Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.
Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.
Equipe de Cytologie Quantitative, Institut Albert Bonniot, La Tronche, France.
J
J
Boutonnat
T
T
Bonnefoix
M
M
Mousseau
D
D
Seigneurin
X
X
Ronot
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D018528:ATP-Binding Cassette Transporters / Q000032:analysis*; D000911:Antibodies, Monoclonal; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D018432:Drug Resistance, Multiple / Q000502:physiology*; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy / Q000378:metabolism; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins / Q000032:analysis*; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein