|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 1014-1022
NEOPLASIA
Arsenic induces apoptosis of multidrug-resistant human myeloid
leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or
Bcl-xL
Charles Perkins,
Caryn N. Kim,
Guofu Fang, and
Kapil N. Bhalla
From the Division of Clinical and Translational Research, Sylvester
Comprehensive Cancer Center, and the Department of Medicine, University
of Miami School of Medicine, Miami, FL.
 |
Abstract |
We investigated the in vitro growth inhibitory and apoptotic effects
of clinically achievable concentrations of
As2O3 (0.5 to 2.0 µmol/L)
against human myeloid leukemia cells known to be resistant to a number
of apoptotic stimuli. These included chronic myelocytic
leukemia (CML) blast crisis K562 and HL-60/Bcr-Abl cells,
which contain p210 and p185 Bcr-Abl, respectively, and HL-60 cell types
that overexpress Bcl-2 (HL-60/Bcl-2), Bcl-xL (HL-60/Bcl-xL), MDR (HL-60/VCR), or MRP (HL-60/AR) protein.
The growth-inhibitory IC50 values for
As2O3 treatment for 7 days against all these
cell types ranged from 0.8 to 1.5 µmol/L. Exposure to 2 µmol/L
As2O3 for 7 days induced apoptosis of all cell
types, including HL-60/Bcr-Abl and K562 cells. This was associated with the cytosolic accumulation of cyt c and preapoptotic mitochondrial events, such as the loss of inner membrane potential
(  m) and the increase in reactive oxygen species
(ROS). Treatment with As2O3 (2 µmol/L)
generated the activities of caspases, which produced the cleavage of
the BH3 domain containing proapoptotic Bid protein and poly
(ADP-ribose) polymerase. Significantly,
As2O3-induced apoptosis of HL-60/Bcr-Abl and
K562 cells was associated with a decline in Bcr-Abl protein levels,
without any significant alterations in the levels of
Bcl-xL, Bax, Apaf-1, Fas, and FasL. Although As2O3 treatment caused a marked increase in the
expression of the myeloid differentiation marker CD11b, it did not
affect Hb levels in HL-60/Bcr-Abl, K562, or HL-60/neo cells. However,
in these cells, As2O3 potently induced
hyper-acetylation of the histones H3 and H4. These findings
characterize As2O3 as a growth inhibiting and
apoptosis-inducing agent against a variety of myeloid leukemia cells
resistant to multiple apoptotic stimuli.
(Blood. 2000;95:1014-1022)
© 2000 by The American Society of Hematology.
| |
Introduction |
Arsenic trioxide (As2O3) is a
clinically active agent against acute promyelocytic leukemia
(APL).1,2 Treatment with clinically achievable
concentrations of As2O3 has been shown to cause
apoptosis and down-regulation of the anti-apoptotic Bcl-2 protein in
the APL NB4 cells.3 At lower concentrations (0.1 to 0.5 µmol/L), As2O3 was also demonstrated to
induce partial differentiation of NB4 cells.4 These
dose-dependent dual in vitro effects may explain the clinical activity
of As2O3 in APL.4
Recently, As2O3 was shown to inhibit growth,
reduce intracellular Bcl-2 levels, and induce apoptosis of several
other myeloid leukemia, multiple myeloma, and HTLV-1-transformed T
cells.5-7 Additionally, low concentrations of an organic
arsenical melarsoprol (0.1 µmol/L), but not
As2O3, were demonstrated to down-regulate Bcl-2
and to mediate apoptosis of B-leukemia cell lines.8 Both Bcl-2 and its homologue Bcl-xL confer resistance against
apoptosis by inhibiting the preapoptotic mitochondrial permeability
transition ( m), the cytosolic accumulation of cytochrome c (cyt
c), and the activation of the executioner caspases of
apoptosis.9-12 The mitochondrial effects of Bcl-2 include
an antioxidant effect.12,13 In this context, it is
noteworthy that the intracellular levels of the antioxidant glutathione
(GSH) were shown to modulate the cytotoxic effects of
As2O3 against lymphoma cells (ie, lower
glutathione levels resulted in enhanced cytotoxicity of
As2O3).14 Collectively, these
findings suggest that Bcl-2 or Bcl-xL may exert an
inhibitory effect on the antileukemic activity of
As2O3. In contrast, the effects of other
multidrug resistance proteins, including the mdr-1 encoded
P-glycoprotein and MRP, on the antileukemic activity of
As2O3 have not been investigated.15
Several reports have indicated that in leukemic blasts, the expression
of CML-associated Bcr-Abl tyrosine kinase also inhibits anti-leukemia
drug-induced mitochondrial m and cyt c release, thereby blocking
the activation of the downstream caspases and apoptosis.16-18 Bcr-Abl expression is known to increase
Bcl-xL levels and the activity of NFkB in myeloid leukemia
cells18,19; the latter has also been implicated in
conferring resistance to apoptosis.20 However, whether
As2O3 can overcome this resistance and trigger the cascade of preapoptotic molecular events in Bcr-Abl-positive cells
has not been reported. In the current report, we have demonstrated that
As2O3 induced the apoptosis of
multidrug-resistant acute myelocytic leukemia cells,
regardless of whether they overexpressed Bcl-2, Bcl-xL,
P-glycoprotein, or MRP. Significantly, we also presented evidence that
clinically achievable concentrations of As2O3
induced preapoptotic mitochondrial events, caspase activity, and
apoptosis of Bcr-Abl-positive cells. In conjunction with these effects,
As2O3 treatment produced a significant decline
in the Bcr-Abl protein levels in HL-60/Bcr-Abl and K562 cells.
Recently, it has been shown that some agents that promote
differentiation, apoptosis, or both of leukemic cells may concomitantly
inhibit the activity of the enzyme histone
deacetylase.21,22 This results in increased acetylation of
histones, which facilitates gene transcription.23 In this
study, we also demonstrated that the concentrations of As2O3 that induce apoptosis clearly increase
the acetylation of the intracellular histones H3 and H4 in
Bcr-Abl-positive leukemic blasts.
| |
Materials and methods |
Reagents
As2O3 and trichostatin A were purchased from
Sigma (St. Louis, MO). As2O3 was dissolved in
1.65 mol/L sodium hydroxide (NaOH) to make a stock solution of 1 mmol/L, which was serially diluted in RPMI 1640. A monoclonal
anti-Bcl-2 antibody was purchased from DAKO (Carpinteria, CA).
Polyclonal anti-Bcl-x and anti-Bax antibodies and monoclonal anti-cyt
c, anti-cIAP, and anti-Bcr-Abl antibodies were purchased from
Pharmingen (San Diego, CA). Rabbit anti-DFF (DNA fragmentation
factor),24 anti-Apaf-1,25 and anti-Bid antisera26 were kindly provided by Dr Xiaodong
Wang (University of Texas Southwestern Medical Center, Dallas, TX).
Cell culture and cell growth inhibition
Human leukemic cells HL-60/neo, HL-60/Bcl-2,
HL-60/Bcl-xL, HL-60/VCR, HL-60/AR, HL-60/Bcr-Abl, and the
erythroid blast crisis CML K562 cells were cultured and passaged as
previously described.15,18,27,28
Growth inhibitory effects of As2O3
Logarithmically growing cells were exposed to various concentrations
and exposure intervals (up to 7 days) of As2O3.
After these treatments with As2O3, aliquots of
cells were withdrawn and cell numbers were determined using a Coulter
particle count and size analyzer (Coulter, Hialeah, FL). Suspension
culture growth inhibition and the 50% inhibitory concentration values
(IC50) for As2O3 were determined as
previously described.29
Flow cytometric analysis of cell-cycle status and apoptosis
The flow cytometric evaluation of the cell-cycle status and
apoptosis was performed according to a modification of a previously described method.30 Briefly, untreated or
As2O3-treated cells were centrifuged, washed in
Hanks' balanced salt solution, and fixed in 70% ethanol. The tubes
containing the cell pellets were stored at  20°C for at least
24 hours. After this, the cells were centrifuged at 800g for 15 minutes, and the supernatant was discarded to remove ethanol
completely. The pellets were resuspended in 40 µL (for
2-3 × 106 cells) of phosphate-citrate buffer at
room temperature for 30 minutes. After this incubation, cells were
washed with 4 to 5 mL phosphate-buffered saline (PBS) and stained with
propidium iodide (PI) solution (20 µg/mL PI and 20 µg/mL RNAse A in
PBS) for 30 minutes. The samples were read on a Coulter Elite flow cytometer using Elite software program 4.0 for 2-color detection. The
percentage of cells in the apoptotic sub-G1 and the G1-S
phase and G2-M phases were calculated using Multicycle software
(Phoenix Flow Systems, San Diego, CA).
Western analyses of proteins
Western analyses of Bcl-2, Bcl-xL, Bax, Bid, Fas
receptor (CD95), Fas ligand (Fas L), Bcr-Abl, DFF, cIAP, and  -actin
were performed using specific antisera or monoclonal antibodies (see above), as described previously.15,18 Horizontal scanning
densitometry was performed on Western blots by using acquisition into
Adobe Photo Shop (Apple, Cupertino, CA) and analysis by the NIH Image Program (National Institutes of Health, Bethesda, MD). The expression of -actin was used as a control.
Histone acetylation analysis
Histones were acid-extracted from whole cells as described
previously21,22; 20 µg-isolated histones were subjected
to SDS-PAGE as above (15% gel). Ponceau stain (Sigma) visualization
was used as a control for the amount of protein loading. Antibodies
that specifically recognize the acetylated forms of histone H3 and H4
(Upstate Biotechnology, Lake Placid, NY) were used to detect hyperacetylated histones.
Measurement of mitochondrial membrane potential and ROS
For As2O3-induced changes in mitochondrial
membrane potential (m) and ROS, 5 × 105
HL-60/neo, HL-60/Bcr-Abl, and K562 cells were incubated with 40 nmol/L
3,3 dihexyloxacarbocyanine iodide or 5 µmol/L
dichlorodihydrofluorescein diacetate, respectively, and were analyzed
by flow cytometry, as described previously.18,31,32
Immunophenotyping for differentiation markers and hemoglobin
production
HL-60/neo, HL-60/Bcr-Abl, and K562 cells were treated with various
concentrations of As2O3 for up to 7 days. Cells
were then washed with PBS and resuspended in 100 µL FACS wash buffer
(PBS, 0.2% NaN3, 0.1% bovine serum albumin, 2% human
AB-positive serum, filtered by suction at 0.45 µm). After 10 µL PE
antihuman CD11b, CD33, CD34, or HLA-DR antibody (Pharmingen, San Diego,
CA) was added,33,34 the cells were incubated in the dark at
4°C for 30 minutes. Samples were then analyzed by flow cytometry.
Alternatively, untreated or As2O3-treated cells
were washed in PBS and intracellular hemoglobin levels were determined
by a previously described method.35
Preparation of S-100 fraction and Western blot analysis for
cytochrome c
Untreated and As2O3-treated cells were
harvested by centrifugation at 1000g for 10 minutes at 4°C.
Cell pellets were washed once with ice-cold PBS and resuspended with 5 vol buffer (20 mmol/L HEPES-KOH, pH 7.5, 10 mmol/L KCl, 1.5 mmol/L
MgCl2, 1 mmol/L sodium EDTA, 1 mmol/L sodium EGTA, 1 mmol/L
dithiothreitol, and 0.1 mmol/L phenylmethylsulfonyl fluoride),
containing 250 mmol/L sucrose. Cells were homogenized with a 22-gauge
needle, and the homogenates were centrifuged at 100,000g for 30 minutes at 4°C (S-100 fraction).18 Supernatants were
collected, and the protein concentrations of S-100 were determined by
the Bradford method (Bio-Rad, Hercules, CA). After that, 20 to 30 µg
S-100 was used for Western blot analysis of cyt c, as described
previously.15,18
Morphology of apoptotic cells
After treatment with or without As2O3,
50 × 103 cells were washed with PBS (pH, 7.3) and
resuspended in the same buffer. Cytospin preparations of the
cell suspensions were fixed and stained with Wright stain. Cell
morphology was determined by light microscopy. In all, 5 different
fields were randomly selected for counting of at least 500 cells. The percentage of apoptotic cells was calculated for each
experiment, as described previously.36
Apoptosis assessment by annexin-V staining
After drug treatment, 5 × 105 to
1 × 106 cells were washed in PBS and resuspended in
100 µL staining solution (containing annexin-V fluorescein and PI in
a HEPES buffer, annexin-V-Fluos Staining Kit; Boehringer-Mannheim,
Indianapolis, IN). After 15-minute incubation at room temperature,
cells were analyzed by flow cytometry. Annexin-V binds to those cells
that express phosphatidylserine on the outer layer of the cell
membrane, and PI stains the cellular DNA of those that have a
compromised cell membrane. This allows for the discrimination of live
cells (unstained with either fluorochrome) from apoptotic cells
(stained only with annexin-V) and necrotic cells (stained with both
annexin-V and PI).37
Statistical analysis
Significant differences between values obtained in a population of
leukemic cells treated with different experimental conditions were
determined by paired Student t test analyses. A 1-way analysis of variance was also applied to the results of the various treatment groups, and post hoc analysis was performed using the Bonferroni correction method.
| |
Results |
Effects of As2O3 on cell proliferation and
apoptosis
Recent reports indicate a broad spectrum of antileukemic
activity for As2O3.5,7 This
prompted us to investigate its efficacy against a variety of human
myeloid leukemia cell types that display a multidrug-resistant
phenotype caused by diverse mechanisms.15,18,27 The growth
inhibitory effects of 1, 2, and 10 µmol/L
As2O3 were determined after 3-, 5-, and 7-day exposure intervals. Figure 1
demonstrates that a dose-dependent growth inhibitory effect of
As2O3 was evident against the control
HL-60/neo, as well as against HL-60/AR, HL-60/Bcl-2,
HL-60/Bcl-xL, HL-60/Bcr-Abl, and K562 cells. After exposure
to clinically achievable concentrations of As2O3
(1 or 2 µmol/L) for 7 days, the relative degree of growth inhibition in the various cell lines was HL-60/neo > HL-60/VCR > HL-60/AR > HL-60/Bcr-Abl > K 562 > HL-60/Bcl-2 > HL-60/Bcl-xL (Figure 1). In these cell lines, the
IC50 values for As2O3 were determined to be between 0.8 and 1.5 µmol/L. Figure
2 shows the percentage of apoptotic cells
observed in the various cell types after exposure to
As2O3 (0.5 to 10 µmol/L) from 1 to 7 days. At the end of a 7-day exposure to 2 µmol/L
As2O3, approximately 30% to 50% of cells
showed either the morphologic features of apoptosis (data not shown) or
the cell-surface phosphatidylserine expression detectable by
annexin-V staining and flow cytometry (Table
1). After exposure to 2 µmol/L
As2O3 for 7 days, there was no significant difference in the apoptotic rate in HL-60/AR or HL-60/VCR versus HL-60/neo cells. However, a lower percentage of apoptotic cells was
observed in identically treated HL-60/Bcl-2,
HL-60/Bcl-xL, HL-60/Bcr-Abl, and K562 cells.
View larger version (23K):
[in this window]
[in a new window]
| Fig 1.
As2O3 (arsenic)-mediated growth
inhibition of K562 and the various HL-60 cell types.
Cells were incubated with the indicated concentrations and exposure
intervals of As2O3. After this treatment, total
number of cells were counted using a Coulter Z2 particle count and size
analyzer. Data represent the mean of 4 independent experiments.
|
|
View larger version (23K):
[in this window]
[in a new window]
| Fig 2.
Effects of As2O3 (arsenic) on the
apoptotic rate of K562 and the various HL-60 cell types.
Cells were treated with the indicated concentrations and exposure
intervals of As2O3, and percentage apoptotic
cells were characterized as those that stained with annexin-V and
excluded PI, using the annexin-V assay kit (see "Materials and
Methods"). Data represent the mean of 3 independent experiments.
|
|
As2O3-induced preapoptotic mitochondrial
events, cytosolic cyt c accumulation, and caspase activities
A previous report18 from our laboratory demonstrates
that the expression of Bcr-Abl in HL-60/Bcr-Abl (p185) and K562 (p210) produces resistance against antileukemic drug-induced mitochondrial m and cyt c release from the mitochondria into cytosol, caspase activation, and apoptosis. In the current study, we compared the effect
of As2O3 on the mitochondrial m and the
release of cyt c and on caspase activation in HL-60/neo versus
HL-60/Bcr-Abl or K562 cells. Figure 3
demonstrates that a 7-day exposure to 2 µmol/L
As2O3 produced similar levels of accumulation
of cyt c in the cytosol of HL-60/neo, HL-60/Bcr-Abl, and
K562 cells. This was associated with the cleavage of p116 poly
(ADP-ribose) polymerase (PARP) into its p85 and p31 fragments and with
the degradation of p45 DNA fragmentation factor (DFF) into its p30 and
p11 fragments (not shown). As has been reported,24 cleavage of PARP and DFF largely results from the generation of caspase-3 activity. DFF is known to be the inhibitory protein for the
endonuclease (caspase-associated DNase), which produces the DNA
fragmentation of apoptosis.38,39 Recently, a pathway has
been elucidated by which the activity of the upstream caspases can
cause the release of cyt c from the mitochondria to the cytosol,
resulting in Apaf-1-mediated sequential cleavage of caspase-9 followed
by caspase-3.40 Bid (p21), a BH3 domain containing
proapoptotic members of the Bcl-2 family, was cleaved
directly by caspase-8, and the C-terminal fragment (p14) acted on the
mitochondria to trigger cyt c release.26,41 As shown in
Figure 3, treatment with 1 or 2 µmol/L As2O3
for 7 days produced p21 Bid cleavage into its p14 fragment in HL-60/neo and in HL-60/Bcr-Abl and K562 cells, though more Bid cleavage occurred
in HL-60/neo cells treated with 1 µmol/L
As2O3. These data indicated that treatment with
As2O3 generated the activity of both the
upstream (caspase-8) and the effector caspase-3.
View larger version (69K):
[in this window]
[in a new window]
| Fig 3.
Molecular events of apoptosis induced by
As2O3 treatment.
HL-60 control (neo) and cells stably transfected with Bcr-Abl, as well
as K562 cells, were treated with the indicated concentrations of
As2O3 for 7 days; cells were then harvested for
the following Western blot analyses: (A) cytosolic levels of cytochrome
c; (B) full-length PARP (116 kd) and 1 of its cleaved fragments (85 kd); (C) DNA fragmentation factor (DFF45) and its cleaved intermediate
fragment (30 kd); (D) Bid proform (21 kd) and its 14-kd-activated
cleaved product.
|
|
Because the activated Bid acted on the mitochondria, we examined
whether As2O3-induced Bid cleavage and the
release of cyt c from mitochondria were associated with m and the
increase in ROS. Figure 4 demonstrates that
the treatment with 1 or 2 µmol/L As2O3
produced more m and the generation of ROS in HL-60/neo versus
HL-60/Bcl-Abl and K562 cells, though significant preapoptotic mitochondrial alterations were also produced in HL-60/Bcr-Abl and K562
cells after exposure to 2 µmol/L As2O3
(Figure 4, panels A1 and B1 versus panels A2, A3, B2, and B3). Reduced
mitochondrial effect of 1 µmol/L As2O3 in
HL-60/Bcr-Abl and K562 cells was consistent with decreased Bid cleavage
in these cells caused by this dose; however, after treatment with
As2O3, the cytosolic accumulation of cyt c was
similar in the 3 cell types (Figure 3). Table
2 also demonstrates the cell-cycle effects
of As2O3 (1 and 2 µmol/L for 7 days)
determined by flow cytometry in the control HL-60/neo versus multidrug
resistant HL-60/Bcr-Abl and K562 cells. As shown, treatment with 1 or 2 µmol/L As2O3 significantly increased the percentage of HL-60/Bcr-Abl and K562 cells accumulated in the G2/M phase of the cell cycle. Although in
HL-60/neo cells this was not obvious by flow cytometry (Table 2),
mitotically arrested HL-60/neo cells were observed by Wright staining
and light microscopy (data not shown). In comparison with HL-60/Bcr-Abl
and K562, a higher percentage of HL-60/neo cells underwent apoptosis
after treatment with As2O3. This could also
partly explain the low percentage of HL-60/neo cells in the
G2/M phase observed after exposure to As2O3. Our data corroborate a recent report
that demonstrates that As2O3-induced mitotic
arrest in myeloid leukemia cells coincides with loss of
viability.34
View larger version (33K):
[in this window]
[in a new window]
| Fig 4.
Reduction of the mitochondrial membrane potential
(m,) (A) and production of ROS (B) in untreated
control or As2O3-treated (7 days) HL-60/neo,
HL-60/Bcr-abl, and K562 cells.
As2O3 treatment increased the percentage of
HL-60/neo, HL-60/Bcr-abl, and K562 cells, which displayed low
m and high ROS production. Data are representative of
3 independent experiments.
|
|
Compared with the apoptotic rate in Table 1, detected by annexin-V
staining, Table 2 shows a lower rate of
As2O3-induced apoptosis, represented by the
percentage of sub-G1 cells containing hypodiploid amounts
of DNA. This is caused by the differences in the 2 methods used to
detect apoptosis. Flow cytometry to detect apoptotic cells with
hypodiploid DNA content may miss those apoptotic cells that undergo
apoptosis in the G2/M-arrested state and do not lose enough DNA from fragmentation to be detected as hypodiploid. Additional confirmation of the apoptosis data shown in Tables 1 and 2
was obtained by performing TUNEL assays on untreated and
As2O3-treated cells using the in situ cell
death detection kit (Boehringer-Mannheim, Indianapolis, IN). TUNEL
assay results (data not shown) were consistent with the flow cytometric
findings of the percentage sub-G1 cells containing
hypodiploid amounts of DNA (Table 2). As2O3
-induced mitochondrial m, ROS, and apoptosis (as detected
by annexin-V staining) were also determined after exposure to
HL-60/neo, HL-60/Bcr-Abl, and K562 cells at time points earlier than 7 days (ie, 24 and 72 hours). As shown in Table
3, (and compared with Tables 1 and 2),
there was a time-dependent increase in the effects of
As2O3 (1 or 2 µmol/L) on mitochondrial
m, ROS, and apoptosis of the 3 cell types. In general, the total
loss of cell viability detected by PI staining occurred after the
mitochondrial effects and annexin-V staining, as has been previously
reported.30,35,36
As2O3 treatment down-regulates Bcr-Abl but
not Bcl-xL in HL-60/Bcr-Abl or K562 cells
Previous reports18 indicate that ectopic expression of
Bcr-Abl in HL-60 cells is associated with a marked down-regulation of
Bcl-2 and an increased expression of Bcl-xL in HL-60 cells. Treatment with 1 or 2 µmol/L As2O3 for 7 days
did not cause any significant alterations in Bcl-2 in HL-60/neo or
Bcl-xL in HL-60/Bcr-Abl or K562 cells (Figure
5A). Bax, Apaf-1, cIAP, Fas L, and Fas
levels in the 3 cell types were also unaffected by treatment with
As2O3 (Figure 5). However, it is important to
note that a dose-dependent decline in p185 Bcr-Abl in HL-60/Bcr-Abl and
p210 Bcr-Abl in K562 was clearly observed (Figure 5A). At the higher
dose levels of As2O3, greater than or equal to
2 µmol/L, this was also observed after exposure to shorter intervals
(48 hours) (data not shown). Because Bcr-Abl expression is known to
exert resistance against apoptosis, the decline in Bcr-Abl levels may
explain why As2O3 treatment induced apoptosis
in HL-60/Bcr-Abl and K562 cells.
View larger version (41K):
[in this window]
[in a new window]
| Fig 5.
Intracellular level of protein modulators of apoptosis
and acetylation of histones H3 and H4.
(A) Western blot analysis of the levels of Bcr-abl, Apaf-1,
Bcl-xL, Bcl-2, Bax, Fas receptor (Fas), and Fas
ligand (FasL) in HL-60/neo, HL-60/Bcr-Abl, and K562 cells.
-Actin was used as a control for equal protein loading. (B)
Western blot analysis of acetylated histones H3 and H4 in response to
treatment with As2O3 (1 or 2 µmol/l for 7 days). (C) Western blot analysis of histone H3 and H4 after treatment
with 2 µmol/L As2O3 for 24 hours.
Hyperacetylation was detected by the use of antibody against acetylated
histone H3 and H4. Histones were acid-extracted from the indicated cell
lines after exposure to As2O3. The histone
deacetylase inhibitor trichostatin A (150 nmol/L, 24 hours) was used as
a positive control.
|
|
As2O3 induces hyperacetylation of histones
Recent reports42 indicate that hybrid polar compounds
and sodium phenylbutyrate, which induce terminal differentiation or apoptosis of leukemic cells, concomitantly induce hyperacetylation of
the histones. Based on this, we determined the effect of
As2O3 on the acetylation status of histones H3
and H4 in HL-60/neo, HL-60/Bcr-Abl, and K562 cells. Immunoblot analysis
in Figure 5B demonstrates that, after treatment with 1 or 2 µmol/L
As2O3 for 7 days, approximately, a 3- to 4-fold
increase in the amount of acetylated histones was observed in
HL-60/neo, HL-60/Bcr-Abl, and K562 cells. This approximated the amount
of histone hyperacetylation seen with treatment of these cells with
trichostatin A, a known inhibitor of histone deacetylase (Figure 5B).
Figure 5C demonstrates that the hyperacetylation of histones H3 and H4
was evident even after a shorter exposure to
As2O3 (2.0 µmol/L for 24 hours).
Effect of As2O3 on the immunophenotype
of myeloid leukemia cells
After exposure to 1 or 2 µmol/L As2O3,
flow cytometric analyses of the cell-surface expression of CD11b, CD33,
CD34, and HLA-DR in HL-60/neo, HL-60/Bcr-Abl, and K562 cells were
performed. Figure 6 demonstrates that
As2O3 markedly increased the percentage of cells expressing of the myeloid differentiation marker CD11b in all
cell types. Taken together with the data in Table 3, these data
demonstrate that there was a progressive increase in the percentage of
cells expressing CD11b in all cell types after exposure to 2 µmol/L
As2O3 from 24 hours to 7 days.
These data do not exclude the possibility that there
is a CD11b-positive subgroup of leukemic cells that is
relatively insensitive to As2O3 and is
selectively expanded during treatment with
As2O3. However, As2O3
treatment did not affect the expression of CD33, CD34, and
HLA-DR (data not shown). Because the differentiation of K562 cells was
shown in a previous study35 to be associated with increased
intracellular levels of hemoglobin, this was determined in the
untreated and the As2O3-treated cells.
Treatment with As2O3 did not induce hemoglobin production or morphologic differentiation in K562, HL-60/Bcr-Abl, or
HL-60/neo cells (data not shown).
View larger version (44K):
[in this window]
[in a new window]
| Fig 6.
As2O3 treatment induces CD11b
expression in HL-60/neo (A), HL-60/Bcr-abl (B), and K562 (C) cells.
Cells were treated with the indicated concentrations of
As2O3 for 7 days, and the percentage of cells
expressing CD11b on the cell surface was determined by
fluorescence-activated cytometry. Data are representative of 3 separate
experiments.
|
|
| |
Discussion |
Data presented here clearly demonstrate that exposure to clinically
achievable concentrations of As2O3 is able to
induce growth inhibition and apoptosis of human myeloid leukemia cells
resistant to multiple apoptotic stimuli. In these cells, apoptosis and
multidrug resistance were shown to be secondary to diverse mechanisms,
including the expression of Bcr-Abl or the overexpression of Bcl-2,
Bcl-xL, MDR, or MRP.15,18,27,29,34
As2O3-induced apoptosis of HL-60/VCR or
HL-60/AR cells was not significantly different from HL-60/neo cells
(P > .05). This suggests that As2O3
is not a substrate for the mdr-1 gene-encoded p-glycoprotein nor is it
exported by MRP. Compared with HL-60/neo, however,
As2O3-induced apoptosis was partially
attenuated in HL-60/Bcl-2, HL-60/Bcl-xL, HL-60/Bcr-Abl, and
K562 cells. Consistent with recent reports, our findings demonstrate that As2O3 (2 µmol/L for 7 days) was able to
induce mitochondrial m and cytosolic accumulation of cyt c in
HL-60/neo, HL-60/Bcr-Abl, and K562 cells.43,44
As2O3 also generated PARP, DFF, and Bid cleavage activities of caspases in these cells.9,22,24 This suggests that As2O3 is able to induce the
cleavage and activity of both the upstream (caspase-8) and the
downstream executioner caspase (such as caspase-3) in HL-60/neo,
HL-60/Bcr-Abl, and K562 cells.45 A recent
report34 indicates that As2O3
inhibits the binding of guanosine triphosphate to tubulin, its
polymerization, and its microtubule formation, resulting in mitotic
arrest of myeloid leukemia cells.34 This led to the
apoptosis of leukemic cells. Our results confirmed that
As2O3 treatment produces mitotic arrest (Table 2) and apoptosis (Table 1) of Bcr-Abl-positive and
Bcr-Abl-negative myeloid leukemia cells. However, as for
other antimicrotubule agents such as paclitaxel and vincristine,
the precise mechanism by which the mitotic arrest induced by
As2O3 is linked to preapoptotic mitochondrial
events, cyt c release, and caspase activity remains to be
elucidated.46
Bcr-Abl expression mediates resistance to apoptosis.47 we
have reported that HL-60/Bcr-Abl and K562 cells are highly resistant to
high-dose ara-C and etoposide-induced mitochondrial m, cytosolic accumulation of cyt c caspase activation, and apoptosis.18
In addition, ara-C and etoposide fail to alter p210 or p185 Bcr-Abl levels in these cells.18 In contrast, treatment with
As2O3 significantly down-regulates Bcr-Abl
levels in both cell types, which may explain why
As2O3 causes mitochondrial m,
accumulation of cyt c in the cytosol, and apoptosis of HL-60/Bcr-Abl
and K562 cells. These findings are consistent with a previous
report48 demonstrating that the abrogation of Bcr-Abl
expression by antisense oligonucleotides selectively eliminates CML
blast cells. In addition, the abrogation of Bcr-Abl activity by a
relatively specific tyrosine kinase inhibitor CGP57 148B recently has
been shown to cause in vitro and in vivo eradication of human Bcr-Abl
positive leukemia cells.49 Although ectopic or endogenous
Bcr-Abl expression is associated with the up-regulation of
Bcl-xL,18,35,47 our current data show that As2O3-mediated declines in Bcr-Abl levels do
not down-regulate Bcl-xL levels. Recently, it has been
shown that Bcl-xL is a caspase-3 substrate and that the
cleavage of Bcl-xL in the loop region releases a c-terminal
product that lacks the BH4 homology domain and induces cell
death.50,51 However, As2O3-induced
apoptosis of HL-60/Bcr-Abl and K562 cells was not seen to be associated
with Bcl-xL cleavage. Furthermore, As2O3
also did not alter Bax, Apaf-1, Fas L, Fas R, and cIAP levels.
Collectively, these findings point to
As2O3-mediated down-regulation of Bcr-Abl as
the key perturbation responsible for facilitating the apoptosis of
HL-60/Bcr-Abl and K562 cells. Bcl-2 or Bcl-xL
overexpression (as in HL-60/Bcl-2 or HL-60/Bcl-xL cells)
also inhibits preapoptotic mitochondrial events.9 However, the relative potency of Bcl-2 or Bcl-xL versus Bcr-Abl for
exerting this effect is unknown. As shown in the current study, though As2O3 does not lower Bcl-2 and
Bcl-xL levels in HL-60/neo, HL-60/Bcl-2, and
HL-60/Bcl-xL, the mitochondrial toxic effects of
As2O3 may be potent enough to overcome the
inhibitory effects of Bcl-2 and Bcl-xL and may induce
apoptosis of HL-60/neo, HL-60/Bcl-2, and HL-60/Bcl-xL
cells. In contrast, As2O3-induced
down-regulation of Bcr-Abl may be necessary for facilitating apoptosis
of HL-60/Bcr-Abl and K562 cells.
Recent studies have firmly established that targeted acetylation of the
internal lysine residues in the amino-terminal tails of histones
relieves nucleosomal repression, which limits the access of the
transcriptional machinery to the DNA template and facilitates
transcriptional activation.23,50 Histone acetylation is a
reversible process.23,52 Histone acetyltransferases
transfer the acetyl moiety from acetyl coenzyme A to the lysine
residues neutralizing the positive charge and increasing
hydrophobicity, whereas histone deacetylases remove the acetyl groups
and reestablish the positive charge in the histones.23,52
Recently, a class of hybrid polar compounds and sodium phenylbutyrate,
which induce terminal differentiation or apoptosis, were shown to
induce concomitantly the hyperacetylation of histones by inhibiting
histone deacetylase.21,22 Our data demonstrate, for the
first time, that clinically relevant concentrations of
As2O3 induce the hyperacetylation of histones H3 and H4. It is unclear whether this is a direct or an indirect effect
mediated through the modulation of transcriptional coactivators or
corepressors that may have histone acetyltransferase or deacetylase activity, respectively.42,53 The effect of these
corepressors and coactivators is selective. Some promoters and
transcription factors are blocked, whereas others are not, by this
recruitment of histone deacetylase or histone
acetyltransferase.42,53,54 As2O3-induced histone hyperacetylation may be
responsible for altering the transcription of a number of genes, which
may collectively mediate As2O3-induced growth
inhibition and apoptosis. Our studies do not establish whether the
As2O3-induced down-regulation of Bcr-Abl is
transcriptionally or posttranscriptionally regulated. If
As2O3 affects the transcription of the bcr-abl
fusion gene, this may also be mediated directly or indirectly by
altered gene-transcription and expression brought about by
As2O3-induced histone hyperacetylation. These
mechanistic issues would have to be resolved by future studies.
In summary, this article highlights the activity of
As2O3 against leukemic cells that are resistant
to apoptotic stimuli either because of the expression of Bcr-Abl or the
overexpression of Bcl-2, Bcl-xL, MDR, or MRP proteins.
These findings, as well as As2O3-induced
declines in Bcr-Abl, suggest that As2O3 should
be investigated for potential in vivo activity against refractory acute
myelocytic leukemia and CML.
| |
Footnotes |
Submitted March 31, 1999; accepted September 20, 1999.
Reprints: Kapil Bhalla, Division of Clinical and Translational
Research, Sylvester Comprehensive Cancer Center University of Miami
School of Medicine (M710), 1550 NW 10th Avenue, Miami, FL 33136;
e-mail: kbhalla{at}med.miami.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Soignet S, Maslak P, Wang ZG, et al.
Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide.
N Engl J Med.
1998;339:1341[Abstract/Free Full Text].
2.
Look AT.
Arsenic and apoptosis in the treatment of acute promyelocytic leukemia.
J Natl Cancer Inst.
1998;90:86[Free Full Text].
3.
Chen GQ, Zhu J, Shi XG, 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-RAR /PML proteins.
Blood.
1996;88:1052[Abstract/Free Full Text].
4.
Chen GQ, Shi XG, Tang W, 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[Abstract/Free Full Text].
5.
Wang ZG, Rivi R, Delva L, et al.
Arsenic trioxide and melarsoprol induce programmed cell death in myeloid leukemia cell lines and function in a PML and PML-RAR independent manner.
Blood.
1998;92:1497[Abstract/Free Full Text].
6.
Rousselo P, Labaume S, Marolleau J, et al.
Arsenic trioxide and melarsoprol induce apoptosis in plasma cell lines and in plasma cells from myeloma patients.
Cancer Res.
1999;59:1041[Abstract/Free Full Text].
7.
Bazarbachi A, El-Sabban M, Nasr R, et al.
Arsenic trioxide and interferon- synergize to induce cell cycle arrest and apoptosis in human T-cell lymphotropic virus type I-transformed cells.
Blood.
1999;93:278[Abstract/Free Full Text].
8.
Konig A, Wrazel L, Warrell R Jr, et al.
Comparative activity of melarsoprol and arsenic trioxide in chronic B-cell leukemia lines.
Blood.
1997;90:562[Abstract/Free Full Text].
9.
Yang J, Liu X, Bhalla K, et al.
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science.
1997;275:1129[Abstract/Free Full Text].
10.
Adams J, Cory S.
The Bcl-2 protein family: arbiters of cell survival.
Science.
1998;281:1322[Abstract/Free Full Text].
11.
Perkins C, Kim C, Fang G, Bhalla K.
Overexpression of Apaf-1 promotes apoptosis of untreated and paxlitaxel- or etoposide-treated HL-60 cells.
Cancer Res.
1998;58:4561[Abstract/Free Full Text].
12.
Green D, Reed J.
Mitochondria and apoptosis.
Science.
1998;281:1309[Abstract/Free Full Text].
13.
Hockenbery D, Oltvai Z, Yin XM, Milliman C, Korsmeyer.
Bcl-2 functions in an antioxidant pathway to prevent apoptosis.
Cell.
1993;75:241[Medline]
[Order article via Infotrieve].
14.
Dai J, Weinberg RS, Waxman S, Jing Y.
Malignant cells can be sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through modulation of the glutathione redox system.
Blood.
1999;93:268[Abstract/Free Full Text].
15.
Huang Y, Ibrado AM, Reed JC, et al.
Co-expression of several molecular mechanisms of multidrug resistance and their significance for paclitaxel cytotoxicity in human AML HL-60 cells.
Leukemia.
1997;11:253[Medline]
[Order article via Infotrieve].
16.
Bedi A, Barber JP, Bedi GC, et al.
BCR-ABL-mediated inhibition of apoptosis with delay of G2/M transition after DNA damage: a mechanism of resistance to multiple anticancer agents.
Blood.
1995;86:1148[Abstract/Free Full Text].
17.
McGahon A, Bissonnette R, Schmitt M, Cotter KM, Green DR, Cotter TG.
BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death.
Blood.
1994;83:1179[Abstract/Free Full Text].
18.
Amarante-Mendes G, Kim C, Liu L, et al.
Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome c and activation of caspase-3.
Blood.
1998;91:1700[Abstract/Free Full Text].
19.
Hamdane M, David-Cordonnier M-H, D'Halluin JC.
Activation of p65 NF-KB protein by p210 BCR - ABL in a myeloid cell line (p210 BCR - ABL activates p65 NF-KB).
Oncogene.
1997;15:2267[Medline]
[Order article via Infotrieve].
20.
Wang CY, Mayo MW, Baldwin JAS.
TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-KB.
Science.
1996;274:784[Abstract/Free Full Text].
21.
Warrell R Jr, He LZ, Richon V, Calleja E, Pandolfi PP.
Accelerated discovery: therapeutic targeting of transcription of acute promyelocytic leukemia by use of an inhibitor of histone deacetylase.
J Natl Cancer Inst.
1998;90:1621[Abstract/Free Full Text].
22.
Richon V, Emiliani S, Verdin E, et al.
A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases.
Proc Natl Acad Sci U S A.
1998;95:3003[Abstract/Free Full Text].
23.
Kuo MH, Allis D.
Roles of histones acetyltransferases and deacetylases in gene regulation.
Bioessays.
1998;20:615[Medline]
[Order article via Infotrieve].
24.
Liu X, Zou H, Slaughter C, Wang X.
DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis.
Cell.
1997;89:175[Medline]
[Order article via Infotrieve].
25.
Zou H, Henzel WJ, Liu X, Lutschg A, Wang X.
Apaf-1, a human protein homologous to C. elegans CED-4, participated in cytochrome c-dependent activation of caspase-3.
Cell.
1997;90:405[Medline]
[Order article via Infotrieve].
26.
Luo X, Budihardjo I, Zou H, Slaughter C, Wang X.
Bid, a Bcl-2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors.
Cell.
1998;94:481[Medline]
[Order article via Infotrieve].
27.
Ibrado AM, Huang Y, Fang G, Liu L, Bhalla K.
Overexpression of Bcl-2 or Bcl-xL inhibits Ara-C-induced CPP32/Yama protease activity and apoptosis of human acute myelogenous leukemia HL-60 cells.
Cancer Res.
1996;56:4743[Abstract/Free Full Text].
28.
Lozzio CB, Lozzio BB.
Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome.
Blood.
1975;45:321[Abstract/Free Full Text].
29.
Bhalla K, Hindenburg A, Taub R, Grant S.
Isolation and characterization of an anthracycline-resistant human leukemic cell line.
Cancer Res.
1985;45:3657[Abstract/Free Full Text].
30.
Ibrado A M, Huang Y, Fang G, Bhalla K.
Bcl-xL overexpression inhibits taxol- induced Yama protease activity and apoptosis.
Cell Growth & Differ
1996;7:1087[Abstract]
31.
Decaudin D, Geley S, Hirsch T, et al.
Bcl-2 and Bcl-xL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents.
Cancer Res
1997;57:62[Abstract/Free Full Text].
32.
Xiang J, Chao DT, Korsmeyer SJ.
BAX-induced cell death may not require interleukin 1 -converting enzyme-like proteases.
Proc Natl Acad Sci U S A.
1996;93:14,559[Abstract/Free Full Text].
33.
Freemerman AJ, Vrana JA, Tombes RM, et al.
Effects of antisense p21 (WAF1/CIP1/MDA6) expression on the induction of differentiation and drug-mediated apoptosis in human myeloid leukemia cells (Hl-60).
Leukemia.
1997;11:504[Medline]
[Order article via Infotrieve].
34.
Li YM, Broome J.
Arsenic targets tubulins to induce apoptosis in myeloid leukemia cells.
Cancer Res.
1999;59:776[Abstract/Free Full Text].
35.
Ray S, Bullock G, Nunez G, et al.
Enforced expression of Bcl-xS induces differentiation and sensitizes chronic myelogenous leukemia-blast crisis K562 cells to 1-B-D-Arabinofuranosylcytosine-mediated differentiation and apoptosis.
Cell Growth Differ.
1996;7:1617[Abstract].
36.
Ray S, Ponnathpur V, Huang Y, et al.
1--D-arabinofuranosylcytosine-, mitoxantrone- and paclitaxel-induced apoptosis in HL-60 cells: improved method for detection of internucleosomal DNA fragmentation.
Cancer Chemother Pharmacol.
1994;34:365[Medline]
[Order article via Infotrieve].
37.
Koopman G, Reutelingsperger CPM, Kuijten GAM, Keehnen RMJ, Pals ST, van Oers MHJ.
Annexin-V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.
Blood.
1994;84:1415[Abstract/Free Full Text].
38.
Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S.
A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.
Nature.
1998;391:43[Medline]
[Order article via Infotrieve].
39.
Tang D, Kidd V.
Cleavage of DFF-45/ICAD by multiple caspases is essential for its function during apoptosis.
J Biol Chem.
1998;273:28,549[Abstract/Free Full Text].
40.
Li P, Nijhawan D, Budihardjo I, et al.
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.
Cell.
1997;91:479[Medline]
[Order article via Infotrieve].
41.
Wang K, Yin XM, Chao DT, Milliman CL, Korsmeyer SJ.
BID: a novel BH3 domain-only death agonist.
Genes Dev.
1996;10:2859[Abstract/Free Full Text].
42.
Luo R, Postigo A, Dean D.
Rb Interacts with histone deacetylase to repress transcription.
Cell.
1998;92:463[Medline]
[Order article via Infotrieve].
43.
Zhu X-H, Shen Y-L, Jing Y-k, et al.
Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations.
J Natl Cancer Inst.
1999;91:772[Abstract/Free Full Text].
44.
Kroemer G, de Thé H.
Arsenic trioxide, a novel mitochondriotoxic anticancer agent?
J Natl Cancer Inst.
1999;91:743[Free Full Text].
45.
Green D.
Apoptotic pathways: the roads to ruin.
Cell.
1998;94:695[Medline]
[Order article via Infotrieve].
46.
Ibrado AM, Kim CN, Bhalla K.
Temporal relationship of CDK1 activation and mitotic arrest to cytosolic accumulation of cytochrome C and caspase-3 activity during Taxol-induced apoptosis of human AML HL-60 cells.
Leukemia.
1998;12:1930[Medline]
[Order article via Infotrieve].
47.
Amarante-Mendes G, McGahon A, Nishioka W, Afar D, Witte O, Green D.
Bcl-2-independent Bcr-Abl-mediated resistance to apoptosis: protection is correlated with up regulation of Bcl-xL.
Oncogene.
1998;16:1383[Medline]
[Order article via Infotrieve].
48.
Szczylik C, Skorski T, Nicolaides N, et al.
Selective inhibition of leukemia cell proliferation by Bcr-Abl antisense oligodeoxynucleotides.
Science.
1991;253:562[Abstract/Free Full Text].
49.
le Courte P, Malogni L, Cleris L, et al.
In vitro eradication of human Bcr/Abl-positive leukemia cells with an ABL kinase inhibitor.
J Natl Cancer Inst.
1999;91:163[Abstract/Free Full Text].
50.
Clem R, Cheng E, Karp C, et al.
Modulation of cell death by Bcl-xL through caspase interaction.
Proc Natl Acad Sci U S A.
1998;95:554[Abstract/Free Full Text].
51.
Fujita N, Nagahashi A, Nagashima K, Rokudai S, Tsuruo T.
Acceleration of apoptotic cell death after the cleavage of Bcl-xL protein by caspase-3-like proteases.
Oncogene.
1998;7:1295.
52.
Imof A, Wolffe A.
Transcription: gene control by targeted histone acetylation.
Curr Biol.
1998;8:422.
53.
Ait-Si-Ali S, Ramirez S, Barre F-X, et al.
Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A.
Nature.
1998;396:184[Medline]
[Order article via Infotrieve].
54.
Boyes J, Byfield P, Nakatani Y, Ogryzko V.
Regulation of activity of the transcription factor GATA-1 by acetylation.
Nature.
1998;396:594[Medline]
[Order article via Infotrieve].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Q.-Y. Zhang, J.-H. Mao, P. Liu, Q.-H. Huang, J. Lu, Y.-Y. Xie, L. Weng, Y. Zhang, Q. Chen, S.-J. Chen, et al.
A systems biology understanding of the synergistic effects of arsenic sulfide and Imatinib in BCR/ABL-associated leukemia
PNAS,
March 3, 2009;
106(9):
3378 - 3383.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. F. Taylor, S. C. McNeely, H. L. Miller, G. M. Lehmann, M. J. McCabe Jr., and J. C. States
p53 Suppression of Arsenite-Induced Mitotic Catastrophe Is Mediated by p21CIP1/WAF1
J. Pharmacol. Exp. Ther.,
July 1, 2006;
318(1):
142 - 151.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Nencioni, L. Wille, G. Dal Bello, D. Boy, G. Cirmena, S. Wesselborg, C. Belka, P. Brossart, F. Patrone, and A. Ballestrero
Cooperative Cytotoxicity of Proteasome Inhibitors and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand in Chemoresistant Bcl-2-Overexpressing Cells
Clin. Cancer Res.,
June 1, 2005;
11(11):
4259 - 4265.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Lunghi, A. Costanzo, M. Levrero, and A. Bonati
Treatment with arsenic trioxide (ATO) and MEK1 inhibitor activates the p73-p53AIP1 apoptotic pathway in leukemia cells
Blood,
July 15, 2004;
104(2):
519 - 525.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. La Rosee, K. Johnson, A. S. Corbin, E. P. Stoffregen, E. M. Moseson, S. Willis, M. M. Mauro, J. V. Melo, M. W. Deininger, and B. J. Druker
In vitro efficacy of combined treatment depends on the underlying mechanism of resistance in imatinib-resistant Bcr-Abl-positive cell lines
Blood,
January 1, 2004;
103(1):
208 - 215.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Chandra, J. Hackbarth, S. Le, D. Loegering, N. Bone, L. M. Bruzek, V. L. Narayanan, A. A. Adjei, N. E. Kay, A. Tefferi, et al.
Involvement of reactive oxygen species in adaphostin-induced cytotoxicity in human leukemia cells
Blood,
December 15, 2003;
102(13):
4512 - 4519.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. W. N. Deininger and B. J. Druker
Specific Targeted Therapy of Chronic Myelogenous Leukemia with Imatinib
Pharmacol. Rev.,
September 1, 2003;
55(3):
401 - 423.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Aron, M. R. Parthun, G. Marcucci, S. Kitada, A. P. Mone, M. E. Davis, T. Shen, T. Murphy, J. Wickham, C. Kanakry, et al.
Depsipeptide (FR901228) induces histone acetylation and inhibition of histone deacetylase in chronic lymphocytic leukemia cells concurrent with activation of caspase 8-mediated apoptosis and down-regulation of c-FLIP protein
Blood,
July 15, 2003;
102(2):
652 - 658.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Sturlan, M. Baumgartner, E. Roth, and T. Bachleitner-Hofmann
Docosahexaenoic acid enhances arsenic trioxide-mediated apoptosis in arsenic trioxide-resistant HL-60 cells
Blood,
June 15, 2003;
101(12):
4990 - 4997.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Liu, S. Hilsenbeck, and Y. Gazitt
Arsenic trioxide-induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9, and synergy with APO2/TRAIL
Blood,
May 15, 2003;
101(10):
4078 - 4087.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kanzawa, Y. Kondo, H. Ito, S. Kondo, and I. Germano
Induction of Autophagic Cell Death in Malignant Glioma Cells by Arsenic Trioxide
Cancer Res.,
May 1, 2003;
63(9):
2103 - 2108.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z.-y. Wang
Ham-Wasserman Lecture: Treatment of Acute Leukemia by Inducing Differentiation and Apoptosis
Hematology,
January 1, 2003;
2003(1):
1 - 13.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Nimmanapalli, E. O'Bryan, M. Huang, P. Bali, P. K. Burnette, T. Loughran, J. Tepperberg, R. Jove, and K. Bhalla
Molecular Characterization and Sensitivity of STI-571 (Imatinib Mesylate, Gleevec)-resistant, Bcr-Abl-positive, Human Acute Leukemia Cells to SRC Kinase Inhibitor PD180970 and 17-Allylamino-17-demethoxygeldanamycin
Cancer Res.,
October 15, 2002;
62(20):
5761 - 5769.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y.-H. Ling, J.-D. Jiang, J. F. Holland, and R. Perez-Soler
Arsenic Trioxide Produces Polymerization of Microtubules and Mitotic Arrest before Apoptosis in Human Tumor Cell Lines
Mol. Pharmacol.,
September 1, 2002;
62(3):
529 - 538.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. V. Blagosklonny, R. Robey, D. L. Sackett, L. Du, F. Traganos, Z. Darzynkiewicz, T. Fojo, and S. E. Bates
Histone Deacetylase Inhibitors All Induce p21 but Differentially Cause Tubulin Acetylation, Mitotic Arrest, and Cytotoxicity
Mol. Cancer Ther.,
September 1, 2002;
1(11):
937 - 941.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Hayashi, T. Hideshima, M. Akiyama, P. Richardson, R. L. Schlossman, D. Chauhan, N. C. Munshi, S. Waxman, and K. C. Anderson
Arsenic Trioxide Inhibits Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment
Mol. Cancer Ther.,
August 1, 2002;
1(10):
851 - 860.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Slack, S. Waxman, G. Tricot, M. S. Tallman, and C. D. Bloomfield
Advances in the Management of Acute Promyelocytic Leukemia and Other Hematologic Malignancies with Arsenic Trioxide
Oncologist,
April 1, 2002;
7(90001):
1 - 13.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. O'Dwyer
Multifaceted Approach to the Treatment of Bcr-Abl-Positive Leukemias
Oncologist,
April 1, 2002;
7(90001):
30 - 38.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Yu, G. Krystal, L. Varticovksi, R. McKinstry, M. Rahmani, P. Dent, and S. Grant
Pharmacologic Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase/Mitogen-activated Protein Kinase Inhibitors Interact Synergistically with STI571 to Induce Apoptosis in Bcr/Abl-expressing Human Leukemia Cells
Cancer Res.,
January 1, 2002;
62(1):
188 - 199.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. J. Anto, A. Mukhopadhyay, K. Denning, and B. B. Aggarwal
Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl
Carcinogenesis,
January 1, 2002;
23(1):
143 - 150.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Dvorakova, C. M. Payne, M. E. Tome, M. M. Briehl, M. A. Vasquez, C. N. Waltmire, A. Coon, and R. T. Dorr
Molecular and Cellular Characterization of Imexon-resistant RPMI8226/I Myeloma Cells
Mol. Cancer Ther.,
January 1, 2002;
1(3):
185 - 195.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. J. Druker, S. G. O'Brien, J. Cortes, and J. Radich
Chronic Myelogenous Leukemia
Hematology,
January 1, 2002;
2002(1):
111 - 135.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. M. Grad, N. J. Bahlis, I. Reis, M. M. Oshiro, W. S. Dalton, and L. H. Boise
Ascorbic acid enhances arsenic trioxide-induced cytotoxicity in multiple myeloma cells
Blood,
August 1, 2001;
98(3):
805 - 813.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Jia, S. M. Srinivasula, F.-T. Liu, A. C. Newland, T. Fernandes-Alnemri, E. S. Alnemri, and S. M. Kelsey
Apaf-1 protein deficiency confers resistance to cytochrome c-dependent apoptosis in human leukemic cells
Blood,
July 15, 2001;
98(2):
414 - 421.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Chen, K. W. Woodburn, C. Shi, D. C. Adelman, C. Rogers, and D. I. Simon
Photodynamic Therapy With Motexafin Lutetium Induces Redox-Sensitive Apoptosis of Vascular Cells
Arterioscler. Thromb. Vasc. Biol.,
May 1, 2001;
21(5):
759 - 764.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. J. Murgo
Clinical Trials of Arsenic Trioxide in Hematologic and Solid Tumors: Overview of the National Cancer Institute Cooperative Research and Development Studies
Oncologist,
April 1, 2001;
6(90002):
22 - 28.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. Nimmanapalli, E. O’Bryan, and K. Bhalla
Geldanamycin and Its Analogue 17-Allylamino-17-demethoxygeldanamycin Lowers Bcr-Abl Levels and Induces Apoptosis and Differentiation of Bcr-Abl-positive Human Leukemic Blasts
Cancer Res.,
March 1, 2001;
61(5):
1799 - 1804.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. Nimmanapalli, M. Porosnicu, D. Nguyen, E. Worthington, E. O’Bryan, C. Perkins, and K. Bhalla
Cotreatment with STI-571 Enhances Tumor Necrosis Factor {{alpha}}-related Apoptosis-inducing Ligand (TRAIL or Apo-2L)- induced Apoptosis of Bcr-Abl-positive Human Acute Leukemia Cells
Clin. Cancer Res.,
February 1, 2001;
7(2):
350 - 357.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. Nimmanapalli, C. L. Perkins, M. Orlando, E. O’Bryan, D. Nguyen, and K. N. Bhalla
Pretreatment with Paclitaxel Enhances Apo-2 Ligand/Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis of Prostate Cancer Cells by Inducing Death Receptors 4 and 5 Protein Levels
Cancer Res.,
January 1, 2001;
61(2):
759 - 763.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
O. Bruserud, B. T. Gjertsen, and T.-s. Huang
Induction of Differentiation and Apoptosis-- A Possible Strategy in the Treatment of Adult Acute Myelogenous Leukemia
Oncologist,
December 1, 2000;
5(6):
454 - 462.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. Wen, N. Ramadevi, D. Nguyen, C. Perkins, E. Worthington, and K. Bhalla
Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L-induced apoptosis of human acute leukemia cells
Blood,
December 1, 2000;
96(12):
3900 - 3906.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. McCabe Jr., K. P. Singh, S. A. Reddy, B. Chelladurai, J. G. Pounds, J. J. Reiners Jr., and J. C. States
Sensitivity of Myelomonocytic Leukemia Cells to Arsenite-Induced Cell Cycle Disruption, Apoptosis, and Enhanced Differentiation Is Dependent on the Inter-Relationship between Arsenic Concentration, Duration of Treatment, and Cell Cycle Phase
J. Pharmacol. Exp. Ther.,
November 1, 2000;
295(2):
724 - 733.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
N. C. Gorin, E. Estey, R. J. Jones, H. I. Levitsky, I. Borrello, and S. Slavin
New Developments in the Therapy of Acute Myelocytic Leukemia
Hematology,
January 1, 2000;
2000(1):
69 - 89.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction