[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
Cancer Research 09/2006; 66(15):7801-9. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
British Journal of Haematology 05/2004; 125(2):128-40. · 4.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.