Characterization:novel genotoxic stress-regulated gene

表征：新型遗传毒性应激调节基因

• 批准号: 7030099
• 负责人: M. SAEED SHEIKH
• 金额: $ 22.8万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030099
• 关键词: DNA damage; RNA interference; SDS polyacrylamide gel electrophoresis; apoptosis; biological signal transduction; calcineurin; calmodulin; cell adhesion molecules; cell growth regulation; colon neoplasms; environment related neoplasm /cancer; gene environment interaction; gene induction /repression; genetic markers; genetic promoter element; genetic transcription; green fluorescent proteins; human tissue; messenger RNA; molecular cloning; neoplasm /cancer genetics; neoplastic process; p53 gene /protein; protein binding; protein protein interaction; protein structure function; small interfering RNA; western blottings

DESCRIPTION (provided by applicant): Cells are constantly exposed to a variety of environmental agents some of which induce DNA damage i.e. genotoxic stress. Cellular responses to genotoxic stress are complex and therefore, more studies are needed to better understand the mechanisms that control such responses. Here we propose to characterize a novel gene, which we have named PIQ (p53-regulated IQ protein) that codes for an IQ motif protein. IQ motifs promote protein interactions with calmodulin (CaM). CaM is a calcium-binding protein that controls several important signaling events. Evidence presented here show that PIQ does indeed bind to CaM. Furthermore, PIQ mRNA is overexpressed in primary colon tumors when compared with matching normal tissues and that PIQ is down-regulated in response to genotoxic stress and p53 activation. PUMA is a key mediator of p53- dependent genotoxic stress-induced apoptosis and our results indicate that PIQ down-regulates PUMA promoter expression. Importantly, PUMA promoter harbors regulatory elements that are predicted to be regulated by CaM-dependent signaling pathways. We, therefore, propose that in unstressed cells, PIQ may negatively regulate PUMA expression by interfering with CaM-dependent signaling events and PIQ downregulation following genotoxic stress serves to promote PUMA upregulation partly via CaM-dependent pathways. Our overall hypothesis is that PIQ is a negative modulator of apoptosis and that higher levels of PIQ expression noted in colon cancer may alter the threshold of apoptosis and consequently contribute in part to colon tumorigenesis. Three specific aims are proposed here to further characterize PIQ in order to elucidate its role in cellular response to genotoxic stress in general and in the development and/or progression of colon cancer in particular. Specific Aim 1 is to investigate the role of PIQ during p53 and genotoxic stress-induced apoptosis. Specific Aim 2 is to investigate the mechanism by which PIQ negatively regulates PUMA promoter expression. Specific Aim 3 is to analyze PIQ expression at the mRNA and protein levels in a larger pool of primary colon tumors and their matching normal tissues and to evaluate a relationship between tumor PIQ-status and the clinicopathologic features.

描述（由申请人提供）：细胞不断暴露于各种环境因素，其中一些环境因素会诱导 DNA 损伤，即基因毒性应激。细胞对基因毒性应激的反应很复杂，因此需要更多的研究来更好地了解控制这种反应的机制。在这里，我们建议表征一个新基因，我们将其命名为 PIQ（p53 调节的 IQ 蛋白），它编码 IQ 基序蛋白。 IQ 基序促进蛋白质与钙调蛋白 (CaM) 的相互作用。 CaM 是一种钙结合蛋白，控制多种重要的信号传导事件。这里提供的证据表明 PIQ 确实与 CaM 结合。此外，与匹配的正常组织相比，PIQ mRNA 在原发性结肠肿瘤中过度表达，并且 PIQ 因基因毒性应激和 p53 激活而下调。 PUMA 是 p53 依赖性基因毒性应激诱导细胞凋亡的关键介质，我们的结果表明 PIQ 下调 PUMA 启动子表达。重要的是，PUMA 启动子含有预计受 CaM 依赖性信号通路调节的调节元件。因此，我们提出，在未受应激的细胞中，PIQ 可能通过干扰 CaM 依赖性信号传导事件来负向调节 PUMA 表达，并且基因毒性应激后 PIQ 下调部分通过 CaM 依赖性途径促进 PUMA 上调。我们的总体假设是，PIQ 是细胞凋亡的负调节剂，结肠癌中较高水平的 PIQ 表达可能会改变细胞凋亡的阈值，从而在一定程度上促进结肠肿瘤的发生。这里提出了三个具体目标来进一步表征 PIQ，以阐明其在细胞对遗传毒性应激的反应中的作用，特别是在结肠癌的发生和/或进展中的作用。具体目标 1 是研究 PIQ 在 p53 和基因毒性应激诱导的细胞凋亡过程中的作用。具体目标2是研究PIQ负向调节PUMA启动子表达的机制。具体目标 3 是分析更大范围的原发性结肠肿瘤及其匹配的正常组织中 mRNA 和蛋白质水平的 PIQ 表达，并评估肿瘤 PIQ 状态与临床病理特征之间的关系。