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The DNA-repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) is a decisive determinant of resistance of tumor cells to methylating and chloroethylating anti-cancer drugs. Therefore, selective inhibition of MGMT in tumors is expected to cause tumor sensitization. Several inhibitors of MGMT have been developed which function in both tumors and normal tissue. To deplete MGMT preferentially in tumors, strategies to target the inhibitor to the tumor tissue need to be developed. Here, we report on the properties of glucose-conjugated MGMT inhibitors that might be useful for tumor targeting since tumor cells frequently over-express glucose transporter. O⁶-Benzylguanine (O6BG), 8-aza-O⁶-benzylguanine, O⁶-(4-bromothenyl)-guanine (O6BTG) and the corresponding spacer-linked β-D-glucose conjugates were analyzed comparatively for MGMT-inhibitory activity. Substitution at the N9 position of the purine moiety resulted generally in a reduction in the efficiency with which the inhibitors blocked MGMT. However, the inhibitory activity of the O6BTG conjugates increased with increasing spacer length, and O6BTG conjugated with a C8 spacer with β-D-glucose was nearly as effective as O6BTG on its own. MGMT was inhibited by the conjugates both in crude cell extracts and upon treatment of intact HeLa cells, indicating efficient uptake of the glucose conjugates into cells. Since the O6BTG-C8-D-glucose conjugate 8-[O⁶-(4-bromothenyl)-guan-9-yl]-octyl-β-D-glucoside was highly efficient at MGMT inhibition in a non-toxic concentration range, the drug might be a useful tool for specific tumor sensitization. © 2001 Wiley-Liss, Inc.

Acquired resistance to antineoplastic agents is a frequent obstacle in tumor therapy. Malignant melanoma cells are particularly well known for their unresponsiveness to chemotherapy; only about 30% of tumors exhibit a transient clinical response to treatment. In our study, we investigated the molecular mechanism of acquired resistance of melanoma cells (MeWo) to the chloroethylating drug fotemustine. Determination of O⁶-methylguanine-DNA methyltransferase (MGMT) activity showed that MeWo cells that acquired resistance to fotemustine upon repeated treatment with the drug display high MGMT activity, whereas the parental cell line had no detectable MGMT. The resistant cell lines exhibit cross-resistance to other O⁶-alkylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine. Acquired resistance to fotemustine was alleviated by treatment with the MGMT inhibitor O⁶-benzylguanine demonstrating that reactivation of MGMT is the main underlying cause of acquired alkylating drug resistance. As compared with control cells, both MGMT mRNA and MGMT protein were expressed at a high level in fotemustine resistant cells. Southern blot analysis proved that the MGMT gene was not amplified. There was also only an insignificant difference in the CpG methylation pattern of the MGMT promoter whereas a clear hypermethylation in the body of the gene was observed in fotemustine resistant cells. The conclusion that hypermethylation is responsible for reactivation of the MGMT gene gained support by the finding that MGMT activity significantly declined and cells reverted (partially) to the parental sensitive phenotype upon treatment with 5-azacytidine. This is the first report of acquired resistance to a chloroethylating antineoplastic drug of melanoma cells due to gene hypermethylation. © 2001 Wiley-Liss, Inc.

Malignant melanoma is well known for its primary unresponsiveness to chemotherapy. The mechanisms conferring this intrinsic resistance are unclear. In this study, we investigated the role of genes involved in DNA repair in a panel of human melanoma cell variants exhibiting low and high levels of resistance to 4 commonly used drugs in melanoma treatment, i.e., vindesine, etoposide, fotemustine and cisplatin. We show that in melanoma cells exhibiting resistance to cisplatin, etoposide and vindesine, the nuclear content of each of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2 and hMSH6 was reduced by 30–70%. A decreased expression level of up to 80% of mRNAs encoding hMLH1 and hMSH2 was observed in drug-resistant melanoma cells selected for cisplatin, etoposide and fotemustine, while vindesine-selected cells showed only moderate reduction. In melanoma cells that acquired resistance to fotemustine, the amount of nuclear MMR proteins was nearly unaltered, whereas the activity of O⁶-methylguanine-DNA methyltransferase (MGMT) was considerably enhanced. Activity of N-methylpurine-DNA glycosylase (MPG) was not significantly altered in any of the drug-resistant melanoma cells. Our data indicate that modulation of both MMR components and MGMT expression level may contribute to the drug-resistant phenotype of melanoma cells. Int. J. Cancer 80:744–750, 1999. © 1999 Wiley-Liss, Inc.

The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) is a main determinant of resistance of cells to the cytostatic effects of O⁶-alkylguanine-generating alkylating agents. The purpose of our study was to assay MGMT activity in cells of lung cancers and to correlate MGMT levels with chemotherapy response to cyclophosphamide (CTX) and cisplatin (DDP). MGMT levels were determined in 14 human lung tumor xenografts. There was a wide variation of MGMT expression in these tumors, ranging from 10 to 984 fmol/mg protein. There was also a wide range in the sensitivity of the xenografts to CTX and DDP, as measured by specific growth delay. When the MGMT levels of the different xenograft lines were compared with the corresponding responses to CTX and DDP, a close correlation was found between MGMT activity and CTX (lin reg., r = –0.83, p < 0.05). The higher the MGMT activity, the less pronounced was the growth-inhibiting effect of CTX. With DDP, no such correlation was found. Our results indicate that the in vivo response of tumors to CTX is related to the level of MGMT expression. Int. J. Cancer 77:919–922, 1998. © 1998 Wiley-Liss, Inc.