O6-alkylguanine DNA Alkyltransferase Levels Research Articles

Recent Approaches to Improve the Antitumor Efficacy of Temozolomide

Temozolomide (TMZ) is an oral anticancer agent approved for the treatment of newly diagnosed glioblastoma in combination with radiotherapy. Moreover, TMZ has shown comparable efficacy with respect to dacarbazine, the reference drug for metastatic melanoma. Due to its favorable toxicity and pharmacokinetic profile, TMZ is under clinical investigation for brain metastasis from solid tumors and refractory leukemias. TMZ interacts with DNA generating a wide spectrum of methyl adducts mainly represented by N-methylpurines. However, its antitumor activity has been mainly attributed to O(6)-methylguanine, since tumor cell sensitivity inversely correlates with the levels of O(6)-alkylguanine DNA alkyltransferase and requires an intact mismatch repair system. Therefore, an increasing number of studies have been performed in order to identify patients who will benefit from TMZ treatment on the basis of their molecular/genetic profile. Unfortunately, resistance to the methylating agent occurs relatively often and strongly affects the rate and durability of the clinical response in cancer patients. Thus, different approaches have been developed to abrogate resistance or to increase the efficacy of TMZ and for many of them investigation is still underway. Herein, we provide an overview on the recent findings of preclinical and clinical studies on TMZ in combination with inhibitors of DNA repair, chemotherapeutic drugs with different mechanisms of action or radiotherapy, anti-angiogenic agents and other biological modulators.

Temozolomide: An Update on Pharmacological Strategies to Increase its Antitumour Activity

Temozolomide (TMZ) is a methylating agent with promising antitumour activity against primary or secondary brain tumours. Through the generation of a reactive intermediate, TMZ interacts with DNA at different base site positions, generating a wide spectrum of methyl adducts represented mainly by N-methylpurines (70%) while, at a lesser extent, by N3-methyladenine (9%) and O6-methylguanine (5%). The antitumour activity of TMZ has been primarily attributed to O6- methylguanine, since tumour cell sensitivity inversely correlates with the levels of O6-alkylguanine DNA alkyltransferase and requires an intact mismatch repair system. Even though the pharmacokinetics properties, favourable toxicity profile and antitumour activity against a broad range of tumour types render TMZ an attractive agent in oncology, resistance to the methylating agent occurs relatively often and strongly affects the rate and durability of clinical response in cancer patients. Thus, different strategies aimed at counteracting resistance and increasing the efficacy of TMZ have been designed and for many of them investigation is still underway. Herein, we provide an update on the latest findings of preclinical and clinical studies on TMZ in combination with resistance or biological modulators and anticancer drugs with different mechanisms of action. Keywords: temozolomide, polymerase, chemotherapy, cancer

Rapid Induction Of Chemoresistance In Melanoma Cells Exposed In Vitro To Temo-Zolomide Alone Or Combined With O6-Benzyl-Guanine

Introduction Temozolomide (TMZ) an antitumor DNA methylating agent, is able to induce O6-methyl-guanine DNA adducts. Its activity is largely conditioned by a fully functional mismatch repair system (MMR). In contrast, the antineoplastic effects of the agent are suppressed by the DNA repair enzyme O6-alkylguanine-DNA-alkyl-transferase (OGAT), that removes methyl adducts. The OGAT inhibitor O6-benzyl-guanine (BG) abrogates resistance to TMZ of malignant cells expressing high OGAT levels (i.e. OGAT-dependent resistance, ODR), but not of MMR-deficient tumors, or of tumors showing other resistance mechanisms unrelated to OGAT (i.e. OGAT-independent resistance, OIR). Aim of the study To establish the onset of ODR or of OIR in melanoma cells treated in vitro with TMZ alone or associated with BG. Methods Three MMR-proficient melanoma cell clones with barely detectable OGAT levels and sensitive to TMZ, originated from 3 different parental lines, were treated with TMZ (50 μM), or TMZ (50 μM)+BG (5 μM) daily for 5 days, for 4 consecutive cycles, and tested for sensitivity to TMZ or TMZ+BG, at the end of each cycle. Results Dramatic increase of chemo-resistance to TMZ alone (i.e. ranging from more than 200% to more than 1200% increase of IC50 of TMZ) was detected in all 3 clones, after as early as 1 cycle of treatment with TMZ alone (i.e. TMZ sublines). This increase was maintained essentially constant for up to 4 treatment cycles. In all TMZ sublines OGAT expression was found to be increased, and BG partially reversed resistance to TMZ, thus suggesting that resistance was, at least in part, of ODR type. A similar phenomenon was observed when the 3 clones were treated with TMZ+BG (i.e. TMZ/BG sublines). However, in this case BG reduced TMZ resistance in only 2 of the 3 TMZ/BG sublines. Consistently with these results, OGAT levels were up-regulated in the 2 TMZ/BG sublines still sensitive to TMZ+BG, but not in the TMZ/BG subline in which the inhibitor failed to alleviate TMZ resistance. Conclusions Treatment of melanomas with TMZ, alone or with the OGAT inhibitor BG, shows an unexpectedly high capacity of inducing early anti-TMZ resistance of both ODR and OIR type. The elucidation of the molecular mechanisms underlying OIR requires further studies. However, the present results provide valuable information concerning the poor therapeutic response to triazenes observed in melanoma patients. Supported by the Italian Ministry of Health.

The effect of O6-alkylguanine-DNA alkyltransferase and mismatch repair activities on the sensitivity of human melanoma cells to temozolomide, 1,3-bis(2-chloroethyl)1-nitrosourea, and cisplatin.

The prognosis of advanced melanoma is generally poor, because this tumor commonly exhibits intrinsic or acquired resistance to chemotherapy. In an attempt to identify the underlying causes of this resistance, we studied the roles played by the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair (MMR) system in the sensitivity of melanoma cells to temozolomide (TMZ), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), or cis-diamminedichloroplatinum(II) (CDDP). To this end, OGAT levels and MMR efficiency of extracts of nine melanoma cell lines and selected clones derived from four of these lines were determined and correlated with the sensitivity of the respective cells to these drugs. The effectiveness of O(6)-benzylguanine (BG), a specific OGAT inhibitor, in potentiating TMZ- or BCNU-mediated cytotoxicity was also evaluated. Our results demonstrate that MMR efficiency and OGAT levels strongly affect melanoma cell sensitivity to TMZ. In MMR-proficient cells, a direct correlation between OGAT levels and TMZ IC(50) values was found. When OGAT activity was inhibited with BG, the sensitivity of these cells to TMZ increased and was then dictated largely by their MMR efficiency. MMR-deficient cells were highly resistant to the drug irrespective of their OGAT levels. Although OGAT activity and MMR status seemed to be the major determinants of melanoma sensitivity to TMZ, this was not the case for BCNU and CDDP; resistance to the latter drugs clearly involves processes other than the two DNA repair pathways analyzed in this study.

DNA Repair Enzymes and Cytotoxic Effects of Temozolomide: Comparative Studies Between Tumor Cells and Normal Cells of the Immune System

O6-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair system (MRS) play a crucial role in the susceptibility of tumor cells to the cytotoxic effects of agents that generate O6-methylguanine in DNA, including the triazene compound temozolomide (TMZ). Studies performed with peripheral blood mononuclear cells (MNC) showed that TMZ was scarcely active on lymphocyte functions not dependent on cell proliferation (e.g. NK activity and cytokine-mediated induction of CD1b molecule in adherent MNC). In contrast, TMZ depressed proliferation and lymphokine activated killer (LAK) cell generation in response to IL-2. In this case, a reasonably good inverse relationship was found between OGAT levels of MNC and their susceptibility to TMZ. This study also analyzed the ratio of the toxic effect of TMZ on MNC and on tumor cells (i.e. “Tumor-Immune Function Toxicity Index”, TIFTI). A particularly favorable TIFTI can be obtained when OGAT levels are extremely high in MNC and markedly low in tumor cells. This holds true for MRS-proficient neo-plastic cells, but not for MRS-deficient tumors. In conclusion, strategies aimed at modulating OGAT and MRS may improve the clinical response to TMZ. However, the use of OGAT inhibitors to potentiate the antitumor activity of TMZ might result in a concomitant increase of the immunosuppressive effects of the drug, thus reducing the relative TIFTI.

Characterization of MLH1 and MSH2 DNA mismatch repair proteins in cell lines of the NCI anticancer drug screen.

The lack of a functional DNA mismatch repair (MMR) pathway has been recognized as a common characteristic of several different types of human cancers due to mutation affecting one of the MMR genes or due to promoter methylation gene silencing. These MMR-deficient cancers are frequently resistant to alkylating agent chemotherapy such as DNA-methylating or platinum-containing compounds. To correlate drug resistance with MMR status in a large panel of human tumor cell lines, we evaluated by Western blot the cellular levels of the two MMR proteins most commonly mutated in human cancers, MLH1 and MSH2, in the NCI human tumor cell line panel. This panel consists of 60 cell lines distributed among nine different neoplastic diseases. We found that in most of these cell lines both MLH1 and MSH2 were expressed, although at variable levels. Five cell lines (leukemia CCRF-CEM, colon HCT 116 and KM12 and ovarian cancers SK-OV-3 and IGROV-1) showed complete deficiency in MLH1 protein. MSH2 protein was detected in all 57 cell lines studied. Absence of MLH1 protein was always linked to resistance to the methylating chemotherapeutic agent temozolomide. This resistance was independent of cellular levels of O6-alkylguanine DNA alkyltransferase. Based on data available for review in the NCI COMPARE database, cellular levels of MLH1 and MSH2 did not correlate significantly with sensitivity to any standard anticancer drug or with any characterized molecular target already tested against the same panel of cell lines. Based on evaluation of 60 tumor cell lines in the NCI anticancer drug screen, MLH1 deficiency was more common than MSH2 deficiency and was always associated with a high degree of temozolomide resistance. These data will enable correlations with other drug sensitivities and molecular targets in the COMPARE database to evaluate linked processes in tumor drug resistance.

Inhibition of O6-Alkylguanine DNA-Alkyltransferase or Poly(ADP-ribose) Polymerase Increases Susceptibility of Leukemic Cells to Apoptosis Induced by Temozolomide

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.

Chemosensitivity to triazene compounds and O6-alkylguanine-DNA alkyltransferase levels: Studies with blasts of leukaemic patients

A clinical pilot study performed by our group showed that dacarbazine can induce a marked reduction of blast cells in patients with acute myelogenous leukaemia (AML). Leukaemic blasts (LB) from responsive patients showed low levels of O6-alkylguanine-DNA alkyltransferase (OGAT). An in vitro study was performed to evaluate OGAT levels and sensitivity to temozolomide (a triazene compound that spontaneously decomposes into the active metabolite of dacarbazine) in a relatively large number of LB samples. OGAT levels varied widely among the LB of different patients, with a mean value higher in acute lymphoblastic leukaemias than in AML. About 25% of LB obtained from patients with AML showed low OGAT activity, in the range corresponding to that observed in leukaemic patients responsive to dacarbazine in vivo. A reasonable inverse correlation was found between OGAT levels and LB sensitivity to temozolomide. Triazenes could have a therapeutic potential in human leukaemias. Moreover, OGAT determination could provide rapid and reliable information about a patient's susceptibility to these antitumor agents.