 p300 cell cycle regulation, 48 characteristics, 46–47 hematopoietic disorders, 54–55 MyoD interaction, 51–52 p53 interaction, 47–48 PC, see Plasma cell pool PCAF, MyoD interaction, 51–52

PDK-1 S6K1 regulation, 15–16 S6K1 Thr389 regulation, 14–15 Peptides MHC complexes, tumor cell recognition, 152–153 tumor-derived, recognition by T cells, 153–158 Peptide vaccines, HPV-induced carcinoma immunotherapy, 131–132 Periphery, B cell homeostasis regulation immune response induction, 202–205 immune response peak, 205–206 Phosphoinositide 3-kinase cell size, 22–24 4E-BP1 regulation, 11–12 liver regeneration, 21–22 mRNA splicing and translation, 20–21 mTOR C-terminal domain homology, 3 mTOR pathway coordination, 24–28 mTOR signal integration, 3 overview, 2–3 pathways in cancer, 28–29 S6K1 regulation overview, 12–15 S6K1 regulation subcellular localization, 15–17 translation initiation factors, 10 PI 3-K, see Phosphoinositide 3-kinase Plasma cell pool, B cells molecular regulators, 215–216 overview, 214–215 Plasmid DNA immunizations, HPV-induced carcinoma immunotherapy, 134–136 Pleiotropic growth factors, hepatocellular carcinoma, 83–85 Polymorphisms, human leukocyte antigen, 122–123 Protein kinase C, S6K1 regulation, 15–17 Protumorigenic changes, hepatocellular carcinoma, 86–87 PTEN, PI 3-K mTOR, translation, 23 overview, 2–3

R RAFT, see Mammalian target of rapamycin Rapamycin and FKBP12 target, see Mammalian target of rapamycin RAPT, see Mammalian target of rapamycin RB, see Retinoblastoma protein

232 Retinoblastoma protein, histone deacetylases, 49–50

S S6K1, see S6 kinase-1 S6K2, see S6 kinase-2 S6 kinase-1 mRNA splicing and translation, 20 PI 3-K, mTOR, translation, 22–24 regulation by PI 3-K and mTOR overview, 12–15 subcellular localization, 15–17 S6K2 regulation similarities, 18–20 5 TOP mRNA, 8 S6 kinase-2, regulation, 18–20 Semliki Forest virus, HPV-induced carcinoma immunotherapy, 128 Serine-65, 4E-BP1, regulation, 11–12 Serum mitogens, mTOR homologs, 7 SFV, see Semliki Forest virus Surrogate markers, T-cell responses in cancer patients, 163–171

T T cell receptor ex vivo T cell avidity, 160–163 HPV-induced cervical cancer, 120 T-cell responses, cancer patients alternate effector functions, 173–176 antitumor restricted, CD8 role, 158–160 biomarkers vs. surrogate markers, 163–171 CD8 role, 158–160 crossreactivity, 172–173 immune effector functions deﬁnition, 149–151 MHC–peptide complexes, 152–153 T-cell avidity measurement, 160–163 tumor-derived peptides, 153–158 T cells ex vivo avidity, measurement tools, 160–163 recognized tumor-derived peptides, 153–158 tumor cell recognition, MHC–peptide complexes, 152–153 TCR, see T cell receptor 5 Terminal oligopyrimidine tract, mRNA, 7–8 TGF, see Transforming growth factors

Index

Th, see T help T help, HPV, 121–122 Therapy, hepatocellular carcinoma, 90–91 Threonine residue 70, 4E-BP1 regulation, 11–12 residue 389, S6K1 regulation, 14–15 5 TOP, see 5 Terminal oligopyrimidine tract Transforming growth factors TGFα, hepatocellular carcinoma, 84 TGFβ hepatocellular carcinoma, 85 HPV-induced cervical cancer, 119–120 Translational control liver regeneration, 21–22 mRNA splicing coordination, 20–21 PI 3-K, mTOR, cell size, 22–24 Translational regulatory pathways mTOR pathway, 3–7 PI 3-K pathway, 2–3 Translation initiation factors, see Eukaryotic initiation factors Translation regulatory pathways capped mRNA and eIF-4E, 8–10 5 TOP mRNA and S6K1, 7–8 translation initiation factors, 10 Tumor antigens, classiﬁcation, 153–158 Tumor cells, T-cell-mediated recognition, MHC–peptide complexes, 152–153 Tumors, dervied peptides, recognition by T cells, 153–158

V Vaccines, HPV-induced carcinoma immunotherapy, 130–132 Vaccinia delivery, HPV-induced carcinoma immunotherapy, 127–128 Vascular endothelial growth factor, hepatocellular carcinoma, 84–85 VEE, see Venezuelan equine encephalitis VEGF, see Vascular endothelial growth factor Venezuelan equine encephalitis, HPV-induced carcinoma immunotherapy, 128 Virus-like particles, HPV-induced carcinoma immunotherapy, 129–130 VLPs, see Virus-like particles

W Wingless, hepatocellular carcinoma, 82–83 Wnt, see Wingless