Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 May 10;91(10):4382–4386. doi: 10.1073/pnas.91.10.4382

Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7.

G W Demers 1, S A Foster 1, C L Halbert 1, D A Galloway 1
PMCID: PMC43789  PMID: 8183918

Abstract

Cellular tumor suppressors p53 and Rb play an important role in controlling cell proliferation. Inactivation of these tumor suppressor proteins can occur by gene mutation or by association with oncoproteins from the small DNA tumor viruses. One function of p53 is in regulating cell cycle check-point control after DNA damage. To dissect the pathways by which p53 and Rb may act, the E6 and E7 oncogenes of human papillomavirus (HPV) types 6 and 16 were introduced into primary human epithelial cells by retroviral transfer vector, and cells were assayed for growth arrest after DNA damage induced by actinomycin D. The E6 or E7 oncogenes from the low-risk HPV6 had no affect on growth arrest, p53 protein levels increased, Rb protein levels decreased, and Rb was predominantly in the hypophosphorylated state similar to vector-infected cells. Either the E6 or the E7 oncogene from the high-risk HPV16 abrogated growth arrest. Cells expressing HPV16 E6 (16E6) were severely reduced in p53 protein levels that did not increase detectably after DNA damage, Rb protein levels did not decrease, and hyperphosphorylated Rb was present. After DNA damage in cells expressing 16E7 p53 levels increased, and Rb protein levels decreased; however, Rb was predominantly in the hyperphosphorylated state. Even though p53 protein levels increased in response to DNA damage in cells expressing 16E7, G1 growth arrest was bypassed. This suggests that the circuitry controlling the growth arrest signal after DNA damage may be interconnected between the p53 and Rb pathways.

Full text

PDF
4382

Images in this article

4383Image
on p.4383
4384Image
on p.4384
4384Image
on p.4384
4385Image
on p.4385

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chen P. L., Scully P., Shew J. Y., Wang J. Y., Lee W. H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell. 1989 Sep 22;58(6):1193–1198. doi: 10.1016/0092-8674(89)90517-5. [DOI] [PubMed] [Google Scholar]
  2. Clarke A. R., Maandag E. R., van Roon M., van der Lugt N. M., van der Valk M., Hooper M. L., Berns A., te Riele H. Requirement for a functional Rb-1 gene in murine development. Nature. 1992 Sep 24;359(6393):328–330. doi: 10.1038/359328a0. [DOI] [PubMed] [Google Scholar]
  3. Craig R. W., Frankfurt O. S., Sakagami H., Takeda K., Bloch A. Macromolecular and cell cycle effects of different classes of agents inducing the maturation of human myeloblastic leukemia (ML-1) cells. Cancer Res. 1984 Jun;44(6):2421–2429. [PubMed] [Google Scholar]
  4. DeCaprio J. A., Ludlow J. W., Figge J., Shew J. Y., Huang C. M., Lee W. H., Marsilio E., Paucha E., Livingston D. M. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. doi: 10.1016/0092-8674(88)90559-4. [DOI] [PubMed] [Google Scholar]
  5. Demers G. W., Halbert C. L., Galloway D. A. Elevated wild-type p53 protein levels in human epithelial cell lines immortalized by the human papillomavirus type 16 E7 gene. Virology. 1994 Jan;198(1):169–174. doi: 10.1006/viro.1994.1019. [DOI] [PubMed] [Google Scholar]
  6. Donehower L. A., Harvey M., Slagle B. L., McArthur M. J., Montgomery C. A., Jr, Butel J. S., Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature. 1992 Mar 19;356(6366):215–221. doi: 10.1038/356215a0. [DOI] [PubMed] [Google Scholar]
  7. Dyson N., Guida P., Münger K., Harlow E. Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins. J Virol. 1992 Dec;66(12):6893–6902. doi: 10.1128/jvi.66.12.6893-6902.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dyson N., Howley P. M., Münger K., Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. doi: 10.1126/science.2537532. [DOI] [PubMed] [Google Scholar]
  9. Ewen M. E., Sluss H. K., Sherr C. J., Matsushime H., Kato J., Livingston D. M. Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell. 1993 May 7;73(3):487–497. doi: 10.1016/0092-8674(93)90136-e. [DOI] [PubMed] [Google Scholar]
  10. Halbert C. L., Demers G. W., Galloway D. A. The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells. J Virol. 1992 Apr;66(4):2125–2134. doi: 10.1128/jvi.66.4.2125-2134.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Halbert C. L., Demers G. W., Galloway D. A. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J Virol. 1991 Jan;65(1):473–478. doi: 10.1128/jvi.65.1.473-478.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  13. Hawley-Nelson P., Vousden K. H., Hubbert N. L., Lowy D. R., Schiller J. T. HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 1989 Dec 1;8(12):3905–3910. doi: 10.1002/j.1460-2075.1989.tb08570.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hinds P. W., Mittnacht S., Dulic V., Arnold A., Reed S. I., Weinberg R. A. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell. 1992 Sep 18;70(6):993–1006. doi: 10.1016/0092-8674(92)90249-c. [DOI] [PubMed] [Google Scholar]
  15. Hudson J. B., Bedell M. A., McCance D. J., Laiminis L. A. Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18. J Virol. 1990 Feb;64(2):519–526. doi: 10.1128/jvi.64.2.519-526.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Imai Y., Matsushima Y., Sugimura T., Terada M. Purification and characterization of human papillomavirus type 16 E7 protein with preferential binding capacity to the underphosphorylated form of retinoblastoma gene product. J Virol. 1991 Sep;65(9):4966–4972. doi: 10.1128/jvi.65.9.4966-4972.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jacks T., Fazeli A., Schmitt E. M., Bronson R. T., Goodell M. A., Weinberg R. A. Effects of an Rb mutation in the mouse. Nature. 1992 Sep 24;359(6393):295–300. doi: 10.1038/359295a0. [DOI] [PubMed] [Google Scholar]
  18. Jenison S. A., Yu X. P., Valentine J. M., Koutsky L. A., Christiansen A. E., Beckmann A. M., Galloway D. A. Evidence of prevalent genital-type human papillomavirus infections in adults and children. J Infect Dis. 1990 Jul;162(1):60–69. doi: 10.1093/infdis/162.1.60. [DOI] [PubMed] [Google Scholar]
  19. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991 Dec 1;51(23 Pt 1):6304–6311. [PubMed] [Google Scholar]
  20. Kessis T. D., Slebos R. J., Nelson W. G., Kastan M. B., Plunkett B. S., Han S. M., Lorincz A. T., Hedrick L., Cho K. R. Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3988–3992. doi: 10.1073/pnas.90.9.3988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7491–7495. doi: 10.1073/pnas.89.16.7491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laiho M., DeCaprio J. A., Ludlow J. W., Livingston D. M., Massagué J. Growth inhibition by TGF-beta linked to suppression of retinoblastoma protein phosphorylation. Cell. 1990 Jul 13;62(1):175–185. doi: 10.1016/0092-8674(90)90251-9. [DOI] [PubMed] [Google Scholar]
  23. Lane D. P., Crawford L. V. T antigen is bound to a host protein in SV40-transformed cells. Nature. 1979 Mar 15;278(5701):261–263. doi: 10.1038/278261a0. [DOI] [PubMed] [Google Scholar]
  24. Lechner M. S., Mack D. H., Finicle A. B., Crook T., Vousden K. H., Laimins L. A. Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription. EMBO J. 1992 Aug;11(8):3045–3052. doi: 10.1002/j.1460-2075.1992.tb05375.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lee E. Y., Chang C. Y., Hu N., Wang Y. C., Lai C. C., Herrup K., Lee W. H., Bradley A. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature. 1992 Sep 24;359(6393):288–294. doi: 10.1038/359288a0. [DOI] [PubMed] [Google Scholar]
  26. Linzer D. I., Levine A. J. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell. 1979 May;17(1):43–52. doi: 10.1016/0092-8674(79)90293-9. [DOI] [PubMed] [Google Scholar]
  27. Livingstone L. R., White A., Sprouse J., Livanos E., Jacks T., Tlsty T. D. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell. 1992 Sep 18;70(6):923–935. doi: 10.1016/0092-8674(92)90243-6. [DOI] [PubMed] [Google Scholar]
  28. Lowe S. W., Ruley H. E. Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis. Genes Dev. 1993 Apr;7(4):535–545. doi: 10.1101/gad.7.4.535. [DOI] [PubMed] [Google Scholar]
  29. Ludlow J. W., DeCaprio J. A., Huang C. M., Lee W. H., Paucha E., Livingston D. M. SV40 large T antigen binds preferentially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell. 1989 Jan 13;56(1):57–65. doi: 10.1016/0092-8674(89)90983-5. [DOI] [PubMed] [Google Scholar]
  30. Mietz J. A., Unger T., Huibregtse J. M., Howley P. M. The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein. EMBO J. 1992 Dec;11(13):5013–5020. doi: 10.1002/j.1460-2075.1992.tb05608.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mihara K., Cao X. R., Yen A., Chandler S., Driscoll B., Murphree A. L., T'Ang A., Fung Y. K. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science. 1989 Dec 8;246(4935):1300–1303. doi: 10.1126/science.2588006. [DOI] [PubMed] [Google Scholar]
  32. Münger K., Phelps W. C., Bubb V., Howley P. M., Schlegel R. The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol. 1989 Oct;63(10):4417–4421. doi: 10.1128/jvi.63.10.4417-4421.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Noda A., Ning Y., Venable S. F., Pereira-Smith O. M., Smith J. R. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res. 1994 Mar;211(1):90–98. doi: 10.1006/excr.1994.1063. [DOI] [PubMed] [Google Scholar]
  34. Raychaudhuri P., Bagchi S., Devoto S. H., Kraus V. B., Moran E., Nevins J. R. Domains of the adenovirus E1A protein required for oncogenic activity are also required for dissociation of E2F transcription factor complexes. Genes Dev. 1991 Jul;5(7):1200–1211. doi: 10.1101/gad.5.7.1200. [DOI] [PubMed] [Google Scholar]
  35. Scheffner M., Werness B. A., Huibregtse J. M., Levine A. J., Howley P. M. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 1990 Dec 21;63(6):1129–1136. doi: 10.1016/0092-8674(90)90409-8. [DOI] [PubMed] [Google Scholar]
  36. Trask D. K., Muller M. T. Stabilization of type I topoisomerase-DNA covalent complexes by actinomycin D. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1417–1421. doi: 10.1073/pnas.85.5.1417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang H. G., Draetta G., Moran E. E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products. Mol Cell Biol. 1991 Aug;11(8):4253–4265. doi: 10.1128/mcb.11.8.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wassermann K., Markovits J., Jaxel C., Capranico G., Kohn K. W., Pommier Y. Effects of morpholinyl doxorubicins, doxorubicin, and actinomycin D on mammalian DNA topoisomerases I and II. Mol Pharmacol. 1990 Jul;38(1):38–45. [PubMed] [Google Scholar]
  39. Weinberg R. A. Tumor suppressor genes. Science. 1991 Nov 22;254(5035):1138–1146. doi: 10.1126/science.1659741. [DOI] [PubMed] [Google Scholar]
  40. Werness B. A., Levine A. J., Howley P. M. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990 Apr 6;248(4951):76–79. doi: 10.1126/science.2157286. [DOI] [PubMed] [Google Scholar]
  41. White R. A., Terry N. H., Meistrich M. L., Calkins D. P. Improved method for computing potential doubling time from flow cytometric data. Cytometry. 1990;11(2):314–317. doi: 10.1002/cyto.990110214. [DOI] [PubMed] [Google Scholar]
  42. Whyte P., Buchkovich K. J., Horowitz J. M., Friend S. H., Raybuck M., Weinberg R. A., Harlow E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988 Jul 14;334(6178):124–129. doi: 10.1038/334124a0. [DOI] [PubMed] [Google Scholar]
  43. Xiong Y., Hannon G. J., Zhang H., Casso D., Kobayashi R., Beach D. p21 is a universal inhibitor of cyclin kinases. Nature. 1993 Dec 16;366(6456):701–704. doi: 10.1038/366701a0. [DOI] [PubMed] [Google Scholar]
  44. de Villiers E. M. Heterogeneity of the human papillomavirus group. J Virol. 1989 Nov;63(11):4898–4903. doi: 10.1128/jvi.63.11.4898-4903.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]
  46. zur Hausen H. Viruses in human cancers. Science. 1991 Nov 22;254(5035):1167–1173. doi: 10.1126/science.1659743. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES