A large number of environmental carcinogens and toxicants are alkylating agents. Human O6-alkylguanine-DNA alkyltransferase (AGT, EC 2.1.1.63) is an important DNA repair protein which specifically repairs O6- alkylguanine in DNA, a major premutagenic lesion produced by environmental alkylating agents. AGT plays a critical role in protecting cells against mutation and toxic response induced by alkylating agents, including alkylating chemotherapeutic drugs. Significant interindividual variations (greater than 10 fold) in human AGT activity levels have been reported. However, little is known about the molecular basis of these variations and their importance in an individual's susceptibility to environmental carcinogenesis and toxicity related to alkylating agents. Recently, we have identified two novel forms of genetic polymorphism of human AGT. One missense alteration occurs at the codon 143 (isoleucine to valine). The codon 143 (Ile143) is adjacent to the alkyl acceptor Cys145 of the AGT active site and is conserved among the mammalian AGT. The other polymorphic alteration is localized in the promoter region of the AGT gene, which creates a new BanI restriction site. Our working hypothesis is that genetic polymorphism of AGT, particularly those occurring near the enzyme active sites or within an important regulatory region, is a major reason for the reported large interindividual variations of AGT activity in humans. As a consequence, AGT genetic polymorphism may play a significant role in determining an individual's susceptibility to environmental alkylating carcinogens and response to alkylating chemotherapeutic drugs. The present proposal will test our hypothesis by focusing on the functional characterization of genetic polymorphism of human AGT (known or to be identified) with the following specific aims: 1. To elucidate the functional significance of AGT genetic polymorphism. Initial work will focus on the codon 143 and BanI polymorphisms we recently identified. This will be accomplished by: (a) biochemical studies on AGT missense polymorphic variants; (b) comparison of the toxic response to alkylating agents between human cells expressing wild-type and missense variant AGT; and (c) transcriptional analysis of the BanI polymorphic variation by a reporter gene assay. 2. To determine the effects of polymorphic alterations on AGT function and expression in humans by measuring AGT activity, protein and mRNA levels in human peripheral lymphocytes and esophageal mucosa (a target tissue of alkylating carcinogens). Samples with extremely high or low AGT activity and expression levels will also be screened for new AGT genetic polymorphisms. The current proposal will contribute to our understanding of AGT, an important environmentally relevant enzyme, and help assess the role of AGT genetic polymorphism in human carcinogenesis and toxicity related to environmental alkylating agents. It could have a significant impact on cancer prevention by identifying susceptible subpopulations in future epidemiological studies.
