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
O(6)-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 O(6)-alkylating agents, such as
N-methyl-N'-nitro-N-nitrosoguanidine. Acquired resistance to
fotemustine was alleviated by treatment with the MGMT inhibitor
O(6)-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.