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. 1985 Sep;5(9):2289–2297. doi: 10.1128/mcb.5.9.2289

Neuroblastoma cells express c-sis and produce a transforming growth factor antigenically related to the platelet-derived growth factor.

E J van Zoelen, W J van de Ven, H J Franssen, T M van Oostwaard, P T van der Saag, C H Heldin, S W de Laat
PMCID: PMC366955  PMID: 2874480

Abstract

Mouse neuroblastoma Neuro-2A cells produce transforming growth factors during exponential growth in a defined hormone-free medium, which, on Bio-Gel columns in 1 M HAc, elute at a molecular size of 15 to 20 kilodaltons (kDa). These neuroblastoma-derived transforming growth factors have strong mitogenic activity, but they do not compete with epidermal growth factor for receptor binding (E. J. J. van Zoelen, D. R. Twardzik, T. M. J. van Oostwaard, P. T. van der Saag, S. W. de Laat, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 81:4085-4089, 1984). In this study approximately 80% of the mitogenic activity was immunoprecipitated by antibodies raised against platelet-derived growth factor (PDGF). Immunoblotting indicated a true molecular size of 32 kDa for this PDGF-like growth factor. Analysis of poly(A)+ RNA from Neuro-2A cells demonstrated the expression of the c-sis oncogene in this cell line, whereas in vitro translation of the RNA yielded a 20-kDa protein recognized by anti-PDGF antibodies. Separation by reverse-phase high-pressure liquid chromatography demonstrated the presence of two distinct mitogenic activities in neuroblastoma-derived transforming growth factor preparations, one of which is antigenically related to PDGF. Both activities had the ability to induce anchorage-independent growth in normal rat kidney cells, both in the presence and in the absence of epidermal growth factor. It is concluded that Neuro-2A cells express c-sis with concomitant production and secretion of a PDGF-like growth factor, which plays a role in the induction of phenotypic transformation on normal rat kidney cells.

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Selected References

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  1. Anzano M. A., Roberts A. B., Meyers C. A., Komoriya A., Lamb L. C., Smith J. M., Sporn M. B. Synergistic interaction of two classes of transforming growth factors from murine sarcoma cells. Cancer Res. 1982 Nov;42(11):4776–4778. [PubMed] [Google Scholar]
  2. Anzano M. A., Roberts A. B., Smith J. M., Lamb L. C., Sporn M. B. Purification by reverse-phase high-performance liquid chromatography of an epidermal growth factor-dependent transforming growth factor. Anal Biochem. 1982 Sep 1;125(1):217–224. doi: 10.1016/0003-2697(82)90405-5. [DOI] [PubMed] [Google Scholar]
  3. Assoian R. K., Grotendorst G. R., Miller D. M., Sporn M. B. Cellular transformation by coordinated action of three peptide growth factors from human platelets. 1984 Jun 28-Jul 4Nature. 309(5971):804–806. doi: 10.1038/309804a0. [DOI] [PubMed] [Google Scholar]
  4. Assoian R. K., Komoriya A., Meyers C. A., Miller D. M., Sporn M. B. Transforming growth factor-beta in human platelets. Identification of a major storage site, purification, and characterization. J Biol Chem. 1983 Jun 10;258(11):7155–7160. [PubMed] [Google Scholar]
  5. Betsholtz C., Westermark B., Ek B., Heldin C. H. Coexpression of a PDGF-like growth factor and PDGF receptors in a human osteosarcoma cell line: implications for autocrine receptor activation. Cell. 1984 Dec;39(3 Pt 2):447–457. doi: 10.1016/0092-8674(84)90452-5. [DOI] [PubMed] [Google Scholar]
  6. Bowen-Pope D. F., Vogel A., Ross R. Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor receptors accompany transformation by a wide spectrum of agents. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2396–2400. doi: 10.1073/pnas.81.8.2396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chua C. C., Geiman D., Ladda R. L. Transforming growth factors released from Kirsten sarcoma virus transformed cells do not compete for epidermal growth factor membrane receptors. J Cell Physiol. 1983 Oct;117(1):116–122. doi: 10.1002/jcp.1041170116. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Dicker P., Pohjanpelto P., Pettican P., Rozengurt E. Similarities between fibroblast-derived growth factor and platelet-derived growth factor. Exp Cell Res. 1981 Sep;135(1):221–227. doi: 10.1016/0014-4827(81)90314-1. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Heldin C. H., Westermark B. Growth factors: mechanism of action and relation to oncogenes. Cell. 1984 May;37(1):9–20. doi: 10.1016/0092-8674(84)90296-4. [DOI] [PubMed] [Google Scholar]
  13. Heldin C. H., Westermark B., Wasteson A. Chemical and biological properties of a growth factor from human-cultured osteosarcoma cells: resemblance with platelet-derived growth factor. J Cell Physiol. 1980 Nov;105(2):235–246. doi: 10.1002/jcp.1041050207. [DOI] [PubMed] [Google Scholar]
  14. Heldin C. H., Westermark B., Wasteson A. Demonstration of an antibody against platelet-derived growth factor. Exp Cell Res. 1981 Dec;136(2):255–261. doi: 10.1016/0014-4827(81)90003-3. [DOI] [PubMed] [Google Scholar]
  15. Johnsson A., Betsholtz C., von der Helm K., Heldin C. H., Westermark B. Platelet-derived growth factor agonist activity of a secreted form of the v-sis oncogene product. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1721–1725. doi: 10.1073/pnas.82.6.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnsson A., Heldin C. H., Wasteson A., Westermark B., Deuel T. F., Huang J. S., Seeburg P. H., Gray A., Ullrich A., Scrace G. The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor. EMBO J. 1984 May;3(5):921–928. doi: 10.1002/j.1460-2075.1984.tb01908.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johnsson A., Heldin C. H., Westermark B., Wasteson A. Platelet-derived growth factor: identification of constituent polypeptide chains. Biochem Biophys Res Commun. 1982 Jan 15;104(1):66–74. doi: 10.1016/0006-291x(82)91941-6. [DOI] [PubMed] [Google Scholar]
  18. Kaplan P. L., Ozanne B. Cellular responsiveness to growth factors correlates with a cell's ability to express the transformed phenotype. Cell. 1983 Jul;33(3):931–938. doi: 10.1016/0092-8674(83)90036-3. [DOI] [PubMed] [Google Scholar]
  19. Kelly K., Cochran B. H., Stiles C. D., Leder P. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell. 1983 Dec;35(3 Pt 2):603–610. doi: 10.1016/0092-8674(83)90092-2. [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. Marquardt H., Hunkapiller M. W., Hood L. E., Todaro G. J. Rat transforming growth factor type 1: structure and relation to epidermal growth factor. Science. 1984 Mar 9;223(4640):1079–1082. doi: 10.1126/science.6320373. [DOI] [PubMed] [Google Scholar]
  22. Marquardt H., Todaro G. J. Human transforming growth factor. Production by a melanoma cell line, purification, and initial characterization. J Biol Chem. 1982 May 10;257(9):5220–5225. [PubMed] [Google Scholar]
  23. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nickell K. A., Halper J., Moses H. L. Transforming growth factors in solid human malignant neoplasms. Cancer Res. 1983 May;43(5):1966–1971. [PubMed] [Google Scholar]
  25. Niman H. L. Antisera to a synthetic peptide of the sis viral oncogene product recognize human platelet-derived growth factor. Nature. 1984 Jan 12;307(5947):180–183. doi: 10.1038/307180a0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Palmiter R. D. Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undergraded polysomes and messenger ribonucleic acid. Biochemistry. 1974 Aug 13;13(17):3606–3615. doi: 10.1021/bi00714a032. [DOI] [PubMed] [Google Scholar]
  29. Rapp U. R., Gunnell M., Marquardt H. Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar. J Cell Biochem. 1983;21(1):29–38. doi: 10.1002/jcb.240210105. [DOI] [PubMed] [Google Scholar]
  30. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  31. Rizzino A., Orme L. S., De Larco J. E. Embryonal carcinoma cell growth and differentiation. Production of and response to molecules with transforming growth factor activity. Exp Cell Res. 1983 Jan;143(1):143–152. doi: 10.1016/0014-4827(83)90116-7. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Roberts A. B., Anzano M. A., Lamb L. C., Smith J. M., Frolik C. A., Marquardt H., Todaro G. J., Sporn M. B. Isolation from murine sarcoma cells of novel transforming growth factors potentiated by EGF. Nature. 1982 Feb 4;295(5848):417–419. doi: 10.1038/295417a0. [DOI] [PubMed] [Google Scholar]
  34. Saris C. J., Franssen H. J., Heuyerjans J. H., van Eenbergen J., Bloemers H. P. Blotting of RNA onto ion exchange paper allowing subsequent characterization by in situ translation in addition to blot hybridization. Nucleic Acids Res. 1982 Aug 25;10(16):4831–4843. doi: 10.1093/nar/10.16.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schwab M., Ellison J., Busch M., Rosenau W., Varmus H. E., Bishop J. M. Enhanced expression of the human gene N-myc consequent to amplification of DNA may contribute to malignant progression of neuroblastoma. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4940–4944. doi: 10.1073/pnas.81.15.4940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Slamon D. J., Cline M. J. Expression of cellular oncogenes during embryonic and fetal development of the mouse. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7141–7145. doi: 10.1073/pnas.81.22.7141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stiles C. D. The molecular biology of platelet-derived growth factor. Cell. 1983 Jul;33(3):653–655. doi: 10.1016/0092-8674(83)90008-9. [DOI] [PubMed] [Google Scholar]
  38. Taparowsky E., Shimizu K., Goldfarb M., Wigler M. Structure and activation of the human N-ras gene. Cell. 1983 Sep;34(2):581–586. doi: 10.1016/0092-8674(83)90390-2. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Wang J. Y., Williams L. T. A v-sis oncogene protein produced in bacteria competes for platelet-derived growth factor binding to its receptor. J Biol Chem. 1984 Sep 10;259(17):10645–10648. [PubMed] [Google Scholar]
  41. 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]
  42. Westin E. H., Wong-Staal F., Gelmann E. P., Dalla-Favera R., Papas T. S., Lautenberger J. A., Eva A., Reddy E. P., Tronick S. R., Aaronson S. A. Expression of cellular homologues of retroviral onc genes in human hematopoietic cells. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2490–2494. doi: 10.1073/pnas.79.8.2490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  44. van Zoelen E. J., Twardzik D. R., van Oostwaard T. M., van der Saag P. T., de Laat S. W., Todaro G. J. Neuroblastoma cells produce transforming growth factors during exponential growth in a defined hormone-free medium. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4085–4089. doi: 10.1073/pnas.81.13.4085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. van Zoelen E. J., van Oostwaard T. M., van der Saag P. T., de Laat S. W. Phenotypic transformation of normal rat kidney cells in a growth-factor-defined medium: induction by a neuroblastoma-derived transforming growth factor independently of the EGF receptor. J Cell Physiol. 1985 May;123(2):151–160. doi: 10.1002/jcp.1041230202. [DOI] [PubMed] [Google Scholar]

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