title: Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells.
abstract: Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS.
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA.|Drug Safety Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Kiyohiro
authors.initials: K
authors.lastname: Hashimoto
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA.
authors.forename: Vyom
authors.initials: V
authors.lastname: Sharma
authors.affiliation: Department of Radiation Genetics, Kyoto University, Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sasanuma
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA.
authors.forename: Xu
authors.initials: X
authors.lastname: Tian
authors.affiliation: Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Takata
authors.affiliation: Department of Radiation Genetics, Kyoto University, Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Swenberg
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000502:physiology*; D016923:Cell Death; D002460:Cell Line; D002645:Chickens; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004249:DNA Damage; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005199:Fanconi Anemia / Q000235:genetics*; D051856:Fanconi Anemia Complementation Group Proteins / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008698:Mesylates / Q000633:toxicity*; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D051856:Fanconi Anemia Complementation Group Proteins; D008698:Mesylates; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes; C002531:isopropylmethanesulfonate
keywords: DT40; FANC; alkylation; isopropyl methanesulfonate; micronucleus