Genome-wide gene expression profiling of stress response in a spinal cord clip compression injury model.

BackgroundThe aneurysm clip impact-compression model of spinal cord injury (SCI) is a standard injury model in animals that closely mimics the primary mechanism of most human injuries: acute impact and persisting compression. Its histo-pathological and behavioural outcomes are extensively similar to human SCI. To understand the distinct molecular events underlying this injury model we analyzed global mRNA abundance changes during the acute, subacute and chronic stages of a moderate to severe injury to the rat spinal cord.ResultsTime-series expression analyses resulted in clustering of the majority of deregulated transcripts into eight statistically significant expression profiles. Systematic application of Gene Ontology (GO) enrichment pathway analysis allowed inference of biological processes participating in SCI pathology. Temporal analysis identified events specific to and common between acute, subacute and chronic time-points. Processes common to all phases of injury include blood coagulation, cellular extravasation, leukocyte cell-cell adhesion, the integrin-mediated signaling pathway, cytokine production and secretion, neutrophil chemotaxis, phagocytosis, response to hypoxia and reactive oxygen species, angiogenesis, apoptosis, inflammatory processes and ossification. Importantly, various elements of adaptive and induced innate immune responses span, not only the acute and subacute phases, but also persist throughout the chronic phase of SCI. Induced innate responses, such as Toll-like receptor signaling, are more active during the acute phase but persist throughout the chronic phase. However, adaptive immune response processes such as B and T cell activation, proliferation, and migration, T cell differentiation, B and T cell receptor-mediated signaling, and B cell- and immunoglobulin-mediated immune response become more significant during the chronic phase.ConclusionsThis analysis showed that, surprisingly, the diverse series of molecular events that occur in the acute and subacute stages persist into the chronic stage of SCI. The strong agreement between our results and previous findings suggest that our analytical approach will be useful in revealing other biological processes and genes contributing to SCI pathology

Lyl1 interacts with CREB1 and alters expression of CREB1 target genes

AbstractThe basic helix-loop-helix (bHLH) transcription factor family contains key regulators of cellular proliferation and differentiation as well as the suspected oncoproteins Tal1 and Lyl1. Tal1 and Lyl1 are aberrantly over-expressed in leukemia as a result of chromosomal translocations, or other genetic or epigenetic events. Protein-protein and protein-DNA interactions described so far are mediated by their highly homologous bHLH domains, while little is known about the function of other protein domains. Hetero-dimers of Tal1 and Lyl1 with E2A or HEB, decrease the rate of E2A or HEB homo-dimer formation and are poor activators of transcription. In vitro, these hetero-dimers also recognize different binding sites from homo-dimer complexes, which may also lead to inappropriate activation or repression of promoters in vivo. Both mechanisms are thought to contribute to the oncogenic potential of Tal1 and Lyl1. Despite their bHLH structural similarity, accumulating evidence suggests that Tal1 and Lyl1 target different genes. This raises the possibility that domains flanking the bHLH region, which are distinct in the two proteins, may participate in target recognition. Here we report that CREB1, a widely-expressed transcription factor and a suspected oncogene in acute myelogenous leukemia (AML) was identified as a binding partner for Lyl1 but not for Tal1. The interaction between Lyl1 and CREB1 involves the N terminal domain of Lyl1 and the Q2 and KID domains of CREB1. The histone acetyl-transferases p300 and CBP are recruited to these complexes in the absence of CREB1 Ser 133 phosphorylation. In the Id1 promoter, Lyl1 complexes direct transcriptional activation. We also found that in addition to Id1, over-expressed Lyl1 can activate other CREB1 target promoters such as Id3, cyclin D3, Brca1, Btg2 and Egr1. Moreover, approximately 50% of all gene promoters identified by ChIP-chip experiments were jointly occupied by CREB1 and Lyl1, further strengthening the association of Lyl1 with Cre binding sites. Given the newly recognized importance of CREB1 in AML, the ability of Lyl1 to modulate promoter responses to CREB1 suggests that it plays a role in the malignant phenotype by occupying different promoters than Tal1

Preclinical Models for Investigation of Herbal Medicines in Liver Diseases: Update and Perspective

Liver disease results from a dynamic pathological process associated with cellular and genetic alterations, which may progress stepwise to liver dysfunction. Commonly, liver disease begins with hepatocyte injury, followed by persistent episodes of cellular regeneration, inflammation, and hepatocyte death that may ultimately lead to nonreversible liver failure. For centuries, herbal remedies have been used for a variety of liver diseases and recent studies have identified the active compounds that may interact with liver disease-associated targets. Further study on the herbal remedies may lead to the formulation of next generation medicines with hepatoprotective, antifibrotic, and anticancer properties. Still, the pharmacological actions of vast majority of herbal remedies remain unknown; thus, extensive preclinical studies are important. In this review, we summarize progress made over the last five years of the most commonly used preclinical models of liver diseases that are used to screen for curative herbal medicines for nonalcoholic fatty liver disease, liver fibrosis/cirrhosis, and liver. We also summarize the proposed mechanisms associated with the observed liver-protective, antifibrotic, and anticancer actions of several promising herbal medicines and discuss the challenges faced in this research field

The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells

Abstract Introduction There is growing evidence that the Wilms' tumor 1 suppressor gene (WT1) behaves as an oncogene in some forms of breast cancer. Previous studies have demonstrated that the N-terminal domain of WT1 can act as a dominant negative through self-association. In the studies presented here we have explored the potential for the zinc finger domain (ZF) of WT1 to also have dominant-negative effects, and thus further our understanding of this protein. Methods Using full-length and ZF-only forms of WT1 we assessed their effect on the WT1 and c-myc promoter using luciferase and chromatin immunoprecipitation assays. The gene expression levels were determined by quantitative real-time RT-PCR, northern blot and western blot. We also assessed the effect of the ZF-only form on the growth of breast cancer cell lines in culture. Results Transfection with WT1–ZF plasmids resulted in a stronger inhibition of WT1 promoter than full-length WT1 in breast cancer cells. The WT1–ZF form lacking the lysine–threonine–serine (KTS) insert (ZF - KTS) can bind to the majority of WT1 consensus sites throughout the WT1 promoter region, while the ZF containing the insert (ZF + KTS) form only binds to sites in the proximal promoter. The abundances of endogenous WT1 mRNA and protein were markedly decreased following the stable expression of ZF - KTS in breast cancer cells. The expressions of WT1 target genes, including c-myc, Bcl-2, amphiregulin and TERT, were similarly suppressed by ZF - KTS. Moreover, WT1–ZF - KTS abrogated the transcriptional activation of c-myc mediated by all four predominant isoforms of WT1 (including or lacking alternatively spliced exons 5 and 9). Finally, WT1–ZF - KTS inhibited colony formation and cell division, but induced apoptosis in MCF-7 cells. Conclusion Our observations strongly argue that the WT1–ZF plasmid behaves as a dominant-negative regulator of the endogenous WT1 in breast cancer cells. The inhibition on proliferation of breast cancer cells by WT1–ZF - KTS provides a potential candidate of gene therapy for breast cancer

Genome-wide gene expression profiling of stress response in a spinal cord clip compression injury model

Abstract Background The aneurysm clip impact-compression model of spinal cord injury (SCI) is a standard injury model in animals that closely mimics the primary mechanism of most human injuries: acute impact and persisting compression. Its histo-pathological and behavioural outcomes are extensively similar to human SCI. To understand the distinct molecular events underlying this injury model we analyzed global mRNA abundance changes during the acute, subacute and chronic stages of a moderate to severe injury to the rat spinal cord. Results Time-series expression analyses resulted in clustering of the majority of deregulated transcripts into eight statistically significant expression profiles. Systematic application of Gene Ontology (GO) enrichment pathway analysis allowed inference of biological processes participating in SCI pathology. Temporal analysis identified events specific to and common between acute, subacute and chronic time-points. Processes common to all phases of injury include blood coagulation, cellular extravasation, leukocyte cell-cell adhesion, the integrin-mediated signaling pathway, cytokine production and secretion, neutrophil chemotaxis, phagocytosis, response to hypoxia and reactive oxygen species, angiogenesis, apoptosis, inflammatory processes and ossification. Importantly, various elements of adaptive and induced innate immune responses span, not only the acute and subacute phases, but also persist throughout the chronic phase of SCI. Induced innate responses, such as Toll-like receptor signaling, are more active during the acute phase but persist throughout the chronic phase. However, adaptive immune response processes such as B and T cell activation, proliferation, and migration, T cell differentiation, B and T cell receptor-mediated signaling, and B cell- and immunoglobulin-mediated immune response become more significant during the chronic phase. Conclusions This analysis showed that, surprisingly, the diverse series of molecular events that occur in the acute and subacute stages persist into the chronic stage of SCI. The strong agreement between our results and previous findings suggest that our analytical approach will be useful in revealing other biological processes and genes contributing to SCI pathology

Alteration of expression of WT1 target genes in zinc finger domain lacking KTS-transfected MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R43</p><p>Breast cancer research : BCR 2007;9(4):R43-R43.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC2206716.</p><p></p> Transient and stable transfectants of MCF-7 cells were analyzed for the expression of Wilms' tumor 1 suppressor gene (WT1) target genes or nontarget genes (horizontal axis) with quantitative real-time PCR assays. The relative expression (vertical axis) of these genes in the transfected MCF-7 cells was calculated as compared with the pcDNAcontrol. Results are the average of three experiments. AREG, amphregulin; BASP1, brain acid soluble protein 1; KTS, lysine–threonine–serine; WT1, Wilms' tumor 1 suppressor gene; ZF, zinc finger domain

Inhibition of transcriptional activity of the WT1 promoter by its zinc finger domain

<p><b>Copyright information:</b></p><p>Taken from "The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R43</p><p>Breast cancer research : BCR 2007;9(4):R43-R43.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC2206716.</p><p></p> Schematic drawing for luciferase reporter constructs. The predominant transcription start site (+1) is indicated by an arrow. Small solid triangles indicate Wilms' tumor 1 suppressor gene (WT1) binding sites. MCF-7 cells and MDA468 cells were co-transfected with plasmids expressing the WT1 proteins (either pcDNAcontrol (CON), aminoterminal-only construct N-WT1, zinc finger domain lacking or with lysine–threonine–serine (ZF - KTS or ZF + KTS) or full-length WT1 vectors A~D), and either the pGL2 vector control or the differential WT1 promoter-driven luciferase constructs (horizontal axis), respectively. Luciferase activity was normalized with β-galactosidase activity and is expressed in relative luciferase activity as compared with the luciferase vector control (vertical axis). Results are the average of three experiments

Abrogation of WT1-mediated c-transactivation by the zinc finger domain lacking KTS in MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R43</p><p>Breast cancer research : BCR 2007;9(4):R43-R43.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC2206716.</p><p></p> The MCF-7 cells were transiently co-transfected with the reporter constructs (either pGL2 vector, XNM/Luc or Xmut/Luc), with expression plasmids of Wilms' tumor 1 suppressor gene (WT1) isoforms (either pcDNAvector, A, B, C or D) and with WT1–zinc finger domain (ZF) (either pcDNAcontrol (CON) or ZF lacking the lysine–threonine–serine insert (ZF-KTS)) as well as β-galactosidase expression plasmid, respectively. Relative luciferase activity (vertical axis) is indicated. Histograms show average of three independent experiments