Survival Signaling After Genotoxic Insult

描述（由申请人提供）：基因毒性损伤后控制细胞存活的关键信号的不适当激活/失活可能有助于自主生长和肿瘤转化。基因毒性损伤的最初后果是细胞周期检查点阻滞，但基因毒性也可能激活细胞凋亡或终末生长阻滞等细胞死亡途径。细胞对基因毒性损伤的生存反应可能产生内在的死亡抵抗；这种选择性生长优势可能允许出现转化表型。某些形式的六价铬[(Cr(VI))]是已知的人类呼吸道致癌物，可作为有用的遗传毒性工具，具有相关的毒理学重要性。我们的初步研究表明，维持蛋白酪氨酸磷酸化（与AKT激活一致）可以克服cr诱导的生长停滞并提高克隆生存。组成型AKT激活在癌变中起重要作用。因此，本建议的总体目标是阐明介导基因毒性损伤后细胞命运决定和存活的协调信号事件。提出的研究的两个主要假设是：1)在Cr（Vl）基因毒性损伤后，AKT的激活改变了细胞命运的平衡，使其朝着存活的方向发展；2)面对Cr（Vl）基因毒性损伤时AKT的激活增加了基因组的不稳定性。为了验证这些假设，我们将采用分子、药理学和遗传学方法，通过使用人类二倍体肺成纤维细胞（HLF）和人类大气道上皮细胞（HLAE）的相关模型系统，研究AKT通路的关键信号成分的参与。AKT作用的分子通路将在Aim 1中描述，而AKT诱导的“覆盖”基因毒素引发的细胞死亡程序的后果将在Aim 2中研究。目的3将确定AKT在对Cr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（Vl）诱导的克隆性死亡的抗性中的作用。我们将在与人类暴露相关的浓度范围内使用可溶性Na2CrO4，并且DNA加合物频率和遗传毒性病变已被充分记录。拟议研究的结果将确定基因毒性损伤后赋予细胞生长优势的分子机制，并为理解Cr（Vl）诱导的肺癌提供新的见解，同时解决与致癌物质暴露及其癌症发病率相关的敏感和特异性分子指标的需求。对AKT存活信号通路的描述可能对确定抗癌治疗中合理药物设计的分子靶点有额外的好处。

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.