JC-1, a sensitive probe for a simultaneous detection of P-glycoprotein activity and apoptosis in leukemic cells.
BACKGROUND
JC-1 probe has been successfully used for the analysis of either apoptosis or P-glycoprotein (P-gp) activity. Therefore, we wanted to see if JC-1 could also simultaneously assess both, P-gp activity and apoptosis, in acute myeloid leukemia (AML) cells.


METHODS
P-gp activity was measured using JC-1 and compared to the results of the Rhodamine 123 (Rh 123) assay in P-gp negative and P-gp positive cell lines, and 12 AML samples. For apoptosis, spontaneous apoptosis, as well as, apoptosis induced by Cytosine Arabinosine and Homoharringtonine were analyzed. Both mitochondrial red fluorescence and cytoplasmic green fluorescence of JC-1 with and without a P-gp inhibitor (Cyclosporine A : CsA) were used for the identification of apoptotic cells, and this was compared to Annexin V/PI staining.


RESULTS
(1) We found a good correlation between JC-1 and Rh 123 in viable cells. Even in a small population of viable cells, P-gp positive cells emitting low red fluorescence, gained on red fluorescence after P-gp inhibition with CsA permitting an evaluation of P-gp activity. (2) We found a good correlation between the Annexin V/PI staining and JC-1 (P < 0.0001) in the assessment of apoptotic cells. Most importantly, the apoptotic cells could be distinguished by the loss of red fluorescence and the increase of green fluorescence without any change after P-gp inhibition with CsA.


CONCLUSIONS
JC-1 can simultaneously evaluate two important parameters involved in drug resistance in AML cells, P-gp activity and apoptosis.
Université Pierre et Marie Curie-Paris 6, UMR S 736, Les Cordeliers, France.
Driss
D
Chaoui
Anne-Marie
AM
Faussat
Patricia
P
Majdak
Ruoping
R
Tang
Jean-Yves
JY
Perrot
Sabine
S
Pasco
Christophe
C
Klein
Jean-Pierre
JP
Marie
Ollivier
O
Legrand
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D000208:Acute Disease; D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis; D001562:Benzimidazoles / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry*; D045744:Cell Line, Tumor; D016572:Cyclosporine / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007951:Leukemia, Myeloid / Q000378:metabolism / Q000473:pathology*; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects; D011419:Propidium / Q000737:chemistry; D020112:Rhodamine 123 / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017304:Annexin A5; D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D020112:Rhodamine 123; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D011419:Propidium; D016572:Cyclosporine