Ohnishi K, Ota I, Takahashi A, Yane K, Matsumoto H, Ohnishi T
Department of Biology, Nara Medical University, Kashihara, Nara 634-8521, Japan.
Apoptosis. 2002 Aug;7(4):367-72. doi: 10.1023/a:1016131614856.
The present study examined whether X-ray- and CDDP-sensitivities depend on p53 gene status in human squamous cell carcinoma of the head and neck (SAS cells) showing dominant negative nature of mutant p53 protein. SAS cells were transfected with a vector carrying a mutant p53 gene (SAS/Trp248 cells) or neomycin resistant gene control vector (SAS/neo cells). Sensitivities of the transfected cells to X-ray or CDDP were measured with colony formation assay. The incidence of apoptosis by X-ray or CDDP was analyzed with Hoechst staining or DNA ladder formation assay. The activation of caspase-3 was estimated as an indicator of apoptosis by the detection of fragmentation of caspase-3 or poly (ADP ribose) polymerase (PARP) with Western blot. SAS/Trp248 cells showed X-ray- and CDDP-resistance due to the dominant negative nature of mutant p53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly in SAS/neo cells after X-ray irradiation or CDDP treatment, but increased only slightly in SAS/Trp248 cells. Fragmentation of caspase-3 and PARP was observed in SAS/neo cells, but almost no such fragmentation was observed in SAS/Trp248 cells after X-ray irradiation or CDDP treatment. The present results strongly suggest that the X-ray- and CDDP-sensitivities of human squamous cell carcinomas are p53-dependent, and that the sensitivities are tightly correlated with the induction of apoptosis through caspase-3 activation. The p53-dependent X-ray- or CDDP-sensitivity was supported by results from p53-null human lung cancer H1299 cells which were transfected with wild-type or mutant p53 gene.
本研究检测了在显示突变型p53蛋白显性负性性质的人头部和颈部鳞状细胞癌（SAS细胞）中，X射线敏感性和顺铂（CDDP）敏感性是否依赖于p53基因状态。用携带突变型p53基因的载体（SAS/Trp248细胞）或新霉素抗性基因对照载体（SAS/neo细胞）转染SAS细胞。用集落形成试验测量转染细胞对X射线或顺铂的敏感性。用Hoechst染色或DNA梯状条带形成试验分析X射线或顺铂诱导的凋亡发生率。通过蛋白质免疫印迹法检测半胱天冬酶-3（caspase-3）或聚（ADP核糖）聚合酶（PARP）的裂解片段，以评估caspase-3的激活作为凋亡的指标。与SAS/neo细胞相比，由于突变型p53的显性负性性质，SAS/Trp248细胞表现出对X射线和顺铂的抗性。X射线照射或顺铂处理后，SAS/neo细胞中DNA梯状条带和凋亡小体的发生率显著增加，但在SAS/Trp248细胞中仅略有增加。X射线照射或顺铂处理后，在SAS/neo细胞中观察到caspase-3和PARP的裂解，但在SAS/Trp248细胞中几乎未观察到此类裂解。本研究结果有力地表明，人鳞状细胞癌对X射线和顺铂的敏感性是p53依赖性的，并且这些敏感性与通过caspase-3激活诱导的凋亡密切相关。p53基因敲除的人肺癌H1299细胞转染野生型或突变型p53基因的结果支持了p53依赖性X射线或顺铂敏感性。
