CPEB and Cell Senescence

描述(申请人提供)：CPEB是一种序列特异性RNA结合蛋白，在脊椎动物发育过程中调节聚腺苷酸化诱导的翻译。虽然不育，但全球CPEB基因敲除小鼠是可行的和正常的大小。然而，来自这些基因敲除(KO)动物的小鼠胚胎成纤维细胞(MEF)是永生的，而来自野生型(WT)动物的MEF在培养7-9代后会衰老。用表达CPEB的病毒感染早期传代的KO MEF可挽救衰老表型；感染传代后期永生化的KO MEF则没有影响。此外，早期传代的WT MEF感染表达CPEB的病毒可诱导细胞提前衰老，而感染永生化的WT MEF则没有影响。这些结果表明CPEB是细胞衰老所必需的。更多的实验表明，i.)CPEB对细胞衰老的调节需要肿瘤抑制基因p53和p19ARF，II。)CPEB介导的翻译控制衰老，以及III。)CPEB KO MEF可能发生部分转化。这项建议的具体目标包括确定CPEB调节小鼠细胞衰老的潜在分子和细胞机制。另有证据表明，CPEB也控制着人类细胞的衰老。我们将采用的许多技术都是我们多年来一直用于研究CPEB调节的翻译的技术，包括RNA-蛋白质相互作用、蛋白质-蛋白质相互作用的分析、Poly(A)尾长的检查和免疫细胞化学。这些拟议的实验直接涉及癌症病因学和衰老过程，这是NIH任务的核心。这项提议的重点是调节细胞衰老的新分子。由于这一过程与癌症和老龄化密切相关，它显然对人类健康具有重要影响。

DESCRIPTION (provided by applicant): CPEB is a sequence-specific RNA binding protein that regulates polyadenylation-induced translation during vertebrate development. While infertile, a global CPEB knockout mouse is viable and normal size. However, mouse embryo fibroblasts (MEFs) derived from these knockout (KO) animals are immortal whereas MEFs derived from wild type (WT) animals, as expected, become senescent after 7-9 passages in culture. Infection of early passage KO MEFs with a virus expressing CPEB rescues the senescence phenotype; infection of late passage immortalized KO MEFs has no effect. Moreover, infection of early passage WT MEFs with a CPEB-expressing virus induces premature senescence while infection of immortalized WT MEFs has no effect. These results demonstrate that CPEB is necessary for cell senescence. Additional experiments indicate that i.) CPEB regulation of cell senescence requires the tumor suppressors p53 and p19ARF, ii.) CPEB-mediated translation controls senescence, and iii.) the CPEB KO MEFs may be partially transformed. The specific aims of this proposal include the determination of the underlying molecular and cellular mechanisms by which CPEB regulates senescence in mouse cells. Additional evidence is presented indicating that CPEB also controls senescence in human cells. Many of the techniques we will employ are those we have used for several years to study CPEB-regulated translation and include an analysis of RNA-protein interactions, protein-protein interactions, examination of poly(A) tail length, and immuno-cytochemistry. These proposed experiments deal directly with cancer etiology and the ageing process, which is central to the mission of NIH. This proposal focuses on new molecules that regulate cell senescence. Because this process is strongly linked to cancer and ageing, it clearly has important implications for human health.