c-Abl regulates Mcl-1 gene expression in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a malignancy characterized by clonal expansion of mature B cells that are resistant to apoptosis. This resistance to apoptosis partly results from Mcl-1 expression because high levels of this protein in CLL cells correlate with poor disease prognosis and resistance to chemotherapy. Thus, understanding the mechanism(s) regulating Mcl-1 expression in CLL cells may be useful in the development of new therapies for this incurable disease. In the present study, we show a strong relationship between c-Abl and Mcl-1 expression in CLL cells. We show that treatment of CLL cells with Abl-specific siRNA or with imatinib, to inhibit c-Abl activity, results in the down-regulation of Mcl-1 protein and mRNA. A major regulator of Mcl-1 gene expression is STAT3. Our data show that CLL cells expressing high levels of c-Abl also show elevated levels of phospho-STAT3, and that STAT3 phosphorylation in CLL cells is dependent on c-Abl activity. However, STAT3 phosphorylation by c-Abl requires activation of nuclear factor-κB, secretion of autocrine interleukin-6, and active protein kinase C. Taken together, our data demonstrate the mechanism involved in c-Abl regulation of Mcl-1 expression in CLL cells, and suggest that c-Abl inhibition has therapeutic application in the treatment of this disease.

Vascular endothelial growth factor stimulates protein kinase CbetaII expression in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a malignant disease of mature B lymphocytes. We have previously shown that a characteristic feature of CLL cells are high levels of expression and activity of protein kinase CbetaII (PKCbetaII), and that this might influence disease progression by modulating signaling in response to B-cell receptor engagement. The aim of the present work was to investigate the factors involved in stimulating PKCbetaII expression in CLL cells. Here we show that the activation of PKCbetaII in CLL cells stimulated with vascular endothelial growth factor (VEGF) can drive expression of the gene for PKCbeta, PRKCB1. We found that this effect of VEGF on PRKCB1 transcription is paralleled by high expression of PKCbetaII protein and therefore probably contributes to the malignant phenotype of CLL cells. Taken together, the data presented in this study demonstrate that VEGF, in addition to its role in providing prosurvival signals, also plays a role in overexpression of PKCbetaII, an enzyme with a specific pathophysiologic role in CLL.

Microparticle-associated endothelial protein C receptor and the induction of cytoprotective and anti-inflammatory effects.

BACKGROUND: The endothelial protein C receptor plays an important role within the protein C pathway in regulating coagulation and inflammation. Recently, we described that endothelial protein C receptor can be released in vitro in microparticulate form from primary endothelial cells by exogenous activated protein C. Activated protein C bound to this endothelial protein C receptor retains anticoagulant activity and we hypothesize that this microparticulate endothelial protein C receptor-activated protein C complex can also cleave endothelial protease-activated receptor 1 to modulate inflammation and increase cell survival. Our main objective was, therefore, to study the effect that microparticle-associated endothelial protein C receptor-activated protein C has on endothelial function. DESIGN AND METHODS: Mini-arrays were used and probed with cDNA obtained from endothelial cells after treatment with microparticle-associated endothelial protein C receptor-activated protein C and results were confirmed by real time polymerase chain reaction. The functional relevance of changes at gene level were further analyzed by endothelial apoptosis and permeability assays, in the presence and absence of specific blockade of endothelial protein C receptor, protein C and protease-activated receptor 1. RESULTS: Gene profiling of endothelial cells stimulated by 40 nmol/L activated protein C on microparticles showed significant changes in anti-apoptotic and inflammatory pathways. This was accompanied by protease-activated receptor 1-dependent anti-apoptotic and barrier protective effects, the latter of which also involved sphingosine 1-phosphate receptor and vascular endothelial growth factor receptor-2/ kinase insert domain receptor. Protein C blockade reversed these effects showing specificity for activated protein C on microparticles. Furthermore, confocal microscopy and enzyme-linked immunosorbent assay of plasma obtained from septic patients during recombinant activated protein C treatment showed evidence of their presence in vivo. CONCLUSIONS: Activated protein C on microparticle-associated endothelial protein C receptor release can induce protease-activated receptor 1-dependent endothelial effects. The mechanisms underlying barrier protection involve sphingosine 1-phosphate receptor and kinase insert domain receptor.

Cell motility in chronic lymphocytic leukemia: defective Rap1 and alphaLbeta2 activation by chemokine.

Chemokine-induced activation of alpha4beta1 and alphaLbeta2 integrins (by conformational change and clustering) is required for lymphocyte transendothelial migration (TEM) and entry into lymph nodes. We have previously reported that chemokine-induced TEM is defective in chronic lymphocytic leukemia (CLL) and that this defect is a result of failure of the chemokine to induce polar clustering of alphaLbeta2; engagement of alpha4beta1 and autocrine vascular endothelial growth factor (VEGF) restore clustering and TEM. The aim of the present study was to characterize the nature of this defect in alphaLbeta2 activation and determine how it is corrected. We show here that the alphaLbeta2 of CLL cells is already in variably activated conformations, which are not further altered by chemokine treatment. Importantly, such treatment usually does not cause an increase in the GTP-loading of Rap1, a GTPase central to chemokine-induced activation of integrins. Furthermore, we show that this defect in Rap1 GTP-loading is at the level of the GTPase and is corrected in CLL cells cultured in the absence of exogenous stimuli, suggesting that the defect is the result of in vivo stimulation. Finally, we show that, because Rap1-induced activation of both alpha4beta1 and alphaLbeta2 is defective, autocrine VEGF and chemokine are necessary to activate alpha4beta1 for ligand binding. Subsequently, this binding and both VEGF and chemokine stimulation are all needed for alphaLbeta2 activation for motility and TEM. The present study not only clarifies the nature of the alphaLbeta2 defect of CLL cells but is the first to implicate activation of Rap1 in the pathophysiology of CLL.

Central role of protein kinase Cepsilon in constitutive activation of ERK1/2 and Rac1 in the malignant cells of hairy cell leukemia.

We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCepsilon in the constitutive activation of Rac1 and ERK in HCs. PKCepsilon and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCepsilon was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCepsilon plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia.

B-cell receptor signaling in chronic lymphocytic leukemia cells is regulated by overexpressed active protein kinase CbetaII.

Signals through the B-cell antigen receptor (BCR) are important for the survival of chronic lymphocytic leukemia (CLL) cells. Therefore, factors that influence these signals have important pathophysiological roles in this disease. One key mediator of BCR signaling is protein kinase C beta (PKCbeta), which regulates the activation of I-kappaB kinases and the deactivation of Bruton tyrosine kinase within the signaling pathways initiated by BCR engagement. The present study demonstrates that overexpression of the PKCbetaII isoform is a feature of CLL cells and that activity of this enzyme strongly correlates with CLL cell response to BCR engagement. Thus, intracellular Ca2+ release and increases in cell survival after BCR cross-linking were significantly greater in CLL patients with low levels than in CLL patients with high levels of active PKCbetaII. Furthermore, BCR-induced Ca2+ fluxes could be restored in CLL patients with high levels of active PKCbetaII by pretreating the cells with the PKCbeta-specific inhibitor LY379196. Conversely, BCR-mediated intracellular Ca2+ release could be inhibited in CLL cells with low levels of active PKCbetaII by pretreatment with the PKC agonist bryostatin. Taken together, these results demonstrate that overexpressed active PKCbetaII plays a role in the regulation and outcome of BCR signals that can be important for the progression of CLL.

c-Abl expression in chronic lymphocytic leukemia cells: clinical and therapeutic implications.

c-Abl is important for normal B-cell development, but little is known about the function of this nonreceptor tyrosine kinase in chronic lymphocytic leukemia (CLL). Therefore, the aim of the present study was to examine the clinical, therapeutic, and pathogenetic importance of c-Abl in this disease. We show that the malignant cells of CLL predominantly express the type 1b splice variant of c-Abl and that the expression of c-Abl protein is higher in CLL cells than in normal peripheral blood B cells. Moreover, we show that the levels of c-Abl protein expression correlate positively with tumor burden and disease stage, and negatively with IgV(H) mutation. We also show that STI-571, an inhibitor of c-Abl kinase activity, induces apoptosis of CLL cells with high c-Abl expression levels through a mechanism involving inhibition of nuclear factor kappaB. We conclude that overexpression of c-Abl is likely to play a pathogenetic role in CLL and that STI-571 may be of potential use in the treatment of this disease.

Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia.

Hairy cells (HCs) are mature malignant B cells that contain a number of constitutively active signaling molecules including GTP-bound Rac1, protein kinase C, and Src family kinases. Because Rac1 is a component of the reactive oxidant species (ROS)-generating NADPH oxidase system, we investigated the role of this GTPase in ROS production in HCs. In this study, we show that ROS production in HCs involves a flavin-containing oxidase dependent on Ca2+, but not on GTP-Rac1 or protein kinase C. This suggests the involvement of the nonphagocytic NADPH oxidase NOX5, an enzyme found in lymphoid tissues, but not in circulating lymphocytes. By using RT-PCR and Southern and Western blotting and by measuring superoxide anion production in membrane fractions in the absence of cytosolic components, we demonstrate for the first time that HCs (but not circulating normal B cells or some other lymphoid cell types) express NOX5. We also demonstrate that inhibition of NADPH oxidase in HCs results in a selective increase in the activity of Src homology region 2 domain-containing phosphatase 1 (SHP-1). Furthermore, SHP-1 in HCs coimmunoprecipitates with tyrosine phosphorylated CD22 and localizes in the same cellular compartment as NOX5. This allows the inactivation of SHP-1 by NOX5-generated ROS and contributes to the maintenance of the constitutive activation of HCs.

B-cell receptor translocation to lipid rafts and associated signaling differ between prognostically important subgroups of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which interaction of the malignant cells with antigen is thought to play a key role. Individual CLL-cell clones markedly differ in their ability to respond to B-cell receptor ligation, but the mechanism underlying the frequent hyporesponsiveness is incompletely understood. Our aim was to further clarify the extent and cause of the B-cell receptor signaling abnormality in CLL and to assign pathophysiologic relevance to the presence or absence of B-cell receptor responsiveness. We show that extracellular signal-regulated kinase-2 phosphorylation, intracellular Ca2+ increases, CD79a phosphorylation, and translocation of the B-cell receptor to lipid rafts in response to ligation with anti-immunoglobulin M (as a surrogate for antigen) are features of CLL cells with relatively unmutated VH genes (<5% deviation from germ line) and a poor prognosis. B-cell receptor stimulation in these cases also promoted cell survival. In clones with mutated VH genes (>5% deviation from germ line), surface immunoglobulin M ligation failed to induce receptor translocation to rafts or to prolong cell survival. This failure of receptor translocation observed in mutated CLL cells was associated with the constitutive exclusion of the B-cell receptor from rafts by a mechanism involving src-dependent interactions between the B-cell receptor and the actin cytoskeleton. We conclude that exposure to antigen promotes the survival of unmutated CLL clones, contributing to the poor prognosis of this group. In contrast, hyporesponsive mutated CLL clones may have developed into a stage where continuous exposure to antigen results in relative tolerance to antigenic stimulation mediated by the exclusion of the B-cell receptor from lipid rafts.

CLL, but not normal, B cells are dependent on autocrine VEGF and alpha4beta1 integrin for chemokine-induced motility on and through endothelium.

Vascular endothelial cell growth factor (VEGF) is a multifunctional cytokine involved in tumor formation. In chronic lymphocytic leukemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and alpha(4)beta(1) integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium-processes important for the invasion of lymphoreticular tissues, a major determinant of disease outcome. In contrast, normal lymphocytes were not dependent on autocrine VEGF or alpha(4)beta(1) for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate alpha(L)beta(2) in response to chemokines, unless VEGF receptor(s) and alpha(4)beta(1) were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility, and that the alpha(L)beta(2) on the malignant cells is functionally altered compared with that of normal B cells in not undergoing activation in response to chemokine alone. Given the importance of cell motility for tissue invasion, the present results provide a rationale for a trial of VEGF and alpha(4) blockade in patients with CLL who have tissue disease.

The role of matrix metalloproteinase 9 in the pathogenesis of chronic lymphocytic leukaemia.

Matrix metalloproteinases (MMPs) are important for the pathogenesis and progression of different tumours. MMPs-2 and -9 are the principal MMPs produced by lymphocytes; these enzymes can degrade a number of matrix proteins but are the two main MMPs that digest type IV collagen, the major component of basement membranes. Therefore, these enzymes are potentially important for tissue invasion and remodelling by malignant lymphocytes. This study showed that chronic lymphocytic leukaemia (CLL) cells produce and secrete variable amounts of pro-MMP-9, but no MMP-2 or tissue inhibitor of metalloproteinase 1 (TIMP-1). The pro-enzyme was found in monomeric and dimeric forms and also complexed with lipocalin. Moreover, a small fraction of secreted monomer became associated with the cell surface and activated upon cell adhesion to insolubilized type IV collagen. High levels of intracellular MMP-9 were associated with advanced (stage C) disease and with poor patient survival. Immunohistochemical studies demonstrated that MMP-9 was associated with areas of tissue invasion and remodelling. The relatively specific MMP-9 inhibitors, Ro31-9790 (3 micromol/l) and TIMP-1, reduced CLL-cell migration through type IV collagen and through endothelial monolayers suggesting that the enzyme may also be important in malignant cell entry and egress to and from involved tissue. Our data raise the possibility that MMP-9 modulation may have therapeutic potential in advanced CLL.

Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function.

Atrolysin A and jararhagin are class P-III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin-like and a cysteine-rich. The metalloproteinase and the disintegrin-like domains of atrolysin A and jararhagin contain peptide sequences that interact with alpha2beta1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine-rich domain of atrolysin A was shown to have similar effects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine-rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of alpha2-expressing K562 cells to this protein. In addition, the peptide effects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the first time, sites in the cysteine-rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological effects of these enzymes with multifunctional domains.

The role of autocrine FGF-2 in the distinctive bone marrow fibrosis of hairy-cell leukemia (HCL).

Bone marrow (BM) fibrosis is a central diagnostic and pathogenetic feature of hairy-cell leukemia (HCL). It is known that fibronectin (FN) produced and assembled by the malignant hairy cells (HCs) themselves is a major component of this fibrosis. It is also known that FN production is greatly enhanced by adhesion of HCs to hyaluronan (HA) via CD44. The aim of the present study was to establish the roles of fibrogenic autocrine cytokines (fibroblast growth factor-2 [FGF-2] and transforming growth factor beta [TGFbeta]) and of different isoforms of CD44 in this FN production. We show that HC adhesion to HA stimulates FGF-2, but not TGFbeta, production and that HCs possess FGF-2 receptor. In a range of experiments, FN production was greatly reduced by blocking FGF-2 but not TGFbeta. Moreover FN, but not FGF-2, secretion was blocked by down-regulation of the v3 isoform of CD44 and by addition of heparitinase. These results show that autocrine FGF-2 secreted by HCs is the principal cytokine responsible for FN production by these cells when cultured on HA. The central role of FGF-2 in the pathogenesis of the BM fibrosis of HCL was supported by our immunohistochemical demonstration of large amounts of this cytokine in fibrotic BM but not in HCL spleen where there is no fibrosis. As regards CD44 isoforms, the present work demonstrates that CD44v3 is essential for providing the heparan sulfate necessary for HC stimulation by FGF-2, whereas the signal for production of the cytokine was provided by HA binding to CD44H, the standard hematopoietic form of the molecule.

Regulation of hairy-cell survival through constitutive activation of mitogen-activated protein kinase pathways.

The hairy cells (HCs) of hairy-cell leukemia are intrinsically activated mature clonal B cells. The aims of this study were to gain further insights into the nature of this activation and to assess its importance for the prolonged HC survival in this chronic disease. We show that HCs contain phosphorylated/activated p38 MAPK, JNK and ERK1/ERK2 (ERK1/2). PKC inhibitors increased the activation of p38 and JNK, but reduced the phosphorylation of ERK1/2. Moreover, PKC inhibition resulted in cell death; cell death was also observed when the activation of ERK1/2 in HCs was abrogated with an inhibitor of MEK1/2 activation. In addition to PKC, active Src kinase was also shown to be involved in the maintenance of Raf-independent ERK activation in HCs. During cell culture on a nonadherent surface, ERK phosphorylation was sustained, while phosphorylation of p38 and JNK decreased. This decrease was not observed in HCs cultured on vitronectin (VN), indicating that p38/JNK activation is probably a consequence of in vivo HC interaction with VN present in abundance in the red pulp of the spleen. Taken together, these results suggest that active p38/JNK make HCs susceptible to apoptosis, but the cells are effectively rescued by ERK activation involving constitutively active PKC and Src. These findings are relevant for the understanding of the prolonged cell survival of HCs and their selective sensitivity to some chemotherapeutic agents.

The role of phospholipases A2 in the stimulation of neutrophil motility by cobra venoms.

Neutrophil (PMN) accumulation frequently occurs at the site of snakebite as part of the inflammatory response to envenoming. We demonstrate here that the venoms of the cobras, Naja naja and N. mossambica, and two purified venom phospholipase A(2)s (PLA(2)s) isolated from the latter venom, stimulate CD11b translocation from the PMN granule store to the plasma membrane and enhance neutrophil motility on collagen-coated surfaces. These effects were partially attenuated by the PLA(2) inhibitor, aristolochic acid, and almost completely abolished by the specific cytosolic PLA(2) inhibitor, methylarachidonylfluorophosphonate (MAFP). Annexin V and inhibitors of collagenase, cyclo-oxygenase and lipo-oxygenase, all inhibited PMN motility to a variable extent. FACS analysis and confocal microscopy showed that Annexin V interfered with binding and rapid endocytosis of the venom PLA(2). These results indicate that venom and venom PLA(2) preparations first caused a non-enzymatic stimulation of PMN leading to the activation of cytosolic PMN PLA(2) and production of arachidonate metabolites involved in stimulation of PMN degranulation and motility. The evidence suggests that venom PLA(2) then interacts with anionic phospholipids exposed on stimulated PMN, becomes endocytosed, and then contributes itself to the production of chemoattractants responsible for PMN accumulation at the site of the snakebite.

Response of hairy cells to IFN-alpha involves induction of apoptosis through autocrine TNF-alpha and protection by adhesion.

Although hairy cell leukemia is uniquely sensitive to interferon-alpha (IFN-alpha), the biologic basis for this phenomenon remains unclear. Here we examine the effects of IFN-alpha on cultured hairy cells (HCs), taking into account the possible modifying influence of cell adhesion. We make the novel observation that therapeutic concentrations of IFN-alpha kill nonadherent HCs by inducing apoptosis. In keeping with the persistence of HCs in tissues during therapy, such killing was inhibited by integrin-mediated adhesion to vitronectin or fibronectin. Exposure of HCs to IFN-alpha resulted in a marked increase in tumor necrosis factor-alpha (TNF-alpha) secretion. Furthermore, blocking antibodies to TNF-RI or TNF-RII protected HCs from IFN-alpha-induced apoptosis, demonstrating that such killing was mediated by TNF-alpha. In the absence of IFN-alpha, exogenous TNF-alpha did not induce HC apoptosis, showing that IFN-alpha sensitized HCs to the proapoptotic effect of autocrine TNF-alpha. This sensitization to TNF-alpha-induced killing was attributable to suppression of IAP (inhibitors of apoptosis) production known to be regulated by the cytoprotective nuclear factor-kappaB-dependent arm of TNF-alpha signaling. Moreover, engagement of the receptors for fibronectin or vitronectin prevented this IFN-alpha-induced down-regulation of IAPs. Understanding of the signals involved in the combined effects of IFN-alpha and TNF-alpha and abrogation of those induced by integrin engagement offers the possibility of sensitizing other malignant cells to IFN-alpha-induced killing and thereby extending the therapeutic use of this cytokine.

The chemokine receptor CCR7 and alpha4 integrin are important for migration of chronic lymphocytic leukemia cells into lymph nodes.

Malignant lymphocyte migration into lymph nodes is an important aspect of chronic lymphocytic leukemia (CLL), yet little is known about the processes involved. Here we demonstrate that CLL cells migrate across vascular endothelium in response to at least 3 chemokines, namely, CCL21, CCL19, and CXCL12. Moreover, transendothelial cell migration (TEM) in response to CCL21 and CCL19 was significantly higher for the malignant B cells of patients who had clinical lymph node involvement as compared with those of patients lacking such organomegaly. Furthermore, the expression of CCR7, the receptor for both CCL21 and CCL19, correlated with clinical lymphadenopathy, and blocking of CCR7 inhibited CLL cell TEM. By using immunohistochemistry we demonstrated that CCL21 and CCL19, but not CXCL12, are located in high endothelial venules and are, therefore, in an appropriate location to induce TEM. Regarding the adhesion receptors involved in TEM, alpha4 (most likely in association with beta1) and alphaLbeta2 were shown to be important in CLL cell TEM in vitro, but only the level of alpha4 expression correlated with the presence of clinical lymphadenopathy. The present studies are the first to shed light on the factors determining CLL cell entry into nodes and define the phenotype of circulating malignant cells likely to determine the pattern of lymph node enlargement in the disease.