技术资料

The human HDAC (histone deacetylase) family,a well-validated anticancer target,plays a key role in the control of gene expression through regulation of transcription. While HDACs can be subdivided into three main classes,the class I,class II and class III HDACs (sirtuins),it is presently unclear whether inhibiting multiple HDACs using pan-HDAC inhibitors,or targeting specific isoforms that show aberrant levels in tumours,will prove more effective as an anticancer strategy in the clinic. To address the above issues,we have tested a number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system,and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1,rhHDAC2,rhHDAC3 and rhHDAC8) and class II HDAC isoforms (rhHDAC4,rhHDAC6,rhHDAC7 and rhHDAC9) was determined. MS-275 was HDAC1-selective,MGCD0103 was HDAC1- and HDAC2-selective,apicidin was HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I HDACs. The hydroxamic acid-derived compounds (trichostatin A,NVP-LAQ824,panobinostat,ITF2357,vorinostat and belinostat) were potent pan-HDAC inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted in increased acetylation of histones,but only pan-HDAC inhibitor treatment resulted in increased tubulin acetylation,which is in agreement with their activity towards the HDAC6 isoform. View Publication

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors,whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed,washed,immunomagnetically depleted of cells expressing glycophorin A and CD14,reacted for flow cytometric detection of ALDH,and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H,10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed,banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid,myeloid and megakaryocytic blood elements. View Publication

The present study explored the sensitivity of leukaemic blasts derived from 30 acute myeloid leukaemia (AML) patients to Bortezomib. Bortezomib induced apoptosis of primary AML blasts: 18/30 AMLs were clearly sensitive to the proapoptotic effects of Bortezomib,while the remaining cases were moderately sensitive to this molecule. The addition of tumour necrosis factor-related-apoptosis-inducing ligand,when used alone,did not induce apoptosis of AML blasts and further potentiated the cytotoxic effects of Bortezomib. The majority of AMLs sensitive to Bortezomib showed immunophenotypic features of the M4 and M5 French-American-British classification subtypes and displayed myelomonocytic features. All AMLs with mutated FLT3 were in the Bortezomib-sensitive group. Biochemical studies showed that: (i) Bortezomib activated caspase-8 and caspase-3 and decreased cellular FLICE [Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme]-inhibitory protein (c-FLIP) levels in AML blasts; (ii) high c-FLIP levels in AML blasts were associated with low Bortezomib sensitivity. Finally,analysis of the effects of Bortezomib on leukaemic cells displaying high aldehyde dehydrogenase activity suggested that this drug induced in vitro killing of leukaemic stem cells. The findings of the present study,further support the development of Bortezomib as an anti-leukaemic drug and provide simple tools to predict the sensitivity of AML cells to this drug. View Publication

BACKGROUND AND PURPOSE: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimeŕs disease (AD) such as hyperphosphorylation of the protein,tau. GSK-3beta expression,enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here,we assess five GSK-3beta inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3beta enzyme activity levels in 12-day old postnatal rats. EXPERIMENTAL APPROACH: Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses,p-tau (Ser(396)) levels in brain tissue was measured by immunoblotting and correlated with GSK-3beta enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau,GSK-3beta activities and cell death were measured in a human neuronal cell line (LUHMES). KEY RESULTS: Lithium and CHIR98014 reduced tau phosphorylation (Ser(396)) in the cortex and hippocampus of postnatal rats,while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3'-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC(50) values obtained in recombinant or cell-based GSK-3beta enzyme activity assays. The inhibitory effect on GSK-3beta activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells,using low microM inhibitor concentrations. CONCLUSIONS AND IMPLICATIONS: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3beta may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis. 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

A newly recognized link between the complement system and adaptive immunity is that decay accelerating factor (DAF),a cell surface C3/C5 convertase regulator,exerts control over T cell responses. Extending these results,we show that cultures of Marilyn TCR-transgenic T cells stimulated with DAF-deficient (Daf1(-/-)) APCs produce significantly more IL-12,C5a,and IFN-gamma compared with cultures containing wild-type APCs. DAF-regulated IL-12 production and subsequent T cell differentiation into IFN-gamma-producing effectors was prevented by the deficiency of either C3 or C5a receptor (C5aR) in the APC,demonstrating a link between DAF,local complement activation,IL-12,and T cell-produced IFN-gamma. Bone marrow chimera experiments verified that bone marrow cell-expressed C5aR is required for optimal differentiation into IFN-gamma-producing effector T cells. Overall,our results indicate that APC-expressed DAF regulates local production/activation of C5a following cognate T cell/APC interactions. Through binding to its receptor on APCs the C5a up-regulates IL-12 production,this in turn,contributes to directing T cell differentiation toward an IFN-gamma-producing phenotype. The findings have implications for design of therapies aimed at altering pathologic T cell immunity. 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

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

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

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

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

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