title: Potentiation of temozolomide and BCNU cytotoxicity by O(6)-benzylguanine: a comparative study in vitro.
abstract: Depletion of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) with O(6)-benzylguanine (O(6)-BG) has been widely shown to enhance 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) activity. This study aimed to determine whether temozolomide, a methylating imidazotetrazinone, would similarly benefit from combination with O(6)-BG. Seven human cell lines were examined with AGT activities ranging from <6 fmol mg-1 protein to >700 fmol mg-1 protein. Comparisons with BCNU were made on both single and multiple dosing schedules, since temozolomide cytotoxicity is highly schedule dependent. In single-dose potentiation studies, cells were preincubated with 100 microM O(6)-BG for 1 h, a treatment found to deplete AGT activity by >90% for 24 h. No potentiation of either temozolomide or BCNU cytotoxicity was observed in two glioblastoma cell lines with <6 fmol mg-1 protein AGT. In all other cell lines studied potentiation of BCNU toxicity by O(6)-BG was between 1.6- and 2.3-fold and exceeded that of temozolomide (1.1- to 1.7-fold). The magnitude of this potentiation was unrelated to AGT activity and the relative potentiation of temozolomide and BCNU cytotoxicity was found to be highly variable between cell lines. In multiple dosing studies two colorectal cell lines (Mawi and LS174T) were treated with temozolomide or BCNU at 24 h intervals for up to 5 days, with or without either 100 microM O(6)-BG for 1 h or 1 microM O(6)-BG for 24 h, commencing 1 h before alkylating treatment. Extended treatment with 1 microM O(6)-BG produced greater potentiation than intermittent treatment with 100 microM O(6)-BG. Potentiation of temozolomide cytotoxicity increased linearly in Mawi with each subsequent dosing: from 1.4-fold (day 1) to 4.2-fold (day 5) with continuous 1 microM O(6)-BG. In contrast, no potentiation was observed in LS174T, a cell line that would appear to be 'tolerant' of methylation. Potentiation of BNCU cytotoxicity increased in both cell lines with repeat dosing, although the rate of increase was less than that observed with temozolomide and continuous 1 microM O(6)-BG in Mawi. These results suggest that repeat dosing of an AGT inhibitor and temozolomide may have a clinical role in the treatment of tumours that exhibit AGT-mediated resistance.
authors.affiliation: Department of Medical Oncology, Charing Cross Hospital, London, UK.
authors.forename: S R
authors.initials: SR
authors.lastname: Wedge
authors.forename: J K
authors.initials: JK
authors.lastname: Porteus
authors.forename: B L
authors.initials: BL
authors.lastname: May
authors.forename: E S
authors.initials: ES
authors.lastname: Newlands
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D002330:Carmustine / Q000494:pharmacology*; D002460:Cell Line; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008780:Methyltransferases / Q000096:biosynthesis; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000077204:Temozolomide; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating; C064976:O(6)-benzylguanine; D006147:Guanine; D003606:Dacarbazine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine; D000077204:Temozolomide