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Originally published online as doi:10.1189/jlb.0905502 on August 2, 2006

Published online before print August 2, 2006
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(Journal of Leukocyte Biology. 2006;80:889-896.)
© 2006 by Society for Leukocyte Biology

Granulocyte macrophage colony-stimulating factor enhances retinoic acid-induced gene expression

Takahisa Shimizu1, Lisa Esaki, Hiroko Mizuno and Ken Takeda

Department of Hygiene-Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan

1 Correspondence: Department of Hygiene-Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki Noda-shi Chiba 278-8510, Japan. E-mail: takahisa{at}rs.noda.tus.ac.jp


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ABSTRACT
 
We reported previously that treatment of human myeloblastic leukemia ML-1 cells with all-trans retinoic acid (ATRA) in combination with GM-CSF enhances the granulocytic differentiation, which is induced only slightly by ATRA alone. To investigate the mechanism underlying this differentiation and the synergistic effect of ATRA and GM-CSF, we used cDNA microarray to examine gene expression profiles of ML-1 cells treated with ATRA and/or GM-CSF. We identified 22 up-regulated genes in ML-1 cells treated with both reagents and examined the expression of these genes in cells treated with ATRA and/or GM-CSF by Northen blot analysis. Comparison of cells treated with both reagents and cells treated with ATRA or GM-CSF alone revealed that expression of nine of the 19 genes was induced synergistically by combined treatment with ATRA and GM-CSF. Expression of most of these genes was increased only slightly by ATRA alone, and this induction was enhanced by the addition of GM-CSF. These results indicate that GM-CSF enhances ATRA-induced gene expression. Moreover, studies with inhibitors of signaling molecules suggested that activation of JAK2 is associated with the synergistic induction of several genes by ATRA and GM-CSF. JAK2 inhibitor suppressed induction of NBT-reducing activity in ML-1 cells treated with both reagents. It is likely that the enhancer effect of GM-CSF on ATRA-induced gene expression leads to the differentiation induced synergistically by ATRA combined with GM-CSF. Further studies of the mechanism underlying this effect may identify better approaches for the treatment of RA-insensitive leukemia.

Key Words: leukemia • differentiation • retinoic acid • GM-CSF • cDNA microarray


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INTRODUCTION
 
Retinoic acid (RA) is a known inducer of cell differentiation into granulocytes. All-trans RA (ATRA), in particular, promotes granulocytic maturation of the human HL-60 cell line [1 ] and of hematopoietic cells from patients with acute promyelocytic leukemia (APL) [2 ]. ATRA is effective in differentiation therapy for APL [3 ]; however, retinoids generally do not promote granulocytic maturation in acute myeloid leukemia (AML) subtypes, with the exception of APL. ML-1 cells, derived from a patient with acute myeloblastic leukemia, are at an earlier stage of differentiation than HL-60 cells and can be easily differentiated into the macrophage but not the granulocytic pathway by various inducers such as TNF [4 ], IFN-{gamma} [5 ], and 12-O-tetradecanoylphorbol-13-acetate (TPA) [6 , 7 ]. We previously succeeded in inducing synergistic differentiation toward granulocytes in ML-1 cells treated with ATRA in combination with GM-CSF [8 ]. Elucidation of the mechanism underlying differentiation in response to ATRA and GM-CSF may lead to development of novel therapies for AML patients.

Cellular responses to ATRA are mediated by two families of transcription factors, the RA receptors (RARs) and the retinoid X receptors. These proteins belong to a superfamily of intracellular receptors, which upon ligand activation, function as dimeric transcription factors to control expression of target genes by binding to specific DNA sequences, termed RA response elements [9 10 11 ]. GM-CSF exerts its actions through binding to a specific receptor consisting of {alpha} and ß subunits, which are members of the type I cytokine receptor family. Transcription factors activated by several signals from the receptor induce expression of target genes, which are thought to be critical for growth and differentiation [12 13 14 15 ]. Little is known, however, about the interactions between ATRA-stimulated signals and GM-CSF-stimulated signals. Comparisons of the global gene expression profiles of ML-1 cells treated with ATRA and/or GM-CSF may provide insights into the synergistic action of ATRA and GM-CSF. Thus, to investigate the mechanism underlying the synergistic action of ATRA and GM-CSF in ML-1 cells, we used a microarray-based approach to analyze changes in gene expression in ML-1 cells treated with one or both of these reagents. We compared the gene expression profiles and found that GM-CSF enhances expression of various genes in response to ATRA. Furthermore, we attempted to identify the signaling pathway associated with the synergistic action of ATRA and GM-CSF.


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MATERIALS AND METHODS
 
Cell culture, reagents, and RNA isolation
ML-1 cells were maintained as suspension cultures in RPMI-1640 medium (Life Technologies Inc., Grand Island, NY) supplemented with 10% heat-inactivated FBS (Filtron Ltd., Victoria, Australia). Incubation was carried out at 37ºC in a humidified 5% CO2 incubator. ATRA (Sigma Chemical Co., St. Louis, MO) was added to the culture medium at the final concentration of 10–7 M, and recombinant human GM-CSF (Sandoz, Wien, Austria) was added to the culture medium at the final concentration of 0.1 ng/ml. In experiments in which signal kinase inhibitors were used, cells were preincubated with the inhibitors for 30 min before addition of the differentiation-induced reagents. PD98059 was from Calbiochem (San Diego, CA). AG490 was from Biomol (Plymouth Meeting, PA). Total cellular RNA was isolated using Isogen (Wako, Osaka, Japan). mRNA was purified using the Oligotex dT30 mRNA pulification kit (Takara, Kyoto, Japan).

cDNA microrray hybridization and gene expression analysis
cDNA probe synthesis, hybridization, and signal analysis were conducted by Incyte Genomics (St. Louis, MO). The cDNA-based human UniGene 1 array was used, which is 100% sequence-verified and contains 9128 independent clones representing 8524 unique genes and expressed sequence tags (ESTs). Initial data analysis was performed with GemTools 2.5. In all cases, cDNAs generated from reagent-treated samples were labeled with Cy5, whereas matching controls were labeled with Cy3. All reported fold changes are a normalized, balanced, differential expression. Spots on the array in which either probe failed the selection criteria as defined by Incyte Genomics were discarded prior to analysis. As described in Results, the cutoff for balanced differential expression (fold change) between the Cy3 and Cy5 probes was 2.0 for all experiments.

Northern blot analysis
mRNA (2 µg) was separated by electrophoresis on 1.2% agarose-formaldehyde gels and transferred to Hybond-N membranes (Amersham Biosciences, Piscataway, NJ). Hybridizations were carried out according to the manufacturer’s instructions. DNA fragments used as probes were amplified by RT-PCR with the following primers: proteinase inhibitor 9 (PI-9), sense 5'-CAGGCTGGTTCTTGTCAATG-3' and antisense 5'-TTCATACAGTCTGGCTTGGTC-3'; myeloid-related protein-14 (MRP-14), sense 5'-GATGAACTCCTCGAAGCTCAG-3' and antisense 5'-TGACAGAGTGCAAGACGATG-3'; transglutaminase 2 (TGM2), sense 5'-AGGTCAATGCCGACGTGGTA-3' and antisense 5'-CTGCCCATGTTCATGCTCTG-3'; 5-lipoxygenase-activating protein (FLAP), sense 5'-CAGAGGACCGGAACACTTG-3' and antisense 5'-GGACATGAGGAACAGGAAGAG-3'; granulocyte chemotactic protein-2 (GCP-2), sense 5'-CGATTGGTAAACTGCAGGTG-3' and antisense 5'-AGCTGTAAACTTCAGGGAGA-3'; cathepsin D, sense 5'-TCCATCCACTGCAAACTGCT-3' and antisense 5'-CTTCTGCTGCATCAGGTTGT-3'; granulin, sense 5'-CCTGCTTCCAAAGATCAGGTA-3' and antisense 5'-CTCACCTCCATGTCACATTTC-3'; inhibitor of DNA binding-1 (ID-1), sense 5'-TCGCATCTTGTGTCGCTGAA-3' and antisense 5'-TTCAGTCGGTGATCATTGTA-3'; pleckstrin, sense 5'-GAGCGTGTTCAATACGTGGA-3' and antisense 5'-ACGCAGTTACCTGTGAAGCA-3'; Src-like adaptor (SLAP), sense 5'-TGCCCTGAATGGAACTACTTT-3' and antisense 5'-CTAGTCTCGCCTCCTCGATT-3'; IFN-{alpha}-inducible protein 6–16 (IFI-6–16), sense 5'-TGCTGCTCTTCACTTGCAGT-3' and antisense 5'-TGCACTCTAGCCTGGACAAT-3'; PI3K{gamma}, sense 5'-ATCATAGCCACTGATCCACT-3' and antisense 5'-TCTGAACTGCAATGGCTCTT-3'; CD11b, sense 5'-GACTCCTACAGGACACAGGT-3' and antisense 5'-TGGTGACCACCATGTAGACA-3'; myosin IE, sense 5'-AAGACCGTCCGGAACAACAA-3' and antisense 5'-CCAATCACATTCATGGCGTG-3'; glutathione S-transferase {omega}-1 (GSTO-1), sense 5'-ACTAGTAAGCAGGGCTGAGA-3' and antisense 5'-GAGTACCTGGATGAAGCATA-3'; zinc finger protein 76 (ZNF76), sense 5'-GGCAGCAAGTTGGAGACAG-3' and antisense 5'-GCTTCTGCAGGTCTCCTGA-3'; zyxin, sense 5'-TGCGAGGGCTGTTACACTGA-3' and antisense 5'-GAGTACCTGGATGAAGCATA-3'; Niemann-Pick disease type C-2 (NPC-2), sense 5'-TGAGCCTGATGGTTGTAAGAG-3' and antisense 5'-TGGAGCAAGTCACTGTTGT-3'; paternally expressed gene-10 (PEG10), sense 5'-TCATCGACTACTCCAATGCT-3' and antisense 5'-AGGACAATTGTCAGCGTAGT-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense 5'-ATCATCAGCAATGCCTCCTG-3' and antisense 5'-CTGCTTCACCACCTTCTTGA-3'.

Western blot analysis
For whole cell extracts, cell lysates were prepared by suspension of 1 x 107 cells in lysis buffer [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM PMSF, 10 ng/ml aprotinin, 10 µg/ml leupeptin, 10 mM ß-glycerophosphate, 1 mM NaF, and 0.1 mM NaVO4] and repetition of freezing and melting. Whole cell extracts were centrifuged to remove insoluble cell debris. Protein concentration was determined by the Bio-Rad (Tokyo, Japan) protein assay. Western blot analysis was carried out according to the instructions of manufacturer-purchased, primary antibody. The denatured cell extracts were analyzed by SDS-PAGE. The proteins were transferred to a polyvinylidene difluoride or nitrocellulose membrane. The blots were blocked with appropriate blocking buffer and then incubated for 2 h at room temperature with one of the following primary antibodies: monoclonal anti-ERK2 (D-2) and anti-phosphorylated-p38 (p-p38; D-8; Santa Cruz Biotechnology, CA); polyclonal anti-active MAPK and anti-active JNK (Promega, Madison, WI); polyclonal anti-p-Jak2 and anti-p-Akt (Cell Signaling Technology, Beverly, MA); and polyclonal anti-Jak2 (Upstate, Charlottesville, VA). The blots were then washed three times and incubated for 1 h with HRP-conjugated secondary antibodies. The proteins were visualized using the ECL detection system (Amersham Biosciences).

NBT-reducing ability
NBT-reducing activity was measured using the method described previously [8 ]. Briefly, 3 x 105 cells were suspended in 96-well microplates with reagents and then incubated at 37ºC. After 1 day of incubation, 0.1% NBT dye and 20 ng TPA were added to each well and incubated at 37ºC for 30 min. The reaction was terminated by adding 50 µl 2N HCl. The medium was discarded, the formazan deposits dissolved by adding DMSO, and the dissolved formazan measured at 595 nm by a spectrophotometer (Microplate Reader Model 550, Bio-Rad).


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RESULTS
 
cDNA microarray analysis of gene expression in ML-1 cells treated with ATRA and/or GM-CSF
RNAs were isolated at 24 h from cells treated with 10–7 M ATRA and/or 0.1 ng/ml GM-CSF and from untreated cells. To examine gene expression profile in ML-1 cells treated with GM-CSF, ATRA, and ATRA plus GM-CSF, one cDNA microarray assay was performed using cDNAs synthesized from these RNAs. A set of microarrays was hybridized with a mixture of Cy3-labeled cDNA corresponding to the control cells and Cy5-labeled cDNA corresponding to the reagent-treated cells. To examine differences in expression patterns after treatment, we counted the genes that showed at least a twofold difference in the Cy5:Cy3 ratio (Table 1 ). We identified 22 up-regulated genes and 145 down-regulated genes in ML-1 cells at 24 h after combined treatment with ATRA and GM-CSF. In these genes, classification of genes changed more than twofold and less than 0.4-fold (except unknown gene) is shown in Table 2 . These 30 differentially expressed genes were assigned to several functional gene categories including nuclear proteins, signal transducers, protein turnover, secreted proteins, enzymes, and structure proteins. Several of the up-regulated genes, including MRP-14, GCP-2, PI3K{gamma}, c-fgr, and CD11b, are known to be involved in granulocytic differentiation. We identified 23 up-regulated genes and 55 down-regulated genes in cells treated with ATRA alone. In contrast, there was little change in gene expression at 24 h after treatment with GM-CSF alone. We first noticed that the number of down-regulated genes was increased by combined treatment with ATRA and GM-CSF. We examined expression of these down-regulated genes by RT-PCR or Northern blot analysis. However, we could not confirm expression change of genes, which suppressed remarkably in microarray analysis, such as uPA (0.08-fold), c-jun (0.19-fold), HSPA8 (0.23-fold), and KIAA1919 (0.29-fold; data not shown). We then examined up-regulated genes. We listed the genes up-regulated in ML-1 cells treated with both reagents along with the Cy5:Cy3 ratios for the ATRA treatment and for the GM-CSF treatment (Table 3 ). We compared the Cy5:Cy3 ratios for these treatments and found that expression of many genes was greater in ML-1 cells that received combined treatment with ATRA and GM-CSF than in cells treated with either reagent alone. This finding indicates the possibility that the expression of various genes was induced synergistically by treatment with ATRA and GM-CSF.


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  		Table 1. Changes in Gene Expression by Treatment with ATRA and/or GM-CSF


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  		Table 2. Classification of Genes Modulated in ML-1 Cells by Treatment with ATRA and GM-CSF for 24 hr


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  		Table 3. Genes Up-Regulated in ML-1 Cells Treated with ATRA and GM-CSF and the Fold Changes by Treatment with ATRA and/or GM-CSF

To confirm the differential gene expression detected by cDNA microarray, we examined expression of genes that were up-regulated in ML-1 cells, which received combined treatment. We examined expression of 20 genes except for ESTs at 24 h after treatment with ATRA and/or GM-CSF by Northern blotting. The expression patterns on Northern blots were similar to those from microarray analyses for various genes (Fig. 1 ). The expression of TGM2, FLAP, cathepsin D, ID-1, pleckstrin, SLAP, and IFI-6–16 genes was induced synergistically by treatment with ATRA and GM-CSF. Moreover, Northern blotting revealed synergistic induction of expression of the MRP-14 and CD11b genes, which was not detected by cDNA microarray analysis. Expression of most of these genes was increased only slightly by ATRA alone, and this induction was enhanced by the addition of GM-CSF. These results indicate that GM-CSF enhances ATRA-induced gene expression. Expression of four genes (granulin, PI3K{gamma}, myosin IE, and ZNF76) was induced by ATRA alone; however, the induction was not enhanced by combined treatment with GM-CSF. Conversely, expression of other genes (PI-9, GCP-2, GSTO-1, zyxin, NPC-2, and PEG10) did not show a twofold or more change by treatment with ATRA and/or GM-CSF. The expression of the c-fgr gene was not detected by Northern blot analysis.


Figure 1
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  		Figure 1. Expression of various mRNAs in ML-1 cells treated with 10–7 M ATRA and/or 0.1 ng/ml GM-CSF. mRNA samples were prepared from cells treated with 10–7 M ATRA and/or 0.1 ng/ml GM-CSF for 24 h. Northern blot analysis with 2 µg mRNA was performed as described in Materials and Methods. Radioactive intensities of the bands on autoradiograms were measured with a BAS1500 image analyzer (Fuji Film, Kanagawa, Japan). Gene expression values were normalized to GAPDH expression. Expression levels of various genes are represented as fold change (F.C.) versus control. The data shown are control (C), GM-CSF (G), ATRA (A), and ATRA plus GM-CSF (A+G).

Analysis of the kinetics of expression of ATRA/GM-CSF-induced genes
ML-1 cells were treated with 10–7 M ATRA and 0.1 ng/ml GM-CSF for different periods of time to examine the kinetics of gene expression induced by ATRA combined with GM-CSF. RNAs were isolated at 1, 3, 6, 24, 48, and 96 h from cells treated with ATRA and GM-CSF, and expression of ID-1, MRP-14, CD11b, and TGM2 mRNAs was analyzed by Northern blotting (Fig. 2 ). ID-1 showed the earliest onset of expression. After combined treatment, ID-1 mRNA was detected at 1 h and peaked at 3 h, followed by a gradual and continuous decrease beginning at 6 h. In contrast, the other genes were expressed at later time-points. CD11b and MRP-14 transcripts were first detected at 3–6 h, and expression levels peaked at 24–48 h of combined treatment. Expression of TGM2 was also increased remarkably after treatment for 24 h. These results suggest that the mechanism that underlies ATRA and GM-CSF-induced expression of ID-1 differs from those of the other genes.


Figure 2
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  		Figure 2. Expression of ID-1, MRP-14, CD11b and TGM2, and GAPDH mRNAs in ML-1 cells treated with 10–7 M ATRA and 0.1 ng/ml GM-CSF. mRNA samples were prepared from cells at the times indicated. Northern blot analysis with 2 µg mRNA was performed as described in Materials and Methods.

Effect of signaling inhibitors on synergistic induction of gene expression and differentiation by ATRA and GM-CSF
GM-CSF uses several signaling pathways, including JAK2/STAT, MAPKs, and PI3K, for induction of its biological actions [12 13 14 15 ]. To determine which pathway is critical for synergistic gene expression induced by combined treatment with ATRA and GM-CSF, we tried to examine the effect of specific inhibitors for each pathway on synergistic gene expression induced by both reagents. We first investigated how signaling molecules known to be activated by GM-CSF are affected in ML-1 cells treated with ATRA and GM-CSF. Activation of each signal molecules was analyzed by Western blotting using antibody against p-ERK, p38, JNK, JAK2, and AKT. As shown in Figure 3A , treatment with both reagents resulted in phosphorylation (activation) of ERK2 (with faster migration on the gel) and JAK2. Activation of ERK and JAK2 became apparent at 5 and 15 min following treatment with ATRA and GM-CSF, respectively. Then, we detected that the activation of these proteins is caused by GM-CSF (Fig. 3B) . We next examined an inhibitory effect of each inhibitor on activation of ERK or JAK2 by both reagents. ML-1 cells were pretreated with PD98059 (an inhibitor of MEK) or AG490 (an inhibitor of JAK2) for 30 min before addition of ATRA and GM-CSF. Pretreatment of the cells with PD98059 or AG490 inhibited reagent-induced phosphorylation of ERK2 or JAK2, respectively (Fig. 3C) . Total protein levels of ERK2 and JAK2 were constant during treatment with these reagents. To investigate the role of ERK and Jak2 in synergistic induction of gene expression by ATRA and GM-CSF, we next examined the effect of PD98059 and AG490 on synergistic induction of expression of five genes (remarkable changed genes) by Northern blotting. As shown in Figure 4 , increased expression of the TGM2, ID-1, MRP-14, and CD11b genes by ATRA and GM-CSF was suppressed by AG490. Expression of FLAP was not affected by AG490. In contrast, PD98059 did not suppress the induction of any of the genes. These findings suggest that the synergistic induction of genes by ATRA and GM-CSF is mediated, at least in part, through activation of JAK2. We finally examined the effect of JAK2 inhibitor on induced differentiation by treatment with ATRA and GM-CSF. We measured NBT-reducing activity as an indicator of differentiation-associated characteristics. As shown in Figure 5 , AG490 inhibited the increase of NBT reduction induced by treatment with ATRA and GM-CSF.


Figure 3
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  		Figure 3. (A) Effect of cotreatment with ATRA and GM-CSF on various signal molecules. Protein samples were prepared from ML-1 cells treated with 10–7 M ATRA and 0.1 ng/ml GM-CSF for the times indicated. (B) Effect of ATRA or GM-CSF on JAK2 and ERK protein; protein samples were prepared from ML-1 cells treated with 10–7 M ATRA and/or 0.1 ng/ml GM-CSF for 15 min. The data shown are control (C), GM-CSF (G), ATRA (A), and ATRA plus GM-CSF (A+G). (C) Effect of PD98059 and AG490 on activated signal molecules. Cells were pretreated for 30 min with PD98059 (20 µM) or AG490 (50 µM) prior to stimulation for 15 or 30 min with 10–7 M ATRA and 0.1 ng/ml GM-CSF (A+G), respectively. Western blot analysis was performed as described in Materials and Methods.


Figure 4
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  		Figure 4. Effects of PD98059 and AG490 on synergistic induction of various genes by ATRA and GM-CSF. Cells were pretreated for 30 min with vehicle (0.1% DMSO), PD98059 (20 µM), or AG490 (50 µM) prior to stimulation for 24 h with 10–7 M ATRA and/or 0.1 ng/ml GM-CSF. Northern blot analysis with 2 µg mRNA was performed as described in Materials and Methods. Gene expression values were normalized to GAPDH expression, and expression levels of various genes are represented graphically as fold change versus control. The data shown are control (C), GM-CSF (G), ATRA (A), and ATRA plus GM-CSF (AG).


Figure 5
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  		Figure 5. Effects of AG490 on induction of NBT-reducing activity by ATRA and GM-CSF. Cells were pretreated for 30 min with vehicle (0.1% DMSO) or AG490 (50 µM) prior to stimulation for 24 h with 10–7 M ATRA and 0.1 ng/ml GM-CSF (A+G). NBT-reducing activity was determined as described in Materials and Methods. Values are the means ± SE of triplicate determinations.


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DISCUSSION
 
The synergistic effects of various reagents, such as hormones, cytokines, and phorbol ester, on differentiation of leukemia cells, have been reported [7 , 8 , 16 , 17 ]. However, the mechanisms that underlie the synergistic actions of these reagents remain unclear. We reported previously that the slight induction of differentiation of human myeloblastic leukemia ML-1 cells by treatment with ATRA alone is enhanced by GM-CSF [8 ]. We also showed changes in expression of several genes during this differentiation [18 19 20 ]. In the present study, we found that GM-CSF enhances expression of various ATRA-induced genes in ML-1 cells. These results suggest that the enhancement of ATRA-induced gene expression by GM-CSF led to the synergistic induction of differentiation of ML-1 cells by ATRA and GM-CSF. We propose that synergistic induction of the expression of differentiation-related genes underlies the synergistic induction of differentiation.

Of the up-regulated genes we found, several are involved in granulocytic differentiation. MRP-14 participates in activation of NADPH oxidase in neutrophils by binding to cytosolic phox proteins [21 ]. Moreover, MRP-14 is reported to function as a stimulator of neutrophil adhesion mediated by the ß2 integrin membrane-activated complex-1 [22 ]. PI3K{gamma} also regulates neutrophil chemotaxis, primarily by controlling the direction of cell migration and the intracellular colocalization of AKT and F-actin to the leading edge [23 ]. ID proteins function as inhibitors of members of the basic helix-loop-helix family of transcription factors and have been demonstrated to play an important role in regulating lymphopoiesis [24 , 25 ]. Recently, Buitenhuis et al. [26 ] have reported that ID-1 and ID-2 play an important role in regulating proliferation during granulocyte differentiation, and ID-1 blocked eosinophil differentiation and induced neutrophil development. We detected induction of expression of these genes related to granulocytic differentiation in the present study. We presume that the expression of these genes is associated with granulocytic differentiation of ML-1 cells in response to ATRA combined with GM-CSF.

It is well known that GM-CSF uses several signaling pathways to mediate its biological actions. GM-CSF activates the MAPK pathway, JAK2/STAT pathway, and PI3K intracellular signaling component via the ß subunit of the GM-CSF receptor [12 13 14 15 ]. Our results suggested that JAK2 plays a critical role in synergistic induction of gene expression in ML-1 cells treated with ATRA and GM-CSF. Regulation of gene expression by JAK2 appears to be mediated primarily by production of a DNA-binding complex containing STAT5 proteins. Moreover, Al-Shami and Naccache [27 ] suggested that stimulation of PI3K activity by GM-CSF is mediated by JAK2. In the present study, we showed that synergistic induction of four genes was suppressed by addition of the JAK2 inhibitor AG490 and that treatment with ATRA and GM-CSF did not result in stimulation of phosphorylation of AKT, which is a known, downstream effector of PI3K. Therefore, the synergistic expression of these genes may be mediated through the JAK2/STAT pathway. In addition, we need to consider the effect of inhibitor on signal pathway by ATRA, as induction of the gene by ATRA alone is also inhibited partially by addition of AG490.

We previously showed that GM-CSF induces expression of RAR{alpha}, and we suggested that GM-CSF stimulates ATRA-induced gene expression via RAR{alpha} [28 ]. Johnson et al. [29 ] reported that GM-CSF regulates the transcriptional activity of RAR in leukemia cells. These findings support the notion that synergistic induction of gene expression in ML-1 cells treated with ATRA and GM-CSF is mediated by the action of GM-CSF on RAR. Many of the genes induced by ATRA combined with GM-CSF in ML-1 cells have been shown previously to be induced by ATRA alone in various cells, for example, MRP-14 [30 ], cathepsin D [31 ], ID-1 [32 ], pleckstrin [33 ], PI3K{gamma} [34 ], c-fgr [35 ], and SLAP [36 ]. However, none of these genes showed expression mediated via RAR. Induction of the c-fgr, cathepsin D, and SLAP genes is not regulated directly by RA [35 36 37 ]. Moreover, expression of MRP-14 is regulated by transcription factors c-myb and C/EBP{alpha} [38 ], and IFI-6–16 is induced by IFN regulatory factor-1 [39 ]. These findings suggest that GM-CSF enhancement of ATRA-induced gene expression is associated, not only with the RAR-mediated signal but also with several other transcription factors and signals. To clarify the mechanism underlying the enhancement by GM-CSF, we must examine the promoter of each gene and identify related transcription factors and cofactors. Furthermore, we need to investigate differences in transcriptional regulation between genes regulated synergistically and genes not regulated in this manner.

The previous studies indicated that treatment with ATRA and GM-CSF induced the expression of several genes remarkably, including p47phox [20 ], gp91phox [20 ], and CD38 [28 ], in ML-1 cells. However, the microarray data showed alterations of 0.82-fold, 1.93-fold, and 1.88-fold in the expressions of the p47phox, gp91phox, and CD38, respectively (data not shown). Moreover, for many of the genes, the microarray analysis understimulated the fold-change seen via subsequent Northern blot analysis (Table 3 and Fig. 1 ). The reason for this is unclear; however, this result is a common finding with array expression studies [40 41 42 ] and may be related in part to differences in hybridization stringency between microarray and Nothern blot analyses.

Our results suggest that GM-CSF enhances the effect of ATRA on leukemia cells via changes in gene expression. Further investigation of the mechanism underlying this action by GM-CSF is critical to identify signals and molecules, which may serve as novel targets for reagents combined with RA. This in turn will be useful for development of differentiation-based therapies for treating various forms of leukemia, in which RA alone is not effective. We expect combination therapies to lead to improved clinical efficacies.


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ACKNOWLEDGEMENTS
 
This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sport and Culture of Japan.

Received September 7, 2005; revised June 16, 2006; accepted June 19, 2006.


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