Abstract
O6-benzylguanine (O6-BG) and 3-aminobenzamide (3-AB) inhibit the DNA repair proteins O6-alkylguanine-DNA alkyltransferase (AGT) and poly(ADP-ribose) polymerase (PARP) respectively. The effect of O6-BG and/or 3-AB on temozolomide and 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) cytotoxicity, was assessed in seven human tumour cell lines: six with an AGT activity of > 80 fmol mg-1 protein (Mer+) and one with an AGT activity of < 3 fmol mg-1 protein (Mer-). Three of the Mer+ cell lines (LS174T, DLD1 and HCT116) were considered to exhibit resistance to methylation by a mismatch repair deficiency (MMR-), each being known to exhibit microsatellite instability, and DLD1 and HCT116 having well-characterised defects in DNA mismatch binding. Potentiation was defined as the ratio between an IC50 achieved without and with a particular inhibitor treatment. Temozolomide or BCNU cytotoxicity was not potentiated by either inhibitor in the Mer- cell line. Preincubation with O6-BG (100 microM for 1 h) was found to potentiate the cytotoxicity of temozolomide by 1.35- to 1.57-old in Mer+/MMR+ cells, but had no significant effect in Mer+/MMR- cells. In comparison, O6-BG pretreatment enhanced BCNU cytotoxicity by 1.94- to 2.57-fold in all Mer+ cell lines. Post-incubation with 3-AB (2 mM, 48 h) potentiated temozolomide by 1.35- to 1.59-fold in Mer+/MMR+ cells, and when combined with O6-BG pretreatment produced an effect which was at least additive, enhancing cytotoxicity by 1.97- to 2.16-fold. 3-AB treatment also produced marked potentiation (2.20- to 3.12-fold) of temozolomide cytotoxicity in Mer+/MMR- cells. In contrast, 3-AB produced marginal potentiation of BCNU cytotoxicity in only three cell lines (1.19- to 1.35-fold), and did not enhance the cytotoxicity of BCNU with O6-BG treatment in any cell line. These data suggest that the combination of an AGT and PARP inhibitor may have a therapeutic role in potentiating temozolomide activity, but that the inhibition of poly(ADP-ribosyl)ation has little effect on the cytotoxicity of BCNU.
Highlights
Temozolomide, a monofunctional methylating imidazotetrazinone, and BCNU, a bifunctional chloroethylnitrosourea, represent two different classes of chemotherapeutic alkylating agent
Temozolomide cytotoxicity can largely be accredited to methylation of 06-guanine (Domoradzki et al, 1984; Margison and O'Connor, 1990) and N3adenine (Karran et al, 1982); the cytotoxicity/mutagenicity of 06-methylguanine being attributed to the induction of futile cycling in the long patch mismatch repair pathway, which results in prolonged DNA strand interruptions and the inhibition of subsequent replication (Ceccotti et al, 1993; Karran et al, 1993)
The low 06-alkylguanine-DNA alkyltransferase (AGT) activity of this cell line combined with an inherent sensitivity to temozolomide alone, would correlate with a Mer-/MMR+ phenotype
Summary
Temozolomide, a monofunctional methylating imidazotetrazinone, and BCNU, a bifunctional chloroethylnitrosourea, represent two different classes of chemotherapeutic alkylating agent. The activity of DNA-alkylating chemotherapy is frequently compromised by the development of resistance; a phenomenon often related to the DNA repair capacity of the tumour cell (Harris et al, 1983; Ludlum, 1990). The main aim of this study, was to determine the relative enhancement of temozolomide or BCNU cytotoxicity by treatment with 06-BG and/or 3-AB in cell lines of varied AGT activity and mismatch repair status
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