ATM and ATR in Chromium-Induced S-Phase Arrest

描述（由申请人提供）：六价铬（Cr [VI]）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。Cr（VI）化合物一旦进入体内就被代谢为五价（Cr [V]）、四价（Cr [IV]）和三价（Cr [III]）铬。Cr（V）和Cr（IV）是高度反应性的中间体，并且已被证明会引起几种类型的DNA损伤。暴露于Cr化合物触发S期细胞周期检查点，这是一种活跃的细胞反应，可以提高细胞存活率，并在DNA损伤的情况下限制可遗传的遗传异常。铬诱导的S期阻滞的生化步骤的表征将有助于提高我们对铬诱导的肿瘤发生机制的理解。提出本申请中的实验的刺激是我们的初步观察，即共济失调毛细血管扩张突变蛋白激酶（ATM）及其相关激酶ATR是铬诱导的S期内阻滞所必需的。ATM和ATR属于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确的进化关系。ATM和ATR对于细胞对DNA损伤的反应都是至关重要的。我们的初步数据表明，暴露于Cr（VI）及其代谢副产物Cr（V）和Cr（IV），诱导可测量的DNA双链断裂。然而，只有Cr（V）和Cr（VI），而不是Cr（IV），可以激活S期内检查点和ATM激酶。Cr诱导的S期阻滞是ATM和ATR依赖的。此外，我们发现，结构维持的染色体蛋白1（SMC 1）在低剂量铬暴露后，磷酸化的ATM依赖的方式。拟议的实验将探讨如何ATM和ATR调节S期逮捕后，暴露于铬和识别分子决定因素的过程。在具体的目标1中，我们将研究NBS 1和BRCA 1，这两者都是响应DNA损伤的ATM底物，是否是ATM依赖性过程所必需的。我们还将研究ATM磷酸化的SMC 1在铬暴露的功能意义。在具体目标2中，我们将研究ATR是否通过暴露于Cr而被激活，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Cr暴露的反应机制以及ATM和ATR在该途径中的作用，作为深入了解致癌，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.