Flow cytometric measurement of glutathione content of human cancer biopsies

DW Hedley,AR Hallahan,EH Tripp

doi:10.1038/bjc.1990.14

Abstract

Rice et al. (1986) have described a flow cytometric method where the non-fluorescent probe monochlorobimane (mBCl) forms a fluorescent adduct with cellular glutathione (GSH) under the action of glutathione-S-transferase. We show here that for EMT6 carcinosarcoma cells there is a close correlation between mean cell fluorescence, expressed as a ratio to that of fluorescence calibration beads, and biochemically determined GSH content over the range 0.2-2.0 fmol cell-1. Single cell suspensions from 14 human cancers were prepared by 23-gauge needle aspiration or mechanical disaggregation of surgical specimens, stained using mBCl and examined by flow cytometry. There was a wide range in individual cell fluorescence, which in contrast to EMT6 cells was not strongly correlated with Coulter volume. By comparing tumour cell fluorescence to that of calibration beads, and assuming that the relationship with GSH content for EMT6 holds for other cells, a mean GSH content of 0.95 fmol cell-1 was derived for nine carcinomas, and 0.21 fmol cell-1 for five non-Hodgkin's lymphomas. Although this semi-quantitation needs further validation, the method used here is rapid, gives an indication of heterogeneity of tumour cell GSH content, and can be applied to fine needle biopsy samples. It therefore shows promise as a means for studying prospectively the relationship of GSH content to clinical drug and radiation sensitivity, and for monitoring the effects of agents such as buthionine sulphoximine which are intended to improve treatment results through tumour cell GSH depletion.

Highlights

Lonising radiation and a variety of clinically useful cytotoxic drugs produce reactive free radicals capable of interacting with essential cellular macromolecules, DNA
Shrieve et al (1988) have previously shown that when EMT6 cells are stained with 40 laM mBCI for 5 min, fluorescence intensity is closely correlated with biochemically determined GSH content, because the staining reaction is strongly catalysed by glutathione-Stransferase
We were able to confirm that > 99% of cytoplasmic fluorescence was of low molecular weight by sonicating EMT6 cells stained with mBCI under the above conditions, fractionating with a Sephadex G25 column, and measuring fluorescence using a spectrofluorimeter

Summary

Introduction

Lonising radiation and a variety of clinically useful cytotoxic drugs produce reactive free radicals capable of interacting with essential cellular macromolecules, DNA. The ubiquitous sulphydryl-containing tripeptide glutathione can protect from this damage by scavenging free radicals, either spontaneously or when catalysed by one of the glutathione-S-transferase isoenzymes. Glutathione is an abundant molecule in cells, cytoplasmic concentrations being typically in the millimolar range, and there is increasing evidence that elevated tumour cell GSH or glutathione-S-transferase activity could be an important cause of radiation and alkylating agent resistance in cancer patients. Results show considerable heterogeneity in cell fluorescence, with carcinomas having significantly higher mean values than lymphomas This method is probably dependent on a number of enzyme activities in addition to cell GSH content, preliminary data are presented to suggest that the FCM method can be made semi-quantitative by reference to a standard biochemical assay of GSH

Methods

Results

Conclusion