Mitomycin C (MC) and 10-decarbamoyl MC Signaling to p53 and Family Members

描述(申请人提供)：肿瘤抑制基因P53是细胞的重要感受器 DNA损伤。细胞应激通过许多不同的信号转导途径传递给P53。我们最近发现，DMC同时激活P53依赖和独立的凋亡途径，而MC只激活P53依赖的途径。了解化疗药物如何向野生型p53发出信号对于癌症治疗至关重要。最近的研究发现，p73和p63是DNA损伤的信号转导途径，它们是协调调节DNA损伤和细胞凋亡反应所必需的蛋白。我们的假设是，DMC可以显著激活一条利用p73和p63的p53非依赖性的凋亡通路。长期目标是确定DMC激活的P53非依赖性信号转导通路(S)。目的1)比较dmc与其他dna损伤药物在细胞系中的细胞毒性和特异性靶基因调控，以及与其他dna损伤药物的比较。 P53。细胞毒性和已知的与诱导细胞凋亡和细胞周期调节相关的选定靶基因的表达将由MC、DMC和一系列特性良好的化疗药物的代表性细胞系的药物剂量曲线决定。多个有效靶点(包括Bax、Fas、Puma、Noxa、GADD45、p21和MDM2)的基因表达水平将使用实时RT-PCR进行监测，目标激酶的激活将通过磷酸盐特异性抗体进行监测。目的2)确定P53靶基因的差异激活是否与染色质上选择性多蛋白复合体的形成有关。染色质免疫沉淀-聚合酶链式反应(ChLP-PCR)利用p53、p73和MDM2特异性抗体，将被用来研究p53、p73和MDM2在已知的凋亡和细胞周期调控靶基因的增强子结合部位的核内相互作用。固定是ChlP-PCR方案的第一步，将保留较高分子量的复合体。目的3)应用Affymetrix DNA微阵列技术检测经P53处理的MC和DMC细胞以及经P53依赖的细胞凋亡抑制因子WISP-1处理前后的基因表达。为了确定细胞凋亡是否可以归因于特定的过度表达基因，将使用siRNA进行功能分析。

DESCRIPTION (provided by applicant): The tumor suppressor p53 is a critical sensor of cellular DNA damage. Cellular stress is signaled to p53 by a number of different signal transduction pathways. We recently identified that DMC activates both p53 dependent and independent apoptotic pathways while MC only activates the p53 dependent pathway. Understanding how chemotherapeutic drugs signal to wild-type p53 is of central relevance to cancer treatment. Determining DNA damage pathways that can signal to p53 family members, p73 and p63 has emerged as equally important with the recent finding that these proteins are required to coordinately regulate the DNA damage apoptotic response. Our hypothesis is that DMC can significantly activate a p53-independent apoptotic pathway that utilizes p73 and p63. The long-range goal is to determine the p53-independent signal transduction pathway(s) activated by DMC. Aim 1) Identify the cytotoxicity and specific target gene regulation, and checkpoint kinases activated, by DMC as compared to other DNA damaging drugs in cell lines, with and without p53. The cytotoxicity and the expression of selected target genes known to associate with the induction of apoptosis and cell cycle regulation will be determined by drug dose curves of representative cell lines with MC, DMC and a battery of well characterized chemotherapeutics. Gene expression levels of multiple validated targets (including Bax, Fas, Puma, Noxa, Gadd45, p21 and mdm2) will be monitored using real-time RT-PCR and target kinase activation will be monitored by phosphor-specific antibodies. Aim 2) Determine if differential activation of p53 target genes associates with alternative multiprotein complex formation on chromatin. Chromatin immuno-precipitation-PCR (ChlP-PCR), utilizing p53 specific, p73 specific and MDM2 specific antibodies, will be used to investigate the nuclear in vivo interaction of p53, p73 and MDM2 at the known enhancer binding sites for the apoptotic and cell cycle regulating target genes. Fixation is the first step in the ChlP-PCR protocol and will preserve higher-molecular weight complexes. Aim 3) Examine gene expression by affymetrix DNA micro-arrays in MC and DMC treated cell lines with and without p53 as well as with and without the p53 dependent apoptosis inhibitor WISP-1. To determine whether apoptosis can be attributed to specific overexpressed genes, siRNA will be used for functional analysis.