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 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.

Synthesis of insulin-like growth factor I using N-methyl pyrrolidinone as the coupling solvent and trifluoromethane sulphonic acid cleavage from the resin.

Insulin-like growth factor I (IGF-I), a protein of 70 amino acid residues and 3 cystine bridges, has been synthesized by two solid phase Boc methods. The first method used N-methylpyrrolidinone as the solvent with single coupling cycles while the second synthesis used dimethylformamide and dichloromethane as the solvents with a double-coupling protocol. In both cases, trifluoroacetic acid/trifluoromethanesulphonic acid cleavage of the peptide from the resin was employed. Purification of the cleavage products followed by removal of the S-acetamidomethyl protecting groups gave reduced peptides which were then oxidized under conditions favouring the formation of the correct disulphide bonds. The purified synthetic IGF-I peptides were full agonists of natural IGF-I in a radioimmunoassay, in an IGF-I radioreceptor assay, in a bioassay which measures the stimulation of protein synthesis in rat L6 myoblasts and in an IGF-binding protein competitive binding assay. Moreover, in each of these assays, the synthetic IGF peptides were found to be at least 70% as potent as natural IGF-I.

A key functional role for the insulin-like growth factor 1 N-terminal pentapeptide.

In order to elucidate the role of the N-terminus of insulin-like growth factor 1 (IGF-1) with respect to its biological properties, we chemically synthesized analogues of IGF-1 truncated by one to five amino acid residues from the N-terminus. In a bioassay that measured the stimulation of protein synthesis in rat L6 myoblasts, the concentrations required to produce a half-maximal response were: IGF-1, 13 ng/ml; des-(1)-IGF-1, 10 ng/ml; des-(1-2)-IGF-1, 13 ng/ml; des-(1-3)-IGF-1, 1.5 ng/ml; des-(1-4)-IGF-1, 5.1 ng/ml; des-(1-5)-IGF-1, 1200 ng/ml. When tested for their abilities to compete with 125I-IGF-1 binding to L6 myoblasts at 3 degrees C, the concentrations required for 50% competition were: IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1, 20 ng/ml; des-(1-3)-IGF-1, 14 ng/ml; des-(1-4)-IGF-1, 40 ng/ml; des-(1-5)-IGF-1, greater than 1000 ng/ml. Receptor-binding experiments at 25 degrees C, however, gave results suggesting that the myoblasts were secreting a binding protein selective for the three longest peptides. This interpretation was confirmed by binding studies with medium conditioned by the L6 myoblasts as well as binding protein purified from MDBK-cell-conditioned medium. In both cases IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1 competed for tracer IGF-1 binding at least 60-fold better than did the three shorter peptides. The results obtained account for the increased potency of des-(1-3)-IGF-1 and des-(1-4)-IGF-1, since their activities are not attenuated by the binding protein, and the relatively lower potency of des-(1-4)-IGF-1 is a consequence of this peptide binding less well to the L6-myoblast receptor.