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Denitrosation of 1,3-Bis(2-chloroethyl)-1-nitrosourea by Class Mu Glutathione Transferases and Its Role in Cellular Resistance in Rat Brain Tumor Cells¹

Department of Biomedical and Environmental Health Sciences, School of Public Health, University of California, Berkeley, California 94720 [M. T. S., C. G. E., P. D-S.]; and the Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-10691, Stockholm, Sweden [V. M. C., M. K. T., B. M.]

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is known to be detoxified by a denitrosation reaction catalyzed by glutathione-dependent enzymes in rat liver cytosol (R. E. Talcott and V. A. Levin, Drug Metab. Dispos., 11: 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 isoenzymes for the denitrosation reaction expressed as the ratio of V_max to K_m were as follows (isoenzyme, V_max/K_m): 1-2, 2.3; 3-3, 12.2; 3-4, 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 immunoblotting 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.

¹ This research was supported by Grant P01-CA13525 from the National Cancer Institute, the National Foundation for Cancer Research, the Swedish Natural Science Research Council, and the Swedish Cancer Society.

² To whom correspondence should be addressed, at School of Public Health, 322 Warren Hall, University of California, Berkeley, California 94720.

Received 7/ 6/88. Revised 12/22/88. Accepted 2/ 3/89.

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