06-Methylguanine-DNA Methyltransferase Activity in Adult ( Â ¿ liornas : Relation to Patient and Tumor Characteristics 1

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 Â± 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 Â± 7.6 versus 1.1 Â±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