ANTHRACYCLINE-INDUCED DNA SEQUENCE SPECIFIC MUTATION

阿霉素和相关的蒽环类类似物仍处于前线 癌症化疗中的药物，尽管有相关的毒性 限制它们的长期临床使用。然而，机制之间的相互作用 导致其毒性的原因尚不清楚。我们实验室最近的工作 已经证明了阿霉素与操作者的选择性相互作用 在uvrB大肠杆菌菌株中的lacI区域，这强烈地暗示了一个 阿霉素与该回文结构的相互作用 这种药物的致突变性。这项建议是为了调查 蒽环类药物对日本血吸虫LACO区诱变特异性的基础 在体外、原核和真核系统中的乳胶操纵子。在……里面 原核细胞，将确定蒽环类药物的突变特异性 抑制子诱导和非诱导下LACO和LacI的i-d区 条件。LACO基因靶标作为单拷贝整合因子的反应 在Laci控制下，将同时在哺乳动物细胞中进行研究。 拟议的研究将把定点突变方法纳入 正向突变分析，它将特别强调 导致遗传物质缺失的过程。推定缺失 启动序列将通过分析体内诱导的 大肠杆菌lacI/laco基因靶点的突变及体外研究 体外实验将包括足迹法来确定结合 阿霉素对线性和回文单链DNA的特异性 阿霉素诱导的DNA修复识别位点的构建 酶、核酸酶SP和uvrABC外切酶以及阿霉素诱导的 可切割的复杂位点。这项工作的总体目标是提供一个 更好地理解DNA序列依赖药物作用的基础 细胞对阿霉素毒性和突变反应中的相互作用 作为研究嵌入剂DNA导向机制的模型 多效性DNA损伤潜力。

Doxorubicin and related anthracycline analogues have remained front-line drugs in chemotherapy of cancer in spite of associated toxicities that limit their long term clinical use. However, the interplay of mechanisms leading to their toxicity remains unclear. Recent work in our laboratory has demonstrated a selective interaction of doxorubicin with the operator region of lacI in a uvrB E. coli strain, which strongly implicates a doxorubicin interaction with this palindromic structure as an intermediate in the mutagenicity of this drug. This proposal is to investigate the basis of the mutagenic specificity of anthracyclines for the lacO region of the lac operon, in vitro and in procaryotic and eukaryotic systems. In procaryotes, mutational specificity of anthracyclines will be determined for lacO and the i-d region of lacI under repressor-induced and uninduced conditions. Response of the lacO gene target as a single copy integrant under lacI control will be simultaneously investigated in mammalian cells. Proposed studies will incorporate site specific mutagenesis approaches into a forward mutational analysis, which will place special emphasis on processes leading to deletions of genetic material. Putative deletion initiating sequences will be identified from analysis of in vivo-induced mutations and in vitro studies in the lacI/lacO gene target of E. coli. Experiments in vitro will include footprinting to determine the binding specificity of doxorubicin for linear and palindromic single stranded DNA constructs, doxorubicin-induced recognition sites for the DNA repair enzymes, nuclease SP and uvrABC exinuclease, as well as doxorubicin-induced cleavable complex sites. The overall aim of this work is to provide a basis for better understanding of the role of DNA sequence dependent drug interactions in the toxic and mutational responses of cells to doxorubicin as a model for study of the DNA directed mechanisms of intercalators with pleiotropic DNA damage potential.