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-

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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

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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 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 ~