Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis (original) (raw)

Abstract

Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To determine the role of TS1 in regulation of endothelial cell phenotype, we stably transfected bEND.3 cells with a human TS1 expression vector. The cells expressing human TS1 were readily identified by their altered morphology and exhibited a slower growth rate and lower saturation density than the parental bEND.3 cells. The TS1-expressing cells also formed aligned cords of cells instead of clumps or cysts in Matrigel. Moreover, while the bEND.3 cells formed large tumors in nude mice within 48 hr, the TS1-expressing cells failed to form tumors even after 1 month. The TS1-transfected cells expressed transforming growth factor beta mRNA and bioactivity at levels similar to those of the parental or vector-transfected bEND.3 cells, indicating that the effects of TS1 expression are not due to the activation of transforming growth factor beta by TS1. TS1 expression resulted in a > 100-fold decrease in net fibrinolytic (urokinase-type plasminogen activator, uPA) activity due to more plasminogen-activator inhibitor 1 and less uPA secretion. TS1 thus appears to be an important regulator of endothelial cell phenotype required for maintaining the quiescent, differentiated state.

6788

Images in this article

Selected References

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

  1. Adams J., Lawler J. Extracellular matrix: the thrombospondin family. Curr Biol. 1993 Mar;3(3):188–190. doi: 10.1016/0960-9822(93)90270-x. [DOI] [PubMed] [Google Scholar]
  2. Bacharach E., Itin A., Keshet E. In vivo patterns of expression of urokinase and its inhibitor PAI-1 suggest a concerted role in regulating physiological angiogenesis. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10686–10690. doi: 10.1073/pnas.89.22.10686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bacharach E., Itin A., Keshet E. In vivo patterns of expression of urokinase and its inhibitor PAI-1 suggest a concerted role in regulating physiological angiogenesis. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10686–10690. doi: 10.1073/pnas.89.22.10686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bagavandoss P., Wilks J. W. Specific inhibition of endothelial cell proliferation by thrombospondin. Biochem Biophys Res Commun. 1990 Jul 31;170(2):867–872. doi: 10.1016/0006-291x(90)92171-u. [DOI] [PubMed] [Google Scholar]
  5. Bautch V. L., Toda S., Hassell J. A., Hanahan D. Endothelial cell tumors develop in transgenic mice carrying polyoma virus middle T oncogene. Cell. 1987 Nov 20;51(4):529–537. doi: 10.1016/0092-8674(87)90122-x. [DOI] [PubMed] [Google Scholar]
  6. Bornstein P. Thrombospondins: structure and regulation of expression. FASEB J. 1992 Nov;6(14):3290–3299. doi: 10.1096/fasebj.6.14.1426766. [DOI] [PubMed] [Google Scholar]
  7. Brooks J. J. Kaposi's sarcoma: a reversible hyperplasia. Lancet. 1986 Dec 6;2(8519):1309–1311. doi: 10.1016/s0140-6736(86)91436-4. [DOI] [PubMed] [Google Scholar]
  8. Canfield A. E., Schor A. M., Schor S. L., Grant M. E. The biosynthesis of extracellular-matrix components by bovine retinal endothelial cells displaying distinctive morphological phenotypes. Biochem J. 1986 Apr 15;235(2):375–383. doi: 10.1042/bj2350375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Corless C. L., Mendoza A., Collins T., Lawler J. Colocalization of thrombospondin and syndecan during murine development. Dev Dyn. 1992 Apr;193(4):346–358. doi: 10.1002/aja.1001930408. [DOI] [PubMed] [Google Scholar]
  10. Dubois N. A., Kolpack L. C., Wang R., Azizkhan R. G., Bautch V. L. Isolation and characterization of an established endothelial cell line from transgenic mouse hemangiomas. Exp Cell Res. 1991 Oct;196(2):302–313. doi: 10.1016/0014-4827(91)90265-v. [DOI] [PubMed] [Google Scholar]
  11. Ensoli B., Barillari G., Gallo R. C. Pathogenesis of AIDS-associated Kaposi's sarcoma. Hematol Oncol Clin North Am. 1991 Apr;5(2):281–295. [PubMed] [Google Scholar]
  12. Frazier W. A. Thrombospondins. Curr Opin Cell Biol. 1991 Oct;3(5):792–799. doi: 10.1016/0955-0674(91)90052-z. [DOI] [PubMed] [Google Scholar]
  13. Good D. J., Polverini P. J., Rastinejad F., Le Beau M. M., Lemons R. S., Frazier W. A., Bouck N. P. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6624–6628. doi: 10.1073/pnas.87.17.6624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hennessy S. W., Frazier B. A., Kim D. D., Deckwerth T. L., Baumgartel D. M., Rotwein P., Frazier W. A. Complete thrombospondin mRNA sequence includes potential regulatory sites in the 3' untranslated region. J Cell Biol. 1989 Feb;108(2):729–736. doi: 10.1083/jcb.108.2.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Iruela-Arispe M. L., Bornstein P., Sage H. Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):5026–5030. doi: 10.1073/pnas.88.11.5026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Iruela-Arispe M. L., Liska D. J., Sage E. H., Bornstein P. Differential expression of thrombospondin 1, 2, and 3 during murine development. Dev Dyn. 1993 May;197(1):40–56. doi: 10.1002/aja.1001970105. [DOI] [PubMed] [Google Scholar]
  17. Kubota Y., Kleinman H. K., Martin G. R., Lawley T. J. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol. 1988 Oct;107(4):1589–1598. doi: 10.1083/jcb.107.4.1589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liotta L. A., Steeg P. S., Stetler-Stevenson W. G. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell. 1991 Jan 25;64(2):327–336. doi: 10.1016/0092-8674(91)90642-c. [DOI] [PubMed] [Google Scholar]
  19. Madri J. A., Pratt B. M. Endothelial cell-matrix interactions: in vitro models of angiogenesis. J Histochem Cytochem. 1986 Jan;34(1):85–91. doi: 10.1177/34.1.2416801. [DOI] [PubMed] [Google Scholar]
  20. Montesano R., Mouron P., Orci L. Vascular outgrowths from tissue explants embedded in fibrin or collagen gels: a simple in vitro model of angiogenesis. Cell Biol Int Rep. 1985 Oct;9(10):869–875. doi: 10.1016/s0309-1651(85)90107-9. [DOI] [PubMed] [Google Scholar]
  21. Montesano R., Pepper M. S., Möhle-Steinlein U., Risau W., Wagner E. F., Orci L. Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene. Cell. 1990 Aug 10;62(3):435–445. doi: 10.1016/0092-8674(90)90009-4. [DOI] [PubMed] [Google Scholar]
  22. Montesano R., Vassalli J. D., Baird A., Guillemin R., Orci L. Basic fibroblast growth factor induces angiogenesis in vitro. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7297–7301. doi: 10.1073/pnas.83.19.7297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Murphy-Ullrich J. E., Schultz-Cherry S., Hök M. Transforming growth factor-beta complexes with thrombospondin. Mol Biol Cell. 1992 Feb;3(2):181–188. doi: 10.1091/mbc.3.2.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. O'Shea K. S., Liu L. H., Kinnunen L. H., Dixit V. M. Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo. J Cell Biol. 1990 Dec;111(6 Pt 1):2713–2723. doi: 10.1083/jcb.111.6.2713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. RayChaudhury A., Frazier W. A., D'Amore P. A. Comparison of normal and tumorigenic endothelial cells: differences in thrombospondin production and responses to transforming growth factor-beta. J Cell Sci. 1994 Jan;107(Pt 1):39–46. doi: 10.1242/jcs.107.1.39. [DOI] [PubMed] [Google Scholar]
  26. Schultz-Cherry S., Murphy-Ullrich J. E. Thrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism. J Cell Biol. 1993 Aug;122(4):923–932. doi: 10.1083/jcb.122.4.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sheibani N., Rhim J. S., Allen-Hoffmann B. L. Malignant human papillomavirus type 16-transformed human keratinocytes exhibit altered expression of extracellular matrix glycoproteins. Cancer Res. 1991 Nov 1;51(21):5967–5975. [PubMed] [Google Scholar]
  28. Taraboletti G., Roberts D., Liotta L. A., Giavazzi R. Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J Cell Biol. 1990 Aug;111(2):765–772. doi: 10.1083/jcb.111.2.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tolsma S. S., Volpert O. V., Good D. J., Frazier W. A., Polverini P. J., Bouck N. Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J Cell Biol. 1993 Jul;122(2):497–511. doi: 10.1083/jcb.122.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weinstat-Saslow D. L., Zabrenetzky V. S., VanHoutte K., Frazier W. A., Roberts D. D., Steeg P. S. Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Cancer Res. 1994 Dec 15;54(24):6504–6511. [PubMed] [Google Scholar]
  31. Williams R. L., Courtneidge S. A., Wagner E. F. Embryonic lethalities and endothelial tumors in chimeric mice expressing polyoma virus middle T oncogene. Cell. 1988 Jan 15;52(1):121–131. doi: 10.1016/0092-8674(88)90536-3. [DOI] [PubMed] [Google Scholar]
  32. Williams R. L., Risau W., Zerwes H. G., Drexler H., Aguzzi A., Wagner E. F. Endothelioma cells expressing the polyoma middle T oncogene induce hemangiomas by host cell recruitment. Cell. 1989 Jun 16;57(6):1053–1063. doi: 10.1016/0092-8674(89)90343-7. [DOI] [PubMed] [Google Scholar]