CPEB and Cell Senescence

描述（由申请人提供）：CPEB是一种序列特异性RNA结合蛋白，在脊椎动物发育期间调节多聚腺苷酸化诱导的翻译。虽然不育，但整体CPEB敲除小鼠是可行的和正常大小的。然而，源自这些敲除（KO）动物的小鼠胚胎成纤维细胞（MEF）是永生的，而源自野生型（WT）动物的MEF，如预期的，在培养物中7-9次传代后变得衰老。用表达CPEB的病毒感染早期传代的KO MEF挽救衰老表型;感染晚期传代的永生化KO MEF没有影响。此外，用表达CPEB的病毒感染早期传代的WT MEFs诱导过早衰老，而感染永生化的WT MEFs没有影响。这些结果表明，CPEB是细胞衰老所必需的。另外的实验表明i.）细胞衰老的CPEB调节需要肿瘤抑制因子p53和p19 ARF，ii.）CPEB介导的翻译控制衰老，和iii.）CPEB KO MEF可以部分转化。该提案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。其他证据表明，CPEB也控制人体细胞的衰老。我们将采用的许多技术是我们多年来用于研究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，这是国家卫生研究院使命的核心。这项提案的重点是调节细胞衰老的新分子。由于这一过程与癌症和衰老密切相关，它显然对人类健康具有重要意义。

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.