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

说明(申请人提供)：丝裂霉素是由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。它的行动模式已经得到了广泛的审查。相比之下，10-脱氨基甲酰丝裂霉素C(DMC)并不是如此密集的研究对象。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产生一种独特的立体异构体链间交联物(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不同。特别是，与MC相反，DMC处理产生的DNA加合物(？-ICL)迅速激活了一条不依赖于p53的信号转导途径。因此，MC-DMC的研究提供了一个理想的模型，用于识别在存在或不存在功能正常的P53通路的情况下，决定细胞信号转导结果的结构特征。这一建议的中心假设是，丝裂素-α和丝裂素-β交联物(？-ICL和？-ICL)局部DNA结构的差异是导致这两种化合物产生不同生化反应的原因。特别是，我们假设DMC通过形成特定的DNA加合物而引发PI3K/Akt/p21细胞死亡途径。为了将MC和DMC加合物结构与p21在β-ICL和β-ICL毒性中的作用联系起来，将实现以下三个目标：1)通过仿生和后齐聚方法合成MC和DMC的α和βDNA交联链。2)通过分析有利的交联途径，测定脱氨甲酰丝裂霉素C(DMC)的序列选择性(CG或GC)。这将揭示药物在DNA双链中的定位：上游或下游。3)最后，通过流式细胞仪检测细胞周期停滞阶段，Western印迹分析p21蛋白的磷酸化状态，免疫细胞化学方法检测p21蛋白的定位，确定p21在MC/DMC及其交联物应答的p53非依赖性信号通路中的作用。我们将研究PI3K/Akt信号通路在p21激活中的作用。将进行聚合酶链式反应阵列，以确定受-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.