ENZYMES AND REACTIONS FOR REPAIR OF DNA

几乎所有活着的生物都进化出DNA烷基转移酶来修复 鸟嘌呤O6-位诱变致癌加合物的研究 DNA通过烷基化试剂，如甲基亚硝脲。这种O6-烷基鸟嘌呤- DNA烷基转移酶(MGMT，以前称为GATase)可以顺便 抗癌药物诱导的细胞毒DNA交联修复前体的研究 氯乙基亚硝脲(Cenus)所有正常人细胞系(Mer+/Mex+) 含有MGMT，而20%-30%的肿瘤细胞系(Mer-/Mex-)缺乏这种作用 并对这些药物的治疗效果过敏 以及简单甲基化试剂的诱变效应。墨西哥人- 亚组肿瘤有望对以下治疗产生有利反应 然而，因为实体瘤总是由正常的 细胞，不可能通过简单的测量来识别粘液瘤 肿瘤活检组织提取液中GAT酶活性的研究这项提议的一个目标 因此，正在建立人类MGMT表达的组织化学检测方法 肿瘤和组织准备。为此，我们正在大力生产 特异性的单抗和多克隆抗体。此外，最近的克隆 人MGMT基因的表达水平的测定使我们能够确定MGMT基因的表达水平 这种制剂中的MGMT基因。初步数据显示，墨西哥- 肿瘤细胞既不表达mgmt蛋白，也不表达mrna，因此我们 假设转录调控对MEX至关重要 表型。我们计划将这些观察扩展到更大的一系列 其他肿瘤系包括人类横纹肌肉瘤和脑瘤 具有MGMT表达谱的异种移植株以及正常 分化的人体组织。我们还将确定基因缺失， 在某些情况下，扩增或重排与MGMT水平相关。 我们将通过确定转录控制机制是否 Mex+和Mex-细胞中MGMT mRNA水平的差异与 随着基因甲基化状态的变化，或随着 顺式作用基因的调控元件，或反式作用的变化 蛋白质转录因子。最终，我们将分离和表征 这样的调节蛋白。正常二倍体细胞从MEX+到Mex+的进展 在病毒诱导的转化和永生过程中将使用MEX-DIME 作为研究MGMT监管的模型。详细分析了 人MGMT分子元件的结构、功能及其调控 活性最终将使人能够预测肿瘤对 氯乙基亚硝脲，以及个人对 致癌。它还可以表明在哪里可以成功 对这些过程进行干预。GRANT=R01CA16265 我们最近的工作为7的转换建立了三条不同的路线， 12-二甲基苯并(A)菲合成致癌前体3，4-二氢二醇 分别在胚胎成纤维细胞、肾上腺皮质和肝脏中。两个新的 在DMBA激活中非常有效的细胞色素P450形式有 在小鼠胚胎成纤维细胞(P450-EF)和大鼠肾上腺中发现 皮质(P450-RAP)。这些细胞色素已被提纯，并具有特异性的抗- 已生成P450 IGG，将用于克隆相应的 使用分别从C3H/10T1/2小鼠胚胎中获得的文库 成纤维细胞和大鼠肾上腺皮质。对P450-EF的相对诱导作用 苯并(A)菲和TCDD建议除了 通过阿受体的刺激。阿受体的参与 将通过使用拮抗剂和同源阿氏反应和 无反应的胚胎成纤维细胞。其他机制，包括蛋白质 稳定性，将进行测试。P450-EF cDNA和抗P450-EF免疫球蛋白 用来确定诱导的机制，最终我们的目标是 获得该基因的5‘-侧翼序列，这将允许定义 融合细胞和肿瘤中调控的分子机制 从转化的胚胎成纤维细胞生长出来的。定义的无血清介质将 用于确定P450-EF是否受特定的细胞外调节 各种因素。特异性c DNA、抗P450抗体和抗体敏感型DMBA 代谢将用于研究P450-EF的分布和调节 (在成纤维细胞、皮肤和乳腺细胞)和P450-RAP(在肾上腺、卵巢、 和睾丸)对各种生长因子和激素作出反应。 不同多环芳烃代谢的特异性及其潜在的天然产物 将检查底物(类固醇、二十烷类化合物、脂肪酸)。在……里面 肝脏，先前的数据引导我们评估了DMBA的两种膜对照 代谢：(1)磷脂酰丝氨酸和磷脂酰丝氨酸激活P450IIC6 细胞色素b5；(2)选择性抑制DMBA代谢 由P450IIB1相对于P450IIB2通过这些或其他膜成分。 为这些P450细胞色素定义的机制将被评估为其他 已知在多环芳烃代谢中活跃的P450(包括P450-EF，P450-RAP， 和P450IA1)。正常和修饰的P450基因在大肠杆菌中的表达 转基因技术将允许对这些基因进行分子解释 DMBA代谢的变化。

Almost all living organisms have evolved DNA alkyltransferases to repair mutagenic and carcinogenic adducts induced at the O6-position of guanine in DNA by alkylating agents such as methylnitrosourea. Such O6-alkylguanine- DNA alkyltransferase (MGMT previously called GATase) can incidentally repair precursors of cytotoxic DNA crosslinks induced by anticancer chloroethylnitrosoureas (CENUs). All normal human cell lines (Mer+/Mex+) contain MGMT whereas 20-30% of tumor cell lines (Mer-/Mex-) lack this activity and are hypersensitive to the therapeutic effects of these drugs as well as to the mutagenic effects of simple methylating agents. The Mex- subset of tumors would be expected to respond favorable to therapy with CENUs; however, because solid tumors are invariably infiltrated by normal cells, it is not possible to identify Mex- tumors by simply measuring GATase activity in extracts of tumor biopsies. A goal of this proposal therefore is to develop histochemical assays for MGMT expression in human tumor and tissue preparations. To this end we are producing highly specific monoclonal and polyclonal antibodies. Moreover the recent cloning of the cDNA for human MGMT enables us to determine the levels of mRNA and the gene for MGMT in such preparations. Preliminary data suggest that Mex- tumor cells express neither the MGMT protein nor the mRNA hence we hypothesize that regulation of transcription is crucial for the Mex phenotype. We plan to extend these observations to a larger series of other tumor lines including human rhabdomyosarcoma and brain tumor xenograft lines having a spectrum of MGMT expression, as well as normal differentiated human tissues. We will also determine if gene deletion, amplification or rearrangement correlate with MGMT levels in some cases. We will examine transcriptional control mechanisms by determining if differences in MGMT mRNA levels in Mex + and Mex - cells are associated with changes in methylation status of the gene, or with changes in cisacting regulatory elements of the gene, or with changes in trans-acting protein transcription factors. Ultimately we will isolate and characterize such regulatory proteins. Progression of normal diploid cells from Mex+ to Mex- during virally induced transformation and immortalization will be used as a model for investigating MGMT regulation. Detailed analysis of the structure, function, and regulation of the molecular elements of human MGMT activity will ultimately enable one to predict tumor resistance to chloroethylnitrosoureas, as well as an individual's susceptibility to carcinogenesis. It may also indicate where one could successfully intervene in these processes. GRANT=R01CA16265 Our recent work has established three distinct routes for conversion of 7, 12-dimethylbenz(a)anthracene to the carcinogenic precursor 3, 4-dihydrodiol in, respectively, embryo fibroblasts, adrenal cortex, and liver. Two new forms of cytochrome P450 which are very effective in DMBA activation have been identified in mouse embryo fibroblasts (P450-EF) and in rat adrenal cortex (P450-RAP). These cytochrome have been purified, and specific anti- P450 IgGs have been generated that will be used to clone the corresponding cDNAs using libraries generated from, respectively, C3H/10T1/2 mouse embryo fibroblasts and rat adrenal cortex. The relative inductions of P450-EF by benzo(a)anthracene and TCDD suggest regulatory mechanisms in addition to stimulation through the Ah-receptor. The participation of the Ah-receptor will be tested by use of an antagonist and also congenic Ah-responsive and non-responsive embryo fibroblasts. Other mechanism, including protein stabilization, will be tested. P450-EF cDNA and anti-P450-EF IgG will be used to determine the mechanism of induction, and ultimately we aim to obtain 5'-flanking sequences for this gene that will permit definition of the molecular mechanism of regulation in confluent cells and in tumors grown from transformed embryo fibroblasts. Defined serum-free media will be used to determine whether P450-EF is regulated by specific extracellular factors. Specific cDNA, anti-P450 IgG, and antibody-sensitive DMBA metabolism will be used to study the distribution and regulation of P450-EF (in fibroblasts, skin, and mammary cells) and P450-RAP (in adrenal, ovary, and testis) in response to a variety of growth factors and hormones. Specificity for metabolism of different PAHs and potential natural substrates (steroids, eicosanoids, fatty acids) will be examined. In liver, previous data leads us to evaluate two membrane controls over DMBA metabolism: (1) activation of P450IIC6 by phosphatidylserine and cytochrome b5; and (2) selective suppression of DMBA metabolism catalyzed by P450IIB1 relative to P450IIB2 by those or other membrane components. Mechanisms defined for these P450 cytochromes will be assessed for other P450s known to be active in PAH metabolism (including P450-EF, P450-RAP, and P450IA1). Expression of normal and modified P450s by use of transfection techniques will allow molecular interpretation of these changes in DMBA metabolism.