技术资料

The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors,which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses,reprogramming by dox addition,selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency,(ii) the duration of transgene activity directly correlates with reprogramming efficiency,(iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming. View Publication

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse,but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed textless10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However,the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses textless20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells,mES cells lacking H2AX,a histone protein involved in the DNA damage response,were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining,H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs),an increase in dsb rejoining rate,and a decrease in Ku70/80. Unlike mouse ES,human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells,they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM,and they add to the growing list of differences in the way rodent and human cells deal with DNA damage. View Publication

Culture materials and grains contaminated with certain isolates of Fusarium moniliforme cause equine leucoencephalomalacia,porcine pulmonary edema syndrome,and liver cancer in rats. The causative agents are thought to be a family of compounds called fumonisins,which bear considerable structural similarity to the long-chain (sphingoid) base backbones of sphingolipids. Incubation of rat hepatocytes with fumonisins inhibited incorporation of [14C]serine into the sphingosine moiety of cellular sphingolipids with an IC50 of 0.1 microM for fumonisin B1. In contrast,fumonisin B1 increased the amount of the biosynthetic intermediate sphinganine,which suggests that fumonisins inhibit the conversion of [14C]sphinganine to N-acyl-[14C]sphinganines,a step that is thought to precede introduction of the 4,5-trans double bond of sphingosine (Merrill,A.H.,Jr. and Wang,E. (1986) J. Biol. Chem. 261,3764-3769). In agreement with this mechanism,fumonisin B1 inhibited the activity of sphingosine N-acyltransferase (ceramide synthase) in rat liver microsomes with 50% inhibition at approximately 0.1 microM and reduced the conversion of [3H]sphingosine to [3H]ceramide by intact hepatocytes. As far as we are aware,this is the first discovery of a naturally occurring inhibitor of this step of sphingolipid metabolism. These findings suggest that disruption of the de novo pathway of sphingolipid biosynthesis may be a critical event in the diseases that have been associated with consumption of fumonisins. View Publication

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patient's disease. These cells could in turn be used for disease modeling,drug discovery,and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state,it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons,the cell type destroyed in ALS. View Publication

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however,the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre-clinical models of metastasis. Therefore,the objective of this study was to develop suitable pre-clinical model systems for studying stem-like cells in breast cancer metastasis,and to test the hypothesis that stem-like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA-MB-435,MDA-MB-231,MDA-MB-468,MCF-7) for expression of prospective CSC markers CD44/CD24 and CD133,and for functional activity of aldehyde dehydrogenase (ALDH),an enzyme involved in stem cell self-protection. We then used fluorescence-activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation,colony-forming ability,adhesion,migration,invasion) and in vivo (tumorigenicity and metastasis). Sub-populations of cells demonstrating stem-cell-like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF-7. When isolated and compared to ALDH(low)CD44(low/-) cells,ALDH(hi)CD44(+)CD24(-) (MDA-MB-231) and ALDH(hi)CD44(+)CD133(+) (MDA-MB-468) cells demonstrated increased growth (P textless 0.05),colony formation (P textless 0.05),adhesion (P textless 0.001),migration (P textless 0.001) and invasion (P textless 0.001). Furthermore,following tail vein or mammary fat pad injection of NOD/SCID/IL2gamma receptor null mice,ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells showed enhanced tumorigenicity and metastasis relative to ALDH(low)CD44(low/-) cells (P textless 0.05). These novel results suggest that stem-like ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells may be important mediators of breast cancer metastasis. View Publication

PURPOSE: The Aurora kinase family plays pivotal roles in mitotic integrity and cell cycle. We sought to determine the effects of inhibiting Aurora kinase on ovarian cancer growth in an orthotopic mouse model using a small molecule pan-Aurora kinase inhibitor,MK-0457. EXPERIMENTAL DESIGN: We examined cell cycle regulatory effects and ascertained the therapeutic efficacy of Aurora kinase inhibition both alone and combined with docetaxel using both in vitro and in vivo ovarian cancer models. RESULTS: In vitro cytotoxicity assays with HeyA8 and SKOV3ip1 cells revealed textgreater10-fold greater docetaxel cytotoxicity in combination with MK-0457. After in vivo dose kinetics were determined using phospho-histone H3 status,therapy experiments with the chemosensitive HeyA8 and SKOV3ip1 as well as the chemoresistant HeyA8-MDR and A2780-CP20 models showed that Aurora kinase inhibition alone significantly reduced tumor burden compared with controls (P valuestextless0.01). Combination treatment with docetaxel resulted in significantly improved reduction in tumor growth beyond that afforded by docetaxel alone (P textlessor= 0.03). Proliferating cell nuclear antigen immunohistochemistry revealed that MK-0457 alone and in combination with docetaxel significantly reduced cellular proliferation (P valuestextless0.001). Compared with controls,treatment with MK-0457 alone and in combination with docetaxel also significantly increased tumor cell apoptosis by approximately 3-fold (Ptextless0.01). Remarkably,compared with docetaxel monotherapy,MK-0457 combined with docetaxel resulted in significantly increased tumor cell apoptosis. CONCLUSIONS: Aurora kinase inhibition significantly reduces tumor burden and cell proliferation and increases tumor cell apoptosis in this preclinical orthotopic model of ovarian cancer. The role of Aurora kinase inhibition in ovarian cancer merits further investigation in clinical trials. View Publication

Tissue fibrosis is a major cause of death in SSc,but therapies that target selectively fibrosis are not yet available for routine clinical use. Recent pre-clinical studies suggest that selective tyrosine kinase inhibitors that target c-Abl,PDGF receptor or Src kinases might be promising targets for anti-fibrotic approaches. Dual inhibition of c-Abl and PDGF receptor by imatinib and nilotinib,and inhibition of Src kinases either selectively by SU6656 or in combination with c-Abl and PDGF by dasatinib exerted potent anti-fibrotic effects. Imatinib,nilotinib,dasatinib and SU6656 reduced dose-dependently the synthesis of extracellular matrix protein in human dermal fibroblasts in vitro and prevented fibrosis in the mouse model of bleomycin-induced skin fibrosis. Clinical data from patients with chronic myelogenous leukaemia suggest that imatinib,nilotinib and dasatinib are well tolerated. Based on the promising pre-clinical data,imatinib is currently evaluated in clinical trials for the treatment of fibrosis in SSc and trials with other tyrosine kinase inhibitors are in preparation. View Publication

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity,a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs,with the former currently undergoing phase I clinical trials. We show here that ST1926,CD437,and active congeners cause DNA damage. Cellular and subcellular COMET assays,H2AX phosphorylation (gamma-H2AX),and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4,an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts,which are selectively resistant to RRMs,do not show any sign of DNA damage after treatment with ST1926,CD437,and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs,increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated,ataxia telangectasia RAD3-related,and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926,indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. View Publication

BACKGROUND AND AIMS: Emerging evidence suggests that highly treatment-resistant tumour-initiating cells (TICs) play a central role in the pathogenesis of pancreatic cancer. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a novel anticancer agent; however,recent studies have shown that many pancreatic cancer cells are resistant to apoptosis induction by TRAIL due to TRAIL-activated nuclear factor-kappaB (NF-kappaB) signalling. Several chemopreventive agents are able to inhibit NF-kappaB,and favourable results have been obtained--for example,for the broccoli compound sulforaphane-in preventing metastasis in clinical studies. The aim of the study was to identify TICs in pancreatic carcinoma for analysis of resistance mechanisms and for definition of sensitising agents. METHODS: TICs were defined by expression patterns of a CD44(+)/CD24(-),CD44(+)/CD24(+) or CD44(+)/CD133(+) phenotype and correlation to growth in immunodeficient mice,differentiation grade,clonogenic growth,sphere formation,aldehyde dehydrogenase (ALDH) activity and therapy resistance. RESULTS: Mechanistically,specific binding of transcriptionally active cRel-containing NF-kappaB complexes in TICs was observed. Sulforaphane prevented NF-kappaB binding,downregulated apoptosis inhibitors and induced apoptosis,together with prevention of clonogenicity. Gemcitabine,the chemopreventive agents resveratrol and wogonin,and the death ligand TRAIL were less effective. In a xenograft model,sulforaphane strongly blocked tumour growth and angiogenesis,while combination with TRAIL had an additive effect without obvious cytotoxicity in normal cells. Freshly isolated patient tumour cells expressing markers for TICs could be sensitised by sulforaphane for TRAIL-induced cytotoxicity. CONCLUSION: The data provide new insights into resistance mechanisms of TICs and suggest the combination of sulforaphane with TRAIL as a promising strategy for targeting of pancreatic TICs. View Publication

Many cellular responses,such as autoimmunity and cytotoxicity,are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Here,we showed that binding of inhibitory natural killer (NK) cell receptors to human leukocyte antigen (HLA) class I on target cells induced tyrosine phosphorylation of the adaptor Crk,concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore,Crk dissociated from the guanine exchange factor C3G and bound to the tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity,providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex,observed here with two types of inhibitory receptors,expands the signaling potential of the large ITIM-receptor family and reveals an unsuspected component of the inhibitory mechanism. View Publication

Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf(-/-)) mice die at day 14.5 of gestation from severe anemia. In this study,we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf(-/-) embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis,transcriptional profiling was performed with RNA from wild-type and Eklf(-/-) early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation,with the critical regulator of the cell cycle,E2f2,at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf(-/-) early erythroid progenitor cells,which showed a delay in the G(1)-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier,EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation. View Publication

During anemia erythropoiesis is bolstered by several factors including KIT ligand,oncostatin-M,glucocorticoids,and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia,however,reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia,both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments,DYRK3-/- progenitors also supported enhanced erythroblast formation,and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo,DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice,in contrast,produced fewer reticulocytes during hemolytic anemia,and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally,as studied in erythroid K562 cells,DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia. View Publication