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The Hypersensitivity of the Chinese Hamster Ovary Variant BL-10 to Bleomycin Killing Is Due to a Lack of Glutathione S-Transferase- Activity¹

Department of Radiation Oncology [A. J. G., A. C., J. W. E., J. M. B.] and Department of Medicine, Division of Oncology [A. D. L.], Stanford University School of Medicine, Stanford, California 94305; Department of Molecular Genetics, University of Cincinnati School of Medicine, Cincinnati, Ohio, 452667 [N. C. D.]; Lankeau Medical Research Center, Lancaster, Philadelphia, Pennsylvania 19151 [T. D. S.]; and Department of Radiation Biology, Fort Collins, Colorado 80523 [C. W.]

As a means to understand the fundamental mechanisms of bleomycin cell killing, we previously isolated 19 bleomycin-sensitive mutants which represent at least six genetically distinct complementation groups (T. D. Stamato, B. Peters, P. Patil, N. Denko, R. Weinstein, and A. Giaccia. Cancer Res., 47: 1588–1592, 1987). One class of mutants represented by the cell line BL-10 displays only hypersensitivity to killing by bleomycin in both acute (16 h) and chronic treatments but no sensitivity to killing by other DNA-damaging agents. Complementation studies between this mutant and human fibroblasts suggested that the human gene which corrects the defect of BL-10 rested on human chromosome 6. It has been reported that the gene for human glutathione S-transferase (GST) also resides on chromosome 6. Measurements of selenium-independent peroxidase (-GST + glutathione peroxidase) activity in wild-type Chinese hamster ovary (CHO) cells, using cumene hydrogen peroxide as a substrate, gave a value of 112 nmol of glutathione oxidized/min/mg protein compared with 88.1 nmol of glutathione oxidized/min/mg protein for BL-10. Measurement of the selenium-dependent peroxidase activity, using H₂O₂ as a substrate, resulted in 65.9 nmol of reduced glutathione oxidized/min/mg protein in CHO and 81.5 nmol of reduced glutathione oxidized/min/mg protein for BL-10. In other words, BL-10 cells did not exhibit a difference in their ability to metabolize both substrates in contrast to CHO cells. This indicates that BL-10 possesses little -GST activity. Transfection of BL-10 cells with a mammalian expression vector containing the -GST gene increases the survival of BL-10 to bleomycin and does not increase the bleomycin resistance of two other bleomycin mutants which lie in different genetic complementation groups. These data strongly implicate a role for -GST in the resistance of cells to bleomycin.

¹ This work was supported by U.S.P.H.S. Grant CA 15201 from the National Cancer Institute, Department of Health and Human Services (to J. M. B.), Grant CA 45277 (to T. D. S.), Grant CA 36447 (to C. A. W.), and a grant supported by funds from the Cigarette and Tobacco tax fund of the state of California through the Tobacco-related Disease Research program of the University of California (to A. D. L.).

² To whom reprint requests should be addressed, at CBRL, GK 115, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305-5468.

Received 2/11/91. Accepted 6/ 5/91.

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