MITOMYCIN C-DNA ADDUCTS IN TUMOR CELLS

拟议研究的长期目标是阐明关键的 丝裂霉素C抗肿瘤作用的细胞机制 (MC)，一种广泛用于临床癌症的天然产品 化疗，并将这些信息转化为改进的使用 MC及其类似物的抗癌活性。四个细胞内变量将 检查它们对细胞毒性的影响和 MC在肿瘤细胞中形成DNA加合物的能力：(I)MC的性质 还原激活MC的酶；(Ii)体内谷胱甘肽水平 细胞；(Iii)细胞内低pH；以及(Iv)单功能 (脱氨甲酰丝裂霉素C)与双功能丝裂霉素(MC)AS 被管理的代理。所有的变量都将在 小鼠乳腺肿瘤细胞株EMT66。完全MC-DNA加合物 在细胞中形成的图案将使用[~3H]标记的MC来确定 以及通过一种方法，用高效液相色谱分析可放射性的种子加合物 这是我们实验室以前开发的。类似的分析将是 使用纯化的MC激活酶在无细胞系统中进行 和EMT6DNA，以评估内源性(细胞非依赖性)效应 以上变量与DNA加合物模式有关。未知数的结构 将测定MC-DNA加合物。MC对细胞的细胞毒作用 随着谷胱甘肽水平的提高以及 丝裂霉素代谢产物2，7-二氨基异丁二烯将在#年研究 结合DNA加合物分析。这项工作有望实现 结果I在完整细胞中鉴定(I)关键的MC激活型苯乙炔和 细胞微环境条件对其活性的调节 和(Ii)MC-DNA交联物的相对细胞毒性重要性 而不是MC-单加合物。所获得的知识应该有助于 改善丝裂霉素在癌症治疗中的使用。

The long-term goal of the proposed studies is to elucidate critical cellular mechanisms involved int he antitumor activity of mitomycin C (MC), a natural product widely employed in clinical cancer chemotherapy, and to translate this information into the improved use of MC and its analogs against cancer. Four intracellular variables will be examined with regards to their effects on the cytotoxicity and DNA-adduct-forming ability of MC in tumor cells: (i) The nature of the enzymes that reductively activate MC; (ii) the glutathione level in the cell; (iii) low intracellular pH; and (iv) monofunctional (decarbamoyl mitomycin C) versus bifunctional mitomycin (MC) as the administered agent. All of the variables will be studies in the EMT66 mouse mammary tumor cell line. Complete MC-DNA adduct patterns formed in the cells will be determined using [3H]-labeled MC and analyzing the radiolableed adducts by HPLC, by a method developed previously in our laboratoy. Analogous analyses will be conducted in cell-free system using purified MC-activating enzymes and EMT6DNA, to evaluate intrinsic (cell-independent) effects of the above variables on DNA adduct patterns. The structures of unknown MC-DNA adducts will be determined. Cytotoxicity of MC to cells with enhanced levels of gluthathione as well as the cytotoxicity of the mitomycin metabolie 2, 7-diaminomitosene will be studied in conjunction with the DNA-adduct analyses. This work is expected to result I identifying in intact cells (i) critical MC-activating enzynes and modulation of their activity by cellular microenvironmental conditions and (ii) the relative cytotoxic importance of MC-DNA cross-links versus MC-monoadducts. The knowledge obtained should contribute to improving the use of the mitomycins in the treatment of cancer.