Flow cytometric measurement of glutathione content of human cancer biopsies

Abstract

Summary 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' . Single cell suspensions from 14 human cancers were prepared by 23-gauge needle aspiration or mechanical disaggregation of surgical specimens, stained using mBCI 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 (Ozols et al., 1987;Lee et al., 1987;Skapek et al., 1988 Clinical samples A total of 14 clinical samples were examined. Seven of these were obtained by 23 gauge needle aspiration from patients who gave verbal informed consent for the procedure, one consisted of peripheral blood lymphoblasts separated by density-gradient centrifugation from a patient with end-stage T-cell acute lymphoblastic leukaemia, and the remainder were fresh surgical specimens which were mechanically disaggregated using crossed scalpel blades. Cell suspensions were filtered through gauze, washed and counted. If necessary, clumps were dispersed by syringing through 25 gauge needles. After adjusting to a final concentration of I x 106ml-', cells were filtered through 70 tm plankton netting and stained using 40 JiM mBCI for exactly 5 min at 2O°C. Flow cytometry was done exactly as for cultured cells, except that the machine was triggered on Coulter volume, set at a threshold level of 300 jtm3 to exclude red blood cells and cellular debris. In addition to fluorescence, the ratio of cell fluorescence to Coulter volume was obtained using the electronics of the FACS Analyser. Results Monochlorobimane staining of EMT6 CellsThe effects of mBCI concentration and time on the staining intensity of EMT6 cells are shown in Figure 1

Monochlorobimane Does Not Selectively Label Glutathione in Peripheral Blood Mononuclear Cells

“…G S H is therefore critical for cellular viability and is generally found in high concentrations (1-10 m M ) in anim al cells. Various pathological states have been associated with low G S H levels including malignancies (1,2), idiopathic pulm onary fibrosis (3), and the ac quired immunodeficiency syndrome (4)(5)(6)(7)(8). G S H m etabo lism has been extensively reviewed (9,10).…”

Novel use of the fluorescent dye 5‐(and‐6)‐chloromethyl SNARF‐1 acetate for the measurement of intracellular glutathione in leukemic cells and primary lymphocytes

“…As discussed earlier, a number of deficiencies exist in the current techniques to rapidly and easily measure GSH levels, particularly on whole cells. One of the flow cytometric methods previously described is based on the conjugation of the fluorescent compound monochlorobimane (mBCl) to GSH catalyzed by GST (27, 28). It was previously assumed that the rate of binding of mBCl to GSH is enhanced up to 1,000‐fold by GST, and that this would allow the use of mBCl concentrations too low to cause nonsignificant binding to other cell molecules containing sulphydryl groups.…”

The Therapeutic Potential of Gallium-Based Complexes in Anti-Tumor Drug Design

“…Upon treatment of the cell lysates with a source of iron, an increased concentration of the metal will replace the gallium center in the protein, thus restoring the ESR signal [31,40]. Consistent with gallium induced reduction of dNTP pools, an S phase blockage was demonstrated in human leukemic cells [29] and a G 2 /M block was observed in bladder carcinoma [42], demonstrating its effects on the cell cycle.…”

“…Gallium, unlike iron, is redox inactive. This property prevents gallium insertion to certain proteins involved with oxygen transport and precludes participation in other redox reactions of biological relevance [29]. However, gallium is able to bind to proteins that require the trivalent form of iron, therefore disrupting normal cellular homeostasis.…”