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

描述（由申请人提供）：丝裂霉素是由链霉菌产生的抗肿瘤抗生素家族。该家族的一个成员丝裂霉素C（MC）目前用于治疗某些癌症。它的作用方式已被广泛研究。相比之下，10-十氨甲酰基丝裂霉素C（DMC）尚未成为如此密集的研究对象。最近发现它与DNA形成与丝裂霉素C（MC）相似或相同的加合物。特别是，DMC产生独特的立体异构体链间交联（β异构体，β-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（β-ICL）产生的DNA加合物快速激活p53非依赖性信号转导途径。因此，研究MC-DMC提供了一个理想的模型，用于确定在存在或不存在功能性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，线粒体-α和线粒体-β交联加合物（β-ICL和β-ICL）的局部DNA结构的差异是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由于形成的特异性DNA加合物而引发PI 3 K/Akt/p21细胞死亡途径。为了将MC和DMC加合物的结构与p21在β- ICL和β-ICL的毒性中的作用相关联，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)通过分析有利的交联途径确定十氨甲酰丝裂霉素C（DMC）β-ICL的序列选择性（CG或GC）。这将揭示药物在DNA双链体中的方向：上游或下游。3)最后，p21在响应MC/DMC的上游p53非依赖性信号通路中的作用以及这些交联将通过使用流式细胞仪验证细胞周期停滞阶段、Western印迹分析以揭示p21磷酸化状态以及免疫细胞化学以定位p21来确定。将检查PI 3 K/Akt信号通路在p21活化中的参与。将进行PCR阵列以鉴定由β-和β-ICLs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.