O 6-Methylguanine-DNAMethyltransferase , O 6-Benzylguanine , and Resistance to Clinical Alkylators in Pediatric Primary BrainTumor Cell Lines

Purpose:Primarybraintumors are the leadingcauseofcancerdeathinchildren.Ourpurpose is (a) to assess the contribution of the DNA repair protein O-methylguanine-DNAmethyltransferase (MGMT) to the resistance of pediatric brain tumor cell lines to clinical alkylating agents and (b) to evaluate variables formaximalpotentiationof cell killingby theMGMTinhibitorO-benzylguanine, currently inclinical trials.Fewsuchdata forpediatricgliomalines,particularly those fromlow-grade tumors, are currentlyavailable. Experimental design:Weusedclonogenicassaysofproliferativesurvival toquantitatecytoxicity of the chloroethylating agent1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the methylating agent temozolomide in11gliomaand fivemedulloblastomalines.Twelve lines arenewlyestablished andcharacterizedhere, nineof themfromlow-gradegliomas includingpilocytic astrocytomas. Results: (a)MGMT is amajor determinant of BCNUresistance and the predominant determinant of temozolomide resistance in both our glioma and medulloblastoma lines. On average, Obenzylguanine reduced LD10 for BCNU and temozolomide, 2.6and 26-fold, respectively, in 15 MGMT-expressing lines. (b) O-Benzylguanine reduced DT (the threshold dose for killing) for BCNU and temozolomide, 3.3and 138-fold, respectively. DT was decreased from levels higher than, to levels below, clinically achievable plasma doses for both alkylators. (c) Maximal potentiation by O-benzylguanine required complete and prolonged suppression of MGMT. Conclusions:Our results support theuseofO-benzylguanine toachieve fullbenefitofalkylating agents,particularly temozolomide, in the chemotherapyofpediatricbrain tumors. Primary brain tumors are the most common solid malignancy of childhood, with 2,200 new cases diagnosed annually (1). The majority (60-70%) are gliomas (astrocytomas, oligodendrogliomas, and ependymomas) histologically similar to those found in adults (2). The remainder consists of diagnostic types uncommon in adults, including medulloblastoma, primitive neuroectodermal tumors, and mixed neuronal-glial tumors (1, 2). Whereas adult tumors occur predominantly in the cerebral hemispheres, half of pediatric cases occur in the cerebellum and brain stem (2). The contemporary standard of care for malignant and many subtotally resected low-grade pediatric brain tumors includes post-operative radiation therapy and/or multiagent chemotherapy. This treatment strategy has produced dramatic increases in 5-year survival rates for the majority of medulloblastomas (3) but has been less effective in improving the prognosis for malignant gliomas and for tumors in infants and young children. In addition, there are no effective therapies for most tumors that recur after previous radiation and chemotherapy. Thus, primary brain tumors are the leading cause of cancer death in children, the overall 5-year survival rate being 50% (1). Chloroethylating agents [e.g., 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] and methylating agents (e.g., temozolomide, procarbazine), used in single-agent or combination regimens, are key components in the chemotherapy of pediatric brain tumors (e.g., refs. 4, 5). Among the most effective drugs for treatment of primary brain tumors, these agents produce a diversity of alkyl adducts in DNA (6–8). Their cytotoxicity has been definitively associated with alkylation at the O atom of guanine (6, 9). Chloroethylating agents introduce O-chloroethylguanine, a precursor of the lethal interstrand cross-link 1-(3-cytosinyl), 2-(1-guanyl)ethane, whereas methylating agents introduce the monoadduct Omethylguanine. Current evidence indicates that persistent interstrand cross-links and O-methylguanine impede DNA replication, resulting in lethal double-strand breaks at collapsed replication forks (10). www.aacrjournals.org Clin Cancer Res 2005;11(7) April 1, 2005 2747 Authors’Affiliations: Division of Neurosurgery; Departments of Surgery and Hematology/Oncology Children’s Hospital and Regional Medical Center, Seattle, Washington; Departments of Neurological Surgery, Pediatrics and Pathology University ofWashington, Seattle,Washington Received10/6/04; revised12/13/04; accepted12/30/04. Grant support: American Cancer Society grants RPG-97-019 CN and RSG 0119101CCE and NIH grants CA70790, CA71937, CA80993, and CA82622. 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 with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Michael Bobola, Division of Neurosurgery, Department of Surgery, Children’s Hospital and Regional Medical Center, Seattle, WA 98105. Phone: 206-987-2046; Fax: 206-987-7311; E-mail: michael.bobola@ seattlechildrens.org. F2005 American Association for Cancer Research. CancerTherapy: Preclinical Research. on April 12, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from A large body of work with brain tumor-derived cell lines and xenografts has shown that the DNA repair protein O-methylguanine-DNA methyltransferase (MGMT) contributes to alkylating agent resistance (e.g., refs. 11–16). MGMT catalyzes the transfer of simple, branched, and halogenated alkyl groups from the O position of guanine in double-stranded DNA to an internal cysteine, yielding guanine and S-alkylcysteine (17). Because the alkyl receptor site is not regenerated, the number of O-alkylguanine adducts that can be removed from DNA in vivo is limited by the number of MGMT molecules and the rate of synthesis of the protein. The majority of adult (18, 19) and pediatric (20) primary brain tumors express MGMT activity. Activity in tumors is elevated 2to >500-fold relative to adjacent normal brain in f65% of adult and pediatric cases (18–20), raising the possibility that MGMT contributes to tumor alkylator resistance in vivo. In accord, low MGMT content, assessed by immunohistochemistry or inferred from the methylation status of the MGMT promoter, has been associated with better clinical outcome following alkylating agent-based chemotherapy in adult gliomas (reviewed in ref. 21). Ablation of MGMT activity with the substrate analogue inhibitor O-benzylguanine (21, 22) enhances the cytotoxicity of methylating and chloroethylating agents in primary brain tumor cell lines (e.g., refs. 11–14) and xenografts (e.g., refs. 15, 16). These preclinical studies are central to the development of treatment regimens to evaluate the efficacy O-benzylguanine in improving response to alkylator-based chemotherapy in adult gliomas (21, 22) and in pediatric brain tumors (e.g., PBTC-005; http://www.cancer.gov). Extensive studies with adult glioma lines have revealed both heterogeneity in benefit conferred by O-benzylguanine, and a requirement for prolonged incubation with O-benzylguanine after alkylator exposure to realize maximal potentiation of cell killing. Comparable evaluation of the efficacy of O-benzylguanine in suppressing alkylator resistance in pediatric brain tumor cells has been less thorough due partly to a lack of cell lines, especially those derived from gliomas. Here we examine the contribution of MGMT to BCNU and temozolomide resistance in 16 pediatric brain tumor-derived cell lines, including 12 lines that we have newly characterized. Nine of the new lines were derived from low-grade gliomas for which there is a paucity of data on alkylator sensitivity and response to O-benzylguanine. Our results show that MGMT is a major determinant of BCNU resistance and the predominant determinant of temozolomide resistance in cultured pediatric glioma and medulloblastoma cells. The data also show that maximal suppression of resistance to both agents requires ablation of MGMT activity with Obenzylguanine not only before but also for a prolonged period after alkylator exposure. Our results support the use of Obenzylguanine to achieve full benefit of alkylating agent chemotherapy for pediatric brain tumors. Materials andMethods Establishment and characterization of cell lines. Tumors and demographic information were obtained from informed patients according to protocols approved by the Institutional Review Board at Children’s Hospital and Regional Medical Center. Diagnosis was obtained from the final neuropathology report. The 12 new cell lines were established as previously described with minor modifications (23). Briefly, tumors were transported from the operating room in icecold DMEM/F12 containing 5% iron-supplemented bovine serum, 100 units/mL penicillin, 100 Ag/mL streptomycin, and 0.25 Ag/mL amphotericin B. After removing large blood vessels and necrotic material, specimens were washed repeatedly with sterile, ice-cold PBS. The tissue was minced with scalpel blades in supplemented medium and serially passed through 18-, 20-, and 22-gauge needles to produce a single cell suspension, as verified by microscopic examination. The cells were pelleted by centrifugation at 800 g and resuspended in 10 mL of 17 mmol/L Tris-HCl (pH 7.2), 140 mmol/L NH4Cl. After incubation at 37jC for 10 minutes to lyse erythrocytes, the cells were washed with PBS. The washed cell pellet was resuspended in PBS and viable cell titer was determined by trypan blue exclusion using a hemacytometer. Supplemented medium was inoculated with z 2 10 viable cells, and cultures were incubated at 37jC in 5% CO2/95% humidified air. Proliferation of tumor cells was evident within 10 to 21 days as foci of overgrown cells. The establishment, phenotypic characterization, and contribution of MGMT to alkylator resistance have been previously reported for the medulloblastoma lines UW228-1, UW228-2, and UW228-3 and the glioma line UW467 (11–13, 23). All lines proliferate as adherent monolayers that have been maintained in continuous culture for >50 to 200 passages and readily form colonies (20-30% plating efficiency). The lines do not display contact inhibition