Targeting cancer cell metabolism is a new promising strategy to fight cancer. Metformin, a widely used antidiabetic agent, exerts antitumoral and antiproliferative action. In this study, the addition of metformin to 2-deoxyglucose (2DG) inhibited mitochondrial respiration and glycolysis in prostate cancer cells leading to a severe depletion in ATP. The combination of the two drugs was much more harmful for cancer cells than the treatment with metformin or 2DG alone, leading to 96% inhibition of cell viability in LNCaP prostate cancer cells. In contrast, a moderate effect on cell viability was observed in normal prostate epithelial cells. At the cellular level, the combination of metformin and 2DG induced p53-dependent apoptosis via the energy sensor pathway AMP kinase, and the reexpression of a functional p53 in p53-deficient prostate cancer cells restored caspase-3 activity. In addition to apoptosis, the combination of metformin and 2DG arrested prostate cancer cells in G 2 -M. This G 2 -M arrest was independent of p53 and correlated with a stronger decrease in cell viability than obtained with either drug. Finally, metformin inhibited 2DG-induced autophagy, decreased beclin 1 expression, and triggered a switch from a survival process to cell death. Our study reinforces the growing interest of metabolic perturbators in cancer therapy and highlights the potential use of the combination of metformin and 2DG as an anticancerous treatment. Cancer Res; 70(6); 2465-75. ©2010 AACR.

Platelets are released by megakaryocytes (MKs) via cytoplasmic extensions called proplatelets, which require profound changes in the microtubule and actin organization. Here, we provide evidence that the Rho/ROCK pathway, a well-known regulator of actin cytoskeleton, acts as a negative regulator of proplatelet formation (PPF). Rho is expressed at a high level during the entire MK differentiation including human CD34 ؉ cells. Thrombopoietin stimulates its activity but at a higher extent in immature than in mature MKs. Overexpression of a dominantnegative or a spontaneously active RhoA leads to an increase or a decrease in PPF indicating that Rho activation inhibits PPF. This inhibitory effect is mediated through the main Rho effector, Rho kinase (ROCK), the inhibition of which also increases PPF. Furthermore, inhibition of Rho or ROCK in MKs leads to a decrease in myosin light chain 2 (MLC2) phosphorylation, which is required for myosin contractility. Interestingly, inhibition of the MLC kinase also decreases MLC2 phosphorylation while increasing PPF. Taken together, our results suggest that MLC2 phosphorylation is regulated by both ROCK and MLC kinase and plays an important role in platelet biogenesis by controlling PPF and fragmentation. IntroductionMegakaryocytes (MKs) are the highly specialized precursor cells that lead to platelet production. MK differentiation is a continuous process characterized by sequential steps. 1 First, MKs increase their ploidy via endomitosis and begin to increase their size. 2 Then, the synthesis of storage organelles is enhanced, as well as the synthesis of plasma membrane to form the demarcation membranes. This cytoplasmic maturation is associated with a marked increase in the MK size. Finally, mature MKs release platelets probably through cytoplasmic fragmentation at the tips of long and thin extensions called proplatelets (PPTs) that contain all the platelet organelles. 3,4 The mechanisms controlling proplatelet formation (PPF) are still incompletely understood. However, PPF is associated with remarkable morphologic changes that require a profound reorganization of the cytoskeleton. 5 Increasing evidence indicates that PPTs arise from the unfolding of demarcation membranes. The microtubule cytoskeleton provides the sliding power to unfold demarcation membranes and thus to induce the pseudopodial elongations corresponding to PPTs. 6 In addition, microtubules permit the organelle transport in the PPTs and maintain the platelet discoid shape. [7][8][9][10] Although not studied in detail, the actin cytoskeleton may also participate in PPF because cytoplasmic polymerized actin is associated with demarcation membranes and actin is highly aggregated in cultured MKs when PPF occurs. 11 In addition, a crucial role of the actin cytoskeleton has been reported in platelet functions since it regulates platelet shape in unstimulated and activated platelets. 12 Evidence suggests that actin cytoskeleton may play important roles during PPT formation at 2 different stages: (1) at early stages,...

The JAK2 617V>F mutation is frequent in polycythemia vera (PV) and essential thrombocythemia (ET). Using quantitative polymerase chain reaction (PCR), we found that high levels of JAK2 617V>F in PV correlate with increased granulocytes and high levels of hemoglobin and endogenous erythroid colony formation. We detected normal progenitors and those that were heterozygous or homozygous for the mutation by genotyping ET and PV clonal immature and committed progenitors. In PV patients, we distinguished homozygous profiles with normal, heterozygous, and homozygous progenitors from heterozygous profiles with only heterozygous and normal progenitors. PV patients with a heterozygous profile had more mutated, committed progenitors than did other PV and ET patients, suggesting a selective amplification of mutated cells in the early phases of hematopoiesis. We demonstrated that mutated erythroid progenitors were more sensitive to erythropoietin than normal progenitors, and that most homozygous erythroid progenitors were erythropoietin independent. Moreover, we observed a greater in vitro erythroid amplification and a selective advantage in vivo for mutated cells in late stages of hematopoiesis. These results suggest that, for PV, erythrocytosis can occur through two mechanisms: terminal erythroid amplification triggered by JAK2 617V>F homozygosity, and a 2-step process including the upstream amplification of heterozygous cells that may involve additional molecular events. (Blood.

Megakaryocyte (MK) is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, which was a process considered to be an incomplete mitosis aborted in anaphase. Here, we used time-lapse confocal video microscopy to visualize the endomitotic process of primary human megakaryocytes. Our results show that the switch from mitosis to endomitosis corresponds to a late failure of cytokinesis accompa-

The megakaryocytic (MK) and erythroid lineages are tightly associated during differentiation and are generated from a bipotent megakaryocyte-erythroid progenitor (MEP). In the mouse, a primitive MEP has been demonstrated in the yolk sac. In human, it is not known whether the primitive MK and erythroid lineages are generated from a common progenitor or independently. Using hematopoietic differentiation of human embryonic stem cells on the OP9 cell line, we identified a primitive MEP in a subset of cells coexpressing glycophorin A (GPA) and CD41 from day 9 to day 12 of coculturing. This MEP differentiates into primitive erythroid (GPA ؉ CD41 ؊ ) and MK (GPA ؊ CD41 ؉ ) lineages. In contrast to erythropoietin (EPO)-dependent definitive hematopoiesis, KIT was not detected during erythroid differentiation. A molecular signature for the commitment and differentiation toward both the erythroid and MK lineages was detected by assessing expression of transcription factors, thrombopoietin receptor (MPL) and erythropoietin receptor (EPOR). We showed an inverse correlation between FLI1 and both KLF1 and EPOR during primitive erythroid and MK differentiation, similar to definitive hematopoiesis. This novel MEP differentiation system may allow an in-depth exploration of the molecular bases of erythroid and MK commitment and differentiation.

IntroductionMorphogen gradients are essential in the patterning of diverse tissues during development. Thus, morphogens stem from a tissue source and act on target cells by modifying programs of gene expression. Members of the hedgehog (Hh) morphogen family include Sonic (Shh), Indian, and Desert hedgehog signaling molecules. 1 Hh precursor is first cleaved and the resulting 22-kDa N-terminal active peptide is modified by addition of a palmitate group and a cholesterol molecule and becomes associated with cholesterol-rich raftlike membrane microdomains. 2,3 Such modifications play a critical role in regulating Hh binding to the plasma membrane and long-range activity. 4,5 Once shed, Hh protein binds to the patched (Ptc) receptor, releasing the latent inhibition of smoothened (Smo), which is the signaling component of the Hh-receptor complex in the target cell. 6 In vivo, Hh proteins play a key role during embryonic development, regulating not only the patterning of the central nervous system, but also cell differentiation and proliferation. Indeed, activation of Shh signaling pathway favors angiogenesis 7,8 and promotes in vitro hematopoietic 9 and thymocyte differentiation. 10 Free diffusion of soluble Hh molecules in the extracellular space has been proposed as a model to explain the formation of morphogen gradients. 11,12 Other studies have shown that membrane fragments, also termed argosomes, transporting morphogens such as wingless, might be used as a vehicle and be involved in tissue patterning, but no mechanistic insight was provided. 13,14 Chen et al 5 have reported that palmitoylation is required for producing Hh multimeric complexes able to form a morphogen gradient. In addition, the effects of Hh morphogens are not only dictated by a temporal gradient, but a spatial gradient seems also necessary. 15,16 Indeed, as recently described by Williams and colleagues 12 different morphogens in different developmental contexts may use different means of transport.Vesiculation or microvesicle (MV) release occurs during activation of virtually all cell types by various agonists, shear stress, or apoptosis. [17][18][19] The release of MVs or apoptotic bodies from senescent cells is a conserved event of the apoptosis execution phase. 20 MVs, also termed microparticles, are small plasma membrane fragments (0.05-1 m) shed by cells after membrane blebbing. At their surface MVs bear antigens characteristic of the cell of origin and carry other membrane and cytoplasmic constituents. They exhibit negatively charged phospholipids at their surface, accounting for their procoagulant character, and carry components responsible for their proinflammatory properties. Elevated levels of circulating MVs have been detected under pathologic situations, such as systemic lupus erythematosus, atherosclerosis, acute coronary syndrome, sepsis, and diabetes. [21][22][23][24][25] Although the majority of in vivo circulating MVs are derived from platelets, the proportions of the different origins can differ. Thus, in For personal use only....

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