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Recent observations indicate that,in several types of human cancer,only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the cancer stem cell" (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues� View Publication

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling,which is implicated in MPD pathogenesis. We developed TG101209,an orally bioavailable small molecule that potently inhibits JAK2 (IC(50)=6 nM),FLT3 (IC(50)=25 nM) and RET (IC(50)=17 nM) kinases,with significantly less activity against other tyrosine kinases including JAK3 (IC(50)=169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC(50) of approximately 200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line,TG101209-induced cell cycle arrest and apoptosis,and inhibited phosphorylation of JAK2V617F,STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore,TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations. View Publication

Novel derivatives of 1,2,4-triazoles are described as potent ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds display VEGFR-2 inhibitory activity comparable to that of Vatalanib and Vandetanib in both homogenous time-resolved fluorescence enzymatic and cellular assays. Several active molecules feature high intrinsic permeability (textgreater30 x 10(-5) cm/min) across Caco-2 cell monolayer. View Publication

A first step in hematopoiesis is the specification of the lymphoid and myeloid lineages from multipotent progenitor cells in the bone marrow. Using a conditional ablation strategy in adult mice,we show that this differentiation step requires Patched (Ptch),the cell surface-bound receptor for Hedgehog (Hh). In the absence of Ptch,the development of T- and B-lymphoid lineages is blocked at the level of the common lymphoid progenitor in the bone marrow. Consequently,the generation of peripheral T and B cells is abrogated. Cells of the myeloid lineage develop normally in Ptch mutant mice. Finally,adoptive transfer experiments identified the stromal cell compartment as a critical Ptch-dependent inducer of lymphoid versus myeloid lineage commitment. Our data show that Ptch acts as a master switch for proper diversification of hematopoietic stem cells in the adult organism. View Publication

OBJECTIVES: Recent data show that Imatinib mesylate (IM) also affects haematopoietic stem cells (HSC),T lymphocytes and dendritic cells that do not harbour constitutively active tyrosine kinases. MATERIALS AND METHODS: We evaluated possible effects of IM on human bone marrow-derived mesenchymal stem cells (MSC) in vitro. RESULTS: Screening the activity of 42 receptor tyrosine kinases revealed an exclusive inhibition of platelet-derived growth factor receptorbeta (PDGFRbeta). Analysis of downstream targets of PDGFRbeta demonstrated IM-mediated reduction of Akt and Erk1/2 phosphorylation. Culture of MSC with IM led to the reversible development of perinuclear multi-vesicular bodies. The proliferation and clonogenicity of MSC were significantly reduced compared to control cultures. IM favoured adipogenic differentiation of MSC whereas osteogenesis was suppressed. The functional deficits described led to a 50% reduction in the support of clonogenic haematopoietic stem cells,cultured for 1 month on a monolayer of MSC with IM. CONCLUSION: In summary,inhibition of PDGFRbeta and downstream Akt and Erk signalling by IM has a significant impact on proliferation and differentiation of human MSC in vitro. View Publication

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research,hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor,Y-27632,to hES cells markedly diminishes dissociation-induced apoptosis,increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore,dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1(+) cortical and basal telencephalic progenitors,as do SFEB-cultured mouse ES cells. View Publication

The aims of our study were to verify whether it was possible to generate in vitro,from different adult human tissues,a population of cells that behaved,in culture,as multipotent stem cells and if these latter shared common properties. To this purpose,we grew and cloned finite cell lines obtained from adult human liver,heart,and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs,obtained from the 3 different tissues,expressed the pluripotent state-specific transcription factors Oct-4,NANOG,and REX1,displayed telomerase activity,and exhibited a wide range of differentiation potential,as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content,and shared a common gene expression signature,compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular,the pathways regulating stem cell self-renewal/maintenance,such as Wnt,Hedgehog,and Notch,were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin. View Publication

After more than 40 years of clinical use,the mechanisms of action of valproate in epilepsy,bipolar disorder and migraine are still not fully understood. However,recent findings reviewed here shed new light on the cellular effects of valproate. Beyond the enhancement of gamma-aminobutyric acid-mediated neurotransmission,valproate has been found to affect signalling systems like the Wnt/beta-catenin and ERK pathways and to interfere with inositol and arachidonate metabolism. Nevertheless,the clinical relevance of these effects is not always clear. Valproate treatment also produces marked alterations in the expression of multiple genes,many of which are involved in transcription regulation,cell survival,ion homeostasis,cytoskeletal modifications and signal transduction. These alterations may well be relevant to the therapeutic effects of valproate,and result from its enhancement of activator protein-1 DNA binding and direct inhibition of histone deacetylases,and possibly additional,yet unknown,mechanism(s). Most likely,both immediate biochemical and longer-term genomic influences underlie the effects of valproate in all three indications. View Publication

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion,stress fiber formation,and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study,we investigated the antiangiogenic effect of fasudil,one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration,viability,and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation,focal adhesion assembly,and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore,fasudil inhibited VEGF-induced phosphorylation of myosin light chain,which is one of the main substrates of ROCK. Therefore,the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis,which resulted in the decrease of cell viability. Moreover,fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore,fasudil might be useful for the treatment of angiogenesis-related diseases,especially cancer. View Publication

Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to beta cells. Generation of glucose-responsive,insulin-producing beta cells from self-renewing,pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol,hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17,and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor,basic fibroblast growth factor,and noggin. Soon thereafter,expression of Ptf1a and Ngn3 was detected,indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development,and the final population contained representatives of the ductal,exocrine,and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells,as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/mug of DNA was measured in the ILCs,representing levels higher than that of human fetal islets. In addition,the hESC-derived ILCs contained numerous secretory granules,as determined by electron microscopy,and secreted human C-peptide in a glucose-dependent manner. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

15-Methyl PGE2 and 16,16-dimethyl PGE2 were found (1) to be 40 and 100 times,respectively,more potent than PGE2 after intravenous administration in inhibiting histamine-stimulated gastric secretion in dogs with a denervated (Heidenhain) gastric pouch,(2) to be active orally and intrajejunally,whereas PGE2 was inactive,and (3) to exert antisecretory activity for longer duration than PGE2. 16,16-Dimethyl PGE2 was about 2.5 times more potent than 15-methyl PGE2. Volume,acid concentration,and output,and pepsin output (but not concentration) were reduced in a dose-dependent manner. In the rat,16,16-dimethyl PGE2 also inhibited gastric secretion and prevented the formation of ulcers produced by various methods: gastric ulcers (Shay,and steroid induced) and duodenal ulcers (secretogogue induced). In this species,1l816-dimethyl PGE2 was 2 to 50 times more potent than PGE2,depending on the endpoint,and was active orally. These prostaglandins appear to inhibit gastric acid secretion by acting directly on the parietal cells,and making these unresponsive to most stimulants. Vomiting was a side effect of the prostaglandin analogues in the dog,but almost exclusively when these were given orally. After intravenous or intrajejunal administration at doses inhibiting gastric secretion by 80%,vomiting was seen only once. These results suggest that 15-methyl PGE2 and 16,16-dimethyl PGE2 may be of value in the treatment of peptic ulcer. View Publication

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover,CaP metastasizes to the bone marrow,which harbors a rich source of lipids stored within adipocytes. Here,we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique,we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition,larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction,the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes,indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction. View Publication