O6-methylguanine-DNA methyltransferase (MGMT) expression in acquired resistance of melanoma cells to temozolomide (TMZ)

O6-METHYLGUANINE-DNA METHYLTRANSFERASE (MGMT) EXPRESSION IN ACQUIRED RESISTANCE OF MELANOMA CELLS TO TEMOZOLOMIDE (TMZ) Alvino E.1, Lacal P.M.2, Castiglia D.2, Caporaso P.2, Caporali S.2, Arcelli D.2, Zambruno G.2, Bonmassar E.2, D'Atri S.2 1Institute of Neurobiology and Molecular Medicine, National Research Council, Rome, Italy; 2Istituto Dermopatico dell'Immacolata (IDI-IRCCS), Rome, Italy. Clinically achievable concentrations of TMZ, an antitumor methylating agent presently in phase II/III clinical trials, exert cytotoxic effects only in cells with a functional mismatch repair (MMR) system and low MGMT activity. Aim of this study was to elucidate TMZ-induced molecular changes leading to acquired resistance to the drug in melanoma cells. Three MMR-proficient melanoma cell clones with low MGMT activity, derived from 3 different cell lines, were treated with 50 mM TMZ (daily for 5 days, to mimic patient exposure to a single cycle of chemotherapy), alone or in the presence of 5 mM O6- benzylguanine (BG), a specific MGMT inhibitor. When exponential growth resumed, the cells were tested for sensitivity to TMZ or TMZ+BG, MGMT activity, and expression of MMR proteins. Both TMZ- and TMZ+BGtreated sublines showed a marked increase of MGMT activity and resistance to TMZ alone, while the expression of MMR proteins was nearly unaltered. The increase of MGMT activity was dependent on increased levels of MGMT mRNA and protein. However, the CpG 34 Cellular and molecular basis of cancer therapya methylation pattern of the MGMT promoter was unaltered in drug-treated sublines. Acquired resistance to TMZ was only partially reversed by BG. Six additional TMZ- or TMZ+BG-treated sublines were generated for each of the three parental melanoma clones. All drug-treated sublines showed increased MGMT activity, with the exception of two sublines in the TMZ+BG group. However, also these two sublines were resistant to TMZ. In conclusion, treatment of melanoma cells with TMZ, alone or combined with BG, rapidly induces drug resistance mainly, but not exclusively, through upregulation of MGMT activity. Studies are in progress to identify genes differentially expressed in the parental clones and drug-resistant sublines possibly involved in MGMT-independent chemoresistance. Supported by the Italian Ministry of Health.