Figure 1.
Figure 1. Nontoxic PK11195 doses can block efflux by Pgp, MRP, and BCRP drug transporters. (A) To identify nontoxic Pgp-modulator doses, DOX40 cells were incubated across micromolar dose ranges with PK11195 (PK; 5 to 150 μM) and the established Pgp-modulator, CSA (0.2 to 5 μM). After 48-hour treatments, flow cytometry was performed in which “live” cell fractions contained cells with high DiOC63 staining (full mitochondrial membrane potential) and low PI fluorescence (high integrity), as illustrated in the top left panel. After other 48-hour treatments, 3H-Tdr was added for an additional 24 hours, and “live” cell fractions were measured as 3H-Tdr counts per minute relative to counts per minute in untreated wells. Treatment-specific live-cell fractions were calculated by subtracting untreated from treated fractions, and summary data from at least 3 assays are shown in the top right panel as means ± SEM. CSA (up to 1 μM) and PK11195 (up to 75 μM) did not significantly reduce live-cell fractions. Effects of CSA and PK11195 dose ranges were also measured in flow cytometry efflux assays after 1-hour MIT (20 μM) exposures. Representative histograms are shown in the bottom left panel. Efflux-blocking ratios were calculated as the MFI in the presence of test agent/MFI in the absence of test agent: “1” represents no efflux blocking, as denoted with a bold, horizontal line in the summary data from at least 3 assays that are shown in the bottom right panel. Also, 75 μM PK11195 blocked MIT efflux significantly more than any CSA dose in DOX40 cells. (B) Nontoxic doses of CSA (1 μM), MK-571 (MK; 25 μM), and GF120918 (GF; 1 μM) were used as Pgp, MRP, and BCRP modulators, respectively, along with dose ranges of PK11195, in MIT efflux assays of 7 additional AML and MM cell lines. Seventy-five μM PK11195 blocked MIT efflux significantly more than CSA in all cell lines tested and blocked MIT efflux significantly more than GF in BCRP-transduced HL60 cells. In all cases, efflux rations were calculated as described in panel A, and summary data from at least 3 assays are shown as means plus or minus SEM.

Nontoxic PK11195 doses can block efflux by Pgp, MRP, and BCRP drug transporters. (A) To identify nontoxic Pgp-modulator doses, DOX40 cells were incubated across micromolar dose ranges with PK11195 (PK; 5 to 150 μM) and the established Pgp-modulator, CSA (0.2 to 5 μM). After 48-hour treatments, flow cytometry was performed in which “live” cell fractions contained cells with high DiOC63 staining (full mitochondrial membrane potential) and low PI fluorescence (high integrity), as illustrated in the top left panel. After other 48-hour treatments, 3H-Tdr was added for an additional 24 hours, and “live” cell fractions were measured as 3H-Tdr counts per minute relative to counts per minute in untreated wells. Treatment-specific live-cell fractions were calculated by subtracting untreated from treated fractions, and summary data from at least 3 assays are shown in the top right panel as means ± SEM. CSA (up to 1 μM) and PK11195 (up to 75 μM) did not significantly reduce live-cell fractions. Effects of CSA and PK11195 dose ranges were also measured in flow cytometry efflux assays after 1-hour MIT (20 μM) exposures. Representative histograms are shown in the bottom left panel. Efflux-blocking ratios were calculated as the MFI in the presence of test agent/MFI in the absence of test agent: “1” represents no efflux blocking, as denoted with a bold, horizontal line in the summary data from at least 3 assays that are shown in the bottom right panel. Also, 75 μM PK11195 blocked MIT efflux significantly more than any CSA dose in DOX40 cells. (B) Nontoxic doses of CSA (1 μM), MK-571 (MK; 25 μM), and GF120918 (GF; 1 μM) were used as Pgp, MRP, and BCRP modulators, respectively, along with dose ranges of PK11195, in MIT efflux assays of 7 additional AML and MM cell lines. Seventy-five μM PK11195 blocked MIT efflux significantly more than CSA in all cell lines tested and blocked MIT efflux significantly more than GF in BCRP-transduced HL60 cells. In all cases, efflux rations were calculated as described in panel A, and summary data from at least 3 assays are shown as means plus or minus SEM.

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