Activation of ERK and P38 by the Addition of Arsenic Trioxide in Flt3-ITD Cells

doi:10.4236/ojbd.2011.12003

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Open Journal of Bloo d Di seases, 2011, 1, 9- 11

doi:10.4236/ojbd.2011.12003 Published Online December 2011 (http://www.SciRP.org/journal/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: *[email protected]

Received September 20th, 2011; revised October 29th, 2011; accepted November 14th, 2011.

ABSTRACT

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.

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