MECHANISM OF METAL CARCINOGENESIS

致癌镍、铬所致的dna -蛋白交联损伤

The DNA-protein crosslink lesion caused by carcinogenic nickel and chromium compounds will be studied. The formation and repair of this lesion will be examined by the alkaline elution technique. Individual proteins crosslinked to the DNA by nickel and chromium will be studied in intact cells and in vitro. To study the proteins crosslinked in intact cultured mammalian cells, DNA will be isolated from cells treated with metal compounds, and proteins intrinsically labeled with 35S methionine that cannot be dissociated from the DNA with high salt and 1% SDS will be analyzed by SDS polyacrylamide gel electrophoresis. The crosslinking reaction occurring in the intact cell will be modeled in vitro by reacting purified or crude nuclear protein fractions with metal and DNA. Order of addition and metal binding studies conducted with this in vitro system will facilitate an understanding of the reaction sequence occurring in the intact cell. The nature of the crosslink reaction will be examined in some detail. The amino acid and the DNA base involved in the metal-bridged crosslinks will be studied. The significance of the nickel or chromium induced DNA-protein lesion will also be examined. DNA containing proteins crosslinked in vitro or in the intact cell will be transfected into NIH 3T3 cells to assess the contribution of this lesion towards the development of transformation. Proteins crosslinked to DNA by nickel or chromium may protect certain DNA sequences from degradation with nucleases. These protected sequences will be examined for enrichment in DNA homologous to specific genomic probes of interest (i.e. oncogenes). The effect of DNA protein crosslinks on RNA and DNA synthesis will also be examined to provide a more complete understanding of the early effects of this lesion on these important cellular processes.