Open Journal of Bloo d Di seases, 2011, 1, 9- 11
doi:10.4236/ojbd.2011.12003 Published Online December 2011 (
Copyright © 2011 SciRes. OJBD
Activation of ERK and P38 by the Addition of
Arsenic Trioxide in Flt3-ITD Cells
Sawami Suzuki1, Hiroko Inaba2, Takashi Satoh1,2, Toshio Okazak i1,2, Shinichiro Takahashi1,2*
1Division of Molecular Hematology, Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan; 2Division of
Hematology, Kitasato University School of Allied Health Sciences, Sagamihara, Japan.
Email: *
Received September 20th, 2011; revised October 29th, 2011; accepted November 14th, 2011.
Flt3-internal tandem duplications (Flt3-ITD) is a prevalent mutation in acute myeloid leukemia (AML). We recently
reported arsenic trioxide (ATO) and Flt3 inhibition synergize to induce apoptosis in Flt3-ITD cells. However, the sig-
naling effect of ATO in these cells has not been elucidated. Here, we demonstrate that the treatment of ATO potently
induces the activa tion of extracellular regulated kina se (ERK)-mitogen activated protein kinase (MAPK), and modestly
activates p38-MAPK in BaF3-Flt3-ITD cells, among other major (PI3-kinase-Akt, c-jun N-terminal kinase [JNK]) sig-
naling pathways examined. In contrast, in BaF3-Flt3-wild type (WT) cells, slight activation of p38, bu t none for others,
was observed. As MAPK kinase (MEK), as well as p38 inhibition is reported to enhance ATO-induced apoptosis in
AML and various hematolog ical malignancies , our results suggest that Flt3 mutation status is important for the effect of
these combinations.
Keywords: Flt3-ITD, ERK, Arsenic Trioxide
1. Introduction
Flt3 is a member of the class III receptor tyrosine
kinase family, and approximately one third of AML
patients have mutation of this gene. The majority of
such mutations are ITD in the juxtamembrane domain
of Flt3, specifically found in AML, which result in
ligand-independent dimerization and tyrosine phos-
phorylation of the receptor [1,2]. This causes constitu-
tive activation of downstream signaling pathways, in-
cluding the Ras/MEK/MAPK pathway, leading to ab-
errant cell growth and differentiation block in leukemia
cells [1]. The MEK/MAPK pathway is an important
signaling cascade involved in the control of hemato-
poietic cell proliferation and differentiation [3]. The
downmodulation of MEK phosphorylation inhibits
proliferation and induces apoptosis of primary AML
blasts [4]. ATO has currently widely used in the treat-
ment of the patients with relapsed or refractory APL
[5]. It was reported that downmodulation of MEK/ERK
phosphorylation significantly enhanced ATO-induced
apoptosis in primary APL blasts [6]. Additionally, it
was reported that not only in APL blasts, but in AML
cases, the combination of ATO with an MEK inhibitor
is very efficient [7,8]. We recently reported that ATO
and Flt3 inhibition synergize to induce apoptosis in
Flt3-ITD cells [8]. In this study, we examined the sig-
naling effect of ATO on Flt3-WT and Flt3 mutated
cells, to clar ify th e me chanis ms of the sp ecif ic effe ct of
ATO on Flt3-ITD cells.
2. Materials and Methods
2.1. Cell Culture
BaF3-Flt3-ITD and BaF3-Flt3-WT cells were recently
established in our laboratory [9]. All experiments using
BaF3-Flt3-ITD cells were performed under IL-3 depriva-
tion because the Flt3-ITD signal is redundant in the
presence of IL-3 stimulation of the cells [10].
2.2. Westernblot
For assay of phosphorylated proteins, 3 × 106 of BaF3-
Flt3-WT and BaF3-Flt3-ITD cells [9] were washed twice
with PBS and resuspended with serum free RPMI and
seeded into 10 cm dishes. Then the cells were exposed
with FL (100 ng/ml) in the presence or absence of ATO
(4 µM). The cells were extracted at indicated time points.
Phosphorylated proteins of ERK, p38, JNK and Akt were
determined by Westernblot as previously described [11].
Activation of ERK and P38 by the Addition of Arsenic Trioxide in Flt3-ITD Cells
3. Results and Discussion
To uncover the signaling difference between Flt3-ITD
and Flt3-WT, we employed BaF3-Flt3-ITD and -WT
cells those we established in our laboratory [9]. As
shown in Figure 1, in BaF3-Flt3-ITD cells, ERK was
potently phosphorylated 30 min after the addition of 4
µM of ATO and its activation was decreased thereafter.
There was a modest effect for the phosphorylation of
p38-MAPK in BaF3-Flt3-ITD cells. In contrast, in
BaF3-Flt3-WT cells, slight activation of p 38-MAPK, bu t
none for others, was observed by the addition of ATO.
It was reported that ATO at clinically achievable con-
centrations (2 - 7 µmol/l) activated p38-MAPK in several
leukemia cell lines or myeloma cells [12,13]. p38-MAPK
has been shown to mediate both proapototic/growth in-
hibitory and antiapoptotic/pro-growth signals in differ-
ent systems, apparently depending on the stimulus and
cell type involved [5,12,14]. Combination of ATO with
p38-MAPK inhibition significantly increased the apop-
tosis and/or growth inhibition induced by ATO treatment
in these cells [12,13]. Therefore, current results indicate
that combination therapy of p38-MAPK inhibitors with
ATO might be effective for both Flt3-ITD and WT cells.
Figure 1. The signaling effect of ATO in BaF3-Flt3-WT and
BaF3-Flt3-ITD cells. 3 × 106 of BaF3-Flt3-WT and BaF3-
Flt3-ITD cells were washed twice with PBS and resuspended
with serum free RPMI and seeded into 10 cm dishes. Then
the cells were exposed with FL (100 ng/ml) in the presence
or absence of ATO (4 µM). The cells were extracted at in-
dicated time points. Phosphorylated proteins of ERK, p38,
Akt and JNK were determined.
-actin served as a loading
control. The results presented are representative of two to
three different experiments.
Common chemotherapeutic drugs usually induce a wide
range of cytotoxic effects on hematopoietic stem cells or
progenitor cells of other tissues. In addition, there are
many serious side effects of chemotherapy. In contrast,
the therapeutic dose of ATO is associated with an ac-
cep ta b l e t ox i city level without bone marrow hypoplasia or
alopecia [15]. Although QT interval prolongation APL
differentiation syndrome are the most serious complica-
tions observed in patients with ATO, ATO is well toler-
ated and toxicities are manageable and reversible. From
these points of view, combinatio n therapy with ATO may
be advantageous in leukemia therapy. It was noted that
ATO is a potent stimulator of ERK and AP-1 transacti-
vational activity and an efficient inducer of c-fos and
c-jun gene expression [16,17]. Induction of c-jun and
c-fos by ATO is also associated with the activation of
JNK [16]. However, a subsequent study indicated that at
a therapeutic dose for AML (<5 µM), ATO dominantly
induces ERK, but not JNK phosphorylation [18], which
is consistent with the current result of our study. As
MEK inhibitors are promising agents for the treatment of
AML [19-22], our results provide an evidence that the
status of Flt3 receptor is responsible for the effect of
MEK inhibition in combination with ATO.
4. Acknowledgements
This work was supported in part by Grants-in-Aid for
Scientific Research (No. 23590687) from the Ministry of
Education, Science and Culture, Japan, Takeda Science
Foundation, and a foundation from Kitasato University
School of Allied Health Sciences (Grant-in-Aid for Re-
search Project, No. 2010-1004, No. 2011-1001). There
are no conflicts of interest.
[1] S. Takahashi, “Downstream Molecular Pathways of Flt3
in the Pathogenesis of Acute Myeloid Leukemia: Biology
and Therapeutic Implications,” Journal of Hematology &
Oncology, Vol. 4, No. 13, 2011, pp. 1-10.
[2] S. Takahashi, “Current Findings for Recurring Mutations
in Acute Myeloid Leukemia,” Journal of Hematology &
Oncology, Vol. 4, No. 36, 2011, pp. 1-11.
[3] M. B. Miranda, T. F. McGuire and D. E. Johnson, “Im-
portance of MEK-1/-2 Signaling in Monocytic and
Granulocytic Differentiation of Myeloid Cell Lines,”
Leukemia, Vol. 16, No. 4, 2002, pp. 683-692.
[4] M. Milella, S. M. Kornblau, Z. Estrov, et al., “Therapeu-
tic Targeting of the MEK/MAPK Signal Transduction
Module in Acute Myeloid Leukemia,” Journal of Clinical
Investigation, Vol. 108, No. 6, 2001, pp. 851-859.
[5] S. Takahashi, “Combination Therapy with Arsenic Tri-
Copyright © 2011 SciRes. OJBD
Activation of ERK and P38 by the Addition of Arsenic Trioxide in Flt3-ITD Cells
Copyright © 2011 SciRes. OJBD
oxide for Hematological Malignancies,” Anti-Cancer
Agents in Medicinal Chemistry, Vol. 10, No. 6, 2010, pp.
[6] P. Lunghi, A. Tabilio, F. Lo-Coco, P. G. Pelicci and A.
Bonati, “Arsenic Trioxide (ATO) and MEK1 Inhibition
Synergize to Induce Apoptosis in Acute Promyelocytic
Leukemia Cells,” Leukemia, Vol. 19, No. 2, 2005, pp.
234-244. doi:10.1038/sj.leu.2403585
[7] P. Lunghi, A. Costanzo, L. Salvatore, et al., “MEK1 In-
hibition Sensitizes Primary Acute Myelogenous Leuke-
mia to Arsenic Trioxide-Induced Apoptosis,” Blood, Vol.
107, No. 11, 2006, pp. 4549-4553.
[8] S. Takahashi, H. Harigae, H. Yokoyama, et al., “Syner-
gistic Effect of Arsenic Trioxide and Flt3 Inhibition on
Cells with Flt3 Internal Tandem Duplication,” Interna-
tional Journal of Hematology, Vol. 84, No. 3, 2006, pp.
256-261. doi:10.1532/IJH97.06076
[9] S. Takahashi, H. Harigae, J. Kameoka, T. Sasaki and M.
Kaku, “AML1B Transcriptional Repressor Function is
Impaired by the Flt3 Internal Tandem Duplication,” Brit-
ish Journal of Haematology, Vol. 130, No. 3, 2005, pp.
428-436. doi:10.1186/1756-8722-4-13
[10] S. Takahashi, H. Harigae, M. Kaku, T. Sasaki and J. D.
Licht, “Flt3 Mutation Activates p21(WAF1/CIP1) Gene
Expression through the Action of STAT5,” Biochemical
and Biophysical Research Communications, Vol. 316, No.
1, 2004, pp. 85-92. doi:10.1186/1756-8722-4-36
[11] M. Hirosawa, M. Nakahara, R. Otosaka, et al., “The p38
Pathway Inhibitor SB202190 Activates MEK/MAPK to
Stimulate the Growth of Leukemia Cells,” Leukemia Re-
search, Vol. 33, No. 5, 2009, pp. 693-699.
[12] J. Wen, H. Y. Cheng, Y. Feng, et al., “P38 MAPK Inhibi-
tion Enhancing ATO-Induced Cytotoxicity Against Mul-
tiple Myeloma Cells,” British Journal of Haematology,
Vol. 140, No. 2, 2008, pp. 169-180.
[13] A. Verma, M. Mohindru, D. K. Deb, et al., “Activation of
Rac1 and the p38 Mitogen-Activated Protein Kinase
Pathway in Response to Arsenic Trioxide,” Journal of
Biological Chemistry, Vol. 277, No. 47, 2002, pp. 44988-
44995. doi:10.1074/jbc.M207176200
[14] P. Lunghi, N. Giuliani, L. Mazzera, et al., “Targeting
MEK/MAPK Signal Transduction Module Potentiates
ATO-Induced Apoptosis in Multiple Myeloma Cells
through Multiple Signaling Pathways,” Blood, Vol. 112,
No. 6, 2008, pp. 2450-2462.
[15] D. Douer and M. S. Tallman, “Arsenic Trioxide: New
Clinical Experience with an Old Medication in Hema-
tologic Malignancies,” Journal of Clinical Oncology, Vol.
23, No. 10, 2005, pp. 2396-2410.
[16] M. Cavigelli, W. W. Li, A. Lin, et al., “The Tumor Pro-
moter Arsenite Stimulates AP-1 Activity by Inhibiting a
JNK Phosphatase,” The European Molecular Biology Or-
ganization Journal, Vol. 15, No. 22, 1996, pp. 6269-
[17] S. Ludwig, A. Hoffmeyer, M. Goebeler, et al., “The
Stress Inducer Arsenite Activates Mitogen-Activated Pro-
tein Kinases Extracellular Signal-Regulated Kinases 1
and 2 via a MAPK Kinase 6/p38-Dependent Pathway,”
Journal of Biological Chemistry, Vol. 273, No. 4, 1998,
pp. 1917-1922. doi:10.1074/jbc.273.4.1917
[18] C. Huang, W. Y. Ma, J. Li, A. Goranson and Z. Dong,
“Requirement of Erk, but Not JNK, for Arsenite-Induced
Cell Transformation,” Journal of Biological Chemistry,
Vol. 274, No. 21, 1999, pp. 14595-14601.
[19] P. Baines, J. Fisher, L. Truran, et al., “The MEK Inhibitor,
PD98059, Reduces Survival but Does Not Block Acute
Myeloid Leukemia Blast Maturation in vitro,” European
Journal of Haematology, Vol. 64, No. 4, 2000, pp. 211-
218. doi:10.1034/j.1600-0609.2000.90139.x
[20] P. Lunghi, A. Tabilio, P. P. Dall'Aglio, et al., “Down-
modulation of ERK Activity Inhibits the Proliferation and
Induces the Apoptosis of Primary Acute Myelogenous
Leukemia Blasts,” Leukemia, Vol. 17, No. 9, 2003, pp.
1783-1793. doi:10.1038/sj.leu.2403032
[21] M. A. Morgan, O. Dolp and C. W. Reuter, “Cell-Cycle-
Dependent Activation of Mitogen-Activated Protein
Kinase (MEK-1/2) in Myeloid Leukemia Cell Lines and
Induction of Growth Inhibition and Apoptosis by Inhibi-
tors of RAS Signaling,” Blood, Vol. 97, No. 6, 2001, pp.
1823-1834. doi:10.1182/blood.V97.6.1823
[22] S. Takahashi, “Inhibition of the MEK/MAPK Signal
Transduction Pathway Strongly Impairs the Growth of
Flt3-ITD Cells,” American Journal of Hematology, Vol.
81, No. 2, 2006, pp. 154-155. doi:10.1002/ajh.20520