Gomez-Manzano C, Fueyo J, Kyritsis A P, McDonnell T J, Steck P A, Levin V A, Yung W K
Department of Neuro-Oncology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
J Natl Cancer Inst. 1997 Jul 16;89(14):1036-44. doi: 10.1093/jnci/89.14.1036.
Alterations of the p53 (also called TP53) gene are one of the most common abnormalities in gliomas. We have previously reported that restoration of wild-type p53 protein function in glioma cells results in programmed cell death (apoptosis). Since p53 functions are mediated by genes that directly control the tumor suppressor effect of the p53 protein, understanding the relationship between p53 and p53-related genes in glioma cells will aid in the design of more rational treatment strategies for brain tumors.
We conducted this study to examine the timing of the p53-mediated events preceding apoptosis. More specifically, we undertook this work to characterize the genetic and cell cycle-related factors that may increase the resistance of glioma cells to p53-induced apoptosis.
Two human glioma cell lines (U-251 MG and U-373 MG) that express mutant p53 protein and two (U-87 MG and EFC-2) that express wild-type p53 protein were used. Replication-deficient adenovirus was utilized as an expression vector to transfer exogenous p53 and p21 complementary DNAs into the glioma cells; control cells were infected with the viral expression vector alone. To monitor gene transfer and the expression of exogenous genes (as well as the expression of endogenous genes), we used western blot analyses and immunohistochemistry analyses. Flow cytometry studies of cellular DNA content were performed to determine the cell cycle phenotype of the glioma cells before and after treatment.
p53-mediated apoptosis was preceded by elevation in the levels of the p21 (cell cycle-related) and Bax (apoptosis-related) proteins. In addition, cell cycle analyses showed that glioma cells were arrested in the G2 phase before undergoing cell death. Transfer of p21 induced a G2 block but did not induce apoptosis. Moreover, coexpression of p21 and p53 prevented glioma cells from undergoing apoptosis. Expression of exogenous p53 in wild-type p53 cells did not induce elevation of Bax levels, arrest in G2 phase, or apoptosis.
Our data confirmed the ability of wild-type p53 to induce apoptosis in p53 mutant glioma cells. In addition, our results document that p21 plays a role in protecting cells from p53-mediated programmed cell death and suggest that p53-mediated apoptosis and p21 induction may represent, at least in certain cases, opposite signals. Finally, our data suggest that over expression of p21 in gliomas may be related to resistance to treatments that induce apoptosis.
p53（也称为TP53）基因改变是胶质瘤中最常见的异常之一。我们之前报道过，在胶质瘤细胞中恢复野生型p53蛋白功能会导致程序性细胞死亡（凋亡）。由于p53功能是由直接控制p53蛋白肿瘤抑制作用的基因介导的，了解胶质瘤细胞中p53与p53相关基因之间的关系将有助于设计更合理的脑肿瘤治疗策略。
我们进行这项研究以检查p53介导的凋亡前事件的时间。更具体地说，我们开展这项工作以表征可能增加胶质瘤细胞对p53诱导凋亡抗性的遗传和细胞周期相关因素。
使用了两种表达突变型p53蛋白的人胶质瘤细胞系（U-251 MG和U-373 MG）以及两种表达野生型p53蛋白的细胞系（U-87 MG和EFC-2）。使用复制缺陷型腺病毒作为表达载体，将外源性p53和p21互补DNA转入胶质瘤细胞；对照细胞仅用病毒表达载体感染。为监测基因转移和外源性基因（以及内源性基因）的表达，我们使用了蛋白质印迹分析和免疫组织化学分析。进行细胞DNA含量的流式细胞术研究以确定治疗前后胶质瘤细胞的细胞周期表型。
p53介导的凋亡之前，p21（细胞周期相关）和Bax（凋亡相关）蛋白水平升高。此外，细胞周期分析表明，胶质瘤细胞在经历细胞死亡之前停滞在G2期。p21的转移诱导了G2期阻滞，但未诱导凋亡。此外，p21和p53的共表达阻止了胶质瘤细胞发生凋亡。在野生型p53细胞中外源性p5۳的表达未诱导Bax水平升高、G2期阻滞或凋亡。
我们的数据证实了野生型p53在p53突变型胶质瘤细胞中诱导凋亡的能力。此外，我们的结果表明p21在保护细胞免受p53介导的程序性细胞死亡中起作用，并表明p53介导的凋亡和p21诱导可能至少在某些情况下代表相反的信号。最后，我们的数据表明胶质瘤中p21的过表达可能与对诱导凋亡治疗的抗性有关。
