GENOTOXICITY IN HUMAN SKIN AND KIDNEY EPITHELIAL CELLS

这项延续拨款的目的是为了描述 人类遗传毒性的分子、细胞和生化过程 肾脏和皮肤上皮细胞。具体来说，我们将解决 DNA修复在保护人类细胞中起关键作用的假说 从环境的细胞毒性、诱变和致癌作用 探员们。在过去的5年里，我们广泛地研究了这些过程 负责修复致突变前/致癌前的O6-烷基鸟嘌呤(O6- 4-烷基胸腺嘧啶(O4-烷基胸腺嘧啶)和嘧啶二聚体 来自人类肾脏和皮肤的提取物和细胞培养。这些 研究提供了对组织和组织修复活动的新见解 培养的细胞群体。随着分子和细胞的出现 技术，我们现在开始关注器官、细胞和基因 对基因毒素暴露的特殊反应。两个器官，人的肾脏和 修复O6-烷基瓜活性不同的皮肤将有助于确定 对基因毒素的器官特异性反应。在这两个组织内和在 从组织培养的细胞，原位杂交技术是 开始在体内鉴定O6-甲基瓜--的细胞多样性 DNA甲基转移酶(O6-MT)基因。这一点尤其重要，因为 O6-MT在O6-烷基瓜加合物的修复过程中被消耗。因此，这就是 系统应建立器官和细胞对 基因毒素。一旦DNA修复在各种细胞类型中的分布 肾脏和皮肤组织的特征，其性质和光谱 O_6-乙基瓜氨酸和O_4-乙基胸苷诱发的突变及DNA修复修饰 将在从这些器官培养的这些细胞类型中确定。这个 突变分析将利用具有良好特征的编码序列 HPRT基因。这些研究将有助于更好地理解 DNA修复在遗传毒性中的分子机制及其与遗传毒性的关系 器官和细胞对细胞毒性、诱变性和 烷化剂的致癌作用。因此，对此的研究 续期补助金申请旨在提供新的信息和 对与DNA和DNA烷基化有关的关键问题的见解 修复过程对人类的遗传毒性。

The objectives of this continuation grant are to characterize the molecular, cellular and biochemical processes of genotoxicity in human kidney and skin epithelial cells. Specifically we will address the hypotheses that DNA repair plays a critical role in protecting human cells from the cytotoxic, mutagenic and carcinogenic effects of environmental agents. Over the past 5 years we have extensively studied the processes responsible for repairing premutagenic/procarcinogenic O6-alkylguanine (O6- alkylGua), O4-alkylthymine (O4-alkylThy) and pyrimidine dimers in cell-free extracts and cell cultures derived from human kidney and skin. These studies have provided new insights into repair activity in tissue and cultured cell populations. With the emergence of molecular and cellular techniques, we are now beginning to focus on the organ, cell and gene specific responses to genotoxin exposure. Two organs, human kidney and skin, of differing activity for repairing O6-alkylGua, will help determine the organ specific responses to genotoxins. Within these 2 tissue and in cells cultured from the tissues, in situ hybridization techniques are beginning to identify, in vivo, the cellular diversity of the O6-methylGua- DNA methyltransferase (O6-MT) mRNA. This is especially important since the O6-MT is consumed during the repair of the O6-alkylGua adduct. Thus this system should establish the organ and cell specific susceptibility to genotoxins. Once the distribution of DNA repair in the various cell types of the kidney and skin tissue is established, the nature and spectra of mutations induced by O6-EthylGua and O4-EthylThy and modified by DNA repair will be determined in these cell types cultured from these organs. The mutational analyses will utilize the well characterized coding sequence of the HPRT gene. These studies will lead to a better understanding of the molecular mechanisms of DNA repair in genotoxicity and its relationship to the organ and cell specific sensitivity to the cytotoxic, mutagenic and carcinogenic effects of alkylating agents. Thus, the studies of this renewal grant application are designed to provide new information and insights into those pivotal questions relating alkylation of DNA and DNA repair processes to human genotoxicity.