MyD118, CR6 and GADD45 in Genotoxic Stress

描述(申请人提供)：基因毒性应激是生活中常见的一个方面 哺乳动物细胞不得不对抗，自相矛盾的是，DNA损伤诱导 诸如伽马射线和烷基化剂等试剂也用于 癌症治疗。很明显，这两个分子基础的初始 恶性细胞对抗癌药物的敏感性增加，以及 治疗耐药性的产生源于遗传性损伤 改变细胞周期停滞和凋亡设定点。了解 介导哺乳动物细胞反应的分子遗传途径 因此，基因毒性和其他类型的环境应激是高度优先的。在……里面 哺乳动物细胞对基因毒性应激的反应已经进化出一种复杂的防御 机制，包括激活01/S和G2/M细胞周期检查点和/或 激活细胞死亡程序。应激反应通路如何相互作用 信号细胞经历细胞周期停滞或程序性细胞死亡 我还是不明白。最近，MyD118/CR6/GADD45家族的核 蛋白质[也称为GADD45f3，GADD45y，Gadd45cx]与 介导哺乳动物细胞对遗传毒性应激的反应，或依赖于 或独立于P53。已有证据表明MyD118/CR6/GADD45显示 与其他蛋白质的物理相互作用的复杂阵列，如增殖细胞核抗原，p21， Cdc2和MEKK4。各MyD1的应激反应函数在多大程度上 18/CR6/GADD45蛋白是独一无二的或与其他蛋白的功能重叠 蛋白质，目前还不清楚。此外，还不了解的是压力的性质是如何 所遇到的刺激、细胞类型、其生理状态及其遗传 组成，尤其是P53的状态，调节MyD18/CR6/GADD45的功能来确定 如果结果是细胞周期停滞、DNA修复和存活或细胞凋亡 细胞死亡。因此，具体目标是： 目的I：阐明MyD118和CR6(以及Gadd4S当 与MyDI18相结合)在正常发育、生长控制和 对原型应激剂的反应。这将通过分析 缺乏一个或多个MyD1的小鼠模型系统的表型 18/CR6/GADD4S基因，并分析细胞(主要是MEF)的表型 从这样的小鼠中获得的，未经处理或经过应激剂处理后的。 目的II：分析myd II 8/CR6/GADD45与cdc2/cyClinB相互作用的作用 1、p21、增殖细胞核抗原和MEKK4在细胞应激反应中的作用，包括细胞周期 停滞、DNA修复、细胞存活和凋亡。MEF空，适用于MyD1 18/CR6/GADD45 将通过编码MyD1的逆转录病毒载体高效感染 18/CR6/GADD4S，重量或相互作用/功能不足，以及压力 将对响应进行分析。细胞凋亡和DNA的体外无细胞系统 修复，使用添加了重组wt或 相互作用/功能缺陷的蛋白质也将被使用。目标三：了解 PS3和P53如何靶向基因；与细胞周期控制有关(p21，14-3-3 Delta) 或细胞凋亡(Bax)，调节MyDI18/CR6/GADD4S应激功能。MEF，Null for P53、p21或Bax，以及14-3-3s缺失的HCT1 16细胞将被感染 用编码MyD118/CR6/GADD45的逆转录病毒载体，并对 将对应力进行分析。贯彻这项研究计划，应该会有结果 加深了对负增长控制的理解，以应对 基因毒性应激，扰乱这些控制因素对致癌作用的影响， 以及癌症治疗中的治疗阻力是如何产生的。

DESCRIPTION (provided by applicant):Genotoxic stress is a common aspect of life that mammalian cells have to contend with, Paradoxically DNA-damage inducing agents, such as gamma-irradiation and alkylating agents, are also used in cancer therapy. It became evident that both the molecular basis for the initial increase in the susceptibility of malignant cells to anti-cancer agents, and the development of treatment resistance originate from genetic lesions that alter cell cycle arrest and apoptotic set points. Understanding the molecular-genetic pathways which mediate the response of mammalian cells to genotoxic and other types of environmental stress is, thus of high priority. In response to genotoxic stress mammalian cells have evolved an intricate defense mechanism, including activation of 01/S and G2/M cell cycle checkpoints &/or activation of a cell death program. How stress response pathways interact to signal cells to undergo either cell cycle arrest or programmed cell death is still not understood. Recently, the MyD1 18/CR6/GADD45 family of nuclear proteins [also termed GADD45f3, GADD45y, Gadd45cx] has been implicated in mediating the response of mammalian cells to genotoxic stress, either dependent or independent of p53. Evidence has accumulated that MyD1 18/CR6/GADD45 display a complex array of physical interactions with other proteins such as PCNA, p21, Cdc2, & MEKK4. To what extent the stress response function of each of the MyD1 18/CR6/GADD45 proteins is unique or overlaps with the functions of the other proteins, is unclear. Also, not understood is how the nature of the stress stimulus encountered, the cell type, its physiological state, and its genetic makeup, notably p53 status, modulate MyD1 18/CR6/GADD45 function to determine if the outcome will be cell cycle arrest, DNA repair and survival or apoptotic cell death. Thus, the specific aims are: Aim I: Elucidate the physiological functions of MyD118 and CR6 (and Gadd4S when in combination with MyDI18) in normal development, growth control and the response to prototype stress agents. This will be done by analyzing the phenotype of mouse model systems deficient for either one or more of MyD1 18/CR6/GADD4S genes, and analyzing the phenotype of cells (primarily MEFs) obtained from such mice, untreated or following treatment with stress agents. AIM II: Dissect the role of MyD II 8/CR6/GADD45 interactions with cdc2/cyclinB 1, p21, PCNA and MEKK4 in cellular stress responses, including cell cycle arrest, DNA repair, cell survival & apoptosis. MEFs null for MyD1 l8/CR6/Gadd45 will be infected at high efficiency with retroviral vectors encoding for MyD1 18/CR6/GADD4S, either wt or interaction/function deficient, and the stress response will be analyzed. In vitro cell free systems for apoptosis and DNA repair, using null cell extracts spiked with recombinant wt or interaction/function deficient proteins, also will be used. AIM III: Understand how pS3 & p53 target genes; implicated in cell cycle control (p21, 14-3-3delta) or apoptosis (Bax), modulate MyDI18/CR6/GADD4S stress functions. MEFs, null for p53, p21, or Bax, and the HCT1 16 cells, null for 14-3-3s, will be infected with retroviral vectors encoding for MyD1 18/CR6/GADD45, and the response to stress will be analyzed. Following through on this research plan, should result in an increased understanding of negative growth control in response to genotoxic stress, how perturbing these controls may contribute to oncogenicity, and how treatment resistance in cancer therapy can arise.