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. 1987 Aug;7(8):2794–2802. doi: 10.1128/mcb.7.8.2794

Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

D S Neufeld 1, S Ripley 1, A Henderson 1, H L Ozer 1
PMCID: PMC367896  PMID: 2823105

Abstract

Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre- and postimmortalization, should be useful in defining molecular changes associated with immortalization.

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Selected References

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

  1. Aaronson S. A. Susceptibility of human cell strains to transformation by simian virus 40 and simian virus 40 deoxyribonucleic acid. J Virol. 1970 Oct;6(4):470–475. doi: 10.1128/jvi.6.4.470-475.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aaronson S. A., Todaro G. J. SV40 T antigen induction and transformation in human fibroblast cell strains. Virology. 1968 Oct;36(2):254–261. doi: 10.1016/0042-6822(68)90142-6. [DOI] [PubMed] [Google Scholar]
  3. Bignone F. A., Panarello C., Gimelli G. G-bands without pretreatment of slides, in chemically defined conditions. Hum Genet. 1983;63(1):63–66. doi: 10.1007/BF00285400. [DOI] [PubMed] [Google Scholar]
  4. Blattner W. A., Lubiniecki A. S., Mulvihill J. J., Lalley P., Fraumeni J. F., Jr Genetics of SV40 T-antigen expression: studies of twins, heritable syndromes and cancer families. Int J Cancer. 1978 Sep 15;22(3):231–238. doi: 10.1002/ijc.2910220303. [DOI] [PubMed] [Google Scholar]
  5. Canaani D., Naiman T., Teitz T., Berg P. Immortalization of xeroderma pigmentosum cells by simian virus 40 DNA having a defective origin of DNA replication. Somat Cell Mol Genet. 1986 Jan;12(1):13–20. doi: 10.1007/BF01560723. [DOI] [PubMed] [Google Scholar]
  6. Carp R. I., Gilden R. V. A comparison of the replication cycles of simian virus 40 in human diploid and African green monkey kidney cells. Virology. 1966 Jan;28(1):150–162. doi: 10.1016/0042-6822(66)90316-3. [DOI] [PubMed] [Google Scholar]
  7. Chang S. E. In vitro transformation of human epithelial cells. Biochim Biophys Acta. 1986;823(3):161–194. doi: 10.1016/0304-419x(86)90001-6. [DOI] [PubMed] [Google Scholar]
  8. Clayton C. E., Murphy D., Lovett M., Rigby P. W. A fragment of the SV40 large T-antigen gene transforms. Nature. 1982 Sep 2;299(5878):59–61. doi: 10.1038/299059a0. [DOI] [PubMed] [Google Scholar]
  9. Clayton C. E., Rigby P. W. Cloning and characterization of the integrated viral DNA from three lines of SV40-transformed mouse cells. Cell. 1981 Aug;25(2):547–559. doi: 10.1016/0092-8674(81)90073-8. [DOI] [PubMed] [Google Scholar]
  10. Colby W. W., Shenk T. Fragments of the simian virus 40 transforming gene facilitate transformation of rat embryo cells. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5189–5193. doi: 10.1073/pnas.79.17.5189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gluzman Y., Frisque R. J., Sambrook J. Origin-defective mutants of SV40. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):293–300. doi: 10.1101/sqb.1980.044.01.033. [DOI] [PubMed] [Google Scholar]
  12. Gluzman Y., Sambrook J. F., Frisque R. J. Expression of early genes of origin-defective mutants of simian virus 40. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3898–3902. doi: 10.1073/pnas.77.7.3898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gotoh S., Gelb L., Schlessinger D. SV40-transformed human diploid cells that remain transformed throughout their limited lifespan. J Gen Virol. 1979 Feb;42(2):409–414. doi: 10.1099/0022-1317-42-2-409. [DOI] [PubMed] [Google Scholar]
  14. Gruss C., Baumann E., Knippers R. DNA binding properties of a mutant T antigen from the simian virus 40-transformed human cell line simian virus 80. J Virol. 1984 Jun;50(3):943–946. doi: 10.1128/jvi.50.3.943-946.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Harlow E., Crawford L. V., Pim D. C., Williamson N. M. Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol. 1981 Sep;39(3):861–869. doi: 10.1128/jvi.39.3.861-869.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Jha K. K., Cacciapuoti J., Ozer H. L. Expression of transformation in cell hybrids. II. Nonsuppression of the transformed phenotype in hybrids between a chemically transformed and nontransformed derivatives of Balb/3T3. J Cell Physiol. 1978 Nov;97(2):147–152. doi: 10.1002/jcp.1040970203. [DOI] [PubMed] [Google Scholar]
  19. La Bella F., Ozer H. L. Differential replication of SV40 and polyoma DNAs in Chinese hamster ovary cells. Virus Res. 1985 Jun;2(4):329–344. doi: 10.1016/0168-1702(85)90029-2. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lewis E. D., Chen S., Kumar A., Blanck G., Pollack R. E., Manley J. L. A frameshift mutation affecting the carboxyl terminus of the simian virus 40 large tumor antigen results in a replication- and transformation-defective virus. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7065–7069. doi: 10.1073/pnas.80.23.7065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Litzkas P., Jha K. K., Ozer H. L. Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension. Mol Cell Biol. 1984 Nov;4(11):2549–2552. doi: 10.1128/mcb.4.11.2549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. MacArthur H., Walter G. Monoclonal antibodies specific for the carboxy terminus of simian virus 40 large T antigen. J Virol. 1984 Nov;52(2):483–491. doi: 10.1128/jvi.52.2.483-491.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Major E. O., Miller A. E., Mourrain P., Traub R. G., de Widt E., Sever J. Establishment of a line of human fetal glial cells that supports JC virus multiplication. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1257–1261. doi: 10.1073/pnas.82.4.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marshak M. I., Varshaver N. B., Shapiro N. I. Induction of gene mutations and chromosomal aberrations by simian virus 40 in cultured mammalian cells. Mutat Res. 1975 Dec;30(3):383–396. [PubMed] [Google Scholar]
  26. Murnane J. P., Fuller L. F., Painter R. B. Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line. Exp Cell Res. 1985 May;158(1):119–126. doi: 10.1016/0014-4827(85)90437-9. [DOI] [PubMed] [Google Scholar]
  27. Nagata Y., Diamond B., Bloom B. R. The generation of human monocyte/macrophage cell lines. Nature. 1983 Dec 8;306(5943):597–599. doi: 10.1038/306597a0. [DOI] [PubMed] [Google Scholar]
  28. Nichols W. W., Girardi A. J., Bradt C. I., Hill R., Cody C. Cytogenetic changes induced in human diploid fibroblasts by tsA58 SV40 at permissive and restrictive temperatures. Mutat Res. 1985 Jun-Jul;150(1-2):327–332. doi: 10.1016/0027-5107(85)90129-0. [DOI] [PubMed] [Google Scholar]
  29. Ozer H. L., Slater M. L., Dermody J. J., Mandel M. Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum. J Virol. 1981 Aug;39(2):481–489. doi: 10.1128/jvi.39.2.481-489.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Petit C. A., Gardes M., Feunteun J. Immortalization of rodent embryo fibroblasts by SV40 is maintained by the A gene. Virology. 1983 May;127(1):74–82. doi: 10.1016/0042-6822(83)90372-0. [DOI] [PubMed] [Google Scholar]
  31. Potter C. W., Potter A. M., Oxford J. S. Comparison of transformation and T antigen induction in human cell lines. J Virol. 1970 Mar;5(3):293–298. doi: 10.1128/jvi.5.3.293-298.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sack G. H., Jr Human cell transformation by simian virus 40--a review. In Vitro. 1981 Jan;17(1):1–19. doi: 10.1007/BF02618025. [DOI] [PubMed] [Google Scholar]
  33. Small M. B., Gluzman Y., Ozer H. L. Enhanced transformation of human fibroblasts by origin-defective simian virus 40. Nature. 1982 Apr 15;296(5858):671–672. doi: 10.1038/296671a0. [DOI] [PubMed] [Google Scholar]
  34. Smith H. S., Owens R. B., Hiller A. J., Nelson-Rees W. A., Johnston J. O. The biology of human cells in tissue culture. I. Characterization of cells derived from osteogenic sarcomas. Int J Cancer. 1976 Feb 15;17(2):219–234. doi: 10.1002/ijc.2910170211. [DOI] [PubMed] [Google Scholar]
  35. Sompayrac L. M., Gurney E. G., Danna K. J. Stabilization of the 53,000-dalton nonviral tumor antigen is not required for transformation by simian virus 40. Mol Cell Biol. 1983 Feb;3(2):290–296. doi: 10.1128/mcb.3.2.290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stein G. H. SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells. J Cell Physiol. 1985 Oct;125(1):36–44. doi: 10.1002/jcp.1041250106. [DOI] [PubMed] [Google Scholar]
  37. Theile M., Scherneck S., Geissler E. Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers. Mutat Res. 1976 Oct;37(1):111–123. doi: 10.1016/0027-5107(76)90059-2. [DOI] [PubMed] [Google Scholar]
  38. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Van Doren K., Gluzman Y. Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants. Mol Cell Biol. 1984 Aug;4(8):1653–1656. doi: 10.1128/mcb.4.8.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zouzias D., Jha K. K., Mulder C., Basilico C., Ozer H. L. Human fibroblasts transformed by the early region of SV40 DNA: analysis of "free" viral DNA sequences. Virology. 1980 Jul 30;104(2):439–453. doi: 10.1016/0042-6822(80)90346-3. [DOI] [PubMed] [Google Scholar]

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