Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems.
Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems.
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发表时间：
1996-02
期刊：
影响因子：
11.2
通讯作者：
P. Perego;M. Giarola;S. Righetti;R. Supino;C. Caserini;D. Delia;M. Pierotti;T. Miyashita;John Calvin
中科院分区：
文献类型：
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作者：
P. Perego;M. Giarola;S. Righetti;R. Supino;C. Caserini;D. Delia;M. Pierotti;T. Miyashita;John Calvin
P53状态可能是肿瘤细胞化疗敏感性的决定因素，然而，其参与细胞对顺铂的耐药性仍然不确定。为了研究p53和顺铂耐药的发展之间的关系，在卵巢癌细胞系统中研究了p53基因的状态，该系统包括两种顺铂耐药变异体（IGF 1 -1/Pt 0.5和IGF 1 -1/Pt 1），其是在顺铂敏感的亲本IGF 1 -1细胞系长时间药物暴露后在体外选择的。在暴露于药物96小时后，IGF 1 -1/Pt 0.5和IGF 1 -1/Pt 1细胞系分别表现出约6和14的耐药性，并且对其他DNA损伤剂（电离辐射和美法仑）具有交叉耐药性。对顺铂的耐药性降低了细胞对顺铂诱导的凋亡的敏感性。外显子5-9的DNA单链构象多态性分析表明，在两个耐药细胞系的外显子8处存在两个突变体等位基因，而亲本IGF 1 -1细胞系表现出野生型p53基因。直接DNA测序显示，这些突变由DNA结合结构域密码子270（T/A）和282（C/T）处的两个核苷酸变化组成。IGF 1/Pt细胞p53蛋白表达水平明显低于IGF 1/Pt细胞。暴露于电离辐射或顺铂后，p53蛋白的积累仅在敏感细胞中显著增强。与此同时，WAF-1蛋白的表达强烈诱导的亲本IGF 1 -1细胞，而WAF-1蛋白仍然检测不到的IGF 1 -1/Pt 1亚系DNA损伤处理后。与这一发现相一致的是，观察到电离辐射在敏感细胞和抗性细胞中引起不同模式的细胞周期扰动。北方印迹分析表明，bax mRNA水平在IGF 1/Pt 1顺铂耐药细胞中显著降低。野生型或突变型p53表达质粒和报告基因质粒，利用bax基因启动子驱动氯霉素乙酰转移酶的转录的共转染试验与p53在这些细胞中调节bax表达的作用一致。两者合计，这些观察结果支持的作用，突变的p53基因在卵巢癌顺铂耐药的发展，作为一个结果的p53的能力丧失的反式激活bax，一个凋亡诱导基因。
P53 status may be a determinant of chemosensitivity of tumor cells; however, its involvement in cellular resistance to cisplatin remains uncertain. To investigate the relationships between p53 and the development of resistance to cisplatin, the p53 gene status was studied in ovarian carcinoma cell systems which included two cisplatin-resistant variants (IGROV-1/Pt 0.5 and IGROV-1/Pt 1) selected in vitro after prolonged drug exposure of the cisplatin-sensitive parental IGROV-1 cell line. IGROV-1/Pt 0.5 and IGROV-1/Pt 1 cell lines exhibited a degree of resistance of approximately 6 and 14, respectively, following 96-h exposure to the drug and were cross-resistant to other DNA-damaging agents (ionizing radiation and melphalan). Resistance to cisplatin paralleled a reduced cell susceptibility to cisplatin-induced apoptosis. DNA single-strand conformation polymorphism analysis of exons 5-9 demonstrated the presence of two mutants alleles at exon 8 in the two resistant cell lines, in contrast to the parental IGROV-1 cell line which exhibited the wild-type p53 gene. Direct DNA sequencing revealed that the mutations consist of two nucleotide changes in the DNA-binding domain at codons 270 (T/A) and 282 (C/T). The consecutive levels of p53 protein were lower in IGROV-1 than in IGROV-1/Pt cells. Following exposure to ionizing radiation or cisplatin, accumulation of the p53 protein was markedly enhanced only in the sensitive cells. Concomitantly, the expression of WAF-1 protein was strongly induced in the parental IGROV-1 cells, whereas WAF-1 protein remained undetectable in the IGROV-1/Pt 1 subline after DNA-damaging treatment. Consistent with this finding is the observation that ionizing radiation caused a different pattern of cell cycle perturbation in sensitive and resistant cells. Northern blot analysis demonstrated a marked reduction in bax mRNA levels in IGROV-1/Pt 1 cisplatin-resistant cells. Cotransfection assays with wild-type or mutant p53 expression plasmids and a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase were consistent with the role of p53 in regulation of bax expression in these cells. Taken together, these observations support a role for mutations of the p53 gene in the development of cisplatin resistance in ovarian cancer as a consequence of loss of the ability of p53 to transactivate bax, an apoptosis-inducing gene.