Certain transition metals, including nickel, chromium, cadmium, and copper, are carcinogenic to humans and/or animals. Their effects include cancer in the progeny of fathers exposed to welding fumes and other metal dusts. However, mechanisms of the carcinogenic activity of these metals remain obscure. In recent years, we have been testing a hypothesis that one such mechanism would involve metal-mediated structural and/or oxidative damage to DNA and nuclear proteins. In 2001/2002 we continued testing the above hypothesis. Our investigations of transition metals' interactions with nuclear proteins were focused on the Ni(II)-assisted hydrolysis of histone H2A. As we have found before in test-tube experiments, Ni(II) bound to the -KTESHHKAKGK motif at the C-terminus of isolated histone H2A causes hydrolytic cleavage of its E-S peptide bond. Besides other effects, this cleavage has the potential of causing chromatin remodeling and deranging gene expression. Therefore, our recent experiments have been aimed at proving (or disproving) the occurrence of such cleavage in living cells exposed to nickel. Analysis of histones isolated from cells of human (HPL1D), rat (NRK-52), or hamster (CHO) origin, cultured with water-soluble and insoluble nickel carcinogens (Ni(II) acetate and nickel subsulfide, respectively), revealed the formation of the expected truncated version of histone H2A. The magnitude of this effect depends on nickel concentration in the culture medium (0.1 - 1 mM) and exposure time (3 - 10 days). Possible secondary results of H2A truncation, noticed in our experiments, included high expression of globins, normally repressed in CHO cells, and extensive modification of histone H2B, a close partner of H2A in chromatin. This may indicate that the structural damage to histone H2A, inflicted by Ni(II), can lead to chromatin remodeling and affect gene expression. Following our search for metal interactions with enzymatic proteins, we continued investigations of the possible role of the tumor suppressor protein Fhit, a diadenosine triphosphate phosphohydrolase, in the mechanisms of metal-induced carcinogenesis. Our studies have been focused on Fhit expression in cells cultured with and/or tranformed by nickel carcinogens. A fifty percent reduction of Fhit protein level was observed in nickel-transformed mouse cells (B200), when compared to their parental NIH 3T3 fibroblasts, and in nickel subsulfide-induced skeletal muscle tumors in mice as compared with normal muscles. These terminal effects prompted us to check the early response of cellular Fhit to nickel. Rat renal tubular epithelial cells NRK-52 were cultured with different doses of nickel subsulfide and their Fhit protein was analyzed by Western blotting with the use of polyclonal anti-Fhit antibodies. FHIT gene transcript was determined by RT-PCR. At 3 days post exposure, Fhit level was reduced in a nickel dose-dependent manner to 10% of the control. In contrast, no changes in FHIT mRNA expression have been found. Therefore, the observed reduction in Fhit levels in nickel-treated cells may be due to either translation suppression, or changes in Fhit protein turnover. Since Fhit is a pro-apoptotic protein, its suppression by nickel may contribute to the survival of cells mutated early after nickel exposure and thus assist in carcinogenesis. Another investigation has been focused on the MTH1 proteins, the oxidized purine nucleoside triphosphate pyrophosphohydrolases (also named 8-oxo-dGTPases), a class of mammalian enzymes preventing incorporation of promutagenic oxidized purine nucleotides, such as 8-oxo-dGTP, 8-oxo-dATP, 2-OH-dATP, 2-OH-ATP, 8-oxo-GTP, and 8-oxo-ATP into DNA or RNA. The basal enzymatic activity of MTH1 proteins has been determined in 59 human cancer cell lines, employed routinely to screen anti-cancer drugs, in order to test if this activity would correlate with patho-physiology of the original tumor, e.g., tissue of origin, histologic type, the dynamics of growth, formation of metastases, and susceptibility to certain treatments, especially with radiation and radio-mimetic drugs. Such correlations, if any, might be used as diagnostic or prognostic markers. In addition, our research provides an experimental basis for collaborative studies on oxidative DNA damage by other chemical carcinogens (see projects ZO1 BC 05352 and ZO1 BC 05488).
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