Bcr-Abl-mediated Raf kinase inhibitor protein suppression promotes chronic myeloid leukemia progenitor cells proliferation

doi:10.4236/scd.2011.13006

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Vol.1, No.3, 54-66 (201 1)

doi:10.4236/scd.2011.13006

S tem Cell Discovery

Bcr-Abl-mediated Raf kinase inhibitor protein

suppression promotes chronic myeloid leukemia

progenitor cells proliferation

Satoki Nakamura1*#, Tomohiro Yagyu2#, Tomonari Takemura2, Lin Tan2, Yasuyuki Nagata2,

Daisuke Yokota2, Isao Hirano2, Kiyoshi Shibata3, Michio Fujie3, Shinya Fujisawa4,

Kazunori Ohnishi1,2

1Department of Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu, Japan;

*Corresponding Author: satonaka@hama-med.ac.jp

2Cancer Center, Hamamatsu University School of Medicine, Hamamatsu, Japan;

3Equipment Center, Hamamatsu University School of Medicine, Hamamatsu, Japan;

4Division of Hematology, Hamamatsu Medical Center, Hamamatsu, Japan.

Received 27 July 2011; revised 18 September 2011; accepted 30 September 2011.

ABSTRACT

The Ras/Raf-1/MEK/ERK pathway is constitutive-

ly activated in Bcr-Abl transformed cells, and Ras

activity enhances the oncogenic ability of Bcr- Abl.

On the other hand, Raf kinase inhibitor protein

(RKIP) inhibits activation of MEK b y Raf-1 and its

downstream signal transduction, resulting in

blocking the MAP kinase pathway. Moreover, Raf-1

has been reported to regu late cell cycle progres-

sion. However, the mechanism by which Bcr-Abl

promotes the cell cycle progression through Ra f- 1

is not completely understood. In the present

study, we found that the expression of RKIP was

suppressed in CML cells, and investigated the

interaction between RKIP and Bcr-Abl in CML

cells. In aldehyde dehydrogenase (A L D H ) hi/ CD34+

cells derived from CML patients, the inhibition of

Bcr-Abl induced RKIP expression and reduced

the phosphorylated-FOXM1 (pFOXM1) status, re-

sulting in inhibited colony formation of Bcr-Abl+

progenitor cells. Moreover, overexpression of

RKIP significantly decreased the colony num-

bers, reduced the pFOXM1 status, and reduced

pFOXM1 target genes such as Skp2, Cdc25B and

KIS, and induced the expression of p27Kip1 and

p21Cip1. Thus, Bcr-Abl represses the expression

of RKIP, and continuously activates FOXM1, re-

sulting in the proliferation of CML pr ogenitor cells

through the c ell cycle modulati on.

Keywords: RKIP; FOXM1; Bcr-Abl; Progenitor

Cells; Cell Cycle

1. INTRODUCTION

Chronic myeloid leukemia (CML) is a hematopoietic

stem cell malignancy that is characterized by the Phila-

delphia chromosome [1,2], a shortened chromosome 22

that is a by-product of a reciprocal chromosomal trans-

location between the long arms of chromosomes 9 and

22 t (9; 22) (q34; q11), resulting in a chimeric Bcr-Abl

oncoprotein with highly deregulated, constitutive tyro-

sine kinase activity [3,4]. The most commonly occurring

from of Bcr-Abl is a 210-kDa protein that is a critical

role in the pathogenesis of CML [5]. Bcr-Abl activates a

variety of signaling pathways, including the Ras/Raf-

1/MEK/ERK [6], PI3K/Akt [7], JAK/STAT [8], and NF-

κB [9] signaling pathways. These signaling pathways play

important role in Bcr-Abl-mediated leukemogenesis. The

Ras pathway regulates multiple biological aspects includ-

ing mitogenesis and differentiation [10,11], and is consti-

tutively activated in Bcr-Abl transformed cells. Moreover,

inhibition of Ras activity represses the oncogenic ability

of Bcr-Abl [6].

Bcr-Abl activates the Ras pathway through the adap-

tor protein Grb2, which associates with the guanine nu-

cleotide exchange factor Sos and binds to the phospho-

rylated tyrosine residue 177 within the Bcr region of Bcr-

Abl. Sos forms a platform for recruitment of Ras, result-

ing in the activation of Ras from GDP- to GTP-bound

state [12,13]. The activated Ras phosphorylates Raf, and

ERK is phosphorylated by MEK, which is phosphoryla-

ted and activated by the activated Raf-1 [14]. ERK then

translocates into the nucleus and promotes cell prolifera-

tion through activation of various transcription factors as

playing important roles in Bcr-Abl-mediated leukemo-

genesis [15]. On the other hand, the anti-apoptotic factor

#These authors contributed equally.

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

5555

of the Bcl-2 family, Mcl-1, is constitutively expressed in

a Ras/Raf-1/MEK-dependent manner via Ras activation in

Bcr-Abl positive cells [16]. Moreover, Raf-1 has been re-

ported to have an important role in regulation of cell cycle

progression [17]. Thus, in Bcr-Abl expressing cells, Ras

pathway plays an important role in both cell proliferation

and survival.

Raf kinase inhibitor protein (RKIP), which is a mem-

ber of the phosphatidylethanolamine binding protein 1

(PEBP1) family, is a ubiquitously expressed and consti-

tutively conserved protein [18]. RKIP inhibits activation

of MEK by Raf-1 and its downstream signal transduc-

tion, resulting in blocking the MAP kinase pathway [19,

20]. It has been reported that RKIP expression is down-

regulated in metastatic prostate cancers and the loss of

RKIP levels promotes the metastatic potential of prostate

cancer cells [21]. Moreover, RKIP has been reported to

be downregulated in malignant melanoma [22], hepato-

cellular carcinoma [23], breast cancer [24], and colorec-

tal cancer [25,26]. These data suggest RKIP might play

as a tumor and/or metastasis suppressor for solid tumors.

However, it has been reported that RKIP expression has

no relationship with ERK phosphorylation or the clinical

course of disease in patients with melanoma or breast

cancer [24,27]. These different observations suggest that

RKIP contributes to the regulation of not only MAPK

signaling including phosphorylated ERK but also other

signal pathways.

In the present study, we investigated the role of RKIP

in CML cell lines and the Bcr-Abl+ hematopoietic pro-

genitor cells derived from CML patients. We found that

Bcr-Abl suppressed the expression of RKIP in CML

cells, and detected changes of the cell cycle regulator

proteins in CML-derived hematopoietic progenitor cells

and CML cell lines. Interestingly, we also found the up-

regulation of p27Kip1 and p21Cip1 through FOXM1 inac-

tivation in RKIP overexpressed-CML cells.

2. MATERIALS AND METHODS

2.1. Reagents

Imatinib mesylate (STI571) and AMN107 were kindly

provided by Novartis Pharmaceuticals (Basel, Switzerland).

BMS354825 was kindly provided by Bristol-Myers Sq-

uibb (New York, NY). Each compound was prepared as a

10 mM stock solution in dimethyl sulfoxide and stored at

–20˚C. Experiments were performed with 1000-fold dilu-

tions of the stock solutions into reaction mixtures.

2.2. Cells and Cell Cultures

Human CML cell lines, K562 and Meg01, and human

acute myeloblastic leukemia (AML) cell lines, U937 and

HL60, were purchased from American Type Culture

Collection (Manassas, VA). We established SHG3 cells

from the bone marrow of a patient with CML (chronic

phase), and YRK2 cells from the bone marrow of patient

with AML M5b (French-American-British (FAB) classi-

fication). These cells were cultured in RPMI 1640 con-

taining 10% heat-inactivated fetal calf serum, 2 mM

L-glutamine, 100 g/mL streptomycin, and 200 U/mL

penicillin (GIBCO-BRL, Gaithersburg, MD) and main-

tained in a humidified 5% CO2 atmosphere at 37˚C.

2.3. Bone Marrow Samples

Prior to participation in the study, patients gave infor-

med consent according to the Declaration of Helsinki.

Samples of normal bone marrow were obtained from 6

healthy volunteers. Bone marrow was also obtained from

12 patients with CML in the first chronic phase. Mo-

nonuclear cells (MNCs) were isolated from bone mar-

row samples by Ficoll-Hypaque density gradient centri-

fuga- tion. CML cells were obtained from patients before

they began treatment with Abl kinase inhibitors.

2.4. Leukemic Blast Cells Purified by ALDH

Activity and CD34 Expression from

Bone Marrow Derived MNCs

For 2-color staining, MNCs were stained with anti-

CD34-phycoerythrin (PE) conjugated antibody (Becton

Dickinson, San Jose, CA) and Aldefluor reagent (Stem-

Co Biomedical, Durham, NC) according to the manu-

facturer’s specifications and separated using fluoresce-

nce-activated cell sorting (FACS) (Becton Dickinson).

Aldefluor substrate (0.625 g/mL) was added to 2 to 7 ×

106 cells/mL suspended in Aldefluor assay buffer and in-

cubated for 20 to 30 min at 37˚C to allow the conversion

of Aldefluor substrate to a fluorescent product that is re-

tained within the cell because of its negative charge. The

ALDHhi cells were gated, and the CD34+ and CD34- ce-

lls in the gated ALDHhi population were sorted on a FACS.

Sorted ALDHhi/CD34+ and ALDHhi/CD34– cell popula-

tions were collected in methylcellulose media (Methocut

H4435; Stem Cell Technologies).

2.5. RT-PCR and Quantitative Real-Time

PCR (QRT-PCR)

Total RNA was extracted from cells by using an

RNeasy system (Qiagen, Tokyo, Japan), and 1 g RNA

was reverse transcribed by using a 1st strand cDNA sy-

nthesis kit (Roche, Indianapolis, IN). PCR was perfor-

med with a DNA thermal cycler (model PTC 200; MJ

Research, Watertown, MA). Oligonucleotide sequences

for each primer were as follows: RKIP, sense 5’-ATG-

CCGGTGGACCTCAGC-3’, antisense 5’-GCTGCTC-

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

GTACAGTTTGGGA-3’; FOXM1, sense 5’-GGAGGA-

AA- TGCCACACTTAGCG-3’, antisense 5’-TAGGAC-

TTCTTGGGTCTTGGGGTG-3’; Bcr-Abl, sense 5’-CGA-

GCGGCTTCACTCAGA-3’, antisense 5’-ACAGCATTC-

CGCTGACCAT-3’, and GAPDH; sense 5’-GAACGGGA-

AGCTCACTGGCATGGC-3’, antisense 5’-TGAGGTCC-

ACCACCCTGTTGCTG-3’. PCR conditions for RKIP,

FOXM1, Bcr-Abl and GAPDH were 28 cycles of denatura-

tion at 96˚C for 30 sec, annealing at 56˚C for 30 sec, and

extension at 72˚C for 30 sec. PCR products were electro-

phoresed in a 1.5% agarose gel containing 500 g/L

ethidium bromide and visualized with UV light. In each

experiment, RT-PCR was performed in duplicate. The

quantitative real-time PCR was performed by using SY-

BER-Green dye on an ABI PRISM 7700 Sequence detec-

tor (Perkin-Elmer/Applied Biosystems, Foster City, CA).

2.6. Plasmid and RNA Interference

The full-length cDNA coding human RKIP and FOXM1

was obtained by RT-PCR from human bone marrow cDNA

(BD Biosciences Clontech, Palo Alto, CA) and cloned into

the eukaryotic expression vector pcDNA3.1/V5-His (In-

vitrogen, Carlsbad, CA). Sequences of recombinant RKIP

and FOXM1 cDNAs were verified by automated sequ-

encing.

The vectors for RNA interference (RNAi) specific to

human RKIP and Bcr-Abl were constructed based on the

piGENE PUR hU6 vector (iGENE Therapeutics, Tsu-

kuba, Japan), according to the manufacturer’s instruc-

tions. We used the following targeting sequences and

oligonucleotides: RKIP shRNA; 5’-AAGGTGGCCTC-

CTTCCGTAAA-3’, Bcr-Abl shRNA #1: sense 5’-GC-

AGAGUUCAAAAGCCCUUdTdT-3’ and antisense 5’-

AAGGGCUUUUGAACUCUGCdTdT-3’ and Bcr-Abl

shRNA #2, sense 5’-AGCAGAGUUCAAAAGCCCU-

dTdT-3’ and antisense 5’-AGGGCUUUUGAACUCU-

GCUdTdT -3’. A scrambled shRNA sequence was sense

5`-UUGUACGGCAUC AGCGUUAdTdT and antisense

5`-UUACGUGAUGCCGUA CAAdTdT and used as a

control. The vector was transfected into cells by using the

Lipofectamine 2000 kit (Life Technologies, Gaithersburg,

MD), according to the manufacturer’s instructions. After

12 h, the same transfection procedure was repeated, and

cells were harvested 48 and 72 h after the initial transfe-

ction. Transfection efficiency was consistently 50% to

60%, as determined by the RT-PCR measurement of RKIP,

FOXM1, and Bcr-Abl mRNA.

2.7. Lentivirus Construction and Production

The full-length RKIP, FOXM1, and b3a2 Bcr-Abl cDNA

(a kind gift from Dr. J. Y. Wang, University of California,

San Diego, CA) was cloned upstream from the internal

ribosomal entry site of replication-deficient, self-inacti-

vating lentiviral vector, pRRLsin-IRES-EGFP. The RKIP,

FOXM1, and Bcr-Abl—containing vectors were termed

LV-RKIP, LV-FOXM1, and LV-Bcr-Abl, respectively, and

the control vector was termed LV-Con. All vector particles,

pseudotyped with the vesicular stomatitis virus G glyco-

protein, were produced using a three-plasmid expression

system as previously described [28]. The cell-free su-

pernatants containing virus particles, LV-RKIP, LV-FOX-

M1, LV-Bcr-Abl, and LV-Con, were separately concentr-

ated by ultracentrifugation at 28,000 rpm for 2 h at 4˚C in

an SW28 rotor. Virus vector stocks were resuspended in

complete DMEM and stored at –80˚C. The virus vector

titer of pRRLsin-IRES-EGFP was derived on a fluores-

cence-activated cell sorter (Becton Dickinson) using

transfected HeLa cells. To calculate titers, the number of

target cells was multiplied by the percentage of eGFP-

positive cells divided by the volume of the input virus

and the titer of each concentrated lentivirus vector stock

(LV-RKIP, LV-FOXM1, LV-Bcr-Abl and LV-Con), which

were 3.2 × 108, 2.4 × 108, 3.1 × 108 and 4.7 × 108 gene-

transducing units per milliter, respectively.

2.8. Immunoprecipitation and Western Blot

Analysis

For immunoprecipitation and immunoblotting, total

cell lysates were prepared from the Abl kinase inhibitor

treated-, DNA-transfected, and shRNA-transfected-cells.

Cell lysates were incubated with the FOXM1, Rb and

Abl-specific monoclonal antibody for 1 hour at 4C,

washed Protein G agarose beads (sc-2002, Santa Cruz,

CA) were added, and incubated overnight at 4C. The

immunoprecipitates were washed 3 times in the lysis

buffer, and proteins were eluted with the SDS sample

loading buffer. Western blot analysis was performed as

previously described [28] using the following antibodies:

anti-RKIP goat polyclonal antibody (sc-5426, Santa Cruz),

anti-FOXM1 rabbit polyclonal antibody (sc-500, Santa

Cruz), anti-c-Abl mouse monoclonal antibody (sc-52990,

Santa Cruz), anti-KIS rabbit polyclonal antibody (AB-

GENT, San Diego, CA), anti-Rb mouse monoclonal an-

tibody (sc-74562, Santa Cruz), anti-phosphotyrosine (p-

Tyr) mouse monoclonal antibody (sc-508, Santa Cruz),

anti-p27Kip1 rabbit polyclonal antibody (sc-527, Santa

Cruz), and anti-p21Cip1 mouse monoclonal antibody (sc-

469, Santa Cruz). To assure equal protein loading, simi-

lar experiments were performed with a mouse mono-

clonal anti-Actin antibody (C-4; ICN, Aurora, OH) as an

internal control. After being washed in Tris-buffered sa-

line Tween, blots were incubated with horseradish peroxi-

dase-conjugated anti-mouse, anti-rabbit, or anti-goat IgG

(Amersham, Arlington Heights, IL) for 1 h and exposed

to X-ray film at room temperature. The signal was de-

tected by chemiluminescence with an ECL detection kit

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

5757

(Amersham).

2.9. Cell Proliferation and Viability Assay

To assess cell proliferation, cells (1 × 104) were see-

ded in 24-well plates and incubated with STI571 (1 M)

at 37˚C. Cells were then transfected with RKIP DNA.

After 24, 48, and 72 hours incubation, cell proliferation

was measured by counting cells using a hemocytometer.

To assess cell viability, the numbers of nonviable cells

were determined by counting the cells that showed try-

pan blue uptake in a hemocytometer and reported as the

percentage of untreated control cells. Each data point

was performed in triplicate, and the results are reported

as mean counts  SD.

2.10. Cell Cycle Analysis

DNA content analysis was performed using propidium

iodide (PI) (Sigma-Aldrich, St. Louis, MO) staining. In

brief, cells were transfected with RKIP DNA. Cells were

cultured in 2 ml complete medium containing 1 × 106

cells at 37˚C. After 7 days incubation, the cells were wa-

shed twice with cold PBS, fixed with 70% ethanol over-

night before treatment with 100 g/ml RNase A, and

then stained with 50 g/ml PI. The relative DNA content

per cell was measured by flow cytometry using an Epics

Elite flow cytometer (Coulter Immunotech, Marseille,

France). The percentage of cells in G1, S, and G2/M ph-

ases, was calculated using ModFit program (Becton Dic-

kinson, San Jose, CA).

2.11. Colony Forming Cell Assa y

ALDHhi/CD34+ cells from CML bone marrow cells

were treated the Abl kinase inhibitors, or transfected with

scrambled shRNA, RKIP shRNA, Bcr-Abl shRNA #1,

RKIP DNA and FOXM1 DNA. Human clonogenic pro-

genitor assays were performed by plating purified popula-

tions of cells at concentrations ranging from 2 × 102 to 7 ×

102 (ALDHhi/CD34+) into methylcellulose media (Metho-

cut H4435). Colonies were enumerated under light mi-

croscopy (Zeiss, Muenchen, Germany) following incuba-

tion at 37˚C, 5% CO2, for 7 to 10 days.

2.12. Collection of Progenitor Cells, and

QRT-PCR and Western Blot Analysis in

the Progenitor Cells

After the colony forming assays, each colony was har-

vested by glass syringe, pooled, and washed. Immunopr-

ecipitation and western blotting were performed using

the interested antibody. An RNeasy system was used to

extract total RNA from approximately 2 × 104 cells from

each colony, and then RT-PCR and QRT-PCR were also

performed using RKIP, FOXM1, Bcr-Abl, and GAPDH

primers.

2.13. Statistical Analysis

Data are representative of at least three experiments

with essentially similar results. These results are expre-

ssed as the means ± SD from three independent experi-

ments. The means were compared by using the Student’s

t test. P values less than 0.01 were considered statisti-

cally significant.

3. RESULTS

3.1. The Abl kinase Inhibitors Induce RKIP

mRNA Expression in CML Cell Lines

RKIP mRNA were constitutively expressed in CML

cell lines (K562, Meg01, and SHG3 cells) (Figure 1(a))

and AML cell lines (U937, HL60, and YRK2 cells)

(Figure 1(b)). In CML cell lines, the expression of RKIP

mRNA was inhibited as compared to AML cell lines.

Interestingly, we found that the expression of RKIP

mRNA increased in the three CML cell lines treated with

Abl kinase inhibitors (STI571, AMN107, or BMS35482

5) for 24 h as compared to untreated cells. However, the

Abl kinase inhibitors did not affect the RKIP mRNA

expression in the AML cell lines, which did not express

Bcr-Abl mRNA. On the other hand, in the CML cell lines

transfected with Bcr-Abl shRNA #1, the RKIP mRNA

expression was significantly increased as compared to

control cells. In the AML cell lines transfected with LV-

Bcr-Abl, the RKIP mRNA expression was reduced as

compared to control cells.

As shown in Figure 1(c), RKIP protein was also con-

stitutively expressed in CML cell line (K562, Meg01 and

SHG3 cells) and in AML cell line (HL60, U937 and

YRK2 cells) (data not shown). The treatment with Abl

kinase inhibitors increased the protein levels of RKIP in

CML cells. Moreover, the RKIP protein level was in-

creased in K562 and Meg01 cells transfected with Bcr-

Abl shRNA #1. Thus, Abl kinase inhibitors or the knock-

down of Bcr-Abl protein induced RKIP protein expres-

sion. The expression of RKIP protein in CML cell lines

was at a lower level compared to that in AML cell lines.

On the other hand, in the AML cell lines, the Abl kinase

inhibitors did not affect the levels of RKIP protein (data

not shown). The Abl kinase inhibitors and knockdown of

Bcr-Abl using shRNA also inhibited the levels of tyro-

sine phosphorylated Bcr-Abl (p-Bcr-Abl) in CML cells.

Thus, Bcr-Abl expression regulates RKIP mRNA and

protein expression.

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

3.2. Effects of RKIP Expression on CML C ell

Proliferation

RKIP expression was induced by the Abl kinase in-

hibitors or the knockdown of Bcr-Abl in CML cells. To

examine the functional importance of RKIP expression,

we transfected K562 cells with RKIP DNA, and assessed

the effects of RKIP on CML cell proliferation (Figure

2(a)). When K562 cells were transfected with RKIP DNA,

the cell proliferation was inhibited compared to untreated

cells, and the Abl kinase inhibitor (STI571) significantly

inhibited the proliferation of untreated K562 cells. The

cell viability of K562 cells transfected with RKIP DNA

or treated with STI571 was almost similar (Figure 2(b)).

On the other hand, the expression of RKIP mRNA was

significantly increased by STI571 treatment (Figure 2(c)).

These results show that the Abl kinase inhibitor induces

RKIP mRNA expression, and RKIP plays an important

role in the inhibition of CML cell proliferation by the

Abl kinase inhibitors.

Next, we examined how RKIP inhibited CML cell pro-

liferation. The analysis of K562 cells at 5 days posttrans-

fection with RKIP DNA indicated an increase in the per-

centage of cells in G1 and decrease in G2/M and S popula-

tion. Moreover, no increase in the sub-G1 population of

(a)

(b)

(c)

(a) & (b) CML cell lines (K562, Meg01, and SHG3) (a) and AML cell lines

(U937, HL60, and YRK2); (b) were untreated or treated with STI571 (0.5

M), ANM107 (5 nM), and BMS354825 (1 nM) for 24 h. CML cells were

harvested 3 days after transfection with control scrambled shRNA #1 or

Bcr-Abl shRNA #1. AML cell lines were harvested 7 days after transfection

with LV-Con or LV-Bcr-Abl. RT-PCR was performed to detect RKIP and

Bcr-Abl mRNAs. GAPDH is shown as an internal control. RT-PCR results

are representative of three independent experiments (left panels). Relative

amounts of mRNAs for RKIP and Bcr-Abl were measured in CML and

AML cells. The expression levels of the target mRNAs were normalized to

the relative ratio of the expression of GAPDH mRNA. The results were

expressed relative to untreated control set at 1. Each RT-PCR assay was

performed at least three times, and the results are expressed as means ± SD.

*P < 0.01 compared with untreated control (right panels); (c) The effects of

the Abl kinase inhibitors and Bcr-Abl shRNA #1 on RKIP expression were

assessed by western blotting. After 24 h treatment with the Abl kinase

inhibitors or 5 days after transfection with control scrambled shRNA or

Bcr-Abl shRNA #1, total cell lysates from K562 (upper left), Meg01 (mid-

dle left) and SHG3 (bottom left) cells were prepared and analyzed by im-

munoblotting. Actin was used as a loading control. Total cell lysates from

treated CML cells were prepared and immunoprecipitation and immunoblot

analyses were performed with, anti-c-Abl and anti-pTyr antibodies (right

panels).

Figure 1. RKIP mRNA protein expression in leukemia cells.

these cells was observed (Figure 2(d)). These results

demonstrate that the overexpression of RKIP induces a

block in G1 phase and no significant increase in apop-

tosis in CML cells. We previously reported that FOX M1

was aberrant expressed and promoted the cell cycle pro-

gression in leukemia cells [28]. Moreover, we also found

that FOXM1 expression was aberrant expressed in CML

cells, and FOXM1 knockdown inhibited CML cell pro-

liferation (data not shown). However, in CML cells, the

Abl kinase inhibitors or Bcr-Abl knockdown did not af-

fect the expression of FOXM1 mRNA and protein.

Therefore, we examined whether RKIP overexpression

reduces the phosphorylation levels of FOXM1 or affects

the expression of the cell cycle regulators, such as

p27Kip1, p21Cip1, and KIS in CML cells. In K562 cells

transfected with RKIP DNA, protein extracts were iso-

lated posttransfected 5 days, and western blot analysis

was performed to measure protein levels of these cell

cycle regulators (Figure 2(e)). The FOXM1 expression

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

5959

was strongly expressed in CML cells. How ever, the over-

expression of RKIP significantly reduced the pFOXM1

status and KIS expression compared to untreated cells. In

contrast, the levels of p27Kip1 and p21Cip1 protein were in-

creased. The similar results were observed in other CML

cell lines (Meg01 and YRK2 cells) (data not shown).

(a)

(b)

(c)

(d)

(e)

(a) and (b). K562 cells were transfected with RKIP DNA. After 5 days, the

cells were treated with the STI571 (0.5 μM) for 24, 48 and 72 h. Cell num-

bers (a) were counted and cell viability (b) was determined. The rate of

K562 cell viability is expressed as the percentage of the corresponding

control; (c). The RKIP DNA–transfected or untransfected K562 cells were

collected at the indicated times after STI571 treatment. For the analysis of

RKIP mRNA expression, quantitative RT-PCR was performed relative to

the GAP DH gene. Each RT-PCR assay was performed at least three times,

and the results are expressed as means ± SD. *P < 0.01 compared with

untreated control cells; (d). Effects of RKIP overexpression on cell cycle in

K562 cells. K562 cells were transfected with RKIP DNA. The cells were

harvested posttransfected 5 days, and FACS analysis was performed to

determine fraction in various cell cycle stages. Data shown as mean  SD.

in triplicate culture and are representative of three independent experiments.

*P < 0.01 compared with untreated control cells; (e). Evaluation of RKIP,

KIS, p27Kip 1, and p21Cip1 protein and FOXM1 phosphorylation levels after

transfection with RKIP DNA into the cells. The cells were transfected with

RKIP DNA, harvested after 5 days, and western blotting was performed by

the interest antibodies for various cell cycle regulator proteins. Actin was

used as a loading control.

Figure 2. Anti-proliferative effects of RKIP in K562 cells.

These results revealed that RKIP inhibited the phos-

phorylation of FOXM1 or KIS expression, and prevented

the cell cycle progression via 27Kip1 and p21Cip1 accumu-

lation resulting in G1 cell cycle arrest in CML cells.

3.3. RKIP Expression in Primary CML

Progenitor Cells

We examined the RKIP mRNA expression in clinical

specimens from CML patients. Hematopoietic progenitor

cells from bone marrow were obtained by flow cytometry

according to ALDH activity using the substrate Aldefluor.

ALDHhi hematopoietic progenitor cells, which include

CD34+, CD133+, c-kit+ or Lin– cells, were selected ac-

cording to side scatter and FITC properties. CD34+ and

CD34– progenitor cells in the ALDHhi population (ALD-

Hhi/CD34+ and ALDHhi/CD34– cells) were then sorted

using FACS. We isolated ALDHhi/CD34+ cells from bone

marrow cells derived from 6 healthy volunteers and 12

CML patients, and representative data from two normal

(#3 and #4) and two CML samples (#10 and #12) are

shown in Figures 3(a) and (b), respectively. In bone

marrow cells derived from healthy volunteers #3 and #4,

the populations of ALDHlow cells were 96.7% and 95.4%,

respectively, and the populations of ALDHhi cells were

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

0.09% and 0.07%, respectively. Within these ALDHhi ce-

lls, the CD34+ cell populations were 78.1% and 80.7%,

and CD34- cell populations were 20.5% and 17.4%, re-

spectively (Figure 3(a)). There were no significant dif-

ferences in the population rates among 6 normal healthy

volunteers. On the other hand, in bone marrow cells de-

rived from CML patients #10 and #12, the populations

of ALDHlow cells were 89.3% and 91.4%, respectively,

and the populations of ALDHhi cells were 4.7% and

2.6%, respectively. Within these ALDHhi cells, the CD

34+ cell populations were 82.6% and 84.1%, and CD34-

cell population were 14.8% and 11.7%, respectively

(Figure 3(b)). There were no significant differences in the

population rates among 12 CML specimens. In ALDHlow,

ALDHhi, ALDHhi/CD34- and ALDHhi/CD34+ populations

derived from CML patients, RKIP mRNA and protein

were slightly expressed as compared to normal ALDHlow,

ALDHhi, ALDHhi/CD34– and ALDHhi/CD34+ cells (data

not shown). Interestingly, in CML (#10 and #12)-derived

ALDHhi/CD34+ cells treated with STI571 or transfected

Bcr-Abl shRNA #1 or #2, RKIP mRNA was strongly

induced compared to untreated cells (Figure 3(c)). In

normal (#3 and #4) ALDHhi/CD34+ cells, the STI571

treatment did not affect the expression levels of RKIP

mRNA. In contrast, the overexpression of Bcr-Abl re-

duced the RKIP mRNA expression. The effects of STI571

on RKIP and Bcr-Abl mRNA levels in ALDHhi/CD34+

cells from CML patients (#10 and #12) were assessed by

quantitative RT-PCR. In ALDHhi/CD34+ cells derived

from all healthy volunteers (n = 6) and all CML patients

(n = 12), there were no significant differences in the lev-

els of RKIP mRNA induction among the other Abl ki-

nase inhibitors (AMN107 or BMS354825). These results

demonstrate that the Bcr-Abl inhibits the RKIP expres-

sion in ALDHhi/CD34+ cells derived from CML pa-

tients.c.

(a)

(b)

(c)

(a) & (b). Selection of ALDHhi/CD34+ hematopoietic progenitor cells from

the bone marrow of two healthy volunteer (#3 and #4) (a) and from two

CML patient (#10 and #12) (b). Region P and Q denote populations of

ALDHhi and ALDHlow cells, respectively (left panels). Region R and S

denote populations CD34+ and CD34- cells in the ALDHhi population (Re-

gion P), respectively. Negative control (light grey region) and CD34-PE

staining (dark grey region) (right panels); (c). The expression of RKIP and

Bcr-Abl mRNA in sorted ALDHhi/CD34+ cells from two healthy volunteers

(#3 and #4) and from two CML patients (#10 and #12) were analyzed after

treatment with STI571 (0.5 μM). After 3 days STI571 treatment or 5 days

after transfection with Bcr-Abl shRNA #1 and #2, the cells were harvested.

The effects of STI571 treatment or Bcr-Abl knockdown on RKIP and

Bcr-Abl mRNA levels in ALDHhi/CD34+ cells were assessed by QRT-PCR.

Representative data are shown (upper panels). The levels of target mRNAs

were normalized to the relative ratio of the expression of GAPDH mRNA

(middle and bottom panels). The results are expressed relative to the un-

treated control, which is set at a value of 1. Each RT-PCR assay was per-

formed at least three times and the results are expressed as means ± SD. *P

< 0.01 compared with untreated control cells.

Figure 3. RKIP expression in hematopoietic progenitor ALDHhi/

CD34+ cells derived from CML patients.

3.4. Induction of RKIP Expression Inhibited

the Colony Formation in CML Derived

ALDHhi/CD34+ Progenitor Cells

We examined the effect of Abl kinase inhibitors or kno-

ckdown of Bcr-Abl on the colony formation of ALD-

Hhi/CD34+ hematopoietic progenitor cells from pretreat-

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

6161

ment a CML patient #10 (Figure 4). The ALDHhi/CD34+

cells, which were untransfected or transfected with Bcr-

Abl shRNA #1, were treated with Abl kinase inhibitors

for 7 to 10 days, and then the colony numbers were cou-

nted. When untreated, the mean colony number was 116

(range, 98 - 127). When treated with STI571, AMN107,

BMS354825, or Bcr-Abl shRNA #1 transfection, the me-

an colony numbers were 11 (6 - 17), 8 (1 - 14), 5 (2 - 11), 4

(0 - 7), respectively (Figure 4(a)). The colony numbers

were remarkably reduced when the cells were cultured

with STI571, AMN107, or BMS354825 and transfected

with Bcr- Abl shRNA #1. The difference between the ef-

fects of Abl kinase inhibitors and the knockdown of Bcr-

Abl was not significant in CML-derived ALDHhi/CD34+

cells. Moreover, in CML-derived ALDHhi/CD-34+ cells tr-

eated with the Abl kinase inhibitors or transfected with

Bcr-Abl shRNA #1, the RKIP protein expression was in-

creased and the phosphorylation levels of FOXM1 were

decreased compared to untreated or untransfected cells.

On the other hand, the Abl kinase inhibitors or Bcr-Abl

knockdown did not affect the expression of FOXM1

mRNA and protein. To assess the function of RKIP ex-

pression on colony formation of the CML derived AL-

DHhi/CD34+ cells, we investigated whether the overex-

pression or suppression of RKIP expression affect the

activity of colony formation. As shown in Figures 4(b)

and 4(c), the overexpression of RKIP significantly de-

creased the colony numbers, and reduced the phosphory-

lation levels of FOXM1. On the other hand, the overex-

pression did not affect the expression of FOXM1 mRNA

and protein. The reduction of RKIP expression slightly

increased the colony numbers by STI571 treatment com-

pared to when not transfected with RKIP shRNA #1.

Moreover, in the RKIP shRNA-transfected ALDHhi/CD34+

cells, the induction of RKIP expression by STI571 was

weakened. There were no significant differences in the

levels of RKIP mRNA induction among CML specimens.

These results demonstrate that the inhibition for Bcr-Abl

expression induced the RKIP expression and reduced the

phosphorylation levels of FOXM1, and the overexpres-

sion of RKIP inhibited the colony formation of ALD-

Hhi/CD34+ cells from CML specimens through the redu-

ction of the pFOXM1 status.

3.5. Induction of RKIP Expression Reduced

the Phosphorylation Levels of FOXM1

in CML-Derived ALDHhi/CD34+

Progenitor Cells

Finally, we examined whether overexpression of RKIP

reduced the phosphorylation levels of FOXM1 in CML

(#10)-derived ALDHhi/CD34+ cells (Figure 5(a)). When

the CML-derived ALDHhi/CD34+ cells were treated with

STI 571 or transfected with RKIP DNA, RKIP expres-

sion was increased, and the phosphorylation levels of

FOXM1 were decreased. Moreover, the phosphorylation

levels of Rb were also decreased. We found that the in-

duction of RKIP expression decreased phosphorylated

FOXM1 and Rb levels in CML derived ALDHhi/CD34+

cells. These results show that activated Rb inhibited the

phosphorylation of FOXM1. Next, we examined whe-

ther the FOXM1 overexpression rescued the effects of

cell cycle genes essential for G1-S progression, includ-

ing Skp2, Cdc25B, KIS, p27Kip1, and p21Cip1 by treat-

ment with STI571 in CML derived ALDHhi/CD34+ cells

(Figure 5(b)). When the CML derived ALDHhi/CD34+

cells were treated with STI 571 or transfected with RKIP

DNA, the phosphorylation levels of FOXM1 and the

expression of Skp2, Cdc25B and KIS protein were de-

creased, and the expression of CDKIs (p27Kip1 and p21Cip1)

protein was increased. On the other hand, in FOXM1

DNA transfected-ALDHhi/CD34+ cells, the reduction of

Skp2, Cdc25B and KIS expression by STI571 treatment

was inhibited, and the induction of p27Kip1 and p21Cip1

expression by STI571 treatment was inhibited. These

results indicate that RKIP promotes cell cycle progress-

sion through the reduction of the pFOXM1 status, and

has an important role in ALDHhi/CD34+ CML cell pro-

liferation.

4. DISCUSSION

We investigated the role of RKIP expression on the cell

growth inhibition of CML cells and CML-derived proge-

nitor cells. In this study, we demonstrated that Bcr-Abl in-

hibited the expression of RKIP and promoted the CML

cell proliferation through the FOXM1 activation.

The Ras/Raf-1/MEK/ERK signaling pathway is known

to be essential for cellular proliferation, differentiation,

apoptosis, survival and migration [29,30]. The deregula-

tion of them leads to various diseases including malig-

nancies [20,31] and developmental disorders [32]. How-

ever, the regulation of Ras/Raf-1/MEK/ERK pathway is

very complex and not clearly understood. It has been

reported that RKIP binds to Raf-1 and disrupt down-

stream of MAP kinase pathway [19], and the overex-

pression of RKIP can act as a competitive inhibitor MEK

[20]. Moreover, RKIP expression has been shown to

downregulated in metastatic prostate cancers and the

loss RKIP levels was suggested to promotes the metas-

tatic potential of prostate cancer cells [21], and it has been

shown to be a decrease of RKIP expression in malignant

melanoma and breast cancer cells [24,33], and the absence

of RKIP expression in melanoma metastases [22].

In cell cycle effects, ERK/MAPK-independent functions

of Raf kinases have been reported. Raf-1 activates the

Cdk25 phosphatase, which results in CDK activation [17].

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

(a) (b)

(c)

(a) The purified ALDHhi/CD34+ cells (3 × 102 to 5 × 102 cells/plate) from a CML patient (#10) were isolated and cultured in

semisolid methylcellulose media (Methocult; H4435). The ALDHhi/CD34+ cells, which were untransfected or transfected

with Bcr-Abl shRNA #1, were treated with the Abl kinase inhibitors, and were enumerated after 7 to 10 days of in vitro cul-

ture. The rate of the progenitor cells was evaluated as the percentage of the corresponding control (top panel). To detect

RKIP and Bcr-Abl protein, western blot was performed. Actinis shown as a loading control. Representative data are shown

(middle panels). For the analysis of RKIP mRNA expression, QRT-PCR was performed relative to the GAPDH gene (middle

panels). The densitometric analysis of pFOXM1 levels in ALDHhi/CD34+ cells from the CML patient (#10). Immunoprecipi-

tation and western blot analysis were performed by using anti-pTyr antibodies and anti-FOXM1 antibodies. Results are rep-

resentative of three independent experiments (bottom panels); (b) ALDHhi/CD34+ cells were transfected with RKIP DNA or

RKIP shRNA and then treated with STI571 (0.5 μM) for 7 to 10 days. The numbers of colony were counted. The rate of the

progenitor cells was evaluated as a percentage of the corresponding control. To detect RKIP and Bcr-Abl protein, western

blot was performed. Actin is shown as a loading control. Representative data are shown (middle panels). For the analysis of

RKIP mRNA expression, QRT-PCR was performed relative to the GAPDH gene (middle panels). The densitometric analysis

of pFOXM1 levels in ALDHhi/CD34+ cells from the CML patient (#10). Immunoprecipitation and western blot analysis were

performed by using anti-pTyr antibodies and anti-FOXM1 antibodies. Results are representative of three independent ex-

periments (bottom panels). Data are shown as the mean  SD in triplicate culture and are representative of three independent

experiments. Data are shown as the mean  SD in triplicate culture and are representative of three independent experiments.

*P < 0.01 compared with untreated control cells; (c) The cells transfected with RKIP DNA or shRNA were viewed using

phase-contrast microscopy after 14 days culture with or without STI571 (0.5 μM). Original magnification  4.

Figure 4. RKIP regulates FOXM1 activation on ALDHhi/CD34+ hematopoietic progenitor cells derived from CML patients.

Openly accessible at

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

6363

(a) (b)

(a) Expression of RKIP, phosphorylated FOXM1, phosphorylated Rb and phosphorylated Bcr-Abl protein in ALD-

Hhi/CD34+ cells isolated from two a CML patient (#10) was analyzed after 40 minutes treatment with STI571 (0.5 μM)

using immunoprecipitation and immunoblot analyses. Representative data are shown. Actin was used as a loading con-

trol; (b) Expression of RKIP, Skp2, Cdc25B, KIS, p27Kip1, and p21Cip1 protein and pFOXM1 in purified ALDHhi/CD34+

cells from one CML patient (#10). ALDHhi/CD34+ cells (3 × 102 to 5 × 102 cells/plate) were untransfected or were

transfected with RKIP DNA and FOXM1 DNA, plated in semisolid methylcellulose media and were then treated with

STI571 (0.5 μM) for 24 hours using immunoblot analyses. Representative data are shown. Immunopretipitation and

western blot analysis was performed by using anti-RKIP, anti-Skp2, anti-Cdc25B, anti-KIS, anti-p27Kip1, anti-p21Cip1,

anti-pTyr antibodies and anti-FOXM1 antibodies. The levels of Actin served as the loading control.

Figure 5. RKIP reduces the phosphorylation of FOXM1 and modulates the cell cycle associated proteins.

The activated CDK4/6 and CDK2 inactivated pRb with

Cyclin D and Cyclin E, respectively, which results in

cell cycle progression from G1/S transition until the en-

try into mitosis. In the present study, we found that tr-

eatment with Abl kinase inhibitors or reduction of Bcr-

Abl induced the expression of RKIP, reduced the phos-

phorylated FOXM1 in CML cells and Bcr-Abl+ proge-

nitor cells from CML patients. Moreover, the overexpre-

ssion of RKIP in CML cells and Bcr-Abl+ progenitor

cells inhibited their proliferation through the reduction

of the pFOXM1 status. These results demonstrate that

RKIP is suppressed by Bcr-Abl and the induction of RK-

IP inhibits the CML progenitor cell proliferation.

In this study, we found at the first time that RKIP in-

hibited the phosphorylation of FOXM1, the central regu-

lator of the cell cycle, resulting in G1 cell cycle arrest via

p27Kip1 and p21Cip1 accumulation in CML progenitor cells.

Expression and phosphorylation of FOXM1 is induced

during the G1 phase of the cell cycle, its expression conti-

nues during S phase and mitosis [34]. FOXM1 activity is

subjected to different levels of regulation [35]. Upon mi-

togenic stimulation, Cyclin D/Cdk4, 6 and Cyclin E/Cdk2

inactivate phosphorylated-Rb (pRb), allowing the cells to

progress to S-phase, but also relieving inhibition of FOX-

M1 by pRb. Furthermore, FOXM1 has been shown to regu-

late transcription of cell cycle genes essential for G1-S and

G2-M progression and chromosome stability and segreg-

ation, including Nek2, KIF20A, CENP-A, CENP-F,

CDc25A, CDc25B, p27Kip1, KIS, Cyclin B, and Cyclin D

[35-39]. FOXM1 has been also reported to contribute to

oncogenesis in basal cell carcinomas, hepatocellular car-

cinomas, and primary breast cancer [40,41]. Moreover,

we have been reported that in acute leukemia cell lines,

the expression of FOXM1 mRNA was significantly hig-

her than normal progenitor cells [28]. The reduction of

FOXM1 expression increased protein expression of

p21Cip1 and p27Kip1. In the clinical samples (127AML (M1;

21, M2; 56, M4; 32, M5; 18)), FOXM1 mRNA was over-

expressed in all AML specimens (127/127; 100%). The

relative folds of FOXM1 gene expression were for AML:

2.14  0.31 (M1), 2.26  0.47 (M2), 1.65  0.42 (M4),

and 1.92  0.38 (M5). In ALDHhi/CD34+ progenitor

cells derived from AML patients, the colony numbers

were remarkably increased by FOXM1 overexpression.

FOXM1 is an essential transcription factor for develop-

ment of acute leukemia cells, and regulate the growth of

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

ALDHhi/CD34+ progenitor cells derived from AML pa-

tients. In this study, we also showed that FOXM1 ex-

pression in CML cells was increased compared to nor-

mal ALDHhi/CD34+ cells. However, the Abl kinase inhi-

bitors or Bcr-Abl knockdown did not affect the expres-

sion of FOXM1 mRNA and protein. On the other hand,

the overexpression of RKIP or Bcr-Abl knockdown re-

duced the pFOXM1 status and KIS expression, and in-

duced p27Kip1 and p21Cip1 expression resulting in inhibition

of CML cells. These results demonstrate that FOXM1 may

be one of target proteins for RKIP.

The ALDHhi/CD34+ progenitor cells derived from CML

patients were significantly reduced when they were

treated with STI571, AMN107, and BMS354825 or tran-

sfected with Bcr-Abl shRNA #1. Moreover, the relative

ex- pression of RKIP mRNA was increased and the

pFOX- M1 status was decreased in the progenitor cells

treated with the Abl kinase inhibitors or transfected with

Bcr-Abl shRNA #1. In ALDHhi/CD34 + progenitor cells

transfected with RKIP DNA, the colony forming activity

was inhibited, and the combination with the Abl kinase

inhibitor showed more strong effects. In contrast, in

ALDHhi/ CD34+ progenitor cells transfected with RKIP

shRNA, the inhibition effects of colony formation activ-

ity by the Abl kinase inhibitors were weakened. These

results indicate that the expression of RKIP induced by

the Abl kinase reduce the pFOXM1 status and col-

ony-forming cells derived from CML progenitor cells.

Moreover, the RKIP overexpression decreased the pho-

sphorylation levels of FOXM1 and Rb proteins, pro-

moted the cell cycle progression via reduction Skp2,

Cdc25B, and KIS proteins expression and induction of

p27Kip1 and p21Cip1 expression in CML-derived ALD-

Hhi/CD34+ progenitor cells. These findings indicate that

Bcr-Abl inhibited the expression of RKIP, and reduced

the phosphorylated FOXM1, resulting in the promotion of

Bcr-Abl+ progenitor cell proliferation.

This study shows for the first time that Bcr-Abl re-

presses the expression of RKIP and that the Abl kinase

inhibitors induced RKIP expression. Moreover, RKIP

reduces the pFOXM1 status. These induced RKIP plays

an important role in cell growth inhibition in CML pro-

genitor cells in vitro.

5. CONFLICT OF INTEREST

None of the authors have competing financial interests

in relation to the work described in this article.

6. ACKNOWLEDGEMENTS

This work was supported by a grant-in-aid for scientific research

(#17590987) from the Ministry of Education, Culture, Sports, Science,

and Technology of Japan.

REFERENCES

[1] Kurzrock, R., Gutterman, J.U. and Talpaz, M. (1988) The

molecular genetics of Philadelphia chromosome-positive

leukemias. New England Journal of Medicine, 319, 990-

998. doi:10.1056/NEJM198810133191506

[2] Rudkin, C.T., Nowell, P.C. and Hungerford, D.A. (1964)

DNA contents of chromosome ph1 and chromosome 21

in human cronic granulocytic leukemia. Science, 144,

1229-1231. doi:10.1126/science.144.3623.1229

[3] Shtivelman, E., Lifshitz, B., Gale, R.P. and Ganaani, E.

(1985) Fused transcript of abl and bcr genes in chronic

myelogenous leukaemia. Nature, 315, 550-554.

doi:10.1038/315550a0

[4] Lugo, T.G., Pendergast, A.M., Muller, A.J. and Witte,

O.N. (1990) Tyrosine kinase activity and transformation

potency of bcr-abl oncogene products. Science, 247,

1079-1082. doi:10.1126/science.2408149

[5] Daley, G.Q., Van, Etten, R.A. and Baltimore, D. (1990)

Induction of chronic myelogenous leukemia in mice by

the P210bcr/abl gene of the Philadelphia chromosome.

Science, 247, 824-830. doi:10.1126/science.2406902

[6] Sawyers, C.L., McLaughlin, J. and Witte, O.N. (1995)

Genetic requirement for Ras in the transformation of fi-

broblasts and hematopoietic cells by the Bcr-Abl onco-

gene. Journal of Experimental Medicine, 181, 307-

313.doi:10.1084/jem.181.1.307

[7] Skorski, T., Bellacosa, A., Nieborowska-Skorska, M.,

Majewski, M., Martinez, R., Choi, J.K., Trotta, R., Wlo-

darski, P., Perrotti, D., Chan, T.O., Wasik, M.A., Tsichlis,

P.N. and Calabretta, B. (1997) Transformation of hema-

topoietic cells by BCR/ABL requires activation of a

PI-3k/Akt-dependent pathway. EMBO Journal, 16, 6151-

6161.doi:10.1093/emboj/16.20.6151

[8] Carlesso, N., Frank, D.A. and Griffin, J.D. (1996) Tyro-

syl phosphorylation and DNA binding activity of signal

transducers and activators of transcription (STAT) pro-

teins in hematopoietic cell lines transformed by Bcr/Abl.

Journal of Experimental Medicine, 183, 811-820.

doi:10.1084/jem.183.3.811

[9] Reuther, J.Y., Reuther, G.W., Cortez, D., Pendergast, A.M.

and Baldwin Jr., A.S. (1998) A requirement for NF-

kappa B activation in Bcr-Abl-mediated transformation.

Genes and Development, 12, 968-981.

doi:10.1101/gad.12.7.968

[10] Nottage, M. and Siu, L.L. (2002) Rationale for Ras and

raf-kinase as a target for cancer therapeutics. Current

Pharmaceutical Design, 8, 2231-2241.

doi:10.2174/1381612023393107

[11] O’Neill, E. and Kolch, W. (2004) Conferring specificity

on the ubiquitous Raf/MEK signalling pathway. British

Journal of Cancer, 90, 283-288.

doi:10.1038/sj.bjc.6601488

[12] Ren, R. (2005) Mechanisms of BCR-ABL in the patho-

genesis of chronic myelogenous leukaemia. Nature Re-

views Cancer, 5, 172-183.doi:10.1038/nrc1567

[13] Pendergast, A.M., Quilliam, L.A., Cripe, L.D., Bassing,

C.H., Dai, Z., Li, N., Batzer, A., Rabun, K.M., Der, C.J.

and Schlessinger, J. (1993) BCR-ABL-induced onco-

genesis is mediated by direct interaction with the SH2

domain of the GRB-2 adaptor protein. Cell, 75, 175-185.

[14] Chang, F., Steelman, L.S., Lee, J.T., Shelton, J.G., Navo-

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

6565

lanic, P.M., Blalock, W.L., Franklin, R.A. and McCubrey,

J.A. (2003) Signal transduction mediated by the Ras/Raf/

MEK/ERK pathway from cytokine receptors to trans-

cription factors: Potential targeting for therapeutic inter-

vention. Leukemia, 17, 1263-1293.

doi:10.1038/sj.leu.2402945

[15] Sattler, M., Mohi, M.G., Pride, Y.B., Quinnan, L.R., Ma-

louf, N.A., Podar, K., Gesbert, F., Iwasaki, H., Li, S., Van

Etten, R.A., Gu, H., Griffin, J.D. and Neel, B.G. (2002)

Critical role for Gab2 in transformation by BCR/ABL.

Cancer Cell, 1, 479-492.

doi:10.1016/S1535-6108(02)00074-0

[16] Aichberger, K.J., Mayerhofer, M., Krauth, M.T., Skvara,

H., Florian, S., Sonneck, K., Akgul, C., Derdak, S., Pickl,

W. F., Wacheck, V., Selzer, E., Monia, B.P., Moriggl, R.,

Valent, P. and Sillaber, C. (2005) Identification of mcl-1

as a BCR/ABL-dependent target in chronic myeloid leu-

kemia (CML): Evidence for cooperative antileukemic

effects of imatinib and mcl-1 antisense oligonucleotides.

Blood, 105, 3303-3311. doi:10.1182/blood-2004-02-0749

[17] Hindley, A. and Kolch, W. (2002) Extracellular signal

regulated kinase (ERK)/mitogen activated protein kinase

(MAPK)-independent functions of Raf kinases. Journal

of Cell Science, 115, 1575-1581.

[18] Serre, L., Pereira, de Jesus, K., Zelwer, C., Bureaud, N.,

Schoentgen, F. and Benedetti, H. (2001) Crystal struc-

tures of YBHB and YBCL from Escherichia coli, two

bacterial homologues to a Raf kinase inhibitor protein.

Journal of Molecular Biology, 310, 617-634.

doi:10.1006/jmbi.2001.4784

[19] Yeung, K., Seitz, T., Li, S., Janosch, P., McFerran, B.,

Kaiser, C., Fee, F., Katsanakis, K.D., Rose, D.W., Mis-

chak, H., Sedivy, J.M. and Kolch, W. (1999) Suppression

of Raf-1 kinase activity and MAP kinase signalling by

RKIP. Nature, 401, 173-177. doi:10.1038/43686

[20] Yeung, K., Janosch, P., McFerran, B., Rose, D.W., Mis-

chak, H., Sedivy, J. M. and Kolch, W. (2000) Mechanism

of suppression of the Raf/MEK/extracellular signal-

regulated kinase pathway by the Raf kinase inhibitor

protein. Molecular Cell Biology, 20, 3079-3085.

doi:10.1128/MCB.20.9.3079-3085.2000

[21] Fu, Z., Smith, P.C., Zhang, L., Rubin, M.A., Dunn, R.L.,

Yao, Z. and Keller, E.T. (2003) Effects of raf kinase in-

hibitor protein expression on suppression of prostate

cancer metastasis. Journal of the National Cancer Insti-

tute, 95, 878-889. doi:10.1093/jnci/95.12.878

[22] Schuierer, M.M., Bataille, F., Hagan, S., Kolch, W. and

Bosserhoff, A.K. (2004) Reduction in Raf kinase inhibi-

tor protein expression is associated with increased Ras-

extracellular signal-regulated kinase signaling in mela-

noma cell lines. Cancer Research, 64, 5186-5192.

doi:10.1158/0008-5472.CAN-03-3861

[23] Schuierer, M.M., Bataille, F., Weiss, T., Hellerbrand, C.

and Bosserhoff, A.K. (2006) Raf kinase inhibitor protein

is downregulated in hepatocellular carcinoma. Oncology

Reports, 16, 451-456.

[24] Hagan, S., Al-Mulla, F., Mallon, E., Oien, K., Ferrier, R.,

Gusterson, B., García, J.J. and Kolch, W. (2005) Reduc-

tion of Raf-1 kinase inhibitor protein expression corre-

lates with breast cancer metastasis. Clinical Cancer Re-

search, 11, 7392-7397.

doi:10.1158/1078-0432.CCR-05-0283

[25] Al-Mulla, F., Hagan, S., Behbehani, A.I., Bitar, M.S.,

George, S.S., Going, J.J., García, J.J., Scott, L., Fyfe, N.,

Murray, G.I. and Kolch, W. (2006) Raf kinase inhibitor

protein expression in a survival analysis of colorectal

cancer patients. Journal of Clinical Oncology, 24, 5672-

5679. doi:10.1200/JCO.2006.07.5499

[26] Zlobec, I., Baker, K., Minoo, P., Jass, J.R., Terracciano, L.

and Lugli, A. (2008) Node-negative colorectal cancer at

high risk of distant metastasis identified by combined

analysis of lymph node status, vascular invasion, and

Raf-1 kinase inhibitor protein exoression. Clinical Can-

cer Research, 14, 143-148.

doi:10.1158/1078-0432.CCR-07-1380

[27] Houben, R., Vetter-Kauczok, C.S., Ortmann, S., Rapp,

U.R., Broecker, E.B. and Becker, J.C. (2008) Phospho-

ERK staining is a poor indicator of the mutational status

of BRAF and NRAS in human melanoma. Journal of

Investigative Dermatology, 128, 2003-2012.

doi:10.1038/jid.2008.30

[28] Nakamura, S., Hirano, I., Okinaka, K., Takemura, T.,

Yokota, D., Ono, T., Shigeno, K., Shibata, K., Fujisawa,

S. and Ohnishi, K. (2010) The FOXM1 transcriptional

factor promotes the proliferation of leukemia cells thr-

ough modulation of cell cycle progression in acute mye-

loid leukemia. Carcinogenesis, 31, 2012-2021.

doi:10.1093/carcin/bgq185

[29] Wellbrock, C., Karasarides, M. and Marais, R. (2004) The

RAF proteins take centre stage. Nature Reviews Mole-

cular Cell Biology, 5, 875-885. doi:10.1038/nrm1498

[30] Dhillon, A.S., Hagan, S., Rath, O. and Kolch, W. (2007)

MAP kinase signaling pathways in cancer. Oncogene, 26,

3279-3290. doi:10.1038/sj.onc.1210421

[31] Roberts, P.J. and Der, C.J. (2007) Targeting the Raf-MEK-

ERK mitogen-activated protein kinase cascade for the

treatment of cancer. Oncogene, 26, 3291-3310.

doi:10.1038/sj.onc.1210422

[32] Schubbert, S., Shannon, K. and Bollag, G. (2007) Hy-

peractive Ras in developmental disorders and cancer. Na-

ture Reviews Cancer, 7, 295-308. doi:10.1038/nrc2109

[33] Li, H.Z., Gao, Y., Zhao, L.X., Liu, Y.X., Sun, B. C., Yang,

J. and Yao, Z. (2009) Effects of Raf kinase inhibitor pro-

tein expression on metastasis and progression of human

breast cancer. Molecular Cancer Research, 7, 832-840.

doi:10.1158/1541-7786.MCR-08-0403

[34] Ye, H., Holterman, A.X., Yoo, K.W., Franks, R.R. and

Costa, R.H. (1999) Premature expression of the winged

helix transcription factor HFH-11B in regenerating

mouse liver accelerates hepatocyte entry into S-phase.

Molecular Cell Biology, 19, 8570-8580.

[35] Laoukili, J., Kooistra, M.R., Bras, A., Kauw, J., Kerkho-

ven, R.M., Morrison, A., Clevers, H. and Medema, R.H.

(2005) FoxM1 is required for execution of the mitotic

programme and chromosome stability. Nature Cell Biol-

ogy, 7, 126-136. doi:10.1038/ncb1217

[36] Wonsey, D.R. and Follettie, M.T. (2005) Loss of the

forkhead transcription factor FoxM1 causes centrosome

amplification and mitotic catastrophe. Cancer Research,

65, 5181-5189. doi:10.1158/0008-5472.CAN-04-4059

[37] Leung, T.W., Lin, S.S., Tsang, A.C., Tong, C.S., Ching,

J.C., Leung, W.Y, Gimlich, R., Wong, G.G. and Yao, K.M.

(2001) Over-expression of FoxM1 stimulates cyclin B1

expression. FEBS Letters, 507, 59-66.

S. Nakamura et al. / Stem Cell Discover y 1 (2011) 54-66

doi:10.1016/S0014-5793(01)02915-5

[38] Wang, X., Kiyokawa, H., Dennewitz, M.B. and Costa,

R.H. (2002) The forkhead box m1b transcription factor is

essential for hepatocyte DNA replication and mitosis

during mouse liver regeneration. Proceedings of the Na-

tional Academy Scie nce of the USA, 99, 16881-16886.

doi:10.1073/pnas.252570299

[39] Petrovic, V., Costa, R.H., Lau, L.F., Raychaudhuri, P. and

Tyner, A.L. (2008) FoxM1 regulates growth factor in-

duced expression of the KIS kinase to promote cell cycle

progression. Journal of Biological Chemistry, 104, 453-

460.

[40] Teh, M.T., Wong, S.T., Neill, G.W., Ghali, L.R., Philpott,

M.P. and Quinn, A.G. (2002) FOXM1 is a downstream

target of Gli1 in basal cell carcinomas. Cancer Research,

62, 4773-4780.

[41] Kalinichenko, V.V., Major, M.L., Wang, X., Petrovic, V.,

Kuechle, J., Yoder, H.M., Dennewitz, M.B., Shin, B.,

Datta, A., Raychaudhuri, P. and Costa, R.H. (2004) Fox-

m1b transcription factor is essential for development of

hepatocellular carcinomas and is negatively regulated by

the p19ARF tumor suppressor. Genes Development, 18,

830-850. doi:10.1101/gad.1200704