Post on 29-May-2020
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: glopez@mdanderson.orgCopyright 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
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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 ).
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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.
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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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