53
Vkws Research 17 (1990) 53-60 Elsevier VIRUS 00598
Adenovirus infection induces amplification of persistent viral DNA sequences (simian virus 40, hepatitis B virus, bovine papillomavirus) in human and rodent cells &kg R. Schlehofer and Harald zur Hausen Institut fir Virusforschung, Deutsches Kkebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg, F. R. G. (Accepted 16 May 1990)
Adenoviruses, types 2 and 12 induce ~p~fication of SV40 DNA sequences in cells of the SV40-transformed human newborn kidney cell line, NB-E. Similarly, integrated hepatitis B virus DNA sequences in the human hepatoma cell line, PLC/ *PR.P/5, and bovine papillomavirus (BPV) DNA sequences in BPV-transformed mouse cells (ID13) are amplified by adenovirus infection. Thus, similar to herpesgroup or vaccinia viruses or DNA damaging agents, adenoviruses are able to mediate selective DNA amplification in addition to their reported mutagenic and chromosome damaging effects. The role of amplification of integrated viral DNA sequences in development and progression of specific tumors (e.g. hepatoeellular carcinoma) remains to be determined.
Adenovirus; SV4O; Hepatitis B virus; DNA ~p~fication;
Bovine pap~lorna~~s
Amplification of specific DNA sequences within cells of hamster and human cell lines is induced by chemical and physical carcinogens and as well by herpesgroup and vaccinia viruses (Schlehofer et al., 1983a, 1986). These viruses are known to
Correspondence to: J.R. Schiehofer, Institut fii Virusforschung, Im Neuenheimer Feld 280, D-6900 Heidelberg, F.R.G. * Supported by the Deutsche Forschungsgemeinschaft. 0168-1702/~/~3.50
0 1990 Ekvier
Deutsches Krebsforschungszentr,
Science Publishers B.V. (Biom~~
Division)
54
induce chromosomal damage and appear to transform rodent cells (Duff and Rapp, 1971; Koziorowska et al., 1971; Albrecht and Rapp, 1973). Selective DNA amplification in addition appears to play a major role in adaptation of cells to DNA damaging agents thus mediating drug resistance (Schimke, 1984a,b; Stark and Wahl, 1984). Furthermore, amplification of oncogene sequences in malignant tissues seems to be correlated with tumor progression (Alitalo and Schwab, 1986; Schwab, 1987). Adenovirus infection leads to specific chromosomal alterations in human cells (zur Hausen, 1967, 1973; Stich, 1973) and to transformation of rodent cells (McBride and Wiener, 1964; Casto, 1973). Furthermore, these virus infections induce mutations in host cell DNA (Stich and Yohn, 1967; Marengo et al., 1981) as also shown for herpes viruses (Schlehofer and zur Hausen, 1982). Therefore it was of interest to analyze whether adenovirus infection induces amplification of specific DNA sequences within appropriate cell culture indicator systems similar to herpesgroup or vaccinia viruses or carcinogens. To this aim we used SV40-transformed human newborn kidney cells [NB-E, (Shein and Enders, 1962)] which are fully permissive for adenovirus replication. The cells were grown in Eagle’s minimal essential medium (Gibco, Karlsruhe, F.R.G.) supplemented with 10% fetal calf serum (Flow, Bonn, F.R.G.) and antibiotics (penicillin G: 100 U/ml, streptomycin: 100 pg/ml; Gibco). NB-E cells were infected with adenovirus type 12 at a multiplicity of infection (MOI) of 0.5 PFU/cell, or mock-infected or treated with the 7,12,dimethyl-benz(a)anthracene [DMBA (final concentration: 0.5 pg/ml dissolved in DMSO), Sigma, Miinchen, F.R.G.), respectively. After five days of incubation at 37°C in a humidified atmosphere containing 5% CO,, cells were trapped by filtration onto nitrocellulose filters (“dispersed cell assay” [Winocour and Keshet, 19801) and hybridized with cloned 32P-labelled SV40 DNA (for details cf. Schlehofer et al., 1983a; 1986). As shown in Fig. 1, infection
Fig. 1. Amplification of SV40 DNA in cells of the SV40- transformed human cell line, NB-E by infection with adenovirus, type 12 (MO1 = 0.5 PFU/cell) or treatment with 7,12,dimethyl-benz.(a)anthracene (DMBA, 0.5 pg/ml): autoradiographs of “dispersed cell assay” analysis of lo6 NB-E cells each, infected with adenovirus type 12 or treatment with DMBA. Control = mock-infected cells. Five days after treatment or infection cells were trypsinized and then trapped onto nitrocellulose filters. The filters were hybridized with 32P-labelled SV40 DNA and autoradiographed.
55
with adenovirus type 12 induced amplification of SV40 DNA in NB-E cells similar to the chemical carcinogen, DMBA. The adenovirus-induced amplification of SV40 DNA sequences was also demonstrated in Southern blots (Southern, 1975) of DNA (extracted with phenol/ chloroform/isoamylalcohol according to standard procedures) of adenovirus type 12-infected cells (MO1 = 0.5 PFUfcell, analysis 5 days post infection) cleaved with the restriction endonucleases, XbaI + BgZII (Boehringer, Mannheim, F.R.G.) which
kb
23.7 123
321
kb
9.5 6.6 4.4
- 23.7 -
9.5
I
- 6.6
I
I
- 4.4 - 2.3 - 2.0
2.3 2.0
I
I
0.5
Fig. 2. Amplification of SV40 DNA in the SV4O-transformed human cell line NB-E by infection with adenovirus type 12 (MO1 = 0.5 PFU/cell) or by DMBA (cf. fig. 1): Autoradiograph of a Southern blot of NB-E cell DNA extracted after 5 days from treated or infected cells (10 cg DNA/lane), separated by agarose gel electrophoresis after cleavage with XbaI + BglII, and hybridized to 32P-labelled SV40 DNA. 1= Mock-infected control cell DNA; 2 = adenovirus type 12-infected cell DNA; 3 = DNA from DMBA-treated cells (in the right panel, the ethidium bromide stained agarose gel is shown for comparison of amounts of DNA kded and separated in each lane prior to blotting) Fig. 3. Amplification of SV40 DNA in the SV40-transformed human cell line, NB-E by infection with adenovirus type 2 (MO1 = 1 PFU/cell). Autoradiograph of a Southern blot of NB-E cell DNA extracted after 5 days from infected cells, prepared as described in Fig. 2. 1= Mock-infected control cell DNA; 2 = adenovirus type 2-infected cell DNA (right panel: ethidium bromide gel for comparison of amounts of DNA (5 pg/lane)
56
do not cut within SV40 DNA (Fig. 2). Similarly to infection with adenovirus type 12, adenovirus type 2 induced amplification of SV40 DNA in NB-E cells (Fig. 3). Cross-hybridization between SV40 DNA and adenovirus DNA could be excluded by hybridizing a Southern blot of adenovirus- infected HeLa cell DNA with 32P-labelled SV40 DNA (data not shown). This is also evident from the lack of hybridization of the SV40 probe to the clearly visible bands of adenovirus DNA (cf. fig. 2, ethidium bromide stain). Recently it was shown by our group that episomal as well as integrated DNA sequences of bovine papillomavirus (BPV) in BPV (type 1)-transformed Cl27 mouse cells (ID13, [Dvoretzky et al., 1980; Law et al., 19811) are amplified by treatment of ID13 cells with chemical or physical carcinogenic agents as well as by infection with herpes simplex virus (Schmitt et al., 1989). As shown in Fig. 4 by a Southern blot of adenovirus type 2-infected ID13 cell DNA cleaved with Sac1 (not cutting within the BPV genome) and hybridized with 32P-labelled cloned BPV type 1 DNA (pD-BPV-l), adenovirus type 2 induced amplification of integrated as well as of episomal BPV DNA (ID 13 cells were cultivated as described above for NB-E cells; adenovirus type 2 infection was at an MO1 of 1 PFU/cell; cellular DNA was extracted 3 days after infection when adenovirus-induced cytopathic changes were observed). Crosshybridization of BPV to adenovirus DNA was excluded as described above for SV40 DNA. Up to now exclusively papovavirus DNA sequences have been shown to be amplified by carcinogenic agents (“initiators”) or virus infections (SV40, lymphotropic papovavirus, polyoma, BPV (see zur Hausen and Schlehofer, 1987 for review). As shown in Fig. 5, DNA sequences of an unrelated virus, hepatitis B virus (HBV), present in the human hepatoma cell line, PLC/ *PRF/S (Alexander et al., 1976; Macnab et al., 1976) are amplified after infection with adenovirus type 2 (Cultivation of cells and infection were the same as described above; PLC/ * PRF/S cells are permissive for adenovirus replication resulting in cytopathic effect). This is shown by hybridization of a Southern blot of cellular DNA cleaved with EcoRI of infected PLC/ * PRF/S cells with 32P-labelled cloned HBV DNA [clone pSh14-3 (Will et al., 1982); Fig. 51. All bands hybridizing to HBV DNA appear to be amplified to a similar extent. Cross-hybridization between HPV and adenovirus DNA was excluded as described above. Our results demonstrate the induction of selective DNA amplification by infections with adenoviruses types 2 and 12. It is interesting to note that adenoviruses share this property with a number of herpesgroup viruses and with vaccinia virus, all coding for their own DNA polymerase (Citarella et al., 1972; Challberg and Englund, 1975; Ostrander and Cheng, 1980). Indeed, in the case of herpes simplex virus infections it has been shown that the viral DNA polymerase plays a key role in the process of DNA amplification (Matz et al., 1984). In addition, it has been demonstrated recently that replicative functions of the viral genome are involved in the induction of selective DNA amplification (Heilbronn and zur Hausen, 1989). It seems to be noteworthy that integrated and episomal sequences of known or putative DNA tumor viruses, but also integrated DNA of adeno-associated viruses become readily amplified by the viral infections outlined above but also by chemical
57
23.7 9.5 6.6
1
I
1 2.3 I 2.0
Fig. 4. Amplification of BPV in cells of the BPV type l-transformed mouse Cl27 cell line, ID13 by infection with adenovirus, type 2 (MO1 = 1 PFU/cell): Autoradiograph of a Southern blot of ID13 cell DNA extracted after 3 days from infected (2) or uninfected (1) cells (5 pg DNA/lane), separated by a&arose gel electrophoresis after cleavage with Sac1 and hybridized to 32P-labelled BPV DNA. Fig. 5. Amplification of HBV DNA with adenovirus, type 2 (MO1 = 0.5 DNA extracted after 2 days from DNA/lane), separated by agarose
in cells of the human hepatoma cell line, PLC/ *F/5 by infection PFU/cell): Autora~o~aph of a Southern blot of PLC,/*F/S cell infected (“Adeno 2”) or m~k-inf~t~ (“Control”) cells (10 pg gel electrophoresis after cleavage with EcoRI, and hybridized to 32P-labelled HBV DNA.
and physical carcinogenic factors (Schlehofer et al., 1983a,b 1986; Yalkinoglu et al., 1988; Schmitt et al., 1989). Since amplification is frequently accompanied by increased expression of the amplified sequences (Kleinberger et al., 1988) this may contribute to the process of transformation. In addition, intramolecular rearrangements or effects acting in cis resulting from additional integration events may also play a significant role. Hum~pathoge~c adenoviruses efficiently transform rodent cells (cf. Flint, 1980 for review). The ~ansform~ cells retain specific viral DNA sequences similar to SV40 and other papova~s-tr~sfo~ed cells. On the other hand tr~sformation
58
by herpes simplex viruses does not seem to require the persistence of specific viral DNA sequences (Galloway and McDougall, 1983). In these instances modifications of the host cell genomes induced by the viruses may be sufficient to result in cell transformation. It will be interesting to analyze whether adenoviruses are able to transform cells by a similar mechanism. This is particularly important since loss of adenovirus sequences from adenovirus-transformed rodent cells has been reported (Kuhlmann et al., 1982). The induced amplification of persisting hepatitis B and bovine papillomavirus sequences deserves special attention. Rearrangements and amplifications of HBV genomes have been frequently noted in primary hepatocellular carcinoma biopsies (Koch et al., 1984; Nagaya et al., 1987). Their role in the process of tumor development is presently not understood. Amplification of integrated papillomavirus DNA sequences is frequently observed in human cervical cancer (Dtirst et al., 1983; Boshart et al., 1984). It is possible that additional viral infections or exposures to chemical or physical carcinogenic factors may result in such amplifications and thus contribute to the malignant conversion (zur Hausen, 1982).
Acknowledgements We gratefully acknowledge the technical assistance of M. Ehrbar. We are indebted to J. Rommelaere and M. Lusky for providing NB-E and ID13 cells, respectively. We thank P. Howley for cloned BPV DNA and and E. Schwarz for SV40 DNA. C.H. Schroder kindly provided us with PLC/*PRF/S cells and with cloned HBV DNA.
References Albrecht, T. and Rapp, F. (1973) Malignant transformation of hamster embryo fibroblasts following exposure to ultraviolet-irradiated human cytomegalovirus. Virology 55, 53-61. Alexander, J.J., Bey, E.M., Geddes, E.W. and Lecatsas, G. (1976) Establishment of a continuously growing cell line from primary carcinoma of the liver. S. Afr. Med. J. 50, 2124-2128. Alitalo, K. and Schwab, M. (1986) Oncogene amplification in tumor cells. Adv. Cancer Res. 47, 235-281. Boshart, M., Gissmann, L., Ikenberg, H., Kleinheinz, A., Scheurlen, W. and zur Hausen, H. (1984) A new type of papillomavirus DNA: its presence in genital cancer biopsies and in cell tines derived from cervical cancer.Eur. Mol. Biol. Organ. J. 3, 1151-1157. Casto, B.C. (1973) Biological parameters of adenovirus transformation. Progr. Exp. Tumor Res. 18, 166-198. Challberg, M.D. and Englund, P.T. (1975) Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus. J. Biol. Chem. 245, 7812-7819. Citarella, R.V., Muller, R., S&aback, A. and Weissbach, A. (1972) Studies on vaccinia virus-directed desoxyribonucleic acid polymerase. J. Virol. 10, 721-729. Duff, R. and Rapp, F. (1971) Properties of hamster embryo fibroblasts transformed in vitro after exposure to ultraviolet- irradiated herpes simplex virus type 2. J. Virol. 8, 496-477. Diirst, M., Gissmann, L., Ikenberg, H. and zur Hausen, H. (1983) A new type of papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsies from different geographic regions. Proc. Natl. Acad. Sci. U.S.A. 60, 3812-3815.
59 Dvoretzky, I., Shober, R., Chattopa~y, SK. and Lowy, D.R. (1980) A quantitative in vitro focus assay for bovine papillomavirus. Virology 103, 369-375. Flint, S.J. (1980) Cell transformation induced by adenoviruses. In: J. Tooze (Ed.), DNA Tumor Viruses, pp. 547-575. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Galloway, D.A. and McDougall, J.K. (1983) The oncogenic potential of herpes simplex viruses: evidence for a “hit-and-run” mechanism. Nature (London) 302, 21-24. Heilbronn, R. and zur Hausen, H. (1989) A subset of herpes simplex virus replication genes induces DNA amp~fi~tion within the host cell genome. J. Viral. 63,3683-3692. Kleinberger, T., Berko Flint, Y., Blank, M., Etkin, S. and Lavi, S. (1988) Carcinogen-induced trans activation of gene expression. Mol. Cell. Biol. 8, 1366-1370. Koch, S., Freytag von Loringhoven, A., Hofschneider, P.H. and Koshy, R. (1984) Amplification and rearrangement in hepatoma cell DNA associated with integrated hepatitis B virus DNA. Eur. Mol. Biol. Organ. J. 3, 2185-2189. Koziorowska, J., Wlodarski, K. and Mazurowa, N. (1971) Transformation of mouse embryo cells by vaccinia virus. J. Natl. Cancer Inst. 46, 225-241. Kuhlmann, I., Achten, S., Rudoplh, R. and Doerfler, W. (1982) Tumor induction by human adenovirus type 12 in hamsters: loss of the viral genome from adenovirus type 1Zinduced tumor cells is compatible with tumor formation. Eur. Mol. Biol. Organ. J. 1, 79-86. Law, M.F., Lowy, D.R., Dvoretzky, I. and Howley, P.M. (1981) Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral sequences. Proc. Natl. Acad. Sci. USA 78, 2727-2731. Macnab, G.M., Alexander, J.J., Lecatsas, E.M., Bey, D. and Urb~o~c~ J.M. (1976) Hepatitis B surface antigen produced by a human hepatoma cell line. Br. J. Cancer. 34, 5099515. Marengo, C., Mbikay, M., Weber, J. and Thirion, J.P. (1981) Adenovirus-induced mutations at the hypoxanthine phospho-ribosyltransferase locus of Chinese hamster cells. J. Virol. 38, 184-190. Matz, B., Schlehofer, J.R. and zur Hausen, H. (1984) Identification of a gene function of herpes simplex virus type I essential for amplification of simian virus 40 DNA sequences in transformed Chinese hamster cells. Virology 134, 328-337. McBride, W.D. and Wiener, A. (1964) In vitro tr~sfo~ation of hamster kidney cells by human adenovirus type 12. Proc. Sot. Exp. Biol. Med. 115, 870.874. Nagaya, T., Nakamura, T., Tokino, T., Tsurimoto, T., Imai, M., Mayumi, T., Kamino, K., Yamamura, K. and Matsubara, K. (1987) The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma. Genes Dev. 1, 773-782. Ostrander, M. AND Cheng, Y.-C. (1980) Properties of herpes simplex virus type 1 and type 2 DNA polymerase. Biochim. Biophys. Acta 609, 32-245. Schimke, R.T. (1984a) Gene ~p~fi~tion in cultured animal cells. Cell 37, 705-713. Schimke, R.T. (198433)Gene amplification, drug resistance, and cancer. Cancer Res. 44,1735-1742. Schlehofer, J.R. and zur Hausen, H. (1982) Induction of mutations within the host cell genome by partially inactivated herpes simplex virus type 1. Virology 122, 471-475. Scblehofer, J.R., Gissmann, L., Matz, B. and zur Hausen, H. (1983a) Herpes simplex virus-induced amplification of SV40 sequences in transformed Chinese hamster embryo ceils. Int. J. Cancer 32, 99-103. Schlehofer, J.R., Heilbronn, R., Georg-Fries, B. and zur Hausen, H. (1983b) Inhibition of initiator-induced SV40 gene amplification in SV40-transformed Chinese hamster cells by infection with a defective parvovirus. Int. J. Cancer. 32, 591-595. Scblehofer, J.R., Ehrbar, M. and zur Hausen, H. (1986) Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus. Virology 152, 110-117. Schmitt J., Schlehofer, J.R., Mergener, K., Gissmann, L. and zur Hausen, H. (1989) Arnpli~~tion of bovine papillomavirus DNA by N-Methyl-N‘-nitr~N-~trosogua~~ne, ultraviolet irradiation, or infection with herpes simplex virus. Virology 172, 73-81 Schwab, M. (1987) Amplification of cellular oncogenes during evolution of tumor cells. In: H. zur Hausen and J.R. Schlehofer (Eds), The Role of DNA Amplification in Carcinogenesis, pp. 178-185. J.B. Lippincott, Philadelphia, PA.
Shein, H.M. and Enders, J.F. (1962) Multiplication and cytopathogenicity of simian vacuolating virus 40 in cultures of human tissues. Proc. Sot. Exp. Biol. Med. 109, 495-500. Southern, EM. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. Stark, G.R. and Wahl, G.M. (1984) Gene amplification. Ann. Rev. B&hem. 53, 447-491. Stich, HF. (1973) Oncogenic and nononcogenic mutants of adenovirus 12: induction of chromosome aberrations and cell divisions. Progr. Exp. Tumor. Res. 18, 260-272. Stich, H.F. and Yohn, D.S. (1967) Mutagenic capacity of adenovirus for mammalian cells. Nature (London) 216, 1292-1294. Will, H., Cattaneo, R., Koch, H.-G., Darai, G. and Schaher, H. (1982) Cloned HBV DNA causes hepatitis in chimpanzees. Nature (London} 299, 740-742. Winocour, E. and Keshet, I. (1980) Indisc~~Rate re~mbination in simian virus 40-infected monkey cells. Proc. Natl. Acad. Sci. USA 77, 4861-4865 YaIkinoglu, A.G., Heilbronn, R., Btirkle, A., Schlehofer, J.R. and zur Hausen, H. (1988) DNA amplification of adeno-associated virus as a response to cellular genotoxic stress. Cancer Res. 48, 3123-3129. Zur Hausen, H. (1967) Induction of specific chromosomal aberrations by adenovirus type 12 in human embryonic kidney cells. J. Viral. 1, 1174-1185. Zur Hausen, H. (1973) Interactions of adenovirus type 12 with host cell chromosomes. Progr. Exp. Tumor Res. 18, 240-259. Zur Hausen, H. (1982) Human genital cancer: synergism between two virus infections or synergism between a virus infection and initiating events ? Lancet ii, 1370 Zur Hausen, H. and Schtehofer, J.R. (1987) The role of DNA amplification in tumor development: prospects from viroiogical studies. In: H. zur Hausen and J.R. Schlehofer (eds), The Rofe of DNA Amplification in Carcinogenesis, pp. l-8.3.B. Lippincott Co,P~ladelp~a, PA. (Received
28 March
1990, revision
received
16 May 1990)
Vkws Research 17 (1990) 53-60 Elsevier VIRUS 00598
Adenovirus infection induces amplification of persistent viral DNA sequences (simian virus 40, hepatitis B virus, bovine papillomavirus) in human and rodent cells &kg R. Schlehofer and Harald zur Hausen Institut fir Virusforschung, Deutsches Kkebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg, F. R. G. (Accepted 16 May 1990)
Adenoviruses, types 2 and 12 induce ~p~fication of SV40 DNA sequences in cells of the SV40-transformed human newborn kidney cell line, NB-E. Similarly, integrated hepatitis B virus DNA sequences in the human hepatoma cell line, PLC/ *PR.P/5, and bovine papillomavirus (BPV) DNA sequences in BPV-transformed mouse cells (ID13) are amplified by adenovirus infection. Thus, similar to herpesgroup or vaccinia viruses or DNA damaging agents, adenoviruses are able to mediate selective DNA amplification in addition to their reported mutagenic and chromosome damaging effects. The role of amplification of integrated viral DNA sequences in development and progression of specific tumors (e.g. hepatoeellular carcinoma) remains to be determined.
Adenovirus; SV4O; Hepatitis B virus; DNA ~p~fication;
Bovine pap~lorna~~s
Amplification of specific DNA sequences within cells of hamster and human cell lines is induced by chemical and physical carcinogens and as well by herpesgroup and vaccinia viruses (Schlehofer et al., 1983a, 1986). These viruses are known to
Correspondence to: J.R. Schiehofer, Institut fii Virusforschung, Im Neuenheimer Feld 280, D-6900 Heidelberg, F.R.G. * Supported by the Deutsche Forschungsgemeinschaft. 0168-1702/~/~3.50
0 1990 Ekvier
Deutsches Krebsforschungszentr,
Science Publishers B.V. (Biom~~
Division)
54
induce chromosomal damage and appear to transform rodent cells (Duff and Rapp, 1971; Koziorowska et al., 1971; Albrecht and Rapp, 1973). Selective DNA amplification in addition appears to play a major role in adaptation of cells to DNA damaging agents thus mediating drug resistance (Schimke, 1984a,b; Stark and Wahl, 1984). Furthermore, amplification of oncogene sequences in malignant tissues seems to be correlated with tumor progression (Alitalo and Schwab, 1986; Schwab, 1987). Adenovirus infection leads to specific chromosomal alterations in human cells (zur Hausen, 1967, 1973; Stich, 1973) and to transformation of rodent cells (McBride and Wiener, 1964; Casto, 1973). Furthermore, these virus infections induce mutations in host cell DNA (Stich and Yohn, 1967; Marengo et al., 1981) as also shown for herpes viruses (Schlehofer and zur Hausen, 1982). Therefore it was of interest to analyze whether adenovirus infection induces amplification of specific DNA sequences within appropriate cell culture indicator systems similar to herpesgroup or vaccinia viruses or carcinogens. To this aim we used SV40-transformed human newborn kidney cells [NB-E, (Shein and Enders, 1962)] which are fully permissive for adenovirus replication. The cells were grown in Eagle’s minimal essential medium (Gibco, Karlsruhe, F.R.G.) supplemented with 10% fetal calf serum (Flow, Bonn, F.R.G.) and antibiotics (penicillin G: 100 U/ml, streptomycin: 100 pg/ml; Gibco). NB-E cells were infected with adenovirus type 12 at a multiplicity of infection (MOI) of 0.5 PFU/cell, or mock-infected or treated with the 7,12,dimethyl-benz(a)anthracene [DMBA (final concentration: 0.5 pg/ml dissolved in DMSO), Sigma, Miinchen, F.R.G.), respectively. After five days of incubation at 37°C in a humidified atmosphere containing 5% CO,, cells were trapped by filtration onto nitrocellulose filters (“dispersed cell assay” [Winocour and Keshet, 19801) and hybridized with cloned 32P-labelled SV40 DNA (for details cf. Schlehofer et al., 1983a; 1986). As shown in Fig. 1, infection
Fig. 1. Amplification of SV40 DNA in cells of the SV40- transformed human cell line, NB-E by infection with adenovirus, type 12 (MO1 = 0.5 PFU/cell) or treatment with 7,12,dimethyl-benz.(a)anthracene (DMBA, 0.5 pg/ml): autoradiographs of “dispersed cell assay” analysis of lo6 NB-E cells each, infected with adenovirus type 12 or treatment with DMBA. Control = mock-infected cells. Five days after treatment or infection cells were trypsinized and then trapped onto nitrocellulose filters. The filters were hybridized with 32P-labelled SV40 DNA and autoradiographed.
55
with adenovirus type 12 induced amplification of SV40 DNA in NB-E cells similar to the chemical carcinogen, DMBA. The adenovirus-induced amplification of SV40 DNA sequences was also demonstrated in Southern blots (Southern, 1975) of DNA (extracted with phenol/ chloroform/isoamylalcohol according to standard procedures) of adenovirus type 12-infected cells (MO1 = 0.5 PFUfcell, analysis 5 days post infection) cleaved with the restriction endonucleases, XbaI + BgZII (Boehringer, Mannheim, F.R.G.) which
kb
23.7 123
321
kb
9.5 6.6 4.4
- 23.7 -
9.5
I
- 6.6
I
I
- 4.4 - 2.3 - 2.0
2.3 2.0
I
I
0.5
Fig. 2. Amplification of SV40 DNA in the SV4O-transformed human cell line NB-E by infection with adenovirus type 12 (MO1 = 0.5 PFU/cell) or by DMBA (cf. fig. 1): Autoradiograph of a Southern blot of NB-E cell DNA extracted after 5 days from treated or infected cells (10 cg DNA/lane), separated by agarose gel electrophoresis after cleavage with XbaI + BglII, and hybridized to 32P-labelled SV40 DNA. 1= Mock-infected control cell DNA; 2 = adenovirus type 12-infected cell DNA; 3 = DNA from DMBA-treated cells (in the right panel, the ethidium bromide stained agarose gel is shown for comparison of amounts of DNA kded and separated in each lane prior to blotting) Fig. 3. Amplification of SV40 DNA in the SV40-transformed human cell line, NB-E by infection with adenovirus type 2 (MO1 = 1 PFU/cell). Autoradiograph of a Southern blot of NB-E cell DNA extracted after 5 days from infected cells, prepared as described in Fig. 2. 1= Mock-infected control cell DNA; 2 = adenovirus type 2-infected cell DNA (right panel: ethidium bromide gel for comparison of amounts of DNA (5 pg/lane)
56
do not cut within SV40 DNA (Fig. 2). Similarly to infection with adenovirus type 12, adenovirus type 2 induced amplification of SV40 DNA in NB-E cells (Fig. 3). Cross-hybridization between SV40 DNA and adenovirus DNA could be excluded by hybridizing a Southern blot of adenovirus- infected HeLa cell DNA with 32P-labelled SV40 DNA (data not shown). This is also evident from the lack of hybridization of the SV40 probe to the clearly visible bands of adenovirus DNA (cf. fig. 2, ethidium bromide stain). Recently it was shown by our group that episomal as well as integrated DNA sequences of bovine papillomavirus (BPV) in BPV (type 1)-transformed Cl27 mouse cells (ID13, [Dvoretzky et al., 1980; Law et al., 19811) are amplified by treatment of ID13 cells with chemical or physical carcinogenic agents as well as by infection with herpes simplex virus (Schmitt et al., 1989). As shown in Fig. 4 by a Southern blot of adenovirus type 2-infected ID13 cell DNA cleaved with Sac1 (not cutting within the BPV genome) and hybridized with 32P-labelled cloned BPV type 1 DNA (pD-BPV-l), adenovirus type 2 induced amplification of integrated as well as of episomal BPV DNA (ID 13 cells were cultivated as described above for NB-E cells; adenovirus type 2 infection was at an MO1 of 1 PFU/cell; cellular DNA was extracted 3 days after infection when adenovirus-induced cytopathic changes were observed). Crosshybridization of BPV to adenovirus DNA was excluded as described above for SV40 DNA. Up to now exclusively papovavirus DNA sequences have been shown to be amplified by carcinogenic agents (“initiators”) or virus infections (SV40, lymphotropic papovavirus, polyoma, BPV (see zur Hausen and Schlehofer, 1987 for review). As shown in Fig. 5, DNA sequences of an unrelated virus, hepatitis B virus (HBV), present in the human hepatoma cell line, PLC/ *PRF/S (Alexander et al., 1976; Macnab et al., 1976) are amplified after infection with adenovirus type 2 (Cultivation of cells and infection were the same as described above; PLC/ * PRF/S cells are permissive for adenovirus replication resulting in cytopathic effect). This is shown by hybridization of a Southern blot of cellular DNA cleaved with EcoRI of infected PLC/ * PRF/S cells with 32P-labelled cloned HBV DNA [clone pSh14-3 (Will et al., 1982); Fig. 51. All bands hybridizing to HBV DNA appear to be amplified to a similar extent. Cross-hybridization between HPV and adenovirus DNA was excluded as described above. Our results demonstrate the induction of selective DNA amplification by infections with adenoviruses types 2 and 12. It is interesting to note that adenoviruses share this property with a number of herpesgroup viruses and with vaccinia virus, all coding for their own DNA polymerase (Citarella et al., 1972; Challberg and Englund, 1975; Ostrander and Cheng, 1980). Indeed, in the case of herpes simplex virus infections it has been shown that the viral DNA polymerase plays a key role in the process of DNA amplification (Matz et al., 1984). In addition, it has been demonstrated recently that replicative functions of the viral genome are involved in the induction of selective DNA amplification (Heilbronn and zur Hausen, 1989). It seems to be noteworthy that integrated and episomal sequences of known or putative DNA tumor viruses, but also integrated DNA of adeno-associated viruses become readily amplified by the viral infections outlined above but also by chemical
57
23.7 9.5 6.6
1
I
1 2.3 I 2.0
Fig. 4. Amplification of BPV in cells of the BPV type l-transformed mouse Cl27 cell line, ID13 by infection with adenovirus, type 2 (MO1 = 1 PFU/cell): Autoradiograph of a Southern blot of ID13 cell DNA extracted after 3 days from infected (2) or uninfected (1) cells (5 pg DNA/lane), separated by a&arose gel electrophoresis after cleavage with Sac1 and hybridized to 32P-labelled BPV DNA. Fig. 5. Amplification of HBV DNA with adenovirus, type 2 (MO1 = 0.5 DNA extracted after 2 days from DNA/lane), separated by agarose
in cells of the human hepatoma cell line, PLC/ *F/5 by infection PFU/cell): Autora~o~aph of a Southern blot of PLC,/*F/S cell infected (“Adeno 2”) or m~k-inf~t~ (“Control”) cells (10 pg gel electrophoresis after cleavage with EcoRI, and hybridized to 32P-labelled HBV DNA.
and physical carcinogenic factors (Schlehofer et al., 1983a,b 1986; Yalkinoglu et al., 1988; Schmitt et al., 1989). Since amplification is frequently accompanied by increased expression of the amplified sequences (Kleinberger et al., 1988) this may contribute to the process of transformation. In addition, intramolecular rearrangements or effects acting in cis resulting from additional integration events may also play a significant role. Hum~pathoge~c adenoviruses efficiently transform rodent cells (cf. Flint, 1980 for review). The ~ansform~ cells retain specific viral DNA sequences similar to SV40 and other papova~s-tr~sfo~ed cells. On the other hand tr~sformation
58
by herpes simplex viruses does not seem to require the persistence of specific viral DNA sequences (Galloway and McDougall, 1983). In these instances modifications of the host cell genomes induced by the viruses may be sufficient to result in cell transformation. It will be interesting to analyze whether adenoviruses are able to transform cells by a similar mechanism. This is particularly important since loss of adenovirus sequences from adenovirus-transformed rodent cells has been reported (Kuhlmann et al., 1982). The induced amplification of persisting hepatitis B and bovine papillomavirus sequences deserves special attention. Rearrangements and amplifications of HBV genomes have been frequently noted in primary hepatocellular carcinoma biopsies (Koch et al., 1984; Nagaya et al., 1987). Their role in the process of tumor development is presently not understood. Amplification of integrated papillomavirus DNA sequences is frequently observed in human cervical cancer (Dtirst et al., 1983; Boshart et al., 1984). It is possible that additional viral infections or exposures to chemical or physical carcinogenic factors may result in such amplifications and thus contribute to the malignant conversion (zur Hausen, 1982).
Acknowledgements We gratefully acknowledge the technical assistance of M. Ehrbar. We are indebted to J. Rommelaere and M. Lusky for providing NB-E and ID13 cells, respectively. We thank P. Howley for cloned BPV DNA and and E. Schwarz for SV40 DNA. C.H. Schroder kindly provided us with PLC/*PRF/S cells and with cloned HBV DNA.
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received
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