Potential Signaling Regulations of Stereisomeric DNA Interstrand Crosslinks Produced by Mitomycins in K562 Cells

Abstract

Mitomycin C (MC) is a DNA alkylating agent broadly used in chemotherapy. MC and Decarbamoyl mitomycin C (DMC), a synthetic MC derivative which lacks the carbamoyl at the C10 position, can form interstrand crosslinks (ICLs) between the exocyclic amines of opposing deoxyguanosine moieties. These ICLs are responsible for the cytotoxicity of mitomycins towards cancer cells since they prevent replication of highly proliferating cells. MC and DMC can crosslink DNA in both 5’‐CpG and 5’‐GpC sequence contexts, and the crosslinking is diastereospecific: At 5’‐CpG, mitomycins produce trans‐ICLs whereas cis‐ICLs are found at 5’‐GpC steps. The major ICL formed by Mitomycin C in culture cells is the trans‐ICL whereas decarbamoylmitomycin C yields the stereoisomeric cis‐ICL preferentially. Our overarching goal is to synthesize oligonucleotides bearing the cisand trans isomeric crosslinks to investigate the relationship between the ICL structure and the cellular response. Our hypothesis is that the trans and cis‐ICLs trigger different cellular responses. There is evidence that the ICL produced by DMC triggers a p53 independent cell death and research uncovering mechanisms through which p53 independent cell death occurs is crucial to identify molecular targets to treat tumors with a mutant p53. These tumors represent at least 50% of all cancers. We present here the different synthetic routes to access cis and trans isomeric interstrand crosslinks formed by mitomycins in their reaction with DNA and preliminary label free proteomic studies to identify the stereoisomeric ICLs molecular targets. Proteomic studies were accomplished from K562 leukemia cells transfected with the purified ICLs for 24 hours and showed several interesting proteins differently regulated by the trans‐ and cis‐ICLs: (1) Histone‐lysine N‐methyltransferase EHMT2 which down‐regulates a cell cycle inhibitory gene expression network (decreased 46% upon treatment with the trans‐ICL, but no change was observed with the cis‐ICL); (2) Target of EGR1 protein 1 which inhibits cell growth rate and cell cycle (increased 69% upon treatment with the trans‐ICL, but decreased 30% with the cis‐ICL); (3) Poly(ADP‐ribose) glycohydrolase DNA repair, involved in DNA repair (decreased 19% upon treatment with the trans‐ICL, but increased 63% with the cis‐ICL); (4) Telomerases Cajal body protein 1, involved in cell cycle regulation (no change observed upon tratment with the trans‐ICL but decreased 75% with the cis‐ICL. In addition, Ingenuity Pathway Analysis was conducted to identify the major molecular pathways impacted by each ICL treatment. In conclusion, we have identified both ICL molecular targets and cellular responses involved in DNA repair and cell cycle regulation in K‐562cell lines. Further validations are needed to confirm the results.