PROTEIN-KINASE-C-DEPENDENT AND PROTEIN-KINASE-C-INDEPENDENT PATHWAYS OF SIGNAL-TRANSDUCTION IN PROSTATE-CANCER CELLS - FIBROBLAST GROWTH-FACTOR UTILIZATION OF A PROTEIN-KINASE-C-INDEPENDENT PATHWAY

To examine the possibility that differences in protein tyrosine phosph orylation contributed to differences in fibroblast growth factor (FGF) responsiveness of clonally derived C3 (modestly responsive) and T5 (h ighly responsive) rat prostate cancer cells, we evaluated the ability of orthovanadate to affect prostate cancer cell thymidine incorporatio n. These analyses showed that C3 cell FCF insensitivity was not attrib utable to enhanced protein phosphotyrosine phosphatase activity. Analy ses of acidic FGF (aFGF)-mediated protein phosphorylation showed mitog en-caused, time-dependent tyrosine phosphorylation of C3 and T5 cell F GF receptors (FGFRs) and other proteins having a mass of 190, 150, 120 , 100, 90, 80, 74, 60/62, 50, 42, or 28 kilodaltons. Although marked d ifferences characterized aFCF mediated intensity of tyrosine phosphory lation, the notable commonality of tyrosine phosphorylation and the ma ss of the phosphorylated proteins suggested that C3 and T5 cells may u se the ras and/or protein kinase C (PKC) pathways for FGF-mediated sig nal transduction. The PKC agonist 12-O-tetradecanpyl-phorbol-13-acetat e (TPA) caused concentration-dependent increases in T5 cell thymidine incorporation. In contrast, TPA did not enhance thymidine incorporatio n of C3 cells or mitogen-sensitive NRK cells included as a nonneoplast ic control. TPA also significantly enhanced T5 cell proliferation, whe reas identical treatment did not affect proliferation of either C3 or NRK cells. Either 12 or 24 h treatment with 200 or 2000 ng/ml TPA caus ed complete PKC alpha and partial PKC delta down-regulation in C3, T5, and NRK cells. Consequently, the failure of TPA to affect C3 or NRK c ell thymidine incorporation or proliferation was not attributable to p otential TPA ineffectiveness in these cells. Survey immunological anal yses showed that all three cell lines lacked PKC beta, PKC eta, and PK C theta. In contrast, T5 cells contained abundant amounts of PKC epsil on, whereas the PKC epsilon content of C3 and NRK cells was near the l imit of detection. TPA treatment of T5 cells evoked only partial PKC e psilon down-regulation. Both aFGF and basic FCF (bFGF) promoted concen tration-dependent enhancement of TPA-pretreated T5 cell thymidine inco rporation, and the effects of combined TPA and either aFCF or bFGF tre atment were additive. Neither aFGF nor bFGF was able to enhance thymid ine incorporation of TPA-pretreated C3 cells beyond the modest effects elicited by FGF treatment of C3 controls. Pretreatment of NRK cells w ith 200 ng/ml TPA did not attenuate the ability of FCFs to enhance NRK cell thymidine incorporation, whereas pretreatment of NRK cells with 2000 ng/ml TPA eliminated this ability of FCFs. Our findings imply tha t effector modulation of C3, T5, and NRK cell proliferation differs si gnificantly. FGF modulation of NRK cell proliferation appears to occur exclusively by a PKC-independent pathway. C3 cell proliferation is no t modulated by FCFR-mediated events effected by either PKC-dependent o r PKC-independent pathways. In marked contrast, T5 cell proliferation appears to be modulated by FGFR-mediated events effected by PKC-depend ent and PKC-independent pathways.