GLUTATHIONE S-TRANSFERASES AND P-GLYCOPROTEIN IN NORMAL RAT HEPATOCYTES AND HEPATOMA-CELLS - ANALYSIS USING FLOW-CYTOMETRY

Using indirect immunofluorescence with fluorescein isothiocyanate-conj ugated antibodies, in combination with flow cytometry (FCM), we have d eveloped a technique to detect the alpha, mu and pi isozymes of GST in cell suspensions from normal rat liver, and in H4IIE cells, a rat hep atoma cell line. Cell suspensions fixed in 1% paraformaldehyde were ob served to require cell membrane permeation with lysolecithin to allow access and binding of antibodies to immunoreactive proteins within the cytoplasm. FCM analysis indicated normal rat hepatocytes to be positi ve for GST alpha and mu, but not GST pi, and the H4IIE cells to be pos itive for all three GST isozymes. Further analysis by FCM for the expr ession of P-glycoprotein (mdr), a membrane-associated protein product of the multidrug resistance gene, showed an association between the pr esence of GST pi and mdr in the two cell types. Thus, mdr was detected in significant amounts in H4IIE cells, but not in rat hepatocytes. Th e method described here has potential applications in screening, sorti ng and further characterisation for GST pi-positive hepatocytes for me chanistic studies during sequential rat liver carcinogenesis, as well as for characterisation of human tumors for the expression of differen t GST isozymes and P-glycoprotein during therapeutic management.