Analysis of Minimal Functions of Simian Virus 40 III. Evidence for “Host Cell Repair” of Oncogenicity and Infectivity of UV-Irradiated Simian Virus 40

The in vitro transforming capacity of simian virus 40 (SV40) for Syrian hamster cells is highly resistant to inactivation by UV light in comparison to infectivity. In the same cell system, we demonstrated a "host cell repair mechanism" sensitive to caffeine which is, to a large extent, responsible for the high resistance to UV inactivation of the transforming capacity of SV40. The survival of infectivity of UV-irradiated SV40 in CV-1 cells was also sensitive to caffeine, again indicating host cell repair. On the other hand, depression of normal cell DNA synthesis by hydroxyurea during the first 24 h postinfection only modestly reduced, and to a similar extent, the transforming capacity of UV-irradiated and nonirradiated SV40.     

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.     

Caffeine, cyclic AMP and postreplication repair of mammalian cell DNA.

The methylxanthines, caffeine and theophylline, inhibit postreplication repair of DNA in mammalian cells. Because they also inhibit cyclic AMP phosphodiesterase, it was thought that there might be some connection between concentrations of cyclic AMP and postreplication repair. We tested this possibility by performing DNA sedimentation experiments with a cyclic AMP-resistant mouse lymphoma cell mutant and its wild-type counterpart. The results show that there is no connection between cellular cyclic AMP concentrations and the rate of postreplication repair. Therefore, it is more likely that caffeine and theophylline inhibit postreplication repair by some other means, such as by binding to DNA.     

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate stimulates simian virus 40 induction by DNA-damaging agents and tumor initiators.

Simian virus 40 (SV40)-transformed Syrian hamster kidney cells produce infectious SV40 virus particles after treatments which damage DNA, such as UV irradiation or mitomycin C treatment. We have found that the induction of SV40 by DNA-damaging agents is greatly stimulated when a typical tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), is present in the medium. Phorbol, which has a molecular structure similar to TPA but does not have any tumor-promoting activity, showed no such stimulatory effect on SV40 induction. This apparent synergistic effect of DNA-damaging agents and tumor promoter (TPA) was more pronounced when a tumor initiator, benzo [a]pyrene or 2-acetamido-fluorene, was combined with TPA. The effect of TPA on UV-triggered SV40 induction was greatly influenced by the timing of TPA addition to the culture medium, which was most efficient when addition of TPA was 5 to 20 h before UV irradiation. The effect of TPA, however, was not observed in SV40 rescue from hamster cells by cell fusion with permissive monkey (C7) cells.     

Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells.

Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells.     

Postreplication repair of DNA in chick cells: studies using photoreactivation.

During replication of DNA after ultraviolet irradiation, gaps are left in the newly-synthesized DNA strands in both bacterial and animal cells and these gaps are subsequently sealed by a process known as postreplication repair. In order to test whether it is the ultraviolet-induced pyrimidine dimers which are responsible for the production of these daughter-strand gaps in animal cells, we have used chick embryo fibroblasts. In these cells the pyrimidine dimers are photoreactivable, i.e. they can be split by an enzymatic process dependent on visible or near ultraviolet light. Our results indicate that chick cells possess a postreplication repair system similar to that in mammalian cells; gaps are produced in the newly-synthesized strands and then filled in. If the ultraviolet-irradiated cells are first photoreactivated to remove most of the dimers, the number of daughter-strand gaps produced is much less than without photoreactivation. This suggests that the dimers are indeed responsible for the formation of many of the gaps in the newly-synthesized DNA. Ultraviolet light also inhibits the overall rate of DNA synthesis. This inhibition is, however, only partly overcome by photoreactivation.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

The co-induction of sister chromatid exchanges and virus synthesis in mammalian cells

The induction of virus synthesis and sister chromatid exchange (SCE) formation was investigated in several mammalian cell lines. Ultraviolet light co-induced the production of virus and SCEs in Simian virus 40 (SV40) transformed hamster cells. Post-irradiation treatment with caffeine enhanced virus induction, though it caused a smaller, less consistent elevation of SCE formation. Co-induction of oncovirus synthesis and SCEs was also observed in three murine cell lines exposed to increasing concentrations of 5-bromodeoxyuridine. These and previous data demonstrate a correlation between the induction of virus synthesis and SCE formation in rodent cells exposed to several agents, although inter-agent variation in the correlation may reflect differences between the two processes.     

Excision of viral DNA from host cell DNA after induction of simian virus 40-transformed hamster cells.

Simian virus 40-transformed hamster cells were induced to produce infectious virus by treatment with mitomycin C or gamma-irradiation. A portion of the simian virus-40 DNA, which is integrated into the host cell genome in uninduced cells, was recovered in a pool of relatively low-molecular-weight DNA early after induction treatment in the absence of DNA replication. The data indicte that excision of the viral genome occurs subsequent to the induction stimulus.     

Induction of Simian Virus 40 Antigen in BSC1 Transformed Cells

Heating to 45 C induced in virus-free clones of simian virus 40 (SV40) transformed BSC₁ cells the synthesis of SV40 viral antigen, as evidenced by immunofluorescence. Up to 3.8% of the cells exhibited viral antigen 72 hr after heating to 45 C for 30 min. Depletion of arginine from the medium of the heated cells enhanced and increased the percentage of cells synthesizing viral antigen to 11%. Cytosine arabinoside completely inhibited the induction of the viral antigen. No infectious virus was recovered from the cells in which synthesis of viral antigen was induced. However, small amounts of infectious SV40 virus were rescued from the BSC₁ transformed cells by fusion with rabbit kidney cells or by treatment with mitomycin C.     

Properties of Simian Virus 40 Rescued from Cell Lines Transformed by Ultraviolet-Irradiated Simian Virus 40

Simian virus 40 (SV40) strains have been rescued from various clonal lines of mouse kidney cells that had been transformed by ultraviolet (UV)-irradiated SV40. To learn whether some of the rescued SV40 strains were mutants, monkey kidney (CV-1) cells were infected with the rescued virus strains at 37 C and at 41 C. The SV40 strains studied included strains rescued from transformed cell lines classified as "good," "average," "poor," and "rare" yielders on the basis of total virus yield, frequency of induction, and incidence of successful rescue trials. Four small plaque mutants isolated from "poor" yielder lines and fuzzy and small plaque strains isolated from an "average" and a "good" yielder line, respectively, were among the SV40 strains tested. Virus strains rescued from all classes of transformed cells were capable of inducing the transplantation antigen, and they induced the intranuclear SV40-T-antigen, thymidine kinase, deoxyribonucleic acid (DNA) polymerase, and cellular DNA synthesis at 37 C and at 41 C. With the exception of four small plaque strains rescued from "poor" yielders, the rescued SV40 strains replicated their DNA and formed infectious virus with kinetics similar to parental SV40 at either 37 or 41 C. The four exceptional strains did replicate at 37 C, but replication was very poor at 41 C. Thus, only a few of the rescued virus strains exhibited defective SV40 functions in CV-1 cells. All of the virus strains rescued from the "rare" yielder lines were similar to parental SV40. Several hypotheses consistent with the properties of the rescued virus strains are discussed, which may account for the significant variations in virus yield and frequency of induction of the transformed cell lines.     

Caffeine-induced enhancement of chromosome damage in human lymphocytes treated with methyl-methanesulphonate, mitomycin C and X-rays

Two hypothese have been put forward in the literature to explain the synergistic effect of caffeine with several mutagens: (1) binding of caffeine to DNA, and (2) inhibition of DNA repair.Autoradiographic studies with ³H- and ¹⁴C-labelled caffeine did not support the binding hypothesis. Caffeine enchanced in a synergistic way the amount of chromatid breaks and exchanged induced in human lymphocytes with methyl-methanesulphonate (MMS), mitomycin C (MC) and X-rays. The results are best explained if caffiene inhibits a post-replication repair process, particularly the filling-in of gaps in the newly synthesized DNA.     

The induction of mitotic gene conversion by chemical and physical mutagens as a function of culture age in the yeast, Saccharomyces cerevisiae

Summary Cultures of yeast progressing from the exponential to the stationary phase of growth show increased resistance to the lethal effects of the chemical mutagens nitrous acid, ethyl methane sulphonate and mitomycin C and increased sensitivity to the lethal effects of UV light. Induced mitotic intragenic recombination produced by gene conversion also shows variation in its response to the growth phase after mutagen treatment. Higher frequencies of recombination per surviving cell were found after nitrous acid and ethyl methane sulphonate treatment of stationary phase cells wherease identical frequencies were produced by UV and mitomycin C treatment in both growth phases.The results were consistent with the hypothesis that the more nitrous acid and ethyl methane sulphonate resistant stationary phase cells were more active in postreplication repair. The sensitivity of exponential phase cells to nitrous acid and ethyl methane sulphonate may result from both increased mutagen uptake and reduced postreplication repair activity. In contrast, irrespective of growth phase all cells surviving UV and mitomycin C treatment appear to have undergone identical levels of post-replication repair.     

Characterization of Postreplication Repair in Mutagen-Sensitive Strains of DROSOPHILA MELANOGASTER

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-³H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.     

The synergistic effects of SV40 and BUdR on induction of gene mutations and chromosomal aberrations in chinese hamster cells

The induction of chromosomal abberations and gene mutations was studied in Chinese hamster cells after separate and combined treatment with BUdR and SV40. Separate treatment of cells with BUdR or virus infection increased the yield of chromosomal aberrations and reversions from glutamine requirement, expressed at 40°C (a ts mutant), to prototrophy. The combined effect of the incorporation of BUdR into one DNA strand, and a subsequent infection by SV40 was additive as regards the percentage of aberrant metaphases. The integration of the analogue into both DNA strands followed by SV40 treatment resulted in a statistically significant increase in the frequency of aberration-carrying metaphases, as compared with the frequency expected if the two agents had acted additively. The same phenomenon was detected when the frequency of reversions to glutamine independence was studied. Hence, the effect of the joint treatment by BUdR incorporated into both DNA strands and SV40 was synergistic. This is known to characterize the effect of BUdR on virus-induced transformation. Therefore, obviously the agent that enhances the malignant transformation of cells by the virus similarly modifies its mutagenic activity.

The results obtained are presumed to confirm the previously advanced hypothesis that the same events following infection might control both the integration of viral DNA into the host-cell chromosome (and hence cell transformation) and virus-induced mutagenesis. The role of repair processes in the synergistic effect of BUdR and SV40 in the yield of reversions to glutamine independence is discussed.     

Dissociated occurrence of single-gene mutation and oncogenic transformation in C3H 10T½ cells exposed to ultraviolet light and caffeine

We examined the relationship of cytotoxicity, mutagenesis, and malignant transformation by measuring in parallel clonogenic survival, mutation to ouabain resistance, and malignant transformation in cultured C3H mouse 10T 1/2 cells. Exposure of caffeine alone for 48 hours was cytotoxic and induced transformation in a dose-dependent manner. However, this same treatment did not induce any detectable ouabain-resistant mutants. When caffeine was present for 48 hours immediately following UV irradiation, alkaline sucrose gradient sedimentation of DNA showed that postreplication repair was inhibited. This inhibition of repair was correlated with reduced survival and inhibition of mutation induction, but the transformation frequencies were either unaltered or potentiated, depending on the UV dose and caffeine concentration. Thus, these experiments demonstrate that gene mutation and malignant transformation in 10T 1/2 cells can be dissociated. We suggest that the mechanism of transformation of 10T 1/2 cells is nonmutagenic in nature.     

Significance of treatment interval and DNA repair in the enhancement of viral transformation by chemical carcinogens and mutagens

Treatment of Syrian hamster embryo cells with diverse classes of chemical carcinogens enhanced transformation by a carcinogenic simian adenovirus, SA7. Optimal enhancement was a function of time of chemical addition in relation to time of virus addition and cell transfer. Aflatoxin B₁ (AFB₁) and the polycyclic hydrocarbons, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz(a)anthracene (DMBA) enhanced SA7 transformation when added prior to virus, but inhibited transformation when added after virus adsorption and cell transfer. The enhancement of SA7 transformation was maximal when cytosine arabinoside, caffeine and 6-acetoxy-benzo(a)pyrene (6-ac-B(a)P) were added after virus, but minimal when added before virus. A third class of chemicals, including β-propiolactone (β-PL), methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (Ac-AAF), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methylazoxymethanol acetate (MAM-ac), enhanced SA7 transformation added before, or after, virus inoculation and cell transfer. All chemicals, which induced changes in DNA sedimentation in alkaline sucrose gradients and unscheduled DNA (repair) synthesis in hamster cells, increased the frequency of SA7 transformation. However, several chemicals such as dibenz(a,h)anthracene (DB(a,h)A), benzo(e)pyrene (B(e)P), cytosine arabinoside, and caffeine enhanced SA7 transformation but did not induce DNA sedimentation changes or repair. Chemicals that cause DNA damage, which can be repaired by hamster cells, may enhance viral transformation by providing additional sites for integration of viral DNA during the repair process. Chemicals that apparently do not induce DNA repair synthesis may enhance viral transformation by incorporation of viral DNA into gaps in cell DNA at sites of unrepaired damage during scheduled DNA synthesis.     

Demonstration of Infectious Deoxyribonucleic Acid in Transformed Cells I. Recovery of Simian Virus 40 from Yielder and Nonyielder Transformed Cells

Deoxyribonucleic acid (DNA) was extracted from virus-free simian virus 40 (SV40)-transformed hamster, mouse, and monkey cells and was inoculated into simian cells in the presence of diethylaminoethyl (DEAE)-dextran; infectious SV40 was recovered by using DNA from cell lines which fail to yield virus by the fusion technique as well as from cell lines which readily yield virus by fusion. The rescued virus was identified as SV40 by three methods: (i) neutralization of plaque formation by specific antiserum; (ii) induction of synthesis of viral-specific antigens detected by immunofluorescence; and (iii) presence of papovavirus particles seen by the electron microscope. Treatment of the transformed cell DNA with deoxyribonuclease or omission of the DEAE-dextran prevented the rescue of virus. Large amounts of transformed cell DNA were required (>10 mug/culture of 10(6) cells) to effect rescue of SV40 by passage through monkey cells. A linear response was obtained between the input of DNA with inocula between 10 and 45 mug of DNA/culture and the yield of SV40 recovered. Biological activity was demonstrable irregularly when the transformed cell DNA was assayed directly in the presence of DEAE-dextran. The DNA induced plaque formation in about 50% of the trials as well as the synthesis of SV40 tumor and viral antigens in rare simian cells. The infectious DNA appeared to be associated with cellular DNA. The infectivity was found in the pellet of precipitated DNA obtained by the Hirt technique and was inactivated by boiling for 15 min. These properties are characteristic of linear cellular DNA and not of free, circular SV40 DNA.     

Spontaneous Virus Production by Clonal Lines of Simian Virus 40-Transformed Cells and Effects of Superinfection by Deoxyribonucleic Acid from Mutant Simian Virus 40 Strains

Small amounts of infectious simian virus 40 (SV40) were recovered from parental cultures of SV40-transformed human embryonic lung (WI38 Va13A) cells, from 12 primary clones, from 17 secondary clones, and from 18 tertiary clones. The cloning experiments demonstrated that the capacity for spontaneous virus production is a hereditary property of WI38 Va13A cells. Infectious virus was not recovered from every clone at every passage. Repeated trials at different passage levels were necessary to detect virus production. Approximately one in 10(5) to 10(6) of the cells of the clonal lines initiated plaque formation when plated on the CV-1 line of African green monkey kidney cells. No increase in infectious center formation was observed after the clonal lines were treated with bromodeoxyuridine, iododeoxyuridine, or mitomycin C or after heterokaryon formation of treated cells with CV-1 cells. The clonal lines of WI38 Va13A cells were susceptible to superinfection by SV40 deoxyribonucleic acid (DNA). To determine whether only those cells which spontaneously produced virus supported the replication of superinfecting SV40 DNA, cultures were infected with DNA from a plaque morphology mutant and a temperature-sensitive mutant of SV40. After infection by SV40 DNA, approximately 100 to 4,400 times more transformed cells formed infectious centers than were spontaneously producing virus. To determine whether the resident SV40 genome or the superinfecting SV40 genome was replicating, infectious centers produced by SV40 DNA-infected WI38 Va13A cells on CV-1 monolayers were picked and the progeny virus was analyzed. Only the superinfecting SV40 was recovered from the infectious centers, indicating that in the majority of superinfected cells the resident SV40 was not induced to replicate.     

Non-mutagenic and mutagenic post-replicative DNA repair in prokaryotic and eukaryotic cells

The review is devoted to mechanisms of repair gaps in DNA daughter strand, formed during the stall of moving replication forks and restart of replication in cells after the action of DNA damaging agents (predominantly--UV light). The repair of daughter DNA, or postreplication DNA repair (PRR), is realized by error-free (non-mutagenic) and error-prone (mutagenic) pathways. The former is a recombination repair, or recombination between two sister duplexes. By this way the major part of postreplication gaps is eliminated. The second way is related with the induction of SOS-response. In Escherichia coli cells mutagenic SOS-response is realized by proteins RecA, UmuD, UmuC, DNA-polymerase III holoenzyme and others. In E. coli some mutagenic enzymes--DNA-polymerase IV (the product of dinB gene) and DNA-polymerase V (the product of umuDC genes) have been recently discovered. In Saccharomyces cerevisiae cells postreplicative translesion synthesis is realized by newly discovered enzymes deoxycytidilmonophosphatetransferase (encoded by REV1 gene), DNA-polymerase zeta (encoded by REV3 gene), DNA-polymerase eta (encoded by RAD30 gene). All the three enzymes share a great homology with UmuC enzyme of E. coli. DNA polymerase eta correctly inserts adenine residues in the daughter strand opposite noncoded thymine residues in cyclobutane pyrimidine dimer. Based on RAD6 gene of S. cerevisiae, human cells hREV1, hREV3 and hRAD30A have been obtained to encode, respectively, deoxycytidiltransferase, DNA-polymerase zeta and DNA-polymerase eta. It has been shown that the defect of PRR DNA in xeroderma pigmentosum variant is associated with DNA-polymerase eta deficiency. This defect is corrected by the extract of intact HeLa cells. The importance of newly discovered enzymes in the system of mechanisms of DNA repair and replication is discussed.