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UBC Theses and Dissertations

Studies on the transformation of mammalian cells by polyoma virus Babiuk, Lorne Allan Ben 1972

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c 13191 STUDIES ON THE TRANSFORMATION OF MAMMALIAN CELLS BY POLYOMA VIRUS by LORNE ALLAN B. BABIUK B.S.A. University of Saskatchewan 19 7^ M. Sc. University of Saskatchewan 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of Microbiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1972 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia Vancouver 8, Canada Abstract Studies were made on the oncogenic properties of a line of polyoma virus-transformed hamster cells. Transplantation of the cells resulted in an increase in oncogenicity. Thus the latent period for the appearance of a tumor was decreased, and as few as five to ten cells were sufficient to produce a tumor in a young hamster. Histological examination revealed marked differences between tumors produced by transplantation of transformed cells and tumors produced by infectious virus. Powassan virus, a group B arbovirus, did not show any oncolytic activity towards tumors induced by polyoma virus-transformed cel l s . A variety of treatments (ultraviolet irradiation; mito-mycin C; heat shocks; and c e l l fusion) proved unsuccessful in attempts to rescue polyoma virus DNA or infectious virus from polyoma virus transformed cel l s . In analogous experi-ments, using simian virus-kO-transformed cultures, the rescue process was followed by readily detectable synthesis of SVkO DNA and infectious virus. The a b i l i t y of heterokaryons (mouse kidney - PyH-1 cells or mouse embryo - 'normal' hamster cells) to support virus multiplication was tested. The presence of hamster nuclei and cytoplasm i n heterokaryons, proved i n h i b i t o r y to polyoma v i r u s m u l t i p l i c a t i o n . This i n h i b i t o r y e f f e c t was more pronounced i f the hamster c e l l s had been transformed with polyoma v i r u s . Evidence has been presented f o r the i n t e g r a t i o n of v i r a l DNA into c e l l u l a r DNA during productive i n f e c t i o n of mouse embryo c e l l s as w e l l as i n BHK-21 c e l l s , a c e l l l i n e which does not support v i r u s m u l t i p l i c a t i o n but i n which a small percentage of the c e l l s can become transformed. Two separate methods were employed to detect v i r a l DNA a s s o c i a t i o n with c e l l u l a r DNA: 1) separation of v i r a l DNA from c e l l u l a r DNA by p r e c i p i t a t i o n of the l a t t e r with s a l t : 2) separation of c e l l u l a r DNA (> 100s) from v i r a l DNA i n a l k a l i n e sucrose gradients. The presence of v i r a l DNA i n the c e l l DNA f r a c t i o n of a l k a l i n e sucrose gradients indi c a t e d that v i r a l DNA was associated i n an a l k a l i n e stable linkage. i i i Table of Contents Page CHAPTER I: GENERAL INTRODUCTION General 1 H i s t o r i c a l 2 P h y s i c a l and Chemical Properties of Polyoma Virus . 1+ Tissue Culture Reactions 7 a. Productive i n f e c t i o n 7 b. Transformation r e a c t i o n 12 Objective l6 References 17 CHAPTER I I : MATERIALS AND METHODS C e l l s U t i l i z e d i n the Study and Their C u l t i v a t i o n . 33 Viruses 36 Preparation of P u r i f i e d Radioactive Polyoma Virus . l+o Uncoating of Polyoma Virus l+i Infectious DNA i+1 Methods of Virus Induction 1+2 a. Mitomycin C treatment 1+2 b. Temperature changes 1+2 c. U l t r a v i o l e t i r r a d i a t i o n 1+3 d. C e l l f u s i o n 1+3 Tumor Production and H i s t o l o g i c a l Studies . . . . . 1+1+ Chromosome Studies 1+5 i v Table of Contents Page CHAPTER I I (continued) Preparation of Antiserum Against Polyoma Capsid Pr o t e i n 1+6 Immunofluorescence Tests f o r Polyoma Capsid P r o t e i n i+7" Autoradiography 1+8 E l e c t r o n Microscopy 1±<Z) E x t r a c t i o n and P u r i f i c a t i o n of V i r a l DNA . . . . 50 E x t r a c t i o n and P u r i f i c a t i o n of Mammalian DNA . . . 51 A l k a l i n e Sucrose Gradient Sedimentation of C e l l DNA and Polyoma DNA 52 DNA-DNA Hy b r i d i z a t i o n 53 References 56 CHAPTER I I I : ONCOGENIC PROPERTIES OF POLYOMA VIRUS TRANSFORMED CELLS Abstract 58 Introduction 59 Results 6 l Tumor Production i n Hamsters by Transformed C e l l s 6 l Tumor Production i n Hamsters by Polyoma Virus . 66 H i s t o l o g i c a l Examination of Tumor Tissue . . . 68 Attempts to Demonstrate an Oncolytic E f f e c t of Powassan Virus 71 Discussion 71+ References 77 V Table of Contents Page CHAPTER IV: THE STATE OF THE VIRAL GENES IN TRANSFORMED CELLS . . . Abstract 79 Introduction 80 Results 82 Attempts to Rescue Infectious Polyoma Virus 82 from Transformed C e l l s 82 V i r a l DNA Synthesis 90 Polyoma Virus M u l t i p l i c a t i o n i n Fused C e l l Cultures 97 Discussion 107 References 113 CHAPTER V: INTEGRATION OF POLYOMA VIRUS DNA INTO MAMMALIAN GENOMES Abstract I l 6 Introduction 117 Results 119 Ass o c i a t i o n of V i r a l DNA with C e l l u l a r DNA . . 119 Evidence that V i r a l DNA i s Covalently Linked to C e l l u l a r DNA -121 Discussion . 3_k2 References li+6 CHAPTER VI: GENERAL DISCUSSION 150 L i s t of Plates Pl a t e Page I I I - I Tumor i n a hamster induced by polyoma 65 v i r u s transformed c e l l s . I l l - I I H i s t o l o g i c a l section of a tumor induced 69 by polyoma v i r u s transformed c e l l s . I l l - I I I H i s t o l o g i c a l section of a tumor induced 70 by polyoma v i r u s . IV - I E l e c t r o n micrograph i l l u s t r a t i n g Sendai 8U v i r u s . IV - I I Polykaryon produced by c e l l fusion. 88 L i s t of Tables S u s c e p t i b i l i t y of hamsters of various ages to tumor production by polyoma v i r u s (IO? pfu/hamster). Test f o r onc o l y t i c e f f e c t of Powassan vi r u s on tumors induced by polyoma v i r u s -transformed c e l l s (PyH-l). Summary of attempts to rescue polyoma v i r u s . E f f e c t of mitomycin C treatment f o r eight hours followed by c e l l f u s i o n with mouse kidney c e l l s . Polyoma v i r u s s p e c i f i c DNA synthesis as determined by DNA-DNA h y b r i d i z a t i o n . Simian virus-kO s p e c i f i c DNA synthesis as determined by DNA-DNA h y b r i d i z a t i o n . Polyoma-virus m u l t i p l i c a t i o n i n fused c e l l s as measured by fluorescence microscopy. L i s t of Tables M u l t i p l i c a t i o n of polyoma v i r u s i n ME + BHK-21 f u s e d - c e l l cultures. M u l t i p l i c a t i o n of polyoma v i r u s i n ME + BHK-21 f u s e d - c e l l cultures Integration of polyoma v i r a l DNA into mouse and hamster genomes as measured by h y b r i d i z a t i o n of DNA extracted from i n f e c t e d c e l l s by H i r t ' s method. Integration of l a b e l l e d polyoma v i r a l DNA into c e l l u l a r DNA. Integration of polyoma v i r a l DNA into c e l l u l a r DNA. The r e l a t i o n s h i p between i n t e g r a t i o n of polyoma v i r u s DNA and m u l t i p l i c i t y of i n f e c t i o n . L i s t of Figures Figures I I I - I Time of appearance of tumors following i n j e c t i o n of polyoma v i r u s transformed c e l l s . IV - I Radiation dose response of Sendai v i r u s to u l t r a v i o l e t i r r a d i a t i o n . IV - I I A l k a l i n e sucrose gradient sedimentation of low molecular weight DNA. IV - I I I C a l i b r a t i o n curve f o r polyoma DNA-DNA hybr i d i z at ion. IV - IV C a l i b r a t i o n curve f o r SVUO-DNA-DNA hy b r i d i z a t i o n . V - I A l k a l i n e sucrose gradient sedimentation of mouse DNA and polyoma DNA. V - I I A l k a l i n e sucrose gradient sedimentation of mixtures of polyoma DNA and BHK-21 c e l l s . V - I I I Thermal denaturation of polyoma DNA-c e l l DNA complexes. L i s t of Figures A l k a l i n e sucrose gradient of mouse embryo and BHK-21 c e l l s i n f e c t e d with methyl-^H-thymidine polyoma v i r u s DNA. A l k a l i n e sucrose gradient of mouse embryo and BHK-21 c e l l s a f t e r i n f e c t i o n with methyl-~H-thymidine l a b e l l e d polyoma v i r u s . Sucrose gradient c e n t r i f u g a t i o n of polyoma v i r u s . ACKNOWLEDGEMENTS I would l i k e to thank Dr. J.J.R. Campbell, Head o f the Department of Microbiology, f o r granting me t h i s opportunity f o r study and research. The guidance and encouragement of Dr. D.M. McLean, Head of the D i v i s i o n of Medical Microbiology and Dr. J.B. Hudson, under whose supervision t h i s research was conducted, i s g r e a t l y appreciated. Thanks are also extended to my committee members; Dr. D. K i l b u r n and Dr. H. Stich, who have provided h e l p f u l advice during the research and t h e s i s w r i t i n g , which I appreciate. The t e c h n i c a l assistance of Miss L. McGrath and Mrs. G. Steele are g r a t e f u l l y acknowledged as i s the typing of t h i s manuscript by Rosemary Morgan. x i i ABBREVIATIONS DNA - Deoxyribonucleic a c i d DNase - Deoxyribonuclease H + E - Hematoxylin and eosin HAU - Hemagglutination u n i t s HBS - Hank's balanced s a l t HI - Hemagglutination i n h i b i t i o n IF - Immunofluorescence ME - Mouse embryo MK - Mouse kidney nm - nanometer PBS - Phosphate buffered s a l i n e SDS - Sodium dodecyl s u l f a t e SSC - Standard s a l i n e c i t r a t e SV^O - Simian v i r u s 1+0 TCD,_0 - Tissue culture dose - The v i r u s dose showing 5 50$ cytopathic e f f e c t Tm - melting temperature - The temperature at which 50$ of the DNA i s denatured UV - U l t r a v i o l e t CHAPTER I GENERAL INTRODUCTION 1 General Polyoma virus is a member of the Papovavirus group (Melnick, 1962). It is a small icosahedral virus containing 72 capsomers (Finch and Klug, 19&5; Mattern et al, 1967) with an overall diameter of 45 nm (Wildy et al, i960). It is similar to the other members of the Papovavirus group which includes: simian virus hO (SV40), papilloma viruses, including the human wart virus, a l l of which contain circular double stranded DNA (Dulbecco and Vogt, 1963; Weil and Vinograd, 19&3; Crawford and Black, 196U; Kleinschmidt et a l , 1965; Vinograd et al, 1965). Each virus is separated from the other members of the Papovaviruses on the basis of i t s different antigenicity and host range. Polyoma virus exists under natural conditions as a 'silent 1 or asymptomatic infection of mice. It is found in healthy mice of several inbred lines (Gross, 1955; Rowe et al, 1959)• The virus has been shown experimentally to induce a broad spectrum of histologically distinct tumors in mice, hamsters, rats, rabbits and guinea pigs (Eddy, 1969). Infection of tissue cultures by polyoma virus may result in one of the following basic reactions: l) productive infection; 2) transformation reaction; or 2 3) abortive infection or lack of a response. Infection of mouse embryo or mouse kidney cultures leads to the production of progeny virus followed by c e l l degeneration and c e l l death (Weisberg, 19^3; Winocour, 19^3; Weil et a l , 1965)• The transformation reaction can occur in c e l l cultures derived from mouse, hamster and rat tissues, with l i t t l e or no pro-duction of progeny virus (Vogt and Dulbecco, i960, 1962; Defendi, 1966). The transformed cells are maintained in the transformed state by the persistence of v i r a l genes (Benjamin, 1966; Martin and Axelrod, 1969). Historical The search for viruses capable of inducing tumors in animals gained impetus, in 1951 when Gross described the induction of leukemia in strain C3H mice that had been inoculated subcutaneously, within l6 hours after birth, with c e l l free extracts of lymphocytes derived from leukemic AKR mice (Gross, 1951)- Instead of developing leukemia, some of the mice injected with the extracts developed tumors on one or both sides of the neck (Gross, 1953). In the same year Stewart also reported the induction of sarcomas in the parotid glands of mice (Stewart, 1953). 3 D i f f i c u l t y in reproducing tumor induction was experi-enced probably due to the small amount of virus in the extracts used. This d i f f i c u l t y was overcome when Stewart reported the cultivation of this oncogenic virus in monkey kidney and mouse embryo cultures (Stewart et al, 1957; Stewart et al, 1958). Mice which had been injected with supernatants obtained from infected tissue cultures produced, within two or three weeks, tumors of the parotid, submaxillary, sublingual and thymic glands, the adrenal medulla and convoluted tubules of the kidney cortex. Due to the fact that the virus was capable of producing a large variety of tumors, Stewart and Eddy proposed the name polyoma for the virus. In later publications i t was designated SE polyoma virus. Since the i n i t i a l isolation of SE polyoma virus a number of other strains have been isolated which are antigenically similar, but differ in their capacity to cause cytopatho-genicity or transformation of c e l l cultures. The BBT2 strain is more cytolytic in tissue culture than the original SE strain. The Toronto strain causes a rapid development of b i l a t e r a l kidney sarcomas and death of the animal (McCulloch, 19595 Axelrad, i960), whereas the SE strain produces pre-dominantly mammary carcinomas and parotid tumors but no kidney tumors. Attempts to plaque-purify polyoma resulted in the isolation of large and small plaque mutants (Gotlieb-k Stematsky and Leventon, i960; Thorne et al, 1965). Temp-erature sensitive mutants are now being u t i l i z e d in elucidating which genes are responsible for specific functions (Fried, 1965a, 1965b; Di Mayorca et al, 1969; Cuzin et al, 1970; Eckhart, 1969, 1971). Physical and Chemical Properties of Polyoma Virus Polyoma virus is an icosabedral particle containing 72 capsomers (Finch and Klug, 1965). The overall size of the virion is kO-k^ nm (Wildy et al, i960) with a density of 1.32k g/cc in cesium chloride (Winocour, 1969). V i r a l particles with lower buoyant density containing smaller amounts of v i r a l DNA have been found after infection at high multi-p l i c i t i e s (Thorne et al, 1968; Blackstein et al, 1969). These defective polyoma viruses may be deletion mutants which replicate under conditions of multiple infection, as has been described for adenoviruses (Burlingham and Doerfler, I969), and bacteriophage lambda (Kellenberger et al, 1961). A similar situation exists for SVkO (Uchida et al, 1968; Yoshike, 1968). Polyoma virus contains 12$ DNA and 88$ protein and no other components (Murakami et al, 1968). Purified DNA 5 as well as intact virus is infectious (Di Mayorca et_ al, 1959)- From the buoyant density (l.7 0 9 g/cc) in CsCl gradients and the melting temperature, Tm, of 89°C (0 .15 M NaCl) i t has been estimated that the DNA of polyoma virus contains about 1+9 mole percent of guanine + cytosine (Weil, 1963). A particularly interesting feature of the DNA extracted from polyoma virus is the variety of forms in which the molecule can exist. Type I is a circular super-coiled duplex (m.w. 3 x 10°" daltons) which sediments at 20s in 1 M NaCl and 0 . 0 1 M Na2HP0^ pH 8 . 0 (Weil and Vinograd, 1963). This form is relatively stable as indicated by the retention of i t s in f e c t i v i t y even after heating at 100°C for 15 minutes (Weil, 1963). Type II DNA is a circular duplex in which one strand is nicked, therefore relaxing the super-coil (Vinograd et al, 1965)• Type II DNA sediments at l 6 s and probably arises from single strand scission of Type I during isolation. Both Type I and II are infectious and are capable of causing morphological transformation of cultured cells (Dulbecco and Vogt, 19°3; Weil and Vinograd, 19^3; Crawford et al, I96U; Bourgaux et al, 1965). Type III DNA is not infectious, and i t has been shown that this com-ponent is heterogeneous in size ( l l - l 4 s ) , and is derived from host c e l l DNA which is excised during infection and is 6 subsequently incorporated, instead of virus DNA, into a portion of the virus particles (Ben-Porat et al, 1966, 19^7; Michel et al, 1967; Winocour, 1969). Polyoma DNA does not appear to be methylated (Kaye and Winocour, 1967) • The polyoma genome consists of almost 1+500 nucleotide pairs, which contain sufficient information for about 1500 amino acids, corresponding to three to eight proteins of a combined molecular - weight totalling 170,000 daltons. Characterization of the number and size of the proteins present in the virion would allow one to determine the amount of genetic information l e f t over for specific virus function other than structural proteins. Early work had indicated the existence of one major protein (Thorne and Warden, 19^7j Kass, 1970) or one major protein and a minor 'internal' protein component (Fine et_ al, 1968). Recently, however, Roblin et a l (1971) have shown by electrophoresis the existence of one major component, P-2, of ^5,000 daltons molecular weight, and five other polypeptides. The sum of the molecular weight of a l l components is about 2^0,000 daltons. This value exceeds the 170,000 daltons for which the virus is capable of coding for, suggesting that some of the minor components may be host c e l l gene products that exist as contaminants in the virus samples, or that they 7 actually play a role as structural components of the virus, although derived from the host. Tissue Culture Reactions (a) Productive infection The biochemistry of polyoma virus multiplication has been studied almost exclusively in primary mouse kidney cultures and mouse embryo cultures since these are the two cultures which give the highest yields of virus. Infection of mouse cultures results in cytopathic effect and release 5 of approximately 10 virus particles per c e l l (Crawford, 1968). Some c e l l cultures derived from hamster, rat and monkey, and continuous mouse cells, occasionally support the growth of a limited quantity of polyoma virus, but show l i t t l e or no cytopathic effect (Eddy, 1969). Carrier cultures have been obtained by infection of L929 c e l l cultures with polyoma virus (Henle et al, 19^3; Hare and Morgan, I96I+). In contact inhibited cells of mouse origin, the infection process has been separated into two distinct phases (Weil et al, 1965; Weil, 1970). Phase one, which 8 is equivalent to an eclipse phase, occurs prior to virus DNA synthesis and may be separated from Phase two, the phase in which virus DNA is synthesized and progeny virus released, by the addition of 5-fluorodeoxyuridine, a DNA synthesis inhibitor (Petursson and Weil, 1968). Figures 1 and 2 may indicate more clearly the sequence of events during productive infection. (Weil, 1970). Induction of cellular DNA synthesis is a specific activation or 'derepression' of the DNA synthesizing apparatus not followed by mitosis (G-ershon, et al, 19^5; Basilico et al, 1966; Hancock and Weil, 1969). Induction of mitochondrial DNA synthesis also occurs in polyoma virus infected cells (Vesco and Basilico, 1971). Cheevers (1972) has proposed that this stimulation of cellular DNA synthesis by polyoma is due to an increase in the number of DNA i n i t i a t i o n sites. The induction of DNA synthesis in polyoma virus i n -fected mouse cells is accompanied by marked increases in the specific activities (2-55 fold) of DNA. polymerase and other enzymes involved in the synthesis of pyrimidine deoxy-ribonucleotides, especially thymidine derivatives (Dulbecco et al, 1965; Hartwell et al, 1965; Kara and Weil, 1967; Kit et al, 1969; Weil et al, 1969). The specific a c t i -v i t i e s of enzymes involved in phosphorylation of purine I P H A S E I I Qct iva l ion of cellular DNA-synthesizing apparatus | P H A S E 2 f 7 ^ L V 5' 5 11 Length of phase I vanes J 37*C 12 - 30 hours in individual cells j 27*C 27i - 5 days R E P L I C A T I O N O F T H E H O S T C E L L C H R O M O S O M E S ( not followed by mitosis ) adsorption of Py virus Py DNA penetrates into nucleus f „early early" Py messenger RNA v host cell factor appearance of intranuclear Py-specific T-antigen „integration"of Py DNA into mouse chromosomal DNA „early"Py messenger RNA transcribed from integrated Py DNA v " »• psychrasensitive event (s) "** onset of viral DNA synthesis i transcription of (polycistronic) „lale"Py messenger RNA transport into cytoplasm synthesis of capsid protein transport into nucleus I assembly of progeny virus I lysis of host cell F I G . 1 . T e n t a t i v e scheme for the lytic cycle of p o l y o m a virus in c o n t a c W n h i b i t e d p r i m a r y mouse kidney tissue culture cells. T h e m a r k e d asj'nchrony of the time course of infection ( F i g . 2 a n d ref. \a-c) is due to the v a r y i n g length, i n i n d i v i d u a l cells, of phase 1 . If P y - i n d u c e d synthesis of v i r a l and cellular D N A is inhibited with 5-fluorodeoxyuridine, little if a n y capsid p r o t e i n 1 " 0 or chromosomal protein 2 is synthesized; however, the events of phase 1 w h i c h l e a d to the appearance of T - a n t i g e n ' " •''•B-12 a n d to the subsequent act ivation of the celluiar D N A -svnthesizine apparatus'" _ c ' 1 2 take place iust as thev do in the absence of the inhibitor. deoxyribonucleotides are not increased (Dulbecco et a l , 1965; Kara and Weil, 1967). Whether these enzymes are coded for by v i r a l genes or by cellular genes has not been deter-mined, but i t would seem impossible for the virus DNA to code for a l l the enzymes (nine in a l l ) with the limited amount of genetic information available. Several research groups have reported differences in the properties of enzymes in extracts of infected and uninfected cells (Hartwell, et al, 1965; Sheinin, 1966). However, the infection of a thymidine-kinase deficient c e l l line,with polyoma,failed to induce thymidine kinase activity, an enzyme normally induced in polyoma virus infected cells, indicating that at least one of the enzymes is c e l l coded ( L i t t l e f i e l d and Basilico, 1966; Basilico, Matsuya and Green, 1969). Polyoma virus DNA replicates semiconservatively (Hirt, I966). Electron micrographs have been presented showing polyoma DNA apparently in different stages of replication (Hirt, 1969). These molecules were circular with two branch points and three branches. Recently Bourgaux et_ al, isolated a replicative intermediate of polyoma DNA, and named i t type II*. This DNA has a sedimentation value of 25s (Bourgaux, 19^9; 1971) and contains single stranded regions susceptible to Neurospora crassa endonuclease (Bourgaux and Bourgaux-Ramoisy, 1971)• A pulse label for five minutes, followed by a 60 minute chase, caused the majority of the •radioactivity in the 25s fraction (type II*) to shift to 20s (type I), as expected for a replicative intermediate. A portion of the 25s v i r a l DNA was stable however and did not convert to a 20s molecule. It has been suggested that this stable fraction of 25s molecules, may represent catenated dimers, which have been observed in electron micrographs (Meinke and Goldstein, 1971). From the above quoted work, a model for the replication of polyoma DNA has been proposed. Since polyoma type I DNA is a circle, the f i r s t step in replication would be to introduce a nick in one of the strands of polyoma DNA type I. To f a c i l i t a t e unwinding of the parental strands and free the topological restraint, i t is l i k e l y that the nick is introduced at or in front of the growing point and is resealed after the synthesis of a few nucleotides. (b) Transformation reaction Infection of cells of hamster origin results in morpho-logical transformation of a small percentage of the cells. Polyoma virus transforming efficiency seldom exceeds 5$ of the cells even i f a very high dose of virus i s used. This efficiency is much lower than the k<yf0 reported for SVkO transformation of a continuous line of mouse cells (3T3) (Black, 1966; Todaro and Green, 1966). Infection of BHK-21 cells could result in 90$ of the cells synthesizing tumor antigen (T antigen) within 2k hours (Meyer, 1971). A small percentage of these T antigen positive cells subsequently became transformed, indicating that the majority of the infected cells were capable of synthesizing T antigen without becoming transformed. The percentage of transformants may be increased by allowing the cells to divide a number of times, immediately after virus infection in order to 'fix' the transformed state (Todaro and Green, 1966). The cells which become transformed differ, from their normal prototype in the following properties: 1. Altered c e l l morphology 2. growth to a higher density in a disoriented multilayer arrangement 3. growth in agar, resulting in clone formation k. increased growth rate 5. increased transplantability i n appropriate animals 6. presence of new antigens including T antigen and transplantation antigen (Habel, 1962, 1965). The genes responsible for the formation of T antigen have not been identified but appear to be v i r a l , since T antigen i s immunologically identical in different host cells transformed by the same virus (Pope and Rowe, 1 9 6 4 ) . Cells transformed by some polyoma strains do not induce detectable transplantation antigens but are tumorigenic, suggesting that this antigen may not be essential for transformation (Hare, I 9 6 7 ) . Changes in the nature and conformation of the c e l l membrane, allowing the cells to be agglutinated by concona-valin A, are also characteristic of transformation. These surface changes may be responsible for loss of contact inhibition (Burger and Noonan, 1970). A similar change in normal 'cells has not been detected following infection with virus mutants which are defective in their transforming a b i l i t y (Benjamin and Burger, 1970). Previous work indicated that the increase in agglutination was due to an unmasking of binding sites; as a result transformed cells bound more conconavalin A and agglutinated (inbar and Sachs, 1 9 6 9 ) • More recent work has shown that both normal and transformed cells bind the same amount of conconavalin A (Cline, 1971; Ozanne, 1971). A possible rearrangement of conconavalin A binding sites, during transformation, has been postulated to be the reason for agglutination during transformation (Nicolson, 1971). Maintenance of the transformed state is probably due to the integration of the entire v i r a l genome, or a part of i t , into the host c e l l OTA (Sambrook et al, 1968; Westphal and Dulbecco, 1968; Gelb, Kohne and Martin, 1971). The integrated v i r a l DNA is functional (Benjamin, 1966). Martin and Axelrod have reported that 20-40$ of the v i r a l genome can be transcribed in transformed cells (Martin and Axelrod, I969). Further evidence, although indirect, for the inte-gration of papovavirus DNA into host c e l l DNA is the presence of both c e l l and v i r a l RNA in the same molecule (Wall and Darnell, 1971). Transformation, and expression of the above mentioned characteristics of transformed cells, do not require the entire virus genome, as shown by the fact that plaque form-ing a b i l i t y is more sensitive to UV inactivation than is transforming a b i l i t y (Basilico and Di Mayorca, 1965; Benjamin, 1965; Laterjet et al, 1967). The presence of an entire virus genome in the transformed cells may allow virus 'rescue' by Sendai virus-mediated fusion of the transformed cells to permissive cells, or by treatment of the transformed cells with various chemicals (Kit et al, 1968; Burns and Black, I969)• Rescue is obtained with relative ease from SVkO transformed cells, but only rarely from polyoma virus-transformed cells (Fogel and Sachs, 1969, 1970). Reversions of cells from the transformed state by the loss of chromosomes, as well as the loss of the cells a b i l i t y to synthesize T antigen, have been reported (Marin and L i t t l e f i e l d , 1968; Marin and Macpherson, 1969; Mac-pherson, 1971). The reverted clones could be retransformed by infection once again with polyoma. These experiments are interpreted to mean that the transformed phenotype depends on the presence of at least some v i r a l genes. Objective The principal objective of this study was to attempt to rescue the v i r a l genome from transformed cells. If rescue could be achieved, then a study of the mechanism of the rescue process would be warranted. If rescue could not be achieved, then i t would be important to determine the reasons for this, and to examine the possible role played by host c e l l factors in preventing complete synthesis of progeny virus particles. References to Chapter I Axelrad, A.A., E.A. McCulloch, A.F. Howatson, A.W. Ham and L. Siminovitch, i 9 6 0 . Induction of tumors in Syrian hamsters by a cytopathogenic virus derived from a C^ H mouse mammary tumor. J. Natl. Cancer Inst. 2k: IO95-IIII. Axelrod, D., K. Habel and E.T. Ballan. 1964. Polyoma genetic material in a virus free polyoma induced tumor. Science 146: 1466-1468. Basilico, C., and G. Di Mayorca. I965. Radiation target size of the l y t i c and transforming a b i l i t y of polyoma virus. Proc. Natl. Acad. Sic. U.S. 5j+: 125-127. Basilico, C , G. Marin, and G. Di Mayorca. 1966. Require-ment for the integrity of the v i r a l genome for the induction of host DNA synthesis by polyoma virus. Proc. Natl. Acad. Sci. U.S. 56: 208-215. Basilico, C, Y. Matsuya, and H. Green, 1969- Origin of the thymidine kinase induced by polyoma virus in productively infected cells. J. V i r o l . 3: l 4 0 - l 4 5 . Benjamin, T.L. 1965. Relative target sites for the i n -activation of the transforming and reproductive a b i l i t i e s of polyoma virus. Proc. Natl. Acad. Sci. U.S. 5^: 121-124. Benjamin, T.L. 1966. Virus-specific RNA in cells productively infected or transformed by Polyoma virus. J. Mol. Biol. 16: 359-373. Benjamin, T.L., and M.M. Burger. 1970. Absence of a mem-brane alteration function in non-transforming mutants of polyoma virus. Proc. Natl. Acad. Sci. U.S. 67: 929-934. Ben-Porat, T., C. Cato, and A.S. Kaplan. 1966. Unstable DNA synthesized by polyoma virus-infected cells. V Virology 30: 74-81. Ben-Porat, T. and A.S. Kaplan. 1967. Correlation between replication and degradation of cellular DNA i n polyoma virus-infected cells. Virology 32: 457-464. Black, P.H., I968. The oncogenic DNA viruses: A. review of in vitro transformation studies. Ann. Rev. Microbiol. 22: 391-426. Black, P.H. 1966. Transformation of mouse c e l l line 3T3 by SV40: Dose response relationship and correlation with SV40 tumor antigen production. Virology 28: 75^-759-Blackstein, M.E., CP. Stanners and A. J. Farmilo. 19&9-Heterogeneity of Polyoma virus DNA. Isolation and characterization of non-infectious supercoiled molecules J. Mol. Biol. 42: 301-313-Bourgaux, P., D. Bourgaux-Ramoisy, and M. Stoker. 19°5-Further studies on transformation by DM from polyoma virus. Virology 25: 364-371. Bourgaux, P., D. Bourgaux-Ramoisy, and R. Dulbecco. 1969. The replication of the ring shaped DNA of polyoma virus I. Identification of the replicative intermediate. Proc. Natl. Acad. Sci. U.S. 64: 701-708. Bourgaux^- P., D. Bourgaux-Ramoisy, and P. Seiler. 1971-The replication of the ring-shaped DNA of polyoma virus II. Identification of molecules at various stages of replication. J. Mol. Biol. 5£: 195-206. Bourgaux, P., D. Bourgaux-Ramoisy. 1971- A symmetrical model for polyoma virus DNA replication. J. Mol. Biol. 62: 513-524. Burlingham, B.T. and W. Doerfler. 1969. Physical character-i s t i c s of incomplete particles df adenovirus types 2 and 12. Bact. P r o c V 208. Burger, M.M. and K.D. Noonan. 1970. Restoration of normal growth by covering of agglutinin sites on tumor c e l l surface. Nature 228: 512-515. Burns, W.H., and P.H. Black, 1969. Analysis of SV40-induced transformation of hamster kidney tissue in vitro. VI. Characteristics of mitomycin C induction. Virology 39 : 625-634. Cheevers, W.P., J. Kowalski, and K. K-I l u . 1972. Synthesis of high molecular-weight cellular DNA in productive polyoma virus infection. J. Mol. Biol. In press. Cline, M.J., and D.C. Livingstone. 1971. Binding of ^H-conconavalin A by normal and transformed cells. Nature New Biology 232: 155-156. Crawford, L.V. 196k. A study of shape papilloma virus DNA. J. Mol. Biol. 8: I189-I495. Crawford, L.V. 1968. DNA viruses of the Adeno, Papilloma and Polyoma groups. In: Molecular Basis of Virology. ACS Monograph l64. Ed. H. Fraenkel-Conrat. 393-434. Crawford, L.V. and P.H. Black. 1964. The nucleic acid of simian virus 40. Virology 24: 388-392. Crawford, L., R. Dulbecco, M. Fried, L. Montegnier, and M. Stoker. I96U. Cell transformation by different forms of polyoma virus DNA. Proc. Natl. Acad. Sci. U.S. 52: 148-152. Cuzin, F., M. Vogt, M. Dieckmann and P. Berg. 1970. Induction of virus multiplication in 3T3 cells transformed by a thermosensitive mutant of polyoma virus. J. Mol. Biol. 47: 317-333. Defendi, V. 1966. Transformation in vitro of mammalian cells by polyoma and simian 40 viruses. Progr. Exptl. Tumor Res. 8: 125-188. Di Mayorca, G., J. Callender, G. Marin and R. Giordano. I969. Temperature-sensitive mutants of polyoma virus. Virology 36: 126-133-Di Mayorca, G.A., B.E. Eddy, S.E. Stewart, W.S. Hunter, C. Friend and A. Bendich. 1 9 5 9 - Isolation of infectious deoxyribonucleic acid from SE polyoma infected tissue cultures. Proc. Natl. Acad. Sci. U.S. 45_: 1805-1808. Dulbecco, R., L.H. Hartwell, and M. Vogt. 1965. Induction of cellular DNA synthesis by polyoma virus. Proc. Natl. Acad. Sci. U.S. 53: 403-410. Dulbecco, R. and M. Vogt. I963. Evidence for a ring structure of polyoma virus DNA. Proc. Natl. Acad. Sci. U.S. 50: 236-243. Eckhart, W. 1969. Complementation and transformation by temperature-sensitive mutants of polyoma virus. Virology 38: 120-125. Eckhart, W. 197L Induced cellular DNA synthesis by 'early' and 'late' temperature sensitive mutants of polyoma virus. Proc. Ray. Soc. London. B. 177: 59-63. Eddy, B.E. 1969. Polyoma virus. In: Virology Monographs. Ed. S. Gard, C. Hallauer and K.F. Meyer. Springer-Verlag, New York, N.Y. Finch, J.T. and A. Klug. I965. The structure of viruses of the papilloma-polyoma type. J. Med. Biol. 13: 1-12. Fine, R., M. Mass, and W.T. Murakami. 1963. Protein composition of polyoma virus. J. Mol. Biol. 36: 167-177. Fogel, M. and L. Sachs. 1969. The activation of virus synthesis i n polyoma-transformed cells. Virology 31: 327-334. Fogel, M. and L. Sachs. 1970. Induction of virus synthesis in polyoma transformed cells by ultraviolet light and mitomycin C. Virology 40: 174-177. Fried, M. 1965a. Induction of temperature-sensitive mutants of polyoma virus. Virology 25_: 667-671. Fried, M. 1965b. C e l l transforming a b i l i t y of a temperature-sensitive-mutant of polyoma virus. Proc. Natl. Acad. Sci. U.S. 53: 486-491. Gelb, L.D., D.E. Kohne and M.M. Martin. 1971. Quantitation of simian virus 40 sequences in African green monkey, mouse and virus transformed c e l l genomes. J. Mol. Biol. 57: 129-145. Gershon, D., P. Hauson, L. Sachs and E. Winocour. 1965. On the mechanism of polyoma virus induced synthesis of cellular DNA. Proc. Natl. Acad. Sci. U.S. 54: 1584-1592. Gotlieb-Stematsky, T. and S. Leventon. i960. Studies on the biological properties of two plaque variants isolated from the SE polyoma virus. Brit. J. Exp. Path. 4 l : 507-519. Gross, L. 1951. 'Spontaneous' leukemia developing in C3H mice following inoculation in infancy with AK leukemia extracts of AK embryos. Proc. Soc. Exp. Biol. (N.Y.) 76: 27-32. Gross, L. 1953. A f i l t e r a b l e agent, recovered from AK leukemic extracts, causing salivary gland carcinomas in C3H mice. Proc. Soc. Exp. Biol. (N.Y.) 83: 4l4-421. Gross, L. 1955. Induction of parotid carcinomas and/or subcutaneous sarcomas in C3H mice with normal C3H organ extracts. Proc. Soc. Exp. Biol. (N.Y.) 88: 362-368. Habel, K. 1962. Polyoma tumor antigen in cells transformed in vitro by polyoma virus. Virology 18: 553-558. Habel, K. 1965. Specific complement-fixing antigens in polyoma tumors and transformed cells. Virology 25: 55-61. Hancock, R., and R. Weil. 1969. Biochemical evidence for induction by polyoma virus of replication of the chromosomes of mouse kidney cells. Proc. Natl. Acad. Sci. U.S. 63: 1144-1150. Hare, J.D. 1967. Transplant immunity to polyoma virus induced tumor cells. IV. A polyoma strain defective in transplant antigen induction. Virology 31: 625-632. Hare, J.D. and H.R. Morgan. 1964. Polyoma vi r u s and L c e l l r e l a t i o n s h i p . I I . A curable c a r r i e r system not dependent on i n t e r f e r o n . J. Natl. Cancer Inst. 33: 765-773. •Hartwell, L.H., M. Vogt and R. Dulbecco. 1965. Induction of c e l l u l a r DNA synthesis by polyoma v i r u s . Increase i n the rate of enzyme synthesis a f t e r i n f e c t i o n with polyoma v i r u s i n mouse kidney c e l l s . V irology 27: 262-272. Henle, G., H.C. Hinze, and W. Henle. 1963. P e r s i s t e n t i n f e c t i o n of L c e l l s with polyoma v i r u s . P e r i o d i c destruction and repopulation of the culture. J. Natl. Cancer Inst. 31: 125-ikl. H i r t , B. 1966. Evidence f o r semi-conservative r e p l i c a t i o n of c i r c u l a r polyoma DNA. Proc. Natl. Acad. S c i . U.S. 55: 997-1004. H i r t , B. 1969. R e p l i c a t i n g molecules of polyoma v i r u s DNA. J. Mol. B i o l . ij£: i k l - l k k . Inbar, M. and L. Sachs. 1969. S t r u c t u r a l difference i n s i t e s on the surface membrane of normal and transformed c e l l s . Nature 223: 710-712. Kass, S.J. 1970. Chemical studies on polyoma and Shope papilloma viruses. J. of V i r o l o g y 5: 381-387. Kaye, A.M., and E. Winocour. 1 9 6 1 . On the 5-methylcytosine found In the DNA extracted from polyoma v i r u s . J. Mol. B i o l . 24: U75-I+76. Kellenberger, G., M.L. Z i c h i c h i , and J. Weigle. 1961. A mutation a f f e c t i n g the DNA content of bacteriophage and i t s lysogenic properties. J. Mol. B i o l . 3"- 399-k08. K i t , S., T. Kurimura, M.L. S a l v i and D.R. Dubbs. 1968. A c t i v a t i o n of i n f e c t i o u s SV40 DNA synthesis i n tra n s -formed c e l l s . Proc. Natl. Acad. S c i . 60: 1233-1246. Kleinschmidt, A.K., S.J. Kass, B.C. Williams, and C.A. Knight. 1965. C y c l i c DNA of Shope papilloma v i r u s . J. Mol. B i o l . 13: 749-756. L a t e r j e t , R., R. Cramer and L. Montagnier. 1967. I n a c t i -v a t i o n by UV, X and 7 r a d i a t i o n s of the i n f e c t i n g and transforming c a p a c i t i e s of polyoma v i r u s . V i r o l o g y 33: 104-111. L I t t l e f i e l d , J.W., and C. B a s i l i c o . 1966. I n f e c t i o n of thymidine k i n a s e - d e f i c i e n t BHK c e l l s with polyoma v i r u s . Nature 211: 250-252. McCulloch, E.A., and F. Howatson, L. Siminovitch, A. A. Axelrad, and A.W. Ham. 1959- A cytopathogenic agent from a mammary tumor i n a C3H mouse that produces tumors i n Swiss mice and hamsters. Nature 183: 1535-1536. Macpherson, L.A. 1971. Reversion in cells transformed by tumor viruses. Proc. Roy. Soc. London B. 177: 41-46. Marin, G. and J.W. L i t t l e f i e l d . 1968. Selection of morphologically normal c e l l lines from polyoma-transformed BHK 21/13 hamster fibroblasts. J. Virol. 2: 69-77. Marin, G. and I.A. Macpherson. 1969. Reversion in polyoma-transformed cells: Retransformation, induced antigens and tumorigenicity. J. Virology. 3: 146-149. Martin, M.A., and D. Axelrod. 19^ 9• Polyoma virus gene activity during l y t i c infection and in transformed animal cel l s . Science l64: 68-70. Mattern, C.F.T., K.K. Takemoto and A.M. DeLava. 1967. Electron microscopic observations on multiple polyoma virus related particles. Virology 32". 378-392. Meinke, W. and D.A. Goldstein. 1971. Studies on the structure and formation of polyoma DNA replicative intermediates. J. Mol. Biol. 6 l : 543-563. Melnick, J.L. 1962. Papova virus group. Science 135: 1128-1130. Meyer, G. 1971- V i r a l genome and oncogenic transformation: Nuclear and plasma membrane events. Adv. Cancer Res. 14: 71-153. Michel, M.R., B. Hirt, and R. Weil. 1967. Mouse cellular DNA enclosed in polyoma v i r a l capsids (pseudovirions) Proc. Natl. Acad. Sci. U.S. 58: 1381-1388. Murakami, W.T., R. Fine, M.R. Harrington and Z. Ben Sassan. 1968. Properties and amino acid composition of polyoma virus purified by zonal centrifugation. J. Mol. 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Distribution of antibody in laboratory mouse colonies. J. exp. Med. 109: 449-462. Sambrook, J., H. Westphal, P.R. Srinivassan, and R. Dulbecco. 1968. The integrated state of v i r a l DNA. i n SV40-trans-formed cells. Proc. Natl. Acad. Sci. U.S. 6_0: 1288-1295. Sheinin, R. 1966. Studies on the thymidine kinase activity of mouse cells infected with polyoma virus. Virology 28: 47-55. Stewart, S.E. 1953. Leukemia in mice produced by a fi l t e r a b l e agent present i n AKR leukemic tissue with notes on a sarcoma produced by the same agent. Anat. Rec. 117: 532. Stewart, S.E., B.E. Eddy, A.M. Gochenour, N.G. Borgese and G. Grubbs. 1957. The induction of neoplasms with a substance released from mouse tumors by tissue culture. Virology 3: 380-400. Stewart, S.E., B.E. Eddy, and N.G. Borgese. 1958. Neoplasms in mice inoculated with a tumor agent carried in tissue culture. J. Nat. Cancer. Inst. 20: 1223-1243. Thorne, H.V., J. Evans and D. Warden. 1968. 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CHAPTER II MATERIALS AND METHODS Cells U t i l i z e d in the Study and Their Cultivation The c e l l types used in this study, and their origin, were as follows: Mouse primary kidney cultures, from 10 day old mice secondary embryo cultures Hamster BHK-21 (Clone 13), continuous line (originally obtained from Microbiological Associates) Monkey CV-1, continuous line (obtained from Dr. Saul Kit) BSC-1, continuous line (obtained from Dr. Bruce Casto) Vero, continuous line (obtained from Flow Laboratories) Human Hep-2 continuous line (obtained from Dr. D. M. McLean) Transformed 1. PyH-1 is a hamster c e l l line transformed Cells by polyoma virus, and was obtained from Microbiological Associates (Cat. Wo. Tk-106). This c e l l line was polyoma virus T antigen positive, and continued to synthesize polyoma specific KNA (Hudson, 1972). 2. HT-20 and HT-23 were derived from tumors produced by transplantation of PyH-1 cells into hamsters. 3. TT-109 is a hamster c e l l line transformed by polyoma virus and was T antigen positive when obtained from Flow Laboratories. k. MKS-A is a mouse kidney c e l l line trans-formed by 150 pfu/cell of SVkO 307 L (Dubbs et al, 1967) and was obtained from Dr. Saul Kit. A l l cells were cultivated i n Dulbecco's modified MEM (Grand Island Biological Co. Cat. No. H-l6) supplemented with p e n i c i l l i n , streptomycin and fungizone ( f i n a l concen-trations per ml of medium respectively 100 units, 100 7 and 0.25 7 ) . Incubation was in plastic dishes at 37°C in a well humidified atmosphere containing 5$ C0 o. Cells were i n i t i a l l y plated with 5$ heat inactivated newborn calf serum, except for primary kidney cultures which received 10$ serum. The various c e l l types were tested for mycoplasma or other extranuclear organisms by autoradiography in the following manner. Sub-confluent monolayers were incubated for k hours i n medium containing 1.0 u.Ci/ml of methyl- H-thymidine (Amersham-Searle, 23 Ci/mmole), followed by a one hour chase in non-radioactive medium. The cultures were fixed in gl a c i a l acetic acid-ethanol (1:3 v/v) for 15 min. and rinsed in water for 2 hours. The cells were exposed to Ilford Irk emulsion (1.5 g. gel to 1 g. water), for 7-10 days, and developed for microscopic examination. Wo extra-nuclear incorporation of thymidine was evident. Ce l l lines derived from hamster tumors were started from either explants or by trypsinization of tumors. If explants were used, the tumor was minced with scalpels u n t i l pieces containing approximately 100-200 cells were obtained. The tissue was washed twice in Hank's Balanced Salt Solution before being placed in beef embryo extract (Gibco Cat. No. 515). The tissue was then transferred to a ste r i l e plasma streak i n a Leighton tube and covered with a coverslip. The plasma clot was allowed to form for ten minutes before the addition of medium containing 20 percent newborn calf serum. The cells grew out within a week and. were then transferred to plastic petri dishes for further cultivation. The explant method was later replaced by trypsinizing chunks of tumor to obtain individual cells. This method was preferred due to the fact that a much larger number of cells would be more easily obtained. The procedure con-sisted of cutting the tumor into pieces approximately 0.25 cm square and washing in Hank's balanced salt solution (HBS). The washed tissue was then treated with 0.25$ trypsin in HBS for twenty minutes with constant stir r i n g . The suspension was decanted and the remaining tissue incubated in HBS for a further t h i r t y minutes. This resulted in a large number of disaggregated cells, which were pooled with the cells from the trypsin treatment and centrifuged in ten percent newborn 5 calf serum. The cells were resuspended to a density of 10 cells per ml, and plated out. In two days the cultures were confluent and ready for transferring. Viruses The polyoma virus strain used (designated wild type) was obtained from Dr. R. Weil of the Swiss Institute for Experimental Cancer Research. The virus was characterized by electron microscopy, specific hemagglutination reactions, growth in specific c e l l cultures, characteristics of the DNA and the density of the virus particles. A l l the character-i s t i c s agreed with the published results for polyoma virus. Primary mouse kidney cultures were grown to confluency in plastic petri dishes (90 mm diameter) and were washed once with phosphate buffered saline (pH 7.2) (Dulbecco and Vogt, 1954) before infecting with O.k ml/dish of polyoma virus (approx. 50 pfu/cell). The virus was allowed to adsorb for two hours with gentle rotation of the plates every fi f t e e n minutes. Following this, the culture was covered with medium containing two percent calf serum. The cultures were examined daily for cytopathic effects and when the cells began to lyze and detach from the plate they were harvested for virus purification (Kohse, et a l , 1971). Plaque assays were performed with secondary mouse embryo cultures in 60 mm Falcon tissue culture dishes. Plaques were counted, following neutral red staining, on the 15th day after infection. Hemagglutination assays were used for routine infect-i v i t y tests because of their relative ease and rapidity. Their reproducibility was sufficient to justify their v a l i d i t y as a quantitative measure of virus. On several 38 occasions the ratio of plaque forming units (pfu) to hema-gglutination units (HAU) was measured, and found to be a approximately 1 x 10 :1.0 in agreement with Crawford (1969). Hemagglutination assays were performed i n microtiter plates, using s e r i a l two-fold dilutions of virus preparation in 0.15 M NaCl, and 1.0$ guinea-pig erythrocytes i n 0.15 M NaCl. Incubation was at k°C. The end point was taken as the highest dilution of virus yielding complete hemagglut inat ion. Powassan virus was derived from a stock obtained from Dr. D. M. McLean of this Department. The virus was passaged twice in polyoma virus transformed cells (PyH-l) and titrated by intracerebral injection into one-week old mice (Connaught). The mice were observed for death up to one week after injection. To study the oncolytic effects of Powassan virus, the virus was either mixed with trans-formed cells and injected subcutaneously into the backs of golden Syrian hamsters, or the virus was injected directly into a tumor, of one to two mm i n diameter. Simian virus kO (SV^O-large plaque strain) was obtained from Dr. Pierre May. It was propagated i n con-fluent cultures of BSC-1 or CV-1 cells, i n an analogous fashion to polyoma. The SYkO was assayed (TCD™) by cytopathogenicity (extreme vacuolation) toward CV-1 cells maintained as monolayers in microtest tissue culture plates (Falcon). Sendai virus was originally obtained from Dr. D. M. McLean. It was propagated in the allantoic cavity of l u -l l day old f e r t i l e chicken eggs. After h& to' 72 hours of infection, the eggs were maintained at k°C for 2 hours to k i l l the embryos. The allantoic f l u i d was then collected and c l a r i f i e d by centrifugation at 600 g for 5 minutes. Virus was pelleted by centrifugation at 29,000 rpm for 60 min. in the Beckman type 30 rotor, and resuspended in one-f i f t h of the original volume of Dulbecco's modified Eagle's medium, supplemented with 10 mg/ml bovine albumin fraction V. The virus was stored at -70°C. Sendai virus was assayed for i n f e c t i v i t y to f e r t i l e eggs, or by hemagglutination of Ufo guinea pig erythrocytes i n microtiter trays as described for polyoma. The microtiter trays were incubated at h°C for 2 hours prior to observation. One hemagglutination unit (HATJ) was defined as the reciprocal of the highest dilution of virus giving complete hemagglutination. Ultraviolet-irradiated Sendai virus was prepared by placing the virus suspension in an open petri dish at a distance of 20 cm from a Sylvania G15T8 germicidal lamp. An exposure of 6 min. reduced the inf e c t i v i t y of the virus 11 2 from 10 egg infectious doses per ml to < 10 , without reducing i t s hemagglutination t i t e r (Figure VI-I). Preparation of Purified Radioactive Polyoma Virus Mouse kidney cultures were infected as previously described. At 2k hours post infection 1-5 uOi/ml of methyl-H-thymidine (Amersham-Searle, specific activity - 1 5 . 6 Ci/ mmole) was added. Infection was allowed to proceed in the presence of the isotope u n t i l about half of the cells had detached (approximately 60 hours after infection). The cells were then harvested by scraping from the dish. The cells plus supernatant were centrifuged at 26,000 rpm in o a Beckman SW27 rotor for 2 hours at 20 C. The virus-con-taining pellet was resuspended in phosphate buffered saline, sonicated for 60 seconds and mixed with solid CsCl to yield a f i n a l density of 1.325 g/c.c. This mixture was then centrifuged for 2k hours at 35,000 rpm in a Beckman SW50.1 rotor at 20°C. Fractions were collected from the bottom of the tube and analyzed for radioactivity and hemagglutin-ation. The peak fractions caontaining f u l l virus particles were pooled and dialyzed against phosphate buffered saline. Uncoating of Polyoma Virus Cells were infected, in the usual manner, with radio-active virus. Following adsorption of virus, the cells were washed three times with phosphate buffered saline, to remove unadsorbed virus. Medium was added and the infection was allowed to proceed. At various times after infection, the medium was removed, the cells were scraped off and r e -suspended in phosphate buffered saline. The cells were lyzed by the addition of sodium deoxycholate (to 0.5$) and sonicated for 20 seconds with the probe of a Bronwill Biosonik II (40$ power setting). The lysate (0.3 mis) was layered onto a 5-30$ sucrose gradient (w/v in phosphate buffered saline) in a SW 50.1 cellulose nitrate tube. The gradient was centrifuged for 20 minutes at 35>000 rpm (at 20°C). Infectious DNA The DNA isolated from polyoma virus, and from fused-c e l l cultures, was assayed for inf e c t i v i t y by plaque formation in secondary mouse embryo cultures, using diethylaminoethyl-dextran (100 |_ig/6o ™* petri dish culture) as described by Warden and Thorne (1968). Methods of Virus Induction a. Mitomycin C treatment: Transformed-cell cultures were exposed to a range of mitomycin C concentrations, from 0.05 M-g/ml to 10 ug/ml. The concentrated stock solution was prepared fresh for each experiment, in phosphate buffered saline (pH 7.2). Treatment was for 8 or 20 hours in the dark. After this time the c e l l sheets were washed and standard medium containing 2.5$ calf serum was added. The cultures were harvested 5 days later for virus assays. In some experiments the mitomycin C treated cells were harvested, by trypsiniz-ation, and fused or co-cultivated with mouse kidney cells as described in a later section, and then assayed for virus 5 days later. b. Temperature changes: Cultures of transformed cells, grown to about 80$ confluency at 37°C, were transferred to hi. 5°C for various periods of time, followed by further incubation at 37°C with or without an intermittent period at h°C. After h days at 37°C the cultures were harvested and assayed for virus. In one case, cultures were exposed to 45°C for 30 min. followed by h days at 37°C. c. Ultraviolet Irradiation: Confluent cultures of trans-formed cells were washed with phosphate-buffered saline (pH 7-2). The phosphate-buffered saline was poured off and the cells were irradiated at a distance of 20 cm. from a Sylvania G15T8 germicidal lamp, for various lengths of time, resulting in a range of c e l l survival from 95$ to 10$. After the irradiation, standard medium containing 2.5$ calf serum was added, and the cultures were incubated at 37°C for four days before harvesting and assaying for virus. d. C e l l Fusion The procedure was essentially that described by Harris (1966). Approximately 1 x 10 cells of the permissive type (CV-1 for SVkO; mouse kidney or mouse embryo for polyoma) were mixed with approximately 5 x 10^ cells of the trans-formed line, and UV-inactivated Sendai virus was added (5,000 to 10,000 HAU). This mixture was incubated at k°C for 15 min., with occasional agitation, to allow adsorption of virus to cells, followed by incubation at 37°C for 20 min. The c e l l aggregates were pelleted by centrifugation at 300 g for two minutes and gently resuspended in growth medium with 10$ calf serum, and plated. After 12 hours, at time sufficient to allow for complete attachment of polykaryons, the medium was removed and replaced with fresh medium con-taining 2$ calf serum. Usually about 6of0 of the plated cells had fused into polykaryons. The cells were harvested (by scraping from the plates) 5 to 7 days after fusion and assayed for virus. For the experiments in which v i r a l DNA was to be extracted from fused-cell cultures, the cultures were incubated with 5 u-Ci/ml of methyl- H-thymidine (Amersham-Searle, specific activity 20 Ci/mmole) between 2k and 29 hours after plating, followed by 45 minutes in non-radio-active medium. For experiments in which the heterokaryons were to be used for autoradiography and immunofluorescence, they were treated as indicated in those specific sections. Tumor Production and Histological Studies Tumors were produced in golden Syrian hamsters by sub-cutaneous injection of either polyoma virus transformed cells, ranging from ten to a million cells per hamster, 7 or by approximately 10 pfu of polyoma virus per hamster. For histological studies, tumors were removed from the animals and were cut into five mm cubes, and placed in Bouin's fixative for twenty-four hours. The tissue 45 was then washed, in 7 0 $ ethanol for a further twenty-four hours (three changes), then dehydrated in absolute alcohol, and embedded in paraffin. Tissue sections of six microns thickness were cut on a Spencer 820 microtome (American Optical), and floated onto water (45°C) to flatten the sections. The sections were then floated onto albumenized slides and allowed to dry for six hours, followed by removal of paraffin, dehydration, and staining in Harris's Hema-toxylin for ten minutes; and counterstained for one minute with one percent aqueous eosin. Finally the sections were allowed to dry and mounted in permount. Chromosome Studies Cells, of which chromosomes numbers were to be deter-mined, were grown to approximately 5 0 $ confluency and then were treated with 0 . 2 |_ig/ml of colcemid for eight to ten hours. The medium was then removed and the c e l l monolayer was treated for fifteen minutes with ten mis of one percent sodium citrate. The mitotic cells were suspended in the solution by gentle pipetting over the monolayer, centrifuged for three minutes at 800 rpm (international centrifuge type CS rotor # 2 4 0 ) , and then resuspended in 0 . 3 ml of fresh sodium citrate. The cells were fixed by the slow addition of ethanol-glacial acetic acid fixative (3:1) with constant gentle shaking. After ten mis of fixative had been added the centrifuge tube was inverted once and centrifuged at low speed. The supernatant was poured off and the cells resuspended in fresh fixative for a further ten minutes, followed by further centrifugation and resuspension of the pellet to the desired c e l l density in fresh fixative. A few drops of this c e l l suspension were placed on a glass slide and allowed to air dry before staining with Giemsa. Preparation of Antiserum against Polyoma Capsid Protein Crude lysates of infected mouse kidney cultures were sonicated for 30 seconds with the probe of a Bronwill Biosonik II (koi power setting). The crude lysate (20,000 HAU/ml) was emulsified with an equal volume of complete Freund's adjuvant. The emulsion was injected into the thigh muscles of six-week-old female mice (0.2 mis per mouse). Ten and 20 days after the intramuscular inoculation, the mice were inoculated intraperitoneally with 0.2 mis of freshly prepared emulsion. Commencing ten days after the last injection, the mice were bled from the eye every second day for one month. Antiserum was also obtained by injecting ascites tumor cells into the peritoneal cavity 10 days after the third injection of polyoma virus. A week later the ascitic f l u i d was removed, cla r i f i e d , and the serum collected. This method yielded a much larger amount of serum per mouse. The serum was heated at 56°C for 30 minutes and stored at -20°C. The hemagglutination inhibition t i t r e of the serum obtained by the f i r s t method was 52,000 Hi/ml and that of the second method was kO,000 Hi/ml, as determined against 8 HAU of polyoma virus. Immunofluorescence Tests for Polyoma Capsid Protein In order to study the intracellular localization of v i r a l capsid protein, cells were grown and infected on glass coverslips (11 x 2k mm), in plastic petri dishes. The infected cells were washed once in phosphate buffered saline (pH 7.2) and fixed in acetone, for 10 minutes at k°C. The coverslips containing the fused cells, were then washed once in phosphate buffered saline and immersed in 95$ ethanol at room temperature for 7 minutes. The coverslips were then washed three times with phosphate buffered saline and incubated with mouse antiserum directed against polyoma capsid protein. Incubation was carried out for 30 minutes at 37°C in a water-saturated atmosphere. After incubation the coverslips were washed four times in phosphate buffered saline and incubated for a further 30 minutes at 37°C with fluorescein labelled 7G immunoglobulin directed against mouse yG immunoglobulin (Hyland, Goat/anti-mouse). The coverslips were washed four times in phosphate buffered saline, dried, mounted with fluormount on glass slides, and observed with a Reichert microscope equipped with a fluorescence attachment. Autoradiography The cells were cultivated on coverslips in plastic petri dishes and incubated with 2 - 5 M-Ci/ml of methyl- H-thymidine (Amersham-Searle, 15.6 Ci/mmole)for the indicated times. Two methods of fixation were employed depending upon the nature of the subsequent operations. If the combined technique of immunofluorescence-autoradiography was to be conducted on the cells, then fixation as described for immunofluorescence was employed (see previous section). If autoradiography alone was analyzed, the cells were fixed at room temperature, for 15 minutes, in a solution of gla c i a l acetic acid - 95$ ethanol (1:3 v/v). The cover-slips were then rinsed for 2 hours with d i s t i l l e d water. The coverslips, containing fixed cells, were dried and mounted with permount onto glass slides. The slides were dipped into Ilford L-k l i q u i d emulsion (1.5 g emulsion to 1 g water) drained, and allowed to dry. The emulsion coated slides were stored for a week at room temperature in the presence of drierite, to allow exposure of the emulsion. After exposure, the photographic emulsion was developed for 12 minutes in Microdiol X:HgO (1:3 v/v) developer. The slides were then washed for 5 minutes in water, fixed for 5 minutes in regular thiosulfate fixative, washed in water, dried and observed. If more cytoplasmic detail was desired the cells were stained through the emulsion with aqueous eosin (l$). Electron Microscopy Carbon coated copper grids containing a formvar support f i l m were floated on a drop of virus for one minute. The grid with the virus was then floated on a drop of 1$ phosphotungstic acid (pH 6.4) for one minute, dried, and observed under a Philips 300 electron microscope. Extraction and Purification of V i r a l DNA Cultures were washed three times with phosphate buffered saline (pH 7.2) and the cells were lyzed by the addition of 1.0 ml of 0.5$ sodium dodecyl sulphate (SDS) in 0.01 M tris-chloride pH 7.5 and 0.01 M disodium-ethylene-diamine-tetracetate. High molecular weight cellular DNA was then precipitated and removed by the technique of Hirt (1967). The low molecular weight DNA fraction was then processed in the manner described by Sambrook and Shatkin (1969). Thus the DNA was extracted with phenol, saturated with 1 M tris-chloride (pH 8.0). The f i n a l aqueous phase was then dialyzed against 0.1 x standard saline citrate (SSC) to remove phenol. Solid CsCl was added to the DNA together with 100 ug/ml ethidium bromide to give a f i n a l density of 1.60 gms/ml. The mixture was centrifuged in the Beckman type 50 rotor at 42,000 rpm for 36 hours. The lower band of v i r a l DNA was collected and the ethidium bromide was removed by two or three extractions with iso-propanol (saturated with CsCl). Finally the DNA was dia-lyzed against 0.1 x SSC and stored at -70°C. Radioactive v i r a l DNA was obtained by adding 5 - 1 0 uCi/ml of methyl- H-thymidine (specific activity 15.6 Ci/ mmole - 49-2 Ci/mmole) to infected mouse kidney cultures 26-32 hours post infection. The labelling period was followed by a 45 min chase after which the v i r a l DNA was extracted and purified as above. Extraction and Purification of Mammalian DNA The procedure used was based upon the methods described by Marmur (1963), Saito and Miura (1963) and Hudson (1971). Tissue homogenates were lyzed by adding ten volumes of t r i s -SDS buffer (0.1 M t r i s ; 0.1 M NaCl, adjusted to pH 9.0 and made 1$ in SDS), i.e. approximately 10 mis per gram of packed cells or wet tissue. Cells from petri dish cultures (9 cm diameter) were lyzed by addition of 1 ml of 0.6$ w/v SDS in 0.01 M tris-chloride pH 7.5 and 0.01 M disodium-ethylene-diamine-tetracetate per petri dish. The viscous lysate was then repeatedly extracted with buffer-saturated phenol, at 4°C, u n t i l the interphase material resulting from low speed centrifugation had diminished to a minimum (usually three to four extractions). Two volumes of ethanol were gently stirred into the f i n a l aqueous phase and the precipitating DNA wound onto a glass rod. This fibrous precipitate was then dissolved in 0.1 x SSC (one-half of the original volume of lysate) and brought to 1.0 x SSC. Pan-creatic ribonuclease (previously heated at 80°C for 15 min. to inactivate possible traces of DNase) was added to 50 fig/ ml and the mixture incubated at 37°C for 30 minutes. This was followed by the addition of fungal protease (Sigma Chemical Co.) at 50 ug/ml for 2 hours at 37°C. The pro-tease had been previously incubated at 37°C for 2 hours to digest possible DNase activity. Sodium dodecyl sulfate was added to 1% and further phenol (saturated with 1 x SSC) extractions performed u n t i l no further interphase material remained upon centrifuging. Following this the DNA was again spooled by ethanol precipitation. The DNA was dissolved in 0.1 x SSC (one-quarter of the original lysate volume), dialyzed against 0.1 x SSC, and centrifuged in CsCl density 1.70 gms/ml in the Beckman No. 50 rotor at 42,000 rpm for 36 hours. The main part of the DNA band was then taken and dialyzed against 0.1 x SSC. Alkaline Sucrose Gradient Sedimentation of C e l l DNA and  Polyoma DNA Mammalian cells (about 5 x 10 ) were harvested by trypsinization and resuspended in 0.25 ml of PBS- (phosphate buffered saline minus divalent cations, pH 7 .2). The cells •were gently layered onto 1.0 ml of lyzing solution (0.5 M NaOH, 0.01 M EDTA 0.2$ SDS) on top of a 36 ml gradient of 10-30$ sucrose (w/v in 0.3 M NaOH, 0.001 M EDTA, 0.01$ SDS) in a Beckman SW27 polyallomer tube. After 12 hours at 20°C, the gradient was centrifuged for 5 hours, at 25,000 rpm, at 20°C. Fractions were collected from the top of the tube, by pumping 50$ sucrose into the bottom of the tube. Controls were conducted in the same manner except that labelled c e l l or virus DNA was used, as indicated in the individual experiments. DNA-DNA Hybridization Viral-specific DNA was measured by the DNA-DNA hybrid-ization technique of Denhardt (1966), using the modifications described by Aloni et a l (1969). Polyoma or SVkO DNA type 1 was denatured in 0.1 x SSC by heating at 98-100°C for 20 minutes. Cellular DNA was denatured in 0.1 x SSC by heating at 98-100°C for 10 minutes. The denatured DNA solutions were then chilled in ice, brought to 3 x SSC and fixed on Millipore HAWP membranes (47 mm diameter) by gravity f i l t r a -tion (Hudson, 1971)• Small circles (6.5 mm diameter) were cut out by means of a hand paper punch, dried at room temperature, and then baked at 80°C for k hours. Before the hybridization reaction, the f i l t e r s were pre-incubated in 3 x SSC, containing 0.04$ bovine serum albumin (Fraction V), at 6o°C for 6 hours. Blank f i l t e r s (without DNA or with other mammalian DNA) were treated identically. The radioactive DNA preparation was then denatured as above and added to the reaction mixture in a small volume (usually 0. 2 ml to a 1. 5 nil mixture containing two DNA-loaded f i l t e r s and one blank f i l t e r ) . This was followed by incubation at 60% for a further 214-hour s. In experiments designed to detect v i r a l DNA integration into host c e l l DNA, the salt concentration for binding the DNA and during'hybridization was increased to 6 x SSC. The radioactive v i r a l DNA was sonicated to fragment i t , prior to denaturation. This was found to increase the efficiency of hybridization. The balance of the hybridization technique was the same as described above. After the incubation period the f i l t e r s were removed and extensively washed with 1 x 10 M t r i s (pH 9.k). Thermal Deraturation of Polyoma DMA-Cell DNA Complexes Nitrocellulose f i l t e r s , containing infected c e l l DNA-t r i t i a t e d polyoma DNA complexes, were washed in 2 x SSC and then were incubated for 1 0 minute periods at successively increasing temperatures. The incubation medium comprised 0 . 5 ml of 2 x SSC. The radioactive DNA eluted at each temperature was precipitated by trichloracetic acid and collected on membrane f i l t e r s for radioactivity measurement. References to Chapter II Aloni, Y., E. Winocour, L. Sachs and J. Torten. 1969. Hybridization between SVl+O DNA and cellular DNA's. J. Mol. Biol, kh: 333-345. Crawford, L.V. 19^9- Purification of polyoma virus. In: Fundamental Techniques in Virology. Edited by K. Habel and N.P. Salzman. Academic Press p. 75. Derihardt, D.T. 1966. A membrane f i l t e r technique for the detection of complementary DNA. Biochem. Biophys. Res. Comm. 23: 61+1-646. Dubbs, D.R., S. Kit, R.A. DeTorres and M. Anken. 1967. Virogenic properties of bromodeoxyuridine sensitive and bromodeoxyuridine resistant simian virus-kO transformed mouse kidney cel l s . J. Vi r o l . 1: 968-979. Dulbecco, R. and M. Vogt. 195*+. Plaque formation and isolation of pure lines with poliomyelitis virus. J. Expt. Med. 99: 167-182. Harris, H., J.F. Watkins, C.E. Ford, and G.I. Schoefl. 1966. A r t i f i c i a l heterokaryons of animal cells from different species. J. Ce l l Sci. 1: 1-30. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse c e l l cultures. J. Mol. Biol. 26: 365-366. 57 Hudson, J.B. 1971. The fixation and retention of v i r a l and mammalian deoxyribonucleic-acids on nitrocellulose f i l t e r s . Can. J. Biochem. 4_9_: 631-636. Hudson, J.B. 1972. Properties of polyoma virus-transformed cells II. Characteristics of the virus specific RNA. Can. J. Micro. 18: 247-254. Kohse, L.M., L. McGrath, and J.B. Hudson. 1971. The recovery of polyoma virus from infected mouse cells: relevance to virus purification. Can. J. Microbiol 17: 747-751. Marmur, J. 1963. A procedure for the isolation of deoxy-ribonucleic acid. In: Methods i n enzymology. Vol. v i . 726-738. Saito, H., and K. Miura. 19&3- Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochem. Biophys. Acta 72: 619-629. Sambrook, J. and A.J. Shatkin. 1969. Polynucleotide ligase activity in cells infected with simian virus 40, polyoma virus, or vaccinia virus. J. Vi r o l . 4: 719-726. Warden, D. and H.V. Thorne. I968. The inf e c t i v i t y of polyoma virus DNA for mouse embryo cells in the presence of diethylaminethyldextran. J. Gen. Virol. 3: 371-377. CHAPTER III ONCOGENIC PROPERTIES OF POLYOMA VIRUS TRANSFORMED CELLS Abstract Studies were made on the oncogenic properties of a line of polyoma virus-transformed hamster cells. Passage of the cells in vivo resulted in an increase i n oncogenicity. Thus the latent period for the appearance of a tumor was decreased, and as few as five to ten cells were now sufficient to produce a tumor in a young hamster. Tumors were invari-ably restricted to the subcutaneous site of injection. In contrast, polyoma virus i t s e l f caused widespread production of tumors throughout the animal. Histological examination of tumor tissue revealed marked differences between tumors caused by virus or virus-transformed cells. It was concluded that l i t t l e or no metastasis of transformed cells could have occurred, and that insignificant rescue of the polyoma virus genome must have been induced in transformed cell-tumors. Powassan virus, a group B arbovirus, did not show any oncolytic activity toward tumors induced by the polyoma virus-transformed ce l l s . Introduction Polyoma virus is known to induce a variety of tumors in rodents, especially hamsters (Eddy, 1969). Multiplication of the virus however, requires cells of murine origin, although hamster cells can support a limited amount of multiplication (Vogt and Dulbecco, i960), probably enough to account for the wide spread of tumors in the hamster. Ce l l lines can be derived from the virus-induced tumors, or from cells transformed i n culture, and such lines frequently show evidence for persistent v i r a l genes, despite their lack of production of virions (Axelrod et_ al, I96I+; Sambrook et_ al, I968). These transformed cells show a high degree of onco-genicity toward hamsters. In contrast to simian-virus k-0 transformed c e l l lines however, l i t t l e success has been achieved in attempts to rescue polyoma virus (Fogel and Sachs, 1970). The studies described here were designed to examine the oncogenic properties of polyoma virus transformed cells (PyH-l), by transplantation. Tumors were also produced by subcutaneous injection of polyoma virus into baby hamsters. Histological studies were conducted to compare the tumors produced by trans-planting transformed cells and the tumors produced by free virus. In addition, Powassan virus, a group B arbovirus, was tested for a possible oncolytic effect on tumors produced by polyoma virus transformed cells. Results Tumor Production in Hamsters by Transformed Cells Subcutaneous injection of transformed cells into hamsters resulted in a rapid production of tumors (Fig. I I I - l ) . The time required to f i r s t detect tumor appearance was directly related to the age of the animal at the time of i n -jection as well as to the dose of cells used. Figure III-I indicates that i f transformed cells were injected into a three-week old hamster, tumors appeared approximately ten days earlier than i f an equivalent number of cells were injected into a five-week old hamster. There was extremely good correlation between the dose of cells injected and the time of tumor appearance within families as well as between different families. Under no circumstances did a hamster with a smaller dose of cells (by one log) show evidence of a tumor before a hamster with a larger dose. No tumors were ever obtained (within an observation period of 5 months), i f 100 cells or less of the PyH-1 c e l l line were injected. Cells derived from the PyH-1 c e l l transplanted tumors were cultivated in vitro (Materials and Methods) and reinjected into three-week old hamsters. Tumors were produced by five Figure III-I: Time of appearance of tumor following injection of polyoma virus transformed cells O O Three-week-old hamsters, PyH-1 cells; o -O five-week-old hamsters, PyH-1 cells; * * three-week-old hamsters, HT-23 cells (in vivo passaged PyH-l). Three separate animals were used for each dose of cells. C e l l numbers were estimated by direct counting of the original suspension in a hemacytometer, followed by appropriate dilution. L o g 1 0 N o . o f c e l l s / h a m s t e r to ten transplanted cells as compared to over 100 cells required to produce a tumor by the PyH-1 c e l l line before transplantation. This increase in oncogenicity required only one in vivo passage. The effect was reproduced in three separate experiments using different families. Tumors produced by the injection of transformed cells occurred only at the site of injection. Tumors never metastasize even i f they were allowed to grow to an extremely large size, e.g. 96 gram tumor i n a 70 gram hamster (Plate I I I - l ) . Surgical removal of part of the encapsidated tumor was expected to increase the probability of metastasis but did not do so in this case. Due to the fact that the tumors were so well encapsidated the entire tumor could be removed, resulting i n an apparently healthy animal. Chromosome studies were conducted on the original transformed c e l l line (PyH'-l) and on c e l l lines recovered after in vivo passage (transplantation). No significant change in the chromosome complement was observed. A l l of the cells tested contained between k-2 and kk chromosomes per c e l l . The increase in the oncogenicity of the in vivo-passaged line (HT-23) did not therefore appear to be due to any selection of cells with different chromosome Plate III-I: Tumor in a hamster induced by polyoma virus transformed cel l s . numbers. These studies also indicate that the transform-ation of hamster cells by polyoma virus did not alter the chromosome complement of the golden Syrian hamster (kk chromosomes). Tumor Production in Hamsters by Polyoma Virus Efficient production of tumors in hamsters by the injection of polyoma virus required the use of animals k8 hours of age or less. Table III-I indicates the relative efficiency of tumor production in hamsters of various ages. If hamsters were less than hQ hours of age at the time of injection of virus they a l l died by six to eight weeks. Death was probably caused by c e l l damage in the organs where tumor production occurred. These sites of tumor production included lungs, l i v e r and kidneys as well as subcutaneous at the site of injection. The growth of tumors in the organs was very rapid, causing a breakdown of the stroma with subsequent internal hemorrhage and thrombosis. A l l animals upon autopsy showed extensive hemorrhage with as much as three to four mis of blood and f l u i d present in the body cavity. The size of tumors in the organs at the time of death varied from minute to five Table III-I: Susceptibility of hamsters of various ages to tumor production by polyoma virus (10 pfu/hamster) A .£> v . 4- No. animals /„ . . . ,„ Age of hamster . . , /Wo. injected* producing tumors/ 2h hours 10/10 (100$) 1+8 hours 13/13 (100$) 100 hours 1/6 ( 16$) 200 hours 0/15 ' ( o$) * Numbers in parentheses indicate the percentage of the injected animals that produced tumors. mm in diameter, whereas the tumors at the site of injection reached ten mm. Hamsters older than k& hours at the time of injection developed tumors at the site of injection as well as other subcutaneous sites, but no tumors were found in internal organs. Due to the lack of involvement of the internal organs of older animals the subcutaneous tumors could be removed surgically. Tumors would then reappear at other subcutaneous sites and the animal could never be completely cured, in contrast to tumors induced by trans-formed cel l s . Histological Examination of Tumor Tissue Histological studies were conducted on tumors produced by injection of transformed cells as well as by the injection of polyoma virus. Plate III-II is a section of a tumor induced by the injection of transformed cells. Very few mitotic figures are seen in the tissue, which is character-i s t i c of benign tumors. A l l the tumors induced by trans-formed cells had this same characteristic appearance. They were extremely well vascularized throughout, allowing the tumors to reach enormous size. Some of the larger tumors showed evidence of necrosis in the centre of the large mass of undifferentiated tissue. 6 9 Plate I I I - I I : H i s t o l o g i c a l section of a tumor induced by polyoma v i r u s transformed c e l l s . H + E s t a i n (Mag. 400x). Plate I I I - I I I : H i s t o l o g i c a l section of a tumor induced by polyoma v i r u s H + E s t a i n (Mag. 400x). Plate III-III is a section of a tumor induced by polyoma virus. The tissue contains a large number of cells with abnormal nuclei and many mitotic figures, which is character-i s t i c of malignant tissue. The tissue appears to be of li v e r origin even though i t was obtained from a lung tumor. It appears that the virus transformed the l i v e r cells, which then metastasized to the lung and developed tumors at this site. Attempts to Demonstrate an Oncolytic Effect of Powassan Virus Certain members of the group B arboviruses have been shown to be oncolytic (Moore, i960). Powassan virus, another member of the group B arboviruses, i s not lethal i n hamsters (McLean, et a l , i960); therefore oncolysis would result in survival of the animal. The virus therefore was f i r s t adapted to grow in the transformed cells, and then used in a series of experiments to determine whether tumor production by polyoma virus transformed cells could be prevented; or i f tumors already established could be caused to regress. Table III-II indicates that the simultaneous injection of 7 Powassan virus, at concentrations as high as 10 LD^QJ A N C ^ polyoma virus transformed cells, did not have any delaying effects on tumor production. In another experiment, Powassan virus was injected directly into the interior of established tumors (1-2 cm in diameter). However, the rate of tumor growth did not decrease, indicating again a lack of oncolytic effect. Table III-II: Test for oncolytic effect of Powassan virus on tumors induced by polyoma virus-transformed cells (PyH-l). No. of transformed cells Powassan virus Tumor appearance Oncolytic injected/hamster d- o s e (days)* effect 10 5 0 30 - 33 10 5 10 7 i o 6 30 - 31 10 5 30 - 32 10 5 10 5 29 - 32 0 0 -* days after injection of transformed cells Discussion Normally v i r a l transformation of cultured cells is associated with chromosomal aberrations (Stich and Yohn, 1965), but in polyoma virus transformed cells there appears to be a lack of any specific aberration or change in any marker chromosome (Defendi and Lehman, 1966). The fact that i n vivo passage of tumor cells resulted in a much higher degree of oncogenicity, with no significant change in chromo-somal complements, suggests that this increase in onco-genicity was not due to a selection of cells, from a hetero-geneous population, with the inherent a b i l i t y to multiply more rapidly. However, this cannot be ruled out. It appears that a l l the cells present underwent this transition from low to high oncogenicity. Polyoma virus transformed cells, upon injection into hamsters, did not produce metastases as do other malignant tumors but were instead encapsulated in a series of layers of fibroblast-like cells. Since the entire tumor was so well contained, surgical removal resulted in an apparently healthy animal. In contrast, surgical removal of tumors produced by virus did not result in a healthy animal. Tumors subsequently appeared at other sites. A possible reason for the i n a b i l i t y to cure a hamster of tumors induced by virus could be that a large number of cells were transformed at various subcutaneous sites. Alternatively, the cells transformed at the site of injection may have been capable of metastasizing. From these studies, i t appears that metastasis occurred at a very early stage after in vivo transformation by polyoma virus, since at later stages the tumors were encapsidated just as were the ones produced by the polyoma virus transformed cel l s . The latter did not show metastasis. Histological examination indicated another striking difference between the tumor tissue induced by polyoma virus transformed cells on the one hand and by polyoma virus on the other. Virus induced tumors consistently showed a larger number of mitotic figures and some anaplasia, features characteristic of malignant tissue, whereas the transformed c e l l tumor tissue was much more uniform and characteristic of benign tumors. Cultures could be derived from the latter much more easily. Viruses are known to be selective in the type of cells in which they multiply and i t has been long hoped that this specificity could be used for the destruction of cancer cell s . One of the problems with v i r a l oncolysis has been the fact that most highly oncolytic viruses are markedly lethal for the host (Lindenmann and Klein, 1967). An ideal situation would he one wherein the virus is oncolytic but has no i l l effects on the host; to satisfy these parameters one realises that the l i s t of virus candidates available is extremely small. A. number of the group B arboviruses (Moore, i960) have been shown to be oncolytic, therefore Powassan virus, a group B arbovirus, which produces a viremia in hamsters but has no i l l effects (McLean, i960), was used in this study to determine i t s oncolytic effect. Although the virus was capable of multiplying i n these tumor cells, after adaptation, i t did not produce any noticeable tumor regression. It was possible that virus multiplication within the tumor cells might cause some antigenic change in the tumor cells, resulting in tumor rejection by the host (Webb and Smith, 1970). Although these studies with Powassan virus have proven to be negative, i t would seem feasible to approach the study of tumor rejection and regression from the aspects of. oncolysis and alteration of the antigenic properties of the tumor cells i n such a way that the natural immunological response could assist in tumor rejection. References to Chapter III Axelrod, D., K. Habel, and E.T. Ballan. 196k. Polyoma genetic material i n a virus free polyoma induced tumor. Science 146: 1466-1468. Crawford, L.V. 1969. Purification of polyoma virus. In: Fundamental techniques in virology. Edited by: K. Habel, and W.P. Salzman, Academic Press, p. 75. Defendi, V. and J.M. Lehman. 1966. Biological characteristics of primary tumors induced by polyoma virus in hamsters. Inst. J. Cancer. 1: 525-540. Eddy, B.E. 19^9 • Polyoma virus. V i r o l . Monogr. 7: I-96. Fogel, M. and L. Sachs. 1970. Induction of virus synthesis in polyoma transformed cells by ultraviolet light and Mitomycin C. Virology 40: 174-177. Lindenmann, J. and P.A. Klein. 1967. Immunological aspects of v i r a l oncolysis. Recent Results Cancer Res. 9 : 1-84. McLean, D.M., L.W. Macpherson, S.J. Walter and G. Funk. i960. Powassan virus: surveys of human and animal sera. Amer. J. Pub. Health 50: 1539-1544. Moore, A.E. i960. The oncolytic viruses. Progr. Exp. Tumor Res. 1: 411-439. Sambrook, J., H. Westphal, P.R. Srinivasan and R. Dulbecco. 1968. The integrated state of v i r a l DNA i n SV40-trans-formed cells. Proc. Nat. Acad. Sci. 60: 1288-1295. Stich, H.F. and D.S. Yohn. 19^5. Viruses and mammalian chromosomes: V. Chromosome aberrations in tumors of Syrian hamsters induced by adenovirus type 12. J. Nat. Cancer Inst. 35j 603-615. Vogt, M. and R. Dulbecco. i960. Virus-cell interaction with a tumor-producing virus. Proc. Nat. Acad. Sci. 46: 365-370. Webb, H.E., and C.E.G. Smith. 1970. Viruses in the treatment of cancer. Lancet, 1: 1206-1208. CHAPTER IV THE STATE OF THE VIRAL GENES IN TRANSFORMED CELLS Abstract A variety of treatments (ultraviolet irradiation; mitomycin C; heat shocks; c e l l fusion) proved unsuccessful in attempts to rescue infectious polyoma virus from virus-transformed cel l s . In order to test the possi b i l i t y that incomplete or otherwise defective v i r a l genomes were r e p l i -cating after giving the above treatments, DNA-DNA hybridiz-ation tests were carried out. It was concluded from these tests that the amount of polyoma DNA synthesis taking place must have been not more than 0.1 per cent of the level found in productively infected mouse cells. In analogous experiments, using simian virus ho (SVkO)-transformed cultures, the rescue process was followed by readily detectable synthesis of SVkO DNA. Thus the block in virus production i n these polyoma virus-transformed cells apparently i s exerted at a step prior to replication of v i r a l DNA. The a b i l i t y of heterokaryons (mouse kidney-PyH-1 cells or mouse embryo-'normal' hamster cells, to support virus multiplication was tested. The presence of hamster nuclei and cytoplasm, in heterokaryons, proved inhibitory to polyoma virus multiplication. This inhibitory effect was more pronounced i f the hamster cells had been transformed by polyoma virus. Introduction Cells transformed by polyoma virus are characterized by the presence of v i r a l specific antigens (reviewed by Black, 1968) and the continuous synthesis of vir a l - s p e c i f i c RNA (Benjamin, 1966; Hudson, 1972). Recently polyoma DNA has been demonstrated to occur, in at least a fraction of the cells of a transformed culture, i n an alkali-stable linkage with host DNA (Sambrook et al , 1968). This had added weight to the concept of analogy with the lysogenic state of bacterial viruses such as lambda (Dulbecco, 1969). Inherent i n this analogy is the prediction that i t should be possible to induce the latent provirus to become autonomous and replicate in an appropriate environment, and indeed this has been accomplished frequently i n the case of simian virus hO (SVkO) from many of i t s transformed cultures (Koprowski et a l , 1967; Watkins and Dulbecco, 1967; Kit et al, 1968). Polyoma virus has rarely been induced or "rescued". In cases where polyoma virus has been rescued, i t has been from 'leaky' transformants, i.e. transformed cells from which small amounts of virus was synthesized and released continuously (Fogel and Sachs, 19°9, 1970). This chapter describes attempts to induce synthesis of virus and/or v i r a l DNA from cultures known to harbour polyoma genetic information. An easily inducible line of SVkO-transformed cells was used as a positive control. Heterokaryons produced by fusion between permissive cells (mouse) and non-permissive cells (hamster) were examined for their a b i l i t y to support polyoma virus multi-plication. The identification of specific c e l l types in a heterokaryon was made by autoradiography, one of the c e l l types having been prelabelled with t r i t i a t e d thymidine. Results Attempts to Rescue Infectious Polyoma Virus from Transformed  C e l l s A v a r i e t y of methods was used i n attempts to induce the production of i n f e c t i o u s polyoma v i r u s , as measured by plaque formation, from the o r i g i n a l l i n e s of transformed c e l l s (PyH-1 and TT 109), and the l i n e s derived from tran s p l a n t a t i o n of PyH-1 c e l l s into hamsters (HT-20 and HT-23). Table IV-I summarizes the e s s e n t i a l r e s u l t s . U l t r a v i o l e t i r r a d i a t i o n r e s u l t e d i n progressively greater c e l l u l a r damage at increas-ing doses, ranging from 10$-90$ c e l l death, but no v i r u s was rescued. A v a r i e t y of temperature shocks was employed (as described i n the Methods and Materials section), r e s u l t i n g i n d i f f e r e n t l e v e l s of c e l l s u r v i v a l , but without y i e l d i n g i n f e c t i o u s v i r u s . Mitomycin-C over a wide range of concen-t r a t i o n , incubated with the transformed c e l l s f o r eit h e r 8 hours or 20 hours, also proved negative. U l t r a v i o l e t i n a c t i v a t e d Sendai v i r u s (Plate IV-I) i r r a d i a t e d c e l l f u s i o n has previously been used to rescue SSfkO v i r u s from SVkO v i r u s transformed c e l l s ( K i t et a l , I968). A t y p i c a l UV i n a c t i v a t i o n curve f o r Sendai v i r u s Table IV-I: Summary of attempts to rescue polyoma virus Treatment Virus Recovered' Ultraviolet (0 - 6k sec.) 0 Heat shock (la.5°C + 45°C) 0 Mitomycin-C (0.05 ng/ml - 10 ng/ml) 0 C e l l fusion (Sendai) 0 Amount of virus recovered as measured by plaque formation on mouse embryo cultures. 8k i s illustrated in Figure IV-I. Ultraviolet irradiation for six minutes, resulted i n almost complete inactivation of virus i n f e c t i v i t y without a detectable decrease in the hema-gglutination t i t r e and presumably the c e l l fusion potential. Plate IV-II illustrates an example of polykaryon production by Sendai virus induced c e l l fusion. A series of c e l l fusion experiments (Table IV-Il) was performed, using primary mouse kidney cells as the permissive c e l l type for polyoma virus. In some cases, mitomycin C was used as an accessory treatment to c e l l fusion. It was considered possible that the mitomycin C might success-f u l l y rescue the polyoma genome, but that an additional requirement for actual multiplication would be the presence of a permissive c e l l component or factor. The results were again negative however. Thus the v i r a l genes present in the PyH-1 cells do not appear to be capable of producing infectious virus particles. Furthermore, the increased tumorgenicity obtained by trans-plantation of the transformed cells (i.e. lines HT 20 and HT 23) was not accompanied by inducibility of the virus. As a control experiment, SV^O-transformed cells of the MKS-A line were fused or co-cultivated, under standard conditions, with SV^O-permissive cells of the CV-1 line. Figure IV-I: Radiation dose response of Sendai virus to ultraviolet irradiation: 1 . 5 mis of Sendai virus suspension, in a 60 mm petri dish, irradiated by a Sylvania germicidal lamp G15T8, at a distance of 20 cm. 88 Plate IV-II; Polykaryon produced by c e l l fusion. Table IV-II: Effect of mitomycin-C treatment for 8 hrs. followed by c e l l fusion with mouse kidney cells. Treatment Virus recovered per culture HT-20 + mouse kidney fused 0 HT-20 + mouse kidney cocultivated 0 HT-20 (Mit. C) + mouse kidney fused 0 HT-23 mouse kidney fused 0 HT-23 (Mit. C) + mouse kidney fused 0 PyH-1 + mouse kidney fused 0 PyH-1 (Mit. C) + mouse kidney fused 0 PyH-1 + mouse kidney cocultivated 0 TT109 + mouse kidney fused 0 TT109 + mouse kidney cocultivated 0 MKSA + CV-1 fused 1 0 5 TCD c n 50 Amount of virus recovered as measured by plaque formation on mouse embryo cultures. Mitomycin C concentration was 0.3 M-g/ml and 0. 5 f-ig/ml. Infectious SVkO was readily rescued, about 10 TCD,_Q per culture in the case of c e l l fusion (Table IV-II). V i r a l DNA Synthesis Despite the absence of infectious virus production, i t was s t i l l conceivable that the polyoma virus transformed cells might be capable of synthesizing v i r a l DNA which was subsequently not incorporated into virions, or that an i n -complete v i r a l genome was replicated, giving rise to non-infectious DNA. To examine such p o s s i b i l i t i e s , cultures of PyH-1 cells (subjected to various treatments) were exposed to methyl- H-thymidine (as described in the Methods and Materials section) and the cells were fractionated into high molecular weight DNA and low molecular weight DNA fractions by the technique of Hirt (1967). By means of this technique, (described i n the Methods and Materials section) i t is possible to separate virus DNA from cellular DNA on the basis of their size differences. In the same experiment, confluent cultures of primary mouse kidney cells were infected with polyoma virus (about 20 pfu/cell), incubated with methyl- H-thymidine, and fractionated in the same manner. Portions of each low molecular weight DNA fraction were centrifuged in alkaline sucrose gradients. Figure TV-II displays the results. The productively infected mouse kidney cells show the typical profile of denatured polyoma DNA type 1 (50s) and the de-natured forms of types II and III DNA ( l 6 s and l 8 s ) . It is evident that the treated PyH-1 cultures show no significant v i r a l DNA synthesis. A similar finding was obtained by ethidium-bromide/CsCl centrifugation. When the DNA extracted from the treated PyH-1 cells was assayed for i n f e c t i v i t y on secondary mouse embryo cultures, no plaques were observed. To confirm this further, and to determine the level of sensitivity with which one can detect v i r a l DNA synthesis, these same DNA samples were tested for v i r a l DNA content by hybridization to polyoma DNA fixed to nitrocellulose f i l t e r s . Table IV-III shows the results, while Figure IV-III displays a calibration curve obtained for the results of hybridization tests with s e r i a l dilutions of the infected mouse kidney samples. From the latter i t can be seen that i f v i r a l DNA replication occurs to the extent of more than 0 . 1 percent of that in productively infected cells, i t should be possible to detect by DNA-DNA hybridization. From Table IV-III, however, i t is evident that none of the treated PyH-1 cultures gave rise to this level of DNA synthesis. Figure IV-II: Alkaline sucrose gradient sedimentation of low molecular weight DNA from: Polyoma-infected mouse kidney cultures - ( 2 0 pfu/cell) ( 0 — — 0 ) and heterokaryon cultures of PyH-1 and mouse kidney cells, (o o ) . The cultures were i n -cubated with 5 M-Ci P e r ml. of ^ -thymidine at 2k hours after infection or fusion, for k hours, followed by a h5 minute incubation in non-radioactive medium. The cells were then washed and lyzed for DNA fractionation as described in the Materials and Methods section. Gradients were made of 5 - 2 0 $ sucrose i n 0.25 M NaOH, 1 0 - 3 M EDTA, and IO - 2 M Tris, pH 12.6. Centrifugation was in the Spinco S W 5 0 . 1 rotor at 4 5 3 0 0 0 rpm for 1 0 0 minutes at 20°C. 3 50 S 1(8-18S Fraction NO. Figure IV-III: Calibration curve for polyoma DNA-DNA hybridization. Polyoma DNA type 1 was isolated from the infected c e l l -extract used for Fig.IV-IE and purified by ethidium bromide/CsCl centrifugation (details in Materials and Methods section). The specific radioactivity was 2 x 10 cpm/ng. Serial dilutions were made and tested for hybridizable radioactivity to unlabelled polyoma DNA on nitrocellulose f i l t e r s , as described in the Materials and Methods section. O A Table IV-III: Polyoma virus specific DNA synthesis as determined by DNA-DNA hybridization Treatment Hybridizable cpm Blank f i l t e r 60 PyH-1 (heat treatment)* 78 PyH-1 + mouse kidney cocultivated 69 PyH-1 + mouse kidney fused 79 Mouse kidney control 63 Mouse kidney infected with polyoma 3,035 virus (30 pfu/cell) * k'fc for 30 minutes 97 Virus may have been rescued and i t s DNA replicated in a small percentage of the cell s . If one c e l l i n 1000 was i n -duced to synthesize DNA at a rate lower than the normal productive infection i t would not be detected by the method of DNA-DNA hybridization. Immunofluorescence could detect whether any cells were producing capsid proteins. No cells synthesized polyoma virus capsid proteins after attempts to induce virus. A similar experiment was performed with the SV40-trans-formed cells (MKS-A), using CV-1 cultures as permissive cells, i n comparison with productive SVkO infection. Table IV-IV shows the hybridization values for the low molecular weight DNA fractions of treated MKS-A cells, and Figure IV-IV shows the corresponding calibration curve. In this case SVkO DNA replication was readily detected following c e l l fusion, and to a lesser extent on co-cultivation, with permissive CV-1 cells. The background counts were lower i n this series of experiments than i n the previous one due to the fact that a low background s c i n t i l l a t i o n counter was employed. Polyoma Virus Multiplication in Fused-Cell Cultures Mouse embryo cells were fused with polyoma virus trans-formed cells (PyH-1) and plated out into monolayer cultures. Figure IV-IV: Calibration curve for SVkO DNA-DNA hybridization. The method was analogous to that described in the legend to Figure IV-III. The specific radioactivity of the SVUO DNA k type 1 was 4 x 1 0 cpm/ug. This DNA had been isolated from SV40-infected BSC-1 cells as described in the Materials and Methods section. 0 I O " 1 10"2 10" D i l u t i o n of S V 4 0 D N A Table IV-IV: Simian virus kO specific DNA synthesis as determined by DNA-DNA hybridization. Treatment Hybridizable cpm Blank 11 CV-1. 10 MKS-A 11 Fused CV-1 + MKS-A k3 CV-1 + MKSTA (cocultivated) 28 MKS-A + MKS-A fused - 1 3 CV-1 infected with 10 TCD,-_ , r o SVkO per c e l l 5 0 k 6 8 CV-1 infected with 0.01 TCD , SV40 per c e l l ^ The cultures contained individual mouse cells, individual PyH-1 cells, heterokaryons and a few homokaryons. Polyoma virus infection of fused c e l l cultures gave variable yields of virus, but was always lower than in mouse embryo cultures which had been infected at the same time. The virus produced i n the fused culture could have arisen from individual mouse cells, mouse homokaryons, or from heterokaryons. A technique combining autoradiography and immunofluor-escence was employed to determine whether virus multiplication occurred only in mouse cells or i f heterokaryons were also capable of supporting virus multiplication. Prior to fusion, PyH-1 cells were labelled with methyl-^H-thymidine to allow identification of their nuclei in the fused c e l l culture. Under the conditions used (details in the Materials and Methods section) more than 95$ of the PyH-1 c e l l nuclei became labelled. No virus multiplication was detected, by immunofluorescence, in the heterokaryons. Individual mouse cells and mouse homokaryons were immunofluorescent positive, indicating that the virus produced in fused c e l l cultures arose from mouse cells only. The lack of virus multiplication i n heterokaryons could have been due to an i n a b i l i t y of the virus to enter and to superinfect the heterokaryons. Polyoma virus transformed cells (PyH-l) are not susceptible to superinfection, possibly-due to a permeability barrier. To alleviate this possi-b i l i t y , mouse embryo cells were infected with polyoma virus prior to fusion with PyH-1 cells. Table IV-V indicates that the process of c e l l fusion and the presence of UV-inactivated Sendai virus, does not have any effect on polyoma virus multiplication. Thus the percentage of immunofluorescent positive mouse cells was similar in mouse homokaryons; in individual mouse cells; and in separate mouse c e l l cultures which had not undergone the c e l l fusion procedure. It was of interest to determine whether inhibition of polyoma virus multiplication i s a general property of hamster cells, or i f i t i s specific only for polyoma virus-transformed hamster ce l l s . Fusion of polyoma virus infected mouse embryo cells to BHK-21 cells, a continuous line of hamster c e l l s ? resulted i n some inhibition of virus synthesis in hetero-karyons (Table IV-VI). The degree of inhibition depended upon the relative numbers of mouse and hamster nuclei in the individual heterokaryons. Thus there appears to be an antagonistic effect between the two types of c e l l . The lower percentage of immunofluorescent positive mouse cells in Table IV-VI & VII, compared to Table IV-V, is due to the fact that the assays were conducted at different times after infection. Table IV-V: Polyoma-virus multiplication in fused cells as measured by fluorescence microscopy io of to t a l cells <f0 of fused cells Type of cells fused fluorescing fluorescing MK + MK k9 53 MK + PyH-1 16 0 Mouse kidney cells infected with polyoma virus (20 pfu/cell) for 6 hours prior to fusion with PyH-1 cells pwhich had previously been labelled for 2k hours with methyl-^H-thymidine (5 u.0i/ml). Forty-eight hours after fusion the cells were assayed for immunofluorescence and autoradiography. Sixty-one percent of mouse kidney cells not exposed to the c e l l fusion technique were immunofluorescent positive. 104 Table TV-VI: Multiplication of polyoma virus in ME + BHK-21 fused-cell cultures i of a Ce l l type type specific c e l l in culture °lo cells of specific type that are immunofluorescent positive 1. Single cells mouse embryo 39 37 BHK-21 16 0 2. Hybrid cells mouse only 14 25 mouse excess 10 5 mouse = hamster 4 0.1 hamster excess 9 0.01 hamster only 8 0 Mouse embryo cells were infected with polyoma virus (20 pfu/ cell) 6 hours prior to fusion with BHK-21 cells. The hamster cells had previously been labelled with methyl-"^H-thymidine for 24 hours (2.5 uCi/ml). Thirty hours after fusion, the cells were assayed for immunofluorescence and autoradiography. A similar result was obtained when BHK-21 cells were infected with virus, and then fused with uninfected mouse embryo cells (Table IV-VIl). An interesting additional finding in this experiment was that a normal percentage of the individual mouse cells were immunofluorescent positive. This indicated that polyoma virus could be reversibly adsorbed to, or persist within BHK-21 cells and subsequently be released back into the medium as infectious virus. Results of Bourgaux (196U) and Figure V-VT of this thesis indicate that whole virus is present within BHK-21 cells for a considerable time after infection. Bourgaux also found that radioactive virus was released back into the medium, from infected ce l l s . To confirm these findings, mono-layers of mouse embryo and BHK-21 cells were infected with methyl-thymidine labelled polyoma virus. After adsorp-tion of the virus, the monolayers were washed three times with phosphate buffered saline. The medium from the infected c e l l cultures was removed at various times after infection. Considerable trichloroacetic acid insoluble radioactive material was found in the medium, indicating the possible presence of virus, which could be infectious. Table IV"-VII: Multiplication of polyoma virus in ME + BHK-21 fused-cell cultures C e l l type $ of a specific c e l l $ c e l l of a specific c e l l type in culture type that are I.F. positive 1. Single cells mouse kk 21 BHK-21 19 0 2. Hybrids mouse only 9 28 mouse excess .15 3 mouse = hamster 2 0.2 hamster excess 8 0.01 hamster only 3 0 M;ethyl- H-thymidine labelled BHK-21 cells were infected with polyoma virus 12 hours prior to fusion with mouse embryo cells. Thirty hours after fusion, the.cells were assayed for immuno-fluorescence and autoradiography. Fusion after 2 hours of infection gave similar results. 107 Discussion The induction of simian virus hO - DNA and - virion synthesis (i.e. 'rescue') i n cells transformed by this virus is a well documented event. In many cases, rescue has been effected by subjecting the transformed cells to ultraviolet irradiation; mitomycin C treatment, or heat shock. Fusion of these cells to permissive cells, however, usually gives greater yields of rescued virus, of the order of 0.1 to 1.0 percent of the amount of virus obtained from productively infected cells (Koprowski et a l , 1967; Watkins and Dulbecco, 1967; Kit et al, 1968). The timing of the onset of SVhO-DNA synthesis is apparently of the same order for the two systems, i.e. about 19 hours post c e l l fusion or post infection (Kit et al, 1968). In contrast, only one report has described successful rescue of polyoma virus from transformed cells, and then only in very low yie l d (Fogel and Sachs, 1969). In the studies described here, a variety of ultraviolet irradiation, mitomycin C and heat treatments was used on two lines of polyoma virus-transformed cells, without production of infectious virus. Fusion of the transformed cells (with or without accessory mitomycin C treatment) with permissive mouse kidney cells, also gave negative results. In the latter experiments, about 70$ of the t o t a l cells in the population were i n polykaryons. Thus, the cell-fusion efficiency was not a limiting factor. Furthermore, analogous fusion experiments, using SVkO-transformed mouse cells (MKS-A) and permissive monkey cells (CV-l), gave yields of more than 10 infectious SVkO particles per culture (Table IV"-II). It was therefore concluded that the lines of polyoma virus transformed cells used in this series of experiments were non-inducible with regard to infectious virus. In addition, the transplanted (in vivo passaged) lines gave negative responses. Burns and Black (1969) had reported that i n vivo passage of non inducible SVk-0-transformed cells resulted in their becoming inducible by mitomycin C. It was conceivable that the 'integrated' v i r a l genes were in fact being 'rescued' or activated by some or a l l of the treatments used on the polyoma virus transformed cells, but that defective virions were synthesized, as i n one reported case for SVkO-transformed cells (Margalith et_ al, 1970), or that incomplete genomes were replicated. To test for such po s s i b i l i t i e s , DNA-DNA hybridization assays were carried out, using extracts of the treated 109 transformed cells, in which newly replicated DNA was labelled 3 by methyl- H-thymidine. By constructing a calibration curve of radioactive polyoma DNA concentration versus hybridizable radioactivity, i t was possible to determine down to what level DNA replication could be detected. This level was about 0.1$ of the level found in productively infected cells. No induction of polyoma DNA synthesis was detected at this level in the treated transformed cel l s . In contrast, induction of SVUO-DNA synthesis was readily detected i n the MKS-A cells after fusing with permissive cells. It is not known whether a large number of cells are involved in induction followed by a low level of virus synthesis, or i f just a few cells are induced to a level comparable to a normal productive infection. No satisfactory'explanation for the failure to rescue polyoma virus can be given at the present time. However, i t can be postulated that the virus genome may be integrated in such a manner or location which prohibits subsequent excision. Another explanation i s that a defective or i n -complete v i r a l genome is present i n the transformed c e l l (evidence for this has been reported (Hudson, 1972). A factor(s) may be present within transformed cells which represses v i r a l multiplication, as has been proposed by other workers (Cassingena and Tournier, 1971). 110 Basilico et a l (1970, 1971)? reported that established hybrid c e l l lines, formed by fusion between a continuous line of mouse 3T3 cells and polyoma virus transformed BHK-21 cells, were able to support polyoma virus multiplication. There was considerable variation i n the chromosome complement of these hybrid c e l l lines, which resulted in differences in the degree to which polyoma virus multiplication could be supported. A progressive inhibitory effect seemed to be associated with an increasing number of hamster chromosomes and a decreasing mouse:hamster ratio. These workers have shown that the hybrid c e l l lines which produced lower than normal virus yields, gave correspondingly lower amounts of v i r a l DNA, suggesting that the block occurred at the level of v i r a l DNA synthesis. The experiments reported here, with mouse embryo-BHK-21 heterokaryons indicated a similar but more pronounced inhibitory effect as a result of an increase in the hamster nuclei:mouse nuclei ratio. This inhibitory effect of BHK-21 cells towards virus multiplication was however much less than the inhibitory effect of PyH-1 c e l l s . In the latter case, the presence of a single PyH-1 nucleus in a hetero-karyon was sufficient to ensure complete inhibition of polyoma virus synthesis, irrespective of the number of mouse nuclei present. The Inhibitory effect of the hamster c e l l could, be interpreted in two ways: l ) a repression due to a specific substance (repressor), produced by the hamster genome or 2) a specific cellular gene product (e.g. an enzyme) is required for virus multiplication. Hamster cells and mouse cells may produce homologous cellular gene products, that of the mouse being suitable for virus multiplication, per-missive 'P', and that of the hamster being inactive 'I' or non-existent. For example, the cellular gene product could be an oligomeric enzyme, which could form from sub-units of the two species, resulting In an inactive hybrid enzyme. Hybrid enzymes have previously been reported to exist i n hybrid cells (Weiss and Ephrussi, 1966; Migeon, 1968). Immediately after c e l l fusion, cellular gene products from both cells would be present. Continued cultivation of a hybrid c e l l for a considerable length of time may result in one of two p o s s i b i l i t i e s : l) loss of a specific gene(s) responsible for 'P' or 'I', or 2) shut off of certain genes by transcriptional control. Repression of the transcription of some genes has been reported in human-mouse hybrid cells ( E l i c e i r i and Green, 1969). If the 'I' gene(s) were shut off then v i r a l multiplication would occur in a hybrid c e l l . I f the "P1 gene(s) were shut off then the hybrid would not support v i r a l multiplication. Whether multiplication would occur in the presence of both gene products would depend on the activities and concentration of each. The extreme degree of inhibition of virus multiplication in PyH-1 mouse hybrids may be due to an extremely active 'I' gene in PyH-1 cells. The p o s s i b i l i t y of the synthesis of a 'repressor' in PyH-1 cells, such as is present in bacterial lysogeny, cannot be ruled out. References to Chapter IV Axelrod, D., K. Habel, and E.T. Ballan. 1964. Polyoma genetic material in a virus free polyoma induced tumor. Science 146: 1466-1468. Basilico, C, Y. Matsuya, and H. Green. 1970. The inter-action of polyoma virus with mouse-hamster somatic hybrid cells. Virol. 4 l : 295-305. Basilico, C. and R. Wang. 1971. Susceptibility to super-infection of hybrids between polyoma 'transformed' BHK and 'normal' 3T3 cells. Nature New Biol. 230: 105-107. Burlingham, B.T., and W. Doerfler. 1971- Three size classes of intracellular adenovirus deoxyribonucleic acid. J. Virol. 7: 707-719-Benjamin, T.L. 1966. Virus-specific RNA in cells productively infected or transformed by polyoma virus. J. Mol. Biol. 16: 359-373. Black, P.H. 1968. The oncogenic DNA viruses: A review of in vitro transformation studies. Annual Rev. Microbiol. 391.426. Bourgaux, P. 1964. The fate of polyoma virus in hamster, mouse and human cells. Virology 23: 46-55-114 Burns, W.H. and P.H. Black. 1969. Analysis of SV40-induced transformation of hamster kidney tissue in vitro. VI. Characteristics of mitomycin C induction. Virology 39: 625-634. Cassingena, R., P. Tournier. 1971. SV40 specific 'repressor' in infected and transformed cells. Proc. Roy. Soc. Lond. B., 177: 77-85. Doerfler, W. 1970. Integration of the deoxyribonucleic acid of adenovirus type 12 into deoxyribonucleic acid of baby hamster kidney cells. J. V i r o l . 6: 652-666. Dulbecco, R. 1969. C e l l transformation by viruses. Science 166: 962-968. E l i c e i r i , G. and H. Green. 1969. Ribosomal RNA synthesis in mouse-human hybrid cells. J. Mol. Biol. 40: 253-260. Fogel, M. and L. Sachs. 1969. The activation of virus synthesis in polyoma-transformed cel l s . Virology 37: 327-334. Fogel, M. and L. Sachs. 1970. Induction of virus synthesis in polyoma transformed cells by ultraviolet light and mitomycin C. Virology 40: 174-177. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cells cultures. J. Mol. Biol. 26: 365-366. Hudson, J.B. 1972. Properties of polyoma virus-transformed cells. II. Characteristics of the virus-specific RNA. Can. J. Microbiol. 18: 247-254. Kit, S., T. Kurmura, M.L., Salvi, and D.R. Dubbs. 1968. Activation of infectious SV40 DNA synthesis in transformed cells. Proc. Natl. Acad. Sci. U.S. 60: 1239-1246. Koprowski, H., F.C. Jensen, Z. Steplewski. 1967. Activation of production of infectious tumor virus SV40 in hetero-karyon cultures. Proc. Natl. Acad. Sci. U.S. 58: 127-133. Margalith, M., E. Margalith, T. , Nasialski, and N. Goldblum. 1970. Induction of simian virus 40 antigen in BSC-1 transformed cells. J. Virol. 5: 305-308. Migeon, B. 1968. Hybridization of somatic cells derived from mouse and Syrian hamster. Evolution of karyotype and enzyme studies. Biochem. Genet. I: 305-322. Sambrook, J., H. Westphal, P.R. Srinivasan, and R. Dulbecco. 1968. The integrated state of v i r a l DNA in SV40-transformed cells. Proc. Natl. Acad. Sci. U.S. 60: 1288-1295. Watkins, J.F., R. Dulbecco. I967. Production of SV40 virus in heterokaryons of transformed and susceptible ce l l s . Proc. Natl. Acad. Sci. U.S. 58: 1396-1403. Weiss, M.C., and B. Ephrussi. 1966. Studies of interspecific (rat X mouse) somatic hybrids. II. Lactate dehydrogenase and (3-glucuronidase. Genetics 54: 1111-1117. CHAPTER V "INTEGRATION" OF POLYOMA VIRUS DNA INTO MAMMALIAN GENOMES Abstract Cells of mouse (secondary embryo cultures), hamster (BHK-21), and human (Hep-2) origin were infected with polyoma virus, and the high molecular weight cellular DNA was isolated, at various times after infection, by sedimentation through alkaline sucrose gradients or by precipitation with salt. Some of the polyoma DNA became associated with the mouse and hamster DNA, but l i t t l e , i f any, with the human DNA. Follow-ing v i r a l DNA replication in the mouse cells, approximately half of the newly synthesized polyoma DNA became c e l l DNA-associated. A number of control experiments were carried out to rule out the possi b i l i t y of artefactual association of v i r a l DNA with the cellular DNA. These included mixing virus particles (containing labelled DNA) or labelled free v i r a l DNA with uninfected or infected cells prior to separation of cellular DNA from v i r a l DNA. In a l l cases the extent of association i n the controls, was considerably less than In the authentic infected c e l l preparations. The amount of polyoma DNA associated with the c e l l DNA increased with increasing virus dose, but less than proportionately, suggest-ing that a limited number of integration sites are available. 117 Introduction A characteristic property of cells transformed by the papovaviru.ses', polyoma and simian virus kO (SVkO), is the persistence of v i r a l DNA, apparently covalently linked to the cellular DNA (Sambrook et al, 1968; Westphal and Dulbecco, 1968). The integrated SVkO and polyoma genes appear to be transcribed contiguously with adjacent host genes giving rise to nuclear transcripts considerably larger than single v i r a l genome size (Lindberg and Darnell, 1970; Wall and Darnell, 1971; Hudson, 1972). The fact that similar large v i r a l specific RNA is synthesized during productive infections with polyoma virus suggested that polyoma DNA might become integrated during the normal course of i t s replication cycle (Hudson et a l , 1970; Acheson et a l , 1971; Petric and Hudson, 1972). To date only indirect evidence for integration during productive infection was available. It was therefore decided to examine and obtain direct evidence for integration of polyoma DNA i n mouse cells; in BHK-21 cells, which do not allow virus DNA replication and in Hep-2 cells. Integration is defined here by the a b i l i t y of v i r a l DNA to associate with cellular DNA in an alkali-stable linkage. Integration of SVl+O DNA and of adenovirus 12 DNA into the DNA of abortively infected cells has been demonstrated recently (Doerfler, 1970; Hirai et al, 1971). Results Association of V i r a l DNA with Cellular DNA Hirt (1967) developed a method for separating high molecular weight cellular DNA from low molecular weight v i r a l DNA, by selective precipitation with salt. Westphal and Dulbecco (1968) employed this method to detect v i r a l DNA association with cellular DNA, i n transformed cells. This same procedure was employed, in the following experiment, to study the kinetics and the degree of v i r a l DNA association with cellular DNA. Cells were infected with polyoma virus in the usual manner (Materials and Methods) and at various times after infection the cells were gently lyzed and the cellular DNA was precipitated with salt (Hirt, 1967). The DNA i n both fractions was purified, and hybridized to methyl-^H-thymidine labelled polyoma type I DNA (Table VI-I). From this experiment, i t is evident that a considerable amount of the input virus DNA was precipitated with the c e l l DNA. Reconstruction experiments were conducted in which methyl-^H-thymidine labelled polyoma DNA was added to the lyzed cells prior to precipitation. Results of these reconstruction experiments indicated that from 15-30$ of the added labelled polyoma DNA could coprecipitate with the c e l l DNA even though Table V-I: Integration of polyoma v i r a l DNA into mouse and hamster genomes as measured by hybridization of DNA extracted from infected cells by Hirt's method Ce l l type Time after Hybridizable counts infection (hours) Pellet (host) Supernatant Mouse embryo 6 3,263 2,670 12 k, 085 k, 455 2k 9,965 7,554 BHK - 21 6 1,580 12 2,428 -2k 6,959 -Hep-2 6 228 2,595 12 336 2, lk6 2k 330 1,96k The DNA was purified by phenol and centrifuged in CsCl. Fixed to nitrocellulose f i l t e r s in 6 x SSC. Hybridization was conducted in 6 x SSC + 0.0k% BSA for 30 hours at 6o°C. Polyoma DNA (Type l ) sp. activity 100,000 cpm/ug was used for the hybridization. i t was not integrated. Due to this high degree of coprecipi tation, the method could not be used satisfactorily as a means of determining the extent of integration. Evidence that V i r a l DNA is Covalently Linked to Cellular DNA In the experiments described here, resolution between cellular DNA and free v i r a l DNA was•effected by alkaline sucrose gradient sedimentation. Figure V-I illustrates the resolution routinely achieved under the standard conditions of sedimentation. A l l forms of free polyoma DNA (53s type 1 l 6 and l 8 s type 11) were completely resolved from the much heavier single stranded cellular DNA. Table V-II shows the result of an experiment designed to examine the extent of association of labelled polyoma DNA of infecting virus with the cellular DNA of mouse and hamster cultures. As a control, an equivalent amount of v i r a l DNA was mixed with uninfected cells just prior to layering onto the gradient. About one percent of the labelled v i r a l DNA become adventitiously associated with the cellular DNA in the control. The experimental values were a l l significantly greater than this value. The 'integration' apparently pre-ceded v i r a l DNA replication i n the mouse cells (which Figure V-I: Alkaline sucrose gradient sedimentation of mouse DNA and polyoma DNA. Mouse embryo cells (about 5 x 10 ), previously incubated for 2k hours in -thymidine (0.25 uOi; specific activity 57 uOi/mmole, Amersham-Searle), were harvested by trypsinization, washed and resuspended in 0.25 ml of PBS (phosphate buffered saline minus divalent cations, pH 7.2). 3 Purified polyoma virus containing methyl- H-labelled DNA, was added to the cells, and the mixture layered gently onto 1.0 ml of lyzing solution (0.5 M NaOH, 0.01 M EDTA, 0.2$ SDS) on top of a 36 ml gradient of 10-30$ sucrose (w/v i n 0 . 3 M • NaOH, 0.001 M EDTA, 0.01$ SDS) in a Beckman SW27 polyallomer tube. After 12 hours at 20°C, the gradient was centrifuged for 5 hours at 25,000 rpm at 20°C. Fractions were collected from the top of the tube by pumping 50$ sucrose into the bottom of the tube. (We are grateful to Branko Palcic of McMaster University for communicating to us the details of this technique for alkaline sucrose gradient sedimentation). 1 2 3 10 20 30 B o t t o m Fraction No. T o o Table V-II: Integration of labelled polyoma v i r a l DNA into cellular DNA Cel l type Time after $ of v i r a l DNA integrated infection (hours) (duplicate tubes) Mouse embryo 6 5.9 2.2 12 6.7 7.1 2k 2.1 k.l BHK-21 6 k.l 6.2 12 6.0 5-3 2k 9.1 6.3 Mouse embryo -polyoma DNA (20,000 cpm) - 0.91 1.13 mixed Cultures containing about 2 x 10 cells were infected with polyoma virus containing 20,000 cpm of ^ H-labelled DNA. The mouse cells took up 15$ of the virus during a 2-hour adsorption period, while the BHK-21 cells took up 8$. Cells were harvested at various times after infection and subjected to alkaline sucrose gradient sedimentation, as described in the legend to Figure V-I. The integration values represent the percentage of the DNA taken up that was recovered in the cellular DNA band. For the control, 20,000 cpm of DNA extracted from the polyoma virus preparation were mixed with uninfected mouse cells prior to layering on a gradient. commences about 12 hours after infection, Hirt, 1967), and occurred at about the same level in the BHK-21 cells, which do not support v i r a l DNA replication (Hudson et a l , 1972), Table V-III illustrates the results of an experiment in which mouse, hamster and human cells were infected with un-labelled polyoma virus. In this case v i r a l DNA was detected by the DNA-DNA hybridization technique. Most of the integration due to parental DNA occurred during the f i r s t 6 hours of infection. The extent of Integration was again similar for BHK and mouse cultures and represented a con-siderable fraction of the total v i r a l DNA within the cells. After the i n i t i a t i o n of v i r a l DNA replication in mouse embryo cells, there was a large increase in the amount of v i r a l DNA associated with the cellular DNA fraction (Table V-III). The quantity of integrated v i r a l DNA at 2k hours post infection (determined by including a hybridization calibration curve) was considerably greater than the original input virus DNA, and therefore must have represented newly synthesized v i r a l DNA. Up to 50$ of the t o t a l cellular content of v i r a l DNA at any given time was apparently integrated. The v i r a l DNA present i n the supernatant, which is not integrated, may exist i n whole virus particles, which have not been uncoated. Uncoating of the virus may be the limiting factor in the kinetics and the degree of integration. Table V-III: Integration of polyoma v i r a l DNA into cellular DNA Ce l l type Time post infection cpm hybridizing v i r a l cellular BHK - 21 6 hours 560 297 12 hours 386 318 24 hours 367 333 Mouse embryo 6 hours 182 549 12 hours 227 1,295 2k hours 5,642 5,768 Hep-2 12 hours 168 73 Cultures were infected with polyoma virus (50 pfu/cell) and samples (about 5 x 10^ cells) taken at various times after infection for alkaline sucrose gradient sedimentation, as described before. Cellular DNA fractions and v i r a l DNA fractions were separately pooled. The DNA-pools were dialyzed against 0.1 x SSC; concentrated; extracted with phenol, followed by ether extraction, and heated at 98°C for 10 min. before fixing to 2.5 cm Millipore HA f i l t e r s in 6 x SSC. The f i l t e r s were cut into O.65 cm diameter discs for DNA-DNA hybridization with sonicated methyl- H-labelled polyoma DNA. The specific activity of the polyoma DNA was 2 x 10^ cpm/ug. Blank f i l t e r s containing 100 ug of mouse embryo DNA (for infected cells) gave an average background value of 100 cpm. This value has been subtracted to give the above hybridization figures. Input radioactivity was 80,000 cpm per reaction mixture. The Hep-2 cells showed a much smaller level of integration, though i t appears to be significant i n this experiment. However in another experiment the level of 'integration' in Hep-2 cells was no higher than a control, test, in which virus DNA was mixed with uninfected Hep-2 cells just prior to l y s i s . Thus, integration may occur in Hep-2 cells but could be limited by a relatively small degree of virus uptake or uncoating. Two additional control tests were performed simultaneously with this experi-ment. Samples of polyoma DNA, unlabelled a), or labelled b), were separately mixed with uninfected BHK-21 cells, and layered onto alkaline sucrose gradients In the usual manner (Figure V-Il). After centrifuging, the distribution of polyoma DNA was determined, by hybridization i n a), and by radioactivity in b). In both cases, approximately 12$ of the v i r a l DNA was recovered in the cellular DNA band. This value is significantly greater than the amount of v i r a l DNA associated with the cellular DNA in the control of Table V-II, but i s nevertheless much less than the experi-mental values depicted in Table V-III. Normally the alkaline sucrose gradient.fractions were pooled into c e l l DNA and v i r a l DNA for the hybridization tests. In one experiment a larger number of fractions was Figure V-II: Alkaline sucrose gradient sedimentation of mixtures of polyoma DNA and BHK-21 cells. Experimental details as before. Samples of uninfected BHK-21 cells (5 x 10 cells) were mixed with a) 2 ug of unlabelled polyoma DNA; b) 5,000 cpm of methyl-TI-labelled polyoma DNA (0.025 M-g). In a) the lowest 10 mis were pooled for the cellular DNA regions and the remainder of the gradient combined i n 5 equal pools for free v i r a l DNA. 129 tested. Figure V-IVa indicates the distribution of v i r a l DM through an entire alkaline sucrose gradient. The con-t r o l in this experiment consisted of infecting mouse embryo cells with unlabelled polyoma virus, 12 hours prior to mixing the cells with labelled polyoma virus and centrifugation (Figure V-IVb). Labelled polyoma virus DNA (in controls) sediments at the same rate, whether i t is mixed with-virus infected cells or with uninfected ce l l s . An increase in the sedimentation rate of v i r a l DNA following infection indicates integration into the cellular DNA. The extent of integration was found to be dependent upon the dose of inoculating virus, as indicated in Table V-IV. Thus with increasing input virus, more integration was detected, i n both mouse and BHK-21 cells, although the increment was less than proportional, suggesting that there may be a limited number of 'integration sites'. Thus at higher multiplicities of infection a greater proportion of the t o t a l v i r a l DNA was present in the free virus DNA region of the gradient. A calibration curve was performed in which f i l t e r s containing various concentrations of v i r a l 3 DNA were hybridized to methyl- H-thymidine labelled polyoma DNA. Assuming that the efficiency of hybridization was the same for free polyoma DNA and integrated virus DNA, Table V-IV: The relationship between integration of polyoma virus DNA and multiplicity of infection Hybridizable cpm at virus multiplicity: C e l l type 10,000 1,000 100' pfu/cell pfu/cell pfu/cell mouse embryo - cellular DNA 6,190 3, 838 1,791 - v i r a l DNA 60,248 7, 289 4,028 BHK - cellular DNA 5,300 1, 099 830 - v i r a l DNA 33,167 2, 737 l , 175 Blank 50 Mouse embryo and BHK-21 cells were infected for 10 hours, with various amounts of polyoma virus. The cells were lyzed on an alkaline sucrose gradient, centrifuged, the DNA purified and hybridizing to polyoma type 1 DNA. (Specific activity 2 x 10 cpm/ug). 132 calculations were made to determine the number of virus genome equivalents integrated per c e l l . The values obtained were 60, 85 and 135 at input multiplicities of 100, 1,000 and 10,000 pfu/cell respectively. As an indication of the specificity of the hybrid formed between labelled polyoma DNA and the putative polyoma DNA sequences in the f i l t e r bound cellular DNA, thermal denatitr-ation of the DNA-DNA hybrids was measured over a range of temperatures. Representative profiles are illustrated in Figure V-III. Similar tests were performed for other times after infection, with no difference in the sharpness of the profiles or in the Tm values. The Tm values (2 x SSC salt concentration) ranged between 80-85°C, a value expected for DNA-DNA complexes containing k<9 mole percent guanine and cytosine (Weil, 1963). Purified virus DNA has been shown to be infectious (Weil, 1963). Therefore, i t was of interest to determine whether Integration of purified virus DNA was similar to the inte-gration of virus DNA derived intracellularly from virus particles. Mouse embryo and BHK-21 cells were infected with labelled purified virus DNA and paral l e l cultures were infected with labelled virus. At various times after infection the cells were layered on an alkaline sucrose gradient, centri-Figure V-III: Thermal denaturation of polyoma DNA-cell DNA complexes. Nitrocellulose f i l t e r s containing infected-cell DNA-tritiated polyoma DNA complexes were washed in 2 x SSC and then were incubated for 10 minute periods at successively increasing temperatures. The incubation medium comprised 0. 5 ml of 2 x SSC. The labelled DNA eluted at each temperature was precipitated by trichloracetic acid and collected on membrane f i l t e r s for radioactivity measurement. The unlabelled DNA on the f i l t e r was in a) denatured polyoma DNA type 1; b) mouse embryo c e l l DNA, 12 hours post infection; c) BHK-21 c e l l DNA, 12 hours post infection; d) Hep-2 c e l l DNA, 12 hours post infection. The 100$ cpm values were respectively: 7,333; 4,035; 2,428; and 336. fuged and fractions were collected. Figures V-IV and V indicate that integration of v i r a l DNA into cellular DNA occurs to the same extent whether purified virus DNA or whole virus was used for infection. An interesting observation was the persistence of v i r a l DNA in the type 1 form (50s in the alkaline gradient) for a considerable length of time after infection of mouse embryo- or BHK-21-cells by whole virus. In contrast, following infection by purified v i r a l DNA, the DNA appeared to convert rapidly to the integrated state or to type II DNA. The experi-ment illustrated in Figure V-VI indicates that the persistence of type 1 DNA i n the former case was due to the slow speed of uncoating the virus. Thus at 36 hours after infection of BHK-21 cells, some virus s t i l l remained uncoated, and would give rise to 50s v i r a l DNA i n the alkaline gradient. Figure V-IV: Alkaline sucrose gradient of mouse embryo and BHK-21 cells infected with methyl-^H-thymidine polyoma virus DNA. Centrifugation was conducted as in Figure V-I. a) mouse embryo cells infected with polyoma virus (10 hrs). DNA extracted and hybridized to methyl-^H-thymidine polyoma type 1 DNA: b) mouse embryo cells mixed with methyl-^H-thymidine polyoma virus DNA and centrifuged: c,e,g) mouse embryo cells Infected with methyl-^-thymidine polyoma DNA for 10, 36, 60 hrs. respectively before centrifugation; d,f,h) same as c,e,g, except BHK-21 cells used instead of mouse embryo cells. 137 138 Figure V-V: Alkaline sucrose gradient of mouse embryo and BHK-21 cells 3 after infection with methyl- H-thymidine labelled polyoma virus. Centrifugation was conducted as in Figure V-I. a) mouse embryo cells infected with polyoma virus 12 hrs. 3 Cells harvested and mixed with methyl- H-polyoma virus layered and centrifuged: b) BHK-21 cells harvested, mixed 3 with methyl- H-polyoma virus, layered and centrifuged. c,e,g. mouse embryo cells infected with methyl-^H-polyoma virus harvested at 10, 36 and 60 hours respectively, layered and centrifuged; d,f,h) same as c,e, and g except BHK-21 cells used instead of mouse embryo. Figure V-VT: Sucrose gradient centrifugation of polyoma virus, a) Cells (BHK-21) were infected with methyl-^H-thymidine labelled polyoma virus. After 36 hours the cells were lyzed with sodium deoxycholate (0.5$) sonicated and layered on a 5-30$ neutral sucrose gradient. Centrifugation was in a Beckman S W 5 0 . 1 rotor at 35,000 rpm for 2 0 minutes (20°C); b) methyl--thymidine labelled polyoma virus centrifuged as in (a). 5 10 15 20 F r a c t i o n No. Discussion Uptake of extracellular DNA into cells was discovered many years ago (Avery et al, l^kk). The f i r s t transfer of DNA to a mammalian c e l l was probably conducted by Szybalska and Szybalski (1962). Since this time a consider-able number of reports has been presented which showed the uptake of functional exogenous DNA (Ayad and Fox, 1968; Meril et al, 1971)• Recently a number of workers have shown that exogenous DNA may enter the c e l l and may become integrated into the chromosomes ( H i l l and Hillova, 1971; Ledoux et a l , 1971). The ab i l i t y of exogenous DNA to enter cells and remain functional, after many c e l l divisions, has resulted i n speculation on the use of exogenous DNA, preferably in a v i r a l coat protein, as a tool in genetic engineering (Osterman et al, 1970; Qasba et a l , 1971). The fact that exogenous DNA is capable of integrating into cellular DNA raises the problem of whether integration of polyoma DNA into cellular DNA is fortuitous or i f i t is a directed type of integration at a specific site as is lambda in E. c o l i (Weil and Signer, 1968). Direct evidence has shown that v i r a l DNA is integrated in the cellular DNA of Papovavirus transformed cells (Sambrook et al, 1968; Westphal and Dulbecco, 1968). Recent indirect evidence has suggested that polyoma virus DNA also integrates into cellular DNA during productive infection (Hudson et a l , 1970; Acheson et a l , 1971; Petric and Hudson, 1972). The experiments reported here present direct evidence for the integration of v i r a l DNA into cellular DNA during productive infection of mouse embryo cells as well as in BHK-21 cells, a c e l l line which does not support virus multiplication but in which a small percentage of the cells can become transformed. Evidence for v i r a l DNA association with cellular DNA was sought by two methods, l) separation of cellular DNA from v i r a l DNA by precipitation of the latter with salt. 2) separation of cellular DNA (> 100s) from v i r a l DNA in alkaline sucrose gradients. The presence of v i r a l DNA in the c e l l DNA fraction of alkaline sucrose gradients indicated that v i r a l DNA was associated in an alkaline stable linkage. Infection of cells with radioactive virus may not indicate true integration. Input v i r a l DNA may become degraded into individual nucleotides and be reincorporated into the cellular DNA. Another problem with using radio-active virus is the presence of pseudovirions. Thus the integration observed may be of mouse DNA but not of virus DNA. Therefore, the technique of DNA-DNA hybridization (using polyoma DNA of high specific activity) was used in the majority of experiments to measure specifically v i r a l DNA integration. The technique of DNA-DNA hybridization does not allow one to make an accurate estimate of the number of genome equiva-lents integrated since the presence of a large excess of cellular DNA might conceivably influence the hybridization efficiency of the v i r a l DNA. The po s s i b i l i t y of artefacts occurring during alkaline sucrose gradient sedimentation has been considered. It is unlikely that whole polyoma virions could survive the alkaline conditions prevailing during c e l l l y s i s and DNA denaturation. Similarly the polyoma-protein complex extracted from infected mouse cells would be expected to dissociate under these conditions (Green et al, 1971)- It was therefore presumed that a l l the v i r a l DNA recovered from the alkaline gradients had been liberated from complexes with proteins. The other remaining p o s s i b i l i t y is the presence of supercoiled oligomers, of virus DNA, which might cosediment with the cellular DNA. Although oligomeric forms of v i r a l DNA have been detected, in small amounts, in infected mouse cells, none of these con-tain more than one supercoiled molecule and hence would be unlikely to sediment with the cellular DNA ( > 1 0 0 s ) (Meinke and Goldstein, 1971). The results presented here strongly argue in favour of the hypothesis that polyoma v i r a l DNA becomes integrated into the cellular DNA during the normal course of infection in mouse cells and i n BHK-21 c e l l s . In the former case a considerable amount of newly synthesized v i r a l DNA also appears to be integrated. Integration in BHK-21 cells, in the absence of v i r a l DNA replication, i s apparently analogous to abortive infections of BHK-21 cells by adenovirus type 12 (Doerfler, 1970) and of Chinese hamster cells by SVkO (Hirai et a l , 1971). It i s tempting to speculate that integration may be a prerequisite for transformation in these cel l s . The integration of newly synthesized polyoma DNA i n mouse cells may be connected with v i r a l induced stimulation of c e l l DNA synthesis (Dulbecco et al, 19&5; Weil et_ a l , 1965). It is conceivable that integration occurs at DNA i n i t i a t i o n sites, of which there is a greatly increased number at later times in the infection cycle (Cheevers, in press). References to Chapter V Acheson, N.H., E. Buetti, K. Scherrer, and R. Weil. 1971. Transcription of the polyoma virus genome. Synthesis and cleavage of giant late polyoma-specific RNA. Proc. Natl. Acad. Sci. U.S. 68: 2231-2235. Avery, O.T., CM. MacLeod and M. McCarty. l^kk. Studies on the c l i n i c a l nature of the substance inducing trans-formation of pneumococcal types. Induction of trans-formation by a deoxyribonucleic acid fraction isolated from pneumococcus type III. ' J. Expt. Med. 79: 137-157-Axelrod, D., K. Habel, and E.T. Ballan. I96I+. Polyoma genetic material i n a virus free polyoma induced tumor. Science ±k6: lk66-lk68. Ayad, S.R. and M. Fox. 1968. DNA uptake by a mutant strain of lymphoma cells. Nature 220 : 35-38. Doerfler, W. 1970. Integration of the deoxyribonucleic acid of adenovirus type 12 into the deoxyribonucleic acid of baby hamster kidney cells. J. V i r o l . 6: 652-666. Dulbecco, R., L.H. Hartwell, and M. Vogt. 19^5- Induction of cellular DNA synthesis by polyoma virus. Proc. Natl. Acad. Sci. U.S. 53: k03. 147 Green, M.H., H.I. Miller, and S. Hendler. 1971. Isolation of a polyoma-nucleoprotein complex from infected mouse-c e l l cultures. Proc. Natl. Acad. Sci. U.S. 68: 1032-1036. H i l l , M. and J. Hillova. 1971. Recombination events between exogenous mouse DNA and newly synthesized DNA strands of chicken cells in culture. Nature New Biology 231: 261-265. Hirai, K., J. Lehman, and V. Defendi. 1971. Integration of simian virus 40 deoxyribonucleic acid into the deoxy-ribonucleic acid of primary infected Chinese hamster cell s . J. V i r o l . 8: 708-715. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse c e l l cultures. J. Mol. Biol. 26: 3^5-366. Hudson, J.B. 1972. Properties of polyoma virus-transformed cells. II. Characteristics of the virus specific RNA. Can. J. Microbiol. 18: 247-254. Hudson, J.B., D. Goldstein, and R. Weil. 1970. A study on the transcription of the polyoma v i r a l genome. Proc. Natl. Acad. Sci. U.S. 65: 226-233. Ledoux, L., R. Huart, and M. Jacobs. 1971. 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The integration state of v i r a l DNA i n SV40 trans-formed cel l s . Proc. Natl. Acad. Sci. U.S. 68: 1288-1295. Smith, H.S., L.D. Gelb, and M. Martin. 1972. Detection and quantitation of simian virus 40 genetic material i n abortively transformed BALB/3T3 clones. Proc. Natl. Acad. Sci. U.S. 69: 152-156. Szybalska, E.H. and W. Szybalski. 1962. Genetics of human cel l ' l i n e s . IV. DNA-mediated heritable transformation of a biochemical t r a i t . Proc. Natl. Acad. Sci. U.S. 48: 2026. Wall and Darnell. 1971. Presence of c e l l and virus specific sequences in the same molecule of nucleus RNA from virus transformed cel l s . Nature New Biology 232: 73-7°". Weil, J. and E.R. Signer. I968. Recombination in bacterio-phage II. Site-specific recombination promoted by the integration system. J. Mol. Biol. 34: 273-279. Weil, R. 1963. The denaturation and the renaturation of the DNA of polyoma virus. Proc. Natl. Acad. Sci. U.S. 4_9: 480-487. Weil, R., M.R. Michel, and G.K. Ruschmann. 1965. Induction of cellular DNA synthesis by polyoma virus. Proc. Natl. Acad. Sci. 53: 1468-1495. Westphal, H. and R. Dulbecco. I968. V i r a l DNA in polyoma-and SV40-transformed c e l l lines. Proc. Natl. Acad. Sci. u.s. 59: 1158-1165. -CHAPTER VI -GENERAL DISCUSSION The disease commonly called cancer encompasses a large variety of diseases. A common feature of a l l cancers is that a c e l l undergoes a permanent genetic change, i n such a manner, that i t escapes the normal control mechanisms of growth and regulation of c e l l division. As a result, a cancer c e l l once initiated, continues to grow and spread throughout the body to form a cancer or tumor. Experimental work designed to study the mechanism of transformation and loss of genetic control of c e l l division makes use of cancers induced a r t i f i c i a l l y by chemicals, radiation or viruses. In recent years, the most widely employed systems used viruses as transforming agents. The primary reason many workers selected Papovaviruses, in the study of transformation, was the fact that these viruses contain a small amount of genetic information (< 10 genes) It was assumed that cancer was induced specifically by the virus genes and by identification of each specific gene function, i t was f e l t the entire mechanism of transformation would be understood. The use of viruses with larger amounts of genetic information would increase the number of gene functions and therefore increase the complexity of the system. In view of the data available at present, concerning the infection process of Papovaviruses, i t is possible to propose a number of hypotheses which may explain v i r u s - c e l l interactions and the mechanism of transformation. 1. New genetic material from infecting virus is integrated into the host c e l l genome resulting in transform-ation. Experimental evidence has shown the presence of integrated virus DNA i n cellular DNA and the studies reported here have shown that integration is one of the i n i t i a l steps in the infection process. Integration appears to precede the induction or 'derepression' of cellular DNA synthesis. It i s not known whether integration at a specific site is alone sufficient to cause derepression. It is quite possible that the integrated virus genome must code for a specific protein which causes the 'derepression' and maintenance of the transformed state may require the persistence of a functional v i r a l gene. 2. The DNA of the infecting virus acts as a carcinogen by stimulating a specific cellular gene (oncogene) which i s normally non-functional or repressed. The role of DNA viruses may end at the i n i t i a l stimulation of the oncogene. The persistence of v i r a l genetic material may be purely a consequence of the fact that the cells were originally infected with the virus. 3. The infecting virus DNA induces mutations i n the c e l l genome. The v i r a l DNA may act as a mutagen, like other less complex chemical carcinogens or mutagens. Mutation of specific cellular genes may be sufficient to lead to a disruption of a specific cellular function and ultimately lead to transformation. No solid evidence is available at present for accepting one hypothesis over the other. However, i t is tempting to speculate that the f i r s t hypothesis is the correct one due to the following arguments. Integration precedes de-repression of the cellular synthetic mechanisms. There is a persistence of integrated v i r a l DNA i n transformed cel l s . The integrated DNA remains functional as shown by the presence of v i r a l specific RNA i n transformed cells. These arguments favour hypothesis number one but cannot rule out the latter two. Whatever the mechanism of transformation i s , i t appears clear that the key to transformation is i n the regulation of c e l l division. 

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