技术资料

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

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

Increasing evidence suggests that the deposition of amyloid plaques,composed primarily of the amyloid-beta protein (Abeta),within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly,the pathogenic mechanisms by which Abeta can alter vascular function may have therapeutic implications. Despite observations that Abeta elicits a number of physiological responses in endothelial cells,ranging from alteration of protein expression to cell death,the Abeta species accountable for these responses remains unexplored. In the current study,we show that isolated soluble Abeta aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast,unaggregated Abeta monomer and mature Abeta fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Abeta aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Abeta aggregates,including Abeta-derived diffusible ligands,oligomers,and protofibrils,and further show that soluble aggregates also selectively exhibit activity in a vascular cell model. View Publication

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL,TEL/PDGFRbeta,FIP1/PDGFRalpha,D816V KIT,NPM/ALK,and FLT3ITD) revealed few common effects on the transcriptome. It is apparent,however,that proteome changes are not directly governed by transcriptome changes. Therefore,we assessed and used a new generation of iTRAQ tagging,enabling eight-channel relative quantification discovery proteomics,to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin,although this did not correspond to changes in motility. However,correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes,illustrating the utility of such a combined approach. View Publication

Recently,a number of clinical trials used either mesenchymal stem cells (MSCs) or natural killer (NK) cells in an attempt to improve the effectiveness of hematopoietic stem cell transplantation (HSCT). In view of the relevant role of both MSCs and NK cells in HSCT,we have recently explored the result of possible interactions between the 2 cell types. We found that activated NK cells could kill MSCs,whereas MSCs strongly inhibited interleukin-2 (IL-2)-induced NK-cell proliferation. In this study,we further analyzed the inhibitory effect exerted by MSCs on NK cells. We show that MSCs not only inhibit the cytokine-induced proliferation of freshly isolated NK cells but also prevent the induction of effector functions,such as cytotoxic activity and cytokine production. Moreover,we show that this inhibitory effect is related to a sharp down-regulation of the surface expression of the activating NK receptors NKp30,NKp44,and NKG2D. Finally,we demonstrate that indoleamine 2,3-dioxygenase and prostaglandin E2 represent key mediators of the MSC-induced inhibition of NK cells. View Publication

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here,we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors,at very low MSC:neutrophil ratios (up to 1:500),significantly inhibited apoptosis of resting and interleukin (IL)-8-activated neutrophils and dampened N-formyl-l-methionin-l-leucyl-l-phenylalanine (f-MLP)-induced respiratory burst. The antiapoptotic activity of MSC did not require cell-to-cell contact,as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC-derived soluble factor responsible for neutrophil protection from apoptosis was IL-6,which signaled by activating STAT-3 transcription factor. Furthermore,IL-6 expression was detected in MSC by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Finally,recombinant IL-6 was found to protect neutrophils from apoptosis in a dose-dependent manner. MSC had no effect on neutrophil phagocytosis,expression of adhesion molecules,and chemotaxis in response to IL-8,f-MLP,or C5a. These results support the following conclusions: (a) in the bone marrow niche,MSC likely protect neutrophils of the storage pool from apoptosis,preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism,and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified. View Publication

By assessing the contribution of deregulated E2F activity to erythroid defects in Rb null mice,we have identified E2f-2 as being upregulated in end-stage red cells,where we show it is the major pRb-associated E2f and the predominant E2f detected at key target gene promoters. Consistent with its expression pattern,E2f-2 loss restored terminal erythroid maturation to Rb null red cells,including the ability to undergo enucleation. Deletion of E2f-2 also extended the life span of Rb null mice despite persistent defects in placental development,indicating that deregulated E2f-2 activity in differentiating erythroblasts contributes to the premature lethality of Rb null mice. We show that the aberrant entry of Rb null erythroblasts into S phase at times in differentiation when wild-type erythroblasts are exiting the cell cycle is inhibited by E2f-2 deletion. E2f-2 loss induced cell cycle arrest in both wild-type and Rb null erythroblasts and was associated with increased DNA double-strand breaks. These results implicate deregulated E2f-2 in the cell cycle defects observed in Rb null erythroblasts and reveal a novel role for E2f-2 during terminal red blood cell differentiation. The identification of a tissue-restricted role for E2f-2 in erythropoiesis highlights the nonredundant nature of E2f transcription factor activities in cell growth and differentiation. View Publication

OBJECTIVE Metformin is an antidiabetic drug commonly used to treat type 2 diabetes. The aim of the study was to determine whether metformin regulates hepatic gluconeogenesis through the orphan nuclear receptor small heterodimer partner (SHP; NR0B2). RESEARCH DESIGN AND METHODS We assessed the regulation of hepatic SHP gene expression by Northern blot analysis with metformin and adenovirus containing a constitutive active form of AMP-activated protein kinase (AMPK) (Ad-AMPK) and evaluated SHP,PEPCK,and G6Pase promoter activities via transient transfection assays in hepatocytes. Knockdown of SHP using siRNA SHP was conducted to characterize the metformin-induced inhibition of hepatic gluconeogenic gene expression in hepatocytes,and metformin-and adenovirus SHP (Ad-SHP)-mediated hepatic glucose production was measured in B6-Lep(ob/ob) mice. RESULTS Hepatic SHP gene expression was induced by metformin,5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR),and Ad-AMPK. Metformin-induced SHP gene expression was abolished by adenovirus containing the dominant negative form of AMPK (Ad-DN-AMPK),as well as by compound C. Metformin inhibited hepatocyte nuclear factor-4alpha-or FoxA2-mediated promoter activity of PEPCK and G6Pase,and the inhibition was blocked with siRNA SHP. Additionally,SHP knockdown by adenovirus containing siRNA SHP inhibited metformin-mediated repression of cAMP/dexamethasone-induced hepatic gluconeogenic gene expression. Furthermore,oral administration of metformin increased SHP mRNA levels in B6-Lep(ob/ob) mice. Overexpression of SHP by Ad-SHP decreased blood glucose levels and hepatic gluconeogenic gene expression in B6-Lep(ob/ob) mice. CONCLUSIONS We have concluded that metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of SHP. View Publication