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ORIGINAL ARTICLE: RESEARCH
Arsenic trioxide cooperates with all-trans retinoic acid to enhancemitogen-activated protein kinase activation and differentiation inPML–RARa negative human myeloblastic leukemia cells
SATYAPRAKASH NAYAK1, MIAOQING SHEN2, RODICA P. BUNACIU2,
STEPHEN E. BLOOM3, JEFFREY D. VARNER1, & ANDREW YEN2
1School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA, 2Department of Biomedical
Sciences, Cornell University, Ithaca, NY, USA, and 3Department of Microbiology and Immunology, Cornell University,
Ithaca, NY, USA
(Received 12 May 2010; revised 4 June 2010; accepted 10 June 2010)
AbstractArsenic trioxide (ATO) synergistically promotes all-trans retinoic acid (ATRA)-induced differentiation of PML–RARanegative HL-60 myeloblastic leukemia cells. In PML–RARa positive myeloid leukemia cells, ATO is known to causedegradation of PML–RARa with subsequent induced myeloid differentiation. We found that ATO by itself does not causedifferentiation of the PML–RARa negative HL-60 cells, but enhances ATRA’s capability to cause differentiation. ATOaugmented ATRA-induced RAF/MEK/ERK axis signaling, expression of CD11b and p47PHOX, and inducible oxidativemetabolism. ATO enhanced ATRA-induced population growth retardation without evidence of apoptosis or enhanced G1/G0 growth arrest. Compared to ATRA-treated cells, the ATRA plus ATO-treated cells progressed more slowly through thecell cycle as detected by a slower rate of accumulation in G2/M following nocodazole treatment. Hoechst/PI staining showedthat low-dose ATO did not induce apoptosis. In summary, our results indicate that ATO in conjunction with ATRA is ofpotential chemotherapeutic use in PML–RARa negative leukemias.
Keywords: HL-60, all-trans retinoic acid, arsenic trioxide, MAPK, differentiation
Introduction
Arsenic has historically been used as a medicinal
agent for a variety of diseases such as psoriasis,
eczema, asthma, malaria, ulcers, syphilis, and leuke-
mia. Arsenic and its derivatives have been used as
antiseptics, antispasmodics, antipyretics, sedatives,
and tonics, etc., especially in traditional Chinese
medicine [1]. The most common compound of
arsenic used in medicine is arsenic trioxide. Arsenic
trioxide (As2O3) or ATO is the active ingredient in
Fowler’s solution developed in the 18th century. It
has been used to treat different malignancies for over
100 years. In the 1930s, before the introduction of
chemotherapy and radiation therapy, arsenic was
used in one of the standard treatments for chronic
myeloid leukemia and other leukemias [1,2]. The
modern use of arsenic trioxide as a therapy for
acute promyelocytic leukemia (APL) originated in
China in the 1990s [3]. In a study at the Shanghai
Institute of Hematology (SIH) in 1999, patients
with relapsed APL were treated with ATO.
Complete remission (CR) was achieved in 9/10 of
the cases treated with ATO alone and 5/5 cases
treated with ATO plus chemotherapy (CT) or
retinoic acid [3–5]. This suggests that ATO is potent
as a single agent in APL, a disease cytogenetically
characterized by a t(15;17) translocation that gives
rise to the PML–RARa fusion protein (promyelocytic
leukemia–retinoic acid receptor a), which is a rogue
transcription factor. This was further proved by
relatively high 2- and 3-year disease-free survival
(DFS) rates seen recently in newly diagnosed
leukemias [6].
Correspondence: Prof. Andrew Yen, Department of Biomedical Sciences, T4-008, Veterinary Research Tower (VRT), Cornell University, Ithaca, NY 14853,
USA. Tel: (607)253-3354. Fax: (607)253-3317. E-mail: [email protected]
Leukemia & Lymphoma, September 2010; 51(9): 1734–1747
ISSN 1042-8194 print/ISSN 1029-2403 online � 2010 Informa UK, Ltd.
DOI: 10.3109/10428194.2010.501535
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All-trans retinoic acid (ATRA) has been used in
differentiation induction therapy for APL since the
early 1980s [7,8]. A recent clinical study of APL
treatment with ATRA differentiation therapy in Japan
resulted in a CR rate of 94%, a 6-year DFS rate of
68.5%, and a 6-year overall survival (OS) rate
of 83.9% [9]. Clinical studies using a combination of
ATRA and ATO showed that the time required to
achieve CR with ATRA–ATO was statistically sig-
nificantly different (25.5 days) from that with ATRA
alone (40.5 days) or ATO alone (31 days). The
median DFS rate was also increased to 20 months for
the combination treatment as compared to ATRA
alone (13 months) and ATO alone (16 months) [10].
Furthermore, the disease burden, determined by the
fold change in PML–RARa, was much shorter in the
combination treatment compared to monotherapy.
Clinical studies thus show a synergistic effect of ATRA
and ATO treatments, motivating interest in the
biochemical cause of this synergy. Both ATRA and
ATO have been found to cause degradation of the
PML–RARa fusion protein, suggesting a mechanism
of action whereby degradation of the transforming
protein relieves the block in differentiation and permits
progression of differentiation along the myeloid line-
age. ATO has been found to bind the PML–RARa and
promote its SUMOylation and degradation [11].
However, ATO may have many other actions, as is
the case for ATRA, and their collaboration may be
attributable to other biochemical mechanisms they
share as targets. It is thus of interest to determine
whether they collaborate in a setting with no PML–
RARa. If so, this may suggest therapeutic potential for
the combination of RA and ATO in tumors other than
PML–RARa positive APL.
HL-60 is a human myeloblastic leukemia cell line
that has been extensively used as a model for
pharmacologically induced differentiation. HL-60
cells undergo either myeloid or monocytic differ-
entiation, and G1/G0 growth arrest, when treated
with ATRA or 1-25-dihydroxyvitamin D3 (vitamin
D3), respectively. Previous studies in our laboratory
have shown that ATRA- or vitamin D3-induced
differentiation and G1/G0 cell arrest require sus-
tained activation of mitogen-activated protein kinase
(MAPK) signaling along the RAF/MEK/ERK axis
[12,13]. ATRA induces the MAPK-dependent ex-
pression of cell surface differentiation markers such
as CD38 and CD11b, as well as functional differ-
entiation markers such as inducible oxidative meta-
bolism that become progressively expressed as the
cells undergo myeloid differentiation.
HL-60 cells are negative for the t(15;17) chromo-
somal translocation that defines APL and gives rise to
the PML–RARa fusion protein. If the ability of ATO
to promote differentiation is solely through
degradation of the PML–RARa fusion protein, then
we should not see any enhanced differentiation for
the combination treatment of ATRA and ATO.
However, if ATO action collaborates with the
mechanism of ATRA-induced differentiation, e.g.
by promoting sustained activation of MAPK, then
addition of ATO to ATRA treatment should
augment the differentiation of HL-60 cells.
Previous studies on the effects of ATO show that
high concentrations of ATO were able to induce
apoptosis in drug-resistant HL-60 cell lines [14].
Experiments by Jiang et al. showed that the effect of
ATO may be mediated by c-Myc down-regulation,
whereas Herst et al. showed that adding ATO led to
apoptosis after 5 days [15,16]. The mechanism of
apoptosis induction was discovered to be via the
death receptor pathway rather than the intrinsic
(mitochondrial) pathway. The present studies using
low doses of ATO showed that, compared to cells
treated with ATRA alone, HL-60 cells treated with a
combination of ATRA and ATO exhibited augmen-
ted differentiation propelled by an enhanced activa-
tion of MAPK signaling. While the addition of ATO
to ATRA treatment enhanced population growth
retardation, there was no corresponding increase
seen in the percentage of cells undergoing G0-
specific cell cycle arrest, but rather an apparent
overall retardation of cell cycle progression. How-
ever, ATO alone did not cause differentiation in HL-
60 cells, as indicated before for PML–RARa positive
cells [17]. The results show that ATO can enhance
the differentiation-inducing effects of ATRA in
PML–RARa negative cells, consistent with a para-
digm whereby ATO enhances the ATRA-induced
MAPK signaling that propels induced differentia-
tion. Since ATO is already Food and Drug Admin-
istration (FDA) approved (Trisenox), along with
ATRA differentiation therapy for APL, a more
thorough understanding of the mechanism of action
of ATO, especially on the MAPK and related
pathways, may be of therapeutic value in treating
other leukemias, or even non-hematologic cancers.
Materials and methods
Cell culture
HL-60 human myeloblastic leukemia cells were
cultured in RPMI 1640 medium supplemented with
5% heat-inactivated fetal bovine serum (Invitrogen,
Carlsbad, CA, USA) in a 5% CO2 humidified
environment at 378C, and were maintained in an
exponential growth phase. All the experimental
cultures were initiated at a density of 0.26106 cells/
mL. Cell viability was monitored by Trypan Blue Stain
(Invitrogen) and consistently exceeded 95%.
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Reagents used
All-trans retinoic acid (Sigma-Aldrich, St. Louis, MO,
USA) was dissolved in 100% ethanol to make a stock
solution concentration of 5 mM. The concentration of
ATRA used in cultures was 1 mM. Arsenic trioxide
(Sigma-Aldrich) was diluted in water to make a stock
concentration of 5 mM. ATO was used at a final
concentration of 2 mM, and added at the same time as
ATRA to the cultures of HL-60 cells. NB4 cells,
however, were treated with 0.25 mM ATRA and
0.5 mM ATO. Mitomycin C (MMC) (Sigma-Aldrich)
was used at a final concentration of 100 mM.
Antibodies for Western analysis against ERK1/2,
phosphorylated ERK-1/2 (Thr-202/Tyr-204), MEK1/
2, phosphorylated MEK1/2 (Ser-217/221), p47PHOX,
and GAPDH (glyceraldehyde 3-phosphate dehydro-
genase), as well as MEK1 inhibitor PD98059, were
obtained from Cell Signaling (Beverly, MA, USA).
PD98059 was dissolved in dimethylsulfoxide (DMSO)
to make a stock solution of 2 mM. PD98059 was
added 16 h before the addition of ATRA and ATO to
make its concentration in culture 1 mM. It was also re-
added along with ATRA and ATO (at 0 h) and finally
at 16 h after the addition of ATRA and ATO. The
detailed procedure for the addition of PD98059 has
been explained elsewhere [12]. Nocodazole was
purchased from Sigma.
Procedure for preparing total cell lysates and Western blots
Total cell lysates were prepared as follows. Cells were
harvested at the 48 h time point, and washed with
phosphate-buffered saline (PBS) and centrifuged
three times to wash away any culture medium. After
washing away the medium, cells were re-suspended
in 100 mL M-PER Mammalian Protein Extraction
Reagent (Invitrogen) buffer mixed with a phospha-
tase and protease inhibitor. Western analysis was
done by resolving the lysates on 12% Tris-HCl gels
(Bio Rad), and the membrane was blocked overnight
with 5% w/v non-fat milk. The membrane was then
probed using primary and secondary antibodies as
described above in ‘Reagents used.’ All Western
blots shown are typical of at least three repeats.
CD38 and CD11b expression studies by flow cytometry
At the 24 h and 48 h time points, 0.56 106 cells
were collected, centrifuged to a pellet, and resus-
pended in 100 mL PBS containing 5 mL allophyco-
cyanin (APC)-conjugated CD11b and 5 mL
phycoerythrin (PE)-conjugated CD38 antibodies.
The cells were then incubated for 1 h in a humidified
atmosphere of 5% CO2 at 378C, and expression
levels were measured by flow cytometry (BD LSR II
flow cytometer; Becton Dickinson, Mountain View,
CA, USA). CD11b expression was measured by
633 nm red laser excitation and emission reflected
from a 735 long-pass dichroic mirror and collected
through a 660/20 band-pass filter. CD38 expression
was measured with 488 nm laser excitation and
emission through a 550 long-pass dichroic mirror
and 576/26 band-pass filter. Gates to determine the
shift in cellular fluorescence intensity were set to
exclude 95% of the control (untreated) cells.
Measurement of inducible oxidative metabolism
Approximately 0.56 106 cells were collected at the
48 h and 72 h time points, centrifuged to a pellet,
and resuspended in 200 mL PBS at 378C containing
5 mmol/L 5- (and 6-) chloromethyl-20,70-dichlorodi-
hydrofluorescein diacetate acetyl ester (H2DCF-DA;
Molecular Probes) without or with 0.2 mg/mL 12-O-
tetradecanoylphorbol-13-acetate (TPA; Sigma).
H2DCF-DA and TPA stock solutions were made
in DMSO at concentrations of 0.2 mg/mL and
0.5 mg/mL, respectively. Cells were incubated for
20 min in a humidified atmosphere of 5% CO2 at
378C. Flow cytometric analysis was done (BD LSR
II flow cytometer) using 488 nm excitation, and
emission from oxidized DCF was collected through a
505 long-pass dichroic mirror and 530/30 band pass
filter. The shift in fluorescence intensity in response
to TPA was used to determine the percentage of cells
capable of inducible oxidative metabolism. Gates to
determine the percentage of positive cells were set to
exclude 95% of the same cells not treated with TPA.
Hoechst/propidium iodide assay to determine extent of
apoptosis in culture
A volume of 250 mL of cell culture at the 72 h time
point was taken and placed in a tube. Then 25 mL of
Hoechst 33342 and 25 mL of propidium iodide
working solutions were added to the cell suspension.
The concentration of Hoechst stock solution was
1 mM, which was diluted 1:4 in sterile water to obtain
the working solution. Propidium iodide stock solution
was prepared at a concentration of 200 mg/mL, which
was diluted 1:9 to obtain the working solution. The
cells were mixed very gently by tapping, and incubated
in a water bath for 15 min at 378C. Next, 25 mL of
stained cells were loaded onto a microscope slide and
placed on the fluorescence microscope for counting.
Round blue cells with no condensation of chromatin
or small masses inside the cells were treated as live,
viable cells. Cells that had small, blue masses inside
them were counted as apoptotic, whereas pink cells
with non-condensed chromatin were treated as necro-
tic. One hundred cells per slide were counted and
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segregated into viable, apoptotic, or necrotic cate-
gories.
Cell cycle analysis
About 0.56106 cells were harvested at 72 h by
centrifugation and resuspended in 200 mL hypotonic
staining solution (at 48C) containing 50 mg/mL propi-
dium iodide (PI), 1 mL Triton X-100, and 1 mg/mL
sodium citrate. Cells were incubated at room tem-
perature for 1 h and analyzed using flow cytometry
(BD LSR II). Excitation was provided by a 488 nm
laser and the emission was collected using a 550 long-
pass dichroic mirror and a 576/26 band-pass filter.
Cell cycle progression and accumulation in G2/M
during mitotic block were measured using 100 ng/
mL nocodazole added to cultures that were un-
treated, or treated with ATRA, ATO, or ATRA plus
ATO for 24 h. The cells were harvested after 0, 6,
12, and 18 h and stained with propidium iodide for
cell cycle analysis. Nocodazole was added to the
cultures at the same time as ATO and ATRA.
Statistical analysis
Statistical analysis was done using MATLAB’s
statistical toolbox. Three independent repeats were
performed in each case. Means of treatment cases
were compared using paired-samples t-test, using the
ttest2 function in MATLAB’s statistical toolbox. The
bar graphs show means of three independent repeats
along with standard errors.
Results
Arsenic trioxide leads to enhanced sustained activation of
ATRA-induced MAPK proteins
It is well known that transient activation of MAPK leads
to proliferation, whereas a sustained activation of
MAPK, especially ERK1/2, has been thought to drive
differentiation in a variety of cell lines, including HL-60
cells [12,13]. It is also known that ATO can activate
MAPK pathways as part of a stress response [18,19].
We studied the effect of ATO on MAPK signaling, in
particular the potential synergistic effects of ATRA and
ATO on MEK and ERK. Treating cells with ATO plus
ATRA enhanced the increase in pMEK1/2 (Ser-217/
221) [Figure 1(A)] and pERK1/2 (Thr-202/Tyr-204)
[Figure 1(B)] induced by ATRA alone (Western blot
for MEK, pMEK, ERK, and pERK was done after
48 h of ATRA/ATO treatment). Interestingly, there
was also increased activation of ERK1/2 (Thr-202/Tyr-
204) in the ATO-alone treatment case compared to the
Figure 1. Western blot analysis of MAPK proteins shows enhanced activation in the combination treatment of ATRA and ATO compared to
ATRA treatment. (A, B) The effect of the different treatments on expression and activation levels of MEK1/2 and ERK1/2, respectively; (C)
the effect in the presence of MEK1 inhibitor PD98059. The left columns show the effect on expression levels at 48 h, whereas the right
columns show the effect on activation of MAPK proteins. The lanes show control (untreated) cells, cells treated with ATRA, ATRA plus
ATO, and ATO only. Increased activation was seen for MEK1/2 as well as ERK1/2 at 48 h. We did not see a large increase in total
expression of MEK1/2 and ERK1/2 for any of the treatment cases (see left column) at 48 h. This suggests that ATO (in combination or
alone) only affects activation (via phosphorylation) of MAPK proteins, without affecting their total expression levels. (C) The effect of adding
MEK1 inhibitor (PD98059) on p-MEK and p-ERK at 48 h time-point is shown.
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control (untreated) case. The ATO-induced increase
could be blunted by addition of a known MEK1
inhibitor, PD98059 [Figure 1(C)]. Western analysis of
ERK1/2 expression showed that there was no increase
in the total expression of ERK1/2 in any of the
treatment cases. The expression levels of ERK1/2
were thus unaffected by treatment, although their
activation increased. Western analysis of total MEK1/
2 [Figure 1(A)] also showed no effect of either ATRA
or ATO treatment on MEK1/2 expression levels.
Similar to ERK, the increased phosphorylated MEK1/
2 with the combination treatment is thus attributable to
increased activation and not to increased expression.
Arsenic trioxide enhances ATRA-induced CD11b but not
CD38 expression
Having established enhanced activation of MAPK
signaling in the combination treatment, we then
proceeded to investigate whether this augmentation
translated into enhanced differentiation in HL-60
cells. A visual inspection of different treatments for
96 h suggested that there was a synergy in the
combination treatment which led to enhanced
differentiation (Figure 2). Cells treated with ATO
alone did not cause any apparent differentiation, but
did exhibit a decrease in growth rate. To investigate
whether combination treatment led to enhanced
differentiation, we started by assessing the cell
surface markers of differentiation, CD38 and
CD11b.
CD38 is a 45 kDa ectoenzyme type II transmem-
brane receptor. The gene is transcriptionally regu-
lated by a retinoic acid response element (RARE) in
its first intron, which confers response to ATRA [20].
It has been shown that CD38 can elicit the MAPK
cascade, and that one component of the signaling
machinery is the c-Cbl adaptor [21]. To determine
the effects of ATO and ATRA on CD38 expression,
we measured expression of CD38 at an early time
point (6 h) and at 24 h after various treatments,
using a PE-conjugated CD38 antibody and flow
cytometry. As seen in Figure 3, treatment with
ATRA induced progressively increasing CD38
Figure 2. Change in cellular morphology induced by different treatments. Wright’s stained images showing morphological changes at 96 h
after different treatments. (A) In the control (untreated) case, mostly round cells were observed. Addition of ATRA led to differentiated cells,
seen here as distorted cells (C). Differentiation was more apparent in the combination treatment of ATRA and ATO, whereas the ATO-alone
treatment case shows a morphology similar to the control case, although a smaller number of cells are seen in this case (D and B,
respectively).
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expression that was indistinguishable from treatment
with ATRA plus ATO. There was no increase in
CD38 expression in the cells treated with ATO
alone. These results indicated that ATO had no
effect on CD38 expression, either in combination
with ATRA or by itself. Since CD38 has retinoic acid
response elements in its promoter, RA-induced
expression will not be affected unless ATO affects
that transcriptional activation.
Another cell surface marker, CD11b, an integrin
receptor subunit, was used to characterize the effect
of ATO on ATRA-induced cell differentiation.
Expression of CD11b typically marks the progression
of ATRA-induced differentiation beyond the stage of
CD38 expression, which is an early marker. CD11b
expression was measured by flow cytometry using an
APC-conjugated CD11b antibody [22,23]. The
effect of ATO and ATRA, alone and in combination,
on CD11b expression was measured at 48 and 72 h.
Compared to untreated control cells, ATRA-treated
cells showed an increase in CD11b expression, as
expected. However, CD11b expression was consis-
tently higher in the case of the ATRA–ATO
combination when compared with the ATRA-alone
case [Figures 3(B) and 3(C)]. The difference
between CD11b expressions was statistically signifi-
cant at both 48 h (p¼ 0.004) and 72 h (p¼ 0.016),
indicating that the differentiation was affected by
ATO during its both early and late progression. In
contrast, the cells treated with ATO alone did not
show an increase when compared with the untreated
control case, indicating that ATO alone does not
affect CD11b expression.
Arsenic trioxide enhances ATRA-induced differentiation
by up-regulating p47PHOX
Inducible oxidative metabolism marked by an
increase in reactive oxygen species (ROS) is one of
the functional markers of differentiation into mature
Figure 3. ATO enhances ATRA-induced CD11b expression, but not CD38 expression. (A) There was no enhancement in CD38 expression
in the combination treatment, but CD11b (B) expression showed a synergistic increase in the ATRA–ATO combined treatment case. CD38
expression was analyzed with PE-conjugated CD38 antibody at 6 h and 24 h and CD11b expression was analyzed with APC-conjugated
CD11b antibody at 48 h and 72 h by flow cytometry. The y-axis shows the percentage of CD38 positive cells (A) and CD11b positive cells
(B) at indicated time points when compared with untreated cells, with gates set to exclude 95% of cells in the control case. The data show
mean values+ standard errors. * and # indicate statistically significant differences compared to control. ## and ** indicate statistically
significant differences compared to ATRA alone. For example, CD11b expression in cells treated with ATRA–ATO was statistically
significantly different from that in cells treated with ATRA alone at both 48 h (p¼0.004) and 72 h (p¼0.016). (C) Representative CD11b
histograms of untreated (control) cells and cells treated with ATRA, ATRA–ATO combination, and ATO alone at 48 h.
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myelo-monocytic cells. p47PHOX is one of the
essential components of the NADPH (reduced
nicotinamide adenine dinucleotide phosphate)–oxi-
dase complex which generates ROS when activated
in differentiated cells. We measured the effect of
ATO and ATRA separately and in combination on
p47PHOX expression. The untreated control cells and
ATO-treated cells did not show p47PHOX expression
at 48 h [Figure 4(A)]. Consistent with the trend seen
in MAPK activation, enhanced expression was seen
in the ATRA–ATO combination treatment com-
pared to treatment with ATRA alone. This result
suggests that ATO synergizes with ATRA to induce
expression of p47PHOX, although ATO by itself does
not induce p47PHOX expression. Also, this enhance-
ment in p47PHOX was abrogated in the presence of
MEK1 inhibitor PD98059 [Figure 4(B)]. This is
consistent with the suggestion that the enhancement
in differentiation in the combined treatment case is
through the MAPK axis, as inhibition of MEK1/2
activation leads to decreased differentiation.
Arsenic trioxide enhances ATRA-induced functional
differentiation in HL-60 and NB4 cells
To confirm the functional significance of the induced
p47PHOX expression, TPA inducible oxidative meta-
bolism, a late functional differentiation marker that
characterizes mature myelo-monocytic cells, was
measured. When treated with ATRA, HL-60 cells
typically exhibit a functional inducible ROS re-
sponse. As before, cells were treated with ATO or
ATRA alone or in combination. Inducible oxidative
metabolism was measured by flow cytometry using
H2DCF-DA, a cell-permeant indicator of reactive
oxygen species that is non-fluorescent until the
acetate groups are removed by intercellular esterases
when oxidation takes place within the cell. As shown
by Figure 5(A), HL-60 cells treated with a combina-
tion of ATRA–ATO showed increased functional
differentiation at both 48 h and 72 h time points
when compared with cells treated with ATRA alone.
For example, at 72 h, 85.3% of cells treated with
ATO plus ATRA were able to produce ROS induced
by TPA, compared to 60.9% of cells treated with
ATRA alone. The enhancement in ROS in the case
of ATO–ATRA was statistically significantly different
from that of ATRA alone with 95% confidence
(p¼ 0.024) at 72 h. In the case of cells treated with
ATO alone, there was no significant increase in ROS
induction upon TPA treatment, indicating that ATO
alone was unable to induce functional differentiation
of HL-60 cells. This is consistent with the inability of
ATO to induce the other differentiation markers
measured. These results indicate that, even though
ATO by itself is unable to induce differentiation of
HL-60 cells, it works synergistically to enhance
ATRA-induced differentiation.
Similar enhancement in differentiation was ob-
tained for NB4 cells with the combination treatment.
There were some key differences, however, between
the responses of HL60 cells and NB4 cells, an APL-
derived cell line. For example, NB4 cells showed
much higher sensitivity toward ATO, and massive
apoptosis was observed in cultures with ATO
concentration more than 0.5 mM. Therefore, a lower
Figure 4. (A) ATO leads to an increase in ATRA-induced expression in p47PHOX. Western analysis of total expression of p47PHOX is shown
at 48 h time point. An up-regulation in expression of p47PHOX is seen in ATRA–ATO treatment when compared with ATRA-alone
treatment. Protein lysates were extracted from untreated cells, and cells treated with ATRA, ATRA–ATO combination, and ATO alone.
Loading control is GAPDH. (B) The effect on p47PHOX upon addition of MEK inhibitor PD98059. As seen from the figure, addition of
PD98059 leads to a decrease in the expression of p47PHOX.
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dose of ATO (0.5 mM) and ATRA (0.25 mM) was
administered to NB4 cells in culture. This dosage
preserved the same ratio of ATRA to ATO as in HL-
60 cells (2:1). Another difference in the responses of
the two cell lines was that almost 100% of the cells
showed terminal differentiation at 72 h in NB4 cells.
Therefore, the inducible oxidative mechanism was
measured at earlier time points, i.e. 24 h and 48 h.
An increase in terminal differentiation was observed
at 24 h and 48 h in the combination treatment case,
when compared to the ATRA-alone case. Using a
one-tailed t-test, we concluded that differentiation in
the ATRA–ATO case was statistically more than that
in the ATO-alone case at 48 h (p¼ 0.046) [Figure
5(B)]. This result suggests that mechanisms involved
in the enhancement of differentiation in the combi-
nation are not HL-60 specific. The APL-derived
NB4 cells were, however, more ATO sensitive.
Arsenic trioxide leads to reduced population growth
HL-60 cells treated with ATO had slower population
growth rates compared to untreated cells as well as
ATRA-treated cells, as seen in Figure 6. Addition of
ATRA alone decreased the population growth rate
compared to untreated cells. However, addition of
ATO had a more drastic effect on the growth rate of
cells than ATRA. Cultures with ATO alone or the
combination treatment of ATRA–ATO grew much
slower than cultures with ATRA alone. Indeed, the
differences in growth rates between control and cell
lines with ATO were statistically significant after just
24 h (p5 0.05). Of note, growth of ATO plus
ATRA-treated populations was slower than that of
ATRA-treated populations. The difference in growth
rates of ATRA-alone and ATRA–ATO treatment
was statistically significant at both 48 h (p¼ 0.006)
Figure 5. Arsenic trioxide enhances ATRA-induced differentiation
of HL-60 cells as well as NB4 cells. (A) The percentage of cells (y-
axis) with capability for inducible oxidative metabolism at
indicated time points. Cells were treated with TPA to induce
oxidative metabolism. Using a functional marker of differentiation,
i.e. generation of ROS, we measured terminal differentiation in
untreated cells, cells treated with ATRA, a combination of ATRA–
ATO, and ATO alone. The percentage of positive cells with TPA,
when gates were set to exclude 95% of control cells (without
TPA), is shown. * indicates statistically significant difference
compared to control. ** indicates statistically significant difference
compared to ATRA alone. There was no statistically significant
difference in the percentage of differentiated cells in the ATRA-
alone versus ATRA–ATO combination treatments at 48 h.
However, at 72 h the difference in DCF positive % cells was
statistically significant between ATRA and ATRA–ATO combina-
tion treatment (p¼0.024). (B) The same is shown for NB4 cells.
There is an enhancement in differentiation seen in NB4 cells also;
however, almost all the cells are terminally differentiated at the
72 h time-point. # indicates that the percentage of DCF positive
cells are more in the ATRA case when compared to control. ##
denotes that the percentage of DCF positive cells was statistically
more (using a one-tailed t-test) in the ATRA–ATO combination
case when compared with the ATRA-alone case at 48 h with 95%
confidence (p¼0.046). The background ROS activity in all
samples was measured by cells treated with DMSO carrier blank.
Figure 6. There is a decrease in cell growth rate upon addition of
ATO. Cell growth rates were measured over a period of 72 h of
treatment. Cells were seeded at an initial concentration of 0.2
million cells/mL. The y-axis shows cell density in the culture in
million cells/mL whereas the x-axis shows time points at which the
cells were counted. Addition of ATRA also leads to decreased
growth rate; however, ATO addition has a more significant effect
by itself or in combination with ATRA. * and ** indicate that the
cell growth rates between ATO and ATRA–ATO combination
treatment cases were statistically significantly different at both 48 h
(p¼ 0.006) and 72 h (p¼ 0.013). { and {{ indicate that growth
rates in cultures with ATO alone also were statistically significantly
different from the control case at 24 h, 48 h, and 72 h time-points.
The ATO cultures did not show an increase in dead cells or debris.
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and 72 h (p¼ 0.013). Microscopic images of the
cultures at 96 h showed morphologically differen-
tiated cells in the ATRA- and ATRA–ATO (Figure
2) combination-treated cultures, whereas cells in the
ATO-alone cultures were not obviously different
from the untreated cells, morphologically. Taken
with the differentiation data, the population growth
results suggest that treatment with ATO alone slows
down population growth without inducing differen-
tiation.
Three possible causes for the decreased growth
were hypothesized and tested. A decrease in popula-
tion growth rate could be due to apoptosis upon
addition of ATO. Alternatively, an increase in cells
arresting in the G1/G0 phase could lead to an overall
decrease in population growth rates due to a
diminishing growth fraction. Finally, an overall
decrease in biosynthetic activity could cause a general
dilatation of the cell cycle and decreased cell-
doubling rates. We performed the Hoechst/propi-
dium iodide assay to seek evidence of apoptosis in
various treatment cases, cell cycle distribution by
flow cytometry for evidence of G0 cell cycle arrest,
and cell cycle transit rates measured by accumulation
in G2/M after mitotic block for evidence of cell cycle
transit slow-down.
Addition of arsenic trioxide with or without ATRA does
not lead to apoptosis
To test the first hypothesis, namely that reduced
growth rates are due to increased apoptosis, we
performed a Hoechst/PI assay to determine the
number of apoptotic and necrotic versus viable cells
in the culture. The Hoechst assay, as developed by
Bloom et al. [24], is a double fluorescence staining
procedure for the simultaneous diagnosis of apopto-
sis and necrosis in cell culture. The procedure uses
the DNA-specific fluorochrome Hoechst 33342, a
vital stain that fluoresces blue when bound to DNA
within the normal chromatin of nuclei. The cells are
also simultaneously stained in propidium iodide,
which necrotic cells cannot exclude and fluoresces
red when bound to DNA. DNA in apoptotic cells is
cleaved into smaller fragments, and there is a loss of
normal chromatin architecture. Chromatin in apop-
totic cells is segregated into variable numbers of
round masses having uniform bright (blue-white)
fluorescence. Normal cells and cells in early stages of
apoptosis take up the Hoechst dye but not the
propidium iodide. Cells having damaged membranes
do take up propidium iodide and stain pink. Necrotic
cells appear swollen and have pinkish nuclei. Figures
7(A), 7(C), and 7(D) show a typical viable cell, an
apoptotic cell, and a necrotic cell, respectively.
Figure 7(B) shows the average number of viable,
apoptotic, and necrotic cells and standard errors
obtained after 72 h of different treatments. Treat-
ment with 100 mM MMC (at 16 h after treatment)
was used as a positive control. The data show that
there is no apparent increase in apoptosis upon the
addition of ATO, when administered with or without
ATRA. The percentage of apoptotic cells seen in
cultures with MMC is consistent with previous data
from Yen et al. [25]. The results are also consistent
with the observations on trypan blue exclusion,
which showed no evidence of induced cell death
when there is arsenic trioxide in the culture. We thus
conclude that the decrease in population growth rates
seen in the ATRA–ATO combination treatment or in
the ATO-alone treatment case is not due to
apoptosis. We next tested whether the decrease in
growth rates was attributable to an increase in the
percentage of cells arresting in the G1/G0 phase.
Arsenic trioxide does not accelerate ATRA-induced G0
arrest
ATRA treatment of HL-60 cells led to myeloid
differentiation as well as G1/G0 arrest. To determine
whether the ATO-induced growth retardation was
attributable to G1/G0 arrest, the effect of ATO on
the percentage of G1/G0 cells was measured by flow
cytometry for untreated cells and cells treated with
ATRA, ATRA–ATO combined, and ATO alone.
Treatment with ATRA caused G1/G0 arrest that was
evidenced by an increase in the percentage of cells in
G1/G0 phase, as seen before [23]. Treatment with
ATO alone, however, did not show an increase in the
percentage of cells arrested, as compared to the
control case, at 72 h [Figure 8(A)]. Treatment with
ATO plus ATRA resulted in an increase in the
percentage of G1/G0 cells, which was indistinguish-
able from that caused by ATRA alone. The effect of
ATO treatment on cell cycle arrest was also
measured at earlier time points (24 h and 48 h,
results not shown), and similar results were obtained.
Thus, although ATO enhances ATRA-induced
differentiation, it does not enhance ATRA-induced
growth inhibition through enhancing G1/G0 arrest.
Arsenic trioxide slows cell cycle transit
To determine whether ATO slowed progression
through the cell cycle, the effect of ATO on the
rate at which cells accumulated in G2/M in cultures
with mitosis blocked was measured. Cells that were
untreated or treated with ATRA, ATO, or a
combination thereof for 24 h were treated with
nocodazole, a microtubule disruptor blocking cells
in M phase. The rate of accumulation of cells in G2/
M and depletion of cells from G1 reflect the rate of
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cell cycle transit. ATO slowed the accumulation of
cells in G2/M and the depletion of G1 cells as
measured by flow cytometry. As shown in Figure 9,
cells treated with a combination of ATO plus ATRA
went through the cell cycle more slowly than cells
that were untreated or treated with just ATRA.
Addition of ATO alone or in combination with
ATRA led to statistically significantly different
percentages of cells (p5 0.05) in G2/M phase
between the control case and treatments containing
ATO, at 12 h as well as 18 h [Figure 9(A)].
Similarly, significant differences were obtained in
the percentages of cells in G1 phase when ATO
treatment was compared with the control case at 18 h
[Figure 9(B)]. Comparing Figures 8 and 9, it is of
interest to note that the rate of transit through the cell
cycle is slower (Figure 9) and the probability of arrest
is higher (Figure 8) in the ATRA plus ATO co-
treatment group. Figure 8 shows arrest after 72 h,
whereas Figure 9 shows the rate of transit during the
first 18 h of treatment.
Discussion
There are a number of potential mechanisms by
which ATO may cause or facilitate leukemic cell
differentiation. A number of regulatory molecules,
pathways, and their downstream cellular outcomes
have been studied. One of the potentially most
significant is the capability of ATO, like ATRA, to
cause degradation of PML–RARa, the fusion protein
resulting from the t(15;17) translocation that is
putatively the seminal cytogenetic cause of APL
[26]. In the NB4 APL cell line, ATO caused
degradation of the PML–RARa fusion protein, but
not the mRNA, in both ATRA sensitive and resistant
variant lines [26]. ATO also affects the spatial
localization and organization of PML and PML–
RARa, altering PML bodies within the nucleus of
NB4 cells [19,26]. Clinical correlates have also been
reported [5]. Degradation of PML–RARa is thus a
prominent potential mechanism of action of ATO.
ATO has also been found to affect signaling
pathways that have been implicated in ATRA-
induced differentiation, as well as growth regulation
and apoptosis. ATO can evoke a cellular oxidative
stress response [27,28]. The glutathione (GSH)
redox system is a critical modulator of cellular
response in oxidative stress. As such it can regulate
apoptosis, a possible sequela of ATO treatment. A
function of the GSH redox system is to scavenge free
radicals. The GSH redox system is targeted by ATO
in various APL cell lines. NB4 cells have one of the
lowest levels of basal GSH in leukemic cell lines, and
Figure 7. Addition of ATO to the culture does not lead to an increase in apoptosis. (A, C, D) Representative viable, apoptotic, and necrotic
cells as seen in the Hoechst/PI assay. (B) The number of apoptotic, necrotic, and viable cells in a representative sample when 100 cells were
counted for the different treatment cases. A positive control in the form of MMC-treated cells was also included in the experiment. As seen in
the figure, addition of ATO did not lead to an increase in apoptosis in the presence or absence of RA.
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were found to be most sensitive to arsenic treatment
[29]. In NB4 cells, ATO treatment modulated the
GSH levels, which sensitized the cells toward
apoptosis [29,30]. There were dose-dependent
growth inhibitory, differentiation enhancing, or
apoptosis enhancing effects of ATO [17,31]. Pre-
vious studies have shown that ATO causes activation
of the stress-activated protein kinase, c-jun N-
terminal kinase (JNK) [28]. This activation is directly
related to apoptosis, as inhibition of p38/JNK was
seen to reduce ATO-induced apoptosis in NB4 cells.
In the case of MAPK signaling, the effect of ATO on
p38 has been widely studied, too. Verma et al.
showed that p38 is phosphorylated and activated by
ATO in a sustained manner, and that this activation
is downstream of redox events activated by ATO [32]
in NB4 cells. Activation of other MAPK proteins,
such as Mkk 3 and Mkk 6, was also seen in NB4 cells
and in chronic myelogenous leukemia (CML)-
derived KT-1 cells, and found to be essential for
p38MAPK activation. Signaling molecules and path-
ways that are in common downstream of ATO and
ATRA may provide a basis for their potential
synergistic interaction. Clinically, a synergistic effect
has been reported for ATRA–ATO combination
treatment, when compared with either ATRA or
ATO monotherapy [10]. Other studies of the
biochemical effects of ATRA–ATO combination
treatment have revealed additive effects in JNK/
p38, tumor necrosis factor (TNF), and cyclic
adenosine monophosphate/protein kinase A (cAMP/
PKA) pathways in APL cell lines [33,34]. For
example, in the cAMP/PKA study, it was found
that cAMP, which can induce monocytic differentia-
tion in HL-60 cells, was synergistically induced by
ATRA and low doses of ATO [33,35].
The results of the present study showed that for the
PML–RARa negative HL-60 cells, ATO did not
induce differentiation, although it did slow cell
proliferation. It did enhance the differentiation
induction capability of ATRA. Although ATO did
not augment ATRA-induced CD38 expression, a
marker of the earliest stages of ATRA-induced
progression along the myeloid lineage, it did enhance
ATRA-induced expression of CD11b, an integrin
receptor subunit that marks further progression of
differentiation, as well as p47PHOX, which is a
component of the NAPDH–oxidase complex used
for the inducible oxidative metabolism characteristic
of mature myeloid cells. ATO also enhanced the
ATRA-induced functional differentiation marker,
inducible oxidative metabolism. While ATO by itself
Figure 8. (A) Arsenic trioxide does not accelerate ATRA-induced G0 arrest. The percentage of G1/G0, S, and G2/M cells at 72 h after
treatment is shown for control (untreated), ATRA, ATRA–ATO combination treated, and ATO-alone treated cells. The y-axis here shows
the percentage of cells in the different cell-cycle phases at 72 h. The x-axis shows the different treatment groups. Cells stained with hypotonic
PI solution were incubated for 1 h and analyzed by flow cytometry. * indicates that treatment with ATRA or ATRA–ATO led to a statistically
significant increase in the percentage of cells in G1/G0 with respect to control, as expected. Similar DNA histograms were generated for
earlier time points also, and we did not see a significant difference in percentages of cells arrested in G1/G0 phase between the ATRA–ATO
case and ATRA-alone case. (B) Representative DNA histograms for untreated (control) cells, cells treated with ATRA, cells treated with
ATRA–ATO combination, and cells treated with ATO alone are shown at 72 h.
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thus could not cause differentiation, it enhanced
ATRA-induced differentiation, indicating that it
affected pathways of importance to propelling differ-
entiation. One such pathway is activation of the RAF/
MEK/ERK axis in MAPK signaling [12,13]. Activa-
tion of this axis was enhanced in cells treated with the
combination of ATRA plus ATO, compared to
ATRA alone. Although phosphorylation of MEK
and ERK betraying activation was enhanced, their
expression levels were not detectably affected. The
activation and expression patterns of MAPK proteins
suggest that the synergy between ATRA and ATO is
at the protein–protein interaction level, rather than at
the transcriptional level. This agrees with a recent
systems-level study which indicated that while the
effects of ATRA involved transcriptional remodeling,
ATO effects were seen at a proteome level, although
the study was done for NB4 cells [34].
The present results also showed that ATO
retarded population growth, and that populations
Figure 9. Addition of ATO leads to a general dilatation in cell cycle. The percentage of cells with G2/M DNA (A) or G1 DNA (B) for cells
that were untreated or treated with ATRA, ATO, or ATRA plus ATO for 24 h and 100 ng/mL nocodazole thereafter for 0, 6, 12, 18 h is
shown. (A) Treatment with ATO (with or without ATRA) leads to a statistically significantly (* and **, p50.05) different percentage of cells
in G2/M phase at 18 h when compared with the control case. # and ## indicate statistically significant differences for 12 h. (B) { and {{indicate that the percentage of cells in G1 phase is statistically different from the control case at 18 h when ATO (with or without ATRA) is
added to the medium. { and {{ show statistically significantly different cases at 12 h, when compared to control. Representative DNA
histograms of control (untreated), ATRA-, ATO-, and ATRA plus ATO-treated populations after 18 h in nocodazole are shown (C).
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treated with ATO plus ATRA were more growth-
inhibited than those treated with ATRA alone. The
viability of cultures detected by trypan blue exclusion
routinely exceeded 95%, and as seen conclusively
from the Hoechst assay there was no increase in
apoptosis upon ATO addition. There was also no
extra accumulation of cells in G0 in ATO plus
ATRA-treated cells compared to ATRA-treated
cells. There was an ATO-induced retardation of
cell cycle progression.
In summary, in PML–RARa negative HL-60
human myeloblastic leukemia cells, ATO causes
MAPK signaling and augments ATRA-induced
differentiation. The effects are more prominent as
the MAPK signal augmentation increases, enhancing
expression of later differentiation markers, but not
the earliest detected. A further potential contributor
to the enhanced differentiation by ATO is the
retardation of growth, possibly slowing down the
self-renewal capacity of these progenitor cells and
favoring differentiation. The results show that ATO
may be of therapeutic use to augment the effects of
ATRA in a PML–RARa negative context.
Acknowledgements
The authors would like to thank Dr. Deanna
Schaefer for her kind help in Wright’s staining of
slides. Additionally, we acknowledge the careful
review of the manuscript by Ryan Tasseff of Prof.
Jeffrey Varner’s research group.
Declaration of interest: This work was supported
in part by grants from NIH (USPS) (CA 033505),
NYSTEM, NY Department of Health, and NSF
CAREER #CBET-0846876.
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