THE ONCOGENE OF BAV-3 AS A MUTAGEN

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1 J. Cell Sci. 78, (1985) 97 Printed in Great Britain The Company of Biologists Limited 1985 THE ONCOGENE OF BAV-3 AS A MUTAGEN L. L. LUKASH 1, N. B. VARSHAVER 2 '*, T. I. BUZHIYEVSKAYA 1 AND N. I. SHAPIRO 2 x Institute of Molecular Biology and Genetics, Ukrainian Academy of Sciences, V24 Zabolotni Street, Kiev 2S2627, U.S.S.R. institute of Molecular Genetics, U.S.S.R. Academy of Sciences, Kurchatov Square, Moscow , U.S.S.R. SUMMARY We studied the mutagenic and carcinogenic effects on mammalian cells of two EcoB.1 DNA fragments of bovine adenovirus type3 (BAV-3) integrated into the pbr325 plasmid. Fragment D located between 3-6 and 19-7 map units, contains the viral oncogene, fragment C, located between 44-3 and 63-7 map units, has no oncogenic activity. The BAV-3 oncogene was shown to increase significantly the frequency of 6-mercaptopurine (6MP)-resistant mutants in Chinese hamster calls. Fragment C, pbr325 without viral sequences and DNA from normal Syrian hamster cells did not have any mutagenic effect. We also looked at the combined action of the viral DNA fragments and the tumour promoter 12- O-tetradecanoylphorbol-13-acetate (TPA), which enhances the transforming effect of carcinogens. TPA was shown to increase the mutant yield on exposure to the viral oncogene but not to induce mutagenic activity in those types of DNA that are unable to transform cells. Probably TPA does not affect the initiation of the mutation process, but acts on later stages just as it affects carcinogenic activity. Thus the results obtained confirm the existence of parallelism between the mutagenic and transforming effects of viral DNA and show that both activities are mapped in the same region of viral DNA - its oncogene. INTRODUCTION One of the ways to study the relationship between viral carcinogenesis and mutagenesis consists of establishing the role of the oncogene in these processes. We have shown earlier that the temperature-sensitive mutant of simian virus 40 (tsa), which is deficient in respect of the oncogene region encoding the large T antigen, stops inducing mutagenesis as well as malignant transformation in Chinese hamster cells at non-permissive temperature (Gorbunova, Varshaver, Marshak & Shapiro, 1982). In other words, in this case one region of the viral genome determines both carcinogenesis and mutagenesis. This provides one further reason for believing that the situation is not accidental and the mechanisms of the two major processes have certain phases in common. Of course it became necessary to find out whether this location of the mutagenic activity was specific to SV40 or characterized other oncogenic viruses as well. With this in mind, we decided to study the mutagenic activity of two restriction DNA fragments of bovine adenovirus type 3 (BAV-3), one of which contained the Author for correspondence. Key words: viral oncogene, induced mutagenesis, tumour promoter TPA.

2 98 L. L. Lukash and others viral oncogene and the other did not. In this way we could find out whether, once again, the induction of mutations was associated with the oncogene's transforming activity or other sequences of the viral genome were also mutagenic. While looking for mutagenic activity in individual regions of viral DNA we did not rule out the possibility that it might be weak. Hence we needed a way of specifically enhancing the mutagenic effect of viral DNA that would affect not just any DNA but only the DNA encoding the mechanism of viral carcinogenesis. We used the tumour promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) for this purpose. MATERIALS AND METHODS Cells and culture conditions I n induced mutagenesis assays we used a continuous hypodiploid clone of Chinese hamster 237 cells (parental line BIId-ii-FAF28) sensitive to purine base analogues (hypoxanthine phosphoribosyltransferase +, HPRT" 1 "), non-permissive for BAV-3 with a modal chromosome number of 18. Cells were cultured in Eagle's medium with 10% bovine serum (inactivated for 90min at S6 C), with penicillin, monomycin and streptomycin. Once a month the cultures were treated with tetracycline to prevent mycoplasmic contamination. Virus and transfection procedure We used clone 3 of the WBR-1 strain of BAV-3, kindly provided by E. S. Zalmanzon from the Institute of Molecular Biology, U.S.S.R. Academy of Sciences. Clone 3 has somewhat reduced oncogenic activity compared with the original WBR-1 strain (Darbyshire, 1966). Viral DNA was isolated as described previously (Charikova, Lukash & Zalmanzon, 1983). To obtain fragments of BAV-3 DNA, it was treated with restriction endonuclease cori. The fragments were cloned in the pbr325 plasmid by V. N. Kalynin at the Institute of Virology, U.S.S.R. Academy of Medical Sciences, which assistance is gratefully acknowledged by the authors. We used two fragments: fragment D, which is located at the left end of viral DNA between 3-6 and 19-7 map units and contains the oncogene; and fragment C, located between 44-3 and 63-7 units and devoid of transforming activity (Igarashi et al. 1978; Kurokawa, Igarashi & Sugino, 1978). The study of the mutagenic activity involved whole intact DNA of BAV-3, viral fragments D and C integrated into the pbr325 plasmid and the pbr32s plasmid without viral DNA. The transfection was performed by a modified calcium phosphate method (Van der Eb & Graham, 1980). Induction of gene mutations to 6MP resistance (HPRT + - HPRT~) Chinese hamster 237 cells were treated with viral DNA and with plasmids containing fragments D and C in concentrations of 1-5, 3-0and6-0/Ug/ml (l'ml per plate). Since the viral DNA fragments constitute about half the recombinant molecule, their concentrations on transfection were 0'7, 1-5 and 3-OjUg/ml. The expression times were 2 and 3 days. The cells were then plated on Petri dishes (SOmm in diameter), at 25 X 10 4 cells per dish, in Eagle's medium with 20% serum and 40^g/ ml6mp (Koch-Light Lab., U.K.). The medium was changed every 3 4 days. From dishes were seeded in the control and each of the experimental series. On the 13th to 14th day of growth under selective conditions the colonies were stained with methylene blue and those containing more than 100 cells were scored on coded dishes. Apart from assays with the pbr325 plasmid containing viral DNA fragments, we performed control experiments on the induction of resistance mutations by the pbr325 plasmid without viral sequences and by DNA fragments from normal Syrian hamster cells of different molecular weight (primary cultures and liver cells). The survival of cells was estimated by the plating efficiency of 200 cells in non-selective medium. Induction of gene mutations by treatment with BAV-3 fragments and TPA combined After transfection by fragments D, C and the plasmid pbr325, the cells were cultivated for 3 days

3 The oncogene ofbav-3 as a mutagen 99 in a full medium, then transferred to a selective medium containing 6MP and TPA at a concentration of 0 - l fig/'ml, which remained in the medium throughout the growth in culture until the time of colony fixation. RESULTS Mutagenic activity ofbav-3 DNA fragments within the pbr325 plasmid As already mentioned, we studied the induction of mutations to 6MP resistance by two EcoRl restriction fragments of BAV-3 DNA: fragment D, which contains the viral oncogene and is located at the left end of the BAV-3 chromosome; and fragment C, which has no oncogenic activity and is located in the middle of the viral genome. As a result it was possible to discover whether or not the regions responsible for the transforming and mutagenic effects coincided. Preliminary experiments had demonstrated that the mutagenic activity of the viral DNA was detected after a 2-day expression time post-transfection, reached a maximum after 3 days and then began to decline. Because of this the mutagenesis induced by BAV-3 DNA fragments was studied with expression times of 2 and 3 days. Table 1 shows the frequency of 6MP-resistant mutants after exposure to viral DNA fragments within the pbr325 plasmid at a concentration of 3 /ig/plate. As seen from the data presented, viral DNA, as well as the plasmid pbr325, did not affect cell survival. The mutation frequency increased after the treatment with fragment D containing the oncogene after both 2 and 3 days of expression time. The increase was statistically significant. During 3 days the cell population doubled four times. Experiments with concentrations of 1-5/ig/plate and 6-0/^g/plate also demonstrated an increase in the mutant yield compared with the control (these data are not shown in Table 1). In five out of seven assays with different concentrations of fragment D the Table 1. Induction of mutations to 6-mercaptopurine resistance by BAV-3 DNA fragments integrated in the pbr325 plasmid Expression time (days) Plating efficiency (%) Frequency of mutants X 10~ 5 (corrected for survival)* Induction X 10" 5 (by difference) Pi Control * C fragment pbr <0-05 Control C fragment pbr <0-01 The concentration of DNA in all the experimental series was 3/xg/ml per plate. fthe mean values for two or three experiments are given. {The significance of the difference was calculated according to the Fischer < > test.

4 100 L. L. Lukash and others Table 2. Induction to mutations to 6-mercaptopurine resistance by fragments of DNA from normal Syrian hamster cells Exp. no. ( DNA Plating efficiency (%) Frequency of mutants X10" 5 (corrected for survival) Induction Xl0~ 5 (by difference) P >0-01 >001 >0-01 >0-01 >0-01 The expression time was 3 days. The concentration of DNA was ljug/ml per plate. increase was statistically significant by Fischer's (j) criterion, while an assessment of the entire experimental series by the Wilcoxon signed rank test showed that the induction was highly significant (P<0-01). At the same time we should note that, compared with native viral DNA (Charikova et al. 1983), fragment D had a lower mutagenic activity. A different result was obtained after the exposure to fragment C: the frequency of mutants was the same as in the control. Nor was there any mutagenic effect in the case of the pbr325 plasmid devoid of viral sequences. Likewise, attempts to induce 6MPresistant mutations by DNA fragments of different molecular weight from normal Syrian hamster cells (primary cultures and liver cells) failed to reveal any mutagenic effect (see Table 2). Thus, the results demonstrate unambiguously that fragment D of BAV-3 DNA, the oncogene, was the only one to possess mutagenic activity under our experimental conditions. Fragment C of the viral DNA, bacterial DNA (pbr325) or the DNA of normal Syrian hamster cells displayed no such activity. Effect of the tumour promotor TPA on the mutagenic activity of BAV-3 DNA fragments Exposure to the tumour promotor TPA after the treatment of cells with carcinogens is known to raise the frequency of malignant transformation sharply in vitro and also that of tumour formation (Fischer, Dorsch-Hasler Weinstein & Ginsberg, 1979; Kopelovich, Bias&Helson, 1979; Mondal, Brankow&Heidelberger, 1976). Besides, treatment with the promoter after the BAV-3 infection has been shown to increase the frequency of 6MP-resistant mutants and tumour formation in mice (Manuilova, Lukash & Shapiro, Genetika, 1985). Proceeding, with knowledge of these facts, we looked at the effect of TPA on the mutagenic activity of our BAV-3 DNA restriction fragments. The results are shown in Tables 3 and 4. They confirm the fact that TPA does not have a mutagenic effect of its own, just as it is known to have no transforming effect (Manuilova et al. 1985). If the cells were treated with TPA we observed slight

5 The oncogene ofbav-3 as a mutagen 101 Table 3. Induction of mutations to 6-mercaptopurine resistance by treatment with BAV-3 oncogene and TPA combined Exp. no. DNA concentration (jug/ml per plate) Plating efficiency (%) Frequency of mutants XlO" 5 (corrected for survival) Induction X10" 5 (by difference) 1. Control TPA + TPA < Control TPA + TPA < Control TPA + TPA <0-01 The expression time was 3 days. The significance of the difference between the frequency of mutants induced by treatment with BAV-3 oncogene and TPA combined and that induced by BAV-3 oncogene. Table 4. Induction of mutations to 6-mercaptopurine resistance by treatment with TPA, BAV-3 DNA C fragment andpbr325 combined Plating efficiency (%) Frequency of mutants X 10" s (corrected for survival)* Induction XlO" 5 (by difference) Control TPA C fragment C fragment + TPA pbr325 pbr325 + TPA <0-05 >0-0S Concentration of DNA was 3^g/ml per plate. The expression time was 3 days. and insignificant variation in the mutation frequency around the control level. In contrast, the mutagenic effect of fragment D (the oncogene) sharply increased if TPA was introduced into the selective medium after the transfection. The frequency of induced mutants increased by a factor of compared with oncogene exposure without TPA. The difference is statistically highly significant. The combined action

6 102 L. L. Lukash and others of TPA and fragment C or the pbr325 plasmid without viral sequences did not increase the yield of mutants. Thus, TPA raised the mutation frequency only if added after exposure to the carcinogenic fragment. DISCUSSION Our experiments with the BAV-3 DNA restriction fragments within the pbr325 plasmid have shown that only the viral oncogene (fragment D), endowed with transforming activity, has a mutagenic effect. Other types of DNA, namely fragment C, bacterial DNA (the pbr325 plasmid) and the DNA of normal Syrian hamster cells, did not induce resistance mutations in Chinese hamster cells. Hence mutagenic activity was inherent only in the DNA that possessed transforming activity. Clearly, a foreign biopolymer/>er se is not mutagenic; in order to induce mutations the foreign DNA must have a specific biological activity, in this case associated with its oncogenic properties. Thus the results of this study confirm the existence of parallelism between the mutagenic and transforming effects of oncogenic viruses and show that both activities are mapped in the same region of the viral genome: its oncogene. It should be noted, however, that the mutagenic effect of the BAV-3 DNA and the oncogene is far less pronounced that that of the intact virus (Charikova et al. 1983; Lukash, Buzhiyevskaya, Varshaver & Shapiro, 1981). To reach the same level of mutagenesis, the number of viral DNA molecules required was 10 4 per cell in transfection experiments and only eight viral particles in the case of the intact virus. There may be various reasons for this. It is possible that viral DNA loses some of its biological activity during isolation. Besides, DNA may be degraded more readily by cell nucleases than intact viral particles. As to the relatively weak effect of the BAV- 3 oncogene, in this case other factors may also be at play. The most important of these is probably the fact that, while excising the oncogene, the EcoRl endonuclease cuts the enhancer off it. Although the exact location of the transcription enhancer in the BAV-3 genome is not known, the considerable homology of adenoviral (Ad) genomes suggests that, just as in Ad5, Ad7 and Ad 12 (Hearing & Shenk, 1983), the enhancer is located to the left of the cori restriction site. An oncogene deprived of the enhancer may have a considerably weakened effect. This hypothesis would probably be tested by studying the mutagenic and transforming activities of a BAV-3 DNA fragment containing the enhancer. Besides, it has recently been demonstrated that the carcinogenic effect of adenoviruses may be influenced by DNA regions far removed from the oncogene, particularly region E4 (Bernardset al. 1984; Shirokiefa/. 1984). The existence of common phases in the processes underlying malignant transformation and mutagenesis induced by oncogenic viruses is confirmed by the analysis of the combined effects of DNA fragments and the TPA promoter. It was established earlier that factors enhancing or weakening the transforming activity of viruses affect mutagenesis in a similar way (Gorbunova et al. 1982; Lukash et al. 1981; Varshaver, Marshak, Gorbunova, Lukash & Shapiro, 1980). The results of the present study confirm this. Indeed, TPA added to the medium after transfection enhanced the oncogene's mutagenic effect but did not induce mutagenic activity in other types of

7 The oncogene ofbav-3 as a mutagen 103 DNA, viral or non-viral, that had no transforming effect on cells. It has already been mentioned that TPA itself does not cause malignant transformation and only acts as a tumour promoter when initiation has already taken place due to some carcinogen. It seems that in mutagenesis also TPA acts at the later stages of the mutation process, though the mechanism of its action is still unknown. The authors are grateful to E. V. Charikova for providing Syrian hamster cells DNA. The excellent technical assistance of I. V. Vavilina and M. G. Petrakova is gratefully acknowledged. REFERENCES BERNARDS, R., DE LEEUW, M. G. W., VAESSEN, M. J., HOUVELING, A. & VAN DER EB, A. J. (1984). Oncogenicity by adenovirus is not determined by the transforming region only. J. Virol. 50, CHARIKOVA, E. V., LUKASH, L. L. & ZALMANZON, E. S. (1983). Mutagenic action of the highly oncogenic bovine adenovirus type 3 (BAV-3). Biol. nauki (U.S.S.R.), N4, DARBYSHIRE, J. H. (1966). Oncogenicity of bovine adenovirus type 3 in hamsters. Nature Lond. 211, FISCHER, P. B., DORSCH-HASLER, K., WEINSTEIN, I. B. & GINSBERG, H. S. (1979). Tumour promoters enhance anchorage-independent growth of adenovirus-transformed cells without altering the integration pattern of viral sequences. Nature, Lond. 281, GORBUNOVA, L. V., VARSHAVER, N. B., MARSHAK, M. I. & SHAPIRO, N. I. (1982). The role of the transforming A gene of SV40 in the mutagenic activity of the virus. Molec. gen. Genet. 187, HEARING, P. & SHENK, T. (1983). The adenovirus type 5 E1A transcriptional control region contains a duplicate enhancer element. Cell 33, IGARASHI, K., SASADA, R., KUROKAWA, T., NIYAMA, Y., TSUKAMOTO, K. & SUGINO, Y. (1978). Biochemical studies on bovine adenovirus type 3. IV. Transformation by viral DNA and DNA fragments. J. Virol. 28, KOPELOVICH, L., BIAS, N. E. & HELSON, L. (1979). Tumour promoter alone induces neoplastic transformation of fibroblasts from humans genetically predisposed to cancer. Nature, Lond. 282, KUROKAWA, T., IGARASHI, K. & SUGINO, Y. (1978). Biochemical studies on bovine adenovirus type 3. III. Cleavage maps of viral DNA by restriction endonucleases cori, BamWl and Hin&\U.J. Virol. 28, LUKASH, L. L., BUZHIYEVSKAYA, T. I., VARSHAVER, N. B. & SHAPIRO, N. I. (1981). Oncogenic adenovirus as mutagen for Chinese hamster cells in vitro. Sotnat. Cell Genet. 7, MANUILOVA, E. S., LUKASH, L. L. & SHAPIRO N. I. (1985). Mutagens and the tumour promoter. Genetics (U.S.S.R.) (in press). MONDAL, S., BRANKOW, D. W. & HEIDELBERGER, CH. (1976). Two-stage chemical oncogenesis in cultures of C3H/10TI/2. Cancer Res. 36, SHIROKI, K., HASHIMOTO, S., SAITO, I., FUKUI, Y., FUKUI, Y., KATO, H. & SHIMOJO, H. (1984). Expression of the E4 gene is required for establishment of soft-agar colony-forming rat cell lines transformed by the adenovirus El gene..?. Virol. 50, VAN DER' EB & GRAHAM, F. L. (1980). Assay of transforming activity of tumor virus DNA. In Methods of Enzymology, vol. 65 (ed. L. Grossman & K. Moldave), pp New York, London: Academic Press. VARSHAVER, N. B., MARSHAK, M. I., GORBUNOVA, L. V., LUKASH, L. L. & SHAPIRO, N. I. (1980). The synergistic effects of SV40 and BUdR on induction of gene mutations and chromosomal aberrations in Chinese hamster cells. Mutat. Res. 70, {Received 31 January Accepted 15 April 1985)

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