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1.
Blood ; 144(13): 1412-1417, 2024 Sep 26.
Article in English | MEDLINE | ID: mdl-39024510

ABSTRACT

ABSTRACT: T-cell lymphoblastic lymphoma (T-LBL) and T-cell acute lymphoblastic leukemia (T-ALL) have common and distinguishing clinical and molecular features. Molecular prognostic factors are needed for T-LBL. We assessed the prevalence and prognostic impact of the T-cell receptor ß (TRB)::NOTCH1 fusion in 192 pediatric patients with T-LBL and 167 pediatric patients with T-ALL, using novel multiplex polymerase chain reaction and genomic capture high-throughput sequencing techniques. The fusion was detected in 12 patients with T-LBL (6.3%) but in none of the patients with T-ALL (P = .0006, Fisher exact test). In T-LBL, the TRB::NOTCH1 fusion was associated with a significantly higher incidence of relapse (67% vs 17% in gene fusion-negative patients, P < .001, Fisher exact test). The breakpoint in TRB was most frequently located in J2-7 (n = 6). In NOTCH1, the breakpoints varied between exon 24 and 27. Consequently, a truncated NOTCH1 with its dimerization, regulation, and signal transduction domains gets controlled by strong TRB enhancer elements. This study reveals a novel recurrent genetic variant with significant prognostic relevance in T-LBL, which was absent in T-ALL. The TRB::NOTCH1 fusion in T-LBL suggests a possible unique pathogenic mechanism divergent from T-ALL. Further studies will validate the role of the TRB::NOTCH1 fusion as prognostic marker in T-LBL and elucidate its pathogenic mechanisms.


Subject(s)
Oncogene Proteins, Fusion , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Receptor, Notch1 , Humans , Child , Receptor, Notch1/genetics , Male , Female , Adolescent , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Oncogene Proteins, Fusion/genetics , Child, Preschool , Prognosis , Infant
2.
BMC Mol Biol ; 7: 48, 2006 Dec 22.
Article in English | MEDLINE | ID: mdl-17187679

ABSTRACT

BACKGROUND: S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca2+-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879-41887). RESULTS: In the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-1 with 1,5-isoquinolinediol (DiQ). CONCLUSION: The candidates, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer.


Subject(s)
Antigens, Nuclear/metabolism , Calgranulin B/genetics , DNA-Binding Proteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Transcription, Genetic/genetics , Amino Acid Sequence , Cell Line , Chromatin Immunoprecipitation , Gene Expression Regulation , Humans , Ku Autoantigen , Molecular Sequence Data , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/chemistry , Protein Binding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
3.
FASEB J ; 19(3): 467-9, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15642721

ABSTRACT

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.


Subject(s)
Calgranulin A/physiology , Calgranulin B/physiology , NADPH Oxidases/metabolism , Phosphoproteins/physiology , rac GTP-Binding Proteins/physiology , Animals , Arachidonic Acid/pharmacology , Calgranulin A/genetics , Calgranulin B/genetics , Cattle , Cell Line, Tumor , Cell-Free System , Enzyme Activation/drug effects , Gene Expression/drug effects , Gene Silencing , Humans , Leukemia, Promyelocytic, Acute , Mice , Mice, Knockout , Neutrophils/enzymology , Neutrophils/physiology , Oligonucleotides, Antisense/pharmacology , Phosphoproteins/genetics , Polymerase Chain Reaction , RNA, Messenger/analysis , Reactive Oxygen Species/metabolism , Recombinant Fusion Proteins , Respiratory Burst , Superoxides/metabolism , Tetradecanoylphorbol Acetate/pharmacology , RAC2 GTP-Binding Protein
4.
Mol Plant Microbe Interact ; 15(2): 102-8, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11876422

ABSTRACT

The necrotrophic pathogen Gibberella pulicaris infects potato tubers through wounds that contain fungitoxic secondary metabolites such as the phytoalexins rishitin and lubimin. In order to colonize tuber tissue, the fungus must possess a mechanism to tolerate potato defense compounds. In this paper, we show that a gene, Gpabc1, that codes an ATP-binding cassette (ABC) transporter is required for tolerance to these phytoalexins and for virulence on potato. The Gpabc1 gene, isolated in the course of a differential cDNA screen, shares high sequence homology with the ABC1 gene of Magnaporthe grisea. G. pulicaris mutants deficient in Gpabc1 were still able to metabolize rishitin but lost their tolerance to this phytoalexin as well as their virulence on potato. These results strongly suggest that the Gpabc1-encoded ABC transporter is necessary for tolerance of G. pulicaris to rishitin and that this tolerance is required for virulence on potato.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Drug Resistance, Multiple , Gibberella/genetics , Gibberella/metabolism , Plant Extracts/pharmacology , Solanum tuberosum/microbiology , Terpenes/pharmacology , ATP-Binding Cassette Transporters/genetics , Genetic Complementation Test , Gibberella/pathogenicity , Plant Diseases/microbiology , Restriction Mapping , Sesquiterpenes , Virulence , Phytoalexins
5.
FEBS Lett ; 585(2): 440-6, 2011 Jan 21.
Article in English | MEDLINE | ID: mdl-21192933

ABSTRACT

S100A8/A9 promotes NADPH oxidase in HaCaT keratinocytes and subsequently increases NFκB activation, which plays important roles in the balance between epidermal growth and differentiation. S100A8/A9-positive HaCaT cells present with a significantly reduced rate of cell division and greater expression of two keratinocyte differentiation markers, involucrin and filaggrin, than control cells. S100A8/A9 mutants fail to enhance NFκB activation, TNFα-induced IL-8 gene expression and NFκB p65 phosphorylation, and S100A8/A9-positive cells demonstrate better cell survival in forced suspension culture than mutant cells. S100A8/A9 is induced in epithelial cells in response to stress. Therefore, S100A8/A9-mediated growth arrest could have implications for tissue remodeling and repair.


Subject(s)
Calgranulin A/genetics , Calgranulin B/genetics , Cell Differentiation , Cell Proliferation , Keratinocytes/cytology , Adaptation, Physiological , Cell Line , Epithelial Cells/cytology , Filaggrin Proteins , Humans , Keratinocytes/metabolism , NF-kappa B/metabolism
6.
J Invest Dermatol ; 127(8): 2001-11, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17429438

ABSTRACT

The calcium- and arachidonic acid (AA)-binding proteins S100A8 and S100A9 are involved in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in phagocytes. They are specifically expressed in myeloid cells, and are also found in epithelial cells in various (patho)physiological conditions. We have investigated the consequences of S100A8/A9 overexpression in epithelial cell lines on reactive oxygen species (ROS) generation and downstream signaling. Epithelial carcinoma HeLa cells, which exclusively express Nox2, showed dramatically increased activation of NADPH oxidase by phorbol 12-myristate 13-acetate after S100A8/A9 gene transfection. HaCaT keratinocytes overexpressing S100A8/A9 showed enhanced, transient ROS generation in response to the calcium ionophore A23187 compared to mock-transfected cells. Polymerase chain reaction analysis revealed mRNA transcripts for Nox1, Nox2, and Nox5 in HaCaT keratinocytes. Detailed transfection studies confirmed that NADPH oxidase activities in Nox1- and Nox5-transfected HeLa cells were enhanced after S100A8/A9 gene complementation. Furthermore, mutational analysis revealed that AA binding and Thr113 phosphorylation are important for S100A8/A9-enhanced activation of NADPH oxidase. Nuclear factor-kappaB (NF-kappaB) activation and interleukin-8 mRNA levels were increased in S100A8/A9-HaCaT keratinocytes, consistent with the view that NF-kappaB is a redox-sensitive transcription factor. Because they are expressed in epithelia under specific conditions, S100A8 and S100A9 might be involved in skin pathogenesis by modulating aspects of downstream signaling.


Subject(s)
Calgranulin A/physiology , Calgranulin B/physiology , Keratinocytes/metabolism , NADPH Oxidases/metabolism , NF-kappa B/metabolism , Calcimycin/pharmacology , Calgranulin A/analysis , Calgranulin B/analysis , Cells, Cultured , Humans , Interleukin-8/biosynthesis , NADPH Oxidases/genetics , Phosphoproteins/genetics , RNA, Messenger/analysis , Reactive Oxygen Species/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transfection , Tumor Necrosis Factor-alpha/pharmacology
7.
Biol Chem ; 383(12): 1895-905, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12553726

ABSTRACT

Protein complexes formed by S100A8 and S100A9 represent the only AA-binding capacity in the human neutrophilic cytosol and are involved in the intracellular arachidonic acid metabolism. The formation of S100A8/A9 protein complexes and the binding of calcium to the complexes are prerequisites for the specific binding of polyunsaturated fatty acids. The present study was undertaken to characterize the fatty acid binding site within the protein complex. Deletions at both termini and point mutations of different basic amino acids especially within the extended C-terminal tail of human S100A9 were introduced. The S100A9 mutant proteins were then analyzed with respect to protein-protein interaction (GST pull down-assay and yeast two-hybrid system) and functional properties (arachidonic acid and calcium binding). The data give strong evidence that the unique C-tail of S100A9 containing the three consecutive histidine residues (His103-His105) represents the region to which the fatty acid carboxy-group is bound to the protein complex. The localization of the AA-binding site within the unique C-tail of S100A9 correlates with the fact that fatty acid binding has not yet been reported for other S100 proteins.


Subject(s)
Arachidonic Acid/chemistry , Amino Acid Sequence , Amino Acids, Basic/chemistry , Calcium/metabolism , Calgranulin A/chemistry , Calgranulin A/genetics , Calgranulin A/metabolism , Fatty Acids/metabolism , Glutathione Transferase/metabolism , Humans , In Vitro Techniques , Molecular Sequence Data , Neutrophils/drug effects , Neutrophils/metabolism , Plasmids/genetics , Point Mutation/genetics , Protein Binding , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Saccharomyces cerevisiae/genetics , Sequence Deletion/genetics , Transformation, Genetic
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