Identification of Potential Cellular Responses Triggered by Stereoisomeric DNA Interstrand Crosslinks Produced by Mitomycins in MCF‐7 Cells

Abstract

Mitomycin C (MC) is a DNA alkylating agent broadly used in chemotherapy. MC and Decarbamoylmitomycin 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 ICL structure and 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 MCF‐7 cells transfected with the purified ICLs for 24 hours. They showed several interesting proteins were differently regulated by the trans‐ and cis‐ICLs: (1) TBC1 domain family member 9 which regulates cell proliferation (increased 41% upon treatment with the trans‐ICL treatment, but decreased 50% with the cis‐ICL); (2) Core histone macro‐H2A.2, involved in DNA repair (decreased 75% upon treatment with the trans‐ICL, but increased 89% with the cis‐ICL); (3) Aurora kinase B which inhibits p21 expression and regulates cell cycle (increased 50% upon treatment with the trans‐ICL, but decreased 73% by the cis‐ICL); and (4) Membrane‐associated tyrosine‐ and threonine‐specific cdc2‐inhibitory kinase which acts a negative regulator of G2 to M transition (decreased 36% upon treatment with the trans‐ICL, but increased 5% 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 now have identified both ICL molecular targets and cellular responses involved in DNA repair and cell cycle regulation in MCF7 cell lines. Further validations are needed to confirm the results.