3-aminobenzamide and/or O6-benzylguanine evaluated as an adjuvant to temozolomide or BCNU treatment in cell lines of variable mismatch repair status and O6-alkylguanine-DNA alkyltransferase activity.

O6-benzylguanine (O6-BG) and 3-aminobenzamide (3-AB) inhibit the DNA repair proteins O6-alkylguanine-DNA alkyltransferase (AGT) and poly(ADP-ribose) polymerase (PARP) respectively. The effect of O6-BG and/or 3-AB on temozolomide and 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) cytotoxicity, was assessed in seven human tumour cell lines: six with an AGT activity of > 80 fmol mg-1 protein (Mer+) and one with an AGT activity of < 3 fmol mg-1 protein (Mer-). Three of the Mer+ cell lines (LS174T, DLD1 and HCT116) were considered to exhibit resistance to methylation by a mismatch repair deficiency (MMR-), each being known to exhibit microsatellite instability, and DLD1 and HCT116 having well-characterised defects in DNA mismatch binding. Potentiation was defined as the ratio between an IC50 achieved without and with a particular inhibitor treatment. Temozolomide or BCNU cytotoxicity was not potentiated by either inhibitor in the Mer- cell line. Preincubation with O6-BG (100 microM for 1 h) was found to potentiate the cytotoxicity of temozolomide by 1.35- to 1.57-old in Mer+/MMR+ cells, but had no significant effect in Mer+/MMR- cells. In comparison, O6-BG pretreatment enhanced BCNU cytotoxicity by 1.94- to 2.57-fold in all Mer+ cell lines. Post-incubation with 3-AB (2 mM, 48 h) potentiated temozolomide by 1.35- to 1.59-fold in Mer+/MMR+ cells, and when combined with O6-BG pretreatment produced an effect which was at least additive, enhancing cytotoxicity by 1.97- to 2.16-fold. 3-AB treatment also produced marked potentiation (2.20- to 3.12-fold) of temozolomide cytotoxicity in Mer+/MMR- cells. In contrast, 3-AB produced marginal potentiation of BCNU cytotoxicity in only three cell lines (1.19- to 1.35-fold), and did not enhance the cytotoxicity of BCNU with O6-BG treatment in any cell line. These data suggest that the combination of an AGT and PARP inhibitor may have a therapeutic role in potentiating temozolomide activity, but that the inhibition of poly(ADP-ribosyl)ation has little effect on the cytotoxicity of BCNU.     

Potentiation of temozolomide and BCNU cytotoxicity by O(6)-benzylguanine: a comparative study in vitro.

Depletion of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) with O(6)-benzylguanine (O(6)-BG) has been widely shown to enhance 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) activity. This study aimed to determine whether temozolomide, a methylating imidazotetrazinone, would similarly benefit from combination with O(6)-BG. Seven human cell lines were examined with AGT activities ranging from <6 fmol mg-1 protein to >700 fmol mg-1 protein. Comparisons with BCNU were made on both single and multiple dosing schedules, since temozolomide cytotoxicity is highly schedule dependent. In single-dose potentiation studies, cells were preincubated with 100 microM O(6)-BG for 1 h, a treatment found to deplete AGT activity by >90% for 24 h. No potentiation of either temozolomide or BCNU cytotoxicity was observed in two glioblastoma cell lines with <6 fmol mg-1 protein AGT. In all other cell lines studied potentiation of BCNU toxicity by O(6)-BG was between 1.6- and 2.3-fold and exceeded that of temozolomide (1.1- to 1.7-fold). The magnitude of this potentiation was unrelated to AGT activity and the relative potentiation of temozolomide and BCNU cytotoxicity was found to be highly variable between cell lines. In multiple dosing studies two colorectal cell lines (Mawi and LS174T) were treated with temozolomide or BCNU at 24 h intervals for up to 5 days, with or without either 100 microM O(6)-BG for 1 h or 1 microM O(6)-BG for 24 h, commencing 1 h before alkylating treatment. Extended treatment with 1 microM O(6)-BG produced greater potentiation than intermittent treatment with 100 microM O(6)-BG. Potentiation of temozolomide cytotoxicity increased linearly in Mawi with each subsequent dosing: from 1.4-fold (day 1) to 4.2-fold (day 5) with continuous 1 microM O(6)-BG. In contrast, no potentiation was observed in LS174T, a cell line that would appear to be ''tolerant'' of methylation. Potentiation of BNCU cytotoxicity increased in both cell lines with repeat dosing, although the rate of increase was less than that observed with temozolomide and continuous 1 microM O(6)-BG in Mawi. These results suggest that repeat dosing of an AGT inhibitor and temozolomide may have a clinical role in the treatment of tumours that exhibit AGT-mediated resistance.     

Phase I trial of temozolomide plus O6-benzylguanine for patients with recurrent or progressive malignant glioma.

PURPOSE: We conducted a two-phase clinical trial in patients with progressive malignant glioma (MG). The first phase of this trial was designed to determine the dose of O6-BG effective in producing complete depletion of tumor AGT activity for 48 hours. The second phase of the trial was designed to define the maximum tolerated dose (MTD) of a single dose of temozolomide when combined with O6-BG. In addition, plasma concentrations of O6-BG and O6-benzyl-8-oxoguanine were evaluated after O6-BG. PATIENTS AND METHODS: For our first phase of the clinical trial, patients were scheduled to undergo craniotomy for AGT determination after receiving a 1-hour O6-BG infusion at 120 mg/m2 followed by a continuous infusion at an initial dose of 30 mg/m2/d for 48 hours. The dose of the continuous infusion of O6-BG escalated until tumor AGT was depleted. Once the O6-BG dose was established a separate group of patients was enrolled in the second phase of clinical trial, in which temozolomide, administered as a single dose at the end of the 1-hour O6-BG infusion, was escalated until the MTD was determined. RESULTS: The O6-BG dose found to be effective in depleting tumor AGT activity at 48 hours was an IV bolus of 120 mg/m2 over 1 hour followed by a continuous infusion of 30 mg/m2/d for 48 hours. On enrolling 38 patients in six dose levels of temozolomide, the MTD was established at 472 mg/m2 with dose-limiting toxicities limited to myelosuppression. CONCLUSION: This study provides the foundation for a phase II trial of O6-BG plus temozolomide in temozolomide-resistant MG.     

O6-benzylguanine potentiates the antitumor effect of locally delivered carmustine against an intracranial rat glioma

Local delivery of carmustine (BCNU) via biodegradable polymers prolongs survival against experimental brain tumors and in human clinical trials. O6-benzylguanine (O6-BG), a potent inhibitor of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), has been shown to reduce nitrosourea resistance and, thus, enhance the efficacy of systemic BCNU therapy in a variety of tumor models. In this report, we demonstrate that O6-BG can potentiate the activity of BCNU delivered intracranially via polymers in rats challenged with a lethal brain tumor. Fischer 344 rats received a lethal intracranial challenge of 100,000 F98 glioma cells (F98 cells have significant AGT activity, 328 fmol/mg protein). Five days later, animals receiving an i.p. injection of O6-BG (50 mg/kg) 2 h prior to BCNU polymer (3.8% BCNU by weight) implantation had significantly improved survival (n = 7; median survival, 34 days) over animals receiving either O6-BG alone (n = 7; median survival, 22 days; P = 0.0002) or BCNU polymer alone (n = 8; median survival, 25 days; P = 0.0001). Median survival for the control group (n = 8) was 23.5 days. Moreover, there was no physical, behavioral, or pathological evidence of treatment-related toxicity. These findings suggest that O6-BG can potentiate the effects of interstitially delivered BCNU and, for tumors expressing significant AGT, may be necessary for the BCNU to provide a meaningful therapeutic benefit. Given the clinical use of BCNU polymers against malignant gliomas, concurrent treatment with O6-BG may provide an important addition to our therapeutic armamentarium.     

Phase I trial of carmustine plus O6-benzylguanine for patients with recurrent or progressive malignant glioma.

PURPOSE: The major mechanism of resistance to alkylnitrosourea therapy involves the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), which removes chloroethylation or methylation damage from the O(6) position of guanine. O(6)-benzylguanine (O(6)-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial of carmustine (BCNU) plus O(6)-BG to define the toxicity and maximum-tolerated dose (MTD) of BCNU in conjunction with the preadministration of O(6)-BG with recurrent or progressive malignant glioma. PATIENTS AND METHODS: Patients were treated with O(6)-BG at a dose of 100 mg/m(2) followed 1 hour later by BCNU. Cohorts of three to six patients were treated with escalating doses of BCNU, and patients were observed for at least 6 weeks before being considered assessable for toxicity. Plasma samples were collected and analyzed for O(6)-BG, 8-oxo-O(6)-BG, and 8-oxoguanine concentration. RESULTS: Twenty-three patients were treated (22 with glioblastoma multiforme and one with anaplastic astrocytoma). Four dose levels of BCNU (13.5, 27, 40, and 55 mg/m(2)) were evaluated, with the highest dose level being complicated by grade 3 or 4 thrombocytopenia and neutropenia. O(6)-BG rapidly disappeared from plasma (elimination half-life = 0. 54 +/- 0.14 hours) and was converted to a longer-lived metabolite, 8-oxo-O(6)-BG (elimination half-life = 5.6 +/- 2.7 hours) and further to 8-oxoguanine. There was no detectable O(6)-BG 5 hours after the start of the O(6)-BG infusion; however, 8-oxo-O(6)-BG and 8-oxoguanine concentrations were detected 25 hours after O(6)-BG infusion. The mean area under the concentration-time curve (AUC) of 8-oxo-O(6)-BG was 17.5 times greater than the mean AUC for O(6)-BG. CONCLUSION: These results indicate that the MTD of BCNU when given in combination with O(6)-BG at a dose of 100 mg/m(2) is 40 mg/m(2) administered at 6-week intervals. This study provides the foundation for a phase II trial of O(6)-BG plus BCNU in nitrosourea-resistant malignant glioma.     

O(6)-(4-bromothenyl)guanine improves the therapeutic index of temozolomide against A375M melanoma xenografts

Tumour resistance to methylating agents is linked to expression of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (ATase). There is considerable interest in improving the efficacy of O(6)-alkylating chemotherapy by prior depletion of ATase. We have tested the ability of a modified guanine base, O(6)-(4-bromothenyl)guanine (4BTG), to inactivate ATase and to enhance the anti-tumour effect of temozolomide in an animal model system. A375M human melanoma xenografts were established in the flanks of nude mice. ATase depletion after a single dose of 4BTG or O(6)-BG (20 mg/kg i.p.) was determined over a 24 hr period. Subsequently, we tested the effect of 4BTG (20 mg/kg i.p. daily) and/or temozolomide (80-175 mg/kg i.p. daily) over a 5-day schedule on tumour growth. 4BTG was an effective inactivator of ATase in tumour, producing complete depletion within 2 hr of dosing. Furthermore, it enhanced the tumour growth delay achieved with temozolomide, increasing the tumour quintupling time by 8.7 days (95% confidence interval 6.1-11.3 days, p < 0.0001). Whilst the delay in tumour growth was indistinguishable from that observed with O(6)-benzylguanine (O(6)-BG) and temozolomide, the 4BTG combination resulted in considerably less toxicity (0/9 vs. 2/9 deaths; 6.84% weight loss vs. 9.48%, p = 0.019). 4BTG is a potent inactivator of ATase and enhances the therapeutic ratio of temozolomide in this model system to a greater extent than O(6)-BG.     

Thresholds of O6-alkylguanine-DNA alkyltransferase which confer significant resistance of human glial tumor xenografts to treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea or temozolomide.

Bis-2-chloroethylnitrosourea (BCNU) or temozolomide (TMZ) were tested alone or in combination with the AGT inhibitors O6-benzyl-2'-deoxyguanosine (dBG) or O6-benzylguanine (BG) against human glial tumor xenografts growing s.c. in athymic mice. Four glioblastoma (SWB77, SWB40, SWB39, and D-54) and one anaplastic oligodendroglioma (SWB61) xenografts having O6-alkylguanine-DNA alkyltransferase (AGT) activities of 75, 45, 10, < 10, and 16 fmol/mg protein, respectively, were used. BCNU at 35 mg/m2 was ineffective against these tumors, although 70 mg/m2 (LD10, 75 mg/m2) produced a marked tumor growth delay (T-C) in D54 but had no effect against SWB40 or SWB77. Coadministration of BG or dBG and BCNU necessitated reduction of the BCNU dose to a maximum of 30 and 35 mg/m2, respectively, because of increased toxicity. Optimized treatment with dBG (250 mg/m2) and BCNU (35 mg/m2) resulted in T-Cs of 30, 29, 11, 16, and 14 days for SWB77, SWB40, SWB39, D-54 and SWB61, respectively. These delays were more pronounced than those induced with optimized, isotoxic treatments with BG (180 mg/m2) and BCNU (30 mg/m2). In comparison to BCNU, TMZ was less toxic, with an LD10 of 400 mg/m2. TMZ (300 mg/m2) was more effective than BCNU against SWB77, SWB40, and SWB61, inducing T-Cs of 23, 53, and 56 days, respectively. BG and dBG enhanced the toxicity of TMZ in athymic mice by decreasing the LD10 from 400 to 200 mg/m2. TMZ (180 mg/m2) with either BG (180 mg/m2) or dBG (250 mg/m2) resulted in T-Cs of 31 and 49 days in SWB77, respectively, as compared with 16 days for TMZ (180 mg/m2) alone. In SWB40, the combination of TMZ with dBG, but not with BG, was significantly more effective than the maximum tolerated dose of TMZ (300 mg/m2) alone. The combination of TMZ with AGT inactivators had no benefit, as compared with TMZ alone, against xenografts with marginal AGT activity. In conclusion, at equimolar doses dBG was less toxic than BG in athymic mice when combined with either BCNU or TMZ. In this regard, BCNU or TMZ can be used at higher doses in combination with dBG than with BG. This study further demonstrates that there is a significant benefit of depleting AGT with nonspecific AGT inhibitors prior to treatment with either BCNU or TMZ in tumors having AGT activity >45 fmol/mg protein.     

Inactivation of O6-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy

DNA adducts at the O6-position of guanine are a result of the carcinogenic, mutagenic and cytotoxic actions of methylating and chloroethylating agents. The presence of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) renders cells resistant to the biological effects induced by agents that attack at this position. O6-Benzylguanine (O6-BG) is a low molecular weight substrate of AGT and therefore, results in sensitizing cells and tumors to alkylating agent-induced cytotoxicity and antitumor activity. Presently, chemotherapy regimens of O6-BG in combination with BCNU, temozolomide and Gliadel are in clinical development. Other ongoing clinical trials include expression of mutant AGT proteins that confer resistance to O6-BG in bone marrow stem cells, in an effort to reduce the potential enhanced toxicity and mutagenicity of alkylating agents in the bone marrow. O6-BG has also been found to enhance the cytotoxicity of agents that do not form adducts at the O6-position of DNA, including platinating agents. O6-BG's mechanism of action with these agents is not fully understood; however, it is independent of AGT activity or AGT inactivation. A better understanding of the effects of this agent will contribute to its clinical usefulness and the design of better analogs to further improve cancer chemotherapy.     

Effect of single and multiple administration of an O 6-benzylguanine/temozolomide combination: an evaluation in a human melanoma xenograft model

The purpose of the present study was to examine the effect of O ⁶-benzylguanine (O ⁶-BG) on the antitumour activity and toxicity of 8-carbamoyl-3-methylimidazo [5, 1-d ] -1,2,3,5-tetrazine-4(3H)-one (temo-zolomide) in a human malignant melanoma xenograft model following single and multiple administration of the combination. O ⁶-BG irreversibly inactivates the DNA-repair protein O ⁶-alkylguanine-DNA alkyltransferase (AGT), which confers resistance to temozolomide. Preadministration of O ⁶-BG (35 mg/kg, i.p.) 1 h prior to temozolomide (i.p.) was examined using single and daily × 5 dosing regimens in athymic mice bearing subcutaneous A375P xenografts. The AGT activity of A375P tumors was 95 ± 8 fmol/mg protein (mean ± SE, n = 4). O ⁶-BG alone completely suppressed xenograft AGT activity within 1 h of administration but had no effect upon tumor growth. O ⁶-BG did not significantly increase the tumor growth delay induced by a single 200-mg/kg dose of temozolomide (P>0.05, two-tailed Mann-Whitney test) but did increase the associated mean body weight loss (P<0.025). In contrast, when the same dose of temozolomide was divided into five equal fractions (40 mg/kg) and given with O ⁶-BG on 5 consecutive days, a comparable increase in toxicity was accompanied by a very significant increase in tumor growth delay (P<0.0025), equivalent to that produced by a 3-fold greater dose of temozolomide alone. O ⁶-BG with temozolomide also produced a greater antitumour effect than an equitoxic dose of temozolomide alone on this schedule (P<0.005). These data indicate that the enhancement of temozolomide antitumour activity by O ⁶-BG preadministration is dependent upon the schedule of drug administration, with multiple dosing of O ⁶-BG + temozolomide producing the greatest effect. The results also suggest that prolonged administration of the combination can lead to an increase in the therapeutic index of temozolomide. Received: 8 September 1996 / Accepted: 8 February 1997     

Effect of O6-benzylguanine on alkylating agent-induced toxicity and mutagenicity. In Chinese hamster ovary cells expressing wild-type and mutant O6-alkylguanine-DNA alkyltransferases

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) has been shown to protect cells from the toxic and mutagenic effect of alkylating agents by removing lesions from the O6 position of guanine. O6-Benzylguanine (BG) is a potent inactivator of AGT, resulting in an increase in the sensitivity of cells to the toxic effects of chemotherapeutic alkylating agents. Chinese hamster ovary (CHO) cells and CHO cells transfected with wild-type AGT (CHOWTAGT) and a mutant AGT [P138 M/V139I/P140K (CHOMIK)] known to be resistant to BG were treated with BG and various alkylating agents. BG treatment alone dramatically decreased AGT activity in CHOWTAGT cells but resulted in no depletion in AGT activity in CHOMIK cells. In the absence of AGT, these cells are highly sensitive to the toxic and mutagenic effects of temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and no further sensitization occurs in the presence of BG. In contrast, CHOWTAGT cells are resistant to temozolomide and BCNU, and treatment with BG resulted in a significantly higher cell killing and mutation frequency. CHOMIK cells were completely resistant to temozolomide or BCNU in the presence and absence of BG. Both cell killing and mutation frequency of 4-hydroperoxycyclophosphamide (4-HC) in CHO, CHOWTAGT, and CHOMIK cells were increased in the presence of BG. 4-HC generates two active metabolites, phosphoramide mustard (PM) and acrolein. BG had no effect on 4hydroperoxydidechlorocyclophosphamide (which generates acrolein and a nonalkylating form of PM) in CHO cells and CHOMIK cells, but enhancement of toxicity was observed with PM in both these cell lines. Therefore, we attribute the enhancement to the PM metabolite of 4-HC. Our results demonstrate that wild-type AGT plays an important role in protecting against the toxic and mutagenic effect of O6 alkylating agents and that a mutant AGT resistant to inactivation by BG effectively prevents BG-enhanced toxicity and mutagenicity induced by these agents. Expression of the AGT protein contributes to resistance of 4-HC. BG also enhances the toxicity of 4-HC and PM by a mechanism that may not involve the AGT repair protein.     

Inhibition of O6-alkylguanine-DNA alkyltransferase in animal and human ovarian tumor cell lines by O6-benzylguanine and sensitization to BCNU

O⁶-Alkylguanine-DNA alkyltransferase (O⁶-AGT) activity in rat ovarian tumor lines O-342 and O-342/DDP was 103.4±18.4 and 240.9±40.2 fmol/mg protein, respectively; thus, cisplatin (DDP) resistance was paralleled by an increase in O⁶-AGT activity by a factor of approximately 2.3. The DDP-resistant line expressed a collateral resistance to BCNU. Both lines could be sensitized to BCNU by O⁶-BG, with sensitization factors of 6.0 and 2.1, respectively. In neither line did depletion of O⁶-AGT have any sensitizing effect towards DDP. In the human ovarian cancer lines SK-OV-3 and OAW 42, O⁶-AGT activity was 337.6±18.2 and 180.0±39.9 fmol/mg protein, respectively; in these lines depletion of O⁶-AGT activity by O⁶-BG treatment resulted in sensitization factors of 3.0 and 4.1, respectively. The increase in sensitivity of ovarian tumor cell lines against a chloroethylating agent by O⁶-AGT depletion and possible pharmacological advantages of regional (i.p.) administration of this combination might be beneficial in advanced ovarian cancer.Abbreviations BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) - CENU 2-chloroethylnitrosourea - DDP cisplatin ED₅₀, the effective dose required to inhibit colony formation or cell proliferation by 50% - O⁶-AGT O⁶-alkylguanine-DNA alkyltransferase - O⁶-BG O⁶-benzylguanine SF sensitization factor     

Effect of temozolomide and dacarbazine on O6-alkylguanine-DNA alkyltransferase activity and sensitivity of human tumor cells and xenografts to 1,3-bis(2-chloroethyl)-1-nitrosourea

We investigated the ability of 5-(dimethyltriazeno)imidazole-4-carboxamide (DTIC, dacarbazine) and an analogue, temozolomide, to deplete cells or tumors of O⁶-alkylguanine-DNA alkyltransferase (AGT) and to enhance the antitumor effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Human colon cancer (HT29) cell survival was decreased by almost 1 log when treated with 500 M temozolomide prior to 150 M BCNU. Administration of the maximal tolerated dose of DTIC (300 mg/kg) to nude mice carrying HT29 xenografts resulted in complete depletion of AGT activity in tumors at 4 h and 16 h. Administration of 150 mg/kg DTIC caused a 76% reduction in AGT activity at 4 h, but only a 28% reduction at 16 h. The maximally tolerated doses of DTIC and BCNU, alone and in combination, were used to treat nude mice bearing HT29 xenografts. No difference in tumor growth occurred when animals were treated with either BCNU alone (50 mg/kg), DTIC alone (300 mg/kg), DTIC (150 mg/kg) followed by BCNU (12.5 mg/kg), or BCNU (25 mg/kg) followed by DTIC (150 mg/kg). These data suggest that methylating agents such as DTIC may be too toxic to be used in combination with BCNU to deplete tumor alkyltransferase levels effectively and increase the therapeutic index of BCNU.This work was supported by National Institutes of Health Grants CA-47228 (M.E.D.) and 5T32-DK-07134 (R.B.M.).     

Phase II trial of carmustine plus O(6)-benzylguanine for patients with nitrosourea-resistant recurrent or progressive malignant glioma.

PURPOSE: We conducted a phase II trial of carmustine (BCNU) plus the O(6)-alkylguanine-DNA alkyltransferase inhibitor O(6)-benzylguanine (O(6)-BG) to define the activity and toxicity of this regimen in the treatment of adults with progressive or recurrent malignant glioma resistant to nitrosoureas. PATIENTS AND METHODS: Patients were treated with O(6)-BG at an intravenous dose of 120 mg/m(2) followed 1 hour later by 40 mg/m(2) of BCNU, with cycles repeated at 6-week intervals. RESULTS: Eighteen patients were treated (15 with glioblastoma multiforme, two with anaplastic astrocytoma, and one with malignant glioma). None of the 18 patients demonstrated a partial or complete response. Two patients exhibited stable disease for 12 weeks before their tumors progressed. Three patients demonstrated stable disease for 6, 12, and 18 weeks before discontinuing therapy because of hematopoietic toxicity. Twelve patients experienced reversible > or = grade 3 hematopoietic toxicity. There was no difference in half-lives (0.56 +/- 0.21 hour v 0.54 +/- 0.20 hour) or area under the curve values (4.8 +/- 1.7 microg/mL/h v 5.0 +/- 1.3 microg/mL/h) of O(6)-BG for patients receiving phenytoin and those not treated with this drug. CONCLUSION: These results indicate that O(6)-BG plus BCNU at the dose schedule used in this trial is unsuccessful in producing tumor regression in patients with nitrosourea-resistant malignant glioma, although stable disease was seen in five patients for 6, 12, 12, 12, and 18 weeks. Future use of this approach will require strategies to minimize dose-limiting toxicity of BCNU such as regional delivery or hematopoietic stem-cell protection.     

Altered O6-alkylguanine-DNA alkyltransferase activity in cell strains originating from mouse skin tumors induced by UV irradiation.

O6-Alkylguanine-DNA alkyltransferase (AGT) activity was assayed in the extracts of 47 cell strains originating from mouse skin tumors induced by UV irradiation. They were also examined for the sensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride by colony formation. The AGT activity (fmol/mg protein) of the tumor cell strains varied widely and the mean +/- SE was 72.5 +/- 9.37, while the AGT activity of the nontumor cell strains was 134 +/- 17. Among 47 strains, 6 strains showed extremely low or no AGT activity, about 5 fmol/mg protein or less, and were hypersensitive to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride. Long-term culture of the tumor cells did not change the AGT activity except in some strains which might have had coexisting normal cells in the population in early passages. All strains showed similar UV sensitivity regardless of AGT activity. This is the first report which demonstrates that about 13% of newly induced tumor cell strains are deficient in AGT activity similar to Mer-/Mex- phenotype that was found in approximately 20% of the established human tumor cell strains.     

Determination of the optimal modulatory dose of O6-benzylguanine in patients with surgically resectable tumors.

PURPOSE: O6-benzylguanine (BG) provides a means to effectively inactivate the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and increase the chemotherapeutic effectiveness of chloroethylating and methylating agents in preclinical and clinical studies. Two different doses of BG have been reported as the optimal biochemical modulatory dose for patients (i.e., 100 and 120 mg/m2). The objective of our study was to compare these doses by measuring AGT in surgically removed specimens after treatment with BG. EXPERIMENTAL DESIGN: BG was administered to patients as an i.v. infusion 16 +/- 4 h before surgical resection of their systemic tumor. AGT activity was measured in the tumor using a methylated DNA substrate. The target end point was defined as > or =11 of 13 patients with undetectable tumor AGT levels (<10 fmol/mg protein). RESULTS: Of the 28 patients enrolled, 25 of whom were analyzed for AGT activity, the most common primary sites of cancer included the colon (n = 11), bladder (n = 3), rectum (n = 4), and stomach (n = 3). Positive (DaOY cells) and negative (Chinese hamster ovary cells) control cell lines were included in each assay. Seven of the 12 patients treated with 100 mg/m2 BG had AGT activity of >10 fmol/mg protein (15-147 fmol/mg protein). Only 2 of the 13 patients treated with 120 mg/m2 BG had AGT activity of >10 fmol/mg protein (11 and 12 fmol/mg protein). CONCLUSIONS: From our surgically removed tissue data, a dose of 120 mg/m2 BG is recommended to deplete systemic tumors of AGT activity.     

Relevance of environmental alkylating agents to repair protein O6-alkylguanine-DNA alkyltransferase: determination of individual and collective repair capacities of O6-methylguanine.

The repair capacity for O6-methylguanine was determined in cell homogenates of peripheral blood lymphocytes of 35 automobile industry workers, exposed to rubber and tires, and of 35 clinical workers, handling cancer chemotherapeutic agents, compared to control groups. lambda-Phage DNA containing one 32P-labeled O6-methylguanine in each BamHI site was used as substrate for the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). The clinical personnel showed in the mean a highly significantly (P = 0.0014, Wilcoxon U test, Mann and Whitney) reduced activity of the repair enzyme [3.28 +/- 0.28 (SEM) fmol AGT/micrograms DNA] as compared to 37 control persons (4.88 +/- 0.32 fmol AGT/micrograms DNA). The mean AGT value of the automobile industry workers (4.40 +/- 0.28 fmol AGT/micrograms DNA) was not significantly different (P = 0.1303) from the mean of 38 examined controls (5.00 +/- 0.28 fmol AGT/micrograms DNA). By dividing these employees into subgroups according to their different work environments (handling of rubber fittings, tire mounting, and tire storage, respectively) the mean AGT value of the 15 tire storage workers was significantly (P = 0.0270) lower (3.80 +/- 0.36 fmol AGT/micrograms DNA) than the mean value of the controls. The interindividual variations in the activity of AGT were 5.1- and 6.5-fold in the control groups and 5.6-fold for the automobile industry workers; the largest variations were found in the group of the clinical personnel with 12.6-fold. No significant correlations between AGT and age, sex, or smoking behavior were observed in any of the groups examined. The decrease in AGT activity will render the afflicted individuals more susceptible to further exposure to methylating agents.     

Inactivation of O6-alkylguanine-DNA alkyltransferase in human peripheral blood mononuclear cells by temozolomide.

O6-alkylguanine-DNA-alkyltransferase (ATase) activity was measured in extracts of peripheral blood mononuclear cells (PMCs) taken from eight patients at various times during 5 days of oral treatment with temozolomide (150 mg m-2, days 1-5). Pretreatment ATase levels ranged from approximately 70 to 600 fmol per mg of protein. Depletion of PMC ATase was seen within 4 h of the first dose of temozolomide and had a median nadir of 52.9% and values ranging from 44.4% to 71.0% of pretreatment levels. There was a correlation between the extent of ATase depletion (pretreatment minus nadir level) and the pretreatment ATase level (r = 0.97). A progressive depletion of ATase was observed during the 5 days of continuous temozolomide therapy with median ATase activities of 66.3%, 52.5%, 39.5%, 30.5% and 28.9% of the pretreatment values at days 2, 3, 4, 5 and 6 respectively. This suggests that the schedule-dependent anti-tumour activity of temozolomide seen in experimental models and clinics may be related to a cumulative depletion of ATase.     

Acetylsalicylic acid (ASA) protects the prostaglandin-cAMP-system of human hypernephroma cells against irradiation-induced alterations.

There is abundant evidence that inhibitors of prostaglandin (PG) biosynthesis might increase the radioresponse of certain tumour cells. This study investigated specific PG binding sites, eicosanoid production as well as intracellular cAMP levels in cultured human hypernephroma cells derived from 11 patients upon nephrectomy. Scatchard analyses of the binding data revealed specific PGE1-, PGE2- as well as PGI2-binding sites (PGE1: Bmax = 755 +/- 206 fmol mg-1 protein, Kd = 3.7 +/- 2.7 nM PGE2: Bmax = 494 +/- 221 fmol mg-1 protein, Kd = 4.2 +/- 2.5 nM; PGI2: Bmax = 693 +/- 164 fmol mg-1 protein, Kd = 6.0 +/- 4.5 nM). Significant (P < 0.01) increase in PG binding sites expressed on human hypernephroma cells (PGE1: Bmax = 1084 +/- 303 fmol mg-1 protein, Kd = 2.8 +/- 1.3 nM; PGE2: Bmax = 663 +/- 309 fmol mg-1 protein, Kd = 2.2 +/- 1.5 nM; PGI2: Bmax = 1021 +/- 391 fmol/protein, Kd = 4.2 +/- 3.6 nM) and inhibition of PG biosynthesis (TXB2: -82.5%, PGE2: -87.5%. PGD2: -80.6%, PGF2: -81.3%) were found after acetylsalicylic acid (ASA)-treatment (0.5 mg 10(-6) cells for 24 h). Following irradiation (60Co, 1.0 Gy/min-1 over 10(min), PG binding sites (PGE1: Bmax = 266 +/- 153 fmol mg-1 protein, Kd = 5.0 +/- 5.0 nM; PGE2: Bmax = 148 +/- 66 fmol mg-1 protein, Kd = 4.7 +/- 3.6 nM; PGI2: Bmax = 325 +/- 194 fmol mg-1 protein, Kd = 6.8 +/- 7.1 nM) were significantly (P < 0.01) diminished. However, irradiation had no significant effect on PG binding sites in ASA-pretreated cells (PGE1: Bmax = 699 +/- 240 fmol mg-1 protein, Kd = 3.5 +/- 1.8 nM; iloprost: Bmax = 766 +/- 452 fmol mg-1 protein, Kd = 3.2 +/- 2.2 nM). Although there was no significant difference in the basal values for cAMP between control and ASA-treated group cells, the PG-induced cAMP-production was less pronounced in the control group. Taken together, the findings suggest that ASA may modify the radioresponse of cultured human hypernephroma cells by preventing the decrease of PG binding sites induced by irradiation.     

The L84F polymorphic variant of human O6-methylguanine-DNA methyltransferase alters stability in U87MG glioma cells but not temozolomide sensitivity

First-line therapy for patients with glioblastoma multiforme includes treatment with radiation and temozolomide (TMZ), an oral DNA alkylating chemotherapy. Sensitivity of glioma cells to TMZ is dependent on the level of cellular O(6)-methylguanine-DNA methyltransferase (MGMT) repair activity. Several common coding-region polymorphisms in the MGMT gene (L84F and the linked pair I143V/K178R) modify functional characteristics of MGMT and cancer risk. To determine whether these polymorphic changes influence the ability of MGMT to protect glioma cells from TMZ, we stably overexpressed enhanced green fluorescent protein (eGFP)-tagged MGMT constructs in U87MG glioma cells. We confirmed that the wild-type (WT) eGFP-MGMT protein is properly localized within the nucleus and found that L84F, I143V/K178R, and L84F/I143V/K178R eGFP-MGMT variants exhibited nuclear localization patterns indistinguishable from WT. Using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] proliferation and clonogenic survival assays, we confirmed that WT cells expressing eGFP-MGMT are resistant to TMZ treatment compared with control U87MG cells, and that each of the polymorphic eGFP-MGMT variants confers similar resistance to TMZ. However, upon exposure to O(6)-benzylguanine (O(6)-BG), a synthetic MGMT inhibitor, the L84F and L84F/I143V/K178R variants were degraded more rapidly than WT or I143V/K178R in a proteasome-dependent manner. Despite the increased O(6)-BG- stimulated protein turnover caused by the L84F alteration, cells expressing L84F eGFP-MGMT did not exhibit altered sensitivity to the combination of O(6)-BG and TMZ compared with WT cells. In conclusion, we demonstrated that the L84F polymorphic variant has altered protein turnover without modifying sensitivity of U87MG cells to TMZ or combined TMZ and O(6)-BG. These findings may provide a clue to determining the clinical significance of MGMT coding-region polymorphisms.     

Influence of O6-benzylguanine on the anti-tumour activity and normal tissue toxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and molecular combinations of 5-fluorouracil and 2-chloroethyl-1-nitrosourea in mice

Previous studies have demonstrated that novel molecular combinations of 5-fluorouracil (5FU) and 2-chloroethyl-1-nitrosourea (CNU) have good preclinical activity and may exert less myelotoxicity than the clinically used nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). This study examined the effect of O6-alkylguanine-DNA-alkyltransferase (ATase) depletion by the pseudosubstrate O6-benzylguanine (BG) on the anti-tumour activity and normal tissue toxicity in mice of three such molecular combinations, in comparison with BCNU. When used as single agents at their maximum tolerated dose, all three novel compounds produced a significant growth retardation of BCNU-resistant murine colon and human breast xenografts. This in vivo anti-tumour effect was potentiated by BG, but was accompanied by severe myelotoxicity as judged by spleen colony forming assays. However, while tumour resistance to BCNU was overcome using BG, this was at the expense of enhanced bone marrow, gut and liver toxicity. Therefore, although this ATase-depletion approach resulted in improved anti-tumour activity for all three 5-FU:CNU molecular combinations, the potentiated toxicities in already dose-limiting tissues indicate that these types of agents offer no therapeutic advantage over BCNU when they are used together with BG.