| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© The Author 2007. Published by Oxford University Press.
ARTICLE |
Lysosomes and Trivalent Arsenic Treatment in Acute Promyelocytic Leukemia
Affiliations of authors: Departments of Pharmacology and Toxicology (SK, BDR, E. Dmitrovsky), Biostatistics and Epidemiology (E. Demidenko), and Medicine (E. Dmitrovsky), Dartmouth Medical School, Hanover, NH; Norris Cotton Cancer Center (E. Demidenko, E. Dmitrovsky), Dartmouth-Hitchcock Medical Center, Lebanon, NH; Department of Biological Sciences, Dartmouth College, Hanover, NH (RDS)
Correspondence to: Sutisak Kitareewan, PhD, 7650 Remsen, Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755 (e-mail: sutisak.kitareewan{at}dartmouth.edu).
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 
(PML/RAR). Patients with APL respond to differentiation therapy with all-trans-retinoic acid, which induces PML/RAR 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/RAR 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/RAR 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/RAR 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.
| CONTEXT AND CAVEATS Prior knowledge All-trans-retinoic acid treatment of APL induces degradation of a fusion protein expressed by leukemia cells and allows the APL cells to differentiate into myelocytes. Arsenite is an effective treatment for APL that stops responding to all-trans-retinoic acid. Study type Preclinical cell line study. Contribution Arsenite treatment of APL cells destabilizes lysosomes, which release proteases into the cytoplasm that can degrade the fusion protein in APL cells. Implications This is a newly described mechanism for arsenite treatment of APL. The effects of arsenite on lysosomes may occur in multiple cell lines, organs, and species. Limitations Only one APL cell line was available for analysis, which limits the generalizability of these results.
|
CiteULike 
Connotea 
Del.icio.us What's this?