Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1995 Jan 1;181(1):9–19. doi: 10.1084/jem.181.1.9

Middle T antigen-transformed endothelial cells exhibit an increased activity of nitric oxide synthase

PMCID: PMC2191838  PMID: 7528781

Abstract

Endothelioma cell lines transformed by polyoma virus middle T antigen (mTa) cause cavernous hemangiomas in syngeneic mice by recruitment of host cells. The production of nitric oxide (NO), as measured by nitrite and citrulline production, was significantly higher in mTa-transformed endothelial cells in comparison with nontransformed control cells. The maximal activity of NO synthase (NOS) was about 200-fold higher in cell lysates from the tEnd.1 endothelioma cell line than in lysates from nontransformed controls, whereas the affinity for arginine did not differ. The biochemical characterization of NOS and the study of mRNA transcripts indicate that tEnd.1 cells express both the inducible and the constitutive isoforms. NOS hyperactivity is not a simple consequence of cell transformation but needs a tissue-specific mTa expression. Since tEnd.1-conditioned medium induces NOS activity in normal endothelial cells, most likely NOS hyperactivity in endothelioma cells is attributable to the release of a soluble factor. This NOS- activating factor, which seems to be an anionic protein, could stimulate tEnd.1 cells to express NOS by an autocrine way. By the same mechanism, tEnd.1 cells could induce NOS in the neighboring endothelial cells, and NO release could play a role in the hemangioma development. Such hypothesis is confirmed by our in vivo experiments, showing that the administration of the NOS inhibitor L-canavanine to endothelioma- bearing mice significantly reduced both the volume and the relapse time of the tumor.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Amber I. J., Hibbs J. B., Jr, Taintor R. R., Vavrin Z. The L-arginine dependent effector mechanism is induced in murine adenocarcinoma cells by culture supernatant from cytotoxic activated macrophages. J Leukoc Biol. 1988 Feb;43(2):187–192. doi: 10.1002/jlb.43.2.187. [DOI] [PubMed] [Google Scholar]
  2. Auger K. R., Carpenter C. L., Shoelson S. E., Piwnica-Worms H., Cantley L. C. Polyoma virus middle T antigen-pp60c-src complex associates with purified phosphatidylinositol 3-kinase in vitro. J Biol Chem. 1992 Mar 15;267(8):5408–5415. [PubMed] [Google Scholar]
  3. Bocchietto E., Guglielmetti A., Silvagno F., Taraboletti G., Pescarmona G. P., Mantovani A., Bussolino F. Proliferative and migratory responses of murine microvascular endothelial cells to granulocyte-colony-stimulating factor. J Cell Physiol. 1993 Apr;155(1):89–95. doi: 10.1002/jcp.1041550112. [DOI] [PubMed] [Google Scholar]
  4. Boyde T. R., Rahmatullah M. Optimization of conditions for the colorimetric determination of citrulline, using diacetyl monoxime. Anal Biochem. 1980 Sep 15;107(2):424–431. doi: 10.1016/0003-2697(80)90404-2. [DOI] [PubMed] [Google Scholar]
  5. Bredt D. S., Snyder S. H. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A. 1990 Jan;87(2):682–685. doi: 10.1073/pnas.87.2.682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bredt D. S., Snyder S. H. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci U S A. 1989 Nov;86(22):9030–9033. doi: 10.1073/pnas.86.22.9030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Busconi L., Michel T. Endothelial nitric oxide synthase. N-terminal myristoylation determines subcellular localization. J Biol Chem. 1993 Apr 25;268(12):8410–8413. [PubMed] [Google Scholar]
  8. Bussolino F., Camussi G., Baglioni C. Synthesis and release of platelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin 1 alpha. J Biol Chem. 1988 Aug 25;263(24):11856–11861. [PubMed] [Google Scholar]
  9. Bussolino F., De Rossi M., Sica A., Colotta F., Wang J. M., Bocchietto E., Padura I. M., Bosia A., DeJana E., Mantovani A. Murine endothelioma cell lines transformed by polyoma middle T oncogene as target for and producers of cytokines. J Immunol. 1991 Oct 1;147(7):2122–2129. [PubMed] [Google Scholar]
  10. Camussi G., Aglietta M., Malavasi F., Tetta C., Piacibello W., Sanavio F., Bussolino F. The release of platelet-activating factor from human endothelial cells in culture. J Immunol. 1983 Nov;131(5):2397–2403. [PubMed] [Google Scholar]
  11. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  12. Curran R. D., Billiar T. R., Stuehr D. J., Hofmann K., Simmons R. L. Hepatocytes produce nitrogen oxides from L-arginine in response to inflammatory products of Kupffer cells. J Exp Med. 1989 Nov 1;170(5):1769–1774. doi: 10.1084/jem.170.5.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Evans T., Carpenter A., Cohen J. Inducible nitric-oxide-synthase mRNA is transiently expressed and destroyed by a cycloheximide-sensitive process. Eur J Biochem. 1994 Jan 15;219(1-2):563–569. doi: 10.1111/j.1432-1033.1994.tb19972.x. [DOI] [PubMed] [Google Scholar]
  14. Furchgott R. F., Vanhoutte P. M. Endothelium-derived relaxing and contracting factors. FASEB J. 1989 Jul;3(9):2007–2018. [PubMed] [Google Scholar]
  15. Garg U. C., Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989 May;83(5):1774–1777. doi: 10.1172/JCI114081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Garlanda C., Parravicini C., Sironi M., De Rossi M., Wainstok de Calmanovici R., Carozzi F., Bussolino F., Colotta F., Mantovani A., Vecchi A. Progressive growth in immunodeficient mice and host cell recruitment by mouse endothelial cells transformed by polyoma middle-sized T antigen: implications for the pathogenesis of opportunistic vascular tumors. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7291–7295. doi: 10.1073/pnas.91.15.7291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Geller D. A., Lowenstein C. J., Shapiro R. A., Nussler A. K., Di Silvio M., Wang S. C., Nakayama D. K., Simmons R. L., Snyder S. H., Billiar T. R. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3491–3495. doi: 10.1073/pnas.90.8.3491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goureau O., Lepoivre M., Becquet F., Courtois Y. Differential regulation of inducible nitric oxide synthase by fibroblast growth factors and transforming growth factor beta in bovine retinal pigmented epithelial cells: inverse correlation with cellular proliferation. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4276–4280. doi: 10.1073/pnas.90.9.4276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gross S. S., Jaffe E. A., Levi R., Kilbourn R. G. Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem Biophys Res Commun. 1991 Aug 15;178(3):823–829. doi: 10.1016/0006-291x(91)90965-a. [DOI] [PubMed] [Google Scholar]
  20. Ignarro L. J. Nitric oxide. A novel signal transduction mechanism for transcellular communication. Hypertension. 1990 Nov;16(5):477–483. doi: 10.1161/01.hyp.16.5.477. [DOI] [PubMed] [Google Scholar]
  21. Isobe K., Nakashima I. Abundant production of nitric oxide from murine macrophages by direct stimulation of tumor cells. Biochem Biophys Res Commun. 1993 Apr 30;192(2):499–504. doi: 10.1006/bbrc.1993.1443. [DOI] [PubMed] [Google Scholar]
  22. Knowles R. G., Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994 Mar 1;298(Pt 2):249–258. doi: 10.1042/bj2980249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Knowles R. G., Palacios M., Palmer R. M., Moncada S. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5159–5162. doi: 10.1073/pnas.86.13.5159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Knowles R. G., Salter M., Brooks S. L., Moncada S. Anti-inflammatory glucocorticoids inhibit the induction by endotoxin of nitric oxide synthase in the lung, liver and aorta of the rat. Biochem Biophys Res Commun. 1990 Nov 15;172(3):1042–1048. doi: 10.1016/0006-291x(90)91551-3. [DOI] [PubMed] [Google Scholar]
  25. Lamas S., Marsden P. A., Li G. K., Tempst P., Michel T. Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6348–6352. doi: 10.1073/pnas.89.14.6348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leibovich S. J., Polverini P. J., Fong T. W., Harlow L. A., Koch A. E. Production of angiogenic activity by human monocytes requires an L-arginine/nitric oxide-synthase-dependent effector mechanism. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4190–4194. doi: 10.1073/pnas.91.10.4190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lejeune P., Lagadec P., Onier N., Pinard D., Ohshima H., Jeannin J. F. Nitric oxide involvement in tumor-induced immunosuppression. J Immunol. 1994 May 15;152(10):5077–5083. [PubMed] [Google Scholar]
  28. Marsden P. A., Schappert K. T., Chen H. S., Flowers M., Sundell C. L., Wilcox J. N., Lamas S., Michel T. Molecular cloning and characterization of human endothelial nitric oxide synthase. FEBS Lett. 1992 Aug 3;307(3):287–293. doi: 10.1016/0014-5793(92)80697-f. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992 Sep;6(12):3051–3064. [PubMed] [Google Scholar]
  31. Palmer R. M., Andrews T., Foxwell N. A., Moncada S. Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes. Biochem Biophys Res Commun. 1992 Oct 15;188(1):209–215. doi: 10.1016/0006-291x(92)92371-4. [DOI] [PubMed] [Google Scholar]
  32. Palmer R. M., Ashton D. S., Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988 Jun 16;333(6174):664–666. doi: 10.1038/333664a0. [DOI] [PubMed] [Google Scholar]
  33. Palmer R. M., Ferrige A. G., Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987 Jun 11;327(6122):524–526. doi: 10.1038/327524a0. [DOI] [PubMed] [Google Scholar]
  34. Palmer R. M., Moncada S. A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem Biophys Res Commun. 1989 Jan 16;158(1):348–352. doi: 10.1016/s0006-291x(89)80219-0. [DOI] [PubMed] [Google Scholar]
  35. Radomski M. W., Palmer R. M., Moncada S. Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. Br J Pharmacol. 1987 Sep;92(1):181–187. doi: 10.1111/j.1476-5381.1987.tb11310.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Radomski M. W., Palmer R. M., Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Natl Acad Sci U S A. 1990 Dec;87(24):10043–10047. doi: 10.1073/pnas.87.24.10043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stuehr D. J., Griffith O. W. Mammalian nitric oxide synthases. Adv Enzymol Relat Areas Mol Biol. 1992;65:287–346. doi: 10.1002/9780470123119.ch8. [DOI] [PubMed] [Google Scholar]
  38. Suschek C., Rothe H., Fehsel K., Enczmann J., Kolb-Bachofen V. Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells. IL-1 beta-mediated induction regulated by tumor necrosis factor-alpha and IFN-gamma. J Immunol. 1993 Sep 15;151(6):3283–3291. [PubMed] [Google Scholar]
  39. Taraboletti G., Belotti D., Dejana E., Mantovani A., Giavazzi R. Endothelial cell migration and invasiveness are induced by a soluble factor produced by murine endothelioma cells transformed by polyoma virus middle T oncogene. Cancer Res. 1993 Aug 15;53(16):3812–3816. [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Xie Q. W., Cho H. J., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Ding A., Troso T., Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science. 1992 Apr 10;256(5054):225–228. doi: 10.1126/science.1373522. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES