Survival Signaling After Genotoxic Insult

基因毒性侮辱后的生存信号

• 批准号: 7285057
• 负责人: SUSAN M CERYAK
• 金额: $ 2.13万
• 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/7285057
• 关键词: DNA damage; 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（由申请方提供）：遗传毒性损伤后控制细胞存活的关键信号的不适当激活/失活可能导致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凋亡或终末生长停滞的细胞死亡途径。对遗传毒性损伤的细胞存活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[（Cr（VI）]是已知的人类呼吸道致癌物，可用作具有相关毒理学重要性的有用遗传毒性工具。我们的初步研究表明，蛋白质酪氨酸磷酸化的维持，这是与AKT激活一致，推翻铬诱导的生长停滞，提高克隆生存。已知组成性AKT活化在癌发生中起重要作用。因此，本提案的总体目标是阐明介导遗传毒性损伤后细胞命运决定和存活的协调信号传导事件。所提出的研究的双重总体假设是：1）在Cr（VI）遗传毒性损伤后，AKT活化使细胞命运的平衡向存活转移;和2）面对Cr（VI）遗传毒性损伤时的AKT活化增加了基因组不稳定性。为了验证这些假设，我们将采用分子，药理学和遗传学方法，通过使用人二倍体肺成纤维细胞（HLF）和人大气道上皮细胞（HLAE）的相关模型系统，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，AKT诱导的基因毒素引起的细胞死亡程序的“覆盖”的后果将在目标2中研究。目的3将鉴定AKT在对Cr（VI）诱导的克隆性死亡具有获得性抗性的亚克隆细胞群体中对Cr（VI）诱导的克隆性死亡的抗性中的作用。我们将使用可溶性Na 2CrO 4，浓度范围与人体暴露相关，DNA加合物频率和遗传毒性病变已得到充分记录。所提出的研究的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，并为理解Cr（VI）诱导的肺癌作用增加新的见解，同时解决对可与致癌剂暴露以及其癌症发病率相关的敏感和特异性分子指标的需求。参与AKT存活信号传导的分子电路的描绘可能具有识别用于抗癌治疗中的合理药物设计的分子靶点的额外益处。