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J. Biol. Chem., Vol. 263, Issue 28, 14107-14114, 10, 1988
DC Shrieve, EA Bump and GC Rice
Monochlorobimane (syn-(ClCH2, CH3)-1,5-diazabicyclo-[3.3.0]-octa-3,6-
dione-2,8-dione; mBCl) forms a fluorescent adduct with glutathione (GSH),
which has been used as a basis for flow cytometric analysis. While mBCl
will react nonspecifically with many different thiols, preferential
derivatization of GSH can be achieved by using a low concentration of mBCl,
since the reaction with GSH is catalyzed by GSH S-transferase, and the
nonenzymatic reaction is very slow (k = 3.3 x 10(-1) M-1 s-1 at 37 degrees
C, pH 7.5). The rate of derivatization of cellular GSH can be 1000 times
greater than predicted from the nonenzymatic reaction rate, although this
factor can vary among cell lines. GSH values obtained by flow cytometry
(FCM) agree well with those obtained by an enzymatic assay, over a wide
range of GSH values, for EMT6/SF cells treated with L-buthionine
sulfoximine to vary GSH content. FCM analysis of the GSH content of cells
obtained by disaggregation of EMT6/SF tumors, grown in BALB/c mice,
revealed a wide variation in single-cell GSH content. The data suggest that
there are distinct subpopulations within these tumors, which can be
partially characterized by GSH content, but may also have other
distinguishing characteristics, such as enhanced sensitivity or resistance
to cytotoxic agents. Heterogeneity in single-cell GSH content was also
observed by FCM analysis of cells obtained by disaggregation of a biopsy of
a human renal cell carcinoma. This result points to the potential value of
FCM analysis of GSH in the identification and characterization of human
tumor subpopulations which may be of clinical significance in the treatment
of cancer by radiation or chemotherapeutic agents.
Heterogeneity of cellular glutathione among cells derived from a murine fibrosarcoma or a human renal cell carcinoma detected by flow cytometric analysis
Department of Radiation Oncology, University of California, San Francisco 94143.
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