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Anthony E. Pegg - One of the best experts on this subject based on the ideXlab platform.

  • brain tumor cell lines resistant to o6 benzylguanine 1 3 bis 2 chloroethyl 1 nitrosourea chemotherapy have o6 alkylguanine dna alkyltransferase mutations
    Molecular Cancer Therapeutics, 2004
    Co-Authors: Manny D Bacolod, Robert C Moschel, Stewart P Johnson, Michael O Colvin, Anthony E. Pegg, Eileen M. Dolan, Nancy S Bullock, Qingming Fang, Paul Modrich, Darell D Bigner
    Abstract:

    The chemotherapeutic activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) may be improved by the addition of O6-Benzylguanine (O6-BG). The reaction of O6-BG with O6-alkylguanine-DNA alkyltransferase (AGT) prevents the repair of O6-chloroethyl lesions caused by BCNU. In clinics, the combination of O6-BG and BCNU is now being tested for the treatment of brain tumors. However, the effectiveness of this drug regimen may be limited by drug resistance acquired during treatment. To understand the possible mechanisms of resistance of brain tumor cells to the O6-BG/BCNU combination, we generated medulloblastoma cell lines (D283 MED, D341 MED, and Daoy) resistant to the combination of O6-BG and BCNU [O6-BG/BCNU resistant (OBR)]. DNA sequencing showed that all of the parent cell lines express wild-type AGTs, whereas every OBR cell line exhibited mutations that potentially affected the binding of O6-BG to the protein as evidenced previously by in vitro mutagenesis and structural studies of AGT. The D283 MED (OBR), Daoy (OBR), and D341 MED (OBR) cell lines expressed G156C, Y114F, and K165T AGT mutations, respectively. We reported previously that rhabdomyosarcoma TE-671 (OBR) also expresses a G156C mutation. These data suggest that the clonal selection of AGT mutants during treatment with O6-BG plus an alkylator may produce resistance to this intervention in clinical settings.

  • 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
    Clinical Cancer Research, 2001
    Co-Authors: Demetrius M Kokkinakis, Robert C Moschel, Clifford S Schold, Dora Bocangel, Anthony E. Pegg
    Abstract:

    Bis-2-chloroethylnitrosourea (BCNU) or temozolomide (TMZ) were tested alone or in combination with the AGT inhibitors O 6 -benzyl-2′-deoxyguanosine (dBG) or O 6 -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 O 6 -alkylguanine-DNA alkyltransferase (AGT) activities of 75, 45, 10, <10, and 16 fmol/mg protein, respectively, were used. BCNU at 35 mg/m 2 was ineffective against these tumors, although 70 mg/m 2 (LD 10 , 75 mg/m 2 ) 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/m 2 , respectively, because of increased toxicity. Optimized treatment with dBG (250 mg/m 2 ) and BCNU (35 mg/m 2 ) 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/m 2 ) and BCNU (30 mg/m 2 ). In comparison to BCNU, TMZ was less toxic, with an LD 10 of 400 mg/m 2 . TMZ (300 mg/m 2 ) 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 LD 10 from 400 to 200 mg/m 2 . TMZ (180 mg/m 2 ) with either BG (180 mg/m 2 ) or dBG (250 mg/m 2 ) resulted in T-Cs of 31 and 49 days in SWB77, respectively, as compared with 16 days for TMZ (180 mg/m 2 ) 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/m 2 ) 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.

  • 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
    Cancer Research, 2000
    Co-Authors: Yingna Cai, Anthony E. Pegg, Susan M. Ludeman, Meng Xuwelliver, Eileen M. Dolan
    Abstract:

    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 4-hydroperoxydidechlorocyclophosphamide (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.

  • alteration of the conserved residue tyrosine 158 to histidine renders human o6 alkylguanine dna alkyltransferase insensitive to the inhibitor o6 benzylguanine
    Cancer Research, 1999
    Co-Authors: Meng Xuwelliver, Sreenivas Kanugula, Jose Leitao, Anthony E. Pegg
    Abstract:

    The DNA repair protein O 6-alkylguanine-DNA alkyltransferase (AGT) protects cells from alkylation damage. O 6-Benzylguanine (BG) is a potent inactivator of human AGT (ED50 of 0.1 μm) that is currently undergoing clinical trials to enhance chemotherapy by alkylating agents. In a screen of AGT mutants randomly mutated at position glycine-160, we found that the double mutant Y158H/G160A protected Escherichea coli from killing by N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) even in the presence of BG and that the AGT activity of this mutant was strongly resistant to BG (ED50 of 180 μm). Because the single mutant G160A was not resistant to BG, this suggested that the presence of the charged histidine residue at position 158 was responsible. This hypothesis was confirmed by the construction of the single mutation Y158H. The Y158H-mutant AGT was slightly less active than wild-type AGT for the repair of methylated DNA in vitro , but it protected E. coli from killing by MNNG even in the presence of BG and had an ED50 for the inactivation by BG of 620 μm. In contrast, mutant Y158F had an ED50 of 0.2 μm. Previous studies (M. Xu-Welliver et al ., Cancer Res., 58: 1936–1945, 1998) have shown that mutant P140K is highly resistant to BG (ED50 of >1200 μm). Models of human AGT suggest that the side chain of the lysine inserted into this mutant is close to tyrosine-158 and that the positively charged lysine side-chain may interfere with BG binding. The double mutants P140K/Y158H and P140K/Y158F resembled P140K and Y158H in being highly resistant to BG, but the use of a sensitive assay for reaction of BG with AGT indicated that their abilities to react were in the order P140K/Y158H < P140K < P140K/Y158F. These results confirm that the presence of a positively charged residue close to the active site of human AGT renders it highly resistant to BG without substantially affecting activity toward methylated DNA substrates. Such mutants may limit the value of BG therapy if they arise in malignant cells during chemotherapy, but the mutant sequences may be useful for gene therapy approaches in which BG-resistant human AGTs are used to prevent hematopoietic toxicity. At least 28 AGT sequences (from 25 species) have now been described. In 25 of these, the position equivalent to 158 in the human AGT is also a tyrosine, and in the other 3, it is a phenylalanine. The importance of an aromatic ring side chain at this position is emphasized by previous studies (S. Edara et al ., Carcinogenesis, 16: 1637–1642, 1995), which show that the replacement by alanine renders human AGT inactive. Our results show that histidine can also substitute for tyrosine at this position.

  • probing of conformational changes in human o6 alkylguanine dna alkyl transferase protein in its alkylated and dna bound states by limited proteolysis
    Biochemical Journal, 1998
    Co-Authors: Sreenivas Kanugula, Karina Goodtzova, Anthony E. Pegg
    Abstract:

    Human O6-alkylguanine-DNA alkyl transferase (hAGT) is a DNA repair protein that protects cells from alkylation damage by transferring an alkyl group from the O6-position of guanine to a cysteine residue in the active site (-PCHR-) of the protein. The structure of the hAGT protein (23 kDa) has been probed by limited proteolysis with trypsin and Glu-C endoproteases and analysis of the polypeptide fragments by SDS/PAGE. The native hAGT protein had limited accessibility to digestion with trypsin and Glu-C in spite of a number of potential cleavage sites. Initial cleavage by trypsin occurred at residue Lys-193 to give a 21 kDa polypeptide fragment, and this polypeptide underwent further cleavage at residues Arg-128 and Lys-165. These trypsin-cleavage sites became more accessible to digestion in the presence of double-stranded DNA (dsDNA), indicating that hAGT undergoes a change in its conformation on binding to DNA. However, the trypsin cutting site at the Arg-128 position was less available for digestion in the presence of single-stranded DNA (ssDNA), suggesting that the hAGT protein has a different conformation when bound to ssDNA compared with dsDNA. When protease digestion was carried out on wild-type protein, preincubated with the low-molecular-mass pseudosubstrate O6-Benzylguanine, increased susceptibility to proteases was observed. A mutant C145A hAGT protein, which cannot repair O6-alkylguanine because the Cys-145 acceptor site in the active site of the protein is changed to Ala, showed identical trypsin cleavage to the wild type, but its digestion was not affected by O6-Benzylguanine. These results suggest that alkylation of hAGT leads to an altered conformation. The acquisition of increased susceptibility to proteases upon DNA binding and alkylation demonstrates that hAGT undergoes considerable conformational changes in its structure upon binding to DNA and after repair of alkylation damage.

Eileen M. Dolan - One of the best experts on this subject based on the ideXlab platform.

  • Phase II Tria l o f Carmust ine Plus O 6-Benzy lguanine for Pat ients With Nitrosourea-Res is tant Recurrent or Progress ive Mal ignant Gl ioma
    2016
    Co-Authors: A. Quinn, Eileen M. Dolan, David A Reardon, Jeremy N Rich, Allan H Friedman, John H Sampson, James M Pluda, Richard Kaplan, Shannon Delaney, Michael O Colvin
    Abstract:

    Purpose: We conducted a phase II trial of carmustine (BCNU) plus the O6-alkylguanine-DNA alkyltransferase inhibitor O6-Benzylguanine (O6-BG) to define the activ-ity and toxicity of this regimen in the treatment of adults with progressive or recurrent malignant glioma resis-tant to nitrosoureas. Patients and Methods: Patients were treated with O6-BG at an intravenous dose of 120 mg/m2 followed 1 hour later by 40 mg/m2 of BCNU, with cycles repeated at 6-week intervals. Results: Eighteen patients were treated (15 with gli-oblastoma multiforme, two with anaplastic astrocy-toma, 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 demon-strated stable disease for 6, 12, and 18 weeks before discontinuing therapy because of hematopoietic toxic-ity. Twelve patients experienced reversible> 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 g/mL/h v 5.0 1.3 g/mL/h) of O6-BG for patients receiving phenytoin and those not treated with this drug. Conclusion: These results indicate that O6-BG plus BCNU at the dose schedule used in this trial is unsuccessful in producing tumor regression in patients with nitro-sourea-resistant malignant glioma, although stable dis-ease 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 re-gional delivery or hematopoietic stem-cell protection

  • Effect of O6-Benzylguanine on nitrogen mustard-induced toxicity, apoptosis, and mutagenicity in Chinese hamster ovary cells. Mol Cancer Ther 2001;1:21–28. [PubMed: 12467235
    2016
    Co-Authors: Yingna Cai, Susan M. Ludeman, Lynette R. Wilson, Aimee B. Chung, Eileen M. Dolan
    Abstract:

    O6-Benzylguanine (BG) inactivates O6-alkylguanine-DNA alkyltransferase (AGT), resulting in an increase in the sensitivity of cells to the toxic effects of O6-alkylating agents. BG significantly enhances the cytotoxicity and decreases the mutagenicity of nitrogen mustards [i.e., phosphoramide mustard (PM), melphalan, and chlorambucil], a group of alkylating agents not known to produce O6-adducts in DNA. The enhancement is observed in cells irrespective of AGT activity. Exposure of Chinese hamster ovary cells to 100 M BG results in enhancement in the cytotoxicity of PM (300 M), chlorambucil (40 M), and melphalan (10 M) by 9-, 7-, and 18-fold, respectively. In contrast, mutation frequency after treatment with 300 M PM is decreased from 259 mutants/106 cells to 22 mutants/ 106 cells when cells are pretreated with BG. The enhancement of toxicity of these bis-alkylating agents appears to involve cross-link formation, because neither cytotoxicity nor mutagenicity of a mono-alkylating PM analogue is significantly altered when combined with BG. Enhanced cytotoxicity and decreased mutagenicity is concomitant with a dramatic increase in the number of cells undergoing apoptosis when BG is combined with PM, melphalan, or chlorambucil at 72–94 h after treatment. Cell cycle analysis demonstrates that BG alone or combined with nitrogen mustards arrests cells in G1 phase of the cell cycle. At 16 h after treatment, 11 and 57 % of cells treated with PM alone or with BG plus PM are in G1 phase, respectively. Our data suggest that treatment with BG causes G1 arrest and drives noncycling cells treated with nitrogen mustards into apoptosis, thus protecting against mutagenic DNA damage introduced by nitrogen mustards

  • phase i trial of polifeprosan 20 with carmustine implant plus continuous infusion of intravenous o6 benzylguanine in adults with recurrent malignant glioma new approaches to brain tumor therapy cns consortium trial
    Journal of Clinical Oncology, 2007
    Co-Authors: Jon D Weingart, Alessandro Olivi, Stephen B Tatter, Stuart A Grossman, Kathryn Carson, Joy D Fisher, Shannon M Delaney, Mark L Rosenblum, Kevin Judy, Eileen M. Dolan
    Abstract:

    Purpose This phase I trial was designed to (1) establish the dose of O6-Benzylguanine (O6-BG) administered intravenously as a continuous infusion that suppresses O6-alkylguanine-DNA alkyltransferase (AGT) levels in brain tumors, (2) evaluate the safety of extending continuous-infusion O6-BG at the optimal dose with intracranially implanted carmustine wafers, and (3) measure the pharmacokinetics of O6-BG and its metabolite. Patients and Methods The first patient cohort (group A) received 120 mg/m2 of O6-BG over 1 hour followed by a continuous infusion for 2 days at escalating doses presurgery. Tumor samples were evaluated for AGT levels. The continuous-infusion dose that resulted in undetectable AGT levels in 11 or more of 14 patients was used in the second patient cohort. Group B received the optimal dose of O6-BG for 2, 4, 7, or 14 days after surgical implantation of the carmustine wafers. The study end point was dose-limiting toxicity (DLT). Results Thirty-eight patients were accrued. In group A, 12 of ...

  • a phase ii trial of o6 benzylguanine and carmustine in patients with advanced soft tissue sarcoma
    Cancer Chemotherapy and Pharmacology, 2006
    Co-Authors: Christopher W Ryan, Eileen M. Dolan, Shannon M Delaney, Bruce Brockstein, Roger E Mclendon, Brian L Samuels, Edem Agamah, Everett E Vokes
    Abstract:

    Purpose: Tumor resistance to alkylating agents such as carmustine (BCNU) has been found to be associated with intracellular expression of O 6 -methylguanine-DNA methyltransferase (MGMT). Administration of O 6-benzylguanine (O 6-BG), a substrate that inactivates MGMT, may help overcome chemotherapy resistance. We performed a phase II study to explore the activity of O 6-BG in combination with BCNU in patients with advanced soft tissue sarcoma. Experimental design: Informed consent was obtained from patients with metastatic soft tissue sarcoma naive to systemic chemotherapy (adjuvant chemotherapy allowed). Patients received O 6 -BG 120 mg/m2 I.V. followed by BCNU 40 mg/m2 I.V. Treatment was repeated every 6 weeks until disease progression or development of unacceptable toxicity. Results: No objective responses were observed in 12 enrolled patients. Four patients exhibited stable disease lasting 11–25+ weeks. The median overall survival was 16.9 months (95% CI, 2.9–NR). The most common grade 3–4 toxicities were neutropenia, thrombocytopenia, and anemia. Depletion of MGMT activity was demonstrated in peripheral blood mononuclear cells. Immunohistochemical estimation of MGMT expression from archival tissue ranged from 20 to 99% positive staining cells. Conclusions: Observed toxicities were consistent with previous studies of O 6-BG plus BCNU. The degree of MGMT expression was variable in this small sample of heterogeneous sarcomas. Further development of this regimen and dose for the treatment of soft tissue sarcoma is not warranted due to the lack of objective responses.

  • brain tumor cell lines resistant to o6 benzylguanine 1 3 bis 2 chloroethyl 1 nitrosourea chemotherapy have o6 alkylguanine dna alkyltransferase mutations
    Molecular Cancer Therapeutics, 2004
    Co-Authors: Manny D Bacolod, Robert C Moschel, Stewart P Johnson, Michael O Colvin, Anthony E. Pegg, Eileen M. Dolan, Nancy S Bullock, Qingming Fang, Paul Modrich, Darell D Bigner
    Abstract:

    The chemotherapeutic activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) may be improved by the addition of O6-Benzylguanine (O6-BG). The reaction of O6-BG with O6-alkylguanine-DNA alkyltransferase (AGT) prevents the repair of O6-chloroethyl lesions caused by BCNU. In clinics, the combination of O6-BG and BCNU is now being tested for the treatment of brain tumors. However, the effectiveness of this drug regimen may be limited by drug resistance acquired during treatment. To understand the possible mechanisms of resistance of brain tumor cells to the O6-BG/BCNU combination, we generated medulloblastoma cell lines (D283 MED, D341 MED, and Daoy) resistant to the combination of O6-BG and BCNU [O6-BG/BCNU resistant (OBR)]. DNA sequencing showed that all of the parent cell lines express wild-type AGTs, whereas every OBR cell line exhibited mutations that potentially affected the binding of O6-BG to the protein as evidenced previously by in vitro mutagenesis and structural studies of AGT. The D283 MED (OBR), Daoy (OBR), and D341 MED (OBR) cell lines expressed G156C, Y114F, and K165T AGT mutations, respectively. We reported previously that rhabdomyosarcoma TE-671 (OBR) also expresses a G156C mutation. These data suggest that the clonal selection of AGT mutants during treatment with O6-BG plus an alkylator may produce resistance to this intervention in clinical settings.

Stanton L Gerson - One of the best experts on this subject based on the ideXlab platform.

  • safety of mgmt gene transfer into hematopoietic progenitors a phase i clinical trial using retroviral gene transfer of g156a mgmt to protect hematopoiesis during bg plus alkylating agent treatment of advanced malignancies
    Blood, 2007
    Co-Authors: Jane S Reese, Karen Lingas, Colin L Sweeney, Joanna M Brell, Stanton L Gerson
    Abstract:

    The G156A mutation of the DNA repair gene O6-methylguanine DNA-methyltransferase (MGMT) confers hematopoietic cell resistance to O6-Benzylguanine (BG) and the DNA-alkylating agents BCNU and temozolomide (TMZ). We conducted a Phase I clinical trial utilizing MGMT-mediated chemoprotection to improve treatment of advanced solid tumors. Here we report the safety and feasibility of this approach. Autologous CD34+ cells were transduced ex vivo with a retroviral vector MFG- G156A MGMT (K. Cornetta, NGVL at Indiana U.), then reinfused into non-myeloablated patients. Eight patients were accrued to the study and 6 patients were infused with a mean of 7.3 × 105 cells/kg obtained post transduction. Of the 6 patients who received cells, all received one cycle of chemotherapy (BG + BCNU or TMZ) 3 days prior to cell infusion and 3 patients received a second cycle of either BG + BCNU or BG + TMZ 4–6 weeks post infusion. No patient experienced grade 2 or greater infusion related toxicity, and one patient went off study prior to receiving cells due to neurological toxicity during cytokine administration. All patients who received G156A MGMT+ cells went off study due to progressive disease, and survival ranged from 2 weeks to 9 months post infusion. No patient showed evidence of replication competent retrovirus (RCR) when assessed for the presence of the viral envelope by quantitative PCR for GALV sequences (performed by the IU Vector Production Facility). The mean vector copy number in post transduction cells (n= 7 patients) was 0.29 copies per genome by quantitative PCR (Q-PCR), with a mean CFU transduction efficiency of 24%, and a mean of 9% CD34 cells expressing the G156A MGMT transgene by flow cytometry. Retroviral insertion sites in pre-infusion CFUs from 2 patients analyzed by linear amplification-mediated (LAM)-PCR were polyclonal, with an average of 1.4 retroviral insertions per positive CFU. Transgene positive cells were detected in vivo in 3 patients. In the first patient, after BG and BCNU at 6 weeks, the transgene was detected in 1 of 100 BM CFU by Q-PCR and one insertion site was detected in this clone by LAM-PCR. The patient went off study at 8 weeks, then received TMZ alone (300 mg/m2/day for 5 days). At 16 weeks, the transgene was detected at low frequency in BM MNCs and granulocytes by Q-PCR and in BM granulocytes as an internal band in LAM-PCR, although no other insertions sites were detectable. In a second patient, the transgene was detected in 2 of 30 BM CFU by PCR and in BM granulocytes and MNCs by LAM PCR at 5 weeks. In a third patient treated with BG + TMZ 4 weeks post cell infusion, 3 of 50 PB CFU were transgene positive by Q-PCR at week 28. These data demonstrate that infusion of retroviral MGMT transduced hematopoietic cells is safe and are the first to show emergence and low level persistence of transduced mutant G156A MGMT cells after nonmyeloablative conditioning in humans. Our future studies include using a lentiviral vector to achieve better levels of gene transfer and reduced risk of insertional mutagenesis.

  • phase ii trial of the o6 alkylguanine dna alkyltransferase inhibitor o6 benzylguanine and 1 3 bis 2 chloroethyl 1 nitrosourea in advanced melanoma
    Clinical Cancer Research, 2005
    Co-Authors: Thomas F Gajewski, Lili Liu, Stanton L Gerson, Jeffrey A Sosman, Eileen Dolan, Shang Lin, Everett E Vokes
    Abstract:

    Purpose: 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) induces DNA damage via a chloroethyl adduct at the O 6 position of guanine, which can be repaired by O 6 -alkylguanine DNA alkyltransferase (AGT) expressed in melanoma. We postulated that the addition of O 6 benzylguanine ( O 6 BG), a potent inactivator of AGT, would improve the clinical response to BCNU in melanoma. Experimental Design: Patients had measurable disease, adequate organ function, and a corrected Diffusing capacity of the lung for carbon monoxide (DLCO) of ≥70% predicted. They were accrued into two cohorts based on prior chemotherapy. O 6 BG (120 mg/m 2 ) was administered i.v. followed by BCNU (40 mg/m 2 ) on an outpatient basis. Peripheral blood mononuclear cells (PBMC) were collected pre- and 18 hours post- O 6 BG to analyze AGT depletion. Treatment was every 6 weeks, and clinical response was assessed after every two cycles. Results: Forty-two patients were enrolled, 22 of these patients were chemotherapy-naive. In the chemotherapy-naive cohort, there was a patient with a complete response (CR), 4 with stable disease (SD), 13 with progressive disease (PD), and 4 nonevaluable patients; the median time to progression was 80 days and the median survival was 211 days. In the prior-chemotherapy cohort, there were no responses, 3 SD, 15 PD, and 2 nonevaluable patients; median time to progression was 54 days and median survival was 120 days. AGT was depleted from PBMC in the 15 patients tested. Grades 3 to 4 myelosuppression was seen in 57% of patients; toxicities were similar between the two cohorts. Conclusions: O 6 BG/BCNU was successfully administered on an outpatient basis and depleted AGT from PBMC. However, significant myelosuppression was observed and the clinical outcome was not improved. Alternative mechanisms of resistance to melanoma cell death need to be investigated.

  • in vivo selection of mgmt p140k lentivirus transduced human nod scid repopulating cells without pretransplant irradiation conditioning
    Journal of Clinical Investigation, 2003
    Co-Authors: Steven P Zielske, Jane S Reese, Karen Lingas, Jon R Donze, Stanton L Gerson
    Abstract:

    Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O 6 -methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and 06-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O 6 -alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.

  • selection for g156a o6 methylguanine dna methyltransferase gene transduced hematopoietic progenitors and protection from lethality in mice treated with o6 benzylguanine and 1 3 bis 2 chloroethyl 1 nitrosourea
    Cancer Research, 1997
    Co-Authors: Brian R Davis, Omer N Koc, Jane S Reese, Keun Myoung Lee, Jane E Schupp, Stanton L Gerson
    Abstract:

    A retroviral gene therapy approach was developed to protect early hematopoietic progenitors from 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a stem cell toxin, and O 6-benzylguanine (BG), an inhibitor of a key BCNU resistance protein, O 6-alkylguanine DNA alkyltransferase (AGT). The retroviral vector MFG was used to transfer the G156A MGMT (ΔMGMT) cDNA, encoding a mutant AGT that is resistant to inhibition by BG, into murine bone marrow-derived hematopoietic progenitors. Following transplantation into lethally irradiated mice, the transduced cells were subjected to in vivo BG and BCNU treatment to examine the ability to enrich for transduced cells expressing ΔAGT. Transplantation of ΔMGMT -transduced cells resulted in δAGT expression in 30% of bone marrow nucleated cells 13 weeks after transplantation. After one cycle of BG and BCNU, ΔAGT expression was observed in 60% of bone marrow cells, and the percentage of colony-forming units (culture; CFU-C) containing proviral sequence increased from 67 to 100%. CFU-C obtained from BG and BCNU-treated ΔMGMT animals up to 23 weeks after transplantation were more resistant to combination BG and BCNU than CFU-C from mice transplanted with lacZ -transduced cells and treated with BG and BCNU or from mice transplanted with ΔMGMT -transduced cells and left untreated. The degree of drug resistance in ΔMGMT -transduced hematopoietic progenitors to BG and BCNU was much greater than we observed previously with wild-type MGMT gene transfer and treatment with BCNU alone. Furthermore, whereas 21 of 22 mice transplanted with ΔMGMT -transduced cells survived in vivo BG and BCNU administration, only 3 of 13 mice transplanted with lacZ -transduced progenitors survived similar drug treatment. Thus, ΔMGMT -transduced murine bone marrow cells selectively survive in vivo BG and BCNU exposure, resulting in prolonged enrichment for the transduced cells and protection from mortality induced by this drug combination.

  • retroviral transduction of a mutant methylguanine dna methyltransferase gene into human cd34 cells confers resistance to o6 benzylguanine plus 1 3 bis 2 chloroethyl 1 nitrosourea
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Jane S Reese, James A Allay, Omer N Koc, Lili Liu, Keun Myoung Lee, Weldon P Phillips, Stanton L Gerson
    Abstract:

    Human CD34 cells express low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Gene transfer of the AGT gene, methylguanine DNA methyltransferase (MGMT), results in only modest BCNU resistance. Recently, an AGT inhibitor, O6-Benzylguanine (BG), entered clinical trials. In preclinical studies, BG potentiated the cytotoxic effect of BCNU in tumors but increased toxicity to normal CD34 cells. We transferred a mutant MGMT containing a glycine-to-alanine mutation at position 156, resulting in marked resistance to BG, into Chinese hamster cells; the K562 cell line and human CD34 cells used the retroviral backbone MFG. In each instance, cells expressed increased AGT and were much more resistant to the combination of BG and BCNU than the parental cells or cells transduced with wild-type MGMT. Furthermore, the transduction efficiency in human CD34 cells was in excess of 70%, and the proportion of CD34 transduced cells resistant to the combination was >30%. Thus, retroviral-mediated transduction of a mutant MGMT into CD34 cells appears to be an effective way to induce selective resistance to a drug combination designed to overcome a significant resistance mechanism to nitrosoureas in tumors.

Darell D Bigner - One of the best experts on this subject based on the ideXlab platform.

  • phase ii trial of temozolomide plus o6 benzylguanine in adults with recurrent temozolomide resistant malignant glioma
    Journal of Clinical Oncology, 2009
    Co-Authors: Jennifer A Quinn, Darell D Bigner, Sara Xiaoyin Jiang, David A Reardon, Annick Desjardins, James J Vredenburgh, Jeremy N Rich, Sridharan Gururangan, Allan H Friedman, John H Sampson
    Abstract:

    Purpose This phase II trial was designed to define the role of O 6 -benzylguanine (O 6 -BG) in restoring temozolomide sensitivity in patients with recurrent or progressive, temozolomide-resistant malignant glioma and to evaluate the safety of administering O 6 -BG in combination with temozolomide. Patients and Methods Patients were accrued into two independent strata on the basis of histology: glioblastoma multiforme (GBM) and anaplastic glioma. Both temozolomide and O 6 -BG were administered on day 1 of a 28-day treatment cycle. Patients were administered a 1-hour O 6 -BG infusion at a dose of 120 mg/m 2 followed immediately by a 48-hour infusion at a dose of 30 mg/m 2 /d. Temozolomide was administered orally within 60 minutes of the end of the 1-hour O 6 -BG infusion at a dose of 472 mg/m 2 . The primary end point was objective response rate. Secondary end points included progression-free survival, overall survival, and safety. Results Sixty-six of 67 patients who enrolled were treated with temozolomide and O 6 -BG. One of 34 patients (3%) with GBM (95% CI, 0.1% to 15%) and five of 32 assessable patients (16%) with anaplastic glioma (95% CI, 5% to 33%) were responders. The most commonly reported adverse events were grade 4 hematologic events experienced in 48% of the patients. Conclusion O 6 -BG when added to a 1-day dosing regimen of temozolomide was able to restore temozolomide sensitivity in patients with temozolomide-resistant anaplastic glioma, but there seemed to be no significant restoration of temozolomide sensitivity in patients with temozolomide-resistant GBM.

  • brain tumor cell lines resistant to o6 benzylguanine 1 3 bis 2 chloroethyl 1 nitrosourea chemotherapy have o6 alkylguanine dna alkyltransferase mutations
    Molecular Cancer Therapeutics, 2004
    Co-Authors: Manny D Bacolod, Robert C Moschel, Stewart P Johnson, Michael O Colvin, Anthony E. Pegg, Eileen M. Dolan, Nancy S Bullock, Qingming Fang, Paul Modrich, Darell D Bigner
    Abstract:

    The chemotherapeutic activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) may be improved by the addition of O6-Benzylguanine (O6-BG). The reaction of O6-BG with O6-alkylguanine-DNA alkyltransferase (AGT) prevents the repair of O6-chloroethyl lesions caused by BCNU. In clinics, the combination of O6-BG and BCNU is now being tested for the treatment of brain tumors. However, the effectiveness of this drug regimen may be limited by drug resistance acquired during treatment. To understand the possible mechanisms of resistance of brain tumor cells to the O6-BG/BCNU combination, we generated medulloblastoma cell lines (D283 MED, D341 MED, and Daoy) resistant to the combination of O6-BG and BCNU [O6-BG/BCNU resistant (OBR)]. DNA sequencing showed that all of the parent cell lines express wild-type AGTs, whereas every OBR cell line exhibited mutations that potentially affected the binding of O6-BG to the protein as evidenced previously by in vitro mutagenesis and structural studies of AGT. The D283 MED (OBR), Daoy (OBR), and D341 MED (OBR) cell lines expressed G156C, Y114F, and K165T AGT mutations, respectively. We reported previously that rhabdomyosarcoma TE-671 (OBR) also expresses a G156C mutation. These data suggest that the clonal selection of AGT mutants during treatment with O6-BG plus an alkylator may produce resistance to this intervention in clinical settings.

  • activity of temozolomide in the treatment of central nervous system tumor xenografts
    Cancer Research, 1995
    Co-Authors: Henry S Friedman, Anthony E. Pegg, Eileen M. Dolan, Stephen T Keir, Darell D Bigner, Susan Marcelli, Joseph J Catino, Clifford S Schold
    Abstract:

    Abstract The activity of 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (temozolomide) in the treatment of a panel of xenografts derived from ependymoma, medulloblastoma, and childhood and adult high-grade glioma was evaluated in athymic nude mice bearing s.c. and intracranial tumors. Temozolomide administered daily for a total of five doses demonstrated marked activity against a panel of Mer+ xenografts despite marginal to moderate activity of 1,3-bis(2-chloroethyl)-1-nitrosourea. The growth delays produced by temozolomide in these xenografts were 1.8–7.5-fold greater than those produced by procarbazine. Although temozolomide demonstrated marginal activity against the Mer+ cell line D341 Med when a 5-day schedule was used, a high-dose 1-day schedule resulted in moderate activity. Temozolomide produced increases in median survival of 1285% (adult glioma D-54 MG), 323% (childhood glioma D-456 MG), and 68% (ependymoma D612 EP). Pretreatment of mice with O6-Benzylguanine increased temozolomide-induced mortality, requiring reduction of the dosage from 1200 to 750 mg/m2 on the single-day regimen. O6-Benzylguanine pretreatment of mice bearing Mer+ D341 Med increased the growth delay of temozolomide, in duplicate experiments, from -3.1 to 4.8 and 1.1 to 4.9 days. These studies suggest that temozolomide may be active in the treatment of a broad spectrum of central nervous system cancers, including Mer+ tumors resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea.

  • enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme
    Journal of the National Cancer Institute, 1992
    Co-Authors: Henry S Friedman, Robert C Moschel, Anthony E. Pegg, Eileen M. Dolan, Darell D Bigner, Jeremy N Rich, Michael G Felker, Clifford S Schold
    Abstract:

    BACKGROUND Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU]). PURPOSE This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-Benzylguanine. METHODS Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-Benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised. RESULTS Pretreatment with O6-Benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-Benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-Benzylguanine. Following therapy with BCNU plus O6-Benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts. CONCLUSION We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-Benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.

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  • phase i trial of single dose temozolomide and continuous administration of o6 benzylguanine in children with brain tumors a pediatric brain tumor consortium report
    Clinical Cancer Research, 2007
    Co-Authors: Alberto Broniscer, Henry S Friedman, Dana Wallace, Clinton F Stewart, Sridharan Gururangan, Tobey J Macdonald, Stewart Goldman, Roger J Packer, Mary K Danks, Tina Young Poussaint
    Abstract:

    Purpose: To estimate the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of escalating doses of temozolomide combined with O 6 -benzylguanine in patients ≤21 years with recurrent brain tumors. Experimental Design: Treatment strata consisted of patients who had previously received no or local radiotherapy (Str1) and patients who had undergone craniospinal radiotherapy or myeloablative chemotherapy (Str2). One-hour i.v. administration of O 6 -benzylguanine at 120 mg/m 2 was followed by 48-h continuous infusion at 30 mg/m 2 /day. Single-dose temozolomide at five dosage levels (267, 355, 472, 628, and 835 mg/m 2 ) was given at least 6 h after completion of O 6 -benzylguanine bolus. Treatment was repeated after recovery from toxicities at least 4 weeks apart for a maximum of 12 courses. Dose escalation followed the modified continual reassessment method. Pharmacokinetic analyses of temozolomide and 5-triazeno imidazole carboxamide (MTIC) were done in 28 patients. Results: A total of 44 and 26 eligible patients were enrolled on Str1 and Str2, respectively. Median age at study entry in each stratum was 8.6 and 11.3 years, respectively. Predominant diagnoses were high-grade/brainstem glioma in Str1 and medulloblastoma in Str2. Whereas the estimated MTDs of temozolomide for Str1 and Str2 were 562 and 407 mg/m 2 , respectively, the doses recommended for phase II investigations are 472 and 355 mg/m 2 , respectively. DLTs were predominantly neutropenia and thrombocytopenia. Three patients with gliomas experienced centrally confirmed partial responses to therapy. Four patients completed all planned therapy. Temozolomide and MTIC exposures were statistically associated with temozolomide dosage. Conclusions: The current schedule of temozolomide and O 6 -benzylguanine is safe and showed modest activity against recurrent brain tumors in children.

  • radiolabeled guanine derivatives for the in vivo mapping of o6 alkylguanine dna alkyltransferase 6 4 18f fluoro benzyloxy 9h purin 2 ylamine and 6 3 131i iodo benzyloxy 9h purin 2 ylamine
    Bioconjugate Chemistry, 2000
    Co-Authors: Ganesan Vaidyanathan, Stewart P Johnson, Henry S Friedman, Donna J Affleck, Christina M Cavazos, Sriram Shankar, Michael Colvin, Michael R Zalutsky
    Abstract:

    Two radiolabeled analogues of 6-benzyloxy-9H-purin-2-ylamine (O6-Benzylguanine; BG) potentially useful in the in vivo mapping of O6-alkylguanine-DNA alkyltransferase (AGT) were synthesized. Fluorine-18 labeling of the known 6-(4-fluoro-benzyloxy)-9H-purin-2-ylamine (FBG; 6) was accomplished by the condensation of 4-[18F]fluorobenzyl alcohol with 2-aminopurin-6-yltrimethylammonium chloride (4) or 2-amino-6-chloropurine in average decay-corrected radiochemical yields of 40 and 25%, respectively. Unlabeled 6-(3-iodo-benzyloxy)-9H-purin-2-ylamine (IBG; 7) was prepared from 4 and 3-iodobenzyl alcohol. Radioiodination of 9, prepared from 7 in two steps, and subsequent deprotection gave [131I]7 in about 70% overall radiochemical yield. The IC50 values for the inactivation of AGT from CHO cells transfected with pCMV-AGT were 15 nM for IBG and 50 nM for FBG. The binding of [18F]6 and [131I]7 to purified AGT was specific and saturable with both exhibiting similar IC50 values (5−6 μM).

  • ubiquitination dependent proteolysis of o6 methylguanine dna methyltransferase in human and murine tumor cells following inactivation with o6 benzylguanine or 1 3 bis 2 chloroethyl 1 nitrosourea
    Biochemistry, 1996
    Co-Authors: Kalkunte S Srivenugopal, Henry S Friedman, Xiaohua Yuan, Francis Aliosman
    Abstract:

    In this study, we investigated the role of ubiquitination in the disposition of the inactivated O6-methylguanine-DNA methyltransferase (MGMT) protein in human (HT-29 and CEM) and murine (ts85) tumor cells. Using a combination of immunoprecipitation and immunoblotting techniques with antibodies against ubiquitin and MGMT, and anti-ubiquitin immunoaffinity chromatography, the MGMT protein was found to coexist with small amounts of its ubiquitinated species in both human and mouse tumor cells, suggesting the presence of endogenous inactivated MGMT. Further, treatment of HT-29 and CEM cells with MGMT-inactivating compounds, O6-Benzylguanine (O6-BG, 20 microM) or 1,3-bis(chloroethyl)-1-nitrosourea (BCNU, 100 microM), resulted in increased levels of ubiquitinated MGMT within 1.5-3 h of drug exposure. Kinetic studies in HT-29 cells treated with O6-BG indicated a slow and gradual conversion of the inactivated MGMT to its polyubiquitinated forms over a course of 3-18 h, with a concomitant disappearance of the parent MGMT protein. We also characterized the previously reported O6-BG-induced degradation of MGMT in HT-29 cell extracts [Pegg et al. (1991) Carcinogenesis 12, 1679-1683] and showed the extracts to be active in conjugation of the MGMT protein with ubiquitin. The proteolysis of O6-BG-inactivated MGMT in HT-29 cell extracts was energy-dependent and was markedly stimulated by ATP and Mg2+ ions. Using the ts85 temperature-sensitive mutant cell line, which expresses a thermolabile ubiquitin-activating enzyme, we observed a differential stability of the inactivated MGMT protein at permissive and nonpermissive temperatures. These results provide conclusive evidence that the MGMT protein, following its inactivation, is degraded via the ubiquitin proteolytic pathway.

  • activity of temozolomide in the treatment of central nervous system tumor xenografts
    Cancer Research, 1995
    Co-Authors: Henry S Friedman, Anthony E. Pegg, Eileen M. Dolan, Stephen T Keir, Darell D Bigner, Susan Marcelli, Joseph J Catino, Clifford S Schold
    Abstract:

    Abstract The activity of 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (temozolomide) in the treatment of a panel of xenografts derived from ependymoma, medulloblastoma, and childhood and adult high-grade glioma was evaluated in athymic nude mice bearing s.c. and intracranial tumors. Temozolomide administered daily for a total of five doses demonstrated marked activity against a panel of Mer+ xenografts despite marginal to moderate activity of 1,3-bis(2-chloroethyl)-1-nitrosourea. The growth delays produced by temozolomide in these xenografts were 1.8–7.5-fold greater than those produced by procarbazine. Although temozolomide demonstrated marginal activity against the Mer+ cell line D341 Med when a 5-day schedule was used, a high-dose 1-day schedule resulted in moderate activity. Temozolomide produced increases in median survival of 1285% (adult glioma D-54 MG), 323% (childhood glioma D-456 MG), and 68% (ependymoma D612 EP). Pretreatment of mice with O6-Benzylguanine increased temozolomide-induced mortality, requiring reduction of the dosage from 1200 to 750 mg/m2 on the single-day regimen. O6-Benzylguanine pretreatment of mice bearing Mer+ D341 Med increased the growth delay of temozolomide, in duplicate experiments, from -3.1 to 4.8 and 1.1 to 4.9 days. These studies suggest that temozolomide may be active in the treatment of a broad spectrum of central nervous system cancers, including Mer+ tumors resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea.

  • enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme
    Journal of the National Cancer Institute, 1992
    Co-Authors: Henry S Friedman, Robert C Moschel, Anthony E. Pegg, Eileen M. Dolan, Darell D Bigner, Jeremy N Rich, Michael G Felker, Clifford S Schold
    Abstract:

    BACKGROUND Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU]). PURPOSE This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-Benzylguanine. METHODS Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-Benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised. RESULTS Pretreatment with O6-Benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-Benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-Benzylguanine. Following therapy with BCNU plus O6-Benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts. CONCLUSION We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-Benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.