POTENTIATION OF TEMOZOLOMIDE AND BCNU CYTOTOXICITY BY O-6-BENZYLGUANINE - A COMPARATIVE-STUDY IN-VITRO

Depletion of the DNA repair protein O-6-alkylguanine-DNA alkyltransfer ase (AGT) with O-6-benzylguanine (O-6-BG) has been widely shown to enh ance 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) activity. This study ai med to determine whether temozolomide, a methylating imidazotetrazinon e, would similarly benefit from combination with O-6-BG. Seven human c ell lines were examined with AGT activities ranging from < 6 fmol mg(- 1) protein to > 700 fmol mg(-1) protein. Comparisons with BCNU were ma de on both single and multiple dosing schedules, since temozolomide cy totoxicity is highly schedule dependent. In single-dose potentiation s tudies, cells were preincubated with 100 mu M O-6-BG for 1 h, a treatm ent found to deplete AGT activity by > 90% for 24 h. No potentiation o f either temozolomide or BCNU cytotoxicity was observed in two gliobla stoma cell lines with < 6 fmol mg(-1) protein AGT. In all other cell l ines studied potentiation of BCNU cytotoxicity 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 fo und to be highly variable between cell lines. In multiple dosing studi es two colorectal cell lines (Mawi and LS174T) were treated with temoz olomide or BCNU at 24 h intervals for up to 5 days, with or without ei ther 100 mu M O-6-BG for 1 h or 1 mu M O-6-BG for 24 h, commencing 1 h before alkylating treatment. Extended treatment with 1 mu M O-6-BG pr oduced greater potentiation than intermittent treatment with 100 mu M O-6-BG. Potentiation of temozolomide cytotoxicity increased linearly i n Mawi with each subsequent dosing: from 1.4-fold (day 1) to 4.2-fold (day 5) with continuous 1 mu M O-6-BG. In contrast, no potentiation wa s observed in LS174T, a cell line that would appear to be 'tolerant' o f methylation. Potentiation of BCNU cytotoxicity increased in both cel l lines with repeat dosing, although the rate of increase was less tha n that observed with temozolomide and continuous 1 mu M O-6-BG in Mawi . These results suggest that repeat dosing of an AGT inhibitor and tem ozolomide may have a clinical role in the treatment of tumours that ex hibit ACT-mediated resistance.