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Quantitative Analysis of Cellular Glutathione by Flow Cytometry Utilizing Monochlorobimane: Some Applications to Radiation and Drug Resistance in Vitro and in Vivo¹

Department of Radiology, Cancer Biology Research Laboratory, Stanford University, Stanford, California 94305 [G. C. R., M. K.]; Joint Center for Radiation Therapy, Harvard Medical School, Boston, Massachusetts 02115 [E. A. B.]; Department of Radiation Oncology, Radiation Oncology Research Lab, CED-200, University of California, San Francisco, California 94143 [D. C. S.]; and SRI International, Menlo Park, California 94025 [W. L.]

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(CICH₂CH₃)-1,5-diazabicycla[3.30]acta-3,6,-diene-2,8-dione], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor cell population with respect to GSH content per cell.

¹ This work was supported in part by NIH grants CA 40324, Ca 32827, CA 42391, CA 38847, and Contract NO1-CM17485 from the National Cancer Institute, Department of Health and Human Services.

Received 4/18/86. Revised 8/ 8/86. Accepted 8/14/86.

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