Malignant melanomas are highly resistant to
chemotherapy. First-line chemotherapeutics used in
melanoma therapy are the methylating agents
dacarbazine (
DTIC) and
temozolomide (TMZ) and the chloroethylating agents
BCNU and
fotemustine. Here, we determined the mode of cell death in 11
melanoma cell lines upon exposure to TMZ and
fotemustine. We show for the first time that TMZ induces apoptosis in
melanoma cells, using therapeutic doses. For both TMZ and
fotemustine apoptosis is the dominant mode of cell death. The contribution of
necrosis to total cell death varied between 10 and 40%. The
O(6)-methylguanine-DNA methyltransferase (MGMT) activity in the cell lines was between 0 and 1100 fmol mg(-1)
protein, and there was a correlation between MGMT activity and the level of resistance to TMZ and
fotemustine. MGMT inactivation by
O(6)-benzylguanine sensitized all
melanoma cell lines expressing MGMT to TMZ and
fotemustine-induced apoptosis, and MGMT transfection attenuated the apoptotic response. This supports that O(6)-alkylguanines are critical lesions involved in the initiation of programmed
melanoma cell death. One of the cell lines (MZ7), derived from a patient subjected to
DTIC therapy, exhibited a high level of resistance to TMZ without expressing MGMT. This was related to an impaired expression of MSH2 and MSH6. The cells were not cross-resistant to
fotemustine. Although these data indicate that methylating drug resistance of
melanoma cells can be acquired by down-regulation of mismatch repair, a correlation between MSH2 and MSH6 expression in the different lines and TMZ sensitivity was not found. Apoptosis in
melanoma cells induced by TMZ and
fotemustine was accompanied by double-strand break (
DSB) formation (as determined by H2AX phosphorylation) and
caspase-3 and -7 activation as well as PARP cleavage. For TMZ, DSBs correlated significantly with the apoptotic response, whereas for
fotemustine a correlation was not found.
Melanoma lines expressing p53 wild-type were more resistant to TMZ and
fotemustine than p53 mutant
melanoma lines, which is in marked contrast to previous data reported for
glioma cells treated with TMZ. Overall, the findings are in line with the model that in
melanoma cells TMZ-induced
O(6)-methylguanine triggers the apoptotic (and necrotic) pathway through DSBs, whereas for chloroethylating agents apoptosis is triggered in a more complex manner.