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© 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).
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ABSTRACT |
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 TopNotes Abstract Context and CaveatsMaterials and MethodsResultsDiscussionReferences |
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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.
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.
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Cells from patients with t(15;17) acute promyelocytic leukemia (APL) express a fusion protein between the promyelocytic leukemia protein and retinoic acid receptor (PML/RAR) that blocks differentiation of promyelocytes and leads to accumulation of leukemic promyelocytes (1). Most patients with APL respond to treatment with all-trans-retinoic acid by degrading the PML/RAR protein through proteasome-dependent (2,3) as well as caspase-dependent (2,4) degradation pathways; this degradation triggers terminal differentiation of APL cells. However, all-trans-retinoic acid treatment causes toxic effects that are often clinically dose limiting (5), and APL can develop resistance to all-trans-retinoic acid (1,5). Therefore, an alternative nonretinoid therapy for APL was needed.
For centuries, arsenic has been used as a therapeutic agent to treat, e.g., ulcer, plague, and malaria, as reviewed (6). Resurgent interest in arsenic-based cancer therapy was generated by reports (7,8) that arsenic induced clinical remission in APL patients, including patients with APL resistant to all-trans-retinoic acid. In these studies, patients were treated with arsenic trioxide (As2O3), a trivalent arsenic (arsenite) that is the biologically and therapeutically active arsenical (9,10). Clinical studies of relapsed APL patients reported complete remission after arsenic therapy (11,12). Studies using transgenic mouse and in vitro APL models showed that most arsenite-induced APL responses occur by activating apoptosis rather than by inducing differentiation of APL blasts (5,13–15). Some evidence for maturation of APL cells was observed at low arsenite concentrations, especially in APL cells that were resistant to arsenite-induced apoptosis (13,14,16). Arsenite also induces the production of reactive oxygen species or the activation of caspase pathways or ubiquitin–proteasome pathways (17–19). It has been hypothesized (1,4,20) that arsenite and other agents active against APL cells act predominantly by triggering degradation of the PML/RAR fusion protein. However, it has been reported that arsenite can also induce apoptosis in some cell lines that do not express PML/RAR (21–24) and that inhibitors of caspase or proteasome function do not fully block arsenite-mediated degradation of PML/RAR (2). Thus, arsenite treatment appears to activate a degradation program that is independent of caspase- or proteasome-dependent pathways.
Possible alternative mechanisms for the degradation of PML/RAR involve the proteases in lysosomes. Lysosomes, which mediate the turnover of normal and abnormal macromolecules and organelles, are involved in endocytosis, exocytosis, inactivation of pathogens, and antigen processing and presentation (25). Lysosomes also participate in certain programmed cell death pathways, including p53-induced apoptosis (26–29). Death receptor activation can induce release of lysosomal cathepsins into the cytosolic compartment (30–32). Oxidative stress, growth factor deprivation, and Fas activation also trigger release of lysosomal cathepsins (33). Lysosomes and their associated enzymes play active roles in carcinogenesis and in antineoplastic responses to pharmacologic agents (34–37), and so lysosomes may be targets for cancer therapy (38).
We have investigated mechanisms of arsenite-induced degradation of the oncogenic APL fusion protein because of previous evidence that proteasome and caspase inhibitors did not fully block arsenite-mediated degradation of PML/RAR (2). We hypothesized that arsenite causes release of lysosomal proteases to confer PML/RAR degradation in NB4 APL cells. We therefore investigated whether arsenite induces lysosomal destabilization and release of lysosomal proteases into the cytosol and whether these proteases in turn degrade PML/RAR.
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Materials and Methods |
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Chemicals and Antibodies
Sodium arsenite, sucrose, digitonin, Pefabloc SC, and porcine esterase were purchased from Sigma-Aldrich (St Louis, MO). Sodium arsenite stock solutions of 10 mM were prepared in sterile water, the concentration was determined by use of inductive coupled plasma–mass spectrometry, and the solution was stored at –20 °C. Fetal bovine serum was purchased from Gemini Bio-Products, Inc (Calabasas, CA). Peroxidase-conjugated antiesterase antibody was purchased from Rockland (Gilbertsville, PA). Cytochrome c (product sc-4270) and anti–cytochrome c (product sc-13560), antiactin (product sc-1615), anti–cathepsin L (product sc-6500), horseradish peroxidase–conjugated donkey anti-goat IgG (product sc-2020), and anti-RAR (product sc-551) antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Horseradish peroxidase–conjugated anti-rabbit IgG, horseradish peroxidase–conjugated anti-mouse IgG, and ECL Plus chemiluminescent western blotting detection reagents were purchased from GE Healthcare UK Limited (Buckinghamshire, U.K.). An antihemagglutinin antibody (product MMS-101R) was obtained from Covance (Berkeley, CA). The proteasome inhibitor ALLN (i.e., N-acetyl-Leu-Leu-Nle-CHO), the caspase inhibitor zVAD (i.e., z-Val-Ala-Asp-CH2F), and the cathepsin L substrate z-Phe-Arg- 7-amido-4-methylcoumarin were purchased from Calbiochem (La Jolla, CA). Protease Arrest protease inhibitor mixture was purchased from GenoTechnology, Inc (St Louis, MO). The lysosome-targeting dye LysoTracker was purchased from Molecular Probes (Eugene, OR). The 12-nm colloidal gold–coupled Affinipure donkey anti-goat antibody was purchased from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA).
Cell Culture
NB4-S1, derived from parental NB4 cells, is a clonal APL cell line that is sensitive to all-trans-retinoic acid (39). Parental NB4 cells were derived from a patient with APL; these leukemic cells contain the t(15;17) chromosomal translocation that encodes the PML/RAR fusion protein (40). NB4-S1 cells were cultured in Advanced RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 2% fetal bovine serum, 4 mM l-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C in an atmosphere of 5% CO2 and 95% air in a humidified incubator. The green monkey kidney fibroblast-like cell line COS-1 was cultured in Advanced Dulbecco's modified Eagle medium (Invitrogen) supplemented with 2% fetal bovine serum, 4 mM l-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C in an atmosphere of 5% CO2 and 95% air in a humidified incubator. The human bronchial epithelial cell line BEAS-2B was cultured in LHC-9 serum-free medium (Biosource, Camarillo, CA), as previously described (41).
Arsenite-Induced Degradation of the Fusion Protein Between Promyelocytic Leukemia Protein and Retinoic Acid Receptor
Sodium arsenite, rather than arsenic trioxide, was used in all experiments because sodium arsenite is readily soluble in aqueous solutions. Both arsenicals are also chemically identical in aqueous solution at neutral pH (42). Clinical pharmacokinetic analyses of arsenite treatment of APL indicate that the peak plasma concentration is in the low (43) to high (44,45) micromolar range.
To investigate arsenite-induced PML/RAR protein degradation, NB4-S1 APL cells were cultured at a density of 0.5 x 106 cells/mL for 2 hours, ALLN or zVAD (each at a final concentration of 100 µM) was added for 30 minutes as indicated, and then sodium arsenite was added to a final concentration of 1 µM. Cells were then cultured for 10 hours, harvested by centrifugation at 1000g for 5 minutes at 4 °C, and washed once with ice-cold phosphate-buffered saline (PBS). Cell pellets were lysed with 250 µL of radioimmunoprecipitation assay (RIPA) buffer (20 mM HEPES at pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1x Protease Arrest). PML/RAR protein degradation was assessed by immunoblot analysis.
To investigate the kinetics of arsenite-induced PML/RAR protein degradation, NB4-S1 cells were cultured with 0.1, 1, or 10 µM sodium arsenite for 2, 4, or 6 hours. Cells were harvested, cell lysates were prepared as described above, and PML/RAR protein degradation was assessed by immunoblot analysis.
For immunoblot analysis, 25 µg of total protein from cell lysates per lane was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were transferred to a nitrocellulose membrane (Optitran BA-S85 reinforced NC, Whatman Schleicher and Schuell, Dassel, Germany), and the membrane was probed with primary rabbit anti-RAR or goat antiactin polyclonal antibodies. Actin was used as a loading control. Primary antibodies were then probed with horseradish peroxidase–conjugated donkey anti-rabbit IgG or horseradish peroxidase–conjugated donkey anti-goat secondary antibodies, respectively, and protein bands were detected with chemiluminescent reagents, followed by exposure to x-ray film. At a low arsenite concentration (1 µM) and/or a short exposure time, degradation of actin was not detected. However, actin was degraded at a high concentration of arsenite (i.e., 10 µM) and/or a longer incubation time. This relative resistance to degradation of actin might reflect the increased sensitivity of abnormal macromolecules, such as PML/RAR, to this lysosomal degradation program.
In Vitro Degradation Assay
To investigate components or pathways in NB4-S1 cells that mediate arsenite-induced PML/RAR degradation, hemagglutinin-tagged PML/RAR was overexpressed in COS-1 cells, which lack PML/RAR. Hemagglutinin-tagged PML/RAR can be readily detected and studied in transfected COS-1 cells. PML/RAR degradation in such COS-1 cell lysates was examined after incubation with arsenite-treated NB4-S1 cell lysates (Fig. 2).
For transfection experiments, 3 x 105 COS-1 cells were transiently transfected with a hemagglutinin-tagged PML/RAR expression vector (3) by use of Effectene, according to the manufacturer's recommended procedures (Qiagen, Valencia, CA), cultured for 24 hours in fresh medium, and then harvested. COS-1 protein extracts were prepared by incubating cells with 250 µL of degradation buffer (50 mM HEPES at pH 7.0, 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 1 mM dithiothreitol, and 1x Protease Arrest inhibitor mixture) for 10 minutes on ice and then centrifuging the mixture for 1 minute at 12 000g and 4 °C to separate the cell pellet from the cell lysate.
For the degradation assay, 10 x 106 NB4-S1 cells were cultured without or with 1 µM sodium arsenite for 72 hours or with 10 µM sodium arsenite for 24 hours. Cells were then washed once with ice-cold PBS, and cell lysates were isolated by incubating NB4-S1 cells with 250 µL of degradation buffer for 1 minute at room temperature followed by centrifugation, as described above, to separate the cell pellet from the cell lysate. The degradation assay for hemagglutinin-tagged PML/RAR protein was performed by mixing 5 µg of total protein from the hemagglutinin-tagged COS-1 cell lysate with 10 µg of total protein from untreated or sodium arsenite–treated NB4-S1 cell lysates. The serine and cysteine proteases inhibitor mixture (2.5x Complete Mini Protease Inhibitor, Roche, Penzberg, Germany) with EDTA (to chelate cations and thus inhibit metalloproteases), 250 µM of ALLN (proteasome inhibitor), or 250 µM zVAD (caspase inhibitor) was added to reaction mixtures as indicated. All reaction mixtures were incubated for 2 hours at 37 °C, and the reactions were terminated with addition of 1x SDS–PAGE loading dye (50 mM Tris–HCl at pH 6.8, 100 mM 2-mercaptoethanol, 2% SDS [wt/vol], 0.1% bromophenol blue, and 10% glycerol). After SDS–PAGE of the reaction mixtures and immunoblot preparation, hemagglutinin-tagged PML/RAR was probed with a monoclonal antibody that recognized the hemagglutinin epitope. Bound antibody was detected with horseradish peroxidase–conjugated anti-mouse IgG and chemiluminescent reagents, followed by exposure to x-ray film. The level of hemagglutinin-tagged PML/RAR detected was used to assess degradation activity in untreated or arsenite-treated NB4-S1 protein lysates. Actin was used as a loading control and detected with an antiactin antibody, as described above.
Lysosomal Staining
NB4-S1 cells were cultured at a density of 0.5 x 106 cells/mL without or with 1 µM sodium arsenite for 2 or 8 hours. The lysosome-targeting dye LysoTracker was then added to a final concentration of 20 nM, and cells were cultured for an additional 30 minutes. Cells were harvested by centrifugation at 1000g for 5 minutes at 4 °C and resuspended in fresh medium. Stained lysosomes were visualized with a Leica confocal scanning spectrophotometer (model TCS SP) equipped with a x63, 1.32 numerical aperture planapochromatic objective. UV, argon, krypton–argon, and helium–neon lasers that provided a broad range of excitation wavelengths were individually coupled to the microscope. To compare the confocal image with overall cell morphology, the cells were imaged by use of differential interference contrast optics. The confocal microscope was set to excite the dye at 586 nm and to record the resulting fluorescent signal at 590 nm to analyze LysoTracker-stained cells. The resulting signal was detected by a tunable spectrophotometer that could be used to discriminate emitted wavelengths. The microscope, laser, spectrophotometer, image capture procedures, and analyses were under computer control. On a given day, settings were not changed from one experiment to another. For image comparisons, all experimental conditions, including radiation exposure, scan rate, sample preparation, and sample temperature, were held constant.
Release of Lysosomal and Mitochondrial Proteins
Immunoblot analysis of lysosomal esterase was used to assess the release of lysosomal proteins after arsenite treatment. NB4-S1 cells were cultured at a density of 0.5 x 106 cells/mL as described above for 2 hours before treatment with 0.1, 1.0, or 10 µM sodium arsenite for 5 or 30 minutes. Cells were then harvested by centrifugation at 1000g for 5 minutes at 4 °C and washed once in ice-cold PBS. Cell pellets were then resuspended in 250 µL of permeabilizing buffer (containing digitonin at 250 µg/mL, 75 mM NaCl, 1 mM NaH2PO4, 8 mM Na2HPO4, 250 mM sucrose, 5 mM EDTA, and 2x Protease Arrest inhibitor mixture) for 30 seconds at room temperature and were then immediately centrifuged at 12 000g for 30 seconds at 4 °C. Cell lysates were isolated, and 50 µg of total protein in the cell lysate was subjected to SDS–PAGE, followed by immunoblot analyses with horseradish peroxidase–conjugated antibodies that specifically recognized esterase or actin (as a loading control). Protein bands were visualized by chemiluminescent-detecting reagents, followed by exposure to x-ray film.
Immunoblot analysis of cytochrome c was used to assess the release of a mitochondrial protein after arsenite treatment. The kinetics of cytochrome c release into the cytosol after treatment of NB4-S1 cells with 1 µM sodium arsenite was determined by culturing cells at the initial density of 0.5 x 106 cells/mL in fresh medium supplemented with 1 µM sodium arsenite every 24 hours for 96 hours. A positive control for release of mitochondrial cytochrome c was obtained by addition of a topoisomerase inhibitor, camptothecin (10 µg/mL, which induces DNA damage and hence apoptosis), to NB4-S1 cell cultures for 4 hours before total protein was isolated in digitonin buffer (digitonin at 200 µg/mL, 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM Pefabloc SC, at pH 7.5). The cell lysates were subjected to SDS–PAGE, followed by immunoblot analyses for cytochrome c and actin (as the loading control) with corresponding antibodies. Bands were visualized by chemiluminescent-detecting reagents, followed by exposure to x-ray film.
Depletion of Lysosomal Cathepsin L
The cellular location of lysosomal cathepsin L was assessed by use of immunogold labeling and transmission electron microscopy. NB4-S1 APL cells were first cultured at 0.5 x 106–1 x 106 cells/mL without (control) or with 1.0, 2.0, 5.0, or 10 µM sodium arsenite for 6 or 24 hours and then fixed for 1 hour in a mixture of 1% glutaraldehyde and 4% paraformaldehyde. Fixed cells were centrifuged at 1000g for 5 minutes at 4 °C, rinsed in PBS for 5 minutes at room temperature, and centrifuged again. Cells were resuspended in 50 mM glycine in PBS to quench any free aldehydes. Samples were then dehydrated by incubation in 50%, 70%, and 85% ethanol (each incubation for 5 minutes), followed by two 30-minute washes in a solution of LR White resin (hard) (Electron Microscope Sciences, Hatfield, PA) and 85% ethanol (2:1 [vol:vol]), followed by two 30-minute washes and one 1-hour wash in LR White resin (hard) solution at room temperature. Cell pellets were placed in gelatin capsules with fresh LR White resin solution, the capsules were sealed, and cells were allowed to settle for 1 hour at room temperature. The resin was polymerized at 50 °C for 24 hours. Samples were sectioned (100 nm thick) and adhered onto 300-mesh nickel transmission electron microscopy grids. Sample grids were then wetted with PBS; blocked for 30 minutes in a solution of 2% bovine albumin, 0.1% fish gelatin, 0.05% Tween-20, and 2% rabbit serum in PBS; and then incubated with anti–cathepsin L antibody (1:10 dilution) at room temperature for 2 hours. Sample grids were washed with PBS for three 5-minute periods and then incubated with a secondary antibody coupled to 12-nm gold particles at room temperature for 1 hour. Sample grids were then washed with PBS for three 5-minute periods, followed by rinsing with water for two 5-minute periods. Samples were then stained with 2% aqueous uranyl acetate for 10 minutes at room temperature, washed with water for three 5-minute periods, counterstained with Reynold's lead citrate solution for 10 seconds, and immediately washed with water for three 5-minute periods. Samples were then examined with a Jeol TEM 1010 transmission electron microscope. Because nonspecific labeling of cells with antibody-conjugated gold particles never showed more than two clustered particles, a level of 10 or more gold-labeled particles was chosen as a stringent selection criterion for clustering. In addition, apoptotic cells (i.e., cells with chromatin compaction, numerous vacuoles, and electron dense micronuclei) were excluded because they would likely lack appreciable detection of gold-labeled cathepsin L clusters. Results are expressed as the percentage of cells with localized cathepsin L.
Lysosomal Cathepsin L Enzyme Activity
Cathepsin L activity in the cytosol was measured in cell lysates of NB4-S1, COS-1, and BEAS-2B cells with a modified procedure (36). Modifications are cell line specific, as described below.
The optimal digitonin concentrations and incubation times were determined for each cell type examined. Because of the low protein content in cell lysate from these cells, cathepsin L activity was measured on the basis of equal numbers of cells initially cultured. For NB4-S1 cells, 2 x 106 cells in 4 mL of medium were cultured with sodium arsenite at a final concentration of 1, 2, 5, or 10 µM for 6 or 24 hours and then harvested to assay cathepsin L activity. Cell pellets were harvested by centrifugation at 1000g for 5 minutes at 4 °C and washed once with ice-cold PBS. The lysate of NB4-S1 cells was prepared by resuspending the cell pellets in 250 µL of the same digitonin buffer used in isolation of cytochrome c for 2 minutes on ice, centrifuging the mixture at 1000g for 30 seconds, and rapidly transferring the cell lysates (i.e., the supernatant) to new assay tubes. Cathepsin L activity was assayed by adding 100 µL of protein lysate to 500 µL of substrate buffer (25 µM z-Phe-Arg-7-amido-4-methylcoumarin, 25 mM sodium acetate, 8 mM EDTA, 8 mM dithiothreitol, and 1 mM Pefabloc SC, at pH 5.0) and incubating the reaction mixture at 30 °C for 10 minutes. The reaction was terminated by addition of 400 µL of stop buffer (150 mM sodium monochloroacetate, 105 mM acetic acid, and 45 mM sodium acetate, at pH 4.3). Relative fluorescence was detected with a fluorescence spectrophotometer (model F-3010; Hitachi, Ltd, Tokyo, Japan). The percentage change (relative to control) in fluorescence units was obtained for an arsenite-treated sample and was corrected for the percentage of viable cells detected at each arsenite concentration.
For analysis of cathepsin L activity in the cell lysates of COS-1 and BEAS-2B cells, 2.5 x 105 COS-1 cells were cultured per well in six-well tissue culture plates for 2 hours or 5 x 105 BEAS-2B cells per well were cultured overnight. Sodium arsenite was added, as indicated, and cells were cultured, as indicated. After treatment, the tissue culture plate was placed on ice, medium was removed, and cells were washed once with ice-cold PBS. Cell lysates were isolated by adding 250 µL of digitonin-containing buffer (digitonin at 20 µg/mL, 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM Pefabloc SC, at pH 7.5) to the cells and incubating the mixture on ice for 5 minutes with periodic agitation. Cell lysates were quickly harvested as described above for NB4-S1 cells, and cathepsin L activities were assayed. Results were corrected for cell line–specific viability, as described below.
Viability Assay
NB4-S1, BEAS-2B, and COS-1 cells were cultured at 5.0 x 105, 2.5 x 105, or 2.5 x 105 cells/mL, respectively, and sodium arsenite was added to a final concentration of 1, 2, 5, or 10 µM. Cells were cultured for 6 or 24 hours and then harvested, and the number of viable cells was scored with trypan blue staining and a hemacytometer. Triplicate trypan blue viability assays were performed for each cell line, and the mean percentages of viable cells at each arsenite concentration for each cell line were obtained. These percentages of viable cells were used to normalize cathepsin L activity in the cell lysates of each cell line for corresponding arsenite concentrations.
Time Course for Caspase 3 and 7 Activity and Cathepsin L Activity
NB4-S1 cells were cultured at 6.25 x 104, 1.25 x 105, 2.5 x 105, 3.75 x 105, or 5.0 x 105 cells/mL for 96, 72, 48, 24, or 6 hours, respectively, with or without 1 µM sodium arsenite. Lysates were isolated as described above with digitonin buffer. Cathepsin L activity was measured using z-Phe-Arg-7-amido-4-methylcoumarin as substrate. Caspase 3 and 7 activity was assessed with the Caspase-Glo 3/7 assay system (Promega, Madison, WI), and activity was measured with a Turner Designs Luminometer TD-20/20 (Sunnyvale, CA) after incubating the cell lysates with the caspase 3 and 7 substrate (the tetrapeptide DEVD, supplied with the Caspase-Glo assay system) at room temperature for 30 minutes. Relative changes of enzyme activities were expressed as percentages of control values.
Statistical Analysis
We used a nonparametric Wilcoxon signed rank test for all group comparisons. Differences with a P value of less than .05 were considered statistically significant. All statistical tests were two-sided. A nonlinear regression with exponential limit-value function of the form a1–a2exp(–a3d) was used to fit the dose–response data (see Fig. 6), where d is the sodium arsenite concentration and a1, a2, and a3 are parameters estimated from the data. All parameters of the curves were statistically significant according to the Z test. Stata Statistics/Data Analysis software (version 4.0, Stata Corporation, College Station, TX) was used for group comparison, and S-Plus (version 6.2, Insightful Inc, Seattle, WA) was used for nonlinear fitting.
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Results |
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Arsenite-Induced Degradation of the Fusion Protein Between Promyelocytic Leukemia Protein and Retinoic Acid Receptor
To confirm that arsenicals trigger PML/RAR degradation in NB4-S1 APL cells (2), cells were incubated for 10 hours with 1 µM arsenite, 100 µM ALLN (an inhibitor of the ubiquitin–proteasome pathway), 100 µM zVAD (an inhibitor of the caspase degradation pathway), or combinations of 1 µM arsenite and either 100 µM ALLN or 100 µM zVAD. Consistent with an earlier report (2), PML/RAR was degraded in arsenite-treated cells, and neither ALLN nor zVAD completely blocked the degradation (Fig. 1). Lactacystin, another proteasome inhibitor, also did not prevent the sodium arsenite–induced degradation of PML/RAR, although it did inhibit the proteasomal degradation of PML/RAR mediated by all-trans-retinoic acid (data not shown). The inability of proteasome or caspase inhibitors to block arsenite-induced degradation of PML/RAR indicated that a pathway other than the proteasome and caspase pathways participates in arsenite-dependent PML/RAR protein degradation.
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In Vitro Degradation of the Fusion Protein Between Promyelocytic Leukemia Protein and Retinoic Acid Receptor
To investigate the mechanism of arsenite-induced degradation of PML/RAR protein, the stability of hemagglutinin-tagged PML/RAR protein expressed in lysates of COS-1 cells was examined after the addition of cell lysates from untreated NB4-S1 cells or from NB4-S1 cells treated with 1 µM arsenite for 72 hours. Lysates from arsenite-treated NB4-S1 APL cells, but not lysates from untreated control cells, degraded hemagglutinin-tagged PML/RAR protein (Fig. 2). Arsenite-mediated degradation of PML/RAR was inhibited by addition of a mixture of serine and cysteine protease inhibitors (Complete Mini Protease Inhibitor Cocktail, Roche), but neither the proteasome inhibitor ALLN nor the caspase inhibitor zVAD blocked degradation of PML/RAR (Fig. 2). A similar degradation profile was observed with lysates isolated from NB4-S1 cells treated with 10 µM arsenite for 24 hours (data not shown). Addition of sodium arsenite directly to the lysates isolated from untreated NB4-S1 APL cells did not induce degradation of the PML/RAR fusion protein (data not shown). Thus, sodium arsenite appears to activate a proteolytic program that involves neither caspase- nor proteasome-dependent pathways but that still degrades PML/RAR. These findings indicate that a previously unrecognized pathway is active in PML/RAR degradation.
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Arsenite and Lysosomal Stability
Because lysosomes contain many proteases, these organelles may be the source of proteases that degrade the fusion protein PML/RAR. The stability of lysosomes in NB4-S1 APL cells in response to arsenite treatment was examined by use of a weak-base lysosome-specific fluorescent dye (LysoTracker) and visualization by confocal microscopy. Relocalization of the LysoTracker into the cytosol indicates destabilization of the lysosomal membranes (46). Increased lysosomal staining was observed as early as 2 hours after treatment of NB4-S1 cells with 1 µM arsenite and lasted for as long as 48 hours (Fig. 3 and data not shown), compared with baseline staining in untreated control NB4-S1 cells. Changes in cell morphology were not observed at 2 or 8 hours, as assessed by differential interference contrast microscopy (Fig. 3).
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Lysosomal Destabilization and Cytochrome c Release from Mitochondria
The intense fluorescent staining of lysosomes in arsenite-treated NB4-S1 APL cells was consistent with lysosomal destabilization and release of lysosomal contents. To further investigate whether arsenite induced release of lysosomal proteases, we assessed release of one such protease, lysosomal esterase, to the cytosol by immunoblot analysis. After treatment of NB4-S1 APL cells with 0.1, 1.0, or 10 µM arsenite for 5 or 30 minutes, lysosomal esterase was detected in a total protein lysate from NB4-S1 APL cells by immunoblot analysis (Fig. 4, A).
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Because release of cytochrome c from the mitochondria precedes caspase-dependent apoptosis (18), we examined the time course of cytochrome c release from arsenite-treated cells. Release of cytochrome c into the cytosol of arsenite-treated cells began 48 hours after arsenite treatment, as compared with 5 minutes for the release of lysosomal esterase (Fig. 4, B). Thus, cytochrome c release occurred much later than the detected changes in lysosomal staining (Fig. 3) and lysosomal esterase release (Fig. 4, A), indicating that the caspase pathway is not involved in the arsenite-induced protein degradation observed shortly after treatment (i.e., 2–6 hours).
Arsenite-Mediated Changes in the Localization of Cathepsin L
The possibility that arsenite destabilized lysosomes and caused lysosomal leakage was tested by use of immunogold labeling and transmission electron microscopy to visualize cathepsin L in arsenite-treated cells. Cathepsin L clusters were observed in lysosomes of untreated cells but not in those of arsenite-treated NB4-S1 APL cells (Fig. 5, A and B). Time course analyses indicated that, after arsenite treatment, the percentage of NB4-S1 cells with cathepsin L clusters decreased with increasing concentrations of sodium arsenite at 6 hours and 24 hours (with linear regression slopes of –2.03% [95% confidence interval {CI} = –4.01 to –0.045; P = .045] and –2.39% [95% CI = –4.54 to –0.024; P = .029] per 1 µM increase in sodium arsenite concentration) (Fig. 5, D). It is important to note that only nonapoptotic cells were scored when the number of cells with cathepsin L clustering was quantified. Inclusion of apoptotic cells (cells with chromatin compaction, numerous vacuoles, and electron dense micronuclei) into the calculation would have markedly decreased the percentage of cells with cathepsin L clustering with increasing sodium arsenite concentration. Cathepsin L clustering was not detected in apoptotic cells, as shown in Fig. 5, C.
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10 gold-labeled particles) were determined. Each point represents data from at least 100 nonapoptotic cells, except for untreated control cells (for which 402 nonapoptotic cells were counted) or cells treated with 10 µM arsenite at 24 hours (for which 200 nonapoptotic cells were counted). Linear regression slopes were –2.03% (95% confidence interval [CI] = –4.01 to –0.045; P = .045) and –2.39% (95% CI = –4.54 to –0.024; P = .029) for 6- and 24-hour treatment groups, respectively.To obtain further support for the hypothesis that cathepsin L delocalization is accompanied by an increase in cathepsin L enzymatic activity in the cytosol of arsenite-treated cells, cathepsin L activity was measured in cell lysates from NB4-S1 cells treated with sodium arsenite at 1, 2, 5, or 10 µM for 6 hours. Cathepsin L activity in the cell lysate from arsenite-treated cells was statistically significantly higher than that of control cells. For example, the cathepsin L activity in cell lysates from cells treated with 1.0 µM arsenite was 26.3% (95% CI = 3.3% to 33%) higher than that of control cells (P<.001). When an exponential limit-value function was used to fit the data as the arsenite concentration increased, cathepsin L activity approached 111% of the control cell lysate activity (i.e., its limiting value) at the rate of 94% per 1 µM of sodium arsenite (Fig. 6, A).
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Arsenite-dependent lysosomal destabilization was also investigated in human bronchial epithelial BEAS-2B cells and green monkey kidney COS-1 cells. Cytosolic activity of cathepsin L 24 hours after arsenite treatment in all three cell lines was statistically significantly higher in arsenite-treated cells than in control cells. For example, the mean cathepsin L activity at 24 hours for 1 µM arsenite treatment, compared with control, was 13.5% (95% CI = 8.5% to 21.3%; P<.001) for COS-1 cells, 40.0% (95% CI = 27.4% to 58.3%; P<.001) for BEAS-2B cells, and 102.5% (95% CI = 78.8% to 133.4%; P<.001) for NB4-S1 cells (Fig. 6, B). NB4-S1 and COS-1 cells had similar cathepsin L activity profiles, with limiting values of 2280% and 137%, respectively (Fig. 6, B), but BEAS-2B cells had a cathepsin L activity profile that increased linearly with arsenite concentration at a rate of 0.33 per 1 µM increase in arsenite concentration (i.e., a 1 µM increase in sodium arsenite concentration increased cathepsin L activity by 33%, compared with that in untreated control cells) (Fig. 6, B). Although cathepsin L activity in NB4-S1 cells at 24 hours after arsenite treatment (Fig. 6, B) was much higher than that at 6 hours after treatment (Fig. 6, A), rates to the limiting value at 5 µM and 10 µM were not statistically significantly different (0.82 versus 0.94, respectively; P = .26).
Arsenite induction of apoptosis in NB4 APL cells has been previously reported (13,14,16), and caspases have been identified as mediators of apoptosis in response to arsenite treatment in various cell types, including APL cells (18,19,21,22). In the protein lysates of NB4-S1 cells treated with 1 µM sodium arsenite, we detected 76% (95% CI = 38.8% to 103%; P<.001) increased caspase 3 and 7 activities 24 hours after arsenite treatment. In contrast, cathepsin L activity began to increase (28.5%, 95% CI = 21.2% to 35.0%; P<.001) after only 6 hours of treatment (Fig. 7). Thus, release of lysosomal cathepsin L into the cytosol of NB4-S1 APL cells preceded that of caspases 3 and 7. Therefore, the kinetics of cathepsin L activity support results of time course and dose–response experiments above, in that arsenite-induced PML/RAR protein degradation was detected as early as 2 hours after treatment with 1 µM arsenite or 6 hours after treatment with 0.1 µM arsenite (Fig. 8).
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Discussion |
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TopNotesAbstractContext and CaveatsMaterials and MethodsResultsDiscussionReferences |
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We have shown that lysosomes appear to be a direct molecular pharmacologic target for arsenite and that arsenite appears to act through a mechanism involving the rapid destabilization of lysosomes (Fig. 3). Several lines of evidence support this conclusion including the release of lysosomal enzymes in arsenite-treated cells (Figs. 4 and 6, A and B) and the delocalization of immunogold labeling of lysosomal cathepsin L (Fig. 5, B and D). Although lysosomal destabilization by arsenite was most evident in APL cells, it was also detected in other cell lines that do not express the PML/RAR
fusion protein (Fig. 6, B).
Cotreatment with arsenite and either a caspase or proteasome inhibitor did not prevent PML/RAR degradation. These findings support the view that a novel protein degradation program was elicited by arsenite treatment of APL cells. Support for the hypothesis that enzymes were released from destabilized lysosomes in arsenite-treated APL cells was obtained by showing an increase in lysosomal cathepsin L activity present in the cell lysates of cells as early as 6 hours after arsenite treatment (Fig. 6, A) and was markedly increased by 24 hours (compare Fig. 6, A and B). Increased cathepsin L activity in the cell lysates of NB4-S1 cells after 6 or 24 hours of sodium arsenite treatment was concomitant with a decline in the number of cells with clusters of cathepsin L (Figs. 5, D, and 6, A and B). These findings established that, after arsenite treatment, cathepsin L appears to move from lysosomes to the cytosol. In addition, a mixture of serine and cysteine protease inhibitors completely blocked PML/RAR degradation (Fig. 2). These results support the hypothesis that lysosomal proteases, including cathepsin L from destabilized lysosomes, degrade the PML/RAR fusion protein in arsenite-treated cells (Fig. 1).
Because lysosomes are ubiquitous, it is possible that lysosomes from cell types other than APL are also targeted by arsenite treatment. To investigate this possibility, we analyzed the human bronchial epithelial cell line BEAS-2B and the green monkey kidney fibroblast-like cell line COS-1 for cytosolic cathepsin L activity after arsenite treatment. Arsenite treatment substantially increased cytosolic cathepsin L activity in BEAS-2B and COS-1 cells, although the increase was less than that observed in NB4-S1 cells (Fig. 6, B). These results indicated that lysosomes in these cell lines were being targeted by arsenite.
This marked increase in cathepsin L activity in arsenite-treated NB4-S1 cells compared with that in BEAS-2B and COS-1 cells may reflect the facts that APL cells are immature promyelocytes that have many lysosomes (47,48) and that primary APL cells appear to accumulate higher concentrations of arsenite than other cell types examined (49). Consistent with this observation, preliminary results with inductive coupled plasma–mass spectrometry found a higher concentration of sodium arsenite accumulated in NB4-S1 cells than in BEAS-2B and COS-1 cells (data not shown). Lysosomes from these three cell types, which arise from different tissues, were differentially sensitive to arsenite treatment, as shown by the leakage of cathepsin L into the cytosol (Fig. 6, B). Thus, arsenite appears to target lysosomes in various types of cells, an effect that should be explored in the future.
The integrity of lysosomes in APL cells after arsenite treatment was assessed by use of the lysosome-specific dye LysoTracker, which fluoresces in the acidic environment of lysosomes. Lysosomal bodies in arsenite-treated NB4-S1 APL cells were stained more intensely than those in untreated NB4-S1 APL cells (Fig. 3). In addition, COS-1 cells treated with 1 µM sodium arsenite for 8 hours also showed more lysosomal staining than untreated COS-1 cells (data not shown). The intense lysosomal staining of arsenite-treated cells might reflect lysosomal membrane destabilization; this destabilization allows acidic lysosomal contents to be released into the intracellular microenvironment (46). Alternatively, arsenite treatment might increase the total number or size of lysosomes within the cells or increase the acidity of lysosomal bodies. However, these effects would not account for lack of lysosomal staining in APL cells observed after exposure to a high concentration (10 µM) of sodium arsenite for 48 hours (data not shown). The absence of lysosomal staining was also observed when NB4-S1 cells were treated with a known lysomorphotropic agent, monensin (50), that can cause collapse of lysosomes (data not shown). After NB4-S1 APL cells were treated with 10 µM sodium arsenite for 48 hours or 50 µM monensin for 8 hours, nuclear condensation (i.e., morphologic evidence of apoptosis) was detected by Hoechst staining, but PML/RAR and actin proteins were not detected, presumably because these proteins had been degraded by released lysosomal proteases (data not shown). Thus, treatment with both low and high arsenite concentrations or with monensin appears to destabilize lysosomes so that lysosomal enzymes can leak into the cytosol.
Because arsenicals have been reported to accumulate in lysosomes (51,52), it is possible that arsenite-induced lysosomal destabilization involves a Fenton-type reaction (53). This arsenite-induced reaction would generate reactive hydroxyl radicals that could not diffuse out of the lysosomes but could destabilize lysosomal membranes so that hydrolytic enzymes would be released that would eventually trigger apoptosis (54).
Detection of lysosomal esterase in the cytoplasm of arsenite-treated cells is consistent with the observation that lysosomal contents were released by destabilized lysosomes in arsenite-treated cells (Fig. 4, A) and is supported by a study (55) of cytosolic oxidation of endothelial cells that detected increased cytosolic esterase within 30 minutes of arsenite treatment. Another study (56) reported increased hydrogen peroxide levels in APL cells after arsenite treatment, as detected by a nonfluorescent dye that must first be cleaved by esterase and then oxidized in the presence of hydrogen peroxide to a fluorescent product. Therefore, accumulation of hydrogen peroxide may be caused by the destabilized lysosomes. In contrast, cytochrome c (an activator of caspase-induced apoptosis) was first detected in the cytosol after 48 hours of arsenite treatment (Fig. 4, B). The kinetics of cytochrome c release is in agreement with the appearance of arsenite-induced apoptosis that occurs in primary APL blasts, APL cell lines, and cells overexpressing the PML/RAR fusion protein (13,16,57,58).
It has been reported (30) that, during induced apoptosis of myeloid leukemic cells, the release of lysosomal contents occurs before mitochondrial changes are detected. In hepatocytes, the lysosomal enzyme cathepsin B appears to mediate apoptosis induced by tumor necrosis factor- (30). In HL-60 cells, the synthetic retinoid CD437 induced release of cathepsin D from lysosomes into the cytosol, leading to apoptosis (59). Release of lysosomal cathepsins into the cytosol of fibroblasts was observed as early as 30 minutes after treatment with an oxidative stress–inducing agent (60). Generation of free radicals and increased oxidative stress have been proposed as mechanisms that could be involved in the release of cathepsins from lysosomes (60), but direct lysosomal destabilization by oxidative stress–inducing agents could not be discounted as a potential mechanism for release of cathepsins by that study.
Caspase 3 has been reported (18,21,22) to mediate arsenite-induced apoptosis in various cell types. However, it has also been noted (57) that arsenite-induced apoptosis in APL cells is independent of Bcl2 and caspase 3. Kinetics of caspase 3 and 7 activities were compared with cathepsin L activity. The activity of caspases 3 and 7 in the cytosol of NB4-S1 cells was relatively low after a 6-hour treatment with 1 µM arsenite, although it increased between 24 and 96 hours after treatment (Fig. 7). In contrast, arsenite-induced PML/RAR degradation was readily detected as early as 2 hours (Fig. 8), which is within the time frame of lysosomal cathepsin L activation (Figs. 6, A, and 7). Several reports (32,38,59–61) have shown that pharmacologic agents can permeabilize lysosomal membranes, releasing cathepsins that induce apoptosis in treated cells. Whether these pharmacologic agents directly destabilize lysosomal membranes or activate expression of other apoptosis regulatory proteins, such as Bax and Bid (61,62), should be investigated.
This study has several potential limitations. Despite identification of lysosomal destabilization in NB4-S1 APL cells cultured with arsenite, determination of whether arsenite-induced lysosomal destabilization also occurs in the in vivo setting of transgenic APL models or during the course of clinical treatment of APL patients was beyond the scope of this study. There is a single widely studied APL cell line (NB4) that carries the PML/RAR fusion gene. Thus, use of only one APL cell line limits the ability to generalize results in APL.
In summary, we have demonstrated that sodium arsenite treatment of APL cells rapidly destabilizes lysosomes, which then release hydrolytic enzymes into the cytosol that trigger PML/RAR protein degradation. Degradation of this oncogenic fusion protein has been shown to lead to apoptosis in APL cells (4,20,63). The destabilization of lysosomes was dependent on the arsenite concentration and on time after treatment. These previously unrecognized effects of arsenite on lysosomes occur in at least three cell lines—NB4-S1, BEAS-2B, and COS-1—derived from different organs and two different species. Thus, arsenite-induced destabilization of lysosomal membranes appears to be directly related to the arsenite treatment. Future studies should be directed toward elucidating the mechanism of arsenite-induced release of lysosomal enzymes and determining whether arsenite treatment of nonhematopoietic tumor cells as well as other hematopoietic tumor cells acts through a similar mechanism.
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NOTES |
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This work was supported by National Institutes of Health (NIH) RO1-CA62275 (E. Dmitrovsky), NIH RO1-CA087546 [GenBank] (E. Dmitrovsky), National Science Foundation (NSF) MCB-9727818 (R. D. Sloboda), NSF MCB-9970048 (R. D. Sloboda), NSF MCB-0418877 (R. D. Sloboda), and NIH RO1-CA39416 (B. D. Roebuck). The authors had full responsibility for the design and conduct of the study, analyses and interpretation of the data, decision to submit the manuscript for publication, and the writing of the manuscript.
The authors would like to thank Dr John Hwa (Dartmouth Medical School) for helpful consultation, Christopher Nitkin (Dartmouth College) and Arielle Rodman (Dartmouth College) for technical assistance, as well as members of the Dmitrovsky laboratory for helpful discussions. We also extend our appreciation to Karen J. Baumgartner for assisting in statistical evaluations and Louisa Howard at the Ripple Electron Microscope facility at Dartmouth College for transmission electron microscopic analyses.
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