Differentiation and cell density upregulate cytochrome c levels in megakaryoblastic cell lines: Implications for analysis of CYCS-associated thrombocytopenia.

Mutations in the cytochrome c gene (CYCS) cause autosomal dominant thrombocytopenia by an unknown mechanism. While attempting to generate megakaryoblastic cell lines exogenously expressing cytochrome c variants, we discovered that endogenous cytochrome c expression increased both upon induction of differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), and as cell density increased. A concomitant increase in cytochrome c oxidase subunit II in response to PMA, but not cell higher cell density, suggests upregulation of the mitochondrial respiratory chain may be a specific feature of differentiation. These results highlight the likely importance of cytochrome c in both differentiating and proliferating cells, and illustrate the unsuitability of megakaryoblastic lines for modeling CYCS-associated thrombocytopenia.

Novel pyrrole carboxamide inhibitors of JAK2 as potential treatment of myeloproliferative disorders.

Compound 1, a hit from the screening of our chemical collection displaying activity against JAK2, was deconstructed for SAR analysis into three regions, which were explored. A series of compounds was synthesized leading to the identification of the potent and orally bioavailable JAK2 inhibitor 16 (NMS-P830), which showed an encouraging tumour growth inhibition in SET-2 xenograft tumour model, with evidence for JAK2 pathway suppression demonstrated by in vivo pharmacodynamic effects.

JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth.

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.

On the origin of matrix metalloproteinase-2 and -9 in blood platelets.

To date, several matrix metalloproteinases (MMPs) have been identified in human platelets. In most research studies, the platelets are obtained using the isolation method from plasma by centrifugation and washing. The metalloproteinase content in the platelets can be affected by the isolation technique and the leukocyte contamination. In this work, we studied the influence of the isolation method on the detection of platelet MMPs and explore the expression of these enzymes in megakaryoblastic MEG-01 cells. We investigated the expression of mRNAs encoding for MMP-2 and -9 in platelets and MEG-01 cells. Using gelatin zymography and western blotting, we examined the expression and release of MMP-2 and 9 by platelets and MEG-01 cells and checked whether the amount of the released MMPs depends on the volume of tested platelet and leukocyte contamination. To investigate the MMP-2 expression profile, we used zymography and flow cytometry. Platelets, in contrast to the MEG-01 cells, neither contain mRNA for MMP-2 nor -9. The platelets contain pro-MMP-2 and release it during the activation. The population of uncontaminated (leukocytes<0.02%) platelets contained no MMP-9 or the active form of MMP-2. We have observed that the activity of MMP-2 in platelet lysate is proportional to their mean volume and that the MMP-2 activity may not be detected if very small platelets are examined. We conclude that the detection of gelatinases in platelets depends on platelet isolation techniques and the degree of leukocyte contamination.

[Src kinases in the process of maturation megakryocyte progenitors]. / Kinazy src w procesie dojrzewania progenitorów megakariocytów.

Src family protein tyrosine kinases play key roles in cell morphology, proliferation, motility, and survival in megakaryocytopoiesis. Six of Src family kinases (Fyn, Lyn, Fgr, Hck, Src and Yes), are present in megakaryocytes (Mks). Src kinases are negative factors of megakaryocytopoiesis induced by thrombopoietin. The inhibitors of Src kinases might be useful as agents inducing maturation of Mks. The experiments with inhibitors of Src kinases used in culture of Mk progenitors and potential megakaryocyte cell lines gave new information about the role of Src kinases in the development of Mks. The pyrrolo-pyrimidyne reagents family and highly selective inhibitor, SU6656, are known and used inhibitors of Src kinases. The presence of inhibitor in ex vivo culture of Mk progenitors blocks proliferation and simultaneously induces the changes in cell morphology, phenotype and ploidy level, indicating the maturation of the cells. The inhibitors of Src kinases also might play the therapeutic role. Dasatinib, dual Bcr-Abl/Src kinase inhibitor, is of high activity and induces hematologic and cytogenetic responses in patients with chronic myelogenous leukaemia in blast crisis.