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Involovement of p16 tumor suppressor gene in cell cycle progression

p16 抑癌基因参与细胞周期进程

基本信息

批准号：
07671773
负责人：
YOKOYAMA Yasuhiro
金额：
$ 1.54万
依托单位：
GIFU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671773/
关键词：
p21WAF1 p16INK4 cell cycle Gene transfer ovarian cancer uterine cancer Cell cycle Cyclin Dependent kinase Ovarian Cancer Uterine cancer Cyclin Gene transter Rotrovivus

项目摘要

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.

我们首先研究了p21和p16在宫颈上皮样癌细胞HeLa、子宫内膜癌细胞石川和卵巢上皮癌细胞2780中的表达。所有三种细胞系均稳定表达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基因序列分析表明，所有细胞系的p16和p21基因均为野生型，而石川细胞的p21基因第31位密码子为精氨酸多态性。其次，通过使用序列分析研究了2780的p53基因，显示其密码子178处缺失。HeLA细胞的p53被乳头状瘤病毒18型的E6蛋白灭活。因此，我们推测这两种细胞系的p21不依赖于p53，将p21基因导入这两种细胞系可能是有意义的。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pXT 1中，并导入这些细胞系中。经G418筛选，获得HeLa细胞和2780细胞亚系。这些亚系表现出明显的细胞生长迟缓和衰减的端粒酶活性。流式细胞仪分析细胞周期表明G0G1期向S期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被病毒癌蛋白失活的癌细胞中，将比p53本身更有效地抑制细胞生长。