ATM and ATR in Chromium-Induced S-Phase Arrest

说明(申请人提供)：六价铬(六价铬)是一种常见的工业废物和环境污染物，是已知的人类致癌物质。铬(VI)化合物一旦进入人体，就会代谢成五价铬(Cr[V])、四价铬(Cr[IV])和三价铬(Cr[III])。铬(V)和铬(IV)是高活性的中间体，已被证明会引起几种类型的DNA损伤。接触铬化合物会触发S期细胞周期检查点，这是一种活跃的细胞反应，可能会提高细胞存活率，并在存在DNA损伤的情况下限制可遗传的遗传异常。刻画铬诱导S期阻滞的生化步骤将有助于我们更好地理解铬诱导的肿瘤发生机制。提出这一实验的动机是我们的初步观察，即肌酸诱导的S期停滞需要共济失调-毛细血管扩张突变蛋白激酶(ATM)及其相关的ATR。ATM和ATR属于一个结构独特的蛋白丝氨酸-苏氨酸激酶家族，其催化结构域与哺乳动物和酵母磷酸肌醇3-激酶的催化结构域具有明显的进化关系。ATM和ATR对于细胞对DNA损伤的反应都是至关重要的。我们的初步数据显示，暴露在铬(VI)及其代谢副产物铬(V)和铬(IV)中，可以诱导可测量的DNA双链断裂。但只有铬(V)和铬(VI)能激活S相内检查点和ATMK，而不能激活铬(IV)。铬诱导的S相停滞既依赖于atm又依赖于atr。此外，我们还发现，在低剂量铬暴露后，染色体蛋白1(SMC 1)的结构维持以一种ATM依赖的方式被磷酸化。拟议的实验将研究atm和atr如何调节暴露于铬后的S相停止，并确定这一过程的分子决定因素。在特定的目标1中，我们将调查NBS 1和BRCA1是否是ATM依赖过程所必需的，这两种物质都是对DNA损伤做出反应的ATM底物。我们还将研究SMC-1 ATM磷酸化在铬暴露反应中的功能意义。在特定的目标2中，我们将研究ATR是否被铬暴露激活，以及SMC 1是否是ATR调节检查点的下游靶点。我们的长期目标是更好地了解哺乳动物细胞对铬暴露的反应机制，以及ATM和ATR在该途径中的作用，作为深入了解癌症发生、细胞生长和细胞死亡的一般机制的基础。

DESCRIPTION (provided by applicant): Hexavalent chromium (Cr [VI]) is a common industrial waste product and an environmental pollutant, and it is a known human carcinogen. Cr (VI) compounds are metabolized to pentavalent (Cr [V]), tetravalent (Cr [IV]), and trivalent (Cr [III]) chromium once they enter the body. Cr (V) and Cr (IV) are highly reactive intermediates and have been shown to cause several types of DNA damage. Exposure to Cr compounds triggers the S-phase cell cycle checkpoint, an active cellular response which may enhance cell survival and limit heritable genetic abnormalities in the presence of DNA damage. Characterization of biochemical steps in Cr-induced S-phase arrest will help improve our understanding of mechanisms of Cr-induced tumorigenesis. The stimulus for proposing the experiments in this application is our preliminary observations that Ataxia-Telangiectasia-Mutated protein kinase (ATM) and its related kinase ATR are required for Cr-induced intra S-phase arrest. ATM and ATR belong to a structurally unique family of protein serine-threonine kinases whose catalytic domains share a clear evolutionary relationship with those of mammalian and yeast phosphoinositide 3-kinases. Both ATM and ATR are critical for cellular response to DNA damage. Our preliminary data has revealed that exposure to Cr (VI), and its metabolic byproducts Cr (V) and Cr (IV), induces measurable DNA double strand breaks. However, only Cr (V) and Cr (VI), but not Cr (IV), can activate intra S-phase checkpoint and ATM kinase. The Cr-induced S-phase arrest is both ATM- and ATR-dependent. Furthermore, we found that the Structural Maintenance of Chromosomal protein 1 (SMC 1) is phosphorylated in an ATM-dependent manner after low dose Cr exposure. The proposed experiments will investigate how ATM and ATR regulate S-phase arrest after exposure to Cr and identify molecular determinants of the process. In specific aim 1, we will investigate whether NBS 1 and BRCA1, both of which are ATM substrates in response to DNA damage, are required for the ATM-dependent process. We will also study the functional significance of ATM phosphorylation of SMC 1 in response to Cr exposure. In specific aim 2, we will study whether ATR is activated by exposure to Cr and whether SMC 1 is a downstream target of ATR in regulating the checkpoint. Our long-term goal is to better understand the mechanisms by which mammalian cells respond to Cr exposure and the role of ATM and ATR in the pathway as a basis for providing insights into general mechanisms of carcinogenesis, cell growth and cell death.