Akashi M, Osawa Y, Koeffler H P, Hachiya M
Division of Radiation Health, National Institute of Radiological Sciences, Chiba, 263-8555 Japan.
Biochem J. 1999 Feb 1;337 ( Pt 3)(Pt 3):607-16.
p21(WAF1) inhibits cyclin-cyclin-dependent kinase (Cdk) complexes, causing cell cycle arrest. p21(WAF1) contains p53-binding sites in its promoter and expression of p21(WAF1) is induced by functional p53. In the present work, we have studied the role of protein kinase C (PKC) in the induction of p21(WAF1) and show that induction of p21(WAF1) expression can occur by activation of PKC in cells having no p53. Human ovarian carcinoma cells, SKOV-3, lack p53 protein and PMA, a potent activator of PKC, did not induce p53. PMA increased the expression of p21(WAF1) mRNA both in these cells and in other cells which do not contain p53 (THP-1 and U937). Treatment of human embryonic fibroblasts, WI38, with PMA also induced the accumulation of p21(WAF1) without affecting p53 levels. However, PMA did not increase levels of p21(WAF1) mRNA in cells where either the PKC or the mitogen-activated protein kinase pathway was blocked. Furthermore, treatment of cells with various phorbol ester derivatives which activate PKC resulted in the induction of p21(WAF1) in SKOV-3 cells. In contrast, phorbol esters which do not activate PKC failed to induce p21(WAF1) expression. PMA increased the transcriptional rate of p21(WAF1) and activated the transcription of a luciferase reporter gene, controlled by the p21 promoter, in SKOV-3 cells with or without a p53 consensus-binding sequence. By contrast, PMA markedly stabilized p21(WAF1) mRNA; the half-life (t1/2) of p21(WAF1) in PMA-treated cells was >8 h compared with <1 h in untreated cells. These findings provide evidence that the PKC pathway induces expression of p21(WAF1) independently of p53. Our present study also suggests that the accumulation of p21(WAF1) transcripts by PMA occurs mainly at post-transcriptional level.
p21（WAF1）抑制细胞周期蛋白 - 细胞周期蛋白依赖性激酶（Cdk）复合物，导致细胞周期停滞。p21（WAF1）在其启动子中含有p53结合位点，并且p21（WAF1）的表达由功能性p53诱导。在本研究中，我们研究了蛋白激酶C（PKC）在p21（WAF1）诱导中的作用，并表明在没有p53的细胞中，PKC的激活可诱导p21（WAF1）表达。人卵巢癌细胞SKOV - 3缺乏p53蛋白，PKC的强效激活剂佛波酯（PMA）并未诱导p53。PMA在这些细胞以及其他不含p53的细胞（THP - 1和U937）中均增加了p21（WAF1）mRNA的表达。用PMA处理人胚胎成纤维细胞WI38也诱导了p21（WAF1）的积累，而不影响p53水平。然而，在PKC或丝裂原活化蛋白激酶途径被阻断的细胞中，PMA并未增加p21（WAF1）mRNA的水平。此外，用各种激活PKC的佛波酯衍生物处理细胞导致SKOV - 3细胞中p21（WAF1）的诱导。相反，不激活PKC的佛波酯未能诱导p21（WAF1）表达。PMA增加了p21（WAF1）的转录速率，并激活了由p21启动子控制的荧光素酶报告基因在有或没有p53共有结合序列的SKOV - 3细胞中的转录。相比之下，PMA显著稳定了p21（WAF1）mRNA；与未处理细胞中小于1小时相比，PMA处理细胞中p21（WAF1）的半衰期（t1/2）大于8小时。这些发现提供了证据表明PKC途径独立于p53诱导p21（WAF1）的表达。我们目前的研究还表明，PMA导致的p21（WAF1）转录本积累主要发生在转录后水平。
