参考文献

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

Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy,although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore,in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells,cetuximab concentration,and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets,respectively,in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore,supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level,cetuximab concentration,and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P textless 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a,and secreted significantly (P textless 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab mediated clinical responses. Cellular and secreted immune profiles and FcgammaR genotypes from patients' lymphocytes may provide clinically useful biomarkers of immune activation in cetuximab treated patients. View Publication

Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC). MET/phosphorylated MET and HGF expression was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry,and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling,migration,viability,and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression,whereas 18 of 20 HNSCC cell lines (90%) expressed MET. HGF overexpression was present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling,cell viability,motility/migration in vitro,and tumor angiogenesis in vivo. Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the semaphorin (T230M/E168D/N375S),juxtamembrane (T1010I/R988C),and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with textgreater10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression,increased gene copy number,and mutations. MET inhibition abrogated MET functions,including proliferation,migration/motility,and angiogenesis. MET is a promising,novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored. View Publication

Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly,the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. Here,we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation,demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides in vivo evidence that Wnt activation in stem cells requires PGE2,and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery. 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

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 human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression,which in turn is driven by promoters,enhancers,insulators and other cis-regulatory DNA sequences for each gene,the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters,enhancers and insulators in the human genome. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast,enhancers are marked with highly cell-type-specific histone modification patterns,strongly correlate to cell-type-specific gene expression programs on a global scale,and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome,significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression. View Publication

Stepwise differentiation from embryonic stem cells (ESCs) to functional insulin-secreting beta cells will identify key steps in beta-cell development and may yet prove useful for transplantation therapy for diabetics. An essential step in this schema is the generation of pancreatic progenitors--cells that express Pdx1 and produce all the cell types of the pancreas. High-content chemical screening identified a small molecule,(-)-indolactam V,that induces differentiation of a substantial number of Pdx1-expressing cells from human ESCs. The Pdx1-expressing cells express other pancreatic markers and contribute to endocrine,exocrine and duct cells,in vitro and in vivo. Further analyses showed that (-)-indolactam V works specifically at one stage of pancreatic development,inducing pancreatic progenitors from definitive endoderm. This study describes a chemical screening platform to investigate human ESC differentiation and demonstrates the generation of a cell population that is a key milepost on the path to making beta cells. View Publication

MicroRNAs (miRNAs) are a newly discovered endogenous class of small noncoding RNAs that play important posttranscriptional regulatory roles by targeting mRNAs for cleavage or translational repression. Accumulating evidence now supports the importance of miRNAs for human embryonic stem cell (hESC) self-renewal,pluripotency,and differentiation. However,with respect to induced pluripotent stem cells (iPSC),in which embryonic-like cells are reprogrammed from adult cells using defined factors,the role of miRNAs during reprogramming has not been well-characterized. Determining the miRNAs that are associated with reprogramming should yield significant insight into the specific miRNA expression patterns that are required for pluripotency. To address this lack of knowledge,we use miRNA microarrays to compare the microRNA-omes" of human iPSCs View Publication

The zinc finger protein growth factor independent-1 (Gfi1) is a transcriptional repressor that is critically required for normal granulocytic differentiation. GFI1 loss-of-function mutations are found in some patients with severe congenital neutropenia (SCN). The SCN-associated GFI1-mutant proteins act as dominant negatives to block granulopoiesis through selective deregulation of a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs,and that deregulated expression of these microRNAs recapitulates a Gfi1 loss-of-function block to granulocyte colony-stimulating factor (G-CSF)-stimulated granulopoiesis. Specifically,bone marrow cells from a GFI1-mutant SCN patient and Gfi1(-/-) mice display deregulated expression of miR-21 and miR-196B expression. Flow cytometric analysis and colony assays reveal that the overexpression or depletion of either miR induces changes in myeloid development. However,coexpression of miR-21 and miR-196b (as seen in Gfi1(-/-) mice and a GFI1N382S SCN patient) completely blocks G-CSF-induced granulopoiesis. Thus,our results not only identify microRNAs whose regulation is required during myelopoiesis,but also provide an example of synergy in microRNA biologic activity and illustrate potential mechanisms underlying SCN disease pathogenesis. View Publication