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.
Statement A lot of chemotherapeutic agents function through the activation of p53 and other apoptotic pathways, however at least 50 percent of all cancers do not have a functional p53-pathway. The proposed research will determine the involvement of p21 in the p53-independent mode of cell death triggered by DMC DNA adducts. This has the potential to offer treatment which could be tailored to cancer cells lacking the p53 transcriptiona factor.
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