Cistulli C A, Kaufmann W K
Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599-7295, USA.
Cancer Res. 1998 May 1;58(9):1993-2002.
The cyclin-dependent kinase inhibitor p21(WAF1/CIP1/SDI1/CAP20) exists in normal human fibroblasts in a quaternary complex with a cyclin, a cyclin-dependent kinase, and proliferating cell nuclear antigen. A model was proposed in which, during p53-mediated suppression of cell proliferation following treatment with 254 nm UV radiation (UVC), the enhanced expression of p21 might inhibit DNA replication by virtue of its interactions with proliferating cell nuclear antigen. To test this model, we examined the mechanisms of inhibition of DNA replication in diploid human fibroblasts that express human papillomavirus type 16 E6, which inactivates p53. E6-expressing cells were defective in G1 checkpoint responses of induction of p21 and G1 arrest after ionizing radiation-induced damage to DNA. Accordingly, E6-expressing cells were resistant to inactivation of single-cell colony formation by ionizing radiation. E6 cells also displayed normal S-phase checkpoint responses of inhibition and recovery of replicon initiation following exposure to ionizing radiation and normal ability to bypass pyrimidine dimers during DNA replication soon after UVC irradiation (i.e., postreplication repair). However, DNA replication 6 h after UVC exposure was significantly inhibited in E6 cells in comparison to isogenic controls. This failure to maintain DNA replication in S-phase cells was associated with enhanced sensitivity to inactivation of single-cell colony formation by UVC. These results indicate that the p53-induced p21 pathway is not involved in the immediate S-phase responses to radiation-induced DNA damage of inhibition of replicon initiation and translesion bypass. However, our results demonstrate that p53 and, conceivably, p21 contribute to the ability of normal human fibroblasts to sustain DNA replication activity and form colonies following UVC irradiation.
细胞周期蛋白依赖性激酶抑制剂p21（WAF1/CIP1/SDI1/CAP20）在正常人成纤维细胞中以四聚体复合物的形式存在，该复合物包含一种细胞周期蛋白、一种细胞周期蛋白依赖性激酶和增殖细胞核抗原。有人提出了一个模型，即在254nm紫外线辐射（UVC）处理后p53介导的细胞增殖抑制过程中，p21表达增强可能通过其与增殖细胞核抗原的相互作用抑制DNA复制。为了验证该模型，我们研究了表达人乳头瘤病毒16型E6（可使p53失活）的二倍体人成纤维细胞中DNA复制抑制的机制。表达E6的细胞在电离辐射诱导DNA损伤后，p21诱导的G1期检查点反应和G1期停滞存在缺陷。因此，表达E6的细胞对电离辐射导致的单细胞集落形成失活具有抗性。E6细胞在暴露于电离辐射后，也表现出正常的S期检查点反应，即复制子起始的抑制和恢复，并且在UVC照射后不久的DNA复制过程中具有绕过嘧啶二聚体的正常能力（即复制后修复）。然而，与同基因对照相比，UVC暴露6小时后E6细胞中的DNA复制受到显著抑制。S期细胞中无法维持DNA复制与对UVC导致的单细胞集落形成失活的敏感性增强有关。这些结果表明，p53诱导的p21途径不参与对辐射诱导的DNA损伤的即时S期反应，即复制子起始抑制和跨损伤修复。然而，我们的结果表明，p53以及可以想象的p21有助于正常人成纤维细胞在UVC照射后维持DNA复制活性和形成集落的能力。
