Brooks G, Goss M W, East J A, Hart I R
Biology of Metastasis Laboratory, Imperial Cancer Research Fund, London, United Kingdom.
J Biol Chem. 1993 Nov 15;268(32):23868-75.
Protein kinase C (PKC) down-regulation has been shown to correlate with the growth of murine melanocytic cells in culture (Brooks, G., Wilson, R. E., Dooley, T. P., Goss, M. W., and Hart, I. R. (1991) Cancer Res. 51, 3281-3288). We now show that PKC alpha, delta, epsilon, and zeta isoforms are present at the protein level in quiescent, non-transformed Mel-ab melanocytes, maintained in the absence of phorbol ester. Proliferation of Mel-ab cells, achieved by incubation in the continual presence of phorbol 12,13-dibutyrate, was associated with a down-regulation of the PKC alpha, delta, and epsilon isozymes. Examination of two transformed syngeneic lines (the B16 murine melanoma and the long terminal repeat Ras.2 line), that grew in the absence of exogenous phorbol esters, showed that PKC alpha protein levels were either partially down-regulated or unaffected, the PKC delta and epsilon isoforms were down-regulated completely, and the levels of PKC zeta protein remained unaltered relative to quiescent Mel-ab cells. Basal levels of total diacylglycerol were elevated 5-fold in B16 melanoma cells compared with levels found in quiescent or proliferating Mel-ab melanocytes and appear to arise largely from the breakdown of phosphatidylinositol phospholipids accompanied by a significant rise in phospholipase C activity. Hourly treatments of quiescent Mel-ab melanocytes with the synthetic diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, for 24 h, resulted in an induction of DNA synthesis which was associated with a significant down-regulation of PKC levels mediated largely via post-translational rather than transcriptional mechanisms. These results show for the first time that specific isoforms of PKC are down-regulated at the protein level during proliferation of murine melanocytic cells and suggest that the constitutive down-regulation of PKC in transformed melanoma cells may arise as a consequence of elevated endogenous phosphatidylinositol-derived diacylglycerol levels.
蛋白激酶C（PKC）的下调已被证明与培养的小鼠黑素细胞的生长相关（布鲁克斯，G.，威尔逊，R.E.，杜利，T.P.，戈斯，M.W.，和哈特，I.R.（1991年）《癌症研究》51，3281 - 3288）。我们现在表明，在不存在佛波酯的情况下维持的静止、未转化的Mel-ab黑素细胞中，PKCα、δ、ε和ζ同工型以蛋白质水平存在。通过在持续存在佛波醇12,13 - 二丁酸酯的情况下孵育实现的Mel-ab细胞增殖与PKCα、δ和ε同工酶的下调相关。对两个在不存在外源性佛波酯的情况下生长的同基因转化系（B16小鼠黑色素瘤和长末端重复序列Ras.2系）的检测表明，PKCα蛋白水平要么部分下调，要么未受影响，PKCδ和ε同工型完全下调，并且相对于静止的Mel-ab细胞，PKCζ蛋白水平保持不变。与静止或增殖的Mel-ab黑素细胞中发现的水平相比，B16黑色素瘤细胞中总二酰基甘油的基础水平升高了5倍，并且似乎主要源于磷脂酰肌醇磷脂的分解，同时磷脂酶C活性显著升高。用合成二酰基甘油类似物1,2 - 二辛酰 - sn - 甘油对静止的Mel-ab黑素细胞进行每小时一次的处理，持续24小时，导致DNA合成的诱导，这与PKC水平的显著下调相关，这种下调主要通过翻译后而非转录机制介导。这些结果首次表明，在小鼠黑素细胞增殖过程中，PKC的特定同工型在蛋白质水平上被下调，并表明转化的黑色素瘤细胞中PKC的组成性下调可能是内源性磷脂酰肌醇衍生的二酰基甘油水平升高的结果。
