The overall objective of this research project is to understand the mechanism by which chromium(VI) compounds, known human lung carcinogens, affect gene expression. The approaches Dr. Wetterhahn plans to use in attacking this problem are: (1) examine the ability of Cr(VI) to affect gene expression in well-defined cultured human lung cells; (2) determine the ability of Cr(VI) to produce reactive oxygen intermediates (ROIs) and oxidative DNA damage; (3) determine the effects of Cr(VI) on the binding of transcription factors known to be activated by oxidative stress to their DNA recognition sequences; and (4) examine the effects of Cr(VI) on oxidative stress-inducible genes. There is mounting evidence that reactive oxygen species may be involved [in] the """"""""promotion"""""""" phase of carcinogenesis. Intracellular metabolism of Cr(VI) may lead to oxidative stress and this may account for the ability of Cr(VI) to act as a complete carcinogen. Dr. Wetterhahn plans to test the hypothesis that Cr(VI) produces ROIs, and thereby affects signal transduction and expression of oxidative stress genes by altering the interaction of transcription factors with promoter regions of these genes.
The specific aims of the proposed research are: (1) Hypothesis: Cr(VI) produces ROIs and oxidative DNA damage. The ability of Cr(VI) to produce ROIs will be determined in human lung cells. Generation of ROIs by Cr(VI) will be determined using fluorescence spectroscopy to measure the increased cellular oxidation of nonfluorescent 2',7'-di- chlorofluorescin diacetate to the fluorescent 2',7'-dichlorofluorescein. DNA damage in the form of Cr-DNA binding, DNA-protein cross-links, DNA strand breaks and 8-oxo-2'-deoxyguanosine will also be determined. (2) Hypothesis: Cr(VI) will increase levels of nuclear proteins which bind to NF-kB and AP-1 response elements. The effect of Cr(VI) on the interaction of transcription factors known to be induced by oxidative stress, i.e., AP-1 and NF-kB, with their recognition elements will be determined in nuclear extracts from human lung cells treated with Cr(VI). Specific DNA-protein interactions will be determined using gel mobility shift assays. (3) Hypothesis: that concentrations of Cr(VI) which alter activation of transcription factors, will induce expression of oxidative stress responsive genes at the level of transcription. The effects of Cr(VI) on the steady-state mRNA levels and transcription rates of genes known to respond to oxidative stress, i.e., 1) CL100 as an example of a gene that is sensitive to low level oxidant stress, 2) heme oxygenase as an example of a gene that is induced by high level oxidative stress and has AP-1 response elements, 3) urokinase-like plasminogen activator as a gene that is responsive to a variety of changes in signal transduction, has NF-kB response elements and whose changes in steady-state mRNA correlate well with the metastatic potential of several cancers, and 4) the constitutive non-inducible beta-tubulin gene, will be determined following treatment of human lung cells with Cr(VI). The effects of Cr(VI) treatment on the steady-state levels of expression of these genes will be determined by solution hybridization and Northern blot analyses. The effects of Cr(VI) on the rates of transcription of these genes will be determined by nuclear """"""""run-off"""""""" transcription assays. The long-range objective of this research project is to understand the mechanism by which Cr(VI) compounds act as carcinogens. The proposed studies should provide evidence for an oxidative damage pathway for Cr(VI) action on gene expression, provide insight into the mechanism by which Cr(VI) causes cancer, and thus provide fundamental insights into the overall mechanism of induction of the neoplastic process by carcinogens known to induce oxidative stress.
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