MECHANISM OF METAL CARCINOGENESIS

致癌性镍、铬引起的DNA-蛋白质交联损伤 将研究化合物。 这种损伤的形成和修复将是 通过碱洗脱技术检查。 单个蛋白质 通过镍和铬与DNA交联，将在完整的 细胞和体外。 为了研究完整培养细胞中的交联蛋白， 在哺乳动物细胞中，DNA将从用金属处理的细胞中分离出来。 化合物和用35 S甲硫氨酸固有标记的蛋白质， 不能与高盐的DNA解离，1%SDS将 通过SDS聚丙烯酰胺凝胶电泳分析。 交联 在完整细胞中发生的反应将在体外模拟， 纯化的或粗的核蛋白级分与金属和DNA。 顺序 使用该体外系统进行的添加和金属结合研究将 有助于理解发生在反应过程中的反应顺序。 完整细胞 交联反应的性质将在一些实施方案中进行检查。 详细 金属桥连的氨基酸和DNA碱基 将研究交叉连接。 镍或铬的重要性 还将检查诱导的DNA-蛋白质损伤。 含DNA蛋白质 将体外交联或在完整细胞中交联的质粒转染入NIH 3 T3 细胞，以评估这种病变对发展的贡献， 转型 通过镍或铬与DNA交联的蛋白质可以 保护某些DNA序列不被核酸酶降解。 这些 受保护的序列将被检查是否富集与 感兴趣的特异性基因组探针（即癌基因）。 DNA的影响 蛋白质交联对RNA和DNA合成的影响也将被研究， 提供对这种病变早期影响的更全面了解 这些重要的细胞过程。

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.