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In the present study, we have investigated the influence of telomerase inhibition in chemosensitivity of melanoma cells to temozolomide (TMZ), a methylating agent with promising antitumor activity against metastatic melanoma. In fact, telomerase, a ribonucleoprotein enzyme expressed in the majority of tumors, is presently considered an attractive target for anticancer therapy, with the double aim of reducing tumor growth and increasing chemosensitivity of cancer cells. Susceptibility to TMZ and to other antitumor agents used for treatment of metastatic melanoma was initially assessed in melanoma lines with different basal levels of telomerase activity. Thereafter, chemosensitivity was investigated after inhibition of telomerase by means of stable transfection of a catalytically inactive, dominant-negative mutant of hTERT (DN-hTERT). This study shows for the first time that: a) susceptibility to TMZ of melanoma lines derived from the same patient did not depend on basal telomerase activity; b) inhibition of telomerase by DN-hTERT resulted in reduced growth rate and increased resistance to TMZ and to the chloroethylating agent carmustine, increased sensitivity to cisplatin, and no change in response to tamoxifen or to a selective N3-adenine methylating agent; c) inhibition of poly(ADP-ribose) polymerase (PARP), an enzyme involved in the repair of N-methylpurines, restored sensitivity of DN-hTERT clones to TMZ. These results indicate that a careful selection of the antitumor agent has to be made when antitelomerase therapy is combined with chemotherapy. Moreover, the data presented here suggest that TMZ + PARP inhibitor combination is active against telomerase-suppressed and slowly growing tumors.

High levels of expression of the DNA repair enzyme O6-alkylguanine DNA-alkyltransferase (OGAT) (EC 2.1.1.63) account for tumor cell resistance to methylating agents. Previous studies suggested that methylating triazenes might have a potential role for the treatment of acute leukemias with low levels of OGAT. In the current study, we transduced the human OGAT cDNA in OGAT-deficient leukemia cell clones. OGAT-transduced cells were more resistant than their OGAT-deficient counterparts to apoptosis triggered by the methylating triazene temozolomide (TZM), as indicated by the results of flow cytometry, terminal deoxynucleotidyl transferase assay, and analysis of DNA fragmentation. Depletion of OGAT activity by O6-benzylguanine increased leukemia cell sensitivity to TZM-mediated apoptosis. Moreover, combined treatment of cells with TZM and benzamide, an inhibitor of the poly(ADP-ribose) polymerase (EC 2.4.2.30), increased the apoptosis induced by the methylating agent. These results demonstrate for the first time that methyl adducts at the O6 position of guanine, which are specifically removed by OGAT, are the principal DNA lesions responsible for the induction of apoptosis on treatment of leukemic cells with the methylating triazene TZM. This study also supports the possible use of TZM for the treatment of acute leukemias and suggests new strategies to increase the susceptibility of tumor cells to methylating triazenes in the clinic.