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.
