[CANCER RESEARCH 49, 2621-2625, May 15, 1989] Denitrosation of l,3-Bis(2-chloroethyl)-l-nitrosourea by Class Mu Glutathione Transferases and Its Role in Cellular Resistance in Rat Brain Tumor Cells1 Martyn T. Smith,2 Celia G. Evans, Penelope Doane-Setzer, Victor M. Castro, M. Kaum Tahir, and Bengt Mannervik Department of Biomédicaland Environmental Health Sciences, School of Public Health, University of California, Berkeley, California 94720 [M. T. S., C. G. E., P. D-SJ; and the Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-1069I, Stockholm, Sweden ¡V.M. C., M. K. T., B. M.] ABSTRACT l,3-Bis(2-chloroethyl)-l-nitrosourea (BCNU) is known to be detoxi fied by a denitrosation reaction catalyzed by glutathione-dependent en zymes in rat liver cytosol (R. E. Talcott and V. A. Levin, Drug Metab. Dispos., //: 175-176, 1983). Using a modification of their procedure, we have measured the ability of different purified rat glutathione transferase isoenzymes to denitrosate BCNU. The catalytic efficiencies of the iso- enzymes for the denitrosation reaction expressed as the ratio of („,„ to Km were as follows (isoenzyme, VmJKm): 1-2, 2.3; 3-3, 12.2; 3^1, 29.2; and 4-4, 26.1. Thus, the class mu isoenzymes containing subunit 4 are by far the best catalysts of the BCNU denitrosation reaction. The class pi transferase 7-7 and class alpha transferases 1-1 and 1-2 demonstrated very weak catalytic activity with BCNU. Determination of the glutathione transferase isoenzyme profiles of 9L rat brain tumor cells and the BCNU-resistant 9L-2 subline by immuno- blotting revealed that although the resistant 9L-2 cells contain lower total glutathione transferase activity than 9L cells, they have elevated levels of the class mu transferases. Also, the class pi transferases were found to be down-regulated in 9L-2 as compared with 9L cells. Thus, the increased resistance of 9L-2 cells to BCNU may, in part, be explained by up-regulation of class mu transferase expression with consequent increased capacity for BCNU detoxication. Further support for this hypothesis comes from the fact that pretreatment of 9L-2 cells with the glutathione transferase inhibitors ethacrynic acid or triphenyltin chloride enhanced the cytotoxic effects of BCNU. These results suggest that the class mu transferases play a role in the resistance of brain tumor cells to BCNU. INTRODUCTION BCNU3 remains the chemotherapeutic agent of choice for human brain tumors. More than 60% of brain tumors do, however, show insensitivity to BCNU (1,2) and often acquire resistance during therapy. Tumor cell resistance is therefore a major cause of treatment failure, as it is for many other anti- tumor agents (3). The cellular capacity to repair O6-alkylguan- ine adducts in DNA has been shown to be a major mechanism of resistance to BCNU (4-6), but additional mechanisms are likely. One such mechanism may be increased drug inactivation in resistant cells. BCNU can be inactivated by a denitrosation reaction that is catalyzed by the cytochrome P-450 monooxygenase system (7) as well as the glutathione transferases (8, 9). The latter are generally much more prevalent in tumor cells than cytochrome P-450 and we therefore postulated that BCNU-resistant cells may have higher levels of glutathione transferase. Surprisingly, we found that the BCNU-resistant 9L-2 subline of the rat 9L gliosarcoma cell line actually had less bulk glutathione trans ferase activity than the sensitive parent line (10). The glutathi- Received 7/6/88; revised 12/22/88; accepted 2/3/89. 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. 'This research was supported by Grant POI -CAI 3525 from the National Cancer Institute, the National Foundation for Cancer Research, the Swedish Natural Science Research Council, and the Swedish Cancer Society. 2To whom correspondence should be addressed, at School of Public Health, 322 Warren Hall. University of California, Berkeley, California 94720. 'The abbreviation used is: BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea. one transferases are, however, a family of different isoenzymes with widely differing substrate specificities (11, 12). More than 10 different rat isoenzymes made up of eight different subunits (numbered 1 through 8) as homo- and heterodimers have been characterized and divided into three classes (alpha, 1-1, 1-2, 2-2, 8-8; mu, 3-3, 3-4, 3-6, 4-4, 4-6, 6-6; and pi, 7-7) (11, 12). The possibility therefore existed that only one form or class of the transferases catalyzed the denitrosation of BCNU and that this form(s) was elevated in resistant cells, even though bulk transferase activity was lower. The experiments described here were performed to test this hypothesis and demonstrate that the class mu glutathione transferases have a high catalytic activity with BCNU and are elevated in BCNU-resistant 9L-2 cells. MATERIALS AND METHODS Chemicals. BCNU was obtained from the Drug Synthesis and Chem istry Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). Ethacrynic acid and triphenyltin chloride were from Sigma Chemical Co., St. Louis, MO. All other reagents were of the highest grade available. Isolation and Purification of Glutathione Transferase Isoenzymes and Antisera. Pure class alpha and mu transferases were prepared in ho mogeneous form from male Sprague-Dawley rat liver essentially as described in (13). Class pi glutathione transferase (7-7) (14) was iso lated using the same technique but with rat hepatoma cells or small intestine as the source. Polyclonal antisera to the different glutathione transferases were raised in rabbits by conventional procedures (13). Assay of BCNU Denitrosation. The formation of nitrite was used as an index of BCNU denitrosation, essentially as described by Talcott and Levin (9). Incubations were conducted in duplicate, at 37°C in 50- ml Erlenmeyer flasks, using 5 ml of phosphate buffer (pH 7.4), 5 mM glutathione, and 250-500 n%/m\ isoenzyme protein. BCNU was added to start the reaction (final concentration, 2.0 mM). One-mi samples were withdrawn at various time points from 15 s to 20 min after BCNU addition. Each sample was extracted twice with 0.5-1.0 ml chloroform to remove intact BCNU. One-hundred-eighty n\ of each extracted aqueous sample was assayed spectrophotometrically for nitrite forma tion according to a modified Bratton-Marshall procedure (9). Additional experiments were performed to obtain kinetic values (i.e., Km, ymn) for each isoenzyme. Incubations were conducted as above, at 37°Cat pH 6.5 for 10 min, with varying substrate concentration (0.4- 2.0 mM BCNU). The resulting Fmaxand Km values were determined from Woolf-Augustinsson-Hofstee plots of the data. Cell Cultures. The 9L and 9L-2 rat gliosarcoma cell lines have been used extensively for the evaluation of chemotherapeutic agents (15) and have well-characterized properties (4, 16). The cell lines were grown to confluence in 75-cm2 tissue culture flasks in Dulbecco's modified Ea gles' medium containing 1 g/liter glucose, 10% fetal calf serum, and 50 Mg/ml gentamicin under 5% CO2/95% air at 37°C. Colony Forming Assay. To determine cytotoxicity, a colony forming efficiency assay was performed essentially as described in (10). Briefly, 50 to 100,000 cells were plated into Flow 4-well plates in medium consisting of 60% fresh medium and 40% medium conditioned by log- phase cells for 2 days prior to filtration. After approximately 18 h of incubation, cells were treated for 15 min with 0.2 mM ethacrynic acid, 0.75 /¿M triphenyltin chloride, or a vehicle control. All cells were then treated for 2 h with various concentrations of BCNU followed by medium replacement. 2621 Research. on February 3, 2015. © 1989 American Association for Cancer cancerres.aacrjournals.org Downloaded from