Role of p21 in the toxicity of MC and DMC DNA Interstrand Crosslinks

摘要 这项提案的总体目标是调查下列结构之间的关系 丝裂霉素C和丝裂霉素形成的立体异构体DNA链间交联物 脱氨酰丝裂霉素C及其药物的分子机制。丝裂霉素C(MC)是 一种抗癌药物，目前用于治疗胃癌、肛门癌和肺癌。立体化学 MC大调ICL的C1‘’处的配置为R(αICL)。相比之下，脱氨甲酰丝裂霉素C(DMC)， 缺少O10氨基甲酰基的MC的衍生物产生S立体异构体ICl(β ICL)。拟议研究的科学前提是ICL构成了分子基础 丝裂霉素的细胞毒性作用。中心假说是局部的差异 α和β-ICL的dna结构决定了不同的生化反应。 由MC和DMC触发。特别是，与MC相反，DMC产生的DNA加合物 治疗(-ICL)迅速激活一条不依赖于P53的细胞死亡途径。因此，研究MC- DMC为识别决定细胞信号转导的结构特征提供了理想的模型 在存在或不存在功能正常的P53通路的情况下的结果。 本项目的意义在于确定了化合物的构效关系 立体异构体DNA交联物。此外，拟议的研究将确定 α和β-ICL作为不同细胞死亡途径的生物信号。关键创新 这个项目的目的是在一定范围内产生立体异构体ICL，这将使研究 使用我们新开发的仿生方法进行生化反应。最后，由于p53肿瘤 抑制子在人类癌症中经常发生突变，需要识别药物和途径 不依赖于P53而导致细胞死亡或细胞周期停滞的机制值得高度重视。 为了将MC和DMC-加合物结构与α-ICL和β-ICL的毒性相关联， 主要目标如下：1)合成MC和DMC DNA加合物。2) P53依赖性和非依赖性MC/DMC DNA加合物反应的测定 使用蛋白质组学的机制。3)确定所涉及的分子信号通路 MC/DMC DNA加合物诱导细胞周期阻滞于G2/M期。 我们的研究将揭示分子和细胞网络受MC/DMC和 α/β-ICL在P53依赖和P53非依赖细胞系中的表达。因此，拟议的工作将 寻找MC和DMC的新的关键分子治疗靶点。

Abstract The overarching goal of this proposal is to investigate the relationship between the structure of stereoisomeric DNA Interstrand Crosslinks (ICLs) formed by Mitomycin C and Decarbamoylmitomycin C and the molecular mechanisms of these drugs. Mitomycin C (MC) is an anticancer drug currently used to treat stomach, anal and lung cancers. The stereochemical configuration at C1’’ of MC major ICL is R (α ICL). In contrast, Decarbamoylmitomycin C (DMC), a derivative of MC lacking the O10 carbamoyl group, generates the S stereoisomeric ICL (β ICL). The scientific premise of the proposed research is that ICLs constitute the molecular basis for the cytotoxic effects of mitomycins. The central hypothesis is that differences in the local DNA structures of the α and β-ICLs are responsible for the distinct biochemical responses triggered by MC and DMC. In particular, contrary to MC, the DNA-adducts generated by DMC treatment (-ICL) rapidly activate a p53-independent cell death pathway. Thus, the study MC- DMC provides an ideal model for identifying structural features determining the cell signaling outcome in the presence or the absence of a functioning p53 pathway. The significance of this project lies in determining the structure-activity relationship for stereoisomeric DNA crosslink adducts. In addition, the proposed research will establish how the α and β-ICLs behave as biological signals to different cell death pathways. The key innovation of this project is to generate stereoisomeric ICLs on a scale which will allow the study of biochemical responses using our newly developed biomimetic method. Finally, since p53 tumor suppressor is frequently mutated in human cancers, the need to identify drugs and pathways that induce cell death or cell cycle arrest independently of p53 deserves substantial attention. In order to correlate MC and DMC-adducts structures with the toxicity of the α-ICL and β-ICL, the following three aims will be pursued: 1) Synthesis of MC and DMC DNA adducts. 2) Determination of p53-dependent and independent MC/DMC DNA adducts response mechanisms using proteomics. 3) Determination of the molecular signaling pathway involved the in G2/M cell cycle arrest triggered by MC/DMC DNA-adducts. Our study will reveal molecular and cellular networks up or downregulated by MC/DMC and the α/β-ICLs in p53-dependent and p53-independent cell lines. The proposed work will therefore identify novel critical molecular therapeutic targets of MC and DMC.