Role of p21 signaling pathway in response to MC & DMC DNA Interstrand Crosslinks

描述（由申请人提供）：丝裂霉素是链霉菌生产的一类抗肿瘤抗生素。该家族的成员之一丝裂霉素C （MC）目前被用于治疗某些癌症。其作用方式已被广泛研究。相比之下，10-decarbamoyl丝裂霉素C （DMC）尚未成为如此深入研究的对象。最近发现它与DNA形成类似或相同的加合物，如丝裂霉素C （MC）。特别是，DMC产生独特的立体异构体链间交联（β异构体，¿-ICL）。虽然两种药物的dna加合物具有共同的结构特征，但对两种药物的生化反应是不同的。特别的是，与MC相反，DMC处理产生的dna加合物（¿-ICL）迅速激活了一个不依赖p53的信号转导通路。因此，MC-DMC研究提供了一个理想的模型，用于识别在p53通路存在或缺失的情况下决定细胞信号转导结果的结构特征。本研究的核心假设是，有丝分裂- α和有丝分裂- β交联加合物（¿-ICL和¿-ICL）的局部DNA结构的差异导致了这两种化合物产生的不同生化反应，特别是，我们假设由于形成的特定DNA加合物，DMC引发了PI3K/Akt/p21细胞死亡途径。为了将MC和DMC-加合物结构与p21在¿-ICL和¿-ICL毒性中的作用联系起来，将实现以下三个目标：1)通过仿生和后寡聚化方法合成MC和DMC的α和β DNA交联。2)通过分析优选的交联途径，确定decarbamoyl mitomycin C （DMC）¿-ICL的序列选择性（CG或GC）。这将揭示药物在DNA双链中的方向：上游或下游。3)最后，通过流式细胞仪验证细胞周期阻滞阶段，Western blot分析揭示p21磷酸化状态，免疫细胞化学定位p21，确定p21在上游p53独立信号通路中响应MC/DMC和这些交联的作用。我们将研究PI3K/Akt信号通路在p21激活中的作用。PCR阵列将用于鉴定受-和- icl调控的基因。

DESCRIPTION (provided by applicant): The mitomycins are a family of antitumor antibiotics made by Streptomyces. One member of this family, mitomycin C (MC), is currently used to treat certain cancers. Its mode of action has been extensively examined. In comparison, 10-decarbamoyl mitomycin C (DMC) has not been the object of such intense investigation. It has recently been found to form similar or identical adducts with DNA as mitomycin C (MC). In particular, DMC generates a unique stereoisomeric interstrand crosslink (beta isomer, ¿-ICL). Although the DNA-adducts of both drugs share common structural features, the biochemical responses to the two drugs are different. In particular, contrary to MC, the DNA-adducts generated by DMC treatment (¿-ICL) rapidly activate a p53-independent signal transduction 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 central hypothesis of this proposal is that the differences in the local DNA structures of the mitosene-alpha and mitosene-beta crosslink adducts ( ¿-ICL and ¿-ICL) are responsible for the different biochemical responses produced by the two compounds in particular, we hypothesize that DMC provokes a PI3K/Akt/p21 cell death pathway due to the specific DNA-adducts formed. In order to correlate MC and DMC-adducts structures with the role of p21 in the toxicity of the ¿- ICL and ¿-ICL, the following three aims will be achieved: 1) Synthesis of the alpha and beta DNA crosslinks of MC and DMC via biomimetic and post-oligomerization methods. 2) Determination of the sequence selectivity (CG or GC) for the ¿-ICL of decarbamoyl mitomycin C (DMC) through analysis of the favored cross-linking pathway. This will reveal the orientation of the drug in the DNA duplex: upstream or downstream. 3) Finally, the role of p21 in the upstream p53-independent signaling pathway in response to MC/DMC and these crosslinks will be determined by using flow cytometer to verify the cell cycle arrest stage, Western blot analysis to reveal the p21 phosphorylation status and immunocytochemistry to localize p21. The involvement of the PI3K/Akt signaling pathway in p21 activation will be examined. PCR array will be performed to identify genes regulated by the ¿- and ¿-ICLs.