The aim of the proposed research is to determine, at the molecular level, the mutagenic potential of reactive oxygen species in a chromosomal gene of human somatic cells. Reactive oxygen species are produced in vivo during normal cellular metabolism and by a wide range of environmental and therapeutic agents. These oxygen metabolites appear to be mutagenic in human cells, and as a result may be an important source of somatic mutations that play a pathogenetically important role in human cancer.
Our specific aims are: 1) We will determine the mutagenicity of the respiratory burst of granulocytes and of superoxide anion generated in vitro in a forward mutation assay system we have developed. Both the dose- and oxygen-species dependence of mutation in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene of HL-60 cells will be determined; 2) We will analyse fifty independent HPRT mutations produced by respiratory burst activity or superoxide using a combination of blot hybridization and, in selected instances, polymerase chain reaction-mediated in vitro gene amplification and direct sequencing; 3) We will construct human phagocyte cell lines having selective defects in the two major pathways used to generate potentially mutagenic reactive oxygen species. These analogues of chronic granulomatous disease and myeloperoxidase deficiency will be constructed by using antisense RNA to suppress production of the cognate endogenous genes in HL-60 cells. Results of the proposed research will provide new information on the mutagenic potential of physiologically important reactive oxygen species in a specific gene of human somatic cells, and thus on the potential contribution of oxygen-induced mutation to the production and progression of human cancer.
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| Chiaverotti, T A; Battula, N; Monnat Jr, R J (1991) Rat hypoxanthine phosphoribosyltransferase cDNA cloning and sequence analysis. Adv Exp Med Biol 309B:117-20 |
