Differential gene expression in retinoic acid-induced differentiation of acute promyelocytic leukemia cells, NB4 and HL-60 cells

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation t(15;17), which results in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor alpha gene (RARalpha). APL can be effectively treated with the cell differentiation inducer all-trans retinoic acid (ATRA). NB4 cells, an acute promyelocytic leukemia cell line, have the t(15;17) translocation and differentiate in response to ATRA, whereas HL-60 cells lack this chromosomal translocation, even after differentiation by ATRA. To identify changes in the gene expression patterns of promyelocytic leukemia cells during differentiation, we compared the gene expression profiles in NB4 and HL-60 cells with and without ATRA treatment using a cDNA microarray containing 10,000 human genes. NB4 and HL-60 cells were treated with ATRA (10(-6)M) and total RNA was extracted at various time points (3, 8, 12, 24, and 48h). Cell differentiation was evaluated for cell morphology changes and CD11b expression. PML/RARalpha degradation was studied by indirect immunofluoresence with polyclonal PML antibodies. Typical morphologic and immunophenotypic changes after ATRA treatment were observed both in NB4 and HL-60 cells. The cDNA microarray identified 119 genes that were up-regulated and 17 genes that were down-regulated in NB4 cells, while 35 genes were up-regulated and 36 genes were down-regulated in HL60 cells. Interestingly, we did not find any common gene expression profiles regulated by ATRA in NB4 and HL-60 cells, even though the granulocytic differentiation induced by ATRA was observed in both cell lines. These findings suggest that the molecular mechanisms and genes involved in ATRA-induced differentiation of APL cells may be different and cell type specific. Further studies will be needed to define the important molecular pathways involved in granulocytic differentiation by ATRA in APL cells.     

ATRA-induced upregulation of Beclin 1 prolongs the life span of differentiated acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) induces clinical remission in APL patients by enhancing the rapid differentiation of APL cells and the clearance of PML-RARα, APL's hallmark oncoprotein. In the present study, we demonstrated that both autophagy and Beclin 1, an autophagic protein, are upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of an APL-derived cell line named NB4 cells. This induction of autophagy is associated with downregulation of Bcl-2 and inhibition of mTOR activity. Small interfering RNA-mediated knockdown of BECN1 expression enhances apoptosis triggered by ATRA in NB4 cells but does not affect the differentiation process. These results provide evidence that the upregulation of Beclin 1 by ATRA constitutes an anti-apoptotic signal for maintaining the viability of mature APL cells, but has no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.     

ATRA-induced upregulation of Beclin 1 prolongs the life span of differentiated acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) induces clinical remission in APL patients by enhancing the rapid differentiation of APL cells and the clearance of PML-RARα, APL’s hallmark oncoprotein. In the present study, we demonstrated that both autophagy and Beclin 1, an autophagic protein, are upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of an APL-derived cell line named NB4 cells. This induction of autophagy is associated with downregulation of Bcl-2 and inhibition of mTOR activity. Small interfering RNA-mediated knockdown of BECN1 expression enhances apoptosis triggered by ATRA in NB4 cells but does not affect the differentiation process. These results provide evidence that the upregulation of Beclin 1 by ATRA constitutes an anti-apoptotic signal for maintaining the viability of mature APL cells, but has no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.     

Annexin A1 mediates the anti‐adhesive effects of dexamethasone during the cell–cell interaction between the all‐trans retinoic acid‐treated acute promyelocytic leukemic cells and endothelial cells

Annexin A1 (AnxA1) is an important anti‐inflammatory mediator during granulocytic differentiation in all trans‐retinoic acid (ATRA) treated acute promyelocytic leukemic (APL) cells. Dexamethasone has been used successfully to prevent complications in ATRA‐treated APL patients, although its mechanism of action is still not clear. In the present study, we have examined the effect of dexamethasone on the modulation of AnxA1 in ATRA‐APL NB4 (ATRA‐NB4) cells, ATRA‐NB4 cells‐derived microparticles (MPs) and its role during cell–cell interaction between ATRA‐NB4 cells and endothelial cells. Our results have shown that dexamethasone can inhibit the percentage of ATRA‐NB4 cells expressing surface AnxA1 and its receptor FPR2/ALX in a time‐dependent manner based on flow cytometric analysis. However, dexamethasone treatment of ATRA‐NB4 cells has no significant effect on the level of AnxA1 mRNA, the total cellular level of AnxA1 protein or the release of AnxA1 from these cells, as determined by RT‐PCR, Western blotting, and ELISA, respectively. Further studies demonstrate that dexamethasone is able to significantly inhibit the adhesion of ATRA‐NB4 cells to endothelial cells, and this anti‐adhesive effect can be inhibited if the cells were pre‐treated with a neutralizing antibody specific for AnxA1. Finally, dexamethasone also enhances the release of AnxA1‐containing MPs from ATRA‐NB4 cells which can in turn prevent the adhesion of the ATRA‐NB4 cells to endothelial cells. We conclude that biologically active AnxA1 originating from dexamethasone‐treated ATRA‐APL cells and their MPs plays an anti‐adhesive effect and this contributes to inhibit the adhesion of ATRA‐APL cell to endothelial cells. J. Cell. Biochem. 114: 551–557, 2013. © 2012 Wiley Periodicals, Inc.     

miR-382-5p modulates the ATRA-induced differentiation of acute promyelocytic leukemia by targeting tumor suppressor PTEN

In acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA) treatment induces granulocytic differentiation and maturation. MicroRNAs play pivotal roles in formation of the leukemic phenotype. Previously, microRNA-382-5p (miR-382-5p) was upregulated in acute myeloid leukemia (AML) with t(15;17). In the present study, we found that miR-382-5p expression was elevated with ATRA-induced differentiation of APL. To investigate the potential functional role of miR-382-5p in APL differentiation, an APL cell line was transfected with miR-382-5p mimics, inhibitors, or negative control (NC). The results showed in APL cell line NB4 that miR-382-5p downregulation upon ATRA treatment was a key event in the drug response. Mechanistic investigations revealed that miR-382-5p targeted the ATRA-regulated tumor suppressor gene PTEN through direct binding to its 3′ UTR. Enforced expression of miR-382-5p or specific PTEN inhibitors inhibited ATRA-induced granulocytic differentiation via regulation of the cell cycle regulator cyclinD1. Conversely, PTEN overexpression promoted differentiation and enhanced sensitivity of NB4 cell line to physiological levels of ATRA. Finally, we found that PTEN overexpression restored PML nuclear bodies (NBs). Taken together, these results demonstrated that up-regulated miR-382-5p in NB4 cell line inhibited granulocytic differentiation through the miR-382-5p/PTEN axis, uncovering PTEN as a critical element in the granulocytic differentiation program induced by ATRA in APL.     

Celastrol Inhibits Lung Infiltration in Differential Syndrome Animal Models by Reducing TNF-α and ICAM-1 Levels while Preserving Differentiation in ATRA-Induced Acute Promyelocytic Leukemia Cells

All-trans retinoic acid (ATRA) is a revolutionary agent for acute promyelocytic leukemia (APL) treatment via differentiation induction. However, ATRA treatment also increases cytokine, chemokine, and adhesive molecule (mainly ICAM-1) expression, which can cause clinical complications, including a severe situation known as differentiation syndrome (DS) which can cause death. Therefore, it is of clinical significance to find a strategy to specifically blunt inflammatory effects while preserving differentiation. Here we report that the natural compound, celastrol, could effectively block lung infiltrations in DS animal models created by loading ATRA-induced APL cell line NB4. In ATRA-treated NB4 cells, celastrol could potently inhibit ICAM-1 elevation and partially reduce TNF-α and IL-1β secretion, though treatment showed no effects on IL-8 and MCP-1 levels. Celastrol’s effect on ICAM-1 in ATRA-treated NB4 was related to reducing MEK1/ERK1 activation. Strikingly and encouragingly, celastrol showed no obvious effects on ATRA-induced NB4 differentiation, as determined by morphology, enzymes, and surface markers. Our results show that celastrol is a promising and unique agent for managing the side effects of ATRA application on APL, and suggest that hyper-inflammatory ability is accompanied by, but not necessary for, APL differentiation. Thus we offered an encouraging novel strategy to further improve differentiation therapy.     

PLC-beta2 monitors the drug-induced release of differentiation blockade in tumoral myeloid precursors

The differentiation therapy in treatment of acute promyelocytic leukemia (APL), based on the administration of all-trans retinoic acid (ATRA), is currently flanked with the use of As2O3, a safe and effective agent for patients showing a resistance to ATRA treatment. A synergy between ATRA and As3O3 was also reported in inducing granulocytic differentiation of APL-derived cells. We have demonstrated that phospholipase C-beta2 (PLC-beta2), highly expressed in neutrophils and nearly absent in tumoral promyelocytes, largely increases during ATRA treatment of APL-derived cells and strongly correlates with the responsiveness of APL patients to ATRA-based differentiating therapies. Here we report that, in APL-derived cells, low doses of As3O3 induce a slight increase of PLC-beta2 together with a moderate maturation, and cooperate with ATRA to provoke a significant increase of PLC-beta2 expression. Remarkably, the amounts of PLC-beta2 draw a parallel with the differentiation levels reached by both ATRA-responsive and -resistant cells treated with ATRA/As2O3 combinations. PLC-beta2 is not necessary for the progression of tumoral promyelocytes along the granulocytic lineage and is unable to overcome the differentiation block or to potentiate the agonist-induced maturation. On the other hand, since its expression closely correlates with the differentiation level reached by APL-derived cells induced to maturate by drugs presently employed in APL therapies, PLC-beta2 represents indeed a specific marker to test the ability of differentiation agents to induce the release of the maturation blockade of tumoral myeloid precursors.     

C/EBPbeta: a major PML-RARA-responsive gene in retinoic acid-induced differentiation of APL cells

In acute promyelocytic leukemia (APL), the translocation t(15;17) induces a block at the promyelocytic stage of differentiation in an all-trans-retinoic acid (ATRA)-responsive manner. Here we report that upon treatment with ATRA, t(15;17) cells (NB4) reveal a very rapid increase in protein level and binding activity of C/EBPbeta, a C/EBP family member, which was not observed in an ATRA-resistant NB4 cell line. We further provide evidence that ATRA mediates a direct increase of C/EBPbeta, only in PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha)-expressing cells. In addition, transactivation experiments indicate that the PML-RARA fusion protein, but not PML-RARA mutants defective in transactivation, strongly transactivates the C/EBPbeta promoter. These results suggest that PML-RARA mediates ATRA-induced C/EBPbeta expression. Finally, we demonstrate the importance of C/EBPbeta in granulocytic differentiation. We show that not only does C/EBPbeta induce granulocytic differentiation of non-APL myeloid cell lines independent of addition of ATRA or other cytokines, but also that C/EBPbeta induction is required during ATRA-induced differentiation of APL cells. Taken together, C/EBPbeta is an ATRA-dependent PML-RARA target gene involved in ATRA-induced differentiation of APL cells.     

Death-associated protein 5 (DAP5/p97/NAT1) contributes to retinoic acid-induced granulocytic differentiation and arsenic trioxide-induced apoptosis in acute promyelocytic leukemia

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells. Here we investigated the role and regulation of death-associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in APL cell differentiation and apoptosis. We found that ATRA markedly induced DAP5/p97 protein and gene expression and nuclear translocation during terminal differentiation of APL (NB4) and HL60 cells but not differentiation-resistant cells (NB4.R1 and HL60R), which express very low levels of DAP5/p97. At the differentiation inducing concentrations, ATO (<0.5 μM), dimethyl sulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate 13-acetate also significantly induced DAP5/p97 expression in NB4 cells. However, ATO administered at apoptotic doses (1–2 μM) induced expression of DAP5/p86, a proapoptotic derivative of DAP5/p97. ATRA and ATO-induced expression of DAP5/p97 was associated with inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, DAP5/p97 expression was upregulated by inhibition of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway via LY294002 and via rapamycin. Finally, knockdown of DAP5/p97 expression by small interfering RNA inhibited ATRA-induced granulocytic differentiation and ATO-induced apoptosis. Together, our data reveal new roles for DAP5/p97 in ATRA-induced differentiation and ATO-induced apoptosis in APL and suggest a novel regulatory mechanism by which PI3K/Akt/mTOR pathway inhibition mediates ATRA- and ATO-induced expression of DAP5/p97. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. B. Ozpolat and U. Akar contributed equally.     

Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis

We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E₂ analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients.     

Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RARα and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.     

Programmed cell death-4 tumor suppressor protein contributes to retinoic acid-induced terminal granulocytic differentiation of human myeloid leukemia cells

Programmed cell death-4 (PDCD4) is a recently discovered tumor suppressor protein that inhibits protein synthesis by suppression of translation initiation. We investigated the role and the regulation of PDCD4 in the terminal differentiation of acute myeloid leukemia (AML) cells. Expression of PDCD4 was markedly up-regulated during all-trans retinoic acid (ATRA)-induced granulocytic differentiation in NB4 and HL60 AML cell lines and in primary human promyelocytic leukemia (AML-M3) and CD34(+) hematopoietic progenitor cells but not in differentiation-resistant NB4.R1 and HL60R cells. Induction of PDCD4 expression was associated with nuclear translocation of PDCD4 in NB4 cells undergoing granulocytic differentiation but not in NB4.R1 cells. Other granulocytic differentiation inducers such as DMSO and arsenic trioxide also induced PDCD4 expression in NB4 cells. In contrast, PDCD4 was not up-regulated during monocytic/macrophagic differentiation induced by 1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl-phorbol-13-acetate in NB4 cells or by ATRA in THP1 myelomonoblastic cells. Knockdown of PDCD4 by RNA interference (siRNA) inhibited ATRA-induced granulocytic differentiation and reduced expression of key proteins known to be regulated by ATRA, including p27(Kip1) and DAP5/p97, and induced c-myc and Wilms' tumor 1, but did not alter expression of c-jun, p21(Waf1/Cip1), and tissue transglutaminase (TG2). Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was found to regulate PDCD4 expression because inhibition of PI3K by LY294002 and wortmannin or of mTOR by rapamycin induced PDCD4 protein and mRNA expression. In conclusion, our data suggest that PDCD4 expression contributes to ATRA-induced granulocytic but not monocytic/macrophagic differentiation. The PI3K/Akt/mTOR pathway constitutively represses PDCD4 expression in AML, and ATRA induces PDCD4 through inhibition of this pathway.     

Over-expression of Mcl-1 impairs the ability of ATRA to induce growth arrest and differentiation in acute promyelocytic leukemia cells

Exposure of acute promyelocytic leukemia (APL) cells to all-trans retinoic acid (ATRA) increases levels of Mcl-1, however, the implication of ATRA-mediated expressions of Mcl-1 in these cells remains to be fully elucidated. This study found that exposure of NB4 and PL-21 cells to ATRA increased levels of Mcl-1 in association with phosphorylation of c-jun N-terminus kinases. Down-regulation of Mcl-1 by a small interfering (siRNA) or an inhibitor of JNK significantly potentiated the ability of ATRA to induce differentiation and apoptosis in these cells. On the other hand, the anti-leukemia effects of ATRA were blunted when Mcl-1 was forced expressed in NB4 and PL-21 cells as well as leukemia cells isolated from individuals with APL. Furthermore, down-regulation of Mcl-1 by an siRNA sensitized non-APL U937 and KG-1 leukemia cells to ATRA-mediated differentiation and apoptosis. Taken together, inhibition of Mcl-1 might be useful to potentiate the action of ATRA in APL as well as non-APL AML cells.     

Triterpenoid CDDO-Im downregulates PML/RARalpha expression in acute promyelocytic leukemia cells

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of diverse human tumor cells. In the present study, we examined the effects of the CDDO imidazolide imide (CDDO-Im) on the NB4 acute promyelocytic leukemia (APL) cell line and primary APL cells. The results show that CDDO-Im selectively downregulates expression of the PML/retinoic receptor alpha fusion protein by a caspase-dependent mechanism and sensitizes APL cells to the differentiating effects of all-trans retinoic acid (ATRA). CDDO-Im treatment of APL cells was also associated with disruption of redox balance and activation of the extrinsic apoptotic pathway. In concert with these results, CDDO-Im sensitizes APL cells to arsenic trioxide (ATO)-induced apoptosis. Our findings indicate that CDDO-Im may be effective in the treatment of APL by: (i) downregulation of PML/RARalpha; (ii) enhancement of ATRA-induced differentiation; and (iii) sensitization of ATO-induced APL cell death.     

The proteasome inhibitor bortezomib enhances ATRA-induced differentiation of neuroblastoma cells via the JNK mitogen-activated protein kinase pathway

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Differentiated human NBs are associated with better outcome and lower stage; induction of differentiation is considered to be therapeutically advantageous. All-trans retinoic acid (ATRA) has been shown to induce the differentiation of neuroblastoma (NB) cell lines. The proteasome inhibitor bortezomib inhibits cell growth and angiogenesis in NBs. Here, we investigated the synergistic effect between bortezomib and ATRA in inducing NB cell differentiation in different NB cell lines. Bortezomib combined with ATRA had a significantly enhanced antiproliferative effect. This inhibition was characterized by a synergistic increase in neuronal differentiation. At the same time, the combination therapy showed little neuronal toxicity which was assessed in primary cultures of rat cerebellar granule cells by the MTT assay, PI staining. The combination of bortezomib and ATRA triggered increased differentiation through the activation of proteins, including RARα, RARβ, RARγ, p-JNK and p21, compared with ATRA treatment alone. Using JNK inhibitor SP600125 to block JNK-dependent activity, the combination therapy-induced neuronal differentiation was partially attenuated. In addition, p21 shRNA had no effect on the combination therapy-induced neuronal differentiation. The in vivo antitumor activities were examined in human NB cell xenografts and GFP-labeled human NB cell xenografts. Treatment of human NB cell CHP126-bearing nude mice with ATRA plus bortezomib resulted in more significant tumor growth inhibition than mice treated with either drug alone. These findings provide the rationale for the development of a new therapeutic strategy for NB based on the pharmacological combination of ATRA and bortezomib.     

Indeno[1,2-c]isoquinolines as enhancing agents on all-trans retinoic acid-mediated differentiation of human myeloid leukemia cells

Induction of differentiation is a new and promising approach to cancer therapy, well illustrated by the treatment of acute myeloid leukemia with all-trans retinoic acid (ATRA). Using combination of ATRA and chemotherapy, adverse effects such as retinoic acid syndrome have decreased, and long-term survival has improved. In this study, we demonstrated that the indeno[1,2-c]isoquinolines markedly enhanced differentiation of human myeloid leukemia HL-60 and NB4 cells when simultaneously combined with a low dose of ATRA. Of the tested compounds, 6-(4-methoxybenzyl)-2,11-dimethyl-6H,11H-indeno[1,2-c]isoquinolin-5-one (IIQ-16), an indeno[1,2-c]isoquinoline derivative, showed the highest differentiation-enhancing activity via a pathway involved with protein kinase C, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. The ability to enhance the differentiation potential of ATRA by IIQ-16 may improve outcomes in the therapy of acute promyelocytic leukemia.     

JWA, a novel signaling molecule, involved in the induction of differentiation of human myeloid leukemia cells

Dysregulation of hematopoietic cellular differentiation contributes to leukemogenesis. Unfortunately, relatively little is known about how cell differentiation is regulated. JWA (AF070523) is a novel all-trans retinoic acid (ATRA) responsible gene that initially isolated from ATRA-treated primary human tracheal bronchial epithelial cells. For the notable performance achieved by ATRA in the differentiation induction therapy, we investigated the role of JWA in the induction of differentiation of human myeloid leukemia cells. Our results showed that JWA was not only regulated by ATRA but also by several other differentiation inducers such as phorbol-12-myristate-13-acetate (TPA), arabinoside (Ara-C), and hemin, involved in the mechanisms of differentiation along different lineages of myeloid leukemia cells arrested at different stages of development. Generally, JWA was up-regulated by these inducers in a time-dependent manner. Inhibition of JWA by RNA interference decreased the induced cellular differentiation. However, in NB4 cells treated with ATRA, dissimilar with others, the expression of JWA was down-regulated, and the induced cellular differentiation could be enhanced by silencing of JWA. Collectively, JWA works as a potential critical molecule, associated with multi-directional differentiation of human myeloid leukemia cells. In NB4 cells, JWA may function as a lineage-restricted gene during differentiation along the monocyte/macrophage-like or granulocytic pathway.