Analysis of the mechanisms involved in the cell cycle arrest at G2-M phases and possible clinical utility in the field of ginecology.

我们观察到，当CaSki人宫颈癌细胞在低于正常37℃的温度下培养时，细胞周期延长和细胞分裂异常的细胞聚集。M期细胞周期进程受损可能是染色体异常分离、非整倍体、细胞生长抑制和细胞死亡的原因。为了探讨这些细胞分裂异常的机制，我们利用基因芯片技术、RT-PCR法和Western blotting分析了培养温度从37℃转变到32℃或30℃后细胞中基因表达的变化。结果表明，在与应激反应相关的因子中，ATF2mRNA在温移后6h和12h分别下降到原来的60%和38%。温移后6h和12h，ATF3的表达分别降至75%和49%。CHOP(C/EBP同源蛋白)和CREB(cAMP反应元件结合蛋白)1基因的表达在6-12小时内被抑制约40%。调控细胞周期的CDK抑制剂p21^&lt；GP1&gt；的mRNA在温移后12小时开始下降，并持续到温移后24小时至72小时。相反，p57^-kip2&gt；在48小时内表达增加1.8倍。P53的表达在6小时内增加一倍，24小时恢复到原来的水平，72小时下降到50%。与纺锤体检查点相关的MAD2的表达在24小时内上调3倍，同时伴随着M期细胞的增加。虽然STK15(Aurora A)的表达在温度变化后12小时下降到27%，但在温度变化48小时后上升到170%。STK12的表达在温移后72小时内稳定增加1.5倍。

We observed elongation of cell cycle and accumulation of cells with anomalies in cell division when CaSki human cervical cancer cells were cultured at temperatures lower than usual 37℃. An impaired cell cycle progression at M phase is a possible cause of abnormal chromosome segregation, aneuploidy, cell growth inhibition, and cell death. To investigate the mechanisms involved in these anomalies in cell division, we analyzed changes in gene expression in the cells after the cultivation temperature shift from 37℃ to 32℃ or 30℃, by using a cDNA microarray technique, RT-PCR and western blotting. The results showed that, among those factors related to stress response, ATF2 mRNA was reduced to 60% and 38%, respectively, of the original revel 6 hrs and 12 hrs after the temperature shift. ATF3 expression also was reduced to 75% and 49%, respectively, 6 hrs and 12 hrs after the temperature shift. Expression of both CHOP (C/EBP-homologous protein) and CREB (cAMP responsive element-binding protein)1 gene was suppressed by approximately 40% in 6 to 12 hrs. mRNA of p21^<GP1>, a Cdk inhibitor modulating cell cycle, started to decrease 12 hrs after the temperature shift, and kept repressed until 24 hrs to 72 hrs after the temperature shift. On the contrary, p57^<kip2> expression increased 1.8 fold in 48 hrs. Expression of p53 doubled in 6 hrs, returned to the original revel at 24 hrs, and fallen to 50% at 72 hrs. Expression of MAD2, a factor related to spindle check point, was up regulated 3 fold in 24 hrs in parallel with an increase of M phase cells. Although STK15 (Aurora A) expression declined to 27% 12 hrs after the temperature shift, it elevated to 170% 48 hrs after the temperature change. STK12 expression steadily raised 1.5 fold within 72 hrs after the temperature shift.