Acquired resistance of melanoma cells to the antineoplastic agent fotemustine is caused by reactivation of the DNA repair gene MGMT

Acquired resistance to antineoplastic agents is a frequent obstacle in tumo r therapy. Malignant melanoma cells are particularly well known for their u nresponsiveness to chemotherapy; only about 30% of tumors exhibit a transie nt clinical response to treatment. In our study, we investigated the molecu lar mechanism of acquired resistance of melanoma cells (MeWo) to the chloro ethylating drug fotemustine, Determination of O-6-methylguanine-DNA methylt ransferase (MGMT) activity showed that MeWo cells that acquired resistance to fotemustine upon repeated treatment with the drug display high MGMT acti vity, 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 w as alleviated by treatment with the MGMT inhibitor O-6-benrylguanine demons trating 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 ce lls. Southern blot analysis proved that the MGMT gene was not amplified. Th ere was also only an insignificant difference in the CpG methylation patter n of the MGMT promoter whereas a clear hypermethylation in the body of the gene was observed in fotemustine resistant cells, The conclusion that hyper methylation is responsible for reactivation of the MGMT gene gained support by the finding that MGMT activity significantly declined and cells reverte d (partially) to the parental sensitive phenotype upon treatment with 5-aza cytidine. This is the first report of acquired resistance to a chloroethyla ting antineoplastic drug of melanoma cells due to gene hypermethylation, (C ) 2001 Wiley-Liss, Inc.