Involovement of p16 tumor suppressor gene in cell cycle progression

我们首先研究了宫颈上皮样癌细胞HeLa细胞、子宫内膜癌细胞Ishikawa细胞和卵巢上皮性癌细胞2780细胞中p21和p16的表达。三种细胞株均稳定表达p21和p16的mRNA和蛋白。然而，这些细胞系的表达水平相对较弱。P21和p16的序列分析表明，所有细胞株的p16和p21均为野生型，而Ishikawa细胞的p21mRNA密码子31为精氨酸多态。其次，对2780株P53基因进行了序列分析，发现其密码子178缺失。HeLa细胞的P53蛋白被18型乳头瘤病毒E6蛋白灭活，因此我们推测这些细胞系中的p21不依赖于P53，p21基因转移到这两个细胞系中可能是有意义的。我们将p21基因亚克隆到真核表达载体pMAMneo和逆转录病毒载体pXT1中，并将其导入这些细胞系。经G418筛选，获得HeLa和2780细胞亚系。这些亚系表现出明显的细胞生长迟缓和端粒酶活性减弱。用流式细胞仪进行细胞周期分析表明，G0G1到S的阻滞是细胞生长迟缓的主要原因。这些结果表明，将p21基因导入p53被病毒癌蛋白灭活的癌细胞，将比p53本身更有效地抑制细胞生长。

We first studies the expression of p21 and p16 in HeLa cell, a epidermoid carcinoma cells of the cervix, Ishikawa cell, a endometrial carcinoma cells, and 2780 cell, an ovarian epithelial carcinoma cell. All three cell lines steadily express mRNA and protein of p21 and p16. The expression levels of these cell lines, however, were relatively weak. Sequence analysis of p21 and p16 of these cell lines revealed that p16 and p21 of all cell line were wild types, though codon 31 of p21 mRNA of Ishikawa cell was arginine polymorphism. Next, p53 gene of 2780 was studied by using sequence analysis, showing deletion in its codon 178. The p53 of HeLA cells is inactivated by E6 protein of papilloma virus type 18. Therefore we supposed that p21 of these cell lines was independent of p53 and gene transfer of p21 into these two cell line may make a sense. We subcloned p21 gene into pMAMneo, a eucaryotic expression vector and pXT1 a retroviral vector and introduced into these cell lines. After G418 selection, we obtained sublines of HeLa and 2780 cells. These sublines showed apparent retardation of cell growth and attenuated telomerase activity. Cell cycle analysis using flowcytometry showed that the blockage from G0G1 to S is major cause of cell growth retardation. These results suggest that introduction of p21 gene into carcinoma cells of which p53 is inactivated by viral oncoproteins will suppress cell growth more efficiently than p53 itself.