ProgrammedCellDeath-4TumorSuppressorProtein ... · at an immature or poorly differentiated state to resume the process of maturation (2). This type of treatment has the advantage

Programmed Cell Death-4 Tumor Suppressor ProteinContributes to Retinoic Acid–Induced TerminalGranulocytic Differentiation of HumanMyeloid Leukemia Cells

Bulent Ozpolat,1 Ugur Akar,1 Michael Steiner,3 Isabel Zorrilla-Calancha,1

Maribel Tirado-Gomez,1 Nancy Colburn,4 Michael Danilenko,3

Steven Kornblau,2 and Gabriel Lopez Berestein1

Departments of 1Experimental Therapeutics and 2Leukemia, The University of Texas M. D. AndersonCancer Center, Houston, Texas; 3Department of Clinical Biochemistry, Faculty of Health Sciences,Ben-Gurion University of the Negev, Beer-Sheva, Israel; and 4Gene Regulation Section,Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland

AbstractProgrammed cell death-4 (PDCD4) is a recently

discovered tumor suppressor protein that inhibits

protein synthesis by suppression of translation

initiation. We investigated the role and the regulation of

PDCD4 in the terminal differentiation of acute myeloid

leukemia (AML) cells. Expression of PDCD4 was

markedly up-regulated during all-trans retinoic acid

(ATRA)–induced granulocytic differentiation in NB4 and

HL60 AML cell lines and in primary human promyelocytic

leukemia (AML-M3) and CD34+ hematopoietic progenitor

cells but not in differentiation-resistant NB4.R1 and

HL60R cells. Induction of PDCD4 expression was

associated with nuclear translocation of PDCD4 in NB4

cells undergoing granulocytic differentiation but not in

NB4.R1 cells. Other granulocytic differentiation inducers

such as DMSO and arsenic trioxide also induced

PDCD4 expression in NB4 cells. In contrast, PDCD4

was not up-regulated during monocytic/macrophagic

differentiation induced by 1,25-dihydroxyvitamin D3 or

12-O-tetradecanoyl-phorbol-13-acetate in NB4 cells or

by ATRA in THP1 myelomonoblastic cells. Knockdown

of PDCD4 by RNA interference (siRNA) inhibited

ATRA-induced granulocytic differentiation and reduced

expression of key proteins known to be regulated by

ATRA, including p27Kip1 and DAP5/p97, and induced

c-myc and Wilms’ tumor 1, but did not alter expression

of c-jun, p21Waf1/Cip1, and tissue transglutaminase (TG2).

Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian

target of rapamycin (mTOR) signaling pathway was

found to regulate PDCD4 expression because inhibition

of PI3K by LY294002 and wortmannin or of mTOR

by rapamycin induced PDCD4 protein and mRNA

expression. In conclusion, our data suggest that PDCD4

expression contributes to ATRA-induced granulocytic

but not monocytic/macrophagic differentiation. The

PI3K/Akt/mTOR pathway constitutively represses

PDCD4 expression in AML, and ATRA induces PDCD4

through inhibition of this pathway. (Mol Cancer Res

2007;5(1):95–108)

IntroductionAcute myeloid leukemia (AML), the most common type of

acute leukemia, is a heterogeneous group of hematologic

malignancies characterized by a differentiation block in

hematopoietic progenitor cells at the early stages of myelopoi-

esis, proliferation of immature blasts, and invasion of bone

marrow. Acute promyelocytic leukemia, a subtype of AML, is

characterized by a t(15;17) translocation involving the genes

encoding promyelocytic leukemia and retinoic acid receptor a.This translocation results in differentiation arrest at the

promyelocytic stage of myeloid cell differentiation (1). Despite

recent improvements in our understanding of terminal cell

differentiation, the molecular mechanisms regulating myeloid

cell differentiation are not fully understood.

Differentiation therapy is based on the concept that

differentiation-inducing agents can force cancer cells arrested

at an immature or poorly differentiated state to resume the

process of maturation (2). This type of treatment has the

advantage of being potentially less toxic than standard

chemotherapy. Induction of differentiation restores a natural

cell death program and inhibits proliferation. Treatment of acute

promyelocytic leukemia with all-trans retinoic acid (ATRA),

the first model of differentiation therapy, has proved extremely

successful in inducing clinical remission in most patients (3).

ATRA, a naturally occurring derivative of vitamin A (retinol), is

a potent inducer of cellular differentiation, growth arrest, and

apoptosis in various tumor cell lines. ATRA induces terminal

differentiation of immature leukemic promyelocytes into

normal mature granulocytes in vitro and in vivo (4, 5). Thus,

Received 5/8/06; revised 11/22/06; accepted 11/27/06.Grant support: Ladies Leukemia League (B. Ozpolat) and National CancerInstitute grant U54 RFA CA096300 (G.L. Lopez Berestein).The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Gabriel Lopez Berestein, Department of ExperimentalTherapeutics, Unit 422, The University of Texas M. D. Anderson Cancer Center,1515 Holcombe Boulevard, Houston, TX 77030. Phone: 1-713-792-8140; Fax:1-713-792-0362. E-mail: [email protected] D 2007 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-06-0125

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this system provides an excellent in vitro model for studying

the molecular events taking place during the terminal differen-

tiation of myeloid cells.

The ATRA-induced granulocytic differentiation program is

a complex process that requires transcriptional and transla-

tional regulation of many specific targets (6-9). We previously

found that ATRA inhibits the translational machinery through

multiple posttranscriptional suppression mechanisms, includ-

ing down-regulation of translation factors and up-regulation of

a repressor of translation initiation, DAP5/p97, in myeloid

progenitor cells during granulocytic differentiation (10).

Currently, the posttranscriptional molecular mechanisms con-

trolling translation initiation and the role of translational

control in terminal myeloid cell differentiation remain largely

unknown.

Programmed cell death 4 (PDCD4) is a recently discovered

tumor suppressor gene that has attracted great interest as an

inhibitor of tumor promoter– induced neoplastic transformation

and as a specific inhibitor of cap-dependent mRNA translation

in vitro and in vivo (11–15). PDCD4 was originally isolated

from a human glioma library and is homologous to the mouse

Pdcd4 (MA-3/TIS/A7-1) gene (16, 17). Human PDCD4 gene

is localized to chromosome 10q24. PDCD4 has been shown to

inhibit the activation of AP-1-dependent transcription, skin

tumorigenesis, and tumor progression in transgenic mice

(12, 18). PDCD4 suppresses translation initiation by specifi-

cally inhibiting the helicase activity of eukaryotic translation

initiation factor 4A (eIF4A), a component of the translation

initiation complex (13, 15). Binding of PDCD4 to eIF4A and

consequent inhibition of translation is required for trans-

repression by PDCD4 of target activities such as AP-1 (15).

PDCD4 is ubiquitously expressed in normal tissues, but its

expression is lost or suppressed in several tumors, including

lung, breast, colon, brain, and prostate cancers (19). Loss of

PDCD4 expression in human lung cancer cells correlates with

tumor progression and poor prognosis (20). The mechanism by

which PDCD4 expression is suppressed is not understood.

Recently, the chicken Pdcd4 gene has been identified as a direct

target of the transcription factor c-Myb, which is essential for

the development of the hematopoietic system, and plays an

important role as a switch that directs hematopoietic progenitor

cells to alternative fates, such as proliferation, differentiation,

and apoptosis (21–23). We therefore hypothesized that PDCD4

plays a role in terminal differentiation and lineage commitment

of human myeloid cells.

In the present study, we show that PDCD4 expression was

markedly induced in AML cell lines and primary promyelocytic

leukemia cells undergoing granulocytic differentiation. Cells

that are undergoing monocytic/macrophagic differentiation and

are resistant to granulocytic differentiation failed to up-regulate

PDCD4 and translocate it into the nucleus after ATRA

treatment. Targeted inhibition of PDCD4 expression by siRNA

resulted in a significant inhibition of ATRA-induced granulo-

cytic differentiation, suggesting that PDCD4 induction contrib-

utes to granulocytic differentiation. We also showed that the

phosphatidylinositol 3-kinase (PI3K)/Akt pathway negatively

regulates induced PDCD4 expression in AML cells and ATRA

induces PDCD4 through inhibition of this pathway. Knock-

down of PDCD4 antagonized the expression of c-myc, p27Kip1,

DAP5, and Wilms tumor 1 (WT1), suggesting that PDCD4 is

involved in the regulation of these downstream proteins.

Overall, PDCD4 may exert its effects on differentiation by

altering the expression of these proteins.

ResultsEvaluation of ATRA-Induced Differentiation

We first examined the surface expression of CD11b, a marker

of myeloid differentiation, in NB4 cells and their differentiation-

resistant derivatives, NB4.R1 cells. Cells were treated with

1 Amol/L ATRA, collected at 12, 24, 48, and 72 h, and analyzed

by fluorescence-activated cell sorting with anti-CD11b antibody

(Fig. 1A). NB4 cells expressed CD11b after ATRA treatment,

whereas NB4.R1 cells lacked surface CD11b expression up to

96 h after ATRA treatment (Fig. 1B), indicating that NB4.R1

cells did not undergo ATRA-induced differentiation. Morpho-

logic changes in the cells were assessed by May-Grunwald-

Giemsa staining, which revealed that the untreated NB4 cells

were predominantly promyelocytes with characteristic cytoplas-

mic granules, large nuclei, and prominent nucleoli. The ATRA-

treated NB4 cells acquired a granulocytic morphology that

included a decreased nuclear/cytoplasmic ratio, appearance

of cytoplasmic granules, chromatin condensation, and loss

of nucleoli (Fig. 1C). To further confirm the induction of

differentiation in NB4 cells, we examined the reorganization

of promyelocytic leukemia nuclear bodies in the cells after 72 h

of ATRA treatment. Immunostaining with anti–promyelocytic

leukemia antibodies revealed a diffusely microspeckled pattern

of promyelocytic leukemias in the nuclei of untreated control

NB4 cells. However, in cells treated with ATRA, the micro-

speckled pattern disappeared and the size and brightness of the

promyelocytic leukemia bodies returned to normal appearance.

In contrast, the normal nuclear organization of promyelocytic

leukemia protein was not seen in ATRA-treated NB4.R1 cells,

indicating that these cells did not differentiate into granulocytes.

ATRA Induces PDCD4 Expression during NB4 andHL60 Cell Differentiation but not in Differentiation-Resistant Cells

Because the translational machinery and protein synthesis

are significantly inhibited in myeloid cells undergoing terminal

differentiation (10, 24–26), we investigated whether PDCD4

expression is induced during the granulocytic differentiation

of myeloid cells. NB4 cells were treated with ATRA at

differentiation-inducing concentrations (0.1 or 1 Amol/L) and

PDCD4 protein levels were examined by Western blot analysis.

ATRA markedly up-regulated the expression of PDCD4 protein

in a time-dependent manner, peaking at 72 h of ATRA

treatment (Fig. 2A and B). To determine whether induction of

PDCD4 expression is regulated by transcriptional or posttran-

scriptional mechanisms, we analyzed PDCD4 mRNA levels in

ATRA-treated NB4 cells by reverse transcription-PCR (RT-

PCR) using specific primers. Induction of PDCD4 mRNA

expression was detectable at 24 h and markedly increased at

48 h of ATRA treatment (Fig. 2C), suggesting that PDCD4

protein expression is regulated at the transcriptional level

during differentiation of promyelocytic cells.

Because differentiation-defective myeloid cells provide a

useful experimental model to study the molecular mechanisms

Ozpolat et al.

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involved in terminal cell differentiation, we compared the

expression of PDCD4 in differentiation-sensitive (NB4 and

HL60) and differentiation-resistant (NB4.R1 and HL60R) cells,

which are unable to undergo ATRA-induced differentiation. In

contrast to the differentiation-sensitive cells, treatment of

NB4.R1 and HL60R cells with ATRA did not induce PDCD4

expression (Fig. 2D and E). ATRA also induced PDCD4 mRNA

expression in HL60 cells but not in HL60R cells treated with

ATRA for up to 96 h (data not shown). We also examined

PDCD4 expression in the primary promyelocytic leukemia cells

isolated from newly diagnosed acute promyelocytic leukemia

patients. A significant up-regulation of PDCD4 protein by

ATRA was observed in two different acute promyelocytic

leukemia patient cells during ATRA-induced granulocytic

differentiation, which was assessed by morphology and surface

expression of myeloid (CD11b) and granulocytic (CD11c)

differentiation markers (Fig. 3A-D), supporting our hypothesis

that PDCD4 expression is induced during granulocytic differ-

entiation of myeloid cells. To determine whether ATRA induces

PDCD4 in normal bone marrow progenitors, we treated CD34+

hematopoietic progenitor cells with ATRA for 72 h. We

observed that these early progenitor cells could also up-regulate

PDCD4 by ATRA treatment (Fig. 3E), suggesting that PDCD4

expression can be regulated in bone marrow microenvironment

by retinoic acid.

PDCD4 Expression Increases during Granulocytic butnot Monocytic/Macrophagic Differentiation

We next investigated whether elevation of PDCD4 expres-

sion is specific to ATRA-induced granulocytic differentiation or

Figure 1. ATRA inducesgranulocytic differentiation inNB4 but not differentiation-resistant NB4.R1 cells. A.Time-dependent expression ofmyeloid differentiation markerCD11b. NB4 cells were treatedwith 1 Amol/L ATRA for up to72 h, stained with monoclonalanti-CD11b antibody, and ana-lyzed by flow cytometry todetect induction of granulocyticdifferentiation. B. ATRA-induced differentiation within96 h in NB4 and NB4.R1 cellsas detected by fluorescence-activated cell sorting (FACS)analysis of CD11b expression.C. ATRA induces morphologicchanges in promyelocytic leu-kemia cells undergoing granu-locytic differentiation. Aftertreatment with ATRA, NB4cells were stained with May-Grunwald-Giemsa dye toreveal formation of myeloper-oxidase-containing granules indifferentiated cells. D. ATRAinduces reorganization of pro-myelocytic leukemia nuclearbodies in NB4 cells but notNB4.R1 cells. Cells were trea-ted with 1 Amol/L ATRA for72 h, stained with monoclonalanti – promyelocytic leukemiaprimary and FITC-labeled sec-ondary antibodies, and ana-lyzed by immunofluorescencemicroscopy. Nuclei werestained with 4¶,6-diamidino-2-phenylindole (blue ).

Role of PDCD4 in Leukemia Cell Differentiation


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also takes place during monocytic/macrophagic differentiation.

NB4 cells were first treated with ATRA and other granulocytic

differentiation-inducing agents, such as arsenic trioxide (27)

and 1% DMSO (28). Granulocytic differentiation induced by

ATRA, arsenic trioxide, or DMSO was associated with marked

up-regulation of PDCD4 (Fig. 4A). As expected, ATRA-

induced PDCD4 expression was detectable at 48 h and

peaked at 72 h of treatment. In contrast, arsenic trioxide at a

differentiation-inducing dose (0.4 Amol/L) did not induce

PDCD4 at early time points, but significant induction of

PDCD4 was observed after 72 h of treatment (Fig. 4A). DMSO,

on the other hand, induced strong PDCD4 expression at 48 h.

These results showed that induction of PDCD4 expression

generalizes to granulocytic differentiation induced by multiple

inducers.

We next treated NB4 cells with phorbol 12-myristate 13-

acetate (PMA; refs. 29, 30) and 1,25-dihydroxyvitamin D3

(31, 32) agents that induce monocytic/macrophagic differenti-

ation. Differentiation-inducing doses of PMA (0.1 Amol/L;

Fig. 4B) and 1,25-dihydroxyvitamin D3 (0.1 Amol/L; Fig. 4C)

did not induce PDCD4 expression. Higher doses (up to 1 Amol/L)

of these compounds also failed to up-regulate PDCD4 expression

in the cells (data not shown). Induction of monocytic/macro-

phagic differentiation by the two agents was confirmed by

assessment of morphologic changes and adhesion to tissue

culture flasks (Fig. 4D).

To confirm the association between PDCD4 induction and

granulocytic differentiation, we investigated PDCD4 expres-

sion in THP1 myelomonocytic AML cells (33, 34), which

undergo monocytic/macrophagic differentiation by ATRA.

Figure 2. ATRA inducesmarked PDCD4 expression inNB4 and HL60 cells but not intheir differentiation-resistantcounterparts. A. Cells weretreated with differentiation-inducing doses (0.1 or 1Amol/L) of ATRA and collectedat the indicated time points.Equal amounts of total celllysates were immunoblottedwith anti-PDCD4 antibody. h-actin was used as a loadingcontrol. B. Bands represent-ing PDCD4 protein expressionin (A) were analyzed by den-si tometry . Resul ts wereexpressed as the relative ratioof PDCD4 to h-actin. C. ATRAinduced PDCD4 mRNA ex-pression in NB4 cells. Follow-ing treatment with 1 Amol/LATRA, RNA was extractedfrom NB4 cells at the indicatedtime points. PDCD4mRNA ex-pression was detected by RT-PCR using PDCD4-specificprimers. D. Bands represent-ing PDCD4 mRNA expressionin (C) were analyzed usingdensitometry. Results wereexpressed as the relativeratio of PDCD4 to h-actin.E. PDCD4 protein expressionis not induced by ATRA inNB4.R1 cells, which are un-able to undergo granulocyticdifferentiation. Cells weretreated with 1 Amol/L ATRAand harvested at the indicatedtime points. NB4 cells wereused as a control. F. ATRAinduces PDCD4 expressionin HL60 but not HL60R cells.HL60 and HL60R cells weretreated with 1 Amol/L ATRAand collected at the indicatedtime points for Western blotanalysis of PDCD4 expression.

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The cells were treated with ATRA (1 Amol/L) for 24, 48, and

72 h, and differentiation was assessed by morphologic

characterization and adherence to plastic tissue culture flasks

(data not shown; ref. 34). Although ATRA effectively

induced monocytic differentiation in THP1 cells, it did not

up-regulate PDCD4 expression (Fig. 4E). These findings

provided further evidence of an association between induction

of PDCD4 expression and granulocytic differentiation of

AML cells.

ATRA Induces Nuclear Translocation of PDCD4 duringGranulocytic Differentiation

The PDCD4 protein contains two basic NH2- and COOH-

terminal domains that may be nuclear localization signals.

Intense nuclear staining of PDCD4 has been found in normal

cells, such as fibroblasts, endothelial cells, and other cells of

normal prostate, colon, breast, and lung tissues, compared with

corresponding tumor cells (19). To elucidate the role of PDCD4

during granulocytic differentiation, we examined its subcellular

Figure 3. ATRA inducesPDCD4 expression in primaryhuman promyelocytic leukemia(AML-M3) and CD34+ normalbone marrow hematopoieticprogenitor cells. The primarypromyelocytic leukemia sam-ples obtained from newly diag-nosed acute promyelocyticleukemia (APL ) patients andnormal bone marrow progeni-tor cells were treated withATRA at indicated time points.Primary acute promyelocyticleukemia cells were divided intwo groups; the first group waslysed for Western blotting forthe detection of PDCD4 ex-pression and the rest of thecells were analyzed for induc-tion of differentiation by exam-ining CD11b and CD11cexpression by fluorescence-activated cell sorting or stainedfor morphologic analysis. A.ATRA induced a significantPDCD4 expression duringgranulocytic differentiation ofacute promyelocytic leukemiacell as indicated by appear-ance of granulocytic morphol-ogy including multilobularnucleus and increases cyto-plasmic to nuclear ratio (B)and up-regulation of differenti-ation markers (CD11b andCD11c; C and D). E. ATRAalso induced PDCD4 expres-sion in normal CD34+ bonemarrow progenitor cells exam-ined at 72 h.



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localization in ATRA-responsive and differentiation-resistant

leukemia cells. To this end, NB4 and NB4.R1 cells were treated

with ATRA for 72 h and stained with anti-PDCD4 antibody.

PDCD4 was located mainly in the cytoplasm in untreated NB4

and NB4.R1 cells, whereas marked nuclear translocation of

PDCD4 was seen in ATRA-treated NB4 but not NB4.R1 cells

(Fig. 5A and B), suggesting that nuclear translocation of

PDCD4 is strongly associated with granulocytic differentiation.

ATRA-Induced Reduction in PI3K/Akt Activity IsAssociated with PDCD4 Induction during GranulocyticDifferentiation

The PI3K/Akt (protein kinase B) pathway and its down-

stream component mammalian target of rapamycin (mTOR)

constitutes a key signaling cascade that links diverse extracel-

lular stimuli to cell proliferation, differentiation, and survival

(35). Because the activity of the PI3K/Akt/mTOR pathway has

been associated with increased proliferation and translation in

cancer cells (36, 37), including AML (38, 39), and PDCD4

functions as a translational suppressor, we hypothesized that the

PI3K/Akt/mTOR pathway negatively regulates PDCD4 expres-

sion and, thus, ATRA induces PDCD4 via inhibition of this

pathway during granulocytic differentiation. We therefore

sought to determine whether the PI3K/Akt/mTOR pathway is

down-regulated during ATRA-induced granulocytic differenti-

ation of NB4 cells. We first examined the phosphorylation

status of Akt during ATRA treatment in NB4 cells. PI3K

activity was reduced by ATRA, as indicated by a reduction in

Figure 4. PDCD4 expres-sion is associated with granu-locytic but not monocytic/macrophagic differentiation inAML cells. A. Granulocyticdifferentiation induced by 1Amol/L ATRA, 0.4 Amol/L ar-senic trioxide, and 1% DMSOwas accompanied by in-creased PDCD4 expression inNB4 cells. Cells were collectedat the indicated time points forWestern blot analysis ofPDCD4 expression. NT, non-treated control cells. B and C.Monocytic/macrophagic differ-entiation induced by 0.1 Amol/LPMA and 0.1 Amol/L 1,25-dihydroxyvitamin D3 did notup-regulate PDCD4 expres-sion in NB4 cells. Equalamounts of total cell lysateswere analyzed by Westernblotting for PDCD4 proteinlevels. h-actin was used as aloading control. D. PMA in-duced morphologic changesassociated with monocytic/macrophagic differentiation inNB4 leukemia cells. E. ATRAdid not induce PDCD4 expres-sion in THP1 myelomonocyticAML cells during monocyticdifferentiation. The cells weretreated with 1 Amol/L ATRA for24, 48, and 72 h and PDCD4expression was detected byWestern blotting.

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phosphorylated (p) Akt (Ser473) levels and the p-Akt/Akt ratio,

reaching maximal inhibition after 48 to 72 h of treatment

(Fig. 6A). These findings suggest that the PI3K/Akt/mTOR

pathway is inhibited during ATRA-induced granulocytic

differentiation. The nadir p-Akt expression corresponded with

the peak PDCD4 expression at 48 to 72 h of ATRA treatment

(Fig. 2A), indicating an inverse association between activation

of PI3K/Akt activity and PDCD4 expression.

PI3K/Akt/mTOR Signaling Pathway Suppresses PDCD4Expression in Leukemia Cells

Because ATRA down-regulates activity of PI3K/Akt

survival pathway under conditions in which it up-regulates

PDCD4 expression, we sought to determine whether the PI3K/

Akt/mTOR pathway plays a role in the regulation of PDCD4 by

ATRA. To that end, we blocked PI3K/Akt/mTOR activity using

specific inhibitors of PI3K (LY294002 and wortmannin; refs.

38, 39) and mTOR (rapamycin; ref. 40) and analyzed PDCD4

levels in the presence and absence of ATRA in NB4 cells by

Western blotting. As expected, ATRA enhanced PDCD4

expression compared with untreated control cells (Fig. 6B),

and treatment of cells with LY294002 or rapamycin enhanced

the of PDCD4 expression alone and produced significant up-

regulation of PDCD4 expression (Fig. 6B). To confirm the

inhibition of PI3K pathway, we examined p-Akt levels in the

cells and found that treatment with LY294002 markedly

reduced p-Akt levels (Fig. 6C). These observations suggest

that the PI3K/Akt/mTOR pathway represses PDCD4 expression

in leukemia progenitors. The inhibition of this pathway by

ATRA and/or by the pathway-specific inhibitors seems to

release suppression of PDCD4.

To determine whether the induction of PDCD4 expression is

mediated at the transcriptional or posttranslational level, we

assessed PDCD4 mRNA expression after treatment with ATRA

and/or the inhibitors. LY294002, wortmannin, and rapamycin

up-regulated PDCD4 mRNA compared with untreated NB4

cells (Fig. 6D). Thus, the ATRA and PI3K/Akt/mTOR pathway

inhibitors seem to regulate PDCD4 at the level of mRNA

expression either by increasing transcription or by inhibiting

mRNA degradation or both in AML cells.

PDCD4 Induction Is Important in Granulocytic Differen-tiation of AML Cells

To elucidate the role of PDCD4 in ATRA-induced granulo-

cytic differentiation of myeloid progenitor cells, we knocked

down PDCD4 expression using PDCD4-specific siRNA. NB4

cells transfected with PDCD4 or nonsilencing (control) siRNA,

or left untreated, were treated with 1 Amol/L ATRA for 72 h,

followed by assessment of the differentiation markers CD11b

and CD11c by flow cytometry. Under these conditions, we

consistently reached a transfection efficiency off60%, without

a significant reduction in cell viability (data not shown). For

Figure 5. ATRA inducesnuclear translocation of PDCD4in differentiation-sensitive butnot in differentiation-resistantcells.NB4 (A) andNB4.R1 cells(B) were treated with 1 Amol/LATRA for 72 h, stained withrabbit anti-PDCD4 primary andFITC-labeled donkeyanti-rabbitsecondary antibodies, and ana-lyzed by immunofluorescencemicroscopy. Nuclei werestained with 4¶,6-diamidino-2-phenylindole (blue).



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CD11b and CD11c, analyses were done including all cells with

or without transfection. As expected, ATRA treatment of cells

transfected with transfection reagent only resulted in no

inhibition of CD11b and CD11c expression in all cells (without

excluding untransfected cells). In contrast, concomitant treat-

ment with ATRA and PDCD4 siRNA resulted in a significant

inhibition of granulocytic differentiation in the cells (P < 0.05),

as indicated by reduced expression of CD11b and CD11c granu-

locytic differentiation markers (Fig. 7A-D) and by morphology

(Fig. 7E), compared with those transfected with control non-

silencing siRNA. These results suggest that PDCD4 expression

contributes to granulocytic differentiation in AML cells.

PDCD4 Mediates Expression of ATRA-Regulated Impor-tant Cellular Proteins

Although indirect transcriptional targets of PDCD4 have

been found, most of the direct targets of PDCD4 have not yet

been identified (14). To identify potential downstream targets of

PDCD4 that may be functionally significant in the mechanism

by which ATRA induces granulocytic differentiation, we

examined the expression of several important cellular proteins

known to be regulated by ATRA in NB4 cells and important in

ATRA-induced growth inhibition and granulocytic differentia-

tion. These proteins include c-jun (7), c-myc, the cyclin-

dependent kinase inhibitors p21Waf1/Cip1 (41) and p27Kip1(42),

WT1 (43), tissue transglutaminase (TG2; refs. 33, 44), and the

novel translational inhibitor DAP5 (10). DAP5 inhibits cap-

dependent and cap-independent mRNA translation by competing

with eIF4G and sequestering eIF4A and eIF3 and is essential for

terminal differentiation (10, 45, 46). WT1 is aberrantly overex-

pressed in majority of AML blasts isolated from patients, a bad

prognostic factor, and inhibited by ATRA during differentiation.

NB4 cells treated with PDCD4 or nonsilencing siRNA, or left

untreated, were incubated with or without ATRA for 72 h,

Figure 6. PI3K/Akt/mTORsignaling pathway repressesPDCD4 expression. A. ATRAinhibits the PI3K/Akt/mTORpathway during granulocyticdifferentiation in NB4 cells.NB4 cells were incubated with1 Amol/L ATRA for up to 72 h orwith 0.1 Amol/L ATRA for up to48 h. Equal amounts of totalcell lysates were analyzed byWestern blotting for phosphor-ylated Akt (Ser473) and Akt. h-actin was used as a loadingcontrol. B. Inhibition of thePI3K/Akt/mTOR pathwayenhances ATRA-inducedPDCD4 expression in NB4cells. The cells were treatedwith PI3K inhibitor (20 Amol/LLY294002) or mTOR inhibitor(20 nmol/L rapamycin) for 72 h,with or without ATRA. Equalamounts of total cell lysateswere analyzed by Westernblotting for PDCD4, p-Akt,and h-actin. C. Inhibition ofPI3K pathway by LY294002inhibits p-Akt. NB4 cells weretreated with LY294002 in thepresence or absence of ATRAfor 48 h. p-Akt was detected byWestern blotting. D. Inhibitorsof the PI3K/Akt/mTOR path-way induce PDCD4 mRNAexpression in NB4 cells. Cellswere treated with PI3K inhib-itors (200 nmol/L wortmannin,20 Amol/L LY294002) or 20nmol/L rapamycin in the pres-ence or absence of 1 Amol/LATRA. The cells were collect-ed and total cellular RNA wasextracted to detect PDCD4mRNA expression by RT-PCR using PDCD4-specificprimers. ATRA markedly in-duced PDCD4 mRNA expres-sion after 24 h of treatment.Bands representing PDCD4mRNA expression in the gelwere analyzed by densitome-try. Results were expressedas relative ratios of PDCD4 toh-actin mRNA.

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followed byWestern blot and RT-PCR analyses. PDCD4 protein

levels were markedly down-regulated within 48 h of transfection

with PDCD4 siRNA (Fig. 8A). In addition, down-regulation in

the levels of p27Kip1 and DAP5, up-regulation of c-myc and

WT1 protein, and no change in p21Waf1/Cip1 levels were

observed in the same samples (Fig. 8A-C). ATRA could not

induce expression of PDCD4 protein when PDCD4 siRNAwas

used, as observed after a 48-h treatment (Fig. 8C, lanes 3 and 4).

However, at higher doses of PDCD4 siRNA, PDCD4 expression

was markedly reduced even when the cells were stimulated with

ATRA. The knockdown of PDCD4 expression was accompa-

nied by a reduction in the ATRA-induced expression of DAP5

and a block in ATRA-induced down-regulation of WT1; in

contrast, the ATRA modulation of c-jun, p21Waf1/Cip1, and TG2

expression remained unchanged (Fig. 8D). RT-PCR analysis

showed that c-myc, DAP5, p27Kip1WT1, and mRNA levels

were not altered by siRNA-induced down-regulation of PDCD4

(Fig. 8E), suggesting that PDCD4 posttrancriptionally regulates

ATRA-modulated expression of these cellular proteins.

DiscussionThe results of the present study show that the PDCD4 is

involved in granulocytic differentiation induced by ATRA.

ATRA-induced PDCD4 expression is mediated by inhibition of

PI3K/Akt/mTOR survival pathway that constitutively represses

PDCD4 expression in AML cells. This study is the first to

implicate PDCD4 in myeloid cell differentiation and reveals a

novel mechanism of ATRA-induced granulocytic differentia-

tion of myeloid cells (Fig. 9).

Recent studies suggested that terminal cell differentiation is

associated with the inhibition of proliferation and repression of

mRNA translation, leading to a decreased rate of protein

synthesis (24, 25). We previously reported significant down-

regulation of the eukaryotic initiation factors, including eIF4A,

eIF4G, eIF2, and eIF5, and up-regulation of a translation

initiation repressor, DAP5/p97, during the granulocytic differ-

entiation induced by ATRA (10). Induction of PDCD4

translational repressor is in agreement with previous studies

and supports the hypothesis that ATRA inhibits translation

initiation, which is a tightly regulated step of translation, and

contributes to the posttranscriptional regulation of genes during

myeloid cell differentiation.

Two lines of evidence obtained in this study suggest that the

expression of PDCD4 is important to granulocytic differenti-

ation of myeloid cells and resistance to retinoic acid-induced

differentiation. First, in contrast to the differentiation-sensitive

NB4 and HL60 cells, the differentiation-resistant NB4.R1 and

HL60R cells did not show up-regulation and nuclear translo-

cation of PDCD4 in response to ATRA (Fig. 2D and E).

Second, down-regulation of PDCD4 reduced the number of

cells undergoing ATRA-induced granulocytic differentiation

(Fig. 7A and B). Our findings are in agreement with a recent

study that showed that PDCD4 is highly expressed in normal

Figure 7. PDCD4 is involved ingranulocytic differentiation of promye-locytic leukemia cells. A and B. NB4cells were transfected with transfectionreagent (TR) alone, PDCD4 siRNA, ornonsilencing control siRNA, followedby ATRA treatment for 72 h. Inductionof granulocytic differentiation was de-termined by flow cytometric analysis ofsurface CD11b and CD11c expressionusing all cells (transfected anduntransfected). Data shown as percentreduction in the number of cells under-going differentiation in transfectionreagent – , PDCD4 siRNA– , controlsiRNA– transfected, and ATRA-treatedcells compared with untransfected con-trol cells treated with ATRA. C. NB4cells after transfection with PDCD4 andcontrol siRNA were stained for mor-phologic analysis of differentiation.Granulocytic phenotype including mul-tilobular nucleus was observed in themajority of control siRNA – treatedcells. In contrast, fewer differentiatedcells were observed in PDCD4 siRNA–treated cells.



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tissues with predominant nuclear localization, but its nuclear

localization is reduced in solid tumors (19, 47), supporting the

hypothesis that lack of nuclear localization of PDCD4 may play

a role in leukemogenesis/carcinogenesis (19). It is also possible

that PDCD4 may interact with promyelocytic leukemia, which

is also localized to nuclear domains and shown to be involved

in translational control (48–52). Many tumor cell types are

undifferentiated or poorly differentiated; deficiency of PDCD4

expression seems to correlate with undifferentiated phenotype

and may contribute to the differentiation block seen in AML

cells.

The PI3K/Akt/mTOR pathway is overactivated in >80% of

AML patients and plays an important role in regulating global

and specific mRNA translation (35, 37). Activation of PI3K has

also been linked with tumorigenesis, metastasis, and resistance

to chemotherapy (53). Activation of PI3K/Akt by growth

factors or mitogens leads to phosphorylation of mTOR, subse-

quent phosphorylation of p70 S6 kinase and eIF4E-binding

protein 1, and activation of translation initiation factor eIF4E,

resulting in an increase in mRNA translation (35, 36).

The present study shows for the first time that the PI3K/Akt/

mTOR pathway represses expression of PDCD4 tumor

Figure 8. PDCD4 regu-lates expression of key cellularproteins. To identify the role ofPDCD4 in regulation of pro-teins, we examined proteinsthat are regulated by ATRAand involved in growth arrestand differentiation in myeloidcells. A. Cell cycle and cyclin-dependent kinase inhibitorprotein p27Kip1 is regulatedby PDCD4. PDCD4 expres-sion was knocked downPDCD4 by siRNA in NB4 cellsand analyzed at 48 h by West-ern blotting for p27Kip1 expres-sion. Inhibition of PDCD4expression by PDCD4 siRNAresulted in down-regulation ofPDCD4 and p27Kip1 proteinexpression, but not house-keeping protein h-actin, sug-ges t i ng tha t PDCD4 isrequired for the expression ofp27Kip1. Right, relative inhibi-tion of PDCD4 by densitomet-ric analysis of the Western blotafter normalizing to actin ex-pression. B. PDCD4 inhibitionleads to induction of c-mycand reduction in DAP5 proteinexpression but no change inp21Waf1/Cip1 cyclin-dependentkinase inhibitor levels. C.WT1expression is up-regulatedby inhibition of PDCD4 bysiRNA. D. The expression ofATRA-modulated proteins, in-cluding DAP5, TG2, WT1, andp21Waf1/Cip1, was determinedin NB4 cells after siRNA-mediated knockdown ofPDCD4 compared with controlcells. Cells were treated withATRA after 48 h of siRNAtransfection. h-actin was usedas a loading control. PDCD4inhibition by siRNA preventedATRA-mediated up-regulationof DAP5 and down-regulationof WT1 proteins. However,PDCD4 deficiency did not alterATRA-induced levels of p21and TG2. E. Knockdown ofPDCD4 by siRNA does notresult in alteration in mRNAlevels of DAP5, c-myc, andWT1 detected by semiquanti-tative RT-PCR analysis.

Ozpolat et al.


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suppressor protein at the transcriptional level, revealing a novel

mechanism of PDCD4 regulation and inactivation in AML. In

addition, a recent report suggested that Akt phosphorylates and

inactivates PDCD4 tumor suppressor function as an inhibitor

of AP-1-mediated transcription.(54). Because this pathway is

crucial to promoting cell growth, survival, and antiapoptotic

responses in AML cells (38, 39), our findings also shed light on

mechanism of antileukemic action of rapamycin, which induces

marked PDCD4 expression in AML cells (39). Inhibitors of

mTOR, such as rapamycin analogues (CCI-779 and RAD001)

have shown promising results in AML, suggesting that

targeting translational pathways is a viable treatment strategy

in AML (39, 55, 56). Inhibitors of mTOR prevent cyclin-

dependent kinase activation, inhibit Rb protein phosphoryla-

tion, and down-regulate cyclin D1, all of which may contribute

to G1 phase arrest (55–59). Therefore, induction of PDCD4 by

inhibition of mTOR by rapamycin analogues provides a novel

rationale for this treatment in AML patients.

The present study shows that ATRA-induced granulocytic

differentiation is associated with the inhibition of PI3K/Akt

activity (Fig. 6). This finding is in agreement with previous

reports that ATRA down-regulates PI3K activity (60–62). The

PI3K pathway has been linked not only with ATRA resistance

but also with ATRA-induced differentiation in promyelocytic

leukemia cells (63–65). We found that ATRA-resistant cells are

unable to enhance PDCD4 expression, thus suggesting that this

pathway may contribute to resistance to ATRA-induced

differentiation through down-regulation of PDCD4. In fact,

inactivation or reduced expression of PDCD4 has been

implicated in drug resistance in breast cancer cells (66), and

knocking down of PDCD4 prevented ATRA-induced differen-

tiation (Fig. 7), supporting this hypothesis.

Although the expression of several proteins, among them

ornithine decarboxylase, cyclin-dependent kinase 4 (18), and

carbonic anhydrase type II (67), has been reported to be down-

regulated by PDCD4 expression, the downstream targets of

PDCD4 have not yet been identified. SiRNA-mediated

knockdown of PDCD4 helped us to identify important cellular

proteins as downstream targets of PDCD4, including c-myc,

p27, DAP5, and WT1. ATRA down-regulates c-myc and WT1

and p27 up-regulates CDC-inhibitor during ATRA-induced

differentiation of NB4 and HL60 cells. Attenuation by PDCD4

of ATRA up-regulation of DAP5 and down-regulation of WT1

and c-myc occurred at the level of protein but not RNA expres-

sion, suggesting that PDCD4 regulates expression of these

proteins involved in granulocytic differentiation (Figs. 8 and 9).

DAP5 is an important mediator of differentiation, and lack of

DAP5 expression prevents ATRA-induced differentiation and

causes resistance to ATRA (10, 45). Because siRNA to PDCD4

attenuates down-regulation of WT1, WT1 may be a direct

translational target of PDCD4, a possibility that requires further

testing.

Overall, the present results suggest that PDCD4-induced

inhibition of translation initiation may play a role in controlling

hyperactivated translation in cancer cells and may lead to

growth inhibition and differentiation in response to the

granulocytic differentiation inducers. A better understanding

of this posttranscriptional mechanism may help identify targets

for new differentiation therapies for cancer.

Materials and MethodsCell Lines, Culture Conditions, and Reagents

The human acute promyelocytic cell line NB4 (M3-type

AML based on French-American-British classification) was

obtained from Dr. Michael Andreeff (The University of Texas

M.D. Anderson Cancer Center, Houston, TX) with permission

of Dr. Michael Lanotte. The NB4.R1 cell line, an ATRA-

resistant derivative of NB4, was generously provided by

Dr. Ethan Dmitrovsky (Norris Cotton Cancer Center, Dart-

mouth Medical School, Hanover, NH; ref. 68). HL60 (M2-type

myeloblastic AML) and THP1 (M5-type myelomonocytic

AML) myeloid leukemia cells were purchased from the

American Type Culture Collection (Manassas, VA). The

granulocytic differentiation-resistant HL60R cell line, an

ATRA-resistant subline of HL60, was provided by Dr. Steven

Collins (Fred Hutchinson Cancer Center, Seattle, WA; ref. 69).

Primary human promyelocytic (AML-M3) cells isolated from

newly diagnosed acute promyelocytic leukemia patients were

provided by the leukemia tissue bank through an Institutional

Review Board protocol. A highly purified population of CD34+

primary human hematopoietic progenitor cells was purchased

from Cambrex (Cambrex Bio Science, Walkersville). The cells

were grown in RPMI 1640 (Life Technologies, Carlsbad, CA)

supplemented with 10% heat-inactivated fetal bovine serum at

37jC under 5% CO2 in a humidified incubator. ATRA, arsenic

trioxide, 1,25-dihydroxyvitamin D3, PMA, and DMSO were

purchased from Sigma (St. Louis, MO). For primary cells, 20%

fetal bovine serum was used. The PI3K-specific inhibitors

LY294002 and wortmannin and the mTOR inhibitor rapamycin

were purchased from Calbiochem (La Jolla, CA).

Cell Treatments and Growth AssaysCells were seeded at 1 � 105/mL in RPMI medium in six-

well tissue culture plates (Costar, Cambridge, MA). After

Figure 9. Model for the role of PDCD4 in mediating ATRA-inducedgranulocytic differentiation. The PI3K/Akt/mTOR signaling pathwaynegatively regulates PDCD4 expression. ATRA inhibits this pathway andenhances PDCD4 expression in myeloid leukemia cells, which in turnleads to granulocytic differentiation. PDCD4 regulates ATRA-modulatedproteins, such as p27Kip1, c-myc, WT1, and DAP5, which are important toinduction of granulocytic differentiation. DAP5, a novel translationalsuppressor, is an important mediator of granulocytic differentiation, andlack of DAP5 expression prevents ATRA-induced differentiation, leading toresistance to ATRA (10, 47).



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dilution with saline from a 10 mmol/L stock, ATRA (Sigma) at

a final concentration of 0.1 or 1 Amol/L was incubated with the

cells for the indicated time points. 1,25-Vitamin D3, PMA, or

arsenic trioxide (dissolved in 5 N NaOH) was added at the

indicated concentrations. Cell viability was determined by

trypan blue (Sigma) exclusion test.

Evaluation of Cellular DifferentiationGranulocytic differentiation of the cells was determined by

examining CD11b and CD11c expression, morphologic

changes, and reorganization of promyelocytic leukemia nuclear

bodies. For the CD11b and CD11c analysis, cells were collected

after 3 to 5 days of treatment with differentiation-inducing

agents and washed with PBS. Cells (5 � 105) in 100 AL of PBS

were incubated for 30 min with FITC-conjugated anti-CD11b

antibody (1:200), phycoerythrin-conjugated anti-CD11b, or

FITC-conjugated immunoglobulin G1 isotype control (Becton

Dickinson, Franklin Lakes, NJ) at room temperature in the

dark, as previously described (34). The cells were then washed

again to remove unbound antibodies and resuspended in

500 AL of PBS. The percentage of CD11b+ and CD11c+

cells was determined by fluorescence-activated cell sorting

analysis (Flow Cytometry and Cellular Imaging Facility, M. D.

Anderson Cancer Center). Morphology was evaluated by

May-Grunwald-Giemsa staining. Briefly, cytospin preparations

of 2 � 105 cells were air-dried, incubated sequentially in

pure May-Grunwald solution (Sigma) for 5 min and 50% May-

Grunwald/water solution for 10 min, and washed with water.

The slides were then incubated in a 20% Giemsa (Sigma)/water

solution for 20 min, washed again with water, air-dried, and

examined under a Nikon microscope.

Immunofluorescence StainingCells were collected from control and ATRA-treated cultures

and washed twice with ice-cold PBS (pH 7.4). Cytospin

preparations of cells were fixed with methanol for 10 min at

room temperature, fixed in cold acetone for 2 min at �20jC,and air-dried. The slides were then washed with PBS, blocked

with 1% bovine serum albumin solution in PBS for 60 min, and

incubated with either anti–promyelocytic leukemia antibody

(Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:50 in

PBS containing 1% bovine serum albumin or anti-PDCD4

antibody (1:200; Santa Cruz Biotechnology) for 45 min at room

temperature. After washing with PBS containing 1% bovine

serum albumin, the slides were incubated with FITC-labeled

goat anti-mouse immunoglobulin G (1:100; Sigma) for 45 min

at room temperature. The cells were then incubated with

blocking buffer containing 1 Ag/mL 4¶,6-diamidino-2-phenyl-

indole for 5 min at room temperature and washed thrice in PBS.

Coverslips were mounted on the slides using a ProLong

antifade kit (Molecular Probes, Carlsbad, CA) to retard fading

and analyzed under a Nikon fluorescence microscope.

Western Blot AnalysisFollowing treatments with differentiation inducers, cells

were collected and centrifuged, and whole-cell lysates were

prepared using a lysis buffer. Total protein concentration was

determined using a detergent-compatible protein assay kit

(Bio-Rad, Hercules, CA). For the inhibition experiments, cells

were preincubated with LY294002, wortmannin, and rapamycin

for 1 to 4 h before treatment with ATRA for the indicated

time periods. Aliquots containing 30 Ag of total protein from

each sample were subjected to SDS-PAGE and electrotrans-

ferred to nitrocellulose membranes. The membranes were

blocked with 5% dry milk in TBST [100 mmol/L Tris-HCl

(pH 8.0), 150 mmol/L NaCl, and 0.05% Tween 20], probed

with primary antibodies diluted in TBST containing 5% dry

milk, and incubated at 4jC overnight. Primary antibodies

against Akt, p-Akt (Ser473), and DAP5 were obtained from Cell

Signaling Technology (Beverly, MA); antibodies against

p27Kip1, p21Waf1/Cip1, WT1, c-myc, and c-jun were obtained

from Santa Cruz Biotechnology. Transglutaminase 2 (TG2)

antibody was purchased from Neomarkers (Fremont, CA).

Serum containing anti-PDCD4 peptide antibodies was

diluted 1:10,000 in TBST (50). After washing, the membranes

were incubated with horseradish peroxidase–conjugated anti-

rabbit secondary antibody (Amersham Life Science, Cleveland,

OH). Mouse anti–h-actin and donkey anti-mouse secondary

antibodies were purchased from Sigma to analyze h-actinexpression for equal loading. The bands were visualized by

enhanced chemiluminescence (KPL, Gaithersburg, MD).

Images were scanned and quantitated using a densitometer

with the Alpha Imager application program (Alpha Innotech,

San Leandro, CA). All experiments were independently

repeated at least thrice.

RNA Isolation and RT-PCR Analysis. Cells were seeded in

six-well plates (1 � 106/mL) and treated with ATRA at a final

concentration of 1 Amol/L or with the specific inhibitors of

PI3K/Akt/mTOR at the indicated concentrations. The cells were

collected at various time points and total cellular RNA was

isolated with TRIzol reagent (Life Technologies). cDNA was

obtained from 5 Ag of total RNA using a Superscript II RT kit

(Life Technologies) as previously described (70). Briefly, 5 ALof the total 20 AL of reverse-transcribed product were used

for PCR in 1� PCR buffer containing 1.5 mmol/L MgCl2,

250 Amol/L deoxynucleotide triphosphates, 0.5 units of Taq

polymerase (Life Technologies), and 100 ng of primers for

PDCD4 (primer I, 5¶-ATGGATGTAGAAAATGAGCAG-3¶;primer II, 5¶-TTAAAGTCTTCTCAAATGCCC-3¶), DAP5

(primer I, 5¶-CAGCAGTGAGTCGGAGCTCTATGG-3¶; prim-

er II, 5¶-GTGGAGAGTGCGATTGCAGAAG-3¶), c-myc

(primer I, 5¶-TCAAGAGGTGCCACGTCTCC-3¶, primer II,

5¶-TCTTGGCAGCAGGATAGTCCTT-3¶) and WT1 (71) or h-actin (Sigma-Genosys, Houston, TX). The following programs

were used for PCR amplification of PDCD4: 1 cycle at 94jCfor 2 min, 25 to 35 cycles, denaturation at 94jC for 1 min,

annealing at 55jC to 65jC for 1 min, and extension at 72jC for

1 min. A cycle of 72jC for 7 min was added to complete the

reaction. The reaction products were analyzed on 2% agarose

gels containing ethidium bromide and cDNA synthesis was

verified by detection of the h-actin transcript.

Targeted Down-Regulation of PDCD4 by siRNAExponentially growing, untreated NB4 cells were harvested

and used for siRNA transfection. Separate aliquots of 2 � 106

cells were transfected with double-stranded siRNA targeting

PDCD4 mRNA or control nonsilencing siRNA (all from

Ozpolat et al.


106



Qiagen, Valencia, CA) using the Amaxa Nucleofector electro-

poration technique (Amaxa, Gaithersburg, MD) according to

the manufacturer’s guidelines. The siRNA sequence (5¶-AAGGUGGCUGGAACAUCUAUU-3¶) targeting PDCD4

was designed using siRNA-designing software (Qiagen).

Untransfected cells, control siRNA–transfected cells, and

transfection reagent alone were used as negative controls.

Forty-eight hours after transfection with siRNA, fresh medium

with or without 1 Amol/L ATRAwas added to the cell cultures.

After treatment, the cells were harvested for Western blot

analysis of PDCD4 protein expression or fluorescence-activated

cell sorting analysis of CD11b expression.

Statistical AnalysisData were expressed as mean F SD of three or more

independent experiments. Statistical analysis was done using

two-tailed Student’s t test for paired data. P < 0.05 was

considered statistically significant.

AcknowledgmentsWe thank Pierette Lo for critical reading and editing of the manuscript.

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2007;5:95-108. Mol Cancer Res Bulent Ozpolat, Ugur Akar, Michael Steiner, et al. Differentiation of Human Myeloid Leukemia Cells

Induced Terminal Granulocytic−Contributes to Retinoic Acid Programmed Cell Death-4 Tumor Suppressor Protein

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ProgrammedCellDeath-4TumorSuppressorProtein ... · at an immature or poorly differentiated state to resume the process of maturation (2). This type of treatment has the advantage

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