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Acute myeloid leukemia (AML) is a highly lethal disease, especially in old patients. Chemoresistance and the absence of host immune responses against autochthonous malignancy play a major role in the poor prognosis of AML. The triazene compounds Dacarbazine and Temozolomide are monofunctional alkylators that donate methyl groups to many sites in DNA, including the O(6)-position of guanine producing O(6)-methylguanine (O(6)-MeG). If not repaired, O(6)-MeG frequently mispairs with thymine during DNA duplication. O(6)-MeG:T mismatches can be recognized by the mismatch repair (MMR) system which activates a cascade of molecular events leading to cell cycle arrest and cell death. If MMR is defective, cells continue to divide and GC → AT transition mutations occur. In preclinical models, such mutations can lead to the appearance of abnormal proteins containing non-self peptides ("chemical xenogenization" CX) that can be recognized by host cell-mediated immunity. Repair of O(6)-MeG is achieved by the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT), which removes the methyl adduct in an autoinactivating stoichiometric reaction. High MGMT levels attenuate the pharmacodynamic effects of triazenes. In the last few years, triazenes, alone or with MGMT inhibitors, have been tested in AML. In view of their potential activity as CX inducers, triazenes could offer the additional advantage of host anti-leukemia immune responses. The present paper describes several studies of leukemia treatment with triazenes and a case of acute refractory leukemia with massive skin infiltration by malignant cells. Treatment with Temozolomide and Lomeguatrib, a potent MGMT inhibitor, produced a huge, although transient, blastolysis and complete disappearance of all skin lesions.

Background: Most DNA-damaging chemotherapeutic agents activate the transcription factor nuclear factor κB (NF-κB). However, NF-κB activation can either protect from or contribute to the growth suppressive effects of the agent. We previously showed that the DNA-methylating drug temozolomide (TMZ) activates AKT, a positive modulator of NF-κB, in a mismatch repair (MMR) system-dependent manner. Here we investigated whether NF-κB is activated by TMZ and whether AKT is involved in this molecular event. We also evaluated the functional consequence of inhibiting NF-κB on tumor cell response to TMZ., Methods: AKT phosphorylation, NF-κB transcriptional activity, IκB-α degradation, NF-κB2/p52 generation, and RelA and NF-κB2/p52 nuclear translocation were investigated in TMZ-treated MMR-deficient (HCT116, 293TLα-) and/or MMR-proficient (HCT116/3-6, 293TLα+, M10) cells. AKT involvement in TMZ-induced activation of NF-κB was addressed in HCT116/3-6 and M10 cells transiently transfected with AKT1-targeting siRNA or using the isogenic MMR-proficient cell lines pUSE2 and KD12, expressing wild type or kinase-dead mutant AKT1. The effects of inhibiting NF-κB on sensitivity to TMZ were investigated in HCT116/3-6 and M10 cells using the NF-κB inhibitor NEMO-binding domain (NBD) peptide or an anti-RelA siRNA., Results: TMZ enhanced NF-κB transcriptional activity, activated AKT, induced IκB-α degradation and RelA nuclear translocation in HCT116/3-6 and M10 but not in HCT116 cells. In M10 cells, TMZ promoted NF-κB2/p52 generation and nuclear translocation and enhanced the secretion of IL-8 and MCP-1. TMZ induced RelA nuclear translocation also in 293TLα+ but not in 293TLα- cells. AKT1 silencing inhibited TMZ-induced IκB-α degradation and NF-κB2/p52 generation. Up-regulation of NF-κB transcriptional activity and nuclear translocation of RelA and NF-κB2/p52 in response to TMZ were impaired in KD12 cells. RelA silencing in HCT116/3-6 and M10 cells increased TMZ-induced growth suppression. In M10 cells NBD peptide reduced basal NF-κB activity, abrogated TMZ-induced up-regulation of NF-κB activity and increased sensitivity to TMZ. In HCT116/3-6 cells, the combined treatment with NBD peptide and TMZ produced additive growth inhibitory effects., Conclusion: NF-κB is activated in response to TMZ in a MMR- and AKT-dependent manner and confers protection against drug-induced cell growth inhibition. Our findings suggest that a clinical benefit could be obtained by combining TMZ with NF-κB inhibitors.

Placenta growth factor (PlGF) and its receptor vascular endothelial growth factor receptor-1 (VEGFR-1) are co-expressed in a large number of human melanoma cell lines. Moreover, a correlation between in vivo PlGF production and melanoma progression has been suggested. To investigate whether PlGF might have a role in protecting melanoma cells from the cytotoxic effects of the anticancer agent temozolomide (TMZ), which is used for the treatment of this malignancy, we stably transfected a doxycycline-inducible PlGF antisense mRNA into a human melanoma cell clone that secretes VEGF-A and PlGF and expresses receptors for both growth factors. Induction of PlGF antisense mRNA in the transfected cells (13443/ASP3 subclone) halved TMZ IC(50), and exogenous addition of PlGF to the culture medium 24 h before TMZ treatment, partially restored IC(50) values to that of control cells. The increased sensitivity of 13443/ASP3 cells upon PlGF antisense mRNA expression was not due to down-regulation of O6-methylguanine-DNA methyltransferase, a DNA repair protein that represents the main mechanism of resistance to TMZ. Since the activity of the transcription factor nuclear factor-κB (NF-κB) has been correlated to melanoma chemoresistance, we investigated whether NF-κB was involved in PlGF-induced melanoma cell resistance to TMZ. Induction of PlGF antisense mRNA in 13443/ASP3 cells halved the levels of active NF-κB and the specific inhibition of this transcription factor increased sensitivity of 13443/ASP3 cells to TMZ. In conclusion, our data strongly suggest that PlGF plays a role in melanoma cell resistance to TMZ through a pathway that involves NF-κB activation.

PHA-848125 is a novel cyclin-dependent kinase inhibitor under Phase I/II clinical investigation. In this study, we describe, for the first time, the effect of PHA-848125 on human melanoma cells in vitro. Seven melanoma cell lines with different sensitivity to temozolomide (TMZ) were exposed to PHA-848125 for 5 days and then assayed for cell growth. In all cases, including TMZ-resistant cells, PHA-848125 IC(50) values were significantly below the maximum plasma concentrations achievable in the clinic. In the most PHA-848125-sensitive cell line, the drug caused a concentration-dependent G(1) arrest. PHA-848125 also impaired phosphorylation of the retinoblastoma protein at CDK2 and CDK4 specific sites, decreased retinoblastoma protein and cyclin A levels, and increased p21(Cip1), p27(Kip1) and p53 expression. Combined treatment with fixed ratios of TMZ plus PHA-848125 was studied in three melanoma cell lines. PHA-848125 was added to the cells 48 h after TMZ and cell growth was evaluated after 3 additional days of culture. Parallel experiments were performed in the presence of O(6)-benzylguanine (BG), to prevent repair of methyl adducts at O(6)-guanine induced by TMZ. Drug combination of TMZ plus BG and PHA-848125 produced additive or synergistic effects on cell growth, depending on the cell line. In the absence of BG, the combination was still more active than the single agents in the cell line moderately sensitive to TMZ, but comparable to PHA-848125 alone in the two TMZ-resistant cell lines. When TMZ plus BG were used in combination with PHA-848125 against cultured normal melanocytes, neither synergistic nor additive antiproliferative effects were observed. Our results indicate that PHA-848125 can have a therapeutic potential in melanoma patients, alone or combined with TMZ. Moreover this agent appears to be particularly attractive on the bases of its effectiveness against TMZ-resistant melanoma cells., ((c) 2009 Elsevier Ltd. All rights reserved.)

The phosphatidylinositol 3-kinase/AKT pathway is activated frequently in human cancer, and it has been implicated in tumor cell proliferation, survival, and chemoresistance. In this study, we addressed the role of AKT in cellular responses to the therapeutic methylating agent temozolomide (TMZ), and we investigated the possible link between TMZ-induced modulation of AKT function and activation of ataxia-telangiectasia and Rad3-related (ATR)- and ataxia telangiectasia mutated (ATM)-dependent signaling pathways. We found that clinically relevant concentrations of TMZ caused activation of endogenous AKT in lymphoblastoid cells, and in colon and breast cancer cells, and that this molecular event required a functional mismatch repair system. Transfection of a dominant-negative kinase-dead form of AKT1 into breast cancer cells abrogated TMZ-induced activation of endogenous AKT, and it markedly enhanced cell sensitivity to the drug. Likewise, exposure of the MMR-proficient cell lines to the AKT inhibitor D-3-deoxy-2-O-methyl-myo inositol 1-[(R)-2-methoxy-3-(octadecyloxy)-propyl hydrogen phosphate] (SH-5) impaired AKT phosphorylation in response to TMZ, and it significantly increased cell chemosensitivity. Furthermore, small interfering RNA (siRNA)-mediated reduction of AKT1 expression in colon cancer cells potentiated the growth inhibitory effects of TMZ. Inhibition of ATM expression in colon cancer cells by siRNA did not impair TMZ-induced activation of AKT, whereas siRNA-mediated inhibition of ATR prevented AKT activation in response to the drug and increased cell chemosensitivity. These results strongly support the hypothesis that clinical benefit could be obtained by combining TMZ with inhibitors of the AKT pathway. Moreover, they provide the first evidence of a novel function of ATR as an upstream activator of AKT in response to DNA damage induced by O(6)-guanine-methylating agents.

Previous studies indicated that dacarbazine and Temozolomide could be highly effective against refractory acute leukaemia. Their activity relies mainly on the generation of methyl adducts at the O(6)-position of guanine in DNA. High levels of O(6)-methylguanine-DNA methyltransferase (MGMT) or a defective mismatch repair (MMR) system, are associated with cellular resistance to triazenes. The MGMT inhibitor, O(6)-(4-bromothenyl)guanine (Lomeguatrib), can restore in vitro sensitivity to Temozolomide in MMR-proficient blasts. In the early 1970s we discovered that, in vivo, triazene compounds induce the appearance of novel transplantation antigens in murine leukaemia ("Chemical Xenogenization", CX). Non-self peptides presented by class I MHC molecules are generated by triazene-induced somatic mutations, affecting retroviral sequences that are detectable in the mouse genome. Moreover, preliminary experiments suggested that human cancer cells can also undergo CX. Therefore, we designed a chemo-immunotherapy strategy in leukaemic patients as follows: (a) cytoreduction and a hypothetical CX phase, i.e. treatment with Lomeguatrib (to suppress MGMT activity) and Temozolomide (to kill sensitive blasts and to presumably induce CX in resistant leukaemic cells); (b) immune response recovery phase using interleukin-2 (to possibly restore an immune response and take advantage of the hypothetical, triazene-induced CX). Here we present the results of pilot study which is in progress in patients with refractory/relapsed acute leukaemia. In all tested cases, Lomeguatrib suppressed MGMT activity in vivo. Six out of eight patients showed partial or complete disappearance of blast cells in peripheral blood or in bone marrow. We observed severe and long-lasting myelosuppression, accompanied by limited non-haematological toxicity. Up to now, two patients are alive (after 9 and 10 months, respectively), four died of opportunistic infections and two of progressive disease. This investigation confirms the potential role of triazenes in leukaemia and highlights the contribution of Lomeguatrib in overcoming drug resistance. Further studies are required to establish whether Temozolomide can induce CX in human leukaemia, and thus offer a new approach to control minimal residual disease.

Hyperthermic isolated limb perfusion (HILP) with L-phenylalanine mustard (L-PAM) represents an effective treatment for locally advanced melanoma of the limbs. However, regional chemotherapy of melanoma still needs to be improved. Temozolomide (TMZ) is a methylating agent that spontaneously decomposes into the active metabolite of dacarbazine, the most effective agent for the systemic treatment of melanoma. Tumor cells with high levels of O6-methylguanine-DNA methyltransferase (MGMT) and/or with a defective DNA mismatch repair (MMR) are resistant to TMZ. Inhibition of MGMT activity increases TMZ sensitivity of MMR-proficient, but not of MMR-deficient cells, while inhibition of base excision repair (BER) potentiates TMZ cytotoxicity in both cell types. Recent studies, performed in an animal model, have shown that TMZ is more effective than L-PAM when applied regionally and that hyperthermia can increase the antitumor activity of TMZ. In this study, three thermoresistant human melanoma cell lines, endowed with different MGMT activity and functional status of the MMR system, were treated with TMZ at 37 degrees C or 41.5 degrees C for 90 min, and then analyzed for cell growth and MGMT activity. Hyperthermia significantly enhanced TMZ cytotoxicity in MMR-proficient cells, either endowed or not with MGMT activity, and in MMR-deficient cells. Endogenous MGMT activity was not affected by hyperthermia that, however, enhanced the enzyme depletion induced by TMZ treatment. Moreover, MGMT recovery after drug removal was delayed in cells that had been treated at 41.5 degrees C. Taken together, these findings confirm the therapeutic potential of a combined treatment of hyperthermia and TMZ. They also suggest that inhibition of BER and/or increased DNA methylation may be involved in the thermal enhancement of TMZ cytotoxicity. Additional studies are necessary to better clarify the mechanisms underlying hyperthermia-induced potentiation of TMZ activity. However, the present investigation provides further support to the development of clinical trials of HILP with TMZ.

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.

Anti-tumour activity of triazene compounds of clinical interest [i.e. dacarbazine and temozolomide (TMZ)] relies mainly on the generation of methyl adducts to purine bases of DNA. Two DNA repair enzyme systems, i.e. the O6-guanine-alkyl-transferase (MGMT) and mismatch repair (MMR), play a predominant role in conditioning the cytotoxic effects of triazenes. In particular, high levels of MGMT associated with target cells are responsible of resistance to triazenes. On the contrary, the presence of MMR is required for the cytotoxic effects of these compounds. Previous studies performed by our group and a more recent clinical investigation reported by Karen Seiter, pointed out that triazene compounds could play an important role in the treatment of refractory acute leukaemia. Leukaemia blasts, especially of lymphoblastic leukaemia, show frequently high levels of MGMT activity. Therefore, it reasonable to hypothesize that combined treatment of leukaemia patients with triazene compounds along with MGMT inhibitors could lead to a better control of the disease. PaTrin-2 (O6-(4-bromothenyl)guanine, PAT) is a potent and scarcely toxic MGMT inhibitor recently introduced in clinical trials. This drug is used in combination with triazene compounds in order to augment their anti-tumour efficacy against neoplastic cells endowed with high MGMT activity. The present report describes, for the first time, pre-clinical in vitro studies on the cytotoxic activity of combined treatment with PAT+TMZ against long-term cultured leukaemia cells and primary leukaemia blasts obtained from patients with acute lymphoblastic leukaemia or acute myeloblastic leukaemia. The results point out that, both in long-term cultured leukaemia cell lines and in primary blast samples, PAT could improve dramatically the sensitivity of malignant cells to the cytotoxic effects of TMZ. This sensitizing effect is detectable when leukaemia cells show resistance mechanisms based on a MGMT-proficient phenotype. On the contrary, when resistance to TMZ is dependent on MMR deficiency, no influence of PAT can be detected in various experimental conditions. In conclusion, these results appear to provide disease-oriented rational basis to design novel clinical protocols for the treatment of acute leukaemia with combined administration of PAT and triazene compounds.

The mammalian mismatch repair (MMR) system has been implicated in activation of the G(2) checkpoint induced by methylating agents. In an attempt to identify the signaling events accompanying this phenomenon, we studied the response of MMR-proficient and -deficient cells to treatment with the methylating agent temozolomide (TMZ). At low TMZ concentrations, MMR-proficient cells were growth-inhibited, arrested in G(2)/M, and proceeded to apoptosis after the second post-treatment cell cycle. These events were accompanied by activation of the ATM and ATR kinases, and phosphorylation of Chk1, Chk2, and p53. ATM was activated later than ATR and was dispensable for phosphorylation of Chk1, Chk2, and p53 on Ser15 and for triggering of the G(2)/M arrest. However, it conferred protection against cell growth inhibition induced by TMZ. ATR was activated earlier than ATM and was required for an efficient phosphorylation of Chk1 and p53 on Ser15. Moreover, abrogation of ATR function attenuated the TMZ-induced G(2)/M arrest and increased drug-induced cytotoxicity. Treatment of MMR-deficient cells with low TMZ concentrations failed to activate ATM and ATR and to cause phosphorylation of Chk1, Chk2, and p53, as well as G(2)/M arrest and apoptosis. However, all these events occurred in MMR-deficient cells exposed to high TMZ concentrations, albeit with faster kinetics. These results demonstrate that TMZ treatment activates ATM- and ATR-dependent signaling pathways and that this process is absolutely dependent on functional MMR only at low drug concentrations.

Resveratrol, a polyphenol present in many plant species, exhibits a wide range of biological and pharmacological activities both in vitro and in vivo. It has been shown to exert a potent chemopreventive effect in carcinogenesis models and to induce cell growth inhibition and apoptosis in human tumour cells, including melanoma cells. Malignant melanoma is considered to be a chemotherapy-refractory tumour, and the commonly used anticancer drugs do not seem to modify the prognosis of metastatic disease. To further evaluate the therapeutic potential of resveratrol in the treatment of melanoma, we selected three human melanoma cell lines with different levels of resistance to temozolomide (TMZ), an antitumour triazene compound. The cell lines were subjected to resveratrol treatment and analysed for cell growth inhibition, cell cycle perturbation and apoptosis induction. We found that resveratrol markedly impaired proliferation of both the TMZ-sensitive M14 and the TMZ-resistant SK-Mel-28 and PR-Mel cell lines. The latter cell line was two-fold more resistant to the drug than M14 and SK-Mel-28 cells. The sensitivity of normal human keratinocytes to resveratrol was found to be significantly higher than that of M14 and SK-Mel-28 cells and similar to that of the PR-Mel cell line. This suggests a possible good in vivo therapeutic index for resveratrol. Our results also show that the growth-inhibitory effect of resveratrol on melanoma cells is mainly due to its ability to induce S-phase arrest and apoptosis. Taken together, our data indicate that resveratrol is an interesting candidate for the treatment of advanced melanoma.

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 neoplastic 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.

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.

Members of hereditary nonpolyposis colon cancer (HNPCC) families harboring heterozygous germline mutations in the DNA mismatch repair genes hMSH2 or hMLH1 present with tumors generally two to three decades earlier than individuals with nonfamilial sporadic colon cancer. We searched for phenotypic features that might predispose heterozygous cells from HNPCC kindreds to malignant transformation. hMSH2(+/-) lymphoblastoid cell lines were found to be on average about 4-fold more tolerant than wild-type cells to killing by the methylating agent temozolomide, a phenotype that is invariably linked with impairment of the mismatch repair system. This finding was associated with an average 2-fold decrease of the steady-state level of hMSH2 protein in hMSH2(+/-) cell lines. In contrast, hMLH1(+/-) heterozygous cells were indistinguishable from normal controls in these assays. Thus, despite the fact that HNPCC families harboring mutations in hMSH2 or hMLH1 cannot be distinguished clinically, the early stages of the carcinogenic process in hMSH2 and hMLH1 mutation carriers may be different. Should hMSH2(+/-) colonocytes and lymphoblasts harbor a similar phenotype, the increased tolerance of the former to DNA-damaging agents present in the human colon may play a key role in the initiation of the carcinogenic process.

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is important in cellular resistance to certain alkylating antitumor agents such as the methylating drug temozolomide (TMZ). To provide a more rational basis for clinical combinations with another commonly used drug, cisplatin, we assessed the modulation of MGMT protein and mRNA levels in the human leukemic cell line Jurkat after treatment with these agents. Cisplatin decreased MGMT activity in a time- and dose-dependent manner, with maximal suppression (50%) observed 24 h after treatment with 25 microM cisplatin. This was probably the result of decreased transcription of the MGMT gene, because there was an earlier nadir of MGMT mRNA levels after cisplatin treatment and neither cisplatin nor DNA reacted with cisplatin in vitro was able to inhibit MGMT activity in an in vitro assay. TMZ alone depleted MGMT activity in a time- and dose-dependent manner with almost complete loss of activity occurring immediately after treatment with 500 microM TMZ. Combinations of cisplatin (12.5 microM) and TMZ (250 microM) caused substantial and prolonged MGMT depletion with recovery to only 30% of pretreatment levels by 48 h. These results suggest that the clinical efficacy of TMZ and cisplatin may be improved by appropriate schedules of combinations of these agents.

Temozolomide (TMZ) is a new cytotoxic triazene compound of clinical interest that is able to generate methyl adducts at the O(6)-guanine of DNA, which can be repaired by O(6)-alkylguanine-DNA alkyltransferase (OGAT). It was previously found that triazene compounds are highly immunosuppressive in mice. In the present study, we investigate whether TMZ could affect immune functions of human competent cells and whether methylation of O(6)-guanine could be involved in the immunosuppressive activity of the drug. Mononuclear cells (MNCs) obtained from peripheral blood of healthy donors were tested for OGAT activity and treated with TMZ alone or combined with the OGAT inhibitor O(6)-benzylguanine. Control or drug-treated MNCs were then assayed for natural killer activity and for the ability to proliferate and to generate cytotoxic effector cells in response to interleukin-2 or allogeneic MT-2 tumor cells. The results show that TMZ inhibited both proliferation and induction of lytic activity in response to interleukin-2 or allogeneic MT-2 cells. Moreover, an inverse correlation was found between the OGAT activity of MNCs and their sensitivity to TMZ. The involvement of O(6)-guanine methylation in the immunosuppressive effects of TMZ was further confirmed by the finding that O(6)-benzylguanine increased the activity of the drug. On the other hand, the natural killer activity of MNCs was only moderately affected by TMZ, and no relationship was observed between OGAT levels and sensitivity to the drug. These data suggest that in patients with tumors who are undergoing TMZ treatment, the drug may impair immune responses involving cell proliferation, depending on OGAT levels of MNCs, and that O(6)-benzylguanine may potentiate this activity.

Postreplicative mismatch repair plays a major role in mediating the cytotoxicity of agents generating O6-methylguanine in DNA. We previously showed that a methylating antitumor triazene compound, temozolomide, induces apoptosis and that the persistence of O6-methylguanine in DNA is required to trigger the process. We wanted to test whether the latter apoptotic signal is dependent on a functional mismatch repair system. To this end, we used two human lymphoblastoid cell lines (i.e., the mismatch repair-proficient TK6 line and its mismatch repair-deficient subline MT1) that are both deficient in O6-methylguanine repair. Temozolomide treatment of TK6 cells brought about efficient cell growth inhibition, G2/M arrest, and apoptosis, as indicated by the results of cytofluorimetric analysis of 5-bromo-2'-deoxyuridine incorporation and DNA content and evaluation of DNA fragmentation. The drug treatment resulted also in the induction of p53 and p21/waf-1 protein expression. In contrast, MT1 cells were highly resistant to the drug and no p53 and p21/waf-1 induction was observed. Importantly, we could show that MT1 cells are not deficient in the p53-dependent apoptosis pathway; treatment with etoposide, a topoisomerase II inhibitor, resulted in p53 and p21/waf-1 protein expression and apoptosis in both cell lines. In conclusion, we demonstrate the existence of a link between a functional mismatch repair system and the trigger of apoptosis in cells exposed to clinically relevant concentrations of temozolomide. The results also suggest that p53 induction in response to O6-guanine methylation involves the mismatch repair system.

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.

Previous studies demonstrated that triazene compounds (TZC) possess antitumor, antimetastatic and immunosuppressive activity, and induce novel antigenic properties in neoplastic cells. Moreover, TZC showed marked antitumor activity in patients with acute myelogenous leukemias (AML). In most cases leukemic blasts with low levels of the repair enzyme O6-alkyl-guanine-DNA alkyltransferase (OGAT) were highly susceptible to TZC. Therefore the cytotoxic effects of TZC against human leukemic cells and the influence of OGAT modulation were investigated. Five leukemia cell lines were treated with the in vitro active derivative of dacarbazine: 5-(3-methyl-1-triazeno) imidazole-4-carboxamide (MTIC), or with temozolomide (TZM), which is readily cleaved to form the linear triazene MTIC in aqueous solution. The results showed that treatment with TZC at concentrations ranging between 62.5 and 250 microM significantly inhibited cell growth of U-937 and K-562 leukemia cell lines, both with undetectable OGAT activity. Growth inhibition was accompanied by DNA fragmentation and reduction of cell volume characteristic of cell undergoing apoptosis. In contrast, Daudi, HL-60 and Jurkat leukemia cell lines, characterized by high levels of the repair enzyme, were resistant to concentrations of TZC up to 500 microM. Treatment of resistant lines with O6-benzylguanine (BG, a specific inhibitor of OGAT) rendered HL-60 and Daudi but not Jurkat cells sensitive to cytotoxic effects and apoptosis mediated by MTIC. The results presented suggest that: (1) apoptosis is involved in cytotoxic activity of TZC; (2) OGAT could have a role in preventing programmed cell death induced by TZC; and (3) treatment with BG could potentiate cytotoxic and apoptotic effects of TZC on leukemic cell lines when high level of OGAT activity is the main factor involved in resistance to TZC.

Background: 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)., Design: 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., Results: 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., Conclusions: 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.

Background and Methods: Preclinical studies performed in our laboratory showed that mouse leukemias become highly immunogenic following in vivo treatment with Decarbazine. This observation led to a successful immunochemotherapy protocol in mice using Dacarbazine plus cytoreductive chemotherapy. Therefore an in vivo and in vitro pilot study was conducted in patients (pts) with resistant or relapsed acute myelogenous leukemia (AML). The DNA-repair enzyme O6-alkylguanine-DNA-alkyl-transferase (OGAT) and the in vitro chemosensitivity to Temozolomide, a Dacarbazine derivative, were evaluated in leukemic blasts., Results: Nine pts received Dacarbazine (0.4-0.8 g/sqm/day) on days 0, 1 and 2. On day 7, noticeable blast reduction occurred in 4 pts: pt 1 was in partial remission on day 21, pt 6 still showed bone marrow leukemia, pt 8 died of sepsis on day 8, pt 9 is still in aplasia on day 25. Low OGAT levels and consistent sensitivity to Temozolomide in vitro were found in the blasts of pts responsive to Dacarbazine. Subsequently, pts 1-7 underwent Ara-C (1g/sqm/day) plus Mitoxantrone (6mg/sqm/day) treatment for 6 days. Four pts entered complete remission after 27-45 days of aplasia. Failures were due to hypoplastic death, absolute drug resistance, or hypoplasia followed by blast cell regrowth., Conclusions: These data point out that Dacarbazine can induce a marked reduction of blast cells as well as severe myelotoxicity in leukemic patients.