Characterization:novel genotoxic stress-regulated gene

描述（由申请人提供）：细胞不断暴露于各种环境因子中，其中一些诱发DNA损伤，即基因毒性应激。细胞对基因毒性应激的反应是复杂的，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，我们提出表征一个新的基因，我们已经命名为PIQ (p53-regulated IQ protein)，它编码一个IQ基序蛋白。IQ基序促进蛋白质与钙调素（CaM）的相互作用。CaM是一种钙结合蛋白，控制着几个重要的信号事件。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表达，PIQ在基因毒性应激和p53激活下下调。PUMA是p53依赖的基因毒性应激诱导细胞凋亡的关键介质，我们的研究结果表明PIQ下调PUMA启动子的表达。重要的是，PUMA启动子含有被预测由cam依赖性信号通路调节的调控元件。因此，我们提出，在非应激细胞中，PIQ可能通过干扰cam依赖的信号事件负调控PUMA的表达，而基因毒性应激后PIQ的下调部分通过cam依赖的途径促进PUMA的上调。我们的总体假设是PIQ是细胞凋亡的负调节因子，结肠癌中较高水平的PIQ表达可能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。本文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。特异性目的1是研究PIQ在p53和基因毒性应激诱导的细胞凋亡中的作用。特异性目的2是研究PIQ负性调控PUMA启动子表达的机制。特异性目的3是分析PIQ mRNA和蛋白水平在更大范围的原发性结肠癌肿瘤及其匹配的正常组织中的表达，并评估肿瘤PIQ状态与临床病理特征之间的关系。

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.