Article Figures & Data
Figures
- FIG. 1.
(A to C) Time- and concentration-dependent intracellular accumulation of 4′-Ed4T radioactive metabolites in CEM cells. CEM cells were incubated in the presence of various concentrations of [3H]4′-Ed4T. At different time intervals, the cells were harvested and extracted with 70% cold methanol. The extracts were subjected to HPLC analyses. The radioactivity detected was calculated as the number of picomoles/106 cells for 4′-Ed4TMP (A), 4′-Ed4TDP (B), and 4′-Ed4TTP (C). The data points represent the means ± standard deviations of triplicate determinations. (D to F) Intracellular persistence of 4′-Ed4T radioactive metabolites in CEM cells. CEM cells were incubated with various concentrations of [3H]4′-Ed4T for 15 h and thoroughly washed to remove the extracellular drug. After further incubation of the cells for 0, 2, 4, 8, 12, and 24 h, cell extracts were prepared and subjected to HPLC analyses. The radioactivity detected was calculated as the numbers of picomoles/106 cells for 4′-Ed4TMP (D), 4′-Ed4TDP (E), and 4′-Ed4TTP (F). The data points represent the means ± standard deviations of triplicate determinations.
- FIG. 2.
Time- and temperature-dependent efflux of 4′-Ed4T (A), AZT (B), and/or their metabolites from CEM cells. CEM cells were preincubated with 1 μM of [3H]4′-Ed4T and [3H]AZT for 15 h. The cells were then washed with PBS and resuspended in fresh medium without drugs. At different time intervals, the cells and media were harvested and extracted with 70% cold methanol. The metabolites in the extracts were determined by the DE-81 disc assay. The amounts of extracellular 4′-Ed4T (A), AZT (B), and/or their metabolites in the medium are shown. (C) Ratio of efflux of 4′-Ed4T, AZT, and/or their metabolites as a percentage of the total metabolites.
- FIG. 3.
Efflux of 4′-Ed4T, AZT, and/or their metabolites from CEM cells. CEM cells were preincubated with 1 μM (A) or 0.1 μM (B) of [3H]AZT or 1 μM of [3H]4′-Ed4T (C) for 15 h. The cells were then washed with PBS and resuspended in fresh medium for another 2 h. The harvested media were concentrated and then extracted with 70% cold methanol. The extracts were subjected to HPLC. The insets in each panel are enlargements of the areas for the monophosphate peaks.
- FIG. 4.
Effect of the nucleoside inhibitor dipyridamole on the efflux and influx of 4′-Ed4T and AZT in CEM cells. For the efflux study, CEM cells were cultured with 2 μM of [3H]AZT or [3H]4′-Ed4T. After 15 h, the cells were washed and resuspended in fresh medium with different concentrations of dipyridamole. The cells and media were harvested after 2 h of incubation and extracted with 70% cold methanol. The metabolites in the extracts were determined by the DE-81 disc assay. (A) Effect of dipyridamole on the efflux of 4′-Ed4T and AZT; (B) effect of dipyridamole on the efflux of AZT nucleoside and its monophosphate metabolite; (C) effect of dipyridamole on the influx of thymidine, 4′-Ed4T, and AZT in CEM cells. For the influx study, CEM cells were incubated with various concentrations of dipyridamole at 37°C for 15 min prior to the uptake assays. [3H]thymidine, [3H]AZT, or [3H]4′-Ed4T was added to the cells for times ranging from 1 to 30 min. Uptake was terminated by the addition of ice-cold PBS containing 20 μM dipyridamole. The cells were washed with the same buffer and solubilized with 1% Sarkosyl. The radioactivity was determined in a liquid scintillation counter.
- FIG. 5.
Effect of downregulation of MRP4 on the efflux of 4′-Ed4TMP and AZTMP. (A) Expression of MRP4 in Tet-On RKO/shMRP4 cells, as detected by Western blotting. Actin was used as the internal control. (B) Doxycycline (Dox)-induced Tet-On RKO/shMRP4 cells were pretreated with 5 μM of [3H]4′-Ed4T or 2 μM of [3H]AZT for 16 h. The cells were then washed with ice-cold PBS and resuspended in fresh medium. The cell and medium extracts were prepared and subjected to the DE-81 disc assay. The radioactivity detected was calculated as the numbers of picomoles of total intracellular or extracellular monophosphate metabolites for 4′-Ed4T and AZT.
