Survival Signaling After Genotoxic Insult

基因毒性侮辱后的生存信号

• 批准号: 6867502
• 负责人: SUSAN M CERYAK
• 金额: $ 28.23万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6867502
• 关键词: DNA damage; RNA interference; RNase protection assay; biological signal transduction; carcinogenesis; cell cycle; cell growth regulation; chemical carcinogen; chromium; environmental exposure; fibroblasts; flow cytometry; gene induction /repression; human tissue; immunocytochemistry; lung neoplasms; phosphorylation; posttranslational modifications; protein tyrosine kinase; respiratory epithelium; serine threonine protein kinase; small interfering RNA; transfection; western blottings

DESCRIPTION (provided by applicant): Inappropriate activation/inactivation of key signals that control cell survival after genotoxic insult can contribute to autonomous growth and neoplastic transformation. An initial consequence of genotoxic injury is cell cycle checkpoint arrest but genotoxicity may also activate cell death pathways of apoptosis or terminal growth arrest. Cellular survival responses to genotoxic insult may produce intrinsic death-resistance; such a selective growth advantage may allow for emergence of a transformed phenotype. Certain forms of hexavalent chromium [(Cr(VI)] are known human respiratory carcinogens that can be employed as useful genotoxic tools with relevant toxicological importance. Our preliminary studies suggest that maintenance of protein tyrosine phosphorylation, which is coincident with AKT activation, overrides Cr-induced growth arrest and enhances clonogenic survival. Constitutive AKT activation is known to play an important role in carcinogenesis. Therefore, the overall objective of this proposal is to elucidate the coordinate signaling events that mediate cell fate determination and survival after genotoxic insult. The dual overarching hypotheses of the proposed studies are that: 1) AKT activation shifts the balance of cell fates, toward survival, after Cr(Vl) genotoxic insult; and 2) AKT activation in the face of Cr(Vl) genotoxic insult increases genomic instability. To test these hypotheses, we will employ molecular, pharmacological and genetic approaches, by using relevant model systems of human diploid lung fibroblasts (HLF), and human large airway epithelial cells (HLAE) and studying the involvement of key signaling components of the AKT pathway. The molecular circuitry of the AKT effect will be delineated in Aim 1, and the consequences of an AKT-induced "override" of the genotoxin-elicited program of cell death will be investigated in Aim 2. Aim 3 will identify the role of AKT in resistance to Cr(Vl)-induced clonogenic lethality in a subclonal population of cells with acquired resistance to Cr-induced clonogenic death. We will use soluble Na2CrO4 at a range of concentrations relevant to human exposure, and for which the DNA adduct frequencies and genotoxic lesions are well documented. Results of the proposed studies will identify molecular mechanism(s) that confer a growth advantage to cells after genotoxic insult, and add new insights to the understanding of Cr(Vl)-induced lung carcinogenesis, while addressing a need for sensitive and specific molecular indices that can be correlated with exposure to carcinogenic agents, as well as with their cancer incidence. Delineation of the molecular circuitry involved in AKT survival signaling may have the added benefit of identifying molecular targets for rational drug design in anti-cancer therapy.

描述(由申请人提供)：在基因毒性损伤后，控制细胞生存的关键信号的不适当激活/失活可能有助于自主生长和肿瘤转化。遗传毒性损伤的最初后果是细胞周期检查点停滞，但遗传毒性也可能激活细胞死亡途径，即凋亡或最终的生长停滞。细胞对基因毒性伤害的生存反应可能产生内在的死亡抗性；这种选择性生长优势可能允许出现转化的表型。某些形式的六价铬[(六价铬)]是已知的人类呼吸道致癌物质，可用作具有相关毒理学意义的有用的遗传毒性工具。我们的初步研究表明，蛋白酪氨酸磷酸化的维持与AKT的激活一致，推翻了铬诱导的生长停滞，并提高了克隆性存活。已知AKT的结构性激活在肿瘤的发生中起重要作用。因此，这项建议的总体目标是阐明协调信号事件，介导细胞命运的决定和遗传毒害后的生存。这些研究的双重假设是：1)AKT的激活改变了细胞命运的平衡，使其在铬(Vl)基因毒性损伤后走向生存；2)面对铬(Vl)的基因毒性损伤，AKT的激活增加了基因组的不稳定性。为了验证这些假设，我们将使用分子、药理学和遗传学的方法，通过使用相关的人二倍体肺成纤维细胞(HLF)和人大气道上皮细胞(HLAE)的模型系统，并研究AKT通路的关键信号成分的参与。AKT效应的分子回路将在目标1中描述，而AKT诱导的基因毒素诱导的细胞死亡程序的后果将在目标2中进行研究。目标3将在对铬诱导的克隆性死亡具有获得性抗性的亚克隆细胞群体中确定AKT在抵抗铬(Vl)诱导的克隆性致死中的作用。我们将在与人类暴露相关的一系列浓度下使用可溶性Na2CrO4，其DNA加合物频率和遗传毒性损伤已有很好的记录。拟议的研究结果将确定在遗传毒性损伤后赋予细胞生长优势的分子机制(S)，并为理解铬(Vl)诱发的肺癌提供新的见解，同时解决与致癌剂暴露及其癌症发病率相关的敏感和特异分子指标的需求。描绘AKT生存信号中涉及的分子回路可能具有额外的好处，即为抗癌治疗中的合理药物设计确定分子靶点。