Spitkovsky D, Schulze A, Boye B, Jansen-Dürr P
Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Heidelberg, Germany.
Cell Growth Differ. 1997 Jun;8(6):699-710.
Treatment of mammalian cells by DNA-damaging agents leads to various cellular responses. At sufficiently high dosage, cisplatin blocks cell proliferation and finally kills cells; this effect is the basis for its widespread use as an anticancer drug. Cisplatin-treated cells arrest in the G1 phase of the cell cycle, most likely due to a signal generated by the stabilization of p53 and the subsequent induction of p21WAF-1/Cip1. We show here that cisplatin-treated mammalian cells accumulate normal levels of cyclin D1 and cyclin E but fail to produce cyclin A. The block to cyclin A gene expression occurs at the level of transcription and is mediated by an E2F binding site in the cyclin A promoter. It is shown here that, upon cisplatin treatment, transcriptionally active free E2F becomes limiting, coincident with the accumulation of hypophosphorylated species of the retinoblastoma protein family. Immunoprecipitation experiments suggest that the loss of free E2F results, at least in part, from the sequestration of E2F-4/DP-1 heterodimers by p107. A role for the kinase inhibitor p21WAF-1/Cip1 in repression of the cyclin A promoter is supported by our finding that ectopic expression of p21WAF-1/Cip1 is sufficient to inhibit transcription from the cyclin A gene, dependent on the E2F site. The data establish the E2F site in the human cyclin A promoter as a key target for the signaling pathway leading to G1 arrest in response to DNA damage by cisplatin and potentially other genotoxic agents.
用DNA损伤剂处理哺乳动物细胞会引发多种细胞反应。在足够高的剂量下，顺铂会阻断细胞增殖并最终杀死细胞；这种效应是其作为抗癌药物广泛应用的基础。顺铂处理的细胞停滞在细胞周期的G1期，最有可能是由于p53稳定化产生的信号以及随后p21WAF-1/Cip1的诱导。我们在此表明，顺铂处理的哺乳动物细胞积累了正常水平的细胞周期蛋白D1和细胞周期蛋白E，但未能产生细胞周期蛋白A。细胞周期蛋白A基因表达的阻断发生在转录水平，并且由细胞周期蛋白A启动子中的E2F结合位点介导。在此表明，在顺铂处理后，转录活性的游离E2F变得受限，这与视网膜母细胞瘤蛋白家族低磷酸化形式的积累同时发生。免疫沉淀实验表明，游离E2F的丧失至少部分是由于p107对E2F-4/DP-1异二聚体的隔离。我们的发现支持了激酶抑制剂p21WAF-1/Cip1在抑制细胞周期蛋白A启动子中的作用，即异位表达p21WAF-1/Cip1足以抑制细胞周期蛋白A基因依赖于E2F位点的转录。这些数据确定了人细胞周期蛋白A启动子中的E2F位点是导致顺铂及可能其他基因毒性剂引起的DNA损伤后G1期停滞的信号通路的关键靶点。
