Hrzenjak M, Shain S A
Department of Obstetrics and Gynecology, University of Texas Health Science Center at San Antonio 78284-7836, USA.
Cell Growth Differ. 1995 Sep;6(9):1129-42.
To examine the possibility that differences in protein tyrosine phosphorylation contributed to differences in fibroblast growth factor (FGF) responsiveness of clonally derived C3 (modestly responsive) and T5 (highly responsive) rat prostate cancer cells, we evaluated the ability of orthovanadate to affect prostate cancer cell thymidine incorporation. These analyses showed that C3 cell FGF insensitivity was not attributable to enhanced protein phosphotyrosine phosphatase activity. Analyses of acidic FGF (aFGF)-mediated protein phosphorylation showed mitogen-caused, time-dependent tyrosine phosphorylation of C3 and T5 cell FGF 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 differences characterized aFGF mediated intensity of tyrosine phosphorylation, the notable commonality of tyrosine phosphorylation and the mass of the phosphorylated proteins suggested that C3 and T5 cells may use the ras and/or protein kinase C (PKC) pathways for FGF-mediated signal transduction. The PKC agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA) caused concentration-dependent increases in T5 cell thymidine incorporation. In contrast, TPA did not enhance thymidine incorporation of C3 cells or mitogen-sensitive NRK cells included as a nonneoplastic control. TPA also significantly enhanced T5 cell proliferation, whereas 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 caused 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 cell thymidine incorporation or proliferation was not attributable to potential TPA ineffectiveness in these cells. Survey immunological analyses showed that all three cell lines lacked PKC beta, PKC eta, and PKC theta. In contrast, T5 cells contained abundant amounts of PKC epsilon, whereas the PKC epsilon content of C3 and NRK cells was near the limit of detection. TPA treatment of T5 cells evoked only partial PKC epsilon down-regulation. Both aFGF and basic FGF (bFGF) promoted concentration-dependent enhancement of TPA-pretreated T5 cell thymidine incorporation, and the effects of combined TPA and either aFGF or bFGF treatment were additive. Neither aFGF nor bFGF was able to enhance thymidine incorporation of TPA-pretreated C3 cells beyond the modest effects elicited by FGF treatment of C3 controls.(ABSTRACT TRUNCATED AT 250 WORDS)
为了研究蛋白质酪氨酸磷酸化差异是否导致克隆衍生的C3（反应适度）和T5（反应高度敏感）大鼠前列腺癌细胞对成纤维细胞生长因子（FGF）反应性的差异，我们评估了原钒酸盐影响前列腺癌细胞胸苷掺入的能力。这些分析表明，C3细胞对FGF不敏感并非归因于蛋白磷酸酪氨酸磷酸酶活性增强。对酸性FGF（aFGF）介导的蛋白质磷酸化分析显示，有丝分裂原引起C3和T5细胞FGF受体（FGFRs）以及其他分子量为190、150、120、100、90、80、74、60/62、50、42或28千道尔顿的蛋白质发生时间依赖性酪氨酸磷酸化。尽管aFGF介导的酪氨酸磷酸化强度存在显著差异，但酪氨酸磷酸化以及磷酸化蛋白质分子量的显著共性表明，C3和T5细胞可能利用ras和/或蛋白激酶C（PKC）途径进行FGF介导的信号转导。PKC激动剂12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）使T5细胞胸苷掺入呈浓度依赖性增加。相比之下，TPA并未增强C3细胞或作为非肿瘤对照的有丝分裂原敏感NRK细胞的胸苷掺入。TPA还显著增强了T5细胞增殖，而相同处理对C3或NRK细胞的增殖均无影响。用200或2000 ng/ml TPA处理12或24小时可导致C3、T5和NRK细胞中PKCα完全下调以及PKCδ部分下调。因此，TPA未能影响C3或NRK细胞胸苷掺入或增殖并非归因于TPA在这些细胞中可能无效。调查性免疫分析表明，所有三种细胞系均缺乏PKCβ、PKCη和PKCθ。相比之下，T5细胞含有大量PKCε，而C3和NRK细胞的PKCε含量接近检测极限。TPA处理T5细胞仅引起PKCε部分下调。aFGF和碱性FGF（bFGF）均促进TPA预处理的T5细胞胸苷掺入呈浓度依赖性增强，TPA与aFGF或bFGF联合处理的效果具有加和性。aFGF和bFGF均无法使TPA预处理的C3细胞胸苷掺入增强超过FGF处理C3对照所引发的适度效果。（摘要截短于250字）
