GLUTATHIONE & RELATED ENZYMES IN CELULAR DRUG RESISTANCE

丝裂霉素C(MMC)通常用于治疗膀胱癌，排名第三 美国男性中流行的恶性疾病。临床疗效观察 MMC的产生往往受到耐药肿瘤细胞的出现的限制 通过机理(S)还没有完全理解。的总体目的 这项研究建议是为了深入了解(S)的发病机制。 对MMC的抵抗，特别强调了解MMC的作用 谷胱甘肽(GSH)和相关的酶在这一过程中。GSH水平将是 比较敏感和耐药的膀胱癌细胞及其相关性 具有MMC细胞毒性。为了进一步证实GSH 水平影响MMC反应，BSO诱导的GSH耗竭对MMC的影响 细胞毒性将通过克隆形成实验和体内实验进行研究。 在裸鼠移植瘤中。此外，对上单元格子集的排序 还将尝试根据GSH含量来寻求GSH之间的相关性 含量和MMC细胞毒性。谷胱甘肽相关酶水平， 特别是包括新型单体形式的GSH过氧化物酶 在这些细胞中确定，并与MMC细胞毒性相关。自.以来 单体过氧化物酶与其他过氧化物酶的相互关系不明显 已知，这种酶将被提纯和鉴定。表达式和 正常人谷胱甘肽S转移酶同工酶的特性 将对膀胱进行调查，以确定组织特异性GST。这些级别 的GST同工酶将在敏感和耐药细胞中进行比较 以查看特定类别的变化-毒性。为了获得 深入了解GST可能导致MMC耐药的机制， GST介导的MMC的催化和非催化解毒 用纯化的GST和原位研究GST在体外的敏感性和抗性 细胞。将进行研究，以确定MMC耐药性是否可以逆转 通过不可逆转的谷胱甘肽转移酶抑制剂。丝裂霉素C诱导的DNA交联会 用碱洗脱法测定敏感细胞和耐药细胞的含量。 参与脱氧核糖核酸交联物去除的酶的程度和水平将 也可以在这些细胞中进行比较。此外，MMC的贡献 诱导型自由基清除剂对ESR产生自由基的影响 克隆形成实验中的光谱和MMC细胞毒性。从长远来看， 拟议的研究可能有助于制定规避的策略 细胞对MMC的抵抗力。

Mitomycin C (MMC) is commonly used to treat bladder cancer, the third most prevalent malignant disease among males in USA. The clinical effectiveness of MMC is often limited by the emergence of drug resistant tumor cells through mechanism(s) not completely understood. The overall purpose of this research proposal is to gain insight into the mechanism(s) of resistance to MMC with particular emphasis on understanding the role of glutathione (GSH) and related enzymes in the process. GSH levels will be compared in sensitive and resistant bladder cancer cells and correlated with MMC cytotoxicity. In order to further substantiate that the GSH levels affect MMC response, effect of BSO induced GSH depletion on MMC cytotoxicity will be studied in vitro in colony forming assay and in vivo in nude mouse xenografts. In addition, sorting of subsets of cells on the basis of GSH content will also be attempted to seek correlation between GSH content and MMC cytotoxicity. The levels of GSH related enzymes, particularly GSH peroxidases including the novel monomeric form will be determined in these cells and correlated with MMC cytotoxicity. Since interrelationship of monomeric peroxidase with other peroxidases is not known, this enzyme will be purified and characterized. Expression and characteristics of GSH S-transferase (GST) isoenzymes of normal human bladder will be investigated to identify tissue specific GSTs. The levels of GST isoenzymes will be compared in sensitive and resistant cells in order to see alterations in specific class-toxicity. In order to gain insight into the mechanism by which GSTs may contribute to MMC resistance, GST mediated catalytic and non-catalytic detoxification of MMC will be studied in vitro using purified GST and in situ in sensitive and resistant cells. Studies will be performed to see if MMC resistance can be reversed by irreversible inhibitors of GST. MMC induced DNA cross-links will be quantitated in sensitive and resistant cells by alkaline elution assay. The extent and levels of enzymes involved in removal of DNA cross-link will also be compared in these cells. In addition, the contribution of MMC induced free radical scavengers on free radical generation by ESR spectroscopy and MMC cytotoxicity in colony forming assay. In long term, the proposed studies may be helpful in devising strategies to circumvent cellular resistance to MMC.