Fajac A, Da Silva J, Ahomadegbe J C, Rateau J G, Bernaudin J F, Riou G, Bénard J
Laboratory of Clinical and Molecular Pharmacology, Institut G. Roussy, Villejuif, France.
Int J Cancer. 1996 Sep 27;68(1):67-74. doi: 10.1002/(SICI)1097-0215(19960927)68:1<67::AID-IJC13>3.0.CO;2-3.
Cisplatin-induced apoptosis and p53 gene status were analyzed in human ovarian carcinoma using a parental IGR-OV1 line and a derived cisplatin-resistant IGR-OV1/DDP subline. Compared with parental cells, cisplatin-resistant cells exhibited a 5-fold higher resistance index and a 2-fold longer doubling time. Cisplatin induced apoptosis in both cell lines, as assessed by cell morphology and the presence of a DNA ladder. However, high concentrations were necessary to induce apoptosis in resistant cells. These cells elicited a 5-fold decrease in the number of platinum atoms bound per nucleotide. IGR-OV1/DDP cells also exhibited enhanced drug efflux and a higher glutathione content. Our data suggest that the levels of cisplatin-DNA lesions are critical for drug sensitivity and apoptosis induction in this in vitro ovarian carcinoma model. Comparative analysis of the p53 gene in sensitive and resistant cells revealed the presence of the same heterozygous mutation in exon 5. A 2-fold increase in p53 mRNA and protein amounts was observed in resistant cells as assessed by Northern and Western blots, respectively. Immunocytochemical staining revealed a higher percentage of p53 stained nuclei in resistant cells. RT-PCR analysis of p53 transcripts showed that both wild-type and mutated alleles were transcribed in sensitive as well as in resistant cells. However, mutated transcripts were 1.5-fold more abundant than wild-type transcripts in sensitive cells, whereas they were 2-fold higher in resistant cells. In addition, mdm-2 protein was over-expressed in resistant cells. Our results address the question of the functionality of p53 protein and its possible role in apoptosis induction in this model. In resistant cells, p53 protein might be inactivated by 2 mechanisms: mutation and complexation with mdm-2 protein. Therefore, the presence of non-functional p53 in resistant cells might be involved in the relative failure of cisplatin-induced apoptosis in these cells.
利用亲本IGR-OV1细胞系和衍生的顺铂耐药IGR-OV1/DDP亚细胞系，对人卵巢癌中顺铂诱导的细胞凋亡和p53基因状态进行了分析。与亲本细胞相比，顺铂耐药细胞的耐药指数高5倍，倍增时间长2倍。通过细胞形态和DNA梯带的存在评估，顺铂在两种细胞系中均诱导细胞凋亡。然而，需要高浓度才能诱导耐药细胞凋亡。这些细胞使每个核苷酸结合的铂原子数量减少了5倍。IGR-OV1/DDP细胞还表现出药物外排增强和谷胱甘肽含量更高。我们的数据表明，在这个体外卵巢癌模型中，顺铂-DNA损伤的水平对于药物敏感性和凋亡诱导至关重要。对敏感细胞和耐药细胞中p53基因的比较分析显示，外显子5存在相同的杂合突变。通过Northern印迹和Western印迹评估，在耐药细胞中观察到p53 mRNA和蛋白量增加了2倍。免疫细胞化学染色显示，耐药细胞中p53染色细胞核的百分比更高。p53转录本的RT-PCR分析表明，野生型和突变型等位基因在敏感细胞和耐药细胞中均有转录。然而，在敏感细胞中，突变转录本比野生型转录本丰富1.5倍，而在耐药细胞中则高2倍。此外，mdm-2蛋白在耐药细胞中过表达。我们的结果解决了p53蛋白的功能及其在该模型中凋亡诱导可能作用的问题。在耐药细胞中，p53蛋白可能通过两种机制失活：突变和与mdm-2蛋白结合。因此，耐药细胞中无功能p53的存在可能与这些细胞中顺铂诱导凋亡的相对失败有关。
