The overall objective of this research project is to understand the mechanism by which carcinogenic chromium(VI) compounds affect gene expression. The approaches we plan to use in attacking this problem are: (1) examine the ability of chromium (VI to differentially affect gene expression in 14 day chick embryos, a simple well-defined in vivo system; (2) compare the effects of chromium (VI) on model genes which have different types of general and specific regulatory elements (GC- boxes, glucocorticoid responsive elements (GREs), cAMP-responsive elements, and metal responsive elements (MREs), and respective specific transcription factors (AP1,SP1, glucocorticoid receptor, cAMP-binding protein, and metal-activated factors): and (3) determine the effect of chromium (VI) on the normal binding of transcription factors to their recognition sequences. Chromium (VI) compounds have been shown to preferentially attack specific DNA sequences and to differentially affect the transcription of inducible vs. non-inducible genes. Therefore, we plan to test the hypothesis that chromium (VI)-induced DNA damage affects gene expression by altering the interaction of transcription factors with promoter regions of the gene. Specifically we propose to test the hypothesis that chromium (VI) interferes with the normal interaction of regulatory protein within the controlling regions of inducible genes and not those of other constitutive genes.
The specific aims of the proposed research are: (1) The effects of chromium (VI)-induced DNA damage on the constitutive and inducible steady-state mRNA levels and transcription rates of metallothionein, phosphoenolpyruvate carboxykinase, and albumin genes will be determined in the liver of chick embryos treated with chromium (VI) in vivo. The effects of chromium (VI) treatment on the basal and inducible steady- state levels of expression of these genes will be determined by solution hybridization and Northern blot analyses. The effects of chromium (VI) on the basal and inducible rates of transcription of these genes will be determined by nuclear """"""""run-off"""""""" transcription assays. (2) The effect of chromium (VI)-induced DNA damage on the interaction of transcription factors with the promoter regions of the metallothionein, phosphoenolpyruvate carboxykinase, and albumin genes will be determined in the liver of 14 day chick embryos treated with chromium (VI) in vivo. The DNA-protein interactions in vivo will be determined using ligation- mediated polymerase chain reaction (PCR)-based genomic footprinting analysis of the promoter regions of these genes. The long-range objective of this research project is to understand the mechanism by which chromium (VI) compounds act as carcinogens. The proposed studies should provide evidence for differential effects of chromium (VI) on important regulatory regions of genes and elucidate critical effects on DNA structure and function, and thus, provide insight into the mechanism by which chromium (VI) initiates cancer. This, in turn, could provide fundamental insights into the overall mechanism of initiation of the neoplastic process by genotoxic carcinogens.
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