Analysis of ZAP70 expression in adult acute lymphoblastic leukaemia by real time quantitative PCR.
ABSTRACT: BACKGROUND: ZAP70 gene expression is associated with poor prognosis in B-cell lymphoproliferative disorders especially chronic lymphocytic leukaemia (CLL) but its role in adult B-ALL has not been established. On diagnostic samples from 76 patients with adult ALL (65 with B-ALL and 11 with T-ALL) ZAP70 mRNA expression levels were studied by real time-quantitative PCR (RT-qPCR) analysis. FINDINGS: A broad distribution of ZAP70 expression was observed in ALL, ranging from 0.002 to 5.3 fold that of the ZAP70 positive Jurkat reference cell line. No association was observed between expression levels and the presence of specific cytogenetic abnormalities. Five cases, including one case of T-ALL, had ZAP70 expression above the level of the Jurkat reference cell line. CONCLUSIONS: Our results confirm the frequent expression of ZAP70 in adult ALL. Limited comparisons made did highlight poor-risk patients with high ZAP70 expression, but due to lack of clinical information on patient samples we were unable to directly assess the impact on disease prognosis. ZAP-70 may be an important laboratory assay in adult ALL and further studies are warranted to study a potential correlation with cytogenetic and other genetic markers.
Project description:The zeta-chain (TCR) associated protein kinase 70kDa (ZAP70) is required for T-cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immunodeficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70y442) that was created using TALENs. In contrast to what has been reported in morphant zebrafish, zap70y442 homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP+ thymic T cells by 5 days post-fertilization that persisted into adult stages. Morphological analysis, RNA sequencing and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70y442 mutant animals. T cell immunodeficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immunodeficiency.
Project description:Comparison of Chronic Lymphocytic Leukemia patients expressing high or low levels of ZAP70 mRNA: prognostic factors and interaction with the microenvironment. Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia (CLL), but mechanisms by which its higher expression leads to a poor outcome remain to be fully explained. In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B-cells from CLL patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Keywords: comparison of poor and good prognosis CLL patient transcriptome regarding ZAP70 expression Overall design: Two groups of seven CLL patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. Total RNA from CD19+ purified cells was exctracted and hybidyzed on Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array. Amplification, hybridization and scanning were done according to standard Affymetrix protocols (www.affymetrix.com). CEL files were ,normalized with RMA method.
Project description:The B-cell receptor (BCR) signaling pathway is of great importance for B-cell survival and proliferation. The BCR expressed on malignant B-CLL cells contributes to the disease pathogenesis, and its signaling pathway is currently the target of several therapeutic strategies. Although various BCR alterations have been described in B-CLL at the protein level, the mRNA expression levels of tyrosine kinases in B-CLL compared to that in normal CD5-high and CD5-low B-lymphocytes remain unknown. In the current study, we measured the mRNA expression levels of CD79A, CD79B, LYN, SYK, SHP1, and ZAP70 in purified populations of CD5-high B-CLL cells, CD5-low B-cells from the peripheral blood of healthy donors, and CD5-high B-cells from human tonsils. Here, we report a clear separation in the B-CLL dataset between the ZAP70-high and ZAP70-low subgroups. Each subgroup has a unique expression profile of BCR signaling components that might reflect the functional status of the BCR signaling pathway. Moreover, the ZAP70-low subgroup does not resemble either CD5-high B-lymphocytes from the tonsils or CD5-low lymphocytes from PBMC (P < 0.05). We also show that ZAP70 is the only gene that is differentially expressed in CD5-high and CD5-low normal B-lymphocytes, confirming the key role of Zap-70 tyrosine kinase in BCR signaling alterations in B-CLL.
Project description:The ?v?3 integrin stimulates the resorptive capacity of the differentiated osteoclast (OC) by organizing its cytoskeleton via the tyrosine kinase, Syk. Thus, Syk-deficient OCs fails to spread or form actin rings, in vitro and in vivo. The Syk family of tyrosine kinases consists of Syk itself and Zap70 which are expressed by different cell types. Because of their structural similarity, and its compensatory properties in other cells, we asked if Zap70 can substitute for absence of Syk in OCs. While expression of Syk, as expected, normalizes the cytoskeletal abnormalities of Syk(-/-) OCs, Zap70 fails do so. In keeping with this observation, Syk, but not Zap70, rescues ?v?3 integrin-induced SLP76 phosphorylation in Syk(-/-) OCs. Furthermore the kinase sequence of Syk partially rescues the Syk(-/-) phenotype but full normalization also requires its SH2 domains. Surprisingly, expression of Zap70 inhibits WT OC spreading, actin ring formation and bone resorptive activity, but not differentiation. In keeping with arrested cytoskeletal organization, Zap70 blocks integrin-activated endogenous Syk and Vav3, SLP76 phosphorylation. Such inhibition requires Zap70 kinase activity, as it is abolished by mutation of the Zap70 kinase domain. Thus, while the kinase domain of Syk is uniquely required for OC function that of Zap70 inhibits it.
Project description:Zeta-associated protein of 70 kD (ZAP70) is a recognized adverse prognostic marker in chronic lymphocytic leukaemia (CLL) associated with enhanced B-cell receptor signalling, significantly more aggressive disease course and poor overall survival. Zeta-associated protein of 70 kD is ordinarily expressed in T cells where it has a crucial role in T-cell receptor signalling, whereas its aberrant expression in CLL leads to enhanced B-cell receptor signalling and significantly more aggressive disease course. Although much is known about the activation of ZAP70 following engagement of T-cell receptor, there are little data on the regulation of ZAP70 gene expression in normal T cells or CLL. To understand the molecular events underpinning epigenetic regulation of ZAP70 gene in CLL, we have defined ZAP70 promoter region and outlined the regions crucial in regulating the gene activity. Following a direct comparison of ZAP70+ and ZAP70- primary CLLs, we show ZAP70 promoter in expressing CLLs to be associated with a spectrum of active histone modifications, some of which are tightly linked to aberrant DNA methylation in CLL. Cross-talk between histone modifications and reduced DNA methylation culminates in transcriptional de-repression of ZAP70. Moreover, treatment with histone deacetylase (HDAC) and DNA methylation inhibitors results in recovery of ZAP70 expression, which provides a possible explanation for the failure of HDAC inhibitors in CLL treatment and might serve as a cautionary warning for their future use in treatment of this leukaemia.
Project description:Chronic lymphocytic leukemia (CLL) can be divided into groups based on biomarkers of poor prognosis. The expression of the tyrosine kinase ZAP-70 (member of the Syk tyrosine kinase family) in CLL cells is associated with shorter overall survival in CLL patients. Currently, there is a lack of targeted therapies for patients with ZAP-70 expression in CLL cells. The tyrosine kinase inhibitor gefitinib has been shown to be effective at induce apoptosis in acute myeloid leukemia through inhibition of Syk. In this study, we sought to test the efficacy of gefitinib in primary human ZAP-70+ CLL cells. We demonstrate that gefitinib preferentially induces cell death in ZAP-70-expressing CLL cells with a median IC50 of 4.5 ?M. In addition, gefitinib decreases the viability of ZAP-70+ Jurkat T leukemia cells but fails to affect T cells from CLL patients. Western blot analysis shows gefitinib reduces both basal and B-cell receptor (BCR)-stimulated phosphorylation of Syk/ZAP-70, ERK, and Akt in ZAP-70+ CLL cells. Moreover, gefitinib inhibits the pro-survival response from BCR stimulation and decreases pro-survival proteins such as Mcl-1. Finally, ZAP-70 expression sensitizes Raji cells to gefitinib treatment. These results demonstrate that gefitinib specifically targets ZAP-70+ CLL cells and inhibits the BCR cell survival pathway leading to apoptosis. This represents the likelihood of tyrosine kinase inhibitors being effective targeted treatments for ZAP-70+ CLL cells.
Project description:T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling.
Project description:Survival of naive T cells requires engagement of TCR with self-peptide major histocompatibility Ags. The signaling pathways required to transmit this survival signal are poorly understood. In this study, we asked whether the tyrosine kinase Zap70 is required to transmit survival signals in naive CD8 T cells. In the absence of Zap70 expression, thymic development is completely blocked. Using a tetracycline-inducible Zap70 transgene (TetZap70), thymic development of Zap70-deficient TCR transgenic F5 mice was restored. Feeding mice doxycycline to induce Zap70 expression resulted in repopulation of the peripheral naive compartment. Zap70 transgene expression was then ablated by withdrawal of doxycycline. Survival of Zap70-deficient naive CD8 T cells depended on host environment. In hosts with a replete T cell compartment, naive T cells died rapidly in the absence of Zap70 expression. In lymphopenic hosts, Zap70-deficient T cells survived far longer, in an IL-7-dependent manner, but failed to undergo lymphopenia-induced proliferation. Analyzing mixed bone marrow chimeras revealed that intact Zap70-dependent signaling was important for integration of recent thymic emigrants into the mature naive compartment. Finally, we asked whether adaptor function conferred by Zap70 tyrosines 315 and 319 was necessary for transmission of homeostatic TCR signals. This was done by analyzing F5 mice expressing mutant Zap70 in which these residues had been mutated to alanines (Zap70(YYAA)). Inducible Zap70 expression rescued thymic development in F5 TetZap70 Zap70(YYAA) mice. However, in the absence of wild-type Zap70 expression, the Zap70(YYAA) mutant failed to transmit either survival or proliferative homeostatic signals.
Project description:BACKGROUND: Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. DESIGN AND METHODS: In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. RESULTS: Twenty-seven genes were differentially expressed with an FDR<10%, and several genes were significant predictors of treatment-free survival (TFS) and/or overall survival; PDE8A and FCRL family genes (down-regulated in ZAP70(+) patients) could predict TFS and overall survival; ITGA4 mRNA (up-regulated in ZAP70(+) patients) could significantly predict overall survival. Importantly, gene set enrichment analysis revealed overrepresentation of adhesion/migration genes. We therefore investigated in vitro adhesion/migration capacity of chronic lymphocytic leukemia cells into a stromal microenvironment or in response to conditioned medium. We showed that ZAP70(+) cells had better adhesion/migration capacities and only ZAP70(+) patient cells responded to microenvironment contact by CXCR4 downregulation. CONCLUSIONS: We concluded that several prognostic factors are the reflection of microenvironment interactions and that the increased adhesion/migratory capacity of ZAP70(+) cells in their microenvironment can explain their better survival and thus the aggressiveness of the disease.
Project description:Activated chemokine receptor initiates inside-out signaling to transiently trigger activation of integrins, a process involving multiple components that have not been fully characterized. Here we report that GM-CSF/IL-3/IL-5 receptor common beta-chain-associated protein (CBAP) is required to optimize this inside-out signaling and activation of integrins. First, knockdown of CBAP expression in human Jurkat T cells caused attenuated CXC chemokine ligand-12 (CXCL12)-induced cell migration and integrin ?4?1- and ?L?2-mediated cell adhesion in vitro, which could be rescued sufficiently upon expression of murine CBAP proteins. Freshly isolated CBAP-deficient primary T cells also exhibited diminution of chemotaxis toward CC chemokine ligand-21 (CCL21) and CXCL12, and these chemokines-induced T-cell adhesions in vitro. Adoptive transfer of isolated naive T cells demonstrated that CBAP deficiency significantly reduced lymph node homing ability in vivo. Finally, migration of T cell-receptor-activated T cells induced by inflammatory chemokines was also attenuated in CBAP-deficient cells. Further analyses revealed that CBAP constitutively associated with both integrin ?1 and ZAP70 and that CBAP is required for chemokine-induced initial binding of the talin-Vav1 complex to integrin ?1 and to facilitate subsequent ZAP70-mediated dissociation of the talin-Vav1 complex and Vav1 phosphorylation. Within such an integrin signaling complex, CBAP likely functions as an adaptor and ultimately leads to activation of both integrin ?4?1 and Rac1. Taken together, our data suggest that CBAP indeed can function as a novel signaling component within the ZAP70/Vav1/talin complex and plays an important role in regulating chemokine-promoted T-cell trafficking.