INVOLVEMENT OF A TRANSFORMING-GROWTH-FACTOR-BETA-LIKE MOLECULE IN TUMOR-CELL-DERIVED INHIBITION OF NITRIC-OXIDE SYNTHESIS IN CEREBRAL ENDOTHELIAL-CELLS

Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor c ells. We have reported that EC219 cells, a rat-brain-microvessel-deriv ed endothelial cell line, produced NO through cytokine-inducible NO sy nthase (iNOS), the induction of which was significantly decreased by ( a) soluble factor(s) secreted by DHD/PROb, an invasive sub-clone of a rat colon-carcinoma cell line. In this study, the DHD/PROb cell-derive d NO-inhibitory factor was characterized. Northern-blot analysis demon strated that the induction of iNOS mRNA in cytokine-activated EC219 ce lls was decreased by PROb-cell-conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A-Se pharose or heparin-Sepharose, the NO-inhibitory activity was found onl y in Con A-unbound or heparin-unbound fractions, respectively, indicat ing that the PROb-derived inhibitory factor was likely to be a nonglyc osylated and non-heparin-binding molecule. Pre-incubation of DHD/PROb- cell supernatant with anti-TGF-beta neutralizing antibody completely b locked the DHD/PROb-derived inhibition of NO production by EC219 cells . Addition of exogenous TGF-beta(1) dose-dependently inhibited NO rele ase by EC219 cells. The presence of active TGF-beta in the DHD/PROb ce ll supernatant was demonstrated using a growth-inhibition assay. Moreo ver, heat treatment of medium conditioned by the less invasive DHD/REG b cells, which constitutively secreted very low levels of active TGF-b eta, increased both TGF-beta activity and the ability to inhibit NO pr oduction in EC219 cells. Thus, DHD/PROb colon-carcinoma cells inhibite d NO production in EC219 cells by secreting a factor identical or very similar to TGF-beta. (C) 1995 Wiley-Liss, Inc.