Lysosomes and Trivalent Arsenic Treatment in Acute Promyelocytic Leukemia

• Published in: JNCI : Journal of the National Cancer Institute Vol. 99; no. 1; pp. 41 - 52
• Main Authors: Kitareewan, Sutisak, Roebuck, B. D., Demidenko, Eugene, Sloboda, Roger D., Dmitrovsky, Ethan
• Format: Journal Article
• Language: English
• Published: Cary, NC Oxford University Press 03.01.2007; Oxford Publishing Limited (England)
• Subjects: Antineoplastic agents; Antineoplastic Agents - pharmacology; Arsenites - pharmacology; Biological and medical sciences; Caspase 3 - metabolism; Caspase 7 - metabolism; Cathepsin L; Cathepsins - metabolism; Cellular biology; Chemotherapy; Cysteine Endopeptidases - metabolism; Dose-Response Relationship, Drug; Humans; Leukemia; Leukemia, Promyelocytic, Acute - drug therapy; Leukemia, Promyelocytic, Acute - metabolism; Lysosomes - drug effects; Lysosomes - metabolism; Medical sciences; Microscopy, Electron, Transmission; Oncogene Proteins, Fusion - drug effects; Oncogene Proteins, Fusion - metabolism; Oncology; Peptide Hydrolases - metabolism; Pharmacology. Drug treatments; Prescription drugs; Proteins; Research Design; Time Factors; Tumor Cells, Cultured; Tumors; Antineoplastic agent; Human; Promyelocytic leukemia; Chemotherapy; Cancerology; Treatment; Acute; Lysosome; Arsenic trioxide; Malignant hemopathy; Trivalent cation; M3 acute myelocytic leukemia
• ISSN: 0027-8874; 1460-2105
• DOI: 10.1093/jnci/djk004

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-trans-retinoic acid, which induces PML/RARα degradation. When resistance to all-trans-retinoic acid develops, an effective treatment is arsenic trioxide (arsenite), which also induces this degradation. We investigated the mechanism of arsenite-induced PML/RARα 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/RARα 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/RARα 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 delocalization. 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 μM arsenite was 26.3% (95% CI = 3.3% to 33%; P<.001), compared with untreated cells. Conclusions In APL cells, arsenite may cause rapid destabilization of lysosomes.