The Effect of the MDM2-p53 Loop on the Sensitivity of Ovarian Cancer Cells to Cisplatin

OBJECTIVE To explore whether MDM2 transfection can alter the MDM2-p53 autoregulatory feedback loop so as to change the sensitivity of ovarian cancer cell lines to cisplatin. METHODS The ovarian cancer cell line A2780 expressing wild-type P53 and the ovarian cancer cell line SKOV-3 with the p53 null type were stably transfected with pCMV-MDM2 or pCMV as a control. The blocked expression of P53 was determined by Western blots. Cytotoxicity was assessed using the MTT assay and the trypan blue exclusion assay. Flow cytometry was used to detect changes in the cell cycle and removal of platinum -DNA adducts was measured by atomic absorption spec-troscopy. RESULTS (1) Parental A2780 and A2780-V cells (IC50= 15.14±1.39 μmol) have similar cisplatin sensitivities, whereas sensitivity to cisplatin in A2780-M cells (IC50=7.98±1.32 μmol) was 2 to 3 fold greater (P=0.001). The trypan blue exclusion assay demonstrated that cisplatin killed a higher percentage of A2780-M cells compared to A2780-V cells. There was no significant change following MDM2 transfection in SKOV-3 cells. (2) After cisplatin treatment, A2780-M cells showed a pronounced S-phase arrest, however, A2780 cells with the intact wild-type P53, arrested primarily at the G2/M transition. (3) Platinum uptake was similar for all of the A2780 cell lines after ciaplatin treatment, but the removal of plat-inum-DNA adducts was reduced in the A2780-M cells compared with A2780-V cells. CONCLUSION MDM2 increases cisplatin cytotoxicity in ovarian cancer cells by blocking the expression of p53 through the MDM2-p53 autoregulatory feedback loop.

3种标记物的检测在卵巢癌诊断中的临床意义

[目的 ]探讨糖蛋白抗原 12 5 (CA12 5 )、恶性肿瘤特异生长因子 (TSGF)、铁蛋白 (FER) 3种标记物联合测定在卵巢癌早期诊断、疗效监测中的临床意义。 [方法 ]采用免疫电化学发光法及放射免疫法测定 78例卵巢恶性肿瘤患者术前、术后 ,37例卵巢良性囊肿及 96例健康体检者血清CA12 5、TSGF、FER。 [结果 ]78例恶性肿瘤患者血清CA12 5、TSGF、FER阳性率分别为 80 .8%、83.3%、5 6 .4 %。联合测定阳性率提高到 91.0 % ,恶性肿瘤组与良性囊肿及健康对照组比较差异显著 (P <0 .0 1) ;良性囊肿组与健康对照组之间无显著差异 (P >0 .0 5 )。有效治疗后 ,三者均可明显下降。若CA12 5持续高值 ,则提示肿瘤残存、转移、预后不良。 [结论 ]CA12 5、TSGF、FER联合测定对早期诊断卵巢癌、术前肿瘤良恶性的判断和术后疗效观察有重要的临床意义。

Activin A对卵巢癌细胞系OVCAR-3的作用探讨

目的检测ActivinA对卵巢癌细胞系OVCAR-3的作用及其作用途径。方法用细胞免疫组织化学方法检测OVCAR-3细胞系膜受体ActRⅡ,用10ng/mL Activin A连续作用7d绘制细胞的生长曲线,设未加Activin A为对照组。用MTT方法检测Activin A对OVCAR-3刺激作用与时间和剂量的关系。West-ern-blot法检测bcl-2蛋白表达。结果OVCAR-3细胞系膜受体ActRⅡ表达阳性,Activin A对细胞增值作用与对照组比较差异有统计学意义,Activin A促进细胞增殖有时间依赖性,在48h达高峰;但无剂量依赖性。通过Western blot检测Activin A作用48h的OVCAR-3细胞Bcl-2蛋白表达情况,发现加Activin A组Bcl-2蛋白表达明显升高,差异有统计学意义。结论Activin A对OVCAR-3细胞系有促细胞增值作用,其促细胞增殖作用途径可能是通过Bcl-2抗凋亡通路发挥作用。

卵巢癌细胞系OVCAR3过表达SND1稳定株的构建

目的:通过慢病毒载体构建过表达SND1的OVCAR3稳定的表达株。方法:重组的pLVX-FLAG-SND1表达载体与包装质粒共同转染293T细胞,获得携带FLAG-SND1的重组慢病毒。病毒感染的OVCAR3细胞经Hygromycin B筛选出稳定表达株。用Western blot方法检测SND1蛋白表达。结果:用病毒感染OVCAR3细胞,药物筛选后获得的过表达稳定株中SND1蛋白表达水平明显增高。结论:利用慢病毒表达载体pLVX-FLAG-SND1成功感染并筛选出稳定表达SND1的OVCAR3细胞株,为进一步研究SND1在卵巢癌中的作用提供了体外细胞系模型。

端粒酶活性与卵巢癌细胞系化学敏感性的关系

目的 探讨端粒酶活性与上皮性卵巢癌细胞系化学敏感性的关系。方法 使用顺铂作用于敏感细胞株A2 780及耐药细胞株AD60。在四甲基偶氮唑盐 (MTT)法测定耐药倍数的基础上 ,应用聚合酶链反应 聚丙烯酰胺凝胶电泳 (PCR -PAGE)方法测定端粒酶活性。结果 经MTT测定卵巢癌耐药细胞株AD60的耐药倍数为 2 4。A2 780端粒酶活性随药物浓度的递增明显降低 ,而AD60降低不明显。结论 端粒酶活性与上皮性卵巢癌细胞对细胞毒性药物的化学敏感性相关。