In contrast to well characterized primary glucocorticoid hormone responses, in which the glucocorticoid receptor interacts directly with viral and cellular target genes to increase their transcriptional rate, secondary glucocorticoid responses remain poorly defined. A hallmark of the secondary response is the requirement for ongoing protein synthesis in order for increased transcription of the secondary response gene to occur. Secondary responses are presumed to be mediated by as yet unidentified transcription factors (transactivators) that are themselves induced or activated by glucocorticoids. We have recently cloned and characterized from the DDT1 tumor cell line a glucocorticoid regulated secondary response gene, hGSTYBX, which is a mu class glutathione S-transferase. This class of gene is directly involved in xenobiotic drug metabolism, multidrug resistance, and is suspected to play a role in hormonal carcinogenesis. Therefore, understanding its regulation may also reveal important information about its role in the neoplastic process. This grant will be structured in two phases to address these issues.
Three specific aims define phase 1.
SPECIFIC AIM 1 is to clone and map the secondary glucocorticoid response element in hGSTYBX and characterize the cognate transactivator protein.
SPECIFIC AIM 2 is to use information obtained in Aim 1 to clone the cDNA of the transactivator protein using concatenated ds oligonucleotide probes representing the secondary response domain to screen appropriate expression libraries. Detailed analysis of cDNA and fusion proteins will be performed. Alternative cloning methods are discussed.
SPECIFIC AIM 3 is to clone the cognate transactivator gene(s) identified in aim 2 and characterize their regulation. Little is known about the regulation of transactivator gene expression particularly during the neoplastic process, therefore this research has the potential to make important contributions in this area. The second phase of the proposal examines the hypothesis that hGSTYBX expression is down regulated during androgen-estrogen induced carcinogenesis. Such down regulation of gene activity may play a decisive role in tumorigenesis.
SPECIFIC AIM 4 will examine hGSTYBX expression by in situ hybridization and immunocytochemistry under a variety of hormonal conditions to determine if the loss of hGSTYBX expression is correlated with carcinogenesis. Understanding the biology and function of this class of gene has the potential to yield great insight into the mechanism(s) of hormonal carcinogenesis involving this 100% reproducible Syrian hamster model for solid tissue tumor formation. The long-term goals of this grant are to describe in detail the genes and related cellular control mechanisms involved in hormonal carcinogenesis. Elucidation of these pathways will ultimately allow definition of novel intervention protocols for the control of cancer.
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