技术资料

TEL-TRKC is a fusion gene generated by chromosomal translocation and encodes an activated tyrosine kinase. Uniquely,it is found in both solid tumors and leukemia. However,a single exon difference (in TEL) in TEL-TRKC fusions is associated with the two sets of cancer phenotypes. We expressed the two TEL-TRKC variants in vivo by using the 3' regulatory element of SCL that is selectively active in a subset of mesodermal cell lineages,including endothelial and hematopoietic stem cells and progenitors. The leukemia form of TEL-TRKC (-exon 5 of TEL) enhanced hematopoietic stem cell renewal and initiated leukemia. In contrast,the TEL-TRKC solid tumor variant (+ TEL exon 5) elicited an embryonic lethal phenotype with impairment of both angiogenesis and hematopoiesis indicative of an effect at the level of the hemangioblasts. The ability of TEL-TRKC to repress expression of Flk1,a critical regulator of early endothelial and hematopoietic cells,depended on TEL exon 5. These data indicate that related oncogenic fusion proteins similarly expressed in a hierarchy of early stem cells can have selective,cell type-specific developmental impacts. View Publication

Polo-like kinases (PLKs) play critical roles throughout mitosis. Here,we report that wortmannin,which was previously thought to be a highly selective inhibitor of phosphoinositide (PI) 3-kinases,is a potent inhibitor of mammalian PLK1. Observation of the wortmannin-PLK1 interaction was enabled by a tetramethylrhodamine-wortmannin conjugate (AX7503) that permits rapid detection of PLK1 activity and expression in complex proteomes. Importantly,we show that wortmannin inhibits PLK1 activity in an in vitro kinase assay with an IC(50) of 24 nM and when incubated with intact cells. Taken together,our results indicate that,at the concentrations of wortmannin commonly used to inhibit PI 3-kinases,PLK1 is also significantly inhibited. View Publication

This letter describes the development of two series of potent and selective allosteric Akt kinase inhibitors that display an unprecedented level of selectivity for either Akt1,Akt2 or both Akt1/Akt2. An iterative analog library synthesis approach quickly provided a highly selective Akt1/Akt2 inhibitor that induces apoptosis in tumor cells and inhibits Akt phosphorylation in vivo. View Publication

Autosomal recessive osteopetrosis (OP) is a rare,lethal disorder in which osteoclasts are absent or nonfunctional,resulting in a bone marrow cavity insufficient to support hematopoiesis. Because osteoclasts are derived from hematopoietic precursors,allogeneic hematopoietic cell transplantation can cure the bony manifestations of the disorder. However,high rates of graft failure have been observed in this population. It is not possible to harvest bone marrow from these patients for reinfusion should graft failure be observed. We report that 8 of 10 patients with OP had high numbers of circulating CD34(+) cells (3% +/- 0.9%). This increased proportion of peripheral CD34(+) cells made it possible to harvest 2 x 10(6) CD34(+) cells per kilogram with a total volume of blood ranging from 8.3 to 83.7 mL (1.3-11.6 mL/kg). In addition,colony-forming assays documented significantly more colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid in the blood of osteopetrotic patients compared with controls; the numbers of colony-forming units approximated those found in control marrow. We conclude that OP patients with high levels of circulating CD34(+) are candidates for peripheral blood autologous harvest by limited exchange transfusion. These cells are then available for reinfusion should graft failure be observed in patients for whom retransplantation is impractical. View Publication

The leukocyte activation marker CD69 is a novel regulator of the immune response,modulating the production of cytokines including transforming growth factor-beta (TGF-beta). We have generated an antimurine CD69 monoclonal antibody (mAb),CD69.2.2,which down-regulates CD69 expression in vivo but does not deplete CD69-expressing cells. Therapeutic administration of CD69.2.2 to wild-type mice induces significant natural killer (NK) cell-dependent antitumor responses to major histocompatibility complex (MHC) class I low RMA-S lymphomas and to RM-1 prostatic carcinoma lung metastases. These in vivo antitumor responses are comparable to those seen in CD69(-/-) mice. Enhanced host NK cytotoxic activity correlates with a reduction in NK-cell TGF-beta production and is independent of tumor priming. In vitro studies demonstrate the novel ability of anti-CD69 mAbs to activate resting NK cells in an Fc receptor-independent manner,resulting in a substantial increase in both NK-cell cytolytic activity and interferon gamma (IFNgamma) production. Modulation of the innate immune system with monoclonal antibodies to host CD69 thus provides a novel means to antagonize tumor growth and metastasis. View Publication

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphoma with poor response to therapy and unfavorable prognosis. Here,we show that retinoic acid (RA) isomers significantly inhibit the proliferation of both primary MCL cultures (n = 7) and established cell lines (Granta 519 and SP-53) as shown by [(3)H]thymidine uptake and carboxyfluorescein diacetate succinimidyl ester labeling coupled with cyclin D1 staining. RA induces cell accumulation in G(0)-G(1) together with a marked up-regulation of p27(Kip1) by inhibiting ubiquitination and proteasome-dependent degradation of the protein. The p21(Cip1) inhibitor was also up-regulated by RA in Granta 519 cells,whereas the expression of cyclin D1 is unaffected. Most of RA-induced p27(Kip1) was bound to cyclin D1/cyclin-dependent kinase 4 complexes,probably contributing to the decreased cyclin-dependent kinase 4 kinase activity and pRb hypophosphorylation observed in RA-treated cells. Experiments with receptor-selective ligands indicate that RA receptor alpha cooperates with retinoid X receptors in mediating RA-dependent MCL cell growth inhibition. Notably,RA isomers,and particularly 9-cis-RA,also inhibited the growth-promoting effect induced in primary MCL cells by CD40 activation alone or in combination with interleukin-4. Immunohistochemical analysis showed that significant numbers of CD40L-expressing lymphoid cells are present in lymph node biopsies of MCL patients. These results therefore further strengthen the possibility that triggering of CD40 by infiltrating CD40L+ cells may continuously promote the growth of MCL cells in vivo. On these grounds,our findings that RA inhibits basal MCL proliferation as well as MCL growth-promoting effects exerted by microenvironmental factors make these compounds highly attractive in terms of potential clinical efficacy in this setting. View Publication

Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications,and knowledge of the surface phenotype of ex vivo-expanded HSCs will be critical to their purification and analysis. Here,we developed a simple culture system for bone marrow (BM) HSCs using low levels of stem cell factor (SCF),thrombopoietin (TPO),insulin-like growth factor 2 (IGF-2),and fibroblast growth factor-1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses,there was a more than 20-fold increase in numbers of long-term (LT)-HSCs after a 10-day culture of total BM cells. Culture of BM side population" (SP) cells� View Publication

Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to derivatives of all three primary germ layers,yet little is known about the signaling cascades that govern their pluripotent character. Because it plays a prominent role in the early cell fate decisions of embryonic development,we have examined the role of TGFbeta superfamily signaling in hESCs. We found that,in undifferentiated cells,the TGFbeta/activin/nodal branch is activated (through the signal transducer SMAD2/3) while the BMP/GDF branch (SMAD1/5) is only active in isolated mitotic cells. Upon early differentiation,SMAD2/3 signaling is decreased while SMAD1/5 signaling is activated. We next tested the functional role of TGFbeta/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extend these findings to show that SMAD2/3 activation is required downstream of WNT signaling,which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly,we show that in ex vivo mouse blastocyst cultures,SMAD2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a crucial role for TGFbeta signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFbeta and WNT signaling in these contexts. View Publication

Experimental evidence indicates that shear stress (SS) exerts a morphogenetic function during cardiac development of mouse and zebrafish embryos. However,the molecular basis for this effect is still elusive. Our previous work described that in adult endothelial cells,SS regulates gene expression by inducing epigenetic modification of histones and activation of transcription complexes bearing acetyltransferase activity. In this study,we evaluated whether SS treatment could epigenetically modify histones and influence cell differentiation in mouse embryonic stem (ES) cells. Cells were exposed to a laminar SS of 10 dyne per cm2/s(-1),or kept in static conditions in the presence or absence of the histone deacetylase inhibitor trichostatin A (TSA). These experiments revealed that SS enhanced lysine acetylation of histone H3 at position 14 (K14),as well as serine phosphorylation at position 10 (S10) and lysine methylation at position 79 (K79),and cooperated with TSA,inducing acetylation of histone H4 and phosphoacetylation of S10 and K14 of histone H3. In addition,ES cells exposed to SS strongly activated transcription from the vascular endothelial growth factor (VEGF) receptor 2 promoter. This effect was paralleled by an early induction of cardiovascular markers,including smooth muscle actin,smooth muscle protein 22-alpha,platelet-endothelial cell adhesion molecule-1,VEGF receptor 2,myocyte enhancer factor-2C (MEF2C),and alpha-sarcomeric actin. In this condition,transcription factors MEF2C and Sma/MAD homolog protein 4 could be isolated from SS-treated ES cells complexed with the cAMP response element-binding protein acetyltransferase. These results provide molecular basis for the SS-dependent cardiovascular commitment of mouse ES cells and suggest that laminar flow may be successfully applied for the in vitro production of cardiovascular precursors. View Publication

Transforming growth factor beta (TGF-beta) signaling pathways regulate a wide variety of cellular processes including cell proliferation,differentiation,extracellular matrix deposition,development,and apoptosis. TGF-beta type-I receptor (TbetaRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TbetaRI is essential for its function. In this study,we have identified a short phospho-Smad peptide,pSmad3(-3),KVLTQMGSPSIRCSS(PO4)VS as a substrate of TbetaRI kinase for in vitro kinase assays. This peptide is uniquely phosphorylated by TbetaRI kinase at the C-terminal serine residue,the phosphorylation site of its parent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by only TbetaRI and not TGF-beta type-II receptor kinase,indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TbetaRI kinase inhibitors. Utilizing pSmad3(-3) as a substrate,we have shown that novel pyrazole compounds are potent inhibitors of TbetaRI kinase with K(i) value as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding site and are typical ATP competitive inhibitors with tight binding kinetics. More importantly,these compounds were shown to inhibit TGF-beta-induced Smad2 phosphorylation in vivo in NMuMg mammary epithelial cells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivity analysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bone morphogenic protein or platelet-derived growth factor signal transduction pathways. Further functional analysis revealed that pyrazoles are capable of blocking the TGF-beta-induced epithelial-mesenchymal transition in NMuMg cells,a process involved in the progression of cancer,fibrosis,and other human diseases. These pyrazoles provide a foundation for future development of potent and selective TbetaRI kinase inhibitors to treat human disease. View Publication