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. 1986 Jul;6(7):2305–2316. doi: 10.1128/mcb.6.7.2305

Regulation of pp60c-src synthesis by inducible RNA complementary to c-src mRNA in polyomavirus-transformed rat cells.

S Amini, V DeSeau, S Reddy, D Shalloway, J B Bolen
PMCID: PMC367783  PMID: 2431289

Abstract

To determine the potential role of pp60c-src in polyomavirus-transformed cells, we constructed a recombinant plasmid with the mouse metallothionein-I promoter upstream of a src gene in an anti-sense orientation. We cotransfected this plasmid into middle tumor antigen-transformed FR3T3 cells with a plasmid containing the neomycin resistance gene, and G418 resistant colonies were selected. Analysis of these cells for pp60c-src expression revealed that 50 of the 200 cellular clones screened were found to have decreased levels of c-src expression when compared with the parental middle tumor antigen-transformed cells. Three independent clones which transcribed the expected 3.6-kilobase src complementary RNA and had levels of pp60c-src kinase activity comparable to that of normal FR3T3 cells were further analyzed. In the presence of Cd2+, these clones grew significantly slower in monolayer cultures than either the parental transformed cells (FR18-1) or FR18-1 cells transfected with the neomycin resistance gene alone. The morphology of these clones in the presence of Cd2+ was distinct from that of either the parental FR18-1 cells or normal FR3T3 cells. The clones expressing the complementary src RNA were found to form fewer colonies in soft agar, form fewer foci on monolayers of normal rat cells, and form tumors more slowly following injection into syngenic rats when compared with parental FR18-1 cells. The results of these studies suggest that the level of pp60c-src kinase activity affects the growth characteristics and transformation properties of polyoma virus-transformed rat cells.

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Selected References

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

  1. Amini S., Lewis A. M., Jr, Israel M. A., Butel J. S., Bolen J. B. Analysis of pp60c-src protein kinase activity in hamster embryo cells transformed by simian virus 40, human adenoviruses, and bovine papillomavirus 1. J Virol. 1986 Jan;57(1):357–361. doi: 10.1128/jvi.57.1.357-361.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolen J. B., Cary K., Scheller A., Basilico C., Israel M. A., Prives C. A subclass of polyomavirus middle tumor antigen binds to DNA cellulose. J Virol. 1986 Apr;58(1):157–164. doi: 10.1128/jvi.58.1.157-164.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolen J. B., Israel M. A. In vitro association and phosphorylation of polyoma virus middle T antigen by cellular tyrosyl kinase activity. J Biol Chem. 1984 Oct 10;259(19):11686–11694. [PubMed] [Google Scholar]
  4. Bolen J. B., Israel M. A. Inhibition of polyoma virus middle T antigen-associated tyrosyl kinase activity by N-ethylmaleimide. J Biol Chem. 1983 Dec 25;258(24):15135–15140. [PubMed] [Google Scholar]
  5. Bolen J. B., Israel M. A. Middle tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with pp60c-src. J Virol. 1985 Jan;53(1):114–119. doi: 10.1128/jvi.53.1.114-119.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bolen J. B., Lewis A. M., Jr, Israel M. A. Stimulation of pp60c-src tyrosyl kinase activity in polyoma virus-infected mouse cells is closely associated with polyoma middle tumor antigen synthesis. J Cell Biochem. 1985;27(2):157–167. doi: 10.1002/jcb.240270209. [DOI] [PubMed] [Google Scholar]
  7. Bolen J. B., Rosen N., Israel M. A. Increased pp60c-src tyrosyl kinase activity in human neuroblastomas is associated with amino-terminal tyrosine phosphorylation of the src gene product. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7275–7279. doi: 10.1073/pnas.82.21.7275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bolen J. B., Thiele C. J., Israel M. A., Yonemoto W., Lipsich L. A., Brugge J. S. Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell. 1984 Oct;38(3):767–777. doi: 10.1016/0092-8674(84)90272-1. [DOI] [PubMed] [Google Scholar]
  9. Brinster R. L., Chen H. Y., Warren R., Sarthy A., Palmiter R. D. Regulation of metallothionein--thymidine kinase fusion plasmids injected into mouse eggs. Nature. 1982 Mar 4;296(5852):39–42. doi: 10.1038/296039a0. [DOI] [PubMed] [Google Scholar]
  10. Carmichael G. G., Schaffhausen B. S., Dorsky D. I., Oliver D. B., Benjamin T. L. Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3579–3583. doi: 10.1073/pnas.79.11.3579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Carter A. D., Felber B. K., Walling M. J., Jubier M. F., Schmidt C. J., Hamer D. H. Duplicated heavy metal control sequences of the mouse metallothionein-I gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7392–7396. doi: 10.1073/pnas.81.23.7392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cartwright C. A., Hutchinson M. A., Eckhart W. Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells. Mol Cell Biol. 1985 Oct;5(10):2647–2652. doi: 10.1128/mcb.5.10.2647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Courtneidge S. A. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. doi: 10.1002/j.1460-2075.1985.tb03805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Courtneidge S. A., Smith A. E. Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature. 1983 Jun 2;303(5916):435–439. doi: 10.1038/303435a0. [DOI] [PubMed] [Google Scholar]
  15. Courtneidge S. A., Smith A. E. The complex of polyoma virus middle-T antigen and pp60c-src. EMBO J. 1984 Mar;3(3):585–591. doi: 10.1002/j.1460-2075.1984.tb01852.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Czernilofsky A. P., Levinson A. D., Varmus H. E., Bishop J. M., Tischer E., Goodman H. Corrections to the nucleotide sequence of the src gene of Rous sarcoma virus. Nature. 1983 Feb 24;301(5902):736–738. doi: 10.1038/301736b0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Graham F. L., van der Eb A. J. Transformation of rat cells by DNA of human adenovirus 5. Virology. 1973 Aug;54(2):536–539. doi: 10.1016/0042-6822(73)90163-3. [DOI] [PubMed] [Google Scholar]
  19. Iba H., Takeya T., Cross F. R., Hanafusa T., Hanafusa H. Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4424–4428. doi: 10.1073/pnas.81.14.4424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Israel M. A., Chan H. W., Rowe W. P., Martin M. A. Molecular cloning of polyoma virus DNA in Escherichia coli: plasmid vector system. Science. 1979 Mar 2;203(4383):883–887. doi: 10.1126/science.217087. [DOI] [PubMed] [Google Scholar]
  21. Izant J. G., Weintraub H. Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA. Science. 1985 Jul 26;229(4711):345–352. doi: 10.1126/science.2990048. [DOI] [PubMed] [Google Scholar]
  22. Izant J. G., Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell. 1984 Apr;36(4):1007–1015. doi: 10.1016/0092-8674(84)90050-3. [DOI] [PubMed] [Google Scholar]
  23. Kim S. K., Wold B. J. Stable reduction of thymidine kinase activity in cells expressing high levels of anti-sense RNA. Cell. 1985 Aug;42(1):129–138. doi: 10.1016/s0092-8674(85)80108-2. [DOI] [PubMed] [Google Scholar]
  24. Kriegler M., Perez C. F., Hardy C., Botchan M. Transformation mediated by the SV40 T antigens: separation of the overlapping SV40 early genes with a retroviral vector. Cell. 1984 Sep;38(2):483–491. doi: 10.1016/0092-8674(84)90503-8. [DOI] [PubMed] [Google Scholar]
  25. Law M. F., Byrne J. C., Howley P. M. A stable bovine papillomavirus hybrid plasmid that expresses a dominant selective trait. Mol Cell Biol. 1983 Nov;3(11):2110–2115. doi: 10.1128/mcb.3.11.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pavlakis G. N., Hamer D. H. Expression of cloned growth hormone and metallothionein genes in heterologous cells. Recent Prog Horm Res. 1983;39:353–385. doi: 10.1016/b978-0-12-571139-5.50013-6. [DOI] [PubMed] [Google Scholar]
  28. Pavlakis G. N., Hamer D. H. Regulation of a metallothionein-growth hormone hybrid gene in bovine papilloma virus. Proc Natl Acad Sci U S A. 1983 Jan;80(2):397–401. doi: 10.1073/pnas.80.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pestka S., Daugherty B. L., Jung V., Hotta K., Pestka R. K. Anti-mRNA: specific inhibition of translation of single mRNA molecules. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7525–7528. doi: 10.1073/pnas.81.23.7525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Raptis L., Lamfrom H., Benjamin T. L. Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts. Mol Cell Biol. 1985 Sep;5(9):2476–2486. doi: 10.1128/mcb.5.9.2476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schaffhausen B. S., Benjamin T. L. Phosphorylation of polyoma T antigens. Cell. 1979 Dec;18(4):935–946. doi: 10.1016/0092-8674(79)90206-x. [DOI] [PubMed] [Google Scholar]
  32. Schaffhausen B., Benjamin T. L. Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro. J Virol. 1981 Oct;40(1):184–196. doi: 10.1128/jvi.40.1.184-196.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schaffhausen B. Transforming genes and gene products of polyoma and SV40. CRC Crit Rev Biochem. 1982;13(3):215–286. doi: 10.3109/10409238209114230. [DOI] [PubMed] [Google Scholar]
  34. Segawa K., Ito Y. Differential subcellular localization of in vivo-phosphorylated and nonphosphorylated middle-sized tumor antigen of polyoma virus and its relationship to middle-sized tumor antigen phosphorylating activity in vitro. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6812–6816. doi: 10.1073/pnas.79.22.6812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shalloway D., Coussens P. M., Yaciuk P. Overexpression of the c-src protein does not induce transformation of NIH 3T3 cells. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7071–7075. doi: 10.1073/pnas.81.22.7071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smith A. E., Smith R., Griffin B., Fried M. Protein kinase activity associated with polyoma virus middle T antigen in vitro. Cell. 1979 Dec;18(4):915–924. doi: 10.1016/0092-8674(79)90204-6. [DOI] [PubMed] [Google Scholar]
  37. Takeya T., Hanafusa H. Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus. Cell. 1983 Mar;32(3):881–890. doi: 10.1016/0092-8674(83)90073-9. [DOI] [PubMed] [Google Scholar]
  38. Templeton D., Eckhart W. Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation. J Virol. 1982 Mar;41(3):1014–1024. doi: 10.1128/jvi.41.3.1014-1024.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Treisman R., Novak U., Favaloro J., Kamen R. Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature. 1981 Aug 13;292(5824):595–600. doi: 10.1038/292595a0. [DOI] [PubMed] [Google Scholar]
  40. Whitman M., Kaplan D. R., Schaffhausen B., Cantley L., Roberts T. M. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature. 1985 May 16;315(6016):239–242. doi: 10.1038/315239a0. [DOI] [PubMed] [Google Scholar]
  41. Yonemoto W., Jarvis-Morar M., Brugge J. S., Bolen J. B., Israel M. A. Tyrosine phosphorylation within the amino-terminal domain of pp60c-src molecules associated with polyoma virus middle-sized tumor antigen. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4568–4572. doi: 10.1073/pnas.82.14.4568. [DOI] [PMC free article] [PubMed] [Google Scholar]

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