ARTICLE
Lysosomes and Trivalent Arsenic Treatment in Acute
Promyelocytic Leukemia
Sutisak Kitareewan, B. D. Roebuck, Eugene Demidenko, Roger D. Sloboda, Ethan Dmitrovsky
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.
J Natl Cancer Inst 2007;99:41–52
Cells from patients with t(15;17) acute promyelocytic leukemia
(APL) express a fusion protein between the promyelocytic leukemia protein and retinoic acid receptor α (PML/RARα) 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/RARα
protein through proteasome-dependent (2,3) as well as caspasedependent (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, includjnci.oxfordjournals.org
ing 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
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: [email protected]).
See “Notes” following “References.”
DOI: 10.1093/jnci/djk004
© The Author 2007. Published by Oxford University Press. All rights reserved.
For Permissions, please e-mail: [email protected].
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CONT E X T A N D C A VEAT S
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.
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/RARα fusion protein. However, it has been reported that
arsenite can also induce apoptosis in some cell lines that do not
express PML/RARα (21–24) and that inhibitors of caspase or proteasome function do not fully block arsenite-mediated degradation
of PML/RARα (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/
RARα 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
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arsenite-mediated degradation of PML/RARα (2). We hypothesized
that arsenite causes release of lysosomal proteases to confer PML/
RARα 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/RARα.
Materials and Methods
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 BioProducts, 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-RARα (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-Arg7-amido-4-methylcoumarin were purchased from Calbiochem (La
Jolla, CA). Protease Arrest protease inhibitor mixture was purchased from GenoTechnology, Inc (St Louis, MO). The lysosometargeting 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/
RARα 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).
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Arsenite-Induced Degradation of the Fusion Protein
Between Promyelocytic Leukemia Protein and Retinoic
Acid Receptor 𝛂
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/RARα protein degradation, NB4-S1 APL cells were cultured at a density of 0.5 × 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 1× Protease Arrest). PML/RARα
protein degradation was assessed by immunoblot analysis.
To investigate the kinetics of arsenite-induced PML/RARα
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/RARα 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-RARα 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/RARα, to this lysosomal degradation program.
In Vitro Degradation Assay
To investigate components or pathways in NB4-S1 cells that mediate arsenite-induced PML/RARα degradation, hemagglutinintagged PML/RARα was overexpressed in COS-1 cells, which lack
PML/RARα. Hemagglutinin-tagged PML/RARα can be readily
detected and studied in transfected COS-1 cells. PML/RARα degradation in such COS-1 cell lysates was examined after incubation
with arsenite-treated NB4-S1 cell lysates (Fig. 2).
For transfection experiments, 3 × 105 COS-1 cells were transiently transfected with a hemagglutinin-tagged PML/RARα
expression vector (3) by use of Effectene, according to the manufacturer’s recommended procedures (Qiagen, Valencia, CA),
jnci.oxfordjournals.org
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 1×
Protease Arrest inhibitor mixture) for 10 minutes on ice and then
centrifuging the mixture for 1 minute at 12000g and 4 °C to separate the cell pellet from the cell lysate.
For the degradation assay, 10 × 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/RARα 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 (2.5× 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 1× 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/RARα 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/RARα 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 × 106 cells/mL without or with 1 μM sodium arsenite for 2 or 8 hours. The lysosometargeting 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 ×63, 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
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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 × 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 2× Protease Arrest inhibitor mixture) for
30 seconds at room temperature and were then immediately centrifuged at 12000g 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 × 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 chemiluminescentdetecting 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 × 106–1 × 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 30minute 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
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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 × 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
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(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 × 105 COS-1 cells were cultured per well in
six-well tissue culture plates for 2 hours or 5 × 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 × 105, 2.5 ×
105, or 2.5 × 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 × 104, 1.25 × 105, 2.5 × 105,
3.75 × 105, or 5.0 × 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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Fig. 1. Arsenite (As)-mediated degradation of the fusion protein between
the promyelocytic leukemia protein and retinoic acid receptor α (PML/
RARα) in the acute promyelocytic leukemia cell line NB4-S1. NB4-S1
cells were untreated (control) or treated with 100 µM N-acetyl-Leu-LeuNle-CHO (ALLN; a proteosome inhibitor) or 100 µM z-Val-Ala-Asp-CH2F
(zVAD; a caspase inhibitor) for 30 minutes, 1 µM sodium arsenite was
added, and cells were incubated for 10 hours. Cell lysates were prepared, and 25 µg of total protein was subjected to sodium dodecyl
sulfate–polyacrylamide gel electrophoresis followed by immunoblot
analyses. PML/RARα and actin proteins were detected with anti-RARα
and antiactin polyclonal antibodies, respectively. Actin was used as the
loading control. This experiment was performed twice, with similar
results obtained in each experiment.
Results
Arsenite-Induced Degradation of the Fusion Protein
Between Promyelocytic Leukemia Protein and Retinoic
Acid Receptor 𝛂
To confirm that arsenicals trigger PML/RARα degradation in NB4S1 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/RARα 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/RARα, although it did inhibit the proteasomal degradation of PML/RARα mediated by all-trans-retinoic
acid (data not shown). The inability of proteasome or caspase inhibitors to block arsenite-induced degradation of PML/RARα indicated
that a pathway other than the proteasome and caspase pathways participates in arsenite-dependent PML/RARα protein degradation.
In Vitro Degradation of the Fusion Protein Between
Promyelocytic Leukemia Protein and Retinoic Acid
Receptor 𝛂
To investigate the mechanism of arsenite-induced degradation of
PML/RARα protein, the stability of hemagglutinin-tagged PML/
RARα 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/
RARα protein (Fig. 2). Arsenite-mediated degradation of PML/
RARα 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/RARα (Fig. 2).
A similar degradation profile was observed with lysates isolated
from NB4-S1 cells treated with 10 μM arsenite for 24 hours
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Fig. 2. In vitro degradation of hemagglutinin (HA)-tagged fusion gene
product between the promyelocytic leukemia protein and retinoic acid
receptor α (PML/RARα) by lysates of arsenite (As)-treated NB4-S1 acute
promyelocytic leukemia cells. COS-1 cells were transfected with a
hemagglutinin-tagged PML/RARα. Transfected COS-1 cell lysate containing hemagglutinin-tagged PML/RARα was incubated with NB4-S1
cell lysates from untreated (control) cells or cells treated with 1 µM
arsenite for 72 hours. Some degradation reaction mixtures were also
treated with a 2.5× protease inhibitor mixture (Roche) and 12.5 mM
EDTA, a proteasome inhibitor (250 µM N-acetyl-Leu-Leu-Nle-CHO
[ALLN]), or a caspase inhibitor (250 µM z-Val-Ala-Asp-CH2F [zVAD]), as
indicated, for 2 hours at 37 °C. Cell lysates were prepared and subjected
to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed
by immunoblot analyses. Hemagglutinin-tagged PML/RARα and actin
proteins were detected with antihemagglutinin and antiactin antibodies, respectively. Actin was used as a loading control. This experiment
was performed three times, with similar results obtained each time.
(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/RARα 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/RARα. These findings indicate that a previously unrecognized pathway is active in PML/RARα degradation.
Fig. 3. Localization of lysosomes in acute promyelocytic leukemia (APL)
cells after arsenite (As) treatment. NB4-S1 cells were either untreated
(control) or treated with 1 µM arsenite for 2 or 8 hours before staining of
lysosomes with LysoTracker. Stained lysosomes (LysoTracker column)
or differential interference contrast (DIC column) imaging were visualized by confocal scanning spectrophotometer. The same representative
NB4-S1 APL cell is shown for both views for the treatment indicated.
Arsenite and Lysosomal Stability
Because lysosomes contain many proteases, these organelles may be
the source of proteases that degrade the fusion protein PML/
RARα. The stability of lysosomes in NB4-S1 APL cells in response
to arsenite treatment was examined by use of a weak-base lysosomespecific 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).
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).
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).
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
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
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Fig. 4. Comparison of arsenite (As)-induced release of lysosomal esterase with that of mitochondrial cytochrome c. A) Esterase release. After
treatment with arsenite as indicated, NB4-S1 cells were harvested, cell
lysates were prepared, and total proteins were subjected to sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblot analysis for esterase. B) Cytochrome c release.
NB4-S1 cells were treated with or without 1 µM arsenite or with camptothecin (10 µg/mL), as indicated. Camptothecin is a topoisomerase
inhibitor that induces DNA damage and apoptosis and is the positive
control for cytochrome c release. Fresh medium containing the indicated components was added every 24 hours for experiments extending over 48–96 hours. At the end of the incubation period, cell lysates
were prepared and subjected to SDS–PAGE, followed by immunoblot
analysis for cytochrome c. For both experiments, actin was used as a
loading control. Each experiment was conducted three times, with similar
results obtained in each experiment.
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.
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).
jnci.oxfordjournals.org
Fig. 5. Arsenite-induced lysosomal cathepsin L delocalization in NB4-S1
acute promyelocytic leukemia cells. NB4-S1 cells were cultured without
(control) or with arsenite for 6 or 24 hours and fixed. Cathepsin L was
identified by use of gold-labeled anti–cathepsin L antibodies through
transmission electron microscopy. A) Cluster (arrow) of cathepsin L in
untreated NB4-S1 cells. B) Unclustered cathepsin L in NB4-S1 cells
treated with 1 µM arsenite for 6 hours. Similar results were obtained
with arsenite at 2, 5, and 10 µM. C) Histologically normal NB4-S1 cell
and an apoptotic NB4-S1 cell (arrow). These cells were treated with
10 µM arsenite for 24 hours. D) Clustering of cathepsin L as a function
of arsenite concentration. Cells were incubated with arsenite as indicated for 6 and 24 hours, and the percentages of NB4-S1 cells with at
least one cathepsin L cluster (defined as ³10 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.
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Fig. 7. Comparison of cytosolic cathepsin L and caspase 3 and 7 activities in arsenite-treated NB4-S1 acute promyelocytic leukemia cells.
Untreated control NB4-S1 cells and cells treated with 1 µM arsenite were
cultured as indicated, cytosolic fractions were prepared, and activities
of cathepsin L and caspases 3 and 7 were determined. Data are expressed as the mean percentage increase from seven experiments over
control as a function of treatment time. Error bars are 95% confidence
intervals.
Fig. 6. Arsenite treatment and activity of lysosomal cathepsin L in the
cell lysate from NB4-S1 acute promyelocytic leukemia, COS-1 green
monkey kidney, and BEAS-2B human bronchial epithelial cells. Cells
were treated with arsenite, as indicated, cell lysates were prepared, and
cathepsin L activity was assayed by use of the cathepsin L substrate zPhe-Arg-7-amido-4-methylcoumarin. The activity was measured on the
basis of an equal number of cells initially cultured. Data are expressed
as the mean percentage increase compared with control, where n =
number of independent experiments. Error bars are 95% confidence
intervals. Nonlinear regression with an exponential limit-value function
was used to fit the arsenite dose–response data for NB4-S1 and COS-1
cells, and a linear function was used for BEAS-2B cells. A) Cytosolic
cathepsin L activity from NB4-S1 cells treated with arsenite at the concentrations indicated for 6 hours. B) Cytosolic cathepsin L activities in
arsenite-treated BEAS-2B, COS-1, and NB4-S1 cells. Cells were treated
for 24 hours with the concentrations of arsenite indicated. All parameters
of the curves are statistically significant according to the Z test (P<.001).
All statistical tests were two-sided.
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 =
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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). NB4S1 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/RARα
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).
Discussion
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
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Fig. 8. Time course and dose–response relationships of arsenite-induced
degradation of fusion protein between the promyelocytic leukemia
protein and retinoic acid receptor α (PML/RARα) in NB4-S1 acute promyelocytic leukemia cells. NB4-S1 cells were cultured with and without
arsenite at the indicated concentrations and times. Cell lysates were
isolated and subjected to sodium dodecyl sulfate–polyacrylamide gel
electrophoresis followed by immunoblot analysis. PML/RARα and actin
proteins were detected with anti-RARα and antiactin polyclonal
antibodies, respectively. Actin was used as a loading control. This
experiment was performed twice, with similar results obtained in each
experiment.
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/RARα
fusion protein (Fig. 6, B).
Cotreatment with arsenite and either a caspase or proteasome
inhibitor did not prevent PML/RARα 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/RARα degradation
(Fig. 2). These results support the hypothesis that lysosomal proteases, including cathepsin L from destabilized lysosomes, degrade
the PML/RARα 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 arsenitetreated 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,
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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 arsenitetreated 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 NB4S1 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/RARα 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 arsenitetreated 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
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occurs in primary APL blasts, APL cell lines, and cells overexpressing the PML/RARα 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-α (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 arseniteinduced 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/RARα 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/RARα
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/
RARα 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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References
(1) Melnick A, Licht JD. Deconstructing a disease: RARα, its fusion partners,
and their roles in the pathogenesis of acute promyelocytic leukemia. Blood
1999;93:3167–215.
(2) Zhu J, Gianni M, Kop E, Honore N, Chelbi-Alix M, Koken M, et al.
Retinoic acid induces proteasome-dependent degradation of retionic acid
receptor α (RARα) and oncogenic RARα fusion protein. Proc Natl Acad
Sci U S A 1999;96:14807–12.
(3) Kitareewan S, Pitha-Rowe I, Sekula D, Lowrey CH, Nemeth MJ,
Golub TR, et al. UBE1L is a retinoid target that triggers PML/RARα
degradation and apoptosis in acute promyelocytic leukemia. Proc Natl
Acad Sci U S A 2002;99:3806–11.
(4) Nervi C, Ferrara FF, Fanelli M, Rippo MR, Tomassini B, Ferrucci PF,
et al. Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARα fusion protein. Blood 1998;92:2244–51.
(5) Chen GQ, Zhu J, Shi XG, Ni JH, Zhong HJ, Si GY, et al. In vitro studies
on cellular and molecular mechanisms of arsenic trioxide (As2O3) in
the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell
apoptosis with downregulation of Bcl-2 expression and modulation of
PML-RARα/PML proteins. Blood 1996;88:1052–61.
(6) Miller WH Jr, Schipper HM, Lee JS, Singer J, Waxman S. Mechanisms
of action of arsenic trioxide. Cancer Res 2002;62:3893–903.
(7) Zhang P, Wang S, Hu L, Shi F, Qiu F, Hong L, et al. Treatment of acute
promyelocytic leukemia with intravenous arsenic trioxide. Chin J Hematol
1996;17:58–60.
(8) Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qiu QY, et al. Use of
arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia
(APL): II. Clinical efficacy and pharmacokinetics in relapsed patients.
Blood 1997;89:3354–60.
(9) Vega L, Styblo M, Patterson R, Cullen W, Wang C, Germolec D. Differential effects of trivalent and pentavalent arsenicals on cell proliferation
and cytokine secretion in normal human epidermal keratinocytes. Toxicol
Appl Pharmacol 2001;172:225–32.
(10) Thomas DJ, Styblo M, Lin S. The cellular metabolism and systemic toxicity of arsenic. Toxicol Appl Pharmacol 2001;176:127–44.
(11) Soignet SL, Maslak P, Wang ZG, Jhanwar S, Calleja E, Dardashti LJ,
et al. Complete remission after treatment of acute promyelocytic leukemia
with arsenic trioxide. N Engl J Med 1998;339:1341–8.
(12) Soignet SL, Frankel SR, Douer D, Tallman MS, Kantarjian H, Calleja E,
et al. United States multicenter study of arsenic trioxide in relapsed acute
promyelocytic leukemia. J Clin Oncol 2001;19:3852–60.
(13) Chen GQ, Shi XG, Tang W, Xiong SM, Zhu J, Cai X, et al. Use of arsenic
trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL):
I. As2O3 exerts dose-dependent dual effects on APL cells. Blood 1997;
89:3345–53.
(14) Lallemand-Breitenbach V, Guillemin MC, Janin A, Daniel MT, Degos L,
Kogan SC, et al. Retinoic acid and arsenic synergize to eradicate leukemic
cells in a mouse model of acute promyelocytic leukemia. J Exp Med
1999;189:1043–52.
(15) Kinjo K, Kizaki M, Muto A, Fukuchi Y, Umezawa A, Yamato K, et al.
Arsenic trioxide (As2O3)-induced apoptosis and differentiation in retinoic
acid-resistant acute promyelocytic leukemia model in hGM-CSF-producing transgenic SCID mice. Leukemia 2000;14:431–8.
(16) Gianni M, Koken MHM, Chelbi-Alix MK, Benoit G, Lanotte M, Chen Z,
et al. Combined arsenic and retinoic acid treatment enhances differentiation and apoptosis in arsenic-resistant NB4 cells. Blood 1998;91:4300–10.
(17) Lallemand-Breitenbach V, Zhu J, Puvion F, Koken M, Honore N,
Doubeikovsky A, et al. Role of promyelocytic leukemia (PML) sumolation
in nuclear body formation, 11S proteasome recruitment, and As2O3induced PML or PML/retinoic acid receptor α degradation. J Exp Med
2001;193:1361–71.
(18) Chen YC, Lin-Shiau SY, Lin JK. Involvement of reactive oxygen species
and caspase 3 activation in arsenic-induced apoptosis. J Cell Physiol
1998;177:324–33.
(19) Kitamura K, Minami Y, Yamamoto K, Akao Y, Kiyoi H, Saito H, et al.
Involvement of CD95-independent caspase 8 activation in arsenic trioxideinduced apoptosis. Leukemia 2000;14:1743–50.
Vol. 99, Issue 1
|
January 3, 2007
(20) Nason-Burchenal K, Allopenna J, Begue A, Stehelin D, Dmitrovsky E,
Martin P. Targeting of PML/RARα is lethal to retinoic acid-resistant
promyelocytic leukemia cells. Blood 1998;92:1758–67.
(21) Akao Y, Nakagawa Y, Akiyama K. Arsenic trioxide induces apoptosis in
neuroblastoma cell lines through the activation of caspase 3 in vitro. FEBS
Lett 1999;455:59–62.
(22) Seol JG, Park WH, Kim ES, Jung CW, Hyun JM, Lee YY, et al. Potential
role of caspase-3 and -9 in arsenic trioxide-mediated apoptosis in PCI-1
head and neck cancer cells. Int J Oncol 2001;18:249–55.
(23) Bajenova O, Tang B, Pearse R, Feinman R, Childs BH, Michaeli J. RIP
kinase is involved in arsenic-induced apoptosis in multiple myeloma cells.
Apoptosis 2004;9:561–71.
(24) Kong B, Huang S, Wang W, Ma D, Qu X, Jiang J, et al. Arsenic trioxide
induces apoptosis in cisplatin-sensitive and -resistant ovarian cancer cell
lines. Int J Gynecol Cancer 2005;15:872–7.
(25) Mullins C, Bonifacino JS. The molecular machinery for lysosome biogenesis. Bioessays 2001;23:333–43.
(26) Jaattela M, Tschopp J. Caspase-independent cell death in T lymphocytes.
Nat Immunol 2003;4:416–23.
(27) Brunk UT, Neuzil J, Eaton JW. Lysosomal involvement in apoptosis.
Redox Rep 2001;6:91–7.
(28) Turk B, Stoka V, Rozman-Pungercar J, Cirman T, Droga-Mazovec G,
Oresic K, et al. Apoptotic pathways: involvement of lysosomal proteases.
Biol Chem 2002;383:1035–44.
(29) Yuan XM, Li W, Dalen H, Lotem J, Kama R, Sachs L, et al. Lysosomal
destabilization in p53-induced apoptosis. Proc Natl Acad Sci U S A
2002;99:6286–91.
(30) Foghsgaard L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M,
et al. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol 2001;153:999–1010.
(31) Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C,
et al. Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest
2000;106:1127–37.
(32) Dietrich N, Thastrup J, Holmberg C, Gyrd-Hansen M, Fehrenbacher N,
Lademann U, et al. JNK2 mediates TNF-induced cell death in mouse
embryonic fibroblasts via regulation of both caspase and cathepsin protease pathways. Cell Death Differ 2004;11:301–13.
(33) Brunk UT, Svensson I. Oxidative stress, growth factor starvation and Fas
activation may all cause apoptosis through lysosomal leak. Redox Rep
1999;4:3–11.
(34) Lane AA, Ley TJ. Neutrophil elastase cleaves PML-RARα and is important for development of acute promyelocytic leukemia in mice. Cell
2003;115:305–18.
(35) Fehrenbacher N, Gyrd-Hansen M, Poulsen B, Felbor U, Kallunki T,
Boes M, et al. Sensitization to the lysosomal cell death pathway upon
immortalization and transformation. Cancer Res 2004;64:5301–10.
(36) Kawasaki G, Kato Y, Mizuno A. Cathepsin expression in oral squamous
cell carcinoma: relationship with clinicopathologic factors. Oral Surg Oral
Med Oral Pathol Oral Radiol Endod 2002;93:446–54.
(37) Erdal H, Berndtsson M, Castro J, Brunk U, Shoshan MC, Linder S.
Induction of lysosomal membrane permeabilization by compounds that activate p53-independent apoptosis. Proc Natl Acad Sci U S A 2005;102:192–7.
(38) Fehrenbacher N, Jaattela M. Lysosomes as targets for cancer therapy.
Cancer Res 2005;65:2993–5.
(39) Nason-Burchenal K, Maerz W, Albanell J, Allopenna J, Martin P,
Moore MAS, et al. Common defects of different retinoic acid resistant
promyelocytic leukemia cells are persistent telomerase activity and nuclear
body disorganization. Differentiation 1997;61:321–31.
(40) Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F,
Berger R. NB4, a maturation inducible cell line with t(15;17) marker isolated
from a human acute promyelocytic leukemia (M3). Blood 1991;77:1080–6.
(41) Langenfeld J, Kiyokawa H, Sekula D, Boyle J, Dmitrovsky E.
Posttranslational regulation of cyclin D1 by retinoic acid: a chemoprevention mechanism. Proc Natl Acad Sci U S A 1997;94:12070–4.
(42) Wetterhahn KE, Dudek EJ, Shumilla JA, Lannon BM, Elmore LC,
Barchowsky A. Possible mechanisms for arsenic-induced proliferative diseases. Proceedings of the Seventh Pacific Basin Conference on Hazardous
jnci.oxfordjournals.org
(43)
(44)
(45)
(46)
(47)
(48)
(49)
(50)
(51)
(52)
(53)
(54)
(55)
(56)
(57)
(58)
(59)
(60)
(61)
(62)
(63)
Waste; 1996; Kuala Lumpur (Malaysia); CONF-9611157; 446–66. Pacific
Basin Consortium of Hazardous Waste Research.
Zhang TD, Chen GQ, Wang ZG, Wang ZY, Chen SJ, Chen Z. Arsenic
trioxide, a therapeutic agent for APL. Oncogene 2001;20:7146–53.
Douer D, Tallman MS. Arsenic trioxide: new clinical experience with an
old medication in hematologic malignancies. J Clin Oncol 2005;23:
2396–410.
Ishitsuka K, Shirahashi A, Iwao Y, Shishime M, Takamatsu Y, Takatsuka Y,
et al. Bone marrow necrosis in a patient with acute promyelocytic leukemia during re-induction therapy with arsenic trioxide. Eur J Haematol
2004;72:280–4.
Ollinger K, Brunk UT. Cellular injury induced by oxidative stress is mediated through lysosomal damage. Free Radic Biol Med 1995;19:565–74.
Boll I. Morphologic phase-contrast cinematographic studies on the behavior of bone marrow cells in vitro. X. Myeloblasts and monoblasts. Blut
1976;32:115–30.
Campbell EJ, Silverman EK, Campbell MA. Elastase and cathepsin G of
human monocytes. Quantification of cellular content, release in response
to stimuli, and heterogeneity in elastase-mediated proteolytic activity.
J Immunol 1989;143:2961–8.
Sahu GR, Jena RK. Significance of intracellular arsenic trioxide for therapeutic response in acute promyelocytic leukemia. Am J Hematol 2005;
78:113–6.
Mullin KA, Foth BJ, Ilgoutz SC, Callaghan JM, Zawadzki JL, McFadden GI,
et al. Regulation of an endoplasmic reticulum membrane protein in a
tubular lysosome in Leishmania mexicana. Mol Biol Cell 2001;12:2364–77.
Fowler BA. General subcellular effects of lead, mercury, cadmium, and
arsenic. Environ Health Perspect 1978;22:37–41.
Berry JP, Galle P. Selenium-arsenic interaction in renal cells: role of lysosomes. Electron microprobe study. J Submicrosc Cytol Pathol 1994;26:
203–10.
Link G, Pinson A, Hershko C. Iron loading of cultured cardiac myocytes
modified sarcolemmal structure and increase lysosomal fragility. J Lab
Clin Med 1993;121:127–34.
Brunk UT, Terman A. The mitochondrial-lysosomal axis theory of aging:
accumulation of damaged mitochondria as a result of imperfect autophagocytosis. Eur J Biochem 2002;269:1996–2002.
Barchowsky A, Dudek EJ, Treadwell MD, Wetterhahn KE. Arsenic
induces oxidant stress and NF-κB activation in cultured aorta endothelial
cells. Free Radic Biol Med 1996;21:783–90.
Jing Y, Dai J, Chalmers-Redman RME, Tatton WG, Waxman S. Arsenic
trioxide selectively induces acute promyelocytic leukemia cell apoptosis via
a hydrogen peroxide-dependent pathway. Blood 1999;94:2102–11.
Sternsdorf T, Puccetti E, Jensen K, Hoelzer D, Will H, Ottmann OG,
et al. PIC-1/SUMO-1-modified PML-retinoic acid receptor α mediates
arsenic trioxide-induced apopotosis in acute promyelocytic leukemia. Mol
Cell Biol 1999;19:5170–8.
Cai X, Shen YL, Zhu Q, Jia PM, Yu Y, Zhou L, et al. Arsenic trioxideinduced apoptosis and differentiation are associated respectively with
mitochrondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia. Leukemia 2000;14:
262–70.
Zang Y, Beard RL, Chandraratna RA, Kang JX. Evidence of a lysosomal
pathway for apoptosis induced by the synthetic retinoid CD437 in human
leukemia HL-60 cells. Cell Death Differ 2001;8:477–85.
Kagedal K, Johansson U, Ollinger K. The lysosomal protease cathepsin D
mediates apoptosis induced by oxidative stress. FASEB J 2001;15:1592–4.
Kagedal K, Johansson AC, Johansson U, Heimlich G, Roberg K,
Wang NS, et al. Lysosomal membrane permeabilization during apoptosisinvolvement of Bax? Int J Exp Pathol 2005;86:309–21.
Guicciardi ME, Bronk SF, Werneburg NW, Yin XM, Gores GJ.
Bid is upstream of lysosome-mediated caspase 2 activation in tumor necrosis
factor alpha-induced hepatocyte apoptosis. Gastroenterology 2005;
129:269–84.
Nason-Burchenal K, Takle G, Pace U, Flynn S, Allopenna J, Martin P,
et al. Targeting the PML/RARα translocation product triggers apoptosis
in promyelocytic leukemia cells. Oncogene 1998;17:1759–68.
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Articles 51
Notes
This work was supported by National Institutes of Health (NIH) RO1CA62275 (E. Dmitrovsky), NIH RO1-CA087546 (E. Dmitrovsky), National
Science Foundation (NSF) MCB-9727818 (R. D. Sloboda), NSF MCB9970048 (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.
52 Articles
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JNCI
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.
Manuscript received February 22, 2006; revised October 19, 2006; accepted
November 17, 2006.
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January 3, 2007