title: The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O(6)-(4-bromothenyl)guanine.
abstract: The O(6)-methylguanine-DNA-methyltransferase (MGMT) inactivator O(6)-benzylguanine (O(6)-beG) is currently under clinical investigation as a potential tumour-sensitising agent. In clinical trials its use has been associated with increased myelotoxicity and a reduced maximum tolerated dose (MTD) for BCNU. Thus the concept of myeloprotection by gene therapy with an O(6)-beG-insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O(6)-(4-bromothenyl)guanine, PaTrin-2), which shows potential advantages over O(6)-beG in terms of higher activity against wild-type MGMT and oral formulation. The use of PaTrin-2 has also been associated with increased myelotoxicity in clinical trials and thus PaTrin-2 may also be a candidate for use in conjunction with mutant MGMT gene transfer in genetic chemoprotective strategies. However, its activity against mutant MGMTs has not been reported. We show here that the P(140)K mutant of MGMT is highly resistant to inactivation by PaTrin-2. Furthermore, we show that a human haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMT(P140K) is highly resistant to the cytotoxic effects of PaTrin-2 in combination with the methylating agent temozolomide, and that cells expressing MGMT(P140K) can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMT(P140K) exhibit haemopoietic resistance to PaTrin-2/temozolomide, which results in in vivo selection of gene-modified cells. All of these effects were comparable to those also achieved using O(6)-beG/temozolomide. Thus our data show that MGMT(P140K) is a suitable candidate for chemoprotective gene therapy where PaTrin-2 is being used in conjunction with temozolomide.
authors.affiliation: Cancer Research UK Gene Therapy Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK.
authors.forename: Lorna B
authors.initials: LB
authors.lastname: Woolford
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Southgate
authors.forename: Geoffrey P
authors.initials: GP
authors.lastname: Margison
authors.forename: Michael D
authors.initials: MD
authors.lastname: Milsom
authors.forename: Leslie J
authors.initials: LJ
authors.lastname: Fairbairn
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D045744:Cell Line, Tumor; D003606:Dacarbazine / Q000009:adverse effects / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005260:Female; D015316:Genetic Therapy; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000009:adverse effects / Q000235:genetics*; D000077204:Temozolomide; D014161:Transduction, Genetic; D023041:Xenograft Model Antitumor Assays
chemical_list: C403290:O(6)-(4-bromothenyl)guanine; D006147:Guanine; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000077204:Temozolomide