Flow cytometric detection of glutathione S-transferase isoenzymes by quantitative immunofluorescence under nonsaturating conditions.
The glutathione (GSH)-glutathione S-transferase (GST) detoxification system is an important element in cellular defence against injurious agents and anticancer drugs. GST isoenzymes may represent biochemical markers of neoplastic transformation, and, possibly, drug resistance is associated with altered GST-isoenzyme levels. The ability to measure GST-isoenzymes in cell populations would be useful for several biological and clinical applications. We have developed an immunofluorescence flow cytometric method for the simultaneous detection of different GST-isoenzymes and of DNA in fixed cells. Due to the impossibility of working under saturating conditions for the anti-GST antibody, a normalizing procedure was developed to permit quantitative analysis of single cells labelled with the anti-GST antibody at high dilution. A theoretical model and experimental data supported the use of this procedure. The method proposed is general and could be applied to other antibodies in order to obtain quantitative data outside saturating conditions. The method was challenged in different applications in order to compare it with other classical techniques. First, we characterized sublines resistant to different anticancer drugs with respect to variations of GST isotypes. In a second application, we studied the intercellular heterogeneity of GST content in mouse renal cells. In addition, GST was determined in aneuploid cells from solid tumor biopsies by separation from diploid cells on the basis of DNA content. Finally, GST distribution during cell-cycle progression was studied in two different cell lines by the biparametric analysis of GST/DNA.
Pifferi
A
A
Politecnico di Milano, Dipartimento di Fisica, Italy.
Filippini
C
C
Capolongo
L
L
Codegoni
A
A
Balconi
G
G
Ubezio
P
P
Tagliabue
G
G
D000818:Animals; D001699:Biometry; D004247:DNA / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000032:analysis*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000032:analysis*; D007939:Leukemia L1210 / Q000201:enzymology; D008214:Lymphocytes / Q000737:chemistry / Q000201:enzymology; D051379:Mice; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D007527:Isoenzymes; D004247:DNA; D005982:Glutathione Transferase