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1.
Sci Rep ; 7(1): 12262, 2017 09 25.
Article in English | MEDLINE | ID: mdl-28947822

ABSTRACT

Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironment, being the BCR pathway one key player in this crosstalk. Among proteins participating, ZAP-70 enhances response to microenvironmental stimuli. MicroRNA-21 (miR-21) is overexpressed in diverse neoplasias including CLL, where it has been associated to refractoriness to fludarabine and to shorter time to progression and survival. To further elucidate the role of ZAP-70 in the biology of CLL, we studied its involvement in miR-21 regulation. MiR-21 expression was higher in CLL cells with high ZAP-70. Ectopic expression of ZAP-70 induced transcription of miR-21 via MAPK and STAT3, which subsequently induced downregulation of tumor suppressors targeted by miR-21. The co-culture of primary CLL cells mimicking the microenvironment induced ZAP-70 and miR-21 expression, as well as downregulation of miR-21 targets. Interestingly, the increase in miR-21 after co-culture was significantly impaired by ibrutinib, indicating that the BCR signaling pathway is involved in its regulation. Finally, survival of CLL cells induced by the co-culture correlated with miR-21 upregulation. In conclusion, stimuli from the microenvironment regulate miR-21 and its targeted tumor suppressor genes via a signaling pathway involving ZAP-70, thus contributing to the cytoprotection offered by the microenvironment particularly observed in CLL cells expressing ZAP-70.


Subject(s)
Apoptosis Regulatory Proteins/biosynthesis , Gene Expression Regulation , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , MicroRNAs/biosynthesis , Molecular Chaperones/biosynthesis , PTEN Phosphohydrolase/biosynthesis , Protein Inhibitors of Activated STAT/biosynthesis , RNA-Binding Proteins/biosynthesis , ZAP-70 Protein-Tyrosine Kinase/biosynthesis , Cells, Cultured , Coculture Techniques , Gene Regulatory Networks , Humans , Leukocytes, Mononuclear/chemistry , Signal Transduction , Tumor Microenvironment
2.
Leuk Res ; 38(4): 509-15, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24480549

ABSTRACT

The analysis of immunoglobulin heavy chain variable (IGHV) region may disclose the influence of antigens in Burkitt's lymphomas (BL). IGHV sequences from 38 patients and 35 cell lines were analyzed. IGHV3 subset genes were the most used and IGHV4-34 gene was overrepresented. IGHV genes were mutated in 98.6% of the cases, 36% acquired potential glycosylation sites, and in 52% somatic-hypermutation-process was ongoing. Binding motifs for superantigens like Staphylococcal protein A and carbohydrate I/i were preserved in 89% of the cases. IGHV analysis of BL cells supports a germinal center origin and points toward a role for superantigens in lymphomagenesis.


Subject(s)
Burkitt Lymphoma/genetics , Cell Transformation, Neoplastic , DNA Mutational Analysis , Germinal Center/pathology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Superantigens/physiology , Adolescent , Adult , Aged , Burkitt Lymphoma/pathology , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/immunology , Child , Child, Preschool , Female , Germinal Center/metabolism , Humans , Male , Middle Aged
3.
PLoS One ; 8(12): e81221, 2013.
Article in English | MEDLINE | ID: mdl-24312539

ABSTRACT

ZAP-70 in chronic lymphocytic leukemia (CLL) is associated with enhanced response to microenvironmental stimuli. We analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells in a xenograft mouse model of disseminated B-cell leukemia. Mice injected with B-cells expressing ZAP-70 showed a prominently higher infiltration of the bone marrow. In vitro analysis of the response of malignant B-cells to CXCL12, the main attracting chemokine regulating trafficking of lymphocytes to the bone marrow, or to bone marrow stromal cells, revealed that ZAP-70 induces an increased response in terms of signaling and migration. These effects are probably mediated by direct participation of ZAP-70 in CXCL12-CXCR4 signaling since CXCR4 stimulation led to activation of ZAP-70 and downstream signaling pathways, such as MAPK and Akt, whereas ZAP-70 did not alter the expression of the CXCR4 receptor. In addition, subclones of primary CLL cells with high expression of ZAP-70 also showed increased migrative capacity toward CXCL12. Neutralization of CXCR4 with a monoclonal antibody resulted in impaired in vitro responses to CXCL12 and bone marrow stromal cells. We conclude that ZAP-70 enhances the migration of malignant B-cells into the supportive microenvironment found in the bone marrow mainly by enhancing signaling and migration after CXCR4 stimulation.


Subject(s)
B-Lymphocytes/metabolism , Bone Marrow/metabolism , Cell Movement , Leukemia, B-Cell/metabolism , Neoplasm Proteins/metabolism , Receptors, CXCR4/metabolism , ZAP-70 Protein-Tyrosine Kinase/metabolism , Animals , B-Lymphocytes/pathology , Bone Marrow/pathology , Chemokine CXCL12/genetics , Chemokine CXCL12/metabolism , Humans , Jurkat Cells , Leukemia, B-Cell/genetics , Leukemia, B-Cell/pathology , Mice , Mice, SCID , Neoplasm Proteins/genetics , Receptors, CXCR4/genetics , Stromal Cells/metabolism , Stromal Cells/pathology , ZAP-70 Protein-Tyrosine Kinase/genetics
4.
Clin Cancer Res ; 18(21): 5924-33, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-22966019

ABSTRACT

PURPOSE: Glucocorticoids are part of the therapeutic armamentarium of chronic lymphocytic leukemia (CLL) where it has been suggested that cells with unmutated IGHV genes exhibit higher sensitivity. The mechanisms by which glucocorticoids are active in CLL are not well elucidated. We aimed to ascertain the activity of dexamethasone in CLL cells according to prognosis and to identify the molecular mechanisms that are influencing the response to this drug. EXPERIMENTAL DESIGN: Sensitivity to dexamethasone was analyzed ex vivo in 50 CLL and compared according to IGHV mutational status and/or ZAP-70 expression. The response was further compared by gene expression profiling (GEP) of selected cases. Expression of genes of interest was validated by quantitative reverse transcriptase PCR. RESULTS: Response to dexamethasone was higher in cases with unmutated IGHV/high ZAP-70 expression, and the levels of induction of the pro-apoptotic Bim protein correlated with the degree of cell death. GEP analysis showed few genes differentially expressed after dexamethasone treatment between mutated and unmutated cases. However, functional annotation analysis showed that unmutated cases had significant enrichment in terms related to apoptosis. Specific analysis of genes of interest conducted in a large series disclosed that in unmutated IGHV cells, FKBP5 expression was higher at baseline and after dexamethasone exposure and that GILZ was more induced by dexamethasone treatment in these cases. CONCLUSIONS: Unmutated IGHV/high ZAP-70 CLL cells exhibit better response to dexamethasone treatment, which is accompanied by a differential expression of genes involved in the glucocorticoid receptor pathway and by an increased induction of genes related to apoptosis.


Subject(s)
Apoptosis , Gene Expression Regulation, Leukemic , Genes, Immunoglobulin Heavy Chain/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Transcriptome , ZAP-70 Protein-Tyrosine Kinase/genetics , Adult , Aged , Aged, 80 and over , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Cluster Analysis , Dexamethasone/pharmacology , Female , Gene Expression Regulation, Leukemic/drug effects , Humans , Male , Membrane Proteins/genetics , Middle Aged , Mutation , Proto-Oncogene Proteins/genetics , RNA Interference , Tacrolimus Binding Proteins/genetics , Tacrolimus Binding Proteins/metabolism , Transcription Factors/genetics
5.
Blood ; 118(16): 4401-10, 2011 Oct 20.
Article in English | MEDLINE | ID: mdl-21865343

ABSTRACT

ZAP-70 in chronic lymphocytic leukemia (CLL) has been associated with enhanced B-cell receptor (BCR) signaling, survival, and migration. We investigated whether ZAP-70 can directly govern migration and the underlying mechanisms. In the ZAP-70 stably transfected Ramos cell line, IgM stimulation, but no IgD, enhanced phosphorylation of ERK1/2, Akt and Syk, and delayed IgM and CD79b internalization. In contrast, in the Raji cell line, where ZAP-70 was constitutively phosphorylated, ERK1/2, but not Akt, was phosphorylated, suggesting that MAPK pathway mediates ZAP-70 effects. BCR stimulation modulated the expression of CCR7, CXCR4, CXCR5, CD44, CD49d, and CD62L, which were up-regulated in ZAP-70-positive CLL primary subclones. The most dramatic change after BCR engagement in ZAP-70-transfected cells was CCR7 up-regulation, this being impaired by ERK1/2 inhibition and translating into both increased signaling and migration toward CCL21. Primary CLL subclones with high ZAP-70 expression showed increased migration toward CCL21. In conclusion, ZAP-70 ectopic expression led to enhanced BCR signaling after IgM stimulation and increased the expression of CCR7 predominantly via ERK1/2, increasing the response and migration toward CCL21. In primary CLL samples, cellular subsets with high ZAP-70 expression had increased expression of adhesion molecules and chemokine receptors in addition to an enhanced ability to migrate toward CCL21.


Subject(s)
B-Lymphocytes/cytology , Burkitt Lymphoma/immunology , Chemokine CCL21/immunology , Chemotaxis , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Receptors, CCR7/immunology , ZAP-70 Protein-Tyrosine Kinase/immunology , B-Lymphocytes/immunology , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Immunoglobulin M/immunology , MAP Kinase Signaling System , Receptors, CCR7/genetics , Tumor Cells, Cultured
6.
Haematologica ; 93(1): 75-82, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18166788

ABSTRACT

BACKGROUND: The aim of this study was to analyze whether in chronic lymphocytic leukemia the cytosolic release of histone H1.2, a new apoptogenic mechanism induced by DNA damage, was associated with the presence of genetic abnormalities and with the response to treatment. DESIGN AND METHODS: Primary tumoral chronic lymphocytic leukemia cells from 25 patients were investigated for histone H1.2 cytosolic release after treatment with genotoxic (fludarabine, mitoxantrone, etoposide, or X-ray radiation) and non-genotoxic (dexamethasone) agents. Cases were analyzed for the presence of poor-risk genetic alterations, particularly deletions at 17p13 and 11q22. Histone H1.2 release was correlated with the presence of genetic abnormalities and with the best clinical response obtained with standard treatments. RESULTS: DNA-damaging agents induced H1.2 release in a p53-dependent manner which was confirmed by the lack of H1.2 release in p53-deleted cases. Non-DNA-damaging agents induced release of H1.2 in both p53-deleted and non-deleted chronic lymphocytic leukemia cases. Moreover, nuclear H1.2 release was observed after genotoxic and non-genotoxic treatment independently of ATM function. From a clinical standpoint, the lack of histone H1.2 release correlated with resistance to genotoxic treatment. CONCLUSIONS: In chronic lymphocytic leukemia cells, histone H1.2 traffic was dependent on the p53-status after genotoxic treatment, but could also be induced after treatments that acted independently of p53. By contrast, histone H1.2 release did not seem to be dependent on ATM function. Nuclear histone H1.2 release appears to be an important element in apoptosis induction in chronic lymphocytic leukemia, particularly in cases with abnormal p53 function resistant to conventional treatment.


Subject(s)
Apoptosis , Chromosome Deletion , Cytosol/metabolism , Gene Expression Regulation, Leukemic , Histones/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Adult , Aged , Cell Nucleus/metabolism , Chromosome Aberrations , DNA Damage , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged
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