Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.
Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.
All-trans retinoic acid (RA) induces granulocytic differentiationbind all-trans RA as well as 9-cis RA, while RXRs bind only of acute promyelocytic leukemia cells both in vivo and in vitro.9-cis RA. [19][20][21] Thus, RARs heterodimerize to form RAR/RXR sociated from induction of differentiation and/or apoptosis of
The pGEX-2T expression vector was used to produce the ligand-binding domain from the human retinoic acid receptor alpha (hRAR alpha LBD) in Escherichia coli. The resulting fusion protein, containing the glutathione S-transferase separated from the truncated receptor (hRAR alpha 186-462) by a thrombin cleavage site, was purified with use of affinity chromatography on immobilized glutathione. A 90% homogeneity was obtained, with a specific activity of 100 pmol/mg and an overall 10% yield. Following purification and thrombin cleavage, a predominant monomeric (stokes radius = 2.3 nm, molecular mass of 32 kDa) [3H]retinoic acid hRAR alpha LBD complex was characterized by high-performance size-exclusion chromatography. The purified hRAR alpha LBD bound retinoic acid with an apparent Kd of 9 nM, a value close to the Kd of the full-length hRAR alpha expressed in COS cells. Kinetic studies at 0 degrees C demonstrate that the association of [3H]retinoic acid and [3H]CD367, a synthetic retinoid, to the overexpressed receptor was extremely rapid (complete in less than 3 min), whereas their dissociation from the receptor was slower, with half-lives of about 40 min at 0 degrees C. Experiments performed at various subzero temperatures allowed a more accurate assay of the association rate constant and indicate that the entropy of activation (delta Sa) is positive, which is characteristic of hydrophobic interactions. The ligand-binding activity was markedly decreased by pretreatment with various sulfhydryl modifying agents. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) appeared to be the most potent, whereas iodoacetamide was the least active. Furthermore, a series of N-alkylmaleimides was shown to inactivate the recombinant receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.