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Originally published online as doi:10.1189/jlb.0606400 on March 8, 2007

Published online before print March 8, 2007
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(Journal of Leukocyte Biology. 2007;81:1599-1608.)
© 2007 by Society for Leukocyte Biology

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

Mattia Rizzi*, Mario P. Tschan*,1, Christian Britschgi*, Adrian Britschgi*, Barbara Hügli*, Tobias J. Grob*, Nicolas Leupin*, Beatrice U. Mueller{dagger}, Hans-Uwe Simon{ddagger}, Andrew Ziemiecki§, Bruce E. Torbett||, Martin F. Fey and Andreas Tobler**

* Experimental Oncology/Hematology,
Medical Oncology, and
** Hematology, Inselspital, and
§ The Tiefenau Laboratories, Department of Clinical Research, and Departments of
{dagger} Internal Medicine and
{ddagger} Pharmacology, University of Bern, Bern, Switzerland; and
|| Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA

1 Correspondence: Experimental Oncology/Hematology, Inselspital and Department of Clinical Research, University of Bern, Murtenstrasse 35, 3010 Bern, Switzerland. E-mail: mtschan@dkf.unibe.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca2+/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34+ progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34+ cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34+ and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBP{epsilon}, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.

Key Words: DAPK2 • DRP-1 • AML • myelopoiesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During hematopoietic differentiation, pluripotent, hematopoietic stem cells differentiate into functionally and morphologically distinct end-stage cells. Differentiation is tightly linked to cell cycle regulation and apoptosis, and it is coordinated through the regulation of different key genes and gene programs. Deregulation of hematopoietic differentiation can result in the development of leukemia. For example, in acute myeloid leukemia (AML), malignant cells typically show maturation arrest at various stages of myeloid differentiation [1 ].

To identify new candidate genes operative in the regulation of myeloid differentiation, we have recently used microarray gene expression analysis of NB4 cells differentiated in tissue culture toward granulocytes in the presence of all-trans retinoic acid (ATRA). A gene, death-associated protein kinase 2 (DAPK2), also known as death-associated protein kinase-related protein 1 (DRP-1), emerged as being up-regulated during NB4 cell differentiation.

DAPK2 belongs to the family of DAPKs, is a Ca2+/calmodulin (CaM)-regulated serine/threonine kinase, and functions as a positive mediator of programmed cell death when overexpressed in several cancer cell lines [2 , 3 ]. To date, four other members of the DAPK family have been identified: DAPK1 [4 , 5 ], DAP-like kinase/zipper-interacting protein kinase [6 , 7 ], DAPK-related apoptosis-inducing protein 1 and 2 (DRAK1 and DRAK2) [8 ]. It is interesting that DAPK1, which displays strong kinase homology to DAPK2, has been shown to be epigenetically silenced in several hematological disorders [9 , 10 ], including AML [11 ], acute lymphoblastic leukemia [12 , 13 ], myelodysplastic syndromes [14 ], lymphomas [15 , 16 ], and myeloma [17 ].

Moreover, there is evidence that kinases, such as members of the serine/threonine protein kinase families protein kinase C (PKC) and MAPKs, are implicated in the process of normal and malignant hematopoiesis. PKC{delta}, for example, was shown to be activated by retinoic acid (RA)-induced differentiation of acute promyelocytic leukemia (APL) cell lines and to form a complex with retinoid acid receptor {alpha}, allowing the regulation of RA-dependent gene transcription [18 ]. The different MAPK groups play critical roles in the pathogenesis of hematological diseases [19 ]. Recently, C/EBP{epsilon}, a member of the C/EBP family involved in neutrophil differentiation [20 ], was shown to be phosphorylated by p38 MAPK, resulting in enhanced transcriptional activity of C/EBP{epsilon} [21 ]. Conversely, the role of CaM-dependent kinases during neutrophil development and function has only been studied minimally. A report by Lawson et al. [22 ] described distinct regulation of CaM kinases during ATRA and human recombinant (hr)G-CSF-induced differentiation of murine hematopoietic cell lines. In addition, a recently identified, novel CaM-dependent kinase I-like kinase (CKLiK) has been linked to granulocyte effector functions [23 ].

Together, our gene expression profiling findings, showing induction of DAPK2 during neutrophil differentiation, observations of a role for DAPK1 and perhaps for other members of this protein family in leukemogenesis [24 ] and the important role of kinases during granulocytic maturation, prompted us to investigate DAPK2 expression levels in primary normal and leukemic myeloid cells. To determine correlates of myeloid function, DAPK2 expression was analyzed during in vitro granulo- and monocytic differentiation using cord blood CD34+ progenitor cells and leukemic cell lines. Ectopic and stable expression of DAPK2 as well as expression of small interfering RNA targeting DAPK2 were used to elucidate the possible role of DAPK2 in myeloid differentiation, particularly during terminal granulocyte maturation. Our results implicate a novel role for DAPK2 in the regulation of normal myelopoiesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture, isolation, and treatment
Six myeloid and seven solid tumor cell lines purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) or American Type Culture Collection (LGC Promochem, Molsheim, France) were maintained in RPMI 1640 or DMEM, supplemented with 10% v/v FCS, 50 U/mL penicillin, and 50 µg/mL streptomycin in a 5% CO2/95% air-humidified atmosphere at 37°C.

Peripheral blood cells were obtained from buffy coats of normal, healthy donors recruited at the General Clinical Research Center at Green Hospital (La Jolla, CA, USA) or at the Inselspital Bern (Switzerland). The Institutional Review Board of The Scripps Research Institute (La Jolla, CA, USA) and the Inselspital approved protocols and the use of all human samples, respectively. PBMC and granulocytes were isolated using a Ficoll gradient (LymphoprepTM, Axon Lab AG, Switzerland) as described previously [25 , 26 ]. To remove red cells, the cells in the bottom layer were treated with erythrocyte lysis buffer (Qiagen AG, Hombrechtikon, Switzerland) to give a fraction enriched with granulocytes (>95% purity). The cells in the middle layer were subjected to different isolation procedures. The monocytic population was isolated using the CD14+ isolation system from the MiniMACS separation unit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) as described by the manufacturer. The purity of each fraction was determined by morphological examination of cytospins stained with May-Grünwald-Giemsa (Merck, Darmstadt, Germany).

To obtain blood-derived macrophages, 2 x 106/mL monocytes were cultured in the presence of 5000 U/mL hrM-CSF (Sigma-Aldrich, Switzerland) for the indicated times in RPMI-1640 medium supplemented with 10% v/v FCS, 50 units/mL penicillin, and 50 µg/mL streptomycin [27 ].

Fresh leukemic blast cells from 100 untreated AML patients at diagnosis were classified according to the French-American-British (FAB) classification and cytogenetic analysis. All leukemia samples had blast counts >90% after separation of mononuclear cells using Ficoll gradient.

Cells from patients with chronic myeloid leukemia in chronic phase (CML-CP) were isolated at diagnosis obtained and treated with erythrocyte lysis buffer (Qiagen AG). Informed consent was obtained from all patients.

In vitro differentiation of leukemic cell lines
For the myeloid differentiation experiments, we used the ATRA-sensitive, human APL cell line NB4 [28 ], the ATRA-resistant NB4-R2 subline, and the human acute myelomonocytic leukemia cell line U937. Cells were seeded at a concentration of 1–2 x 105 cells/mL and treated with ATRA and PMA as indicated. Cell culture reagents, media, and differentiation-inducing agents were purchased from Sigma-Aldrich.

Cell differentiation was assessed morphologically and by CD11b cell surface antigen expression. For the morphological observation, cytospins were stained with May-Grünwald-Giemsa (Merck), evaluated by light microscopy (Nikon E800, Japan), captured with a digital camera DMX1200, and recorded by Leika software. For reasons of simplicity, the morphologic phenotypes were classified into broad stages of differentiation: promyelocytes and myelocytes, metamyelocytes, and banded and segmented neutrophils. For immunophenotyping, 5 x 105 cells were collected, washed, and incubated with monoclonal mouse antihuman PE-labeled anti-CD11b antibody for 30 min at 4°C. Mouse anti-IgG1{kappa} was used for isotope control. Fluorescence intensity was detected by FACScan flow cytometer (Becton Dickinson, Basel, Switzerland) and analyzed using FlowJo Software. Data were based on examination of at least 20,000 cells/sample.

In vitro differentiation of cord blood-derived CD34+ progenitor cells
Cord blood samples were isolated from placentas acquired from mothers with normal, full-term deliveries at Scripps Memorial Hospital. CD34+ progenitor cells were isolated with the MiniMACS separation unit (Miltenyi Biotec GmbH), as described by the manufacturer and expanded in IMDM supplemented with 10% FBS, 1% penicillin/streptomycin, 50 ng/mL stem cell factor, 50 ng/mL IL-3, and 10 ng/mL IL-6. After expansion, cells were further cultured with 10 ng/mL hrG-CSF for neutrophil or with 3000 U/mL hrM-CSF for monocytic differentiation. Cell differentiation was evaluated as described above.

Lentiviral vector cloning, lentivirus preparation, and transduction
The open reading frames of wild-type and kinase-inactive K52A mutant of human DAPK2 were amplified using the Expand High Fidelity PCR system (Roche Diagnostics, Rotkreuz, Switzerland) and the following primers: forward 5'-GGATCCGCCGCCACCATGGACTACAAGGACGACGATGACAAGATGTTCCAGGCCTCAATGA-3' and reverse 5'-GAGGAGGAGCAGTACCTCCTAACTCGAG-3'; as templates for the amplification of human wild-type and kinase-inactive DAPK2 cDNAs in elongation factor-1X(EF) mammalian vector (pEF-BOS), plasmids were used (kindly provided by Dr. Taro Kawai Research Institute for Microbial Diseases, Osaka University, Osaka, Japan). BamHI and XhoI sites were added to the amplification primers for subcloning as well as a KOZAK consensus sequence 5' of the ATG start signal for efficient expression in mammalian cells. The PCR products were cloned into the pCR-XL-topoisomerase (TOPO) vector (TOPO telomerase activity cloning kit, Invitrogen, Basel, Switzerland), and the recombinant clones were sequenced. The gel-purified BamHI/XhoI-digested fragments were subcloned further into the CGW lentiviral transfer vectors 5' of an internal ribosomal entry site-enhanced green fluorescent protein (EGFP) reporter (sequence upon request) by standard cloning techniques (RAPid DNA ligation kit, Roche Diagnostics). MISSIONTM lentiviral clones expressing short hairpin RNAs (shRNAs), targeting DAPK2 (NM_014326.x-557, -675, and -1735) and a nontargeting shRNA control (SHC002) were purchased from Sigma-Aldrich. These vectors contain a puromycin, antibiotic-resistance gene for selection of transduced mammalian cells.

Lentivirus production and titer determination were done as described [27 ]. Briefly, the third-generation, self-inactivating glycoprotein of vesicular stomatitis virus (VSV-G)-pseudotyped virion particles were produced by transient cotransfection of the packaging plasmids pMD.G (VSV-G), pMDLg/p.RRE (gag and pol), and pRSV-Rev (Rev gene), as well as the lentiviral transfer vector. Viral supernatant was harvested 48 h later, and viral titers were determined by infecting 293T cells. NB4 cells were transduced twice overnight in the presence of 8 µg/mL polybrene using lentivirus at a multiplicity of infection of 10, thereafter washed and maintained as described above.

Real-time quantitative RT-PCR (RQ-PCR)
Total RNA was extracted using the RNeasy mini kit and the RNase-free DNase set, according to the manufacturer’s protocol (Qiagen AG). Briefly, 1–2 µg total RNA in 10 µl was reverse-transcribed using random primers (Roche Diagnostics) and Moloney murine leukemia virus RT (Promega, Catalys AG, Wallisellen, Switzerland). PCR and fluorescence detection were performed using the ABI PRISM® 7700 sequence detection system (Applied Biosystems, Rotkreuz, Switzerland) according to the manufacturer’s protocol in a reaction volume of 25 µl containing 1x TaqMan® Universal PCR master mix (Applied Biosystems) and 15–25 ng cDNA.

For quantification of DAPK2, lactoferrin (LF), G-CSF receptor (G-CSFR), and C/EBP{epsilon} cDNA, 1x Taqman® gene expression assays, were used (Applied Biosystems, Assay ID Hs00204888_m1, Hs00158924_m1, Hs00167918_m1, and Hs00357657_m1, respectively). For the housekeeping gene porphobilinogen deaminase (PBGD), we used 5'-GGCAATGCGGCTGCAA-3' and 5'-GGGTACCCACGCGAATCAC-3' at 300 nM and 5'-FAM-CTCATCTTTGGGCTGTTTTCTTCCGCCT-TAMRA-3' at 200 nM. All measurements were performed in duplicates, and the arithmetic mean of the comparative threshold cycle (Ct) values was used for calculations: Target gene mean Ct values were normalized to the respective housekeeping gene (PBGD) mean Ct values (internal reference gene, {Delta}Ct) and then to the experimental control ({Delta}{Delta}Ct). Obtained values were exponentiated (2–{Delta}{Delta}Ct) to be expressed as n-fold changes in regulation compared with the experimental control ({Delta}{Delta}Ct method of relative quantification) [29 ].

Protein extraction and Western blotting
Whole cell extracts were prepared using the nuclear extract kit (Active Motif, Rixensart, Belgium), and 10–25 µg protein was loaded on a 10% denaturing polyacrylamide gel. Blots were incubated with the primary antibodies in TBS 0.05% Tween-20/2.5% milk overnight at 4°C, incubated with secondary HRP-coupled antimouse or -rabbit antibody at 1:5–10,000 for 1 h at room temperature, and analyzed chemoluminescently using the ECL detection kit (Amersham, Freiburg, Germany). Used primary antibodies were anti-DAPK2 rabbit polyclonal antibody (psc2323, ProSci, Inc., Poway, CA, USA), 1:500, anti-EGFP rabbit polyclonal antibody (Molecular Probes, Basel Switzerland), and anti-ß-actin mouse mAb (Sigma-Aldrich), 1:10,000.

Freshly isolated granulocytes and monocytes were lysed as described elsewhere to prevent protein degradation from the leukocyte-proteases [30 ]. Briefly, freshly isolated cells (5x106) were incubated at 99°C for 5 min, and then 5x sample buffer (10% SDS, glycerol, Tris-HCl, 1 M, pH 6.8, H2O, bromophenol blue, and 20% ß-ME) and DTT (final concentration 5 mM) were added, again incubated for 5 min at 99°C, and loaded on a SDS-PAGE gel. A prestained standard (Dual Color Standards, Bio-Rad, Hercules, CA, USA) was included in each run.

Statistical analysis
Analyses were performed using the Mann-Whitney rank sum or Kruskal-Wallis one-way test (SigmaStat 3.0). Graphs were done using the software SigmaPlot (8.0).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Disparate DAPK2 expression during mono- and granulocytic differentiation of leukemic cell lines
DAPK2 mRNA was found to be up-regulated in an oligonucleotide microarray analysis designed to identify genes whose expression is regulated by ATRA-induced granulocytic differentiation. Our microarray analysis of Day 5 ATRA-treated NB4 cells demonstrated that DAPK2 levels were induced ninefold as compared with nontreated cells. A detailed description of the gene expression studies will be reported elsewhere. To confirm our microarray results, we used RQ-PCR to quantitatively evaluate DAPK2 expression during ATRA-mediated in vitro differentiation of NB4 leukemic cells. DAPK2 mRNA levels increased 110-fold 1 day after ATRA treatment and increased to 589-fold by Day 6 with respect to baseline levels. In contrast, an ATRA-resistant NB4 subline, NB4-R2, displayed only a minor DAPK2 increase of 27- and 34-fold on Days 1 and 6, respectively (Fig. 1A ). This increase of DAPK2 mRNA in NB4 was paralleled by an increase of DAPK2 protein levels (Fig. 1B) . Furthermore, ATRA induced a significant, dose-dependent increase of DAPK2 mRNA expression with sevenfold induction with 10 nM, 160-fold with 100 nM, and 514-fold with 1000 nM ATRA, as compared with the baseline levels (Fig. 1C) . The dose-response of ATRA-induced DAPK2 mRNA was paralleled by the expression of CD11b as measured by flow cytometry (data not shown).


Figure 1
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  		Figure 1. Disparate DAPK2 expression during mono- and granulocytic differentiation of leukemic cell lines. (A) DAPK2 mRNA increase during ATRA-mediated NB4 cell differentiation to granulocytes. NB4 and ATRA-resistant NB4-R2 cells (1x105 cells/mL) were cultured with 1 µM ATRA for 6 days. RNA was extracted at every time-point indicated. Relative levels of DAPK2 transcripts were determined using RQ-PCR and normalized to PBGD expression. (B) DAPK2 protein expression in ATRA-treated NB4 cells was detected by Western blot analysis as described. ß-Actin was used as a loading control. (C) NB4 cells (1x105 cells/mL) were cultured during 4 days in the presence of increasing concentrations of ATRA, as indicated, and relative changes in DAPK2 transcripts were determined. (D) U937 cells were treated for the indicated times with 1 µM ATRA for granulocytic differentiation or with 16 nM PMA for monocytic differentiation, and DAPK2 mRNA expression was measured. mRNA expression data are expressed as n-fold regulation compared with Day 0 and are representative of at least three separate experiments. Where shown, data are given as mean ± SD of three separate experiments.

 
To investigate further whether changes in levels of DAPK2 transcripts are specific to the neutrophil pathway and not just limited to NB4 cells, we took advantage of the U937 cell line as an alternative leukemic cell line differentiation model. Differentiation of U937 cells during 6 days with 1 µM ATRA induced a similar pattern of DAPK2 mRNA increase (530-fold) as in NB4 cells. In contrast, DAPK2 expression during monocytic differentiation using 16 nM PMA was significantly less pronounced (Fig. 1D) . Monocytic differentiation was confirmed by flow cytometry analysis of CD11b surface expression and by morphology (data not shown).

Taken together, these results demonstrate that DAPK2 is associated with ATRA-induced granulocytic differentiation in selected hematopoietic cell lines.

DAPK2 expression in normal primary and leukemic myeloid cells
DAPK2 has been shown previously to be highly expressed in heart, lung, skeletal muscle, colon, breast, spleen, and leukocytes [2 , 3 ]; however, expression in various hematopoietic cell lineages has not been analyzed. As our findings from cell-line differentiation experiments suggest an involvement of DAPK2 in the myeloid maturation process, it is necessary to determine whether similar patterns of DAPK2 transcript and protein expression changes occur in primary hematopoietic cells. DAPK2 mRNA expression levels were measured in freshly isolated cord blood CD34+ progenitor cells (CD34+; n=6), granulocytes (G; n=9), and cultured macrophages derived from peripheral blood (M; n=8; Fig. 2A , left panel). The results are presented relative to the values from the PBGD housekeeping gene. Human peripheral blood granulocytes showed a significantly higher expression of DAPK2 mRNA (median 119.4, range 14.9–207.9) than cultured macrophages (median 0.023, range 0.004–0.031, P<0.001) or human cord blood-derived CD34+ progenitor cells (median 0.024, range 0.011–0.049, P<0.001; Fig. 2A , left panel). This difference in DAPK2 expression was also reflected at the protein level. DAPK2 protein was highly expressed in granulocytes (G1 and G2) and was absent in monocyte extracts (M1 and M2) as shown by Western blotting (Fig. 2B) .


Figure 2
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  		Figure 2. High DAPK2 expression in normal granulocytes and CML-CP versus low expression in monocytes/macrophages and in undifferentiated CD34+ progenitor cells and AML blasts. (A) RQ-PCR for DAPK2 mRNA expression in human primary myeloid cells. DAPK2 Ct values were normalized to the Ct values of the housekeeping gene PBGD ({Delta}Ct) and exponentiated for relative DAPK2 expression (2–{Delta}Ct). Each value is shown as a dot, and the horizontal line represents the median. Human peripheral blood granulocytes (median 119.4, range 14.9–207.9; n=9) show significantly higher expression of DAPK2 mRNA ({dagger}, Mann-Whitney Rank Sum test, P<0.001) compared with all other samples. CML-CP (median 13.2, range 3.8–348.5; n=9) show significantly higher DAPK2 mRNA expression ({dagger}{dagger}, Mann-Whitney Rank Sum test, P<0.001) compared with monocytes/macrophages (median 0.023, range 0.004–0.031; n=8), human cord blood-derived CD34+ progenitor cells (CD34+, median 0.024, range 0.011–0.049; n=6), and AML blasts (median 0.044, range 0.0027–0.66; n=100). (B) DAPK2 protein expression analyzed by Western blot analysis in granulocytes (G1 and G2) and monocytes/macrophages (M1 and M2) with ß-actin as loading control. (C) DAPK2 mRNA expression by RQ-PCR in 100 AML patients of all FAB subtypes. Relative DAPK2 mRNA expression to the housekeeping gene PBGD was calculated. Median and each value are shown as in A. Differences among different AML subtypes were not significant (Kruskal-Wallis One-Way, P=0.720).

 
To study DAPK2 expression in malignant cells, we first evaluated various myeloid and solid cancer cell lines (Table 1 ). DAPK2 mRNA expression was low in most cell lines derived from AMLs (median 0.02, range <0.01–0.32) as compared with the solid tumor cell lines (median 0.15, range 0.06–0.41). Particularly low DAPK2 expression was seen in cells blocked at the promyelocytic stage of differentiation (DAPK2/PBGD ratio <0.01). In addition, we have investigated DAPK2 expression in fresh, primary AML and CML-CP from untreated patients. RQ-PCR revealed that blast cells from AML showed low DAPK2 expression (median 0.044, range 0.0027–0.66, n=100) comparable with transcript levels found from CD34+ cord blood progenitor cells (median 0.024, range 0.011–0.049, P<0.091). DAPK2 levels were significantly higher, however, in CML-CP samples (median 13.2, range 3.8–348.5, n=9, P<0.001) and in granulocytes (median 119.4, range 14.9–207.9, P<0.001; Fig. 2A , right panel). DAPK2 levels in granulocytes were significantly higher than in any other cells tested (P<0.001). A more detailed analysis of AML subtypes according to the FAB classification and cytogenetic subtypes revealed no significant differences in DAPK2 mRNA expression among the different subtypes (Kruskal-Wallis test, P=0.720; Fig. 2C ).


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  		Table 1. RQ-PCR Analysis of DAPK2 mRNA Expression in Various Human Myeloid and Nonmyeloid Cell Lines and Primary Cells

 
In summary, our results obtained from hematopoietic cell lines and primary cells suggest that DAPK2 expression is abundant in cells differentiated toward granulocytes (as shown for primary granulocytes and CML-CP) and low in undifferentiated, normal and leukemic hematopoietic cells (as shown for cord blood CD34+ progenitor cells and AML blasts) and in monocytes/macrophages.

DAPK2 mRNA expression during myeloid differentiation of cord blood CD34+ progenitor cells
To further confirm the distinct DAPK2 levels in granulocytes versus macrophages, we isolated CD34+ progenitor cells from cord blood samples obtained from two healthy donors. CD34+ cells were differentiated in vitro to neutrophils using hrG-CSF or to monocytes/macrophages using hrM-CSF. During neutrophil differentiation, DAPK2 mRNA levels increased from 1.4- to 3.4-fold at Day 6 and from 9.4- to 13.5-fold at Day 18 (Fig. 3A ), and the majority of the cells showed a granulocytic phenotype (Fig. 3B) . However, CD34+ cells, differentiated to monocytes/macrophages using hrM-CSF, displayed little change in DAPK2 mRNA expression at Day 24. Moreover, when CD14+ monocytes isolated from two healthy donors were induced to differentiate to macrophages using hrM-CSF, DAPK2 mRNA expression was reduced rather than increased (Fig. 3C) . These data demonstrate that DAPK2 is up-regulated exclusively during granulocytic but not during monocytic maturation.


Figure 3
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  		Figure 3. DAPK2 mRNA is induced exclusively during granulocytic differentiation of CD34+ progenitor cells. (A) Cord blood-derived CD34+ cells from two healthy donors (CB1, CB2) were expanded in vitro and differentiated to neutrophils using hrG-CSF and to monocytes/macrophages using hrM-CSF RQ-PCR analysis of DAPK2, and the housekeeping gene PBGD was performed as described. Data are expressed as n-fold increase over untreated, control cells. (B) Morphological assessment of granulocytic differentiation with May-Grünwald-Giemsa staining (x60 original magnification/1.4 oil). To assess the monocytic differentiation, adherent cells were observed directly under the light microscope. (C) RQ-PCR analysis of DAPK2 mRNA in freshly isolated CD14+ monocytes of two different donors (Mo1, Mo2) differentiated toward macrophages in the presence of 5000 U/mL hrM-CSF for 12 days. DAPK2 mRNA expression of the untreated control cells was arbitrarily set to 100%, and DAPK2 values of hrM-CSF-treated cells are shown as relative values as compared with control cells.

 
DAPK2 expression enhances, whereas DAPK2 knockdown dampens, maturation of ATRA-induced NB4 cells toward granulocytes
Given the association of DAPK2 with neutrophil differentiation, we next asked whether expression of exogenous, wild-type DAPK2 enhances neutrophil differentiation. To accomplish our goals, a lentiviral vector expression system was used to ectopically and stably express the DAPK2 genes. Expression of DAPK2 gene cassette and EGFP from the same vector using an internal ribosomal entry site ensured that cells expressing DAPK2 could be identified by virtue of EGFP expression. One week after vector transduction of NB4 cells with wild-type DAPK2 (DAPK2.WT) or kinase-inactive DAPK2 K52A mutant (DAPK2.K52A), ≥90% of the cells were GFP-positive. NB4 cells transduced with an empty DAPK2 gene cassette vector expressing only EGFP served as a negative control. All three transduced cell lines could be maintained in culture over several weeks without apparent changes in the amount or the signal intensity of EGFP. To verify expression of the different DAPK2 transgenes, Western blot analysis was performed on transduced cell lines. Wild-type and kinase-inactive DAPK2 proteins were found to be expressed in cells transfected with the various DAPK2 vectors, but cells transduced with empty control vector remained negative (Fig. 4A , left panel). Induction of endogenous DAPK2 was observed in the control cells upon 4 days of ATRA treatment (Fig. 4A , right panel).


Figure 4
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  		Figure 4. Wild-type DAPK2 is needed for definitive granulocytic maturation of NB4 cells. (A) Confirmation of DAPK2 transgene expression in stable NB4 cells and induction of endogenous DAPK2 upon ATRA treatment. (Left panel) Protein expression of DAPK2 constructs was detected by Western blotting with EGFP as a loading control (Lanes 1, empty vector; 2, DAPK2.K52A; 3, DAPK2.WT). (Right panel) The same blot was overexposed to show endogenous DAPK2 induction during differentiation of empty vector control cells. All experiments were repeated at least three times. (B) Flow cytometry analysis of the myeloid marker CD11b. NB4 cells stably expressing empty vector (Empty), inactive DAPK2 mutant (K52A), or wild-type DAPK2 (WT) were induced to differentiate toward granulocytes with 1 µM ATRA for 2 and 4 days. Changes in differentiation were evaluated at each indicated time-point by measuring CD11b surface expression. Mean of fluorescent intensities (MFI) were calculated from histograms of CD11b expression and are as indicated. (C) RQ-PCR analysis of myeloid differentiation markers LF, G-CSFR, and C/EBP{epsilon} at Days 4 and 6 of ATRA treatment. Data are expressed as n-fold increase over control vector-positive cells at Day 0. Results are given as mean ± SD of two independent experiments measured in duplicate. (D) RQ-PCR analysis as in C for NB4 cells stably expressing empty vector, wild-type, or K52A mutant DAPK2. Data are expressed as n-fold increase over empty vector control cells of the same day; *, P < 0.05. (E) Morphological assessment by May-Grünwald-Giemsa (x60 original magnification/1.4 oil). Three hundred cells per slide were classified into promyelocyte/myelocyte, metamyelocyte, and mature neutrophil stages of differentiation after 6 days of treatment with 1 µM ATRA. Results are given as percent fraction of the total number of cells counted ± SD. (F) Flow cytometry analysis of the myeloid marker CD11b in NB4 DAPK2 knockdown cells. DAPK2 shRNA-expressing lentiviral vectors (557, 675, and 1735) as well as a control shRNA-expressing lentiviral vector (SHC) were used for stable knockdown. All vectors cotransferred a puromycin antibiotic resistance, and puromycin-resistant cell populations were obtained by treating the cells for 10 days with 1.5 µg/ml puromycin. Differentiation and CD11b analysis was done as described in B, and CD11b MFI values of nontreated versus treated cells are indicated. DAPK2 knockdown after 4 days of ATRA treatment was measured by RQ-PCR, and DAPK2 expression of the control shRNA-transduced cells was set to 100%.

 
No obvious cellular markers of myeloid maturation were seen induced upon expression of wild-type DAPK2; however, upon treatment of transduced cells with 1 µM ATRA, differentiation was enhanced when compared with the controls. The MFI of the myeloid-specific cell surface marker CD11b at Day 0 was similar in all three cell lines bearing the different DAPK2 gene cassettes. However, after induction for 2 and 4 days with 1 µM ATRA, cells stably expressing wild-type DAPK2 showed significantly enhanced CD11b expression (MFI of 104 at Day 2 and 174 at Day 4) when compared with cells expressing kinase-inactive DAPK2 K52A (MFI of 59 at Day 2 and 82 at Day 4) or control vector (MFI of 66 at Day 2 and 95 at Day 4; Fig. 4B ). In addition, we measured mRNA levels of three genes, LF, G-CSFR, and C/EBP{epsilon}, all known to be induced upon ATRA treatment. The NB4 cells expressing the control vector showed a 3600-, 15-, and 14-fold increase of LF, G-CSFR, and C/EBP{epsilon}, respectively, from Days 0 to 6 of ATRA treatment (Fig. 4C) . Exogenous expression of wild-type DAPK2 upon ATRA treatment additionally increased mRNA levels of these genes. During 4/6 days of treatment with 1 µM ATRA in cells expressing wild-type, DAPK2 LF mRNA expression was significantly increased 7-/9.6-fold, G-CSFR 1.4-/2-fold, and C/EBP{epsilon} 1.3-/2-fold, as compared with the cells containing the control vector (Fig. 4D , Mann-Whitney Rank Sum test, P<0.05). The cells expressing the control vector or kinase-inactive DAPK2 K52A did not demonstrate increased levels of the granulocyte maturation marker program genes as compared with the control cells. The enhanced differentiation effect of wild-type DAPK2 expression was also evident morphologically after 6 days of treatment with 1 µM ATRA. NB4 DAPK2.WT cells displayed a significantly smaller fraction of promyelocytes/myelocytes: 33.5% compared with 50% in cells with the control vector and 69% in DAPK2.K52A-containing cells. Moreover, a significantly greater fraction of mature neutrophils was present in cells expressing ectopic DAPK2, 37%, as compared with cells expressing the control vector, 20%, or cells containing DAPK2.K52A, 7% (Fig. 4E) . It is interesting that neither increased apoptosis nor cell cycle changes were observed in any of the cells bearing the three different vectors during 4 days of ATRA treatment, as assessed by 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI)-stained nuclei (Supplemental Fig. 1).

To further probe the function of DAPK2 in neutrophil differentiation, we used shRNA to DAPK2 message to knockdown DAPK2 in NB4 cells and investigated the effects on ATRA-induced differentiation. Lentiviral vectors expressing three different shRNAs targeting different regions of the DAPK2 mRNA (Clones 557, 675, and 1735) and a nontargeting ShRNA (SHC) were used to transduce NB4 cells. Expression of a puromycin-resistance gene from the same delivery vector allowed for puromycin selection of transduced cells. DAPK2 knockdown in puromycin-selected cell populations was confirmed by RQ-PCR. Upon ATRA treatment, all three DAPK2 knockdown cells displayed significantly lower CD11b surface expression as compared with the nontargeting, shRNA-expressing control cells (Fig. 4F) . After 4 days of ATRA treatment, DAPK2 was decreased 70–80% in cells expressing the DAPK2 shRNAs. Assessment of the myeloid differentiation markers LF,G-CSFR, and C/EBP{epsilon} showed significantly lower levels expressed in DAPK2 knockdown as compared with the control cells (Supplemental Fig. 2).

Taken together, our results demonstrate a functional role for DAPK2 in ATRA-induced, granulocytic maturation.

DAPK2 expression in NB4 cells can be restored by demethylating therapy
In many cancers, low expression of DAPK1, the closest homologue of DAPK2, has been shown to be caused by promoter methylation [9 ]. Furthermore, treatment of DAPK1-negative cell lines with demethylating agents, such as 5'-aza-2'deoxycytidine (5'-AZA-dC), restored DAPK1 expression [31 ]. To test whether low or absent DAPK2 expression in leukemic NB4 cells may be a result of promoter methylation, we treated these cells with 5'-AZA-dC. It is interesting that treatment of NB4 cells with increasing amounts of 5'-AZA-dC during 2 days showed a dose- and time-dependent increase of DAPK2 expression (Fig. 5 ). These findings, together with the low DAPK2 expression in primary leukemias, suggest that low DAPK2 expression is, at least partially, a result of promoter silencing.


Figure 5
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  		Figure 5. Treatment of NB4 cells with demethylating agent 5'-AZA-dC restores DAPK2 expression. NB4 cells were cultured during 2 days in the presence of increasing concentrations of 5'-AZA-dC as indicated, and relative changes in DAPK2 transcripts were determined using RQ-PCR at time-points indicated. Relative DAPK2 mRNA levels were normalized to PBGD expression, and results are expressed as n-fold regulation compared with Day 0. Data are given as mean ± SD of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report for the first time that DAPK2 may play a role in human granulocytic maturation. The arguments supporting this claim and demonstrated in our experiments are: distinct granulocyte-pathway-specific DAPK2 up-regulation in vitro and in vivo; significant differences in DAPK2 expression between human immature CD34+ hematopoietic progenitor cells versus mature granulocytes; low expression of DAPK2 in AML but high expression in CML-CP; stable cell line transfection experiments providing evidence for the role of DAPK2 as an enhancer of ATRA-induced granulocytic differentiation; and DAPK2 knockdown attenuates terminal neutrophil maturation.

DAPK2 belongs to the CaM-dependent serine/threonine kinase subfamily of death-associated protein kinases, previously shown to induce apoptosis when overexpressed in nonhematopoietic cell lines [2 , 3 ]. A number of preliminary observations reported in the literature have suggested a critical link between various CaM kinases and myeloid differentiation. CaM kinase subtypes (CaMKI, CaMKIV, and particularly, CaMKK{alpha}) have been shown to be regulated differentially during ATRA- and hrG-CSF-dependent neutrophil maturation [22 ]. Furthermore, Verploegen et al. [23 ] recently identified a CKLiK with granulocyte-restricted expression and function. In the hematopoietic system, increased DAPK2 expression was reported during the differentiation of erythroid progenitor cells toward late-stage erythrocytes, most likely via Epo-receptor signaling [32 ]. Our results are consistent with these previous observations and suggest further that DAPK2 may be critical for terminal differentiation of selected hematopoietic lineages.

It is interesting that the DAPK2 gene maps to chromosome 15q22, a region known to be altered in some hematopoietic malignancies [33 ]. In addition, DAPK1, which shares high kinase domain homology with DAPK2, has previously been shown to be silenced in many hematological disorders [9 , 10 ], among them AML [11 ], acute lymphoblastic leukemia [12 , 13 ], myelodysplastic syndrome [14 ], non-Hodgkin’s lymphoma [15 , 16 ], and myeloma [17 ], through promoter hypermethylation. Furthermore, DAPK1 was reported to be overexpressed in CD34+/CD38– progenitor cells from patients with AML as compared with CD34+/CD38– cells from healthy donors, thereby suggesting a contribution of DAPK1 to the pathogenesis of early leukemic (stem) cell characteristics [24 ]. To elucidate a possible implication of DAPK2 in leukemogenesis, we first compared DAPK2 expression in myeloid and nonmyeloid malignant cells. We found that myeloid cell lines, particularly, cells blocked at the promyelocyte stage, showed weak DAPK2 mRNA expression compared with the solid tumor cell lines. Furthermore, RQ-PCR examination of 100 primary AML and nine CML-CP samples revealed that blast cells from AML show similar expression levels as immature CD34+ cord blood progenitor cells but significantly lower than CML-CP and granulocytes.

As AML blasts represent an immature hematopoietic phenotype, and cells from CML-CP patients are a mixture of fully differentiated (granulocytes) and immature blood cells, our DAPK2 expression results in normal myeloid and leukemic cells suggest that DAPK2 expression levels correlate tightly with the stage of cell differentiation toward granulocytic phenotype. It is possible that the low DAPK2 expression observed in AML blasts may contribute to the differentiation block seen in AML. The mechanism leading to suppression of DAPK2 expression is unclear to date. Analogous to DAPK1 silencing, it is conceivable that it might be mediated through epigenetic silencing. Our data showing a time- and dose-dependent increase of DAPK2 mRNA upon treatment with the demethylating agent 5'-AZA-dC further support this possibility. However, further studies are needed to confirm that DAPK2 promoter methylation is indeed the major silencing mechanism for DAPK2 in leukemia in light of our findings demonstrating a lower DAPK2 increase upon 5'-AZA-dC as compared with ATRA treatment.

Stable, lentiviral vector-mediated expression of wild-type DAPK2 enhanced ATRA-induced granulocytic differentiation of NB4 cells, as shown by morphology and CD11b expression. Furthermore, up-regulation of mRNA levels of key regulator genes for terminal differentiation, such as C/EBP{epsilon} [20 , 34 , 35 ], the G-CSFR [36 , 37 ], and the secondary granule protein LF [38 39 40 ], were also enhanced. A kinase-inactive DAPK2 K52A mutant showed no such effects, thus demonstrating that functional DAPK2 is required. In line with these results, we further show that shRNA-mediated knockdown of DAPK2 expression attenuates terminal granulocytic differentiation. Therefore, our findings underscore a role for the involvement of DAPK2 in pathways responsible for terminal maturation of neutrophils and of genes important for neutrophil effector functions. Although DAPK2 may activate important downstream effectors of hematopoietic differentiation and neutrophil function, as of yet, no DAPK2 targets have been described so far apart from the myosin light chain gene [2 , 3 ].

It is at present unclear which signaling pathway(s) require DAPK2 to mediate the biological effects seen in our studies of hematopoietic cells. A recently proposed role for DAPK2 in autophagy or Type II cell death in nonhematopoietic cells [41 ] may provide insight into DAPK2 function in myeloid cells. Autophagy is a cellular process characterized by the sequestration of portions of the cytoplasm or cytoplasmic organelles into double membrane-bounded vesicles, autophagosomes, which subsequently fuse with the lysosomal system of the same cells, resulting in degradation of the vesicle contents. This process is in principle a cytoplasmic homeostasis pathway, eliminating damaged organelles and promoting turnover of stable macromolecules to support cellular anabolic needs, particularly during starvation. It is interesting that autophagy has also been implicated in cellular architecture changes occurring in differentiation and development, in malignant transformation, and recently, in innate and adaptive immunity [42 43 44 ]. It is presently unclear which role, if any, autophagy plays in granulocyte differentiation and/or function or leukemogenesis; however, the observed participation of DAPK2 in granulocytic maturation warrants further investigation.

In conclusion, our findings strongly implicate a role for DAPK2 in neutrophil maturation and in regulating terminal differentiation programs. Moreover, the significantly lower DAPK2 mRNA levels in AML suggest that alterations of DAPK2 expression may predispose for leukemogenesis.


   ACKNOWLEDGEMENTS
 
These studies were supported by grants from the Swiss National Foundation 3100-067213 (to A. T. and M. F. F.) and 3100AO-100445 (to B. U. M.); the Marlies-Schwegler Foundation and the Bernese Foundation of Cancer Research (to M. F. F. and A. T.); the Werner and Hedy Berger-Janser Foundation of Cancer Research (to M. F. F.); and National Institutes of Health DK54938 and AI49165 (to B. E. T. and M. P. T.). We thank Drs. T. Kawai and A. Kimchi for providing DAPK2 plasmids and K. M. Fischer for technical support with stem cell differentiation.

Received June 14, 2006; revised December 23, 2006; accepted January 24, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Olsson, I., Bergh, G., Ehinger, M., Gullberg, U. (1996) Cell differentiation in acute myeloid leukemia Eur. J. Haematol. 57,1-16[Medline]
  2. Kawai, T., Nomura, F., Hoshino, K., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Akira, S. (1999) Death-associated protein kinase 2 is a new calcium/calmodulin-dependent protein kinase that signals apoptosis through its catalytic activity Oncogene 18,3471-3480[CrossRef][Medline]
  3. Inbal, B., Shani, G., Cohen, O., Kissil, J. L., Kimchi, A. (2000) Death-associated protein kinase-related protein 1, a novel serine/threonine kinase involved in apoptosis Mol. Cell. Biol. 20,1044-1054[Abstract/Free Full Text]
  4. Deiss, L. P., Feinstein, E., Berissi, H., Cohen, O., Kimchi, A. (1995) Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the {gamma} interferon-induced cell death Genes Dev. 9,15-30[Abstract/Free Full Text]
  5. Cohen, O., Feinstein, E., Kimchi, A. (1997) DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity EMBO J. 16,998-1008[CrossRef][Medline]
  6. Kogel, D., Plottner, O., Landsberg, G., Christian, S., Scheidtmann, K. H. (1998) Cloning and characterization of Dlk, a novel serine/threonine kinase that is tightly associated with chromatin and phosphorylates core histones Oncogene 17,2645-2654[CrossRef][Medline]
  7. Kawai, T., Matsumoto, M., Takeda, K., Sanjo, H., Akira, S. (1998) ZIP kinase, a novel serine/threonine kinase which mediates apoptosis Mol. Cell. Biol. 18,1642-1651[Abstract/Free Full Text]
  8. Sanjo, H., Kawai, T., Akira, S. (1998) DRAKs, novel serine/threonine kinases related to death-associated protein kinase that trigger apoptosis J. Biol. Chem. 273,29066-29071[Abstract/Free Full Text]
  9. Katzenellenbogen, R. A., Baylin, S. B., Herman, J. G. (1999) Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies Blood 93,4347-4353[Abstract/Free Full Text]
  10. Ng, M. H. (2002) Death associated protein kinase: from regulation of apoptosis to tumor suppressive functions and B cell malignancies Apoptosis 7,261-270[CrossRef][Medline]
  11. Ekmekci, C. G., Gutierrez, M. I., Siraj, A. K., Ozbek, U., Bhatia, K. (2004) Aberrant methylation of multiple tumor suppressor genes in acute myeloid leukemia Am. J. Hematol. 77,233-240[CrossRef][Medline]
  12. Matsushita, C., Yang, Y., Takeuchi, S., Matsushita, M., Van Dongen, J. J., Szczepanski, T., Bartram, C. R., Seo, H., Koeffler, H. P., Taguchi, H. (2004) Aberrant methylation in promoter-associated CpG islands of multiple genes in relapsed childhood acute lymphoblastic leukemia Oncol. Rep. 12,97-99[Medline]
  13. Gonzalez-Gomez, P., Bello, M. J., Arjona, D., Alonso, M. E., Lomas, J., Aminoso, C., de Campos, J. M., Sarasa, J. L., Gutierrez, M., Rey, J. A. (2003) CpG island methylation of tumor-related genes in three primary central nervous system lymphomas in immunocompetent patients Cancer Genet. Cytogenet. 142,21-24[CrossRef][Medline]
  14. Voso, M. T., Scardocci, A., Guidi, F., Zini, G., Di Mario, A., Pagano, L., Hohaus, S., Leone, G. (2004) Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes Blood 103,698-700[Abstract/Free Full Text]
  15. Rossi, D., Capello, D., Gloghini, A., Franceschetti, S., Paulli, M., Bhatia, K., Saglio, G., Vitolo, U., Pileri, S. A., Esteller, M., Carbone, A., Gaidano, G. (2004) Aberrant promoter methylation of multiple genes throughout the clinico-pathologic spectrum of B-cell neoplasia Haematologica 89,154-164[Abstract/Free Full Text]
  16. Nakatsuka, S., Takakuwa, T., Tomita, Y., Hoshida, Y., Nishiu, M., Yamaguchi, M., Nishii, K., Yang, W. I., Aozasa, K. (2003) Hypermethylation of death-associated protein (DAP) kinase CpG island is frequent not only in B-cell but also in T- and natural killer (NK)/T-cell malignancies Cancer Sci. 94,87-91[CrossRef][Medline]
  17. Ng, M. H., To, K. W., Lo, K. W., Chan, S., Tsang, K. S., Cheng, S. H., Ng, H. K. (2001) Frequent death-associated protein kinase promoter hypermethylation in multiple myeloma Clin. Cancer Res. 7,1724-1729[Abstract/Free Full Text]
  18. Kambhampati, S., Li, Y., Verma, A., Sassano, A., Majchrzak, B., Deb, D. K., Parmar, S., Giafis, N., Kalvakolanu, D. V., Rahman, A., Uddin, S., Minucci, S., Tallman, M. S., Fish, E. N., Platanias, L. C. (2003) Activation of protein kinase C {delta} by all-trans-retinoic acid J. Biol. Chem. 278,32544-32551[Abstract/Free Full Text]
  19. Platanias, L. C. (2003) MAP kinase signaling pathways and hematologic malignancies Blood 101,4667-4679[Abstract/Free Full Text]
  20. Lekstrom-Himes, J. A. (2001) The role of C/EBP({epsilon}) in the terminal stages of granulocyte differentiation Stem Cells 19,125-133[Abstract/Free Full Text]
  21. Williamson, E. A., Williamson, I. K., Chumakov, A. M., Friedman, A. D., Koeffler, H. P. (2005) CCAAT/enhancer binding protein {epsilon}: changes in function upon phosphorylation by p38 MAP kinase Blood 105,3841-3847[Abstract/Free Full Text]
  22. Lawson, N. D., Zain, M., Zibello, T., Picciotto, M. R., Nairn, A. C., Berliner, N. (1999) Modulation of a calcium/calmodulin-dependent protein kinase cascade by retinoic acid during neutrophil maturation Exp. Hematol. 27,1682-1690[CrossRef][Medline]
  23. Verploegen, S., Ulfman, L., van Deutekom, H. W., van Aalst, C., Honing, H., Lammers, J. W., Koenderman, L., Coffer, P. J. (2005) Characterization of the role of CaMKI-like kinase (CKLiK) in human granulocyte function Blood 106,1076-1083[Abstract/Free Full Text]
  24. Guzman, M. L., Upchurch, D., Grimes, B., Howard, D. S., Rizzieri, D. A., Luger, S. M., Phillips, G. L., Jordan, C. T. (2001) Expression of tumor-suppressor genes interferon regulatory factor 1 and death-associated protein kinase in primitive acute myelogenous leukemia cells Blood 97,2177-2179[Abstract/Free Full Text]
  25. Tschan, M. P., Vonlanthen, S., Cajot, J. F., Peters, U. R., Oppliger, E., Betticher, D. C., Yarbrough, W. G., Fey, M. F., Tobler, A. (2001) Different p16INK4a and p14ARF expression patterns in acute myeloid leukemia and normal blood leukocytes Leuk. Lymphoma 42,1077-1087[Medline]
  26. Colotta, F., Peri, G., Villa, A., Mantovani, A. (1984) Rapid killing of actinomycin D-treated tumor cells by human mononuclear cells. I. Effectors belong to the monocyte-macrophage lineage J. Immunol. 132,936-944[Abstract]
  27. Tschan, M. P., Fischer, K. M., Fung, V. S., Pirnia, F., Borner, M. M., Fey, M. F., Tobler, A., Torbett, B. E. (2003) Alternative splicing of the human cyclin D-binding Myb-like protein (hDMP1) yields a truncated protein isoform that alters macrophage differentiation patterns J. Biol. Chem. 278,42750-42760[Abstract/Free Full Text]
  28. Lanotte, M., Martin-Thouvenin, V., Najman, S., Balerini, P., Valensi, F., Berger, R. (1991) NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3) Blood 77,1080-1086[Abstract/Free Full Text]
  29. Livak, K. J., Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-{Delta} {Delta} C(T)) method Methods 25,402-408[CrossRef][Medline]
  30. Sallmyr, A., Miller, A., Gabdoulkhakova, A., Safronova, V., Henriksson, G., Bredberg, A. (2004) Expression of DNA-dependent protein kinase in human granulocytes Cell Res. 14,331-340[CrossRef][Medline]
  31. Kissil, J. L., Feinstein, E., Cohen, O., Jones, P. A., Tsai, Y. C., Knowles, M. A., Eydmann, M. E., Kimchi, A. (1997) DAP-kinase loss of expression in various carcinoma and B-cell lymphoma cell lines: possible implications for role as tumor suppressor gene Oncogene 15,403-407[CrossRef][Medline]
  32. Wojchowski, D. M., Menon, M. P., Sathyanarayana, P., Fang, J., Karur, V., Houde, E., Kapelle, W., Bogachev, O. (2006) Erythropoietin-dependent erythropoiesis: new insights and questions Blood Cells Mol. Dis. 36,232-238[CrossRef][Medline]
  33. Roulston, D., Espinosa, R., III, Nucifora, G., Larson, R. A., Le Beau, M. M., Rowley, J. D. (1998) CBFA2(AML1) translocations with novel partner chromosomes in myeloid leukemias: association with prior therapy Blood 92,2879-2885[Abstract/Free Full Text]
  34. Morosetti, R., Park, D. J., Chumakov, A. M., Grillier, I., Shiohara, M., Gombart, A. F., Nakamaki, T., Weinberg, K., Koeffler, H. P. (1997) A novel, myeloid transcription factor, C/EBP {epsilon}, is upregulated during granulocytic, but not monocytic, differentiation Blood 90,2591-2600[Abstract/Free Full Text]
  35. Yamanaka, R., Kim, G. D., Radomska, H. S., Lekstrom-Himes, J., Smith, L. T., Antonson, P., Tenen, D. G., Xanthopoulos, K. G. (1997) CCAAT/enhancer binding protein {epsilon} is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing Proc. Natl. Acad. Sci. USA 94,6462-6467[Abstract/Free Full Text]
  36. Lotem, J., Sachs, L. (2002) Cytokine control of developmental programs in normal hematopoiesis and leukemia Oncogene 21,3284-3294[CrossRef][Medline]
  37. Avalos, B. R. (1996) Molecular analysis of the granulocyte colony-stimulating factor receptor Blood 88,761-777[Free Full Text]
  38. Cowland, J. B., Borregaard, N. (1999) The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules J. Leukoc. Biol. 66,989-995[Abstract]
  39. Teng, C. T. (2002) Lactoferrin gene expression and regulation: an overview Biochem. Cell Biol. 80,7-16[CrossRef][Medline]
  40. Gregoire, C., Welch, H., Astarie-Dequeker, C., Maridonneau-Parini, I. (1998) Expression of azurophil and specific granule proteins during differentiation of NB4 cells in neutrophils J. Cell. Physiol. 175,203-210[CrossRef][Medline]
  41. Inbal, B., Bialik, S., Sabanay, I., Shani, G., Kimchi, A. (2002) DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death J. Cell Biol. 157,455-468[Abstract/Free Full Text]
  42. Gozuacik, D., Kimchi, A. (2004) Autophagy as a cell death and tumor suppressor mechanism Oncogene 23,2891-2906[CrossRef][Medline]
  43. Levine, B., Klionsky, D. J. (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy Dev. Cell 6,463-477[CrossRef][Medline]
  44. Deretic, V. (2005) Autophagy in innate and adaptive immunity Trends Immunol. 26,523-528[CrossRef][Medline]



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