scholarly journals PML-RAR Binds to the +7kb Enhancer of CEBPE and Inhibits Its Expression

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-43
Author(s):  
Pavithra Shyamsunder ◽  
Shree Pooja Sridharan ◽  
Pushkar Dakle ◽  
Zeya Cao ◽  
Vikas Madan ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Regulatory Element ◽  
Promyelocytic Leukemia ◽  
Luciferase Reporter ◽  
Myeloid Differentiation ◽  
Atra Treatment ◽  
Granulocytic Differentiation ◽  
Nb4 Cells ◽  
Acute Myeloid

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The disease is identified by distinctive morphology and is distinguished by a balanced reciprocal translocation between chromosomes 15 and 17. This aberration leads to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, leading to the resultant chimeric onco-fusion protein PML-RARA, which is detectable in more than 95% patients and disturbs proper promyelocytic differentiation. All-trans retinoic acid (ATRA) can induce granulocytic differentiation in APL and is used to treat APL patients. Genes containing PML-RARA-targeted promoters are transcriptionally suppressed in APL and most likely constitute a major mechanism of transcriptional repression occurring in APL. A growing body of evidence points to the role of distal regulatory elements, including enhancers, in the control of gene expression. In order to understand the unique sets of enhancers that might be under the control of PML-RAR and crucial for granulocytic differentiation of NB4 cells, we analysed the enhancer landscape of control and ATRA treated NB4 cells. H3K9Ac mapping identified a repertoire of enhancers that were gained in NB4 cells treated with ATRA. Closer investigation of these enhancer elements revealed enrichment of H3K9Ac signals around major drivers of myeloid differentiation. Of note, we identified a gain in enhancer signature for a region about 7kb downstream of the CEBPE gene. Our previous studies identified a novel enhancer for CEBPE in murine hematopoietic cells, which was 6 downstream of CEBPE core promoter. It appears that the +7kb region we identified in human APL cells may be analogous to the murine enhancer. We also observed that PML-RAR binds this +7kb region and ATRA treatment of NB4 cells displaced binding of PML-RAR from the + 7kb region, suggestive of a transcriptional repressive effect of PML-RAR at such enhancer elements. To test the transcription regulating potential of this +7kb region, we used catalytically inactive Cas9 fused to Krüppel associated box (KRAB) domain (dCas9-KRAB). We designed three guide RNAs covering this regulatory region. The sgRNAs effectively repressed expression of CEBPE accompanied by lowered granulocytic differentiation of these guide RNA targeted NB4 cells after ATRA treatment. To explore transcription factor (TF) occupancy at this +7 kb region, we analysed public available ChIP-seq datasets for hematopoiesis-specific factors. Analysis revealed that the +7kb region was marked by an open chromatin signature, accompanied by binding of a majority of hematopoietic TFs around this putative regulatory element with concurrent binding of EP300. Strikingly we noticed binding of CEBPA, CEBPB and CEBPE at this regulatory element. To assess whether binding of these members of the CEBP family of TFs is functionally relevant, luciferase reporter and electrophoretic mobility shift assays (EMSA) were performed. Co expression of the CEBP TFs led to significant induction of luciferase expression, and this data was further confirmed using EMSA assays. Based on these observations, we propose that PML-RAR blocks granulocytic differentiation by occupying this +7kb enhancer of CEBPE, hinders binding of other cell type/lineage specific TFs, and blocks CEBPE expression. When cells are stimulated with ATRA, PML-RAR is displaced from the CEBPE enhancer, allowing for efficient binding of myeloid-specific TFs. This results in increased CEBPE expression, which in turn promotes efficient granulocytic differentiation. The findings from our study expands our current understanding of the mechanism of differentiation therapy, the role of onco-fusion proteins in inhibiting myeloid differentiation, and may provide new therapeutic approaches to many acute myeloid leukemias. Disclosures Ong: National University of Singapore: Other: Royalties.

PML/RARa Represses Transactivation of PSMB10 Via a PU.1-Dependent Manner In Acute Promyelocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3866-3866
Author(s):  
Xianwen Yang ◽  
Ping Wang ◽  
Xujie Zhao ◽  
Huahua Zhu ◽  
Sai-Juan Chen ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Retinoic Acid ◽  
Immune System ◽  
Acute Promyelocytic Leukemia ◽  
Target Genes ◽  
Promyelocytic Leukemia ◽  
Class I ◽  
Luciferase Reporter ◽  
Atra Treatment ◽  
Nb4 Cells

Abstract Abstract 3866 Immunoproteasome is a special form of proteasome which contains three unique interferonγ (IFNγ) induced catalytic subunits, i.e. PSMB8, PSMB9 and PSMB10. Immunoproteasome plays a pivotal role in generating certain peptide antigens for MHC class I presentation. Dysregulation of the immunoproteasome system may contribute to the pathogenesis of certain types of malignancies, including leukemia. Our previous study has identified the target genes of PML/RARa, the initiating factor of acute promyelocytic leukemia (APL) on the genome-wide scale, and demonstrated that PML/RARa could selectively target PU.1-regulated genes, which is a critical mechanism for the pathogenesis of APL. PSMB10, encoding an important composition of immunoproteasome, is one of the identified target genes which are regulated by PML/RARa in this manner. Here we revealed the detailed transcriptional regulation mechanism of PSMB10 in APL. Chromatin immunoprecipitation (ChIP)-PCR assay showed that PML/RARa and PU.1 could bind to the PSMB10 promoter in APL cells, including patient derived NB4 cells and Zn-treated PR9 cells. Re-ChIP assay further demonstrated that PML/RARa and PU.1 co-existed on the same DNA fragment of the PSMB10 promoter, which provided the possibility that PML/RARa and PU.1 could co-regulate the PSMB10 promoter. Using a transient luciferase reporter system, we found that PU.1 transactivated the PSMB10 promoter and PML/RARa repressed the PU.1-dependent transactivation. All-trans retinoic acid (ATRA) could relief the repression caused by PML/RARa. To further demonstrate that the PU.1 site (-37bp∼-29bp) and related retinoic acid response elements (RAREs) (-555bp∼-549bp, -258bp∼-252bp) were essential for PML/RARa to function as an effective repressor, we prepared a series of mutant constructs, including the PU.1-site mutant, the construct mutated on both RARE half (RAREh) sites and two constructs respectively mutated on one of the two RAREh sites, and then transfected them into myeloid U937 cells. From the results of luciferase reporter assays, we found that both PU.1 site and RAREh sites played important roles in PML/RARa-mediated transcriptional repression, moreover, the second RAREh site (-258bp∼-252bp) contributed more than the first one (-555bp∼-549bp). Through electrophoretic mobility shift assay (EMSA), we further determined that PML/RARa could interact with PU.1 through protein-protein interaction, and then bind to the PU.1 site on the PSMB10 promoter. Recent study has shown that ATRA treatment could induce the production of anti-PML/RARa in APL mouse, which implicates that ATRA plays an important role in activating immune system. As the essential elements for immune response, HLA class I antigens (A, B & C) present peptides, which are produced from digested proteins degraded by immunoproteasome, to the surface of antigen-presenting cells. We thus utilized real time RT-PCR to measure the expression of PSMB10 and HLA-A/B/C during ATRA-induced NB4 cells differentiation. We found the levels of PSMB10 and HLA-A/B/C expression were up-regulated in ATRA-treated NB4 cells. These results suggested that the enhanced expression of PSMB10 availed immunoproteasome restoration, which benefited the reactivation of immune system during ATRA treatment therapy. Our results not only demonstrate the detailed transcriptional regulation of PSMB10 in APL but imply the potential function of PSMB10 during ATRA treatment as well. Disclosures: No relevant conflicts of interest to declare.

CD14 Mediates Phagocytic Activity during the Granulocytic Differentiation Process in Acute Promyelocytic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4955-4955 ◽  
Author(s):  
Hui-Chi Hsu ◽  
Wen-Hui Tsai ◽  
Yu-Chieh Lin
Keyword(s):  
Phagocytic Activity ◽  
Promyelocytic Leukemia ◽  
Time Dependent ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
Atra Treatment ◽  
Granulocytic Differentiation ◽  
Nb4 Cells ◽  
Pre Treatment

Abstract All-trans retinoic acid (ATRA) can induce acute promyelocytic leukemia (APL) cells differentiation into mature granulocytes. CD14 and Toll-like receptor 4 (TLR-4) play an important role in the phagocytic activity of macrophage, however, their role during granulopoiesis is still unclear. In this study, we determined the role of CD14/TLR-4 in the development of phagocytic activity in NB4 APL cells after induction into the process of granulocytic differentiation by ATRA. Flow cytometry analysis demonstrate that, during ATRA treatment for 6 days, the phagocytic activity of NB4 cells in engulfing either fluorescein-latex beads or idarubicin-induced apoptotic cells increased in a time-dependent manner, and the level of CD14 expression on NB4 cells was also significantly increased in a time dependent manner, though its level was only minimally expressed in ATRA-untreated NB4 cells. However, TLR-4 was constitutionally expressed in ATRA-untreated cells and its level did not changed significantly during the first 5 days of ATRA treatment. Further study demonstrates that the phagocytic activity of ATRA-NB4 cells was significantly inhibited by pre-treating cells with antibodies specific to either CD14 or TLR-4 before phagocytosis assay. In exploring the role of CD14/TLR4 associated signal transduction mediators, NF-κB and IRF-3, we further demonstrate that the phagocytic activity of ATRA-NB4 cells in engulfing beads was significantly inhibited when cells were pretreated with either a NF-κB inhibitor (BAY 11-7082) or an IRF-3 inhibitor (SP600125). However, this activity in engulfing apoptotic cells was only significantly inhibited by pretreatment with BAY11-7082, but not by pre-treatment with SP600125. Finally, our results indicate that the level of CD14(+) microparticles (MPs) released by ATRA-NB4 cells was significantly enhanced when those cells were induced into the process of apoptosis by pre-treatment with idarubicin. Moreover, by incubation with MPs derived from apoptotic ATRA-NB4 cells, the phagocytic activity of living ATRA-NB4 cells in engulfing apoptotic cells was significantly enhanced, and this phagocytic activity was also significantly inhibited by pre-treating MPs with antibody specific to CD14 before phagocytic assay. We conclude that CD14 contributes to the phagocytic activity of APL cells during the process of granulocytic differentiation. Disclosures No relevant conflicts of interest to declare.

APX3330 inhibition of the redox function of ape-1/ref-1 (Ref-1) in promyelocytic leukemia cells enhances retinoic acid (ATRA) induced myeloid differentiation and limits cell proliferation as an approach to the prevention of the retinoic acid syndrome (RAS)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14613-e14613
Author(s):  
K. A. Robertson ◽  
E. S. Colvin ◽  
M. R. Kelley ◽  
M. L. Fishel
Keyword(s):  
Flow Cytometry ◽  
Retinoic Acid ◽  
Cell Growth ◽  
Real Time ◽  
Real Time Pcr ◽  
Mapk Pathway ◽  
Promyelocytic Leukemia ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation

e14613 Background: ATRA + chemotherapy has improved the treatment of promyelocytic leukemia(APL). However, 25% of ATRA treated APL patients experience toxicities that comprise the RAS (life-threatening respiratory distress, edema, renal failure, hypotension, coagulopathy and rising blast count). One approach to prevent RAS is to limit blast proliferation and enhance myeloid differentiation. Ref-1 is a DNA repair protein that functions in redox regulation of cellular proteins, such as Fos, Jun, p53, and NFkB. HL60 myeloid leukemia cells are promyeloblasts that respond to ATRA with granulocytic differentiation/growth arrest. Prior studies suggest Ref-1 redox control is integral to ATRA-induced differentiation. To define the role of the redox function of Ref-1, we used the Ref-1 specific drug, APX3330, to block Ref-1 redox function and examined the response of HL60 cells to ATRA. Methods: Cell growth assessed using trypan blue. Differentiation was evaluated by morphology and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis assayed with propidium iodide flow cytometry. To assess activation of the MAPK pathway, BLR-1 expression was determined by real time PCR. Results: 1) APX3330 blockade of Ref-1 redox function resulted in limited cell growth yet a profound increase in differentiation and a moderate increase in apoptosis. 2) dose dependent studies with ATRA showed a similar degree of differentiation in cells treated with 10 μM ATRA to cells treated with APX3330 + 0.01 μM ATRA; allowing HL60 cells + APX3330 to give a similar response to a 1000 fold lower dose of ATRA. APX3330 alone did not induce differentiation and induced only minimal apoptosis but in combination with ATRA, increased the number of cells in G1/G0 phase significantly. 3) APX3330 + ATRA increased BLR-1 expression significantly by real time PCR suggesting enhanced activation of the MAPK pathway. Conclusions: APX3330 + ATRA limits HL60 growth and dramatically enhances terminal granulocytic differentiation. These finding may provide a therapeutic approach for prevention of the RAS. No significant financial relationships to disclose.

scholarly journals Retinoic acid is required for and potentiates differentiation of acute promyelocytic leukemia cells by nonretinoid agents

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2122-2129 ◽  
Author(s):  
A Chen ◽  
JD Licht ◽  
Y Wu ◽  
N Hellinger ◽  
W Scher ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Drug Exposure ◽  
Primary Cultures ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Granulocytic Differentiation ◽  
Nb4 Cells

Abstract Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

PKCδ Regulates Eukaryotic Initiation Factor eIF2α through PKR during Retinoic Acid-Induced Myeloid Cell Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1928-1928
Author(s):  
Bulent Ozpolat ◽  
Ugur Akar ◽  
Magaly Barria ◽  
Gabriel Lopez-Berestein
Keyword(s):  
Retinoic Acid ◽  
Cell Differentiation ◽  
Protein Kinase ◽  
Translation Initiation ◽  
Myeloid Leukemia ◽  
Mrna Translation ◽  
Leukemia Cells ◽  
Granulocytic Differentiation ◽  
Nb4 Cells

Abstract Dysregulation of mRNA translation can contribute to malignant transformation. Translation initiation is a rate limiting step of mRNA translation and protein synthesis and plays a critical role in regulation of cell growth, proliferation and differentiation. We previously reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms during terminal cell differentiation detected by proteomic analysis (Harris et al, Blood, 104 (5) 2004). Here we investigated the regulation of translation initiation and the role of eIF2α during terminal differentiation of myeloid leukemia cells. We found that ATRA and other granulocytic differentiation inducing agents, such as dimethyl sulfoxide (DMSO), arsenic trioxide (ATO) induce phosphorylation of eIF2α on serine 51 in promyelocytic leukemia (NB4) cells, indicating the suppression of translation initiation. However, monocytic/macrophagic differentiation of NB4 cells by phorbol 12-myristate 13-acetate (phorbol ester, PMA), or by ATRA in U937 and THP-1 myelomonoblastic myeloid leukemia (AML) cells, was not accompanied with induction of eIF2α phosphorylation. ATRA, ATO or DMSO-induced granulocytic differentiation closely correlated with induction of expression and phosphorylation/activation of protein kinase C-delta (PKCδ) on threonin 505 and serine 643 in NB4 cells. The specific PKCδ inhibitor, rottlerin, markedly inhibited ATRA-induced expression and phosphorylation (serin 51) of eIF2a in NB4 cells. Rottlerin reduced phosphorylation of eIF2α expression not only in the leukemia cells but also in solid tumor cells such as breast (MCF7) and pancreatic (Panc28) cancer cells. Because protein kinase R (PKR) has been shown to inhibit mRNA translation by inducing phosphorylation of eIF2α, we also examined whether this pathway is involved in ATRA-induced phosphorylation of eIF2α and whether it is downstream of PKCδ. We observed that ATRA induces expression and phosphorylation/activation of PKR in NB4 cells. Rottlerin inhibited ATRA-induced expression and activity of PKR , suggesting that activity of PKR is regulated by PKCδ in response to ATRA in NB4 cells. Overall, our data suggest that retinoic acid suppresses translation initiation through PKCδ/PKR/eIF2α pathway during granulocytic but not monocytic differentiation of acute myeloid leukemia cells. These results revealed a novel role of ATRA in granulocytic cell differentiation of myeloid cells. Because malignant cells usually have hyperactivated mRNA translation, targeting translational factors/regulators of initiation may offer new strategies for the treatment of myeloid leukemia cells.

Death Associated Protein 5 (DAP5/p97/NAT1) Contributes to Retinoic Acid-Induced Granulocytic Differentiation and Arsenic Trioxide Induced Apotosis through Inhibition of PI3K/Akt/mTOR Pathway in Acute Promyelocytic Leukemia Cells: A Novel Mechanism.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2836-2836
Author(s):  
Bulent Ozpolat ◽  
Ugur Akar ◽  
Isabel Zorilla-Calancha ◽  
Pablo Vivas-Mejia ◽  
Gabriel Lopez-Berestein
Keyword(s):  
Retinoic Acid ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Translation Initiation ◽  
Promyelocytic Leukemia ◽  
Mtor Pathway ◽  
Granulocytic Differentiation ◽  
Induced Expression ◽  
Nb4 Cells ◽  
Induced Apoptosis

Abstract All-trans Retinoic Acid (ATRA) is a naturally occurring metabolite of retinol (vitamin A)and acts as a potent inducer of cellular differentiation and growth arrest in acute promyelocytic leukemia (APL), a type of acute myeloid leukemia (M3-AML). APL is characterized by translocation t(15;17), fusing PML (promyelocytic leukemia) and RARα (retinoic acid receptor) genes, leding to expression of PML/RARα receptor protein and differentiation block. Arsenic trioxide (ATO) induces (<0.5 μM) differentiation at low doses and apoptosis at high doses (>1 μM) in APL cells. Currently, both ATRA and ATO are successfully used in the treatment of APL in the clinic. However, the molecular mechanisms of myeloid differentiation and apoptosis induced by these agents are not fully understood. We previously reported that ATRA inhibits the translation initiation through multiple mechanisms, including upregulation of translation initiation inhibitors, DAP5/p97 and PDCD4 tumor suppressor protein. Here we investigated the role and regulation of death associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in myeloid (granulocytic and monocytic) cell differentiation and apoptosis. We found that ATRA (1 μM) induced a marked DAP5/p97 protein and mRNA expression during granulocytic differentiation of NB4 and HL60 cells but not in differentiation-resistant cells, which express very low levels of DAP5/p97. DAP5/p97 was translocated into nucleus during the differentiation of NB4 cells induced ATRA. At differentiation inducing doses, ATO, dimethysulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate-13-acetate also induced a significant DAP5/p97 expression in NB4 cells. However, ATO at apoptotic doses, but not ATRA, induced DAP5/p86, a proapoptotic form of DAP5/p97. ATRA and ATO -induced expression of DAP5/p97 was associated with inhibition of phosphaditylinositol 3-kinase (PI3K)/Akt pathway, which is known to stimulate cap-dependent translation of mRNAs. To show direct link between PI3K/Akt/mTOR pathway and DAP5 expression, we treated cell with PI3K and mTOR inhibitors LY294002 and by rapamycin, respectively. We found that inhibition of PI3K/Akt/mTOR pathway upregulated DAP5/p97 expression in NB4 cells. Finally, knockdown of DAP5/p97 expression by small interfering RNA significantly inhibited ATRA-induced granulocytic differentiation detected by expression of CD11b and ATO-induced apoptosis in NB4 cells detected by Annexin V assay (p<0.05). In conclusion, our data suggest that DAP5/p97 plays a role in ATRA-induced differentiation and ATO-induced apoptosis in APL cells. Our data demonstrated for the first time that DAP5/p97 is constitutively suppressed by of PI3K/Akt/mTOR pathway, and ATRA and ATO-induced expression of DAP5 is mediated by the inhibition of this survival pathway, suggesting a novel mechanism of DAP5 regulation and a role of translational control in induction of differentiation and apoptosis. Figure Figure

Gene Expression Analysis of Bexarotene Differentiated Acute Myeloid Leukemias Identifies Known and Novel Gene Targets.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1199-1199
Author(s):  
Patricia Vanessa Sanchez ◽  
Reid P Bissonnette ◽  
Donald E Tsai ◽  
Martin Carroll
Keyword(s):  
Transcription Factor ◽  
Retinoic Acid ◽  
Cell Differentiation ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Myeloid Differentiation ◽  
Induced Differentiation ◽  
Pparγ Agonist ◽  
Acute Myeloid

Abstract Despite advances in understanding the molecular pathogenesis of acute myeloid leukemia (AML), therapy for relapsed disease remains inadequate with high mortalities. Clinicians at the University of Pennsylvania have demonstrated that the FDA approved retinoid X receptor (RXR) agonist bexarotene (Targretin™) stimulates leukemic cell differentiation in a subset patents with relapsed AML leading to clinical responses. This underscores the importance of identifying the mechanism by which bexarotene induces differentiation in AML in order to enhance the efficacy of this therapeutic approach. To understand the role of bexarotene and RXR receptors in leukemic cell differentiation, we initially utilized a pharmacogenetic approach to study the effects of bexarotene on AML cell lines using combinations of bexarotene with other differentiation induction agents. These studies demonstrate that bexarotene induces myeloid differentiation in MOLM14, HL60, THP-1, and NB4 cell lines but not in the myeloblastic cell line KG1a. Combination treatment of AML cell lines with bexarotene in combination with all trans retinoic acid (ATRA) enhanced differentiation suggesting that the mechanism of action for bexarotene is through RARα (retinoic acid receptor)/RXRα heterodimer stimulation. Consistent with this, differentiation induced by the drug combination was effectively blocked by the RAR antagonist, LG100815 and partially blocked by the RXR antagonist, LG101208. In contrast, bexarotene does not cooperate with valproic acid, theophylline, the PPARγ agonist rosiglitazone, or the LXR agonist T0901317. Preliminary data from quantitative RT-PCR and Affymetrix microarray analysis of bexarotene responsive AML cell lines at 3, 6, 12, and 96 hours post treatment has identified a subset of genes potentially regulated by bexarotene. CEBPε, a transcription factor known to play a critical role in granulopoiesis and PIM-1, a known oncogenic transcription factor, were among the genes that were significantly upregulated after bexarotene treatment of AML cells. Analysis of the functional role of C/EBPε in retinoid induced differentiation will be presented. Overall, this data supports the hypothesis that bexarotene, like ATRA, induces myeloid differentiation through activation of a RAR/RXR heterodimeric partner. However, other data suggests the presence of RAR independent pathways of signaling. LG100268, a pure RXR agonist induced myeloid differentiaton although not as robustly as bexarotene. Analysis of RAR and RXR mRNA expression in AML cell lines demonstrates that bexarotene does not induce expression of RARβ or p21, known targets induced by ATRA during myeloid differentiation. Chromatin immunoprecipitation assays demonstrate RXRα occupancy at RARβ and p21 promoter regions containing retinoid response elements (RARE). However, expression of these genes does not correlate with bexarotene-induced differentiation. This data suggests that although their expression has been linked to ATRA responsiveness, induction of RARβ and p21 expression is not necessary for retinoid induced myeloid differentiation. In summary, bexarotene induces myeloid differentiation through RAR dependent and independent pathways. Further analysis of the signaling events necessary for induction of myeloid differentiation by bexarotene may allow for improved selection of patients with AML who will respond to bexarotene.

scholarly journals Retinoic acid is required for and potentiates differentiation of acute promyelocytic leukemia cells by nonretinoid agents

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2122-2129 ◽  
Author(s):  
A Chen ◽  
JD Licht ◽  
Y Wu ◽  
N Hellinger ◽  
W Scher ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Drug Exposure ◽  
Primary Cultures ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Granulocytic Differentiation ◽  
Nb4 Cells

Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

Impact of Protein Phosphatase PP2A on ATRA-Induced Activation of FoxO3a In Acute Promyelocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2492-2492
Author(s):  
Yasuhiko Sakoe ◽  
Kumi Sakoe ◽  
Haruo Shimazaki ◽  
Keita Kirito ◽  
Norio Komatsu
Keyword(s):  
Retinoic Acid ◽  
Cell Growth ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Cellular Responses ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Atra Treatment ◽  
Granulocytic Differentiation

Abstract Abstract 2492 Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia caused by reciprocal translocations of the long arms of chromosomes 15 and 17, which prevent cellular differentiation into mature neutrophils. The translocation of the promyelocytic leukemia (PML) gene on chromosome 15 and a retinoic acid receptor α (RARα) gene on chromosome 17 generates a PML-RARα fusion protein that inhibits PML-dependent apoptotic pathways in a dominant negative fashion. This fusion protein also blocks granulocytic differentiation by direct transcriptional inhibition of retinoic acid target genes. All-trans retinoic acid (ATRA) arrests cell growth, granulocytic differentiation, and apoptosis in APL cells via proteasome-dependent degradation of PML-RARα fusion protein and subsequent PML-nuclear body (NB) formation. Although PML is the essential component of PML-NBs and functions as a tumor suppressor, disruption of PML-NBs by the PML-RARα fusion protein inhibits endogenous PML tumor-suppressive functions in APL cells. Therefore, degradation of PML-RARα fusion protein and reorganization of PML-NBs during ATRA treatment are regarded as critical cellular responses, similar to the cell growth arrest and apoptosis of leukemia cells. Recently we demonstrated that FoxO3a (also named FKHRL1), a member of the Forkhead family of transcription factors, is a key molecule for the ATRA-induced cellular responses in APL cells (Blood 2010; 115: 3787–3795). In this study, we investigated the mechanism by which FoxO3a is activated by ATRA treatment in a human promyelocytic leukemia cell line NB4. Okadaic acid, a potent PP2A inhibitor, cancelled ATRA-induced dephosphorylation of AKT and its downstream molecule FoxO3a in NB4 cells. Knockdown of endogenous PP2A by siRNA significantly enhanced phosphorylation of both AKT and FoxO3a. These results suggested that PP2A is involved in ATRA-induced dephosphorylation of AKT and FoxO3a. Concomitantly, PP2AC, a catalytic subunit of PP2A, was dephoshorylated at tyrosine 307, and phosphatase activity of PP2A increased after ATRA treatment. Co-immunoprecipitation assay revealed that PP2A constitutively and directly binds to FoxO3a. Using artificial oligopeptides, we demonstrated that enhanced PP2A activity by ATRA directly dephosphorylates phosphothreonine 32 on FoxO3a. In addition, we found that 14-3-3 epsilon binded to phosphorylated FoxO3a in the cytoplasm in the absence of ATRA. After ATRA treatment, however, dephosphorylated FoxO3a dissociated from 14-3-3 epsilon and moved into the nucleus. Confocal microscopic analysis revealed that PP2A-FoxO3a complex partially co-localized with PML-NBs in the nucleus after ATRA treatment. Together, PML orchestrates nuclear networking with PP2A and FoxO3a for ATRA-induced granulocytic differentiation and apoptosis of APL cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effects of Fifty-Hertz Electromagnetic Fields on Granulocytic Differentiation of ATRA-Treated Acute Promyelocytic Leukemia NB4 Cells

2018 ◽  
Vol 46 (1) ◽  
pp. 389-400 ◽  
Author(s):  
Alfredo Errico Provenzano ◽  
Stefano Amatori ◽  
Maria Gemma Nasoni ◽  
Giuseppe Persico ◽  
Sergio Russo ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Electromagnetic Fields ◽  
Cellular Proliferation ◽  
Biological Effects ◽  
Promyelocytic Leukemia ◽  
Ros Generation ◽  
Granulocytic Differentiation ◽  
Nb4 Cells ◽  
Oxidative Balance

Background/Aims: Life on Earth is constantly exposed to electromagnetic fields (EMFs) and the effects induced by EMFs on biological systems have been extensively studied producing different and sometimes contradictory results. Extremely low-frequency electromagnetic fields (ELF-EMFs) have shown to play a role in regulating cell proliferation and differentiation, although how EMFs influence these processes remains unclear. Human acute promyelocytic leukemia (APL) cells are characterized by the arrest of differentiation at the promyelocytic stage due to epigenetic perturbations induced by PML/RARα fusion protein (Promyelocytic Leukemia protein - PML/Retinoic Acid Receptor alpha - RARα). Therapeutic administration of all-trans retinoic acid (ATRA) re-establishes the leukemogenic mechanism re-inducing the normal differentiation processes. Methods: We studied the effects of ELF-EMFs (50 Hz, 2 mT) on the ATRA-mediated granulocytic differentiation process of APL NB4 cells (a cell line established from the bone marrow of a patient affected by the acute promyelocytic leukemia) by monitoring cellular proliferation and morphology, nitrob lue tetrazolium (NBT) reduction and the expression of differentiation surface markers. Finally, we investigated mechanisms focusing on reactive oxygen species (ROS) generation and related molecular pathways. Results: ELF-EMF exposure decreases cellular proliferation potential and helps ATRA-treated NB4 cells to mature. Furthermore, the analysis of ROS production and the consequent extracellular signal regulated kinases (ERK1/2) phosphorylation suggest that a changed intracellular oxidative balance may influence the biological effects of ELF-EMFs. Conclusions: These results indicate that the exposure to ELF-EMF promotes ATRA-induced granulocytic differentiation of APL cells.

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