Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents.

Abstract

O6-Alkylguanine-DNA alkyltransferase was rapidly and irreversibly inactivated by exposure to O6-benzylguanine or the p-chlorobenzyl and p-methylbenzyl analogues. This inactivation was much more rapid than with O6-methylguanine: incubation with 2.5 microM O6-benzylguanine led to more than a 90% loss of activity within 10 min, whereas 0.2 mM O6-methylguanine for 60 min was required for the same reduction. O6-Benzylguanine was highly effective in depleting the alkyltransferase activity of cultured human colon tumor (HT29) cells. Complete loss of activity was produced within 15 min after addition of O6-benzylguanine to the culture medium and a maximal effect was obtained with 5 microM. In contrast, at least 100 microM O6-methylguanine for 4 hr was needed to get a maximal effect, and this reduced the alkyltransferase by only 80%. Pretreatment of HT29 cells with 10 microM O6-benzylguanine for 2 hr led to a dramatic increase in the cytotoxicity produced by the chemotherapeutic agents 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) or 2-chloroethyl(methysulfonyl)methanesulfonate (Clomesone). Administration of O6-benzylguanine to mice at a dose of 10 mg/kg reduced alkyltransferase levels by more than 95% in both liver and kidney. These results indicate that depletion of the alkyltransferase by O6-benzylguanine may be used to investigate the role of the DNA repair protein in carcinogenesis and mutagenesis and that this treatment may be valuable to increase the chemotherapeutic effectiveness of chloroethylating agents.

Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids

“…As MGMT is consumed by the demethylation reaction, temozolomide is thought to deplete its own detoxification mechanism. Furthermore, cotreatment of tumours with O 6 -benzylguanine, another otherwise nontoxic inhibitor of MGMT, increases the sensitivity of MGMT positive cells towards temozolomide (Dolan et al, 1990;Tentori et al, 1997).…”

Inhibition of telomerase activity in malignant glioma cells correlates with their sensitivity to temozolomide

“…Therefore, the sensitivity of tumour cells to TMZ is correlated with the activity of the DNA repair protein O 6 -alkylguanine alkyltransferase (AGT), which removes alkyl groups at the O 6 position of guanine. Cells with low levels of AGT are sensitive to TMZ, while cells expressing high levels of AGT are more resistant (Dolan et al, 1990;Wedge et al, 1996). O 6 -benzylguanine (O 6 -BG), a potent inhibitor of AGT-mediated resistant, enhances the activity of TMZ in tumour cells with high levels of AGT (Dolan et al, 1990;Wedge et al, 1996).…”

Tailored temozolomide therapy according to MGMT methylation status for elderly patients with acute myeloid leukemia

“…For these reasons, inactivation of MGMT activity in combination with temozolomide has been proposed as a method to increase the sensitivity of resistant tumors [17,18]. Unfortunately, most MGMT inhibitors tested have failed to improve clinical activity in temozolomide-resistant tumors [19].…”