Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1997 Sep;71(9):6629–6640. doi: 10.1128/jvi.71.9.6629-6640.1997

Adenovirus type 12 early region 1B 54K protein significantly extends the life span of normal mammalian cells in culture.

P H Gallimore 1, P S Lecane 1, S Roberts 1, S M Rookes 1, R J Grand 1, J Parkhill 1
PMCID: PMC191941  PMID: 9261385

Abstract

The life span of normal human cells in culture is extended by two to four total life spans following retrovirus-mediated transfer of the adenovirus type 12 E1B 54,000-molecular-weight protein (54K protein). This extension of the in vitro growth potential was accomplished without any of the obvious changes in morphology or growth properties that are usually associated with viral transformation. These 54K+ cells escape the normal senescence checkpoint (M1) and show a very extended secondary growth phase. The 54K+ human cells eventually enter crisis (M2), which does not appear to be due to either telomere attrition or the activation of the senescence-associated proteins p21SdilCipIWaf1 and p16INK4A. Even in the absence of telomerase activity, high-molecular-weight heterogeneous telomeres are produced and maintained in both 54K+ adult dermal fibroblasts and embryo kidney cells, indicating that the 54K protein may interfere with the normal metabolism of telomeric structures during cell division. These findings are discussed with reference to the known ability of the 54K protein to influence p53 function.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. Babiss L. E., Ginsberg H. S., Darnell J. E., Jr Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport. Mol Cell Biol. 1985 Oct;5(10):2552–2558. doi: 10.1128/mcb.5.10.2552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bai L., Mihara K., Kondo Y., Honma M., Namba M. Immortalization of normal human fibroblasts by treatment with 4-nitroquinoline 1-oxide. Int J Cancer. 1993 Feb 1;53(3):451–456. doi: 10.1002/ijc.2910530317. [DOI] [PubMed] [Google Scholar]
  3. Banks L., Matlashewski G., Crawford L. Isolation of human-p53-specific monoclonal antibodies and their use in the studies of human p53 expression. Eur J Biochem. 1986 Sep 15;159(3):529–534. doi: 10.1111/j.1432-1033.1986.tb09919.x. [DOI] [PubMed] [Google Scholar]
  4. Bannister A. J., Kouzarides T. The CBP co-activator is a histone acetyltransferase. Nature. 1996 Dec 19;384(6610):641–643. doi: 10.1038/384641a0. [DOI] [PubMed] [Google Scholar]
  5. Bayreuther K., Rodemann H. P., Hommel R., Dittmann K., Albiez M., Francz P. I. Human skin fibroblasts in vitro differentiate along a terminal cell lineage. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5112–5116. doi: 10.1073/pnas.85.14.5112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bond J. A., Wyllie F. S., Wynford-Thomas D. Escape from senescence in human diploid fibroblasts induced directly by mutant p53. Oncogene. 1994 Jul;9(7):1885–1889. [PubMed] [Google Scholar]
  7. Bond J., Haughton M., Blaydes J., Gire V., Wynford-Thomas D., Wyllie F. Evidence that transcriptional activation by p53 plays a direct role in the induction of cellular senescence. Oncogene. 1996 Nov 21;13(10):2097–2104. [PubMed] [Google Scholar]
  8. Bryan T. M., Englezou A., Gupta J., Bacchetti S., Reddel R. R. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J. 1995 Sep 1;14(17):4240–4248. doi: 10.1002/j.1460-2075.1995.tb00098.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bryan T. M., Reddel R. R. SV40-induced immortalization of human cells. Crit Rev Oncog. 1994;5(4):331–357. doi: 10.1615/critrevoncog.v5.i4.10. [DOI] [PubMed] [Google Scholar]
  10. Byrd P. J., Chia W., Rigby P. W., Gallimore P. H. Cloning of DNA fragments from the left end of the adenovirus type 12 genome: transformation by cloned early region 1. J Gen Virol. 1982 Jun;60(Pt 2):279–293. doi: 10.1099/0022-1317-60-2-279. [DOI] [PubMed] [Google Scholar]
  11. Byrd P. J., Grand R. J., Breiding D., Williams J. F., Gallimore P. H. Host range mutants of adenovirus type 12 E1 defective for lytic infection, transformation, and oncogenicity. Virology. 1988 Mar;163(1):155–165. doi: 10.1016/0042-6822(88)90242-5. [DOI] [PubMed] [Google Scholar]
  12. Byrd P. J., Grand R. J., Gallimore P. H. Differential transformation of primary human embryo retinal cells by adenovirus E1 regions and combinations of E1A + ras. Oncogene. 1988 May;2(5):477–484. [PubMed] [Google Scholar]
  13. Byrd P., Brown K. W., Gallimore P. H. Malignant transformation of human embryo retinoblasts by cloned adenovirus 12 DNA. Nature. 1982 Jul 1;298(5869):69–71. doi: 10.1038/298069a0. [DOI] [PubMed] [Google Scholar]
  14. Counter C. M., Avilion A. A., LeFeuvre C. E., Stewart N. G., Greider C. W., Harley C. B., Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 1992 May;11(5):1921–1929. doi: 10.1002/j.1460-2075.1992.tb05245.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Counter C. M., Botelho F. M., Wang P., Harley C. B., Bacchetti S. Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes. J Virol. 1994 May;68(5):3410–3414. doi: 10.1128/jvi.68.5.3410-3414.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Counter C. M., Hirte H. W., Bacchetti S., Harley C. B. Telomerase activity in human ovarian carcinoma. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2900–2904. doi: 10.1073/pnas.91.8.2900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. DiPaolo J. A. Relative difficulties in transforming human and animal cells in vitro. J Natl Cancer Inst. 1983 Jan;70(1):3–8. [PubMed] [Google Scholar]
  18. Dimri G. P., Lee X., Basile G., Acosta M., Scott G., Roskelley C., Medrano E. E., Linskens M., Rubelj I., Pereira-Smith O. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9363–9367. doi: 10.1073/pnas.92.20.9363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Duncan E. L., Whitaker N. J., Moy E. L., Reddel R. R. Assignment of SV40-immortalized cells to more than one complementation group for immortalization. Exp Cell Res. 1993 Apr;205(2):337–344. doi: 10.1006/excr.1993.1095. [DOI] [PubMed] [Google Scholar]
  20. Fukasawa K., Choi T., Kuriyama R., Rulong S., Vande Woude G. F. Abnormal centrosome amplification in the absence of p53. Science. 1996 Mar 22;271(5256):1744–1747. doi: 10.1126/science.271.5256.1744. [DOI] [PubMed] [Google Scholar]
  21. GIRARDI A. J., JENSEN F. C., KOPROWSKI H. SV40-INDUCED TRANFORMATION OF HUMAN DIPLOID CELLS: CRISIS AND RECOVERY. J Cell Physiol. 1965 Feb;65:69–83. doi: 10.1002/jcp.1030650110. [DOI] [PubMed] [Google Scholar]
  22. Gallimore P. H., Byrd P. J., Whittaker J. L., Grand R. J. Properties of rat cells transformed by DNA plasmids containing adenovirus type 12 E1 DNA or specific fragments of the E1 region: comparison of transforming frequencies. Cancer Res. 1985 Jun;45(6):2670–2680. [PubMed] [Google Scholar]
  23. Gallimore P. H., Grand R. J., Byrd P. J. Transformation of human embryo retinoblasts with simian virus 40, adenovirus and ras oncogenes. Anticancer Res. 1986 May-Jun;6(3 Pt B):499–508. [PubMed] [Google Scholar]
  24. Goldstein S. Replicative senescence: the human fibroblast comes of age. Science. 1990 Sep 7;249(4973):1129–1133. doi: 10.1126/science.2204114. [DOI] [PubMed] [Google Scholar]
  25. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  26. Grand R. J., Grant M. L., Gallimore P. H. Enhanced expression of p53 in human cells infected with mutant adenoviruses. Virology. 1994 Sep;203(2):229–240. doi: 10.1006/viro.1994.1480. [DOI] [PubMed] [Google Scholar]
  27. Grand R. J., Lecane P. S., Owen D., Grant M. L., Roberts S., Levine A. J., Gallimore P. H. The high levels of p53 present in adenovirus early region 1-transformed human cells do not cause up-regulation of MDM2 expression. Virology. 1995 Jul 10;210(2):323–334. doi: 10.1006/viro.1995.1349. [DOI] [PubMed] [Google Scholar]
  28. Grand R. J., Lecane P. S., Roberts S., Grant M. L., Lane D. P., Young L. S., Dawson C. W., Gallimore P. H. Overexpression of wild-type p53 and c-Myc in human fetal cells transformed with adenovirus early region 1. Virology. 1993 Apr;193(2):579–591. doi: 10.1006/viro.1993.1166. [DOI] [PubMed] [Google Scholar]
  29. Grand R. J., Owen D., Rookes S. M., Gallimore P. H. Control of p53 expression by adenovirus 12 early region 1A and early region 1B 54K proteins. Virology. 1996 Apr 1;218(1):23–34. doi: 10.1006/viro.1996.0162. [DOI] [PubMed] [Google Scholar]
  30. HAYFLICK L., MOORHEAD P. S. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961 Dec;25:585–621. doi: 10.1016/0014-4827(61)90192-6. [DOI] [PubMed] [Google Scholar]
  31. HAYFLICK L. THE LIMITED IN VITRO LIFETIME OF HUMAN DIPLOID CELL STRAINS. Exp Cell Res. 1965 Mar;37:614–636. doi: 10.1016/0014-4827(65)90211-9. [DOI] [PubMed] [Google Scholar]
  32. Harley C. B., Futcher A. B., Greider C. W. Telomeres shorten during ageing of human fibroblasts. Nature. 1990 May 31;345(6274):458–460. doi: 10.1038/345458a0. [DOI] [PubMed] [Google Scholar]
  33. Harley C. B. Telomere loss: mitotic clock or genetic time bomb? Mutat Res. 1991 Mar-Nov;256(2-6):271–282. doi: 10.1016/0921-8734(91)90018-7. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Harvey M., McArthur M. J., Montgomery C. A., Jr, Butel J. S., Bradley A., Donehower L. A. Spontaneous and carcinogen-induced tumorigenesis in p53-deficient mice. Nat Genet. 1993 Nov;5(3):225–229. doi: 10.1038/ng1193-225. [DOI] [PubMed] [Google Scholar]
  36. Hastie N. D., Dempster M., Dunlop M. G., Thompson A. M., Green D. K., Allshire R. C. Telomere reduction in human colorectal carcinoma and with ageing. Nature. 1990 Aug 30;346(6287):866–868. doi: 10.1038/346866a0. [DOI] [PubMed] [Google Scholar]
  37. Huschtscha L. I., Holliday R. Limited and unlimited growth of SV40-transformed cells from human diploid MRC-5 fibroblasts. J Cell Sci. 1983 Sep;63:77–99. doi: 10.1242/jcs.63.1.77. [DOI] [PubMed] [Google Scholar]
  38. Kaighn M. E., Camalier R. F., Bertolero F., Saffiotti U. Spontaneous establishment and characterization of mouse keratinocyte cell lines in serum-free medium. In Vitro Cell Dev Biol. 1988 Aug;24(8):845–854. doi: 10.1007/BF02623657. [DOI] [PubMed] [Google Scholar]
  39. Kano Y., Little J. B. Mechanisms of human cell neoplastic transformation: relationship of specific abnormal clone formation to prolonged lifespan in X-irradiated human diploid fibroblasts. Int J Cancer. 1985 Sep 15;36(3):407–413. [PubMed] [Google Scholar]
  40. 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]
  41. Kaur P., McDougall J. K. HPV-18 immortalization of human keratinocytes. Virology. 1989 Nov;173(1):302–310. doi: 10.1016/0042-6822(89)90247-x. [DOI] [PubMed] [Google Scholar]
  42. Kim N. W., Piatyszek M. A., Prowse K. R., Harley C. B., West M. D., Ho P. L., Coviello G. M., Wright W. E., Weinrich S. L., Shay J. W. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994 Dec 23;266(5193):2011–2015. doi: 10.1126/science.7605428. [DOI] [PubMed] [Google Scholar]
  43. Klingelhutz A. J., Barber S. A., Smith P. P., Dyer K., McDougall J. K. Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells. Mol Cell Biol. 1994 Feb;14(2):961–969. doi: 10.1128/mcb.14.2.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Klingelhutz A. J., Foster S. A., McDougall J. K. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature. 1996 Mar 7;380(6569):79–82. doi: 10.1038/380079a0. [DOI] [PubMed] [Google Scholar]
  45. Levy M. Z., Allsopp R. C., Futcher A. B., Greider C. W., Harley C. B. Telomere end-replication problem and cell aging. J Mol Biol. 1992 Jun 20;225(4):951–960. doi: 10.1016/0022-2836(92)90096-3. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Mak I., Mak S., Benchimol S. Expression of the cellular p53 protein in cells transformed by adenovirus 12 and viral DNA fragments. Virology. 1988 Mar;163(1):201–204. doi: 10.1016/0042-6822(88)90248-6. [DOI] [PubMed] [Google Scholar]
  48. Merrick R. M., Grand R. J., Brown J. C., Gallimore P. H. The use of beta-galactosidase fusion proteins encoding the early region 1 transforming proteins of adenovirus type 12 to examine the humoral response in tumour-bearing animals. J Gen Virol. 1991 Apr;72(Pt 4):955–960. doi: 10.1099/0022-1317-72-4-955. [DOI] [PubMed] [Google Scholar]
  49. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  51. Murnane J. P., Sabatier L., Marder B. A., Morgan W. F. Telomere dynamics in an immortal human cell line. EMBO J. 1994 Oct 17;13(20):4953–4962. doi: 10.1002/j.1460-2075.1994.tb06822.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Pilder S., Moore M., Logan J., Shenk T. The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs. Mol Cell Biol. 1986 Feb;6(2):470–476. doi: 10.1128/mcb.6.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sarnow P., Ho Y. S., Williams J., Levine A. J. Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells. Cell. 1982 Feb;28(2):387–394. doi: 10.1016/0092-8674(82)90356-7. [DOI] [PubMed] [Google Scholar]
  55. Shay J. W., Wright W. E., Brasiskyte D., Van der Haegen B. A. E6 of human papillomavirus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts. Oncogene. 1993 Jun;8(6):1407–1413. [PubMed] [Google Scholar]
  56. Stacey M., Gallimore P. H., McConville C., Taylor A. M. Rearrangement of the same chromosome regions in different SV40 transformed human skin keratinocyte lines is associated with tumourigenicity. Oncogene. 1990 May;5(5):727–739. [PubMed] [Google Scholar]
  57. Steegenga W. T., Van Laar T., Shvarts A., Terleth C., Van der Eb A. J., Jochemsen A. G. Distinct modulation of p53 activity in transcription and cell-cycle regulation by the large (54 kDa) and small (21 kDa) adenovirus E1B proteins. Virology. 1995 Oct 1;212(2):543–554. doi: 10.1006/viro.1995.1512. [DOI] [PubMed] [Google Scholar]
  58. Tahara H., Sato E., Noda A., Ide T. Increase in expression level of p21sdi1/cip1/waf1 with increasing division age in both normal and SV40-transformed human fibroblasts. Oncogene. 1995 Mar 2;10(5):835–840. [PubMed] [Google Scholar]
  59. Van den Elsen P., Houweling A., Van der Eb A. Expression of region E1b of human adenoviruses in the absence of region E1a is not sufficient for complete transformation. Virology. 1983 Jul 30;128(2):377–390. doi: 10.1016/0042-6822(83)90264-7. [DOI] [PubMed] [Google Scholar]
  60. Wang S. S., Zakian V. A. Telomere-telomere recombination provides an express pathway for telomere acquisition. Nature. 1990 May 31;345(6274):456–458. doi: 10.1038/345456a0. [DOI] [PubMed] [Google Scholar]
  61. White E., Cipriani R., Sabbatini P., Denton A. Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins. J Virol. 1991 Jun;65(6):2968–2978. doi: 10.1128/jvi.65.6.2968-2978.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Whittaker J. L., Byrd P. J., Grand R. J., Gallimore P. H. Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines. Mol Cell Biol. 1984 Jan;4(1):110–116. doi: 10.1128/mcb.4.1.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wiesmüller L., Cammenga J., Deppert W. W. In vivo assay of p53 function in homologous recombination between simian virus 40 chromosomes. J Virol. 1996 Feb;70(2):737–744. doi: 10.1128/jvi.70.2.737-744.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wright W. E., Pereira-Smith O. M., Shay J. W. Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts. Mol Cell Biol. 1989 Jul;9(7):3088–3092. doi: 10.1128/mcb.9.7.3088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wynford-Thomas D., Bond J. A., Wyllie F. S., Jones C. J. Does telomere shortening drive selection for p53 mutation in human cancer? Mol Carcinog. 1995 Mar;12(3):119–123. doi: 10.1002/mc.2940120302. [DOI] [PubMed] [Google Scholar]
  66. Yew P. R., Berk A. J. Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein. Nature. 1992 May 7;357(6373):82–85. doi: 10.1038/357082a0. [DOI] [PubMed] [Google Scholar]
  67. Yew P. R., Liu X., Berk A. J. Adenovirus E1B oncoprotein tethers a transcriptional repression domain to p53. Genes Dev. 1994 Jan;8(2):190–202. doi: 10.1101/gad.8.2.190. [DOI] [PubMed] [Google Scholar]
  68. Zacksenhaus E., Bremner R., Jiang Z., Gill R. M., Muncaster M., Sopta M., Phillips R. A., Gallie B. L. Unraveling the function of the retinoblastoma gene. Adv Cancer Res. 1993;61:115–141. doi: 10.1016/s0065-230x(08)60957-4. [DOI] [PubMed] [Google Scholar]
  69. Zantema A., Schrier P. I., Davis-Olivier A., van Laar T., Vaessen R. T., van der EB A. J. Adenovirus serotype determines association and localization of the large E1B tumor antigen with cellular tumor antigen p53 in transformed cells. Mol Cell Biol. 1985 Nov;5(11):3084–3091. doi: 10.1128/mcb.5.11.3084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. de Ronde A., Sol C. J., van Strien A., ter Schegget J., van der Noordaa J. The SV40 small t antigen is essential for the morphological transformation of human fibroblasts. Virology. 1989 Jul;171(1):260–263. doi: 10.1016/0042-6822(89)90534-5. [DOI] [PubMed] [Google Scholar]
  71. 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]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES