Up-regulation Of Gene Transcription Research Articles

The same pocket in menin binds both MLL and JUND but has opposite effects on transcription

Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.

P3-17-01: ApoE and Its Receptors (LRP8, VLDLR) Function as Growth Signals for Triple-Negative Breast Cancer and Represent a Novel Therapeutic Target.

Abstract Triple-negative breast cancers (TNBC) lack the expression of estrogen, progesterone, and the human epidermal growth factor 2 receptors and have limited treatment options. We hypothesized that genes that are frequently amplified or overexpressed in these cancers are functionally important for the growth and survival of triple-negative breast cancer. Materials/Methods: We identified genes overexpressed in TNBC in 3 different human breast cancer gene expression data sets (n=294, n=286, n=198). We assessed the functional importance of the consistently overexpressed genes using siRNA screen on 18 breast cancer cell lines (10 ER-, 8 ER+) in vitro. We targeted each gene with 4 different siRNA constructs separately and each experiment was performed in triplicate. The genes with the greatest TNBC-specific inhibitory effect after siRNA down regulation were selected for further mechanistic and signal transduction studies. Results: 684 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in all 3 data sets. siRNA suppression of 161 of these genes inhibited cell growth significantly more in the ER-negative compared to ER-positive cells by at least 1 of the 4 siRNAs, 27 genes showed similar effect with 2 or more siRNAs and for 2 genes 3 of the 4 siRNAs showed preferential growth inhibition in ER-negative cells. These two genes were VLDLR (very low-density lipoprotein receptor) and LRP8 (low density lipoprotein receptor-related protein 8). We validated the siRNA screen results and confirmed down regulation of the mRNA and protein levels for VLDLR and LRP8 in 4 different ER-negative cell lines and showed that siRNA inhibition can be rescued by co-transfection of the receptor genes. Reanalysis of gene expression data also indicated expression of the LRP8 and VLDLR ligands, Reelin or ApoE, in both breast cancer tissues and in cell lines. We next demonstrated that exposure to Reelin and ApoE stimulates the growth of ER-negative cells in vitro. The stimulatory effect of ApoE was isoform dependent ApoE2 (Cys112/Cys158;) has the lowest receptor binding affinity and showed no growth stimulation, ApoE3 (Cys112/ Arg158) had modest 50–60% growth stimulation and ApoE4 (Arg112/ Arg158) had the greatest stimulatory effect (300-400%). Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. ApoE4 stimulation results in the transcriptional upregulation of genes involved in proliferation, metabolism, and inflammatory signaling pathways. ApoE4 stimulation increases expression of proteins involved in MAPK/ERK pathway, DNA damage repair, and inflammation. Conclusions: We show that theApoE - LRP8/VLDLR ligand receptor system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro. Interestingly, allelic imbalance favoring ApoE4 expression (E3/E4 or E4/E4) has been linked to higher risk of developing early onset breast cancer, which is primarily TNBC. We propose that inhibitors of LRP8/VLDLR signaling may be clinically useful therapeutic or preventive agents for TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-01.

Expression of a metallothionein A1 gene of Pisum sativum in white poplar enhances tolerance and accumulation of zinc and copper

Metallothioneins (MT) play an important role in heavy metal detoxification and homeostasis of intracellular metal ions in plant. In this study, two transgenic lines expressing MT type 2 gene ( PsMT A1 ) from Pisum sativum, a regenerated non transformed line NT and clone AL22, selected as heavy metal tolerant, were characterized in presence of the heavy metals for the ability to accumulate zinc and copper and to activate antioxidative enzyme defences: superoxide dismutase, catalase, ascorbate peroxidase. The levels of expression of MT type 2 gene assessed by RT-qPCR confirmed the gene over-expression in transgenic lines and evidenced in NT and AL22 the up-regulation of gene transcription by zinc and copper. Transgenic poplar lines during heavy metal stress displayed increased ability to translocate and accumulate zinc and copper compared with NT and AL22. The antioxidant enzyme defence was differently activated in response to metals in the transgenic lines without a significant increase of ROS. These results suggested that PsMT A1 could play a role in ROS scavenging leading to enhanced metal tolerance and increased zinc and copper sequestration in root and leaf.

PFOS prenatal exposure induce mitochondrial injury and gene expression change in hearts of weaned SD rats

Xenobiotics exposure in early life may have adverse effects on animals’ development through mitochondrial injury or dysfunction. The current study demonstrated the possibility of cardiac mitochondrial injury in prenatal PFOS-exposed weaned rat heart. Pregnant Sprague-Dawley (SD) rats were exposed to perfluorooctane sulfonate (PFOS) at doses of 0.1, 0.6 and 2.0 mg/kg/d and 0.05% Tween 80 as control by gavage from gestation days 2–21. The dams were allowed to give nature delivery and then heart tissues from weaned (postnatal day 21) offspring rats were analyzed for mitochondrial injury through ultrastructure observation by electron microscope, global gene expression profile by microarray, as well as related mRNA and proteins expression levels by quantitative PCR and western blot. Ultrastructural analysis revealed significant vacuolization and inner membrane injury occurred at the mitochondria of heart tissues from 2.0 mg/kg/d dosage group. Meanwhile, the global gene expression profile showed significant difference in level of some mRNA expression associated with mitochondrial function at 2.0 mg/kg/d dosage group, compared to the control. Furthermore, dose-response trends for the expression of selected genes were analyzed by quantitative PCR and western blot analysis. The selected genes were mainly focused on those encoding for proteins involved in energy production, control of ion levels, and maintenance of heart function. The down-regulation of mitochondrial ATP synthetase (ATP5E, ATP5I and ATP5O) implicated a decrease in energy supply. This was accompanied by down-regulation of gene transcripts involved in energy consumption such as ion transporting ATPase (ATP1A3 and ATP2B2) and inner membrane protein synthesis (SLC25A3, SLC25A4, SLC25A10, SLC25A29). The up-regulation of gene transcripts encoding for uncoupling proteins (UCP1 and UCP3), epidermal growth factor receptor (EGFR) and connective tissue growth factor (CTGF), was probably a protective process to maintain heart function. The results indicate PFOS prenatal exposure can induce cardiac mitochondrial injury and gene transcript change, which may be a significant mechanism of the developmental toxicity of PFOS to rat.

Transcriptome Analysis of the Barley–Deoxynivalenol Interaction: Evidence for a Role of Glutathione in Deoxynivalenol Detoxification

Trichothecenes are a major group of toxins produced by phytopathogenic fungi, including Fusarium graminearum. Trichothecenes inhibit protein synthesis in eukaryotic cells and are toxicologically relevant mycotoxins for humans and animals. Because they promote plant disease, the role of host responses to trichothecene accumulation is considered to be an important aspect of plant defense and resistance to fungal infection. Our overall objective was to examine the barley response to application of the type B trichothecene deoxynivalenol (DON). We found that DON is diluted by movement from the application site to acropetal and basipetal florets. A susceptible barley genotype converted DON to DON-3-O-glucoside, indicating that UDP-glucosyltransferases capable of detoxifying DON must exist in barley. RNA profiling of DON-treated barley spikes revealed strong upregulation of gene transcripts encoding ABC transporters, UDP-glucosyltransferases, cytochrome P450s, and glutathione-S-transferases. We noted that transcripts encoding cysteine synthases were dramatically induced by DON, and that toxin-sensitive yeast on glutathione- or cysteine-supplemented media or carrying a gene that encodes a cysteine biosynthetic enzyme exhibit DON resistance, suggesting that preventing glutathione depletion by increasing cysteine supply could play a role in ameliorating the impact of DON. Evidence for nonenzymatic formation of DON-glutathione adducts in vitro was found using both liquid chromatography-mass spectrometry and nuclear magnetic resonance analysis, indicating that the formation of DON-glutathione conjugates in vivo may reduce the impact of trichothecenes. Our results indicate that barley exhibits multiple defense mechanisms against trichothecenes.

The ubiquitin-conjugating enzyme HR6B is required for maintenance of X chromosome silencing in mouse spermatocytes and spermatids

BackgroundThe ubiquitin-conjugating enzyme HR6B is required for spermatogenesis in mouse. Loss of HR6B results in aberrant histone modification patterns on the trancriptionally silenced X and Y chromosomes (XY body) and on centromeric chromatin in meiotic prophase. We studied the relationship between these chromatin modifications and their effects on global gene expression patterns, in spermatocytes and spermatids.ResultsHR6B is enriched on the XY body and on centromeric regions in pachytene spermatocytes. Global gene expression analyses revealed that spermatid-specific single- and multicopy X-linked genes are prematurely expressed in Hr6b knockout spermatocytes. Very few other differences in gene expression were observed in these cells, except for upregulation of major satellite repeat transcription. In contrast, in Hr6b knockout spermatids, 7298 genes were differentially expressed; 65% of these genes was downregulated, but we observed a global upregulation of gene transcription from the X chromosome. In wild type spermatids, approximately 20% of the single-copy X-linked genes reach an average expression level that is similar to the average expression from autosomes.ConclusionsSpermatids maintain an enrichment of repressive chromatin marks on the X chromosome, originating from meiotic prophase, but this does not interfere with transcription of the single-copy X-linked genes that are reactivated or specifically activated in spermatids. HR6B represses major satellite repeat transcription in spermatocytes, and functions in the maintenance of X chromosome silencing in spermatocytes and spermatids. It is discussed that these functions involve modification of chromatin structure, possibly including H2B ubiquitylation.

The Submergence Tolerance Regulator SUB1A Mediates Crosstalk between Submergence and Drought Tolerance in Rice

Submergence and drought are major constraints to rice (Oryza sativa) production in rain-fed farmlands, both of which can occur sequentially during a single crop cycle. SUB1A, an ERF transcription factor found in limited rice accessions, dampens ethylene production and gibberellic acid responsiveness during submergence, economizing carbohydrate reserves and significantly prolonging endurance. Here, we evaluated the functional role of SUB1A in acclimation to dehydration. Comparative analysis of genotypes with and without SUB1A revealed that SUB1A enhanced recovery from drought at the vegetative stage through reduction of leaf water loss and lipid peroxidation and increased expression of genes associated with acclimation to dehydration. Overexpression of SUB1A augmented ABA responsiveness, thereby activating stress-inducible gene expression. Paradoxically, vegetative tissue undergoes dehydration upon desubmergence even though the soil contains sufficient water, indicating that leaf desiccation occurs in the natural progression of a flooding event. Desubmergence caused the upregulation of gene transcripts associated with acclimation to dehydration, with higher induction in SUB1A genotypes. SUB1A also restrained accumulation of reactive oxygen species (ROS) in aerial tissue during drought and desubmergence. Consistently, SUB1A increased the abundance of transcripts encoding ROS scavenging enzymes, resulting in enhanced tolerance to oxidative stress. Therefore, in addition to providing robust submergence tolerance, SUB1A improves survival of rapid dehydration following desubmergence and water deficit during drought.

Autophagy Induced by NGAL Protein in Esophageal Carcinoma Cells*

Previous studies suggest that NGAL (neutro phil gelatinase-associated lipocalin) is involved in the transformation and development of esophageal carcinoma. Alteration of NGAL expression can trigger the change of cellular morphology in esophageal carcinoma cells. However, the mechanisms remain unclear. To get a better understanding of NGAL function in esophageal carcinoma, NGAL protein was expressed in methylotrophic yeast, Pichia pastoris, and purified by chromatography. EC1.71 cells expressed high levels of NGALR (NGAL receptor) and EC109 cells expressed low levels of NGALR were used as cells model. The trafficking and the possible function of NGAL protein were then analyzed in the esophageal carcinoma cells. The results showed that 5-FAM-labeled recombinant NGAL protein could internalize into the EC1.71 and EC109 cells. Furthermore, the internalized NGAL protein could induce the alteration of cellular morphology, resulting in generation of autophagosome, transcriptional up-regulation of genes associated with autophagy and increase of phospho-ERK1/2 (p-ERK1/2). Interestingly, the treatment with the NGAL protein did not affect the intracellular iron level. These data indicate that induced autophagy by exogenous NGAL protein is a mechanism that internalized NGAL plays important roles in esophageal carcinoma cells, independent with NGAL-mediated iron transport process, while ERK1/2 signal pathway is involved in activation of autophagy by exogenous NGAL protein.

Histone deacetylase inhibitors induce apoptosis, histone hyperacetylation and up-regulation of gene transcription in Schistosoma mansoni

In order to explore the conservation/divergence of transcriptional regulation in the platyhelminth parasite Schistosoma mansoni, we are studying the structures and functions of transcriptional mediators and in particular histone-modifying enzymes. Reversible histone acetylation changes chromatin structure and modulates gene transcription. The removal of acetyl residues from histones and other proteins is catalyzed by histone deacetylases (HDACs) that are under increasing study as therapeutic targets, both in cancer and parasitic diseases. In order to determine the extent and importance of histone acetylation in S. mansoni, we tested the effects of three histone deacetylase inhibitors (HDACi) on both larval and adult worms in culture. Trichostatin A (TSA), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) inhibited global HDAC activity at all life-cycle stages. TSA and VPA, but not SAHA, caused mortality of schistosomula and adults, with TSA showing the most rapid effect. Moreover, TSA caused an increase in apoptosis in schistosomula shown by the TUNEL assay and an increase in caspase 3/7 activity. Both TSA and VPA were shown to cause an increase in general levels of protein acetylation in schistosomes; more particularly of histone 4 whereas histone 3 acetylation was less affected. In the case of TSA treatment this histone hyperacetylation was correlated with the increased expression of caspases 3 and 7 transcripts. Finally, quantitative chromatin immunoprecipitation showed that the proximal promoter region of the S. mansoni caspase 7 gene was hyperacetylated on histone H4 after TSA treatment.

Mitogen-activated protein kinase signaling is necessary for the maintenance of skeletal muscle mass

The signal transduction cascades that maintain muscle mass remain to be fully defined. Herein, we report that inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in vitro decreases myotube size and protein content after 3-day treatment with a MEK inhibitor. Neither p38 nor JNK inhibitors had any effect on myotube size or morphology. ERK1/2 inhibition also upregulated gene transcription of atrogin-1 and muscle-specific RING finger protein 1 and downregulated the phosphorylation of Akt and its downstream kinases. Forced expression of enhanced green fluorescent protein-tagged MAPK phosphatase 1 (MKP-1) in soleus and gastrocnemius muscles decreased both fiber size and reporter activity. This atrophic effect of MKP-1 was time dependent. Analysis of the reporter activity in vivo revealed that the activities of nuclear factor-kappaB and 26S proteasome were differentially activated in slow and fast muscles, suggesting muscle type-specific mechanisms may be utilized. Together, these findings suggest that MAPK signaling is necessary for the maintenance of skeletal muscle mass because inhibition of these signaling cascades elicits muscle atrophy in vitro and in vivo.

Loss of Caveolin-3 Induces a Lactogenic Microenvironment that Is Protective Against Mammary Tumor Formation

Here, we show that functional loss of a single gene is sufficient to confer constitutive milk protein production and protection against mammary tumor formation. Caveolin-3 (Cav-3), a muscle-specific caveolin-related gene, is highly expressed in muscle cells. We demonstrate that Cav-3 is also expressed in myoepithelial cells within the mammary gland. To determine whether genetic ablation of Cav-3 expression affects adult mammary gland development, we studied the phenotype(s) of Cav-3(-/-)-null mice. Interestingly, Cav-3(-/-) virgin mammary glands developed lobulo-alveolar hyperplasia, akin to the changes normally observed during pregnancy and lactation. Genome-wide expression profiling revealed up-regulation of gene transcripts associated with pregnancy/lactation, mammary stem cells, and human breast cancers, consistent with a constitutive lactogenic phenotype. Expression levels of three key transcriptional regulators of lactation, namely Elf5, Stat5a, and c-Myc, were also significantly elevated. Experiments with pregnant mice directly showed that Cav-3(-/-) mice underwent precocious lactation. Finally, using orthotopic tumor cell implantation, we demonstrated that virgin Cav-3(-/-) mice were dramatically protected against mammary tumor formation. Thus, Cav-3(-/-) mice are a novel preclinical model to study the protective effects of a lactogenic microenvironment on mammary tumor onset and progression. Our current studies have broad implications for using the lactogenic microenvironment as a paradigm to discover new therapies for the prevention and/or treatment of human breast cancers.

Upregulation of Molecules Associated With T-Regulatory Function by Thymoglobulin Pretreatment of Human CD4+ Cells

This study evaluated the immunologic effects of thymoglobulin in modulating human CD4+ cells. Human CD4+ cells were purified from peripheral blood mononuclear cells by negative selection method. CD4+ cells were pretreated with thymoglobulin and incubated for 72 hr. Cells and culture supernatants were collected and studied by real-time quantitative polymerase chain reaction, fluorescence activated cell scanning, multiplex cytokine assay, and mixed lymphocyte reaction (MLR). Thymoglobulin pretreated CD4+ cells demonstrated up-regulation of gene transcripts for CTLA-4, OX40, forkhead box P3 (Foxp3), CD25, IFN-gamma, IL-10, and IL-2 as determined by real-time quantitative polymerase chain reaction. Fluorescence-activated cell scanning analysis demonstrated that CD4+ cells, pretreated with thymoglobulin, up-regulated CD25 expression on their surface, and the surface expression of CTLA-4 and OX40 and the expression of intracellular Foxp3 were observed in these CD4+CD25+ cells. Additionally, MLR demonstrated that thymoglobulin-pretreated cells partially inhibited proliferation of untreated autologous CD4+ cells in response to allogeneic cells. The high levels of IFN-gamma, IL-10, IL-2, and IL-4 were detected by multiplex cytokine assay in supernatants collected from cultures of thymoglobulin-pretreated CD4+ cells. The lymphocyte proliferation of allogeneic MLR was also partially blocked in the presence of supernatants from cultures of thymoglobulin-pretreated CD4+ cells. This study demonstrates that the unique effects of thymoglobulin in modulating CD4+ cells may be an important mechanism for its action in inducing immunosuppression and transplant tolerance.

Molecular Characterization of Hypoxia-Hypothermia–Conditioned Human Endothelial Cells and Their Interaction With Human Monocytes

This in vitro study was designed to characterize the molecular profiling of human endothelial cells (ECs) during the early phase of hypoxia-hypothermia (HH) conditioning and to evaluate their interactions with allogeneic monocytes. The HH-conditioned ECs were analyzed using real-time quantitative polymerase chain reaction (RT-PCR). A cell adhesion assay was performed to assess adhesion of purified allogeneic monocytes as well as CD4- and CD8-positive T cells to HH-conditioned ECs with or without blocking antibodies specific for CD15s and CD162. Uptake of EC membrane by monocytes with or without scavenger receptor blockade was examined using fluorescence-activated cell scanning. The RT-PCR revealed up-regulation of gene transcripts for inflammatory cytokines, monocyte-associated growth factors, costimulatory, and apoptosis-related molecules in HH-conditioned ECs. Analysis using fluorescence-activated cell scanning showed minimal CD54 up-regulation in HH-conditioned ECs. We noted low-level adhesion of CD4- or CD8-positive cells to resting and HH-conditioned ECs. High-level adhesion of monocytes to HH-conditioned ECs was observed when compared with resting ECs. Blockade of CD15s and CD162 dramatically reduced monocyte adhesion to normal and HH-conditioned ECs. Monocytes but not T cells showed uptake of EC membranes during their interactions with HH-conditioned ECs, which was inhibited by scavenger receptor blockade. These data characterized the molecular features of ECs during early HH-conditioning. The EC transcripts related to monocyte recruitment and interaction between monocytes and HH-conditioned ECs dominated the early post-HH condition. Blockade of CD15s and CD162 prevented monocyte adhesion to ECs. These findings suggest that the initial interaction between monocytes and HH-conditioned ECs has a central role during the early phase of reperfusion injury.

Transcription of the chicken Grin1 gene is regulated by the activity of SP3 and NRSF in undifferentiated cells and neurons

The NMDA (N-methyl-D-aspartate) receptors are important in the regulation of neuronal development, synaptic plasticity, learning and memory, and are involved in several brain pathologies. The NR1 subunit is essential for the assembly of functional receptors, as it forms the calcium-permeable ion channel and contains the obligatory co-agonist binding site. Previous studies have shown that NR1 gene (Grin1) expression is up-regulated during neuronal differentiation and its expression is widespread in the central nervous system. We have previously cloned the chicken Grin1 gene and 1.9 kb of the 5'-regulatory region. In the present study, we analysed the molecular mechanisms that regulate chicken Grin1 gene transcription in undifferentiated cells and neurons. By functional analysis of chicken Grin1-luciferase gene 5'-regulatory region constructs, we demonstrate that the basal promoter is delimited within 210 bp upstream from the main transcription initiation site. DNA-protein binding and functional assays revealed that the 5'-UTR (untranslated region) has one consensus NRSE (neuron-restrictive silencing element) that binds NRSF (neuron-restrictive silencing factor), and one SP (stimulating protein transcription factor) element that binds SP3, both repressing Grin1 gene transcription in undifferentiated P19 cells (embryonic terato-carcinoma cells) and PC12 cells (phaeochromocytoma cells). The promoter region lacks a consensus TATA box, but contains one GSG/SP (GSG-like box near a SP-consensus site) that binds SP3 and up-regulates gene transcription in embryonic chicken cortical neurons. Taken together, these results demonstrate a dual role of SP3 in regulating the expression of the Grin1 gene, by repressing transcription in the 5'-UTR in undifferentiated cells as well as acting as a transcription factor, increasing Grin1 gene transcription in neurons.

Synthesis and Biological Evaluation of Novel 2,4-Diaminoquinazoline Derivatives as SMN2 Promoter Activators for the Potential Treatment of Spinal Muscular Atrophy

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by death of motor neurons in the spinal cord that is caused by deletion and/or mutation of the survival motor neuron gene ( SMN1). Adjacent to SMN1 are a variable number of copies of the SMN2 gene. The two genes essentially differ by a single nucleotide, which causes the majority of the RNA transcripts from SMN2 to lack exon 7. Although both SMN1 and SMN2 encode the same Smn protein amino acid sequence, the loss of SMN1 and incorrect splicing of SMN2 have the consequence that Smn protein levels are insufficient for the survival of motor neurons. The therapeutic goal of our medicinal chemistry effort was to identify small-molecule activators of the SMN2 promoter that, by up-regulating gene transcription, would produce greater quantities of full-length Smn protein. Our initial medicinal chemistry effort explored a series of C5 substituted benzyl ether based 2,4-diaminoquinazoline derivatives that were found to be potent activators of the SMN2 promoter; however, inhibition of DHFR was shown to be an off-target activity that was linked to ATP depletion. We used a structure-guided approach to overcome DHFR inhibition while retaining SMN2 promoter activation. A lead compound 11a was identified as having high potency (EC50 = 4 nM) and 2.3-fold induction of the SMN2 promoter. Compound 11a possessed desirable pharmaceutical properties, including excellent brain exposure and long brain half-life following oral dosing to mice. The piperidine compound 11a up-regulated expression of the mouse SMN gene in NSC-34 cells, a mouse motor neuron hybrid cell line. In type 1 SMA patient fibroblasts, compound 11a induced Smn in a dose-dependent manner when analyzed by immunoblotting and increased the number of intranuclear particles called gems. The compound restored gems numbers in type I SMA patient fibroblasts to levels near unaffected genetic carriers of SMA.

Transcriptional analysis of the common carp ( Cyprinus carpio L.) immune response to the fish louse Argulus japonicus Thiele (Crustacea: Branchiura)

In the present study we investigated changes in transcription levels of a panel of selected immune relevant genes in peripheral blood leucocytes (PBL) and skin samples collected from carp exposed to larval Argulus japonicus. We show that in skin up-regulation of gene transcription of the chemokine CXCa, and to a lesser extent the chemokine receptor CXCR1 and the cytokine TNFα, are good indicators of parasite-induced skin damage at 2 days post-parasite exposure. Up-regulation of gene transcription corresponded well with an increase in leucocytes, probably neutrophilic granulocyte numbers in skin samples collected at the sites of infection. We show that time-point controls are essential when studying gene expression, especially in peripheral blood leucocytes (PBL). In addition, we demonstrate that non-infected control samples isolated from the skin of infected fish are suitable autologous controls, at least until after larval A. japonicus have undergone their first moult and begun to demonstrate increased mobility over their host's integument. The observed results are indicative of A. japonicus affecting the skin as a whole organ, particularly after the parasites' first moult, a phenomenon which has a great impact on correct skin sampling for RNA isolation.

A Molecular Signature of Gastric Metaplasia Arising in Response to Acute Parietal Cell Loss

Loss of gastric parietal cells is a critical precursor to gastric metaplasia and neoplasia. However, the origin of metaplasia remains obscure. Acute parietal cell loss in gastrin-deficient mice treated with DMP-777 leads to the rapid emergence of spasmolytic polypeptide/trefoil factor family 2 (TFF2)-expressing metaplasia (SPEM) from the bases of fundic glands. We now sought to characterize more definitively the pathway for emergence of SPEM. Emerging SPEM lineages in gastrin-deficient mice treated with DMP-777 were examined for immunolocalization of TFF2, intrinsic factor, and Mist1, and morphologically with electron microscopy. Emerging SPEM was isolated with laser-capture microdissection and RNA was analyzed using gene microarrays. Immunohistochemistry in mouse and human samples was used to confirm up-regulated transcripts. DMP-777-induced SPEM was immunoreactive for TFF2 and the differentiated chief cell markers, Mist1 and intrinsic factor, suggesting that SPEM derived from transdifferentiation of chief cells. Microarray analysis of microdissected SPEM lineages induced by DMP-777 showed up-regulation of transcripts associated with G1/S cell-cycle transition including minichromosome maintenance deficient proteins, as well as a number of secreted factors, including human epididymis 4 (HE4). HE4, which was absent in the normal stomach, was expressed in SPEM of human and mouse and in intestinal metaplasia and gastric cancer in human beings. Although traditionally metaplasia was thought to originate from normal mucosal progenitor cells, these studies indicate that SPEM evolves through either transdifferentiation of chief cells or activation of a basal cryptic progenitor. In addition, induction of metaplasia elicits the expression of secreted factors, such as HE4, relevant to gastric preneoplasia.

Anti-Acute Myeloid Leukemia Activity of Chaetocin, a Novel Epigenetic Drug Inhibitor Inducing Oxidative Stress.

Recent studies have highlighted the importance of epigenetic modifications in the pathogenesis of Acute Myeloid Leukemia (AML). This results have been confirmed by the activity of new drug like DNA demethylating agents and histone deacetylase (HDAC) inhibitors in both in vivo and in vitro studies. Recently, Chaetocin, a natural fungal compound, has been identified as the first specific inhibitor of the histone methyltransferase SU(VAR)3–9 which plays a role in heterochromatin gene silencing. In this study, we decided to evaluate Chaetocin as a therapeutic agent in AML in vitro and to explore the related mechanisms. We show that Chaetocin induce dramatic cell death at nanomolar concentrations in U937 and HL60 (97.2% ± 0.4 and 91.6% ± 9 cell death at 100 nM chaetocin, respectively), and to a lesser extend in K562 (67.3% ± 1.6 cell death at 100 nM chaetocin), cell cultures. Cell death occurred at 24 h incubation time which correlated with induction of apoptosis as assessed by Annexin V/7-AAD staining and activation of downstream executioner caspase-3/7. Using transcription low-density array and quantitative RT- PCR, Chaetocin was showed to up-regulate gene transcription such as of the cell cycle inhibitor p21/WAF1 consistent with a role for the targeted SU(VAR)3–9 in heterochromatin gene silencing. In agreement with the recent report of Chaetocin being a promising new antimyeloma agent acting via imposition of oxidative stress, intracellular levels of oxidative species were increased in Chaetocin treated U937 cells in a time- and dose-dependent manner that correlated with induction of cell death. Furthermore, incubation of cells with N-acetyl cysteine, a cell-permeable precursor of intracellular glutathione reductant, prevented chaetocin-induced accumulation of oxidative species, transcription of selected genes (e.g. p21/WAF1), activation of caspase-3, and cell death. Finally, Chaetocin was found to increase the antileukemia activity of HDAC inhibitors and Aracytin, and thus appears as a promising agent for further study as a potential anti-AML therapeutic. Preliminary results obtained in vivo in xenograft models and ex vivo, using blasts of a panel of patients with AML, will be presented.

UPREGULATED ENDOTHELIN SYSTEM IN DIABETIC VASCULAR DYSFUNCTION AND EARLY RETINOPATHY IS REVERSED BY CPU0213 AND TOTAL TRITERPENE ACIDS FROM FRUCTUS CORNI

1. The aims of the present study were to examine whether: (i) upregulation of the endothelin (ET) pathway is involved in impairment of vascular relaxation and early retinopathy in diabetic rats; and (ii) vascular and retinal abnormalities respond to the total triterpene acid (TTA) isolated from Fructus Corni compared with responses to the novel endothelin receptor antagonist CPU0213 and aminoguanidine (AMG), a special antagonist for advanced glycation end-products (AGE) and inducible nitric oxide synthase (iNOS). 2. Male Sprague-Dawley rats were randomized into five groups, namely a normal control and four diabetic groups, which included an untreated diabetic group and groups treated with AMG (100 mg/kg, i.g.), CPU0213 (30 mg/kg, s.c.) or TTA (50 mg/kg, i.g.). Diabetes was induced by single injection of streptozotocin (60 mg/kg, i.p.) on the 1st day. The mRNA expression of prepro-endothelin-1 (ppET-1), endothelin-converting enzyme (ECE) and iNOS in the thoracic aorta and mRNA for ET(A) receptors and iNOS in the retina were detected by reverse transcription-polymerase chain reaction. Vasorelaxation to acetylcholine (ACh) and functional assessment of nitric oxide (NO) bioavailability was determined in the thoracic aorta. 3. We observed upregulated mRNA expression of iNOS, ppET-1 and ECE in the thoracic aorta and upregulated mRNA for the ET(A) receptor and iNOS in the retina in the untreated diabetic group. Vasodilatation mediated by ACh and NO bioavailability were markedly reduced in the thoracic aorta compared with the normal control group. These abnormalities were essentially reversed by TTA, CPU0213 or AMG, with the exception with that AMG did not modify vasodilatation to ACh. 4. These data suggest that upregulation of gene transcription of the ET system mediates depressed vasorelaxation, NO bioavailability and changes in iNOS and ET(A) receptors that reflect early retinopathy in diabetic rats. Total triterpene acid, in terms of pharmacological properties resembling the endothelin receptor antagonist CPU0213, is effective in normalizing expression of the ET system and iNOS in early diabetic retinopathy and vasculaopathy.

Activity-dependent neuroprotective protein (ADNP) differentially interacts with chromatin to regulate genes essential for embryogenesis

Complete deficiency in activity-dependent neuroprotective protein (ADNP) results in neural tube closure defects and death at days 8.5–9.5 of gestation in the mouse (E8.5–9.5). To elucidate ADNP associated pathways, Affymetrix 22,690-oligonucleotide-based microarrays were used on ADNP knockout and control mouse embryos (E9) separated completely from extra embryonic tissue. Marked differences in expression profiles between ADNP-deficient embryos and ADNP-expressing embryos were discovered. Specifically, a group of dramatically up-regulated gene transcripts in the ADNP-deficient embryos were clustered into a family encoding for proteins enriched in the visceral endoderm such as apolipoproteins, cathepsins and methallotionins. In contrast, a down regulated gene cluster associated with ADNP-deficiency in the developing embryo consisted of organogenesis markers including neurogenesis ( Ngfr, neurogenin1, neurod1) and heart development ( Myl2). The pluripotent P19 cells were used for ADNP-chromatin-immunoprecipitation, showing direct interactions with multiple relevant gene promoters including members of the up-regulated as well as the down-regulated gene clusters. A comparison between non-differentiated and neuro-differentiated P19 cells revealed increased chromatin interaction of ADNP with chromatin from differentiated cells. These results place ADNP at a crucial point of gene regulation, repressing potential endoderm genes and enhancing genes associated with organogenesis/neurogenesis.