技术资料

An investigation was conducted to demonstrate that neurodazine (Nz) and neurodazole (Nzl),two imidazole-based small molecules,promote neuronal differentiation in both neuroblastoma and fibroblast cells. The results show that differentiated cells generated by treatment with Nz and Nzl express neuron-specific markers. The ability of Nz and Nzl to induce neurogenesis of neuroblastoma and fibroblast cells was found to be comparable to those of the known neurogenic factors,retinoic acid and trichostatin A. In addition,the cells differentiated by Nz and Nzl are observed to express different isoforms of glutamate receptors. The results of signaling pathway studies reveal that two substances enhance neurogenesis in neuroblastoma cells by activating Wnt and Shh signaling pathways and neurogenesis in fibroblast cells by mainly activating the Wnt signaling pathway. Observations made in the present study suggest that Nz and Nzl will serve as chemical tools to generate specific populations of neuronal cells from readily available and simply manageable cells. View Publication

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either,replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array,we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition,activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2,IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely,based on epigenetic markers studied here,non-stimulated cells infected by HIV-1,showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover,non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations,it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection. View Publication

BACKGROUND In colorectal carcinoma (CRC),activation of the Raf/MEK/ERK signaling pathway is commonly observed. In addition,the commonly used 5FU-based chemotherapy in patients with metastatic CRC was found to enrich a subpopulation of CD26(+) cancer stem cells (CSCs). As activation of the Raf/MEK/ERK signaling pathway was also found in the CD26(+) CSCs and therefore,we hypothesized that an ATP-competitive pan-Raf inhibitor,Raf265,is effective in eliminating the cancer cells and the CD26(+) CSCs in CRC patients. METHODS HT29 and HCT116 cells were treated with various concentrations of Raf265 to study the anti-proliferative and apoptotic effects of Raf265. Anti-tumor effect was also demonstrated using a xenograft model. Cells were also treated with Raf265 in combination with 5FU to demonstrate the anti-migratory and invasive effects by targeting on the CD26(+) CSCs and the anti-metastatic effect of the combined treatment was shown in an orthotopic CRC model. RESULTS Raf265 was found to be highly effective in inhibiting cell proliferation and tumor growth through the inhibition of the RAF/MEK/ERK signaling pathway. In addition,anti-migratory and invasive effect was found with Raf265 treatment in combination with 5FU by targeting on the CD26(+) cells. Finally,the anti-tumor and anti-metastatic effect of Raf265 in combination with 5FU was also demonstrated. CONCLUSIONS This preclinical study demonstrates the anti-tumor and anti-metastatic activity of Raf265 in CRC,providing the basis for exploiting its potential use and combination therapy with 5FU in the clinical treatment of CRC. View Publication

BACKGROUND Acute myeloid leukemia (AML) is initiated and maintained by a subset of self-renewing leukemia stem cells (LSCs),which contribute to the progression,recurrence and therapeutic resistance of leukemia. However,the mechanisms underlying the maintenance of LSCs drug resistance have not been fully defined. In this study,we attempted to elucidate the mechanisms of LSCs drug resistance. METHODS We performed reverse phase protein arrays to analyze the expression of anti-apoptotic proteins in the LSC-enriched leukemia cell line KG-1a. Immuno-blotting,cell viability and clinical AML samples were evaluated to verify the micro-assay results. The characteristics and transcriptional regulation of survivin were analyzed with the relative luciferase reporter assay,mutant constructs,chromatin immuno-precipitation (ChIP),quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR),and western blotting. The levels of Sp1,c-Myc,phospho-extracellular signal-regulated kinase (p-ERK),phospho-mitogen and stress-activated protein kinase (p-MSK) were investigated in paired CD34+ and CD34- AML patient samples. RESULTS Survivin was highly over-expressed in CD34 + CD38- KG-1a cells and paired CD34+ AML patients compared with their differentiated counterparts. Functionally,survivin contributes to the drug resistance of LSCs,and Sp1 and c-Myc concurrently regulate levels of survivin transcription. Clinically,Sp1 and c-Myc were significantly up-regulated and positively correlated with survivin in CD34+ AML patients. Moreover,Sp1 and c-Myc were further activated by the ERK/MSK mitogen-activated protein kinase (MAPK) signaling pathway,modulating survivin levels. CONCLUSION Our findings demonstrated that ERK/MSK/Sp1/c-Myc axis functioned as a critical regulator of survivin expression in LSCs,offering a potential new therapeutic strategy for LSCs therapy. View Publication

The extent,regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific CtextgreaterU RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins,including many that are involved in pathogenesis of viral diseases. APOBEC3A,which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons,mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of CtextgreaterU RNA editing in mammals. View Publication

Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study,we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging,we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next,we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain,we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective,potent and secretable variant of a TRAIL,S-TRAIL,and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore,the incorporation of pro-drug converting enzyme,herpes simplex virus thymidine kinase,into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications. View Publication

Host defense peptides have recently gained much interest as novel anti-infectives owing to their ability to kill bacteria and simultaneously modulate host cell responses. The cationic host defense peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE),derived from the C terminus of human thrombin,inhibits proinflammatory responses in vitro and in vivo,but the mode of action is unclear. In this study,we show that GKY25,apart from binding bacterial LPS,also interacts directly with monocytes and macrophages in vitro,ex vivo,and in vivo. Moreover,GKY25 inhibits TLR4- and TLR2-induced NF-κB activation in response to several microbe-derived agonists. Furthermore,GKY25 reduces LPS-induced phosphorylation of MAPKs p38α and JNK1/2/3. FACS and electron microscopy analyses showed that GKY25 interferes with TLR4/myeloid differentiation protein-2 dimerization. The results demonstrate a previously undisclosed activity of the host defense peptide GKY25,based on combined LPS and cell interactions leading to inhibition of TLR4 dimerization and subsequent reduction of NF-κB activity and proinflammatory cytokine production in monocytes and macrophages. View Publication

BACKGROUND Accumulating evidence suggests that some long noncoding RNAs (lncRNAs) are involved in certain diseases,such as cancer. The lncRNA,CCDC26,is related to childhood acute myeloid leukemia (AML) because its copy number is altered in AML patients. RESULTS We found that CCDC26 transcripts were abundant in the nuclear fraction of K562 human myeloid leukemia cells. To examine the function of CCDC26,gene knockdown (KD) was performed using short hairpin RNAs (shRNAs),and four KD clones,in which CCDC26 expression was suppressed to 1% of its normal level,were isolated. This down-regulation included suppression of CCDC26 intron-containing transcripts (the CCDC26 precursor mRNA),indicating that transcriptional gene suppression (TGS),not post-transcriptional suppression,was occurring. The shRNA targeting one of the two CCDC26 splice variants also suppressed the other splice variant,which is further evidence for TGS. Growth rates of KD clones were reduced compared with non-KD control cells in media containing normal or high serum concentrations. In contrast,enhanced growth rates in media containing much lower serum concentrations and increased survival periods after serum withdrawal were observed for KD clones. DNA microarray and quantitative polymerase chain reaction screening for differentially expressed genes between KD clones and non-KD control cells revealed significant up-regulation of the tyrosine kinase receptor,KIT,hyperactive mutations of which are often found in AML. Treatment of KD clones with ISCK03,a KIT-specific inhibitor,eliminated the increased survival of KD clones in the absence of serum. CONCLUSIONS We suggest that CCDC26 controls growth of myeloid leukemia cells through regulation of KIT expression. A KIT inhibitor might be an effective treatment against the forms of AML in which CCDC26 is altered. View Publication

BACKGROUND: Epithelial-mesenchymal transition (EMT) is involved in important malignant features of cancer cells,like invasion,metastatic potential,anti-apoptotic and stem-cell like phenotypes. Among several transcription factors,SNAI2/SLUG is supposed to play an essential role for EMT. METHODS: Paraffin embedded tumor samples from 63 patients with metastatic non-small cell lung cancer,enrolled in a randomized phase II trial,were prospectively collected,53 samples qualified for further analysis. Automated RNA extraction from paraffin and RT-quantitative PCR was used for evaluation of SNAI2/SLUG,estrogen receptor 1 (ESR1) and matrix-metalloproteinases (MMP) mRNA expression. RESULTS: Clinical features like age,gender,performance status,histological subtype and stage were similarly distributed among SNAI2/SLUG positive and negative patients. SNAI2/SLUG was significantly,inversely correlated with ESR1 mRNA expression (p textless 0.0001). In contrast,MMP2 (p = 0.387),MMP7 (p = 0.396) and MMP9 mRNA expression (p = 0.366) did not correlate with SNAI2/SLUG. Patients with high SNAI2/SLUG expression (grouped by median expression) had a worse outcome. Median overall survival in patients with high SNAI2/SLUG expression was 5.7 months versus 11.6 months with low SNAI2/SLUG expression (p = .038). Inversely,patients with high ESR1 expression (grouped by median expression) had an improved median OS with 10.9 months vs. 5.0 months in the low expression group (p = .032). In multivariate analysis,SNAI2/SLUG2 (p = .022) and ESR1 (p = .017) separately were independent prognostic factors for survival. CONCLUSION: SNAI2/SLUG is prognostic of patients' outcome. The strong inverse correlation with ESR1 indicates a significant impact of estrogen receptor pathway regarding these malignant features. View Publication

Insulin-like growth factor 1 (IGF-1) enhances cellular proliferation and reduces apoptosis during the early differentiation of bone marrow derived mesenchymal stem cells (BMSCs) into neural progenitor-like cells (NPCs) in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). BMSCs were differentiated in three groups of growth factors: (A) EGF + bFGF,(B) EGF + bFGF + IGF-1,and (C) without growth factor. To unravel the molecular mechanisms of the NPCs derivation,microarray analysis using GeneChip miRNA arrays was performed. The profiles were compared among the groups. Annotated microRNA fingerprints (GSE60060) delineated 46 microRNAs temporally up-regulated or down-regulated compared to group C. The expressions of selected microRNAs were validated by real-time PCR. Among the 46 microRNAs,30 were consistently expressed for minimum of two consecutive time intervals. In Group B,only miR-496 was up-regulated and 12 microRNAs,including the let-7 family,miR-1224,miR-125a-3p,miR-214,miR-22,miR-320,miR-708,and miR-93,were down-regulated. Bioinformatics analysis reveals that some of these microRNAs (miR-22,miR-214,miR-125a-3p,miR-320 and let-7 family) are associated with reduction of apoptosis. Here,we summarize the roles of key microRNAs associated with IGF-1 in the differentiation of BMSCs into NPCs. These findings may provide clues to further our understanding of the mechanisms and roles of microRNAs as key regulators of BMSC-derived NPC maintenance. View Publication

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Unintended exposure to teratogenic compounds can lead to various birth defects; however current animal-based testing is limited by time,cost and high inter-species variability. Here,we developed a human-relevant in vitro model,which recapitulated two cellular events characteristic of embryogenesis,to identify potentially teratogenic compounds. We spatially directed mesoendoderm differentiation,epithelial-mesenchymal transition and the ensuing cell migration in micropatterned human pluripotent stem cell (hPSC) colonies to collectively form an annular mesoendoderm pattern. Teratogens could disrupt the two cellular processes to alter the morphology of the mesoendoderm pattern. Image processing and statistical algorithms were developed to quantify and classify the compounds' teratogenic potential. We not only could measure dose-dependent effects but also correctly classify species-specific drug (Thalidomide) and false negative drug (D-penicillamine) in the conventional mouse embryonic stem cell test. This model offers a scalable screening platform to mitigate the risks of teratogen exposures in human. View Publication