参考文献

Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here,we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules,we have identified SB203580,a specific p38 MAP kinase inhibitor,as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations textless10 microM,induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers,so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly,transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly,cardiomyogenesis was strongly inhibited at SB203580 concentrations textgreater or =15 microM. Thus,modulation of the p38MAP kinase pathway,in combination with factors released by END2 cells,plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process. View Publication

Over the past 5 years,the Raf kinase family has emerged as a promising target for protein-directed cancer therapy development. The goal of this review is to first provide a concise summary of the data validating Raf proteins as high-interest therapeutic targets. The authors then outline the mode of action of Raf kinases,emphasizing how Raf activities and protein interactions suggest specific approaches to inhibiting Raf. The authors then summarize the set of drugs,antisense reagents and antibodies available or in development for therapeutically targeting Raf or Raf-related proteins,as well as existing strategies combining these and other therapeutic agents. Finally,the authors discuss recent results from systems biology analyses that have the potential to increasingly guide the intelligent selection of combination therapies involving Raf-targeting agents and other therapeutics. View Publication

BACKGROUND: The enhancement of circulating endothelial progenitor cells (EPCs) obtained by exercise training can be beneficial to patients with cardiac disease. Changes in the levels and differentiation of CD34(pos)/KDR(pos) EPCs,as well as the plasma concentration of vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1 EPC-mobilizing cytokines,were evaluated in patients with chronic heart failure after 8 weeks of supervised aerobic training (SAT) and 8 weeks of subsequent discontinued SAT (DSAT). METHODS AND RESULTS: The levels of circulating EPC and EPC differentiation potential of 22 patients who underwent SAT were studied by fluorescence-activated cell sorter analysis and colony forming-unit assay,respectively. The plasma levels of VEGF and SDF-1 were measured by enzyme-linked immunosorbent assay. In response to SAT,the levels of both EPC and VEGF/SDF-1 markedly increased (P textless .001 vs baseline) but returned to the baseline levels after DSAT. A similar change was observed with the EPC clonogenic potential,but on DSAT the baseline level was incompletely attained. CONCLUSIONS: In response to SAT,patients with chronic heart failure show enhanced EPC levels and clonogenic potential that is mirrored by increased plasma VEGF and SDF-1 levels. DSAT can interfere with the maintenance of training-acquired VEGF/SDF-1-related EPC levels and clonogenic potential. View Publication

Thiazolidinediones (TZD) are widely prescribed for the treatment of Type 2 diabetes. Increased loss of bone mass and a higher incidence of fractures have been associated with the use of this class of drugs in post-menopausal women. In vitro studies performed in rodent cell models indicated that rosiglitazone (RGZ),one of the TZD,inhibited osteoblastogenesis and induced adipogenesis in bone marrow progenitor cells. The objective of the present study was to determine for the first time the RGZ-dependent shift from osteoblastogenesis toward adipogenesis using a human cell model. To this purpose,bone marrow-derived mesenchymal stem cells were characterized and induced to differentiate along osteogenic and adipogenic lineages. We found that the exposure to RGZ potentiated adipogenic differentiation and shifted the differentiation toward an osteogenic phenotype into an adipogenic phenotype,as assessed by the appearance of lipid droplets. Accordingly,RGZ markedly increased the expression of the typical marker of adipogenesis fatty-acid binding protein 4,whereas it reduced the expression of Runx2,a marker of osteoblastogenesis. This is the first demonstration that RGZ counteracts osteoblastogenesis and induces a preferential differentiation into adipocytes in human mesenchymal stem cells. View Publication

Aberrant expression of Jagged1 and Notch1 are associated with poor outcome in breast cancer. However,the reason that Jagged1 and/or Notch overexpression portends a poor prognosis is unknown. We identify Slug,a transcriptional repressor,as a novel Notch target and show that elevated levels of Slug correlate with increased expression of Jagged1 in various human cancers. Slug was essential for Notch-mediated repression of E-cadherin,which resulted in beta-catenin activation and resistance to anoikis. Inhibition of ligand-induced Notch signaling in xenografted Slug-positive/E-cadherin-negative breast tumors promoted apoptosis and inhibited tumor growth and metastasis. This response was associated with down-regulated Slug expression,reexpression of E-cadherin,and suppression of active beta-catenin. Our findings suggest that ligand-induced Notch activation,through the induction of Slug,promotes tumor growth and metastasis characterized by epithelial-to-mesenchymal transition and inhibition of anoikis. View Publication

Reversine (RV) is the synthetic purine identified from a protein kinase-based screen of purine mimetics and it has been shown to induce muscle myoblast differentiation into progenitor cells that can be further converted into other cell lineages. Since protein kinases play a pivotal role in cell cycle control,we hypothesize that RV might affect the proliferation of cancer cells. Herein we report that RV inhibited growth of cultured human tumor cells,respectively,PC-3,HeLa,CWR22Rv1,and DU-145 cells,and induced accumulation of polyploidal cells with textgreater or =4N DNA content. However,RV was without effect on growth of normal prostate epithelial cells. RV-treated PC-3 cells showed enlarged nuclei and an estimated 100-fold increase in cell size. Moreover,PC-3 cells treated with RV for 2-4 days were accompanied by a marked increase in the expression of p21(WAF1),a modest elevation in the levels of cyclin D3 and CDK6 and concomitantly,also a substantial reduction in cyclin B and CDK1. These results suggest that RV may induce polyploidy and increase in cell size by up-regulating p21(WAF1) and cyclin D3/CDK6,while simultaneously suppressing the expression of cyclin B and CDK1. View Publication

We have shown that epigallocatechin-3-gallate (EGCG),a polyphenolic compound from green tea,inhibits growth and induces apoptosis in human pancreatic cancer cells. However,the preclinical potential of EGCG in a suitable mouse model has not been examined. In this study,we examined the molecular mechanisms by which EGCG inhibited growth,invasion,metastasis and angiogenesis of human pancreatic cancer cells in a xenograft model system. EGCG inhibited viability,capillary tube formation and migration of HUVEC,and these effects were further enhanced in the presence of an ERK inhibitor. In vivo,AsPC-1 xenografted tumors treated with EGCG showed significant reduction in volume,proliferation (Ki-67 and PCNA staining),angiogenesis (vWF,VEGF and CD31) and metastasis (MMP-2,MMP-7,MMP-9 and MMP-12) and induction in apoptosis (TUNEL),caspase-3 activity and growth arrest (p21/WAF1). EGCG also inhibited circulating endothelial growth factor receptor 2 (VEGF-R2) positive endothelial cells derived from xenografted mice. Tumor samples from EGCG treated mice showed significantly reduced ERK activity,and enhanced p38 and JNK activities. Overall,our data suggest that EGCG inhibits pancreatic cancer growth,invasion,metastasis and angiogenesis,and thus could be used for the management of pancreatic cancer prevention and treatment. View Publication

Under standard culture conditions,tumor cells are exposed to 20% O(2),whereas the mean tumor oxygen levels within the tumor are much lower. We demonstrate,using low-passaged human tumor cell cultures established from glioma,that a reduction in the oxygen level in these cell cultures dramatically increases the percentage of CD133 expressing cells. View Publication

Few epitopes are available for vaccination therapy of patients with squamous cell carcinoma of the head and neck (SCCHN). Using a tumor-specific CTL,aldehyde dehydrogenase 1 family member A1 (ALDH1A1) was identified as a novel tumor antigen in SCCHN. Mass spectral analysis of peptides in tumor-derived lysates was used to determine that the CTL line recognized the HLA-A*0201 (HLA-A2) binding ALDH1A1(88-96) peptide. Expression of ALDH1A1 in established SCCHN cell lines,normal mucosa,and primary keratinocytes was studied by quantitative reverse transcription-PCR and immunostaining. Protein expression was further defined by immunoblot analysis,whereas ALDH1A1 activity was measured using ALDEFLUOR. ALDH1A1(88-96) peptide was identified as an HLA-A2-restricted,naturally presented,CD8(+) T-cell-defined tumor peptide. ALDH1A1(88-96) peptide-specific CD8(+) T cells recognized only HLA-A2(+) SCCHN cell lines,which overexpressed ALDH1A1,as well as targets transfected with ALDH1A1 cDNA. Target recognition was blocked by anti-HLA class I and anti-HLA-A2 antibodies. SCCHN cell lines overexpressing ALDH1 had high enzymatic activity. ALDH1A1 protein was expressed in 12 of 17 SCCHN,and 30 of 40 dysplastic mucosa samples,but not in normal mucosa. ALDH1A1 expression levels in target cells correlated with their recognition by ALDH1A1(88-96) peptide-specific CD8(+) T cells. Our findings identify ALDH1A1,a metabolic antigen,as a potential target for vaccination therapy in the cohort of SCCHN subjects with tumors overexpressing this protein. A smaller cohort of subjects with SCCHN,whose tumors express little to no ALDH1A1,and thus are deficient in conversion of retinal to retinoic acid,could benefit from chemoprevention therapy. View Publication

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed adhesion and neuritogenic protein whose processing has previously been shown to be regulated by reproductive hormones including the gonadotropin luteinizing hormone (LH) in human neuroblastoma cells. We report for the first time the expression of AbetaPP in human embryonic stem (hES) cells at the mRNA and protein levels. Using N- and C-terminal antibodies against AbetaPP,we detected both the mature and immature forms of AbetaPP as well as truncated variants ( approximately 53kDa,47kDa,and 29kDa) by immunoblot analyses. Expression of AbetaPP is regulated by both the stemness of the cells and pregnancy-associated hormones. Addition of human chorionic gonadotropin,the fetal equivalent of LH that is dramatically elevated during pregnancy,markedly increased the expression of all AbetaPP forms. These results indicate a critical molecular signaling link between the hormonal environment of pregnancy and the expression of AbetaPP in hES cells that is suggestive of an important function for this protein during early human embryogenesis prior to the formation of neural precursor cells. View Publication