Certain transition metals, including nickel, chromium, cadmium, and copper, are carcinogenic to humans and/or animals. They induce cancer of the respiratory tract in persons exposed to metal-containing aerosols and also increase the incidence of childhood malignancies in progeny of the exposed fathers. However, mechanisms of the carcinogenic activity of these metals remain obscure. In recent years, we have been testing a hypothesis that the mechanisms would involve metal-mediated structural and/or oxidative damage to chromatin and some enzymatic proteins. In 2002/2003 we continued testing the above hypothesis. Our investigations of transition metals' interactions with chromatin components were focused on 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-terminal tail of isolated histone H2A mediates hydrolytic cleavage of its E-S peptide bond. In vivo, such cleavage has the potential of causing chromatin remodeling and gene expression derangement. Therefore, our recent experiments have been aimed at proving the occurrence of such cleavage in living cells. Analysis of histones isolated from cells of human, rat, or hamster origin, cultured with nickel carcinogens did, in fact, reveal the formation of the expected truncated version of histone H2A. Moreover, extensive modification of histone H2B, a close partner of H2A in chromatin, was also observed. Using various chromatografic, mass-spectrometric, and amino acid-sequencing techniques, we have identified two types of the H2B modifications: (1) ubiquitination, and (2) loss of a 17-mer peptide from its N-terminal tail. The nature of the other detectable modifications of this histone and the biological significance of nickel interactions with both histones are being investigated. One effect of these interactions may include derepression of gene expression. The latter might be signalled by high expression of alpha-globin found in nickel-exposed hamster ovary (CHO) cells. Globin genes are normally silent in these cells. Following our search for toxic metals' effects on enzymatic proteins, we continued investigations of possible role of the tumor suppressor protein Fhit, a diadenosine triphosphate phosphohydrolase, in the mechanisms of metal-induced carcinogenesis. Since Fhit is a pro-apoptotic protein, its suppression by nickel may contribute to the survival of cells mutated by nickel and thus assist in carcinogenesis. As we found before, Fhit' s enzymatic activity was inhibited by Cu(II), Ni(II) and other toxic metals. Also, Fhit expression was markedly lower in cells cultured with and/or tranformed by nickel carcinogens to malignant phenotype, and in nickel-induced skeletal muscle tumors in mice as compared with normal cells and tissues. At least a part of this decrease was due to nickel effect on the translation of Fhit mRNA, observed in cells at early imes after the exposure. In FY2003, we have finished a 2-year bioassay in rats aimed at determination of FHIT gene expression in target tissues of nickel carcinogenesis, kidney and muscle, during the entire course of carcinogenesis, from 3-days after treatment to the time of tumor development. Thus far, immunohistochemistry revealed absence or presence of only very low levels of Fhit protein in nickel-induced sarcomas. The premalignant tissues remain to be investigated. In addition, we have designed, expressed in bacteria, and purified a recombinant Fhit protein having the Cys-39 residue near the enzymatic active site replaced with Ala-39. This mutation caused no change in enzymatic activity of Fhit, but made this enzyme resistant to oxidative inhibition by metals like copper. This finding may indicate that the wild-type Fhit is redox-regulated. Another investigation has been focused on the MTH1 proteins, the oxidized purine nucleoside triphosphate pyrophosphohydrolases (also named 8-oxo-dGTPases), a class of mammal
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