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. 2005 Jul 28;106(10):3584–3593. doi: 10.1182/blood-2005-02-0711

Figure 3.

Figure 3.

PK11195 blocks MIT efflux in primary AML cells. To further assess the clinical relevance of our cell-line data, we performed efflux assays in primary AML cell samples. (A) Efflux blockade in Pgp-selective DiOC23 and ABC transporter-general MIT efflux assays was measured as increased dye retention. Efflux-blocking effects are presented as ratios, as in Figure 1. PK11195 and CSA effects were correlated in both assays, as noted in the figure. (B) In additional MIT efflux assays of 8 efflux-competent AML samples, MK-571 and Ko143 were used as specific MRP and BCRP modulators along with PK11195 and CSA. PK11195 again substantially blocked MIT efflux in all of these, and Ko143 measurably blocked MIT efflux in 4 samples, suggesting that these functionally expressed BCRP. MK-571 measurably blocked MIT efflux one, apparently MRP-expressing sample. PK11195 blocked efflux substantially more effectively than CSA in 3 samples. PK11195, CSA, and Ko143 also significantly increased MIT retention in repeated assays of normal CD34+CD38- hematopoietic progenitor cells (P = .01, P < .005, and P < .05, respectively), and PK11195 and CSA significantly increased MIT retention in more mature CD34+CD38+ normal cells (P = .05 in each case). Three immunolabeled NBM samples were analyzed, and summary data are shown as means ± SEM. KG1a cells were assayed along with primary cells in every assay; summary data from at least 5 assays are shown as means ± SEM. (C) 3H-PK11195 binding assays were performed in additional aliquots of 11 primary AML cell samples. Specific 3H-PK11195 binding was determined by competition with 1000-fold excess unlabeled PK11195, and competed counts are shown with means plus or minus SEMs for 7 samples (Pgp+ AMLs) in which both CSA and PK11195 increased DiOC23 retention and for 4 samples without efflux capacity (Pgp- AMLs). 3H-PK11195 binding was significantly higher in Pgp-positive AMLs than in Pgp-negative AMLs (P < .05), and Pgp-expressing DOX6 and DOX40 MM cells also bound very high levels of 3H-PK11195, but Pgp-expressing VCR and KG1a cells bound relatively low levels of PK11195 (means ± SEM are shown from at least 3 assays for each cell line). 3H-PK11195 binding was competed by excess of another pBR ligand, FGIN-1-27 (FGIN), but excess unlabeled CSA did not compete for 3H-PK11195 binding in any assay of Pgp-expressing cells.