Acute myeloid leukemia cells with low P-glycoprotein expression and high Rhodamine 123 efflux capacity display high clonogenicity

This study was designed to correlate the clonogenic capacity of acute myeloid leukemia (AML) cells with P-glycoprotein (P-gp) expression level and P-gp-mediated efflux capacity. Fifty AML cell samples were tested far P-gp expression using MRK16 monoclonal antibody and flow cytometry. Among them, 12 samples were selected for sorting experiments according to the following two criteria: their clonogenic capacity in methyl-cellulose in the presence of 5637 conditioned medium, and the heterogeneity of P-gp distribution in leukemic cells. For each of these 12 samples, leukemic cells which displayed the highest P-gp expression level (P-gp(++)) and P-gp(-) leukemic cells were sorted after MRK16 staining and seeded into methylcellulose for primary clonogenic assay. In each case, the number of CFU-L in the P-gp(-) fraction was significantly higher than that of the P-gp(++) fraction (P < 0.01); the median number of CFU-L for 10(5) seeded cells being 147 (range 3-1855) and 495 (range 60-4100) for P-gp(++) and P-gp(-) populations, respectively. Furthermore, in order to correlate clonogenic capacity and P-gp function, AML cells were stained with rhodamine 123 (Rh 123), washed and then sorted after 4 h incubation at 37 degrees C in Rh 123-free media an the basis of their residual fluorescence intensity before plating. For each of six samples, we found that the number of CFU-L in the AML cell fraction which displayed the most efficient Rh 123 efflux capacity (Rh 123(dull)) was significantly higher compared to that of the AML cell fraction which displayed high residual fluorescence signal (Rh 123(bright)) (P = 0.05); the median number of CFU-L for 10(5) seeded cells being 1025 (range 250-2240) and 296 (range 11-838) for Rh 123(dull) and Rh 123(bright) populations, respectively. Altogether this study suggests that, for an individual AML cell population, the clonogenic fraction is preferentially recruited in AML cells which display low P-gp expression and high P-gp-mediated efflux capacity.