GROWTH CONTROL IN AGING FIBROBLASTS

老化成纤维细胞的生长控制

基本信息

批准号：
6168044
负责人：
EUGENIA WANG
金额：
$ 25.83万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-03-01 至 2003-04-30
项目状态：
已结题

项目摘要

Our effort to identify candidate genes responsible for the nonproliferating senescent phenotype led us to characterize a 57 kDa nonproliferation-specific nuclear protein, statin, which is phosphorylated by an associated 45 kDa serine/threonine kinase (p45), and forms a complex with this enzyme and another unphosphorylated protein of 110 kDa (p110), identified so far as the retinoblastoma gene product, RB. We hypothesize that in nongrowing cells p57 statin may function as a sequester to prevent the phosphorylation of the associated p110 RB; because the lack of p34(cdc2) kinase alone does not block RB phosphorylation in early G1, we suggest that this p45 kinase may be the missing enzyme needed for this step. During early G1 phase, rapid statin loss releases p45 kinase to phosphorylate RB; in nongrowing cells such as senescent fibroblasts, permanent statin presence sequesters p45, and along with the absence of p34(cdc2) is part of the mechanism producing a complete blockade of RB phosphorylation, with cascade effects culminating in inability of DNA synthesis. We suggest that a "two-hit RB antiphosphorylation" model may pertain in nongrowing cells; this proposal aims to investigate how statin functions to block RB phosphorylation, and whether molecular manipulations might remove this blockade in nonreplicating cells, or implement it in their replicating counterparts. Specific aims include: (1.) investigating the specific cellular action regulating the binding reaction of p57 statin, p45 statin kinase, and p110 RB in nongrowing cells; (2.) purifying p57 and p45 from senescent cells, for protein microsequencing and functional studies; (3.) molecular cloning, sequencing and characterization of p57; (4.) investigating the molecular regulation of p57 expression during in vitro aging; (5.) early functional analysis of p57 and p45 in RB phosphorylation in early G1 phase; and (6.) early functional analysis of the presence of p57 and absence of cdc2 kinase in RB phosphorylation in senescent cells. Our hypothesis that statin may function as the sequester of the kinase needed for early RB phosphorylation provides the first handle to begin the investigation of kinases involved in early G1 phase, and offers an intriguing and workable model in the continuing research quest to understand the molecular mechanisms controlling the cessation of proliferation in in vitro aged human fibroblasts. Answers to this ques- tion will add to our knowledge of cell cycle control, a fundamental mechanism pivotal to the wellbeing of significant processes such as terminal differentiation and cellular aging.

我们努力寻找负责的候选基因非增殖衰老表型使我们表征了 57 kDa 抗增殖特异性核蛋白他汀类药物由相关的 45 kDa 丝氨酸/苏氨酸激酶 (p45) 磷酸化，并且与该酶和另一种未磷酸化的蛋白质形成复合物 110 kDa (p110)，目前已鉴定为视网膜母细胞瘤基因产物 RB。我们假设，在非生长细胞中，p57 他汀类药物可能充当隔离以防止相关 p110 RB 的磷酸化；因为单独缺乏 p34(cdc2) 激酶并不能阻断 RB G1 早期的磷酸化，我们认为该 p45 激酶可能是缺少此步骤所需的酶。在 G1 早期，快速他汀类药物损失释放 p45 激酶以磷酸化 RB；在非生长细胞中，例如衰老的成纤维细胞，他汀类药物的永久存在会隔离 p45，以及随着 p34(cdc2) 的缺乏，它是产生 a 的机制的一部分。完全阻断 RB 磷酸化，级联效应达到顶峰无法合成DNA。我们建议“两次打击RB” 抗磷酸化”模型可能适用于非生长细胞；该提议旨在研究他汀类药物如何发挥阻断 RB 磷酸化的作用，以及分子操作是否可以消除这种封锁非复制细胞，或在其复制细胞中实现它。具体目标包括：（1.）研究特定的细胞作用调节 p57 他汀类药物、p45 他汀类药物激酶的结合反应，非生长细胞中的 p110 RB； (2.)从衰老中纯化p57和p45 细胞，用于蛋白质微测序和功能研究； (3.) 分子 p57 的克隆、测序和表征； (4.) 调查体外衰老过程中p57表达的分子调控； (5.) 早 G1早期RB磷酸化中p57和p45的功能分析阶段; (6.) p57 存在的早期功能分析和衰老细胞中 RB 磷酸化中 cdc2 激酶的缺失。我们的假设他汀类药物可能起到所需激酶的螯合剂的作用为早期 RB 磷酸化提供了第一个开始的手柄研究参与早期 G1 期的激酶，并提供在持续的研究探索中有趣且可行的模型了解控制停止的分子机制体外老化人成纤维细胞的增殖。这个问题的答案—— 化将增加我们对细胞周期控制的了解，这是一个基本的机制对于重要过程的健康至关重要，例如终末分化和细胞衰老。