Glutathione S-transferases have the uncommon characteristic of being able to scavenge and detoxify reactive, electrophilic molecules within the environment of living cells. Experimental results from past work indicate that this ability to conjugate electrophiles to glutathione is essential for the sustained life of a cell. What is now needed to move this research area forward is: i) an understanding of how endogenous GST gene expression is regulated, especially induction, so that chemopreventative molecules can be rationally designed and put into practice, and ii) an understanding of the parameters which determine the extent to which GST/GSH conjunction can be used to capture and detoxify electrophiles in cells, and by so doing, decrease the associated cancer risk.
The Aims of our proposed work are designed to bring the concept of preemptive glutathione conjugation of electrophiles to fruition.
Our Aims are to: 1) identify how sequence variation in Antioxidant Responsive Elements (AREs) affects inducibility of the dependent, chemoprotective genes, and hence, affects cancer risk, 2) determine the identify of key regulation transcription factor that binds to AREs and regulates the Chemoprotective Response, and 3) develop a comprehensive understanding of the parameters which determine the level of GST/GSH-conferred resistance in mammalian cells. We will use several experimental approaches for these studies, including: analysis of amplified mouse genomic sequences containing AREs, relating ARE DNA sequence to inducibility in an in vitro functional assay as well as inducibility in mouse live; oligonucleotide-based affinity purification of ARE-BP-1, the key ARE-binding transcription factor and cloning of its cDNA; expression of recombinant genes which encode wild-type or mutant, gain-of-function GST isoforms from our current, the GSH biosynthesis enzyme gamma-glutamyl-cysteinesynthase (gammaGCS) or the membrane glutathione-conjugate pump (MRP) in cultured mammalian cells; and the production of two gene-replacement strains of mice, one carrying a knockout of its endogenous GST Yc/1 gene, and one carrying a three codon replacement mutations in the GST Yc/1 gene to confer a gain-of-function phenotype in catalyzing the conjugation of toxic nitrogen mustards. Through these studies, we will work to acheive an understanding of how expression of GST genes is regulated and how endogenous and recombinant GSTs can be used to protect cells from electrophiles.
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