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CaSki人宫颈癌细胞在低于正常温度37℃的条件下，细胞周期延长，细胞积累，细胞分裂异常。在M期受损的细胞周期进程是染色体异常分离、非整倍体、细胞生长抑制和细胞死亡的可能原因。为了探讨这些细胞分裂异常的机制，我们利用cDNA芯片技术、RT-PCR和western blotting分析了培养温度从37℃转变为32℃和30℃后细胞中基因表达的变化。结果表明，在应激反应相关因子中，ATF2 mRNA在温度变化后6小时和12小时分别降低到原来水平的60%和38%。温度变化后6 h和12 h， ATF3的表达量分别下降到75%和49%。CHOP （C/ ebp同源蛋白）和CREB （cAMP响应元件结合蛋白）1基因的表达在6 ~ 12小时内均被抑制约40%。调节细胞周期的Cdk抑制剂p21^<GP1> mRNA在温度变化后12小时开始下降，并在温度变化后24 ~ 72小时保持抑制。相反，p57^<kip2>的表达在48小时内增加了1.8倍。p53的表达在6小时内翻倍，24小时后恢复到原来的水平，72小时时下降到50%。纺锤体检查点相关因子MAD2的表达在24 h内随着M期细胞数量的增加而上调3倍。STK15 （Aurora A）在温度变化后12 h的表达量下降到27%，而在温度变化后48 h的表达量上升到170%。STK12表达量在温度变化后72 h内稳定升高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.