G6PD通过细胞周期蛋白D1/D2调控人黑色素瘤细胞周期的进程

G6PD Regulates the Course of Cell Cycle through Cyclin D1/D2 in Human Melanoma Cells

葡萄糖-6-磷酸脱氢酶(G6PD)在人皮肤黑色素瘤A375细胞中处于高表达与高活性状态,但G6PD在黑色素瘤发生发展过程中的作用及其具体机制尚不明确.本文在前期运用siRNA方法构建G6PD敲减的黑色素瘤A375稳转细胞(A375-G6PDΔ)基础上,构建表达载体pBabe-puro-G6PDWT在A375-G6PDΔ细胞中过表达野生型的G6PD基因,从而构建G6PD表达恢复的稳转细胞(A375-G6PDΔ-G6PDWT).3株细胞A375-WT、A375-G6PDΔ和A375-G6PDΔ-G6PDWT经G6PD酶活性测定、MTT测定、克隆形成实验、流式细胞仪分析细胞周期和Western印迹检测.结果显示,A375-G6PDΔ-G6PDWT细胞的G6PD蛋白表达量(0.847±0.080)及其活性(0.394±0.029)分别是A375-G6PDΔ的3.28倍(P<0.01)和7.34倍(P<0.01),分别是A375-WT细胞的91.57%和2.12倍(P<0.05).与A375-WT细胞相比,A375-G6PDΔ细胞G0/G1期细胞数增加,S期细胞数减少,增殖指数PI降低了25.70%(P<0.05),细胞周期蛋白D1/D2、细胞周期蛋白E表达分别下降37.4%、54.3%(P<0.01)和17.3%;而A375-G6PDΔ-G6PDWT细胞呈现G1/S期阻滞解除,细胞周期蛋白D1/D2蛋白分别恢复到A375-WT细胞的89.5%和87.6%,细胞周期蛋白E表达未见恢复,呈现生长增殖和克隆形成率的恢复并接近于A375-WT细胞.结果提示,G6PD通过细胞周期蛋白D1/D2调控人皮肤黑色素瘤A375细胞G1期向S期转换的进程,这为黑色素瘤发病机制的研究提供了新的思路.

We previously found that Glucose-6-phosphate dehydrogenase（G6PD） was highly expressed with high activity in the human melanoma A375 cell（A375-WT）.How G6PD affects the tumorigenesis and progression of human melanoma was not entirely known.To elucidate the relationship between G6PD and human melanoma,we constructed the stable cell line（A375-G6PDΔ-G6PDWT） with over expression of wide type human G6PD gene in the A375-G6PDΔ cell（G6PD gene knock down in the A375-WT with siRNA） using expression vector pBabe-puro-G6PDWT.Western blotting and ultraviolet spectrophotometry showed that protein amount（0.847 ± 0.080） and activity（0.394 ± 0.029） of G6PD in A375-G6PDΔ-G6PDWT cells increased by 3.28 times（P0.01） and 7.34 times（P0.01）,respectively,as those of the A375-G6PDΔ cells,accompanied by restorations of growth,proliferation and cloning efficiency and are close to those of A375-WT cells.In comparison with the A375-WT cells,the A375-G6PDΔ cells were characterized by an increasing of G0/G1 phase cells,a reduction of S phase cells and a 25.70%（P0.05）decrease in proliferation index with flow cytometry analysis,and 37.4% descend of cyclin D1（P0.01）,54.3% decrease of cyclin D2（P0.01） and 17.3% reduction of cyclin E with Western blotting,respectively.Further study showed that G1/S phase arrest was relieved in the A375-G6PDΔ-G6PDWT,and cyclin D1 and D2 restored 89.5% and 87.6% of A375-WT cells,respectively,without cyclin E restoration.The results indicate that G6PD regulates the course of G1 phase to S phase in the A375 cell cycle through the expression changes of cyclin D1/D2 protein,which may be a new clue in the study of melanoma tumorigenesis,and the role and mechanism of G6PD in melanoma cell growth and proliferation need to be further investigated.