Esposito F, Cuccovillo F, Vanoni M, Cimino F, Anderson C W, Appella E, Russo T
Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Naples, Italy.
Eur J Biochem. 1997 May 1;245(3):730-7. doi: 10.1111/j.1432-1033.1997.00730.x.
p21(waf1/cip1) gene expression is induced by DNA damage in cells with wild-type p53 and contributes to the arrest of cell growth. It was demonstrated that under many experimental conditions, including oxidative stress, p21(waf1/cip1) expression can be induced through p53-independent pathways. Since most of these experimental conditions induce the phosphorylation of mitogen-activated protein kinase (MAPK) and thus its activation, we evaluated p21(waf1/cip1) mRNA levels in cells exposed to an oxidative stress, induced by diethylmaleate (Et2Mal), and in which the MAPK pathway was blocked. The expression of a dominant-negative mutant of MEK, the MAPK kinase that phosphorylates and activates MAPK, and of a dominant-negative [Asn17]Ras mutant prevented the Et2Mal-induced accumulation of p21(waf1/cip1) mRNA. Similarly, the expression of MEK- and of [Asn17]Ras mutants decreased the 12-O-tetradecanoyl-phorbol 13-acetate (TPA)-mediated p21(waf1/cip1) induction. Furthermore, TPA-induced and serum-induced p21(waf1/cip1) mRNA accumulation was blocked by pretreating the cells with the antioxidant compound N-acetylcysteine, suggesting that oxidative stress is involved in these responses. p21(waf1/cip1) mRNA levels reached a maximum within 2 h of adding Et2Mal or TPA; however, the rate of transcription from a p21(waf1/cip1)-promoter construct did not increase during this period. In contrast, cells treated with actinomycin D show an increase of p21(waf1/cip1) mRNA stability after Et2Mal treatment. This result suggests that the increase in p21(waf1/cip1) mRNA at early times results from post-transcriptional regulatory events. Longer exposure to TPA may activate p21(waf1/cip1) gene transcription through an Sp1-dependent mechanism, while Et2Mal treatment gradually inhibits p21(waf1/cip1) gene transcription through oxidative changes that affect Sp1 binding to DNA.
在具有野生型p53的细胞中，DNA损伤可诱导p21（waf1/cip1）基因表达，这有助于细胞生长停滞。研究表明，在包括氧化应激在内的许多实验条件下，p21（waf1/cip1）表达可通过不依赖p53的途径诱导产生。由于这些实验条件大多会诱导丝裂原活化蛋白激酶（MAPK）磷酸化从而使其激活，我们评估了暴露于由马来酸二乙酯（Et2Mal）诱导的氧化应激且MAPK途径被阻断的细胞中p21（waf1/cip1）mRNA水平。磷酸化并激活MAPK的MAPK激酶MEK的显性负性突变体以及显性负性[Asn17]Ras突变体的表达，可阻止Et2Mal诱导的p21（waf1/cip1）mRNA积累。同样，MEK和[Asn17]Ras突变体的表达降低了12-O-十四烷酰佛波醇-13-乙酸酯（TPA）介导的p21（waf1/cip1）诱导。此外，用抗氧化剂化合物N-乙酰半胱氨酸预处理细胞可阻断TPA诱导和血清诱导的p21（waf1/cip1）mRNA积累，这表明氧化应激参与了这些反应。添加Et2Mal或TPA后2小时内，p21（waf1/cip1）mRNA水平达到最大值；然而，在此期间，来自p21（waf1/cip1）启动子构建体的转录速率并未增加。相反，用放线菌素D处理的细胞在Et2Mal处理后显示p21（waf1/cip1）mRNA稳定性增加。这一结果表明，早期p21（waf1/cip1）mRNA的增加是转录后调控事件的结果。长时间暴露于TPA可能通过依赖Sp1的机制激活p21（waf1/cip1）基因转录，而Et2Mal处理则通过影响Sp1与DNA结合的氧化变化逐渐抑制p21（waf1/cip1）基因转录。
