FLOW CYTOMETRIC DETECTION OF GLUTATHIONE-S-TRANSFERASE ISOENZYMES BY QUANTITATIVE IMMUNOFLUORESCENCE UNDER NONSATURATING CONDITIONS

The glutathione (GSH)-glutathione S-transferase (GST) detoxification s ystem is an important element in cellular defence against injurious ag ents and anticancer drugs, GST isoenzymes may represent biochemical-ma rkers of neoplastic transformation, and, possibly, drug resistance is associated with altered GST-isoenzyme levels, The ability to measure G ST-isoenzymes in cell populations would be useful for several biologic al and clinical applications, We have developed an immunofluorescence now cytometric method for the simultaneous detection of different GST- isoenzymes and of DNA in fixed cells, Due to the impossibility of work ing under saturating conditions for the anti-GST antibody, a normalizi ng procedure was developed to permit quantitative analysis of single c ells labelled with the anti-GST antibody at high dilution, A theoretic al model and experimental data supported the use of this procedure, Th e method proposed is general and could be applied to other antibodies in order to obtain quantitative data outside saturating conditions, Th e method was challenged in different applications in order to compare it with other classical techniques, First, we characterized sublines r esistant to different anticancer drugs with respect to variations of G ST isotypes, In a second application, we studied the intercellular het erogeneity of GST content in mouse renal cells. In addition, GST was d etermined in aneuploid cells from solid tumor biopsies by separation f rom diploid cells on the basis of DNA content, Finally, GST distributi on during cell-cycle progression was studied in two different cell lin es by the biparametric analysis of GST/DNA. (C) 1995 Wiley-Liss, Inc.