Bortezomib administered prior to temozolomide depletes MGMT, chemosensitizes glioblastoma with unmethylated MGMT promoter and prolongs animal survival.

BACKGROUND: Resistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6-methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ. METHODS: Cell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan-Meier method. RESULTS: Pre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood-brain barrier, diminished proteasome activity and significantly prolonged animal survival. CONCLUSION: BTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.

Repair gene O6 -methylguanine-DNA methyltransferase is controlled by SP1 and up-regulated by glucocorticoids, but not by temozolomide and radiation.

Therapy of malignant glioma relies on treatment with the O6 -methylating agent temozolomide (TMZ) concomitant with ionizing radiation followed by adjuvant TMZ. For the treatment of recurrences, DNA chloroethylating drugs are also used. The main killing lesion induced by these drugs is O6 -alkylguanine. Since this damage is repaired by O6 -methylguanine-DNA methyltransferase (MGMT), the repair enzyme represents a most important factor of drug resistance, limiting the therapy of malignant high-grade gliomas. Although MGMT has been shown to be transcriptionally up-regulated in rodents following genotoxic stress, it is still unclear whether human MGMT is subject to up-regulation. Here, we addressed the question whether MGMT in glioma cells is enhanced following alkylating drugs or ionizing radiation, using promoter assays. We also checked the response of glioma cell lines to dexamethasone. In a series of experiments, we found no evidence that the human MGMT promoter is significantly up-regulated following treatment with TMZ, the chloroethylating agent nimustine or radiation. It was activated, however, by dexamethasone. Using deletion constructs, we further show that the basal level of MGMT is mainly determined by the transcription factor SP1. The high amount of SP1 sites in the MGMT promoter likely prevents transcriptional up-regulation following genotoxic stress by neutralizing inducible signals. The regulation of MGMT by miRNAs plays only a minor role, as shown by DICER knockdown experiments. Since high dose dexamethasone concomitant with temozolomide is frequently used in glioblastoma therapy, induction of the MGMT gene through glucocorticoids in MGMT promoter unmethylated cases might cause further elevation of drug resistance, while radiation and alkylating drugs seem not to induce MGMT at transcriptional level.

In human glioblastomas transcript elongation by alternative polyadenylation and miRNA targeting is a potent mechanism of MGMT silencing.

Favorable outcome after chemotherapy of glioblastomas cannot unequivocally be linked to promoter hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene encoding a DNA repair enzyme associated with resistance to alkylating agents. This indicates that molecular mechanisms determining MGMT expression have not yet been fully elucidated. We here show that glioblastomas are capable to downregulate MGMT expression independently of promoter methylation by elongation of the 3'-UTR of the mRNA, rendering the alternatively polyadenylated transcript susceptible to miRNA-mediated suppression. While the elongated transcript is poorly expressed in normal brain, its abundance in human glioblastoma specimens is inversely correlated with MGMT mRNA expression. Using a bioinformatically guided experimental approach, we identified miR-181d, miR-767-3p, and miR-648 as significant post-transcriptional regulators of MGMT in glioblastomas; the first two miRNAs induce MGMT mRNA degradation, the latter affects MGMT protein translation. A regression model including the two miRNAs influencing MGMT mRNA expression and the MGMT methylation status reliably predicts The Cancer Genome Atlas MGMT expression data. Responsivity of MGMT expressing T98G glioma cells to temozolomide was significantly enhanced after transfection of miR-181d, miR-767-3p, and miR-648. Taken together, our results uncovered alternative polyadenylation of the MGMT 3'-UTR and miRNA targeting as new mechanisms of MGMT silencing.

The expression of O(6) -methylguanine-DNA methyltransferase in human oral keratinocytes stimulated with arecoline.

BACKGROUND: O(6) -methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that can protect cells from carcinogenic effects of alkylating agents by removing adducts from the O(6) position of guanine. Evidences indicated that areca quid chewing may increase the risk of oral squamous cell carcinoma (OSCC). This study was to investigate the role of MGMT expression in OSCCs and the normal oral tissues. METHODS: Thirty-two OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by the immunohistochemistry for MGMT. Primary human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot. Nicotine, an important component of cigarette smoke, was added to find the possible regulatory mechanisms. RESULTS: Significant association was observed between low MGMT expression and advanced clinical stage of OSCCs and lymph node metastasis (P = 0.03). MGMT expression was significantly higher in patients only chewing areca quid than patients both chewing areca quid and smoking (P = 0.028). Arecoline was found to elevate MGMT expression in a dose- and time-dependent manner. The addition of nicotine was found to enhance arecoline-induced MGMT expression. CONCLUSION: Our results indicate that MGMT could be used clinically as a predictive marker for tumor processing, the potential for lymph node metastasis as well as advanced clinical stage. MGMT expression was significantly upregulated by arecoline in HOKs. Nicotine has a synergistic effect of arecoline-induced MGMT expression. The cigarette smoking may act synergistically in the pathogenesis of OSCC in areca quid chewers via the upregulation of MGMT.

Phase I study of temozolomide and laromustine (VNP40101M) in patients with relapsed or refractory leukemia.

PURPOSE: Although alkylators are known to be effective against some myeloid leukemias, resistance is often mediated via O6-alkylguanine-DNA alkyltransferase (AGT). Temozolomide's inhibition of AGT may sensitize leukemia cells to the novel alkylator laromustine. We conducted a phase I translational study to evaluate the toxicities and estimate the maximum tolerated dose (MTD) of laromustine when administered with temozolomide (TMZ) in patients with hematologic malignancies. PATIENTS AND METHODS: TMZ was delivered twice daily for 5 doses followed by a single infusion of laromustine. The target TMZ dose was the dose that would reliably result in > 90% AGT depletion. Once the target TMZ dose was identified, the laromustine dose was escalated. A total of 35 patients with relapsed/refractory leukemia were treated. RESULTS: Treatment with TMZ 300 mg for 5 doses resulted in > 90% depletion of AGT levels in 5 of 6 patients. The MTD of the combination was established at TMZ 1500 mg and laromustine 300 mg/m2. Three of the 7 patients assayed from cohort 1 achieved > 90% depletion of AGT activity (range, 77%-100% depletion; median, 88%). Five of 6 patients enrolled in cohort 2 achieved > 90% depletion of AGT activity (range, 92%-100% depletion; median, 93.5%). This established that the 300-mg dose of TMZ (1500 mg total) would be maintained in subsequent cohorts. The majority of adverse events were primarily hematologic, with infectious and pulmonary complications also noted. Three (9%) of the patients with previous refractory disease achieved a complete remission, and 5 (14%) of the patients achieved a morphologic, leukemia-free, but persistent hypocellular bone marrow status. CONCLUSION: Laromustine in combination with TMZ is tolerable and manageable at doses that predictably suppress AGT. Reliable TMZ-induced inhibition of AGT was observed in doses that are clinically tolerable. Evidence of antitumor effect was observed with this combination, suggesting that further efficacy studies should be performed.

Loss expression of O6-methylguanine DNA methyltransferase by promoter hypermethylation and its relationship to betel quid chewing in oral squamous cell carcinoma.

OBJECTIVE: O(6)-methylguanine-DNA methyltransferase (MGMT) ameliorates mutagenic, carcinogenic, and cytotoxic adducts from O(6)-methylguanine in DNA through a direct reversal mechanism. Decreased expression of MGMT has been reported in a variety of human malignant tumors. The purpose of this study was to clarify the correlation of MGMT expression levels in oral squamous cell carcinoma (OSCC) with promoter hypermethylation and with betel quid chewing and cigarette smoking. STUDY DESIGN: MGMT protein expression in 63 cases of oral squamous cell carcinoma by immunohistochemistry was investigated. Methylation status of the MGMT was analyzed by methylation-specific PCR. Correlation with clinicopathologic parameters was then tested by statistical analysis. RESULTS: MGMT immunohistochemistry revealed nuclear staining in normal epithelium, whereas 47 (75%) of 63 OSCC tumors were devoid of MGMT expression and this was related to tumor cell differentiation. Furthermore, the association between loss of MGMT expression and promoter hypermethylation was significant. Lacking protein expression for MGMT in OSCC was also associated with the use of betel quid. CONCLUSIONS: The results suggest that the absence of MGMT expression, which would seem to be associated with promoter hypermethylation, is related to betel quid chewing and, thus, in turn, might be a significant event in oral carcinogenesis.

A phase I trial of lomeguatrib and irinotecan in metastatic colorectal cancer.

BACKGROUND: Expression of the DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT) correlates with resistance to irinotecan in colorectal cancer cell lines. This phase I study evaluated the maximum tolerated dose (MTD) of lomeguatrib, an inactivating pseudosubstrate of MGMT, in combination with irinotecan in patients with metastatic colorectal cancer and assessed the safety, toxicity and clinical pharmacology of combination treatment. PATIENTS AND METHODS: Patients with metastatic colorectal cancer received lomeguatrib (10-80 mg PO) on days 1-5 with irinotecan (250-350 mg/m(2) IV) on day 4 of a 21-day cycle. RESULTS: Twenty-four patients, pre-treated with a median of 2 lines of chemotherapy, received 104 cycles of treatment. The MTD was defined as 80 mg/day lomeguatrib with 300 mg/m(2) irinotecan. The main toxicities observed were neutropaenia and diarrhoea. Lomeguatrib of 80 mg/day produced complete MGMT depletion in all available peripheral blood mononuclear cells (PBMCs) and paired tumour biopsies (one patient). There was no pharmacokinetic interaction between the drugs. In 22 patients assessable for tumour response, one achieved a partial response and 16 had stable disease. CONCLUSION: This study defined a tolerable dose of irinotecan in combination with lomeguatrib in patients with metastatic colorectal cancer. Combination treatment gave a similar response rate to irinotecan monotherapy in this heavily pre-treated patient group.

O(6)-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib.

We evaluated the pharmacodynamic effects of the O(6)-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O(6)-methylguanine (O(6)-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O(6)-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O(6)-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O(6)-meG in PBMC DNA during treatment.

O (4)-benzylfolic acid inactivates O (6)-alkylguanine-DNA alkyltransferase in brain tumor cell lines.

PURPOSE: The DNA repair protein, O (6)-alkylguanine-DNA alkyltransferase (AGT), is a primary source of tumor resistance to agents such as temozolomide and chloroethylnitrosoureas that form DNA lesions at the O (6)-position of guanines. To increase the efficacy of these drugs, pseudosubstrate inactivators of AGT such as O (6)-benzylguanine have been developed. A novel inactivator of AGT, O (4)-benzylfolic acid (O(4)-BFA), has been reported which is more potent and water soluble than O (6)-benzylguanine. Previous studies have suggested that uptake of O(4)-BFA is mediated by the folate receptor (FR), and, thus, its use may be limited to cells expressing FR. METHODS: We measured AGT activity in cell extracts from a panel of brain tumor cells exposed to O(4)-BFA. Inactivation of AGT by O(4)-BFA was measured in cells grown without folic acid as well as in cells grown in folic acid-containing media. Competitive binding studies were performed using purified FR to determine its affinity for O(4)-BFA. RESULTS: The observed IC(50) for O(4)-BFA in brain tumor cell lines ranged from 0.2 to 1.3 microM for cells grown in media containing 2.3 microM folic acid. At this concentration, folic acid would saturate the FR and the FR would be unable to take up O(4)-BFA. When cells were grown in folic acid free media, there was at most a 50% decrease in the observed IC(50)s, indicating that the FR was not essential for O(4)-BFA uptake. Competitive binding studies using purified FR confirmed that the IC(50) for O(4)-BFA is approximately 180 times greater than folic acid, i.e., it has a very weak affinity for FR. CONCLUSION: These results indicate that O(4)-BFA has potentially broad use as an inactivator of AGT as its use is not limited to tumors expressing high levels of FR.

Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms.

PURPOSE: In this study, we investigated the mechanisms by which temozolomide enhances radiation response in glioblastoma cells. EXPERIMENTAL DESIGN: Using a panel of four primary human glioblastoma cell lines with heterogeneous O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression, normal human astrocytes, and U87 xenografts, we investigated (a) the relationship of MGMT status with efficacy of temozolomide-based chemoradiation using a panel of in vitro and in vivo assays; (b) underlying mechanisms by which temozolomide enhances radiation effect in glioblastoma cells; and (c) strategies to overcome resistance to radiation + temozolomide. RESULTS: Temozolomide enhances radiation response most effectively in glioblastomas without detectable MGMT expression. On concurrent radiation + temozolomide administration in MGMT-negative glioblastomas, there seems to be decreased double-strand DNA (dsDNA) repair capacity and enhanced dsDNA damage compared either with radiation alone or with sequentially administered temozolomide. Our data suggest that O(6)-benzylguanine can enhance the antitumor effects of concurrent radiation + temozolomide in MGMT-positive cells by enhancing apoptosis and the degree of dsDNA damage. O(6)-Benzylguanine was most effective when administered concurrently with radiation + temozolomide and had less of an effect when administered with temozolomide in the absence of radiation or when administered sequentially with radiation. Our in vivo data using U87 xenografts confirmed our in vitro findings. CONCLUSIONS: The present study shows that temozolomide enhances radiation response most effectively in MGMT-negative glioblastomas by increasing the degree of radiation-induced double-strand DNA damage. In MGMT-positive glioblastomas, depletion of MGMT by the addition of O(6)-benzylguanine significantly enhances the antitumor effect of concurrent radiation + temozolomide. These are among the first data showing mechanisms of synergy between radiation and temozolomide and the effect of MGMT.

Mode of action of the chloroethylating and carbamoylating moieties of the prodrug cloretazine.

Cloretazine is an antitumor sulfonylhydrazine prodrug that generates both chloroethylating and carbamoylating species. The cytotoxic potency of these species was analyzed in L1210 leukemia cells using analogues with chloroethylating or carbamoylating function only. Clonogenic assays showed that the chloroethylating-only agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) produced marked differential cytotoxicity against wild-type and O6-alkylguanine-DNA alkyltransferase-transfected L1210 cells (LC10, 1.4 versus 31 micromol/L), indicating that a large portion of the cytotoxicity was due to alkylation of DNA at the O-6 position of guanine. Consistent with the concept that O-6 chloroethylation of DNA guanine progresses to interstrand cross-links, the comet assay, in which DNA cross-links were measured by a reduction in DNA migration induced by strand breaks, showed that cloretazine and 90CE, but not the carbamoylating-only agent 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), produced DNA cross-links and that cloretazine caused more DNA cross-links than 90CE at equimolar concentrations. Cell cycle analyses showed that 90CE and 101MDCE at concentrations of 5 and 80 micromol/L, respectively, produced similar degrees of G2-M arrest. 90CE produced selective inhibition of DNA synthesis after overnight incubation, whereas 101MDCE caused rapid and nonselective inhibition of RNA, DNA, and protein syntheses. Both 90CE and 101MDCE induced phosphorylation of histone H2AX, albeit with distinct kinetics. These results indicate that (a) differential expression of O6-alkylguanine-DNA alkyltransferase in tumor and host cells seems to be responsible for tumor selectivity exerted by cloretazine; (b) 101MDCE enhances DNA cross-linking activity; and (c) 90CE induces cell death at concentrations lower than those causing alterations in the cell cycle and macromolecular syntheses.

Differential competitive resistance to methylating versus chloroethylating agents among five O6-alkylguanine DNA alkyltransferases in human hematopoietic cells.

P140K-MGMT and G156A-MGMT genes encode two O(6)-benzylguanine-resistant O(6)-alkylguanine DNA alkyltransferase proteins that confer a high degree of O(6)-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or O(6)-benzylguanine and temozolomide resistance to primary hematopoietic cells. In this study, we directly compared these and three other O(6)-benzylguanine-resistant MGMT genes for their ability to protect the human erythroleukemia cell line, K562, using a direct competitive selection strategy to identify the mutation that conferred the greatest degree of protection from O(6)-benzylguanine and either BCNU or temozolomide. MFG retroviral vector plasmids for each of these mutants [G156A-MGMT (ED(50) for O(6)-benzylguanine, 60 micromol/L); and P140K-MGMT, MGMT-2 (S152H, A154G, Y158H, G160S, L162V), MGMT-3 (C150Y, A154G, Y158F, L162P, K165R), and MGMT-5 (N157T, Y158H, A170S; ED(50) for benzylguanine, >1,000 micromol/L)] were mixed, and the virus produced from Phoenix cells was transduced into K562 cells. Stringent selection used high doses of O(6)-benzylguanine (800 micromol/L) and temozolomide (1,000 micromol/L) or BCNU (20 micromol/L) administered twice, and following regrowth, surviving clones were isolated, and the MGMT transgene was sequenced. None of the mutants was lost during selection. Using temozolomide, the enrichment factor was greatest for P140K-MGMT (1.7-fold). Using BCNU selection, the greatest enrichment was observed with MGMT-2 (1.5-fold). G156A-MGMT, which is the least O(6)-benzylguanine-resistant MGMT gene of the mutants tested, was not lost during selection but was selected against. The optimal mutant MGMT useful as a drug resistance gene may depend on whether a methylating or chloroethylating agent is used for drug selection.

Role of O6-alkylguanine-DNA alkyltransferase in protecting against 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-induced long-term toxicities.

O6-alkylguanine-DNA alkyltransferase (AGT) protects from the mutagenic and toxic lesions induced by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and in many tumors, AGT overexpression provides a means of resistance. To circumvent this, O6-benzylguanine, an inactivator of AGT, has been developed and is currently in clinical development with BCNU; however, the potential long-term toxicities associated with this treatment are unknown. With the inactivation of AGT by O6-benzylguanine, a higher number of toxic and mutagenic O6-alkylguanine lesions introduced by methylating or chloroethylating agents would be expected. In this study, cohorts of mice were treated with vehicle, O6-benzylguanine (30 mg/kg), BCNU alone (low dose of 15 mg/kg or high dose of 50 mg/kg), or O6-benzylguanine (30 mg/kg) plus BCNU (15 mg/kg) and followed for 12 months post-treatment. Mice treated with O6-benzylguanine plus BCNU or high-dose BCNU died significantly earlier (p < 0.0001) than mice in the other three cohorts with a median survival of 8.3 (O6-benzylguanine plus BCNU) and 7.9 months (high-dose BCNU). Histopathologic sections of tissues revealed that the most common morphological diagnosis in animals treated with O6-benzylguanine plus BCNU (15 mg/kg) or BCNU (50 mg/kg) was cytomegaly in the lung with greater severity observed in mice receiving the combination O6-benzylguanine plus BCNU. Four of five mice analyzed in this cohort had alveolar histiocytosis, with one also having alveolar edema. In contrast, liver and kidney toxicity was only observed in mice treated with BCNU (50 mg/kg). These results suggest that O6-benzylguanine enhances long-term pulmonary toxicity associated with BCNU in mice.

Role of O6-methylguanine-DNA methyltransferase and effect of O6-benzylguanine on the anti-tumor activity of cis-diaminedichloroplatinum(II) in oral cancer cell lines.

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) modulates the effectiveness of alkylating agents. However, the relationship between MGMT and the sensitivities to other agents has not been explored. In the present study, the association between MGMT expression and the cellular sensitivity to the platinum agent, CDDP was examined in four human oral cancer cell lines. CDDP depleted MGMT protein and mRNA levels in all four cell lines. Two cell lines with low MGMT expression were sensitive to an alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine and CDDP, whereas two other cell lines with high MGMT expression were resistant to both agents. Furthermore, the addition of the MGMT inhibitor, O6-benzylguanine (O6-BG), invariably enhanced CDDP sensitivity. CDDP depleted MGMT expression, and CDDP sensitivity was enhanced by O6-BG. These results provide valuable information about the relationship between MGMT expression and CDDP sensitivity in oral cancer chemotherapy.

Gene expression profile induced by BCNU in human glioma cell lines with differential MGMT expression.

Chemotherapy with the alkylating agent BCNU (1,3-bis (2-chloroethyl)-1-nitrosourea) is the most commonly used chemotherapeutic agent for gliomas. However, the usefulness of this agent is limited because tumor cell resistance to BCNU is frequently found in clinical brain tumor therapy. The O6-methylguanine-DNA methyltransferase protein (MGMT) reverses alkylation at the O6 position of guanine and we have reported the role of MGMT in the response of brain tumors to alkylating agents. However, the different mechanisms underlying the patterns related to MGMT remain unclear. To better understand the molecular mechanism by which BCNU exerts its effect in glioma cell lines according MGMT expression, we used microarray technology to interrogate 3800 known genes and determine the gene expression profiles altered by BCNU treatment. Our results showed that treatment with BCNU alters the expression of a diverse group of genes in a time-dependent manner. A subset of gene changes was found common in both glioma cell lines and other subset is specific of each cell line. After 24 h of BCNU treatment, up-regulation of transcription factors involved in the nucleation of both RNA polymerase II and III transcription initiation complexes was reported. Interestingly, BCNU promoted the expression of actin-dependent regulators of chromatin. Similar effects were found with higher BCNU doses in MGMT+ cell line showing a similar mechanism that in MGMT-deficient cell with standard doses. Our data suggest that human glioma cell lines treated with BCNU, independently of MGMT expression, show changes in the expression of cell cycle and survival-related genes interfering the transcription mechanisms and the chromatin regulation.

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 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.

Formation of DNA adducts and induction of lacI mutations in Big Blue Rat-2 cells treated with temozolomide: implications for the treatment of low-grade adult and pediatric brain tumors.

Temozolomide (TMZ) is a chemotherapeutic agent used in the treatment of high-grade brain tumors. Treatment of patients with alkylating chemotherapeutic agents has been established to increase their risk for acute myelogenous leukemia. The formation of DNA adducts and induction of mutations are likely to play a role in the etiology of therapy-related acute myeloid leukemia. To evaluate this issue for TMZ, we have measured the formation of DNA adducts and induction of lacI mutations in Big Blue Rat-2 cells treated with TMZ. Treatment of Big Blue Rat-2 cells with either 0, 0.5, or 1 mM TMZ resulted in lacI mutant frequencies of 9.1 +/- 2.9 x 10(-5), 48.9 +/- 12 x 10(-5), and 89.7 +/- 40.3 x 10(-5), respectively. Comparison of the mutant frequencies demonstrated that 0.5 and 1 mM TMZ treatments increased the mutant frequencies by 5.3- and 9.8-fold and that this increase was significant (P < 0.001). Sequence analysis of the lacI mutants from the TMZ treatment group demonstrated that they were GC-->AT transitions at non-CpG sites, which is significantly different from the mutation spectrum observed in the control treatment group. Treatment of Big Blue Rat-2 cells with various concentrations of TMZ produced a linear increase in the levels of N7-methylguanine and O(6)-methylguanine. The lacI mutation spectrum induced by TMZ treatment is consistent with these mutations being produced by O(6)-MeG. This study establishes TMZ has significant mutagenic potential and suggests that careful consideration in the use of TMZ for the treatment of low-grade adult and pediatric brain tumors should be given.

Molecular and cellular influences of butylated hydroxyanisole on Chinese hamster V79 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine: antimutagenicity of butylated hydroxyanisole.

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O(6)-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity.

Sequential tumor biopsies in early phase clinical trials of anticancer agents for pharmacodynamic evaluation.

PURPOSE: In the setting of target-based anticancer drug development, it is critical to establish that the observed preclinical activity can be attributed to modulation of the intended target in early phase trials in human subjects. This paradigm of target modulation allows us to determine a Phase II or III dose (optimal biochemical/biological modulatory dose) that may not necessarily be the maximum tolerated dose. A major obstacle to target-based (often cytostatic) drug development has been obtaining relevant tumor tissue during clinical trials of these novel agents for laboratory analysis of the putative marker of drug effect. EXPERIMENTAL DESIGN: From 1989 to present, we have completed seven clinical trials in which the end point was a biochemical or biological modulatory dose in human tumor tissues (not surrogate tissue). Eligibility enrollment required that patients have a biopsiable lesion either with computerized tomography (CT) guidance or direct visualization and consent to sequential (pre and posttreatment) biopsies. RESULTS: A total of 192 biopsies were performed in 107 patients. All but 8 patients had sequential pre and posttreatment biopsies. Seventy-eight (73%) of the 107 patients had liver lesion biopsies. In eight patients, either one or both biopsies contained insufficient viable tumor tissue or no tumor tissue at all for analysis. Of a total of 99 patients in whom we attempted to obtain paired biopsies, a total of 87 (88%) were successful. Reasons for failure included patient refusal for a second biopsy (n = 2), vasovagal reaction with first biopsy precluding a second biopsy (n = 1), subcapsular hepatic bleeding (n = 1), and most commonly obtaining necrotic tumor, fibrous, or normal tissue in one of the two sequential biopsies (n = 8). CONCLUSIONS: This is the first and largest reported series demonstrating that with adequate precautions and experience, sequential tumor biopsies are feasible and safe during early phase clinical trials.

Temozolomide: the effect of once- and twice-a-day dosing on tumor tissue levels of the DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase.

Temozolomide (TMZ) is a methylating agent of the imidotetrazine class, whose cytotoxic product is O(6)-methylguanine DNA adducts, which initiate a futile recycling of the mismatch repair pathway causing DNA strand breaks and apoptotic cell death in mismatch repair proficient cells. The DNA repair protein O(6)-alkylguanine DNA alkyltransferase (AGT) repairs these adducts in a suicide manner and reduces the cytotoxic action of TMZ. An antitumor threshold is reached when sufficient adducts are formed by TMZ to inactivate AGT. In this study, we evaluated the relation between TMZ dosing and AGT depletion in patients with deep visceral tumors and in peripheral blood mononuclear cells (PBMCs) to determine whether the dose of TMZ was sufficient to inactivate AGT and lead to therapeutic efficacy. To do so, we compared single dose therapy with a novel twice daily regimen in a laboratory correlate-driven Phase I dose escalation study. p.o. bolus dose TMZ 200 mg/m(2) daily times five was compared with the same bolus on day 1 followed by nine doses at 12-h intervals of 50, 75, 90, or 100 mg/m(2). Dose-limiting toxicity in the bid regimen (grade IV thrombocytopenia and neutropenia) was seen at 100 mg/m(2), cumulative dose 1100 mg/m(2), and the maximum tolerated dose was 1010 mg/m(2). The degree of tumor tissue AGT activity depletion measured in biopsies before and on day 5 of therapy varied widely, between 0 (in 3 patients) and 99% (in 1), with the majority of patients (10 of 15) having 52-84% tumor AGT depletion. In contrast, AGT activity in PBMCs fell rapidly during TMZ administration to undetectable levels in all dosage groups on day 5 but did not correlate with tumor AGT depletion. TMZ pharmacokinetics were dose proportional; no accumulation occurred >5-day period in the bid regimen. Two partial responses were seen, lasting 3 and 4 months. Five additional patients achieved prolonged stabilization of disease for 4-6 monthly cycles. This is the first study to document that at maximum tolerated doses, TMZ depletes PBMC AGT but only partially and variably depletes visceral tumor AGT in most patients, even during twice daily dosing. Drug combinations or schedules designed to maximally deplete tumor AGT might improve TMZ efficacy.