Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1993 May;12(5):1847–1852. doi: 10.1002/j.1460-2075.1993.tb05833.x

Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.

V Band 1, S Dalal 1, L Delmolino 1, E J Androphy 1
PMCID: PMC413405  PMID: 8387914

Abstract

Normal mammary epithelial cells are efficiently immortalized by the E6 gene of human papillomavirus (HPV)-16, a virus commonly associated with cervical cancers. Surprisingly, introduction of the E6 gene from HPV-6, which is rarely found in cervical cancer, or bovine papillomavirus (BPV)-1, into normal mammary cells resulted in the generation of immortal cell lines. The establishment of HPV-6 and BPV-1 E6-immortalized cells was less efficient and required a longer period in comparison to HPV-16 E6. These HPV-6- and BPV-1 E6-immortalized cells demonstrated dramatically reduced levels of p53 protein by immunoprecipitation. While the half-life of p53 protein in normal mammary epithelial cells was approximately 3 h, it was reduced to approximately 15 min in all the E6-immortalized cells. These results demonstrate that the E6 genes of both high-risk and low-risk papilloma viruses immortalize human mammary epithelial cells and induce a marked degradation of p53 protein in vivo.

Full text

PDF
1852

Images in this article

Selected References

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

  1. Baker S. J., Markowitz S., Fearon E. R., Willson J. K., Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990 Aug 24;249(4971):912–915. doi: 10.1126/science.2144057. [DOI] [PubMed] [Google Scholar]
  2. Band V., De Caprio J. A., Delmolino L., Kulesa V., Sager R. Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells. J Virol. 1991 Dec;65(12):6671–6676. doi: 10.1128/jvi.65.12.6671-6676.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Band V., Sager R. Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1249–1253. doi: 10.1073/pnas.86.4.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Band V., Zajchowski D., Kulesa V., Sager R. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements. Proc Natl Acad Sci U S A. 1990 Jan;87(1):463–467. doi: 10.1073/pnas.87.1.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Band V., Zajchowski D., Swisshelm K., Trask D., Kulesa V., Cohen C., Connolly J., Sager R. Tumor progression in four mammary epithelial cell lines derived from the same patient. Cancer Res. 1990 Nov 15;50(22):7351–7357. [PubMed] [Google Scholar]
  6. Barbosa M. S., Schlegel R. The E6 and E7 genes of HPV-18 are sufficient for inducing two-stage in vitro transformation of human keratinocytes. Oncogene. 1989 Dec;4(12):1529–1532. [PubMed] [Google Scholar]
  7. Barbosa M. S., Vass W. C., Lowy D. R., Schiller J. T. In vitro biological activities of the E6 and E7 genes vary among human papillomaviruses of different oncogenic potential. J Virol. 1991 Jan;65(1):292–298. doi: 10.1128/jvi.65.1.292-298.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Beckmann A. M., Daling J. R., Sherman K. J., Maden C., Miller B. A., Coates R. J., Kiviat N. B., Myerson D., Weiss N. S., Hislop T. G. Human papillomavirus infection and anal cancer. Int J Cancer. 1989 Jun 15;43(6):1042–1049. doi: 10.1002/ijc.2910430615. [DOI] [PubMed] [Google Scholar]
  9. Bedell M. A., Jones K. H., Laimins L. A. The E6-E7 region of human papillomavirus type 18 is sufficient for transformation of NIH 3T3 and rat-1 cells. J Virol. 1987 Nov;61(11):3635–3640. doi: 10.1128/jvi.61.11.3635-3640.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bártek J., Bártková J., Vojtesek B., Stasková Z., Rejthar A., Kovarík J., Lane D. P. Patterns of expression of the p53 tumour suppressor in human breast tissues and tumours in situ and in vitro. Int J Cancer. 1990 Nov 15;46(5):839–844. doi: 10.1002/ijc.2910460515. [DOI] [PubMed] [Google Scholar]
  11. Crawford L. V., Pim D. C., Lamb P. The cellular protein p53 in human tumours. Mol Biol Med. 1984 Aug;2(4):261–272. [PubMed] [Google Scholar]
  12. Crook T., Tidy J. A., Vousden K. H. Degradation of p53 can be targeted by HPV E6 sequences distinct from those required for p53 binding and trans-activation. Cell. 1991 Nov 1;67(3):547–556. doi: 10.1016/0092-8674(91)90529-8. [DOI] [PubMed] [Google Scholar]
  13. Crook T., Wrede D., Tidy J., Scholefield J., Crawford L., Vousden K. H. Status of c-myc, p53 and retinoblastoma genes in human papillomavirus positive and negative squamous cell carcinomas of the anus. Oncogene. 1991 Jul;6(7):1251–1257. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Hubbert N. L., Sedman S. A., Schiller J. T. Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes. J Virol. 1992 Oct;66(10):6237–6241. doi: 10.1128/jvi.66.10.6237-6241.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Kasher M. S., Roman A. Characterization of human papillomavirus type 6b DNA isolated from an invasive squamous carcinoma of the vulva. Virology. 1988 Jul;165(1):225–233. doi: 10.1016/0042-6822(88)90676-9. [DOI] [PubMed] [Google Scholar]
  20. Kaur P., McDougall J. K. Characterization of primary human keratinocytes transformed by human papillomavirus type 18. J Virol. 1988 Jun;62(6):1917–1924. doi: 10.1128/jvi.62.6.1917-1924.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  22. Lamb P., Crawford L. Characterization of the human p53 gene. Mol Cell Biol. 1986 May;6(5):1379–1385. doi: 10.1128/mcb.6.5.1379. [DOI] [PMC free article] [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. Levine A. J., Momand J. Tumor suppressor genes: the p53 and retinoblastoma sensitivity genes and gene products. Biochim Biophys Acta. 1990 Jun 1;1032(1):119–136. doi: 10.1016/0304-419x(90)90015-s. [DOI] [PubMed] [Google Scholar]
  26. Lowy D. R., Rands E., Scolnick E. M. Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. J Virol. 1978 May;26(2):291–298. doi: 10.1128/jvi.26.2.291-298.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Malkin D., Li F. P., Strong L. C., Fraumeni J. F., Jr, Nelson C. E., Kim D. H., Kassel J., Gryka M. A., Bischoff F. Z., Tainsky M. A. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science. 1990 Nov 30;250(4985):1233–1238. doi: 10.1126/science.1978757. [DOI] [PubMed] [Google Scholar]
  28. Matlashewski G., Schneider J., Banks L., Jones N., Murray A., Crawford L. Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells. EMBO J. 1987 Jun;6(6):1741–1746. doi: 10.1002/j.1460-2075.1987.tb02426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Münger K., Werness B. A., Dyson N., Phelps W. C., Harlow E., Howley P. M. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 1989 Dec 20;8(13):4099–4105. doi: 10.1002/j.1460-2075.1989.tb08594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. O'Brien W., Stenman G., Sager R. Suppression of tumor growth by senescence in virally transformed human fibroblasts. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8659–8663. doi: 10.1073/pnas.83.22.8659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Perez-Reyes N., Halbert C. L., Smith P. P., Benditt E. P., McDougall J. K. Immortalization of primary human smooth muscle cells. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1224–1228. doi: 10.1073/pnas.89.4.1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rando R. F., Groff D. E., Chirikjian J. G., Lancaster W. D. Isolation and characterization of a novel human papillomavirus type 6 DNA from an invasive vulvar carcinoma. J Virol. 1986 Jan;57(1):353–356. doi: 10.1128/jvi.57.1.353-356.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Runnebaum I. B., Nagarajan M., Bowman M., Soto D., Sukumar S. Mutations in p53 as potential molecular markers for human breast cancer. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10657–10661. doi: 10.1073/pnas.88.23.10657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Scheffner M., Münger K., Byrne J. C., Howley P. M. The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5523–5527. doi: 10.1073/pnas.88.13.5523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Scheffner M., Münger K., Huibregtse J. M., Howley P. M. Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins. EMBO J. 1992 Jul;11(7):2425–2431. doi: 10.1002/j.1460-2075.1992.tb05307.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Schiller J. T., Vass W. C., Lowy D. R. Identification of a second transforming region in bovine papillomavirus DNA. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7880–7884. doi: 10.1073/pnas.81.24.7880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sedman S. A., Hubbert N. L., Vass W. C., Lowy D. R., Schiller J. T. Mutant p53 can substitute for human papillomavirus type 16 E6 in immortalization of human keratinocytes but does not have E6-associated trans-activation or transforming activity. J Virol. 1992 Jul;66(7):4201–4208. doi: 10.1128/jvi.66.7.4201-4208.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Steger G., Pfister H. In vitro expressed HPV 8 E6 protein does not bind p53. Arch Virol. 1992;125(1-4):355–360. doi: 10.1007/BF01309654. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Yelton D. E., Desaymard C., Scharff M. D. Use of monoclonal anti-mouse immunoglobulin to detect mouse antibodies. Hybridoma. 1981;1(1):5–11. doi: 10.1089/hyb.1.1981.1.5. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES