Abstract
To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage- independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage- independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.
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- Adkins B., Leutz A., Graf T. Autocrine growth induced by src-related oncogenes in transformed chicken myeloid cells. Cell. 1984 Dec;39(3 Pt 2):439–445. doi: 10.1016/0092-8674(84)90451-3. [DOI] [PubMed] [Google Scholar]
- Alema S., Tato F., Boettiger D. myc and src oncogenes have complementary effects on cell proliferation and expression of specific extracellular matrix components in definitive chondroblasts. Mol Cell Biol. 1985 Mar;5(3):538–544. doi: 10.1128/mcb.5.3.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Anthony P. P. Precursor lesions for liver cancer in humans. Cancer Res. 1976 Jul;36(7 Pt 2):2579–2583. [PubMed] [Google Scholar]
- Balmain A., Ramsden M., Bowden G. T., Smith J. Activation of the mouse cellular Harvey-ras gene in chemically induced benign skin papillomas. Nature. 1984 Feb 16;307(5952):658–660. doi: 10.1038/307658a0. [DOI] [PubMed] [Google Scholar]
- Boschek C. B., Jockusch B. M., Friis R. R., Back R., Grundmann E., Bauer H. Early changes in the distribution and organization of microfilament proteins during cell transformation. Cell. 1981 Apr;24(1):175–184. doi: 10.1016/0092-8674(81)90513-4. [DOI] [PubMed] [Google Scholar]
- Burr J. G., Dreyfuss G., Penman S., Buchanan J. M. Association of the src gene product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3484–3488. doi: 10.1073/pnas.77.6.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carley W. W., Barak L. S., Webb W. W. F-actin aggregates in transformed cells. J Cell Biol. 1981 Sep;90(3):797–802. doi: 10.1083/jcb.90.3.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christopherson W. M. Dysplasia, carcinoma in situ, and microinvasive carcinoma of the uterine cervix. Hum Pathol. 1977 Sep;8(5):489–501. doi: 10.1016/s0046-8177(77)80110-x. [DOI] [PubMed] [Google Scholar]
- 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]
- Cross F. R., Garber E. A., Pellman D., Hanafusa H. A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation. Mol Cell Biol. 1984 Sep;4(9):1834–1842. doi: 10.1128/mcb.4.9.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
- David-Pfeuty T., Nouvian-Dooghe Y. Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells. J Cell Biol. 1990 Dec;111(6 Pt 2):3097–3116. doi: 10.1083/jcb.111.6.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davis S., Lu M. L., Lo S. H., Lin S., Butler J. A., Druker B. J., Roberts T. M., An Q., Chen L. B. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. doi: 10.1126/science.1708917. [DOI] [PubMed] [Google Scholar]
- Day D. W. The adenoma-carcinoma sequence. Scand J Gastroenterol Suppl. 1984;104:99–107. [PubMed] [Google Scholar]
- Farber E. The multistep nature of cancer development. Cancer Res. 1984 Oct;44(10):4217–4223. [PubMed] [Google Scholar]
- Gilmer T. M., Annab L. A., Oshimura M., Barrett J. C. Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene. Mol Cell Biol. 1985 Jul;5(7):1707–1713. doi: 10.1128/mcb.5.7.1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hamaguchi M., Grandori C., Hanafusa H. Phosphorylation of cellular proteins in Rous sarcoma virus-infected cells: analysis by use of anti-phosphotyrosine antibodies. Mol Cell Biol. 1988 Aug;8(8):3035–3042. doi: 10.1128/mcb.8.8.3035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hamaguchi M., Hanafusa H. Association of p60src with Triton X-100-resistant cellular structure correlates with morphological transformation. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2312–2316. doi: 10.1073/pnas.84.8.2312. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hamaguchi M., Xiao H., Uehara Y., Ohnishi Y., Nagai Y. Herbimycin A inhibits the association of p60v-src with the cytoskeletal structure and with phosphatidylinositol 3' kinase. Oncogene. 1993 Mar;8(3):559–564. [PubMed] [Google Scholar]
- Hanahan D. Transgenic mice as probes into complex systems. Science. 1989 Dec 8;246(4935):1265–1275. doi: 10.1126/science.2686032. [DOI] [PubMed] [Google Scholar]
- Hennings H., Shores R., Wenk M. L., Spangler E. F., Tarone R., Yuspa S. H. Malignant conversion of mouse skin tumours is increased by tumour initiators and unaffected by tumour promoters. Nature. 1983 Jul 7;304(5921):67–69. doi: 10.1038/304067a0. [DOI] [PubMed] [Google Scholar]
- Hjelle B., Liu E., Bishop J. M. Oncogene v-src transforms and establishes embryonic rodent fibroblasts but not diploid human fibroblasts. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4355–4359. doi: 10.1073/pnas.85.12.4355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hunter T. Cooperation between oncogenes. Cell. 1991 Jan 25;64(2):249–270. doi: 10.1016/0092-8674(91)90637-e. [DOI] [PubMed] [Google Scholar]
- Hölttä E., Auvinen M., Andersson L. C. Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype. J Cell Biol. 1993 Aug;122(4):903–914. doi: 10.1083/jcb.122.4.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Inoue H., Isaka M., Takeda S., Hakura A. Simple system for isolation of cellular and viral mutants for transformation by retrovirus. J Med Virol. 1991 Dec;35(4):246–249. doi: 10.1002/jmv.1890350407. [DOI] [PubMed] [Google Scholar]
- Inoue H., Owada M. K., Yutsudo M., Hakura A. A rat mutant cell clone showing temperature-dependent transformed phenotypes with functional expression of the src gene product. Virology. 1989 Jan;168(1):57–66. doi: 10.1016/0042-6822(89)90403-0. [DOI] [PubMed] [Google Scholar]
- Jove R., Hanafusa H. Cell transformation by the viral src oncogene. Annu Rev Cell Biol. 1987;3:31–56. doi: 10.1146/annurev.cb.03.110187.000335. [DOI] [PubMed] [Google Scholar]
- Kamps M. P., Buss J. E., Sefton B. M. Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4625–4628. doi: 10.1073/pnas.82.14.4625. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kamps M. P., Sefton B. M. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988 Apr;2(4):305–315. [PubMed] [Google Scholar]
- Kanner S. B., Reynolds A. B., Wang H. C., Vines R. R., Parsons J. T. The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO J. 1991 Jul;10(7):1689–1698. doi: 10.1002/j.1460-2075.1991.tb07693.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krueger J. G., Wang E., Garber E. A., Goldberg A. R. Differences in intracellular location of pp60src in rat and chicken cells transformed by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4142–4146. doi: 10.1073/pnas.77.7.4142. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Land H., Chen A. C., Morgenstern J. P., Parada L. F., Weinberg R. A. Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts. Mol Cell Biol. 1986 Jun;6(6):1917–1925. doi: 10.1128/mcb.6.6.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Land H., Parada L. F., Weinberg R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 1983 Aug 18;304(5927):596–602. doi: 10.1038/304596a0. [DOI] [PubMed] [Google Scholar]
- MacAuley A., Pawson T. Cooperative transforming activities of ras, myc, and src viral oncogenes in nonestablished rat adrenocortical cells. J Virol. 1988 Dec;62(12):4712–4721. doi: 10.1128/jvi.62.12.4712-4721.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marshall C. J. Tumor suppressor genes. Cell. 1991 Jan 25;64(2):313–326. doi: 10.1016/0092-8674(91)90641-b. [DOI] [PubMed] [Google Scholar]
- Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. doi: 10.1093/nar/18.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nigg E. A., Sefton B. M., Singer S. J., Vogt P. K. Cytoskeletal organization, vinculin-phosphorylation, and fibronectin expression in transformed fibroblasts with different cell morphologies. Virology. 1986 May;151(1):50–65. doi: 10.1016/0042-6822(86)90103-0. [DOI] [PubMed] [Google Scholar]
- Parsons J. T., Weber M. J. Genetics of src: structure and functional organization of a protein tyrosine kinase. Curr Top Microbiol Immunol. 1989;147:79–127. doi: 10.1007/978-3-642-74697-0_3. [DOI] [PubMed] [Google Scholar]
- Ruley H. E. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature. 1983 Aug 18;304(5927):602–606. doi: 10.1038/304602a0. [DOI] [PubMed] [Google Scholar]
- Sabe H., Okada M., Nakagawa H., Hanafusa H. Activation of c-Src in cells bearing v-Crk and its suppression by Csk. Mol Cell Biol. 1992 Oct;12(10):4706–4713. doi: 10.1128/mcb.12.10.4706. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sandgren E. P., Quaife C. J., Pinkert C. A., Palmiter R. D., Brinster R. L. Oncogene-induced liver neoplasia in transgenic mice. Oncogene. 1989 Jun;4(6):715–724. [PubMed] [Google Scholar]
- Schaller M. D., Borgman C. A., Cobb B. S., Vines R. R., Reynolds A. B., Parsons J. T. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. doi: 10.1073/pnas.89.11.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Seidel-Dugan C., Meyer B. E., Thomas S. M., Brugge J. S. Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src. Mol Cell Biol. 1992 Apr;12(4):1835–1845. doi: 10.1128/mcb.12.4.1835. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spandidos D. A., Wilkie N. M. Malignant transformation of early passage rodent cells by a single mutated human oncogene. Nature. 1984 Aug 9;310(5977):469–475. doi: 10.1038/310469a0. [DOI] [PubMed] [Google Scholar]
- Turner C. E., Glenney J. R., Jr, Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol. 1990 Sep;111(3):1059–1068. doi: 10.1083/jcb.111.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
- Wu H., Parsons J. T. Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex. J Cell Biol. 1993 Mar;120(6):1417–1426. doi: 10.1083/jcb.120.6.1417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wulf E., Deboben A., Bautz F. A., Faulstich H., Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4498–4502. doi: 10.1073/pnas.76.9.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]