Foreign DNA in Mammalian Systems

Walter Doerfler

Foreign DNA in Mammalian Systems

8WILEY-VCH

Walter Doerfler

Foreign DNA in Mammalian Systems

8WILEY-VCH Weinheim ' New York . Chichester . Brisbane . Singapore * Toronto

Prof. Dr. med. Walter Doerfler Institut fur Genetik Universitat zu Koln Weyertal 121 D-50931 Koln Germany

This book was carefully produced. Nethertheless, author and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Cover illustration: FISH of spread chromosomes from the Ad12-transformed hamster cell line T637. A mixture of the biotinylated Pstt-fragment D probe of Ad12 DNA and the digoxygenin-labeled IAPI cellular DNA probe was applied. Chromosomal DNA was counterstained with DAPI (4'-6-diamidino2-phenylindole dihydrochloride). The arrowhead designates Ad12 DNA. Intensely pink signals visualize multiple copies of IAP retrotransposons (Heller et al., 1905). This photograph was taken hy Petra Wilgenbus, then at the lnstitut fur Genetik in Koln (from Heller et al., 1995). For technical reasons, Figures 3, 10, 14,16,21,29,30,35 and 37 are reproduced as color plates between pages 65-75.

Library of Congress Card No. applied for A catalogue record for this book is available from the British Library

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0WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany), 2000 Printed on acid-free and chlorine-free paper All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book. even when not specifically marked as such are not to be considered unprotected by law. Composition: Kuhn & Weyh, D-79111 Freiburg Printing: betz-druck GmbH. D-64291 Darmstadt Bookbinding: Wilh. Osswald + Co., D-67433 Neustadt Printed in the Federal Republic of Germany

Dedicated to David S. Hogness, Stanford University Medical School, Stanford CA, in memoriam Igor Tamm, Rockefeller University, New York NY, and Wolfram Zillig, Max-Planck-Institut fur Biochemie Munchen-Martinsried, my former mentors in Molecular Biology, and to Kathryn and in memoriam Hilton S. Read, Thomasville G k the Ventnor Foundation, who introduced many of us to tht United States of America.

The joy of science is in the j c not in the arrival,

Preface It is unlikely that the established genomes of present-day organisms remaii pletely stable. Occasionally, foreign DNA can gain entry into individual c an organism. Foreign DNA is defined as genetic material that derives another organism of the same or a different species. The natural environn heavily “contaminated” with foreign DNA. Mammals, like all other orga are constantly exposed to foreign DNA in their environments, most freq through their daily food supply. By necessity the gastrointestinal tract (mammalian) organisms is permanently in contact with foreign DNA. Vim? ubiquitous parasites and well equipped with mechanisms that have evolve1 millenia to introduce their genomes into established organisms. The del plant and animal organisms is a further abundant source of foreign genoml their fragments which are ready to be processed in the long-term cycle of b cal reutilization. So far, next to nothing is known about defense mechz against the intrusion of foreign DNA in mammals. At least in cells growing ture, the uptake and genomic fixation via integration of foreign DNA can r be demonstrated. For a number of reasons, investigations on the phenomer mechanisms involved in the interaction of foreign DNA with mammaliai and organisms will prove important. During the evolution of organisms thes cesses have likely played a crucial role, although this presumption is diffic prove. For the following fields of research the topics of uptake and fate of fc DNA are particularly relevant: Gene transfer and gene therapy, the generat transgenic organisms including the problematic “knock-in” and “knocl experiments, gene technology and biotechnology, viral oncology, and DNA nes. In fact, almost all areas of research in experimental biology and moll medicine are related to a fascinating array of problems emanating fro1 uptake, fixation and eventual fate of foreign DNA. In these experiments, fc DNA is introduced into cells or organisms under different regimens 01 various investigative or therapeutic aims. For more than three decades, my laboratory has pursued problems closel! ed to the fate of foreign DNA in mammalian cells and organisms. In parti many aspects of the integration of adenovirus DNA in the genomes of ma1 ian cells in culture and in adenovirus type 12 (Adl2)-induced tumor cells been investigated. The phenomena and mechanisms recorded in the cou these studies resemble very closely those encountered when foreign DN, been genomically fixed in mammalian cells by various protocols of gene tra Thus, mammalian cells avail themselves of a limited number of reactions they have to cope with foreign DNA. Nevertheless, several molecular rr

VIII

Prefuce

nisms of entry into the nucleus of the cell and of insertion into the established genome of recipient cells may exist. The outcome of the insertional recombination reaction is very similar in any of these events: the integration of a fragment or of an entire foreign DNA molecule(s) that has found access to the chromatin of the recipient cell. In the long-term planning of projects aimed at elucidating mechanisms of foreign DNA integration in mammalian cells, it appeared sensible to concentrate on one system and to study it in detail. Upon the entry of foreign DNA into the mammalian cell nucleus, two routes of further processing exist, the episomal persistence of free DNA in the linear or circular form or insertion into the host genome by covalent linkage. We cannot predict a priori what factors determine this two-way decision. The presence of an origin of DNA replication on the foreign DNA molecule functional in mammalian cells, the stage in the cell cycle or the recombinatorial capacity of the recipient cell can influence this decision. Convenient access to the world literature via the electronic media with a multitude of literature retrieval programs renders the composition of conventional reviews less attractive. Therefore, the aim of this book is not the compilation of the vast literature on this or related topics. I intend instead to present a book which will incorporate relevant data from many laboratories. A t the same time, a balanced account and interpretation of work performed in my own laboratory will form the core of this book. A book as this one will not only be of interest to specialists in the field, but to a wider audience of researchers in molecular biology and medicine. Since almost all fields of biology and experimental medicine have to utilize the techniques of gene transfer, the fate of the DNA transfected into mammalian cells can be of paramount importance for the design and success of many experimental approaches. In part of this treatise, I have addressed the more general, less theoretical needs and interests of researchers who use concepts and techniques of molecular biology for applied research goals. Even towards the close of this century in which science has made outstanding contributions to all fields of human endeavor acceptance of gene technology and gene therapy by the general public remains a vexing problem in some countries. The documentation that foreign DNA naturally enters into and can persist in the gastrointestinal tracts of organisms may help the lay public to evaluate the everyday facts of life more realistically. Improved information could mitigate their apprehensions of and bias against modern biology and medicine. As in any field. education and research are the keys to the understanding and the appreciation of the phantastic array of complexities that life offers to all of us every day. Koln/Weissenburg, October 1999

Walter Doerfler

Contents

Preface VII

1 Introduction 1 1.1

Further reading 3

2 ForeignDNA 2.1 2.2 2.3

5

Foreign DNA in the environment 5 Uptake and modes of persistence of foreign Db cells 6 Further reading 11

3 Methods to detect integrated foreign DNA 3.1 3.2 3.3 3.4 3.5 3.6 3.7

13

Parameters of foreign DNA integration 13 The fluorescent in situ hybridization method 1 Detection of foreign DNA by Southern DNA ti DNA hybridization 15 Detection of foreign DNA sequences by the pol reaction (PCR) 20 Recloning of junction fragments and the detern nucleotide sequences 22 Equilibirum sedimentation in alkaline CsCl gral DNA hybridization 24 Further reading 26

4 The adenovirus paradigm 27 4.1 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.2 4.3 4.3.1 4.3.2 4.3.3

An introduction to the adenovirus system 27 Clinical background 27 Virion structure 28 Classification 29 Multiplication during productive infection 30 Abortive infection 32 A n example: Ad12 DNA integration in the Ad1 hamster cell line T637 32 The state of the viral DNA in different cell systc Productive infection 34 Abortive infection 34 Transformation of cells in culture 35

X

Content.7

4.3.4 4.3.5 4.3.6 4.4 4.5 4.6 4.7

Tumor induction by Ad12 in newborn hamsters 36 Tumor cells in culture 36 Loss of previously integrated Ad12 DNA sequences 37 Adenovirus infection, DNA transfection or DNA microinjec Integration of adenovirus DNA in human cells - significance human somatic gene therapy 40 Studies on integrative recombination of adenovirus DNA in free system 41 Further reading 43

5 Conclusions derived from a survey of junction sites 47 5.1 5.2 5.3 5.4

On the characteristics of junction sequences 47 Persistence of integrated foreign DNA - a novel functional t insertional mutagenesis 51 Adenovirus DNA: chromosomal association - covalent genc integration 52 Further reading 55

6 Adenovirus-inducedtumor cells and revertants 57 6.1 6.2 6.2.1 6.2.2 6.3

Clonal origin of Ad12-induced tumors 57 Stability - instability 57 Hit-and-run mechanism of viral oncogenesis? 60 General implications of a hit-and-run mechanism 61 Further reading 61

7 Comparisons with other viral systems 63 7.1 7.2 7.3 7.4 7.5 7.6 7.6.1 7.6.2 7.6.3 7.7

Integration of viral DNA 63 Transcription of integrated viral genomes 64 Virus-induced tumors 79 Replication and integration of the retroviral genome 82 Endogenous retroviral genomes 84 The viral archetype: integration of bacteriophage h DNA t The most important regulatory functions in the phage h genc Control of transcription at the right operator ORof phage h D N A 90 A closer look at the integration and excision of the bacteriol hgenome 92 Further reading 95

8 Non-viral systems 97 8.1 8.2

Exchange of genetic information with extracellular DNA in pneumococci 98 IS elements and transposons 98

Contents

8.3 8.4 8.5 8.6 8.7

XI

Thoughts on the mechanism of foreign DNA integration 99 Expression of integrated foreign DNA 100 Fixation of foreign DNA in transgenic animals 101 Critical evaluation of the results obtained with transgenic animals 103 Further reading 104

9 Patterns of DNA methylation in the human and in viral genomes 105 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.7.1 9.7.2 9.7.3 9.7.4 9.7.5 9.7.6 9.7.7 9.8 9.8.1 9.8.2 9.8.3 9.8.4 9.8.5 9.9

Introduction 105 Methods available for the study of DNA methylation 106 Patterns of DNA methylation 109 A long-term signal for promoter inactivation 111 DNA methylation, an important parameter in genetic imprinting 114 Chromatin structure and patterns of DNA methylation 115 Patterns of DNA methylation in selected segments of the human genome 116 Tumor necrosis factors a and p 116 Human gene for the interleukin-2 receptor a chain (IL-2Ra) 116 Prader-Labhart-WilWAngelman syndrome region on human chromosome 15qll-13 117 Alu sequences 118 Wide range of human randomly selected DNA sequences 118 Selected human genes in different Hodgkin’s lymphoma and leukemia cell lines and in normal human lymphocytes 119 R E T protooncogene 119 Patterns of methylation in viral DNA 120 Human adenovirus types 2 and 12 120 Frog virus 3 123 Autographa californica nuclear polyhedrosis virus (AcNPV) 123 Human cytomegalovirus 124 General considerations 124 Further reading 125

10 Consequences of foreign DNA integration and persistence

129

De ylovo methylation of integrated foreign DNA 129 De ylovo methylation of targeted versus randomly integrated foreign genes 131 10.3 The insertion of foreign DNA into an established mammalian genome can alter patterns of methylation in cellular genome segments 134 10.3.1 Introduction 134 10.3.2 Integration of Ad12 DNA or bacteriophage h DNA into the hamster genome: consequences for the stability of the targeted genome 135 10.3.3 Alterations in cellular methylation patterns 137

10.1 10.2

Contents

XI1

10.3.4 Foreign DNA integration and cellular chromatin structure 141 10.3.5 General implications 142 10.4 De novo methylation of foreign DNA - a hypothetical ancient defense mechanism 143 10.5 Malignancy - a chromatin disease? 144 10.6 Further reading 146

11 Uptake of foreign D N A from the environment: the gastrointestinal tract and the placenta as portals of entry 147 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8

Summary 147 Foreign DNA is abundant in our environment 147 The epithelia of the gastrointestinal tract are constantly exposed to large amounts of foreign DNA and proteins 148 Foreign DNA orally ingested by mice reaches peripheral white blood cells, spleen and liver via the intestinal epithelia and can be covalently linked to mouse DNA 149 The fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus 152 Possible functional consequences 154 Concerns for and fears of foreign DNA by the public 155 Further reading 156

12 Relevance in applied molecular biology: an overview 159 12.1 12.2 12.3 12.4

Gene transfer to mammalian cells via artificial chromosomes 159 Human somatic gene therapy 160 Injection of promoter-fused gene constructs into animals and DNA vaccines 162 Further reading 163

13 Future research 165 13.1 13.2 13.3 13.4 13.5 13.6

The mechanism of the uptake of foreign DNA by mammalian cells 165 The fate of foreign DNA upon injection into animals 165 Response against foreign DNA 166 Mechanisms of foreign DNA integration into the host genome 166 The secrets of de novo methylation 166 Alterations of methylation in cellular DNA segments upon foreign DNA insertion 167

14 Studies on the biological significance of D N A methylation by using adenovirus D N A as a model 169 14.1

De novo methylation of integrated adenovirus DNA 169

Contents

14.2 14.3 14.4 14.5

Inverse correlations between the extent of (promoter) methylation and the state of promoter activity 170 Reconstruction experiments: gene transfer and transient or long-term expression of unmethylated or premethylated promoter-indicator gene constructs 170 Release of the inhibitory effect of promoter methylation by gene products of the adenovirus E l region or by the presence of a strong enhancer from human cytomegalovirus in the construct 171 Bibliography: DNA methylation in the adenovirus and related systems 171

Acknowledgments Index

XI11

177

Color plates 65-75

174

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

1 Introduction After intensive research on the fate of foreign DNA in mammalian systems, many questions have become more amenable to analyses at the molecular level and can now be phrased more precisely and investigated with refined concepts and very sensitive novel technology. It is the aim of this monograph not only to attempt a summary of the current status of research in this challenging area but to emphasize that this field offers many important applications in molecular biology and biomedical research with interesting possibilities for further basic work. Foreign DNA can enter mammalian cells via different routes. Viral infections provide a highly specialized and efficient way into selected host cells and organisms. Many viral systems utilize the integration of their genomes into the host’s chromosomal DNA as a means to fix their genomes permanently in an environment that provides a milieu in which the viral genomes can replicate and/or become transcribed under optimized conditions. Under these premises, some viral genomes have cohabited with the host genome for millions of years. The ubiquity of multiple copies of endogenous retroviral genomes in all mammalian, possibly in many vertebrate genomes investigated for the presence of such companion genomes attests to the success of this strategy. Can one imagine a more intimate association of foreign genomes with that of the host than parasitic insertion into the genome of ones prey or predator? Foreign, food-ingested DNA is not completely degraded in the gastrointestinal tract but can persist in fragmented form in the intestinal tract at least of mammals. This discovery has demonstrated the possibility of foreign DNA getting access to mammalian organisms by overcoming a presumed, but realistically not existing, barrier in the gastrointestinal tract against the penetration by macromolecules. Experiments in which highly sensitive molecular and histological tools have been applied have proven the entry of food-ingested DNA into the organism and into various organs of the mouse. In pregnant animals, food-ingested DNA is capable of transgressing the placental barrier and can be traced in isolated cell clusters in the fetus. Urgent questions remain to be answered about the frequency of these events, the long-term fate of the intruding foreign DNA - integration versus degradation -, the existence of defense mechanisms against the uptake and expression of foreign DNA, and about the consequences for natural mutagenesis and oncogenicity. This route of entry is probably more frequently travelled by foreign DNA species than the highly specialized one of viral infections. Since mammalian organisms feed themselves by devouring other animals or plants, persistence of DNA from the sacrificed prey might be considered the ultimate genetic instrument of the weaker against the stronger competitor in nature. In this way, genetic equilibrium and genetic information can be maintained that have evolved over millenia in a biological system that is based on competition. It is also important to investigate how readily mammalian cells in culture and inside living organisms, e.g., the intestinal epithelia, are capable of taking up for-

2

I Introduction

eign DNA from the environment without artificial manipulations of the cells as utilized in various currently practiced transfection protocols. How does the DNA penetrate the cytoplasmic membrane? Do proteins complexed with DNA facilitate the uptake? How is the DNA transported to the nuclear and chromatin compartments, and how readily can it recombine with the cellular DNA? Answers to some of these questions will contribute to an improved understanding of interactions of cells and organisms with their environment which provides frequent access to foreign DNA. DNA molecules in fragmented form tend to recombine with other DNA molecules, particularly via free DNA termini, and to seek the most appropriate milieu for their efficient replication. Little is known about the perhaps equally efficient mechanisms of protection against DNA degradation by complexing DNA with proteins and by the formation of aggregates that improve the foreign DNA’s chances to penetrate through cytoplasmic membranes. It is pure conjecture to consider that precursor DNA molecules might have played an important role during evolution very early in the history of life. The elucidation of the fate and the gamut of interactions of foreign DNA has gained practical importance because in many fields of applied molecular biology foreign DNA is artificially introduced into mammalian cells and organisms. Transfection experiments, the generation of transgenic organisms, different regimens aiming at somatic gene therapy in humans or the development of DNAvaccines, all depend on the introduction and preferably the genomic fixation of foreign DNA. This monograph might, therefore, help also those who are primarily interested in the application of the techniques of molecular biology. An improved understanding of the mechanisms and problems underlying these techniques will further their utilization. Is it still realistic for one author to analyze such a diverse array of complex biological problems? Notwithstanding long-term active participation in research in this field, the views of one author will, by necessity, be limited and sometimes biased. I cannot expect my colleagues in the field to agree with me on all accounts. The propensity and necessity for disagreement and discourse are essential daily experiences and the gist of science. There are, however, advantages for a single author to address the problems posed by investigations on the fate of foreign DNA in mammalian systems. The novices and the non-experts will find it easier to familiarize themselves with the concepts and experimental approaches in this fast growing field in a coherently written treatise. The facility to insert and to accept foreign genetic information are essential features in evolving biological systems. Parasites like viruses, which are known to constantly evolve, teach us essential lessons about processes that are less easily recognized in multicellular organisms. Instantaneous consequences of foreign DNA insertion into their genomes do not become readily apparent as they do in viral genomes. However, with progressing age all organisms will be affected by the insertion of foreign genes, although we hitherto have not had the technology to investigate these events at the single cell level in multicellular organisms: the problem of the needle in a haystack.

1.1 Further reading

3

1.1 Further reading Coffin, J.M. (1996) Retroviridae: the viruses and their replication. In: Fields Virology, 3rd Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.). Lippincott-Raven Publishers, Philadelphia, New York, pp. 1767-1847. Doerfler, W., Gahlmann, R., Stabel, S., Deuring, R., Lichtenberg, U., Schulz, M., Eick, D., Leisten, R. (1983) On the mechanism of recombination between adenoviral and cellular DNAs: the structure of junction sites. Curr. Topics Microbiol. Immunol. 109,193-228. Roth, D.B., Wilson, J.H. (1986) Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction. Mol. Cell. Biol. 6,4295-4303. Roth, D.B., Wilson, J.H. (1988) Illegitimate recombination in mammalian cells. In: Genetic Recombination (Kucherlapati, R., Smith, G., Eds.). ASM, Washington, D.C., pp. 621-653. Schubbert, R., Renz, D., Schmitz, B., Doerfler, W. (1997) Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc. Natl. Acad. Sci. U S A94,961-966.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

2 Foreign DNA 2.1 Foreign DNA in the environment Table 1. Large amounts of foreign DNA are part of our environment

1. Uptake of food (humans) (a) Uptake per day: 100 mg-1 g DNA (minimum) (b) Excretion per day: 1 mg-10 mg, about 1% of ingested DNA (c) Sewage: 1 kg-10 kg DNA per day per lo6 inhabitants 2. Infections with viruses and microorganisms 3. Human burials per year 100 g DNA per human: 88 tons DNA per year in Germany'

4. Plants in the environment (a) Pollen in the spring (b) Foliage and fruits in the fall Possibly tons of foreign DNA

5. Sexual activites 10-30 mg DNA per year; 5-15 kg DNA per year per lo6 inhabitants 6. Recombinant DNA in research laboratories Per laboratory ng to yg DNA per experiment2

1. to 5. Relavant for million of years 6. Recombinant DNA since 1972

'

In 1996 approximately 880.000 people died in Germany. Humans consist of vidual. One human cell contains about lo-' pg of DNA (order of magnitude): lo2 g DNA per individual. One nanogram (ng) equals I o-' g; 1 microgram (pg) 1o - ~ g.

cells per indix lo-' pg =

'

Table 1 lists some of the sources of foreign DNA in the environment. For all living organims foreign DNA can be considered part of the natural ecosystem. Reliable data on the quantitation of foreign DNA in the environment are, however, not available. In fact, considerations about foreign DNA in the environment have not attracted much interest among molecular biologists. This neglect is all the more surprising, since the realization that foreign DNA is abundant in the environment provides an important argument against real apprehensions or politically motivated reservations about gene technology. All organisms constantly expose their integument-covered outer and their intestinal inner surfaces to the environment and potentially to foreign DNA. It is a safe prediction that the life-sustaining ingestion of food by all organisms presents the most frequent and intense exposure to foreign DNA of many different derivations. For their food supplies all organisms are dependent on the uptake of other organisms or their products which contain considerable amounts of foreign DNA.

6

2 Foreign DNA

Since foreign DNA is a frequent and ubiquitous companion with enormous genetic potential, one would expect defense mechanisms against the uptake and persistence of foreign DNA in all organisms. Does the immune system play a role in this defense? Traditional immunology has paid rather limited attention to the interactions of the immune system with foreign DNA. The size and topological arrangement of immune complexes in the intestinal tract and its vicinity suggest that foreign DNA potentially persisting in the gastrointestinal organs would be met by a very potent defensive system. In recent years, virus research has made a seminal contribution to our understanding of possible defense responses against foreign DNA through the demonstration that the injection of DNA coding for foreign, often viral, gene products can elicit an effective immune reaction against these proteins and thus against virus infection. It is completely unknown by what mechanism this response operates. It is conceivable that the injection of foreign genes under the control of a strong promoter could lead to the synthesis of sufficient amounts of the viral gene products to raise antibodies in the conventional way. There is yet no proof for this plausible mechanism. Whatever the mechanism, the described discovery has opened the new field of DNA vaccines with considerable practical potential. Expanding on these ideas and on the demonstration of foreign DNA penetrating into the organism from the gastrointestinal tract, the thought of oral DNA vaccines appears obvious. Robust research will be required to evaluate this possibility.

2.2 Uptake and modes of persistence of foreign DNA in mammalian cells Experimental evidence from work with mammalian cells in culture suggests that foreign DNA can be taken up under certain also naturally occurring conditions. The mechanism of this uptake is not understood. Apparently, not any DNA can penetrate the cytoplasmic membrane of mammalian cells. Uptake seems to be facilitated when the DNA is complexed with basic proteins. It is unknown whether specific or unspecific receptors in the cytoplasmic membranes are capable of or necessary for mediating the interaction with DNA or with DNA-protein complexes. There is an important field for further research to investigate to what extent cells in the organism are capable of taking up foreign DNA. The transfer of foreign DNA from the cytoplasm to the nucleus may take place via nuclear pores. For viral DNA molecules that reach the nucleus in the course of the natural infection cycle, analyses using the electronmicroscope demonstrate transfer through nuclear pores (Fig. 1). The uptake of foreign adenovirus DNA through the cytoplasmic and nuclear membranes of human HeLa cells has been demonstrated in electron micrographs by autoradiography of 3H thymidine-labeled adenovirus DNA that was directly added to the maintenance medium of HeLa cells in culture (Fig. 2).

2.2 Uptake and modes of persistence of foreign D N A in mammalian cells

Cell Surlace Binding

Endocylosir

Acid-Dependent

n

‘Lw

8045%-‘ Endosome

7

Binding to Nuclear Pore Complex, Disassembly 01 Caprid. DNA Import

D 75%

‘‘-‘

Ac#drhedEndosome

0

100 %

Dissociated 1 Fiber b90 %) 2 Protan llla (SO %) 3 Proteln vlll (70 %)

Degraded 4 Protein VI (80 50 %, DIssoClNed 5 Penlon base 180 40%) 6 Protein IX (70 %)

Dissociated 7 DNA (40 Yo) lmporfsd DNA Hexon

Nuclear Envelope Pare Complex

II

Figure 1. (a) Electron microscopy of adenovirus-infected human HeLa cell. Viral core material (nucleoprotein) in the process of release to a nuclear pocket: 40 min. after infection x 1S0,OOO(Morgan et al., 1969). (b) Sequence and efficiencies of the stepwise uncoating of adenovirus 2 during cell entry. Internalization via coated pits occurs with an efficiency of 8O%-8S% and a half time of 10 min. Efficient penetration of about 90% from endosomes happens after 15 min, and viruses are found at nuclear pore complexes after 3 5 4 5 min. About 40% of the originally cell-bound viruses release their DNA into the nucleus. The efficiencies of the individual dismantling events are indicated in parentheses. Bars represent the fibers, closed small circles represent the vertex complex, and the closed hexagon depicts the viral chromosome (Lonberg-Holm and Philipson, 1969).

8

2 Foreign D N A

2.2 Uptake and modes of persistence of foreign DNA in mammalian cells

9

Figure 2. Electron microscopy and autoradiography of KB cells exposed to %labeled Ad2 DNA. KB cells growing in monolayers were directly exposed to 2.3 pg of "-labeled Ad2 DNA. Adsorption at 37°C was allowed to proceed for 30 min (a), 6 h (b), and 24 h (e, d), respectively. At the end of the incubation period, the inoculum was removed and the cells were immediately fixed with 2 ml of 1.5% glutaraldehyde. Samples were prepared for electron microscopy and autoradiography. Magnification 22,000 x (a), 24,600 x (b), 15,400 x ( c ) 13,300 x (d). N: Nucleus, C: Cytoplasm, M: Mitochondrion (Groneberg et al., 1975).

10

2 Foreign D N A

Several methods to introduce foreign DNA artificially into mammalian cells have been developed: Ca2' phosphate transfection, electroporation, lipofection, complexing DNA with Superfect, based on chemically activated dendrimer molecules, microinjection and other techniques. For the purpose of generating transgenic animals or plants, oocytes, zygotes or plant protoplasts are usually microinjected; transfection methods have also been applied, though less frequently. These methods have been designed and optimized for particular experimental purposes and have proven useful for the in general limited experimental projects for which they have been designed. A problem of far greater biological and medical importance is the uptake of foreign DNA by cells of the organism without the use of artificial procedures. Here we enter an area of astounding uncertainty. The natural uptake of foreign DNA, e.g., by cells of the skin, the gastrointestinal or genital tracts of organisms has been studied only in very rare instances. Apparently, organisms are considered protected by unknown natural defenses, although the exposure on the described surfaces of organisms is unavoidable and frequent. For these reasons, it would be highly desirable to learn more about the fate of foreign DNA molecules at these potential portals of entry into established organisms. Once foreign DNA molecules have penetrated the cytoplasmic membrane, there must still be possibilities to degrade the DNA, even when it has reached the nucleus of the cell. Again, these defenses are almost completely unknown. Extent and specificities of DNase activities in different cellular compartments have not been sufficiently investigated to evaluate their role in the defense against foreign DNA. Upon entry into the nucleus, the persistence of foreign DNA becomes dependent on either its insertion into the host genome or on the establishment of an episomal state with the capability for replication synchronized with the cellular genome. Episomal replication and thus persistence can be facilitated under the precise regulation of an origin of DNA replication in the viral genome that responds to cellular replication factors. We do not understand which factors contribute to the decision between loss of the foreign DNA and persistence by one of the two possible mechanisms of permanent fixation in the host nucleus. Among viral systems two different DNA-containing viruses, human adenovirus or the herpesvirus Epstein-Achong-Barr (EBV) virus, utilize one of the two different pathways of persistence. Adenoviruses can insert their DNAs by covalent linkage into the host cell genomes, whereas EBV DNA, in many instances, persists as an episome in a supercoiled circular form of DNA in the nucleus of infected or transformed cells. There is evidence mainly from experiments using the fluorescent in situ hybridization technique (see below) that some of the persisting EBV genomes might also become integrated into the host genome. The EBV genome carries an origin of replication that apparently can be recognized and activated by host cellular replication factors in a way that permits episomal persistence and synchronized replication. These mechanisms are only partly understood at the molecular level. Bovine papilloma virus type 1 (BPV-1) DNA replicates in cells of the lower stratum of the epidermis, including basal cells, once per cell cycle during S-phase

2.3 Further reading

11

and in synchrony with the host cell DNA. The DNA copies appear to be distributed equally to the progeny cells. In this way persistence and latent infection are maintained in the stem cells of the epidermis. In more differentiated epithelial cells bursts of viral DNA synthesis are observed in the absence of cellular DNA replication. The DNA of at least some of the papilloma viruses, e.g., of human papilloma viruses 16 and 18 have been shown to be integrated into the genomes of human cancer cells. Adenoviruses, on the other hand, encode essential elements of their replication machinery, as DNA polymerase, single-strand DNA binding protein, and terminal viral protein, in their own genomes and need a number of cellular nuclear factors for replication. Thus, replication of the adenoviral DNA and viral multiplication are rendered very efficient so that the infected permissive cells usually serve as factories for viral propagation and succumb to infection. Probably as a consequence of extensive association of adenoviral genomes with the chromosomes of the infected cells, a subpopulation of the viral genomes might become integrated, but in the dying cell population it is difficult to detect a small number of host genome-associated viral DNA molecules. A completely different situation is observed in hamster cells which are non-permissive for the replication of human adenovirus type 12 (Ad12-) which is completely blocked in early steps of its replication cycle prior to viral DNA replication. In this system, viral DNA is linked, though perhaps only transiently, to cellular DNA. Most of the cells survive viral infection and a small subpopulation of the infected cells become transformed to tumor cells or to tumorlike cells. Investigations on these systems permit the description of many of the phenomena of foreign DNA persistence, although we do not understand yet the enzymatic and molecular mechanisms that determine the type of persistence or the extent and duration of foreign DNA fixation. The following factors may influence the outcome of the encounter between the cell and the invading viral DNA: The genetic repertoire of the foreign DNA under investigation, the cell type and the cellular functions available for the processing of the incoming foreign DNA molecules, the stage in the cell cycle at which the foreign DNA reaches the nucleus, the interaction of cellular proteins with the foreign DNA molecules, growth conditions or location and function of cells in an established organism that become exposed to the foreign DNA. It is apparent already from this brief introductory discussion that most of the relevant questions arising in this field require further study.

2.3 Further reading Doerfler, W. (1968) The fate of the DNA of adenovirus type 12 in baby hamster kidney cells. Proc. Nutl. Acud. Sci. U S A60,636-643. Doerfler, W. (1991) Abortive infection and malignant transformation by adenoviruses: integration of viral DNA and control of viral gene expression by specific patterns of DNA methylation. Adv. Virus Res. 39,89-128.

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2 Foreign DNA

Doerfler, W., Schubbert, R. (1998) Uptake of foreign DNA from the environment: the gastrointestinal tract and the placenta as portals of entry. Wiener Klin. Wochenschrift110,4044. Graham, EL., van der Eb, A.J. (1973) Transformation of rat cells by DNA of human adenovirus 5. Virology54,536-539. Groneberg, J., Brown, D.T., Doerfler, W. (1975) Uptake and fate of the DNA of adenovirus type 2 in KB cells. Virology64,115-131. Kieff, E. (1996) Epstein-Barr virus and its replication. In: Fields Virology, 3rd Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.), Lippincott-Raven Publishers, Philadelphia, New York, pp. 2343-2396. Lonberg-Holm, K., Philipson, L. (1 969) Early events of virus-cell interaction in an adenovirus system. J. Virol. 4,323-338. Morgan, C., Rosenkranz, H.S., Mednis, B. (1969) Structure and development of viruses as observed in the electron microscope. V. Entry and uncoating of adenovirus. J. Virol. 4,777-796. Schroer, J., Holker, I., Doerfler, W. (1997) Adenovirus type 12 DNA firmly associates with mammalian chromosomes early after virus infection or after DNA transfer by the addition of DNA to the cell culture medium. J. Virol. 71, 7923-7932. Taubes, G. (1997) Salvation in a snippet of DNA. Science 278,1711-1714. Torres, R.M., Kiihn, R. (1997) ES cell culture and transfection. In: Laboratory Protocols for Conditional Gene Targeting. Oxford University Press, Oxford, pp. 73-79. Wienhues, U., Hosokawa, K., Hoveler, A., Siegmann, B., Doerfler, W. (1987) A novel method for transfection and expression of reconstituted DNA-protein complexes in eukaryotic cells. D N A 6,Sl-89. zur Hausen, H. (1967) Association of adenovirus type 12 deoxyribonucleic acid with host cell chromosomes. J. Virol. 2,218-223.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

3 Methods to detect integrated foreign DNA 3.1 Parameters of foreign DNA integration The genomic fixation of foreign DNA by integration into the host cell’s genome is the most reliable way of fixing the newly acquired genetic information in the nuclear environment. By integrative recombination, the foreign DNA is covalently linked via a phosphodiester bond to the recipient host DNA molecule. The best studied examples of foreign DNA integration are those of bacteriophage lambda DNA into the genome of Escherichia coli (E. coli) (see Section 7.6) or of the genomes of tumor viruses, e.g., of retroviruses (see Section 7.4) or of human adenoviruses (Chapters 4,5)into the genomes of cells oncogenically transformed by these viruses. The enzymatic mechanisms facilitating the integration reactions have been studied in considerable detail for the integration of bacteriophage lambda DNA and of retroviral DNA. The consequences of foreign DNA insertion into established mammalian genomes have been studied to a limited extent, except for the possible functions of the products that have been expressed from integrated foreign (viral) genes. This latter aspect has attracted much interest particularly in the field of viral oncology. Some of the viral gene products transcribed from the integrated viral genomes are capable of interacting with specific cellular factors that are presumed to regulate cellular replication and growth. It is thought that cellular replication could be deregulated in this way leading to the oncogenic phenotype. When foreign DNA has been genomically fixed upon transfection and selection of transgenic cell clones, it has frequently been the aim of the experimental procedure to express genes encoded in the transfected foreign DNA which are transcribed under the control of an artificially inserted strong promoter. The modes of transcriptional regulation in the thus genomically fixed foreign genes are not understood. It is left more or less to chance or trial and error whether the foreign DNA continues to be expressed for longer periods of time. A similar empirical approach is often chosen in the generation of transgenic animals. For the molecular biologist, many problems with a wide spectrum of practical applicability remain to be investigated. The solution of these problems depends on the development of new techniques which are refined enough to follow DNA molecules intracellularly at the single cell level. Today, some of these possibilities can be approached with the presently available technology. How can the usually miniscule amounts of foreign genes be detected in the vast arrays of the mammalin cells? A number of sensitive techniques have helped facilitate this task.

14

3 Methods to detect integrated foreign D N A

3.2 The fluorescent in situ hybridization (FISH) method DNA-DNA hybridization experiments with intensely labeled DNA probes continue to present the most sensitive techniques for the visualization of specific DNA molecules in a vast background of unrelated DNA sequences. With the FISH method the foreign DNA can in fact be visuslaized by fluorescent UV light microscopy. Interphase nuclei or chromosome spreads on glass slides are prepared for these hybridization reactions. The specific DNA sequences in these structures are made accessible to hybridizing labeled DNA probes upon fixation in methanol-acetic acid and the controlled pretreatment with proteolytic enzymes. The DNA to be detected is nonradioactively labeled with nucleotides to which biotin has been covalently linked. Usually biotinylated deoxycytidine triphosphate (16-dUTP) is incorporated into the DNA probe in conventional nick translation reactions; occasionally digoxygenated-UTP is also used. After completion of the hybridization reaction and extensive washing to remove the unhybridized DNA probe in order to minimize unspecific background binding of the labeled DNA, the nuclei or chromosome preparations are treated with avidinfluorescein isothiocyanate in blocking solution, e.g., 3% milk powder in a NaC1-sodium citrate solution. The biotin-avidin recognition is one of the most sensitive and specific reactions in biochemistry. When digoxygenin has been used to label the DNA probe, an anti-digoxigenin-rhodamin conjugate is applied for its detection. Two different DNA segment probes labeled with biotinylated or with digoxigenin-labeled UTP allow the simultaneous visualization of two different DNA sequences in the same reaction. The method can be rendered even more sensitive by complementing the described procedures with a second sandwich type reaction using fluorescein- or rhodamin-labeled antibodies against avidin or digoxygenin, respectively. The slides are then examined and photographed under UV-light in a suitable microscope. For higher resolution, the FISH technique can be combined with the method of stretched chromosome preparations. By exposing chromosomes to moderate centrifugal fields at 70 to 80 x g for 4 min, the chromosomes are extended and foreign DNA molecules or endogenous genes can be distinguished in far greater detail (Fig. 3a, see color plates, for an example). This method permits the direct visualization of foreign DNA in association with both chromatids of the chromosome that has served as the target for the insertional recombination reaction. Symmetrically distributed hybridization signals on both chromatids provide good evidence for the integrated state of the foreign DNA but not stringent proof. It is necessary to reclone the foreign DNA in covalent linkage to the host DNA and to determine the nucleotide sequence across the junction site to firmly establish the integrated state (see Section 3.5). Nevertheless, the critical application of the less rigorous-in-proof FISH technique can provide useful information and reassures the investigator that recloning experiments are indicated and promise more direct information.

3.3 Detection of foreign DNA by Southern D N A transfer

15

The photograph in Fig. 3a demonstrate the presence of several Ad12 genomes in an integrated state on one of the stretched chromosomes of the Adl2-transformed Syrian hamster cell line T637. One intact Ad12 genome comprises 34,125 nucleotide pairs, and the analysis by a different set of hybridization techniques has provided an estimate of the number of Ad12 genome equivalents in this particular hamster cell line. There are in the order of 20 copies of viral DNA which may, however, not all be intact. The FISH method is capable of tracing much smaller amounts of foreign DNA, i.e., of fragments of one Ad12 DNA. In the revertant cell line TR12, which has been derived from the T637 cell line, most of the foreign Ad12 DNA molecules have been lost presumably by an excision event. Only about one Ad12 genome persists in this revertant cell line and can also be readily detected by the FISH method (Fig. 3b). Obviously, the FISH technique has the advantage of following foreign DNA molecules on chromosomes at the single cell and chromosome levels. Moreover, by combining the FISH method with appropriate chromosome banding protocols or with the cohybridization of selected cellular DNA probes, the integrated foreign DNA can be localized on the genetic map of the recipient host genome. With the FISH technique it is not necessary to grow up a clonal cell population to millions of cells in order to extract sufficient amounts of DNA for further molecular analyses. During these steps of cellular expansion, the localization and quantity of integrated foreign DNA might be subject to alterations and thus not reflect accurately the original state of foreign DNA persistence. The major limitation of the procedure, however, lies in its nonquantitative character. It is impossible to correlate the intensities of hybridization signals to the size or number of foreign genomes integrated at a particular chromosomal site. The data presented in Fig. 3a-b demonstrate that signal intensities generated by about 20 integrated foreign DNA genomes are indistinguishable from those due to about one integrated Ad12 genome.

3.3 Detection of foreign DNA by Southern DNA transfer and DNA-DNA hybridization In all fields of research in molecular biology, the most frequently applied method for the detection of foreign DNA in a vast background of cellular DNA is the Southern DNA transfer (blot) hybridization technique. At the time of its publication in 1975 and ever since, this procedure has been considered a breakthrough in molecular biology and has been applied in innumerable experiments and variations. As with many important discoveries, the idea behind the technique is simple. The cellular genome is cleaved with a suitable restriction endonuclease and the millions of DNA fragments of varying sizes are separated by electrophoresis in a carrier, agarose or polyacrylamide, gel. The gel concentration is chosen according to the expected size range of DNA fragments that has to be analyzed. Upon the appropriate electrophoretic separation, the DNA fragments in the gel

16

3 Methods to detect integrated foreign D N A

are denatured by alkali treatment. For some analytical purposes, prior to denaturation the DNA is further fragmented in each gel compartment by a brief exposure to acid facilitating a more efficient transfer of the higher molecular mass fragments. This latter procedure leads to partial depurination and breakage of DNA. Excessive depurination might endanger the specficity of the DNA-DNA hybridization reaction. Depurination has, therefore, to be applied with caution. The denatured DNA fragments are subsequently transferred to a nitrocellulose or nylon membrane. Some of these membranes are positively charged for optimal transfer to and fixation of DNA on the carrier membranes. Transfer is accomlished by suction of a neutral or alkaline buffer solution through the gel and subsequently through the membrane in a type of sandwich arrangement in the order gel-membrane-stacks of filter paper. The filter paper drives the flow of buffer through the stacked components of the system. A vertical downward transfer procedure has proved most efficient for a nearly quantitative transfer for the denatured DNA fragments onto the membranes (Fig. 4). The transferred DNA fragments are more stably attached to the membrane carriers by a baking step at 80 "C which is sometimes reinforced by the irradiation of the filter with ultraviolet light. Specific DNA sequences in the mass of membrane-bound DNA can be detected and actually visualized by hybridizing the membrane-bound DNA to radioactively "P-labeled specific DNA fragments, whose sequences are searched in the mass of cellular DNA. The hybridization probe is frequently clone-purified. Areas on the membrane which are devoid of affixed DNA are covered by protein usually by bovine serum albumine or bovine milk proteins to minimize unspecific binding of the labeled DNA probe to empty filter regions. Probe hybridization can be rendered more efficient and purportedly more specific by adding dextran sulfate to the hybridization reaction. After hybridization of the labeled specific DNA probe, the filters are extensively washed under stringent conditions, usually at 68 "C at low salt concentration and in the presence of 0.1% sodium dodecylsulfate (SDS). The filter is then dried and autoradiographed on X-ray film. After sufficient exposure, the location of the specifically hybridized DNA probe can be directly observed on the autoradiograms. For many Southern blot hybridization experiments coelectrophoresis of size- and sequence-defined marker DNA fragments has proven extremely helpful for the interpretation of the

Figure 4. Schematic representation of the downward DNA transfer technique (Koetsier et al., 1993).

3.3 Detection of foreign D N A by Southern DNA transfer

h

TI011 -

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.-

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17

Figure 5. Patterns of Ad12 DNA integration in the DNA of different Adl2-induced hamster tumors. The DNA from individual tumors as indicated was extracted and cleaved with Hind111 or PstI, and the fragments were separated by electrophoreses on 0.8% agarose gels. Subsequently, the DNA was transferred to Qiagen nylon-plus membranes. As size and quantity markers, 5- and 10-genome equivalents of authentic Ad12 virion DNA per diploid hamster genome were similarly treated and coelectrophoresed. The DNA was then hybridized to 32P-labeled Ad12 DNA (a) or the cloned left-terminal Pstl-C (b) or the cloned rightterminal BamHI-E (c) fragment of Ad12 DNA. The authentic Hind111 or PstI fragments of Ad2 virion marker DNA were indicated in the margins of the Ad12 DNA autoradiogram (a). Off-size fragments, which did not comigrate with any of the authentic marker Ad12 virion DNA fragments and were due to junction fragments between Ad12 and hamster cell DNAs or to partly rearranged Ad12 DNA, were designated by arrowheads in a*. The PstI, BamHI and Hind111 restriction maps of Ad12 DNA are shown (a, bottom); shaded areas, the terminal Ad12 DNA fragments used as hybridization probes (HilgerEversheim and Doerfler, 1997).

18

3 Methods t o detect integrated foreign D N A

data. The DNA probe is often radioactively "P-labeled by nick translation. More recently, nonradioactive labeling protocols have also been applied but appear to be less sensitive in the detection of specific DNA sequences in the analyzed DNA. In Fig. 5 , the results of a Southern transfer hybridization experiment have been reproduced. The presence of integrated Ad12 DNA in an Adl2-induced hamster tumor is demonstrated. For the evaluation of the Southern transfer autoradiograms a number of parameters have to be considered. 0

0

0

The amount of foreign DNA present in the cellular DNA can be estimated by photometrically comparing the signal intensities due to foreign DNA in the mass of cellular genomic DNA with signal intensities in a series of lanes on the electropherogram in which predetermined amounts of the unlabeled foreign DNA probe have been coelectrophoresed. Scanning with a phosphoimager can be a useful procedure for more precise quantitation. The method is not fully quantitative but provides reasonable estimates of the amount of foreign DNA present per cell. For the detection of single copy quantities of foreign DNA per cell, 10 to 30 pg of cellular DNA have to be cleaved and analyzed by electrophoresis to generate interpretable signals on the autoradiograms. Since one mammalian cell contains in the order of several times lo4 pg of chromosomal DNA, a single cell clone whose content and array of foreign DNA has to be analyzed has to be expanded to at least lo7 to 10' cells to generate sufficient amounts of DNA for Southern blot hybridization analyses. During cell replication the amount and distribution of foreign DNA might change and these possible changes cannot be controlled or detected. This problem constitutes one of the major drawbacks of the Southern transfer hybridization technique for the analyses of foreign DNA in mammalian cells or in cells of other species. On the other hand, with this method the physical arrangement and identification of foreign DNA molecules in the recipient genome can be by far more precisely approached than with other procedures. In Adl2-induced hamster tumor cells, we have not detected changes in integration patterns of Ad12 DNA in about 75 to 96 cell doublings. Similarly, in the Adl2-transformed hamster cell line T637 the integration pattern of viral DNA have not changed over a period of two decades. Possibly, the danger of alterations of insertion patterns of foreign DNA upon the continuous cultivation of cells are negligible. The method also provides the possibility to distinguish between integrated and episomally persisting foreign genomes. By using integrated adenovirus DNA as an example, I will explain the experimental strategy (see the autoradiogram in Fig. 5). The integrated adenovirus DNA is covalently linked to the cellular DNA, often via the termini of the viral genome. By cleaving the total intranuclear DNA of the adenovirus DNA-carrying cell with a restriction endonuclease that cleaves the viral DNA a few times, adenovirus DNA-containing fragments are generated which are derived from the interior parts of the viral genome. In addition, there are the fragments that carry the junction sites between viral and cellular DNA (Fig. 6 for a schematic presentation). These so-called junction fragments do not comigrate with any of the virion

3.3 Detection of foreign D N A by Southern D N A transfer

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Figure 6. Analysis of foreign DNA in mammalian cells by restriction endonuclease analysis, Southern blotting and DNA-DNA hybridization. As an example, Ad12 DNA integrated in hamster cell DNA was chosen. The DNA was cleaved with a restriction endonuclease, e.g., EcoRI, and fragments were fractionated by agarose gel electrophoresis. Ad1 2-virion DNA was used as internal standard. Upon hybridization of the Southern blot with 32P-labeled Ad12 DNA or the cloned terminal fragments (A or C fragment) of Ad12 DNA, the colinearly arranged internal viral DNA fragments and the off-size terminal Ad12 DNA fragments, which were linked to cellular DNA, were visualized by autoradiography. Schematic representation of experimental procedure (Doerfler, 1982).

DNA fragments, which have been generated by cleaving virion DNA, i.e., viral DNA extracted from purified virus particles and which are coelectrophoresed as size markers on the same Southern transfer gel. The internal fragments from the integrated viral genomes do, however, comigrate with the internal virion DNA fragments. The terminal viral junction fragments assume an offsize position with respect to any of the virion marker DNA fragments due to their linkage to cellular DNA. These off-size fragments can serve as the source for the isolation of the cellular DNA sequences to which the foreign viral DNA molecule has been linked. For further details, see Fig. 6.

20

3 Methods to detect integrated foreign D N A

In this type of analysis, viral DNA persisting in the nonintegrated free form would give rise only to bona fide virion DNA fragments. Episomally persisting circular DNA would be represented by the internal viral DNA fragments and the linkage product of the two terminal DNA fragments. In transformed or tumor cells with persisting adenovirus DNA, we have never encountered free or episomally persisting forms of viral DNA. For other viral genomes, e.g., the often episomally persisting Epstein-Achong-Barr Virus DNA, a different persistence pattern is found. 0 In many instances of cells or organisms with integrated or episomally persisting foreign DNA genomes the organization of the foreign DNA molecule can be rearranged, partly deleted, partly amplified or altered by integration in multiple tandem or pseudo-tandem arrays. Under such conditions, the analyses can become very difficult and even ambigous and the application of the Southern transfer hybridization technique will not always provide unequivocally interpretable results on the arrangement of multiple integrated foreign DNA molecules. In these instances, it will be necessary to reclone the integrate and determine the entire nucleotide sequence. Only in rare instances such a major sequencing project would be justified. a Integrated or episomally persisting foreign DNA molecules can also be lost upon serial subculturing of cells or in subsequent generations of transgenic animals. These losses can be restricted only to subpopulations of cells. These circumstances call for the careful subcloning of cells and the detailed analyses of subclone populations (see Section 6.2). 0 Integrated foreign DNA genomes frequently become de lzovo methylated. Their investigation requires a completely different approach using methylation-sensitive restriction endonucleases and the Southern blot hybridization method or, more importantly, application of the genomic sequencing technique (see Section 9.2). The latter technique facilitates the investigation of each deoxycytidine residue in a nucleotide sequence and to decide whether it represents a deoxycytidine or a 5-methyl deoxycytidine residue.

3.4 Detection of foreign DNA sequences by the polymerase chain reaction (PCR) Foreign DNA sequences persisting in mammalian or other cells and tissues can be traced in even minute amounts by PCR. By using the appropriate oligodeoxyribonucleotides as primers to initiate the expansion reaction and multiple cycles of amplification, possibly supplemented by a second round of amplification with a nested set of primers, foreign DNA segments defined by their authentic nucleotide sequence can be easily detected (Fig. 7). Frequent reproduction of the results and inclusion of several standard control reactions - negative controls omitting any DNA from the reaction, positive controls with the cloned foreign DNA mole-

3.4 Detection of foreign DNA sequences by the polymerase chain reaction (PCR)

21

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Figure 7. Schematic representation of the principles used in the polymerase chain reaction (PCR). PCR results in amplification of a selected region of a DNA molecule. Regions of 5 kb and more can be amplified without difficulty, and longer amplifications - up to 40 kh - are possible using modifications to the standard technique. The reaction is carried out by mixing together the target DNA molecule, which can be present in extremely small amounts, with nucleotides, two synthetic oligonucleotide primers, and a thermostable DNA polymerase that is resistant to denaturation by heat treatment. Usually Taq polymerase from the bacterium Thermus aquaticus is used. The two primers must anneal to the target DNA on either side of the region to be amplified, which means that the sequences of these borders must he known so that the appropriate oligonucleoti-

22

3 Methods to detect integruted ,foreign D N A

cule on a plasmid as an internal standard-protect against artifacts with the highly sensitive PCR method. At the same time, the nucleotide sequence of the amplified DNA can be directly redetermined. We have so far avoided applying the PCR method to the investigation of junction fragments of integrated foreign DNA. Theoretically, analyses with randomly selected primers or cleavage and recircularization of the junction fragments in highly diluted DNA solutions have been recommended for this purpose. These protocols may not be reliable enough to seriously attempt their application, although in some instances they may have provided useful information. Thus the PCR method is extremely useful to detect small amounts of persisting foreign DNA. For the study of the integrated state of foreign DNA molecules, however, I have preferred a different, admittedly more laborious, approach.

3.5 Recloning of junction fragments and the

determination of their nucleotide sequence With this technique the definite proof for the integrated state and information on the detailed arrangement of foreign DNA molecules in the recipient genome can be obtained. The details of the procedure are explained schematically in Fig. 8. First, the foreign DNA is identified in the mass of genomic DNA by Southern DNA transfer hybridization (Southern blotting) as described above. We have found it most efficient to choose the restriction endonuclease for this analysis in a way that the foreign DNA can be localized in preferrably one specific DNA fragment. The recipient host DNA has therefore to be cleaved with a restriction endonuclease that produces one or a limited number of junction DNA fragments between foreign DNA and host DNA. Subsequently, the thus generated DNA fragments are fractionated by velocity sedimentation in a sucrose density gradient. Electrophoretic separation on a gel matrix is also possible. The enriched junction fragment is then ligated into the appropriately precleaved DNA of a bacteriophage lambda vector. The lambda DNA is in vitro packaged and lambda plaques are subsequently screened for the presence of the foreign DNA fragment in the junction DNA segment. The separately precloned foreign DNA fragment des can be made. The oligonucleotides prime the synthesis of new complementary polynucleotides which are made in the 5’ to 3’ direction by copying the template DNA in the 3’ to 5’ direction. Because the Taq polymerase is thermostable, the reaction mixture can be heated to 90°C without destroying the enzyme activity. At this temperature, the new strands detach from the template DNA. When the mixture is cooled down again, more primers anneal to the template DNA and also to the new strands, and the Taq polymerase carries out a second cycle of DNA synthesis. The PCR can be continued for 30 to 40 cycles before the enzyme eventually becomes inactivated or the primers or nucleotides are used up. A single starting molecule can be amplified into tens of millions of identical fragments, representing a few micrograms of DNA. (T.A. Brown, 1999).

3.5 Recloning of junction fragments and the determination of their nucleotide sequence D

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off size fragment 1. Restriction enzyme cleavage and Southern blot

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Figure 8. Schematic presentation of major steps in the cloning of junction sequences. Details of the cloning procedure have been described in the text (Doerfler et al., 1983).

is 32P labeled by nick translation and is used as hybridization probe on the filtertransferred lambda plaques. Positive plaques are isolated and rescreened two to three more times with the same probe to ascertain the authenticity of the isolated plaque. Confirmed positive plaques are multiplied, lambda DNA is prepared from these isolates and the nucleotide sequence across the junction is determined by using the known segments in the DNA sequence of the foreign DNA fragment for the localization of the oligodeoxyribonucleotide primers. The nucleotide sequence is then analyzed electronically for the detection of the known foreign DNA sequence. The adjacent, previously unknown host DNA sequence is investigated for the occurrence of similar or previously described cellular DNA sequences. In this way, deletions in the foreign DNA sequence during integration

24

3 Method7 to detect integruted foreign D N A

can be detected. Moreover, the cloned cellular junction DNA sequence at the recipient site of integration can now be used to clone the preinsertion sequence from cellular DNA of the recipient cells prior to the insertion of foreign DNA by a similar approach. Comparisons of the nucleotide sequences found in the preinsertion and the foreign-DNA-occupied sites facilitate determination of deleted cellular DNA sequences. The integrated state of the foreign DNA can best be proven by demonstrating, e.g., by FISH that the foreign and the preinsertion DNA sequences in the junction DNA fragment actually reside at the same site on one of the chromosomes of the cell carrying the foreign DNA. Hence, an involved series of procedures has to be critically applied and interpreted so that one can ascertain the integrated state of the foreign DNA.

3.6 Equilibrium sedimentation in alkaline CsCl gradients and DNA-DNA hybridization Early after the infection of productively infected human or abortively infected hamster cells with human Ad12, a considerable amount of the viral DNA becomes associated with the host chromosomes possibly in an integrated state. In order to document the integration, hamster cells that had been grown in the presence of 5-bromodeoxyuridine (5-BrdU) were used in infection experiments using Ad12 labeled in its DNA moiety with 'H-thymidine. In this way it is possible to separate the 5-BrdU-substituted heavy-density cellular DNA from the normal low-density 'H-labeled viral DNA by equilibrium sedimentation in alkaline CsCl gradients (Fig. 9). By alkali-treatment at pH values >13, the DNA remains single-stranded. The data demonstrate that already 16-21 h postinfection considerable amounts of the parental viral DNA have shifted to the heavy density stratum of cellular DNA. Since Ad12 DNA is subject to a complete block of replication in hamster cells, the parental viral DNA can remain only in the low density stratum unless it has become covalently linked to the heavy cellular DNA. This interpretation has been confirmed by demonstrating that the 'H-labeled DNA in the heavy cellular density stratum hybridizes to Ad12 DNA (Fig. 9). Moreover, when the total intracellular DNA in this experiment is physically sheared to smaller fragments of DNA of approximately 1 to 2 kb by ultrasonic treatment, the 'H-labeled Ad12 DNA in the heavy density position is shifted to the low, Ad12 DNA-specific or to an intermediate density stratum. This shift is due to the separation of the light viral DNA from the heavy cellular DNA to which it had been covalently linked by integration. Since these data have been derived from alkaline CsCl gradients at a pH value of above 13, covalent linkage between cellular and viral DNA has been demonstrated. This experiment constitutes rigorous prove for the integrated state of viral DNA in the host genome. Of course, much evidence adduced in subsequent years and projects has corroborated this inter-

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Figure 9. Equilibrium sedimentation patterns in alkaline CsCl of DNA from 3H-Ad12-infected5BUBHK21 cells (a, b) and from mock-infected SBU-BHK21 cells (c, d) before (a, c) and after (b, d) DNA fragmentation. SBU-BHK21 cells were infected with 'H-Adl2. At 42 h after infection, the cells were washed and lysed. Mock-infected SBU-BHK21 cells were grown in medium containing 2 pCi of 'H-thymidine per ml. The DNAwas extracted and analyzed by equilibrium sedimentation in alkaline CsCl density gradients. To each gradient 5.1 pg of ''C-labeled Ad12 DNAwas added as density marker. (a) DNA from AdlZinfected SBU-BHK21 cells was sedimented to equilibrium without prior fragmentation. The horizontal bars indicate the fractions of cellular (C) and viral (V) DNA which were pooled in similar experiments and shown by the DNA-DNA hybridization procedure to contain Ad12 DNA. The arrows indicate the position of the peak of the ODzh0in each of the graphs. (b) DNA extracted from the Adl2-infected cells was first fragmented by ultrasonic treatment; then 14Clabeled Ad12 DNAwas added and the mixture was sedimented to equilibirum. (c) DNA from mockinfected SBU-BHK21 cells which had been labeled with 'H-thymdine was centrifuged to equilibrium in an alkaline CsCl density gradient. (d) DNA from mock-infected 5BU-BHK21 cells was fragmented by ultrasonic treatment; then ''C-labeled Ad12 DNA was added and the mixture was centrifuged to equilibrium (Doerfler, 1970).

pretation. It is surprising that such a large amount of the infecting viral DNA becomes linked to cellular DNA. The results of analyses of the state of parental Ad12 DNA in abortively infected hamster cells by FISH have further strengthened the interpretation of integrated viral genomes in the hamster cell genome and have also shown surprisingly large amounts of Ad12 DNA in association with the hamster chromosomes (Fig. 10, see color plates). Association and insertion are probably transient: Alternatively, the cells carrying integrated for-

26

3 Methods t o detect integruted foreign DNA

eign (viral) DNA may have a growth disadvantage in comparison to cells devoid of insertions of foreign genomes, hence, replicate less efficiently and are lost from the total cell population.

3.7 Further reading Brown, T.A. (1999) Genomes. BIOS Scientific Publishers, Oxford, U. K. Doerfler, W. (1968) The fate of the DNA of adenovirus type 12 in baby hamster kidney cells. Proc. Natl. Acad. Sci. U S A60,636-643. Doerfler, W. (1969) Nonproductive infection of baby hamster kidney cells (BHK21) with adenovirus type 12. Virology 38,587-606. Doerfler, W. (1970) Integration of the deoxyribonucleic acid of adenovirus type 12 into the deoxyribonucleic acid of baby hamster kidney cells. J. Virol.6,652-666. Haaf, T., Ward, D.C. (1994) Structural analysis of a-satellite DNA and centromer proteins using extended chromatin and chromosomes. Hum. Mol. Genet. 3,697709. Knoblauch, M., Schroer, J., Schmitz, B., Doerfler, W. (1996) The structure of adenovirus type 12 DNA integration sites in the hamster cell genome. J. ViroZ. 70, 3788-3796. Koetsier, P.A., Schorr, J., Doerfler, W. (1993) A rapid optimized protocol for downward alkaline Southern blotting of DNA. BioTechniques 15,260-262. Lichter, P., Tang, C.-J.C., Call, K., Hermanson, G., Evans, G.A., Housman, D., Ward, D.C. (1990) High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247,6449. Nevins, J.R., Vogt, P.K. (1996) Cell transformation by viruses. In: Fields Virology, 3rd Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.) , Lippincott-Raven Publishers, Philadelphia, New York, pp. 301-343. Rigby, P.W.J., Dieckmann, M., Rhodes, C., Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. B i d . 113,237-251. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, GT., Mullis, K.B., Erlich, H. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Schroer, J., Holker, I., Doerfler, W. (1997) Adenovirus type 12 DNA firmly associates with mammalian chromosomes early after virus infection or after DNA transfer by the addition of DNA to the cell culture medium. J. Virol. 71,7923-7932. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. B i d . 98,503-517. Sutter, D., Westphal, M., Doerfler, W. (1978) Patterns of integration of viral DNA sequences in the genomes of adenovirus type 12-transformed hamster cells. Cell 14,569-585.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

4 The adenovirus paradigm Studies on the molecular biology of mammalian cells have frequently profited from results adduced from work with the adenovirus system. In the author’s experimental work the adenovirus system has helped to investigate the integration of foreign DNA into the mammalian genome. It is therefore adequate to present an overview of this system. Some of the data on adenoviral DNA integration may be of relevance uniquely to this viral system, in particular where they relate to problems of viral oncogenesis. In these studies, adenovirus-infected and transformed cells have been studied. But in many aspects, this system appears to be representative for the mechanisms governing the recombination of foreign DNA with the mammalian genome. When the DNA of bacteriophage h or of bacterial plasmids is transfected into mammalian cells, the patterns of integration of these foreign DNA molecules are essentially indistinguishable from those observed after the infection of mammalian cells with adenoviruses or after the transfection of adenovirus DNA. Apparently, the mode of introduction and the type of foreign DNA molecules reaching the nucleus of the recipient cell do not decisevely affect the mechanism of foreign DNA integration. However, it may be prudent to refrain from generalizing conclusions at a time when we have only limited information about this complex problem.

4.1 An introduction to the adenovirus system The adenoviruses are common pathogens in humans and animals. Moreover, several strains have been the subject of intensive research and are used as tools in mammalian molecular biology. More than 100 serologically distinct types of adenoviruses have been identified including > 50 types that infect humans. The family Adenoviridae is divided in two genera, the mammalian adenoviruses (mastadenoviruses) and the avian adenoviruses (aviadenoviruses). The adenoviruses are named after the human adenoids, from which they were first isolated. Based on the genome organization and the high AT content of the viral DNA, a third genus of adenoviruses, the AT adenoviruses, has been proposed (M. Benko, Budapest, personal communication).

4.1.1 Clinical background The main target for human adenoviruses is the respiratory tract. Various adenoviruses can also induce acute follicular conjunctivitis, epidemic keratoconjunctivitis, and less frequently cystitis and gastroenteritis. In infants, the most common clinical manifestations of adenovirus infections are acute febrile pharyngitis and pharyngeal-conjunctival fever. In military recruits, acute respiratory disease is the

28

4 The adenovirus paradigm

predominant form of adenovirus pathology with adenovirus pneumonia as a not infrequent complication. Except for outbreaks in military compounds and occasionally among children, adenovirus infections do not occur epidemically. The virus is probably transmitted via droplets of respiratory or ocular secretions. Several of the adenoviruses can cause respiratory and conjunctival diseases. In addition, a few types of human adenoviruses induce tumors, probably of neuroblastome-like character, in newborn hamsters and other rodents and can transform certain rodent and human cells in culture. There is currently no evidence that adenoviruses are oncogenic in humans but the possibility remains of interest.

4.1.2 Virion structure The adenovirus particle consists of an icosahedral protein shell surrounding a protein core that contains the linear double-stranded DNA genome. The shell which is 70 to 100 nm in diameter is made up of 2.52 structural capsomeres. The 12 vertices of the icosahedron are occupied by units called pentons each of which has a slender projection called a fiber. The 240 capsomeres that make up the 20 faces and the edges of the icosahedron are called hexons because they form hexagonal arrays. The shell also contains some additional minor polypeptide elements (Fig. 11). The core particle comprises two major proteins, polypeptide V and polypeptide VII and a minor arginine-rich protein termed p. A 5.5 kDa protein is covalently attached to the 5'-ends of the DNA. virion

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On the genetic map of the prototype adenovirus (Fig. 12), human adenovirus type 2 (Ad2), the genome is divided into early functions (ElA, ElB, E2A, E2B, E3, and E4 regions) expressed first during viral replication and late functions (L1 to LS regions) which are usually expressed, with the exception of L1, after the early functions and after the beginning of viral DNA replication. The late genes encode the viral structural proteins. In the case of Ad2, DNA replication begins 6 to 8 hours following infection of cultured human cells. The VA segment of the genome codes for small RNAs (VAI and VAII RNAs) which are about 160 nucleotides long and are not translated. VA RNAs regulate translation of viral mRNAs. The VA RNAs are transcribed by eukaryotic RNA polymerase 111. The genome also codes for a tripartite RNA leader sequence that is spliced onto all the late viral mRNAs. In 1977, RNA splicing was discovered in adenovirusinfected cells; yet another example of adenovirus research pioneering eukaryotic molecular biology. Both strands of the double-stranded DNA code for specific viral functions. The termini of the DNA molecule carry inverted repeat sequences so that denatured single strands can form circular DNA molecules.

4.1.3 Classification At present > SO types of human adenoviruses have been identified, five additional candidate types are under investigation (Table 2). The genomes of different adenoviruses are genetically distinct and vary somewhat in size.

30

4 The adenovirus paradigm

Table 2. Human adenovirus types

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12,18,31

3,7,11, 14, 16,21,34,35 1,2,5,6 8,9,10, 13, 15, 17,19,20,22-30,32,33,36-39,4247 4

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4.1.4 Multiplication during productive infection Host cells differ in their permissivity for adenovirus types. In permissive cells, the virus multiplies productively and kills the host cell. Other cells are semipermissive allowing replication at low efficiency whereas in some cell types replication is blocked and the infection is abortive. As discussed below, in some abortive infections all or part of the genome may be integrated into the host DNA resulting in latent infection which may lead to oncogenic transformation. Adenoviruses and the picornavirus coxsackievirus both use an immunoglobuline-type cellular surface protein as receptor for attachment. There is evidence that different domains of this receptor are used by the two different viruses. The knob-like structure of the fiber protein is essential for receptor-anchoring. The virion enters host cells either by attaching to the cytoplasmic membrane and is then engulfed into the cytoplasm in a membrane-bound vesicle (viropexis) or by directly penetrating the cytoplasmic membrane. The viral DNA is gradually uncoated and enters the nucleus of the cell most probably as a nucleoprotein complex that still contains viral core proteins (Fig. 1). The viral DNA is transcribed and replicates in the nucleus of the host cell. The viral mRNA undergoes processing in the nucleus and/or during transport through the nuclear membrane into the cytoplasm where it is translated on polysomes into viral proteins. These proteins return to the nucleus where new virions are assembled. The mass of newly synthesized virus particles can assume crystal-like arrangements. The bulk of the virions are not readily released from the nucleus and the cell. There is evidence that extracellular Ad1 2 virions have a considerably higher specific infectivity than intracellular virions. During active viral release, the newly synthesized virions may receive properties conferring high infectivity toward the host cells. These final steps of virion maturation are not understood at the biochemical level. The initiation of adenovirus DNA replication is atypical in that the P-hydroxyl group of a serine residue in the precursor to the terminal protein (pTP) - an 80 to

4.1 A n introduction to the adenovirus system

31

87 kDa polypeptide - acts as a primer in DNA replication. Viral DNA replication can proceed bidirectionally and by single-strand displacement from either end of the DNA duplex. The adenovirus-encoded DNA polymerase, pTP, the adenovirus E2A single-stranded DNA binding protein, and several host proteins nuclear factors I to 111- catalyze viral DNA replication. Most of the adenovirus genes are transcribed by the DNA-dependent RNA polymerase I1 of the host cell in a complex transcriptional program. This program is regulated by the nucleotide sequences and the structure of the viral promoters and by a large number of cell-encoded transcription factors that recognize specific upstream and downstream nucleotide sequence motifs in the promoters. Genes in the E1A region of the adenovirus genome are the first to be transcribed. A 289 amino acid protein encoded in the E1A region is a transactivator that is essential for the activation of all other viral genes. This immediate-early function can also activate or inactivate certain cellular genes. These investigations on the adenovirus system led to the discovery of transcriptional transactivation. The jointly controlled early E2A and E2B regions code for proteins that are essential for viral DNA replication. Among the E3-encoded functions, one is a 25,000(19,000)-molecular-weightglycoprotein responsible for the interaction with the cell membrane-associated proteins of the major histocompatibility complex. The E3 region-encoded functions appear not to be required for viral replication in cell culture but essential for the interaction with the intact defense system of an organism and for the modulation of host defense functions. The late viral L1 region can also be transcribed early in the infection cycle probably to a limited extent. Genes encoded in the L1 region of Ad5 DNA are essential for virion assembly. Expression of all the late viral functions is under the control of the major late promoter (MLP) components which are located at about 17,20 and 27 map units on the viral genome (Fig. 12). The gene encoding the fiber structural protein can also be controlled via the x, y, and z leaders. The regulation of promoter activity in all biologic systems is dominated by the interactions of promoter sequence motifs with specific factors. These (protein) factors in turn bind to a host of further proteins, cofactors, that determine the structure of transcription complexes. Viral promoters are conditioned to the factors present in specific host cells. Enhancers and silencers are quantitative modulators of promoter function. Both act independently of position and orientation and can exert their influence over relatively long distances. Enhancers strengthen promoter activity whereas silencers have a negative effect abrogating or diminishing promoter function. Enhancer and silencer elements can be species-specific and have first been recognized in work on viral systems. The VAI and VAII RNAs are transcribed by RNA polymerase 111. VAI RNA is an important translational activator of host cell and viral messenger RNAs (mRNAs) late after infection. VAI RNA prevents activation of a protein kinase that is responsible for the phosphorylation and ensuing inhibition of the cellular eIF-2 translation factor. This kinase can be induced by interferon. VAI RNA thus can be viewed as part of a viral defense mechanism against interferon.

32

4 The adennvirits parudigm

4.1.5 Abortive infection Virus infection of a host cell can be blocked at many different steps, thus leading to an incomplete or abortive cycle. Depending on the permissivity of the host cell, different types of adenovirus-host cell interactions can be distinguished. Many cultured human epitheloid cell lines are productively infected by human adenoviruses. Rat cells are semipermissive (e.g., for Ad5), and permit viral replication only at low efficiency. The outcome of an adenovirus infection depends on the animal species, cell type and virus type involved. For example, hamster cells are abortively infected with human Ad12. The viral DNA is transported to the nucleus. There is massive chromosomal association of Ad12 DNA and some of these molecules are integrated into the host cell genome. Both in productively and in abortively infected cells, the viral DNA gravitates towards and becomes transiently associated with the host cell chromosomes as demonstrated by fluorescent in situ hybridization. In the Adl2-hamster cell abortive system, most of the early viral genes are transcribed but the late genes remain silent in the host cells. Ad12 DNA replication in hamster cells cannot be detected with the most sensitive techniques. The major late promoter of Ad12 DNA is inactive in both uninfected and Adl2-infected hamster cells whereas it functions in infected human cells. Ad2 cannot replicate in monkey cells; in this case the translation of some of the late viral mRNAs is deficient. The adenovirus genome persists - perhaps for a very long time - in cells of the human tonsils. It is not known how adenovirus replication in this human organ is restricted.

4.2 An example: Ad12 DNA integration in the Ad12transformed hamster cell line T637 As described in Chapter 3, there are several methods to demonstrate the integrated state of foreign DNA in the recipient host genome. The T637 cell line has been generated by infecting BHK21 hamster cells with Ad12 and upon selection for the transformed cell phenotype with antibodies from hamsters bearing Ad12induced tumors. We have used this cell line for many investigations on viral DNA integration, because the biological properties and the Ad12 integration patterns in these cells have been stable over several decades of subculturing. In Fig. 3 the results of the FISH analyses have been presented which document the insertion of multiple copies of Ad12 DNA at a single chromosomal site. This site has been cloned and the nucleotide sequence across one of the sites of junction between Ad12 and cellular DNA has been determined (Fig. 13). Thus the chromosomally integrated state has been ascertained by t w o independent methods. In this way we have analyzed the persistence of the viral genome in a considerable number of adenovirus-transformed cells and in Adl2-induced tumors or tumor cell lines.

4.2 An example: Ad12 DNA integration in the Adl2-transformed hamster cell line T637

33

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Figure 13. Structure (a) and nucleotide sequence (b) at the osl junction site between Ad12 DNA and hamster cell DNA in the tumor T191. (a) The capital letters refer to the PstI fragments of Ad12 DNA (map in Fig. 5). the A indicates that parts of the sequence were deleted. The nucleotide segments of Ad12 DNA represented in the osl clone were identified by nucleotide numbers from the authentic Ad12 DNA sequence. XbaI subclones are included in the map as well as repetitive sequence motifs (m). The nucleotide sequence was determined (see panel b) for the segment spanned by the dashed arrow in (a). (b) Nucleotide sequence at and adjacent to the site of linkage between viral and cellular DNAs (double-headed vertical arrow). The Ad12 sequence is in italics. Homologies to known hamster DNA sequences and patchy homologies (4 to 13 nucleotides long) between viral and cellular DNAs (horizontal arrows A to D) are designated. A-rich sequences are in bold type. Cellular sequences with homologies to hamster Alu sequences are underlined. Dashed arrows indicate direct repeats of Alu elements (Knoblauch et al., 1996).

34

4 The Lirlenovirus paradigm

4.3 The state of the viral DNA in different cell systems Depending on the genetics of the cellular system and on the virus type, adenoviruses have different modes of interaction with their host cells.

4.3.1 Productive infection Human cells are productively infected with the virus and it replicates actively in these cells. We have adduced several lines of evidence that the viral DNA can become covalently linked to cellular DNA even in productively infected human cells. Since the productively infected human cells succumb to infection it is impossible to pursue the integrated state of the viral genome in subsequently established clonal cell lines. Hence, the demonstration of covalent linkage between viral and cellular DNAs in productively infected cells has been difficult. The most convincing case for this covalent linkage has been made for a symmetric recombinant (SYREC) between adenoviral and cellular DNAs that has been naturally generated during productive infection. This recombinant is packaged into virions that are produced due to the concomitant coinfection of the cells with authentic intact viral genomes and the symmetric recombinant viral genome which carries the packaging signal (see Table 3 ) . Moreover, FISH analyses provide microscopic images of the association of Ad12 DNA with the chromosomes of productively infected human cells. These pictures are indistinguishable from those documenting the integrated state of this viral genome in the Ad12-transformed hamster cell line T637 (Fig. 14, see color plates). The association and/or integration of Ad12 DNA with the host chromosomes in productively infected cells could be transient in this virus-cell system, particularly at late times after viral infection with progressing cell damage and destruction.

4.3.2 Abortive infection Ad12 infects Syrian hamster cells in culture abortively. Virions do enter Syrian hamster cells. Similarly, the infection of Chinese hamster cells in culture is nonproductive, probably because Ad12 cannot enter Chinese hamster cells. Ad12 DNA replication is blocked completely in Syrian hamster cells; early viral genes are transcribed but the transcription and translation of the late functions are again completely blocked. A mitigator sequence in the first intron of the major late transcript of Ad12 DNA is possibly contributing to this efficient late transcriptional inactivation. Ad12 DNA associates extensively with the chromosomes of hamster cells early after infection (Fig. 14). An integrated state for some of this DNA has been demonstrated by physical methods and by DNA-DNA hybridization studies using high molecular mass cellular DNA from abortively Ad12-

4.3 The state o f the viral DNA in different cell systems

35

Table 3. Properties of the symmetric recombinant (SYREC) DNA”

Originated during serial passage of Ad12 on human KB cells at high multiplicities of infection. Encapsidated into Ad12 virions. SYREC DNA-containing virions have lower buoyant density in CsCl density gradients than Ad12 DNA virions. Upon denaturation and renaturation, SYREC DNA molecules fold back into half length DNA molecules (EM study). The left terminal 2081 nucleotides of Ad12 DNA flank the SYREC DNA molecule on either end. The bulk of the nucleotide sequence is made up of a huge palindrome of cellular DNA. The SYREC-Ad12 DNA is encapsidated into virions because it carries the left terminal packaging signal of adenovirus DNA. The cellular DNA in SYREC DNA is not methylated, the same cellular DNA sequences in the cellular genome are highly methylated. The cellular DNA sequences in SYREC DNA are in part of the unique, in part of the repetitive type. The observations on SYREC DNA have helped the design of the “third generation” adenovirus vectors. a

(Deuring et al., 1981; Deuring and Doerfler, 1983)

infected cells. Details of these analyses using equilibrium centrifugation methods in alkaline CsCl density gradients have been explained in Fig. 9 and in Section 3.6. Since the interaction of Ad12 virions with Syrian hamster cells is non-productive the infected cells continue to grow. Upon continuous cultivation of the cells, the previously integrated and free viral genomes are gradually lost from the host nuclei. This loss may be the consequence of a transient state of viral DNA integration and/or selection against cells with foreign DNA integrates. It is conceivable that the cells carrying integrated or more loosely associated viral genomes suffer from a selective growth disadvantage in comparison to the cells devoid of integrated viral genomes and are then eliminated from the replicating cell population. The site of viral DNA integration and/or association with the cellular genome is not specific but different in each cell in both the productive and the abortive systems. Therefore, some of the techniques applied to investigate the integrated covalently linked state of the foreign DNA molecules such as Southern transfer hybridization cannot be sensibly applied.

4.3.3 Transformation of cells in culture Although at an extremely low rate, Syrian hamster or mouse cells in culture can be transformed with adenoviruses into tumor-like cells. The most reliable criterion for cell transformation in culture is the capacity of the transformed cells to

36

4 The adenovinw purudigm

form tumors upon the reinjection into animals. Most of the adenovirus-transformed cells show this phenomenon. Since adenovirus-transformed cells are clonal cell lines the site of viral DNA integration is identical in all cells of an individual tumor. The integrated state is therefore readily proven by several of the analytic procedures described in Chapter 3. In our studies, the cell line T637 has often been used as the prime experimental reference for the integrated state of the Ad12 genome.

4.3.4 Tumor induction by Ad12 in newborn hamsters After the subcutaneous injection of nanogram to microgram amounts of Ad12 virions into newborn Syrian hamsters (Mesocricetus auratus), undifferentiated tumors possibly of neuroblastoma-like origin are generated at the site of virus injection. So far, metastases of these tumors remote from the site of virus injection have not been observed even when the tumors have attained considerable mass. A large number of - in part - clonal tumors can be induced after intraperitoneal injection of Ad12 into newborn Syrian hamsters. Some of these tumors are attached to the peritoneal surface of the intraabdominal organs, probably due to the intraperitoneal distribution of tumor cells. We have studied a large number of independently elicited tumors and demonstrated the integrated state of Ad12 DNA in each of these tumor cells by FISH, Southern transfer hybridization and, in some cases, by the molecular cloning of the sites of junction between cellular and viral DNAs. These tumors are characterized by their clonal origins and by different chromosomal locations of the site of integration of the viral (foreign) DNA in each individual tumor clone. Sometimes several tumors arise in one animal. Each of these tumors has clonal characteristics and again shows independent sites of viral DNA integration.

4.3.5 Tumor cells in culture The Adl2-induced hamster tumors can often be successfully explanted into culture and can be cultivated for long periods of time. The characteristics described previously in Sections 4.3.3 and 4.3.4 hold also for these tumor cell lines. In general, we have observed that the integrated state of the viral genome and the site of its insertion remain stable over many cell generations at least in the vast majority of the cells investigated. Hence, primary tumors and established cell lines from these tumors d o not demonstrably differ in the characteristics of their integrated viral (foreign) genomes.

4.3 The vote ofthe viral D N A in clijycerent cell systems

31

4.3.6 Loss of previously integrated Ad12 DNA sequences While stability of viral (foreign) DNA integrates in transformed and tumor cells appears to be the rule it is not without exceptions. Upon prolonged propagation of these cells in culture, revertants can arise often with an altered cell morphology. These revertants are not frequent but they have often been detected after the freezing and thawing of the original cell line. Perhaps the revertants possess some growth advantage compared to the original transformed or tumor cell line. Initially by Southern transfer hybridization, the loss of previously integrated viral genomes could be documented. This loss can be partial or complete. In one instance, only about one half of a viral D N A copy has been preserved as integrate. Even the recloned cell lines, completely devoid of Adl2-DNA by the criterion of Southern blot-hybridization, have retained their oncogenic potential when reinjected into weanling Syrian hamsters. The revertant cell lines apparently free of integrated viral DNA as determined by Southern transfer hybridization continue to carry minute fragments of viral DNA integrates which are revealed only by the polymerase chain reaction and by using oligodeoxyribonucleotide primers chosen from different parts of the viral genome (Fig. 15). These investigations have been performed with carefully recloned cell populations over several recloning cycles. Different cell clones exhibit completely different patterns of minute amounts of Ad12 DNA fragments persisting in the cellular genome. Hence, it is likely that the loss of originally integrated foreign (viral) DNA proceeds in a gradual cell clone-typical manner such that different cell clones eventually carry remnants from very different parts of the original viral genome. After many recloning steps, we have not found subclones absolutely devoid of Ad12 DNA. Tiny segments, e.g., derived from the right terminus of the Ad12 genome have been found to persist in some of these clones which are, however, devoid of any other part of the Ad12 genome. Even such cell populations have retained their oncogenic potential when reinjected into weanling Syrian hamsters. Obviously, in these cells the persistence of the entire viral genome or of its left terminus is not essential for the maintenance of the transformed phenotype. Early viral functions encoded in the left terminal DNA sequences of the adenoviral genome are presumed to be important in the process of oncogenic transformation of rodent cells. It is doubtful whether the minute amounts of tiny viral DNA fragments can play a role in contributing to the oncogenic potential of these revertants. Could the almost complete loss of previously integrated viral DNA and the persisting oncogenic phenotype in these cell lines present evidence for a hit and run mechanism? As will be discussed in Section 10.3, alterations in the methylation and transcription patterns in Ad12induced tumor cells and in Ad1 2-transformed cells have been demonstrated. I pursue the possibility that these alterations might be important for the process of oncogenic transformation.

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4.4 Adenovirus infection, DNA trnnsfection or DNA rnicroinjectron

39

4.4 Adenovirus infection, DNA transfection or DNA microinjection Most data on the integration of adenoviral DNA have been derived from studies on transformed or tumor cells that have been generated by the infection with intact virions. This way of virus-host interaction resembles natural conditions more closely than transfection or microinjection. We have also investigated the integration of adenovirus DNA or of the DNA of bacteriophage h (see Section 10.3.2) after DNA transfection and genomic fixation. In these experiments the standard cotransfection of the gene for neomycin phosphotransferase and G418 (gentamycin) selection procedures have been employed. The fate of the transfected DNA was followed by DNA-DNA hybridization of the DNA from the transformed cells to ascertain the fixation of the foreign DNAs by the integration in the cellular genomes. According to the parameters tested the patterns of integration of multiple copies of the foreign DNA and their fixation predominantly at a single chromosomal site paralleled the findings with adenovirus-transformed cells or Adl2-induced tumor cells. In the tumor cells, the foreign DNA has been introduced into the cellular genome after virion infection. The transfer of foreign DNA both by virus infection and by transfection of foreign DNA lead to apparently identical patterns of foreign DNA integration. The integration of adenovirus DNA, bacteriophage h DNA or plasmid DNA leads to indistinguishable patterns of insertion as determined by present technology. In a much more limited study, we have followed DNA constructs, e.g., of a fusion product between the E2A late Ad2 promoter and the prokaryotic gene for the enzyme chloramphenicol acetyltransferase after transfection into hamster cells or after microinjection into mouse oozytes. Upon genomic fixation and selection in the hamster cells or after rearing founder and progeny generations of mice, respectively, the integration patterns of the foreign DNA have been determined. Again, in all aspects tested at the time the integration patterns resembled those obtained for integrated Ad12 DNA in transformed or tumor cells. As will be described in Chapter 11, the fate of orally applied bacteriophage M13 DNA or of plasmid DNA containing the gene for the green fluorescent protein (GFP) has been followed in mice. The foreign DNA enters the organism and can be recovered in fragmented form from several organs of the animals including intestinal wall, spleen, liver, and peripheral white blood cells. The foreign DNA can become covalently linked to mouse DNA under these conditons. The cellular nucleotide sequences found adjacent to the M13 DNA recovered in nuclear DNA from the spleen of M13 DNA-fed mice exhibit 70% to 80% homology to authentic mouse DNA segments which might represent mouse pseudogenes. Since only minute amounts of orally introduced foreign DNA can be recuperated from mouse organ DNA a detailed analysis of the patterns of foreign DNA integration could not be performed. The information available argues in favor of similarities in the mode of integration as described for the more classical systems. There is also transfer of food-

40

4 The udetioviriis paradigm

ingested foreign DNA into various organ systems of fetuses and newborns when pregnant mice have been fed with test DNAs (see Section 11.5). These data support the notion that type of foreign DNA and mode of application to and introduction into the cells do not affect the patterns of foreign DNA integration. Of course, patterns of distribution in the recipient host genome do not necessarily reflect the mechanisms by which the foreign DNA has become covalently inserted. These mechanism(s) appear to be flexible and capable of handling many types of foreign DNAs or DNA fragments. The processing of foreign DNA in mammalian DNA may be the task of ancient cellular mechanisms.

4.5 Integration of adenovirus DNA in human cells significance for human somatic gene therapy Studies on the fate of adenovirus DNA in productively infected human cells in culture have provided conclusive evidence for the generation of a high molecular mass form of adenovirus DNA that is firmly associated with cellular DNA. This high molecular mass form of viral DNA can best be found only in actively dividing human cells in culture. Moreover, FISH analyses in the same experimental system have demonstrated the Ad12 DNA to be in a configuration indistinguishable from that of hona fide integrated Ad12 DNA in transformed hamster cells. Thus Ad12 DNA, probably Ad2 DNA as well, can be covalently linked to cellular DNA in productively infected human cells. Since in the cell culture system used for these experiments essentially all productively adenovirus-infected human cells cease to replicate and eventually die, it is impossible to pursue the integrated state of adenovirus DNA in human cells in clonal cell populations in culture. All available evidence renders it likely that adenovirus DNA integration and/or chromosomal association is not specific for a particular cellular site. Linkage can be established at many different loci. The generation of naturally occurring symmetric recombinants (SYREC) between the left terminus of Ad12 DNA and a large palindromic segment of cellular DNA provides further support for the notion that Ad12-cellular DNA recombinants can be generated in Ad12infected human cells. The recombinant SYREC DNA molecules have been found encapsidated into virions due to the presence of the authentic packaging signal in the Ad12 terminal DNA sequence as part of the SYREC molecule. Although humans are the natural host of adenoviruses, the persistence of adenovirus DNA in the human organism has been investigated much less intensely and very little definitive information is available. Adenovirus DNA (fragments) persist in human tonsils and perhaps in peripheral white blood cells. The physical configuration of the adenoviral DNA in these organs is not known. Cells in the human organism obviously differ in many ways from human HeLa cells in culture.

4.6 Studies on integrative recombination of adenovirus DNA in a cell-free system

41

Since adenoviruses have been injected as vectors for foreign genes in somatic gene therapy experiments in animals and humans, the problem of adenovirus DNA integration in human cells and organs becomes important, particularly for therapeutic regimens. Chromosomal integration might prove advantageous for the long-term fixation and expression of foreign therapeutically important genes. The prolonged expression over weeks and months of foreign genes inserted into currently used adenovirus vectors in experimental animals argues for but does not prove, chromosomal insertion. O n the negative side, the chromosomal integration of adenovirus genomes used as vectors for therapeutic genes into the host genome would be affiliated with the problematic consequences of foreign DNA insertion as insertional mutagenesis or alterations in cellular methylation and transcription patterns (Chapter 10). In that respect, vector DNA integration would be less desirable for gene therapeutic programs. In gene therapy, adenovirus DNA persistence and expression have been studied frequently in organs like liver, brain or muscle in which cell divisions are rare events. Possibly because of the scarcity of rapidly dividing cells that could provide the enzymatic repertoire and the cellular chromatin structure most conducive to insertional recombination events between viral and celluar DNAs, adenovirus DNA integration in organs could be a rare event. In actual fact, experiments clearly designed to answer these complex but practically relevant problems are notoriously absent from the literature. Frequently echoed claims that adenovirus DNA did not integrate lack scientific credibility.

4.6 Studies on integrative recombination of adenovirus DNA in a cell-free system We have tried to mimic events during the integration of foreign (viral) DNA into the host genome by developing a cell-free system from nuclear extracts of BHK21 hamster cells. A preinsertion sequence from Syrian hamster BHK21 cells (p7), into which Ad12 DNA had integrated previously during the generation of one of the Adl2-induced hamster tumors, CLACl, and specific fragments of Ad12 DNA have been chosen as recombination partners in this system. The AdlZinduced hamster tumor cell line CLACl had been generated by injecting Ad12 into newborn Syrian hamsters. The nuclear extracts have been highly purified by standard chromatographic procedures. In this cell free system a considerable number of recombinants between Ad12 DNA and cellular DNA have so far been generated. In all of these recombinants, the nucleotide sequence across the sites of junction between the viral and cellular DNA components have been determined. The junction sites in these recombinants in several ways resemble those in naturally generated integrates in transformed or tumor cell lines in that patchy or short sequence homologies have been observed in almost all instances between the viral and cellular sequences close to the junction site or between the cellular DNA replaced by viral DNA sequences at

42

4 T h e ndenoviriis paradigm

the junction sites. This similarity in the structure of in vitro and in vivo generated recombinants argues in favor of the reliability of the cell free system. At least in part, the cell free extracts may be equipped with the enzymatic functions responsible for the integrative recombination process in Ad12-infected hamster cells. Integrative recombination between the nucleotide 20,885 to 24,053 (PstI-D) fragment of Ad12 DNA and the plasmid pBR322-cloned hamster preinsertion DNA sequence p7 has been studied in a cell-free system. Nuclear extracts from uninfected BHK21 hamster cells have been fractionated by a series of chromatographic procedures. The most highly purified protein fraction contains a limited number of protein bands and is still capable of catalyzing the cell-free recombination reaction. The recombination-active proteins range in sizes between 40 and 90/91 kDa. The purified proteins in this fraction have been characterized by determining the N-terminal amino acid sequence, by Western blotting or by their enzymatic activity. The following protein components have been tentatively identified: a homolog of human single-strand DNA binding protein (HSSB), replication factor C, a ligase and helicase activity, nucleosome assembly protein 1 (NAPl), protein disulfide isomerase (PDI) and glucose response protein 78 (GRP78) (K. Fechteler, G. Multhaup, K. Beyreuther, and W. Doerfler, unpublished experiment). The Rad51 protein, the hamster homolog of the RecA/Rad51 protein of E.coli/yeast has also been found and plays a crucial role in recombination in these organisms. The cellular recombination system apparently suffices to catalyze cell-free recombination between Ad12 and hamster cell DNA, but Ad12-specific proteins may modify or enhance the reaction directly or via the modification of cellular factors. In the course of the reaction described, two linear molecules are joined. The recombination reaction takes place between short sequence homologies. In all analyzed single reactions leading to an in vitro recombinant molecule at least one of the interacting short sequence homologies has been found close to a DNA terminus. In most instances, this short sequence homology lies within the first twenty nucleotides and always within the first sixty nucleotides. Corresponding short sequence homologies in the reaction partner can be remote from the DNA terminus. Sequences inbetween these homologies are deleted in the recombinants. The size of the deletion in any single recombinant is dependent on the location of the short sequence homologies. The positions of the short sequence homologies and therefore the sites of recombination are not restricted to the preinsertion site p7, they can also be found in the backbone of the plasmid carrying the p7 preinsertion sequences. Nevertheless, 12 out of 20 in vitro-generated recombinants show linkage of Ad12 DNA to the p7 sequence. Further unknown sequence motifs required for the preinsertion sequence might contribute to this site selection. Additional nucleotides have not been found in any of the in v i m recombinants. The recombinant sequence has been derived exclusively from the PstI-D fragment of Ad12 DNA and the p7pBR322 hamster preinsertion sequences. Sequence alterations of the original sequences have not been found at the sites of junctions. The protruding single-strands generated by the restriction enzymes PstI and EcoRI are not preserved in the recombinants.

4.7 Further reuding

43

The short sequence homologies described here show a high degree of variation. Moreover, the reaction is not limited to substrates with short sequence homologies close to the DNA termini. The interacting short sequence homologies can also be found at a distance of several kilobases from the DNA terminus. Thus, the chances for the formation of junctions between two separate linear DNA molecules are enhanced. These characteristics of the recombinants are reminiscent of a strand invasion model of recombination. Some of the described properties of the in vitro recombinants are also found in the junction sites cloned from adenovirus-transformed cell lines or Adl2-induced hamster tumor cell lines. Of course, the in vivo situation is much more complex, and it is likely that several different mechanisms can be involved in the process of integration of adenoviral DNA into the cellular genome. By comparing the in vitro and the in vivogenerated junction sites, the following similarities are apparent: 0

0

Deletions of (terminal) viral and cellular nucleotides at the sites of junction. Frequent occurrence of short sequence homologies beween viral and displaced or preserved cellular DNA sequences. The terminal sequences of adenoviral DNA are frequently involved in the formation of junctions.

These data support the notion that this cell-free system developed for investigations on the integrative recombination between viral and cellular DNAs resembles the in vitro reaction in a number of important parameters. Apparently, the biological system selected for studies on the integration of foreign (adenovirus) DNA might not play a decisive role in the outcome of the reaction. On the other hand, the current state of the analytical tools available for these experiments is still limited, and more refined methodology may reveal more details and possibly differences in different systems. Uptake and insertion of foreign DNA can be considered an ancient cellular mechanism that proceeds monotonously but has retained a great deal of mechanistic flexibility. Obviously, short patchy sequence homologies could greatly enhance the probability of insertional recombination at appropriate sites.

4.7 Further reading Akusjarvi, G., Pettersson, U., Roberts, R.J. (1986) Structure and function of the adenovirus-2 genome. In: Adenovirus DNA: The Viral Genome and its Expression. Developments in Molecular Virology, Vol. 8 (Doerfler, W., Ed.). Martinus Nijhoff Publishing, Boston, Dordrecht, pp. 53-95. Bergelson, J.M., Cunningham, J.A., Droguett, G., Kurt-Jones, E.A., Krithivas, A., Hong, J.S., Horwitz, M.S., Crowell, R.L., Finberg, R.W. (1997) Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275,1320-1323.

44

4 The adenovirus puradigrn

Brown, D.T., Westphal, M., Burlingham, B.T., Winterhoff, U., Doerfler, W. (1975) Structure and composition of the adenovirus type 2 core. J. Virol. 16,366-387. Cook, J.L., Lewis, A.M., Jr. (1979) Host response to adenovirus 2-transformed hamster embryo cells. Cancer Res. 39,1455-1461. Deuring, R., Doerfler, W. (1983) Proof of recombination between viral and cellular genomes in human KB cells productively infected by adenovirus type 12: structure of the junction site in a symmetric recombinant (SYREC). Gene 26, 283-289. Deuring, R., Klotz, G., Doerfler, W. (1981) An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA. Proc. Natl. Acad. Sci. U S A 78,3142-3146. Doerfler, W. (1969) Non-productive infection of baby hamster kidney cells (BHK21) with adenovirus type 12. Virology 38,587-606. Doerfler, W. (Ed.) (1995) The molecular repertoire of adenoviruses. Current Topics in Microbiology and Immunology 199/I-III. Springer Verlag, Berlin, Heidelberg, New York, Tokyo. Doerfler, W. (1996) Adenoviruses. In: Medical Microbiology, 4th. Edition (Baron, S., Ed.), pp 813-821. Doerfler, W., Bohm, P. (Eds.) (1986) Developments in Molecular Virology, Vol. 8. Adenovirus DNA: The Viral Genome and its Expression. Martinus Nijhoff Publishing, Boston. Eick, D., Stabel, S., Doerfler, W. (1980) Revertants of adenovirus type 12-transformed hamster cell line T637 as tools in the analysis of integration patterns. J. Virol. 36,4149. Fechteler, K., Tatzelt, J., Huppertz, S., Wilgenbus, P., Doerfler, W. (1995) The mechanism of adenovirus DNA integration: studies in a cell-free system. Curr. Topics Microbiol, Zmmunol. 199/II, 109-137. Green M., Wold, W.S.M., Mackey, J.K., Ridgen, P. (1979) Analysis of human tonsil and cancer DNAs and RNAs for DNA sequences of group C (serotypes 1, 2,5, and 6) human adenoviruses. Proc. Natl. Acad. Sci. U S A 76,6606-6610. Groneberg, J., Sutter, D., Soboll, H., Doerfler, W. (1978) Morphological revertants of adenovirus type 12-transformed hamster cells. J. Gen. Virol. 40, 635645. Johansson, K., Persson, H., Lewis, A.M., Pettersson, U., Tibbetts, C., Philippon, L. (1978) Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2. J. Virol. 27,628-639. Knoblauch, M., Schroer, J., Schmitz, B., Doerfler, W. (1996) The structure of adenovirus type 12 DNA integration sites in the hamster cell genome. J. Virol. 70, 3788-3796. Miiller, U., Doerfler, W. (1987) Fixation of unmethylated or the 5’-CCGG-3’ methylated adenovirus late E2A promoter-CAT gene construct in the genome of hamster cells: gene expression and stability of methylation patterns. J. Virol. 61.371 0-3720.

4.7 Further reading

45

Neumann, R., Genersch, E., Eggers, H.J. (1987) Detection of adenovirus nucleic acid sequences in human tonsils in the absence of infectious virus. Virus Rex 7, 93-97. Pfeffer, A., Schubbert, R., Orend, G., Hilger-Eversheim, K., Doerfler, W. (1999) Integrated viral genomes can be lost from adenovirus type 12-induced hamster tumor cells in a clone-specific, multistep process with retention of the oncogenic phenotype. Virus Res. 59, 113-127. Schroer, J., Holker, I., Doerfler, W. (1997) Adenovirus type 12 DNA firmly associates with mammalian chromosomes early after virus infection or after DNA transfer by the addition of DNA to the cell culture medium. J. Virol. 71, 7923-7932. Strohl, W.A. (1969) The response of BHK21 cell to infection with type 12 adenovirus. Virology 39,642-652. Strohl, W.A. (1973) Alterations in hamster cell regulatory mechanisms resulting from abortive infection with an oncogenic adenovirus. Progr. Exp. Tumor Rex 18,199-239. Tatzelt, J., Fechteler, K., Langenbach, P., Doerfler, W. (1993) Fractionated nuclear extracts from hamster cells catalyze cell-free recombination at selective sequences between adenovirus DNA and a hamster preinsertion site. Proc. Natl. Acud. Sci. USA 90,7356-7360. Wronka, G., Fechteler, K., Schmitz, B., Doerfler, W. Integrative recombination between adenovirus type 12 DNA and mammalian DNA: purification of a cell-free system and analyses of in vitro-generated recombinants. Submitted. Zock, C., Doerfler, W. (1990) A mitigator sequence in the downstream region of the major late promoter of adenovirus type 12 DNA. EMBO J. 9,1615-1623.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

5 Conclusions derived from a survey of junction sites These studies have been performed over a long period and the results from biologically quite different systems have been included in this summary: Junctions between adenovirus DNA and cellular DNA from adenovirus-transformed cells, Adl2-induced tumors and tumor cell lines derived from these tumors, SYREC DNA, a naturally occurring symmetric recombinant between Ad12 DNA and cellular DNA from Ad12 productively infected human cells, junctions between transfected foreign DNA and host cell DNA, junctions between mouse-like DNA and bacteriophage M13 DNA from spleen cells of mice that had been fed with M13 DNA. The M I 3 DNA had reached the mouse organism via gastrointestinal uptake of foreign DNA (see Chapter 11). In Table 4 junction sequences and their computer-generated structures have been summarized. Of course, the stem-loop designs suggested by computer analyses remain hypothetical and cannot be verified in vivo by current technology.

5.1 On the characteristics of junction sequences The conditions under which sites of insertional recombination in mammalian cells have been investigated have been outlined in the previous chapters. The general validity of the characteristics deduced have to be treated with a certain caveat, since only a relatively small number of nucleotide sequences at junction sites have so far been determined. Table 4. (From Knoblauch et al., 1996). T191 (-1 )

HA1217 CLAC3

T1111(2)

CLACl

CBA-12-1-T

T637

BHK21

(os2)

I.DNA

-

Junction site

Prelneertion aite

48 0

0

0

0

0

0

5 Concliirion~drrrveti from a yurvey oflirncrion sites

In most instances multiple copies of foreign DNA have been inserted at a single chromosomal locus. It is not known whether these multimers are generated before integration by joining foreign DNA molecules in a “packelesome” (M. Wigler). Alternatively, postinsertional amplifications could explain the tandem integrates which frequently are not true tandems - “one after or behind another”, as defined by Webster - but are often separated by nucleotide sequences of unknown derivations, possibly of cellular or rearranged viral origin (see Fig. 3a). The foreign DNA can be inserted into unique or into repetitive DNA sequences. Hard facts about the frequently cited insertional mutagenesis are difficult to find, perhaps because mutagenesis events that affect cellular functions seriously might not be compatible with cell survival and hence escape detection. In Section 10.3, a functional type of mutagenesis effect will be discussed: In the wake of foreign DNA insertion, cellular DNA methylation patterns can be altered, and these changes likely affect the cellular transcriptional profile. The inserted foreign DNA molecule can be intact or can be integrated with minor deletions of a few nucleotides at the termini. Integrates with major deletions have also been observed. Similarly, on the cellular side the target sequence can accept the foreign DNA molecule without the loss of a single nucleotide. Alternatively, larger segments of cellular DNA can be deleted during or after integration of foreign DNA. Occurrence and extent of deletions depend on unknown events and mechanisms. The nucleotide sequences at several sites of covalent linkage between viral (foreign) and cellular DNAs have been determined. In this way the covalent nature of this linkage has been unequivocally established. At many, but not all, of these sites patchy or more extensive nucleotide sequence identities or homologies have been observed. Sometimes, these patchy homologies exist between the terminal viral DNA sequence that has been deleted, and the cellular nucleotide sequence that replaced the viral DNA, e.g., at the junction site in the Adl2-induced hamster cell line CLAC1. It is likely that these sequence identities play a major guiding role for the mechanism of insertional recombination between foreign and cellular recipient DNA. This mechanism is, however, not completely dependent on the availability of nucleotide sequence homologies, but they probably facilitate the insertion event considerably. There are junction sites without sequence homologies. Many of the cellular nucleotide sequences that have served as targets for the insertion of Ad12 DNA have been shown to be transcriptionally active even in cells that have never been exposed to foreign DNA and/or viral infections. It is at least plausible to postulate that actively transcribed domains of the cellular genome with a specific open or uncoiled chromatin structure could be particularly suitable sites for the recombination with foreign DNA in the nucleus of the cell. The question has also been raised whether sites of chromosomal instability or breaks could serve as preferred loci of foreign (viral) DNA insertion. There is

5.1 O n the characteristics of junction sequences

49

some evidence from the study of integration sites of the genomes of papillomaviruses and of plasmid DNA that insertion sites can be located in the vicinity of fragile sites. Even in the best documented cases, however, the experimental evidence for this notion has been adduced at the level of cytogenetic studies. To my knowledge, these investigations have not been supported by studies at the nucleotide level. It appears, therefore, premature to speculate about the possibility that fragile sites might serve as preferential integration sites for foreign DNA. On the other hand, it has been well documented that the infection with certain DNA viruses, like adenoviruses, can create new fragile sites in the mammalian genome. It will certainly be worth investigating further and particularly at the nucleotide level, to what extent fragile sites, whatever their molecular structure might turn out to be, allow preferred insertional recombination with foreign (viral) DNA. When the cellular nucleotide sequences between different sites of linkage in Adl2-induced tumor cell lines or in adenovirus-transformed cell lines are compared, no nucleotide sequence homologies or similarities between different junction sites can be detected. The existence of a specific or preferred cellular nucleotide sequence motif for the insertion of adenovirus or foreign DNA is therefore unlikely. The insertion of foreign DNA into established mammalian genomes hence seems to follow a mechanism akin to non-homologous recombination in which short sequence homologies can assume an auxiliary function. Of course, in enzymatic reactions as complex as insertional recombination categorizations in simplistic mechanistic models are unrealistic and not useful for the design of further experiments. At present, I favor an insertional recombination model for foreign DNA in mammalian cells that accounts for the non-specific sites of integration and the utilization of nucleotide sequence homologies that hapharzardly exist between the foreign and the recipient cellular genomes. We have also demonstrated that the chromosomal localizations of integrated Ad12 genomes in Adl2-induced tumors and in the cell lines derived from these tumors are different for each tumor, even among multiple tumors that are not infrequently induced in the same animal. This observation is consistent with the notion of non-specific sites of foreign DNA integration, also after the induction of Ad12 DNA-containing tumors in newborn hamsters. Upon explantation of cells from the tumor and serial passage of these cells in culture, the site and pattern of integration appear to be stable over many cell generations. The integrated foreign DNA often becomes de novo methylated in specific patterns. This de novo methylation is initiated at specific sites in the inserted viral genomes, even when multiple copies of Ad12 DNA have been integrated. The study of the parameters affecting the de novo methylation of foreign DNA requires further work. An understanding of the mechanism and preconditions for de n o w methylation may hold the key to the elucidation of perplexing phenomena during embryonal development, imprinting, and the reshuffling of transcriptional patterns in tumor cells. Work on integrated viral

so

0

0

5 Conclusions derived from a wrvey of junction sites

genomes will help in solving this challenging task. The de novo methylation of integrated foreign DNA in transgenic cells or organisms can help explain the frequently reported finding that transgenes active at early stages after insertion often become gradually inactivated. Of course, factors other than the de novo methylation might contribute to this inactivation. Specific patterns of cellular DNA methylation, as they exist in different segments of established cellular genomes in cell lines or in organisms, can be altered as a consequence of foreign DNA insertion into an established mammalian genome. These patterns can substantially differ between an organism and a cell line derived from one of its organs or from a virus-induced tumor. Extensive changes of cellular DNA methylation patterns have been documented when these patterns have been compared between BHK21 or primary hamster cells and Ad12-transformed hamster cells or several Adl2-induced hamster tumor cell lines. These alterations in cellular methylation patterns might, in part, be due to the transformed phenotype of the cell. Evidence is emerging that the insertion of foreign DNA by itself, e.g., of bacteriophage h DNA, elicits similar, though less extensive, changes (see Section 10.3). In general, the integrated state of foreign (viral) DNA is stable over many cell generations or many generations of transgenic organisms. However, at unknown frequency and by unknown mechanisms the integrated foreign DNA can be lost partly or in toto. We have studied revertants of Adl2-induced tumor cell lines that apparently have lost all or most of the multiple, previously integrated viral genome copies. However, the cells apparently devoid of integrated viral DNA do retain their oncogenic phenotype in newborn or weanling hamsters (see Chapter 6). There are intricate questions as to the persistence of tiny viral DNA fragments in an integrated state in subpopulations of cells in the total cell population of a tumor cell line. How stable are these miniscule DNA fragments and how reliably can they be determined, e.g., by the polymerase chain reaction?

It is interesting to note that many of the observations made with intact integrated Ad12 genomes in cells that have been transformed by virion infection, or in Adl2-induced tumor cells that developed after the injection of intact virions in newborn hamsters, are closely paralleled in cells that have been transformed after the transfection of Ad12 DNA fragments from the left viral DNA terminus. Thus, the mode of transfer of foreign DNA into mammalian cells and the length of the viral DNA seem to have little or no influence on the mode and mechanism of foreign DNA integration.

5.2 Persistence of integrated foreign DNA

51

5.2 Persistence of integrated foreign DNA - a novel functional type of insertional mutagenesis The persistence of adenovirus DNA in an integrated state in hamster or other rodent cells is frequently associated with the transformed or oncogenic phenotype. In virus-transformed and virus-induced tumor cells the integrated adenovirus genomes continue to be expressed. In general, the early viral genes are transcribed and translated. Some of the early adenovirus proteins interact specifically with cellular proteins. The 289 amino acid protein encoded in the adenovirus E1A region can bind to the gene product of the retinoblastoma gene, a cell cycle and transcriptional regulator. The human retinoblastoma (RB) gene has originally been identified in patients with retinoblastomas in whose genome both RB alleles are deleted or mutated. Similarly, the 55 kDa protein encoded in the E1B region of the adenovirus genome has been shown to complex the p.53 and similar cellular antioncogene proteins. The cellular p53 gene has originally been found by the capability of the p.53 gene product to coprecipitate with the large T antigen of simian virus 40 (SV40) in SV40-transformed rodent cells. A strong case has been made for the notion that these specific interactions of certain adenovirus proteins with cellular antioncogenes or tumor suppressor genes might interfere with the regulation of cell growth in a way that the cells override apoptotic harnesses and are shifted onto a path of unlimited replications, thus leading to tumor development. It remains to be established that the sequestration of one or several tumor suppressor genes in the cell would in fact suffice to transform a cell to the oncogenic phenotype. Cellular growth regulation seems to be subject to and safeguarded by several alternative pathways. Viral (foreign) DNA integration in transformed or virus-induced tumor cells can have one or several of the following consequences for the cell whose genome has been targeted for foreign DNA insertion. Insertion of foreign (viral) DNA leads to the stable and permanent genomic fixation of genes or DNA segments from sources outside the targeted cell or organism. Depending on the type of foreign genes inserted or the site of insertion and its sequence environment, the foreign DNA can be expressed, silenced, subjected to cellular regulation or influence itself the regulation of neighboring cellular genes. The DNA sequences in the targeted cellular genome that are directly affected by the insertion event could be silent or without an essential function. In that case insertion might not have a direct topical effect. When a functional genetic center of the cellular genome is hit, however, the cell might not survive or change its biological properties. Integrated foreign DNA in mammalian genomes frequently becomes de n o w methylated by the DNA methyltransferase systems of the cell. Since de n o w methylation is a frequent event in development and selective gene inactiva-

52

0

0

5 Conclusions derived from u survey of junction sites

tion, this system offers possibilities to study the mechanisms of de novo methylation. As a new thought, we pursue the possibility that the insertion of foreign DNA into an established mammalian genome alters patterns of cellular DNA methylation both at sequences close to and remote from the integrate. Such changes have been demonstrated in Adl2-transformed cells and in Ad12induced tumor cells. The insertion of non-transforming DNA, e.g., of that of bacteriophage lambda can elicit similar changes, although to a lesser extent. Alterations of patterns in cellular DNA methylation have been shown to be associated with altered transcription patterns of the cellular genes affected. This model of the consequences of foreign DNA integration predicts transeffects on regions of the cellular genome which are located remote from the site of insertion. Alterations of the transcriptional program of the cell would amount to a novel type of insertional epigenetic mutagenesis which is not limited to genes at the sites of the foreign DNA integrates but involves larger parts of the genome. The consequences of foreign DNA insertion on the overall chromatin structure of the targeted cell have not yet been investigated. Most frequently, foreign DNA molecules are inserted as multiple copies in pseudo-tandem arrays. With Ad12 as an example, up to 30, 50 or more copies of the viral DNA can thus be genomically fixed. With a genome length of 34.125 nucleotide pairs, 30 integrated copies of Ad12 DNA amount to the addition of > 1 megabase to the highly organized genome of the cell. It is doubtful that perturbations of this magnitude can remain without functional sequelue.

Inserting foreign DNA into an established genome is likely to have consequences whose extent and nature will depend on the size and the genetics of the integrates and on the genetic functions at the cellular site of insertion. In the pursuit of unlocking the puzzles of oncogenic transformation it will be cogent to consider more than one possibility in the face of a gamut of mechanisms which are all only incompletely understood.

5.3 Adenovirus D N A chromosomal association covalent genomic integration Upon the infection of permissive human or of totally non-permissive hamster cells with Ad12, a large number of the incoming viral genomes become associated with the chromosomes in either host cell system (Fig. 10,14,16, see color plates). Similar observations have been made when viral DNA is transfected into cells by the Ca2+ phosphate precipitation method or when the viral DNA or a complex between the viral DNA and the 5'-terminally linked terminal binding protein (tp) are simply added to the growth medium of the cells. The bulk of the viral DNA colocalizing with the chromosomes at early and late times after infection or after

5.3 AdenovirusDNA: chromosomal association

- covalent

genomic integration

53

different means of gene transfer might be only transiently linked to chromatin. In a large number of cells, this step is possibly the first one on the way to bona fide integration in perhaps only some of the cells. A subpopulation of the viral genomes will then become truly integrated by covalent linkage. What is the nature of the chromosomal association and how does the viral DNA penetrate the chromatin structure? Does the terminally linked adenoviral protein play a role in this pathway? By applying an analytical modification of the conventional FISH technique a closer insight into Ad12 DNA linkage to chromosomes and the host genome has been obtained. Chromosomes from Ad12 productively infected human HeLa cells and - for comparison - from the Adl2-transformed hamster cell line T637 with about 20 copies of Ad12 DNA covalently linked to cellular DNA were subjected to low-speed centrifugation. In this way, the chromosomes were mechanically stretched prior to being analyzed for the presence of Ad12 genomes by FISH. The data in Fig. 14 present a direct visual comparison of chromosome fibers from productively infected human cells and from Adl2-transformed cells with fibers from both cell types carrying viral DNA signals. The physical states of the viral genomes in both cell types appear to be identical or very similar. Multiple copies of viral DNA signals alternate with cellular DNA stretches in an alternating pattern. For the Adl2-transformed cell line T637, the direct demonstration of this pearl-threaded pattern of Ad12 DNA integration independently confirms the previously documented model that consecutive viral DNA genomes at the site of insertion are separated by DNA other than Ad12 DNA, most likely cellular DNA. In restriction analyses performed earlier, we had shown that one viral terminus in T637 DNA is not linked directly to the next one but that cellular DNA must be interposed between individual Ad12 integrates (Fig. 17). The massive association of viral DNA with human or hamster cell chromosomes in infected cells resists a number of chemical and physical agents, including chromosome stretching by centrifugation, employed to test the resilience of the chromosomal linkage of Ad12 DNA. During the replication of cells, this linkage may be broken. Moreover, it is conceivable that even covalently integrated viral DNA molecules can be released from cellular DNA linkage. Mechanism and frequency of these associationhtegration events are unknown. Circumstantial evidence suggests that linkage often appears unstable at early times after insertion, possibly because in an infected or transfected mass-culture of cells those cells are functionally selected for survival in which the integrates had been inserted at sites not essential for cell survival. Adenovirus-transformed cells have Ad12 DNA integration patterns that are stable over decades in culture, although rare revertants can arise that have lost all or most of the integrated viral DNA.

54

5 Conclusions derived from n survey of junction sites

Msp I

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Figure 17a. Design of an experiment to determine the mode of integration of Ad12 DNA in Adl2-transformed hamster cells. Arrows indicate sites of cleavage of the MspI restriction endonuclease (only a few of these sites are indicated). Crosses designate label in ”P-deoxyribonucleotides incorporatcd into DNA.

5.4 Further reading

EcoRI

55

Eco R I

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Figure 17b. Possible models for the integration of Ad12 genomes in the DNA of transformed hamster cells. A and C refer to the terminal EcoRI fragments A and C of Ad12 DNA. H I and H2 represent specific hamster sequences of the repetitive type. HI could also contain rearranged viral DNA sequences. Arrows indicate cleavage sites of the EcoRI restriction endonuclease. (a) Integration into repetitive DNA, or postintegrational amplification. The cellular sequences separate viral genomes in a regular spacer array. (b) Integration into repetitive hamster DNA. (c) True tandems of Ad12 DNA integrated into cellular DNA. (d) This model suggests that Ad12 DNA is integrated via a circular intermediate. The circular molecule of Ad12 DNA is stabilized is assumed by the terminal adenovirus protein (P). Recombination with the host DNA (w) to occur inside the right terminal EcoRI fragment A of Ad12 DNA. (From Stabel et al., 1980)

5.4 Further reading Doerfler, W., Bohm, I? (Eds.) (1995) The Molecular Repertoire of Adenoviruses. Current Topics in Microbiology and Immunology, vol. 199/1-111. Springer-Verlag, Berlin, Heidelberg, New York, Tokyo. Doerfler, W., Gahlmann, R., Stabel, S., Deuring, R., Lichtenberg, U., Schulz, M., Eick, D., Leisten, R. (1983) On the mechanism of recombination between adenoviral and cellular DNAs: the structure of junction sites. Curu. Topics Microbiol. Immunol. 109,193-228.

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5 Conclusions derived from a ,survey of junction sites

Fujinaga, K., Yoshida, K., Yamashita, T., Shimizu, Y. (1984) Organization, integration, and transcription of transforming genes of oncogenic human adenovirus types 12 and 7. Curr. Topics Microbiol. Immunol. 110,53-72. Horwitz, M.S. (1996) Adenoviruses. In: Fields Virology, 3rd Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Mehick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.), Lippincott-Raven Publishers, Philadelphia, New York, pp. 2149-2171. Knoblauch, M., Schroer, J., Schmitz, B., Doerfler, W. (1996) The structure of adenovirus type 12 DNA integration sites in the hamster cell genome. J. Virol. 70, 3788-3796,1996. Kucherlapati, R., Smith, G.R. (Eds.) (1988) Genetic recombination. American Society for Microbiology, Washington. Schroer, J., Holker, I., Doerfler, W. (1997) Adenovirus type 12 DNA firmly associates with mammalian chromosomes early after virus infection or after DNA transfer by the addition of DNA to the cell culture medium. J. Virol. 71, 7923-7932,1997. Shenk, T. (1996) Adenoviridae: the viruses and their replication. In: Fields Virology, 3rd Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.), Lippincott-Raven, Philadelphia, New York, pp. 21 11-2148. Stabel, S., Doerfler, W., Friis, R.R. (1980) Integration sites of adenovirus type 12 DNA in transformed hamster cells and hamster tumor cells. J. Virol.36,22-40.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

6 Adenovirus-induced tumor cells and revertants 6.1 Clonal origin of Adl2-induced tumors Adl2-induced tumors that have been generated by the subcutaneous or intraperitoneal injection of the virus into newborn hamsters are of clonal origin. By the fluorescent in situ hybridization (FISH) technique, the viral DNA has been shown to be integrated at one chromosomal site which has been different in 60 different tumors analyzed. Only in one tumor, viral DNA has been found integrated on two chromosomes. Moreover, when the integration patterns in the 60 different tumors have been investigated by Southern blot hybridization after cleavage of the tumor cell DNA with PstI or HindII, distinct and unique patterns have been observed for each tumor (Fig. 5 ) . Upon passage of the tumor cells in culture for up to 75 to 96 generations, integration patterns or unique chromosomal locations have remained unaltered. In each tumor, all cells carried integrated Ad12 DNA at the same chromosomal site. The data demonstrate that in Ad12 tumor induction in hamsters the tumor cells are of clonal origin. In each tumor cell population, the site of viral DNA insertion is different. As discussed in Section 5.1, on the basis of nucleotide sequence analyses at the sites of junction between viral and cellular DNAs, there is no evidence for the existence of specific sites of viral DNA integration in Adl2-induced tumors or in adenovirus-transformed cells. After the subcutaneous injection of Ad12 virions and tumor formation at the site of injection, the formation of metastases has not been observed, except for the identification of tumor cells in some of the local lymph vessels. When Ad12 is administered intraperitoneally, extensive tumor formation spreads across the entire peritoneal cavity involving the surface of many abdominal organs. Some of these tumors exhibit identical integration patterns and may also have clonal origins. However, in this system several clonal tumors develop which show different integration patterns for Ad12 DNA.The dissemination of these tumors in the peritoneal cavity may be due to the special anatomy there or to cell culture-like conditions in the abdominal compartment.

6.2 Stability - instability Upon serial passage of adenovirus-transformed cells or of Adl2-induced tumor cells in culture, unknown events, possibly enhanced by freezing and thawing of the cells, can lead to the destabilization of the integrated adenovirus genomes. It is conceivable that, at early stages after the integration event, a metastable association of the bulk of the integrated viral genomes with the host genome, perhaps

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6 Adenovirus-induced tumor cells and reverlants

akin to that demonstrated in Fig. 14, prevails which is converted to a more stable configuration with an increasing number of cell divisions. The differences between metastable as compared to the stable states of foreign DNA integration may be related to chromatin structure and to the covalent bond between viral and recipient cellular DNAs. Upon continuous passage, and in particular after freezing and thawing, we have repeatedly observed that Adl2-induced hamster tumor cell lines have been found free of Ad12 DNA sequences as determined by the conventional Southern blot hybridization, and yet these cell lines retain their full oncogenic potential when reinjected into newborn or weanling Syrian hamsters. When carefully recloned sublones of these revertant cell lines have been investigated for the continued presence of small amounts of Ad12 DNA by using the polymerase chain reaction, traces of small fragments of viral DNA can still be detected. In different subclones, different persistence patterns of fragments have been found (Fig. IS). I surmise that, after the loss of the bulk of the previously integrated Ad12 genomes, small fragments of Ad12 DNA can persist in subpopulations of the cells in highly variable distribution patterns. Apparently, the excision events of integrated foreign DNA molecules proceed in a non-systematic way leaving different remnants of the previously integrated foreign DNA genomes in different members of the cell population. Again, there is no definite information about the rules or the mechanisms by which integrated foreign DNA can be eliminated from the transgenic cell or organism. In a cell population, there seem to be constant excision events following a mosaic pattern in a subgroup of the population. The fact that Adl2-induced tumor cells can lose practically all viral DNA segments and still retain the oncogenic phenotype, raises questions about the possibility of a hit and run mechanism of DNA viral oncogenesis. If one accepted the possibility of such an ephemeral sequence of events, failure to detect viral, e.g., adenoviral DNA sequences in human tumors might not vindicate the conclusion that human tumors have no relationship to the infection with any of the adenoviruses. It will be necessary to subclone cells from human tumors and search for small fragments of adenoviral or other viral DNAs by the polymerase chain reaction.

Figure 18. PCR analyses of DNA preparations from subclones of cell lines HI 11l(1) (a, b) and CLACl (c) for the presence of Ad12 DNA sequences. Total cellular DNA was isolated from subclones as designated. By using the PCR primer oligodeoxyribonucleotides C4.1 and C4.2 from the PstI-C and D1.l and D3.2 from the PstI-D fragments, DNA samples were screened for the presence of Ad12 DNA sequences. PCR products were fractionated by electrophoresis on 1% agarose gels, Southern blotted and hybridized to Ad12 DNA which was "P-labeled by nick translation. DNAs from the uncloned line H1111(1) (a) and from the revertant TR12 (a-c) were also studied and found positive. The lanes designated H20 contained PCR samples derived from reactions in which DNA was omitted and replaced by H20. The PstI fragments of Ad12 DNA were co-electrophoresed as size markers. (b) PCR analyses in Ad12 DNA segments from the E4 region. The numbers I to V1 refer to primer pairs as indicated (see right margin) (Pfeffer et a]., 1999).

6.2 Stability - instability

59

flu

6 Adenovirii.\-inu’iiced tunlor cells and revemirits

6.2.1 Hit-and-run mechanism of viral oncogenesis? Integrated Ad12 DNA can be lost from Adl2-transformed cells or Adl2-induced hamster tumor cells under conditions that are not understood in detail. At least in a few examples, the total or near-total loss of integrated foreign (Ad12) genomes, in particular of all sequences from the left terminus of the Ad12 genome, was still compatible with the maintenance of the oncogenic phenotype of the revertant cells. Investigations by PCR on the presence of tiny segments of Ad12 DNA in some of the originally Adl2-induced hamster tumor cells have revealed that individual cells from this initially clonal population can lose the Ad12 integrates at different rates. While some of the cells in the total cell population have lost most or actually all traces of viral DNA integrates, others may still contain small Ad12 DNA segments. These interpretations, which are based on the analyses of cell populations derived from clonal tumor cells, argue in favor of an excision mechanism that proceeds in a gradual and step-wise fashion to eliminate the foreign DNA integrates. At present, it is unknown whether these excisions are due to random events or result from an active recognition and defense-excision mechanism against integrated foreign DNA in the mammalian genome. In at least some of the Adl2-induced tumor cells, the continued persistence of integrated Ad12 DNA sequences is not a precondition for the maintenance of the oncogenic phenotype. Cells devoid of Ad12 DNA except for tiny segments from the right viral DNA terminus continue to be oncogenic in hamsters. A total population of between lo2 to lo7 cells are routinely injected into hamsters to test for oncogenicity. The presence of the viral genome and its products is not necessary for the maintenance of the transformed phenotype. The essential parameters in viral transformation are perhaps to be sought in the reorganization of the cellular genome. Viral gene products might initially assume an auxiliary function in eliciting these processes. In this context, it is important to recall that in those revertants of cell line T637, which have lost all but one or a few copies of the integrated Ad12 DNA, the remaining Ad12 DNA sequences are more heavily methylated than they are in the parent T637 cell line. This finding implies that levels of methylation in the integrated foreign DNA might somehow be related to the ability of cellular defense mechanisms to recognize recently inserted DNA as foreign. We have also demonstrated that the alterations in cellular DNA methylation patterns in the Adl2-transformed hamster cell line T637 persist in the revertant TR3 in which Ad12 DNA cannot be detected any longer by Southern transferhybridization experiments. It is, therefore, likely that alterations in cellular transcription patterns and/or in cellular genome organization that have been initiated by Ad12 DNA integration and expression patterns of viral genome products are permanently imprinted onto the cellular genome so that the oncogenic phenotype persists. These considerations are consistent with the hit-and-run mechanism of viral oncogenesis discussed above. The viral genome enters the cell, becomes chromosomally integrated and viral genome products are produced. Cellular DNA methylation and transcription patterns are .altered, and the cell is trans-

6.3 Flirther reading

61

formed to the oncogenic phenotype, possibly also concomitant with marked changes in cellular chromatin structure. Upon the loss of the integrated viral DNA in some of the cells, the oncogenic phenotype persists along with all changes in cellular structures and genetic activities, although the viral genome has left the scene of its previous activities.

6.2.2 General implications of a hit-and-runmechanism When cells originally transformed by Ad12 to tumor cells can maintain their oncogenic properties in spite of the loss of viral DNA from the cellular genome, the long-standing assumption that the human adenoviruses have no role in human oncogenesis will have to be reconsidered. Obviously, failure to find adenovirus DNA sequences in human malignancies - even if confirmed with up-todate technology - will not rule out a potential role of the virus or its genome since it could have been eliminated from the human malignant cells early after the original transformation event. In natural oncogenesis in humans, there may be decades between the time of tumor cell generation, e.g., by a mechanism related to virus infection, and the time the tumor becomes clinically manifest and can be diagnosed and analyzed. Of course, the finding of viral genomes persisting in human malignant cells per se lacks significance with respect to human oncogenesis, since the viral genomes could be passengers with growth advantages in some of the human tumor cells, thus permitting detection in the human tumors. The role of viruses in human oncogenesis definitely remains a possibility as a cofactor as frequently discussed, but final convincing proof has remained extremely difficult to adduce.

6.3 Further reading Hilger-Eversheim, K., Doerfler, W. (1997) Clonal origin of adenovirus type 12induced hamster tumors: nonspecific chromosomal integration sites of viral DNA. Cancer Res. 57,3001-3009. Kuhlmann, I., Achten, S., Rudolph, R., Doerfler, W. (1982) Tumor induction by human adenovirus type 12 in hamsters: loss of the viral genome from adenovirus type 12-induced tumor cells is compatible with tumor formation. EMBO J. 1,7946. Pfeffer, A., Schubbert, R., Orend, G., Hilger-Eversheim, K., Doerfler, W. (1999) Integrated viral genomes can be lost from adenovirus type 12-induced hamster tumor cells in a clone-specific, multistep process with retention of the oncogenic phenotype. Virus Res. 59,113-127.

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6 Adenovirus-induced tumor cells and revertunts

Nevins, J.R., Vogt, P.K. (1996) Cell transformation by viruses. In: Fields Virology, 3rd Edition (Fields, B.M., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.), Lippincott-Raven Publishers, Philadelphia, New York, pp. 301-343.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

7 Comparisons with other viral systems There are many other mammalian virus systems that have been well analyzed as potential tumor viruses. In this chapter, a brief survey of data relevant for the topic of this treatise will be presented summarizing work with the simian virus 40 (SV40), polyoma virus (PyV), human and bovine papilloma viruses (HPV and BPV), the Epstein-Achong-Barr virus (EBV), human hepatitis B virus (HBV), and retrovirus systems. I have used the adenovirus system as an example to outline important aspects of foreign DNA integration. There is a wealth of outstanding work that has been performed with the other viral systems mentioned. A separate chapter will be devoted to the integration of bacteriophage lambda DNA into the Escherichia coli system - the prototype of all work on viral DNA integration.

7.1 Integration of viral DNA With some qualifications, integration of the viral genomes into the host cell genomes has been documented for SV40, PyV, HPV, EBV, HBV, and retroviral DNAs in particular in transformed and tumor cells. For the retroviral DNA and for HBV DNA, integration is part of their replication cycle and a requirement for viral gene transcription. The enzymatic mechanism of retroviral DNA integration and the role of the viral genome-encoded and virion-encapsidated integrase have been investigated in considerable detail. This aspect of retroviral DNA integration will be addressed in Section 7.4. In terms of many other aspects of viral DNA integration, parallels to the integration of adenoviral DNA can be found. There is no evidence for specific sites of mammalian or avian viral DNA integration for any of these systems. Like integrated adenovirus DNA, other viral genomes can become de ylovo methylated upon integration into the host genome. For several of the papilloma viruses and for the EBV genomes, persistence in the episomal state as supercoiled circular viral DNA in the nucleus of the infected cell has been demonstrated. Multiple copies of viral DNA in this conformation can be found in productively infected as well as in transformed and tumor cells. With the exception of some of the HPVs, BPVs, and HBV in certain cell systems, the episomal state is the predominant mode of persistence in the host cell nucleus. However, even in these systems a few of the multiple copies of EBV, HPV, BPV, or HBV DNA might well be chromosomally integrated. This issue has received limited attention, although there are recent reports employing the FISH technique which provide evidence for chromosomal association of EBV DNA. In human malignancies, e.g., cervical uterine carcinomas, HPV16 and HPV18 DNAs, in bovine esophageal carcinomas BPV3 DNA, or in human hepatic carcinomas HBV DNA have been demonstrated in the integrated form. Thus, viral DNA integration appears to be a general phenomenon for many, if not all,

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7 Comparisons with other virnl systems

of the oncogenic viruses, although in some systems the integration problem has been obscured and more difficult to assess due to the large copy number of free episomal viral DNA in the nucleus.

7.2 Transcription of integrated viral genomes In general, the integrated viral genomes of the DNA viruses are expressed, sometimes selectively in that the early viral genes are transcribed, whereas the late viral genes can be silenced and frequently hypermethylated resulting in longterm gene inactivation. It is not understood how this selective viral gene inactivation is regulated. Of course, it guarantees the absence of late viral gene products, perhaps also of viral genome replication in the free non-integrated state. In this way, the latent state of viral persistence in the affected cell is maintained and viral replication is forestalled which would likely destroy the cell. This system, therefore, offers a superb model to study the de novo methylation of integrated foreign DNA and its dependence on gene regulation. These studies open the possibility to determine whether de novo methylation preceeds gene inactivation or vice versa. Such investigations necessitate the isolation of cells early after the integration of foreign DNA and require a method to measure DNA methylation which is sufficiently sensitive to analyze small amounts of cells and DNA. Only the genomic sequencing protocol meets these requirements. Extensive studies along these lines have so far not been performed. Most frequently, the expression of integrated viral genes has been investigated in DNA-virus-transformed cells and in virus-induced tumors. The focus in these experiments has usually been on the problem of oncogenic transformation, less on the control of gene expression in integrated foreign DNA. There is ample evidence to link viral transformation and the presence of specific viral gene products in transformed and tumor cells. In Fig. 19, the transcription and expression of the SV4O/PyV, the HPV14, and the HBV genomes are schematically described as examples of viral genes being transcribed from integrated viral genomes in different virus-transformed cell systems. Recently, evidence has been adduced that in HPV-transformed cell lines, which carry multiple copies of integrated viral DNA, only one or very few of these copies are transcribed. For the retroviral genome, the integration of the proviral DNA into the host genome is part of the viral replication cycle. The scheme in Fig. 20 explains the mode of viral replication. In brief, the single-stranded viral RNA genome is reverse transcribed by the virion-associated reverse transcriptase into a DNARNA hybrid molecule. By the combined action of RNase H, DNA polymerase, and integrase activities, which all reside in the virus particle, the DNA-RNA hybrid molecule is converted to double-stranded DNA which is subsequently inserted into the genome of the host cell. This reaction is catalyzed by the viral genome-encoded integrase. Again, there is no evidence for the occurrence of specific sites of insertional recombination between the retroviral and the host gen-

Color plates

65

Figure 3. (a) Laser scan microscopy of mechanically stretched chromosomes of Adl2-transformed hamster T637 cell. There are about 20 copies of viral DNA integrated (Schroer et al., 1997). (b) FISH visualization of - 1 copy of integrated Ad12 DNA in the revertant cell line TR12 of cell line T637 (Photograph by J. Schroer, Institut fur Genetik, Koln).

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Color plutes

Figure 10. Association of Ad12 DNA with metaphase chromosomes in cultured hamster BHK2I cells (a to d) or in the Ad12-transformed BHK21 cell line T637 (e to h). Cells were mock infected with PBS (no association) (a and e) or Ad12 infected (10’ PFUkell) and analyzed at 2 (b and f), 6 (c and g), or 24 (d and h) hpi. There is symmetric Ad12 DNA association with both chromatids, which is indicative of genomic integration in Adl2-infected cells (b to d). In the Adl2-transformed T637 cells, the strong symmetric signal was due to 10 to 20 copies of integrated Ad12 DNA (e to h). Magnification, x 1,250.

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67

Figure 14. Laser scan microscopy. Mechanically stretched chromosomes still retain the associated Ad12 DNA in Adl2-infected HeLa cells (a, c) or in Adl2-transformed T637 hamster cells with about 20 integrated Ad12 genomes (d, f) (Schroer et al., 1997).

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Color plates

Figure 16. Association of Ad12 DNA with metaphase chromosomes in cultured human HeLa cells (a-d) or in primary HUCF (e-h) after mock infection with PBS (lack of association) (a, e) or 2 h (b, f), 6 (c, g), or 24 h (d, h) after Ad12 infection (10’ PFUkell). Ad12 DNA molecules were detected by the FISH method using Ad12 DNA or cloned Ad12 DNA fragments as hybridization probes.. Magnification, x 1,250 (Schroer et al., 1997).

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69

Figure 21. Karyotype of a BHK21 cell (derived from a male Syrian hamster) as seen in a FISH experiment. Chromosomes were ordered by size. In this hybridization experiment a mixture of probes IAPI to IV was used (Meyer zu Altenschildesche et al., 1996).

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Color plates

Figure 29. (a) Increases in DNA methylation in the IAPI DNA segment in Ad12-transformed hamster cell lines in comparison with BHK21 cells. The nuclear DNAs from cell lines as indicated were isolated by standard procedures and cleaved with MspI (M), HpaII (H) or HhaI (Hh); the fragments were separated by electrophoresis on a 1% agarose gel, transferred to a Hybond N' nylon membrane, and hybridized to the "P-labeled IAPI probe. HhaI or HpaII cleavage patterns differed strikingly in Adl2-transformed cell lines from those in uninfected or Adl2-infected BHK21 cells (left). As a control, the same blot was freed from the IAPI probe by boiling in 0.1% SDS for 30 min, and the DNA was then hybridized with "Plabeled Ad12 DNA (right, data shown only for T637 DNA). Hybridization probes are designated at bottom. On the right margin the MspI cleavage pattern of authentic Ad12 virion DNA and the sizes in kbp of the MspI fragments of Ad12 DNA are indicated. (b) Increases in DNA methylation in MHC class I, DNA segment I in Adl2-transformed hamster cell lines in comparison with BHK21 cells. For details, see (a). (c) FISH of spread chromosomes from the Adl2-transformed hamster cell line T637. A mixture of biotinylated PstI-fragment D probe of Ad12 DNA and digoxygeninlabeled IAPI cellular DNA probe was applied (bottom). Chromosomal DNA was counterstained with DAPI (4'-6-diamidino-2phenylindole dihydrochloride). Arrowheads designate Ad12 DNA. Intenselly yellow (center) and pink signals (bottom) visualize multiple copies of IAP retrotransposons (see Fig. 21) (Heller (c) et al., 1995).

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Figure 30. FISH analyses of the chromosomal location of the integrated h genomes and the integrated pSV2neo plasmids in a series of BHK21 cell lines rendered transgenic for h DNA and pSV2neo DNA. (a) Control BHK21 cell devoid of foreign DNA, (b) BHK2147, (c) BHK21h17, (d) BHK21-h18*; (e, f) BHK21-hI5. (a, f) Biotinylated h DNA alone was used as hybridization probe; (b-e) a mixture of biotinylated h and pSV2neo DNAs was used for hybridization. The finding of a single signal demonstrated that both transgene DNAs were located at one chromosomal site, which was different for each cell line (Remus et al., 1999).

Color plates

13

Figure 35. Histological sections through cecum wall (a, b, and e-i) or through liver (c, d) from mice that had been fed 50 kg of M13 DNA (a, b, d, and e-i) or TE-buffer (c). Tissue samples were prepared as described. M13 DNA was identified by FISH. Tissues were counterstained in propidium iodide solution. (a, b) Peyer’s patch inside the cecum wall S h after feeding, (e-i) cecum epithelia from mice 3-5 h after feeding, (c) liver from a TE-fed control mouse, (d) liver from an animal 6 h after feeding M13 DNA (a: x SO, b-i: x 1250) (Schubbert et al., 1997).

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4 Figure 37. Detection by the FISH technique of the M13 (a, c, d, e, f) or pEGFP-Cl (b, g) test DNA in various tissues and organs of fetuses (a, b, g) or newborn mice (c, d, e, f) whose mothers had been fed daily with 50 pg of test DNA during pregnancy. (a-f) In sections of 10- (a) or 14 (b)-day old fetuses, signals of plasmid DNA are detectable in brain (c) or eye tissue (d) as yellow-green dots in nuclei of clusters of cells. In sections of liver tissue ( e ) ,cells in the endothelium of blood vessels are positive for foreign DNA, in sections of testis tissue (f) epithelial cells are positive. (g) Both chromatids of one chromosome in a fetal mouse cell in culture carry pEGFPC1 DNA ingested by pregnant mice. The chromosome spread analyzed was derived from a cell preparation that had been cultured from a 16-day mouse fetus. Its mother had received 50 pg of pEGFP-C1 DNA daily for 14 days during pregnancy. Magnification 1200 x, additional magnification for reproduction: 2 x (a-f) and 3 x (g) (Schubbert et al., 1998).

7 Comparisons with other viral systems

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Figure 19a. The regulatory regions of SV40 and PyV. Shown are the locations of the core origin of viral DNA replication and the locations of auxiliary sequences (aux) that enhance viral DNA replication. Within each core origin region are located a TA tract, with a strand bias for Ts and As, the palindromic region that serves as an origin recognition element (ORE), and a region with a purine (PU)/pyrimidine (PY) strand bias, within which bidirectional DNA replication is initiated. Also shown are sites in which large T antigen binds to viral DNA; pentanucleotides involved directly in binding are indicated by arrowheads that point the direction of the pentanucleotide, 5' GAGGC 3'. Sites within this region at which transcription is initiated for production of viral early and late mRNAs are also indicated. The sites marked EE for SV40 are those used early after infection; those marked LE are used to produce early mRNAs following the onset of viral DNA replication. Additional sites for production of both SV40 and PyV late mRNAs are located further downstream for both SV40 and PyV. For SV40 the locations that serve as enhancers and of the three nearly perfect 21-bp GC-rich repeats of the two 72-bp repeats are also shown. Within these repeats are six sites to which transcription factor Spl can bind (Cole C.N. in Fields et al., 1996).

omes. As described for the adenoviral genome, the retroviral progenome can integrate at many different cellular sites. The scheme in Fig. 20 also outlines the complex structural changes that the retroviral genomes undergo in the course of replication. Upon viral DNA integration, the regulated transcription of the viral genome commences. In fact, integration is an absolute requirement for the transcription of the retroviral genome. It is unknown whether the transcription of the

7.2 Transcription of integrated viral genomes

I7

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Infected Differentiated Epithelial Cell Infected Suprabasal + Epithelial Cell Cellular

b Basal Squamous Epithelial Cell

Infected Basal Epithelial Cell

Figure 19b. Replication cycle of a papillomavirus. In order to establish a wart or papilloma, the virus must infect a basal epithelial cell. Our knowledge is limited about the initial steps in the replication cycle such as attachment (l),uptake (Z), endocytosis (3),transport to the nucleus and uncoating of the viral DNA (4). Early region transcription (5), translation of the early proteins (6), and steady-state viral DNA repliction (7) all occur in the basal cell and in the infected suprabasal epithelial cell. Events in the viral life cycle leading to the production of virion particles occur in the differentiated keratinocyte: vegetative viral DNA replication (8), transcription of the late region (9), production of the capsid proteins L1 and L2 (lo), assembly of the virion particles (ll),nuclear breakdown (12) and release of virus (13) (Howley, P.M. in Fields et al., 1996).

integrated viral genomes proceeds with equal efficiency at any location in the host genome. The integration reaction per se is very efficient, probably due to the specificity of the viral genome-encoded integrase. The frequency of retroviral DNA integration transcends by far that of the integration of DNA virus genomes or of foreign DNA molecules that entered the host cell without the mediation of viral infection. For that reason, retroviruses have been considered preferred tools as vectors for gene transfer in gene therapeutic regimens. Of course, other factors less favorable in considering retroviruses have also to be weighed in selecting appropriate vector systems. It has frequently been a problem that integrated foreign genes cease to be expressed, possibly because they are recognized as foreign and silenced by var-

78

7 Comparisons with other viral systems

Vesicular transport

ddin9

--_---

@

Plus strand synthesis

8

+

1

Minus strand synthesis

Figure 19c. Hepadnaviral life cycle (Ganern, D. in Fields et al., 1996).

Adsorption to specific receptor

Figure 20. Overview of retrovirus replication (Coffin, J.M. in Fields et al., 1996).

7.3 Virus-induced tumors

79

ious mechanisms. The mechanism of de n o w methylation has often been invoked to explain this loss of function. Other mechanims may also play a role. In virustransformed cells, the transcription of the early genes appears frequently to be quite stable, probably because in the process of selecting transformed or tumor cells with an aggressive growth and invasion profile, cell populations have been chosen that carry the foreign DNA in a cellular genome position compatible not only with cell survival but also with the oncogenic phenotype and the expression of the decisive viral genes. It can, therefore, be misleading to draw generalizing conclusions about integrated foreign genes and their continued transcription in the target cells or organisms. Except for functionally selected transgenic cells or organisms, the most readily recognized consequence of foreign gene insertion appears to be de n o w methylation and gene inactivation. These observations are an important message for gene therapeutic strategies.

7.3 Virus-induced tumors Studies on viral oncogenesis can be subdivided into three distinct categories. (i) Viruses can transform cells in culture. This system has been used in many experimental approaches towards work on the mechanism of viral tumorigenesis, although there is no proof that the events in cell transformation in culture are identical to those in virus-induced tumorigenesis in experimental animals. (ii) Results from this latter system probably provide a more realistic insight into problems of viral tumorigenesis. However, there seem to be many parallels to the events in cell culture systems. (iii) Lastly, problems of viral oncogenesis in humans have occupied many laboratories and a huge body of information has been gathered on this topic. In most instances, viral infections as with EBV in Burkitt lymphoma, in nasopharyngeal carcinoma or perhaps in Hodgkin’s lymphoma, with HPVs in human skin or genital malignancies, or with HBV in primary hepatocellular carcinoma, to name but a few well investigated examples, have been considered important cofactors in the tumorigenic process. A major problem in unequivocally linking viral infections to human malignancies is the usually long period of latency, often of several decades, between viral infection and the occurrence of the malignant disease in humans. The decade-long periods of latency between virus infection and the initiation of malignant disease might be understood better if one considered a state of equilibrium between the replicative potential of transformed cells in an organism and the defenses that this organism can utilize against the tumor disease. The contribution that viral infections make to the oncogenic pathway is difficult to assess. Many revealing and plausible models have been proposed. Viral infection often has been considered a cofactor in tumorigenesis, although the molecular nature of other factors has remained uncertain. The role assigned to transforming viral encoded proteins has ranked high in identifying viral functions in viral oncogenic transformation. In the light of the concepts discussed in this

80

7 Comparisons with other viral systems

book, the notion of viral DNA integration and the ensuing structural and functional reorganizations of the cellular genome assume additional importance that have so far been less frequently considered. Malignancies could be viewed as chromatin diseases originating from profound alterations in the structure and function of large segments of the genome. The huge number of genes affected in their transcriptional patterns in tumor cells could thus be explained in a more rational way. The older, more traditional concept postulates that alterations in expression patterns in a particular gene that happened to be investigated at a given time, might be causally related to tumorigenesis. As an alternative, the notion is proposed that as a consequence of severe structural alterations in the genome, namely in the wake of the integration of several copies of foreign genomes, the transcription and expression patterns of a multitude of cellular genes is changed, and thus the cell is switched to the pathway of oncogenic transformation. With the appreciation of the impressive complexities of tumor diseases, it becomes doubtful that, apart from a few exceptions, changes in the expression levels of a single gene could possibly account for the far-reaching destructive potential of oncogenic transformation. In animal models, it is usually much more straightforward to link virus infection to the generation of tumors. In the prototype of experiment in viral oncology, the subcutaneous injection of avian retroviruses like Rous sarcoma virus leads to the induction of sarcomas in chicken. The types of tumors that can be induced by viruses are summarized in Table 5. All tumor-inducing RNA viruses are retroviruses. Tumor induction with these viruses is in general rapid and highly efficient. For the DNA viruses, longer periods of latency are characteristic. In the Adl2-hamster tumor model, tumors are induced within 30 to 50 days in more than 70% of the hamsters surviving virus injection. Tumor induction is considered a multistep process requiring multiple genetic changes in a cell and keen competitive interactions of the transformed cells against multiple stages of cellular defense mechanisms directed towards cells with the transformed cell phenotype. In the adenovirus system, e.g., the subcutaneous injection of small amounts, of as little as 10' plaque forming units per animal of purified Ad12 into newborn hamsters elicits the growth of tumors, probably of neuroblastoma-like origin, at the sites of virus application in 60% to 70% of the surviving Syrian hamsters (Mesocricetus uurutus). It appears that these tumors do not metastasize. Upon intraperitoneal injection, tumors are disseminated over the entire peritoneal cavity with even higher efficiency as compared to subcutaneous tumor production approaching 90% to 100%. Intra- or retroperitoneal organs, like liver or kidney, can be enmeshed by tumor growth. It is not known whether the intraperitoneally expanding tumors can cause genuine metastases (U. Hohlweg and W. Doerfler, unpublished experiments).

7.3 Virus-induced tumors Table 5. Virus-induced tumors

Taxonomic grouping

Examples

Tumor types

Mouse mammary tumor virus

Mammary carcinoma T cell lymphoma

Murine leukemia virus Gross leukemia virus Moloney leukemia virus Graffi leukemia virus Friend leukemia virus Moloney sarcoma virus Kirsten sarcoma virus Harvey sarcoma virus Feline leukemia viruses Gardner-Amstein feline sarcoma virus McDonough feline sarcoma virus Simian sarcoma virus

Leukemia, lymphoma, sarcoma, various other malignancies and pathologic conditions

Avian leukosis and sarcoma viruses Rous sarcoma virus Rous-associated viruses (RAV) Avian leukosis viruses Avian myeloblastosis virus Avian erythroblastosis virus Mill-Hill 2 virus Myelocytoma virus MC29

Sarcoma, B cell lymphoma, myeloid and erythroid leukemia, various carcinomas and other tumors

Human T-lymphotropic virus Bovine leukemia virus

T cell leukemia” B cell lymphoma

Some types

Various solid tumors

I I. RNA viruses Mammalian B type

t-

HTLV-BLV

I II. DNA viruses

“

~

Hepadnaviridae

Hepatitis B

Hepatocellular carcinoma”

Herpesviridae

EBV

Burkitts’ lymphoma (African)”, nasopharyngeal carcinomas”

Papovaviridae Polyomaviruses Papillomaviruses

sv40, PyV HPV, Shope papillomavirus

Various solid tumors Papillomas, carcinomasa

Shope fibroma

Myxomas, fibromas

Human tumors

81

82

7 Comparisons with other virul systems

7.4 Replication and integration of the retroviral genome For the replication of the retroviral genome the insertion of the proviral genome into the host genome plays an essential role. An overview of the retroviral replication cycle is depicted in Fig. 20 and can be summarized as follows. After the attachment of the viral particle to specific cell membrane proteins, the core of the virion enters the cell. For each type of retrovirus, a different cellular surface protein serves as receptor and each virus uses a single receptor, e.g., the CD4 protein, an immunoglobulin superfamily molecule (human immunodeficiency virus), a basic amino acid transporter (murine leukemia virus), a phosphate transporter (feline leukemia virus), a low density lipoprotein-related receptor (avian leukemia sarcoma virus) etc. Upon the penetration of the virion through the cell wall, the viral RNA genome is transcribed by the reverse transcriptase in the viral core into a DNA copy which is transported in association with viral proteins into the nucleus. The viral DNA is then integrated into the host genome as provirus at non-specific sites. Possibly integration occurs completely randomly but this notion is difficult to prove. There is no evidence for the existence of preferred loci of retroviral DNA integration. The cellular DNA-dependent RNA polymerase I1 transcribes the integrated proviral DNA into viral RNA which in turn is processed into new virion RNAs or into messenger RNAs. The latter catalyzes the synthesis of viral proteins and new virions are assembled and released by budding from the cell surface. Some of the virion proteins in the capsid are subsequently cleaved proteolytically. Each of these steps of viral replication has been studied in detail. In the present context the discussion will be limited to viral DNA integration. It has been well documented that the integration of retroviral DNA is an essential part of retroviral replication and viral genome transcription. The proviral genome is integrated into the cellular genome in an orientation colinear with that of the virion genome. In this process, the viral DNA usually loses two nucleotides on each terminus. The terminal DNA sequences in all retroviral genomes are 5'T G.......CA-3'. The cellular DNA sequences flanking the integrated viral genome usually are not grossly altered, except that, depending on the type of retrovirus, between 4 to 6 nucleotides abutting the proviral DNA at the cellular target site are duplicated. The 2 to 10 terminal nucleotides in the long terminal repeat (LTR) of the viral DNA molecule provide one of the essential insertion signals for the integration mechanism, since mutations in the six terminal nucleotides affect integration efficiency. Thus, on the side of the viral genome integration is a highly sequence-specific event. In accordance with the integration of DNA viral genomes or of non-viral foreign genomes into the mammalian genome, no specificities in nucleotide sequences or chromosomal locations have been detected on the cellular sites of retroviral DNA integration. Specific nucleosome structures, sites of transcriptional activity, methylated cellular sequences or bends in the chromosomal DNA might constitute preferred targets for the insertion of retro-

7.4 Replication and integration of the retroviral genome

83

viral DNA. Occasional reports on the selection of presumably specific cellular targets have described such insertion sites in specific retrovirally elicited tumors. In these instances, the allegedly specific insertion sites, e.g., within or close to protooncogenes may have been selected due to a specific oncogenesis event. In contrast, in infected cells no preferences of any kind have been observed. The mechanism of retroviral DNA integration has been investigated by using a cell-free system. Integrative recombination has been studied in crude extracts of retrovirus-infected cells or with purified or recombinant virus-encoded integrase (int) protein and specific oligodeoxyribonucleotides as recombination substrates. Apparently, integration complexes consisting of DNA and the int protein only are sufficient to catalyze the reaction. This cell-free in vitro system can catalyze the integration reaction and seems devoid of side reactions such as circle formation or autointegration. Autointegrations are not apparent in the integration reactions in living cells either. It is not known how autointegration is avoided in the naturally occurring integration reaction. There has been a debate on whether the linear or a circular form of the retroviral DNA serve as the substrates for integration. At present, it appears more likely that the viral integrase operates with linear viral DNA as the integration precusor. Within the integrase molecule, the same protein domain is thought to act in the cleavage and strand transfer reactions. A free hydroxyl of the attacking integrate is directed towards a phosphodiester bond in the target sequence leading to the transesterification at an internucleotide bond. The int protein is also responsible for the trimming of the termini of the viral DNA. The integration reaction does not require ATP or an energy-regenerating system. At least in vitro, the integrase can also effect the excision of the integrated proviral genome. In living cells, however, the provirus is perfectly stable, and an occasional loss of the to lo-’ per cell generation is proviral genome at an estimated frequency of attributed to cellular recombinational events rather than to a true excision event catalyzed by the viral int system. In brief, the retroviral integration pathway can be summarized as follows. 0

0

0

0

0

Upon viral DNA synthesis, the viral core that contains linear viral DNA and several viral proteins, including integrase and reverse transcriptase, is transferred to the nucleus. Two 3’-terminal nucleotides are cleaved from the viral DNA generating a free 3’-OH group. In the strand transfer reaction, both viral termini are simultaneosly linked to cellular DNA. The cellular repair enzymes are responsible for filling nucleotide gaps. Thus the repair enzymes remove the mismatched nucleotides at the 5‘-end and ligate the remaining termini. Replication and transcription of the viral genome proceed starting from the integrated proviral genome.

The study of retroviral DNA integration has elucidated many interesting details on how foreign DNA can be permanently fixed in the mammalian genome and

84

7 Comparisons with other viral systems

has facilitated a better understanding of retroviral replication mechanisms. Undoubtedly, this system is special in several respects, particularly in that the machinery to catalyze the insertion of the viral genome is virally encoded. In contrast, the insertion of adenovirus DNA or any other foreign DNA seems to be dependent largely on cellular recombinatorial mechanisms. The morphology of adenoviral or other DNA integrates and their cellular environment exhibits numerous similarities to the retroviral parallels, although distinct differences exist in detail. Comparisons between both systems offer gratifying insights into the capability of mammalian cells to cope with the influx of foreign genetic information. The sites of retroviral DNA integration have also .been studied for their isochore characteristics. These sites belong to the isochores richest in guanine plus cytosine, e.g., for Rous sarcoma virus genomes in hamster cells. These cellular DNA sequences are the most active in transcription and recombination.

7.5 Endogenous retroviral genomes Retroviral-like DNA sequences have been discovered in most mammalian genomes. Since these sequences often contain poly-adenosine-poly-thymidinetails, it is thought that they have been generated from retroviral RNA and transcribed into DNA and retrotransposed into the host genome. These endogenous retroviral genomes are species-specific, they are present in large copy numbers, and evolutionary comparisons have suggested that these retrotransposons have been companions of the mammalian genomes for millions of years. Biologically, these endogenous sequences can be considered highly relevant since they have succeeded in becoming part of the target host genome and have remained there over evolutionary periods. Whether they serve a function for the host is a complete enigma. These DNA sequences are frequently transcribed into RNA. Since most of the endogenous retroviral genomes have become defective or mutated, infectious virus particles are rarely produced. In Chapter 10.3, the intracisternal A particle (IAP) genomes will be discussed as a target for changes in DNA methylation patterns in Adl2-transformed and in h DNA-transgenic hamster cells. The name IAP derives from virus-like particles detected in cytoplasmic vesicles (cisternae) in the cells of several rodent species by electron microscopy. The genomes of these particles have later been identified at a copy number of 900 per haploid genome as endogenous retroviral DNA in all hamster or other rodent cells. The distribution of these IAP genomes on the hamster chromosome is very striking in that many of the 44 hamster chromosomes carry multiple IAP copies frequently on their short arms (Fig. 21, see color plates). It is unknown how these distribution patterns arose. Endogenous retroviral genomes together with a large amount of different repetitive DNA sequences make up the bulk of the mammalian genome. It will remain a major challenge for future research to determine the role of these repe-

7.6 The viral archetype: integration of bacteriophage h D N A

85

titive sequences in the organization of the mammalian genome and its evolution. Billions of nucleotide pairs that are replicated in each cell division and have remained stable - at least to some extent - over millions of years certainly deserve more attention by curious investigators than they have previously received.

7.6 The viral archetype: integration of bacteriophage h DNA The phenomenon of lysogeny in bacteriophage h has been discovered by Lwoff, Siminovitch and Kjellgaard in 1950. When Escherichia coli bacteria are irradiated with ultraviolet light, they can lyse after about 90 minutes and liberate bacteriophage h. These cells are termed lysogenic. In the lysogenic state in the absence of UV irradiation, no phage is produced and the lysogenic cells are immune to superinfection with phage h. In 1962, Allan Campbell has presented his, at the time revolutionary and creative, model for the integration of h DNA that is based essentially on genetic data (Fig. 22). The genetic distance between two genes of Escherichia coli, gal and bio, has been shown to be larger in bacteriophage h-lysogenic strains than in non-lysogenic strains, because the genome of bacteriophage h consisting of 48,514 nucleotide pairs has been linearly inserted between the two bacterial genes. In the process of insertion, the gene sequence of h DNA is circularly permuted, since the precursor for insertion is a circular molecule of phage DNA. The lysogenic state of the cell and the prophage configuration of the viral genome are maintained by one of the most important regulatory proteins encoded in the h phage genome in its CI gene, the h repressor which blocks the expression of all other viral genes during the lysogenic state. A brief summary of the bacteriophage h system, particularly on the elements of viral DNA integration and lysogeny, has been included in this chapter because the seminal work on bacteriophage h to this day remains one of the intellectual highlights and a prime model for regulatory mechanisms in molecular biology and, in particular, for foreign DNA integration into established host chromosomes. Many of the concepts on oncogenic viral transformation have been decisively influenced by the wealth of detailed information in the bacteriophage h system. For an understanding of the essential biology of this bacteriophage, the main genetic features of the viral DNA have to be recapitulated. The circular genetic map in Fig. 23a presents a simplified version which summarizes the most important genetic viral functions. Immediately after the infection of Escherichia coli cells by phage h, the viral genome is circularized. The ends of the linear phage h DNA carry the cos sites, cohesive termini or “sticky ends” consisting of single-stranded DNA comprising 12 nucleotides which are complementary in sequence between the two termini. The circular DNA molecule, which is initially formed by base pairing

86

7 Cornpurisons with other viral s y s t e m m

POP'

Figure 22. The Campbell (1962) model of prophage insertion and excision. Conversion of linear to circular h DNA (straight lines) occurs by basepairing and ligation of short complementary single-stranded segments at each end. Circular h DNA inserts into the E. coli chromosome by crossing over at the att sites (rectangles). The hatched regions within the att sites represent the crossover region (0),and the solid and open regions represent the unique sequences or arms of bacterial (B and B ) or phage (P and P') origin, respectively. Insertion and excision are promoted by the proteins indicated alongside the arrows (Weisberg and Landy in Hendrix et al., 1983).

between the cos sites on either DNA terminus, is then covalently closed by a host cell ligase. For a didactic overview, the functions on the genetic map can be subdivided into the regulatory region, the regions for recombination and integration carrying about 10 genes, the two viral genes for DNA replication, the region encoding the three genes involved in cell lysis, and the genes responsible for the synthesis of the structural proteins of the virion, the phage head (10 genes) and tail (12 genes) (see map in Fig. 23a). In contrast to animal viruses, bacteriophage h is liberated from the cell by lysing the cell wall. There are two important modes in which phage h is able to interact with its host Escherichia coli, the one leading to viral replication and subsequent cell lysis, or alternatively to the lysogenic response. The latter involves silencing of all viral functions, except for the cI-gene that is responsible for the synthesis of the h repressor which shuts off the transcription of all other viral genes. New viral particles are, therefore, not produced. Moreover, in the process of lysogenization the viral genome becomes integrated at a specific site into the host genome. Depending on growth conditions, quality of medium and the availability of a series of host factors, the lytic response can be initiated. In its course, all classes of viral genes are sequentially activated, commencing with the early functions, in particular the overriding activity of the cro gene, and progressing to the transcription of

7.6 The viral archetype: integration of bacteriophage h DNA

87

Figure23a. The h chromosome. In general, genes of related function are grouped together. The genes within each of these groups are as a rule regulated coordinately. On this map, six control genes are named individually, as are two sites, att (attachment site) and cos (cohesive ends).

Figure 23b. The action of N. When no N protein is present, polymerase ignores the Nut site and falls off the DNA, releasing the mRNA, when it reaches the stop signal. But in the presence of N, polymerase becomes a juggernaut as it passes over Nut and ignores the stop signal (Ptashne, 1986).

88

7 Comparisons with other viral systems

all late viral functions which encode the structural proteins of the virus as well as functions facilitating cell lysis. Under this lytic regimen, the cI gene is inactivated, the antagonistic cro gene dominates events, integration of the viral genome is impossible, and about 100 phage particles per cell are produced and liberated upon cell lysis. The decision between the two pathways, the lytic and the lysogenic one, is effected by a single protein, the product of the cII gene. In rich medium under optimal growth conditions, the lytic response is favored and many new phage particles can be assembled. Under limiting growth conditions for the host cells, it makes sense for the phage to lysogenize the cell, to go into eclipse, store its genome in the most carefully guarded location of the cell, its genome, and await more suitable conditions for phage replication. As is commonly observed in viral genomes, there is a strictly regulated hierarchical and developmental schedule that drives viral gene expression. In the immediate early phase of the infection cycle, the host RNA polymerase initiates transcription from the viral promoters PL and PR (Fig. 23b) which leads to the expression of the very early genes N and cro, respectively. In the subsequent early phase, N as a positive regulator helps to activate transcription of viral genes cIII, xis and int in the recombinationhtegration region of the genome, which are located to the left of N (Fig. 23). N also activates transcription to the right of cro, i.e., of genes cII, 0, P, and Q. Late after infection, transcriptional patterns differ fundamentally depending on whether the decision has been switched to the lytic or the lysogenic mode. Under conditions of predominance of the lytic response, late in the lytic pathway, the product of gene Q drives transcription of the genes for lysis and for phage heads and tails. New phage particles are produced and the cell lyses. In contrast, late during the lysogenic interaction the product of gene cII leads to the transcription of genes CI and int. The product of gene cI, the h repressor, firmly blocks the phage promoters PR and PL maintaining the lysogenic state. The int gene product is essential for the site specific integration of the circularized phage genome into the host chromosome. After the establishment of the lysogenic state, only the cI-encoded repressor is synthesized which controls its own transcription from the promoter for the maintenance of represion ( P R M , Fig. 24a). There are about 100 repressor molecules, mainly as dimers, in each lysogenic cell. The abundance of these repressor molecules in the cell is responsible for its immunity to superinfection with phage h because incoming phage DNA molecules are immediately repressed by the reservoir of active phage repressor molecules. It is experimentally possible to overcome the lysogenic state by superinfecting lysogenic cells with a multiplicity of h phage in excess of 100 particles per cell. The repressor molecules in the cell are then titrated by incoming phage genomes, and the cell can be switched to the lytic mode.

7.6 The viral archetype: integration of bacteriophage h DNA

89

7.6.1 The most important regulatory functions in the phage h genome The product of gene N, whose mechanism of function is not entirely understood, activates transcription from the viral promoters PR and PL. In the presence of N gene product, the host RNA polymerase can overcome the stop signal between gene N and cIII and does not stop at the nut sites (nut : N utilization) but continues to transcribe genes to the left and right. The nut sites are located between PL and N and to the right of cro (Fig. 23b). The product of gene Q recognizes the Qut (Q utilization) site and initiates transcription at PR,(Fig. 23a). A single long transcript of the genes in R and the head and tail genes is produced. The product of gene CIis the h repressor - a protein of 236 amino acids. Its Nterminus of 92 amino acids binds specifically to the operators of h DNA, its Cterminus with 105 amino acids enables the repressor to form dimers and to interact with RNA polymerase. The two terminal domains are connected by a stretch of 40 amino acids.

Figure 24a. Repressor binding to the three sites in OK.0 ~ binds 1 repressor about 10 times more tightly than does OR2 or OR3,so repressor first binds to 0 ~ 1A. second repressor very quickly binds to OR2,but OK3continues to bind weakly, and is filled only at higher repressor concentrations.

90

7 Comparisons with other viral systems

Figure 24b. Order of binding of Cro dimers for sites in OK.The affinity of site 0 ~ for 3 Cro is about tenfold higher than that for 01<2or OK1.After the first Cro dimer has filled OK3,the second dimer binds to either OKl or to OR2.The order with which Cro fills the sites is opposite to that with which repressor fills the sites. Thus on a wild-type O R , the affinity for Cro is OK3> OK2 > OR1, whereas that for repressor is O R 1 > OR2 > OK3(Ptashne, 1986).

The product of gene CYO consisting of 66 amino acids in the form of a dimer binds competitively with repressor to the phage operators and activates the lytic pathway of phage replication. The product of the cII gene is reponsible for establishing lysogeny. The cII product promotes transcription of cI, the gene for the repressor, from the promoter PRE(repression establishment; Fig. 23a) and of gene int from the promoter Pint. Thus cII controls the expression of the two key players in establishing lysogeny, repressor and integrase. Bacterial proteases, which are abundant when the cells actively replicate, regulate the levels of the cII gene product. The product of gene cIII protects the cII gene product from degradation by bacterial proteases.

7.6.2 Control of transcription at the right operator OR of phage h DNA The structure of the operator OR is remarkable in that it consists of three 17 nucleotide pair segments (Fig. 24) with diametrically different affinities for the binding of the gene products of CI (repressor) and c m (effector of rightward, late transcription). The h repressor has the highest affinity for the 0 ~ sequence 1 and decreasing affinities for the OR2 and OR3sites (OR1 > OR2 > OR3). The product of gene cro has the reverse affinity distribution (OR1 < 0 ~ <2 0 ~ 3 ) When . operator segments OR1 and OR2 are each occupied by repressor dimers, there is

7.6 The viral archetype: integration of bacteriophage h DNA

91

a positive influence on the binding of the host RNA polymerase which starts transcription from P R M (Fig. 24a), the promoter for the maintenance of repression, and transcription of the CI gene. The occupation of the operator sites 1 and 2 by repressor dimers keeps the cro gene product from binding to the operator. At high repressor concentrations, OR3 is also blocked by a repressor dimer which inhibits RNA polymerase binding and shuts off repressor synthesis in an autoregulatory loop. In lysogens, 90% of all operator sites 2 and 3 are occupied by repressor molecules. As mentioned earlier in this chapter, lysogeny has been discovered by irradiating bacteria with ultraviolet (UV) light which leads to the induction of phage synthesis and lysis of the bacteria. This phenomenon has been termed UV induction of lysogenic bacteria. In its course, the lysogenic mode is flipped to the lytic pathway. UV irradiation of cells causes DNA damage which induces the SOS pathway in bacteria. This pathway activates a specific set of genes, among them RecA, a gene of great importance in the recombination of different DNA molecules. RecA inactivates the h repressor by cleavage in the 40 amino acid connector between the N- and C-termini of the repressor molecule. Now the OR sites are liberated from the repressor, repressor synthesis decreases drastically, because repressor dimers are required to keep its synthesis up. The polymerase can now bind to PR and initiate transcription of cro. Dimers of cro bind to the three operator sites, most avidly to OR3 thus further inhibiting RNA polymerase to bind to P R M and stopping repressor synthesis. From there on, the lytic functions are sequentially transcribed and the lysogenic state is irreversibly switched to the lytic pathway. After infection with phage h, the decision between the lytic and lysogenic pathways is made essentially by the levels of the cII product which are determined by environmental factors. When the bacteria grow in rich medium, protease levels in the cell are high, and the cII product is rapidly degraded. Repressor is not synthesized, the products of genes Q and cro are made and lytic growth ensues. When cells grow poorly under limiting environmental conditions, protease levels in the cell are low, the cII product is protected by that of cIII and transcription of CI and int are activated (Fig. 25a). The repressor turns off all genes and the integrase inserts the viral genome into the host chromosome.

stop

'

I

a

'int Figure 25a. CII-stimulated transcription of int. The promoter for the int gene, P,,,, lies within the xis gene. Therefore Int but not Xis production is stimulated by CII.

92

7 Comparisons with other viral systems

A

Figure 25b. Retroregulation. The mRNA copy of sib forms a hairpin that attracts the bacterial enzyme RNase 111, which cleaves the hairpin. Other bacterial RNase molecules then cut the mRNA, beginning at the cleavage site (Ptashne, 1986).

7.6.3 A closer look at the integration and excision of the bacteriophage h genome The lysogenic response of Escherichia coli upon infection with bacteriophage h involves dominance by the CI gene product and the inactivation of all viral functions, except cI. Integration, i.e., site specific recombination of the circular viral genome with the h integration site between the gal and hio genes on the Escherichia coli genome leads to the linear insertion of the phage genome in a circularly permuted orientation. Thus, the viral int gene is eventually juxtaposed to the bacterial gal gene and the viral sib gene the bio gene of the host (Fig. 25). The nucleotide sequence in the bacterial genome that serves as target for the site specific insertional recombination, the prototype of homologous recombination, comprises about 40 nucleotide pairs with homologies to an equivalent site in the phage att site which consists of about 240 bp (Fig. 26). The common core sequence for attB (bacterial) and attP (phage) is 15 bp long (Fig. 26). For historic reasons these sites have been termed attachment ( a f t ) sites, because initially it was thought that the viral and cellular genomes would become attached to each other at these sites by an at the time not defined mechanism. In Fig. 25 the decisive events governing h DNA integration and excision are schematically depicted. For integration the integrase int function is required; excision is effected by xis and int. In establishing the lysogenic pathway, cII activates the promoter Pintfor the transcription of gene int which lies inside the xis

- -

-

0 -\ 8'

AGGCMCTTCGGACWMMTATG~TTGMCTCGCTTTGC

B

-20

(w),

as (+) site their arm; for and Figure indicated: designated of deduced forthe strand only 26. Xis each respective the C the arm-type from protein and exchangetop Functionally 15-bp recognition C‘ the strand symbols. in binding DNA (w), and is attPcommon the Within sites important shown sequence. and sequencedesignated B core for overlap for each Intand region The the X. B’ regions region (Weisberg in protein amino protected DNA that in remainder )attB; -( is the acid and between the protected region of identidal sequences IHF Landy, sequence attP. the themin against nttBof binding 1983). and designated attP consensus attP (oooo), the and sites (bottom) nuclease and two P1 in attB isattB. designated and attP recognition digestion (H); P2 terminator slightly The in the 0; DNA bythe shorter sequence P the location codons binding arm sequences is ofthan junction-type and each designatedthethe correspond indicatedF”1, are of staggered to (=)theP 2 H1 sequence shown and binding the and cut proteins P’3 H2sites shown. carboxyl along in in sites is the fordouble-stranded the with P’ Several (curved indicated P Int terminus the arm; arm of protein of byandand solidthefrom relative the thethe H (-), -20 arrow) features inf extent in to the polarity binding usedare+20, gene of F”

7.7 Further reading

95

gene. Thus, xis transcription from Pintis impossible. Low amounts of PL-directed transcription of xis and int at this stage do not suffice to activate excision in the presence of excess amounts of int. The PL-transcribed mRNA carries a sib transcript which renders this RNA unstable, a process termed retroregulation of mRNA activity (Fig. 25b). The circularized viral DNA molecule recombines with its attP site at the homologous bacterial attB site. The gene order in the phage genome is permuted in the process. The attB and attP sequences are now located Sit the ends of the viral genome, sib and int are separated and can no longer be transcribed into the same mRNA. Upon the induction of lysogenic bacteria, e.g., by UV light, the CI encoded repressor is inactivated by Rec-A-mediated cleavage, as described above. Consequently CYO will dominate transcription from the PL promoter. The thus produced long transcript from the integration region leads to the production of sufficient amounts of xis and int which together initiate the excision of the integrated h genome. Since in the prophage genome the int and sib DNA segments are widely separated, the long leftward transcript from the prophage genome does not carry sib information. Hence, the message is stable and xis and int production abounds. The lytic pathway is activated, phage is produced and the cell lyses. In rare instances (lop6), h DNA can become integrated at randomly located cellular sites lacking the specific attB nucleotide sequence. This type of insertional recombination has been termed illegitimate or non-homologous. This brief review of one of the most fascinating and best studied viral systems can only provide an introduction into the system and transmit an idea on how site-specific integrative recombination is directed by nucleotide sequence homologies and regulated by viral and cellular functions. A more detailed description of the h system can be found in two excellent books on the subject: Marc Ptashne’s “A Genetic Switch”, 1992; and Lambda 11, Cold Spring Harbor Laboratory, 1983.

7.7 Further reading Brown, P.O. (1990) Integration of retroviral DNA. In: Swanstrom, R., Vogt, P.K., eds., Retroviruses: strategies of replication. Springer-Verlag,New York, pp. 1948. Campbell, A. (1962) Episomes. Adv. Genet. 11,101-145. Coffin, J.M. (1996) Retroviridae: the viruses and their replication. In: Fields Virology, 3rd. Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.), Lippincott-Raven Publishers, Philadelphia, New York, pp. 1767-1847. Falk, K.I., Szekely, L., Aleman, A., Ernberg, I. (1998) Specific methylation patterns in two control regions of Epstein-Barr virus latency: the LMP-1-coding upstream regulatory region and an origin of DNA replication (oriP). J. Virol. 72.2969-2974.

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Heller, H., Kammer, C., Wilgenbus, P., Doerfler, W. (1995) Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage h) DNA is associated with enhanced methylation of cellular DNA segments. Proc. Natl. Acad. Sci. USA 92,5515-5519. Hendrix, R.W., Roberts, J.W., Stahl, F.W., Weisberg, R.A. (Eds.) (1983) Lambda 11. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Howley, P.M. (1996) Papillomaviridae: the viruses and their replication. In: Fields Virology, 3rd. Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.), LippincottRaven Publishers, Philadelphia, New York, pp. 2045-2076. Kieff, E. (1996) Epstein-Barr virus and its replication. In: Fields Virology, 3rd. Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.), Lippincott-Raven Publishers, Philadelphia, New York, pp. 2343-2396. Meyer zu Altenschildesche, G., Heller, H., Wilgenbus, P., Tjia, S.T., Doerfler, W. (1996) Chromosomal distribution of the hamster intracisternal A-particle (TAP) retrotransposons. Chromosoma 104,341-344. Minarovits, J., Hu, L.F., Minarovits-Kormuta, S., Klein, G., Ernberg, 1. (1994) Sequence-specific methylation inhibits the activity of the Epstein-Barr virus LMP 1 and BCR2 enhancer-promoter regions. Virology 200,661-667. Ptashne, M. (1986) A genetic switch. Gene control and phage h. Blackwell Scientific Publications, Palo Alto, Carlton, Oxford, and Cell Press, Cambridge. Rickinson, A.B., Kieff, E. (1996) Epstein-Barr virus. In: Fields Virology, 3rd. Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.), Lippincott-Raven Publishers, Philadelphia, New York, pp. 2397-2446. Rynditch, A., Kadi, F., Geryk, J., Zoubak, S., Svoboda, J., Bernardi, G. (1991) The isopycnic, compartmentalized integration of Rous sarcoma virus sequences. Gene 106,165-172. Sambrook, J., Westphal, H., Srinivasan, P.R., Dulbecco, R. (1968) The integrated state of viral DNA in SV40-transformed cells. Proc. Nutl. Acad. Sci. U S A 60, 2188-2195. Shah, K.V., Howley, P.M. (1996) Papillomaviruses. In: Fields Virology, 3rd. Edition (Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E., Eds.), Lippincott-Raven Publishers, Philadelphia, New York, pp. 2077-2109. Weisberg, R.A., Landy A. (1983) Site-specific recombination in phage lambda. In: Hendrix et al., Lambda IT, pp. 21 1-250.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

8 Non-viral systems Any foreign DNA can be transfected or injected into mammalian cells and can be genomically fixed in the recipient genome. Apparently, fixation is facilitated only in a subset of the transfected or injected cells. The cells transgenic for the foreign DNA are routinely selected by cotransfection of a selectable marker. In a typical experiment, the foreign DNA to be introduced into the host genome is cotransfected with a plasmid, pSV2ne0, that carries the gene for the enzyme neomycin phosphotransferase under the control of the early promoter of the DNA virus SV40. The transfected cells are subsequently exposed to neomycin or its analog G418 (geneticin) which eliminates all cells that do not inactivate the drug by phosphorylation with the enzyme neomycin phosphotransferase. Detailed cytogenetic or molecular analyses of the integrated genomes by restriction and Southern blot hybridization experiments reveal that the two different types of DNA molecules, foreign DNA and pSV2neo DNA, are frequently integrated en bloc and in multiple copies at the same chromosomal location in the mammalian genome. Possibly, the two different DNA molecules are ligated or recombined with each other prior to the integration reaction. This interpretation is likely but has not been rigorously proven. In many aspects, integrates of the described configuration resemble integrated adenovirus DNA, possibly because in both systems foreign DNA has been inserted by similar or identical cellular mechanisms of insertional recombination. Many questions about extent, timing and mechanisms involved in insertional recombination can presently not be answered, e.g., on the mode of uptake through the cytoplasmic and nuclear membranes, transport and fixation of foreign DNA in mammalian genomes. Can purified DNA enter a mammalian cell? We have demonstrated in several series of experiments that adenovirus DNA with the terminal protein still covalently bound to the viral DNA molecule can enter the cell and can be transported very efficiently to the nucleus where it becomes associated extensively with the chromosomes of the recipient cell. When foreign DNA is complexed with a basic protein, like protamin, the DNA is transported very effectively into the nucleus but it is not expressed, whereas the same DNA transfected with the Ca2' phosphate precipitation technique into the same cells elicits expression of the transfected gene, although the overall uptake of DNA can be considerably lower than in the DNA-protein complex-transfected cells (see Section 4.4). Apparently, efficiency of uptake and of expression are unrelated parameters regulated by different, presently unknown, mechanisms.

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8.1 Exchange of genetic information with extracellular DNA in pneumococci In studies on the molecular biology of mammalian systems, comparisons to prokaryotic organisms have often opened new experimental vistas. In prokaryotic systems, e.g., for the DNA of pneumococci the pathways of DNA uptake and insertional recombination, which are intimately linked, have been elucidated in considerable detail. When actively growing cultures of pneumococci (Streptacoccus pneumoniae) reach a critical, but rather low, cell density they become competent for the uptake of foreign DNA from their environment. This capability has been termed the quorum-sensing effect. The biochemical conditions conducive to this exchange are poorly understood. Aside from an optimal Ca2' ion concentration of 1 mM, a specific competence stimulating peptide (CSP) of 17-amino acids length that is spontaneously produced by the pneumococci has recently been identified. An ATP-binding cassette (ABC) transporter removes the leader from the propeptideif CSP and Translocates the latter across the cytoplasmic membrane. A number of closely linked genes have been mapped in the pneumococcal genome that are all related to these mechanisms. How the DNA is actually taken up and transported is still poorly understood. There is evidence that the uptake of foreign DNA in this bacterial system is accompanied by the activation of genes responsible for homologous recombination. Thus, uptake and recombination of foreign DNA seem to be coordinated processes. It will be interesting to investigate whether comparable mechanisms exist also in mammalian cells. Systematic studies on this important topic are urgently needed.

8.2 IS elements and transposons Mobile genetic elements have originally been discovered in maize by Barbara McClintock. Their precise molecular nature has been identified in E. coli. The E. coli genome can carry up to 20 transposons of different types. IS sequences frequently encode an enzyme, the transposase which catalyzes the insertion of the transposon. Transposition can be conservative, i.e., an IS element is excised and translocated from one position to another one. In replicative transposition, the IS sequence is replicated, and the newly synthesized copy is inserted at a remote site in the cellular genome. There are many different types of transposons varying in size and gene content. Almost invariably, they carry the gene for the transposase. As a consequence of their insertion at many different sites in the genome, transposons can lead to insertional mutagenesis and/or changes in the distance between endogenous genes as typically exemplified by the integration of bacteriophage h DNA (Section 7.6). In mammalian systems, retrotransposition can play a role. Repetitive DNA sequences in the genome are transcribed into RNA, this RNA is reverse tran-

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scribed into DNA which in turn is retrotransposed into a different part of the genome. The biological function of transposons and retrotransposons is not understood. Their role in mutagenesis and during evolution appears evident but is difficult to prove except for rare cases.

8.3 Thoughts on the mechanism of foreign DNA integration It is conceivable that in mammalian cells mechanisms comparable to those just described exist. Modes of uptake, fixation and expression of foreign DNA by mammalian cells and the mechanisms underlying these events have not yet been actively explored. In the literature, several possible mechanisms for the integration of foreign DNA into the mammalian genome have been considered. In most cases the evidence for one or the other enzymatic mechanism is circumstantial. Some of the proposed schemes are plausible but not proven. Much of the reasoning has been deduced from the structure of integrative recombination sites between foreign and mammalian cellular DNAs. Frequently, viral systems have played a major role in studying this problem. Here is a survey of parameters discussed that might contribute to an improved understanding of foreign DNA integration: 0

0 0 0

0 0 0 0 0

0

0

0 0

Specific chromatin structures at the sites of DNA transcription or origins of DNA replication. Chromosome breaks. Alu sequences or other repetitive elements. Preinsertional ligation of foreign DNA; concatenate formation. Extrachromosoma1 concatemers may be due to rounds of homologous recombination. Preinsertional linkage of cotransfected DNA molecules of different types. In contrast, it has also been postulated that single copies of foreign DNA are integrated and become amplified subsequent to insertion. ADP-ribosylation. Deletions at sites of insertion. Insertional mutagenesis. Single-strand invasion at or close to the site of insertion. Utilization of patchy or short sequence homologies between the recombination partners. “Illegitimate recombination”, sometimes followed by rearrangements. In rare cases homologous recombination: How is the decision between these alternatives reached? Double-strand or single-strand breaks, end-to-end ligation. Retroviral integration mechanism (see Section 7.4). A serious, usually ignored problem: The mode of selection of the surviving cells employed after foreign gene transfection.

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Occasionally, loss and rearrangement of nucleotide sequences during integration of the integrates and/or the cellular target sites. Recognition sites for topoisomerase I, E. coli Chi site, meiotic recombination hot spots etc. Problem of stability of integrated foreign DNA sequences. Which factors play a role? Specific sequence motifs at sites of insertion. Structural motifs, hairpins, repeats, inverted repeats etc. Level of DNA methylation at site of insertional recombination. Do the same mechanisms apply after transfection or microinjection of cells to produce transgenic organisms? Opportunistic repair-ligation systems. Mode of introducing the foreign DNA into the cell, size of foreign DNA, number of copies injected or transfected. Type of cells used in experiments. Cells in culture, oocytes or cells in resting tissues in animals. Time of DNA transfer during the cell cycle. Is active cell replication required for foreign DNA integration? It is uncertain whether only one specific mechanism exists that governs the insertion of foreign DNA into the mammalian genome in transfected or microinjected cells. The historic terms of “homologous” and of “illegitimate” recombination will not suffice to explain the complexity of these mechanims. It will be essential to consider mechanisms that account for the variability and flexibility of the events associated with insertional recombination. This variability and flexibility might have been a decisive advantage during evolution. A study of the literature on the integration of foreign DNA into the genomes of mammalian cells reveals that many of the details elucidated in the integration of adenovirus DNA are very much alike, if not identical, to the integration of foreign DNA in general, in rendering cells or animals transgenic. Distinct differences are, of course, found in retroviral DNA integration or in targeted integration with sophisticated but artifactual selection systems. In the natural encounter of mammalian cells or organisms with foreign DNA - be it through viral infections or with foreign DNA ingested via the gastrointestinal tract - targeting and selection are certainly not the rule.

8.4 Expression of integrated foreign DNA Upon the fixation of foreign DNA in cells or transgenic organisms, the expression of the genomically fixed genes plays an important role and is frequently the goal of foreign DNA (gene) transfection and fixation. Foreign gene expression has been most intensely studied in viral oncology, in gene transfer and gene therapy experiments, and in transgenic organisms. The question arises of whether foreign gene expression in these systems is a controlled or a haphazardly occurring pro-

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cess. In random integration - the expected mode in mammalian cells - expression is usually not controlled, unless insertion leads by chance to the influence of cellular promoters or additional control elements. In random integration or in gene targeting experiments, initially successful transcription frequently ceases after some time. There is no safe method to predict whether a transfection and genomic fixation series of experiments will be followed by continuous gene expression or by gradually waning transcription followed by the complete silencing of the transgenes. There is practically no information on the rules that determine transgene transcriptional control. Most likely, the type of foreign DNA inserted and the site of foreign DNA insertion are among the essential factors to decide about the transcriptional fate of the foreign genes. What role does the de novo methylation of recently integrated foreign DNA play as a long-term signal for gene inactivation? The mechanisms of de novo methylation of integrated foreign DNA also affect the continued expression of foreign transgenes. It is a prime goal of basic research in mammalian molecular biology to investigate these mechanisms, since transgenic research depends on an improved understanding of these complex interdependencies. Hence this field of research assumes a crucial role in all biological applications of transgenic research, since expression of the genes newly introduced into the genome of an organism or of cells is the major aim of most projects in these fields of application. Effects of the expression of integrated foreign genes on the cellular phenotype are equally poorly understood. A very recently investigated area of molecular biology is the intrusion of foreign DNA into mammalian, and possibly other, organisms with the daily food supply via the gastrointestinal tract (Chapter 11).We do not yet know to what extent this DNA persists for long periods of time and whether this DNA can be transcribed and whether genes acquired via this route can be expressed into their products. DNA vaccines represent excellent examples that demonstrate the long-term persistence and successful expression of foreign DNA injected into mammalian organisms. Apparently, specific defense mechanisms or the degradation of foreign DNA by DNases in the tissues play only a minor role or are countered by steric complexities of the foreign DNA molecules associated with proteins or by a compartmentalization in the organism that has as yet not been investigated. Surprisingly sparse solid information is available on the fate of foreign DNA in the cell or in an organism.

8.5 Fixation of foreign DNA in transgenic animals The generation of transgenic animals, in particular of transgenic mice, has become one of the most frequently practiced techniques in biomedical research. This procedure has been routinely applied in the pursuance of two major goals. Firstly, foreign or authentic genes of the animal can be fixed in the genome of the recipient by homologous or by heterologous recombination in order to introduce

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new genetic information or to repair genetic defects in the recipient. Thus, gene therapeutic regimes take advantage of and are, at least partly, based on this possibility. Secondly, genes of the organism can be “knocked out” by homologously recombining constructs that carry sizable deletions of the gene and intact foreign genes that can be used to select those cells that carry the appropriately deleted gene under investigation. Typically, embryonal stem cells of the mouse are the targets for these “knock out” experiments. As a corollary, “knock in” experiments using the same gene that is introduced into animals carrying the “knock out”, are frequently performed to prove that the effect of the deletion of a gene can be compensated or amended by reinserting the intact gene. This latter approach, when successful, serves as proof that the gene under investigation is actually responsible for the observed “knock out” phenotype. By the insertion of the Cre/Lox gene casette, that has originally been developed in prokaryotic systems, into the gene construct-combination introduced into the mouse genome by homologous recombination and selection, the “knock out” can be elicited conditionally or only in specific organs of the animal that has developed from the transgenic mouse stem cells. This treatise is not the place to describe this impressive technology in detail. The “knock out, knock in” technology utilizes the homologous recombination mechanism which under natural conditions is an extremely rare event in mammalian cells. Hence rigorous selection procedures are required to select those cells in which the transfected or injected DNA has been inserted into the homologous positon. Without such selection schemes, the foreign DNA becomes integrated by heterologous recombination into many different targets in different experiments. The characteristics of these recombination events and of the integrated foreign DNA are practically identical to those of integrated viral DNA as described previously (see Chapters 4,5). Upon the transfer of foreign DNA into mammalian cells, it is unknown what cellular factors influence the type of recombination that ensues, homologous or heterologous. In mammalian cells heterologous recombination seems to be the most frequent recombination mechanism. Judging from the data adduced on the integration of adenovirus DNA into the genome of mammalian cells, the mechanism of heterologous integrative recombination is supported by the presence of shorter or longer sequence homologies, termed patch homologies, between viral and cellular DNAs. In that sense, differences between the mechanisms of homologous and heterologous recombination become mechanistically blurred and a strict distinction between clear alternatives is not realistic. Nevertheless, when cloned cellular genes are transfected for integrative recombination experiments without selection, the DNA is integrated randomly at many different sites, although in a single recombination event the insertion is frequently observed at only one site. In yeast, in contrast, homologous recombination is by far the predominant mode of integrative recombination, a fact that has greatly facilitated the genetic analysis in this model organism. The reason for this fundamental difference between the yeast and mammalain systems in this respect is not understood.

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8.6 Critical evaluation of the results obtained with transgenic animals In molecular medicine, the “knock-out’’ technique in transgenic mice has brought forward a wealth of very valuable information on the involvement of specific murine genes in the pathogenesis of certain diseases. Moreover, the deletion of specific mouse genes has often aided in the identification of their function. Upon the isolation of new mammalian genes and the determination of their nucleotide sequence, the question about the function of such genes calls for computer comparisons to known genes, particularly in the yeast genome. When information is not forthcoming from these sources or too limited, “knock-out’’ experiments in the mouse genome are usually initiated. These experiments, though labor- and time-intensive, have often provided answers to the quest for functional information. Success, however, is not guaranteed. In some instances, complex phenotypes are generated after the “knock-out’’ of a single gene. It is difficult to obtain reliable quantiative evaluations on the percentage of successful experiments in which the generation of a “knock-out” animal has contributed to the identification of the function of the investigated gene. Experiments that fail to provide straightforward information are often, not published. Complex phenotypes are difficult to evaluate and not likely to meet the favors of success-oriented referees of scientific journals with a growing tendency to select contributions that conform to established scientific models. A problem that is shunned by analysts of “knock-out’’ or “knock-in’’ animals or of transgenic organisms in general pertains to additional alterations in the recipient genome as a consequence of the insertion of DNA blocks of considerable lengths. It is unlikely that the integration of several kilobase to megabase pairs of foreign DNA would not have far-reaching effects on the organization and transcription patterns of a large number of cellular genes. By using DNA array technology which allows to assess transcriptional patterns of thousands of genes in a cell, an organ or in an animal, it is already becoming apparent that in transgenic animals the transcription of large numbers of genes can be altered. As detailed in Section 10.3, the insertion of foreign DNA into an established mammalian genome can alter patterns of DNA methylation in the cellular genome at sites remote from the location of insertion. Since patterns of DNA methylation and transcription are functionally tightly linked, the insertion of foreign DNA could lead to significant alterations in the cellular transcriptional programs. Hence it cannot be expected that changes in the functional program of a transgenic organism or of a transgenic cell can be unequivocally assigned to the premeditated “knock-out” or “knock-in’’ strategy. Additional factors have to be considered that are usually ignored because it is very difficult to ascertain changes in cellular transcription programs due to foreign DNA insertion, particularly as long as one does not search for them. Moreover, such changes may not invariably occur and it remains so far impossible to predict at what sites in the cellular genome they are most likely to be expected. In any event, methods to investigate alterations in

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cellular methylation and transcription patterns have to be refined, and concepts about the possible consequences of foreign DNA insertion will have to be developed by work on suitable model systems. With the advent and more frequent application of DNA array technologies these shortcomings will be quickly remedied. As will be described in the following chapters of this book, we have used cells transgenic for adenovirus DNA or bacteriophage lambda DNA as well as mice transgenic for specific constructs or for lambda DNA to initiate studies on changes in cellular DNA methylation patterns in selected cellular genes.

8.7 Further reading Doring, H.P., Starlinger, I? (1986) Molecular genetics of transposable elements in plants. Annu. Rev. Genet. 20,175-200. HAvarstein, L.S., Coomaraswamy, G., Morrison, D.A. (1995) An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Nutl. Acad. Sci. USA 92, 11140-1 1144. HAvarstein, L.S., Gaustad, P., Nes, I.F., Morrison, D.A. (1996) Identification of the streptococcal competence-pheromone receptor. Mol. Microbiol. 21, 863869. Kucherlapati, R., Smith, G.R. (Eds.) (1988) Genetic recombination. American Society for Microbiology, Washington. Schroer, J., Holker, I., Doerfler, W. (1997) Adenovirus type 12 DNA firmly associates with mammalian chromosomes early after virus infection or after DNA transfer by the addition of DNA to the cell culture medium. 1. Vim/. 71, 7923-7932. Thomas, K.R., Capecchi, M.R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51,503-512. Tomasz, A. (1965) Control of the competent state in pneumococcus by a hormone-like cell product: an example of a new type of regulatory mechanism in bacteria. Nature 208,155-159. Torres, R.M., Kuhn, R. (1997) In: Laboratory Protocols for Conditional Gene Targeting. Oxford University Press, Oxford, pp. 73-79. Wienhues, U., Hosokawa, K., Hoveler, A., Siegmann, B., Doerfler, W. (1987) A novel method for transfection and expression of reconstituted DNA-protein complexes in eukaryotic cells. D N A 6,81-89.

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9 Patterns of DNA methylation in the human and in viral genomes We have studied patterns of DNA methylation in functionally interesting parts of the human genome as well as in several viral genomes that resided free or integrated in the host cells’ genome in infected or virally transformed cells, respectively. The objective of this project has been to gain a better understanding of the biological function of the fifth nucleotide, 5-methyldeoxycytidine, mainly in mammalian DNA and in the genomes of mammalian viruses. We have also pursued questions about changes in DNA methylation patterns in transformed and tumor cells and in a number of human diseases. At a first level of analysis, considerable evidence has been adduced over the last 20 years that specific patterns of 5-methyldeoxycytidine distribution in the promoter and upstream regions of mammalian and integrated viral genes are instrumental in the long-term inactivation of their promoters. This basic concept has been extended to interrelationships between DNA methylation and the genetic imprinting of limited segments of the human genome. As complex, cell type-specific patterns of DNA methylation in mammalian genomes are recognized, the notion gains increasing probability that these patterns permeating the genome are tied to a more profound purpose, the structure of the genome inside chromatin. Current state-of-the-art work on DNA methylation requires that all 5-methyldeoxycyticine residues in a DNA sequence of interest should be determined by applying genomic sequencing techniques. We have found the bisulfite protocol of this method to be highly reliable when performed under appropriate and controlled conditions.

9.1 Introduction Much like the star-spangled firmament exhibits a non-random distribution of celestial bodies that is due to astronomical history, the distribution patterns of the fifth nucleotide 5-methyldeoxycytidine in mammalian genomes reflect their evolutionary past and are highly specific and potentially different for each gene in each cell type. At present, we lack a convincing explanation for this complexity. In recent years, a few attempts to provide quick and easy answers have been publicized, but the experimental base for such hypotheses is often weak because the underlying analyses have omitted to determine the existing patterns by the genomic sequencing technique which facilitates the recognition of all 5-methyldeoxycytidine residues in a DNA sequence. In many reports, methylation-sensitive restriction enzymes have been utilized to analyze methylation patterns. While this approach is valuable to obtain an overview of existing patterns, it is necessarily restricted to only a subset of the existing 5’-CG-3’ dinucleotide sequences and will miss any 5-methyldeoxycytidine residues outside nucleotide sequences recognized by methylation-sensitive restriction endonucleases. The

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conventional investigations using restriction enzymes encompass only a small fraction of the 5-methyldeoxycytidine residues in a sequence (see Ad12 DNA example below). Of course, it is difficult, time consuming, and labor-intensive to detect all 5-methyldeoxycytidine residues in a nucleotide sequence and, depending on the questions asked, restriction enzyme analyses still have their place but they can contribute to the understanding of the biological significance of methylation patterns merely in a limited way. It is remarkable to observe that large scale programs, like the Human Genome Project, do not even discuss the inconvenient fact that they will miss 20% of the nucleotide information in the genome, i.e., a major aspect of its genetic complexity. Although the present protocols for genomic sequencing, have been much improved mainly by the bisulfite technique, a more advanced procedure is still required before one can hope to decipher methylation patterns in vast segments of mammalian, especially the human, genome. As so often in molecular biology, research on viral systems has helped to sharpen the definition of problems and to facilitate novel experimental approaches toward their solution. We have first recognized the importance of methylation patterns and the interdependencies between sequence-specific promoter methylation and longterm gene inactivation by studying integrated adenovirus genomes in mammalian cells. Subsequent to their covalent insertion into the mammalian genome, adenoviral genomes become subject to cellular control mechanisms and to de novo methylation. In contrast, adenovirus DNA molecules in the virion or its intracellular forms in productively or abortively infected cells residing free in the nucleus never become methylated. On the other hand, the genome of frog virus 3, an iridovirus, is methylated in all 5’-CG-3’ dinucleotides, and its late promoters are fully active both in fish and mammalian cells, notwithstanding their complete methylation status. We have learned that it is necessary to investigate each biological system in depth and that it can be misleading to extrapolate the results adduced in one system to generalizing conclusions on different systems. In this chapter, I will first outline some of the problems currently facing the investigator on the biological significance of DNA methylation and will then summarize our own findings on DNA methylation patterns in the human genome and juxtapose this discussion with observations on patterns in integrated and free viral genomes in mammalian cells.

9.2 Methods available for the study of DNA methylation In molecular genetics, the task has been posed to determine the sites of DNA methylation precisely. Chemical methods, e.g., total hydrolysis of DNA and the subsequent analysis of the amount of 5-methyldeoxycytidine relative to deoxycytidine by two dimensional electrophoresis and chromatography or by HPLC

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(high performance liquid chromatography) permit the exact quantitation of the modified nucleotide in DNAs of different species or from different tissue sources. However, the requirement to be fulfilled is more demanding. In genetics, the position of the modified nucleotide in its sequence context and its significance as a functional signal have to be elucidated. The position of each 5-methyldeoxycytidine in a nucleotide sequence has to be exactly determined. Genomic sequencing methods have been developed, since the conventional cloning and sequencing strategies cannot be applied for a simple reason. The propagation of a cloned mammalian DNA segment of interest in a prokaryotic host would obviously erase the specific mammalian DNA methylation pattern. Methods of biological relevance have, therefore, to be based on the direct assessment of 5-methyldeoxcytidine residues in the mammalian nucleotide sequence. In practical terms, it is advisable to analyze integrated foreign DNA and the sequences in the environment of the inserted foreign DNA in its target host first by restriction with methylation-sensitive restriction endonucleases. Cleavage of the DNA by the isoschizomeric restriction endonuclease pair HpaII and MspI in two different reactions, followed by gel electrophoretic separation of the DNA fragments and identification of the restriction froducts of interest by Southern blotting and DNA-DNA hybridization with a P-labeled probe, provides a reliable survey of the extent of DNA methylation in all 5’-CCGG-3’ sites in this sequence. The 5’-CCGG-3’ sequences are cut by MspI regardless of the presence of 5-methyldeoxycytidine in the 5’-CG-3’ dinucleotide, whereas HpaII is inhibited by 5-methyldeoxycytidine in the same position. Hence HpaII generates fewer DNA fragments of higher molecular mass and Msp cuts the same sequence at all 5’-CCGG-3’ sites to fragments of lower molecular mass. Obviously, this approach can be informative only when a considerable number of 5’-CCGG-3’ sequences occurs in the DNA segment to be investigated. Although the results of this type of analysis can only be of limited value, they nevertheless yield an overview of the extent of DNA methylation. Frequently, though not invariably, the pattern of methylation in 5’-CCGG-3’ sequences reflects to some degree that in all 5’-CG-3’ sequences. However, depending on the nucleotide sequence, 5’CCGG-3’ sequences represent only a small subset of all 5’-CG-3’ dinucleotides in a DNA segment. In the nucleotide sequence of Ad12 DNA, e.g., only 5 % of all 5’-CG-3’ sites are part of a 5’-CCGG-3’ sequence and only 17% of all 5’-CG-3’ sequences can be recognized by HpaII (5’-CCGG-3’) and HhaI (5’-GCGC-3’) cleavage. By using additional 5-methyldeoxycytidine-sensitiverestriction endonucleases, as HhaI (5’-GCGC-3’), BssHII (5’-GCGCGC-3’), ClaI (5’-ATCGAT30, EagI (5’-CGGCCG-3’), and others, this approach can be further refined. However, the fact must not be overlooked that many 5’-CG-3’ dinucleotides are not part of a restriction site. Genomic sequencing protocols of DNA are all based on deoxycytidine residue-specific chemical reactions. Under defined conditions, hydrazine or bisulfite react specifically with deoxycytidine residues but cannot modify 5-methyldeoxycytidine residues. In my laboratory, we have experienced the bisulfite protocol to yield reliable data when appropriately applied and controlled. Bisulfite converts

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>97% of all deoxycytidine residues in a sequence to uracil but fails to attack 5methyldeoxycytidine. Details of the genomic sequencing protocol with the use of bisulfite as the specific reactant are described in Table 6. In brief, the total genomic DNA is completely denatured using strong alkali, subsequently reacted with bisulfite, and the DNA segment to be investigated is then amplified by the PCR, whereby all uracil (fomerly deoxycytidine) residues are converted to deoxyadenosine/thymidine residues. Subsequently, individual PCR-amplified molecules are cloned and the nucleotide sequence in many individual clones is determined by using primers specific for sites in the cloning vector. Since the nucleotide sequence of the DNA segment to be investigated has to be known beforehand, complete conversion of all non-methylated deoxycytidine residues to thymidine residues can be readily ascertained. Further controls, which we have found to be absolutely mandatory, include analyses of the DNA segment under investigation as a cloned sequence that has been propagated in a methylation-deficient prokaryotic host (negative control). The same DNA segment previously in vitro premethylated by the M-HpaII (5'-CCGG-3') or the M-SssI (5'-CG-3') DNA methyltransferase is also analyzed by using the same bisulfite protocol (positive control). One advantage of this method is the positive display it provides for all Table 6. Bisulfite treatment (Marc Munnes, Institut fur Genetik, Koln).

Cytosine

Uracil

t PCR -b Cloning-bSequencing genomic DNA

bisulfite treated DNA 5'-Aj\ 1

+ + u 1 Jiu . ) +

1

PCR product

.) t .)

-3'

9.3 Patterns of DNA methylation

109

deoxycytidine residues that have been converted to thymidine residues. In an earlier developed protocol, the reaction of deoxycytidine residues with hydrazine led to the elimination of the deoxycytidine nucleotides and, in the subsequent DNA sequencing reaction, to the absence of deoxycytidine signals in the sequencing ladder. Sometimes the interpretation of data gleaned with this older method has been ambiguous or misleading, particularly in CG-rich regions because of sequence compressions and poor readability of the sequencing gels. The positive display of deoxycytidine residues after their conversion to thymidine residues by the bisulfite protocol obviates this problem. We consider it essential to clone and sequence individual PCR-amplified molecules and not to sequence the bulk of the PCR products directly and cumulatively. There is very considerable heterogeneity of DNA methylation at many sites which is sufficiently high to obscure the results of direct sequencing and render them unreliable. Methylation polymorphisms and heterogeneities at individual sites are a fact that must be incorporated into any useful interpretation on the biological significance of DNA methylation patterns. In particular repetitive DNA sequences in the mammalian genomes can exhibit remarkable polymorphisms with respect to the methylation of individual 5’-CG-3’ dinucleotide sites. As an example the methylation pattern for a segment of the retrotransposon sequence of the intracisternal A particle (IAP) genomes in the hamster genome, as determined by the described method, is presented (Table 7). The presently available protocols for genomic sequencing and the precise elucidation of DNA methylation patterns are still too labor- and cost-intensive to permit the analyses of extended genome segments or the realistic inclusion into the Human or similar Genome Projects. However, I wish to emphasize that at the time of the completion of any of these projects, their protagonists will become painfully aware of the fact that they have ignored “20%” of the nucleotides (5methyldeoxycytidine) occurring in human (mammalian) DNA. Improved methods for the rapid determination of 5-methyldeoxycytidine in the natural sequence context are urgently needed, before one can hope to assess the actual patterns of 5-methyldeoxycytidine distribution in a meaningful way.

9.3 Patterns of DNA methylation There is evidence that very specific patterns of DNA methylation exist in eukaryotic genomes. These patterns appear to be stable and specific for each segment of the genome. Moreover, these patterns can be different from cell type to cell type in an organism and may change when a particular cell type is explanted and carried in serial culture but such alterations are not always observed. Frequently, when patterns of DNA methylation in the human genome have been precisely assessed by using one of the genomic sequencing protocols, patterns in specific cell types have been found to be interindividually conserved and independent of ethnic origin among individuals. The role that alterations in these patterns of

9 Putterns of DNA methylation in the human and in viral genornes

110

Table 7. Normalized levels of DNA methylation at individual 5‘-CG-3’ dinucleotides in the IAPI (p3p4) segment of the BHK2l cell line (reference), in cell line T637, and in four different nontransgenic BHK21 subclonal cell lines’

DNA methylation levels in cell line:

5’-CG-3‘ position

1 2 3 4 5 6c 7 8 qd 10 1Id 12c 13 14 15 16 17 18 20 21 22 28 29 30 31 32 33c 34

BHK21 (68Ih

T637 (43)

BHK21-1 (50)

BHK21-2 (60)

BHK21-3 (54)

BHK21-4 (60)

1.000 1.000 1.ooo 1.000 1.000 1.ooo 1.000 1.000 1.ooo

1.445 1.255 1.154 1.276 1 ,050 1.149 1.094 0.999 1.062 1.086 1.058 0.977 1.146 1.231 1.048 1.083 1.279 1.728 1.123 1.534 1.182 1.085 0.842 1.247 1.337 1.050 1.194 1.213

0.850 0.931 0.874 1.050 1.014 0.996 0.899 0.900 0.894 0.894 1.076 0.843 1.106 1.061 0.917 0.785 0.777 1.395 0.788 0.810 0.912 0.986 0.738 1.107 0.906 0.909 0.891 0.933

0.602 0.840 0.831 0.841 1.002 1.010 0.987 0.823 0.847 0.996 1.014 0.628 1.018 0.963 0.998 0.787 0.569 0.966 0.915 0.704 0.523 0.775 0.611 1.089 0.825 1.006 0.992 0.999

1.062 1.148 0.822 0.855 1.027 1.010 0.946 0.910 0.892 1.107 1.120 0.870 1.123 0.994 0.940 0.920 0.678 1.111 0.946 1.230 1.043 0.890 0.861 1.063 1.047 0.948 0.982 1.090

0.837 1.091 0.822 0.855 1.027 1.010 0.938 0.901 1.013 0.880 1.125 0.915 1.039 0.930 0.962 0.983 0.976 1.277 0.899 1.112 0.818 0.901 0.927 1.116 0.986 0.945 1.049 1.035

1.000

1.ooo 1.000 1.000

1.ooo 1.000 1 .000 1.000 1.000 1.000 1.ooo 1.000 1.000 1.ooo 1.ooo 1.000 1.ooo 1.000 1.000

Experimental details and methods of calculation are described in the legends to Fig. 31 and Table 9. However, the data here are derived from completely independent sequence determinations of cloned PCR products in the IAPI p3-p4 DNA segment (Fig. 31). The subclonal cell line BHK21-4 had been mock transfected, with foreign DNA omitted from the transfection mixture. In the genornic sequencing experiments, the number of clones as indicated under each cell line were sequenced. Percent values of clones methylated in a 5’-CG-3’ position were normalized to the respective values for the reference cell line BHK21 whose values in a given 5’CG-3’ psition were arbitrarily set to 1.000. The number of clones individually sequenced are given in parentheses. ‘ HhaI (5’-GCGC-3’) sites. H ~ I (s-ccGG-~’) I sites. a

’

9.4 A long-term signal for promoter inactivation

111

DNA can play in pathogenesis is only beginning to be appreciated. There is much evidence to support the interpretation that in transformed and tumor cells patterns can be profoundly altered. However, it is unknown whether these changes are cause or consequence, or likely both, of the underlying pathology. We have recently reported that the insertion of foreign DNA in an established genome, possibly in part in conjunction with the ensuing transformation or independently of it, can lead to marked alterations of DNA methylation patterns in that genome. Before we can even speculate on the significance of these patterns and the role their alterations might have in pathogenesis, improved methods will have to be developed and a lot of work will have to be invested to assess these patterns in all parts of the genome and in the different cell types of an organism.

9.4 A long-term signal for promoter inactivation There is ample experimental evidence from many different biological systems that supports the concept that sequence-specific patterns of promoter methylation are a signal for long-term gene inactivation. A summary of many examples in the literature is presented in Table 8. This frequently applicable rule is not without exceptions. Frog virus 3 promoters are completely methylated in all 5’CG-3’ dinucleotides and fully active, both in fish and in mammalian cells. The promoter and 5’-upstream region of the human R E T protooncogene on chromosome 10q21 is completely devoid of 5-methyldeoxycytidine in cells and tissues that do not express that gene. There are, of course, multiple elements that exert regulatory functions on a mechanism as crucial for cell survival as controlled gene activity. The methylation status of a promoter appears an important, but only one of several crucial parameters which, when needed, can be overruled in its effects by a number of other factors. By using adenovirus promoters as a model system, we have demonstrated that the 289 amino acid product of the E1A region of adenovirus type 2, a paradigm gene transactivator or the strong enhancer of the cytomegalovirus can reactivate a methylation-silenced promoter upon transfection into cells in culture. A major research goal will therefore be to understand the interplay of a given methylation status of a promoter with protein-binding motifs in the promoter, the function of these promoter-binding proteins, and the overall chromatin structure in promoters and their sequence environment. New technologies will have to be developed to help elucidate the still elusive chromatin structure and its role in transcriptional regulation.

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9 Patterns of D N A methylation in the human and in viral genomes

Table 8. Summary of experiments investigating promotor (gene) methylation and transcriptional inactivation of promoters”

Species

Gene

Location

Effecth

Type of evidence‘

Human

Alpha-fetoprotein Alu Apolipoprotein-E Beta-amyloid protein precursor B-cell tyrosine kinase c-myc Cyclin D2 Cytochrome P-450 2Et E-selectin Estrogen receptor Fragile X mental retardation 1 Gamma globin Glucose-6-phosphate dehydrogenase HLA class I loci A, B, G. E Hypoxanthine phosphoribosyltransferase IFN-gamma IGF2 and H19 Lactate dehydrogenase Leukosalin Major breakpoint cluster region mb-1 Multiple tumor suppressor 1 Myeloperoxidase myoD 06-Methylguanine-DNA methyltransferase Parathyroid hormone-related peptide Phosphoglycerate kinase pi-class glutathion S-transfcrase Platelet-derived growth factor A-chain Pro alpha 1(I) collagen Proenkephalin Proliferating ccll nuclear antigen Retinoblastoma tumor suppressor p-actin Sea urchin retroposon family -1/-2 Thymidine kinase Tumor necrosis factor-alpha von Hippel-Landau

5’-region Promoter Gene 5‘-region Gene Gene Gene Gene Promoter Gene Promoter Promoter 5‘-region Gene Gene Promoter Gene Gene 5’-region Gene 5’-region Gene 5’-region 5’-region Promoter Promoter 5‘-region Promoter Promoter Promoter 5‘-region Gene 5‘-region Gene Promoter Promoter 5‘-region 5’-region

Inactivated Inactivated No effect Inactivated Inactivated Questionbable Inactivated Questionable Inactivated Inactivated Inactivated Inactivated Questionable Inactivated Inactivated Inactivated Questionable Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated No effect Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated

A1 A1 A1 Al, C A1 A l , A2 C A1 B Al, C A2, C A1 A1 A1 A2 Al, C Al A1 A1 A1 A1 Al, C A1 A1 A1 AI, C C A1 B, C B, C B Al, C A1 B B, C A1 B A1

9.4 A long-term signal for promoter inuctivation

1 I3

Table 8. Continued

Species

Gene

Location

Effecth

Type of evidence‘

Mouse

Adenine phosphoribosyltransferase Alpha 1(I) collagen c-alb thymidine kinase c-Ha-rasVal 12 oncogene H1Y HRD-transgene Hypoxanthine phosphoribosyltransferase IFN-gamma Insulin-like growth factor 2 Insulin-like growth factor 2-receptor Metallothionein I promoter myoD Ovine growth hormone Phosphoglycerate kinase Sex-limited protein Sea urchin retroposon family -I/-2 U2AF1 -rs 1 Xist

Gene Promoter Promoter S.3’-region Promoter Gene Gene Promoter 5’-region 5’-region Promoter Gene Gene 5’-region %region Promoter Promoter Promoter

Inactivated Inactivated Inactivated No effect Inactivated Inactivated Inactivated Inactivated No effect Questionable Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated N o effect Inactivated

Al, C B B A1 Al, B A1 A2 Al, C A1 A1 Al, B C A1 A2 B B, C A1 A2

Rat

Apolipoprotein-E Gamma-crystallin Gamma-glutamyl transpeptidase Glial fibrillary acidic protein Pro alpha 2(I) collagen Prolactin Testis-specific H2B histone

Gene 5’-region 5’-region 5‘-region Promoter Promoter Promoter

No effect Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated

B C A1 B B A2

Dog Chicken Xenopus

Thyroglobulin Pepsinogen Ribosomal genes

Promoter Promoter Promoter

Inactivated Inactivated Questionable

B A1 A1

Virusesd Ad2

E2A late

5’-region

Inactivated

Ad12

ElA

Inactivated

Cau MV CRPV EBV EBV EBV FV3 HCMV

35s Different BCR2 BNLFl BamHI W promoter L1140 Early promoter

5’-region, Promoter Gene 5‘-region 5‘-region 5’-region Promoter 5’-region Coding

A l , A2, B B

Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated Inactivated

C A1 B B C B B

A1

9 Patterns of DNA methylation in the human and in viral genomes

114

Table 8. Continued

Species HIV-1 HPV-18 HSV SFV-3 SV40

Gene

Location

Effect’

Type of evidence‘

LTR URR Thymidine kinase Different Early promoter

Complete Complete 5’,3’-regions Complete Promoter

Inactivated Inactivated Inactivated Inactivated Inactivated

B B B C B

Complete

Questionable

C

Ciliate Colpoda General ~

a

’

The data shown in this compilation concern genes investigated after 1989. (Table from Munnes and Doerlfer, 1997). The term “inactivated” refers to the effect of promoter inactivation due to DNA methylation. In some cases (see footnote c), treatment of cells with 5-aza-2’-deoxycytidine has led to the reactivation of the gene. See evidence C. These interpretations are based on inverse correlations between promoter methylation and gene inactivation analyzed with (At) restriction enzymes or (A2) the method of genomic sequencing, or (B) on the premethylation of promoter-indicator gene constructs whose activity has subsequently been assessed upon transfection into mammalian cells. In some experiments, previously silent and methylated genes have been reactivated by 5-am-2’-deoxycytidine (C). Ad2, human adenovirus tpye 2; Cau MV, cauliflower mosaic virus; CRPV, cottontail rabbit papillomavirus; EBV, Epstein-Achong-Barr virus; FV3, frog virus 3; HCMV, human cytomegalovirus; HIV-I , human immunodeficiency virus 1; HPV-18, human papillomavirus 18; HSV, herpes simplex virus; SFV-3, simian foamy virus type 3; SV40, simian virus 40.

9.5 DNA methylation, an important parameter in genetic imprinting In a small percentage of the large number of genes in the human or mouse genome, the two alleles on the autosomes of the genome have been found to be functionally non-equivalent. Some genes are exclusively expressed from the maternally or the paternally inherited chromosome, whereas the majority of genes seems to be transcribed off both chromosomes. The functionally non-homologous regions on the two allelic chromosomes have been termed imprinted. In females, a large set of genes on one of the two X chromosomes become randomly inactivated. A number of studies has demonstrated conclusively that there are striking differences in the extent of DNA methylation between the two X chromosomes and between allelic regions in imprinted segments on the human or mouse autosomes. Since transcriptionally inactive segments of mammalian or integrated viral genomes have been shown to exhibit specific and extensive patterns of DNA methylation, this epigenetic signal is a likely factor in the molecular mechanism of imprinting. Additional parameters, possibly linked as well to DNA methylation, like chromatin structure, unequal timing of repli-

9.6 Chromatin structure and patterns of D N A methylation

115

cation, specific interactions among the homolog chromosomes and others, are being intensely investigated. In spite of very detailed studies on patterns of DNA methylation in imprinted segments of the mammalian genome, the enigma of the phenomenon remains enticing. What factors influence the decision on where de rzovo methylation is to be initiated, what regulates the spreading of DNA methylation? During embryonic development, a preexisting pattern of DNA methylation is apparently erased, later reestablished. Several laboratories have reported on different types of DNA demethylating enzymes. Two different mechanisms have been envisioned. The direct removal of a methyl group from the pyrimidine ring has been considered unlikely. Alternatively, a 5-methyldeoxycytidine residue may be excised and replaced by a deoxycytidine residue in a repair-like excision and replacement event. It remains puzzling how the DNA methyltransferase system “remembers” the right pattern, particularly in imprinted regions of the genome, and how this pattern can be reset parent- and sex-specifically during embryonic development. “Imprinting centers” have been postulated to play a role in the decisions on controlled and specific de ~ O V Omethylation. However, the biochemical signal of such centers remains hitherto undefined. Of course, erasure and reappearance of a given pattern of DNA methylation might proceed segment by segment in a stepwise manner and not all at once across huge parts of the chromosome. This progression could be reminiscent of the gradual spreading of de rzovo methylation in mammalian genomes after the insertion of foreign DNA. It is conceivable that the two chromosomal homologs exchange information also relevant to DNA methylation patterns upon physical contact during mitosis or meiosis. It is assumed that major parts of the mammalian (human) genomes are not imprinted, i.e., that both alleles are functional. However, this notion has not yet been rigorously investigated. It is unknown whether both alleles can in fact be equally transcribed at all times, in all cell types and at different developmental stages. The problem of imprinting will have to be investigated at a much wider scope.

9.6 Chromatin structure and patterns of DNA methylation It remains a major research goal to link these two important aspects of genome organization structurally and functionally. Complex patterns in either parameter can likely be understood only in context with each other. Progress in this direction will most probably depend on the development and adaptations of new technologies. Ideally a technique simultaneously displaying both features would help to elucidate suspected interdependencies. Recently, attempts have been made to link highly methylated DNA with the deacetylated state of histones. In this configuration, histones are thought to bind tightly to DNA and thus block transcription.

116

9 Patterns o,f D N A methylalion in the human arid in viral genomes

9.7 Patterns of DNA methylation in selected segments of the human genome We have determined the distribution of all 5-methyldeoxycytidine residues in several gene and promoter segments of the human genome by using the genomic sequencing method. Patterns of DNA methylation can be reliably determined by this method, since it is essential to assess the location of each 5-methyldeoxycytidine residue in a sequence. One of the goals of studying several parts of the human genome by this technology has been to obtain a more precise appreciation of the complexity of DNA methylation patterns, their stability, cell-type specificity, and interindividual conformity. I will briefly summarize some of the observations adduced from the human DNA methylation patterns determined in this laboratory.

9.7.1 Tumor necrosis factors a and p The genes for tumor necrosis factors (TNF) a and p - in particular, their 5’upstream and promoter regions - have been investigated in DNA isolated from human lymphocytes, granulocytes, and sperm. The results are characterized by a remarkable interindividual concordance of DNA methylation in specific human cell types. The patterns are identical in the DNA from one cell type for different individuals even of different ethnic origins but different in the DNA from different cell types. As an example, in the DNA from human granulocytes of 15 different individuals between the ages of 20 to 48 years and of both sexes, 5-methyldeoxycytidine residues have been localized by the genomic sequencing technique in three identical sequence positions in the 5’-upstream region and in one downstream position of the gene encoding TNF-a. The promoter of this gene is free of 5-methyldeoxycytidine and TNF-a is expressed in human granulocytes. The TNF-P promoter is methylated in granulocytes from 9 different individuals and TNF-P is not expressed. In human lymphocytes, the main source of TNF-a, the TNF-a promoter is free of 5-methyldeoxycytidine residues. All 5’-CG-3’ sites studied in the TNF-a and -p genes are however methylated in DNA from human sperm. In human cell lines HL-60, Jurkat, and RPMI 188, the extents of DNA methylation in TNF-a and -p genes have also been studied.

9.7.2 Human gene for the interleukin-2 receptor a chain (IL-2Ra) The gene for the human interleukin-2 receptor a chain (IL-2Ra) is expressed only in stimulated, not in resting, human T lymphocytes. We have investigated the promoter and exon 1region, the nucleotide -300 to +300 region of this gene with respect to the

9.7 Patterns of DNA merhylation in selected segments of the human genome

117

extent of DNA methylation at the 5‘-CG-3‘ sequences and its changes upon lymphocyte induction. By using RNA transfer analyses and the in vivo footprinting technique, we have confirmed that, upon stimulation of lymphocytes by phytohemagglutinin (PHA) plus interleukin-2 (IL-2), the IL-2Ra gene can be induced to be transcribed. The region of the IL-2Ra gene, as analyzed for 5’-CG-3’ methylation by the genomic sequencing method or a polymerase chain reaction-based method subsequent to HpaII or HhaI cleavage of the DNA, does not seem to be significantly methylated in most cell types tested, except for the cytidine residue in position +198 which is partly methylated. In the DNA of cells from a chronic B cell lymphatic leukemia, 5’-CCGG-3‘ sequences in the exon 1 region are almost completely unmethylated. It is striking that the absence of DNA methylation in this promoter and exon 1segment also extends to cell types not directly associated with the immune response and even to continuous cell lines. The gene for the IL-2Ra is not expressed in quiescent human lymphocytes but can be activated by the stimulation of lymphocytes. It, therefore, seems plausible that the promoter of this gene is not permanently inactivated by DNA methylation.

9.7.3 Prader-Labhart-Willingelman syndrome region on human chromosome 15qll-13 A deletion at 15qll-ql3 or uniparental disomy of chromosome 15 leads to the Prader-Labhart-Willi syndrome (PWS) or the Angelman syndrome (AS) because this region contains genes which are expressed exclusively from the paternally (PWS) or the maternally (AS) inherited chromosome, respectively. We have applied the bisulfite protocol of genomic sequencing to study all 5’-CG-3’ dinucleotides around the chromosome 15-located exon 1 of SNRPN and at the D15S63 locus. There is a start site for alternative SNRPN transcripts possibly involved in imprint switching during gametogenesis. At least 17 PCR products derived from single chromosomes of normal individuals as well as PWS and AS patients have been sequenced. Cytosine residues outside 5’-CG-3’ dinucleotides are always unmethylated. However, 96% of all of the 23 5’-CG-3’dinucleotides around SNRPN exon 1are methylated on the maternal chromosome and completely devoid of methylation on the paternal chromosome. This finding is in contrast to the D15S63 locus, where only the two CfoI/HhaI sites are methylated on the maternal chromosome at the same frequency as seen for the SNRPN segment. At the other five 5’-CG-3’ dinucleotides, differential methylation is less pronounced, i.e., 45-70% of the 5’-CG-3’ dinucleotides on the maternal chromosome and 5-14% on the paternal chromosome are methylated. The systematic investigation of 5‘-CG-3’ patterns have provided the basis for a new PCR-based methylation test to diagnose PWS and AS.

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9 Patterns of D N A methylation in the human and in viral genornes

9.7.4 Alu sequences We have also investigated DNA methylation in human Alu sequences, both in general and in specific Alu sequences associated with the genes for a1 globin, tissue plasminogen activator (tPA), adrenocorticotropic hormone (ACTH) and angiogenin. We studied DNAs from lymphocytes, granulocytes, brain, heart muscle and sperm, and from the human HeLa and KB cell lines by using cleavage with methylation-sensitive restriction enzymes combined with Southern blot hybridization and by using genomic sequencing. (i) In differentiated primary human cells, Alu elements are often highly methylated even when they are in very 5’-CG-3’-rich regions. (ii) There are distinct differences in the levels of methylation in the specific Alu sequences. (iii) Alu elements in the DNA of haploid spermatozoa are much less methylated than in diploid cells. (iv) The results of cell-free transcription experiments with Alu elements suggest that the in vitro transcription of Alu elements can be inhibited by 5‘-CG-3’ methylation. (v) The patterns of methylation observed in the Alu elements and in the surrounding sequences are characterized by cell type specific interindividual concordance.

9.7.5 Wide range of randomly selected human DNA sequences Patterns of DNA methylation at 5’-CCGG-3’ and 5‘-GCGC-3‘ sequences have been determined in about 570 kb, equivalent to about 0.02% of the human genome, by using HpaII and HhaI restriction endonucleases, respectively, and randomly selected cosmid clones of human DNA as hybridization probes. Many of these human DNA sequences are of the repetitive type. The DNAs from human lymphocytes, from a mixture of all blood cells or from several established human cell lines (HeLa, KB, 293, or DEV) have been included in these analyses. In the segments of the human genome investigated, the patterns of DNA methylation are characterized by completely or partly methylated 5’-CCGG-3’ or by partly methylated 5’-GCGC-3’ sequences. Even among individuals of different ethnic origins, East-Asian or Caucasian, these patterns of DNA methylation are indistinguishable by the method applied. The cytokine-dependent stimulation of human lymphocytes to replicate in culture does not affect the stability of these patterns. In the same DNA sequences from several human cell lines, much lower levels of DNA methylation have been observed.

9.7 Patterns of DNA methylation in selected segments of the human genome

119

9.7.6 Selected human genes in different Hodgkin’s lymphoma and leukemia cell lines and in normal human lymphocytes We have performed a study of DNA methylation patterns in human lymphocytes and in human cell lines of different malignant origins. Several of the protooncogenes, parts of the genes for tumor necrosis factors a and p, the insulin receptor and lamin C have been used as hybridization probes. We have relied to some extent on the documented observation that the methylation patterns at 5’CCGG-3’ (HpaIUMspI) sequences yield at least a reflection of patterns at all 5‘CG-3‘ sequences. Three main types of patterns have been observed. Some of the probed segments are completely unmethylated; others are fully methylated, most of the areas are partly methylated exhibiting complex patterns at the 5’-CCGG-3’ sites. In different tumor cell lines, different DNA methylation patterns are apparent for the same DNA probes. Comparisons of the methylation patterns in a given DNA segment between DNA from primary normal human lymphocytes and DNA from different tumor cell lines reveal changes, increases or decreases, in these patterns in several instances.

9.7.7 RET Protooncogene In a large proportion of familial and sporadic cases of Hirschsprung disease (HSCR), mutations in the RET (rearranged during transfection) protooncogene have been described. We have investigated the structure of the RET gene promoter and have analyzed a region of approximately 1000 nucleotides in its promoter and 5’-upstream segments for the occurrence of 5-methyldeoxycytidine residues by using the bisulfite protocol of the genomic sequencing method. This technique has an estimated sensitivity of about 93%. Not a single 5methyldeoxycytidine residue could be detected in the control region of the RET gene that seems to be silenced or exhibits low activity in many adult tissues. In these experiments, the DNAs of peripheral white blood cells (PWBC) from 4 healthy individuals, from 7 patients with familial HSCR, as well as DNAs from different human tissues and from a human embryonic kidney (HEK) cell line have been included. In a DNA segment starting 790 nucleotides upstream of the transcriptional start site of the RET gene, a few 5-methyldeoxycytidine residues have been identified. This region possibly constitutes the transition site from an unmethylated promoter to a more extensively methylated region in the human genome. The data presented are remarkable in that a highly 5’-CG-3’-enriched segment of the human genome with 49 5’-CG-3’ dinucleotide pairs in 400 bp within the putative promoter region is completely devoid of 5-methyldeoxycytidine residues, although this control region is not actively transcribed in most adult human tissues. By the hybridization of a PCR-amplified RET protooncogene cDNA probe harboring exons 9 to 15 to a membrane (Clontech) containing poly-A selected RNAs from 50 different

120

9 Patterns of D N A methylation in the human and in viral genomes

human tissues, weak RET protooncogene expression in many of the neural cellderived tissues has been detected. RNAs extracted from many other human tissues do not share sequence homologies to this ”P-labeled probe. Mechanisms other than DNA methylation obviously play the crucial role in the inactivation of the RET gene promoter in these tissues. We have also demonstrated by the in vitro premethylation of a RET promoter-chloramphenicol acetyltransferase (CAT) gene construct and transient transfection experiments into neuroblastoma cells that the transcriptional activity of the RET promoter is decreased by HpaII (5’-CCGG-3’) methylation and abolished by SssI (5’-CG-3’) methylation. Hence the RET promoter region is sensitive to this regulatory signal. However, in vivo, DNA methylation of the promoter region seems not to be the predominant regulatory mechanism for the RET protooncogene. Possibly, in adults the RET gene can be occasionally activated.

9.8 Patterns of methylation in viral DNA In molecular biology, viral systems have often helped elucidate complex mechanisms in pro- or eukaryotic cell systems and organisms. We have investigated a number of different viruses for the patterns of DNA methylation in their genomes. In general, viral DNAs replicate free in the nuclear, sometimes in the cytoplasmic,compartment of infected cells. In rare instances, the viral genome can become integrated into the genome of the host cell and can persist in that state for many cell generations. As an integrate, the viral genome becomes subject to cellular mechanisms of regulation and modification. In our laboratory, the following viral genomes have been studied for their methylation patterns, human adenovirus types 2 and 12, the iridovirus frog virus 3, human cytomegalovirus, and the insect baculovirus Autogrupha culifornica nuclear polyhedrosis virus (AcNPV).

9.8.1 Human adenovirus types 2 and 12 The DNA incorporated into mature adenovirions of either type is not detectably methylated. We have applied different methods to the study of methylation in viral genomes, i.e., the complete hydrolysis of viral DNA and the two-dimensional separation of the products by electrophoresis and chromatography, analyses with methylation-sensitive restriction enzymes, and different protocols of the genomic sequencing procedure. None of these methods revealed any 5methyldeoxycytidine residues in adenovirion DNA. Similarly, the intranuclear free, non-integrated adenovirus DNA in productively infected human (Ad2 and Ad12) or in abortively infected hamster cells (Ad12) lacks detectable levels of 5methyldeoxycytidine residues. Other human DNA virus genomes, e.g., the genome of the Epstein-Achong-Barr virus (EBV) have been shown to be methy-

9.8 Patterns of methylation in viral D N A

121

lated in their intracellular episomally persisting forms. It is conceivable that the free adenoviral DNA that is only partly dependent on cellular proteins for its replication and supplies itself major parts of its replication machinery encoded in the viral genome, i.e., the DNA-binding protein, the adenovirus DNA polymerase, and the preterminal viral protein, never becomes associated with the cellular DNA replication apparatus that is thought to contain the DNA methyltransferase systems. By virtue of its self-sufficiency, adenovirus DNA appears to be synthesized in an intranuclear compartment distinct from cellular DNA replication and may, for that reason, escape de novo methylation in mammalian cells whose genomes are methylated in highly specific patterns. In this context, it is interesting to mention that many kilobases of cellular DNA sequences that have become inserted into the Ad12 genome inside mature virions in a symmetric recombinant viral genome (SY REC), generated naturally in productively infected cells, are not methylated, whereas the same cellular DNA segments located in the cellular genome are heavily methylated. This finding is consistent with the notion that DNA sequences in the virion, regardless of their actual nucleotide sequence, are not subject to DNA methylation, whereas the same sequences in the context of the cellular genome are methylated. Thus, the intranuclear compartment apparently can influence the accessibility of a DNA sequence to the DNA methyltransferase systems of the cell. A completely different situation arises when the adenoviral genome becomes integrated into the genome of mammalian cells as has been demonstrated in adenovirus-transformed cells or in Adl2-induced tumor cells (see Chapters 4, 5). Integrated as part of the cellular genome, adenoviral DNA becomes de novo methylated in highly specific patterns (see Chapters 4,lO). This de novo methylation is initiated in distinct segments of the viral genome and begins to spread from these initially very size-limited loci across the entire viral genome of some 34 to 35 kbp, depending on the type of adenovirus DNA integrated (Fig. 27). The mechanisms underlying this complete de novo methylation are still enigmatic but may hold the clue for many different types of de novo DNA methylation in mammalian systems, e.g., also for a better understanding of the process of imprinting in specific parts of mammalian genomes. It is likely that the initiation and extent of de novo methylation of integrated adenovirus genomes is not solely, perhaps not even mainly, dependent on the nucleotide sequence of adenovirus DNA. Other factors as site of integration, chromatin structure and copy number of integrates, and of the DNA sequences at the insertion site of foreign DNA may play important roles in eliciting the modification of the inserted foreign DNA. It has been postulated that the de novo methylation of integrated foreign DNA in mammalian genomes can be interpreted to be part of a cellular defense mechanism against the activity of integrated foreign genes. There is evidence that the insertion of foreign DNA into an established mammalian genome can lead to alterations at the site of viral DNA integration, as documented for retroviral DNA and for integrated adenovirus DNA. We have also observed major alterations of cellular DNA methylation patterns in genome segments located remote from the integration site of foreign DNA. These

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40

40

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I

+l I

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-40 I

-60

-80

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BstXI -120

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4-J

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5‘-C-3‘

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Figure 27. Initiation and spreading of de n o w DNA methylation in integrated foreign DNA. Fully and partially methylated or unmethylated 5’-CG-3’ dinucleotides in the late E2A promoter of Ad2 DNA were presented for the transformed cell lines HEI, HE2, uc2, uc20, or for cell lines mc23 and mc40 in different passages (p), as shown by genomic sequencing. The scale refers to Transcription . proceeds nucleotide numbers in the late E2A promoter relative to the cap site (4) to the left.The 5’-CCGG-3’ sequences are at nucleotides +6 (+I) and +24 (+II), which have been premethylated in v i m prior to transfection in the generation of cell ines mc23 and mc40. Horizontal lines represent the late E2A promoter segment in individual cell lines. The S’-CG-3’ sequences in this segment (+I11 to -XI) are represented by vertical bars: (0) unmethylated; completely methylated; and (m) 5’-CG-3’ sequences which are methylated in only some of the integrated promoter copies. The bars above the horizontal line designate 5’-CG-3‘ dinucleotides in the top strand of the promoter sequence, the bars below the line represent the same dinucleotides in the bottom strand. In the E2A region of Ad2 DNA the bottom strand is the transcribed (minus) strand, the top strand the non-transcribed complement (Toth et ai., 1990).

(m)

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123

changes have been observed in adenovirus-transformed cells or in Adl2-induced tumor cells as well as in hamster cells transgenic for bacteriophage h DNA. We are pursuing the possibility that the insertion of foreign DNA into mammalian genomes can per se contribute to the alteration of cellular methylation patterns, possibly via impacts on the cells chromatin structure (see Sections 10.3.3, 10.3.4).

9.8.2 Frog virus 3 Both the virion DNA and the intracellular frog virus 3 (FV3) DNA late after infection are extensively methylated, apparently in all 5’-CG-3’ dinucleotides as determined by the genomic sequencing technique in parts of the genome. The viral DNA replicates in the nucleus of fish or mammalian cells and remains undermethylated only for a very short time immediately after replication. When late viral gene expression commences, the intranuclear viral DNA in both fish and mammalian cells has become extensively de novo methylated. Nevertheless, late viral gene expression proceeds at a maximal rate. Obviously the activity of the late FV3 promoters in infected cells is not sensitive to 5’-CG-3’ methylation. In reconstitution experiments, in which the promoter of the FV3 gene L1140 has been fused to the chloramphenicol acetyltransferase gene and in which this construct has been transfected into fish or mammalian cells, the activity of this promoter has been shown to be fully retained even after the premethylation of all 5‘CG-3‘ dinucleotides. This finding is consistent with the in vivo activity of this fully 5’-CG-3’ methylated promoter inside the FV3 genome in infected cells. In contrast, the activity of the same FV3 promoter is seriously affected when only the eight 5’-CCGG-3’ (HpaII) sequences of this promoter have become methylated. These findings suggest that specific patterns of methylation at specific sites and not the presence or absence of 5-methyldeoxycytidine residues or the absolute number of 5-methyldeoxycytidine residues influence the activity profiles of eukaryotic and/or viral promoters. So far, there is no evidence for the occurrence of integrated forms of FV3 DNA.

9.8.3 Autographa califomica nuclear polyhedrosis virus (AcNPV) There is only very limited information on the status of methylation in the genome of this virus. The experimental data available suggest that the virion genome and the intracellular forms of this viral DNA are not methylated in the sequences investigated. It is concluded that the occurrence of 5-methyldeoxycytidine residues in viral genomes depends on a large number of parameters and entirely on the biological system under investigation. Patterns of DNA methylation in cellu-

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9 Patterns of D N A methylation in the human and in viralgenomes

lar or in viral genomes can be considered to serve as specific modulators of specific protein-DNA interactions and co-determinants of chromatin or chromatinlike structures, as is the case in the more complex virions. These patterns are also a reflection of the intracellularhntranuclear localization of viral genomes but also of the type of virus investigated. The functional significance of a given pattern of promoter methylation can not be predicted as the example of the FV3 genome documents. For the time being, it will be mandatory to analyze carefully many different patterns of DNA methylation. The functional consequences of promoter methylation connot be predicted but have to be determined experimentally for each promoter.

9.8.4 Human cytomegalovirus (HCMV) The virion DNA and the intranuclear viral DNA in infected human fibroblasts up to 263 h after infection have been investigated by cleavage with a number of methylation-sensitive restriction endonucleases. There is no evidence for methylation in the DNA of the AD169 strain of HCMV in the virion or in productively infected cells at any of thes restriction sites. Genomic sequencing experiments have not yet been done.

9.8.5 General considerations We have determined the exact patterns of DNA methylation in various parts of the human genome by the bisulfite or the hydrazine protocol of the genomic sequencing method. The data are, of course, still very limited. The results attest to the enormous complexity of the naturally occurring patterns which actually defy simplistic models that have prematurely been proposed in the literature. Often inverse correlations between levels of promoter methylation and gene activity exist, as have been frequently described. However, inactive very 5’-CG3’-rich promoters can be completely unmethylated, e.g., the promoter of the R E T protooncogene, or of the TNF-a and -p genes. Specific patterns of DNA methylation may serve more than one biological function depending on site and specific nucleotide sequence. 5’-CG-3’-rich sequences can be highly methylated or be devoid of any 5-methyldeoxycytidine residues. Inserted foreign genes are often de novo methylated but the extent of this modification depends on several factors. The frog virus system, though perhaps exotic, defies all simple model ideas. I consider DNA methylation patterns a primary imprint on the genome which is able to modulate DNA-protein interactions positively or negatively depending on the protein. In this way, the first layer of protein interactions with the genomic DNA can be determined upon which the construction of chromatin rests through several rounds of protein-protein interactions.

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9.9 Further reading Allfrey, V.G. (1970) Changes in chromosomal proteins at times of gene activation. Fed. Proc. 29,1447-1460. Allfrey, V.G., Chen, T.A. (1991) Nucleosomes of transcriptionally active chromatin: isolation of template-active nucleosomes by affinity chromatography. Methods Cell Biol. 35,315-335. Behn-Krappa, A., Doerfler, W. (1993) The state of DNA methylation in the promoter and exon 1 regions of the human gene for the interleukin-2 receptor a chain (IL-2Ra) in various cell types. Human Mol. Genet. 2,993-999. Buiting, K., Saitoh, S., Gross, S., Dittrich, B., Schwartz, S., Nicholls, R.D., Horsthemke, B. (1995) Inherited microdeletions in the Angelman and PraderWilli syndromes define an imprinting centre on human chromosome 15. Nature Genetics 9,395-400. Church, G.M., Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. U S A81,1991-1995. Clark, S.J., Harrison, J., Paul, C.L., Frommer, M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22,2990-2997. Deuring, R., Klotz, G., Doerfler, W. (1981) An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA. Proc. Natl. Acad. Sci. U S A 78,3142-3146. Doerfler, W. (1983) DNA methylation and gene activity. Annu. Rev. Biochem. 52, 93-124. Doerfler, W. (1991) Patterns of DNA methylation - evolutionary vestiges of foreign DNA inactivation as a host defense mechanism - a proposal. Biol. Chem. Hoppe-Seyler 372,557-564. Feinberg, A.P., Vogelstein, B. (1983) Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301,89-92. Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L., Paul, C.L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. U S A89,1827-1831. Heller, H., Kammer, C., Wilgenbus, P., Doerfler, W. (1995) Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage 1)DNA is associated with enhanced methylation of cellular DNA segments. Proc. Natl. Acad. Sci. U S A 92,5515-5519. Holliday, R. (1987) The inheritance of epigenetic defects. Science 238,163-170. Holliday, R., Pugh, J.E. (1975) DNA modification mechanisms and gene activity during development. Science 187,226-232. Jahner, D., Jaenisch, R. (1985) Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity. Nature 315,594-597. Jost, J.P., Saluz, H.P. (Eds.) (1993) DNA Methylation. Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Boston, Berlin.

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Knebel-Morsdorf, D., Achten, S., Langner, K.-D., Riiger, R., Fleckenstein, B., Doerfler, W. (1988) Reactivation of the methylation-inhibited late E2A promoter of adenovirus type 2 by a strong enhancer of human cytomegalovirus. Virology 166,166-174. Kochanek, S., Toth, M., Dehmel, A., Renz, D., Doerfler, W. (1990) Interindividual concordance of methylation profiles in human genes for tumor necrosis factors a and p. Proc. Natl. Acad. Sci. U S A87,8830-8834. Kochanek, S., Radbruch, A., Tesch, H., Renz, D., Doerfler, W. (1991) DNA methylation profiles in the human genes for tumor necrosis factors a and p in subpopulations of leukocytes and in leukemias. Proc. Natl. Acad. Sci. U S A 88, 5759-5763. Kochanek, S., Renz, D., Doerfler, W. (1993) DNA methylation in the Alu sequences of diploid and haploid primary human cells. EMBO J. 12,1141-1151. Langner, K.-D., Vardimon, L., Renz, D., Doerfler, W. (1984) DNA methylation of three 5’-C-C-G-G-3’ sites in the promoter and 5‘ region inactivates the E2a gene of adenovirus type 2. Proc. Natl. Acad. Sci. U S A81,2950-2954. Lichtenberg, U., Zock, C., Doerfler, W. (1988) Integration of foreign DNA into mammalian genome can be associated with hypomethylation at site of insertion. Virus Res. 11,335-342. Munnes, M., Doerfler, W. (1997) DNA methylation in mammalian genomes: Promoter activity and genetic imprinting. Encyclopedia of Human Biology, 2”d edition, vol. 3, Academic Press, pp. 435-446. Munnes, M., Patrone, G., Schmitz, B., Romeo, G., Doerfler, W. (1998) A 5’-CG3’-rich region in the promoter of the transcriptionally frequently silenced RET protooncogene lacks methylated cytidine residues. Oncogene 17,2573-2584. Munnes, M., Schetter, C., Holker, I., Doerfler, W. (1995) A fully 5’-CG-3’ but not a 5’-CCGG-3’ methylated late frog virus 3 promoter retains activity. J. Virol. 69,2240-2247. Orend, G., Kuhlmann, I., Doerfler, W. (1991) Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells. J. Virol. 65,4301-4308. Pfeifer, G.P., Steigerwald, S.D., Hansen, R.S., Gartler, S.M., Riggs, A.D. (1990) Polymerase chain reaction-aided genomic sequencing of an X chromosomelinked CpG island: Methylation patterns suggest clonal inheritance, CpG site autonomy and an explanation of activity state stability. Proc. Natl. Acad. Sci. U S A87,8252-8256. Razin, A., Cedar, H. (1991) DNA methylation and gene expression. Microbiol. Rev.55,451-458. Remus, R., Kammer, C., Heller, H., Schmitz, B., Schell, G., Doerfler, W. (1999) Insertion of foreign DNA into an established mammalian genome can alter the methylation of cellular DNA sequences. J. Virol. 73,1010-1022. Sutter, D., Doerfler, W. (1980) Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression. Proc. Natl. Acad. Sci. U S A77,253-256.

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Sutter, D., Westphal, M., Doerfler, W. (1978) Patterns of integration of viral DNA sequences in the genomes of adenovirus type 12-transformed hamster cells. Cell 14, 569-585. Vardimon, L, Kressmann, A., Cedar, H., Maechler, M., Doerfler, W. (1982) Expression of a cloned adenovirus gene is inhibited by in vitro methylation. Proc. Natl. Acad. Sci. USA79,1073-1077. Weisshaar, B., Langner, K.-D., Juttermann, R., Muller, U., Zock, C., Klimkait, T., Doerfler, W. (1988) Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by E1A (13s) functions. J. Mol. Biol. 202,255-270. Willis, D.B., Granoff, A. (1980) Frog virus 3 DNA is heavily methylated at CpG sequences. Virology 107,250-257. Zeschnigk, M., Schmitz, B., Dittrich, B., Buiting, K., Horsthemke, B., Doerfler, W. (1997) Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Human Mot. Genet. 6,387-395.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

10 Consequences of foreign DNA integration and persistence The mammalian genome has evolved over a long period of time and the genomes of the contemporary species can be considered stable for the moment at which they are analyzed. The addition of foreign DNA by insertion into an established genome cannot remain without consequences. Unfortunately only limited information is available on the nature and extent of these sequelae. The stability and protected state of the existing genomes has been taken for granted, although it is not reasonable to assume that the assault by intruding foreign DNA would not continue indefinitely. The realization that foreign DNA can be added relatively easily by viral infection, by DNA transfection of mammalian cells or can be constantly taken up via the gastrointestinal tract with the daily food supply has raised novel questions about the outcome of the encounters of mammalian cells and organisms with foreign genetic material. In practical terms, many experimentalists have been alerted to the consequences of foreign DNA insertion only after their favorite constructs genomically fixed in transgenic cells or organisms ceased or failed to be expressed. Questions about site of insertion, insertional mutagenesis or de novo methylation of the transgenes have then become relevant for those striving for the optimization of foreign gene expression. The current enthusiasm about the potential of gene therapy will also have to meet the challenge of reality in the interaction of integrated foreign genes in the natural environment of the mammalian cell with its defenses and recombinational mechanisms. In this chapter the following topics will be discussed: 0 0

0

0

0

De novo methylation of integrated foreign DNA (10.1) De novo methylation of targeted versus randomly integrated foreign DNA (10.2) The insertion of foreign DNA into an established mammalian genome can alter patterns of methylation in cellular genome segments (10.3) De novo methylation of foreign DNA - a hypothetical ancient defense mechanism (10.4) Malignancy - a chromatin disease? (10.5)

10.1 De novo methylation of integrated foreign DNA The de n o w methylation of foreign DNA subsequent to its integration into established eukaryotic genomes appears to be a general phenomenon that has been observed in mammalian as well as in plant cell systems. An early demonstration of this de novo methylation stems from experiments in which the integrated

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10 Consequences of foreign DNA integration rind persistence

Ad12 DNA in Adl2-transformed cells or in Adl2-induced tumor cells could not be cleaved with methylation-sensitive enzymes like HpaII or HhaI. Viral DNA isolated from purified virions or from productively infected human cells lacks 5methyldeoxycytidine in its nucleotide sequence. Upon the insertion into hamster genomes, however, the viral DNA that can be integrated at many different sites is rapidly de novo methylated in precise patterns which are often very similar in independently derived tumor cell lines. D e novo methylation is initiated at distinct 5’-CG-3’ dinucleotides in the integrated viral genomes, and the modification then spreads across the entire viral genome. It is unknown which factors in the mammalian genome or which signals in the integrated foreign viral DNA are decisive to direct and regulate the initiation and the spreading of de novu methylation. In what way could de novo methylation be linked to integrative recombination and which DNA methyltransferases and regulatory cofactors participate in these reactions? Chromatin structure is thought to be an important element in influencing de novo methylation and probably vice versa So far, the chromatin structure of the integrated foreign DNA and of the neighboring cellular DNA sequences in comparison to their structure in the established genome devoid of foreign integrates has not yet been studied in sufficient detail. The methylation of cytidine residues in mammalian and other eukaryotic DNAs is the only known modified nucleotide with functional significance. The influence of 5-methyldeoxycytidine residues in specific promoter positions on promoter activity has been well documented. The mechanism of this in general negative effect on promoter activity is only partly understood: Structural alterations in the promoter region, impaired or enhanced binding of transcription factors or cofactors or changes in the acetylation of histones have been implicated in the inactivation of promoters due to the methylation of promoter sequences. Several experimental designs have been used to document the effect of DNA methylation on promoter function (Table 8). Inverse correlations between promoter activity and extent of promoter methylation have been observed in many different systems. Moreover, it has been demonstrated that inactive mammalian promoters or promoters in integrated viral genomes can be reactivated by growing the cells in the presence of 5-deoxyazacytidine, a known inhibitor of the DNA methyltransferase system of the cell which reduces the levels of DNA methylation in growing cells. Numerous studies have been published in which many different promoter-indicator gene constructs have been in vitro premethylated at specific 5’-CG-3’ sequences (Table 8). Subsequently, these constructs have been transfected into mammalian cells, and their activities have been compared with those of unmethylated or mock-methylated constructs. Almost invariably, the premethylated constructs have proved inactive or less active when methylated in the functionally correct positions. This approach has usually served to identify the rnethylation-sensitive 5’-CG-3’ sequences in a promoter.

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10.2 De novo methylation of targeted versus randomly integrated foreign genes For many studies in molecular biology it will be of interest to elucidate the mechanisms of regulation of de n o w methylation in foreign DNA that has been inserted into an established mammalian genome, haphazardly or by experimental design. All transfection and genomic fixation experiments and approaches towards gene therapy have to deal with the problems of foreign DNA methylation and possible gene inactivation. In developmental biology, genetic imprinting, X chromosome inactivation, tumor biology, alterations of DNA methylation play an important role. One of the major unresolved questions on the mechanism of de nova methylation addresses the factors determining sites and extent of foreign DNA methylation upon the insertion of foreign DNA into the recipient genome. The following parameters have been studied, though not in sufficient detail: 0 0

0 0 0 0

The site of foreign DNA insertion in the recipient genome. The site of recombination in the inserted foreign DNA. The size of the insert and the copy number of integrates. The nucleotide sequence of and specific motifs in the foreign DNA. The timing of the insertion event relative to the cell cycle. Promoter strength and/or regulatory elements, e. g., enhancers in the inserted foreign DNA.

We have devised a series of experiments in which the murine BLK (B lymphocyte tyrosine kinase) gene on chromosome 14 was reinserted into one of its authentic endogenous genomic sites by homologous recombination and selection of mouse ES cell clones with the reinserted DNA in the correct position. ES clones with the BLK DNA inserted into randomly targeted positions have also been analyzed as controls. Foreign DNA sequences have been placed inside the endogenous BLK gene in the constructs used in the electroporation-driven transfection experiments. Three different constructs have been transfected in different experiments. In all of them the neomycin gene has the same phosphoglycerate kinase promoter. The luciferase gene, however, has a different promoter and/or enhancer sequence in each construct. The AdBLK plasmid carries the luciferase gene under the control of the Ad2 E2A late promoter, and the ES clones derived from these constructs have been designated A clones. In the plasmid SVBLK, the SV40 early promoter is located 5’ of the luciferase gene and the SV40 enhancer sequences are located 3‘. These ES clones have been termed S clones. The DNA in all of the A clones becomes de nova methylated in both the luciferase and neomycin genes, while the homologously recombined S clones remain hypomethylated even after 30 passages. Another construct has been made, SVpBLK, which is identical to SVBLK, except that it contains the SV40 promoter without the enhancer sequences at the 3’ end. Unlike the original S clones, these homologously recombined clones become de n o w methylated within t h e luciferase and neomycin sequences. These results indicate that the enhancer ele-

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ment, containing the 21 bp repeats, can inhibit de novo methylation within the entire luciferase and neomycin regions. In mammalian genomes, the mechanism of de novo methylation of integrated foreign DNA may affect in particular multiple copies of integrated foreign DNA which are often found after random integration events. The DNAs from all the clones with randomly integrated foreign DNA in our study, regardless of the nature of the integrated foreign DNA sequences, become highly methylated. These sequences can be recognized as foreign and may activate the host defense, the de novo methylation reaction. Thus, the enhancer element that is present in some of the clones with randomly integrated foreign DNA, does not suffice to inhibit de novo methylation after random integration. Therefore, our data indicate that the location of the foreign DNA in the genome and perhaps the copy number of inserted foreign genomes play a major role in the regulation of de novo methylation. Our data support the following interpretations (Fig. 28): 0

0

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The homologously recombined foreign DNA (the BLK gene) sequences become remethylated in patterns very similar to the authentic pre-existing cellular patterns of DNA methylation in the endogenous alleles. Thus, position and vicinity effects including sequence and structure of the inserts must be of considerable importance in determining de n o w methylation patterns. Randomly integrated foreign DNA becomes heavily de novo methylated. In randomly integrated constructs the nature of the promoter or the presence of the SV40 enhancer elements appear irrelevant for the de novo methylation of the inserted foreign genes. Thus, the role of these elements in determining de novo methylation may not be of fundamental importance. Upon homologous site-directed insertion, the extents of de novo methylation of the foreign luciferase and neomycin genes reintegrated jointly with the BLK sequence depend on the promoter sequence present in these constructs. The early SV40 promoter with the enhancer sequences seems to prohibit de novo methylation, and the luciferase gene is expressed in these transgenic ES cells. In contrast, constructs under the control of the E2A late promoter of Ad2 DNA or under the SV40 promoter without the enhancer elements become extensively de novo methylated even in the authentic BLK position, and luciferase expression is reduced. Removal and addition of the selective drug G418 (geneticin) do not alter the methylation levels of the examined DNA sequences in the ES clones that contain the Ad2 E2A late promoter. After the homologous recombination into the BLK gene, nucleotide sequences in the transgenic foreign DNA can affect de novo methylation. One can ponder the possibility that promoters with their transcriptional potential in transgenic sequences within a specific chromatin arrangement interact with the cellular machinery that is responsible for de novo methylation or its inhibition.

The mechanism of de novo methylation of foreign DNA inserted into an established mammalian genome cannot yet be explained in detail. De novo methylation may be related to an ancient cellular defense system targeted against foreign

10.2 D e n o v o methylation of targeted versus randomly integrated foreign genes Reinsertionat authentic site by homologous integration

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b) Strong SV40 promoter + enhancer

Figure 28. Responses to the integration of foreign DNA depending on the site of insertion: targeted vs. random integration. See text on pages 131-134.

DNA.We have investigated some of the factors influencing d e novo methylation when an authentic cellular D N A sequence is reinserted into the mouse genome at different locations. The experimental approach chosen exposes the authentic unmethylated BLK sequence to the d e novo methylation system in two different ways. In one set of experiments, the BLK D N A has been reinserted into its original chromosomal location by homologous recombination. Alternatively, upon random integration by heterologous recombination, the BLK and adjacent sequences are located at several randomly selected sites where they are apparently recognized as foreign by the de novo methylation machinery. In the latter

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context, promoter strength and presence or absence of the SV40 enhancer sequence seem to be of no importance in determining the extent of de novo methylation. As a working model, we pursue the possibility that the enzymatic process of de n o w methylation recognizes and is dependent upon the specific chromatin structure at the site of an individual gene or DNA segment. Thus, the endogenous BLK DNA at its authentic site or the same DNA inserted at randomly chosen locations would look very different to the DNA methyltransferase system. When the BLK gene and adjoining sequences in the construct are homologously reinserted into one of the authentic allelic BLK positions on mouse chromosome 14, the pre-existing chromatin configuration can be reconstituted. Under these conditions, the de novo methylation system then imprints the methylation pattern specific for the BLK locus that had pre-existed prior to the transfection of ES cells. In contrast after random integration, when the BLK construct arrives in an alien position with a locus-specific, but not BLK-typical chromatin arrangement, a different pattern of de novo methylation is imposed upon the integrated DNA which is recognized as foreign in this location with a heterologous sequence context. Of course, this model still leaves the question unanswered of how de n o w methylation and chromatin structure arc interdependent. Differences in the accessibility of individual 5’-CG-3’ dinucleotides to the DNA methyltransferase system may be one of the important parameters.

10.3 The insertion of foreign DNA into an established mammalian genome can alter patterns of methylation in cellular genome segments 10.3.1 Introduction Established mammalian and other eukaryotic genomes allow the insertion of foreign DNA sequences both naturally, e.g., upon virus infections or via the gastrointestinal tract, and under experimental conditions exemplified by the genomic fixation of foreign DNA after the application of various transfection or microinjection protocols. While investigating the oncogenic transformation of hamster cells after infection with human Ad12, we have studied the insertion of Ad12 DNA into the hamster cell genome and some of its consequences. Among the sequelae of foreign DNA insertion, we have concentrated on the de novo methylation of the integrated foreign DNA (Sections 10.1, 10.2) and on alterations in the patterns of methylation in several cellular genes and DNA segments. The integration of foreign (Ad12) DNA into the hamster cell genome is not nucleotide sequence- or chromosomal site-specific. Ad12 DNA, h DNA and probably any other foreign DNA can be inserted in multiple copies at many dif-

10.3 The insertion of foreign DNA into an established mammalian genome

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ferent sites, sometimes partly fragmented, often and as a rule, at a single chromosomal location (see Section, 4.3.4,6.1). The described alterations in cellular DNA methylation patterns upon foreign DNA insertion are perhaps a reflection of more far-reaching changes in chromatin structure in the affected cells. Implications of DNA methylation for chromatin structure and vice versa have been investigated in several laboratories. We have observed that up to >50 genome equivalents of Ad12 DNA, i.e., an integrate of foreign DNA measuring 1 to 2 megabase pairs in total length, have been inserted into the established hamster cell genome. The addition of such a large segment of foreign DNA could well lead to structural rearrangements in the cellular genome. The extent and nature of such structural chromatin changes are unknown but analyses on alterations of DNA methylation patterns might be a useful experimental indicator.

10.3.2 Integration of Ad12 DNA or bacteriophage h DNA into the hamster genome: consequences for the stability of the targeted genome The insertion of Ad12 DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments (Fig. 29, see color plates; Fig. 31, Table 9). Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these changes have played a role in the generation or the maintenance of the Ad12-transformed phenotype. We have also isolated clonal BHK21 hamster cell lines that carry in their genomes bacteriophage h and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copies of integrated h DNA, which often colocalize with the PSV2neo DNA, usually at a single chromosomal site as determined by the FISH technique (Fig. 30, see color plates). In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage h DNA frequently becomes de novo methylated much like Ad12 DNA or any other foreign DNA. In some of the thus generated hamster cell lines, the levels of DNA methylation in the retrotransposon genomes of the endogenous intracisternal A particles (IAP) are increased in comparison to the non-h-DNA-transgenic BHK21 cell lines. These changes in the methylation patterns of the IAPI-segment have been documented by restriction analyses using methylation-sensitive endonucleases followed by Southern transfer hybridization and phospho imager quantitation. The results of genomic sequencing experiments using the bisulfite protocol yielded additional evidence for alterations in the patterns of DNA methylation in selected segments of the IAP sequences. In these experiments, the nucleotide sequences in >330 PCRgenerated cloned DNA molecules were determined (Fig. 31, Table 9).

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10 Consequences of foreign D N A integration and persistence

The possibility existed that the hamster BHK21 cell genomes represented mosaics with respect to DNA methylation in the IAPI segment. Hence, some of the cells with altered methylation patterns observed after h DNA integration might have existed prior to h DNA integration and been selected by chance. A total of 66 individual BHK21 cell clones from the BHK21 cell stock were recloned up to three times, and the DNAs of these cell populations were analyzed for differences in IAPI methylation patterns. None were found. These patterns were identical among the individual BHK21 cell clones and identical to the patterns of the originally used BHK21 cell line. Similar results were obtained in 9 clones isolated from BHK21 cells mock-transfected by the Ca2+phosphate precipitation procedure omitting DNA from the transfection mixture. In four clonal sublines of BHK21 cells, genomic sequencing by the bisulfite protocol of 33.5 PCR-generated clones revealed S’-CG-3’ methylation levels in the IAPI segment that were comparable to those of the uncloned BHK21 cell line (Table 7). We conclude that the observed changes in the DNA methylation patterns in BHK21 cells with integrated h DNA are unlikely to pre-exist or to be caused by the transfection procedure. Our data support the interpretation that the insertion of foreign DNA into a pre-existing mammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure.

Figure 31. (a) Alterations in DNA methylation at 28 5’-CG-3’ sites in the p3 and p4 segment (see b) of the IAPI retrotransposon sequence in the Ad12-transformed hamster cell line T637 and in two h DNA-transgenic BHK21 hamster cell lines, BHK21-h7 and A10. The numbering of the 5’-CG-3’ dinucleotides corresponds to that in Table 9. During the genomic sequencing experiments, 123 DNA clones from the reference BK21 cell line, 79 DNA clones from the Adl2-transformed T637 cell line, 58 DNA clones from the BHK21-h7 cell line and 73 clones from the BHK21-210 cell line were sequenced. The percentage values represent the average of methylated 5’-CG-3’ dinucleotides at each site for DNA from each of the cell lines. In many of the clones, 5’-CG-3’ positions 23 to 27 were in a deleted segment as part of a naturally occurring polymorphism in this region compared to the published nucleotide sequence. Furthermore, 5’-CG-3’ positions 19 and 35 were altered to 5’-TG-3’ in many clones and were therefore omitted from this analysis (Remus et al., 1999). (b) Methylation analyses of the IAPI segments in the BHK21 and T637 cell lines and in the h DNA-transgenic clonal BHK21 cell lines BHK2Lh7 and BHK21-hlO by using the bisulfite protocol of the genomic sequencing procedure. A map of subclone I in the IAP retrotransposon in hamster cells is shown. The locations of the primers used in the amplification step of the genomic sequencing procedure following the bisulfite reaction with clonal DNAs are designated with horizontal arrows. Several segments were genomically sequenced. The data shown in Tables 7 and 9 were derived from the primer p3-p4-flanked subsegment of the IAPI region, which contains 28 5’-CG-3‘ dinucleotide sequences (vertical lines) in the BHK21 cells used in our experiments (35 sites in the published sequence). HpaII (stars) and HhaI (circles) sites are indicated. Numbers refer to the published nucleotide sequence. (Remus et al., 1999).

10.3 The insertion offoreign DNA into an established mammalian genome

137

10.3.3 Alterations in cellular DNA methylation patterns A series of studies has been performed to determine whether changes in DNA methylation paterns of cellular genes and DNA segments observed in Ad12transformed hamster cells are due to the transformed state of the cells, to the insertion of foreign DNA into an established mammalian genome, to a combination of both events or to pre-existing mosaics in the methylation patterns in the cellular IAP segments. The data obtained support the interpretation that the integration of non-transcribed bacteriophage h DNA in the hamster genome is assoa

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10 Consequences offoreign DNA integration and persistence

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Table 9. Normalized levels of DNA methylation at individual 5’-CG-3’ dinucleotides’

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ciated with changes of DNA methylation in some of the clonal transgenic BHK21 cell lines, notably in the IAPI retrotransposon sequences. Other hitherto undetected segments of the genome may also be affected. It is conceivable that the repetitive IAP sequences with about 900 copies per genome, which have been integrated into the hamster genome probably several million years ago, may be particularly susceptible to alterations in DNA methylation patterns subsequent to the insertion of foreign DNA (Fig. 32). Moreover, the sites and the extent of changes in methylation patterns of cellular DNA segments may depend on the

10.3 The insertion of foreign D N A into an established mammalian genome

139

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Figure 32. Foreign DNA is integrated into an existing mammalian genome and causes perturbations in the organization of chromatin. Hypothetical scheme. Explanation of symbols: +trans effects on DNA methylation; chromatin perturbations; ? unmethylated,T methylated 5’-CG-3’ dinucleotide.

140

10 Con~eqiienceso j foreign DNA integration and persistence

size and location of the foreign DNA integrates. In this respect, there could be a hierarchy of endogenous sequences ranging from ancient segments of cellular DNA via retrotransposons added a few million years ago, as compared to more recently integrated foreign DNA within the last decades, as in our examples of Ad12 DNA or bacteriophage h DNA. Alterations in DNA methylation can be most convincingly documented with a method that permits the analysis of each individual 5’-CG-3’ dincleotide in a sequence, e.g., by the bisulfite protocol of the genomic sequencing technique. The data in Table 9 have been adduced from genomic sequencing studies extending over a DNA stretch of 522 nucleotide pairs and encompassing 28 5’-CG-3’ dinucleotides in the IAPI DNA region. These results are based on the genomic sequencing of over 330 individual DNA molecules and demonstrate that alterations in DNA methylation patterns in cellular DNA segments upon the integration of foreign DNA can be documented by applying the genomic sequencing technique. This result has been strengthened by the genomic sequencing data adduced from the DNA of four clonal sublines of non-transgenic BHK21 cell lines. The levels of DNA methylation in the same IAPI segment in these sublines varied to some extent but were close to those in the original BHK21 cell line (Table 7). At the sites of foreign DNA integration in the BHK21-h cell lines, h DNA colocalizes with pSV2neo DNA, ususally at one chromosomal insertion site. Since these cell lines have been selected for resistance against G418, the pSV2neo DNA has to be transcribed in these cells only during the selection period, whereas h DNA transcription has never been detected in the transgenic cell lines tested. When considering possible mechanisms by which foreign DNA integration may lead to alterations of cellular DNA methylation patterns and possibly of chromatin structure, at least transiently, the transcriptional activity in the pSV2neo part of the transgenic DNA might be debated. However, by the time that cellular methylation patterns were determined, (3418 selection had been discontinued. Moreover, pSV2neo-specific signals could not be detected by RNA blot experiments. More sensitive methods might detect some transcripts. The infection of BHK21 cells with Ad12 and the concomitant transcription of early Ad12 genes does not elicit detectable changes in cellular DNA methylation patterns. Minor changes in a subpopulation of the cells cannot be ruled out. In the BHK21.Ad12 system, there is a complete block of Ad12 DNA replication. Arrays of newly synthesized Ad12 DNA thus cannot be formed. Moreover, it is unlikely that the transfection protocol by itself is capable of altering cellular DNA methylation patterns. It is impossible to rule out categorically the existence of mosaics in IAP methylation patterns in a small subset of the BHK21 hamster cells. However, in a total of 75 isolated subclonal cell lines we have investigated, uniform and completely stable methylation patterns have been observed in the IAPI segments, whereas among the h DNA-transgenic cell lines about 13 out of 77 exhibited changes in the methylation of the investigated IAPI segment. Genomic sequencing in the same IAPI segment has not revealed major differences in DNA methylation in four BHK21 DNA subclones (Table 7 ). Hence, two lines of

10.3 The insertion of foreign DNA into an established mammalian genome

141

evidence derived from several sublones of BHK21 cells - restriction patterns with HpaII and HhaI (not shown) and genomic sequencing data (Table 7) - argue against the existence of mosaics with respect to methylation in the IAPI DNA segment. Therefore, I favor the interpretation that the alterations observed in the methylation patterns of the IAPI segments upon integration of bacteriophage h DNA have been induced by the integration of foreign DNA. In the Adl2-transformed hamster cell lines, notably in the cell line T637, which has been directly derived from BHK21 cells by Ad12 transformation, the increases in DNA methylation patterns have been more pronounced, probably because of the transformed phenotype of these cells. These increases in DNA methylation are stable in the TR3 revertant of the Adl2-transformed cell line T637 which has lost all of the multiple copies of integrated Ad12 DNA, as detected by Southern blotting. There is evidence from several lines of studies that the transformed or oncogenic phenotype of mammalian cells is frequently associated with alterations in cellular DNA methylation patterns. Upon freezing and thawing and long term cultivation of the h DNA-transgenic BHK21 cell lines, the alterations in IAP DNA methylation patterns are no longer apparent. The cells with altered patterns might have selective growth disdvantages and thus disappear from the cell population. The integration of h DNA into the hamster cell genome is not known to transform cells to the oncogenic phenotype. However, it is conceivable that specific integration events with the concomitant alterations in cellular DNA methylation and expression patterns are, however rarely, capable of eliciting the oncogenic transformation events.

10.3.4 Foreign DNA integration and cellular chromatin structure We have set out to investigate whether foreign DNA insertion into an established hamster cell genome can be related to detectable structural changes in cellular chromatin. Under these premises, different cellular DNA segments might be exposed more directly t o the cellular DNA methyltransferase systems from which they are protected prior to the insertion of foreign DNA. Since both after viral infection and after the transfection of foreign DNA, multiple copies of the foreign DNA can become inserted into the recipient genome, major structural perturbations due to the addition of large DNA blocks may ensue. Preliminary data are consistent with this interpretation but need further refinement. Since chromosomes have a unique topological relationship among each other in the interphase nucleus, structural perturbations caused by the insertion of foreign DNA at one site might be transmitted in a site-specific manner to those parts of neighboring chromosomes that lie in the immediate vicinity. Depending on the sites of foreign DNA insertion, different genomic segments might be subject to changes in methylation patterns, because in the nucleus of a

142

I0 Consequences of foreign DNA integration and persistence

living cell different segments of individual chromosomes have unique and highly specific spatial interchromosomal relationships. Thus by integrating foreign DNA arrays at a given site, specific genome neighborhoods of that site would be primarily affected. Insertions at a different site would affect other regions of the genome (Fig. 32). Probably only a limited number of such alterations in cellular chromatin structure will be compatible with cell survival. This consideration might explain that multiple copies of foreign DNA can be observed at only one site per clonal cell line in most instances. Two integration sites per genome have been detected only rarely. It will also be interesting to investigate to what extent signal transmissions via the nuclear matrix upon insertion of foreign DNA will be able to affect the methyltransferase systems of the cell.

10.3.5 General implications In many experimental procedures, the insertion of foreign DNA into an established mammalian or plant genome has become everyday practice, usually with the goal to express a foreign gene or to eliminate or to restore the function of an endogenous gene. The possibly more far-reaching sequelae of such manipulatons have frequently not been contemplated. Changes in cellular DNA methylation patterns as a consequence of foreign DNA insertion would call for a precise, case by case analysis of alterations in cellular DNA methylation and transcription patterns with the potential for functional consequences. It has been known for almost two decades that DNA methylation and transcription patterns are functionally related. In adenovirus-transformed cells or in Adl2-induced tumor cells, altered transcription patterns in 5 out of 40 investigated, in part randomly selected, cellular DNA segments and genes have been described. The experimental findings reported here can be considered in a wider general context when interpreting experiments in viral oncogenesis or with transgenic organisms. 0

0

0

Studies on the mechanism of viral oncogenic transformation. The process of viral oncogenic transformation might be causally related to changes in chromatin structure and in cellular DNA methylation and transcription patterns. Studies on transgenic organisms. The most obvious interpretations of data gleaned from the experiments with transgenic organisms may not always coincide with reality. The insertion of foreign DNA or of DNA with a “foreign” pattern of methylation in these experiments could influence the activity of many more genes than of the ones directly affected by the “knock-in’’ or the “knock-out’’ procedures. Schemes developed for human somatic gene therapy. The stable insertion of foreign DNA into the human genome could have far-reaching consequences with undesirable side effects which will have to be carefully weighed in all such therapeutic manipulations.

10.4 De novo methylation of foreign DNA - u hypothetical ancient defense mechanism

143

10.4 De novo methylation of foreign DNA a hypothetical ancient defense mechanism The proposals in this chapter are based on experimental work on the integration of foreign DNA in mammalian cells, on the establishment of specific de novo patterns of DNA methylation, and on the inhibition of transcription by the sequence-specific methylation of promoter sequences. It is suggested that eukaryotic cells have developed several mechanisms of defense against the uptake, integration, and continued expression of foreign DNA. In the course of evolution and continuing at present, cells have been exposed to foreign DNA, entire genomes or fragments of them. A particularly problematic organ system in that respect is the digestive tract in higher organisms (see Chapter 11). The defense mechanisms are thought to be the following: 0 0

0

Degradation and/or excretion of foreign DNA. Excision and loss of previously integrated DNA from the host genome. Targeted long-term inactivation of foreign genes by sequence-specific methylation. Genes whose products could be advantageous to the transformed cells might be somehow selectively excluded from this silencing mechanism.

Patterns of DNA methylation are cell type-specific and reflect in a complex way the specific functional requirements of different cell types. At least for humans, a high degree of interindividual concordance of DNA methylation at the nucleotide and the kilobase pair levels has been described in some, but not all, parts of the genome. In other segments of the human genome, patterns of DNA methylation may show individual differences. It is not known whether equivalent genes or DNA segments are methylated in comparable patterns among different species. Most likely there are differences in methylation patterns between unique and repetitive DNA sequences, between exons and introns. In the course of evolution, foreign DNA may have frequently been incorporated and methylated in a certain pattern, e.g., as the consequence of the mentioned defense mechanism. The patterns of DNA methylation, which exist in today’s species, may be the vestiges of evolution and the remnants of multiple methylation, perhaps demethylation and mutation events. Of course, details of these processes can no longer be traced. Methylation patterns are associated with the long-term inactivation of DNA sequences whose function has not been required in, or might even have been harmful to the specific cell type or organism. These methylation and inactivation steps have been different in each cell type dependent on the functional specificities of each cell type. Hence, it would be expected that the existing DNA methylation patterns are cell type-specific. This hypothesis may also explain the finding that not all inactive genes are heavily methylated. Clearly, a number of genes will be inactive in a certain cell type most of the time, but may have to be occasionally activated. In that case, a longterm mechanism of inactivation, as that mediated and finalized by DNA methylation, would be inappropriate, since there are other effective ways of keeping a

144

10 Consequences of foreign D N A integration and persistence

gene silent. These hypotheses leave the question still unanswered, how the DNA methyltransferase system of the host cell can distinguish between DNA sequences that have to be inactivated for a long time and have to be methylated and those whose activity will occasionally be required and which must not be methylated. Of course, a host of DNA binding proteins may have the ability to influence positively or negatively the methylation of DNA. The possibility exists that an established pattern of DNA methylation in a given species can be reevaluated, perhaps even changed during (early) embryonic/fetal development. Demethylation and remethylation events are thought to occur during that phase in an organism’s life, perhaps even in adult life.

10.5 Malignancy - a chromatin disease? Cell death and uncontrolled cell replication that can lead to the development of malignant tumors in living organisms, are among the most actively investigated problems in biology and medicine. A multitude of mechanisms has been discussed and experimentally pursued. Each individual type of tumor disease has its very specific properties, mechanisms of generation and origin. A general principle to explain the generation of all tumors has not evolved and is not to be realistically expected. Similarly, it is unlikely, except perhaps in very rare cases, that changes in a single gene or alterations in the available levels of a single gene product would account for the multitude of changes encountered in tumors. Current notions center rather on mechanisms responsible for the induction of several crucial changes in the transcriptional patterns or in the chromatin structure of mammalian cells that in combination can tip the balance in favor of uncontrolled growth. Alterations in the functions of oncogenes and tumor suppressor genes are interesting indicators along these lines but probably only in rare cases the sole cause for malignant transformation. Cell division and growth are controlled by a multitude of genes. How many of these functions have to be eliminated or altered before these controls are decisively derailed, and the generation of the malignant phenotype ensues? The insertion of viral (foreign) DNA into mammalian genomes might play a decisive role in the generation of the malignant phenotype during viral oncogenesis. Based on the results described on the insertion of different types of foreign DNA into cellular DNA and the possible consequences on the alterations of cellular DNA methylation and transcription patterns, the following scenario is proposed. Depending on the site of insertion of foreign, viral or any other type of DNA, methylation patterns can be altered in specific cellular DNA sequences. In case these changes affected promoter or other regulatory DNA sequences, cellular transcriptional patterns are altered at several or many different sites in the genome thus leading to a reprogramming of the cellular transcriptional program. Possibly at different sites, chromatin structures in the cellular genome could also be grossly affected as a consequence of the insertion of large complexes of for-

10.5 Malignancy - a chromatin disease?

145

eign DNA. This possibility has so far not been unequivocally documented; more experimental work will be required to investigate such changes. After foreign DNA insertion, mechanisms can be set in motion that provide a significantly altered transcription program (Fig. 33). Cells in each organ or tissue might respond with an organ- or tissue-specific mode of rescheduling their transcriptional patterns as a consequence of foreign DNA insertion, perhaps as a reflection of their ontogenetic inactivation patterns during development and differentiation. Depending on the ensemble of changes provoked, a transformed cell type could arise. In rare instances, a similar chain of events might be initiated when food-ingested foreign DNA becomes integrated into the host genome as described in Chapter 11. It remains a matter of conjecture to speculate whether “spontaneously” occurring tumors are related to this type of mechanism. Since our results indicate that foreign DNA is constantly taken up into the organism and can, in rare instances, become integrated into the DNA of various organ sysInsertion, de novo methylation of integrates

1

1 Perturbationof cellular genome at site of insertion

2

Alteration of cellular methylation and transcription in TRANS

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Altered cellular transcription pattern Altered cellular chromatin structure

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Figure 33. Insertion of foreign DNA into an established mammalian genome leads to the alteration of patterns of DNA methylation at remote sites in this cellular genome. As a consequence, patterns of cellular DNA transcription are altered: a novel type of functional insertional mutagenesis.

146

10 Consequences of foreign D N A integration und persistence

tems, the possibility of oncogenic events in the wake of food ingestion over long periods of time has to be considered. Of course, the transformation of individual cells to the oncogenic phenotype in the animal is only the first step towards tumor growth. The constant survey mechanisms by multiple defense systems in a mammalian organism would have to be overcome before a transformed cell succeeds in developing into an invasively growing malignant tumor.

10.6 Further reading Doerfler, W. (1983) DNA methylation and gene activity. Annu. Rev. Biochem. 52, 93-124. Doerfler, W. (1991) Patterns of DNA methylation - evolutionary vestiges of foreign DNA inactivation as a host defense mechanism - A proposal. Biol. Chem. Hoppe-Seyler 372,557-564. Doerfler, W. (1995) The insertion of foreign DNA into mammalian genomes and its consequences: a concept in oncogenesis. Adv. Cancer Rex 66,313-344. Doerfler, W. (1996) A new concept in (adenoviral) oncogenesis: integration of foreign DNA and its consequences. BBA Rev. Cancer 1288, F79-F99. Heller, H., Kammer, C., Wilgenbus, P., Doerfler, W. (1995) Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage h) DNA is associated with enhanced methylation of cellular DNA segments. Proc. Natl. Acud. Sci. USA 92,5515-5519. Hertz, J., Schell, G., Doerfler, W. (1999) Factors affecting de novo methylation of foreign DNA in mouse embryonic stem cells. J. Biol. Chem. 274,24232-24240. Jost, J.P., Saluz, H.P. (1993) DNA Methylation: Molecular Biology and Biological Significance. Birkhhauser Verlag, Basel, Boston, Berlin. Munnes, M., Doerfler, W. (1997) DNA methylation in mammalian genomes: promoter activity and genetic imprinting. Encyclopedia of Human Biology, Vol. 3, Academic Press, pp. 435446. Razin, A., Szyf, M., Kafri, T., Roll, M., Giloh, H., Scarpa, S., Carotti, D., Cantoni, G.L. (1986) Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation. Proc. Nutl. Acad. Sci. USA 83,2827-2831. Remus, R., Kammer, C., Heller, H., Schmitz, B., Schell, G., Doerfler, W. (1999) Insertion of foreign DNA into an established mammalian genome can alter the methylation of cellular DNA sequences J. Virol. 73,1010-1022. Rosahl, T., Doerfler, W. (1992) Alterations in the levels of expression of specific cellular genes in adenovirus-infected and -transformed cells. VirusRex 26,71-90. Sutter, D., Doerfler, W. (1980) Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression. Proc. Natl. Acad. Sci. U S A77,253-256. Sutter, D., Westphal, M., Doerfler, W. (1978) Patterns of integration of viral DNA sequences in the genomes of adenovirus type 12-transformed hamster cells. Cell 14. 569-585.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

11 Uptake of foreign DNA from the environment: the gastrointestinal tract and the placenta as portals of entry 11.1 Summary Foreign DNA is part of our environment. Considerable amounts of foreign DNA of very different origins are constantly ingested with the daily food supply. In a series of experiments, we have fed the DNA of bacteriophage M13 or of the cloned gene for the green fluorescent protein (GFP) from Aeguorea victoria as test DNAs to mice and have shown that fragments of these DNAs survive in small amounts (1-2%) the passage through the gastrointestinal (GI) tract. Foodingested foreign DNA reaches peripheral white blood cells, spleen and liver via the intestinal epithelia and cells in the Peyer’s patches of the intestinal wall. Food-ingested foreign DNA can become covalently linked to DNA with 7 0 4 0 % homology to known mouse genes, probably to pseudogenes. When M13 DNA is fed to pregnant mice, the test DNA can be detected in cells in various organs of the fetuses and of newborn animals, but never in all cells of the mouse fetus. The test DNA has also been documented in association with both chromatids of chromosomes in fetal cells. This finding also supports the notion that food-ingested foreign DNA can be integrated into the host genome. It is likely that the foreign DNA is transferred by the transplacental route and not via the germ line. The consequences of foreign DNA uptake for mutagenesis and oncogenesis have not yet been investigated.

11.2 Foreign DNA is abundant in our environment For millions of years, the exterior and interior environments, i.e., the gastrointestinal tract of all organisms have been exposed to large amounts of foreign DNA. Table 1 lists frequent sources of foreign DNA in the natural surroundings of all organisms. Since exact data are not available, the amounts of DNA presented are estimates. The fate of foreign DNA in the ecosystem has so far not been investigated, although it has been shown that the DNA molecule is very stable and can persist in fragmented form in organisms, e.g., in Egyptian mummies for thousands of years. DNA fragments with free ends are known to be highly recombinogenic. Upon uptake into cells, these fragments are therefore likely to recombine with genomic DNA. The genomes of all species have evolved over long time periods to the status existing today. It is an oversimplification to assume that genomes, like that of humans, encompassing 3 x lo9 to 4 x lo9 nucleotide pairs with an estimated num-

148

11 Uptake of foreign DNA from the environment

ber of some lo5 genes would be stable forever. As all other organisms, humans have to ingest large amounts of foreign DNA with their food supplies and are frequently infected by viruses and microorganisms introducing their foreign genomes in biologically intricate ways and highly active forms. Each fall, the environment is inundated with tons of foreign plant DNA in many parts of the world. For millions, if not billions, of years, the exposure to foreign DNA has been a daily experience for Homo supiens and her/his ancestors. Defense systems attempt to minimize the invasion of foreign organisms. However, little information has been available on whether related or totally different defense systems can also deal with the ingestion of foreign DNA, or whether specific mechanisms exist to counter the influx of foreign genes.

11.3 The epithelia of the gastrointestinal tract are constantly exposed to large amounts of foreign DNA and proteins The surface epithelia of the GI tract are optimized for the contact with and the resorption of molecules that are ingested with the daily food supply and, upon resorption by these epithelia, serve as energy source and/or building blocks for the organism. Macromolecules, as DNA or proteins, may not be completely degraded to nucleotides or amino acids, respectively. Thus, there is an excellent chance that foreign DNA or proteins can be intruding into the organism via the epithelial lining of the gastrointestinal tract by a mechanism termed macromolecular uptake. With a resorptive surface of possibly several hundred square meters, the intestinal surface represents the organ in mammalian organisms with the highest frequency of exposure to foreign genes and proteins. For millions of years, the epithelia of the GI tracts in humans and their ancestors have been exposed to huge amounts of animal and plant tissues and the DNAs contained in and constantly liberated from them by the enzymatic machinery of the GI tract. Thus, the daily caloric and structural replacement requirements of all organisms can be ascertained. For good reasons, the Peyer’s patches in the intestinal wall and the regional lymph nodes in the mesenteria of the intestines belong to the largest lymphatic organs in the mammalian organism. The cellular monolayer systems of the GI tract epithelia are thus functionally protected by the extensive lymphatic apparatus in the intestinal wall and in the mesenteric lymph nodes. We have started to analyze in some detail the genetic consequences of this huge gene technology experiment to which humans, their ancestors and practically all organisms have been naturally exposed for millions of years. As it were, we are all obligatory “cannibals” who are entirely dependent on the consumption of other organisms for their daily food supplies. In the process, all genes occurring in animal or plant tissues are offered to the GI tract in any imaginable random combination. When these DNA molecules are endonucleolytically cleaved,

11.4 Foreign DNA orally ingested by mice

149

highly recombinogenic DNA fragments are generated which are ideally suited for the recombination with the genomes of the host organisms. These possibilities have hitherto escaped careful genetic analyses.

11.4 Foreign DNA orally ingested by mice reaches peripheral white blood cells, spleen and liver via the intestinal epithelia and can be covalently linked to mouse DNA The DNA of bacteriophage M13 has no genetic homology to mouse DNA or to DNA that can be reisolated from the feces of mice. M13 DNA is therefore suited to serve as indicator DNA to follow the fate of foreign, orally ingested DNA in the murine intestinal system. The length of the M13 test DNA used in our experiments measures 7250 nucleotide pairs. Only the double-stranded, circular or EcoRI-linearized form of this DNA has been administered to mice by pipette feeding or by the addition to the food pellets. The following questions have been investigated: 0

0

0

Is the foreign M13 DNA completely degraded to mononucleotides in the GI tract of mice or can M13 DNA fragments be recovered from the GI tract or from the feces? Is M13 DNA taken up with the food supply by the epithelia of the GI tract of mice, and does this DNA reach the organism? In what form does this foreign DNA persist in the mouse organism? Can food-ingested foreign DNA taken up by pregnant animals be transferred to the organisms of the fetus or of newborn animals?

The experiments described here have been performed in the author’s laboratory during the past 12 years by three independently trained doctoral students and have been confirmed by the results of numerous control experiments. The fate of the foreign M13 DNA in the animals was followed by several methods. In 84 animals fragments of M13 DNA were detected in the contents of the small intestine, the cecum (until 18 h), the large intestine, or the blood (Fig. 34). In 254 animals, M13 DNA fragments of up to 976 bp were found in blood 2-8 h after feeding. In buffer-fed control animals, M13 DNA could not be detected. M13 DNA fragments were traced by the polymerase chain reaction (PCR) in peripheral leukocytes and located by fluorescent in situ hybridization in about 1 out of 1000 white cells between 2 and 8 h, and in spleen or liver cells up to 24 h after feeding, but not later. M13 DNA was found by the fluorescent in situ hybridization (FISH) technique in the columnar epithelial cells, in the leukocytes in Peyer’s patches of the cecum wall, in liver cells (Fig. 35, see color plates), and in B cells, T cells as well as macrophages from spleen. These findings suggested

11 Uptake of foreign D N A from the environment

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Figure 34. Quantitation of MI3 foreign DNA detectable by dot blot hybridization in different segments of the intestine (a-c) or in peripheral blood (d).50 pg of M13 DNA (7250 bp) was pipette-fed to 2-6( 12)-month-old mice. As indicated, at times after feeding, the DNA was extracted from the contents of the small intestine (a), the cecum (b), the large intestine (c), or from blood (d). The persisting M13 DNA was identified by dot blot hybridization. Aliquot portions of the extracted DNA were fixed on a positively charged Qiagen nylon membrane and hybridized to "P-labeled M I3 DNA followed by autoradiography. In reconstitution experiments, amounts of M13 DNA ranging from 5 ng to 1 pg were also bound to the membranes. Subsequently, autoradiographically localized dots were excised, and the amounts of membrane-bound 32P label in these spots were determined by Cerenkov counting. Considering the fraction of the total reextracted DNA from gut segments or blood applied to the dot blot matrix, percentages (scales on right) of the 50 pg of orally administered M13 test DNA were calculated (hatched bars). The scales in cpm and the solid bars on the left referred to the actual '*P Cerenkov cpm for reference M13 DNA (Schubbert et al., 1997).

transport of foreign DNA through the intestinal wall and Peyer's patches to peripheral blood leukocytes and into several organs. Upon extended feeding, M13 DNA could be recloned from total spleen DNA into a bacteriophage h vector. Among about 2.5 x lo7 plaques, one plaque was isolated that contained a 1299 nucleotide pair fragment (nt 4736-6034) of sequence-identified M13 DNA. This fragment was covalently linked to an 80 nucleotide long DNA segment with 70% homology to the mouse IgE receptor gene. The DNA from another h plaque also contained mouse DNA, bacterial DNA, and rearranged h DNA. Two additional

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previously termination the in adenovirus M13 4640-5433 Nucleotide 2726-5521 4736-6034 2660-3300 GenBank lambda 3355-5030 with between type sequence method 50 data 2 Fg DASH11 of using DNA integrated coordinates sources M13 a vector in Model DNA integrated foreignonline. the were and 377 in and The its

81 Left Table 70% 100% (80IgEUnknown The The (138 Yo(120 100% Clp (175 sequences 1997). Similar thyroid minute 10. indentified flanking homologyprotease homologyhomology All 9 receptor homology M13 non-homologously nucleotide cases tonucleotides)nucleotides nucleotides) experimental nucleotides) hormone 12 are nucleotide of nucleotide DNA to geneto to geneto nucleotide alisted DNAE. E. mouse mouse fewsequences M31045)coli (82598)(M59317) receptor coli in recovered (U15542) sequence recombined procedures beta sequences sequencea nucleotides were gene2 parentheses. from for SV40of were the compared between these sequenceh Nucleotides and spleen 5‘-GATATAT-3’ unknown determined to of M13 pBR322 therecloning of mice DNA origin 5’-GATTTAAAAGGTGGA-3’ by unknown fed DNA EMBL the and analyses being 24-72 and and chain origin h adjacent for using inserted NCBL

a

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11 Uptake of foreign D N A from the environment

plaques contained M13 DNA fragments of at least 641 (nt 2660-3300) or 794 (nt 4640-5433) nucleotide pairs. More recently, additional h clones were isolated that contained both M13 and mouse DNA. One plaque contained M13 DNA linked to mouse DNA with about 80% homology to mouse DNA (Table 10). The results of these recloning experiments demonstrate that food-ingested foreign DNA cannot only find access to the nuclei of cells in the organism but can, probably in rare instances, become covalently linked to authentic mouse DNA. Linkage as shown here has probably been to mouse pseudogenes. At least for the experiments carried out with mice, the conclusions drawn from these results are unequivocal. Foreign DNA is not completely degraded to mononucleotides in the G I tract of mice. The intestinal epithelia are not a complete barrier against the intrusion of highly recombinogenic DNA fragments. Small amounts of the DNA fragments present in the intestinal tract can reach several organ systems via the intestinal epithelia and can become covalently linked to DNA with a high degree of homology to bona fide mouse genes, probably pseudogenes. This foreign DNA integration appears to be a rare event. The amount of 50 pg of M13 DNA administered is not unrealistic, since it can be calculated that mice ingest milligram quantities of foreign DNA with their daily food supplies. Intentionally naked foreign DNA has been chosen for these experiments, since we have argued that survival of DNA in this unprotected configuration renders the persistence of intracellular DNA in various food materials even more likely. Of course, it is conceivable that food-ingested naked DNA might become complexed with proteins during the passage through the intestinal tract and will be taken up in this configuration by the intestinal wall cells. More recently, a plant-specific gene - the 5-ribulose-biphosphate-carboxylase gene - has been followed in mice fed with soybean leaves. This gene of the light cycle is absent from the mammalian genome. The data indicate that this gene can be traced in the contents of different parts of the G I tract. Fragments of this gene have been found in spleen and liver. Thus intranuclear genomic DNA from plants also seems to gain access to the mouse organism (U. Hohlweg and W. Doerfler, unpublished results).

11.5 The fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus As discussed in the previous section we have shown that, when administered orally to mice, bacteriophage M13 DNA, as a paradigm for foreign DNA without homology to the mouse genome, can persist in fragmented form in the G I tract, penetrate the intestinal wall, and reach the nuclei of leukocytes, spleen and liver cells. Similar results were obtained when a plasmid containing the gene for the green fluorescent protein (pEGFP-C1) was fed to mice (Fig. 36). In spleen the

11.5 The fate of orally ingested foreign DNA in mice ...

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Figure 36. (a) The intact CMV promoter-GFP gene construct can still be detected by PCR as a 1277411product in DNA from liver, spleen and kidney of mice that had been fed with the construct 3-8 h previously. (b) The map of the construct and the locations of primer oligodeoxyribonucleotides used for PCR are indicated (Schubbert et a]., 1998).

foreign DNA was detected in covalent linkage to DNA with a high degree of homology to mouse genes, perhaps pseudogenes, or to authentic E. coli DNA (Table 10). The lengths of the recloned M13 DNA sequences in these isolates measure between 0.6 and 2.8 bp and would therefore be detectable by the FISH method at the individual cell level. We have extended these studies to the offspring of mice that were fed regularly during pregnancy with a daily dose of 50 pg of M13 or pEGFP-C1 DNA. Using the polymerase chain reaction (PCR) or the FISH method, foreign DNA, orally ingested by pregnant mice, can be discovered in various organs of fetuses and of newborn animals. The M13 DNA fragments have a length of about 830 bp. In various organs of the mouse fetus, clusters of cells contain foreign DNA as revealed by FISH (Fig. 37). The foreign DNA is invariably located in the nuclei. We have never found all cells of the fetus to be transgenic for the foreign DNA. This distribution pattern argues for a transplacental pathway rather than for germline transmission which might be expected only after long-time feeding regimens. In rare cells of three different fetuses, whose mothers have been fed with foreign DNA during gestation, the foreign DNA was detected by FISH in association with both chromatids (Fig 37g). This finding also argues for the integrated state of the foreign DNA. Is maternally ingested foreign DNA a potential mutagen for the developing fetus?

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11 U pfake of foreign DNA from the environment

The ingested foreign DNA can thus transgress not only the intestinal but also the placental barrier and reach the nuclei of cells of fetuses or newborns from female mice that have been fed the test DNA daily for longer periods during pregnancy. FISH (Fig. 37, see color plates) and PCR data (Fig. 36) reveal the presence of the test DNA in the nuclei of clusters of cells in the fetus or in newborn mice. Some of this DNA is associated with chromosomes and is probably integrated into host DNA. The length of the persisting M13 DNA fragments extends to maximally 830 bp. The patterns of distribution of foreign DNA molecules in the offspring organisms are consistent with transplacental transfer. Germline transmission would probably have been difficult to detect under the experimental regimen chosen. The intimate association of the foreign test DNA with both chromatids in cells of the offspring (Fig. 37g) and the long-term persistence of foreign DNA in newborn animals for up to 3 months after birth further support a mode of persistence by genomic integration and raise novel questions about the genetic effects that this intrusion might have on the fetus. It will be a challenging task to investigate the possible consequences of foreign DNA insertion into the genomes of the recipient organisms.

11.6 Possible functional consequences The uptake of foreign DNA via the GI tract can be interpreted as an ancient mechanism. The consequences of the ingestion of considerable amounts of foreign DNA for the mammalian organism have not yet been investigated and will have to be evaluated with caution. Realistically, the constant uptake of foreign genes opens practically unlimited recombinatorial possibilities of foreign DNA fragments in the host organism. Mutagenic or oncogenic consequences for individual cells that have served as targets for the foreign DNA fragments are probably rare but these possibilities cannot be excluded. The exposure of organisms to foreign DNA fragments in the GI tract is probably more frequent than the infection with foreign genes via viral or microbial vectors. Viruses and microorganisms have evolved highly specialized and effective mechanisms to introduce their genomes into specific cells of the human organism. For DNA fragments, on the other hand, even upon the formation of complexes with proteins in the gastrointestinal tract, access to the nuclei of specific human cells may be much harder to obtain. It is tempting to argue that only tumor viruses would be in a position to introduce specific “transforming genes” like oncogenes into the genomes of mammalian cells and to contribute thus to the oncogenic transformation of cells. However, with the large amounts of foreign DNA that are constantly ingested by all organisms including humans, fragments of all possible genes potentially involved in the oncogenic process reach the organism via the GI tract. Thus there are no convincing arguments that would render viral infections more likely candidates for mutagenic or oncogenic events in an organism than the ingestion of foreign DNA fragments.

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Defense mechanisms of the organism against the attacks by foreign DNA have not been sufficiently investigated. Apparently antibodies against DNA can be raised. The observations that food-ingested DNA can frequently be detected in peripheral white blood cells or in spleen cells, and that the foreign DNA can be retrieved only with difficulties later than 24 h after administering it, suggest the existence of efficient mechanisms for the elimination of foreign DNA from the mammalian organism. It has been postulated that the de novo methylation of integrated foreign DNA by the DNA methyltransferase systems of the host constituted an ancient defense mechanism against long-term foreign gene activities that have intruded into an established genome (see Section 10.4).

11.7 Concerns for and fears of foreign DNA by the public With few exceptions, the general public is not well informed about the most elementary facts of molecular biology and genetics, even less about the natural occurrence of foreign DNA in our environment. Effective campaigns to inform the public will be paramount to improve the acceptance of biotechnology or gene therapy programs, some of which will be of great value for medicine and agriculture in the very near future. For millions of years, humans and their ancestors have been exposed to innumerable combinations of genes ubiquitous in our food derived from plants and animals. In view of these facts, the arguments of some of the opponents of gene technology must appear misplaced. To alleviate apprehensions in the public, it would be cogent to designate “manipulated” products as such, although the practical implementation might be difficult. In the long run, scientists will have to consider it one of their foremost obligations to the public to teach modern biology in a way that people without training in science can giean the necessary information. This will not be an easy task, mainly because the concept of adult education is still in its infancy. There is, however, an excellent chance for those at school age if education in biology were to be made obligatory for all types and grades of schools. An understanding of the elementary concepts in modern biology should be rated as essential for the general public as literacy and the basic rules of arithmetic. With that background information, everyone could then realistically judge biological and medical problems that concern all of us daily at an ever increasing rate. The observation that food-ingested foreign DNA is not completely degraded to mononucleotides in the GI tract and gains access to different cell systems in the mammalian organism and even penetrates the placental barrier has prompted positive and sceptical interpretations: 1. On the scientifically plausible side, it can be argued that the uptake of foodassociated DNA and exposure of all organisms to DNA from their environment is a fact of life and has been daily experience for millions of years. Randomly gen-

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erated fragments of any gene or of all repetitive sequences from plant or animal sources have been available for uptake by the GI tracts of mammals and had the chance for integrative recombination with the mammalian genome for long periods of time during evolution. It is unknown whether these mechanisms have played a role in the evolution of the mammalian genome. Since these events have been a reality for millennia and cannot be altered by scientific interference, it is unrealistic to portray threatening scenarios about the uptake of “natural” or “manipulated” food. If one wanted to derive nutritional recommendations from these results, one would advise against the uptake of food rich in nuclei and DNA, e.g., of parenchymatous organs like liver etc., in particular during pregnancy. Unfortunately precise quantitations of DNA in the main nutritional staples are not available. Such data will be important to obtain before nutritional schemes can be formulated that are based on realistic genetic concepts. 2. Skeptical interpretations attempt to turn this argument around. Uptake of foreign DNA from the G I tract and the uncontrollable integration of foreign DNA fragments into the recipients’ genome and even transfer into the fetal genome are portrayed as highly alarming. Presumably, this chain of events, although logically it has to be accepted, is exploited by anti-genetic dilletants and crusaders skillfully fusing these, to the lay public, unpleasant thoughts with their ideologically motivated reasoning against genetically manipulated plants and food. Of course, it requires a public that is susceptible to ideological seduction. Unfortunately, in the 80s and 90s of the passing century, a tiny political minority numerable enough to gain political influence and trained in ideological agitation has caused considerable damage. The consequences of poorly-advised decisions taken in politics and industry will be painfully felt in the development of medicine and the labor market of the future in Germany. Realistically and fortunately, such minority views have been ignored and not even noticed world-wide. The concepts and techniques of molecular genetics have penetrated and irreversibly altered all fields of biological research, medicine, agriculture and nutrition.

11.8 Further reading Mathiowitz, E., Jacob, J.S., Jong, Y.S.,Carino, G.P., Chickering, D.E., Chaturvedi, P., Sanctos, C.A., Vijayaraghavan, K., Montgomery, S., Bassettt, M., Morrell, C., (1997) Biologically erodable microspheres as potential oral drug delivery system. Nature 386,410-414. Paabo, S., Gifford, J.A., Wilson, A.C. (1988) Mitochondria1 DNA sequences from a 7000 year old brain. Nucleic Acids Rex 16,9775-9787. Schubbert, R., Lettmann, C., Doerfler, W. (1994) Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice. Mol. Gen. Genet. 242,495-504. Schubbert, R., Renz, D., Schmitz, B., Doerfler, W. (1997) Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen and liver via the intes-

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tinal wall mucosa and can be covalently linked to mouse DNA. Proc. Natl. Acad. Sci. U S A 94,961-966. Schubbert, R., Hohlweg, U., Renz, D., Doerfler, W. (1998) On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus. Mol. Gen. Genet. 259,569-576. Tsukamoto, M., Ochiya, T., Yoshida, S., Sugimura, T., Terada, M. (1995) Gene transfer and expression in progeny after intravenous DNA injection into pregnant mice. Nature Genetics 9,243-248.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

12 Relevance in applied molecular biology: an overview For many applications of the concepts and techniques of molecular genetics, the fixation of foreign DNA in mammalian cells and their continued expression are of considerable interest. The replacement of defective genes or the addition of novel genes, e.g., in tumor therapy are the essential goals of gene therapy. Most of the basic problems emanating from the realization of these goals have not yet been solved and will necessitate more basic research. A t the time of this writing, none of the proposed regimens of gene therapy has proved successful when critically evaluated. Although the fixation and expression of foreign genes in many transgenic organisms has been accomplished by heterologous or homologous recombination after rigorous selection, the long-term consequences of these genetic alterations have hardly been investigated and have often not even been considered. The visions of gene therapy cannot become reality without the appreciation of many problems and basic research towards acceptable solutions.

12.1 Gene transfer to mammalian cells via artificial chromosomes The decisive structural and functional requirements for the stability and activity of mammalian chromosomes are only partly understood. Research on artificial chromosomes, therefore, promises benefits for both a better appreciation of elements in chromosome structure and function and the development of improved techniques for the transfer of foreign genes into mammalian cells. The latter aspect pertains to goals in experimental work on the function of foreign genes in mammalian cells and in human somatic gene therapy as well as the generation of transgenic cells and organisms. A number of sequence requirements have to be fulfilled in artificial chromosomes to ascertain their mitotic stability, particularly in the absence of selective markers: presence of the authentic telomere sequence and its functional DNA component, the a-satellite centromere sequence, and of an origin of DNA replication in addition to the genomic DNA sequences intended for transfer. For the construction of artificial chromosomes, sequences from yeast or bacterial artificial chromosomes (YACs or BACs) are frequently employed. Transfer to mammalian, frequently human, cells is often accomplished by lipid-mediated transfection. Other transfer methods are under investigation. Mitotic stability of human artificial chromosomes (HACs) in human cells has been reported for at least 100 generations; HACs appear, however, to be less stable in mouse cells. There might be species-specific factors controlling chromosome replication and segregation. By using the FISH technique, DNA sequences

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from HACs have been detected mainly in the HAG, and not - or to a much lesser extent - in any of the endogenous human chromosomes. However, HAC vector integration into the host cell chromosome - obviously at non-specific locations - have also been reported and tentatively been ascribed to problems of the telomeres. Further problems arising in research on HACs derive from epigenetic effects on centromere function, from de novo methylation of the transferred sequences, a little researched aspect, and from the formation of dicentric chromosomes. Obviously, there is an interesting field of investigation which holds promise also for more “natural” ways of foreign gene transfer in human somatic gene therapy.

12.2 Human somatic gene therapy Within the framework of this book, it cannot be intended to give a full-fledged account of all the possible approaches and goals of human somatic gene therapy. There are basically two aims: (i) The replacement of defective genes in individual organs in human genetic disease. (ii) The introduction of genes or genetic elements into tumor cells or into tumors in the “hope” to forestall further tumor growth or to activate host defense mechanisms against tumor spread and invasion. In spite of considerable investments of time, energy and ingenuity, not to speak of financial resources, current concepts and techniques have not yet spawned realistically workable approaches. Harold Varmus’ committee, the Matulski-Orkin report of 1995, and their recommendations continue to be highly relevant: Back to basic research to develop novel schemes for gene transfer and fixation under conditions of continued gene activity. Viruses may not be the ideal vector systems, although we can learn from viruses how to enter a cell and how to launch foreign genes into the chromosomes of the cells. Although one can easily understand that clinicians are eagerly awaiting new methods, we may serve the clinicians and their patients best by cautiously developing sensible tools. Overly optimistic predictions raise unwarranted hopes and can only hurt the burgeoning field of human somatic gene therapy. Retroviral mediated gene transfer offers the advantage to transduce a large portion of the target cells. Ex vivo gene transfer experiments have frequently utilized these vectors. There are however limitations. As discussed in Section 7.4, each retrovirus requires a specific receptor to enter the cell. In particular, hematopoietic stem cells may lack the appropriate viral receptor. Moreover, the transduced cells have to divide for proviral integration to proceed. Another problem derives from the fact that retroviral genomes integrate at many different cellular sites with the possible consequences discussed in Chapter 10. Thus retroviral vectors may have their realms of application, however, many unresolved problems remain: 0 Choice of the appropriate vector that can transfer the foreign gene constructs into all cells of an organ or of a tumor. Ideally, the vector should not elicit an

12.2 Human somatic gene therapy

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antibody response. Hence viruses may be of limited value as vectors or may have to be reconstructed in their protein coats. There are non-viral vectors which are often based on lipid formulations containing the foreign DNA-promoter constructs. High frequency DNA transfer has been reported in some of these systems. Fixation of the foreign DNA in the cellular genome, without the inserted genes being recognized as foreign and subjected to de n o w methylation and inactivation. The integration of foreign DNA in the human genome might lead locally to insertional mutagenesis or to trans effects of perturbations in the genome; e.g., alterations in the methylation and transcriptional patterns of cellular genes could be a problem. In case the vector and the therapeutic genes persisted in a non-integrated episoma1 state, long-term expression and gene regulation must be ascertained.

Adenoviruses as obligatory parasites and gene packages with long-standing experience in the mammalian and human organisms have also been developed as possible vectors for human somatic gene therapy. The activation of all late adenoviral gene functions, and hence the replication of adenoviruses, is dependent on the functions encoded by the adenoviral E l region (Fig. 12). The human cell line 293 carries the adenoviral El region integrated into the cellular genome and expresses the E l functions constitutively. This cell line permits the replication of E l deletion mutants of adenoviruses. Such deletion mutants of the virion can be reconstructed to carry foreign genes intended for gene transfer in human somatic gene therapy. Such an adenovirus construct has been used in 1994 to transfer the cystic fibrosis transmembrane regulator (CFTR) gene in a spray formulation to the bronchial system of cystic fibrosis (CF) patients. CF, a frequent and severe human genetic disease, is caused by mutations in the CFTR gene. After the application of the recombinant CFTR-adenovirus, the biochemical properties of the bronchial secretions of CF patients improved, viscosity decreased, however, the adenovirion proved toxic to the bronchial epithelia. Moreover, antibodies against adenoviruses increased dramatically. First clinical trials had therefore to be discontinued. Another segment of the adenovirus genome that can be replaced by foreign genes is the E3 region (Fig. 12), because the viral genes therein encoded are not required for viral replication in cell culture. In Chapter 4.5 the naturally occurring Adl2-cellular DNA recombinant SYREC has been described. On either genome terminus it carries the left terminal 2081 nucleotides of Ad12 DNA and in the center a huge palindrome of cellular DNA encompassing some 30 kb. Replication of SYREC is helper adenovirus dependent. SYREC molecules are packaged into Ad12 virions because the left terminal viral sequences provide the essential, &-acting, packaging signal. The construction of the third generation of adenoviral vectors is based on this observation of basic research in virology. These new adenovirus vector molecules consist of the viral DNA termini only and large segments of human genomic DNA, e.g., the human gene for dystrophin to treat the Duchenne or the Becker

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form of muscular dystrophy. Another large human gene that has been included is that of a-antitrypsin. In the genome of the adenovirus helper used in these experimente the packaging signal has been deleted. Hence, in a mixed transfection of human cells in culture, the helper genome provides all the viral capsid proteins. The helper genome itself, however, cannot be packaged and only the recombinant adenovirus genome becomes encapsidated. The vector virus can be further purified by equilibrium centrifugation in CsCl density gradients, such that the final vector virus preparation is 99.9% pure. Many important questions remain: Does the vector molecule integrate? What is the toxicity of these vector preparations? Is an antigenic response elicited? The idea that subviral particles may efficiently transfer the viral genome to the nucleus without eliciting toxicity and antigenic reactions also deserves further study. The future will hopefully witness a multitude of approaches from many different laboratories employing viral and non-viral vector systems.

12.3 Injection of promoter-fused gene constructs into animals and DNA vaccines The intramuscular injection of a cloned influenza virus gene under promoter control has elicited a remarkable antibody response against influenza virus in experimental animals. Since DNA with the nucleotide sequence for a viral gene product, and not the protein, had been applied, the term DNA vaccines was created. In the meantime, a considerable number of experiments was performed and presently human trial vaccinations against influenza virus, malaria, colon cancer, hepatitis B virus, herpes virus, HIV, cutaneous T cell lymphoma and others were initiated. It is still too early to evaluate the eventual success of this new vaccination approach. However, it was a challenge to hitherto accepted principles that the injection of foreign DNA with the encoded gene under the control of the strong immediate early cytomegalovirus promoter permitted to circumvent the injection of the antigen itself. Usually intramuscular injection has been used, but subcutaneous and other loci of injection also seem possible. It is not clear yet whether the inclusion of the coding nucleotide sequence into a bacterial plasmid could be essential for vaccination efficacy against the encoded protein. There is evidence that certain CG-rich bacterial DNA sequences might have an adjuvant effect in vaccinations by stimulating the immune response through an as yet unidentified mechanism. The mode of action of DNA vaccines is essentially unknown. State and fate of the injected DNA constructs have not been investigated in sufficient detail. Perhaps the ubiquitous dendritic cells in the organism might take up the DNA and serve as the first target in the immune response, but other cell types could also play a major role. What loci do the applied DNA constructs have to reach, what conformation to assume in order to evoke the antibody response? Does the

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injected DNA have to become integrated? Does the foreign DNA primarily reach the immune system? How quickly is the injected DNA degraded? Many questions for fundamental research remain. The answers to these questions will be decisive in order to develop this experimental approach into a functioning vaccination regimen. It is rewarding to note that the invention of a possibly new mode of vaccination again emanated from studies on viral systems, in this case the influenza virus, as so frequently happened in molecular biology. In following the fate of foreign DNA in the GI tract of mice (see Chapter 11), uptake of fragments of this DNA via the intestinal epithelia and intrusion into various organ systems was demonstrated. Obviously, the G I tract, with possibly the largest immune system in the mammalian organism, would be an excellent site for the application of DNA vaccines and their presentation to the immune system. In practical terms, the problem of degradation of DNA would have to be solved by including the DNA into an appropriate carrier formulation before this path of application could become efficient for routine vaccinations. Via the intestinal tract, the DNA would have immediate access to a huge defense repertoire in the Peyer’s patches and the mesenterial lymph nodes. As all organisms have experienced exposure to foreign DNA in their GI systems for millions of years, the defense system might have been well adapted to foreign DNA. Perhaps, the ability of mammals to respond to foreign genes with an antibody response is just part of that ancient defense system against foreign DNA that all organisms have inherited. It is time to question some of the cherished concepts in immunology with foreign proteins and small peptides being the main structures recognizable by the defense systems. The recognition that foreign DNA can persist in the gastrointestinal tract and intrude into various organ systems might also affect our thinking about the function and the targets of the cellular and humoral defense systems.

12.4 Further reading Cotton, M. (199.5) Adenovirus-augmented, receptor-mediated gene delivery and some solutions to the common toxicity problems. Curr. Topics Microbiol. Zmmunol. 199/111,283-295. Crystal, R.G., McElvaney, N.G., Rosenfeld, M.A., Chu, C.S., Mastrangeli, A., Hay, J.G., Brody, S.L., Jaffe, H.A., Eissa, N.T., Danel, C. (1994) Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nature Genetics 8,42-51. Friedman, T. (1996) Human gene therapy - an immature gene, but certainly out of the bottle. Nature Medicine 3, 144-147. Hacker, H., Mischak, H., Miethke, T., Liptay, S., Schmid, R., Sparwasser, T., Heeg, K., Lipford, G.B., Wagner, H. (1998) CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by nonspecific endocytosis and endosomal maturation. EMBO J. 17,6230-6240.

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Haddada, H., Cordier, L., Perricaudet, M. (1995) Gene therapy using adenovirus vectors. Curr. Topics Microbiol. Immunol. 1991111,297-306. Harrington, J.J., Van Bokkelen, G., Mays, R.W., Gustashaw, K., Willard, H.F. (1 997) Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nature Genetics 15,345-355. Henning, K.A., Novotny, E.A., Compton, S.T., Guan, X.Y., Liu, P.P., Ashlock, M.A. (1999) Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and singlecopy DNA sequences. Proc. Natl. Acad. Sci. U S A 96,592-597. Krieg, A.M. (1999) CpG DNA: a novel immunomodulator. Trends Microbiol. 7, 6465. Krieg, A.M., Yi, A.K., Schorr, J., Davis, H.L. (1998) The role of CpG dinucleotides in DNA vaccines. Trends Microbiol. 6,23-27. Marshall, E. (1995) Less hype, more biology needed for gene therapy. Science 270,1751. Mulligan, R.C. (1993) The basic science of gene therapy. Science 260,926-932. Schiedner, G., Morral, N., Parks, R.J., Wu, Y., Koopmans, S.C., Lanston, C., Graham, EL., Beaudet, A.L., Kochanek, S. (1998) Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nature Genetics 18,180-183. Schweighofer, T., Berger, M., Buschle, M., Schmidt, W., Birnstiel, M.L. (1996) Adenovirus-enhanced receptor-mediated transfer infection for the generation of tumor vaccines. Cytokines Mol. Ther. 2,185-191. Taubes, G. (1997) Salvation in a snippet of DNA. Science 278,1711-1714. Willard, H.F. (1998) Centromeres: the missing link in the development of human artificial chromosomes. Curr. Opin. Genet. Dev. 8,219-225.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

13 Future research Directions of future research in molecular biology are impossible to predict. From the results and problems presented in this book a number of research topics are obvious that require further investigations.

13.1 The mechanism of the uptake of foreign DNA by mammalian cells The mechanism of the uptake of foreign DNA by mammalian cells is not understood. What receptors or portals of entry do mammalian cells provide to interact with naked DNA or with DNA that has been complexed with proteins or precipitated by Ca2' phosphate? The pathway through the cytoplasmic membrane, the cytoplasm and through the nuclear membrane into the nucleus need be determined. There are two branches of research that might be pursued. First, how can DNA fragments survive digestion in the GI tract and become incorporated into the nuclei of the intestinal epithelia, and how does food-ingested foreign DNA reach cells in the Peyer's patches. Secondly, detailed work on the interaction of foreign DNA with the cytoplasmic membranes of mammalian cells in culture might open the most sensible first approach towards the study of this problem. Complex formation of DNA with other macromolecules like proteins, lipids or carbohydrates might be an important protection against degradation by DNases and, at the same time, a precondition for uptake by mammalian cells. Hence, it will be interesting to isolate such complexes from the intestinal tract or from cell culture media and to analyze the constituents of these complexes and the kinetics of their formation.

13.2 The fate of foreign DNA upon injection into animals Surprisingly, DNA vaccines are effective after the parenteral administration of a strong promoter-gene construct into animals used to raise antibodies. Presumably, the gene product is synthesized in the animal and subsequently, antibodies are generated by the conventional route. While this suggested pathway appears likely and plausible, it is far from being proven. Many questions about the fate of foreign DNA injected into animals remain. How quickly is foreign DNA degraded and to what extent does the route of application influence the rate of degradation? Is foreign DNA, naked or after complex formation with other macromolecules, taken up by cells of the immune system, or which cell system can preferentially interact with foreign DNA? Do serum proteins bind foreign

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DNA? There is evidence that DNA with a certain 5’-CG-3’ density and distribution, e.g., bacterial DNA sequences, can stimulate the immune system, possibly as part of a defense mechanism against foreign DNA. Upon the methylation of these sequences, the effect on the immune system appears to be eliminated. In preliminary experiments (U. Hohlweg and W. Doerfler, unpublished results) we have shown that foreign DNA injected intramuscularly into mice can be recovered as DNA fragments in different organs and in the intestinal contents of the animals.

13.3 Response against foreign DNA There may be cellular and humoral antibody responses against foreign DNA. Are there serum proteins or peripheral cellular systems that can recognize and dispose of DNA sequences in the serum or in other compartments of the organism? Moreover, upon the integration of viral (foreign) DNA into the host genome, the foreign DNA sequences are efficiently de novo methylated. Again, this mechanism and the mode of selection of sites to be de novo methylated are not understood.

13.4 Mechanisms of foreign DNA integration into the host genome In yeast cells homologous, site-specific recombination is the predominant mode of interaction with transfected DNA molecules. In contrast, in mammalian cells foreign DNA does not recombine at specific, homologous sites with the recipient genome. Although sequence homologies that may exist serendipitously, can be utilized to direct the site of insertion. Foreign DNA inserts at many different chromosomal sites in mammalian cells. The enzymatic tools for insertional recombination are essentially unknown. It is tempting to speculate that the existence of this, perhaps ubiquitous, cellular facility to incorporate foreign DNA reflects “ancient” evolutionary processes. The term “ancient” is probably inadequate, since evolution has not stopped in the 20th century.

13.5 The secrets of de novo methylation Enzymatic mechanism, site of initiation, mode and extent of spreading DNA methylation starting from the locus of initiation, dependence on site of foreign DNA insertion, modulation by chromatin structure, and others will be challen-

13.6 Alterations of methylation in cellular D N A segments upon foreign D N A insertion

167

ging topics for future research. While cell culture systems are problematic to use for these studies, because these mechanisms may be different in comparison to an intact organism, they will facilitate many investigations on a more realistic time scale vis-8-vis transgenic animals. In transgenic animals the additional parameters of genetic environment, cell type and developmental stage may affect de n o w methylation.

13.6 Alterations of methylation in cellular DNA segments upon foreign DNA insertion Perturbations and destabilizations of the recipient cellular genome upon foreign DNA insertion have been studied to a limited extent. A precondition for these investigations would be a detailed study of the structure of chromatin with refined methods. It is likely that the site of foreign DNA integration will affect type and extent of genome alterations.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

14 Studies on the biological significance of DNA methylation by using adenovirus DNA as a model There are numerous examples for the pioneer role of research in the adenovirus system that have led to the discovery of fundamental mechanisms in the molecular biology of mammalian cells. Here I will present a brief summary of the work that my laboratory has contributed to the rapidly evolving field of DNA methylation in the course of more than twenty years.

14.1 De novo methylation of integrated adenovirus DNA As stated in Sections 4 and 9, the DNA extracted from purified virions or the free intranuclear adenovirus DNA in infected cells is devoid of methylation. Upon the insertion into the host chromosomes as in adenovirus-transformed or in Ad12induced tumor cells, the viral DNA becomes de novo methylated. As the mechanism of de novo methylation is still unresolved, the adenoviral system has offered the possibility for further studies on that problem. D e novo methylation is initiated at certain sites on the integrated viral genome and spreads from there in both directions. The same phenomenon is observed when constructs consisting of the E2A late promoter of Ad2 and the chloramphenicol acetyltransferase (CAT) gene as activity indicator are integrated into mammalian cells in culture or into the mouse germ line. In these experiments the extent of de novo methylation varies possibly depending on the site of integration. When the same constructs in the premethylated state were genomically fixed, some of these integrates maintained the methylation patterns previously imposed, others became partly un- or hypomethylated. Since in each experiment the same foreign nucleotide sequence had been integrated, the insertion site and its chromatin structure of different complexity must play a major role in the establishment and/or the maintenance of DNA methylation patterns. The extent of methylation in foreign DNA inserted into the mouse genome at the blastocyst stage can also be influenced by the genetic background of the mouse strain used. Hence, except for the site of DNA integration, a multitude of genetic functions of the recipient cell seem to participate in the formation of unique patterns of DNA methylation.

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14 Studies on the biological significance of DNA methylation by using adenovirus ...

14.2 Inverse correlations between the extent of (promoter) methylation and the state of promoter activity Studies on integrated Ad12 and Ad2 DNA and on specific adenoviral promoters were the first to establish this inverse relationship which was later confirmed for hundreds of mammalian promoters. A particularly convincing case could be made for the late E2A promoter in Ad2-transformed hamster cells which carried partly deleted Ad2 genomes in an integrated state. In the cell line HEI, this promoter is methylated at all 5’-CCGG-3’ sites and at additional 5’-CG-3’ dinucleotides and is inactive. In contrast, in cell lines HE2 and HE3 the same promoter is unmethylated and active. Table 8 in Section 9.4 presents an exemplary, but by no means complete list of mammalian promoters for which such inverse correlations have also been documented.

14.3 Reconstruction experiments: gene transfer and transient or long-term expression of unmethylated or premethylated promoterindicator gene constructs In studies on adenovirus promoters this since then often repeated approach has been initiated. While this type of experiment can obviously not exactly mimic the conditions under which a viral promoter functions in the natural, chromosomally integrated state, valuable information was obtained to assess the role of specificsite promoter methylation in long-term gene inactivation. Details of the adenovirus promoter work can be retrieved from the bibliography in Section 14.5. In one set of early experiments, the E2A late Ad2 promoter-E2A gene construct was either premethylated in three 5’-CCGG-3’ (HpaII) sequences in the promoter or in eleven such sites in the remainder of the gene. Upon microinjection into Xenopus laevis oocytes and measurement of the transcriptional activity of either construct, the promoter-methylated construct was inactive, the gene body-methylated construct, however, retained activity. This transcriptional activity was initiated at the authentic promoter sites. Similar results were obtained with methylated or unmethylated constructs consisting of the Ad2 E2A late promoter and the CAT indicator gene after transfection into mammalian cells in culture. The inhibiting effect could also be demonstrated in a transcription system by using cell-free extracts. Transient expression upon transfection or expression upon genomic fixation of the transfected constructs in mouse blastocysts and eventually in transgenic mice or in mammalian cells in culture yielded similar results. After genomic fixation, problems

14.5 Bibliography: D N A methylation in the adenovirus and related systern.v

171

of de novo methylation or of the stability of a preimposed methylation pattern had to be assessed and complicated the situation in some of the experiments. In a different line of investigations,evidence was adduced that the methylation of 5’-CG-3’ sites in a promoter could alter the structure of DNA as demonstrated by effects on the electrophoretic migration rate in appropriate gel systems.

14.4 Release of the inhibitory effect of promoter methylation by gene products of the adenovirus E l region or by the presence of a strong enhancer from human cytomegalovirus in the construct The inhibition of the Ad2 E2A late promoter by site-specific methylation was partly or completely released under one of the following conditions: (1) coexpression of the E l gene of Ad2 or Ad5 in the cells used for promoter-construct transfection or (2) presence of the strong early gene enhancer of human cytomegalovirus DNA in the construct carrying the methylated E2A late promoter. In either case, structural alterations in the methylated promoter and/or interference with transcription factor binding or its inhibition might account for these reversal effects. The notion of a structural modulation gains credence by the observation that the methylation of all 5’-CG-3’ dinucleotides in the cytomegalovirus enhancer abolishes its positive action on E2A late promoter activity. The construct was then completely inactive.

14.5 Bibliography: DNA methylation in the adenovirus and related systems Achten, S., Behn-Krappa, A., Jucker, M., Sprengel, J., Holker, I., Schmitz, B., Tesch, H., Diehl, V., Doerfler, W. (1991) Patterns of DNA methylation in selected human genes in different Hodgkin’s lymphoma and leukemia cell lines and in normal human lymphocytes. Cancer Res. 51,3702-3709. Dobrzanski, P., Hoeveler, A., Doerfler, W. (1988) Inactivation by sequence-specific methylations of adenovirus promoters in a cell-free system. J. Virol. 62, 3941-3946. Doerfler, W. (1983) DNA methylation and gene activity. Annu. Rev. Biochem. 52, 93-124. Doerfler, W. (1995) The insertion of foreign DNA into mammalian genomes and its consequences: a concept in oncogenesis. Adv. Cancer Res. 66,313-344.

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14 Studies on the biological significance of D N A methylation by using adenovirus .

Doerfler, W. (1996) A new concept in (adenoviral) oncogenesis: integration of foreign DNA and its consequences. B B A Rev. Cancer 1288, F79-F99. Gunthert, U., Schweiger, M., Stupp, M., Doerfler, W. (1976) DNA methylation in adenovirus, adenovirus-transformed cells, and host cells. Proc. Natl. Acad. Sci. U S A73,3923-3927. Heller, H., Kammer, C., Wilgenbus, P., Doerfler, W. (1995) Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage 1)DNA is associated with enhanced methylation of cellular DNA segments. Proc. Natl. Acad. Sci. U S A 92,5515-5519. Hermann, R., Hoeveler, A., Doerfler, W. (1989) Sequence-specific methylation in a downstream region of the late E2A promoter of adenovirus type 2 DNA prevents protein binding. J. Mol. Biol. 210,411-415. Knebel, D., Doerfler, W. (1986) N6-Methyldeoxyadenosine residues at specific sites decrease the activity of the E1A promoter of adenovirus type 12 DNA. J. Mol. Biol, 189,371-375. Knebel-Morsdorf, D., Achten, S., Langner, K.-D., Ruger, R., Fleckenstein, B., Doerfler, W. (1988) Reactivation of the methylation-inhibited late E2A promoter of adenovirus type 2 by a strong enhancer of human cytomegalovirus. Virology 166, 166-174. Knust, B., Bruggemann, U., Doerfler, W. (1989) Reactivation of a methylationsilenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation. J. Virol. 63,3519-3524. Kochanek, S., Toth, M., Dehmel, A., Renz, D., Doerfler, W. (1990) Interindividual concordance of methylation profiles in human genes for tumor necrosis factors a and p. Proc. Natl. Acad. Sci. U S A87,8830-8834. Kochanek, S., Radbruch, A., Tesch, H., Renz, D., Doerfler, W. (1991) DNA methylation profiles in the human genes for tumor necrosis factors a and j3 in subpopulations of leukocytes and in leukemias. Proc. Nutl. Acad. Sci. U S A 88, 5759-5763. Kochanek, S., Renz, D., Doerfler, W. (1993) DNA methylation in the Alu sequences of diploid and haploid primary human cells. EMBO J. 12, 11411151. Koetsier, P.A., Mangel, L., Schmitz, B., Doerfler, W. (1996) Stability of transgene methylation patterns in mice: position effects, strain specificity and cellular mosaicism. Transgen. Res. 5,235-244. Kruczek, I., Doerfler, W. (1982) The unmethylated state of the promoter/leader and 5’-regions of integrated adenovirus genes correlates with gene expression. E M B O J. 1,409-414. Langner, K.-D., Vardimon, L., Renz, D., Doerfler, W. (1984) DNA methylation of three 5‘ C-C-G-G 3’ sites in the promoter and 5’ region inactivates the E2a gene of adenovirus type 2. Proc. Natl. Acad. Sci. U S A 81,2950-2954. Langner, K.-D., Weyer, U., Doerfler, W. (1986) Trans effect of the El region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5’-CCGG-3’ methylation. Proc. Natl. Acad. Sci. U S A 83, 15981602

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Muiznieks, I., Doerfler, W. (1994) The topology of the promoter of RNA polymerase II-and III-transcribed genes is modified by the methylation of 5’-CG-3’ dinucleotides. Nucleic Acids Res. 22,2568-2575. Muiznieks, I., Doerfler, W. (1998) DNA fragments with specific nucleotide sequences in their single-stranded termini exhibit unusual electrophoretic mobilities. Nucleic Acids Res. 26, 1899-1 905. Muller, U., Doerfler, W. (1987) Fixation of unmethylated or the 5’-CCGG-3’ methylated adenovirus late E2A promoter-cat gene construct in the genome of hamster cells: gene expression and stability of methylation patterns. J. Virol. 61,3710-3720. Munnes, M., Schetter, C., Holker, I., Doerfler, W. (1995) A fully 5’-CG-3’ but not a 5’-CCGG-3’ methylated frog virus 3 promoter retains activity. J. Virol. 69, 2240-2247. Munnes, M., Patrone, G., Schmitz, B., Romeo, G., Doerfler, W. (1998) A 5’-CG3’-rich region in the promoter of the transcriptionally frequently silenced R E T protooncogene lacks methylated cytidine residues. Oncogene 17,2573-2584. Orend, G., Kuhlmann, I., Doerfler, W. (1991) Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells. J. Virol. 65,4301-4308. Orend, G., Knoblauch, M., Kammer, C., Tjia, S.T., Schmitz, B., Linkwitz, A., Meyer zu Altenschildesche, G., Maas, J., Doerfler, W. (1995) The initiation of de novo methylation of foreign DNA integrated into a mammalian genome is not exclusively targeted by nucleotide sequence. J. Virol. 69,1226-1242. Remus, R., Kammer, C., Heller, H., Schmitz, B., Schell, G., Doerfler, W. (1999) Insertion of foreign DNA into an established mammalian genome can alter the methylation of cellular DNA sequences. J. Virol. 73,1010-1022. Schumacher, A., Buiting, M., Zeschnigk, M., Doerfler, W., Horsthemke, B. (1998) Methylation analysis of the PWS/AS region does not support an enhancer competition model of genomic imprinting on human chromosome 15. Nature Genet, 19,324-325. Sutter, D., Doerfler, W. (1979) Methylation of integrated viral DNA sequences in hamster cells transformed by adenovirus 12. Cold Spring Harbor Symp. Quant. Biol. 44,565-568. Sutter, D., Doerfler, W. (1980) Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression. Proc. Natl. Acad. Sci. U S A 77,253-256. Sutter, D., Westphal, M., Doerfler, W. (1978) Patterns of integration of viral DNA sequences in the genomes of adenovirus type 12-transformed hamster cells. Cell 14, 569-585. Toth, M., Lichtenberg, U., Doerfler, W. (1989) Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns. Proc. Natl. Acad. Sci. U S A86,3728-3732. Toth, M., Miiller, U., Doerfler, W. (1990) Establishment of de novo methylation patterns. Transcription factor binding and deoxycytidine methylation at CpG

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of

D N A methylation by using adenovirus ..

and non-CpG sequences in an integrated adenovirus promoter. J. Mol. Biol. 214,673-683. Vardimon, L., Neumann, R., Kuhlmann, I., Sutter, D., Doerfler, W. (1980) DNA methylation and viral gene expression in adenovirus-transformed and -infected cells. Nucleic Acids Res. 8, 2461-2473. Vardimon, L., Kuhlmann, I., Doerfler, W., Cedar, H. (1981) Methylation of adenovirus genes in transformed cells and in vitro: influence on the regulation of gene expression? Eur. J. Cell Biol. 25, 13-15. Vardimon, L., Kressmann, A., Cedar, H., Maechler, M., Doerfler, W. (1982) Expression of a cloned adenovirus gene is inhibited by in vitro methylation. Proc. Natl. Acad. Sci. USA 79,1073-1077. Vardimon, L., Gunthert, U., Doerfler W. (1982) In vitro methylation of the BsuRI (5’-GGCC-3’) sites in the E2a region of adenovirus type 2 DNA does not affect expression in Xenopus laevis oocytes. Mol. Cell. Biol.2,1574-1580. Weisshaar, B., Langner, K.-D., Juttermann, R., Muller, U., Zock, C., Klimkait, T., Doerfler, W. (1988) Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by El A (13s) functions. 1. Mol. Biol. 202,255-270. Zeschnigk, M., Schmitz, B., Dittrich, B., Buiting, K., Horsthemke, B., Doerfler, W. (1997) Imprinted segments in the human genome: different DNA methylation patterns in the Prader-WiWAngelrnan syndrome region as determined by the genomic sequencing method. Hum. Mol. Genet. 6,387-395.

Acknowledgments This review is indebted to the work of many former and present associates of my laboratory and to collaborating colleagues who have contributed to these studies between 1966 and 1999: Sabine Achten, Knut Baczko, Annett Behn-Krappa, Dennis Brown, Ute Briiggemann, Harold Burger, Byron Burlingham, Yvette Chardonnet (INSERM Lyon), Renate Deuring, Pawel Dobrzanski, Dirk Eick, Helmut Esche, Ellen Fanning, Katja Fechteler, Reinhold Gahlmann, Jurgen Groneberg, Hilde Heller, Ratf Hermann, Jennifer Hertz, Dagmar Heuss-Neitzel, Kristina Hilger-Eversheim, Monika Hirsch-Kauffmann, Arnd Hoeveler, Urte Hohlweg, Bernhard Horsthemke (Universitatsklinikum, Essen), Rolf Jessberger, Ruth Jiittermann, Keiichi Hosokawa (Kawasaki Medical School, Kurashiki, Japan), Horst Ibelgaufts, Christina Kammer, Albrecht K. Kleinschmidt (New York University, later Ulm University), Thomas Klimkait, Gunther Klotz (Ulm University), Dagmar Knebel-Morsdorf, Margit Knoblauch, Birgit Knust, Stefan Kochanek, Inge Kruczek, Ingrid Kuhlmann, Klaus-Dieter Langner, Reiner Leisten, Clarissa Lettmann, Ursula Lichtenberg, Ulla Lundholm, Knut Muller, Uli Muller, Marc Munnes, Reiner Neumann, Gertraud Orend, Juan Ortin, Anja Pfeffer, Ralph Remus, Doris Renz, Thomas Rosahl, Karl-Heinz Scheidtmann, Gudrun Schell, Christian Schetter, Sabine Schirm, Birgit Schmitz, Jorg Schroer, Rainer Schubbert,

Acknowledgments

175

Manfred Schulz, Beate Siegmann, Jorg Sprengel, Silvia Stabel, Anna StarzinskiPowitz, Diane Sutter, Fuyuhiko Tamanoi (UCLA, Los Angeles), Jorg Tatzelt, Miklos Toth, Lily Vardimon, Bernd Weisshaar, Monika Westphal, Ulrike Weyer, Ulla Wienhues, Petra Wilgenbus, Ute Winterhoff, Gerd Wronka, Michael Zeschnigk, Christiane Zock. I particularly thank Petra Bohm for expert editorial work and helpful comments on the manuscript, and Karin Denibowsky from WILEY-VCH for help and encouragement. In experimental science, the best concepts and projects remain futile without the necessary financial resources. Since 1966 my research has been supported at different times by the National Institutes of Health (Institute of Allergy and Infectious Disease), the Public Health Research Council of the City of New York (Career Scientist Award, 1969/71), the American Cancer Society, the Rockefeller University, New York, NY, the Swedish Cancer Society, the Deutsche Forschungsgemeinschaft through different programs, notably through the Sonderforschungsbereiche SFB74 and SFB274, the Center for Molecular Medicine Koln, the Fritz Thyssen-Stiftung, the Wilhelm Sander-Stiftung, the Volkswagen-Stiftung, the Fonds der Chemischen Industrie, the Alexandervon-Humboldt-Stiftung, and last but not least the Land Nordrhein-Westfalen through the Universitat zu Koln.

Foreip DNA 21Mammalian Sptems Walter Doerfle copyright 0 WILEY VCH Vrrlag Gmbll. 2000

Index abortive infection 32,34 by adenoviruses 32 AcNPV (Autographa californica nuclear polyhedrosis virus) 123 Ad12 (adenovirus type 12) 120 - extracellular virions 30 - transformed hamster T637 cell 71 Ad12 DNA 34,60 - association of 68 - integrated 60 - replication block 34 Adl2-induced hamster tumors 17 Adl2-transformed hamster cell line T637 71 Ad2 (adenovirus type 2) 29, 120 - genetic map of 29 adenoviral vectors, third generation of 161 adenovirion 28 - model of the type 2 28 adenovirus DNA 6 - integration of de n o w methylated 169 - uptake of foreign 6 adenovirus infection 39 adenovirus system - DNA methylation in 171 adenovirus system and neuroblastoma-like tumors 80 adenovirus type 12 see Ad12 adenovirus type 2 see Ad2 adenoviruses 27 ff, 32,40,161 - abortive infection 32 - classification of 29 - clinicaI background of 27 - vectors 161 - integration of DNA in human cells 40 - virion structure of 28 adenoviruses and coxsackievirus receptor 30 ADP-ribosylation 99 Alu sequences 99,118 Angelman syndrome 117 antibody response 162, I66 - against foreign DNA 166 applied molecular biology 159 array 103 - DNA technology 103 artificial chromosomes 1.59 association of viral DNA with human or hamster cell chromosomes 53 ATP-binding cassette (ABC) transporter 98 attB 94 f attP 94 f attachment 92 -

( a f t )sites 92 Autographa californica nuclear polyhedrosis virus see AcNPV -

bacteriophage 39,85,87,149 DNA of M13 149 - orally applied M13 DNA 39 bacteriophage h 8.5 - chromosome 87 - genome 92 - vectors22 biological significance of DNA methylation 169 bisulfite 107 - protocol 107 BLK gene 132 BPV-1 (bovine papilloma virus type 1) 10 -

cancer 11 human cells 11 capsomeres 28 cell-free system 41 - from nuclear extracts of BHK2I hamster cells 41 cellular chromatin structure and foreign DNA integration 141 cellular DNA methylation patterns 137 - alterations of 137 CG-rich bacterial DNA stimmulating the immune response 162 chromatin 48,.52, 115,139, 141,144 - cellular structure 141 - organization of 139 - structure 48,.52,11.5, 144 - structure and patterns of DNA methylation 11.5 - structure in mammalian cells 144 chromosomal 32,48,49,.52,72,1.52 - association of Ad12 DNA 32 - association of Ad12 DNA with metaphase 66 - association of adenovirus DNA 52 - association of orally ingested foreign DNA 1.52f - localizations of integrate Ad12 genomes 49 - location of the integrated h genomes and the integrated pSV2neo plasmids 72 - sites of instability 48 chromosome breaks 99 chromosomes 66 f - maternal 117 -

178 -

CI -

Index

paternal 117 89 product of gene 89

CII 88, yo - product of gene 88, YO clonal origin 57 - Adl2-induced tumors 57 cocatenate formation YY cohesive termini 85 competence stimulating peptide 98 consequences of foreign DNA integration 12Y cos 85 - sites 85 - covalent linkage 10,34,48, IS3 CreiLox 102 - gene casette 102 Cro YO - dimersY0 cro YO - product of gene YO cystic fibrosis transmembrane regulator (CTFR) 161 cytidine 130 - methylation of residues 130

de novo DNA methylation 49,64,51, 121 f, 129,131 f, 143f, 166 - an ancient defense mechanism 129 - and loss of function 79 - in mammalian genomes 115 - of integrated adenovirus DNA 169 - of integrated foreign DNA 20, 129 f defense mechanism 6,143, 155 deoxycytidine residues 109 dinucleotides 107,110, 138, 140 - analysis of individual 5’-CG-3’ 140 - S’-CG-3’ 107 - levels of DNA methylation at individual 5’-CG-3’ 110, 138 DNA-DNA hybridization 24 DNA fragmented by ultrasonic treatment 25 h DNA integrated and randomly located cellular sites 95 DNA methylation and transcription patterns 142 DNA methylation patterns 60, 115 ff - and chromatin structure 115 DNA methyltransferase system 115 DNA microinjection 39 DNA transfection 39 downward DNA transfer 16

enhancer 132 SV40 clement 132 environment 5,147 - foreign DNA in the 5 - plants in the 5 - uptake of foreign DNA from 147 epithelia of the gastrointestinal tract 148 Epstein-Achong-Barr virus (EBV) 10 equilibrium sedimentation in alkaline CsCl -

24,25

sedimentation patterns 25 expression of integrated foreign DNA 100

-

fate of foreign DNA 1 feline leukemia virus 82 FISH method (fluorescent in situ hybridization method) 14,24 food-ingested foreign DNA 1,147 foreign DNA - and the public 155 - in our environment 147 - insertion and changes in cellular methylation patterns 134 - portals of entry 147 FV3 (frog virus 3) 123 gastrointestinal 147, 1.54 tract 147 f - uptake of foreign DNAvia the tract 154 gene lSY,160 - human somatic therapy 40,142,160 - retroviral mediated transfer 160 - transfer 159 genetic imprinting 114 - and DNA methylation 111 genomic 51,107,140 - fixation of genes or DNA segments 51 - sequencing 107,140 -

HBV (human hepatitis B virus) 63,79 HCMV (human cytomegalovirus) 124 hepadnaviral life cycle 78 heterologous recombination 102, 133 hexons 28 hit-and-run mechanism 60 Hodgkin’s lymphoma 119 homologous recombination 92,102, 132, 133 homologously recombined foreign DNA 132 HPV (human papilloma virus) 63,7Y human 159 - mitotic stability of artificial chromosomes 159

early functions 29 EBV (Epstein-Achong-Barr virus) 63.79 endogenous retroviral genomes 84

skin or genital malignancies 75, human cytomegalovirus see HCMV Human Genome Project 109

-

Index

Human hepatitis B virus see HBV human immunodeficiency virus 82 human papilloma virus see HPV human somatic gene therapy 40, 142 hybridization 15,24 - DNA-DNA 15.24 IAP 84,138,140 - methylation patterns 140 - repetitive sequences 138 IAPI DNA segment 71,136 IAPI to IV 69 11-2Ra 116 “illegitimate” recombination 99 immediate early function 31 inactivation 143 - of DNA sequences 143 induction of lysogenic bacteria 95 infection 88 - immediate early phase of the cycle 88 - with viruses 5 insertion 135, 141 - of Ad12 DNA into the hamster genome 135 - sites of foreign DNA 141 insertional mutagenesis 99 insertional site specific recombination 92 integrase 83 - virus-encoded (int)protein 83 integrated 37,57 - Ad12 DNA 60 - destabilization of the adenovirus genomes 57 - loss of previously Ad12 DNA sequences 37 integrated and episomally persisting foreign genomes 18 integrated foreign DNA 50,99 - IOSS of 50 integration 13, 17,32,40,54,55,63,82,99, 133.166 - Ad12 DNA 32 - and excision of bacteriophage h genome 92 - into the host genome 166 - mechanism of foreign DNA 99 - of Ad12 DNA in Adl2-transformed hamster cells 54 - of adenovirus DNA in human cells 40 - of retroviral DNA 82 - of viral DNA 63 - parameters of foreign DNA 13 - patterns of Ad12 DNA 17 - possible models of Ad12 genomes in the DNA of transformed hamster cells 55 - responses to the of foreign DNA 133 integration reaction 83

179

integrative recombination 41 f interleukin-2 receptor CI chain see 11-2Ra intracisternal A particle see IAP IS elements 98 inverse correlations 170 junction 22,23.33,41,47 - cloning of sequences 23 - fragments 22 - sequences 47 - site bctween Ad12 DNA and hamster cell DNA 33 - sites 41 knock in experiments l02f, 142 knock out experiments 102 f, 142 laser scan microscopy 65 late 29.31 - functions 29 - viral functions 31 leukemia 119 - cell lines 119 long-term signal for promoter inactivation 11 I luciferase gene 131 lymphatic organs in the mammalian organism 148 lymphocytes 119 lysis 86 - cell 86 lysogenic 86,88, 91 - interaction 88 - response 86 - UV induction of bacteria 91 lysogeny 85 lytic pathway 86,88 lytic and lysogenic pathways - decision between 91 M13 149,150 - bacteriophage DNA of 73, 149 - foreign DNA in different segments of the intestine 150 major late promoter (MLP) 31 malignancy - a chromatin disease? 144 maternal chromosome 117 mechanism of viral oncogenic transformation 142 methods to introduce foreign DNA 10 methylation 49,50 ff. 105 ff. 109 f, 120 ff, 134ff, 140, 143f, 166f - and promoter activity 170 - at site of insertional recombination 100 - biological significance of 169

180

Index

cellular DNA patterns 137 concordance of DNA 143 - de novo 49,51,64, 121 f, 131 f, 143f, 166 - de novo, of integrated adenovirus DNA 169 - IAP patterns 140 - in cellular DNA segments 167 - levels of DNA at individual 5'-CG-3' dinucleotides 110, 138 - methods available for the study of DNA 106 - of DNA in the adenovirus system 171 - patterns SO, 105,109, 135 - patterns in cellular DNA 52 - patterns of in cellular genome segments 134 - patterns of in viral DNA 120 5-methyldeoxycytidine 105,109, 130 microinjection into mouse oocytes 39 mitigator sequence of Ad12 DNA 34 mitotic stability of human artificial chromosomes 159 mosaics 136 - DNA-methylation 136 mouse pseudogenes 153 multimers 48 murine leukemia virus 82 mutagenesis 48,5 I , 145 - functional insertional 145 - insertional 48.51 -

N gene of h DNA 87,89 - product of gene 89 - protein 87 neomycin 131 - gene 131 non-homologous recombination 4Y non-viral systems 97 oncogenesis 60,7Y, 144 viral 60,79, 144 oncogenic 37,s I , 58, 154 - phenotype 51,58 - process 154 - transformation of rodent cells 37 operator OR 90 oral ingestion of foreign DANN 149 organs 153 - foreign DNA in various of fetuses 153

-

p53 51 cellular gene 5 I P,", 91 papillomavirus 77 - replication cycle of 77 patchy or short sequence homologies 99 -

paternal chromosome 117 patterns of DNA methylation 50, 105, 109, 115ff, 135 PCR (polymerase chain reaction) 20 f, 58 - analyses58 - schematic representation 21 pentons 28 persistence 10,51, 58, 64 - of foreign DNA 10 - of integrated foreign DNA 51 - pathways of 10 - patterns58 - viral 64 Peyer's patches 148 placental barrier 1.54 pneumococci 98 polymerase chain reaction see PCR polypeptides 28 - viral 28 Prader-Labhart-Willi syndrome 117 premethylated promoter-indicator gene constructs 170 productive infection 34 promoter 31, 111 f, 130 - activity31 - inactivation 111, 130 - transcriptional inactivation of 112 promoter methylation 112, 170 f - and promoter activity 170 - release of inhhibitory effect 171 prophage86 - insertion and excision 86

Q gene of h DNA 89 product of gene 89

-

randomly integrated foreign DNA 131 f recloning of junction fragments 22 recognition sites for topoisomerase I 100 recombinants 41 - between Ad12 DNA and cellular DNA 41 recombination 42,48 f - active proteins 42 - mechanism of insertional 48 - non-homologous 49 regulatory functions in phage h 89 release of inhibitory effect of promoter methylation 171 repetitive 84 - DNA sequences 84 - elements 99 replication 11,64,77f, 82 - cycle of a papillomavirus 77 - factors I1 - of the retroviral genome 82

Index overview of retrovirus 78 - viral cycle 64 h repressor 89,91 repressor molecules 88 responses to the integration of foreign DNA 133 restriction 107 - methylation-sensitive endonucleases 107 RET protooncogene 111,119 retroregulation 92 retrotransposon 84,136 - sequence 136 retroviral63,82 - DNA63 - endogenous genomes 84 - integration mechanism 99 - progenome 76 - replication cycle 82 revertant cell lines 37 revertants 37,60 5-ribulose biphosphate-carboxylase gene 152 Rous sarcoma virus 80 -

short sequence homologies 42 f single copy quantities of foreign DNA 18 SNRPN transcripts 117 somatic gene therapy 41,160f Southern DNA transfer 15 splicing of RNA 29 stability 37 - integrated foreign DNA 100 - of viral DNA integrates 37 stability-instability 57 sticky ends 85 strechted chromosomes 15,65 - mechanically stretched 67 SV40 76 - regulatory regions 76 SYREC (symmetric recombinants) 34,35,40, 121,161 Syrian hamster (Mesocricetus uurutus) 36 targeted 135 stability of the genome 135 transcription 64

-

181

- of integrated viral genomes 64 transfection 39 - into hamster cells 39 transfer 6 - to the nucleus 6 transformation 35 - of cells in culture 35 transformed phenotype 135 transgenic organisms 101,142 - critical evaluation of 103 transplacental transfer 154 transport 150 - of foreign DNA through the intestinal wall and Peyer’s patches 150 transposons 98 tumor 41,36,57,79f, 116,144 - Ad12-induced 36,57 - cell line CLACl 41 - cells in culture 36 - development of malignant 144 - induction 36 - necrosis factors 116 - virus-induced 79

uncoating of adenovirus 7 uptake 6,147,154,165 - of food 5 - of foreign DNA 6 - of foreign DNA by mammalian cells 165 - of foreign DNA from the environment 147 - of foreign DNA via the gastrointestinal tract 154 UV induction of lysogenic bacteria 9 1 vaccines 162 f, 165 DNA 162,165 VAI RNA 31 vectors 160,161 - adenovirus 161 - retroviral 160 viral DNA 34 - state of 34 virus-induced tumors 81 visualization of foreign DNA 14 -

X chromosome inactivation 114