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Originally published In Press as doi:10.1074/jbc.M307077200 on September 29, 2003

J. Biol. Chem., Vol. 278, Issue 52, 52651-52659, December 26, 2003
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Restoration of C/EBP{alpha} Expression in a BCR-ABL+ Cell Line Induces Terminal Granulocytic Differentiation*

Sigal Tavor{ddagger}, Dorothy J. Park{ddagger}, Sigal Gery, Peter T. Vuong, Adrian F. Gombart, and H. Phillip Koeffler§

From the Division of Hematology Oncology, Cedars-Sinai Medical Center, School of Medicine, UCLA, Los Angeles, California 90048

Received for publication, July 2, 2003 , and in revised form, September 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factor C/EBP{alpha} plays a critical role in the process of granulocytic differentiation. Recently, mutations that abrogated transcriptional activation of C/EBP{alpha} were detected in acute myeloid leukemia patient samples. Moreover, the progression of chronic myelogenous leukemia (CML) to blast crisis in patients was correlated with down-modulation of C/EBP{alpha}. The KCL22 cell line, derived from BCR-ABL+ CML in blast crisis, expressed wild-type C/EBP{epsilon} protein but not a functional C/EBP{alpha}, -{beta}, and -{gamma}. Restoration of C/EBP{alpha} expression in KCL22 cells triggered a profound proliferative arrest, a block in the G2/M phase of the cell cycle and a gradual increase in apoptosis. Within 3 days of inducing expression of C/EBP{alpha}, a remarkable neutrophilic differentiation of the KCL22 blast cells occurred as shown by morphologic changes, induction of expression of CD11b, primary, secondary, and tertiary granule proteins, and granulocyte colony-stimulating factor receptor. Using high density oligonucleotide microarrays, the gene expression profile of KCL22 cells stably transfected with C/EBP{alpha} was compared with that of empty vector, and we identified genes not previously known to be regulated by C/EBP{alpha}. These included the up-regulation of those genes important for regulation of hematopoietic stem cell homing, granulocytic differentiation, and cell cycle, whereas down-regulation occurred for genes coding for signaling molecules and transcription factors that are implicated in regulation of proliferation and differentiation of hematopoietic cells. Our study showed that restoration of C/EBP{alpha} expression in BCR-ABL+ leukemic cells in blast crisis is sufficient for rapid neutrophil differentiation suggesting a potential therapeutic role for ectopic transfer of C/EBP{alpha} in acute phase of CML.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factor CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha})1 has been implicated as an inhibitor of cell proliferation and a regulator of differentiation in various cell types, including adipocytes, hepatocytes, and myeloid cells. Within hematopoiesis, C/EBP{alpha} is essential for development of the neutrophil lineage (1). The C/EBP{alpha} knockout mice display a complete maturational block of the granulocytic pathway (2), and deletion of C/EBP{alpha} in the bone marrow induces accumulation of myeloblasts (3). Conditional expression of C/EBP{alpha} in U937 myelomonoblastic leukemia cells leads to their partial granulocytic differentiation over a 2-week period (4). Consistent with C/EBP{alpha} promoting neutrophilic differentiation, mutations that abrogated transcriptional activation of C/EBP{alpha} were recently found in samples of acute myeloid leukemia (AML) (5, 6). Taken together, interruption of C/EBP{alpha} function results in an early block in myeloid differentiation.

Chronic myelogenous leukemia (CML) is a clonal hematopoietic stem cell disorder caused by the BCR-ABL fusion oncogene. In the chronic phase of the disease, the leukemic cells retain the ability to differentiate into mature granulocytes; however, after a period of 3–5 years, transformation to the acute fatal stage invariably occurs characterized by aggressive proliferation of immature cells and block in differentiation. The molecular events underlying the transition from the chronic phase to the blast crisis are still poorly understood (7–12). The BCR-ABL-positive KCL22 cell line is composed of myelomonocytic blast cells established from an individual with blast transformation of CML. In contrast to normal myeloid progenitor cells, we found that KCL22 cells have no detectable expression of C/EBP{alpha} protein.

To study the possible role of C/EBP{alpha} in blastic transformation of CML, we stably transfected KCL22 cells with C/EBP{alpha} under the control of a zinc-inducible promoter. Brief ectopic expression of C/EBP{alpha} triggered these BCR-ABL-positive cells to undergo growth arrest, develop neutrophilic morphologic changes, up-regulate levels of G-CSF receptor, and express primary, secondary, and tertiary granule genes. In addition, we used high density oligonucleotide microarray analysis to identify novel C/EBP{alpha} target genes and to gain further insight into the process of leukemogenesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—All-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (RA) (Sigma, St. Louis, MO) were dissolved in 95% ethanol to a stock concentration of 10–2 mol/liter and stored at –70 °C. Hexamethylene bisacetamide (HMBA) was purchased from Sigma.

Plasmids and Transfections—The zinc-inducible C/EBP{alpha} expression vector (pMT{alpha}) was constructed by inserting a full-length human C/EBP{alpha} cDNA (a kind gift from Dr. D. G. Tenen, Harvard Institute of Medicine, Boston, MA) at the HindIII and KpnI sites of MTCB6+ (pMT) (a kind gift from F. J. Rauscher III, The Wistar Institute, Philadelphia, PA). The KCL22 cells (ATCC, Rockville, MD) were transfected with either pMT{alpha} (KCL22-pMT{alpha}) or pMT (KCL22-pMT, as a control). Both plasmids carry neomycin resistance gene as a selection marker. Using an Electro Square Porator T820 electroporation apparatus (BTX, San Diego, CA), a total of 2.5 x 107 KCL22 cells were electroporated with 30 µg of linearized plasmids at 310 V for 35 ms. Selection with G418 at 1 mg/ml was started 48 h after electroporation to obtain stably transfected cells. Multiple polyclonal and monoclonal cultures were screened for zinc-inducible C/EBP{alpha} expression by Western blot analysis.

Western Blot Analysis—Total cell lysate (30 µg) was electrophoresed on 10–20% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred by electroblotting to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA). The membranes were incubated for 1 h with rabbit polyclonal C/EBP{alpha},-{beta}, or-{gamma} antibodies (0.2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA) or C/EBP{epsilon} (13) antibody (0.9 µg/ml) followed by a secondary horseradish peroxidase-conjugated donkey anti-rabbit antibody (Amersham Biosciences). Detection was performed using the SuperSignal chemiluminescence substrate (Pierce, Rockford, IL).

RNA Isolation and Reverse Transcription-PCR—KCL22-pMT and KCL22-pMT{alpha} cells were cultured in medium supplemented with ZnSO4 (100 µM) for up to 14 days, and total RNA was harvested at different time points. Three micrograms of total RNA was treated with RNase-free DNase I (1 unit, Promega, Madison, WI) to eliminate genomic DNA contamination and reverse-transcribed using SuperScript II (Invitrogen) according to the manufacturer's protocol. Semi-quantitative RT-PCR was performed using the following conditions: an initial denaturation step at 94 °C for 5 min followed by 25–35 cycles, 94 °C for 30 s, 56 °C for 40 s, and 72 °C for 1 min. RT-PCR for 18 S was utilized as an internal control to ensure equal loading of samples. Sequences of the primers will be provided upon request. Reaction products were visualized on ethidium bromide-stained agarose gels, and images of PCR products were captured using AlphaImager 2000 Gel Documentation software.

Real-time PCR was performed to ensure quantitative nature of gene expressions either using SYBR Green I or a gene-specific TaqMan probe (Applied Biosystems, Foster City, CA). For SYBR Green protocol, RT-PCR reactions were carried out using HotStarTaq DNA polymerase (Qiagen), 50 ng of cDNA for all genes of interest (500 to 5 ng in serial dilutions for standard curves) or 1 pg for 18 S (10 to 0.1 pg for standard curve), and SYBR Green I in a 1:60,000 dilution in triplicate. Melting curve analysis was carried out as described previously (14). PCR conditions were: a 95 °C initial activation for 15 min was followed by 45 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s and fluorescence determination at the melting temperature of the product for 20 s on an ICycler detection system (Bio-Rad, Hercules, CA). Determination of C50 cycles and quantification were done as previously described (14). Similarly, real-time PCR for RIN1 gene was carried out using a Taq-Man probe according to the manufacturer's protocol. Concentrations of primers and TaqMan probes (Applied Biosystems, Foster City, CA) (sequences will be provided upon request) were 300 and 100 nM, respectively. All PCR experiments were performed multiple times using both polyclonal and monoclonal cell cultures to ensure reproducibility.

Cell Proliferation and Morphologic Analysis—KCL22-pMT and KCL22-pMT{alpha} cells (5 x 104) were grown in RPMI with 10% fetal bovine serum either with or without ZnSO4 (100 µM), and the mean number of viable cells in triplicate experiments was determined on days 0, 1, 3, and 5 using trypan blue exclusion. The cellular morphology was studied after Wright-Giemsa staining of cytospin slides.

Cell Cycle Analyses and Apoptosis Assays—Cell cycle analyses were performed on KCL22-pMT and KCL22-pMT{alpha} cells incubated for 0, 3, 6, and 9 days with ZnSO4 (100 µM). The cells were fixed in cold ethanol, stained with 50 µg/ml propidium iodide, 1 mg/ml RNase, and 0.1% Nonidet P-40 and analyzed by FACScan and CELLFit programs (BD Biosciences). Apoptosis was detected by terminal deoxynucleotidyltransferase-mediated UTP end-labeling technique using in situ Cell Death Detection kit (Roche Molecular Biochemicals) according to the manufacturer's protocol.

Oligonucleotide Microarray Expression Analysis—KCL22-pMT and KCL22-pMT{alpha} cells were cultured in media containing 100 µM ZnSO4 (Sigma) for 12 h for the induction of C/EBP{alpha}. Three independent cultures were carried out for the microarray experiments. Total RNA extracted by TRIzol was purified using the RNeasy system (Qiagen, Valencia, CA) according to the manufacturer's instruction. 8 µg of purified total RNA from each sample was used to prepare biotinylated cRNA probes, and 15 µg of labeled cRNA from each sample was used for hybridization to HuGeneFL Array (Affymetrix, Inc., Santa Clara, CA) at Microarray Core, University of California, Los Angeles. Following the hybridization, arrays were washed and stained with streptavidin-phycoerythrin and scanned on a Hewlett Packard scanner. The measured fluorescence intensity values were captured using GeneChip software (Affymetrix), and the data were normalized by global scaling to a target value of 2500 and to the average fluorescence intensity for the entire microarray (15).

Microarray Data Analysis—Data generated by GeneChip software (Affymetrix) were exported to GeneSpring software version 4.2 (Silicon Genetics, Inc., San Carlos, CA) for further analyses. Pairwise comparisons were performed to examine C/EBP{alpha} gene effect in triplicate. Gene lists were generated by selecting genes with at least 2-fold expression change and the raw intensity value of at least 1000 in the experimental sample in triplicate experiments. For -fold expression change calculations, average difference values below the detection limit of 10, including the negative expression values, were set at an arbitrary "11" to capture the genes that are not expressed in one sample but switched on in the other or vice versa. Mean -fold changes were calculated using a simple division of raw expression values between experimental sample and control. p values by Student's t test in pairwise comparisons are also reported (Tables I and II).


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  		TABLE I
C/EBP {alpha} up-regulated genes in KCL22 cells

 


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  		TABLE II
C/EBP {alpha} down-regulated genes in KCL22 cells

 
Northern Blot Analysis—Total RNA was extracted using TRIzol (Invitrogen, Gaithersburg, MD) according to the manufacturer's instructions, electrophoresed on a denaturing formaldehyde gel, and blotted in 20x SSC overnight to a nylon membrane. Blots were hybridized overnight at 42 °C in Ultra-Hyb buffer (Amersham Biosciences, Arlington Heights, IL) and [{alpha}-32P]dATP-labeled full-length C/EBP{alpha} cDNA or partial cDNA probes for CXCR4, C3aR1, annexin 1, LITAF, cyclin D2, annexin 1, BTG2, IFI15, CASP2, and RIPK1 domain-containing adaptor with death domain (CRADD), monocyte/neutrophil elastase inhibitor (ELANH2), and glyceraldehyde-3-phosphate dehydrogenase (loading control). The blots were washed twice with 2x SSC and 0.1% SDS followed by two washes with 0.2x SSC and 0.1% SDS for 20 min each. The probes were generated by PCR, and PCR products were sequenced to ensure gene specificity. Sequences of the primers used will be provided upon request.

Immunofluorescence Analysis—Cells were washed once with phosphate-buffered saline, resuspended in 50 µl of staining buffer (RPMI media with 1% fetal bovine serum, 0.1% sodium azide), and incubated with a phycoerythrin-conjugated murine monoclonal antibody against human CXCR4 (R&D, Minneapolis, MN) for 30 min at 4 °C in the dark. After incubation, cells were washed in staining buffer, fixed with 2% paraformaldehyde, and analyzed by flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of C/EBP Family Members in KCL22 Cells—Previously, we observed a lack of C/EBP{alpha} DNA binding activity in the KCL22 cells (leukemic cell line derived from a patient with CML in myeloid blast crisis) (16). Analysis of protein expression of the C/EBP family members ({alpha}, {beta}, {epsilon}, and {gamma}) by Western blotting detected expression of C/EBP{beta} and -{epsilon}, but no expression of C/EBP{alpha} and -{gamma} (data not shown). We previously showed that C/EBP{beta} in these cells had a mutation (17) and could not bind to C/EBP DNA binding sequences (16).

The lack of detectable C/EBP{alpha} protein lead us to hypothesize that restoration of C/EBP{alpha} expression would promote granulocytic differentiation of KCL22 cells. The human C/EBP{alpha} cDNA expression vector under the control of a zinc-inducible metallothionein promoter was stably transfected into KCL22 cells. The protein levels of C/EBP{alpha} were measured in cells from different clones of KCL22-pMT{alpha} cultured in either zinc-containing or -deficient media for 16 h. C/EBP{alpha} protein levels in cells from two independent clones (#1 and #2) are shown in Fig. 1A. Using real-time RT-PCR, we analyzed C/EBP{alpha} mRNA expression levels in KCL22-pMT{alpha} cells (clone #1) and in purified normal CD34+ bone marrow cells. Results showed that levels of C/EBP{alpha} mRNA are 50-fold higher in the KCL22-pMT{alpha} cells compared with normal bone marrow cells (Fig. 1B).



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  		FIG. 1.
C/EBP{alpha} expression in stably transfected KCL22 cells. A, Western blot analysis for C/EBP{alpha} from whole cell lysates of KCL22 transfected with empty vector (pMT) and C/EBP{alpha} (pMT{alpha}; clones #1 and #2). The cells were grown in media with (+) and without (–) zinc (100 µM) for 16 h, and Western blots were hybridized with either C/EBP{alpha} or glyceraldehyde-3-phosphate dehydrogenase (control for protein loading) antibodies. B, real-time RT-PCR analysis of C/EBP{alpha} from pMT and pMT{alpha} (clone #1) cells with (+) and without (–) zinc (100 µM, 16 h). RNA from normal human CD34+ bone marrow cells (BM) was used as a control. The results are expressed in arbitrary units as a ratio of C/EBP{alpha} transcripts/18 S transcripts.

 
ATRA, 9-cis-RA, and HMBA Inhibited Proliferation of KCL22 Cells without Triggering Differentiation—We examined whether KCL22 cells have the ability to differentiate by chemical reagents that are known to trigger differentiation in other AML cell lines. The KCL22 cells were cultured for up to 14 days in the presence of either ATRA (10–7 to 10–5 mol/liter), 9-cis-RA (10–7 to 10–5 mol/liter), or HMBA (4 mmol/liter). Cell proliferation was markedly inhibited by the different treatments in a dose-dependent manner (Fig. 2). However, none of the treatments led to the differentiation of KCL22 cells as measured by morphologic changes of differentiation or up-regulation of expression of neutrophil-specific genes (collagenase and lactoferrin; data not shown).



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  		FIG. 2.
Inhibition of cell proliferation of KCL22 cells by ATRA, 9-cis-RA, and HMBA. KCL22 cells (1 x 104) were cultured either in the absence (No treatment) or presence of 9-cis-RA (10–7 to 10–5 M) (A), ATRA (10–7 to 10–5 M) (B), or HMBA (4 mM) (C). Viable cells were counted at days 0, 1, 3, 5, and 7 after trypan blue exclusion to examine the rate of cell proliferation. Three independent experiments were performed, and the mean values ± S.E. are shown.

 
C/EBP{alpha} Expression in BCR-ABL-positive Cells Rapidly and Dramatically Induced Granulocytic Differentiation—Overexpression of C/EBP{alpha} in the 32D cl3 myeloblasts was shown to trigger neutrophil differentiation (18). However, cotransfection of BCR-ABLp210 in the murine nonmalignant 32D cl3 cells prevented differentiation of these cells (18). Using KCL22 cells stably transfected with zinc-inducible C/EBP{alpha} expression plasmid, we addressed the question of whether restoring C/EBP{alpha} expression can trigger differentiation in human CML blast cells that express BCR-ABL. Multiple clones of KCL22-pMT and KCL22-pMT{alpha} cells were grown in media either with or without ZnSO4 (100 µM) for up to 14 days. Cell morphology was examined daily by cytospin and staining with Wright-Giemsa. Most remarkably after only 3 days in the presence of ZnSO4, many of the KCL22-pMT{alpha} cells morphologically matured to myelocytes, metamyelocytes, bands, and neutrophils (>90%) (Fig. 3A). Most of the cells had a reduction in their nuclear to cytoplasmic ratio with more condensed nuclear chromatin, and many had segmented nuclei typical of mature granulocytes. In contrast, KCL22-pMT cells grown in the presence of ZnSO4 (Fig. 3A) as well as KCL22-pMT{alpha} and KCL22-pMT cells grown without ZnSO4 had the characteristic appearance of myeloblasts, indistinguishable from untransfected KCL22 cells (data not shown).



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  		FIG. 3.
Conditional expression of C/EBP{alpha} rapidly induces granulocytic maturation. A, pMT (KCL22 transfected with empty vector) and pMT{alpha} (C/EBP{alpha} transfected KCL22 clones #1 and #2) were cultured in media containing ZnSO4 (100 µM) for induction of C/EBP{alpha} expression. Shown are representative morphologic changes seen at 3 day of zinc incubation (Wright-Giemsa-stained cytospin slides under light microscopy). B, total RNA was harvested from KCL22-pMT{alpha} cells before and after stimulation with ZnSO4 (100 µM) for 3 and 7 days. Expression of genes associated with the various neutrophil granules as well as CD11b were assessed by RT-PCR. a, amplification products were gel-separated and stained with ethidium bromide in semiquanti tative RT-PCR, or b, real-time RT-PCR of selected genes 3 days after either without (–) or with ({alpha}) zinc induction of C/EBP{alpha} in KCL22 (pMT{alpha}). C, pMT{alpha} and pMT cells were cultured in the presence of ZnSO4, harvested at 0, 5, 12, 24, and 50 h, and total RNA was prepared. RNA from normal human neutrophils (N) was used as a positive control. a, ethidium bromide staining of semiquantitative RT-PCR products of G-CSF receptor (G-CSFR, 25 and 32 cycles). b, real-time RT-PCR analysis of G-CSF receptor (G-CSFR) expression at 0, 5, and 12 h of stimulation with zinc, as well as 0 and 50 h of stimulation with zinc and positive control (N). The results are expressed in arbitrary units as a ratio of G-CSFR transcripts/18 S transcripts.

 
Induction of expression of C/EBP{alpha} also induced a remarkable up-regulation of neutrophil-specific genes in the KCL22 cells. KCL22-pMT and KCL22-pMT{alpha} cells were cultured either with or without zinc, and total RNA was extracted daily and analyzed by RT-PCR. In the absence of zinc, the transcript levels of MPO, collagenase, lactoferrin, CAP18, gelatinase, and CD11b were negligible or very low. Within 3 days of culture in zinc-containing media, KCL22-pMT{alpha} cells dramatically increased their levels of each of these granulocyte-related genes (Fig. 3B). In addition, the mRNA expression level of granulocyte colony-stimulating factor receptor (G-CSFR), a known direct target of C/EBP{alpha}, also markedly increased in the presence of zinc (Fig. 3C). Real-time RT-PCR was also performed on selected genes to ensure quantitative induction of gene expressions (Fig. 3, B and C).

C/EBP{alpha} Inhibited Cell Growth and Induced Apoptosis of KCL22 Cells—The proliferative rate of KCL22-pMT and KCL22-pMT{alpha} cells was evaluated by daily viable cell counts. The KCL22-pMT cells proliferated rapidly regardless of either the presence or absence of zinc (Fig. 4A and data not shown). In contrast, the proliferative rate of KCL22 cells induced to express C/EBP{alpha} was remarkably flat (Fig. 4A).



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  		FIG. 4.
C/EBP{alpha} slows proliferation in KCL22 CML blast cells. A, KCL22 cells (5 x 104/ml) stably transfected with either the empty expression vector (pMT) or the C/EBP{alpha} construct (pMT{alpha}), were grown in media with ZnSO4 (100 µM) and counted at days 0, 1, 3, and 5 after trypan blue exclusion. The results represent the mean ± S.D. of three experiments. Experiments with different clones of pMT{alpha} showed similar results (data not shown). B, KCL22-pMT and KCL22-pMT{alpha} cells were cultured with zinc (100 µM) for 3 days, harvested, stained with PI, and analyzed by flow cytometry for cell cycle analysis. The percentage values represent the mean of three independent experiments.

 
Effect of C/EBP{alpha} ectopic expression on the cell cycle of the KCL22 CML cells was examined. After 3 days of culture in the presence of zinc, the KCL22-pMT{alpha} cells had an increased number of cells (30.2% ± 3.7) in the G2/M phase and a decreased number of cells (25.2% ± 3.8) in the S phase of the cell cycle as compared with KCL22-pMT cells containing the empty vector (14.4% ± 1.7 (G2/M) and 39.6% ± 3 (S phase)) (Fig. 4B). Furthermore, increased apoptosis was observed in KCL22-pMT{alpha} cells at 6 days of culture in zinc-supplemented media (36 ± 4%), in contrast to the low level of apoptosis in the KCL22-pMT cells (4 ± 2.1%) cultured under identical conditions (Fig. 5, A and B).



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  		FIG. 5.
Restoration of C/EBP{alpha} expression in KCL22 cells induces apoptosis. KCL22-pMT and KCL22-pMT{alpha} were cultured for up to 6 days in the presence of zinc and were tested by terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay for apoptosis on days 0, 2, 3, 5, and 6. A, shown are representative images at 6 days using fluorescence microscopy. B, the percent apoptotic cells were quantified by counting the positive cells using fluorescent microscopy. The results represent the mean percent apoptotic cells ± S.D. in two independent experiments.

 
Expression Analysis of C/EBP{alpha}-regulated Genes—High density oligonucleotide array analysis was performed to identify potential C/EBP{alpha} target genes during neutrophil differentiation. We compared global gene expression in KCL22-pMT and KCL22-pMT{alpha} cells after 12-h stimulation with 100 µM ZnSO4 in triplicate experiments. After 12–16 h of stimulation with zinc, the level of C/EBP{alpha} protein was prominent in the KCL22-pMT{alpha} cells (Fig. 1 and data not shown). In addition, RT-PCR analysis demonstrated that by 6–12 h, the level of transcripts for G-CSFR, a known direct target of C/EBP{alpha}, began to increase significantly (Fig. 2C) (19). Therefore, we chose the 12-h time point to harvest the RNA to enrich for genes potentially regulated directly by C/EBP{alpha}.

To compile a list of C/EBP{alpha}-responsive genes, we selected those genes that were either induced or repressed at least 2-fold and had a raw intensity value of at least 1000 in the experimental sample in all three independent experiments. Of 5600 genes interrogated, 52 of them were induced, and 33 genes were repressed by ectopic expression of C/EBP{alpha} in the KCL22 CML blast crisis leukemia cells (Tables I and II).

We classified these genes into different functional groups (Tables I and II). Genes up-regulated by C/EBP{alpha} included those involved in chemotaxis and cell motility (CXCR4, TIM) and response by myeloid cells to inflammatory stimuli (C3AR1, LITAF, PTX3, annexin 1, and S100A9). In general, these genes are integral either to commitment to the myeloid lineage or important in granulocyte function. Furthermore, genes that play important roles in apoptosis (BCL2A1 and BIK) and cell cycle regulation (cyclin D2, BTG1, BTG2) were up-regulated by C/EBP{alpha}. For genes down-regulated by C/EBP{alpha}, a large group coding for transcriptional factors (GATA2, MYC, HKR3, LYL1, and FKHR), interferon-induced proteins (IFI27, OAS2, G1P3, and IFI15), and cell signaling (connexin 43 and RIN1) were down-regulated in the KCL22 CML cells.

The reliability of the chip data was confirmed by repeating the zinc induction of the cells and isolating the RNA and examining it for the expression of genes by Northern blot analysis (Fig. 6A). In the majority of cases, the Northern blot analyses confirmed the microarray results, including the markedly enhanced expression of CXCR4, cyclin D2 (CCND2), BTG2, C3aR1, LITAF, and annexin 1 as well as the decreased expression of CRADD and ELANH2. In addition, expression of CXCR4 protein was analyzed using FACS and was found to be up-regulated in the KCL22-pMT{alpha} as compared with the KCL22-pMT cells (Fig. 6B). The enhanced levels of protein expression paralleled the changes of RNA expression identified by microarray analysis.



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  		FIG. 6.
Northern blots and FACS analysis of C/EBP{alpha} target genes. Expression data from the oligonucleotide microarray analyses were confirmed by Northern blotting. Total RNA harvested from pMT and pMT{alpha} KCL22 cells cultured in the presence of zinc-supplemented media (100 µM) for 12 h was used for Northern blot analysis. We examined selected genes that had at least 2-fold change in expression levels and had a raw intensity value of at least 1000 in the experimental sample. A, Northern blot analysis of C/EBP{alpha}-up-regulated genes. In B: a, Northern blot analysis of C/EBP{alpha}-down-regulated genes. b, real-time PCR analysis of RIN1 expression using TaqMan probe either without (–) or with ({alpha}) zinc induction (12 h) of C/EBP{alpha} KCL22-pMT{alpha} cells. C, FACS analysis of CXCR4 and Fc{gamma}RI surface expression on pMT and pMT{alpha} cells at 24 h of exposure to ZnSO4 (100 µM).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Disruption of C/EBP{alpha} gene was found in up to 8% of AML samples. These alterations included deletions, insertions, and point mutations that impaired the function of C/EBP{alpha} (5, 6). Studies have also suggested that the AML-1/ETO fusion product of [t(8;21)] myeloid leukemia-suppressed expression of C/EBP{alpha}, and the PML/RAR{alpha} fusion product of acute promyelocytic leukemia [t(15;17)] might trap C/EBP{alpha} in the cytoplasm preventing it from functioning normally in the nucleus (20, 21). These observations indicate that alteration of this crucial myeloid transcription factor may have a role in the pathogenesis of AML. Recently, Perrotti et al. (12) showed that, in blast crisis of CML, C/EBP{alpha} protein expression is suppressed by hnRNPE2. We hypothesized that the BCR-ABL-positive CML blast crisisderived cell line KCL22, which does not express detectable levels of C/EBP{alpha} protein, might have, as a result, a block in differentiation. To test this hypothesis, KCL22 cells were stably transfected with a zinc-inducible C/EBP{alpha} construct. Dramatic morphologic changes consistent with terminal granulocytic differentiation occurred within 3 days of inducing expression of C/EBP{alpha}. These morphologic changes occurred concomitantly with the appearance of primary (MPO), secondary (collagenase, lactoferrin, and CAP18), and tertiary (gelatinase) granule gene expression as well as an arrest in cell proliferation and induction of apoptosis. Treatment of KCL22 cells with various differentiation-inducing reagents did not trigger differentiation of these cells in the absence of C/EBP{alpha} expression. Taken together, we showed that ectopic expression of C/EBP{alpha} in BCR-ABL+, C/EBP{alpha}-deficient cells, is sufficient for their dramatic induction of terminal granulocytic differentiation.

The level of C/EBP{alpha} mRNA in the C/EBP{alpha}-induced KCL22 cells was significantly higher compared with levels of C/EBP{alpha} in normal myeloid progenitor cells. Nonetheless, the results presented in this study probably reflect the physiologic effects of C/EBP{alpha}, because they agree with pervious studies performed in animal models and CML patients, suggesting that loss of C/EBP{alpha} expression contributes to transformation (2, 12). Interestingly, ectopic expression of C/EBP{alpha} in U937 cells led to granulocytic differentiation at 17 days (4, 22); in stark contrast, only 3 days were needed for terminal maturation of C/EBP{alpha}-induced KCL22 CML blast cells. The high induction levels of C/EBP{alpha} expression in the stably transfected KCL22 may partly explain the rapid differentiation observed in these cells.

To elucidate the molecular events that could lead to such a rapid and terminal differentiation and to discover potential new C/EBP{alpha} targets genes, we utilized oligonucleotide microarrays. These provided a powerful tool to assess the alteration of expression of thousands of genes in a very controlled environment. Analysis of our microarray data identified many potential new C/EBP{alpha} target genes that had previously been implicated to play a role in regulating mature neutrophil function, including chemotaxis, degranulation, phagocytosis, adhesion, and response to inflammatory mediators. Many of them were not identified in prior studies involving overexpression of C/EBP{alpha} (4, 22). This may reflect that we utilized a BCR-ABL+ cell line that completely lacks C/EBP{alpha} and C/EBP{gamma} expression and has a mutation of C/EBP{beta} (17). Thus, this cell line affords the opportunity to dissect functional activities of the various members of the C/EBP family.

A prominent group of up-regulated genes are involved in the modulation and structure of the cytoskeleton such as CXCR4 and TIM. The chemokine receptor CXCR4, which is also a coreceptor for the human immunodeficiency virus, controls stem cells migration, homing, and egression from bone marrow in response to its ligand, the stroma-derived factor-1 (SDF-1) (23, 24). In the KCL22 cell line, CXCR4 mRNA and protein were undetectable at baseline, and a prominent increase in CXCR4 expression occurred after induction of C/EBP{alpha} expression in these cells. A previous study showed a significantly lower expression of CXCR4 protein in CML-derived bone marrow CD34+ cells as compared with those from normal bone marrow progenitors and those from CML patients treated with alpha interferon and STI571 treatment (25). This may relate to effects of hnRNPE2 on C/EBP{alpha}. In light of the growing evidence that CXCR4 and similar proteins (26, 27) are critical for myeloid trafficking and migration of metastatic tumor cells, a further understanding of their regulation by C/EBP{alpha} will be important.

Among the down-regulated genes by C/EBP{alpha}, many encode transcription factors important for control of proliferation and differentiation of various tissues. The GATA1 and GATA2 transcriptional factors are expressed in erythroid, mast cell, megakaryocytic lineage, and early progenitor cells. Enforced expression of either factor in 416B, an early myeloid cell line, blocked their myeloid differentiation and induced their megakaryocytic differentiation (28). Notch1 inhibits myeloid differentiation in 32D mouse progenitor cells by sustained GATA2 expression (29). Furthermore, GATA2 interacts with PU.1 and represses its transcriptional activity, including enhancement of myeloid differentiation (30). These studies demonstrated that suppression of GATA2 is essential for myeloid differentiation. Here, we showed that ectopic expression of C/EBP{alpha} in KCL22 cells down-regulated the GATA2 transcript level by 2.6-fold. Further studies of the role that C/EBP{alpha} plays in the regulation of GATA2 expression will provide a better understanding in the involvement of lineage-specific transcription factors in mediating differentiation.

The MYC is a basic helix-loop-helix leucine zipper protein that dimerizes with its partner MAX, and the MYC-MAX heterodimer helps to induce differentiation (31). In contrast, overexpression of MYC, as is seen in many tumors, promotes proliferation (32). In addition, cells transformed by BCR-ABL have high levels of MYC, whereas the overexpression of a dominant negative mutant of MYC suppresses transformation (33). Results obtained using the C/EBP{alpha}-inducible U937 myeloid cell line suggested that MYC expression is decreased by C/EBP{alpha} blocking the transcriptional activity of E2F, which regulates MYC expression (22). In the present study, we found that induction of C/EBP{alpha} expression in the KCL22 CML blast cells was associated with repression of MYC expression (3-fold). This suggests that C/EBP{alpha} could inhibit proliferation using a pathway that intersects with the secondary signals stimulated by BCR-ABL. The up-regulation of MYC by BCR-ABL is an important factor contributing to blocking myeloid differentiation. Therefore, the down-regulation of MYC expression by C/EBP{alpha} is probably an important event promoting the differentiation of KCL22.

The chimeric oncogene BCR-ABL depends on the ABL-encoded tyrosine kinase activity (34). RIN1 protein can interact with different signaling molecules, including Ras and c-ABL, and is also a substrate of BCR-ABL. RIN1 mRNA was down-regulated (2.4-fold) after the expression of C/EBP{alpha} in KCL22. Our data, together with the finding that overexpression of RIN1 increased the leukemogenic activity of BCR-ABL in mice (35) suggest the central role of RIN1 signaling in CML pathogenesis.

In summary, our data indicate that restoring C/EBP{alpha} expression in KCL22 cells is sufficient to induce rapid granulocytic maturation. In addition, we have identified numerous potential novel target genes and pathways regulated by C/EBP{alpha}. It will be necessary to determine if the regulation of these genes is a direct or indirect effect of C/EBP{alpha} or due to the induction of differentiation of these cells. At the same time that this manuscript was submitted, Perrotti et al. (12) found low or undetectable levels of C/EBP{alpha} protein in the BM mononuclear cells from patients with CML blast crisis, which was in contrast to cells during the chronic phase of the disease, which had C/EBP{alpha}. If the C/EBP{alpha} gene is mutated or its protein expression is suppressed by interaction with hnRNPE2 (12), we hypothesize that the loss of normal C/EBP{alpha} expression may contribute to transformation. Understanding the role C/EBP{alpha} or its downstream targets in blast crisis cells of CML in conjunction with developing new delivery systems to place C/EBP{alpha} into these cells, may provide novel therapeutic approaches to this disease.


   FOOTNOTES
 
* This work was supported in part by a Grant-in-Aid from the Israel Humanitarian Fund Research (to S. T.) as well as by the National Institutes of Health, the Joseph Troy Fund, the Ko-So Foundation, and the Parker Hughes Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both authors contributed equally to this work. Back

§ Holds the Mark Goodson endowed chair for Cancer Research. To whom correspondence should be addressed: Cedars-Sinai Medical Center, UCLA School of Medicine, 8700 Beverly Blvd., Rm. 5433, Los Angeles, CA 90048. Tel.: 310-423-4502; Fax: 310-423-0443; E-mail: koeffler{at}cshs.org.

1 The abbreviations used are: C/EBP{alpha}, CCAAT/enhancer binding protein {alpha}; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; G-CSF, granulocyte colony-stimulating factor receptor; G-CSFR, G-CSF receptor; ATRA, all-trans-retinoic acid; RA, 9-cis-retinoic acid; HMBA, hexamethylene bisacetamide; RT, reverse transcription; FACS, fluorescence-activated cell sorting; MPO, myeloperoxidase. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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