Posttranslational Regulation of Myc Function in Response to Phorbol Ester/Interferon-γ–Induced Differentiation of v-Myc–Transformed U-937 Monoblasts

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3900-3912 ◽  
Author(s):  
Fuad Bahram ◽  
Siqin Wu ◽  
Fredrik Öberg ◽  
Bernhard Lüscher ◽  
Lars-Gunnar Larsson
Keyword(s):  
Dna Binding ◽  
Phorbol Ester ◽  
Tumor Development ◽  
Constitutive Expression ◽  
Interferon Γ ◽  
Binding Activity ◽  
Response To Treatment ◽  
Induced Differentiation ◽  
Dna Binding Activity ◽  
Ifn Γ

The transcription factors of the Myc/Max/Mad network are important regulators of cell growth, differentiation, and apoptosis and are frequently involved in tumor development. Constitutive expression of v-Myc blocks phorbol ester (TPA)-induced differentiation of human U-937 monoblasts. However, costimulation with interferon-γ (IFN-γ) and TPA restores terminal differentiation and G1cell-cycle arrest despite continuous expression of v-Myc. The mechanism by which TPA + IFN-γ counteract v-Myc activity has not been unravelled. Our results show that TPA + IFN-γ treatment led to an inhibition of v-Myc– and c-Myc–dependent transcription, and a specific reduction of v-Myc:Max complexes and associated DNA-binding activity, whereas the steady state level of the v-Myc protein was only marginally affected. In contrast, TPA + IFN-γ costimulation neither increased the expression of Mad1 or other mad/mnt family genes nor altered heterodimerization or DNA-binding activity of Mad1. The reduced amount of v-Myc:Max heterodimers in response to treatment was accompanied by partial dephosphorylation of v-Myc and c-Myc. Phosphatase treatment of Myc:Max complexes lead to their dissociation, thus mimicking the effect of TPA + IFN-γ. In addition to modulation of the expression of Myc/Max/Mad network proteins, posttranslational negative regulation of Myc by external signals may, therefore, be an alternative biologically important level of control with potential therapeutic relevance for hematopoietic and other tumors with deregulated Myc expression.

Posttranslational Regulation of Myc Function in Response to Phorbol Ester/Interferon-γ–Induced Differentiation of v-Myc–Transformed U-937 Monoblasts

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3900-3912 ◽  
Author(s):  
Fuad Bahram ◽  
Siqin Wu ◽  
Fredrik Öberg ◽  
Bernhard Lüscher ◽  
Lars-Gunnar Larsson
Keyword(s):  
Dna Binding ◽  
Phorbol Ester ◽  
Tumor Development ◽  
Constitutive Expression ◽  
Interferon Γ ◽  
Binding Activity ◽  
Response To Treatment ◽  
Induced Differentiation ◽  
Dna Binding Activity ◽  
Ifn Γ

Abstract The transcription factors of the Myc/Max/Mad network are important regulators of cell growth, differentiation, and apoptosis and are frequently involved in tumor development. Constitutive expression of v-Myc blocks phorbol ester (TPA)-induced differentiation of human U-937 monoblasts. However, costimulation with interferon-γ (IFN-γ) and TPA restores terminal differentiation and G1cell-cycle arrest despite continuous expression of v-Myc. The mechanism by which TPA + IFN-γ counteract v-Myc activity has not been unravelled. Our results show that TPA + IFN-γ treatment led to an inhibition of v-Myc– and c-Myc–dependent transcription, and a specific reduction of v-Myc:Max complexes and associated DNA-binding activity, whereas the steady state level of the v-Myc protein was only marginally affected. In contrast, TPA + IFN-γ costimulation neither increased the expression of Mad1 or other mad/mnt family genes nor altered heterodimerization or DNA-binding activity of Mad1. The reduced amount of v-Myc:Max heterodimers in response to treatment was accompanied by partial dephosphorylation of v-Myc and c-Myc. Phosphatase treatment of Myc:Max complexes lead to their dissociation, thus mimicking the effect of TPA + IFN-γ. In addition to modulation of the expression of Myc/Max/Mad network proteins, posttranslational negative regulation of Myc by external signals may, therefore, be an alternative biologically important level of control with potential therapeutic relevance for hematopoietic and other tumors with deregulated Myc expression.

Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells

2004 ◽  
Vol 92 (6) ◽  
pp. 1282-1295 ◽  
Author(s):  
Anna Grynfeld Smith ◽  
Nikita Popov ◽  
Martha Imreh ◽  
Håkan Axelson ◽  
Marie Henriksson
Keyword(s):  
Dna Binding ◽  
Neuroblastoma Cells ◽  
Binding Activity ◽  
Human Neuroblastoma Cells ◽  
Induced Differentiation ◽  
Human Neuroblastoma ◽  
Dna Binding Activity

scholarly journals Recombinant Gamma Interferon Stimulates Signal Transduction and Gene Expression in Alveolar Macrophages In Vitro and in Tuberculosis Patients

2003 ◽  
Vol 71 (4) ◽  
pp. 2058-2064 ◽  
Author(s):  
Rany Condos ◽  
Bindu Raju ◽  
Antony Canova ◽  
Ben-Yang Zhao ◽  
Michael Weiden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Dna Binding ◽  
Alveolar Macrophages ◽  
Binding Activity ◽  
Tuberculosis Patients ◽  
Dna Binding Activity ◽  
Ifn Γ ◽  
Hiv 1

ABSTRACT Tuberculosis is the seventh leading cause of morbidity and mortality in the world, with eight million cases per year. Animal and human studies demonstrate an enrichment of CD4 cells at sites of disease, with a more favorable clinical course when there is a Th1 response with the presence of gamma interferon (IFN-γ). We previously treated patients who had multidrug-resistant tuberculosis with recombinant IFN-γ (rIFN-γ) in aerosol form and were able to convert smear-positive cases to smear negative with 12 treatments over 1 month. We hypothesized that rIFN-γ would induce signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) binding activity in alveolar macrophages (AM). AM treated in vitro showed clear upregulation of STAT-1 and IRF-1 by rIFN-γ. STAT-1 was not activated and IRF-1 was only weakly induced after 1 day of infection by Mycobacterium tuberculosis TN913. In bronchoalveolar lavage (BAL) cells obtained from 10 of 10 tuberculosis patients 10 ± 2 days post-antituberculosis treatment, there was no detectable STAT-1 or IRF-1 DNA-binding activity. After 4 weeks of treatment with rIFN-γ aerosol in addition to the antituberculosis drugs, 10 of 10 patients had increased STAT-1, IRF-1, and/or IRF-9 DNA-binding activity in BAL cells from lung segments shown radiographically to be involved and in those shown to be uninvolved. Symptoms and chest radiographs improved, and amounts of macrophage inflammatory cytokines and human immunodeficiency virus type 1 (HIV-1) viral loads (in five of five HIV-1-coinfected patients) declined in the second BAL specimens. rIFN-γ aerosol induces signal transduction and gene expression in BAL cells and should be evaluated for efficacy in a randomized, controlled clinical trial.

scholarly journals Phenotypic Analysis of Random hnsMutations Differentiate DNA-Binding Activity from Properties offimA Promoter Inversion Modulation and Bacterial Motility

1999 ◽  
Vol 181 (3) ◽  
pp. 941-948 ◽  
Author(s):  
Gina M. Donato ◽  
Thomas H. Kawula
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Constitutive Expression ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Phenotypic Analysis ◽  
Mutator Strain ◽  
Dna Binding Activity ◽  
And Function

ABSTRACT H-NS is a major Escherichia coli nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in hns result in constitutive expression of theproU and fimB genes, increased fimApromoter inversion rates, and repression of the flhCDmaster operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned hns gene into a mutator strain and collected a series of mutant alleles that failed to repress proUexpression. Each of these isolated hns mutant alleles also failed to repress fimB expression, suggesting that H-NS-specific repression of proU and fimBoccurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type fimA promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various hnsmutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.

CRTF is a novel transcription factor that regulates multiple stages of Dictyostelium development

Development ◽  
2001 ◽  
Vol 128 (13) ◽  
pp. 2569-2579 ◽  
Author(s):  
Xiuqian Mu ◽  
Seth A. Spanos ◽  
Joseph Shiloach ◽  
Alan Kimmel
Keyword(s):  
Dna Binding ◽  
Constitutive Expression ◽  
Binding Activity ◽  
Receptor Subtype ◽  
Continuous Exposure ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Expression Pathways ◽  
Mobility Shift Assay ◽  
Camp Receptor

During aggregation, Dictyostelium establish nanomolar oscillation waves of extracellular cAMP, but as development progresses, cells become responsive to higher, non-fluctuating concentrations of cAMP. The regulation of the promoter responsible for expression of cAMP receptor subtype 1, CAR1, during aggregation reflects these signaling variations. Transcription of CAR1 from the early, aggregation promoter is activated by cAMP pulsing, but is repressed by continuous exposure to micromolar concentrations of cAMP. Deletion and mutation analyses of this promoter had defined an element essential for cAMP-regulated expression, and mobility shift assay, DNA crosslinking and DNase I footprinting experiments had identified a nuclear protein (CRTF) with zinc-dependent sequence binding specificity. In our study, CRTF was purified to homogeneity, peptides were sequenced and full-length cDNAs were obtained. The deduced CRTF protein is ∼100 kDa with a C-terminal, zinc finger-like motif required for DNA binding; CRTF purified from cells, however, represents only a 40 kDa C-terminal fragment that retains DNA-binding activity. As might have been predicted if CRTF were essential for the regulation of CAR1, crtf-null strains fail to develop under standard conditions or to exhibit induced expression of CAR1 or other cAMP-regulated genes. Furthermore, crtf-nulls also fail to sporulate, even under conditions that bypass the dependence on early cAMP signaling pathways. In addition, early developmental events of crtf-null strains could be rescued with exogenous cAMP treatment, constitutive expression of CAR1 or co-development with wild-type cells; however, these treatments were insufficient to promote sporulation. This suggests a cell-autonomous role for CRTF during late development that is separate from its capacity to control CAR1 expression. Finally, ablation of CRTF promotes a precocious induction of certain cAMP-dependent gene expression pathways. We suggest that CRTF may function to help insulate distinct pathways from simultaneous and universal activation by cAMP. CRTF, thus, exhibits multiple complex and independent regulatory functions during Dictyostelium development.

The Mechanism of Constitutive NF-kB Activity in Myeloma Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4740-4740
Author(s):  
Jakia Amin ◽  
Ken-ichiro Otsuyama ◽  
Abul Islam ◽  
Karim Shamsasenjan ◽  
Shamim Mohd Iqbal ◽  
...  
Keyword(s):  
Dna Binding ◽  
Western Blot ◽  
Cell Lines ◽  
Target Gene ◽  
Target Genes ◽  
Constitutive Expression ◽  
Myeloma Cell ◽  
Binding Activity ◽  
Myeloma Cells ◽  
Dna Binding Activity

Abstract [Purpose] NF-kB has a key function in the transformation, proliferation and invasion of cancer cells as well as in resistance to radiotherapy and chemotherapy. Although constitutive NF-kB activation has been reported in many human tumors, the underlying factors and mechanisms responsible for constitutive NF-kB activation in myeloma cells has not been known. The purpose of this study was to determine the mechanism of constitutive NF-kB activity in myeloma cell lines and quantification of NF-kB activity in primary myeloma cells by expression of CD54 (a NF-kB target gene). [Methods] We checked the constitutive expression of NF-kB family proteins by western blot analysis and possible dimer formation of different NF-kB family members by Immunoprecipitation-western blot reaction. Constitutive NF-kB DNA-binding activity and dimers that are responsible for NF-kB activity were analyzed by electrophoretic mobility shift assay (EMSA). Moreover, expression of different NF-kB target genes was done by RT-PCR. [Results and discussion] Constitutive NF-kB activity was determined in myeloma cell lines and our results suggested that p50/RelB, p50/p50, p50/p52 dimers might be responsible for this. We also analyzed several NF-kB target gene expressions and found that the intensity of CD54 expression was positively correlated with total NF-kB DNA binding activity. Although there is no method to quantified NF-kB activity, it can be determine in terms of its target genes e.g. CD54. Therefore, this study provide the frame work for understanding the molecular mechanism of constitutive activation of NF-kB and would help to quantify NF-kB activation in primary myeloma cells.

scholarly journals Constitutive expression of Bc1-3 in thymocytes increases the DNA binding of NF-kappaB1 (p50) homodimers in vivo.

1996 ◽  
Vol 16 (4) ◽  
pp. 1342-1348 ◽  
Author(s):  
J H Caamaño ◽  
P Perez ◽  
S A Lira ◽  
R Bravo
Keyword(s):  
Dna Binding ◽  
Biological Activities ◽  
Constitutive Expression ◽  
Transgenic Animals ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Nf Kappab

Previous studies have indicated that Bcl-3 interacts through its ankyrin repeats with the transcriptional factors NF-kappaB1 (p50) and NF-kappaB2 (p52), affecting their biological activities. To further investigate the role of Bcl-3 in vivo and its association with the NF-kappaB proteins, we have generated transgenic mice constitutively expressing Bcl-3 in thymocytes. The results indicate that Bcl-3 is associated with endogenous p50 and p52 in nuclear extracts from transgenic animals. Remarkably, constitutive expression of Bcl-3 in these cells augments the DNA binding activity of p52 homodimers. This effect could be reproduced in vitro and is blocked by anti-Bcl-3 antibodies. We have also shown that Bcl-3 is phosphorylated in thymocytes and that its dephosphorylation greatly decreases the effect on p50 homodimers.

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