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. 1981 Nov;1(11):994–1006. doi: 10.1128/mcb.1.11.994

Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.

S Chen, M Verderame, A Lo, R Pollack
PMCID: PMC369721  PMID: 6287215

Abstract

Normal fibroblasts display two distinct growth controls which can be assayed as requirements for serum or for anchorage. Interaction of mouse 3T3 fibroblasts with simian virus 40 (SV40) thus generates four classes of transformed cells. We have examined viral gene expression in these four classes of cell lines. Immunoprecipitation of [35S]methionine-labeled cell extracts with an antiserum obtained from tumor-bearing hamsters detected the SV40 large T and small t proteins (94,000 molecular weight [94K], 17K) and the nonviral host 54K protein in all cell lines tested. A tumor antigen with an apparent molecular weight of 100,000 was also found in some, but not all, lines. Similar "super T" molecules have been found by others in many rodent transformed lines. We carried out an analysis of the relation of phenotype to relative amounts of these proteins in cell lines of the four classes, using the Spearman rank correlation test. The amount of the 100K T antigen relative to the 94K T antigen or to total viral protein was well correlated with the ability to form colonies in semisolid medium. No significant correlation was found between quantities of labeled 94K T antigen, 54K host antigen, or 17K t antigen and either serum or anchorage independence. Mouse cells transformed with the small t SV40 deletion mutant 884 synthesized a 100K T antigen, suggesting that small t is not required for the production of this protein. The 100K T antigen migrated more slowly than lytic T. Since mixtures of extracts from cells expressing and lacking the 100K T antigen yielded the expected amount of this protein, it is unlikely that the 100K T derives from the 94K protein by a posttranslational modification.

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Selected References

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

  1. Ahmad-Zadeh C., Allet B., Greenblatt J., Weil R. Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1097–1101. doi: 10.1073/pnas.73.4.1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alwine J. C., Dhar R., Khoury G. A small RNA induced late in simian virus 40 infection can associate with early viral mRNAs. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1379–1383. doi: 10.1073/pnas.77.3.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alwine J. C., Khoury G. Simian virus 40-associated small RNA: mapping on the simian virus 40 genome and characterization of its synthesis. J Virol. 1980 Dec;36(3):701–708. doi: 10.1128/jvi.36.3.701-708.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barrett J. C., Crawford B. D., Mixter L. O., Schechtman L. M., Ts'o P. O., Pollack R. Correlation of in vitro growth properties and tumorigenicity of Syrian hamster cell lines. Cancer Res. 1979 May;39(5):1504–1510. [PubMed] [Google Scholar]
  5. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Botchan M., Topp W., Sambrook J. The arrangement of simian virus 40 sequences in the DNA of transformed cells. Cell. 1976 Oct;9(2):269–287. doi: 10.1016/0092-8674(76)90118-5. [DOI] [PubMed] [Google Scholar]
  7. Bouck N., Beales N., Shenk T., Berg P., di Mayorca G. New region of the simian virus 40 genome required for efficient viral transformation. Proc Natl Acad Sci U S A. 1978 May;75(5):2473–2477. doi: 10.1073/pnas.75.5.2473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brugge J. S., Butel J. S. Role of simian virus 40 gene A function in maintenance of transformation. J Virol. 1975 Mar;15(3):619–635. doi: 10.1128/jvi.15.3.619-635.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang C., Simmons D. T., Martin M. A., Mora P. T. Identification and partial characterization of new antigens from simian virus 40-transformed mouse cells. J Virol. 1979 Aug;31(2):463–471. doi: 10.1128/jvi.31.2.463-471.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen T. R. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res. 1977 Feb;104(2):255–262. doi: 10.1016/0014-4827(77)90089-1. [DOI] [PubMed] [Google Scholar]
  11. Crawford L. V., Cole C. N., Smith A. E., Paucha E., Tegtmeyer P., Rundell K., Berg P. Organization and expression of early genes of simian virus 40. Proc Natl Acad Sci U S A. 1978 Jan;75(1):117–121. doi: 10.1073/pnas.75.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Denhardt D. T., Crawford L. V. Simian virus 40 T-antigen: identification of tryptic peptides in the C-terminal region and definition of the reading frame. J Virol. 1980 May;34(2):315–329. doi: 10.1128/jvi.34.2.315-329.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Deppert W., Henning R. SV40 T-antigen-related molecules on the surfaces of HeLa cells infected with adenovirus-2-SV40 hybrids and on SV40-transformed cells. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):225–234. doi: 10.1101/sqb.1980.044.01.026. [DOI] [PubMed] [Google Scholar]
  14. Doerfler W., Stabel S., Ibelgaufts H., Sutter D., Neumann R., Groneberg J., Scheidtmann K. H., Deuring R., Winterhoff U. Selectivity in integration sites of adenoviral DNA. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):551–564. doi: 10.1101/sqb.1980.044.01.057. [DOI] [PubMed] [Google Scholar]
  15. Dubrow R., Pardee A. B., Pollack R. 2-amino-isobutyric acid and 3-O-methyl-D-glucose transport in 3T3, SV 40-transformed 3T3 and revertant cell lines. J Cell Physiol. 1978 May;95(2):203–211. doi: 10.1002/jcp.1040950210. [DOI] [PubMed] [Google Scholar]
  16. Eckhart W., Hutchinson M. A., Hunter T. An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell. 1979 Dec;18(4):925–933. doi: 10.1016/0092-8674(79)90205-8. [DOI] [PubMed] [Google Scholar]
  17. Edwards C. A., Khoury G., Martin R. G. Phosphorylation of T-antigen and control T-antigen expression in cells transformed by wild-type and tsA mutants of simian virus 40. J Virol. 1979 Feb;29(2):753–762. doi: 10.1128/jvi.29.2.753-762.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Erikson R. I., Collett M. S., Erikson E., Purchio A. F., Brugge J. S. Protein phosphorylation mediated by partially purified avian sarcoma virus transforming-gene product. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 2):907–917. doi: 10.1101/sqb.1980.044.01.098. [DOI] [PubMed] [Google Scholar]
  19. Fanning E., Nowak B., Burger C. Detection and characterization of multiple forms of simian virus 40 large T antigen. J Virol. 1981 Jan;37(1):92–102. doi: 10.1128/jvi.37.1.92-102.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Feldman R. A., Hanafusa T., Hanafusa H. Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus. Cell. 1980 Dec;22(3):757–765. doi: 10.1016/0092-8674(80)90552-8. [DOI] [PubMed] [Google Scholar]
  21. Giacherio D., Hager L. P. A poly(dT)-stimulated ATPase activity associated with simian virus 40 large T antigen. J Biol Chem. 1979 Sep 10;254(17):8113–8116. [PubMed] [Google Scholar]
  22. Gluzman Y., Davison J., Oren M., Winocour E. Properties of permissive monkey cells transformed by UV-irradiated simian virus 40. J Virol. 1977 May;22(2):256–266. doi: 10.1128/jvi.22.2.256-266.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Griffin J. D., Spangler G., Livingston D. M. Enzymatic activities associated with the SV40 large T antigen. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):113–122. doi: 10.1101/sqb.1980.044.01.013. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Hayward W. S., Neel B. G., Astrin S. M. Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature. 1981 Apr 9;290(5806):475–480. doi: 10.1038/290475a0. [DOI] [PubMed] [Google Scholar]
  26. Hiscott J. B., Defendi V. Viral and cellular control of the SV40-transformed phenotype. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):343–352. doi: 10.1101/sqb.1980.044.01.039. [DOI] [PubMed] [Google Scholar]
  27. Hiscott J., Murphy D., Defendi V. Amplification and rearrangement of integrated SV40 DNA sequences accompany the selection of anchorage-independent transformed mouse cells. Cell. 1980 Nov;22(2 Pt 2):535–543. doi: 10.1016/0092-8674(80)90363-3. [DOI] [PubMed] [Google Scholar]
  28. Houweling A., van den Elsen P. J., van der Eb A. J. Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA. Virology. 1980 Sep;105(2):537–550. doi: 10.1016/0042-6822(80)90054-9. [DOI] [PubMed] [Google Scholar]
  29. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Häyry P., Defendi V. Use of mixed hemagglutination technique in detection of virus-induced antigen(s) on SV40-transformed cell surface. Virology. 1968 Oct;36(2):317–321. doi: 10.1016/0042-6822(68)90153-0. [DOI] [PubMed] [Google Scholar]
  31. Kaplan P. L., Topp W. C., Ozanne B. Simian virus 40 induces the production of a polypeptide transforming factor(s). Virology. 1981 Jan 30;108(2):484–490. doi: 10.1016/0042-6822(81)90455-4. [DOI] [PubMed] [Google Scholar]
  32. Ketner G., Kelly T. J., Jr Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1102–1106. doi: 10.1073/pnas.73.4.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kress M., May E., Cassingena R., May P. Simian virus 40-transformed cells express new species of proteins precipitable by anti-simian virus 40 tumor serum. J Virol. 1979 Aug;31(2):472–483. doi: 10.1128/jvi.31.2.472-483.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Linzer D. I., Maltzman W., Levine A. J. The SV40 A gene product is required for the production of a 54,000 MW cellular tumor antigen. Virology. 1979 Oct 30;98(2):308–318. doi: 10.1016/0042-6822(79)90554-3. [DOI] [PubMed] [Google Scholar]
  36. Mark D. F., Berg P. A third splice site in SV40 early mRNA. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):55–62. doi: 10.1101/sqb.1980.044.01.008. [DOI] [PubMed] [Google Scholar]
  37. May E., Kress M., Daya-Grosjean L., Monier R., May P. Mapping of the viral mRNA encoding a super-T antigen of 115,000 daltons expressed in simian virus 40-transformed rat cell lines. J Virol. 1981 Jan;37(1):24–35. doi: 10.1128/jvi.37.1.24-35.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. McCormick F., Chaudry F., Harvey R., Smith R., Rigby P. W., Paucha E., Smith A. E. T antigens of SV40-transformed cells. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):171–178. doi: 10.1101/sqb.1980.044.01.020. [DOI] [PubMed] [Google Scholar]
  39. McCormick F., Harlow E. Association of a murine 53,000-dalton phosphoprotein with simian virus 40 large-T antigen in transformed cells. J Virol. 1980 Apr;34(1):213–224. doi: 10.1128/jvi.34.1.213-224.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Murphy R. F., Pearson W. R., Bonner J. Computer programs for analysis of nucleic acid hybridization, thermal denaturation, and gel electrophoresis data. Nucleic Acids Res. 1979 Aug 24;6(12):3911–3921. doi: 10.1093/nar/6.12.3911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Oey J., Vogel A., Pollack R. Intracellular cyclic AMP concentration responds specifically to growth regulation by serum. Proc Natl Acad Sci U S A. 1974 Mar;71(3):694–698. doi: 10.1073/pnas.71.3.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pintel D., Bouck N., di Mayorca G. Separation of lytic and transforming functions of the simian virus 40 A region: two mutants which are temperature sensitive for lytic functions have opposite effects on transformation. J Virol. 1981 May;38(2):518–528. doi: 10.1128/jvi.38.2.518-528.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pollack R. E., Burger M. M. Surface-specific characteristics of a contact-inhibited cell line containing the SV40 viral genome. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1074–1076. doi: 10.1073/pnas.62.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pollack R., Vogel A. Isolation and characterization of revertant cell lines. II. Growth control of a polyploid revertant line derived from SV40-transformed 3T3 mouse cells. J Cell Physiol. 1973 Aug;82(1):93–100. doi: 10.1002/jcp.1040820111. [DOI] [PubMed] [Google Scholar]
  45. Prives C., Beck Y., Shure H. DNA binding properties of simian virus 40 T-antigens synthesized in vivo and in vitro. J Virol. 1980 Feb;33(2):689–696. doi: 10.1128/jvi.33.2.689-696.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Prives C., Gilboa E., Revel M., Winocour E. Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen. Proc Natl Acad Sci U S A. 1977 Feb;74(2):457–461. doi: 10.1073/pnas.74.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Prives C., Gluzman Y., Winocour E. Cellular and cell-free synthesis of simian virus 40 T-antigens in permissive and transformed cells. J Virol. 1978 Feb;25(2):587–595. doi: 10.1128/jvi.25.2.587-595.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Risser R., Pollack R. A nonselective analysis of SV40 transformation of mouse 3T3 cells. Virology. 1974 Jun;59(2):477–489. doi: 10.1016/0042-6822(74)90457-7. [DOI] [PubMed] [Google Scholar]
  49. Seif R., Martin R. G. Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells. J Virol. 1979 Dec;32(3):979–988. doi: 10.1128/jvi.32.3.979-988.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sleigh M. J., Topp W. C., Hanich R., Sambrook J. F. Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells. Cell. 1978 May;14(1):79–88. doi: 10.1016/0092-8674(78)90303-3. [DOI] [PubMed] [Google Scholar]
  51. Smith A. E., Smith R., Paucha E. Characterization of different tumor antigens present in cells transformed by simian virus 40. Cell. 1979 Oct;18(2):335–346. doi: 10.1016/0092-8674(79)90053-9. [DOI] [PubMed] [Google Scholar]
  52. Smith H. S., Gelb L. D., Martin M. A. Detection and quantitation of simian virus 40 genetic material in abortively transformed BALB-3T3 clones (mice-diploid cells-virus equivalents). Proc Natl Acad Sci U S A. 1972 Jan;69(1):152–156. doi: 10.1073/pnas.69.1.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Smith H. S., Scher C. D., Todaro G. J. Induction of cell division in medium lacking serum growth factor by SV40. Virology. 1971 May;44(2):359–370. doi: 10.1016/0042-6822(71)90267-4. [DOI] [PubMed] [Google Scholar]
  54. Soprano K. J., Jonak G. J., Galanti N., Floros J., Baserga R. Identification of an SV40 DNA sequence related to the reactivation of silent rRNA genes in human greater than mouse hybrid cells. Virology. 1981 Feb;109(1):127–136. doi: 10.1016/0042-6822(81)90477-3. [DOI] [PubMed] [Google Scholar]
  55. Soule H. R., Butel J. S. Subcellular Localization of simian virus 40 large tumor antigen. J Virol. 1979 May;30(2):523–532. doi: 10.1128/jvi.30.2.523-532.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Soule H. R., Lanford R. E., Butel J. S. Antigenic and immunogenic characteristics of nuclear and membrane-associated simian virus 40 tumor antigen. J Virol. 1980 Feb;33(2):887–901. doi: 10.1128/jvi.33.2.887-901.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Steinberg B. M., Pollack R. Anchorage independence: analysis of factors affecting the growth and colony formation of wild-type and dl 54/59 mutant SV40-transformed lines. Virology. 1979 Dec;99(2):302–311. doi: 10.1016/0042-6822(79)90009-6. [DOI] [PubMed] [Google Scholar]
  58. TODARO G. J., GREEN H., GOLDBERG B. D. TRANSFORMATION OF PROPERTIES OF AN ESTABLISHED CELL LINE BY SV40 AND POLYOMA VIRUS. Proc Natl Acad Sci U S A. 1964 Jan;51:66–73. doi: 10.1073/pnas.51.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tegtmeyer P. Function of simian virus 40 gene A in transforming infection. J Virol. 1975 Mar;15(3):613–618. doi: 10.1128/jvi.15.3.613-618.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Tevethia S. S., Flyer D. C., Tevethia M. J., Topp W. C. Biology of simian virus 40 (SV40) transplantation antigen (TrAg). VII. Induction of SV40 TrAg in nonpermissive mouse cells by early viable SV40 deletion (o.54/0.59) mutants. Virology. 1980 Dec;107(2):488–496. doi: 10.1016/0042-6822(80)90315-3. [DOI] [PubMed] [Google Scholar]
  61. Tevethia S. S., Greenfield R. S., Flyer D. C., Tevethia M. J. SV40 transplantation antigen: relationship to SV40-specific proteins. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):235–242. doi: 10.1101/sqb.1980.044.01.027. [DOI] [PubMed] [Google Scholar]
  62. Tjian R., Robbins A., Clark R. Catalytic properties of the SV40 large T antigen. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):103–111. doi: 10.1101/sqb.1980.044.01.012. [DOI] [PubMed] [Google Scholar]
  63. Tjian R., Robbins A. Enzymatic activities associated with a purified simian virus 40 T antigen-related protein. Proc Natl Acad Sci U S A. 1979 Feb;76(2):610–614. doi: 10.1073/pnas.76.2.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Todaro G. J., Green H. High frequency of SV40 transformation of mouse cell line 3T3. Virology. 1966 Apr;28(4):756–759. doi: 10.1016/0042-6822(66)90261-3. [DOI] [PubMed] [Google Scholar]
  65. Vogel A., Pollack R. Isolation and characterization of revertant cell lines. IV. Direct selection of serum-revertant sublines of SV40-transformed 3T3 mouse cells. J Cell Physiol. 1973 Oct;82(2):189–198. doi: 10.1002/jcp.1040820207. [DOI] [PubMed] [Google Scholar]

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