作　　者： ; ; ; ;

机构地区： 北京大学药学院

出　　处： 《生物化学与生物物理进展》 2008年第5期512-520,共9页

摘　　要： ING1(inhibitor of growth 1)是一个候选抑癌基因家族,p47ING1a、p33ING1b和p24ING1c是其三种剪接异构体.通过MTT法和流式细胞术研究ING1a、ING1b和ING1c对HeLa细胞增殖的影响,结果发现三者均可将HeLa细胞阻滞于G0/G1期并抑制细胞生长.采用PCR方法构建ING1a和ING1b的PHD结构域缺失体1a!C和1b!C,进而使ING1a,ING1b、ING1c、1a△C和1b△C在HeLa细胞中过表达.采用Western blot检测上述HeLa细胞中p16INK4a、PTEN/p27Kip1和p53/p21Waf1的表达变化,结果发现ING1a、ING1b、ING1c和1a△C均可促进p16INK4a的表达,其中ING1c的促进作用最为显著,1b△C则略微抑制了p16INK4a的蛋白质表达.利用荧光素酶分析初步确定1a△C可增强p16INK4a启动子活性而促进p16INK4a的转录,1b△C则抑制了p16INK4a启动子活性.上述结果阐释了ING1家族各成员对HeLa细胞增殖的影响及其分子机制,从而确定了各异构体功能的异同及它们所调控的重要基因.首次发现除p53/p21Waf1通路外,ING1家族各异构体还可通过上调p16INK4a和PTEN的表达而抑制肿瘤细胞生长,并且ING1a的PHD结构域删除体可以增强p16INK4a的转录,这为研究ING1家族抑制肿瘤细胞生长的分子机制提供了新的线索. ING1 family is a candidate for tumor suppressor, which has three splicing isoforms named p47INGla, p33INGlb, and p24INGlc. Study of the effect of different isoforms of ING1 on HeLa cells proliferation and its molecular mechanism would help further identifying the functional relationship of ING1 isoforms, and finding important genes regulated by ING1. Cell growth curve and cell cycle analysis were used to observe the effect of INGla, INGlb, and INGlc on HeLa cells growth, and the result indicated that they could all inhibit HeLa cells growth by arresting cell cycle at G0/G1 phase. PCR method was used to construct the PHD domain deletions of ING1a and ING1b. ING1a, ING1b, ING1c and the PHD domain deletions 1a△C and 1b△C were then overexpressed in HeLa cells, p16^INK4a, PTEN/p27^Kip1 and p53/p21^Waf1 protein levels were detected by Western blot. The result showed that ING1a, ING1b, ING1c, and 1a△C except for 1b△C induced p16^INK4a protein expression, in which ING1 c had the most powerful effect. Luciferase assay identified that overexpression of pcDNA3.1 （＋）- 1 a△C facilitated p16^INK4a transcription through enhancing p16^INK4a promoter activity, while pcDNA3.1 （＋）- 1 b△C repressed the p16^INK4a promoter activity. In a word, it was found for the first time that except for the p53/p21^Waf1 pathway, three splicing isoforms of ING1 family could also inhibit HeLa cells proliferation though upregulation of p16^INK4a and PTEN, and the PHD domain deletion of INGla enhanced p16^INK4a transcription. These findings provide new clews to further study on the mechanisms of ING1 family suppressing cancer cells growth.

关 键 词： 结构域

领　　域： [生物学] [生物学]