Lysosomes and trivalent arsenic treatment in acute promyelocytic leukemia.
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 alpha (PML/RAR alpha). Patients with APL respond to differentiation therapy with all-trans-retinoic acid, which induces PML/RAR alpha 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 alpha 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 alpha 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 alpha 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 microM 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 microM 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.
Kitareewan
Sutisak
S
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755, USA. sutisak.kitareewan@dartmouth.edu
Roebuck
B D
BD
Demidenko
Eugene
E
Sloboda
Roger D
RD
Dmitrovsky
Ethan
E
D000970:Antineoplastic Agents / Q000494:pharmacology*; D018053:Arsenites / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy* / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D015514:Oncogene Proteins, Fusion / Q000187:drug effects / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D012107:Research Design; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D018053:Arsenites; D015514:Oncogene Proteins, Fusion; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D002403:Cathepsins; D010447:Peptide Hydrolases; D053148:Caspase 3; D053179:Caspase 7; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C015001:arsenite