技术资料

Loss of neurofibromin or interferon consensus sequence binding protein (Icsbp) leads to a myeloproliferative disorder. Transcription of NF1 is directly controlled by ICSBP. It has been postulated that loss of NF1 expression resulting from loss of transcriptional activation by ICSBP contributes to human hematologic malignancies. To investigate the functional cooperation of these 2 proteins,we have established Icsbp-deficient mice with Nf1 haploinsufficiency. We here demonstrate that loss of Icsbp and Nf1 haploinsufficiency synergize to induce a forced myeloproliferation in Icsbp-deficient mice because of an expansion of a mature myeloid progenitor cell. Furthermore,Nf1 haploinsufficiency and loss of Icsbp contribute synergistically to progression of the myeloproliferative disorder toward transplantable leukemias. Leukemias are characterized by distinct phenotypes,which correlate with progressive genetic abnormalities. Loss of Nf1 heterozygosity is not mandatory for disease progression,but its occurrence with other genetic abnormalities indicates progressive genetic alterations in a defined subset of leukemias. These data show that loss of the 2 tumor suppressor genes Nf1 and Icsbp synergize in the induction of leukemias. View Publication

Transgenic expression of just four defined transcription factors (c-Myc,Klf4,Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral,lentiviral,adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis,they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent,and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision,we show that the individual PB insertions can be removed from established iPS cell lines,providing an invaluable tool for discovery. In addition,we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies. View Publication

PURPOSE: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study,we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab,a compound used for the targeted therapy of breast cancer. EXPERIMENTAL DESIGN: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. RESULTS: In HER2-overexpressing carcinoma cell lines,cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling,as shown by the reduction of HER2 cell surface expression and lower SFE following gamma-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines,as indicated by the significant decrease in SFE and the loss of serial transplantability,following treatment of HER2-overexpressing xenotransplants. CONCLUSIONS: Here,we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression,thereby representing a critical survival pathway of tumor-initiating cells. View Publication

BACKGROUND: Omega 3 fatty acids have been found to inhibit proliferation,induce apoptosis,and promote differentiation in various cell types. The processes of cell survival,expansion,and differentiation are of key importance in the regulation of hematopoiesis. We investigated the role of omega 3 fatty acids in controlling the frequency of various myeloid progenitor cells in the bone marrow of mice. Increased progenitor cell frequency and blocked differentiation are characteristics of hematopoietic disorders of the myeloid lineage,such as myeloproliferative diseases and myeloid leukemias. RESULTS: We found that increasing the proportion of omega 3 fatty acids relative to the proportion of omega 6 fatty acids in the diet caused increased differentiation and reduced the frequency of myeloid progenitor cells in the bone marrow of mice. Furthermore,this had no adverse effect on peripheral white blood cell counts. CONCLUSION: Our results indicate that omega 3 fatty acids impact hematopoietic differentiation by reducing myeloid progenitor cell frequency in the bone marrow and promoting progenitor cell differentiation. Further exploration of this discovery could lead to the use of omega 3 fatty acids as a therapeutic option for patients that have various disorders of hematopoiesis. View Publication

The Disrupted in Schizophrenia 1 (DISC1) gene is disrupted by a balanced chromosomal translocation (1; 11) (q42; q14.3) in a Scottish family with a high incidence of major depression,schizophrenia,and bipolar disorder. Subsequent studies provided indications that DISC1 plays a role in brain development. Here,we demonstrate that suppression of DISC1 expression reduces neural progenitor proliferation,leading to premature cell cycle exit and differentiation. Several lines of evidence suggest that DISC1 mediates this function by regulating GSK3beta. First,DISC1 inhibits GSK3beta activity through direct physical interaction,which reduces beta-catenin phosphorylation and stabilizes beta-catenin. Importantly,expression of stabilized beta-catenin overrides the impairment of progenitor proliferation caused by DISC1 loss of function. Furthermore,GSK3 inhibitors normalize progenitor proliferation and behavioral defects caused by DISC1 loss of function. Together,these results implicate DISC1 in GSK3beta/beta-catenin signaling pathways and provide a framework for understanding how alterations in this pathway may contribute to the etiology of psychiatric disorders. View Publication

Adult hematopoietic stem cells (HSCs) are routinely used to reconstitute hematopoiesis after myeloablation; however,transplantation efficacy and multilineage reconstitution can be limited by inadequate HSC number,or poor homing,engraftment,or self-renewal. Here we report that mouse and human HSCs express prostaglandin E2 (PGE2) receptors,and that short-term ex vivo exposure of HSCs to PGE2 enhances their homing,survival,and proliferation,resulting in increased long-term repopulating cell (LTRC) and competitive repopulating unit (CRU) frequency. HSCs pulsed with PGE2 are more competitive,as determined by head-to-head comparison in a competitive transplantation model. Enhanced HSC frequency and competitive advantage is stable and maintained upon serial transplantation,with full multilineage reconstitution. PGE2 increases HSC CXCR4 mRNA and surface expression,enhances their migration to SDF-1 in vitro and homing to bone marrow in vivo,and stimulates HSC entry into and progression through cell cycle. In addition,PGE2 enhances HSC survival,associated with an increase in Survivin mRNA and protein expression and reduction in intracellular active caspase-3. Our results define novel mechanisms of action whereby PGE2 enhances HSC function and supports a strategy to use PGE2 to facilitate hematopoietic transplantation. View Publication

Splitomicin is derived from beta-naphthol and is an inhibitor of Silent Information Regulator 2 (SIR2). Its naphthoic moiety might be responsible for its inhibitory effects on platelets. The major goal of our study was to examine possible mechanisms of action of splitomicin on platelet aggregation in order to promote development of a novel anti-platelet aggregation therapy for cardiovascular and cerebrovascular diseases. To study the inhibitory effects of splitomicin on platelet aggregation,we used washed human platelets,and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml),collagen (2 microg/ml),arachidonic acid (AA) (0.5 mM),U46619 (2 microM) or ADP (10 microM). Splitomicin inhibited platelet aggregation induced by thrombin,collagen,AA and U46619 with a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when splitomicin (150 microM) was combined with PGE1 (0.5 microM). It did not further increase cAMP when combined with IBMX. This data indicated that splitomicin increases cAMP by inhibiting activity of phosphodiestease. In addition,splitomicin (300 microM) attenuated intracellular Ca(++) mobilization,and production of thromboxane B2 (TXB2) in platelets that was induced by thrombin,collagen,AA or U46619. The inhibitory mechanism of splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca(++) mobilization,TXB2 formation and ATP release. View Publication

A primary goal of cancer immunotherapy is to improve the naturally occurring,but weak,immune response to tumors. Ineffective responses to cancer vaccines may be caused,in part,by low numbers of self-reactive lymphocytes surviving negative selection. Here,we estimated the frequency of CD8(+) T cells recognizing a self-antigen to be textless0.0001% ( approximately 1 in 1 million CD8(+) T cells),which is so low as to preclude a strong immune response in some mice. Supplementing this repertoire with naive antigen-specific cells increased vaccine-elicited tumor immunity and autoimmunity,but a threshold was reached whereby the transfer of increased numbers of antigen-specific cells impaired functional benefit,most likely because of intraclonal competition in the irradiated host. We show that cells primed at precursor frequencies below this competitive threshold proliferate more,acquire polyfunctionality,and eradicate tumors more effectively. This work demonstrates the functional relevance of CD8(+) T cell precursor frequency to tumor immunity and autoimmunity. Transferring optimized numbers of naive tumor-specific T cells,followed by in vivo activation,is a new approach that can be applied to human cancer immunotherapy. Further,precursor frequency as an isolated variable can be exploited to augment efficacy of clinical vaccine strategies designed to activate any antigen-specific CD8(+) T cells. View Publication

Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high,and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk,yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast,we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing,including E2F transcription factors,chromatin licensing and DNA replication factor 1 (Cdt1),and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone,showing that progesterone influences epithelial cell lineage differentiation. Therefore,we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers. View Publication

The potential for directed differentiation of human-induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs),we found that once specified to a neural lineage,human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS-derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS-derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS-derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. View Publication

Identification of rational therapeutic targets is an important strategy to improve the cure rate of diffuse large B-cell lymphoma (DLBCL). We previously showed that inhibition of the phosphodiesterase 4B (PDE4B) unleashes cyclic-AMP (cAMP) inhibitory effects toward the PI3K/AKT pathway and induces apoptosis. These data raised important considerations as to which upstream regulators mediate cAMP inhibition of PI3K/AKT,and how identifying this signaling route could be translated into clinical initiatives. We found that in normal and malignant B cells,cAMP potently inhibit the phosphorylation and activity of the tyrosine kinase SYK. Using genetic models of gain- and loss-of-function,we demonstrated the essential role for PDE4B in controlling these effects in DLBCL. Furthermore,we used a constitutively active SYK mutant to confirm its central role in transducing cAMP effects to PI3K/AKT. Importantly,given SYK credentials as a therapeutic target in B-cell tumors,we explored the role of PDE4B in these responses. In multiple DLBCL models,we found that genetically,hence specifically,inhibiting PDE4B expression significantly improved the efficacy of SYK inhibitors. Our data defined a hitherto unknown role for cAMP in negatively regulating SYK and indicate that combined inhibition of PDE4B and SYK should be actively pursued. View Publication

Induction of pluripotent stem cells from human fibroblasts has been achieved by the ectopic expression of two different sets of four genes. However,the mechanism of the pluripotent stem cell induction has not been elucidated. Here we identified a marked heterogeneity in colonies generated by the four-gene (Oct3/4,Sox2,c-Myc,and Klf4) transduction method in human neonatal skin-derived cells. The four-gene transduction gave a higher probability of induction for archetypal pluripotent stem cell marker genes (Nanog,TDGF,and Dnmt3b) than for marker genes that are less specific for pluripotent stem cells (CYP26A1 and TERT) in primary induction culture. This tendency may reflect the molecular mechanism underlying the induction of human skin-derived cells into pluripotent stem cells. Among the colonies induced by the four-gene transduction,small cells with a high nucleus-to-cytoplasm ratio could be established by repeated cloning. Subsequently established cell lines were similar to human embryonic stem cells as well as human induced pluripotent stem (iPS) cells derived from adult tissue in morphology,gene expression,long-term self-renewal ability,and teratoma formation. Genome-wide single-nucleotide polymorphism array analysis of the human iPS cell line indicates that the induction process did not induce DNA mutation. ?? 2008 Elsevier B.V. All rights reserved. View Publication