Reversal of malignant transformation induced by the oncogenic virus SV40. I. Induction of reversal to normal type for the contact inhibition trait

The possibility of induction by the oncogenic DNA-containing virus SV40 of reversions to normal phenotype as regards contact inhibition ("flat" revertants), was studied in spontaneously transformed chinese hamster fibroblasts. Negative selection was used for detection of revertants. The method adopted allowed to study the mutagenic activity of the virus, while excluding its transforming effect. In all experiments the frequency of revertants after infection exceeded that in control series. The value of induction varied from 1.2 to 28.4 X 10(-6). The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) known to increase the frequency of mutations induced by carcinogens in vitro, displayed no enhancing effect on the frequency of revertants induced by SV40. The lack of enhancement of virus-induced reversions after TPA treatment might be explained by the lack of the transforming effect of SV40 in the system studied. Some of the normal "flat" colonies were T-antigen positive, i. e. the viral oncogene was expressed. The role of mutations induced by SV40 in cellular genes controlling malignancy is discussed.     

Reversibility of malignant transformation as affected by the oncogenic virus SV40. II. The characteristics of virus-induced revertant clones

Fifteen revertant clones exhibiting contact inhibition, one of the typical characteristics of normal cells, were studied after treatment of spontaneously transformed Chinese hamster fibroblasts with SV40. The clones proved to be partial revertants, as regards to other properties of the normal phenotype--loss of the ability to grow in a medium with a low serum content and anchorage-dependence. Viral DNA was detected in all revertant clones. The expression of T-antigen--the product of viral oncogene, was observed in 13 of 15 revertants analyzed. The study of SV40 "rescued" from several revertants in permissive monkey cells has shown that the virus is non-defective. In 7 clones, reversion was accompanied with polyploidization. In the cases, reversion could be due to changes in the balance between oncogenes and suppressor genes (anti-oncogenes). The possibility of induction by SV40 of mutations in anti-oncogenes suppressing the expression of both cellular and viral oncogenes is discussed. It is suggested that reversion to the normal phenotype in clones with a near-diploid karyotype could result from such virus-induced suppressor mutations.     

Integration of SV40 DNA into the cell genome and viral mutagenesis

Integration of DNA of a temperature-sensitive SV40 mutant (tsA239) into the cell genome was studied. The viral A gene (the oncogene) encodes the tumour T antigen which is ts in the mutant and is devoid of mutagenic and transforming activity under non-permissive conditions (40 degrees C). Clones of Chinese hamster cells infected by tsA239 mutant were analysed. Those infected by wild-type SV40 served as controls. As shown by dot-hybridization, SV40 DNA was detected in cells of 14 out of 18 clones infected by tsA mutant and incubated at 40.5 degrees C, and in all 20 clones infected by tsA mutant and incubated under permissive conditions (33 degrees C), the difference between the two groups being insignificant (p greater than 0.05). By means of blot-hybridization it was established that viral DNA was integrated into the cell genome of all 12 clones analysed, belonging to the three experimental series: infection by tsA mutant, incubation at 40.5 and 33 degrees C, infection by wt SV40, incubation at 40.5 degrees C. The number of integration sites ranged from one to four in different clones. Integration of SV40 DNA in tandems was observed. The data presented allow to conclude that integration per se does not play a crucial role in determining the mutagenic and transforming effect of the virus. Obviously, what matters is the activity of viral oncogene product - the T antigen.     

Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers.

The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.     

The role of the transforming a gene of SV40 in the mutagenic activity of the virus

Summary The mutagenic activity of the tsA239 mutant of SV40 which synthetizes a defective T antigen at 40°C was investigated in Chinese hamster cells under permissive and nonpermissive temperature. At 33°C the virus increased the yield of 6-mercaptopurine-resistant colonies after 2 days expression time by a factor of 1.6–4 as compared with the control and raised the frequency of aberrant metaphases after the same time by a factor of 1.9–3.4.In the same experiments, with the same initially infected population of Chinese hamster cells, at 40°C tsA SV40 did not induce either gene mutations or chromosome aberrations at the same early stage after infection. Presumably the activity of the A gene of SV40 is necessary not only for the transforming but also for the mutagenic effect of the virus.Abbreviations SV40 Simian virus 40 - BAV3 bovine adenovirus 3 - 6MP 6-mercaptopurine     

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.     

Role of the oncogene in the mutagenic activity of bovine adenovirus type 3

The mutagenic and carcinogenic effect of two EcoRI-fragments of bovine adenovirus type 3 (BAV-3) DNA inserted into pBR325 has been studied. The C fragment (located between 3,6 and 19,7 map units) contains the viral oncogene, the C fragment (between 44,3 and 63,7 map units) displays no transforming activity. It has been established that oncogene BAV-3 statistically true increases the yield of mutants resistant to 6-mercaptopurine (6MP) in Chinese hamster cells. The C fragment, pBR325 without viral sequences and DNA fragments of different molecular weights from normal Syrian hamster cells have no mutagenic effect. The control over tumor formation in syngenic mice after injection of C3H10T 1/2 and D. C fragments and pBR325 treatment exposed a parallelism between the mutagenic and transforming effect. The study of the combined effect of viral DNA fragments and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) which increases the transforming activity of different carcinogens, shows that the promoter increases the frequency of mutants after viral oncogene treatment and does not induce mutagenic activity of those types of DNA which are unable to transform the cells.     

Induction of gene mutations and chromosomal aberrations by simian virus 40 in cultured mammalian cells

• Induction of gene mutations by SV40 was studied in aneuploid human and Chinese hamster cells. In Chinese hamster cells SV40-induced chromosome aberrations were also studied.

• SV40 penetrated into the cells of both lines and induced synthesis of the T antigen. The efficiency of infection in Chinese hamster cells was tested additionallby their ability to form colonies in medium lacking the serum growth factor. The maximal number of cells with growth factor independence was observed on the first day after infection. When hamster cells had been maintained in “factor-free medium” for the first two passages after infection a sub-line was isolated, which synthesized T antigen 60 days after exposure to SV40. This was considered to be an indirect proof of the integration of viral genome into host chromosome.

• A significant increase in the frequency of chromosomal aberrations was detected in SV40-infected Chinese hamster cells. It was observed on the first and second days after treatment. The most numerous were the chromosome and chromatid breaks, which were distributed randomly in 5 morphological groups according to the chromosome length.

• SV40-induced mutations of resistance to 8-AG and 6-MP in human and Chinese hamster cells respectively were detected, when cells were plated in selective medium one to five days after infection. Induction was detected in all the 4 experiments with human cells and in 9 out of 11 experiments with Chinese hamsters cells. Induction was highly significant according to the Wilcoxon test (P>0.99), when the results of all experiments carried out in human and Chinese hamster cells were summarized. Resistance was stable after prolonged cultivation of 13 isolated clones under non-selective conditions.

• It is suggested that viral genome integration, gene mutations and chromosomal aberrations may have common molecular mechanisms. The role of gene mutations in virus-induced carcinogenesis is discussed.

The plasmid pEJ6.6 carrying the activated c-Ha-ras-1 oncogene increases the mutation frequency in Chinese hamster cells]

The induction of gene mutations and chromosome aberrations by the plasmid pEJ6.6 carrying the activated c-Ha-ras-1 oncogene from human bladder carcinoma was studied in cultured Chinese hamster cells. Both an increase in the frequency of gene mutations to 6-mercaptopurine resistance and of chromosome aberrations was observed after pEJ6.6 treatment as compared to control series (pBR322). Thus the results of experiments carried out show that the pEJ6.6 plasmid possesses a mutagenic activity.     

A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome.

Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of six HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensible for SV40 DNA amplification. Our results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.     

Carcinogen-Mediated Amplification of Specific DNA Sequences

A model experimental system based on SV40-transformed Chinese hamster embryo cells and a highly sensitive in situ hybridization procedure was designed. Exposure of the cells to different categories of chemical and physical carcinogens resulted in the induction of SV40 DNA synthesis in the treated cells. Although the carcinogen-mediated amplification of SV40 DNA sequences is regulated by the viral “A” gene, neither infectious virus nor complete viral DNA molecules were rescued from the treated cells. A heterogenous collection of DNA molecules containing SV40 sequences was generated following treatment with DMBA. Restriction enzyme analysis of the amplified DNA molecules in the Hirt supernatant revealed that not all sequences in the integrated SV40 inserts are present. The possibility that the amplification of SV40 sequences is a reflection of a general gene amplification phenomenon mediated by carcinogens is discussed.     

Induction and expression of mutations in mammalian cells in the absence of DNA synthesis and cell division

The induction of mutations by the alkylating agent ethyl methanesulfonate (EMS) was determined with Chinese hamster ovary cells maintained in serum-free medium to arrest DNA synthesis and cell division. The arrested cultures were treated with EMS and maintained in serum-free medium for various time intervals post-treatment before serum containing medium was added to initiate DNA synthesis and cell division. The concentration-dependent increase in 6-thioguanine-resistant mutants in the arrested cultures was similar to that found with exponentially dividing cultures when serum was added to the arrested cultures immediately after the EMS treatment; the time course of phenotypic expression was also similar with both cultures. In addition, maintenance of the arrested cultures in serum-free medium for up to 18 days post-treatment resulted in no change in the mutant frequency. This suggests that the mutagenic damage is not removed in these arrested cultures. Furthermore, maintenance of the arrested state for increasing time intervals before serum addition results in decreases in the time necessary for maximum phenotypic expression. Cultures maintained in serum-free medium for 16 days after mutation treatment show complete expression of the mutations with no need for subculture. This last result suggests that the mutagenic damage induced by EMS in Chinese hamster ovary cells is not removed and that this damage results in both the induction and expression of mutation in the absence of DNA replication.     

Genetic nature of one of the traits of malignant cell transformation in vitro

The genetic events controlling the ability of transformed cells to grow in a medium with a low serum content (ser+) were studied. A hypodiploid clone of Chinese hamster cells with normal serum requirements (49a5ser-) was used as starting material. The results of the fluctuation tests have shown that serum-independence is a random spontaneous event. Its rate of occurrence is 1-2 . 10(-5). The concomitant study of a gene mutation (resistance to 6-mercaptopurine) revealed similar characteristics with respect to the distribution of the number of mutants in replicate cultures and the mutation rate. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the oncogenic SV40 virus significantly increased the frequency of ser+ colonies. In the majority of clones isolated in a medium with 1% serum (11 spontaneous and 7 induced by MNNG), the ser+ character proved to be stable after different periods of cultivation without selective pressure. The degree of serum-independence varied in different clones. The results suggest that the ability to grow in a medium with a low serum content originates, in most cases, from a mutation event.     

Differential effect of ultraviolet light on the induction of simian virus 40 and a cellular mutator phenotype in transformed mammalian cells

UV irradiation of simian virus 40 (SV40)-transformed human and hamster cells induced them both to express a mutator phenotype and to produce SV40. The mutator could also be activated indirectly by transfecting unirradiated cells with UV-damaged calf thymus DNA. In contrast, UV-damaged exogenous DNA failed to rescue SV40 from unirradiated transformed cells. These results suggest that the expression of transforming viruses and of cellular mutator functions is regulated by at least partially independent mechanisms. Unlike the activation of a cellular mutator phenotype, the rescue of SV40 from virus-transformed mammalian cells by UV light might require that the integrated viral DNA and/or specific cellular sequences are directly damaged.     

Amplification of oncogenes and integrated SV40 sequences in mammalian cells by the decay of incorporated iodine-125

Iodine-125, in the form of 5-[125I]iododeoxyuridine (I-UdR), was incorporated into the DNA of SV40 transformed Chinese hamster embryo cells. Disintegration of the 125I led to increased cell killing with increasing dose as measured by the colony-forming ability of single cells. The D37 (the dose at which 37% of the cells survive) amounts to 95 decays per cell, corresponding to 0.66 Gy. Variations in the copy number of specific DNA sequences was measured by using dispersed cell blotting with sensitive DNA hybridizations. A 13-fold amplification of the viral DNA sequences (SV40) and a twofold amplification of two cellular oncogenes of the ras-family (Ki-ras and Ha-ras) were found. Other cellular genes, like the alpha-actin gene, were not amplified, and no variation in gene copy number was detected after incubation of cells with cold I-UdR. We suggest the observed gene amplifications are induced by the densely ionizing radiation emitted by the decay of the incorporated 125I atoms.     

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.     

DNA crosslinking, sister-chromatid exchange and specific-locus mutations

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.     

Enhancement by caffeine of N-methyl-N-nitrosourea-induced mutations and chromosome aberrations in Chinese hamster cells

N-Methyl-N-nitrosourea (MNU) increased the induction of mutations to 8-azaguanine resistance in Chinese hamster cells in a dose-dependent manner. Mutations were only observed with toxic concentrations of MNU. Since a plot of the fraction of cells surviving alkylation against the extent of methylation of DNA exhibited a shoulder it followed that there was a threshold level of DNA reaction which did not lead to mutations possibly due to efficient repair of DNA damage. Post-alkylation incubation in medium containing caffeine decreased cell survival while at the same time it increased the induced mutation frequency. Mutation frequency was increased whether caffeine was present for 48 h or for a further 12 days in the presence of the selective agent 8-azaguanine. MNU caused chromatid aberrations in Chinese hamster cells and these reached a value of 15% of the treated cells by 48 h after methylation. Post-alkylation incubation in caffeine increased the percentage of cells showing chromosomal damage to a maximum of 86% of treated cells by 40 h after alkylation. A large proportion of cells exhibited completely fragmented or shattered chromosomes. The proportion of cells showing the presence of micronuclei also dramatically increased following incubation of methylated cells in caffeine. These results are discussed in terms of the possibility that damage to DNA is responsible for the lethal, mutagenic and cytological effects of MNU in Chinese hamster cells, and that there is a caffeine sensitive step(s) in the repair of the DNA damage which is responsible for these effects.     

Analysis of Minimal Functions of Simian Virus 40 II. Enhancement of Oncogenic Transformation in Vitro by UV Irradiation

After light UV irradiation (5,000 to 10,000 ergs/mm²) “complete” and “defective” simian virus 40 (SV40) showed an enhancement of oncogenic transformation capacity in Syrian hamster kidney cells in vitro up to 180 and 270% of the controls, respectively. Simultaneously with the enhancement of transformation, an increase in T-antigen induction was observed in CV-1 cells infected with light UV-irradiated SV40; infectivity, however, was correspondingly reduced by 1 log₁₀. After strong UV irradiation (10,000 to 80,000 ergs/mm²) of “complete” and “defective” SV40, transformation capacity in vitro proved to be the most resistant viral function. It was only slightly reduced in comparison with a 4 to 5 log₁₀ reduction of infectivity. T-antigen induction of SV40 was also equally resistant to strong UV irradiation. We found no evidence of “multiplicity reactivation” involved in the high resistance of transformation capacity of SV40 after UV irradiation. Syrian hamster kidney cells transformed in vitro by UV-irradiated SV40 contained the SV40-specific T-antigen and showed the same morphology and growth characteristics as cells transformed by non-irradiated “complete” or “defective” SV40. They induced malignant tumors after subcutaneous inoculation into Syrian hamsters.