Background: CpG methylation in the O-6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies.Methods: We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP).Results: Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P <= 0.001) and continuous (P <= 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P <= 0.005) and longer PFS (P <= 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P <= 0.005), and among unmethylated tumors with less than median activity (P <= 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs.Conclusions: Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas.General significance: MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND 4.0 license (http://creativecommons.org/licenses/by/4.0/).

Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during and after radiotherapy has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O6-methylguanine (O6-meG) mediates GBM cell killing. Moreover, low or absent expression of O6-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O6-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O6-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O6-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O6-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.

Gliomas are the most frequent adult primary brain tumor, and are invariably fatal. The most common diagnosis glioblastoma multiforme (GBM) afflicts 12,500 new patients in the U.S. annually, and has a median survival of approximately one year when treated with the current standard of care. Alkylating agents have long been central in the chemotherapy of GBM and other gliomas. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O(6)-alkylguanine adducts from DNA, promotes resistance to anti-glioma alkylators, including temozolomide and BCNU, in GBM cell lines and xenografts. Moreover, MGMT expression assessed by immunohistochemistry, biochemical activity or promoter CpG methylation status is associated with the response of GBM to alkylator-based therapies, providing evidence that MGMT promotes clinical resistance to alkylating agents. These observations suggest a role for MGMT in directing adjuvant therapy of GBM and other gliomas. Promoter methylation status is the most clinically tractable measure of MGMT, and there is considerable enthusiasm for exploring its utility as a marker to assign therapy to individual patients. Here, we provide an overview of the biochemical, genetic and biological characteristics of MGMT as they relate to glioma therapy. We consider current methods to assess MGMT expression and discuss their utility as predictors of treatment response. Particular emphasis is given to promoter methylation status and the methodological and conceptual impediments that limit its use to direct treatment. We conclude by considering approaches that may improve the utility of MGMT methylation status in planning optimal therapies tailored to individual patients.

Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers radiation resistance in human cells. Here we examined the association between Ap endo activity and response to radiotherapy in pediatric ependymomas, tumors for which treatment options are limited and survival rates are only about 50%. We assayed Ap endo activity in 36 ependymomas and expression of Ape1/Ref‐1, the predominant Ap endo activity in humans, in 44 tumors by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity or expression with progression‐free survival or with overall survival. Activity varied 13‐fold and was not associated with tumor or patient characteristics. In univariate models with Ap endo activity entered as a continuous variable, the hazard ratio for progression increased by a factor of 2.18 for every 0.01 unit increase in activity ( p ≤ 0.003) in 24 grade II ependymomas. Risk for death increased by a factor of 1.89 ( p ≤ 0.02) in the same population. The fraction of Ape1/Ref‐1 immunopositive cells varied widely within individual tumors and was not associated with either progression‐free or with overall survival. Suppressing Ap endo activity in pediatric ependymoma cells significantly increased radiation sensitivity, suggesting that the association of activity with radiation response reflected, at least in part, repair of radiation‐induced DNA lesions. Our data indicate that Ap endo activity is predictive of outcome following radiotherapy, and suggest that Ape1/Ref‐1 promotes radiation resistance in pediatric ependymomas. Our findings support the use of inhibitors of Ap endo activity to overcome resistance.

Concurrent treatment with the methylating agent temozolomide during radiotherapy has yielded the first significant improvement in the survival of adult glioblastomas (GBM) in the last three decades. However, improved survival is observed in a minority of patients, most frequently those whose tumors display CpG methylation of the O6-methylguanine (O6-meG)-DNA methyltransferase (MGMT) promoter, and adult GBMs remain invariably fatal. Some, although not all, preclinical studies have shown that temozolomide can increase radiosensitivity in GBM cells that lack MGMT, the sole activity in human cells that removes O6-meG from DNA. Here, we systematically examined the temozolomide dose dependence of radiation killing in established GBM cell lines that differ in ability to remove O6-meG or tolerate its lethality. Our results show that minimally cytotoxic doses of temozolomide can produce dose-dependent radiosensitization in MGMT-deficient cells, MGMT-proficient cells, and MGMT-deficient cells that lack mismatch repair, a process that renders cells tolerant of the lethality of O6-meG. In cells that either possess or lack MGMT activity, radiosensitization requires exposure to temozolomide before but not after radiation and is accompanied by formation of double-strand breaks within 45 minutes of radiation. Moreover, suppressing alkyladenine-DNA glycosylase, the only activity in human cells that excises 3-methyladenine from DNA, reduces the temozolomide dose dependence of radiosensitization, indicating that radiosensitization is mediated by 3-methyladenine as well as by O6-meG. These results provide novel information on which to base further mechanistic study of radiosensitization by temozolomide in human GBM cells and to develop strategies to improve the outcome of concurrent temozolomide radiotherapy. Mol Cancer Ther; 9(5); 1208–18. ©2010 AACR.

Human alkyladenine-DNA glycosylase (AAG) initiates base excision repair (BER) of alkylated and deaminated bases in DNA. Here, we assessed the mutability of the AAG substrate binding pocket, and the essentiality of individual binding pocket amino acids for survival of methylation damage. We used oligonucleotide-directed mutagenesis to randomize 19 amino acids, 8 of which interact with substrate bases, and created more than 4.5 million variants. We expressed the mutant AAGs in repair-deficient Escherichia coli and selected for protection against the cytotoxicity of either methylmethane sulfonate (MMS) or methyl-lexitropsin (Me-lex), an agent that produces 3-methyladenine as the predominant base lesion. Sequence analysis of 116 methylation-resistant mutants revealed no substitutions for highly conserved Tyr(127)and His(136). In contrast, one mutation, L180F, was greatly enriched in both the MMS- and Me-lex-resistant libraries. Expression of the L180F single mutant conferred 4.4-fold enhanced survival at the high dose of MMS used for selection. The homogeneous L180F mutant enzyme exhibited 2.2-fold reduced excision of 3-methyladenine and 7.3-fold reduced excision of 7-methylguanine from methylated calf thymus DNA. Decreased excision of methylated bases by the mutant glycosylase could promote survival at high MMS concentrations, where the capacity of downstream enzymes to process toxic BER intermediates may be saturated. The mutant also displayed 6.6- and 3.0-fold reduced excision of 1,N(6)-ethenoadenine and hypoxanthine from oligonucleotide substrates, respectively, and a 1.7-fold increase in binding to abasic site-containing DNA. Our work provides in vivo evidence for the substrate binding mechanism deduced from crystal structures, illuminates the function of Leu(180) in wild-type human AAG, and is consistent with a role for balanced expression of BER enzymes in damage survival.

The DNA repair protein O-6-methylguanine-DNA methyltransferase (MGMT) is a cardinal defense against the mutagenic and carcinogenic effects of alkylating agents. We have reported evidence that absence of detectable MGMT activity (MGMT(-) phenotype) in human brain is a predisposing factor for primary brain tumors that affects ca. 12% of individuals [J.R. Silber, A. Blank, M.S. Bobola, B.A. Mueller, D.D. Kolstoe, G.A. Ojemann, M.S. Berger, Lack of the DNA repair protein O-6-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary brain tumors, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 6941-6946]. We report here that MGMT- phenotype in the brain of children and adults, and the apparent increase in risk of neurocarcinogenesis, may arise during gestation. We found that MGMT activity in 71 brain specimens at 6-19 weeks post-conception was positively correlated with gestational age (P <= 0.0015). Moreover, the proportion of specimens exhibiting MGMT- phenotype (MGMT content < 0.42 fmol/10(6) cells or 255 molecules/cell) declined progressively from 76% (16/21) at 6-8 weeks to 13% (1/8) at 15-19 weeks. All liver specimens that accompanied MGMT- brain (15/15) had measurable MGMT activity, demonstrating that the phenotype was not systemic in these cases. In contrast to MGMT, apurinic endonuclease, DNA polymerase and lactate dehydrogenase activities were found in every brain extract assayed, and showed no significant relationship with gestational age. The observed gestational pattern has at least two implications for neurocarcinogenesis. (1) Early in development, brain tissue that has MGMT- phenotype and is rapidly proliferating may be especially vulnerable to alkylation-induced mutations, including mutations that lead to brain tumors. (2) Persistence of prenatal MGMT deficiency into postnatal life in a subpopulation of individuals may increase brain tumor risk. Our findings provide possible mechanistic insight into epidemiologic data associating maternal alkylating agent exposure with brain tumor incidence. (c) 2007 Elsevier B.V. All rights reserved.

Purpose: Defining the cytotoxicity of individual adducts in DNA is necessary for mechanistic understanding of human brain tumor resistance to therapeutic alkylating agents and for design of DNA repair-related antiresistance strategies. Our purpose is to characterize the sensitivity of human glioma cells to methyl-lexitropsin (Me-lex), a sequence-specific alkylator that produces 3-methyladenine (3-meA) as the predominant (>90%) DNA lesion. Experimental Design: We quantitated the Me-lex cytotoxicity of 10 human glioma cell lines that differ in O6-methylguanine (O6-meG)-DNA methyltransferase (MGMT) and mismatch repair activity. We used antisense suppression of alkyladenine DNA glycosylase (AAG) and Ape1 to assess the contribution of 3-meA and abasic sites to lethality and measured abasic sites. Results: (a) The LD10 for Me-lex varied widely among the cell lines. (b) MGMT-proficient lines were more resistant than MGMT-deficient lines, an unexpected finding because Me-lex produces very little O6-meG. (c) Suppression of AAG increased Me-lex killing and reduced abasic site content. (d) Suppression of Ape1 increased Me-lex killing and increased abasic site content. (e) Ablation of MGMT had no effect on Me-lex cytotoxicity. Conclusions: (a) Me-lex is cytotoxic in human glioma cells and AAG promotes resistance, indicating that 3-meA is a lethal lesion in these cells. (b) Abasic sites resulting from 3-meA repair are cytotoxic and Ape1 promotes resistance to these derivative lesions. (c) A factor(s) associated with MGMT expression, other than repair of O6-meG, contributes to Me-lex resistance. (d) Me-lex may have clinical utility in the adjuvant therapy of gliomas. (e) AAG and Ape1 inhibitors may be useful in targeting alkylating agent resistance.

The DNA repair protein 06-methylguanine-DNA methyltransferase (MGMT) confers resistance to therapeutic methylating and chloroethylating agents in human brain tumor-derived cell lines. In this work, we assayed MGMT activity in 152 adult gliomas to establish correlates with patient and tumor characteristics. We also assayed MGMT in histologically normal brain adjacent to 87 tumors to characterize changes in activity accompanying neurocarcinogenesis. MGMT activity was detect able in 76% (115 of 152) of tumors, ranging -300-fold from 0.30 to 89 tinol/IO'1 cells (180-57,000 molecules/cell). Mean activity was 6.6 ± 13 fmol/106 cells and varied 4-fold among diagnostic groups. The mean for oligodendrogliomas was 2-fold lower (/' < 0.03), and for mixed oligodendroglioma-astrocytomas, the mean was 4-fold lower (P < 0.006) than for astroglial tumors. Twenty-five % of gliomas had no detectable MGMT activity (Mer~ phenotype; <0.25 fmol/106 cells or 150 molecules/cell). Glioma MGMT was inversely correlated with age (P < 0.01), consistent with the observed age dependence in the progenitor tissue of brain tumors (J. R. Silber et al., Proc. Nati. Acad. Sci. USA, 93: 6941-6946, 1996). Neither MGMT activity nor proportion of Mer~ tumors differed by sex. Glioma MGMT was correlated with degree of aneuploidy (P < 0.006) but not with fraction of S-phase cells. Mean activity in tumors was 5-fold higher than in adjacent histologically normal brain (5.0 ± 7.6 versus 1.1 ±1.9 fmol/106 cells; P < 0.001). Notably, elevation of tumor activity was observed in 62% of tissue pairs, ranging from 2-fold to > 105-fold. Moreover, 64% of Mer~ normal tissue was accompanied by Mer+ tumor. These observations indicate that expression of MGMT activity is fre quently activated and/or increased during human neurocarcinogenesis, and that the enhancement is not related to proliferation per se. Signifi cantly, enhanced MGMT activity may heighten the resistance of brain tumors to therapeutic alkylating agents. INTRODUCTION The DNA repair protein MGMT4 is a primary defense against cytotoxic 06-alkylguanine adducts (1). MGMT exerts its protective effect by transferring alkyl adducts from the O6 atom of guanine in DNA to an internal cysteine, yielding guanine and 5-alkylcysteine. Although the preferred substrate of MGMT is O6-methylguanine in double-stranded DNA, the protein removes larger alkyl groups at progressively slower rates. MGMT also prevents the formation of chloroethylnitrosourea-induced interstrand cross-links by reacting with the monoadduct precursors O6-chloroethylguanine and /V',O6ethanoguanine (2, 3). Importantly, the alkyl receptor site is not regen erated, thereby limiting the number of O6-alkylguanine adducts that Received 9/11/97; accepted 1/13/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Grants CA 707090 and OIG R35-CA39903 from the NIH. Grants EDT-53 and CN842I4 from the American Cancer Society, and a grant from the American Federation for Aging Research. Additional support was from the John Gallagher Fund, the Doris Schiffman Fund, and the Brain Tumor Research Fund of the Department of Neurological Surgery, University of Washington, and the Neurooncology Gift Fund and Jessie's Perfect Peach Fund of Children's Hospital and Medical Center. 2 To whom requests for reprints should be addressed, at Department of Neurological Surgery, Box 356470, University of Washington School of Medicine, Seattle, WA 981956470. Phone: (206)685-8642; Fax: (2061543-8315; E-mail: jrsilber@u.washington.edu. ' Present address: Department of Neurosurgery, University of California San Fran cisco, San Francisco, CA 94143-0112. 4 The abbreviation used is: MGMT, O*1-methylguanine-DNA methyltransferase. can be removed in vivo to the number of MGMT molecules and the rate of synthesis of the protein (1). Alkylating agents with proven clinical effectiveness against pri mary brain tumors (4) include nitrosourea and imidazotetrazinone derivatives, which produce relatively high yields of O6-methylguanine (5, 6) and 06-chloroethylguanine (7). Work from numerous laborato ries has demonstrated a role for MGMT in conferring alkylating agent resistance in cell lines and xenografts derived from a variety of human tumors, including gliomas (reviewed in Refs. 1 and 8). For example, depletion of MGMT activity with the substrate analogue inhibitor O6-benzylguanine (9) increases the rate of killing of human gliomaderived cell lines by clinically used methylating and chloroethylating agents (10-12). Similarly, the response of human glioma xenografts to these agents is markedly improved when MGMT is depleted with O6-benzylguanine (8). Most human tumors, including those from brain, express MGMT activity, suggesting that MGMT contributes to alkylator resistance in vivo. We reported previously a 200-fold range of detectable activity among 60 pediatrie and adult brain tumors (13). Importantly, 27% had no detectable activity (Mer~ or methyl repair-deficient status), sug gesting that an appreciable fraction of brain tumors may have height ened sensitivity to alkylating agents as a consequence of lacking MGMT. In the present study, we expand our initial findings (13) by assaying activity in 152 adult primary brain tumors and, for 87 cases, in adjacent, histologically normal brain. Our objective is to delineate biological mechanisms underlying brain tumor alkylator resistance by: (a) seeking correlates of glioma MGMT activity with tumor and patient characteristics; and (b) examining the effects of tumorigenesis on activity in normal brain. Our analysis revealed previously unreported associations between glioma MGMT activity and tumor and patient characteristics. It also demonstrated that tumorigenesis in brain is most often accompanied by an increase in MGMT activity, resulting in the majority of cases from loss of Mer phenotype in normal progenitor tissue. This increase may have significance for clinical response to alkylating agents. MATERIALS AND METHODS Tissue. Tumors were resected at the University of Washington Medical Center from 1991 to 1996. Subcortical normal brain adjacent to tumor was obtained from 87 patients. The specimens included 22 tumors and 63 samples of normal brain whose MGMT activity was reported earlier ( 13, 14). Diagnosis was obtained from the final neuropathology report, which included flow cytometry analysis of ploidy for 94 tumors and fraction of proliferating cells for 97 tumors. Normal tissue was microscopically free of infiltrating tumor, endothelial proliferation, edema, and gliosis. Demographic information and course of adjuvant therapy was obtained from medical records. Immediately upon resection, tissue was placed in ice-cold sterile DMEM supplemented with 15% fetal bovine serum and transported to the laboratory within minutes. We have successfully established cell lines from tumors held overnight on ice in supplemented DMEM. We have also found identical MGMT activities in aliquots of brain tumor and normal brain processed either immediately upon arrival in the laboratory or after being held overnight on ice in supplemented medium, demonstrating that our protocol for transporting specimens preserves cellular viability and MGMT activity. To determine cell number, a small piece of tissue (0.05-0.1 g) was finely minced with scalpel

PURPOSE:Primary brain tumors are the leading cause of cancer death in children. Our purpose is (a) to assess the contribution of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) to the resistance of pediatric brain tumor cell lines to clinical alkylating agents and (b) to evaluate variables for maximal potentiation of cell killing by the MGMT inhibitor O6-benzylguanine, currently in clinical trials. Few such data for pediatric glioma lines, particularly those from low-grade tumors, are currently available.EXPERIMENTAL DESIGN:We used clonogenic assays of proliferative survival to quantitate cytoxicity of the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the methylating agent temozolomide in 11 glioma and five medulloblastoma lines. Twelve lines are newly established and characterized here, nine of them from low-grade gliomas including pilocytic astrocytomas.RESULTS:(a) MGMT is a major determinant of BCNU resistance and the predominant determinant of temozolomide resistance in both our glioma and medulloblastoma lines. On average, O(6)-benzylguanine reduced LD10 for BCNU and temozolomide, 2.6- and 26-fold, respectively, in 15 MGMT-expressing lines. (b) O6-Benzylguanine reduced DT (the threshold dose for killing) for BCNU and temozolomide, 3.3- and 138-fold, respectively. DT was decreased from levels higher than, to levels below, clinically achievable plasma doses for both alkylators. (c) Maximal potentiation by O6-benzylguanine required complete and prolonged suppression of MGMT.CONCLUSIONS:Our results support the use of O6-benzylguanine to achieve full benefit of alkylating agents, particularly temozolomide, in the chemotherapy of pediatric brain tumors.

The Werner syndrome (WS) protein (WRN), a DNA helicase/exonuclease, is required for genomic stability and avoidance of cancer. Current evidence suggests that WRN is involved in the resolution of stalled and/or collapsed replication forks. This function is indicated, in part, by replication defects in WS cells and by hypersensitivity to agents causing major structural aberrations in DNA that block replication. We show here that antisense suppression of WRN in two human glioma cell lines reproduces hallmarks of the drug cytotoxicity profile of WS cells, namely, hypersensitivity to 4-nitroquinoline 1-oxide, camptothecin and hydroxyurea. We also show that antisense-treated cells are hypersensitive to methyl-lexitropsin, a site-specific alkylating agent that produces mainly N3-methyladenine, a cytotoxic and replication-blocking lesion. Antisense-treated cells are hypersensitive to O6-methylguanine adducts as well, but only when repair by O6-methylguanine–DNA methyltransferase is lacking. Our results illustrate the drug sensitivity caused by deficiency of WRN in a uniform genetic background. They extend the WRN DNA damage sensitivity spectrum to methyl base adducts that can result in blocked replication, and suggest that WRN may be required for resumption of processive replication when incomplete repair of DNA damage leaves blocking lesions at forks. The evidence that highly disparate lesions fall within the purview of WRN, and that abrogating DNA repair can reveal dependence on WRN, suggests that WRN may protect the genome from the lethal, mutagenic and carcinogenic effects of widely diverse DNA damage arising from endogenous processes and environmental agents.

Purpose: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair enzyme that cleaves DNA at cytotoxic abasic sites caused by alkylating agents and radiation. We have observed that human glioma cells deficient in Ap endo activity are hypersensitive to clinically used alkylators (Silber et al., Clin Cancer Res 2002;8:3008.). Here we examine the association of glioma Ap endo activity with clinical response after alkylating agent-based chemotherapy or after radiotherapy. Experimental Design: Cox proportional hazards regression models were used to analyze the relationship of Ap endo activity with time to tumor progression (TTP). Results: In a univariate model with Ap endo activity entered as a continuous variable, the hazard ratio (HR) for progression after alkylator therapy in 30 grade III gliomas increased by a factor of 1.061 for every 0.01 increase in activity (P = 0.013). Adjusting for age, gender, extent of resection, and prior treatment strengthened slightly the association (HR = 1.094; P = 0.003). Similarly, the HR for progression after radiotherapy in 44 grade II and III tumors increased by a factor of 1.069 (P = 0.008). Adjusting for the aforementioned variables had little effect on the association. In contrast, we observed no association between activity and TTP in grade IV gliomas after either alkylator therapy in 34 tumors or radiotherapy in 26 tumors. Conclusions: Our data suggest that Ap endo activity mediates resistance to alkylating agents and radiation and may be a useful predictor of progression after adjuvant therapy in a subset of gliomas.

Alkylating agents are standard components of adjuvant chemotherapy for gliomas. We provide evidence here that Ape1/Ref-1, the major mammalian apurinic/apyrimidinic endonuclease (Ap endo), contributes to alkylating agent resistance in human glioma cells by incising DNA at abasic sites. We show that antisense oligonucleotides directed against Ape1/Ref-1 in SNB19, a human glioma cell line lacking O(6)-methylguanine-DNA-methyltransferase, mediate both reduction in Ape1/Ref-1 protein and Ap endo activity and concurrent reduction in resistance to methyl methanesulfonate and the clinical alkylators temozolomide and 1,3-(2-chloroethyl)-1-nitrosourea. An accompanying increase in the level of abasic sites indicates that the DNA repair activity of Ape1/Ref-1 contributes to resistance. Conversely, we also show that exposure of SNB19 cells to HOCl, a generator of reactive oxygen species (ROS), results in elevated Ape1/Ref-1 protein and Ap endo activity, enhanced alkylator resistance, and reduced levels of abasic sites. Given current evidence that heightened oxidative stress prevails within brain tumors, the finding that ROS increase resistance to clinical alkylators in glioma cells may have significance for the response of gliomas to alkylating agent-based chemotherapy. Our results may also be relevant to the design of therapeutic regimens using concurrent ionizing radiation (a generator of ROS) and alkylating agent-based chemotherapy.

Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers resistance to ionizing radiation and alkylating agents in human cell lines. The major Ap endo in human cells is Ape1, an abundant multi-functional protein also known as Ref-1, Hap-1, and Apex. In this work, we assayed Ap endo activity in human adult gliomas to establish correlates with tumor characteristics, and in histologically normal brain adjacent to tumors to characterize changes in activity accompanying neurocarcinogenesis. To our knowledge, this is the first available analysis of Ap endo activity in human brain tumors. Mean activity in 84 gliomas of different diagnostic types and grades was 0.072 +/- 0.095 fmol abasic sites incised/cell/min, ranging approximately 550-fold from 0.00077 to 0.42. The mean for high-grade gliomas was 3.5-fold greater than for low-grade tumors (P < or = 4.0 x 10(-5)), a difference observed within all diagnostic types. Activity was correlated with the fraction of S-phase cells in diploid gliomas (P < or = 0.02), suggesting that proliferation could be a determinant of activity in these tumors. Activity was also correlated with S-phase fraction in the majority of aneuploid gliomas (P < or = 0.03). Moreover, within the aneuploid tumors, there was a significant relationship between activity and the fraction of aneuploid cells (P < or = 4.0 x 10(-4)). In the 58 cases analyzed, mean activity was 7.3-fold higher in gliomas than in adjacent histologically normal brain (0.070 +/- 0.10 versus 0.0096 +/- 0.012 fmol/cell/min; P < or = 3.0 x 10(-5)). Increased tumor activity was observed in 93% of tumor/normal pairs, indicating that elevation of Ap endo activity is characteristic of human gliomagenesis. The elevation was large within most pairs, being 13-fold on average and > or = 10-fold in 43% of cases. A concomitant increase in Ape1 protein was observed by Western blotting in the subset of tumor/normal pairs examined. A clinically important consequence of the increase in Ap endo activity that accompanies neurocarcinogenesis may be enhanced resistance to the radiotherapy and alkylating agent-based chemotherapy that are mainstays of adjuvant therapy for malignant gliomas.

Phe(667) in the conserved O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) is known to be important for discrimination against dideoxy-NTPs. We show here that Phe(667) is also important for base selection fidelity. In a forward mutation assay at high polymerase concentration, wild type pol I catalyzed frequent A --> T and G --> T transversions and -1 frameshifts at nonreiterated sites involving loss of a purine immediately downstream of a pyrimidine, The mutants F667L and A661E,I665T, F667L exhibited large decreases in A --> T and G --> T transversions, and the triple mutant displayed reduction in the aforementioned -1 frameshifts as well. Kinetic analysis showed that the F667L and A661E,I665T, F667L polymerases discriminated against synthesis of A:A mispairs more effectively and catalyzed less extension of A:A mispairs than the wild type enzyme. These data indicate that Phe(667) functions in maintaining the error frequency and spectrum, and the catalytic efficiency, of wild type pol I. We also found that the strong general mutator activity conferred by the single A661E substitution was entirely suppressed in the A661E, I665T,F667L polymerase, exemplifying how interactions among O-helix residues can contribute to fidelity. We discuss the mutator and anti-mutator mutations in light of recently obtained three-dimensional structures of T, aquaticus pol I.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) contributes to the resistance of human brain tumor cell lines and xenografts to methylating and chloroethylating agents. We assayed MGMT in 174 newly diagnosed or recurrent gliomas to (a) quantitate changes in MGMT activity associated with alkylating agent-based chemotherapy; and (b) assess the contribution of MGMT to clinical outcome. Glioma MGMT activity ranged 300-fold, averaging 3,800+/-7,200 molecules/cell. Twenty-four percent of tumors lacked detectable activity [Methyl repair-deficient (Mer-) phenotype, defined here as <151 molecules/cell or <0.25 fmol/10(6) cells]. Tumors treated with surgery alone and tumors recurring after surgery and radiotherapy did not differ significantly in frequency of the Mer- phenotype (29% versus 24%). However, the frequency of the Mer- phenotype among tumors recurring after surgery, radiation, and alkylating agent-based chemotherapy was 7-fold lower than in tumors treated with surgery alone (4.3% versus 29%; P < or = 0.02) and 6-fold lower than in tumors recurring after surgery and radiation (4.3% versus 24%; P < or = 0.05). In contrast to gliomas, there was no relationship of alkylating agent-based therapy with the frequency of the Mer- phenotype in paired histologically normal brain. These data suggest that alkylating agents, either alone or synergistically with radiotherapy, selectively kill Mer- glioma cells in situ. Importantly, Mer- and Mer+ tumors did not differ in time to tumor progression following treatment with alkylating agents, indicating that although Mer- glioma cells may be differentially killed by alkylators, factors other than Mer phenotype were the principal determinants of time to clinical progression. Nonetheless, our results support the possibility that complete ablation of glioma MGMT with substrate analogue inhibitors could improve the efficacy of alkylating agent-based chemotherapy.

Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging. The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer 1 , 2 . In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture 3 . Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translations 4 and at the molecular level by multiple large deletions 5 . The Werner syndrome gene ( WRN ) has recently been cloned 6 . The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases. Such homology does not necessarily mean that WRN encodes an active helicase. For example, the Saccharomyces cerevisiae RAD26 gene protein 7 and the human transcription-repair coupling factor CSB (Cockayne syndrome B) 8 are highly homologous to known helicases, yet neither encodes an active helicase. Moreover, the Bloom's syndrome gene ( BLM )9, discovered before WRN , is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase. Here we report that the WS protein does indeed catalyze DNA unwinding.

Exposure to exogenous alkylating agents, particularly N-nitroso compounds, has been associated with increased incidence of primary human brain tumors, while intrinsic risk factors are currently unknown. The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a major defense against the carcinogenicity of N-nitroso compounds and other alkylators. We report here that in 55% (64/117) of cases, histologically normal brain tissue adjacent to primary human brain tumors lacked detectable MGMT activity [methyl excision repair-defective (Mer-) status]. The incidence of Mer- status in normal brain tissue from brain tumor patients was age-dependent, increasing from 21% in children 0.25-19 years of age to 75% in adults over 50. In contrast, Mer- status was found in 12% (5/43) of normal brain specimens from patients operated for conditions other than primary brain tumors and was not age-dependent. The 4.6-fold elevation in incidence of Mer- status in brain tumor patients is highly significant (chi2 = 24; p < or = 0.001). MGMT activity was independent of age in the lymphocytes of brain tumor patients and was present in lymphocytes from six of nine tumor patients whose normal brain specimen was Mer-. DNA polymerase beta, apurinic/apyrimidinic endonuclease, and lactate dehydrogenase activities were present in all specimens tested, including Mer- specimens from brain tumor patients. Our data are consistent with a model of carcinogenesis in human brain in which epigenetically regulated lack of MGMT is a predisposing factor and alkylation-related mutagenesis is a driving force.

We have analyzed the sensitivity of 14 human medulloblastoma- and glioma-derived cell lines to the clinically used methylating agents temozolomide and streptozotocin. The cell lines responded similarly to these agents, displaying a 3-fold range in cytotoxicity, assessed as the 10% survival dose (LD(10)). The contribution of O-6-methylguanine-DNA methyltransferase (MGMT) to resistance, measured as reduction in the LD(10) by O-6-benzylguanine (O-6-BG), varied among the lines by 1 order of magnitude for both agents. However, in all MGMT-expressing lines, O-6-BG eliminated a threshold dose that accounted for up to one-half of the LD(10). The effect of O-6-BG on the rate of killing varied 13-fold for temozolomide and 14-fold for streptozotocin. Some lines displayed two subpopulations with different rates of killing, with one subpopulation that comprised 20-60% of cells showing essentially no dependence of the rate of killing on MGMT, O-6-BG increased the range of the LD(10) for both agents, The persistent, heightened variability in cytotoxicity in the absence of MGMT, the lack of correlation between MGMT content of the lines and cytoxicity (LD(10)), and the lack of correlation between MGMT content and the contribution of MGMT to resistance (O-6-BG-mediated reduction of the LD(10)) reflect the operation of resistance mechanisms other than MGMT. We also analyzed sensitivity to methyl methanesulfonate, observing little dependence of resistance on MGMT and persistent variability in cytotoxicity in the presence of O-6-BG. We discuss the implications for clinical use of methylators and O-6-BG.