Biological Significance of p16 gene inactivation on glioma progress

结果表明，在大多数p53野生型(WT)胶质母细胞瘤中，p14(ARF)基因经常缺失，提示p14(ARF)失活在P53-WT胶质瘤的发生发展中起重要作用。P14(ARF)蛋白与MDM2结合，抑制MDM2介导的P53降解。这些结果表明，p14(ARF)的修复可以抑制P53-WT胶质瘤细胞的生长。因此，为了阐明p14(ARF)失活在脑胶质瘤中的生物学意义，我们将p14(ARF)cDNA载体导入p14(ARF)阴性的胶质瘤细胞。本研究以人胶质母细胞瘤细胞系U87 MG和A1207(p14/p16缺失，p53-WT)、T98G(p14/p16-缺失，p53-突变)、LNZ308(p14/p16-WT，p53-空)为研究对象。用脂质体法将p14或p16表达载体与绿色荧光蛋白(GFP)表达载体按4：1比例共导入细胞。用GFP间接检测转基因细胞，并用流式细胞仪进行分析。转移p14(ARF)基因后，U87 MG和A1207细胞发生G2/M期阻滞，但未引起G1期阻滞和细胞凋亡，但外源性p14(ARF)对T98G和LNZ308细胞周期分布无明显影响。除LNZ308细胞外，其他3个p14/p16缺失细胞系均被外源p16诱导G1期停滞。这些结果表明，p14(ARF)失活通过将细胞周期停滞在G2/M期，促进了P53-WT胶质瘤细胞的进一步恶性转化。

It was shown that the p14 (alternating reading frame : ARF) gene was frequently deleted in the majority of p53 wild-type (WT) glioblastomas, suggesting that p14 (ARF) inactivation plays an important role in p53-WT glioma progression. p14(ARF) protein binds to MDM2 and inhibits MDM2-mediated degradation of p53. These findings suggest that the restoration of p14 (ARF) can suppress the growth of p53-WT glioma cells. Therefore, to clarify the biological significance of p14 (ARF) inactivation in glioma, we introduced a p14 (ARF) cDNA plasmid vector into p14 (ARF)-negative glioma cells. Human glioblastoma cell lines, U87MG and A1207 (p14/p16-deleted, p53-WT), T98G (p14/p16-deleted, p53-mutated), LNZ308 (p14/p16-WT, p53-null), were used in this study. Expression constructs (p14 or p16 cDNA) and green fluorescence protein (GFP) expression vector at a 4:1 ratio were co-introduced into cells using the lipofection method. Transfected cells were indirectly detected by the presence of GFP and analyzed with flow cytometry. Although the transfer of the p14 (ARF) gene induced G2/M arrest but not G1 arrest or apoptosis in U87MG and A1207 cells, cell cycle distributions of T98G and LNZ308 cells were not significantly changed by exogenous p14 (ARF). G1 arrest was observed in three p14/p16-deleted cell lines other than LNZ308 cells by exogenous p16. These results suggest that p14 (ARF) inactivation contributes to further malignant transformation of p53-WT glioma cells by representing cell cycle arrest at G2/M.