Mitomycin C and Decarbamoyl mitomycin C

Research project 1

“Role of p21 in the upstream p53-independent signaling pathway in response to MC and DMC DNA Interstrand Crosslinks.”

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, β-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 (β-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 research work is that differences in the local DNA structures of the mitosene-alpha and mitosene-beta crosslink adducts (α-ICL and β-ICL) are responsible for 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.

crosslinks
Figure 1: Mitomycin C (MC) and Decarbamoyl Mitomycin C (DMC) interstrand cross links (ICLs)

In order to correlate MC and DMC-adducts structures with the role of p21 in the toxicity of the α-ICL and β-ICL, we are working toward the completion of the following three aims:
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 β-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 and DMC, and in response to the crosslinks themselves will be determined by using western blot analysis and immunocytochemistry. PCR array will be performed to identify genes regulated by the α- and β-ICLs. MCF-7 (p53 dependent) and K562 (p53 mutant) cell lines will be utilized.

pathways
Figure 2: Hypothetical p21 signaling pathways in normal and p-53-deficient cell lines.

We have already accomplished the following aims and established the following conclusions regarding the mechanisms of MC and DMC DNA-adducts specific biological responses:

1) Synthesis of the alpha ICL-MC DNA adduct in an 18 MER sequence.
2) Synthesis and of the trans and cis monoadducts at the nucleoside level.
(Champeil E., Cheng S. Y., Huang B.T., Conchero-Guisan M., Martinez T., Paz M., Sapse A.M. “Synthesis of Mitomycin C and Decarbamoylmitomycin C N2 deoxyguanosine-adducts” Bioorganic Chemistry, 65 2016 90 and Champeil E., Paz M., Lukasiewicz E., Kong W., Watson S., Sapse A.M.“Synthesis of a major mitomycin C DNA adduct via a triaminomitosene”. Bioorganic and Medicinal Chemistry Letters, 22, 2012 7198)
3) Cell cycle distribution patterns of MCF-7 and K562 cells treated with MC and DMC and with the α-ICL
4) Measure of the activation of p21WAF1/CIP1 in MCF-7 and K562 cells under MC and DMC treatment.
5) We found that the activation of p21WAF1/CIP1 by MC and DMC in MCF-7 is p53 independent.
6) We found that the α-ICL itself can activate p21WAF1/CIP1, but not halt the cell cycle at G1/S phase in MCF-7.

Research project 2

“Synthesis of dG-N2-MC and dG-N2-2,7-DAM”

Our laboratory also been involved in collaborative projects with Prof. Ashis Basu at the University of Connecticut regarding Translesion Synthesis (TLS) Efficiency of Mitomycin C-DNA adducts.
Mitomycin C forms six DNA adducts, including mono-adducts and both inter and intra-strand cross-links. 2,7-Diaminomitosene (2,7-DAM) is a major metabolite of MC in tumor cells, which also alkylates DNA. We have synthesized a 12 MER oligonucleotide site selectively modified by the major MC adduct (i.e. dG-N2-MC) using bio-mimetic synthesis and the major adduct formed by 2,7-DAM (dG-N2-2,7-DAM) in the same 12 MER sequence by post-oligomerization synthesis.

Translesion synthesis (TLS) efficiencies of dG-N2-MC and dG-N2-2,7-DAM formed by MC and 2,7-DAM have been compared by constructing single-stranded plasmids containing these adducts and replicating them in human embryonic kidney (HEK) 293T cells. This work has been done at the University of Connecticut. It was found that, unlike pol κ, three TLS polymerases : pol η, pol ζ, and Rev1 cooperatively perform the error-prone TLS of these adducts.

dGN2MC
Figure 3: Synthesis of dG-N2-MC and dG-N2-2,7-DAM