P15INK4B高表达对人黑色素瘤细胞G1/S进程的阻滞及与MAPK信号相关性之初探

利用TdR-N_2O同步法分别获得P15高表达的MLIK6和表达空载体的MLC2的M期细胞和G_1期细胞,~3H-TdR掺入结果显示,与对照组细胞MLC2相比,实验组细胞MLIK6从G_1期进入S期时间延长2h,并且掺入强度明显减弱,DNA合成被抑制。进一步观察了P15~(INK4B)对G_1/S相关调控蛋白的影响,在M期细胞释放8h(晚G_1期细胞)后,与对照组MLC2细胞相比,实验组MLIK6细胞中CyclinD1,CyclinE,Cdk4,C-Myc蛋白水平均降低。相反,P27~(KIP1)的表达却上升。同时探讨了MAPK信号在P15~(INK4B)阻抑A375细胞G_1/S转换中的作用与相关性,结果显示晚G_1期的MLIK6细胞中ERK1,ERK2水平变化不大,而具有活性的P-ERK1和P-ERK2均表现出下降。上述实验表明,P15~(INK4B)可能通过作用于G_1期相关的周期调节蛋白和抑制ERK1和ERK2活性,阻滞G_1/S转换与抑制DNA合成。

EFFECT OF P15INK4B EXPRESSION ON THE INHIBITION OF G1/S PROGRESSION AND THE RELATION TO MAPK SIGNAL PATHWAY IN HUMAN MELANOMA CELLS

The synchronized M phase cells of MLIK6 and MLC 2 were harvested by the method of thymidine(TdR)and N2O arresting. The result of 3H-TdR incorporation indicated that the transition of Gi/S of MLIK6 cells was delayed 2h compared with the control MLC 2 cells and the incorporation rate decreased obviously. Furthermore, the effect of P15INK4Bexpression on the some G1 phase related regulatory protein was studied. When the synchronized cells in M phase were released for 8 h( in late G] phase), the levels of CyclinD1, CyclinE, Cdk4 and C-Myc were decreased obviously in MLIK6 cells. But the expression of P27 'increased as compared with that of control MLC 2 cells. At the same time, the relationship between overexpression of P151NK4Band MAPK signal in transition from G1 to S phase of A375 cells at late G1 phase was studied, comparing with the control MLC 2 cells, the level of ERK1 and ERK2 almost have no change in MLIK6 cells, but that of P-ERK1 and P-ERK2(activated form)were decreased markedly. The results suggest that P15INK4Bdelayed G,/S transition and inhibited DNA synthesis by affecting G1/S phase related regulatory proteins and inhibiting the activity of ERK1 and KRK2.