Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53.
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.
Naumann
S C
SC
Department of Toxicology, University of Mainz, Mainz, Germany.
Roos
W P
WP
Jöst
E
E
Belohlavek
C
C
Lennerz
V
V
Schmidt
C W
CW
Christmann
M
M
Kaina
B
B
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D024161:Collagen Type XI / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D053843:DNA Mismatch Repair / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000378:metabolism*; D003606:Dacarbazine / Q000008:administration & dosage / Q000031:analogs & derivatives; D004789:Enzyme Activation / Q000187:drug effects; D000068338:Everolimus; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D009336:Necrosis; D010766:Phosphorylation / Q000187:drug effects; D020123:Sirolimus / Q000008:administration & dosage / Q000031:analogs & derivatives; D000077204:Temozolomide; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
C499319:COL11A2 protein, human; D024161:Collagen Type XI; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D003606:Dacarbazine; D000068338:Everolimus; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D020169:Caspases; D045643:DNA Repair Enzymes; D020123:Sirolimus; D000077204:Temozolomide