Vol. 65, No. 1
JOURNAL OF VIROLOGY, Jan. 1991, p. 479-482 0022-538X/91/010479-04$02.00/0 Copyright © 1991, American Society for Microbiology
Rat Primary Embryo Fibroblast Cells Suppress Transformation by the E6 and E7 Genes of Human Papillomavirus Type 16 in Somatic Hybrid Cells MICHIO MIYASAKA,* YASUNARI TAKAMI, HIROKAZU INOUE, AND AKIRA HAKURA
Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan Received 15 June 1990/Accepted 8 October 1990
The E6 and E7 genes of human papillomavirus type 16 (HPV-16) transform established lines of rat cells but not rat cells in primary culture irrespective of the expression of the two genes. The reason for this difference
between the susceptibilities of cell lines and primary cells was examined by using hybrid cells obtained by somatic cell fusion of rat cell lines transformed by the E6 and E7 genes of HPV-16 and freshly isolated rat embryo fibroblast cells. In these hybrid cells, transformed phenotypes, including colony formation in soft agar, saturation density, and tumorigenicity in nude mice, were suppressed, whereas hybrid cells between E6/E7-transformed cell lines and normal rat cell lines retained these transformed phenotypes. RNA analysis showed that the E6 and E7 genes were transcribed in both types of hybrid cells. These results suggest that primary rat cells possess intracellular functions that cause posttranscriptional suppression of induction of the transformed phenotypes by the E6 and E7 genes of HPV-16.
panel). Similar amounts of ZE67 viral mRNA (5.5 kb) were expressed in REF-ZE67 and 7-ZE67 cells. The amounts of 3-actin mRNA in REF-ZE67 and 7-ZE67 cells were also similar. These results show that rat primary cells are resistant at the posttranscriptional level to transformation by the E6 and E7 genes of HPV-16, whereas the established cell line, No. 7, is easily transformed by these genes. There are two possible explanations for the different responses of the primary cells and an established cell line to the E6 and E7 genes: (i) primary cells may have some tumor suppressor(s) for the E6 and E7 genes or (ii) cell lines may have some factor(s) that promotes transformation by the E6 and E7 genes. To test for a possible suppressor(s) of transformation by the E6 and E7 genes of HPV-16, we used primary rat (REF) cells or normal (untransformed) rat cell lines (No. 7 and No. 20) fused with rat cell lines transformed by ZE67 recombinant retrovirus (7-ZE67 and 20-ZE67). As controls, we used combinations of the normal cells (No. 7 x No. 20) and of the transformed cells (7-ZE67 x 20-ZE67). No. 7 is a hypoxanthine-guanine phosphoribosyltransferasedeficient strain of the rat cell line F2408 (10), and No. 20 is a thymidine kinase-deficient strain of F2408. No. 7 and No. 20 were infected with ZE67 virus, and infected cells were selected with G418 (400 ,ug/ml). Then 100 to 200 G418-resistant colonies of each strain were pooled and designated 7-ZE67 and 20-ZE67. All of these cells were cultured in Dulbecco's modified Eagle minimal essential medium (DMEM) supplemented with 5% fetal calf serum (FCS). The parental cells were mixed and treated with 50% polyethylene glycol. Equal numbers (105) of polyethylene glycol-treated cells were inoculated into liquid selective medium containing hypoxanthine-aminopterin-thymidine (HAT) or HAT plus G418 (400 ,ug/ml) and soft agar selective medium. The number of surviving colonies in liquid medium (a) was scored after 10 days, and that of growing colonies in soft agar medium (b) was scored after 3 weeks (Table 2). The bla ratio indicates the efficiency of colony formation of the hybrid cells in soft agar. The hybrids of primary cells and E6/E7-transformed cell lines (REF x 7-ZE67 and REF x
The E6 and E7 open reading frames (ORFs) of human
papillomavirus type 16 (HPV-16) or HPV-18 were shown to transform established rodent cell lines (1, 14, 29). Although these ORFs immortalized human (12, 17, 26) or rodent (15) primary cells, they did not alone transform any of these primary cells. The different responses of primary and established cells to the E6 and E7 ORFs suggest the involvement of cellular factors in addition to the transforming genes of HPV in malignant transformation of primary cells. This situation resembles in vivo carcinogenesis associated with HPV, because only a small proportion of HPV-infected individuals develop cancer (6) after long latent periods (often 20 to 50 years) (31), and chemical or physical carcinogens are often observed to lead to carcinogenesis synergistically with HPV infection (30). Therefore, for an understanding of the mechanism of in vivo carcinogenesis by HPV, it is important to examine the reason for the different responses of primary and established cells to the E6 and E7 genes. To test the transforming ability of the E6 and E7 ORFs of HPV-16, an established line of rat cells (No. 7) (13) and primary rat embryo fibroblast (REF) cells were infected with murine recombinant retrovirus containing the E6 and E7 ORFs of HPV-16 and the bacterial neo gene (ZE67) (3, 29). The infected cells were selected with the neomycin analog G418 (0.4 ,ug/ml), G418-resistant colonies were pooled, and their transformation was examined. No. 7 cells infected with ZE67 virus (7-ZE67) produced colonies in soft agar with high efficiency (35.0%) and induced tumors in nude mice (Table 1). On the other hand, REF cells infected with ZE67 virus (REF-ZE67) produced no colonies in soft agar and did not induce tumors, although they were immortalized. Uninfected No. 7 and REF cells did not show any transformed phenotypes. Southern analysis showed that like 7-ZE67 cells, REF-ZE67 cells contained proviral DNA of ZE67 (Fig. 1, right panel). Expression of viral mRNA was also examined by Northern (RNA) blot hybridization (Fig. 1, left *
Corresponding author. 479
480
J. VIROL.
NOTES
TABLE 1. Transformed phenotypes of REF and No. 7 cells infected with ZE67 virus Colony formation i soft soft agar aar (%)a Cells in Cells
(%)~
0 0 0 35.0
REF REF-ZE67 No. 7 7-ZE67
No. ofin tumors miceb nude
Immortalization
0 0 0 5
+ + +
a A total of 5 x i01 cells were inoculated into 0.35% agar containing DMEM supplemented with 10% FCS; after incubation for 3 weeks, colonies of more than 0.125-mm diameter were scored. b A total of 105 cells were injected subcutaneously into each of five 7- to 8-week-old BALB/c nude mice, and the development of tumors was examined 5 weeks later.
20-ZE67) showed low bla ratios (0.01), whereas the hybrids of normal cell lines and E6/E7-transformed cell lines (No. 20 x 7-ZE67 and No. 7 x 20-ZE67), like the hybrid of the two transformed parents 7-ZE67 x 20-ZE67, showed high bla ratios (around 0.5). The bla ratio of No. 7 x No. 20 was low (0.03), like those of the REF x E6/E7-transformed cell lines. These results suggest that the transforming ability of E6/E7transformed cell lines was suppressed by cell fusion with primary (REF) cells but not by cell fusion with untransformed cell lines (No. 7 and No. 20). Hybrid colonies in liquid selective medium were pooled and used for further assays of transformed phenotypes such as anchorage-independent growth, saturation density, and tumorigenicity in nude mice. To confirm the stability of hybrid cells, we also analyzed the karyotypes of the hybrid cells REF x 20-ZE67, No. 7 x 20-ZE67, No. 7 x No. 20, and 7-ZE67 x 20-ZE67. All of the hybrids examined contained approximately doubled numbers of chromosomes (75 to 82; averages for all parental cell lines, 38 to 42) (data not
RNA
DNA
REF No.7 E67 E67
REF No7 E67 E67
ZE67
mRNIA .mlNA
-*
ZE67
prov iral
DNA
TABLE 2. Colony formation by hybrid cells in liquid and soft agar selective media Parental cell combination
REF x 7-ZE67 REF x 20-ZE67 No. 20 x 7-ZE67 No. 7 x 20-ZE67 No. 7 x No. 20 7-ZE67 x 20-ZE67
No. of colonies/disha Soft agar (b) Liquid (a)
687 202 180 210 700 360
Ratio,
bla
0.01 0.01 0.46 0.51 0.03 0.55
8 2 83 107
19 197
a Surviving colonies 10 days after inoculation of 105 cells into liquid selective medium containing HAT (No. 7 x No. 20, No. 20 x 7-ZE67, No. 7 x 20-ZE67, and 7-ZE67 x 20-ZE67) or HAT plus G418 (REF x 7-ZE67 and REF x 20-ZE67) were scored. Colonies of more than 0.125-mm diameter were scored 21 days after inoculation of 10' cells into 0.35% agar selective medium.
shown). These hybrid cells were cultured in liquid selective medium and used for further assays. In soft agar, the REF x 20-ZE67 and No. 7 x No. 20 hybrids produced scarcely any colonies (0.1 and 0.4%, respectively) (Table 3). On the other hand, No. 7 x 20-ZE67 and 7-ZE67 x 20-ZE67 formed colonies with high efficiencies (40.2 and 34.7%, respectively) (Table 3). These results are consistent with those in Table 2. Another phenotype of transformation, loss of contact inhibition, was also tested by measuring the saturation density in culture plates. The saturation densities of the REF x 20-ZE67 and No. 7 x No. 20 hybrids were approximately one-third those of No. 7 x 20-ZE67 and 7-ZE67 x 20-ZE67 (Table 3). The tumorigenicities of these hybrid cells were also tested in nude mice. Inocula of 105 hybrid cells were injected subcutaneously into groups of five animals. The REF x 20-ZE67 and No. 7 x No. 20 hybrids formed no tumors within 10 weeks, whereas the No. 7 x 20-ZE67 and 7-ZE67 x 20-ZE67 hybrids formed tumors within 2 to 3 weeks in all animals (Table 3 and Fig. 2). These results suggest that primary REF cells have suppressor functions against the transformed phenotypes of rat cell lines transformed by the E6 and E7 genes of HPV-16 both in vitro and in vivo but that these functions are lost or inactivated in the rat cell line F2408. In this study, the E6 and E7 ORFs were inserted into a retroviral vector and expressed as retroviral mRNA. To determine whether suppression of transformed phenotypes in REF cells occurred at the transcriptional level, we compared the steady-state levels of ZE67 retroviral mRNA in these hybrid cells. For this analysis, cellular RNA was TABLE 3. Transformed phenotypes of hybrid cells
It-Actin rRNA
FIG. 1. DNA and RNA analyses of REF and No. 7 cells infected with ZE67 virus. (Left) RNA analysis. Cellular RNA was isolated from cells by the guanidinum isothiocyanate-cesium chloride method (4). A 20-,ug sample of RNA was separated by electrophoresis and transferred to a nylon filter. The filter was hybridized with a labeled E6/E7 DNA fragment and a human P-actin DNA probe (18). (Right) DNA analysis. Isolation of genomic DNA and Southern blot hybridization were performed as described elsewhere (2, 21). Samples of genomic DNAs (20 ,ug) were digested with SacI, which cuts the long terminal repeat, separated by electrophoresis, and transferred to a nylon filter. The filter was hybridized with an HPV-16 E6/E7 DNA probe (SphI-KpnI; 7467 to 884) (29) radiolabeled by the multiprime DNA labeling system (8).
Hybrid REF x 20-ZE67 No. 7 x 20-ZE67 No. 7 x No. 20 7-ZE67 x 20-ZE67
in soft agar (%)a
(105 cells/cm2)'
No. ofin tude nude mice'
0.1 40.2 0.4 34.7
0.81 2.7 0.98 2.2
0 5 0 5
Colony formation Saturation density
a A total of 5 x 103 cells were inoculated into 0.35% agar containing DMEM supplemented with 10% FCS; after incubation for 3 weeks, colonies of more than 0.125-mm diameter were scored. b Measured as the maximum number of cells recovered after initial plating of 5 x 104 cells per 35-mm dish and incubation at 37°C. ' A total of 105 cells were injected subcutaneously into each of five 7- to 8-week-old BALB/c nude mice, and the development of tumors was examined 5 weeks later.
VOL. 65, 1991
0
NOTES
;
ZEE)
FIG. 2. Tumorigenicities of hybrid cells. Tumors induced in nude mice were photographed 8 weeks after injection of 105 cells of REF x 20-ZE67 (a) and No. 7 x 20-ZE67 (c) hybrids.
isolated and subjected to Northern blot hybridization with a 32P-labeled E6/E7 DNA fragment of HPV-16 as a probe. The hybrid REF x 20-ZE67, in which transformed phenotypes are suppressed, expressed about the same amount of viral mRNA as did the hybrid 7-ZE67 x 20-ZE67, although the amount of viral mRNA in the hybrid 20-ZE67 x No. 7 was a little higher than that in hybrid REF x 20-ZE67 (Fig. 3). Endogenous 1-actin mRNA was also expressed at similar levels in all of the hybrids. These results indicate that suppression of transformed phenotypes in the hybrid REF x 20-ZE67 was not caused by suppression of transcription of viral mRNA containing the E6 and E7 ORFs. In this study, we found that in somatic hybrids, primary REF cells suppress the transformed phenotypes of rat cell lines transformed by the E6 and E7 ORFs of HPV-16. Expression of viral mRNA containing the E6 and E7 ORFs was not suppressed in these hybrids, suggesting that the primary cells contain some functional suppressor(s) of transformation by the transforming genes of HPV-16 that acts at the posttranscriptional level. Previously we demonstrated functional dissociation of the transforming genes of HPV-16; the E6 ORF governs the tumorigenicity in nude mice, and the E7 ORF governs cell growth properties such as colony formation in soft agar and saturation density (29). Primary
b
a
C
d
kb
81ZE67 Z
4.43.3-
2.1-
_
1
3
-~~~9act in
Expression of ZE67 viral mRNA in hybrid cells. Northhybridization was performed as described for Fig. 1. Cellular RNA from the hybrids REF x 20-ZE67 (lane a), No. 7 x 20-ZE67 (lane b), No. 7 x No. 20 (lane c), and 7-ZE67 x 20-ZE67 (lane d) was separated by electrophoresis and transferred to a nylon filter. The filter was hybridized with an HPV-16 E6/E7 DNA probe and a human ,-actin DNA probe. FIG. 3.
ern blot
481
cells suppressed all of these transformed phenotypes (Table 3), suggesting that these suppressor functions act against the transforming activities of both the E6 and E7 genes. However, established lines of normal rat cells (No. 7 and No. 20, derived from line F2408) did not suppress the gene activities in E6/E7-transformed cell lines. These results support the hypothesis that loss or inactivation of tumor suppressors leads to malignant transformation synergistically with the transforming genes of HPV-16. However, there are also reports that rat primary cells were transformed when the E7 ORF of HPV-16 and an activated c-Ha-ras (16, 25) or fos (19) oncogene were cotransfected. One possible explanation for this observation is that overexpression of an activated oncogene such as the ras gene may overcome the effect of the suppressor functions in primary rat cells. This possibility is supported by the finding that rat embryo cells immortalized by the E6 and E7 ORFs do not show transformed phenotypes irrespective of the expression of viral mRNA and still have the ability to suppress E6/E7-mediated transformation in somatic hybrid cells (12a). After long-term cultivation of the immortalized cells, a small proportion of the cells became transformed. During this progression, the amounts of viral mRNA and E7 protein did not change, but the expression of c-K-ras mRNA and its product increased remarkably (12b). There are reports of suppressor functions in human cells against HPV-mediated carcinogenesis. Studies on hybrids of HeLa (a cervical tumor-derived, HPV-18-positive cell line) and normal human fibroblast cells have indicated that the tumorigenic phenotype of HeLa cells is suppressed in these hybrids (22, 23) and that rare tumorigenic segregants from the normal parental hybrid cells have lost specific chromosomes (24). However, these hybrids retained most of the characteristic in vitro growth properties of HeLa cells, such as anchorage independence, density-dependent inhibition of growth, and reduced requirement for serum growth factors. This tumor-suppressing function in nontumorigenic hybrids has been suggested to depend on host-specific factors present in vivo but not in vitro (20, 31). Therefore, the tumor suppressor for HeLa cells is evidently different from our suppressive factor(s) in primary rat cells. Recently, a product of a tumor suppressor, the retinoblastoma susceptibility (RB) gene (11), was shown to associate with HPV-16 E7 protein (7), adenovirus Ela protein (28), and simian virus 40 large T antigen (5). The p53 protein, another possible tumor suppressor gene product (9), was also demonstrated to associate with E6 protein (27). These findings suggest functional relationships among the transforming genes of DNA tumor viruses and tumor suppressor genes in malignant transformation. In this report, we demonstrated the existence of a tumor suppressor(s) against transformation by the E6 and E7 genes of HPV-16 in rat cells. For an understanding of the mechanism of carcinogenesis by HPV, it is important to identify and characterize these tumor-suppressor genes. This work was supported by grants from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan.
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3. Cepko, C. L., B. E. Roberts, and R. C. Mulligan. 1984. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell 37:1053-1062. 4. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:52945299. 5. DeCaprio, J. A., J. W. Ludlow, J. Figge, J.-Y. Shew, C.-M. Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D. M. Livingston. 1988. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 54:275-283. 6. de Villiers, E.-M., D. Wagner, A. Schneider, H. Wesch, H. Miklaw, J. Wahrendorf, U. Papendick, and H. zur Hausen. 1987. Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet ii:703-706. 7. Dyson, N., P. M. Howley, K. Munger, and E. Harlow. 1989. The human papillomavirus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934-937. 8. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 9. Finlay, C. A., P. W. Hinds, and A.-J. Levine. 1989. The p53 proto-oncogene can act as a suppressor of transformation. Cell 57:1083-1093. 10. Freeman, A. E., H. J. Igel, and P. J. Price. 1975. In vitro transformation of rat embryo cells: correlations with the known tumorigenic activities of chemicals in rodents. Carcinogenesis In Vitro 11:107-116. 11. Friend, S. H., R. Bernards, S. Rogelj, R. A. Weinberg, J. M. Rapaport, D. M. Albert, and T. P. Dryja. 1986. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature (London) 323:643-646. 12. Hawley-Nelson, P., K. H. Vousden, N. L. Hubbert, D. R. Lowy, and J. T. Schiller. 1989. HPV 16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 12: 3905-3910. 12a.Inoue, H., et al. Unpublished data. 12b.Inoue, H., G. Kondoh, C. R. Kamakura, M. Yutsudo, and A. Hakura. Virology, in press. 13. Inoue, H., M. Yutsudo, and A. Hakura. 1983. Rat mutant cells showing temperature sensitivity for transformation by wild-type Moloney murine sarcoma virus. Virology 125:242-245. 14. Kanda, T., A. Furuno, and K. Yoshiike. 1988. Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells. J. Virol. 62:610-613. 15. Kanda, T., S. Watanabe, and K. Yoshiike. 1988. Immortalization of primary rat cells by human papillomavirus type 16 subgenomic DNA fragments controlled by the SV40 promoter. Virology 165:321-325. 16. Matlashewski, G., J. Schneider, L. Banks, N. Jones, A. Murray, and L. Crawford. 1987. Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells. EMBO J. 6:1741-1746. 17. Munger, K., W. C. Phelps, B. Bubb, P. M. Howley, and R.
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Schlegel. 1989. The E6 and E7 genes of human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol. 63:4417-4421. Nakajima-lijima, S., H. Hamada, P. Reddy, and T. Kakunaga. 1985. Molecular structure of the human cytoplasmic P-actin gene: interspecies homology of sequences in the introns. Proc. Natl. Acad. Sci. USA 82:6133-6137. Phelps, W. C., C. L. Yee, K. Munger, and P. M. Howley. 1988. The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus Ela. Cell 53:539-547. Schwarz, E., A. Schneider-Gadicke, and H. zur Hausen. 1987. Human papillomavirus type-18 transcription in cervical carcinoma cell lines and in human cell hybrids, p47-53. In B. M. Steinberg, J. L. Brandsma, and L. B. Taichman (ed.), Cancer cells 5. Papillomaviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Stanbridge, E. J. 1976. Suppression of malignancy in human cells. Nature (London) 260:17-20. Stanbridge, E. J., C. J. Der, C. J. Doersen, R. Y. Nishimi, D. M. Peehl, B. E. Weissman, and J. E. Wilkinson. 1982. Human cell hybrids: analysis of transformation and tumorigenicity. Science 215:252-259. Stanbridge, E. J., R. R. Flandermeyer, D. W. Daniels, and W. A. Nelson-Rees. 1981. Specific chromosome loss associated with the expression of tumorigenicity in human cell hybrids. Somatic Cell Genet. 7:699-712. Storey, A., D. Pim, A. Murray, K. Osborn, L. Banks, and L. Crawford. 1988. Comparison of the in vitro transforming activities of human papillomavirus types. EMBO J. 7:1815-1820. Watanabe, S., T. Kanda, and K. Yoshiike. 1989. Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7. J. Virol. 63:965-969. Werness, B. A., A. J. Levine, and P. M. Howley. 1990. Association of human papillomavirus type 16 and 18 E6 proteins with p53. Science 248:76-79. Whyte, P., K. J. Buchkovich, J. M. Horowitz, S. H. Friend, M. Raybuck, R. A. Weinberg, and E. Harlow. 1988. Association between an oncogene and an antioncogene: the adenovirus Ela proteins bind to the retinoblastoma gene product. Nature (London) 334:124-129. Yutsudo, M., Y. Okamoto, and A. Hakura. 1988. Functional dissociation of transforming genes of human papillomavirus type 16. Virology 166:594-597. zur Hausen, H. 1977. Human papillomaviruses and their possible role in squamous cell carcinomas. Curr. Top. Microbiol. Immunol. 78:1-30. zur Hausen, H. 1986. Intracellular surveillance of persisting viral infections: human genital cancer results from deficient cellular control of papillomavirus gene expression. Lancet ii:489-491.
JOURNAL OF VIROLOGY, Jan. 1991, p. 479-482 0022-538X/91/010479-04$02.00/0 Copyright © 1991, American Society for Microbiology
Rat Primary Embryo Fibroblast Cells Suppress Transformation by the E6 and E7 Genes of Human Papillomavirus Type 16 in Somatic Hybrid Cells MICHIO MIYASAKA,* YASUNARI TAKAMI, HIROKAZU INOUE, AND AKIRA HAKURA
Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan Received 15 June 1990/Accepted 8 October 1990
The E6 and E7 genes of human papillomavirus type 16 (HPV-16) transform established lines of rat cells but not rat cells in primary culture irrespective of the expression of the two genes. The reason for this difference
between the susceptibilities of cell lines and primary cells was examined by using hybrid cells obtained by somatic cell fusion of rat cell lines transformed by the E6 and E7 genes of HPV-16 and freshly isolated rat embryo fibroblast cells. In these hybrid cells, transformed phenotypes, including colony formation in soft agar, saturation density, and tumorigenicity in nude mice, were suppressed, whereas hybrid cells between E6/E7-transformed cell lines and normal rat cell lines retained these transformed phenotypes. RNA analysis showed that the E6 and E7 genes were transcribed in both types of hybrid cells. These results suggest that primary rat cells possess intracellular functions that cause posttranscriptional suppression of induction of the transformed phenotypes by the E6 and E7 genes of HPV-16.
panel). Similar amounts of ZE67 viral mRNA (5.5 kb) were expressed in REF-ZE67 and 7-ZE67 cells. The amounts of 3-actin mRNA in REF-ZE67 and 7-ZE67 cells were also similar. These results show that rat primary cells are resistant at the posttranscriptional level to transformation by the E6 and E7 genes of HPV-16, whereas the established cell line, No. 7, is easily transformed by these genes. There are two possible explanations for the different responses of the primary cells and an established cell line to the E6 and E7 genes: (i) primary cells may have some tumor suppressor(s) for the E6 and E7 genes or (ii) cell lines may have some factor(s) that promotes transformation by the E6 and E7 genes. To test for a possible suppressor(s) of transformation by the E6 and E7 genes of HPV-16, we used primary rat (REF) cells or normal (untransformed) rat cell lines (No. 7 and No. 20) fused with rat cell lines transformed by ZE67 recombinant retrovirus (7-ZE67 and 20-ZE67). As controls, we used combinations of the normal cells (No. 7 x No. 20) and of the transformed cells (7-ZE67 x 20-ZE67). No. 7 is a hypoxanthine-guanine phosphoribosyltransferasedeficient strain of the rat cell line F2408 (10), and No. 20 is a thymidine kinase-deficient strain of F2408. No. 7 and No. 20 were infected with ZE67 virus, and infected cells were selected with G418 (400 ,ug/ml). Then 100 to 200 G418-resistant colonies of each strain were pooled and designated 7-ZE67 and 20-ZE67. All of these cells were cultured in Dulbecco's modified Eagle minimal essential medium (DMEM) supplemented with 5% fetal calf serum (FCS). The parental cells were mixed and treated with 50% polyethylene glycol. Equal numbers (105) of polyethylene glycol-treated cells were inoculated into liquid selective medium containing hypoxanthine-aminopterin-thymidine (HAT) or HAT plus G418 (400 ,ug/ml) and soft agar selective medium. The number of surviving colonies in liquid medium (a) was scored after 10 days, and that of growing colonies in soft agar medium (b) was scored after 3 weeks (Table 2). The bla ratio indicates the efficiency of colony formation of the hybrid cells in soft agar. The hybrids of primary cells and E6/E7-transformed cell lines (REF x 7-ZE67 and REF x
The E6 and E7 open reading frames (ORFs) of human
papillomavirus type 16 (HPV-16) or HPV-18 were shown to transform established rodent cell lines (1, 14, 29). Although these ORFs immortalized human (12, 17, 26) or rodent (15) primary cells, they did not alone transform any of these primary cells. The different responses of primary and established cells to the E6 and E7 ORFs suggest the involvement of cellular factors in addition to the transforming genes of HPV in malignant transformation of primary cells. This situation resembles in vivo carcinogenesis associated with HPV, because only a small proportion of HPV-infected individuals develop cancer (6) after long latent periods (often 20 to 50 years) (31), and chemical or physical carcinogens are often observed to lead to carcinogenesis synergistically with HPV infection (30). Therefore, for an understanding of the mechanism of in vivo carcinogenesis by HPV, it is important to examine the reason for the different responses of primary and established cells to the E6 and E7 genes. To test the transforming ability of the E6 and E7 ORFs of HPV-16, an established line of rat cells (No. 7) (13) and primary rat embryo fibroblast (REF) cells were infected with murine recombinant retrovirus containing the E6 and E7 ORFs of HPV-16 and the bacterial neo gene (ZE67) (3, 29). The infected cells were selected with the neomycin analog G418 (0.4 ,ug/ml), G418-resistant colonies were pooled, and their transformation was examined. No. 7 cells infected with ZE67 virus (7-ZE67) produced colonies in soft agar with high efficiency (35.0%) and induced tumors in nude mice (Table 1). On the other hand, REF cells infected with ZE67 virus (REF-ZE67) produced no colonies in soft agar and did not induce tumors, although they were immortalized. Uninfected No. 7 and REF cells did not show any transformed phenotypes. Southern analysis showed that like 7-ZE67 cells, REF-ZE67 cells contained proviral DNA of ZE67 (Fig. 1, right panel). Expression of viral mRNA was also examined by Northern (RNA) blot hybridization (Fig. 1, left *
Corresponding author. 479
480
J. VIROL.
NOTES
TABLE 1. Transformed phenotypes of REF and No. 7 cells infected with ZE67 virus Colony formation i soft soft agar aar (%)a Cells in Cells
(%)~
0 0 0 35.0
REF REF-ZE67 No. 7 7-ZE67
No. ofin tumors miceb nude
Immortalization
0 0 0 5
+ + +
a A total of 5 x i01 cells were inoculated into 0.35% agar containing DMEM supplemented with 10% FCS; after incubation for 3 weeks, colonies of more than 0.125-mm diameter were scored. b A total of 105 cells were injected subcutaneously into each of five 7- to 8-week-old BALB/c nude mice, and the development of tumors was examined 5 weeks later.
20-ZE67) showed low bla ratios (0.01), whereas the hybrids of normal cell lines and E6/E7-transformed cell lines (No. 20 x 7-ZE67 and No. 7 x 20-ZE67), like the hybrid of the two transformed parents 7-ZE67 x 20-ZE67, showed high bla ratios (around 0.5). The bla ratio of No. 7 x No. 20 was low (0.03), like those of the REF x E6/E7-transformed cell lines. These results suggest that the transforming ability of E6/E7transformed cell lines was suppressed by cell fusion with primary (REF) cells but not by cell fusion with untransformed cell lines (No. 7 and No. 20). Hybrid colonies in liquid selective medium were pooled and used for further assays of transformed phenotypes such as anchorage-independent growth, saturation density, and tumorigenicity in nude mice. To confirm the stability of hybrid cells, we also analyzed the karyotypes of the hybrid cells REF x 20-ZE67, No. 7 x 20-ZE67, No. 7 x No. 20, and 7-ZE67 x 20-ZE67. All of the hybrids examined contained approximately doubled numbers of chromosomes (75 to 82; averages for all parental cell lines, 38 to 42) (data not
RNA
DNA
REF No.7 E67 E67
REF No7 E67 E67
ZE67
mRNIA .mlNA
-*
ZE67
prov iral
DNA
TABLE 2. Colony formation by hybrid cells in liquid and soft agar selective media Parental cell combination
REF x 7-ZE67 REF x 20-ZE67 No. 20 x 7-ZE67 No. 7 x 20-ZE67 No. 7 x No. 20 7-ZE67 x 20-ZE67
No. of colonies/disha Soft agar (b) Liquid (a)
687 202 180 210 700 360
Ratio,
bla
0.01 0.01 0.46 0.51 0.03 0.55
8 2 83 107
19 197
a Surviving colonies 10 days after inoculation of 105 cells into liquid selective medium containing HAT (No. 7 x No. 20, No. 20 x 7-ZE67, No. 7 x 20-ZE67, and 7-ZE67 x 20-ZE67) or HAT plus G418 (REF x 7-ZE67 and REF x 20-ZE67) were scored. Colonies of more than 0.125-mm diameter were scored 21 days after inoculation of 10' cells into 0.35% agar selective medium.
shown). These hybrid cells were cultured in liquid selective medium and used for further assays. In soft agar, the REF x 20-ZE67 and No. 7 x No. 20 hybrids produced scarcely any colonies (0.1 and 0.4%, respectively) (Table 3). On the other hand, No. 7 x 20-ZE67 and 7-ZE67 x 20-ZE67 formed colonies with high efficiencies (40.2 and 34.7%, respectively) (Table 3). These results are consistent with those in Table 2. Another phenotype of transformation, loss of contact inhibition, was also tested by measuring the saturation density in culture plates. The saturation densities of the REF x 20-ZE67 and No. 7 x No. 20 hybrids were approximately one-third those of No. 7 x 20-ZE67 and 7-ZE67 x 20-ZE67 (Table 3). The tumorigenicities of these hybrid cells were also tested in nude mice. Inocula of 105 hybrid cells were injected subcutaneously into groups of five animals. The REF x 20-ZE67 and No. 7 x No. 20 hybrids formed no tumors within 10 weeks, whereas the No. 7 x 20-ZE67 and 7-ZE67 x 20-ZE67 hybrids formed tumors within 2 to 3 weeks in all animals (Table 3 and Fig. 2). These results suggest that primary REF cells have suppressor functions against the transformed phenotypes of rat cell lines transformed by the E6 and E7 genes of HPV-16 both in vitro and in vivo but that these functions are lost or inactivated in the rat cell line F2408. In this study, the E6 and E7 ORFs were inserted into a retroviral vector and expressed as retroviral mRNA. To determine whether suppression of transformed phenotypes in REF cells occurred at the transcriptional level, we compared the steady-state levels of ZE67 retroviral mRNA in these hybrid cells. For this analysis, cellular RNA was TABLE 3. Transformed phenotypes of hybrid cells
It-Actin rRNA
FIG. 1. DNA and RNA analyses of REF and No. 7 cells infected with ZE67 virus. (Left) RNA analysis. Cellular RNA was isolated from cells by the guanidinum isothiocyanate-cesium chloride method (4). A 20-,ug sample of RNA was separated by electrophoresis and transferred to a nylon filter. The filter was hybridized with a labeled E6/E7 DNA fragment and a human P-actin DNA probe (18). (Right) DNA analysis. Isolation of genomic DNA and Southern blot hybridization were performed as described elsewhere (2, 21). Samples of genomic DNAs (20 ,ug) were digested with SacI, which cuts the long terminal repeat, separated by electrophoresis, and transferred to a nylon filter. The filter was hybridized with an HPV-16 E6/E7 DNA probe (SphI-KpnI; 7467 to 884) (29) radiolabeled by the multiprime DNA labeling system (8).
Hybrid REF x 20-ZE67 No. 7 x 20-ZE67 No. 7 x No. 20 7-ZE67 x 20-ZE67
in soft agar (%)a
(105 cells/cm2)'
No. ofin tude nude mice'
0.1 40.2 0.4 34.7
0.81 2.7 0.98 2.2
0 5 0 5
Colony formation Saturation density
a A total of 5 x 103 cells were inoculated into 0.35% agar containing DMEM supplemented with 10% FCS; after incubation for 3 weeks, colonies of more than 0.125-mm diameter were scored. b Measured as the maximum number of cells recovered after initial plating of 5 x 104 cells per 35-mm dish and incubation at 37°C. ' A total of 105 cells were injected subcutaneously into each of five 7- to 8-week-old BALB/c nude mice, and the development of tumors was examined 5 weeks later.
VOL. 65, 1991
0
NOTES
;
ZEE)
FIG. 2. Tumorigenicities of hybrid cells. Tumors induced in nude mice were photographed 8 weeks after injection of 105 cells of REF x 20-ZE67 (a) and No. 7 x 20-ZE67 (c) hybrids.
isolated and subjected to Northern blot hybridization with a 32P-labeled E6/E7 DNA fragment of HPV-16 as a probe. The hybrid REF x 20-ZE67, in which transformed phenotypes are suppressed, expressed about the same amount of viral mRNA as did the hybrid 7-ZE67 x 20-ZE67, although the amount of viral mRNA in the hybrid 20-ZE67 x No. 7 was a little higher than that in hybrid REF x 20-ZE67 (Fig. 3). Endogenous 1-actin mRNA was also expressed at similar levels in all of the hybrids. These results indicate that suppression of transformed phenotypes in the hybrid REF x 20-ZE67 was not caused by suppression of transcription of viral mRNA containing the E6 and E7 ORFs. In this study, we found that in somatic hybrids, primary REF cells suppress the transformed phenotypes of rat cell lines transformed by the E6 and E7 ORFs of HPV-16. Expression of viral mRNA containing the E6 and E7 ORFs was not suppressed in these hybrids, suggesting that the primary cells contain some functional suppressor(s) of transformation by the transforming genes of HPV-16 that acts at the posttranscriptional level. Previously we demonstrated functional dissociation of the transforming genes of HPV-16; the E6 ORF governs the tumorigenicity in nude mice, and the E7 ORF governs cell growth properties such as colony formation in soft agar and saturation density (29). Primary
b
a
C
d
kb
81ZE67 Z
4.43.3-
2.1-
_
1
3
-~~~9act in
Expression of ZE67 viral mRNA in hybrid cells. Northhybridization was performed as described for Fig. 1. Cellular RNA from the hybrids REF x 20-ZE67 (lane a), No. 7 x 20-ZE67 (lane b), No. 7 x No. 20 (lane c), and 7-ZE67 x 20-ZE67 (lane d) was separated by electrophoresis and transferred to a nylon filter. The filter was hybridized with an HPV-16 E6/E7 DNA probe and a human ,-actin DNA probe. FIG. 3.
ern blot
481
cells suppressed all of these transformed phenotypes (Table 3), suggesting that these suppressor functions act against the transforming activities of both the E6 and E7 genes. However, established lines of normal rat cells (No. 7 and No. 20, derived from line F2408) did not suppress the gene activities in E6/E7-transformed cell lines. These results support the hypothesis that loss or inactivation of tumor suppressors leads to malignant transformation synergistically with the transforming genes of HPV-16. However, there are also reports that rat primary cells were transformed when the E7 ORF of HPV-16 and an activated c-Ha-ras (16, 25) or fos (19) oncogene were cotransfected. One possible explanation for this observation is that overexpression of an activated oncogene such as the ras gene may overcome the effect of the suppressor functions in primary rat cells. This possibility is supported by the finding that rat embryo cells immortalized by the E6 and E7 ORFs do not show transformed phenotypes irrespective of the expression of viral mRNA and still have the ability to suppress E6/E7-mediated transformation in somatic hybrid cells (12a). After long-term cultivation of the immortalized cells, a small proportion of the cells became transformed. During this progression, the amounts of viral mRNA and E7 protein did not change, but the expression of c-K-ras mRNA and its product increased remarkably (12b). There are reports of suppressor functions in human cells against HPV-mediated carcinogenesis. Studies on hybrids of HeLa (a cervical tumor-derived, HPV-18-positive cell line) and normal human fibroblast cells have indicated that the tumorigenic phenotype of HeLa cells is suppressed in these hybrids (22, 23) and that rare tumorigenic segregants from the normal parental hybrid cells have lost specific chromosomes (24). However, these hybrids retained most of the characteristic in vitro growth properties of HeLa cells, such as anchorage independence, density-dependent inhibition of growth, and reduced requirement for serum growth factors. This tumor-suppressing function in nontumorigenic hybrids has been suggested to depend on host-specific factors present in vivo but not in vitro (20, 31). Therefore, the tumor suppressor for HeLa cells is evidently different from our suppressive factor(s) in primary rat cells. Recently, a product of a tumor suppressor, the retinoblastoma susceptibility (RB) gene (11), was shown to associate with HPV-16 E7 protein (7), adenovirus Ela protein (28), and simian virus 40 large T antigen (5). The p53 protein, another possible tumor suppressor gene product (9), was also demonstrated to associate with E6 protein (27). These findings suggest functional relationships among the transforming genes of DNA tumor viruses and tumor suppressor genes in malignant transformation. In this report, we demonstrated the existence of a tumor suppressor(s) against transformation by the E6 and E7 genes of HPV-16 in rat cells. For an understanding of the mechanism of carcinogenesis by HPV, it is important to identify and characterize these tumor-suppressor genes. This work was supported by grants from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan.
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