Control of cell differentiation during proliferation — II. Myeloid differentiation and cell cycle arrest of HL-60 promyelocytes preceded by nuclear structural changes

1985 ◽  
Vol 9 (1) ◽  
pp. 51-71 ◽  
Author(s):  
Andrew Yen ◽  
Sara L. Reece ◽  
Kevin L. Albright
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
Cell Cycle Arrest ◽  
Structural Changes ◽  
Myeloid Differentiation ◽  
Cycle Arrest

scholarly journals Adenosine Signaling Perturbs Erythropoiesis and Promotes Myeloid Differentiation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1992-1992
Author(s):  
Mahmoud Mikdar ◽  
Marion Serra ◽  
Elia Colin ◽  
Yves Colin Aronovicz ◽  
Caroline Le Van Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Tissue Injury ◽  
Myeloid Differentiation ◽  
Hematopoietic Progenitors ◽  
Extracellular Adenosine ◽  
Cycle Arrest ◽  
Erythroid Precursors ◽  
Adenosine Signaling

Abstract Background Adenosine is a major signaling nucleoside that activates cellular signaling pathways through a family of four different G protein-coupled adenosine receptors (ARs), A 1, A 2A, A 2B, and A 3. At steady state conditions, extracellular levels of adenosine remain low (10 to 200 nM) either through its rapid cellular uptake by specialized nucleoside transporters, mainly through the equilibrative nucleoside transporter 1 (ENT1), or its degradation by adenosine deaminases. However, the extracellular levels of adenosine can be rapidly elevated up to 100 μM in response to hypoxia, inflammation, or tissue injury. Under pathophysiological conditions, adenosine signaling is involved in modulating inflammation, fibrosis, and ischemic tissue injury. In sickle cell disease (SCD), adenosine signaling is enhanced and contributes to the pathophysiology of the disease. Despite the importance of adenosine signaling in regulating cell proliferation, and stem cell regeneration, as well as in red blood cell functions and adaptation to hypoxia, very little is known about its implication in hematopoiesis, and more specifically during erythropoiesis. Here, we aimed to investigate the effects of high extracellular adenosine on the erythroid commitment and differentiation of hematopoietic progenitors, and to decipher the implication of ARs in these processes. Results To investigate the role of high extracellular adenosine in regulating erythroid commitment and differentiation of hematopoietic progenitors, we performed ex vivo erythropoiesis of healthy CD34 + cells in the presence or absence of increased extracellular adenosine concentrations ranging from 10 to 200 µM. Our results showed that adenosine decreases erythroid proliferation in a dose dependent manner. High adenosine levels (>50μM) inhibited the proliferation of erythroid precursors and increased apoptosis via a cell cycle arrest in G1. Accordingly, western blots revealed the accumulation of p53 and its downstream target p21, a well-known mediator of G1 cell-cycle arrest, in adenosine-treated cells. Moreover, adenosine treatment led to the persistence of a non-erythroid GPA neg subpopulation expressing myeloid markers (CD18, CD11a, CD13, CD33). May-Grünwald Giemsa staining of this subset revealed granular cells at different stages of differentiation. The culture of FACS-sorted CD36 + and CD36 - cells suggested that this adenosine-induced GPA neg population originates from the survival of CD36 - myeloid progenitors even in the presence of erythropoietin. Importantly, these effects were specific to adenosine as neither guanosine, uridine nor cytidine affected the proliferation and differentiation of erythroid precursors. Furthermore, we have recently shown that ENT1-mediated adenosine uptake is essential for optimal erythroid differentiation. Therefore, we suggested that elevated extracellular adenosine perturbs erythropoiesis via its signaling upon ARs activation. To confirm this hypothesis, we assessed the effect of ARs activation during erythropoiesis. Given that A 2B and A 3 are the only known ARs expressed in human hematopoietic progenitors and erythroid precursors, we used BAY60-6583 and CI-IB-MECA, two highly selective agonists for A 2B and A 3 receptors, respectively. Both BAY60-6583 and CI-IB-MECA increased apoptosis and decreased erythroblast maturation and enucleation, while only Cl-IB-MECA led to the upregulation of CD33 and CD11a myeloid markers and promoted the differentiation of a GPA neg myeloid subpopulation. Conclusion Overall, our results place adenosine signaling as a new player in hematopoiesis regulation. Adenosine signaling via A 3 perturbs erythropoiesis and promotes the survival and differentiation of myeloid progenitors even in an erythroid favoring environment. While the activation of A 2B hampers optimal erythropoiesis without impacting the myeloid differentiation. As both ineffective erythropoiesis and increased leucocyte counts are reported in SCD, and given the detrimental role of high adenosine levels in its pathophysiology, further studies are ongoing to address the impact of adenosine signaling on hematopoiesis in this disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Elevated EGR2 Expression Provides a Potential Link Between Cell Cycle Arrest and Induced Differentiation in Myeloid Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2733-2733
Author(s):  
Joshua B. Bland ◽  
Jose R. Peralta ◽  
William T. Tse
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Progenitor Cells ◽  
Myeloid Differentiation ◽  
Phenotypic Effect ◽  
Induced Differentiation ◽  
Hl60 Cells ◽  
Cycle Arrest ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

Abstract Similar to many immature cell types, myeloid progenitor cells need to exit cell cycle to undergo terminal differentiation, but the mechanism linking the two is still unclear. Elucidating this mechanism could lead to the development of new differentiation therapies to treat myeloid leukemia. Recent studies have suggested that the processes regulating myeloid differentiation and cell cycle progression together constitute a positive feedback loop where each process reciprocally affects the other. To study the relationship between these processes, we examined early cellular and molecular events associated with induced differentiation of the HL60 human promyelocytic leukemia cells. We treated HL60 cells with 3 classical inducers of differentiation (vitamin D3 analog EB1089 (EB), all-trans retinoic acid (ATRA), and dimethyl sulfoxide (DMSO)), along with PD0332991 (PD), a selective cyclin D-dependent kinase 4/6 inhibitor that caused G1-phase-specific cell-cycle arrest. We evaluated differentiation of the treated cells by flow cytometric analysis of CD11b (integrin αM) and CD71 (transferrin receptor) expression. In untreated HL60 cells, a baseline subset of 3-5% of cells exhibits a differentiated, CD11b+CD71- phenotype. Exposure to the various inducers revealed a progressive increase in the percentage of CD11b+CD71- cells with time, such that by day 4 of treatment, it has increased to 50-90% in the treated samples, indicating that all 4 agents tested were effective in inducing myeloid differentiation. To understand how differentiation induced by each agent affects cell cycle progression, the cell cycle status of the induced cells were evaluated by a BrdU-incorporation assay after a 30-minute pulse of BrdU labeling. Uninduced cells exhibited a baseline cell cycle phase distribution of 64%-28%-8% (G1-S-G2/M phases). After 1 day of induction, EB-treated sample showed no changes in the distribution (58%-33%-9%), but ATRA, DMSO and PD-treated samples showed significant changes, with an increase of cell numbers in G1 phase and decrease in S phase (74%-18%-8%, 79%-13%-8%, and 93%-4%-3%, respectively). These results reveal that an early induction of G1 arrest was caused by treatment with ATRA, DMSO and PD, but not EB, and that the cell cycle arrest occurred before major changes in the myeloid phenotype were observed. To determine how the cell cycle perturbation relates to changes in the underlying genetic regulatory network, we examined by quantitative RT-PCR analysis the expression of several transcription factors associated with myeloid differentiation. PU.1 and CEBPA were found to be expressed at high levels but these levels did not change upon treatment with the inducing agents. Similarly, the expression levels of GFI1 and EGR1 did not change significantly with induction. In contrast, the expression level of EGR2 (Early Growth Response 2) was found to be low initially but became elevated upon treatment with 3 of the 4 inducers. EGR2 is a zinc finger transcription factor implicated in the control of a switch between pro- and anti-proliferation pathways. EGR2 has been shown to regulate the transition between differentiation states of Schwann cells, induction of anergic and regulatory T cells, growth and survival of osteoclasts, and proliferation and apoptosis of acute myeloid leukemia blasts. We found that EGR2 expression, after 1 day of treatment with ATRA, DMSO or PD, was increased by 5.2 ± 0.9, 7.6 ± 1.9, 5.8 ± 0.9 folds, respectively, whereas treatment with EB led to no significant change (1.5 ± 0.2 fold). We evaluated whether simultaneous treatment of the cells with 2 inducers would result in an additive effect. Treatment of HL60 cells with a combination of ATRA/DMSO, ATRA/PD, or DMSO/PD increased the percentage of CD11b+CD71- cells to 55%, 70% and 25% after just 1 day of treatment. In line with the enhanced phenotypic effect, the expression level of EGR2 was further elevated to 7.7 ± 1.4, 15.4 ± 3.5, and 11.3 ± 3.4 folds, respectively, when the cells were treated with the above inducer combinations, indicating a tight association between EGR2 expression and the phenotypic effect. In summary, our data suggest that elevated expression of EGR2 is an early event in the induction of myeloid differentiation in HL60 cells. Because of its known role in cell cycle regulation, EGR2 could function as a mechanistic link between cell cycle arrest and induced differentiation in myeloid progenitor cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Silencing of miR-124 induces neuroblastoma SK-N-SH cell differentiation, cell cycle arrest and apoptosis through promoting AHR

FEBS Letters ◽  
2011 ◽  
Vol 585 (22) ◽  
pp. 3582-3586 ◽  
Author(s):  
Tsui-Chin Huang ◽  
Hsin-Yi Chang ◽  
Cheng-Yu Chen ◽  
Pei-Yi Wu ◽  
Hsinyu Lee ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
Cell Cycle Arrest ◽  
Cycle Arrest

scholarly journals Icariin induces cell differentiation and cell cycle arrest in mouse melanoma B16 cells via Erk1/2-p38-JNK-dependent pathway

Oncotarget ◽  
2017 ◽  
Vol 8 (59) ◽  
pp. 99504-99513 ◽  
Author(s):  
Dan Wang ◽  
Wenjuan Xu ◽  
Xiaoyu Chen ◽  
Jichun Han ◽  
Lina Yu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
Cell Cycle Arrest ◽  
Mouse Melanoma ◽  
Melanoma B16 ◽  
Cycle Arrest ◽  
Dependent Pathway

Role of Different C/EBPα Mutations in AML Transformation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1343-1343
Author(s):  
Oscar Quintana-Bustamante ◽  
S. Lan-Lan Smith ◽  
Jude Fitzgibbon ◽  
Dominique Bonnet
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation ◽  
Self Renewal ◽  
Cycle Arrest

Abstract Acute Myeloid Leukemia (AML) is characterized by an abnormal hematopoietic differentiation and uncontrolled cell proliferation. Mutations in several transcription factors (TFs) have been implicated in the development of leukemia. One of these TFs is CCAAT/enhancer-binding protein-α (C/EBPα). In normal hematopoiesis, C/EBPα plays a central role to coordinate myeloid differentiation and growth arrest. C/EBPα is mutated in approximately 9% of AML; these mutations take place either in C or N terminal domains of the protein, although there are several familial cases of AML where both types of mutations have been found. We use C and/or N terminal C/EBPα mutations from one case of sporadic AML to investigate the role of each mutation in leukemic transformation (Smith et al., 2004, N Engl J Med 351, 2403–2407). Human lineage negative (Lin-) umbilical cord blood were transduced with lentiviral vectors carrying the wild type C/EBPα (WT), N terminal mutated C/EBPα (N-ter) or N and C terminal mutated (NC-ter) C/EBPα cloned from this sporadic case of AML. We observed differences in proliferation of transduced Lin- in vitro: WT C/EBPα expression resulted in G0 cell cycle arrest causing a progressive extinction of the transduced cells overtime; N-ter cells showed a higher proliferative advantage over untransduced cells. The NC-ter CEBPα cells like untransduced cells kept their levels throughout culture. Furthermore, when induced into myeloid differentiation in vitro, WT C/EBPα cells were mainly inducing fully mature granulocytes whereas N-ter C/EBPα was not able to induce terminal granulocytic differentiation; in contrast NC-ter C/EBPα did not increase myeloid differentiation. Additionally, their ability to form Colony Forming Units (CFUs) in primary, secondary and tertiary replating was also tested: WT transduced cells gave rise to few primary CFUs; contrary, N and NC-ter could generate both primary and secondary CFUs, but only NC-ter cells were able to produce CFUs in tertiary replating, indicating its ability to maintain undifferentiated hematopoietic progenitors in vitro. These results were confirmed using Long-Term Culture Initiating Cells (LTC-IC) where the NC-ter mutated cells showed the highest LTC-IC after 5 weeks. Finally, in vivo transplantation in NOD/SCID/β2mnull indicated that NC-ter mutated cells engraft better than WT and N-ter 8 week post- transplant. Serial transplantation experiments are underway to evaluate their self-renewal capacity. Our results confirmed some known functions of WT C/EBPα in human hematopoiesis, such as inducing myeloid differentiation and cell cycle arrest. On the other hand, we showed new functions for the C/EBPα mutants. The N-ter C/EBPα mutation caused an increase in cell proliferation and blockage of terminal granulocytic differentiation, whereas the NC-ter C/EBPα mutation increased the self-renewal capacity of progenitor/stem cells without having an influence on myeloid differentiation. This work provides further insight into the mechanisms by which different C/EBPα mutations induce AML.

TTC5, a TPR Motif Cofactor Protein Required for Myeloid Leukaemogenesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 59-59
Author(s):  
James T Lynch ◽  
Tim Somervaille
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Cell Cycle Arrest ◽  
Critical Role ◽  
Short Latency ◽  
Gene Set Enrichment Analysis ◽  
Myeloid Differentiation ◽  
Published Data ◽  
Down Regulation ◽  
Cycle Arrest

Abstract Abstract 59 Identification of genes and cellular pathways that are critical for the function of leukemia stem cells (LSC) could lead to novel therapeutic approaches. We present data which demonstrates that TTC5 (also called Strap) is required in acute leukaemogenesis. In a targeted lentiviral shRNA knockdown (KD) screen of chromatin regulatory factors, we identified the EP300 co-factor TTC5 as required for survival of human THP-1 MLL-AF9 acute monoblastic leukaemia cells. TTC5 is a tetratricopeptide repeat (TPR) motif protein known to be a stress responsive co-factor and activator of the histone acetyltransferase EP300. Using two separate shRNAs targeting different parts of the transcript, TTC5 KD in THP-1 cells resulted in G1 cell cycle arrest and the initiation of myeloid differentiation (as evidenced by down regulation of KIT/CD117 and up regulation of the monocyte differentiation marker CD36 over 48–72 hours) prior to apoptosis. Colony formation in semi-solid culture was completely abolished. TTC5 KD also induced apoptosis and loss of colony forming potential in other myeloid leukaemia cell lines including Fujioka, Kasumi-1, NB4, HL60, K562 and Mono-Mac-1. In xenotransplantation assays, TTC5 KD THP-1 cells failed to initiate AML in contract to control cells infected with a non-targeting control (NTC) hairpin, which induced short latency AML (range 51–72 days). Likewise, lentivirally infected TTC5 KD primary human acute lymphoblastic leukaemia cells from a patient with a t(4;11) translocation, the cytogenetic hallmark of MLL-AF4, failed to induce lethal leukemia when xenografted into neonatal mice, in contrast to NTC cells which induced lethal ALL in recipients of median latency 117 days. Separately, TTC5 KD induced proliferative arrest, differentiation and death in in vitro analyses of primary human AML cells which exhibited MLL associated translocations or trisomy 11 (frequently associated with a partial tandem duplication of MLL) (n=4 patients). These data suggest a critical role for TTC5 in human leukaemic haematopoiesis. To confirm our conclusions, we extended our investigations in a murine model of human leukaemic haematopoiesis initiated by MLL-AF9. TTC5 KD MLL-AF9 AML cells formed significantly fewer colonies in methylcellulose versus control cells and failed to initiate AML in secondary transplantation experiments versus control cells which induced short latency disease (48 days). To investigate the mechanism by which TTC5 knockdown initiates the cell cycle arrest and myeloid differentiation phenotype observed in THP-1 cells, we performed exon array analyses 48 hours following lentiviral infection. Gene set enrichment analysis (GSEA) demonstrated co-ordinate down regulation of an MLL leukemia stem cell maintenance program as well as genes bound by the transcription factor MYB. In contrast there was co-ordinate up regulation of genes known to be positively regulated by PU.1 or IRF8, two myeloid transcription factors that promote differentiation. These significant transcriptional changes occurred in the absence of change in the levels of MYB or PU.1 transcripts, arguing that TTC5 may be critical in regulating the function of these transcription factors rather than modulating their expression. Published data from the FANTOM4 consortium identified MYB and, to a lesser extent, PU.1 as key transcriptional regulators of CD36 expression (Suzuki et al., 2008, Nature Genetics). We confirmed up regulation of CD36 upon MYB knockdown in THP-1 cells. CD36 up regulation was not observed when either MYC or EP300 was knocked down, suggesting that TTC5 is required for MYB function and loss of TTC5 perturbs MYB activity, initiating a differentiation program. The mechanism by which TTC5 regulates MYB activity is under investigation but appears independent of EP300. We propose that in addition to the role of TTC5 in the stress response, it is a critical cofactor protein that regulates haematopoietic transcription factors and is required for human leukaemogenesis. Disclosures: No relevant conflicts of interest to declare.

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