Concin Nicole, Stimpfl Margit, Zeillinger Christa, Wolff Uwe, Hefler Lukas, Sedlak Jan, Leodolter Sepp, Zeillinger Robert
Department of Obstetrics and Gynaecology, Molecular Oncology Group, University of Vienna Medical School, Vienna, Austria.
Int J Oncol. 2003 Jan;22(1):51-7.
We investigated the cell cycle and apoptotic response to irradiation in 4 human ovarian carcinoma cell lines, i.e., PA-1, Caov-3, SK-OV-3, and ES-2. Cell lines were also analysed for their p53 and Bax expression to address the relationship with cell cycle and apoptotic response. Apoptosis was examined by flow cytometric measurement of annexin V binding and by determination of cytoplasmic histone-associated DNA fragments with a photometric enzyme immunoassay. Cell cycle analyses were performed on the basis of flow cytometry. p53 and Bax protein expression was examined by immunocytochemistry in untreated cells and after irradiation. p53 cDNA sequencing and a functional yeast-based assay (FASAY) were performed to determine the p53 mutational status. All cell lines exhibited a dose-dependent G2/M arrest. No arrest in G1 was seen. A strong correlation was found between the G2/M arrest and the induction of apoptosis. PA-1, the only cell line found to express wild-type p53, showed the highest susceptibility to accumulate in G2/M and the strongest apoptotic response after irradiation. In this cell line irradiation resulted in an unequivocal accumulation of p53 protein and in an increased expression of Bax protein. Caov-3, lacking wild-type p53, showed upregulation of Bax expression after irradiation. Caov-3 proved to be relative sensitive to apoptosis compared to SK-OV-3 and ES-2. These two cell lines were found to be p53 mutated in sequence analysis and irradiation had no effect on the expression of p53. No change in Bax expression was seen in ES-2, while SK-OV-3 exhibited decreased Bax protein levels after irradiation. Our data suggest that the G2/M arrest is an important component of the pathway leading from irradiation-induced DNA damage to apoptosis in the examined cell lines. The G2/M arrest and associated apoptosis found in the examined cell lines does not necessarily require wild-type p53, although wild-type p53 and possibly Bax may contribute to a maximum response to irradiation. Two independent mechanisms, p53-dependent and p53-independent, are suggested in the examined cell lines.
我们研究了4种人卵巢癌细胞系（即PA-1、Caov-3、SK-OV-3和ES-2）对辐射的细胞周期和凋亡反应。还分析了这些细胞系的p53和Bax表达，以探讨其与细胞周期和凋亡反应的关系。通过流式细胞术检测膜联蛋白V结合情况以及用光度酶免疫测定法测定细胞质组蛋白相关DNA片段来检测凋亡。基于流式细胞术进行细胞周期分析。通过免疫细胞化学检测未处理细胞及辐射后细胞中的p53和Bax蛋白表达。进行p53 cDNA测序和基于酵母的功能检测（FASAY）以确定p53突变状态。所有细胞系均表现出剂量依赖性的G2/M期阻滞。未观察到G1期阻滞。发现G2/M期阻滞与凋亡诱导之间存在强相关性。PA-1是唯一被发现表达野生型p53的细胞系，在照射后显示出在G2/M期积累的最高敏感性和最强的凋亡反应。在该细胞系中，辐射导致p53蛋白明确积累以及Bax蛋白表达增加。缺乏野生型p53的Caov-3在辐射后显示Bax表达上调。与SK-OV-3和ES-2相比，Caov-3被证明对凋亡相对敏感。在序列分析中发现这两种细胞系p53发生突变，辐射对p53表达无影响。ES-2中未观察到Bax表达变化，而SK-OV-3在辐射后Bax蛋白水平降低。我们的数据表明，G2/M期阻滞是所检测细胞系中从辐射诱导的DNA损伤到凋亡的通路的重要组成部分。在所检测细胞系中发现的G2/M期阻滞和相关凋亡不一定需要野生型p53，尽管野生型p53以及可能的Bax可能有助于对辐射产生最大反应。在所检测细胞系中提示存在两种独立机制，即p53依赖性和p53非依赖性机制。
