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NDRG1 contributes to retinoic acid-induced differentiation of leukemic cells
Leukemia Research 33 (2009) 1108–1113
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Leukemia Research journal homepage: www.elsevier.com/locate/leukres
NDRG1 contributes to retinoic acid-induced differentiation of leukemic cells Su Chen a,1 , Yu-Hui Han a,1 , Ying Zheng a , Meng Zhao b , Hua Yan a , Qiao Zhao a , Guo-Qiang Chen a,b , Dao Li a,∗ a Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine, No. 280, Chong-Qing South Road, Luwan, Shanghai 200025, China b Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences-Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, China
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Article history: Received 21 August 2008 Received in revised form 17 October 2008 Accepted 18 October 2008 Available online 28 November 2008 Keywords: NDRG1 Leukemia Differentiation ATRA C/EBP PU.1
a b s t r a c t N-Myc downstream-regulated gene 1 (NDRG1) protein has been shown to be up-regulated during leukemic cell differentiation induced by some differentiation-inducing agents such as all-trans retinoic acid (ATRA). However, the potential role of up-regulated NDRG1 in the event is greatly unknown. In this work, we show that inducible NDRG1 expression can drive leukemic U937 cells to undergo differentiation, while the knock-down of NDRG1 expression by specific small interfering RNA significantly antagonizes ATRA-induced differentiation of leukemic cells, proposing the role of NDRG1 in leukemic cell differentiation. Furthermore, our work shows that CCAAT/enhancer-binding protein beta (C/EBP) and PU.1, which are important hematopoiesis-related transcription factors, may act as downstream effectors of NDRG1 in leukemic cell differentiation. Taking together, this study provides direct evidence for the role of NDRG1 protein in myeloid leukemic cell differentiation. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction N-Myc downstream-regulated gene (NDRG) belongs to a family of closely related genes that are down-regulated by c-myc or the N-Myc/Max complex. The family includes at least four members, respectively, named NDRG1-4 [1–3]. NDRG1, also known as Drg1, Cap43, RTP/rit42 and Proxy-1, is expressed as a 43-kDa protein composed of 394 amino acids. It was first recognized as a gene whose mutation was linked to a demyelinating neuropathy, and which mapped to human chromosome 8q24. NDRG1 is highly conserved among multicellular organisms, and is expressed ubiquitously in tissues. It has been reported that NDRG1, the expression of which can be induced by various cellular stress signals, is involved in many normal and pathologic cellular activities, such as cellular responses to heavy metals, hypoxia, DNA damage, proliferation and growth arrest, neoplasia, tumor progression and metastasis [4]. The regulation of NDRG1 expression during cancer cell differentiation is also explored. For example, NDRG1 protein is induced during the in vitro differentiation of the HT29-D4 colon carcinoma cell line [5], androgen-induced differentiation of the prostatic adenocarcinoma cell line LNCaP [6], calcium switch or serum starvation-induced
∗ Corresponding author. Fax: +86 21 64154900. E-mail address: [email protected] (D. Li). 1 These authors should be considered to contribute equally to this work. 0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.10.016
differentiation of keratinocytes [7], and the maturation of mouse bone marrow-derived mast cells into cells with a connective tissue mast cell-like phenotype induced by the presence of stem cell factor and coculturing with fibroblasts [8]. Additionally, NDRG1 knock-out mice exhibit abnormal peritoneal mast cells [9]. Acute myeloid leukemia (AML), a class of prevalent hematopoietic malignancies, is characterized by complete or partial blockage at different stages of the differentiation of myeloid progenitor cells, together with deregulated proliferation and a survival advantage of hematopoietic progenitors. These pathophysiologic changes have been attributed to the acquired genetic abnormality, especially to different types of specific reciprocal chromosome translocations [10,11]. Accordingly, differentiation induction is a potentially attractive strategy for AML therapy. For this strategy, a typical example is the successful application of differentiation-inducing agent all-trans retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL), a unique subtype of AML. Hence, it has been attracting great attentions to understand the mechanisms by which ATRA and other agents induce leukemic cell differentiation, and significant progress has been made [12,13]. Using a differential display method to identify differentiation-related genes in human myelomonocytic leukemic U937 cells, Piquemal et al. [14] found that NDRG1/Drg1 expression was up-regulated by the differentiation-inducing agents phorbol-12-myristate-13acetate (PMA), ATRA and 1,25-(OH)2 vitamin D3 . However, the possible role of the up-regulated NDRG1 protein in leukemic cell
S. Chen et al. / Leukemia Research 33 (2009) 1108–1113
differentiation remains unclear. In this study, we find that inducible NDRG1 expression can drive leukemic U937 cells to undergo differentiation, while ATRA-induced differentiation of leukemic cells is significantly antagonized by the knock-down of NDRG1 expression by specific small interfering RNA (siRNA), proposing the role of NDRG1 in leukemic cell differentiation. Furthermore, CCAAT/enhancer-binding protein beta (C/EBP) and PU.1, the important transcriptional factors for hematopoiesis, may act as downstream effectors of NDRG1 in leukemic cell differentiation.
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2.4. Design and transfection of siRNA Three pairs of complementary oligonucleotides against NDRG1 were synthesized by Invitrogen (Shanghai, China), annealed and ligated into pSilencer 3.1-H1-neo vector (Ambion, Austin, TX, USA). Target sequences of siRNA against NDRG1 were 5 -GCA TTA TTG GCA TGG GAA C-3 for siRNA #1, 5 -ACT ATT GTG CAC AAG TCT T-3 for siRNA #2 and 5 -TAG TGA CAT GCA GGC ACCT-3 for siRNA #3. These vectors and negative controls (NC) were transfected into U937 cells using the Bio-Rad gene-Pulser II (Bio-Rad) with square-wave electroporation at 0.17 kV and 960 F. Forty-eight hours later, 1000 g/ml of G418 (Calbiochem, Germany) were added to the medium and stable transformants were selected by testing targeted proteins by Western blot.
2. Materials and methods 2.5. Western blot 2.1. Cell lines and treatment The human promonocytic leukemic cell line U937 and APL cell line NB4 were cultured in RPMI 1640 medium (Sigma–Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT) in 5% CO2 /95% air humidified atmosphere at 37 ◦ C. U937T cells (kindly provided by Dr. D.G. Tenen at Harvard Institute of Medicine, Harvard Medical School, Boston, MA) were cultured in RPMI1640 medium supplemented with 10% FBS, 1 mg/ml tetracycline (Sigma–Aldrich) and 0.5 mg/ml puromycin (Sigma–Aldrich). For experiments, cells were originally seeded at 2.0–5.0 × 105 cells/ml, and were treated with or without ATRA, PMA and dimethyl sulfoxide (DMSO) (Sigma–Aldrich).
Total cellular protein extracts were loaded onto an 8–10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis, and then transferred to ECLnitrocellulose (Amersham, Buckinghamshire, UK). After blocking with 5% nonfat milk in Tris-buffered saline, membranes were incubated with antibodies against human NDRG1 (Zymed, CA), C/EBP␣ (Cell Signaling Technology, Beverly, MA), C/EBP and PU.1 (Santa Cruz Biotechnology, CA) as well as -tubulin monoclonal antibody (Oncogene, CA) or actin (Cell Signaling) at 4 ◦ C overnight, followed by HRPlinked secondary antibody (Cell Signaling) for 1 h at room temperature. Detection was then performed by chemiluminescence phototope-HRP kit, according to the manufacturer’s instructions (Cell Signaling).
2.2. Cell differentiation assay
3. Results
For cell morphology, cells were collected onto slides by cytospin (Shandon, Runcorn, UK), and examined by microscope after Wright’s staining. Cell differentiation-related antigens CD11c and CD14 were measured by flow cytometry (Beckman-Coulter, Miami, FL) as described previously [15].
3.1. NDRG1 protein is up-regulated during ATRA, PMA and DMSO-induced differentiation of leukemic cells
2.3. Establishment of U937T transformants with inducible NDRG1 expression Plasmid pBI-NDRG1-EGFP was generously provided by Dr. P. Liang [16]. EGFP and NDRG1 were located on opposite sides of the tetracycline (Tet) response element in the plasmid, and were controlled by tetracycline. To generate a cell line with stable inducible expression of NDRG1 protein, pBI-NDRG1-EGFP and pTRE2hyg (BD Biosciences, San Diego, CA) with a eukaryotic selection marker were co-transfected into U937T cells, which were stably transfected with a pUHD-tTA (tetracycline responsive transcription activator) under the control of a tetracycline-inducible promoter. To this aim, U937T cells were resuspended at 5 × 107 cells/ml in 200 l of RPMI 1640 medium without FBS. Eight micrograms of linearized pBI-NDRG1-EGFP and 2 mg of pTRE2hyg were mixed and transferred to electroporation cuvettes with a 0.4 cm gap (Bio-Rad, Hercules, CA). Electroporation was performed using a Gene-Pulser II (BioRad) at 0.17 kV and 960 F. The samples were then transferred to complete RPMI 1640 medium containing 1 g/ml tetracycline and 0.5 g/ml puromycin and incubated at 37 ◦ C in 5% CO2 /95% air humidified atmosphere. Twenty-four hours later, 500 g/ml of hygromycine (BD Biosciences) was added to the medium. Following tetracycline withdrawal, positive polyclonal populations were initially evaluated by fluorescence microscopy (Olympus BX-51, Olympus Optical, Japan) for EGFP and then for expression of NDRG1 protein. The cells were maintained in RPMI 1640 medium supplemented with 10% FBS, with 1 g/ml tetracycline, 0.5 g/ml puromycin, and 500 g/ml of hygromycine. To test the inducibility of NDRG1 expression, the transfected cells were incubated in the presence or absence of 1 g/ml tetracycline.
Leukemic cell lines U937 and NB4 were treated with the appropriate concentrations of the differentiation-inducing agents ATRA (10−7 M), PMA (10−7 M) and DMSO (1%) for 3 days. As described previously [17], these treatments significantly induced cell differentiation, as evaluated by morphologic examination and flow cytometry assay for CD11c and CD14 antigens (data not shown). In consistence with a previous report [14], ATRA and PMA significantly increased NDRG1 protein expression in both NB4 and U937 cells (Fig. 1A). DMSO also produced a similar but weaker effect on NDRG1 expression, which was paralleled to its weaker differentiation-inducing ability in these two cell lines (data not shown). We also tested the time-course effect of ATRA on NDRG1 expression. As depicted in Fig. 1B, increased NDRG1 protein expression was detected before 12 h and its level was further increased at 24 h after ATRA treatment. 3.2. NDRG1 induction triggers differentiation of myeloid leukemic cells The above observation suggests that increased NDRG1 protein expression is an early event during ATRA-induced differentiation.
Fig. 1. Effects of three differentiation-inducing agents on NDRG1 expression in leukemia cell lines. NB4 and U937 cells were treated with 10−7 M ATRA, 10−7 M PMA and 1% DMSO for 3 days (A) or 10−7 M ATRA for hours as indicated (B). NDRG1 was detected by Western blots with -tubulin as a control. All experiments were repeated at least three times with the same results.
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Fig. 2. NDRG1 induction triggers differentiation of myeloid leukemic cells. (A) NDRG1-transfected U937T cells were treated with or without tetracycline (tet) for days as indicated, and NDRG1 protein was detected by Western blots. (B and C) After incubation in medium with (+) or without (−) tet for 4 days, U937T cells were collected onto slides, stained by Wright’s staining and observed under a microscope (B, magnification ×1000). CD11c and EGFP were detected by flow cytometry (C). All experiments were repeated at least three times with the same results.
To investigate the potentially direct role of NDRG1 in the differentiation of myeloid leukemic cells, an inducible NDRG1-expressing U937T cell line was generated using a tetracycline-off system. pBINDRG1-EGFP and pTRE2hyg were co-transfected into U937T cells,
which contain stably transfected pUHD-tTA whose expression is under the control of tetracycline. It should be pointed out that U937 cells were selected because transfection is more difficult in NB4 cells, although the clinical application of a differentiation
Fig. 3. Suppression of NDRG1 expression by siRNA antagonizes ATRA-induced differentiation of U937 cells. (A) U937 cells were stably transfected with siRNA #1–3 against NDRG1 or negative control vector (NC), and NDRG1 protein was detected by Western blot with -tubulin as a loading control. (B and C) U937 siRNA #2 and NC cells were treated with or without 0.1 M ATRA for 48 h. Cells were collected onto slides by cytospin, stained by Wright’s staining and observed under a microscope (B, magnification ×1000) or CD11c+ cells were measured by flow cytometry (C). The values represent mean ± S.D. of triplicate independent experiments, which were repeated more than three times with the same results.
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Fig. 4. NDRG1 regulates expression of PU.I and C/EBP proteins. U937 siRNA #2 and NC cells were treated with 0.1 M ATRA for indicated hours (A) or NDRG1 transfected U937T cells and control cells were incubated for days, as indicated, after removal of tetracycline (B). The indicated proteins were detected, using -tubulin as a loading control. All experiments were repeated at least three times with the same results.
therapeutic approach is best described in APL. As shown in Fig. 2A, NDRG1 protein began to increase at day 2 and reached a maximum at day 4 after tetracycline removal. When NDRG1 had been induced for 4 days, U937T cells exhibited differentiation-related morphological changes such as reduced cell size, condensed chromatin and decreased nucleus/cytoplasm ratio, with smaller and distorted nuclei (Fig. 2B). Furthermore, NDRG1 induction remarkably increased CD11c expression, indicating that NDRG1 induction can induce U937 cells to undergo differentiation (Fig. 2C). 3.3. Suppression of NDRG1 expression by siRNA antagonizes ATRA-induced differentiation of U937 cells Three pairs of siRNAs against NDRG1 mRNA and a NC siRNA were transfected into the parental U937 cells. After selection with G418, we found that siRNA #2 was the most effective one against NDRG1. As shown in Fig. 3A, this siRNA significantly suppressed expression of NDRG1 protein. Intriguingly, the silencing of NDRG1 expression by siRNA #2, but not by the other two ineffective siRNAs and NC transfection, significantly antagonized ATRA-induced differentiation of U937 cells, as assessed by morphology (Fig. 3B) and decreased percentage of CD11c-positive cells (Fig. 3C). These results further suggest a contribution of NDRG1 to the ATRA-induced differentiation of myeloid leukemic cells. 3.4. NDRG1 contributes to regulation of C/EBPˇ and PU.1 expression Finally, we examined the possible effects of NDRG1 on the expression of C/EBP␣, C/EBP and PU.1, three important transcrip-
tional factors for hematopoiesis [10,18,19]. To this end, U937 cells with siRNA #2 or NC transfection were treated with 10−7 M ATRA for varying lengths of time, and the expression of these proteins was detected, using -tubulin as a loading control. In line with previous reports [20,21], ATRA treatment up-regulated expression of C/EBP and PU.1 but not C/EBP␣ protein. More interestingly, suppression of NDRG1 expression by specific siRNA significantly inhibited both basal and ATRA-up-regulated expression of C/EBP and PU.1 (Fig. 4B). Conversely, induction of NDRG1 protein in U937T transformants after tetracycline removal significantly increased the expression of PU.1 and C/EBP proteins. These results indicate that NDRG1 contributes to the regulation of C/EBP and PU.1 expression. 4. Discussion It is well known that NDRG1 expression is regulated by stress signals and by the cell growth and differentiation-related environment [1]. We previously compared the global protein expression profiles of human leukemic U937 cells incubated in air and under 2% O2 , or treated with the hypoxia-mimetic agent cobalt chloride (CoCl2 ), which effectively induces AML cells to undergo differentiation through hypoxia-inducible factor-1␣ [15,22,23]. Moreover, NDRG1 is up-regulated in both 2% O2 - and CoCl2 -treated U937 cells [24]. In line with a previous report [14], we showed here that three differentiation-inducing agents, ATRA, PMA and DMSO, could significantly increase NDRG1 expression in both leukemic NB4 and U937 cell lines. More importantly, this induction appeared to be rapid, occurring in the early phase of differentiation. Based on these findings, we further explored the possible role of up-
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regulated NDRG1 protein in leukemic cell differentiation. For this purpose, we generated an inducible NDRG1-expressing U937T cell line using a tetracycline-off system. This cell line had the advantage of not having been directly selected for NDRG1 expression during its establishment; it had not been preselected for features such as reduced proliferation or enhanced differentiation/apoptotic potential prior to the analysis of NDRG1 expression. In this transformant, NDRG1 was tightly regulated and induced only upon tetracycline withdrawal. Using this transformant, we found that NDRG1 induction significantly induced U937T cells to undergo differentiation, as evidenced by morphological features and increased CD11c expression. To take account of possible artifacts in over-expressing cells, we also stably transduce siRNAs specifically against NDRG1 into U937 cells. Our results demonstrated that silencing of NDRG1 expression by siRNA antagonized ATRA-induced differentiation of U937 cells. All these data strongly suggest that up-regulated NDRG1 protein contributes to ATRA-induced differentiation of myeloid leukemic cells. It is well known that tightly regulated expression of many transcription factors is crucial for normal hematopoiesis, and that their genetic alterations can lead to leukemogenesis. For instances, changes in concentration of the Ets family transcriptional factor, PU.1, play a role in directing cell fate during hematopoiesis [25]. This transcriptional factor is expressed at its highest level in granulocytic cells, and plays a crucial role during myeloid differentiation [26]. In line with these facts, PU.1 knock-out mice lack mature myeloid cells [27], and decreased PU.1 expression induces leukemia in mice [28]. Mutations of the PU.1 gene are also found in some AML patients [29]. Furthermore, conditional expression of APL-specific t(15;17) translocation-generated PML-RAR␣ (for promyelocytic leukemiaretinoic acid receptor ␣) suppresses PU.1 expression [21], and t(8;21) leukemia-related AML1-ETO fusion protein inactivates the function of PU.1 [30]. Intriguingly, ATRA treatment restores PU.1 expression during induction of neutrophil differentiation of APL cell lines and primary cells [21]. Members of the C/EBP family are also implicated in the growth and differentiation of the myeloid lineage [31], mediated by their ability to bind DNA (often containing the sequence CCAAT) to control gene expression, and/or by protein–protein interactions [32]. Three members of this family, C/EBP␣, C/EBP and C/EBP, have been shown to be involved at different stages of myeloid development [33,34]. More recently, ATRA treatment was reported to induce a very rapid increase in the protein level and binding activity of C/EBP in PML-RAR␣-expressing cells (NB4), but not in a NB4-derived ATRA-resistant cell line [20]. In this work, we found that NDRG1 induction could up-regulate the expression of PU.1 and C/EBP proteins. Conversely, suppression of NDRG1 expression by specific siRNA significantly inhibited both basal and ATRA-induced expression of these two proteins. These results indicate that C/EBP and PU.1 proteins may be down-stream effectors of NDRG1, and that they might mediate NDRG1-related leukemic cell differentiation. However, the mechanisms whereby NDRG1 regulates C/EBP and PU.1 expression remain to be further investigated. Conflict of interest The authors declare that they have no potential conflicts of interest. Acknowledgements We appreciate the help of Drs. D.G. Tenen and P. Liang in generously providing us with U937T cells and plasmid, respectively. This work was supported in part by grants from the Ministry of Science and Technology (NO2002CB512806), the National Natural Science
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Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
NDRG1 contributes to retinoic acid-induced differentiation of leukemic cells Su Chen a,1 , Yu-Hui Han a,1 , Ying Zheng a , Meng Zhao b , Hua Yan a , Qiao Zhao a , Guo-Qiang Chen a,b , Dao Li a,∗ a Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine, No. 280, Chong-Qing South Road, Luwan, Shanghai 200025, China b Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences-Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, China
a r t i c l e
i n f o
Article history: Received 21 August 2008 Received in revised form 17 October 2008 Accepted 18 October 2008 Available online 28 November 2008 Keywords: NDRG1 Leukemia Differentiation ATRA C/EBP PU.1
a b s t r a c t N-Myc downstream-regulated gene 1 (NDRG1) protein has been shown to be up-regulated during leukemic cell differentiation induced by some differentiation-inducing agents such as all-trans retinoic acid (ATRA). However, the potential role of up-regulated NDRG1 in the event is greatly unknown. In this work, we show that inducible NDRG1 expression can drive leukemic U937 cells to undergo differentiation, while the knock-down of NDRG1 expression by specific small interfering RNA significantly antagonizes ATRA-induced differentiation of leukemic cells, proposing the role of NDRG1 in leukemic cell differentiation. Furthermore, our work shows that CCAAT/enhancer-binding protein beta (C/EBP) and PU.1, which are important hematopoiesis-related transcription factors, may act as downstream effectors of NDRG1 in leukemic cell differentiation. Taking together, this study provides direct evidence for the role of NDRG1 protein in myeloid leukemic cell differentiation. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction N-Myc downstream-regulated gene (NDRG) belongs to a family of closely related genes that are down-regulated by c-myc or the N-Myc/Max complex. The family includes at least four members, respectively, named NDRG1-4 [1–3]. NDRG1, also known as Drg1, Cap43, RTP/rit42 and Proxy-1, is expressed as a 43-kDa protein composed of 394 amino acids. It was first recognized as a gene whose mutation was linked to a demyelinating neuropathy, and which mapped to human chromosome 8q24. NDRG1 is highly conserved among multicellular organisms, and is expressed ubiquitously in tissues. It has been reported that NDRG1, the expression of which can be induced by various cellular stress signals, is involved in many normal and pathologic cellular activities, such as cellular responses to heavy metals, hypoxia, DNA damage, proliferation and growth arrest, neoplasia, tumor progression and metastasis [4]. The regulation of NDRG1 expression during cancer cell differentiation is also explored. For example, NDRG1 protein is induced during the in vitro differentiation of the HT29-D4 colon carcinoma cell line [5], androgen-induced differentiation of the prostatic adenocarcinoma cell line LNCaP [6], calcium switch or serum starvation-induced
∗ Corresponding author. Fax: +86 21 64154900. E-mail address: [email protected] (D. Li). 1 These authors should be considered to contribute equally to this work. 0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.10.016
differentiation of keratinocytes [7], and the maturation of mouse bone marrow-derived mast cells into cells with a connective tissue mast cell-like phenotype induced by the presence of stem cell factor and coculturing with fibroblasts [8]. Additionally, NDRG1 knock-out mice exhibit abnormal peritoneal mast cells [9]. Acute myeloid leukemia (AML), a class of prevalent hematopoietic malignancies, is characterized by complete or partial blockage at different stages of the differentiation of myeloid progenitor cells, together with deregulated proliferation and a survival advantage of hematopoietic progenitors. These pathophysiologic changes have been attributed to the acquired genetic abnormality, especially to different types of specific reciprocal chromosome translocations [10,11]. Accordingly, differentiation induction is a potentially attractive strategy for AML therapy. For this strategy, a typical example is the successful application of differentiation-inducing agent all-trans retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL), a unique subtype of AML. Hence, it has been attracting great attentions to understand the mechanisms by which ATRA and other agents induce leukemic cell differentiation, and significant progress has been made [12,13]. Using a differential display method to identify differentiation-related genes in human myelomonocytic leukemic U937 cells, Piquemal et al. [14] found that NDRG1/Drg1 expression was up-regulated by the differentiation-inducing agents phorbol-12-myristate-13acetate (PMA), ATRA and 1,25-(OH)2 vitamin D3 . However, the possible role of the up-regulated NDRG1 protein in leukemic cell
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differentiation remains unclear. In this study, we find that inducible NDRG1 expression can drive leukemic U937 cells to undergo differentiation, while ATRA-induced differentiation of leukemic cells is significantly antagonized by the knock-down of NDRG1 expression by specific small interfering RNA (siRNA), proposing the role of NDRG1 in leukemic cell differentiation. Furthermore, CCAAT/enhancer-binding protein beta (C/EBP) and PU.1, the important transcriptional factors for hematopoiesis, may act as downstream effectors of NDRG1 in leukemic cell differentiation.
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2.4. Design and transfection of siRNA Three pairs of complementary oligonucleotides against NDRG1 were synthesized by Invitrogen (Shanghai, China), annealed and ligated into pSilencer 3.1-H1-neo vector (Ambion, Austin, TX, USA). Target sequences of siRNA against NDRG1 were 5 -GCA TTA TTG GCA TGG GAA C-3 for siRNA #1, 5 -ACT ATT GTG CAC AAG TCT T-3 for siRNA #2 and 5 -TAG TGA CAT GCA GGC ACCT-3 for siRNA #3. These vectors and negative controls (NC) were transfected into U937 cells using the Bio-Rad gene-Pulser II (Bio-Rad) with square-wave electroporation at 0.17 kV and 960 F. Forty-eight hours later, 1000 g/ml of G418 (Calbiochem, Germany) were added to the medium and stable transformants were selected by testing targeted proteins by Western blot.
2. Materials and methods 2.5. Western blot 2.1. Cell lines and treatment The human promonocytic leukemic cell line U937 and APL cell line NB4 were cultured in RPMI 1640 medium (Sigma–Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT) in 5% CO2 /95% air humidified atmosphere at 37 ◦ C. U937T cells (kindly provided by Dr. D.G. Tenen at Harvard Institute of Medicine, Harvard Medical School, Boston, MA) were cultured in RPMI1640 medium supplemented with 10% FBS, 1 mg/ml tetracycline (Sigma–Aldrich) and 0.5 mg/ml puromycin (Sigma–Aldrich). For experiments, cells were originally seeded at 2.0–5.0 × 105 cells/ml, and were treated with or without ATRA, PMA and dimethyl sulfoxide (DMSO) (Sigma–Aldrich).
Total cellular protein extracts were loaded onto an 8–10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis, and then transferred to ECLnitrocellulose (Amersham, Buckinghamshire, UK). After blocking with 5% nonfat milk in Tris-buffered saline, membranes were incubated with antibodies against human NDRG1 (Zymed, CA), C/EBP␣ (Cell Signaling Technology, Beverly, MA), C/EBP and PU.1 (Santa Cruz Biotechnology, CA) as well as -tubulin monoclonal antibody (Oncogene, CA) or actin (Cell Signaling) at 4 ◦ C overnight, followed by HRPlinked secondary antibody (Cell Signaling) for 1 h at room temperature. Detection was then performed by chemiluminescence phototope-HRP kit, according to the manufacturer’s instructions (Cell Signaling).
2.2. Cell differentiation assay
3. Results
For cell morphology, cells were collected onto slides by cytospin (Shandon, Runcorn, UK), and examined by microscope after Wright’s staining. Cell differentiation-related antigens CD11c and CD14 were measured by flow cytometry (Beckman-Coulter, Miami, FL) as described previously [15].
3.1. NDRG1 protein is up-regulated during ATRA, PMA and DMSO-induced differentiation of leukemic cells
2.3. Establishment of U937T transformants with inducible NDRG1 expression Plasmid pBI-NDRG1-EGFP was generously provided by Dr. P. Liang [16]. EGFP and NDRG1 were located on opposite sides of the tetracycline (Tet) response element in the plasmid, and were controlled by tetracycline. To generate a cell line with stable inducible expression of NDRG1 protein, pBI-NDRG1-EGFP and pTRE2hyg (BD Biosciences, San Diego, CA) with a eukaryotic selection marker were co-transfected into U937T cells, which were stably transfected with a pUHD-tTA (tetracycline responsive transcription activator) under the control of a tetracycline-inducible promoter. To this aim, U937T cells were resuspended at 5 × 107 cells/ml in 200 l of RPMI 1640 medium without FBS. Eight micrograms of linearized pBI-NDRG1-EGFP and 2 mg of pTRE2hyg were mixed and transferred to electroporation cuvettes with a 0.4 cm gap (Bio-Rad, Hercules, CA). Electroporation was performed using a Gene-Pulser II (BioRad) at 0.17 kV and 960 F. The samples were then transferred to complete RPMI 1640 medium containing 1 g/ml tetracycline and 0.5 g/ml puromycin and incubated at 37 ◦ C in 5% CO2 /95% air humidified atmosphere. Twenty-four hours later, 500 g/ml of hygromycine (BD Biosciences) was added to the medium. Following tetracycline withdrawal, positive polyclonal populations were initially evaluated by fluorescence microscopy (Olympus BX-51, Olympus Optical, Japan) for EGFP and then for expression of NDRG1 protein. The cells were maintained in RPMI 1640 medium supplemented with 10% FBS, with 1 g/ml tetracycline, 0.5 g/ml puromycin, and 500 g/ml of hygromycine. To test the inducibility of NDRG1 expression, the transfected cells were incubated in the presence or absence of 1 g/ml tetracycline.
Leukemic cell lines U937 and NB4 were treated with the appropriate concentrations of the differentiation-inducing agents ATRA (10−7 M), PMA (10−7 M) and DMSO (1%) for 3 days. As described previously [17], these treatments significantly induced cell differentiation, as evaluated by morphologic examination and flow cytometry assay for CD11c and CD14 antigens (data not shown). In consistence with a previous report [14], ATRA and PMA significantly increased NDRG1 protein expression in both NB4 and U937 cells (Fig. 1A). DMSO also produced a similar but weaker effect on NDRG1 expression, which was paralleled to its weaker differentiation-inducing ability in these two cell lines (data not shown). We also tested the time-course effect of ATRA on NDRG1 expression. As depicted in Fig. 1B, increased NDRG1 protein expression was detected before 12 h and its level was further increased at 24 h after ATRA treatment. 3.2. NDRG1 induction triggers differentiation of myeloid leukemic cells The above observation suggests that increased NDRG1 protein expression is an early event during ATRA-induced differentiation.
Fig. 1. Effects of three differentiation-inducing agents on NDRG1 expression in leukemia cell lines. NB4 and U937 cells were treated with 10−7 M ATRA, 10−7 M PMA and 1% DMSO for 3 days (A) or 10−7 M ATRA for hours as indicated (B). NDRG1 was detected by Western blots with -tubulin as a control. All experiments were repeated at least three times with the same results.
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Fig. 2. NDRG1 induction triggers differentiation of myeloid leukemic cells. (A) NDRG1-transfected U937T cells were treated with or without tetracycline (tet) for days as indicated, and NDRG1 protein was detected by Western blots. (B and C) After incubation in medium with (+) or without (−) tet for 4 days, U937T cells were collected onto slides, stained by Wright’s staining and observed under a microscope (B, magnification ×1000). CD11c and EGFP were detected by flow cytometry (C). All experiments were repeated at least three times with the same results.
To investigate the potentially direct role of NDRG1 in the differentiation of myeloid leukemic cells, an inducible NDRG1-expressing U937T cell line was generated using a tetracycline-off system. pBINDRG1-EGFP and pTRE2hyg were co-transfected into U937T cells,
which contain stably transfected pUHD-tTA whose expression is under the control of tetracycline. It should be pointed out that U937 cells were selected because transfection is more difficult in NB4 cells, although the clinical application of a differentiation
Fig. 3. Suppression of NDRG1 expression by siRNA antagonizes ATRA-induced differentiation of U937 cells. (A) U937 cells were stably transfected with siRNA #1–3 against NDRG1 or negative control vector (NC), and NDRG1 protein was detected by Western blot with -tubulin as a loading control. (B and C) U937 siRNA #2 and NC cells were treated with or without 0.1 M ATRA for 48 h. Cells were collected onto slides by cytospin, stained by Wright’s staining and observed under a microscope (B, magnification ×1000) or CD11c+ cells were measured by flow cytometry (C). The values represent mean ± S.D. of triplicate independent experiments, which were repeated more than three times with the same results.
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Fig. 4. NDRG1 regulates expression of PU.I and C/EBP proteins. U937 siRNA #2 and NC cells were treated with 0.1 M ATRA for indicated hours (A) or NDRG1 transfected U937T cells and control cells were incubated for days, as indicated, after removal of tetracycline (B). The indicated proteins were detected, using -tubulin as a loading control. All experiments were repeated at least three times with the same results.
therapeutic approach is best described in APL. As shown in Fig. 2A, NDRG1 protein began to increase at day 2 and reached a maximum at day 4 after tetracycline removal. When NDRG1 had been induced for 4 days, U937T cells exhibited differentiation-related morphological changes such as reduced cell size, condensed chromatin and decreased nucleus/cytoplasm ratio, with smaller and distorted nuclei (Fig. 2B). Furthermore, NDRG1 induction remarkably increased CD11c expression, indicating that NDRG1 induction can induce U937 cells to undergo differentiation (Fig. 2C). 3.3. Suppression of NDRG1 expression by siRNA antagonizes ATRA-induced differentiation of U937 cells Three pairs of siRNAs against NDRG1 mRNA and a NC siRNA were transfected into the parental U937 cells. After selection with G418, we found that siRNA #2 was the most effective one against NDRG1. As shown in Fig. 3A, this siRNA significantly suppressed expression of NDRG1 protein. Intriguingly, the silencing of NDRG1 expression by siRNA #2, but not by the other two ineffective siRNAs and NC transfection, significantly antagonized ATRA-induced differentiation of U937 cells, as assessed by morphology (Fig. 3B) and decreased percentage of CD11c-positive cells (Fig. 3C). These results further suggest a contribution of NDRG1 to the ATRA-induced differentiation of myeloid leukemic cells. 3.4. NDRG1 contributes to regulation of C/EBPˇ and PU.1 expression Finally, we examined the possible effects of NDRG1 on the expression of C/EBP␣, C/EBP and PU.1, three important transcrip-
tional factors for hematopoiesis [10,18,19]. To this end, U937 cells with siRNA #2 or NC transfection were treated with 10−7 M ATRA for varying lengths of time, and the expression of these proteins was detected, using -tubulin as a loading control. In line with previous reports [20,21], ATRA treatment up-regulated expression of C/EBP and PU.1 but not C/EBP␣ protein. More interestingly, suppression of NDRG1 expression by specific siRNA significantly inhibited both basal and ATRA-up-regulated expression of C/EBP and PU.1 (Fig. 4B). Conversely, induction of NDRG1 protein in U937T transformants after tetracycline removal significantly increased the expression of PU.1 and C/EBP proteins. These results indicate that NDRG1 contributes to the regulation of C/EBP and PU.1 expression. 4. Discussion It is well known that NDRG1 expression is regulated by stress signals and by the cell growth and differentiation-related environment [1]. We previously compared the global protein expression profiles of human leukemic U937 cells incubated in air and under 2% O2 , or treated with the hypoxia-mimetic agent cobalt chloride (CoCl2 ), which effectively induces AML cells to undergo differentiation through hypoxia-inducible factor-1␣ [15,22,23]. Moreover, NDRG1 is up-regulated in both 2% O2 - and CoCl2 -treated U937 cells [24]. In line with a previous report [14], we showed here that three differentiation-inducing agents, ATRA, PMA and DMSO, could significantly increase NDRG1 expression in both leukemic NB4 and U937 cell lines. More importantly, this induction appeared to be rapid, occurring in the early phase of differentiation. Based on these findings, we further explored the possible role of up-
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regulated NDRG1 protein in leukemic cell differentiation. For this purpose, we generated an inducible NDRG1-expressing U937T cell line using a tetracycline-off system. This cell line had the advantage of not having been directly selected for NDRG1 expression during its establishment; it had not been preselected for features such as reduced proliferation or enhanced differentiation/apoptotic potential prior to the analysis of NDRG1 expression. In this transformant, NDRG1 was tightly regulated and induced only upon tetracycline withdrawal. Using this transformant, we found that NDRG1 induction significantly induced U937T cells to undergo differentiation, as evidenced by morphological features and increased CD11c expression. To take account of possible artifacts in over-expressing cells, we also stably transduce siRNAs specifically against NDRG1 into U937 cells. Our results demonstrated that silencing of NDRG1 expression by siRNA antagonized ATRA-induced differentiation of U937 cells. All these data strongly suggest that up-regulated NDRG1 protein contributes to ATRA-induced differentiation of myeloid leukemic cells. It is well known that tightly regulated expression of many transcription factors is crucial for normal hematopoiesis, and that their genetic alterations can lead to leukemogenesis. For instances, changes in concentration of the Ets family transcriptional factor, PU.1, play a role in directing cell fate during hematopoiesis [25]. This transcriptional factor is expressed at its highest level in granulocytic cells, and plays a crucial role during myeloid differentiation [26]. In line with these facts, PU.1 knock-out mice lack mature myeloid cells [27], and decreased PU.1 expression induces leukemia in mice [28]. Mutations of the PU.1 gene are also found in some AML patients [29]. Furthermore, conditional expression of APL-specific t(15;17) translocation-generated PML-RAR␣ (for promyelocytic leukemiaretinoic acid receptor ␣) suppresses PU.1 expression [21], and t(8;21) leukemia-related AML1-ETO fusion protein inactivates the function of PU.1 [30]. Intriguingly, ATRA treatment restores PU.1 expression during induction of neutrophil differentiation of APL cell lines and primary cells [21]. Members of the C/EBP family are also implicated in the growth and differentiation of the myeloid lineage [31], mediated by their ability to bind DNA (often containing the sequence CCAAT) to control gene expression, and/or by protein–protein interactions [32]. Three members of this family, C/EBP␣, C/EBP and C/EBP, have been shown to be involved at different stages of myeloid development [33,34]. More recently, ATRA treatment was reported to induce a very rapid increase in the protein level and binding activity of C/EBP in PML-RAR␣-expressing cells (NB4), but not in a NB4-derived ATRA-resistant cell line [20]. In this work, we found that NDRG1 induction could up-regulate the expression of PU.1 and C/EBP proteins. Conversely, suppression of NDRG1 expression by specific siRNA significantly inhibited both basal and ATRA-induced expression of these two proteins. These results indicate that C/EBP and PU.1 proteins may be down-stream effectors of NDRG1, and that they might mediate NDRG1-related leukemic cell differentiation. However, the mechanisms whereby NDRG1 regulates C/EBP and PU.1 expression remain to be further investigated. Conflict of interest The authors declare that they have no potential conflicts of interest. Acknowledgements We appreciate the help of Drs. D.G. Tenen and P. Liang in generously providing us with U937T cells and plasmid, respectively. This work was supported in part by grants from the Ministry of Science and Technology (NO2002CB512806), the National Natural Science
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