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

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.

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

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 (P＜0.01) and 7.34 times (P＜0.01)，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%（P＜0.05）decrease in proliferation index with flow cytometry analysis, and 37.4% descend of cyclin D1(P＜0.01), 54.3% decrease of cyclin D2 (P＜0.01) 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.