Lysosomes and trivalent arsenic treatment in acute promyelocytic leukemia

Abstract

Background Cells from patients with t(15;17) acute promyelocytic leukemia (APL) express the fusion protein between the promyelocytic leukemia protein and retinoic acid receptor α (PML/RARα). Patients with APL respond to differentiation therapy with all-frans-retinoic acid, which induces PML/RARa degradation. When resistance to all-frans-retinoic acid develops, an effective treatment is arsenic trioxide (arsenite), which also induces this degradation. We investigated the mechanism of arsenite-induced PML/RARa degradation. Methods NB4-S1 APL cells were treated with clinically relevant concentrations of arsenite. Lysosomes were visualized with a lysosome-specific dye. Lysosomal protein esterase was measured by immunoblot analysis. Lysosomal cathepsin L was detected by immunogold labeling and transmission electron microscopy, and its activity was measured in cytosolic cellular fractions. In vitro degradation assays of PML/RARa in cell lysates were performed with and without protease inhibitors and assessed by immunoblot analysis. Only nonparametric two-sided statistical analyses were used. The nonparametric Wilcoxon test was used for group comparison, and the nonlinear regression technique was used for analysis of dose-response relationship as a function of arsenite concentration. Results Arsenite treatment destabilized lysosomes in APL cells. Lysosomal proteases, including cathepsin L, were released from lysosomes 5 minutes to 6 hours after arsenite treatment. PML/RARa was degraded by lysate from arsenite-treated APL cells, and the degradation was inhibited by protease inhibitors. At both 6 and 24 hours, substantially fewer arsenite-treated APL cells, than untreated cells, contained cathepsin L clusters, a reflection of cathepsin L derealization. Cells with cathepsin L clusters decreased as a function of arsenite concentration at rates of -2.03% (95% confidence interval [CI] = -4.01 to -.045; P =.045) and -2.39% (95% CI = -4.54 to -.024; P =.029) in 6- and 24-hour treatment groups, respectively, per 1.0 μM increase in arsenite concentration. Statistically significantly higher cytosolic cathepsin L activity was detected in lysates of arsenite-treated APL cells than in control lysates. For example, the mean increase in cathepsin activity at 6 hours and 1.0 JM arsenite was 26.3% (95% CI = 3.3% to 33%; P