GENE EXPRESSION IN SV40-TRANSFORMED HUMAN FIBROBLASTS

SV40 转化的人类成纤维细胞中的基因表达

• 批准号: 6267166
• 负责人: HARVEY L OZER
• 金额: $ 11.95万
• 依托单位: ALLEGHENY UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 1998-11-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6267166
• 关键词: DNA damage; Drosophilidae; aging; apoptosis; cell cycle; cell senescence; cell transformation; fibroblasts; gene expression; gene therapy; genetic library; laboratory rabbit; mitogens; oncogenes; simian virus 40; temperature sensitive mutant; tissue /cell culture; virus antigen

Human diploid fibroblasts (HF) have a limited lifespan in culture, ending in a postmitotic state of senescence, which has been documented as a model for cellular aging. One approach toward an understanding of senescence is to examine mechanisms of overcoming it. The protein large T antigen (LT) encoded by the DNA virus SV40 transiently overcomes the growth arrest in senescent cells, extends the lifespan of cells beyond senescence, and, less frequently, permanently overcomes senescence generating immortal cells. We propose to investigate each of the three SV40-cell interactions and their interrelationships. Our working model is that viral gene expression (i.e. LT) promotes cellular proliferation and alterations in cellular genes which are responsible for overcoming senescence in a stable manner. We will particularly assess the role of cell death through apoptosis in limiting the growth of SV40-transformed HF in all three cell states. To facilitate this analysis, we have developed a series of matched pre-immortal and immortal SV40-transformed cell lines (SV/HF) including ones with a temperature-dependent growth behavior (SVtsA/HF and AR 5). Several questions will be addressed in this proposal in an effort to identify effector genes involved in overcoming senescence. We will determine whether the reported ability of LT to induce cellular DNA synthesis (DS) but not mitosis in senescent cells is actually due to a defect in DS, leading to a checkpoint arrest in G2. SV40 TL is able to induce mitosis upon transformation but its effect is temporary. We will evaluate the role of LT, p53, and apoptosis at the end of the extended lifespan of SVtsA/HF-C, termed crisis. In an effort to define novel functions responsible for crisis, we will analyze cDNA libraries generated from SVtsA/HF-C mRNAs overexpressed in crisis as compared to early in the period of extended lifespan. To assess the mitogen role of LT in extended lifespan and immortalization we will attempt to replace LT function by exploiting lines which are temperature-dependent for growth (AR5) into which c-myc has been introduced alone and together with other genes such as ras, and p53. Rare clones of AR5/cmyc which have overcome the temperature defect will also be characterized to determine the cellular basis for this effect. In conclusion, these studies should clarify the role of known factors and define unknown factors in overcoming senescence.

人二倍体成纤维细胞（HF）的文化寿命有限，结束 在衰老后衰老状态中，已被记录为 细胞衰老的模型。 一种理解的方法 衰老是要检查克服它的机制。 蛋白质大 由DNA病毒SV40编码的T抗原（LT）瞬时克服了 衰老细胞的生长停滞，将细胞的寿命延长 衰老，较少的频率，永久性超越衰老 产生不朽的细胞。 我们建议调查三个 SV40细胞互动及其相互关系。 我们的工作模型 是病毒基因表达（即LT）促进了细胞增殖 以及负责克服的细胞基因的改变 以稳定的方式衰老。 我们将特别评估 细胞死亡通过细胞凋亡限制了SV40转化的生长 HF在所有三个细胞状态下。 为了促进这种分析，我们有 开发了一系列匹配的弱不病和不朽的SV40转换 细胞系（SV/HF），包括具有温度依赖生长的细胞系 行为（SVTSA/HF和AR 5）。 将在 该建议是为了识别涉及的效应基因 克服衰老。 我们将确定LT报告的诱导细胞的能力是否 DNA合成（DS），但在衰老细胞中没有有丝分裂实际上是由于 DS缺陷，导致G2中的检查站逮捕。 SV40 TL能够 在转化时诱导有丝分裂，但其作用是暂时的。 我们 将评估LT，p53和凋亡在结束时的作用 SVTSA/HF-C的延长寿命称为危机。 为了定义 负责危机的新功能，我们将分析cDNA图书馆 与SVTSA/HF-C mRNA产生的危机过表达 在延长寿命的初期。 评估有丝分裂原的作用 LT在延长的寿命和永生化中，我们将尝试更换 LT功能通过利用温度依赖的线 生长（AR5）单独引入了C-MYC并与 其他基因，例如Ras和p53。 AR5/CMYC的稀有克隆 克服温度缺陷也将被表征以确定 该作用的细胞基础。 总之，这些研究应该 阐明已知因素的作用并定义未知因素 克服衰老。