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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2844-2853
Phenylarsine Oxide Blocks Interleukin-1 -Induced Activation of
the Nuclear Transcription Factor NF- B, Inhibits
Proliferation, and Induces Apoptosis of Acute Myelogenous Leukemia
Cells
By
Zeev Estrov,
Sunil K. Manna,
David Harris,
Quin Van,
Elihu H. Estey,
Hagop M. Kantarjian,
Moshe Talpaz, and
Bharat B. Aggarwal
From the Departments of Bioimmunotherapy, Molecular Oncology, and
Leukemia, The University of Texas M. D. Anderson Cancer Center,
Houston, TX.
 |
ABSTRACT |
Arsenic compounds have recently been shown to induce high rates of
complete remission in patients with acute promyelocytic leukemia (APL).
One of these compounds, As2O3, induces
apoptosis in APL cells via a mechanism independent of the retinoic acid pathway. To test the hypothesis that arsenic compounds may be effective
against other forms of acute myelogenous leukemia (AML), we studied the
membrane-permeable arsenic compound phenylarsine oxide (PAO). Because
interleukin-1 (IL-1) plays a key role in AML cell proliferation,
we first tested the effect of PAO on OCIM2 and OCI/AML3 AML cell lines,
both of which produce IL-1 and proliferate in response to it. We
found that PAO inhibited the proliferation of both OCIM2 and OCI/AML3
cells in a dose-dependent fashion (0.01 to 0.1 µmol/L) and that
IL-1 partially reversed this inhibitory effect. We then measured
IL-1 levels in these cells by using an enzyme-linked immunosorbent
assay and Western immunoblotting and found that PAO almost completely
abolished the production of IL-1 in these AML cells, whereas it did
not affect the production of IL-1 receptor antagonist. Because PAO
inhibits activation of the transcription factor NF- B and because
NF-B modulates an array of signals controlling cellular survival,
proliferation, and cytokine production, we also studied the effect of
PAO on NF-B activation in AML cells and found that PAO suppressed
the IL-1-induced activation of NF-B. Because inhibition of
NF-B may result in cellular apoptosis, we also tested whether PAO
may induce apoptotic cell death in AML cells. We found that PAO induced apoptosis in OCIM2 cells through activation of the cystein protease caspase 3 and subsequent cleavage of its substrate, the DNA repair enzyme poly (ADP-ribose) polymerase. The PAO-induced apoptosis was
caspase dependent, because it was completely blocked by the caspase
inhibitor Z-DEVD-FMK. Finally, we tested the effect of PAO on fresh AML
marrow cells from 7 patients with newly diagnosed AML and found that
PAO suppressed AML colony-forming cell proliferation in a
dose-dependent fashion. Taken together, our data showing that PAO is an
effective in vitro inhibitor of AML cells suggest that this compound
may have a role in future therapies for AML.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ACUTE MYELOGENOUS leukemia (AML) is a
clonal hematologic malignancy characterized by abnormal proliferation
of myeloid leukemia cells. Despite extensive clinical research with
numerous combinations of cytotoxic agents, the overall prognosis of
patients with AML remains poor (reviewed in Estey1). Thus,
the search for more effective agents continues.
Arsenic compounds, such as arsenic trioxide
(As2O3) and arsenic disulfide,2
which are occasionally used in traditional Chinese medicine, were found
to induce complete remission lasting for varying lengths of time in
more than 70% of patients with acute promyelocytic leukemia
(APL).3 In a recent study by Chen et al,4
As2O3 induced apoptosis in APL cells. Because
this apoptosis induction occurred independently of the retinoid
pathway,4 we therefore hypothesized that arsenic compounds
may effectively inhibit other forms of AML.
To test our hypothesis, we used phenylarsine oxide (PAO). This arsenic
compound, which is structurally different from the arsenicals mentioned
above, is a membrane-permeable molecule that inhibits phosphotyrosine
phosphatase (PTPase).5 Inhibition of PTPase by agents such
as PAO, in turn, inactivates the transcription factor NF-B in
various cell types, including hematopoietic cells.6 NF-B
modulates the effects of various transcription factors responsible for
the proliferation of normal myeloid and leukemia cells,7 and its activation induces the expression of various cytokines, including interleukin-1 (IL-1; reviewed in Seibenlist et
al8). IL-1 itself is a proinflammatory cytokine that
plays a major role in stimulating the proliferation of AML
cells.9-12 Thus, because PAO seemed to possess properties
sufficient to inhibit AML cells, we studied its effects on AML cell
lines and on fresh bone marrow (BM) cells from 7 patients with newly
diagnosed AML.
Consequently, we found that PAO inhibited the proliferation of both AML
cell lines and fresh AML marrow blast colony-forming cells and that PAO
also inhibited the IL-1-induced activation of NF-B and activated
caspase 3, thus inducing apoptotic cell death in AML cells.
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MATERIALS AND METHODS |
Cell lines.
The AML cell lines OCI/AML313 and OCIM214 were
kindly provided by M.D. Minden (Ontario Cancer Institute, Toronto,
Ontario, Canada). OCI/AML3 was established from an AML patient and
OCIM2 from a patient with erythroleukemia. Both cell lines proliferate in the presence of culture medium and fetal calf serum (FCS) without exogenous growth factors. The leukemia cell lines HL60 and K562 were
obtained from the American Type Culture Collection (ATCC; Rockville,
MD). All cells were maintained in RPMI-1640 culture medium (GIBCO,
Grand Island, NY) supplemented with 10% FCS (Flow Laboratories,
McLean, VA).
Subjects.
BM aspirates were obtained from 7 AML patients with high marrow blast
counts (see Table 1 for clinical data). All
studies were performed with the patients' informed consent and were
approved by the Human Experimentation Committee of our institution.
Cell line clonogenic assay.
Clonogenic assays were performed as previously described.15
Briefly, OCI/AML3, OCIM2, HL60, and K562 cells (2 to 4 × 104 cells/mL) were cultured in 0.8% methylcellulose (Fluka
Chemical Corp, Ronkonkoma, NY), 10% FCS, and RPMI-1640 medium in the
presence of PAO (Aldrich, Milwaukee, WI), which was dissolved in
dimethyl sulfoxide (DMSO) at a concentration of less than 0.1%, with
or without 10 ng/mL recombinant human (rh) IL-1 (molecular weight 17,500; Boehringer Mannheim Biochemicals, Indianapolis,
IN). The culture mixture was placed in 35-mm Petri dishes
(Nunc Inc, Naperville, IL) in duplicate or triplicate and maintained at
37°C with 5% CO2 in air in a humidified atmosphere.
Colonies were counted after 7 days by using an inverted microscope. A
colony was defined as a cluster of more than 40 cells.
Enzyme-linked immunosorbent assay (ELISA).
ELISAs were performed with IL-1 and IL-1 receptor antagonist
(IL-1RA) ELISA kits (Cistron Biotechnology [Pine Brook, NJ] and
Amersham Life Science [Arlington Heights, IL], respectively) as
previously described.16 Cell lysates and standard dilutions of either IL-1 or IL-1RA were added to test wells in duplicate and
incubated for 2 hours at 37°C. The test wells were then washed 3 times with phosphate-buffered saline (PBS), incubated with rabbit IL-1 antiserum for 2 hours, washed as previously described, and incubated for 30 minutes with goat antirabbit IgG conjugated to horseradish peroxidase. The test wells were vigorously washed, and a
substrate (o-phenylenediamine dissolved in 3% hydrogen
peroxide solution) and 4 N sulfuric acid were added. The color
intensity was read within 15 minutes at a wavelength of 490 nm with a
microplate autoreader (Model EL-309; Biotek, Winooski, VT). The average
net optical densities (OD) of the standard IL-1 and IL-1RA
concentrations were then plotted, and the amount of the corresponding
cytokine in each sample was determined by interpolation from the
standard curve.
Western immunoblotting for detection of IL-1.
Cell lysates were assayed for protein concentration with the BCA
Protein Assay Reagent kit (Pierce Chemical Co, Rockford, IL). Each set
of paired samples was then adjusted to have the same protein
concentration. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis was performed by using a
modification of the method of Laemmli.17 In brief, antigens were dissolved in Laemmli sample buffer at room temperature.
Electrophoresis was conducted at a constant wattage (10 W) in running
buffer cooled to 4°C. Stacking gels contained 4% (wt/vol)
acrylamide, and separating gels contained 12% (wt/vol) acrylamide.
Approximately 200 µL of sample protein was loaded into each of the
appropriate lanes. Proteins separated by SDS-PAGE were then transferred
to nitrocellulose membranes overnight at 30 V in a cooled (4°C)
reservoir containing transfer buffer (25 mmol/L Tris, 192 mmol/L
glycine, and 20% methanol, pH 8.3).18 Nitrocellulose
membranes were then removed from the blot apparatus and placed in a
Ponceau S staining solution (0.5% Ponceau S and 1% glacial acetic
acid in H2O) for 5 minutes to verify the equal loading of
protein in control and treated samples.19
After equal loading of protein was verified, the membranes were then
rinsed for an additional 10 minutes and immunoscreened. In brief, the
membranes were blocked in Blotto (5% dried milk dissolved in 50 mmol/L PBS) for at least 1 hour at room temperature. The membranes
were then washed 3 times in PBS plus 0.5% Tween 20. Next, the
membranes were incubated for 1 hour with polyclonal rabbit
anti-IL-1 antibodies (Endogen Inc, Boston, MA) or with normal
rabbit IgG (used as a control) diluted 1:200 in PBS containing 0.5%
Tween 20. After incubation, the membranes were subjected to three
15-minute rinses in PBS containing 0.5% Tween 20. Bound antibody was
detected with the ECL Western Blotting Detection System (Amersham Corp,
Arlington Heights, IL). The membranes were incubated with antirabbit
horseradish peroxidase-labeled antibody at a concentration of 1:2,000
in PBS plus 0.5% Tween 20 at room temperature for 1 hour. After this
incubation, the membranes were washed in PBS containing 0.5% Tween 20, and bound antibody was detected according to the ECL protocol. The
chemiluminescence of the membranes was detected by exposure to X-OMAT
AR5 x-ray film (Kodak, Rochester, NY) in stainless steel cassettes
(Sigma Chemical Co, St Louis, MO).
Electrophoretic mobility shift assay (EMSA) of NF-B
activation.
OCIM2 cells (2 × 104 cells/mL) were incubated
in the presence of increasing concentrations of IL-1 at 37°C.
Nuclear extracts were then prepared according to the method of Shreiber
et al.20 Briefly, 2 × 106 cells were
washed with cold PBS and suspended in a tube with 0.4 mL of lysis
buffer (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol [DTT], 0.5 mmol/L
phenylmethylsulfonyl fluoride [PMSF], 2.0 µg/mL leupeptin, 2.0 µg/mL aprotinin, and 0.5 mg/mL benzamidine). The tube was then placed
on ice, where the cells were allowed to swell for 15 minutes.
Afterward, 12.5 µL of 10% Nonidet P-40 was added. The tube was then
vigorously vortexed for 10 seconds to homogenize its contents and then
centrifuged for 30 seconds in a microcentrifuge. The nuclear pellet was
resuspended in 25 µL of ice-cold nuclear extraction buffer (20 mmol/L
HEPES, pH 7.9, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF, 2.0 µg/mL leupeptin, 2.0 µg/mL
aprotinin, and 0.5 mg/mL benzamidine), and the tube was
incubated on ice for 30 minutes with intermittent mixing. This nuclear
extract (NE) was then centrifuged for 5 minutes in a microcentrifuge at 4°C, and the supernatant was either used immediately or stored at
 70°C for later use. The protein content was measured by the method of Bradford.21
To determine NF-B activation, EMSAs were performed as previously
described.22 Briefly, 4 to 5 µg of NE was incubated with 16 fmol of 32P-end-labeled 45-mer double-stranded NF-B
oligonucleotides from the human immunodeficiency virus-1 long terminal
repeat (HIV-LTR; 5'-TTGTTACAAGGGACTT-TCCGCTGGGGGACTTTTCCAGGGAGGCGTGG-3')
in the presence of 1 to 2 µg of poly(dI-dC) in a binding buffer (25 mmol/L HEPES, pH 7.9, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 1% Nonidet
P-40, 5% glycerol, and 50 mmol/L NaCl) for 20 minutes at 37°C (we
used HIV-LTR-containing NF-B binding sites in this assay because
myeloid cells are targets for HIV). The DNA-protein complexes formed
were separated from free oligonucleotides on a 4.5% or 7.5% native polyacrylamide gel using a buffer containing 50 mmol/L Tris, 200 mmol/L
glycine, pH 8.5, and 1 mmol/L EDTA, after which the gel was dried. A
mutated oligonucleotide probe was used to examine the specificity of
NF-B's binding to the DNA. For supershift assays, NE was incubated
with the antibodies for 15 minutes at room temperature before analyzing
the NF-B by EMSA. Radioactive bands were visualized with a Phosphor
Imager (Molecular Dynamics, Sunnyvale, CA) using Imagequant software.
TdT-mediated dUTP nick-end labeling (TUNEL) assay for detection of
apoptosis.
The apoptosis detection system Fluorescein (Promega, Madison, WI) was
used to perform TUNEL assays.23 Briefly, 4%
formaldehyde-treated cytospun cells were made permeable with 0.2%
Triton-100 in PBS. After washing, slides were treated with
equilibration buffer (supplied with kit) and then incubated with a TdT
buffer (prepared according to the manufacturer's instructions) for 60 minutes. The staining reaction was terminated by treating the slides
with 2× SSC for 15 minutes. After washing, the slides were
treated with an anti-fade solution and then mounted on slides with
glass coverslips and rubber cement. The slides were analyzed using a
fluorescence microscope.
Western immunoblotting for detection of caspase 3 and PARP proteins.
Cell lysates (from 5 × 105 cells) were used as
described above. The following antibodies were used to detect the
relevant proteins: monoclonal mouse antihuman CPP32 (Transduction
Laboratories, Lexington, KY) to detect uncleaved caspase 3, polyclonal
rabbit antihuman CPP32 (PharMingen, San Diego, CA) to detect cleaved
caspase 3, and mouse antihuman PARP (Upstate Biotechnology, Lake
Placid, NY) to detect PARP. Normal mouse IgG and normal rabbit serum
were used as a control. To confirm detection of uncleaved caspase 3, Jurkat cells (ATCC) were used; to confirm detection of cleaved caspase
3 and PARP, 3T3 cells (ATCC) and HeLa cell (ATCC) nuclear extracts were
used, respectively. Bound antibody was detected according to the ECL
protocol (Amersham Life Science) as described above.
Adherent-cell fractionation.
Low-density BM mononuclear cells obtained by Ficoll-Hypaque (Pharmacia,
Piscataway, NJ) fractionation were incubated in plastic tissue-culture
dishes or flasks (Falcon Plastics; Becton Dickinson, Oxnard, CA) with
10% FCS in  -medium (GIBCO). The fractionation procedure was
repeated until no cells adhered to the tissue-culture dishes.
Nonadherent cells harvested in this way contained less than 3%
monocytes, as confirmed by the following techniques: (1) microscopic
differential counting of at least 100 cells prepared with Wright's
stain and (2) nonspecific (-naphthyl butyrate) esterase staining and
immunocytochemical analysis with CD14 monoclonal antibodies (Becton
Dickinson) to identify monocyte-promonocyte cells, as previously
described.24,25
T-cell depletion.
T cells were depleted from the nonadherent fraction by negative
immunomagnetic selection.26 In a modification
of this technique, nonadherent BM cells were incubated with CD3
monoclonal antibodies (Becton Dickinson) at a concentration of 1 µg/106 cells in PBS with 0.25% FCS for 30 minutes at
4°C. The labeled cells were washed 3 times and then incubated with
goat antimouse IgG-conjugated immunomagnetic beads (Advanced Magnetics,
Cambridge, MA) at 4°C for 60 minutes in an end-over-end rotation at
a 20:1 bead:cell ratio. Immunomagnetic bead-rosetted cells were removed with a magnetic particle concentrator (Advanced Magnetics), and unrosetted cells remaining in suspension were harvested by a Pasteur pipette. In some experiments, this procedure was repeated twice. The
T-lymphocyte-depleted population contained less than 3% CD3 cells as
assessed by an immunocytochemical technique performed on cytospun
cells.24,25
AML blast colony assay.
A previously described method was used to assay AML blast colony
formation.27,28 Briefly, 1 × 105 nonadherent T-cell-depleted BM cells were plated in
0.8% methylcellulose in -medium supplemented with 10% FCS and 50 ng/mL recombinant human granulocyte-macrophage colony-stimulating
factor (rhGM-CSF; Immunex Corp, Seattle, WA). PAO was
dissolved in DMSO and added at the initiation of the cultures at
concentrations ranging from 0.01 to 0.1 µmol/L in the absence or
presence of 10 U/mL of IL-1. The cultures were incubated in 35-mm
Petri dishes in duplicate or triplicate for 7 days at 37°C in a
humidified atmosphere of 5% CO2 in air. AML blast colonies
were microscopically evaluated on day 7 of culture. A blast colony was
defined as a cluster of 20 or more cells. Individual colonies were
plucked, smeared on glass slides, and stained to confirm their leukemic
cell composition. (That the AML blast colony assay identifies blasts
rather than normal progenitors had been previously demonstrated by
cytogenetic analysis of colonies obtained using this
assay.29)
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RESULTS |
PAO inhibits leukemia cell line colony proliferation.
We began studying the effect of PAO on the proliferation of OCI/AML3
and the OCIM2 cell lines and found that PAO suppressed their
colony-forming cell growth in a dose-dependent fashion at concentrations ranging from 0.01 to 0.1 µmol/L
(Fig 1, upper panel). Although the OCI/AML3
cells were more sensitive to PAO than were OCIM2 cells, both cell lines
were much more sensitive to PAO than either HL60 or K562 cells. Indeed,
the growth of OCI/AML3 and OCIM2 cells was almost completely abolished
by PAO at concentrations of 0.05 and 0.1 µmol/L, respectively,
whereas HL60 and K562 cells were only partially inhibited (27% and
61%, respectively) by 0.1 µmol/L (Fig 1, lower panel). The DMSO that
was used to dissolve PAO had no effect on the proliferation of either
cell line.
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| Fig 1.
Effect of PAO on AML cell line colony proliferation. The
upper panel shows the effect of PAO on OCIM2 and OCI/AML3 cells, and
the lower panel shows the effect on HL60 and K562 cells. Each data
point represents the mean colony number ± SD in triplicate
cultures.
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IL-1 partially reverses the inhibitory effect of PAO.
Because OCI/AML3 and OCIM2 cells, unlike HL60 and K562 cells,
proliferate in response to IL-1,12,30 we also tested
whether IL-1 could affect the inhibitory effect of PAO. We found
that 10 ng/mL IL-1 added at the initiation of culture partially
reversed PAO's suppressive effect (Fig 2).
This is in keeping with previous studies in which we found that (1)
OCI/AML3 and OCIM2 cells produce large quantities of IL-1, which
maximally stimulates their proliferation in an autocrine fashion; (2)
the addition of exogenous IL-1 could not significantly stimulate
their further growth; and (3) IL-1 antibodies could suppress their
growth.12,30 Our current results therefore suggested that
PAO might suppress IL-1 production by OCI/AML3 and OCIM2 cells.
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| Fig 2.
Effect of PAO and IL-1 on OCIM2 and OCI/AML3 colony
proliferation. Each data point represents the mean colony number ± SD
in triplicate cultures. PAO was added to each culture at a final
concentration of 0.08 µmol/L and IL-1 at 10 ng/mL.
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PAO inhibits IL-1 protein production.
In light of the results given above and published data indicating that
PAO inhibits NF-B,6 a binding site known to be present
in the IL-1 promoter,31,32 we hypothesized that PAO inhibits IL-1. To test this idea, we first incubated the cells with
PAO and measured IL-1 protein levels in lysates of OCI/AML3 and
OCIM2 cells. Using ELISAs, we found that 24 hours of incubation with
0.1 µmol/L PAO significantly reduced the production of IL-1 but
not of IL-1RA protein by both cell types
(Fig 3). These results indicated that the
effect of PAO on IL-1 production is specific and does not result
from a general suppression of protein synthesis. Because the ELISA
detects both the uncleaved and cleaved forms of IL-1, we also used
Western immunoblotting to measure active (cleaved) IL-1 in OCIM2
line, whose morphology and origin do not resemble those of APL, and
found that PAO significantly suppressed the production of mature
IL-1 (Fig 4).
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| Fig 3.
Effect of PAO on the production of IL-1 and IL-1RA by
OCIM2 and OCI/AML3 cells. Cells were incubated in the presence or
absence of 1.0 µmol/L PAO for 24 hours. The amount of IL-1 (upper
panel) and IL-1RA (lower panel) produced by these cells was then
assessed by ELISA as described in Materials and Methods.
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| Fig 4.
Effect of PAO on the production of mature IL-1 by
OCIM2 cells. Cells were incubated in the presence and absence of 0.1 µmol/L PAO. The amount of mature IL-1 protein produced by these
cells was then assessed by Western immunoblotting. The arrow points to
the 17.5-kD mature IL-1 protein. Lane C shows control mature IL-1
protein, lane 1 shows protein from cells incubated in tissue culture
media with DMSO, and lane 2 shows protein from cells incubated with
PAO.
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PAO inhibits IL-1-induced NF-B activation.
Because IL-1 is known to activate NF-B,8 we sought to
determine whether IL-1 activates NF-B in OCIM2 cells. We did so by incubating the cells for 1 hour in the presence of increasing concentrations of IL-1 and then testing them for NF-B activity by
EMSA. As shown in Fig 5A, NF-B
activation increased with IL-1 dose, reaching a maximum at 10 ng/mL
IL-1. Next, we examined the effect of increasing concentrations of
PAO on IL-1-induced NF-B activation. For this, OCIM2 cells were
treated for 1 hour with 0.1, 0.3, and 1.0 µmol/L PAO and then for 1 more hour with the addition of 10 ng/mL of IL-1 to activate NF-B.
As shown in Fig 5B, PAO abolished the IL-1-induced NF-B
activation in a dose-dependent manner, with maximum inhibition
occurring at a PAO concentration of 1.0 µmol/L (the minor activation
of NF-B induced by 1.0 µmol/L of PAO was not significant by a
quantitative analysis, as found in our previous
study33).
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| Fig 5.
(A) Activation of NF-B by IL-1. OCIM2 cells (2 × 106/mL) were incubated at 37°C with increasing
concentrations of IL-1 for 1 hour. Nuclear extracts were then
prepared and assayed for NF-B as described above. (B) Inhibition of IL-1-induced NF-B activation by PAO. Cells were
treated with PAO for 1 hour and then with IL-1 for 1 hour. Nuclear
extracts were prepared and assayed for NF-B activity as described in
Materials and Methods.
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The specificity of the NF-B band in the EMSAs was demonstrated by
its ability to compete with a cold oligo but not with an oligo
containing a mutated NF-B site. Thus, these results show that PAO
blocked IL-1-induced NF-B activation.
PAO induces apoptosis in OCIM2 cells.
Because a lack of NF-B activation may abolish cellular proliferation
and lead to apoptotic cell death34,35 and the arsenic compound AS2O3 can induce apoptosis in APL
cells,4 we hypothesized that PAO might have a similar
effect on OCIM2 cells. To test this idea, OCIM2 cells at the peak of
their growth were washed and then incubated in PBS in the presence and
absence of 0.1 µmol/L PAO for 4, 6, and 8 hours. Using the TUNEL
assay, we found that PAO induced apoptosis in these AML cells and that
longer exposure to this compound increased the number of cells
undergoing apoptotic cell death
(Fig 6).
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| Fig 6.
Induction of apoptosis by PAO. OCIM2 cells were incubated
in the absence (A) and presence of PAO for 4 (B), 6 (C), and 8 (D)
hours. Apoptotic cells appear yellow.
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PAO induces apoptosis by cleaving caspase 3.
To determine the mechanism by which PAO induces apoptosis, OCIM2 cells
were incubated in the absence and presence of 0.06, 0.08, 0.1, 0.4, 0.6, and 0.8 µmol/L of PAO for 4 hours and then harvested for Western
immunoblot analysis as described above. As shown in
Fig 7, PAO downregulated the expression of
uncleaved PARP protein in a dose-dependent fashion. Because caspase
activation seems to be an essential step in PARP cleavage and cellular
apoptosis36-38 and because caspase 339-41
appears to be involved in apoptosis induced in leukemia
cells,42-44 we measured the levels of uncleaved and cleaved
caspase 3 in OCIM2 AML cells. As shown in
Fig 8, we found that the incubation of
OCIM2 cells with 0.08 and 0.1 µmol/L of PAO upregulated the levels of
the biologically active (cleaved) caspase 3 and the inactivated
(cleaved) form of the DNA-repair enzyme PARP,36-38 thereby
activating the apoptotic cascade. To further investigate whether
caspase activation is essential for PAO-induced apoptosis, we incubated
OCIM2 cells with 0.1 µmol/L of PAO with and without 50 µmol/L of
the caspase inhibitor Z-DEVD-FMK.45,46 Using the TUNEL
assay, we found that Z-DEVD-FMK completely blocked PAO-induced
apoptosis (Fig 9).
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| Fig 7.
Effect of PAO on PARP protein expression. OCIM2 cells
were incubated in the absence and the presence of increasing
concentrations of PAO. The results shown here were obtained after
incubating OCIM2 cells for 4 hours.
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| Fig 8.
Effect of PAO on caspase 3 and PARP cleavage. OCIM2 cells
were incubated without PAO (lane C) and with it at a concentration of
0.08 µmol/L (lane 2) or 0.1 µmol/L (lane 1) for 8 hours. Cleavage
of caspase 3 was already detected after 4 hours. Increments in the
levels of cleaved caspase 3 (A) and cleaved PARP (B) are depicted.
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| Fig 9.
Effect of Z-DEVD-FMK on PAO-induced apoptosis. OCIM2
cells were incubated for 6 hours without any drug (A), with 0.1 µmol/L of PAO (B), with 50 µmol/L of Z-DEVD-FMK (C), and with both
PAO and Z-DEVD-FMK (D). Apoptotic cells appear yellow.
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PAO inhibits fresh AML blast colony-forming cell proliferation.
We then studied the effect of PAO on the proliferation of fresh AML
marrow blast colony-forming cells. For this, we used diagnostic BM
cells from 7 AML patients whose clinical characteristics are depicted
in Table 1. As shown in Fig 10, PAO
inhibited AML blast colony-forming cell growth in a dose-dependent
manner in all of the samples studied. Similar to its effect on AML cell
lines, IL-1, when added at the initiation of culture, partially
reversed the inhibitory effect of PAO
(Fig 11).
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| Fig 10.
Effect of PAO on fresh AML blast colony-forming cells.
After adherent cell fractionation of adherent cells and depletion of T
lymphocytes, remaining cells were cultured in a clonogenic assay with
PAO at concentrations ranging from 0.01 to 0.1 µmol/L. AML colonies
are presented as the percentage of control (the mean number of colonies
obtained in the absence of PAO). Data from patients no. 1 through 6 are
depicted.
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| Fig 11.
Effect of PAO and IL-1 on proliferation of AML
colony-forming cells. Data from triplicate cultures of marrow samples
from patient no. 6 (Expt I) and patient no. 7 (Expt II) are depicted.
PAO (0.1 µmol/L) and IL-1 (10 ng) were added at the initiation of
culture.
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| |
DISCUSSION |
Throughout history, arsenic compounds have been useful therapeutic
agents against many human ailments.47 The antileukemic properties of arsenic have been known since the mid
1800s.47 Arsenicals together with irradiation were the
treatment of choice for chronic myelogenous leukemia until busulfan was
introduced in 1953.48 However, in the late 20th
century, research has concentrated on the toxic effects of
arsenic compounds. Several investigators have shown a relationship
between ingestion of or exposure to various arsenicals and the
occurrence of lung,49 skin,50
bladder,51 and hepatocellular52 cancer, and
numerous studies have demonstrated that arsenic compounds are
environmental carcinogens.53 Yet, recent reports from China
show that As2O3 and arsenic disulfide can
induce complete remission of APL2,3,54,55 via pathways different from those used by retinoids.4,56 These studies have prompted other investigators to explore the effects of various arsenicals on APL and other leukemias.57-59
The arsenical PAO is a membrane-permeable PTPase inhibitor that is
active in hematopoietic cells.5,60 At high concentrations it causes nonspecific leakage in mitochondria. PAO also has been shown
to inhibit early elevations in cytosolic calcium
concentrations61 and to interfere with the insulin
transduction pathway.62 Because PAO is also known to
inhibit the activation of NF-B6 in hematopoietic cells,
we therefore sought to investigate its effects on AML.
NF-B is a ubiquitous transcription factor and a major regulator of
the immune system through its induction of expression of various
inflammatory cytokines including IL-1.32,33 NF-B exists in the cytoplasm as a heterotrimeric complex with the inhibitor IB (reviewed in Seibenlist et al63). Within minutes
of activation by inflammatory agents such as IL-1, IB
undergoes phosphorylation, ubiquitination, and proteolytic degradation,
thus releasing the NF-B p50-p65 complex for translocation from the
cytoplasm to the nucleus. Whereas the activation of NF-B induces
cellular proliferation8 and protects cells from apoptotic
cell death,35,64 its inhibition enhances spontaneous
apoptosis34 or apoptosis induced by various stimuli such as
irradiation or cytotoxic drugs.35
Several growth factors regulate hematopoietic cell survival by
interfering with apoptotic signals.65-67 One of these is
the cytokine IL-1, a proinflammatory protein that has been
implicated in early events in hematopoiesis. It induces the production
of various cytokines and synergizes with several growth factors in stimulating hematopoietic progenitor multiplication.31,68
In addition, as we and others have found, IL-1 plays an important role in AML cell proliferation (reviewed in Estrov et
al68). Suppression of IL-1 production or inhibition of its
interaction with the corresponding cellular receptors significantly
inhibits AML progenitor cell growth.9-12,30,69 Furthermore,
the activation of NF-B appears to be an important step in the
molecular events leading to IL-1 production8,31,32 and,
as a result, also appears to stimulate leukemia cell proliferation.
In this light, we assumed that an effective NF-B inhibitor such as
PAO might either suppress the production of IL-1, inhibit the direct
NF-B-mediated leukemia cell proliferation,7 or both. In
a previous study70 we have already demonstrated that PAO
can block the NF-B-dependent expression of various adhesion molecules, thus suggesting that PAO inhibits the activity of NF-B. Now, in our current study, we have found that PAO inhibits the proliferation of HL60 and K562 and, more significantly, of the IL-1-responsive OCI/AML3 and OCIM2 cells, that PAO suppressed the
growth of the IL-1-responsive lines in a dose-dependent manner, and
that IL-1 partially reverses this inhibitory effect. Together, these
results suggest that at least part of the PAO-induced suppression observed in the present study was mediated through PAO's inhibition of
IL-1 production. Indeed, incubation of the OCI/AML3 and OCIM2 cell
lines in the presence of PAO almost completely abolished the production
of IL-1 but not IL-1RA protein. In addition, PAO significantly
inhibited the IL-1-induced NF-B activity. Whereas IL-1
activated NF-B in these cells, PAO suppressed it in a dose-dependent fashion. Thus, PAO inhibited both IL-1 production and the
IL-1-mediated activation of NF-B, resulting in an additional
reduction in the production of IL-1.
Because NF-B activation suppresses the signals for cell death and
inhibition of NF-B may result in apoptotic cell
death,34,35 we tested the effect of PAO on the induction of
apoptosis. We found that treatment of leukemia cells with PAO induced
apoptotic cell death. Our results agree with those of Jimi et
al,71 who recently found that inhibition of NF-B by
oligodeoxynucleotides to p65 and p50 abolished the IL-1-induced
survival of osteoclasts.
Because most cell types require activation of a specific proteolytic
cascade if apoptosis is to occur, we also wondered whether PAO might
help activate that cascade in AML cells. In particular, we chose to
study the effect of PAO on caspase 3. Caspase 3 is a key executioner of
apoptosis39-41 whose activation downstream in the apoptotic
cascade is essential for leukemia cell apoptosis.33,42,43 Moreover, the activation of caspase 3 results in the cleavage of
cellular substrates critical for cell survival, such as PARP and
lamins, which, in turn, precipitates the morphological changes characteristic of apoptosis (reviewed in Cohen72). This
approach of ours found support in the work of Zhang et
al,73 who had already shown that arsenic trioxide
downregulates the expression of bcl-2, an antiapoptotic protein known
to inhibit the activation of caspase 3. As hoped, we found that PAO
activated caspase 3 and consequently cleaved PARP. Interestingly,
Barkett et al74 have recently reported that caspase 3 cleaves human I-B- in vitro at a conserved Asp-Ser sequence, thus
creating a dominant inhibitor that prevents the activation of NF-B
and thereby adding another death signal.
Similar to its effect on AML cell lines and comparable to the effect of
other IL-1 inhibitors,10-12,30 PAO suppressed AML progenitor proliferation and had its inhibitory effect partially reversed by IL-1. These results indicate that the inhibition of
IL-1 production is part of PAO's inhibitory mechanism in AML cells.
Taken together, our data suggest that PAO, either through inhibition of
NF-B, suppression of IL-1 production, or both, may eliminate
leukemia cells and so prove to be an effective agent in the treatment
of AML.
| |
ACKNOWLEDGMENT |
The authors thank Jude Richard for editing the manuscript.
| |
FOOTNOTES |
Submitted October 9, 1998; accepted June 9, 1999.
Supported in part by National Cancer Institute Grant No. PO1CA 55164 and by the Clayton Foundation for Research.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Zeev Estrov, MD, Department of
Bioimmunotherapy, Box 302, University of Texas M. D. Anderson Cancer
Center, 1515 Holcombe Blvd, Houston, TX 77030.
| |
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Q. Sun, S. Zachariah, and P. M. Chaudhary
The Human Herpes Virus 8-Encoded Viral FLICE-inhibitory Protein Induces Cellular Transformation via NF-{kappa}B Activation
J. Biol. Chem.,
December 26, 2003;
278(52):
52437 - 52445.
[Abstract]
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Z. Estrov, S. Shishodia, S. Faderl, D. Harris, Q. Van, H. M. Kantarjian, M. Talpaz, and B. B. Aggarwal
Resveratrol blocks interleukin-1{beta}-induced activation of the nuclear transcription factor NF-{kappa}B, inhibits proliferation, causes S-phase arrest, and induces apoptosis of acute myeloid leukemia cells
Blood,
August 1, 2003;
102(3):
987 - 995.
[Abstract]
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Q. Sun, H. Matta, and P. M. Chaudhary
The human herpes virus 8-encoded viral FLICE inhibitory protein protects against growth factor withdrawal-induced apoptosis via NF-kappa B activation
Blood,
March 1, 2003;
101(5):
1956 - 1961.
[Abstract]
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A. C. Bharti, N. Donato, S. Singh, and B. B. Aggarwal
Curcumin (diferuloylmethane) down-regulates the constitutive activation of nuclear factor-kappa B and Ikappa Balpha kinase in human multiple myeloma cells, leading to suppression of proliferation and induction of apoptosis
Blood,
February 1, 2003;
101(3):
1053 - 1062.
[Abstract]
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C. D. Chen and C. L. Sawyers
NF-{kappa}B Activates Prostate-Specific Antigen Expression and Is Upregulated in Androgen-Independent Prostate Cancer
Mol. Cell. Biol.,
April 15, 2002;
22(8):
2862 - 2870.
[Abstract]
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P. Fiumara, V. Snell, Y. Li, A. Mukhopadhyay, M. Younes, A. M. Gillenwater, F. Cabanillas, B. B. Aggarwal, and A. Younes
Functional expression of receptor activator of nuclear factor kappa B in Hodgkin disease cell lines
Blood,
November 1, 2001;
98(9):
2784 - 2790.
[Abstract]
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A. Stucki, A.-S. Rivier, M. Gikic, N. Monai, M. Schapira, and O. Spertini
Endothelial cell activation by myeloblasts: molecular mechanisms of leukostasis and leukemic cell dissemination
Blood,
April 1, 2001;
97(7):
2121 - 2129.
[Abstract]
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F. Chen, Y. Lu, Z. Zhang, V. Vallyathan, M. Ding, V. Castranova, and X. Shi
Opposite Effect of NF-kappa B and c-Jun N-terminal Kinase on p53-independent GADD45 Induction by Arsenite
J. Biol. Chem.,
March 30, 2001;
276(14):
11414 - 11419.
[Abstract]
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ABSTRACT
INTRODUCTION