Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 May;8(5):2089–2096. doi: 10.1128/mcb.8.5.2089

Partially transformed, anchorage-independent human diploid fibroblasts result from overexpression of the c-sis oncogene: mitogenic activity of an apparent monomeric platelet-derived growth factor 2 species.

C W Stevens 1, W H Brondyk 1, J A Burgess 1, T H Manoharan 1, B G Häne 1, W E Fahl 1
PMCID: PMC363389  PMID: 3290648

Abstract

A human c-sis cDNA in an expression vector was introduced into human diploid fibroblasts by transfection or electroporation. Fibroblast clones showing an aberrant, densely packed colony morphology were isolated and found to overexpress a 3.6-kilobase sis mRNA species and associated immunoprecipitable platelet-derived growth factor (PDGF) 2 proteins. Parallel analyses in cell clones of sis mRNA expression and colony formation in agar indicated that, above a threshold, a linear, positive correlation existed between sis overexpression and acquired anchorage independence. The sis-overexpressing cells formed transient, regressing tumor nodules when injected into nude mice, consistent with the finite life span which they retained. Protein products generated from the transfected c-sis construct in two overexpressing clones were immunoprecipitated with anti-human PDGF antibodies. One clone contained an apparent PDGF dimer of 21 kilodaltons; the second clone contained only an apparent PDGF monomer of 12 kilodaltons, which was shown to account for all of the mitogenic activity present in the cells, essentially all of which was concentrated in the membrane fraction. The results demonstrate a clear link between sis overexpression and acquisition of a partially transformed, anchorage-independent phenotype, and when combined with previous observations of sis overexpression in human tumors, clearly implicate sis overexpression as a genetic mechanism which contributes to human cell transformation.

Full text

PDF
2091

Images in this article

Selected References

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

  1. 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]
  2. Betsholtz C., Heldin C. H., Nister M., Ek B., Wasteson A., Westermark B. Synthesis of a PDGF-like growth factor in human glioma and sarcoma cells suggests the expression of the cellular homologue to the transforming protein of simian sarcoma virus. Biochem Biophys Res Commun. 1983 Nov 30;117(1):176–182. doi: 10.1016/0006-291x(83)91557-7. [DOI] [PubMed] [Google Scholar]
  3. Betsholtz C., Johnsson A., Heldin C. H., Westermark B., Lind P., Urdea M. S., Eddy R., Shows T. B., Philpott K., Mellor A. L. cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumour cell lines. Nature. 1986 Apr 24;320(6064):695–699. doi: 10.1038/320695a0. [DOI] [PubMed] [Google Scholar]
  4. Clarke M. F., Westin E., Schmidt D., Josephs S. F., Ratner L., Wong-Staal F., Gallo R. C., Reitz M. S., Jr Transformation of NIH 3T3 cells by a human c-sis cDNA clone. 1984 Mar 29-Apr 4Nature. 308(5958):464–467. doi: 10.1038/308464a0. [DOI] [PubMed] [Google Scholar]
  5. Deuel T. F., Huang J. S., Huang S. S., Stroobant P., Waterfield M. D. Expression of a platelet-derived growth factor-like protein in simian sarcoma virus transformed cells. Science. 1983 Sep 30;221(4618):1348–1350. doi: 10.1126/science.6310754. [DOI] [PubMed] [Google Scholar]
  6. Doolittle R. F., Hunkapiller M. W., Hood L. E., Devare S. G., Robbins K. C., Aaronson S. A., Antoniades H. N. Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor. Science. 1983 Jul 15;221(4607):275–277. doi: 10.1126/science.6304883. [DOI] [PubMed] [Google Scholar]
  7. Eva A., Robbins K. C., Andersen P. R., Srinivasan A., Tronick S. R., Reddy E. P., Ellmore N. W., Galen A. T., Lautenberger J. A., Papas T. S. Cellular genes analogous to retroviral onc genes are transcribed in human tumour cells. Nature. 1982 Jan 14;295(5845):116–119. doi: 10.1038/295116a0. [DOI] [PubMed] [Google Scholar]
  8. Fry D. G., Milam L. D., Maher V. M., McCormick J. J. Transformation of diploid human fibroblasts by DNA transfection with the v-sis oncogene. J Cell Physiol. 1986 Aug;128(2):313–321. doi: 10.1002/jcp.1041280225. [DOI] [PubMed] [Google Scholar]
  9. Garrett J. S., Coughlin S. R., Niman H. L., Tremble P. M., Giels G. M., Williams L. T. Blockade of autocrine stimulation in simian sarcoma virus-transformed cells reverses down-regulation of platelet-derived growth factor receptors. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7466–7470. doi: 10.1073/pnas.81.23.7466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gazit A., Igarashi H., Chiu I. M., Srinivasan A., Yaniv A., Tronick S. R., Robbins K. C., Aaronson S. A. Expression of the normal human sis/PDGF-2 coding sequence induces cellular transformation. Cell. 1984 Nov;39(1):89–97. doi: 10.1016/0092-8674(84)90194-6. [DOI] [PubMed] [Google Scholar]
  11. Giese N. A., Robbins K. C., Aaronson S. A. The role of individual cysteine residues in the structure and function of the v-sis gene product. Science. 1987 Jun 5;236(4806):1315–1318. doi: 10.1126/science.3035718. [DOI] [PubMed] [Google Scholar]
  12. Hall A., Marshall C. J., Spurr N. K., Weiss R. A. Identification of transforming gene in two human sarcoma cell lines as a new member of the ras gene family located on chromosome 1. Nature. 1983 Jun 2;303(5916):396–400. doi: 10.1038/303396a0. [DOI] [PubMed] [Google Scholar]
  13. Hannink M., Sauer M. K., Donoghue D. J. Deletions in the C-terminal coding region of the v-sis gene: dimerization is required for transformation. Mol Cell Biol. 1986 Apr;6(4):1304–1314. doi: 10.1128/mcb.6.4.1304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Huang J. S., Huang S. S., Deuel T. F. Transforming protein of simian sarcoma virus stimulates autocrine growth of SSV-transformed cells through PDGF cell-surface receptors. Cell. 1984 Nov;39(1):79–87. doi: 10.1016/0092-8674(84)90193-4. [DOI] [PubMed] [Google Scholar]
  15. Igarashi H., Rao C. D., Siroff M., Leal F., Robbins K. C., Aaronson S. A. Detection of PDGF-2 homodimers in human tumor cells. Oncogene. 1987 Mar;1(1):79–85. [PubMed] [Google Scholar]
  16. Josephs S. F., Ratner L., Clarke M. F., Westin E. H., Reitz M. S., Wong-Staal F. Transforming potential of human c-sis nucleotide sequences encoding platelet-derived growth factor. Science. 1984 Aug 10;225(4662):636–639. doi: 10.1126/science.6740330. [DOI] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. 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]
  19. Leal F., Williams L. T., Robbins K. C., Aaronson S. A. Evidence that the v-sis gene product transforms by interaction with the receptor for platelet-derived growth factor. Science. 1985 Oct 18;230(4723):327–330. doi: 10.1126/science.2996133. [DOI] [PubMed] [Google Scholar]
  20. Leof E. B., Proper J. A., Goustin A. S., Shipley G. D., DiCorleto P. E., Moses H. L. Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor beta: a proposed model for indirect mitogenesis involving autocrine activity. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2453–2457. doi: 10.1073/pnas.83.8.2453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McCormick J. J., Kateley-Kohler S., Maher V. M. Factors involved in quantitating induction of anchorage independence in diploid human fibroblasts by carcinogens. Carcinog Compr Surv. 1985;9:233–247. [PubMed] [Google Scholar]
  22. Nistér M., Heldin C. H., Wasteson A., Westermark B. A glioma-derived analog to platelet-derived growth factor: demonstration of receptor competing activity and immunological crossreactivity. Proc Natl Acad Sci U S A. 1984 Feb;81(3):926–930. doi: 10.1073/pnas.81.3.926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Owen A. J., Pantazis P., Antoniades H. N. Simian sarcoma virus--transformed cells secrete a mitogen identical to platelet-derived growth factor. Science. 1984 Jul 6;225(4657):54–56. doi: 10.1126/science.6328659. [DOI] [PubMed] [Google Scholar]
  24. Pavelic Z. P., Slocum H. K., Rustum Y. M., Creaven P. J., Karakousis C., Takita H. Colony growth in soft agar of human melanoma, sarcoma, and lung carcinoma cells disaggregated by mechanical and enzymatic methods. Cancer Res. 1980 Jul;40(7):2160–2164. [PubMed] [Google Scholar]
  25. Pavelic Z. P., Slocum H. K., Rustum Y. M., Creaven P. J., Nowak N. J., Karakousis C., Takita H., Mittelman A. Growth of cell colonies in soft agar from biopsies of different human solid tumors. Cancer Res. 1980 Nov;40(11):4151–4158. [PubMed] [Google Scholar]
  26. Peres R., Betsholtz C., Westermark B., Heldin C. H. Frequent expression of growth factors for mesenchymal cells in human mammary carcinoma cell lines. Cancer Res. 1987 Jul 1;47(13):3425–3429. [PubMed] [Google Scholar]
  27. Potter H., Weir L., Leder P. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7161–7165. doi: 10.1073/pnas.81.22.7161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Robbins K. C., Antoniades H. N., Devare S. G., Hunkapiller M. W., Aaronson S. A. Structural and immunological similarities between simian sarcoma virus gene product(s) and human platelet-derived growth factor. Nature. 1983 Oct 13;305(5935):605–608. doi: 10.1038/305605a0. [DOI] [PubMed] [Google Scholar]
  29. Robbins K. C., Leal F., Pierce J. H., Aaronson S. A. The v-sis/PDGF-2 transforming gene product localizes to cell membranes but is not a secretory protein. EMBO J. 1985 Jul;4(7):1783–1792. doi: 10.1002/j.1460-2075.1985.tb03851.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sugden B., Marsh K., Yates J. A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus. Mol Cell Biol. 1985 Feb;5(2):410–413. doi: 10.1128/mcb.5.2.410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sutherland B. M., Bennett P. V. Transformation of human cells by DNA transfection. Cancer Res. 1984 Jul;44(7):2769–2772. [PubMed] [Google Scholar]
  32. Sutherland B. M., Cimino J. S., Delihas N., Shih A. G., Oliver R. P. Ultraviolet light-induced transformation of human cells to anchorage-independent growth. Cancer Res. 1980 Jun;40(6):1934–1939. [PubMed] [Google Scholar]
  33. Waterfield M. D., Scrace G. T., Whittle N., Stroobant P., Johnsson A., Wasteson A., Westermark B., Heldin C. H., Huang J. S., Deuel T. F. Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature. 1983 Jul 7;304(5921):35–39. doi: 10.1038/304035a0. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES