A single cycle of treatment with temozolomide, alone or combined with O(6)-benzylguanine, induces strong chemoresistance in melanoma cell clones in vitro: role of O(6)-methylguanine-DNA methyltransferase and the mismatch repair system.
Clinically achievable concentrations of temozolomide (TMZ) produce cytotoxic effects only in mismatch repair (MMR)-proficient cells endowed with low O6-methylguanine-DNA methyltransferase (MGMT) activity. Aim of the present study was to investigate the molecular mechanisms underlying acquired resistance of melanoma cells to TMZ and the effect of O6-benzylguanine (BG), a specific MGMT inhibitor, on the development of a TMZ-resistant phenotype. Three MMR-proficient melanoma cell clones with low or no MGMT activity were treated daily for 5 days with 50 micromol/l TMZ, alone or in combination with 5 micromol/l BG. Parental clones and sublines established after one or four cycles of treatment were analyzed for sensitivity to TMZ or TMZ+BG and for other parameters. The sublines established after one cycle of TMZ or TMZ+BG exhibited a marked increase in MGMT activity and resistance to TMZ alone. BG only partially reversed acquired resistance to the drug. In some cases, alterations in the MMR system accounted for MGMT-independent resistance to TMZ. Up-regulation of MGMT activity was associated with either demethylation of the MGMT promoter or hypermethylation of the body of the gene, and partially reversed by 5-aza-2'-deoxycytidine. The sublines established after four cycles of TMZ or TMZ+BG did not show a further increase in resistance to TMZ alone. However, two out of three sublines established after TMZ+BG treatment exhibited increased resistance to TMZ+BG. In conclusion, our data demonstrate that a single cycle of TMZ is sufficient to induce high levels of drug resistance in melanoma clones, principally, but not exclusively, via up-regulation of MGMT expression. Exposure to TMZ+BG favors the development of MGMT-independent mechanisms of TMZ resistance.
Alvino
Ester
E
Institute of Neurobiology and Molecular Medicine, Tor Vergata Research Area, National Research Council, I-00133 Rome, Italy.
Castiglia
Daniele
D
Caporali
Simona
S
Pepponi
Rita
R
Caporaso
Patrizia
P
Lacal
Pedro Miguel
PM
Marra
Giancarlo
G
Fischer
Franziska
F
Zambruno
Giovanna
G
Bonmassar
Enzo
E
Jiricny
Joseph
J
D'Atri
Stefania
S
D000970:Antineoplastic Agents / Q000494:pharmacology*; D020137:Base Pair Mismatch / Q000502:physiology*; D019175:DNA Methylation; D004260:DNA Repair; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000201:enzymology* / Q000235:genetics*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic; D000077204:Temozolomide; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; C064976:O(6)-benzylguanine; D006147:Guanine; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000077204:Temozolomide