John C. Cawley

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.

Autocrine VEGF mediates the antiapoptotic effect of CD154 on CLL cells.

CD154 is an important regulator of chronic lymphocytic leukaemia (CLL)-cell survival. In CLL, high serum levels of VEGF are a feature of advanced disease, and we and others have previously shown that CLL cells produce and secrete this growth factor. Since CD154 stimulates VEGF production in other cell types, and VEGF is known to promote cell survival, we examined whether the cytoprotection of CLL cells by CD154 involves VEGF. We report for the first time that treatment of CLL cells with CD154 results in increased VEGF production and demonstrate involvement of NF-κB in this process. Moreover, we show that CD154-induced CLL-cell survival is reduced by anti-VEGF-neutralising antibody and by inhibiting VEGF receptor (VEGFR) signalling with SU5416. However, addition of exogenous VEGF alone or blocking secreted autocrine VEGF had little or no effect on CLL-cell survival. We therefore conclude that CLL-cell cytoprotection in the presence of CD154 requires combined signalling by both CD40 and VEGFR. This combined signalling and resulting cytoprotection were shown to involve NF-κB activation and increased survivin production. In conclusion, our findings identify autocrine VEGF as an important mediator of the antiapoptotic effect of CD40 ligation, and thus provide new insights into CLL-cell rescue by CD154 in lymphoreticular tissues.

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 μmol/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.

Purine analogues kill resting lymphocytes by p53‐dependent and ‐independent mechanisms

To resolve the controversy concerning the role of p53 in the killing of resting lymphocytes by purine nucleoside analogues, we examined the cytotoxic effects of chlorodeoxyadenosine, fludarabine and deoxycoformycin (plus deoxyadenosine) on unstimulated spleen cells from p53‐knockout versus wild‐type mice.

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.

Heterogeneity of CLL: high CD23 antigen and αIFN receptor expression are features of favourable disease and of cell activation

Summary The relationship between α‐interferon receptor (αIFNR) numbers, B‐cell‐antigen expression and clinical stage was determined in 35 cases of typical chronic lymphocytic leukaemia (CLL). αIFNR numbers were shown to be low in patients with advanced disease and high in those with a more favourable prognosis. The B‐cell activation antigen CD 23 was similarly related to stage, being high in more favourable disease. Also, αIFNR expression was directly related to CD 23 positivity, but αIFN binding was not inhibited by CD 23 monoclonal antibody. There was no correlation between CD 19, 20, 22 and 24 antigen expression and either αIFNR numbers or clinical stage.

Akt is activated in chronic lymphocytic leukemia cells and delivers a pro-survival signal: the therapeutic potential of Akt inhibition.

Background The aims of the present study were to ascertain the activation status of Akt in the primary cells of chronic lymphocytic leukemia and to investigate the effects of specific Akt inhibition on chronic lymphocytic leukemia-cell survival. Design and Methods Anti-phospho-Akt (Ser473 or Thr308) antibodies and western blotting were used to establish the activation status of Akt. The effects of two different, specific small-molecule inhibitors (A-443654 or Akti-1/2) or small interfering RNA on cell survival and downstream targets of Akt were assessed. Apoptosis was determined by fluorescence-activated cell sorting analysis of phosphatidylserine exposure and by measurement of PARP cleavage. The phosphorylation status of GSK-3 and MDM2, two immediate downstream substrates of Akt, levels of the anti-apoptotic proteins BCL2 and MCL1, and expression of p53 and p21 were all measured by western blotting. Results Fully activated Akt was demonstrable in all chronic lymphocytic leukemia clones examined (n=26). These results were validated with extensive controls and it was shown that a harsh method of cell extraction is needed for detection of the active enzyme. Specific inhibition of Akt induced extensive apoptosis of chronic lymphocytic leukemia cells, which was associated with both a rapid loss of MCL1 through proteasomal degradation and increased expression of p53. Moreover, the Akt inhibitors, at concentrations that induced extensive apoptosis in chronic lymphocytic leukemia cells, had little or no effect on normal peripheral blood mononuclear cells. Conclusions Chronic lymphocytic leukemia clones consistently contain activated Akt which plays a pivotal role in maintaining cell survival. Inhibition of the Akt pathway may be of potential value as a novel therapeutic strategy in chronic lymphocytic leukemia.

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.

Mechanism of action of α interferon in chronic granulocytic leukaemia: evidence for preferential inhibition of late progenitors

The effect of α interferon (α IFN) on colony forming unit, granulocyte‐macrophage (CFU‐GM) formation by normal bone marrow (BM) as compared with chronic granulocytic leukaemia (CGL) BM and peripheral blood (PB) was tested in semi‐solid assay systems employing either 5637CM or recombinant granulocyte‐macrophage colony stimulating factor (GM‐CSF) to support growth. αIFN (> 125 U/ml) caused consistent inhibition (P=0.02) of day‐7(late progenitor) colonies, but had little or no effect on either day‐7 clusters or day‐14 colonies/clusters. This selective effect on day‐7 colonies was quantitatively similar for both normal and CGL (P < 0.5). Similar results were obtained whether or not the mononuclear preparations were depleted of potential accessory cells, suggesting that the αIFN‐suppression is directly mediated. Morphological examination of colonies and clusters showed that IFN had no effect on cell maturation and that colony inhibition is not, therefore, a consequence of blocked maturation. Since the late‐progenitor compartment is preferentially expanded in CGL, we suggest that our demonstration that αIFN selectively inhibits this compartment is relevant to the clinical effects of the cytokine in the disease.

Platelets prime PMN via released PF4 : mechanism of priming and synergy with GM-CSF

Summary. Platelet‐PMN interactions have been extensively studied and a spectrum of possible effects has been demonstrated. However, the physiological relevance of many of the observed in vitro phenomena remains obscure. Here we report a novel, and potentially pathophysiologi‐cally important, mechanism by which platelets can enhance PMN reactivity. We first observed that addition of platelets to PMN suspensions enhanced the chemilumines‐cence response of PMN to FMLP. This enhancement occurred without augmentation of superoxide generation and did not involve mutual platelet‐PMN adhesion. The soluble material responsible was biochemically and immunologically identified as PF4 derived from platelet α‐granules. The α‐granule release was shown to be selective and required minimal platelet stimulation. Since the PF4 effect did not influence NADPH oxidase activation, it differed markedly from that of other priming agents such as GM‐CSF. Further studies showed that the PF4 effect was attributable entirely to the surface translocation and secretion of primary granule myeloperoxidase. There was marked synergy between PF4 and GM‐CSF and both were required for maximal potentiation of PMN reactivity. These results demonstrate that PF4 and GM‐CSF employ different pathways in PMN priming. The ease with which platelets could release PF4 at sites of vessel‐wall damage and inflammation suggests that platelet‐PMN interaction via PF4 is likely to be of major pathophysiological importance.