1: Biochem Biophys Res Commun. 2003 Oct 3;309(4):910-6. Effect of cysteamine on redox-sensitive thiol-containing proteins in the duodenal mucosa. Khomenko T, Deng X, Jadus MR, Szabo S. Pathology and Laboratory Medicine Service, Diagnostic and Molecular Medicine Health Care Group, VA Medical Center, Long Beach, CA 90822, USA. Recent studies from our laboratory demonstrated that Egr-1 is upregulated in the rat duodenal mucosa during cysteamine-induced duodenal ulceration and that antisense egr-1 oligonucleotide aggravates the duodenal ulcers. This study was aimed to determine the effects of cysteamine on redox-sensitive Egr-1 transcriptional activity and on other thiol-containing proteins such as redox factor-1 (Ref-1) and thioredoxin (Trx). Here we demonstrate for the first time that cysteamine increases the expression and nuclear translocation of Egr-1, Ref-1, and Trx, and activates binding of Egr-1 to DNA. Moreover, we also show that Egr-1 forms a complex with other redox-sensitive transcription factors (e.g., AP-1, AP-2, NFATc, Sp1, PAX-5, MTF-1, c-Myb, and CREB) in rat duodenal mucosa and that cysteamine enhances the formation of these complexes. The antioxidant ebselen markedly elevated the nuclear Ref-1 expression and Egr-1/DNA binding, and decreased the ulcerogenic effect of cysteamine as did catalase. Thus, redox-sensitive signaling systems seem to play an important role in cysteamine-induced duodenal ulceration. PMID: 13679060 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Immunol Lett. 1999 May 3;68(1):187-95. Signalling mechanisms and the role of calcineurin in erythropoiesis. Magocsi M, Apati A, Gati R, Kolonics A. Department of Cell Metabolism, National Institute of Haematology and Immunology, Budapest, Hungary. Erythropoietin (Epo) is the principal regulator of the production of circulating erythrocytes by controlling the proliferation, the differentiation and the survival of the erythroid progenitor cells. Early down-regulation of c-myb expression in erythroleukemia cells is a common feature of the action of Epo and chemical inducers of differentiation such as DMSO. Previously we have shown that in our Epo-responsive murine erythroleukemia cell line ELM-I-1, [Ca2+]i increasing agents can mimic the effect of Epo on c-myb expression and activate nuclear signal transduction processes involved in the induction of hemoglobin synthesis. These results also indicated that the Ca2+-induced down-regulation of c-myb expression and hemoglobin synthesis are mediated by the Ca2+/calmodulin dependent serine/threonine-specific protein phosphatase PP2B, calcineurin, but the Epo induced processes are not mediated by PP2B. In spite of this, we demonstrated in this paper that in ELM-I-1 cells the Epo-induced down-regulation of c-myb expression and hemoglobin production can be effectively enhanced by the simultaneously added [Ca2+]i-increasing agent, cyclopiazonic acid (CPA). This observation further supports the existence of at least two independent signalling pathways in the mechanism of Epo and [Ca2+]i increasing agents and the strong correlation between c-myb expression and hemoglobin production in differentiating cells. Although the c-AMP-response element binding protein (CREB) could be the common target of both calcium-dependent and -independent dephosphorylation, our results do not support the involvement of CREB in the regulation of c-myb gene expression. In addition to the calcineurin mediated down-regulation of c-myb expression, we have found a negative regulatory effect in the Ca2+-mediated transcriptional activation of certain genes. In response to [Ca2+]i-increasing agents in ELM-I-1 cells, both, egr-1 and c-fos mRNA expression increased significantly after the inhibition of calcineurin by cyclosporine A. Cyclosporin A exerted stimulatory effects on the egr-1 and c-fos expression also at lower (150-400 nM) intracellular Ca2+ levels. This potential co-regulation of c-myb, egr-1 and c-fos expression by calcineurin suggests that the negative modulation of egr-1 and c-fos expression may also be important for the induction of erythroid differentiation by [Ca2+]i-increasing agents. This negative modulation may also contribute to the Epo-induced differentiation in the case of a moderate increase of [Ca2+]i. PMID: 10397175 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Biochem J. 1998 Nov 1;335 ( Pt 3):505-11. Stimulation of the Ca2+-mediated egr-1 and c-fos expression in murine erythroleukaemia cells by cyclosporin A. Schaefer A, Magocsi M, Fandrich A, Marquardt H. Department of Toxicology, Hamburg University Medical School and Fraunhofer Department of Toxicology and Environmental Medicine, Grindelallee 117, D-20146 Hamburg, Germany. aschaefe@uke.uni-hamburg.de The Ca2+-induced expression of the primary response genes egr-1 and c-fos was investigated in the murine erythroleukaemia cell line ELM-I-1. Exposure of the cells to the Ca2+-ionophore A23187 led to a rapid transient rise in egr-1 and c-fos mRNA production followed by an increase in Egr-1 and c-Fos protein levels as well as an increase in Egr-1 and activator protein 1 (AP-1) DNA-binding activity. Preincubation of the cells with KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinases, strongly decreased the Ca2+-mediated expression of egr-1 and c-fos. In contrast, treatment with cyclosporin A, which inhibits the Ca2+/calmodulin-dependent protein phosphatase 2B or calcineurin, increased both egr-1 and c-fos mRNA production and the DNA-binding activity of the Egr-1 and AP-1 transcription factors in response to the intracellular Ca+ concentration ([Ca2+]i)-increasing agents A23187 or cyclopiazonic acid. Enhancement of the Ca2+-induced c-fos and egr-1 expression by cyclosporin A was correlated with the capability of this agent to inhibit calcineurin phosphatase activity in ELM-I-1 cells. Studies on the phosphorylation state and DNA-binding activity of the cAMP response element-binding protein (CREB) did not demonstrate an early Ca2+-dependent activation of this transcription factor, suggesting that the regulation of c-fos and egr-1 expression by Ca2+ is not linked to CREB in the haematopoietic ELM-I-1 cells. The results indicate that calcineurin exerts negative regulatory effects on both egr-1 and c-fos expression in murine erythroleukaemia cells, in addition to the calcineurin-mediated down-regulation of c-myb expression observed previously in this cell system. This study therefore emphasizes the important role of calcineurin as a negative modulator of gene expression in certain cell types. PMID: 9794788 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Blood. 1998 Sep 15;92(6):1957-66. The zinc finger transcription factor Egr-1 activates macrophage differentiation in M1 myeloblastic leukemia cells. Krishnaraju K, Hoffman B, Liebermann DA. Fels Institute for Cancer Research and Molecular Biology, and Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, USA. We previously have shown that the zinc finger transcription factor Egr-1 blocked granulocytic differentiation of HL-60 cells, restricting differentiation along the monocytic lineage. Egr-1 also was observed to block granulocyte colony-stimulating factor (G-CSF)-induced differentiation of interleukin-3 (IL-3)-dependent 32Dcl3 hematopoietic precursor cells, endowing the cells with the ability to be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) for terminal differentiation along the macrophage lineage. To better understand the function of Egr-1 as a positive modulator of monocytic differentiation, in this work we have studied the effect of ectopic expression of Egr-1 on the murine myeloblastic leukemic cell line M1, which is induced for differentiation by the physiological inducer IL-6. It is shown that, unlike in HL-60 and 32Dcl3 cells, ectopic expression of Egr-1 in M1 cells resulted in activation of the macrophage differentiation program in the absence of differentiation inducer. This included the appearance of morphologically differentiated cells, decreased growth rate in mass culture, and cloning efficiency in soft agar, and expression of endogenous c-myb and c-myc mRNAs was markedly downregulated. Untreated M1Egr-1 cells also exhibited cell adherence, expression of Fc and C3 receptors, and upregulation of the myeloid differentiation primary response genes c-Jun, junD, and junB and the late genetic markers ferritin light-chain and lysozyme. Ectopic expression of Egr-1 in M1 cells also dramatically increased the sensitivity of the cells for IL-6-induced differentiation, allowed a higher proportion of M1 cells to become terminally differentiated under conditions of optimal stimulation for differentiation, and decreased M1 leukemogenicity in vivo. These findings demonstrate that the functions of Egr-1 as a positive modulator of macrophage differentiation vary, depending on the state of lineage commitment for differentiation of the hematopoietic cell type. Copyright 1998 by The American Society of Hematology. PMID: 9731053 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Anticancer Res. 1996 Nov-Dec;16(6B):3483-9. Deoxyadenosine-resistant mouse leukemia L1210 cell lines with alterations in early response genes and p53. Cory JG, He AW, Cory AH. Department of Biochemistry, East Carolina University School of Medicine, Greenville, NC 27858, USA. L1210 cell lines selected for resistance to deoxyadenosine exhibit altered steady-state levels of the mRNA for the early response genes and p53. In the deoxyadenosine-resistant cell lines (Y8 and ED2), the levels of the mRNAs for p53 and c-jun were markedly decreased while the steady-state levels for mRNAs for c-myc, c-fos and jun B were elevated in the Y-8 and ED2 cell lines. The levels of the mRNAs for PCNA and c-myb were the same in the wild type and mutant cell lines. The levels of the mRNAs for krox-24 were extremely low in the wild type and mutant cell lines. Cycloheximide (CHX) treatment of the cells resulted in the increase in the mRNA levels for c-jun, jun B, krox 24 and p53 in the Y-8 and ED2 cell lines. The time courses and the extents of the increases in the mRNA levels following CHX treatment were not the same for all of these mRNAs. The level of p53 RNA increased with no lag following CHX treatment while the levels of the mRNAs for c-myc, c-jun and krox-24 increased after a one-hour lag period. The level of the mRNA for p53 and c-myc increased 20- and 7-fold, respectively while the mRNA level for knox-24 increased 80-fold following CHX treatment. The Y8 and ED2 cell lines that lack steady-state levels of p53 show decreased sensitivity to cisplatin and increased frequency of gene amplification as measured by PALA resistance in a manner similar to other cell lines lacking p53. On the other hand, the ED2 and Y8 cell lines do not show a G1-block in response to PALA treatment. The cell lines appear to offer an experimental system in which to study the interactions between/among these early response genes and the p53-dependent and independent pathways. PMID: 9042210 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Stem Cells. 1994 Jul;12(4):352-69. Differentiation primary response genes and proto-oncogenes as positive and negative regulators of terminal hematopoietic cell differentiation. Liebermann DA, Hoffman B. Fels Institute for Cancer Research and Molecular Biology, Temple University, School of Medicine, Philadelphia, Pennsylvania 19140. By genetically manipulating hematopoietic cells of the myeloid lineage, including both normal cells and differentiation inducible leukemic cell lines, evidence was obtained to indicate that myeloid differentiation primary response (MyD) genes and proto-oncogenes, which are known to control cell growth, function as positive and negative regulators of terminal hematopoietic cell differentiation, which is associated with inhibition of cell growth, and, ultimately programmed cell death (apoptosis). Interferon regulatory factor-1 (IRF-1), an MyD gene induced by Interleukin 6 (IL-6) or Leukemia Inhibitory factor (LIF), plays a role in growth inhibition associated with terminal differentiation. Leucine zipper transcription factors of the fos/jun family, also identified as MyD genes, function as positive regulators of hematopoietic cell differentiation, increasing the propensity of myeloblastic leukemia cells to be induced for differentiation in vitro, and reducing the aggressiveness of their leukemic phenotype in vivo. The zinc finger transcription factor EGR-1, an MyD gene specifically induced upon macrophage differentiation, was shown to be essential for and to restrict differentiation along the macrophage lineage. Finally, evidence has been accumulating to indicate that the novel MyD genes--MyD116, MyD118 and gadd45 (a member in the MyD118 gene family)--play a role in growth arrest and apoptosis of hematopoietic cells, as well as other cell types. The proto-oncogenes c-myc and c-myb, known to regulate cellular growth, were shown to function as negative regulators of terminal differentiation. Both c-myc and c-myb are normally expressed in proliferating myeloblasts and suppressed following induction of differentiation. Deregulated and continuous expression of c-myc was shown to block terminal myeloid differentiation at an intermediate stage in the progression from immature blasts to mature macrophages, whereas deregulated and continuous expression of c-myb blocked the terminal differentiation program at the immature myeloblast stage. By manipulating myc function in conditional (differentiation inducible) mutant myeloblastic leukemia cell lines, expressing a chimeric mycer transgene, it was shown that there is a window during myeloid differentiation, after the addition of the differentiation inducer, when the terminal differentiation program switches from being dependent on c-myc suppression to becoming c-myc suppression independent, and where activation of c-myc has no apparent effect on mature macrophages. These myeloblastic leukemia cell lines provide a powerful tool to increase our understanding of the role of c-myc in normal hematopoiesis and in leukemogenesis, while also providing a strategy to clone myc target genes.(ABSTRACT TRUNCATED AT 250 WORDS) Publication Types: Review PMID: 7951003 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------