Induced Changes In Expression Research Articles

Abstract 192: Mechano-growth Factor (MGF) and Cerebral Vasospasm (CV) Post Aneurysmal Subarachnoid Hemorrhage (aSAH)

Cerebral vasospasm (CV) and related ischemic injury is a major contributor to death and disability after aneurysmal subarachnoid hemorrhage (aSAH). Our overall goal is to understand molecular mechanisms of action of endogenous mechano-growth factor (MGF), a splice variant of insulin-like growth factor 1 (IGF-1), in the brain. MGF was found to be neuroprotective after stroke in a gerbil model of ischemic injury and may serve as a potential therapeutic target to prevent CV-induced brain damage. In our studies, we characterized hypoxia induced changes in MGF expression in different brain regions, commonly affected by CV, at different time points (from 2 hours to 7 days) post 4h hypoxia treatments (6, 8,10 or 12% oxygen). The brain regions analyzed were motor cortex, hippocampus, striatum and hypothalamus. In addition, we characterized changes in MGF expression in microparticle (MP) fractions isolated from cerebrospinal fluid (CSF) extracted from CV patients. Microparticle fractions were isolated from CSF samples using serial ultracentrifugation. We used real time RT-PCR, western blots, and enzyme immunosorbent assay to quantify changes in gene expression and protein levels. In the animal model, our results showed that there were time, dose and brain region specific changes in MGF expression that correlated with changes in the expression of heme oxygenase 1 (HO-1) and biliverdin reductase A, which are molecules involved in neuroprotection, and caspases, apoptotic markers. These results suggest that changes in endogenous MGF due to hypoxia may activate neuroprotective pathways in the brain. In CSF MPs isolated from CV patients, we observed an increase in MGF expression that correlated with the CV onset window and with an increase in HO-1, suggesting that similar pathways are activated post-CV in humans and post-hypoxia in animals. These data indicate that endogenous MGF may play a role in CV onset and may be a neuroprotective target in CV papteins. However, further studies are required to elucidate the molecular mechanisms of MGF and its exact role in CV development and patient outcome.

Genome-wide DNA methylome reveals the dysfunction of intronic microRNAs in major psychosis.

BackgroundDNA methylation is thought to be extensively involved in the pathogenesis of many diseases, including major psychosis. However, most studies focus on DNA methylation alteration at promoters of protein-coding genes, despite the poor correlation between DNA methylation and gene expression.MethodsWe analyzed differentially methylated regions and differentially expressed genes in patients with schizophrenia and bipolar disorder and normal subjects. Gene expression and DNA methylation were analyzed with RNA-seq and MeDIP-seq of post-mortem brain tissue (brain region BA9) cohort in five schizophrenia, seven bipolar disorder cases and six controls, respectively.ResultsHere, we performed a large-scale integrative analysis using MeDIP-seq, coupled with RNA-seq, on brain samples from major psychotic and normal subjects and observed obvious discrepancy between DNA methylation and gene expression. We found that differentially methylated regions (DMRs) were distributed across different types of genomic elements, especially introns. These intronic DMRs were significantly enriched for diverse regulatory elements, such as enhancers and binding sites of certain transcriptional factors (e.g., Pol3). Notably, we found that parts of intronic DMRs overlapped with some intragenic miRNAs, such as hsa-mir-7-3. These intronic DMR-related miRNAs were found to target many differentially expressed genes. Moreover, functional analysis demonstrated that differential target genes of intronic DMR-related miRNAs were sufficient to capture many important biological processes in major psychosis, such as neurogenesis, suggesting that miRNAs may function as important linkers mediating the relationships between DNA methylation alteration and gene expression changes.ConclusionsCollectively, our study indicated that DNA methylation alteration could induce expression changes indirectly by affecting miRNAs and the exploration of DMR-related miRNAs and their targets enhanced understanding of the molecular mechanisms underlying major psychosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0139-4) contains supplementary material, which is available to authorized users.

Biological therapy induces expression changes in Notch pathway in psoriasis.

Psoriasis is a chronic inflammatory skin disease, characterized by hyperproliferation of keratinocytes and by skin infiltration of activated T cells. To date, the pathophysiology of psoriasis has not yet been fully elucidated. The Notch pathway plays a determinant role in cell fate determination, proliferation, differentiation, immune cell development and function. Many biological agents, used in the treatment of psoriasis, include TFN-α inhibitors, such as etanercept, adalimumab, and anti IL-12/IL-23 p40 antibody, such as ustekinumab. This study aimed to determine mRNA expression levels by real-time RT-PCR, and protein expression levels, analysed by Western blot and immunohistochemistry, of some components of the Notch pathway, such as NOTCH1, NOTCH2, JAGGED1, and HES1 after biological treatments in psoriatic patients. mRNA and protein levels of NOTCH1, NOTCH2, JAGGED1 and HES1 were upregulated in skin samples from untreated psoriatic patients compared with normal controls. Biological therapy showed to downregulate differently the protein expression levels of the molecules under study. Our study suggests that Notch pathway components might be a potential therapeutic target against psoriasis.

Comparative analysis of root transcriptomes from two contrasting drought-responsive Williams 82 and DT2008 soybean cultivars under normal and dehydration conditions.

The economically important DT2008 and the model Williams 82 (W82) soybean cultivars were reported to have differential drought-tolerant degree to dehydration and drought, which was associated with root trait. Here, we used 66K Affymetrix Soybean Array GeneChip to compare the root transcriptomes of DT2008 and W82 seedlings under normal, as well as mild (2 h treatment) and severe (10 h treatment) dehydration conditions. Out of the 38172 soybean genes annotated with high confidence, 822 (2.15%) and 632 (1.66%) genes showed altered expression by dehydration in W82 and DT2008 roots, respectively, suggesting that a larger machinery is required to be activated in the drought-sensitive W82 cultivar to cope with the stress. We also observed that long-term dehydration period induced expression change of more genes in soybean roots than the short-term one, independently of the genotypes. Furthermore, our data suggest that the higher drought tolerability of DT2008 might be attributed to the higher number of genes induced in DT2008 roots than in W82 roots by early dehydration, and to the expression changes of more genes triggered by short-term dehydration than those by prolonged dehydration in DT2008 roots vs. W82 roots. Differentially expressed genes (DEGs) that could be predicted to have a known function were further analyzed to gain a basic understanding on how soybean plants respond to dehydration for their survival. The higher drought tolerability of DT2008 vs. W82 might be attributed to differential expression in genes encoding osmoprotectant biosynthesis-, detoxification- or cell wall-related proteins, kinases, transcription factors and phosphatase 2C proteins. This research allowed us to identify genetic components that contribute to the improved drought tolerance of DT2008, as well as provide a useful genetic resource for in-depth functional analyses that ultimately leads to development of soybean cultivars with improved tolerance to drought.

PP2A inhibitors arrest G2/M transition through JNK/Sp1- dependent down-regulation of CDK1 and autophagy-dependent up-regulation of p21.

Protein phosphatase 2A (PP2A) plays an important role in the control of the cell cycle. We previously reported that the PP2A inhibitors, cantharidin and okadaic acid (OA), efficiently repressed the growth of cancer cells. In the present study, we found that PP2A inhibitors arrested the cell cycle at the G2 phase through a mechanism that was dependent on the JNK pathway. Microarrays further showed that PP2A inhibitors induced expression changes in multiple genes that participate in cell cycle transition. To verify whether these expression changes were executed in a PP2A-dependent manner, we targeted the PP2A catalytic subunit (PP2Ac) using siRNA and evaluated gene expression with a microarray. After the cross comparison of these microarray data, we identified that CDK1 was potentially the same target when treated with either PP2A inhibitors or PP2Ac siRNA. In addition, we found that the down-regulation of CDK1 occurred in a JNK-dependent manner. Luciferase reporter gene assays demonstrated that repression of the transcription of CDK1 was executed through the JNK-dependent activation of the Sp1 transcription factor. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified an element in the CDK1 promoter that responded to the JNK/Sp1 pathway after stimulation with PP2A inhibitors. Cantharidin and OA also up-regulated the expression of p21, an inhibitor of CDK1, via autophagy rather than PP2A/JNK pathway. Thus, this present study found that the PP2A/JNK/Sp1/CDK1 pathway and the autophagy/p21 pathway participated in G2/M cell cycle arrest triggered by PP2A inhibitors.

Transcriptomic analysis provides insight into high-altitude acclimation in domestic goats

Domestic goats are distributed in a wide range of habitats and have acclimated to their local environmental conditions. To investigate the gene expression changes of goats that are induced by high altitude stress, we performed RNA-seq on 27 samples from the three hypoxia-sensitive tissues (heart, lung, and skeletal muscle) in three indigenous populations from distinct altitudes (600m, 2000m, and 3000m). We generated 129Gb of high-quality sequencing data (~4Gb per sample) and catalogued the expression profiles of 12,421 annotated hircine genes in each sample. The analysis showed global similarities and differences of high-altitude transcriptomes among populations and tissues as well as revealed that the heart underwent the most high-altitude induced expression changes. We identified numerous differentially expressed genes that exhibited distinct expression patterns, and nonsynonymous single nucleotide variant-containing genes that were highly differentiated between the high- and low-altitude populations. These genes have known or potential roles in hypoxia response and were enriched in functional gene categories potentially responsible for high-altitude stress. Therefore, they are appealing candidates for further investigation of the gene expression and associated regulatory mechanisms related to high-altitude acclimation.

Tomato root exudates induce transcriptional changes of Meloidogyne hispanica genes

Meloidogyne hispanica is a polyphagous root-knot nematode of emerging importance that has the ability to infect a broad range of plants, and tomato ( Solanum lycopersicum L.) crops can be severely damaged. This study investigated whether tomato root exudates regulate the expression of five candidate parasitism genes previously identified and sequenced in M. Hispanica . These were; calreticulin ( crt-1 ), cathepsin L cysteine protease ( cpl-1 ), β -1,4 endoglucanase-1 ( eng-1 ), fatty acid retinol binding protein ( far-1 ) and venom allergen-like protein ( vap-1 ). One thousand M. hispanica second-stage juveniles (J2) were exposed overnight to tomato root exudates, obtained from the root systems of 4-week-old plants during 4 h of agitation in sterilized distilled water, and the relative expression of the parasitism genes was determined by quantitative real-time PCR. The cpl-1 , crt-1 , far-1 and vap-1 genes were differentially up-regulated ( P <0.05) in the pre-parasitic J2 after exposure to tomato root exudates, while expression of eng-1 was largely unaffected ( P =0.05) by the treatment. This results suggests that tomato root exudates induce changes in the expression of candidate parasitism genes. Gene silencing by RNAi will elucidate the exact function of these candidate parasitism genes in nematode penetration and survival. Identification of the plant signal molecules in the tomato root exudates responsible for the up-regulation of these parasitism genes may lead to the development of novel approaches for the management of M. hispanica .

A transcriptomics-based hepatotoxicity comparison between the zebrafish embryo and established human and rodent in vitro and in vivo models using cyclosporine A, amiodarone and acetaminophen

The zebrafish embryo (ZFE) is a promising alternative, non-rodent model in toxicology, which has an advantage over the traditionally used models as it contains complete biological complexity and provides a medium to high-throughput setting. Here, we assess how the ZFE compares to the traditionally used models for liver toxicity testing, i.e., in vivo mouse and rat liver, in vitro mouse and rat hepatocytes, and primary human hepatocytes. For this comparison, we analyzed gene expression changes induced by three model compounds for cholestasis, steatosis, and necrosis. The three compounds, cyclosporine A, amiodarone, and acetaminophen, were chosen because of their relevance to human toxicity and these compounds displayed hepatotoxic-specific changes in the mouse in vivo data. Compound induced expression changes in the ZFE model shared similarity with both in vivo and in vitro. Comparison on single gene level revealed the presence of model specific changes and no clear concordance across models. However, concordance was identified on the pathway level. Specifically, the pathway “regulation of metabolism – bile acids regulation of glucose and lipid metabolism via FXR” was affected across all models and compounds. In conclusion, our study with three hepatotoxic model compounds shows that the ZFE model is at least as comparable to traditional models in identifying hepatotoxic activity and has the potential for use as a pre-screen to determine the hepatotoxic potential of compounds.

The Long Non-Coding RNA PCAT-1 Promotes Prostate Cancer Cell Proliferation through cMyc

Long non-coding RNAs (lncRNAs) represent an emerging layer of cancer biology, contributing to tumor proliferation, invasion, and metastasis. Here, we describe a role for the oncogenic lncRNA PCAT-1 in prostate cancer proliferation through cMyc. We find that PCAT-1–mediated proliferation is dependent on cMyc protein stabilization, and using expression profiling, we observed that cMyc is required for a subset of PCAT-1–induced expression changes. The PCAT-1–cMyc relationship is mediated through the post-transcriptional activity of the MYC 3′ untranslated region, and we characterize a role for PCAT-1 in the disruption of MYC-targeting microRNAs. To further elucidate a role for post-transcriptional regulation, we demonstrate that targeting PCAT-1 with miR-3667-3p, which does not target MYC, is able to reverse the stabilization of cMyc by PCAT-1. This work establishes a basis for the oncogenic role of PCAT-1 in cancer cell proliferation and is the first study to implicate lncRNAs in the regulation of cMyc in prostate cancer.

Inherited cancer predisposition sensitizes colonic mucosa to address Western diet effects and putative cancer-predisposing changes on mouse proteome

Human epidemiological evidence and previous studies on mice have shown that Western-style diet (WD) may predispose gut mucosa to colorectal cancer (CRC). The mechanisms that mediate the effects of diet on tumorigenesis are largely unknown. To address putative cancer-predisposing events available for early detection, we quantitatively analyzed the proteome of histologically normal colon of a wild-type (Mlh1+/+) and an Mlh1+/− mouse after a long-term feeding experiment with WD and AIN-93G control diet. The Mlh1+/− mouse carries susceptibility to colon cancer analogous to a human CRC syndrome (Lynch syndrome). Remarkably, WD induced expression changes reflecting metabolic disturbances especially in the cancer-predisposed colon, while similar changes were not significant in the wild-type proteome. Overall, the detected changes constitute a complex interaction network of proteins involved in ATP synthesis coupled proton transport, oxidoreduction coenzyme and nicotinamide nucleotide metabolic processes, important in cell protection against reactive oxygen species toxicity. Of these proteins, selenium binding protein 1 and galectin-4, which directly interact with MutL homolog 1, are underlined in neoplastic processes, suggesting that sensitivity to WD is increased by an Mlh1 mutation. The significance of WD on CRC risk is highlighted by the fact that five out of six mice with neoplasias were fed with WD.

PP2A inhibitors suppress migration and growth of PANC-1 pancreatic cancer cells through inhibition on the Wnt/β-catenin pathway by phosphorylation and degradation of β-catenin.

Cantharidin is an active constituent of mylabris, a traditional Chinese medicine, and presents strong anticancer activity in various cell lines. Cantharidin is a potent and selective inhibitor of serine/threonine protein phosphatase 2A (PP2A). Our previous studies revealed the prospect of application of cantharidin, as well as other PP2A inhibitors, in the treatment of pancreatic cancer. However, the mechanisms involved in the anticancer effect of PP2A inhibitors have not been fully explored. The Wnt/β-catenin pathway is involved in cell migration and proliferation and participates in the progression of pancreatic cancer. If β-catenin is phosphorylated and degraded, the Wnt/β-catenin pathway is blocked. PP2A dephosphorylates β-catenin and keeps the Wnt/β-catenin pathway active. In the present study, we found that PP2A inhibitor treatment induced phosphorylation and degradation of β-catenin. The suppression on the migration and growth of PANC-1 pancreatic cancer cells could be attenuated by pretreatment with FH535, a β-catenin pathway inhibitor. Microarray showed that PP2A inhibitor treatment induced expression changes in 13 of 138 genes downstream of the β-catenin pathway. Real-time PCR further confirmed that FH535 attenuated the expression changes induced by PP2A inhibitors in 6 of these 13 candidate genes. These 6 genes, VEGFB, Dkk3, KRT8, NRP1, Cacnalg and WISP2, have been confirmed to participate in the migration and/or growth regulation in previous studies. Thus, the phosphorylation- and degradation-mediated suppression on β-catenin participates in the cytotoxicity of PP2A inhibitors. Our findings may provide insight into the treatment of pancreatic cancer using a targeting PP2A strategy.

Comment on ‘A novel 5‐enolpyruvoylshikimate‐3‐phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide’ by Wang et al. (2014)

Comment on ‘A novel 5‐enolpyruvoylshikimate‐3‐phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (<i><scp>O</scp>ryza sativa</i>) without herbicide’ by Wang <i>et al</i>. (2014)

Neurotrophic Signaling Factors in Brain Ischemia/Reperfusion Rats: Differential Modulation Pattern between Single-Time and Multiple Electroacupuncture Stimulation.

Electroacupuncture (EA) treatment has been widely used for stroke-like disorders in traditional Chinese medicine. However, the underlying mechanisms remain unclear. Our previous studies showed that single-time EA stimulation at “Baihui” (GV 20) and “Shuigou” (GV 26) after the onset of ischemia can protect the brain against ischemic injury in rats with middle cerebral artery occlusion (MCAO). Here, we further investigated the differential effects between multiple EA and single-time EA stimulation on ischemic injury. In the present study, we found that both single-time EA and multiple EA stimulation significantly reduced MCAO-induced ischemic infarction, while only multiple EA attenuated sensorimotor dysfunctions. Also, with PCR array screening and ingenuity gene analysis, we revealed that multiple EA and single-time EA stimulation could differentially induce expression changes in neurotrophic signaling related genes. Meanwhile, with western blotting, we demonstrated that the level of glia maturation factor β (GMFβ) increased in the early stage (day 1) of reperfusion, and this upregulation was suppressed only by single-time EA stimulation. These findings suggest that the short-term effect of single-time EA stimulation differs from the cumulative effect of multiple EA, which possibly depends on their differential modulation on neurotrophic signaling molecules expression.

Benzo[a]pyrene increases DNA double strand break repair in vitro and in vivo: A possible mechanism for benzo[a]pyrene-induced toxicity

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon and carcinogen that is released into the environment through natural and anthropogenic sources. BaP toxicity is dependent on its metabolism by cytochrome P450s to the reactive metabolite benzo[a]pyrene diol epoxide (BPDE), which is strongly associated with increased mutation frequency. BaP can also be metabolized to benzo[a]pyrene quinones that can undergo redox cycling and induce oxidative stress. The purpose of this study was to examine if BaP exposure induces DNA double strand breaks (DSBs) and subsequently activate DNA DSB repair pathways in the CHO 3–6 cell line and pKZ1 mouse model. In vitro assessment of homologous recombination (HR) showed significantly increased HR frequency following exposure to 10μM of BaP. In vivo evaluations of BaP-induced DNA DSB repair demonstrated positive staining for intrachromosomal recombination events, which are associated with non-homologous end joining (NHEJ), in the lung and thymus of exposed animals that were statistically significant in the thymus when quantified by Western blotting. Gene expression analyses from mouse tissues showed significantly decreased expression of ATM and Xrcc6 in BaP-treated liver and lung. In addition, BaP exposure significantly reduced the expression of Xrcc5, p53, and DNA-PKcs in lung. Taken together, our results demonstrate that BaP increases DNA DSB repair in vitro and in vivo, and induces expression changes in DNA repair pathway genes. As repair of DNA DSBs is not error-free, aberrant DNA repair may be contributing to the mechanism of BaP-induced toxicity.

Cytarabine-Induced Gene Expression Signatures in AML Patients and Its Association with Clinical Outcome.

AbstractAbstract 2470Acute myeloid leukemia (AML) is the second most common form of childhood leukemia and has the one of the worst prognoses of all major childhood cancers. Development of resistance to the nucleoside analog cytarabine (ara-C) is one of the biggest challenges that clinicians face. Studies of diagnostic gene expression profiles have attempted to determine the molecular mechanisms that underlie drug resistance. In the present study, we evaluated cytarabine-induced gene expression changes in n=24 AML patients by using the Affymetrix U133A array. We measured gene expression in bone marrow samples obtained at diagnosis and 24 hours after initiation of cytarabine infusion. We identified genes with significant change in expression post cytarabine treatment (after adjusting for arm) and explored the association of cytarabine induced expression changes with minimal residual disease after one course (MRD), and event-free survival. We found significant change in the expression level of 11 genes at p≤0.001 (6 genes down-regulated and 5 up-regulated) and 146 with p≤0.01 (89 genes down-regulated and 57 up-regulated). Pathway analysis tool (IPA) identified Cancer, Tumor Morphology, Cellular Movement (score 32) and Cell Morphology, Nervous System Development and Function, Tissue Morphology (score 29) as top networks. The most significant pathways influenced by ara-C treatment included NRF2-mediated oxidative stress signaling, Hif 1a signaling, Aryl hydrocarbon receptor and acute phase signaling. The top genes with ∼ 3 fold increase in expression included VAX2, PMP22, GMFG, PTP4A1, TIMP3, CHMP2A, GPR56, NFIB and KLRB1, and top genes with ∼ 3 fold decrease in expression included CER1, PREB, CDK13 and CDK10, UVRAG, FGF17, ZNF768, YLPM1, SIK3, PRICKLE3, MAP3K7.We also identified 34 genes whose expression change was significantly associated (p<0.001) with clinically beneficial or detrimental pattern of associations using MRD and EFS as clinical endpoints. Additionally 292 genes showed such a pattern of associations at p≤0.01. Analysis of these genes by Ingenuity Pathway identified Cancer, Tissue Development, Connective Tissue Disorders networks (score 43) and DNA Replication, Recombination, and Repair, Nucleic Acid Metabolism, Small Molecule Biochemistry (score 34) as top two networks. Interestingly of the top 34 gene expression changes (p<0.001), all but one gene demonstrated induction in expression post ara-C. Some of the most interesting genes with increase in expression (post ara-C vs. baseline) that were associated with clinical outcome included transcriptional regulators as Runx-related transcription factors RUNX2, and RUNX1T1, which plays role in BMP and TGF-b signaling pathways; NFKB2, which plays role in apoptosis, cell proliferation, differentiation and survival; SMARCA1, which is involved in cell differentiation and remodeling; MKL1 (megakaryoblastic leukemia (translocation) 1), which has been implicated in M7 AML and homeo box proteins HOXC4, HOXD9 and kinases such as.Overall our results provide insight into cytarabine-induced gene expression differences in AML patients and the association of these expression differences with clinical outcome. Understanding the pathways that are altered in patients once treatment is initiated can provide opportunities to enhance our understanding of drug action and further improve treatment outcome by adjusting the combination therapies. Disclosures:No relevant conflicts of interest to declare.

Is chemically dispersed oil more toxic to Atlantic cod (Gadus morhua) larvae than mechanically dispersed oil? A transcriptional evaluation

BackgroundThe use of dispersants can be an effective way to deal with acute oil spills to limit environmental damage, however very little is known about whether chemically dispersed oil have the same toxic effect on marine organisms as mechanically dispersed oil. We exposed Atlantic cod larvae to chemically and mechanically dispersed oil for four days during the first-feeding stage of development, and collected larvae at 14 days post hatch for transcriptional analysis. A genome-wide microarray was used to screen for effects and to assess whether molecular responses to chemically and mechanically dispersed oil were similar, given the same exposure to oil (droplet distribution and concentration) with and without the addition of a chemical dispersant (Dasic NS).ResultsMechanically dispersed oil induced expression changes in almost three times as many transcripts compared to chemically dispersed oil (fold change >+/−1.5). Functional analyses suggest that chemically dispersed oil affects partly different pathways than mechanically dispersed oil. By comparing the alteration in gene transcription in cod larvae exposed to the highest concentrations of either chemically or mechanically dispersed oil directly, the chemically dispersed oil affected transcription of genes involved nucleosome regulation, i.e. genes encoding proteins participating in DNA replication and chromatin formation and regulation of cell proliferation, whereas the mechanically dispersed oil most strongly affected genes encoding proteins involved in proteasome-mediated protein degradation. Cyp1a was the transcript that was most strongly affected in both exposure groups, with a 60-fold induction in the two high-exposure groups according to the RT-qPCR data, but no significant difference in transcriptional levels was observed between the two treatments.ConclusionsIn summary, dispersants do not appear to add to the magnitude of transcriptional responses of oil compounds but rather appear to lower or modify the transcriptional effect on cod larvae.

ERK signaling pathway mediated epithelial-mesenchymal transition induced by SiO₂ in human bronchial epithelial cells

To determine the role of extracellular signal regulated kinase (ERK) signaling pathway in SiO₂ induced epithelial-mesenchymal transition (EMT) in human bronchial epithelial cells (HBEC) in vitro. HBEC were treated with SiO₂ (0-300 μg/mL) for 72 h or pretreated with U0126 (0-30 μmol/L) for 1 h and then treated with 200 μg/mL SiO₂ for 72 h. Western blot was used to detect the protein expression of E-cadherin and α-smooth muscle actin (α-SMA). The activity of ERK was examined by mitogen-activated protein kinase (MAPK) activity assay kit in HBEC exposing to SiO₂ (200 μg/mL) for 0-8 h. The expression of E-cadherin decreased gradually in SiO₂ -stimulated HBEC, and the effect was most significant at 300 μg/mL (P<0.01). The expression of α-SMA increased and the effect was most evident at 200 μg/mL (P<0.01). With SiO₂ treatment, the activity of ERK was upregulated significantly. The phosphorylation of ERK increased at 30 min and decreased after 1 h. U0126 significantly inhibited SiO₂ -induced expression changes in E-cadherin and α-SMA. At 30 μmol/L, the effect was most evident(P<0.01). ERK signaling pathway mediated EMT induced by SiO₂ in HBEC.

NMDA Mediated Contextual Conditioning Changes miRNA Expression

We measured the expression of 187 miRNAs using quantitative real time PCR in the hippocampal CA1 region of contextually conditioned mice and cultured embryonic rat hippocampal neurons after neuronal stimulation with either NMDA or bicuculline. Many of the changes in miRNA expression after these three types of stimulation were similar. Surprisingly, the expression level of half of the 187 measured miRNAs was changed in response to contextual conditioning in an NMDA receptor-dependent manner. Genes that control miRNA biogenesis and components of the RISC also exhibited activity induced expression changes and are likely to contribute to the widespread changes in the miRNA profile. The widespread changes in miRNA expression are consistent with the finding that genes up-regulated by contextual conditioning have longer 3′ UTRs and more predicted binding sites for miRNAs. Among the miRNAs that changed their expression after contextual conditioning, several inhibit inhibitors of the mTOR pathway. These findings point to a role for miRNAs in learning and memory that includes mTOR-dependent modulation of protein synthesis.

Abstract 2179: Bile acids downregulate the squamous differentiation program of esophageal cells

Abstract Background: The lumen of the normal esophagus is covered by a nonkeratinized, stratified squamous epithelium. However, in individuals with prolonged gastroesophageal reflux disease the squamous epithelium is gradually replaced by a more acid-resistant metaplastic columnar epithelium known as Barrett's Esophagus (BE). BE is the strongest known risk factor for development of esophageal adenocarcinoma. Understanding the molecular mechanisms underlying the formation of BE is fundamental to the development of new and effective prevention and treatment strategies for patients with BE. Methods: We performed a global analysis of gene expression in normal squamous esophageal cells in response to bile acids or acid exposure. Differentially expressed genes were classified into major biological functions based on analysis using pathway and interaction network software programs. Array data were verified by quantitative PCR and western blot both in-vitro and in human biopsies from esophageal normal squamous and columnar epithelia. Results: Bile acids modulated expression of 218 genes (153 up and 65 down) and acid 112 genes (31 up and 81 down). Interestingly, genes involved in squamous differentiation formed the largest functional group of differentially expressed genes and consisted of 40 genes downregulated by bile acids exposure. Bile acids decreased expression of key squamous differentiation genes such as KRT10, KRTDAP and GRHL1 which was confirmed at both the mRNA and protein level as was increased expression of CDX2, a transcription factor key to the intestinal epithelial phenotype. Bile acids induced expression changes were also seen in genes involved in the pathways of cell adhesion, DNA repair, oxidative stress, cell cycle, Wnt signaling and lipid metabolism. Expression of DSG1, a target gene of GRHL1, and DKK1, a Wnt inhibitor, were confirmed to be respectively downregulated and upregulated by bile acids. Further, expression data from human columnar epithelial biopsies revealed low or no expression of KRT10, KRTDAP, DSG1 and GRHL1 compared to squamous epithelia. Finally, network analysis suggested that bile acids exposure modulated the transcription activity of CREB and bile acids induced phosphorylation of CREB was confirmed experimentally. Conclusion: We report for the first time that bile acids inhibit the squamous differentiation program of esophageal cells while inducing columnar differentiation via CDX2 expression possibly via CREB and Wnt signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2179. doi:10.1158/1538-7445.AM2011-2179

Abstract LB-331: Protein expression signatures of epidermal growth factor receptor inhibition

Abstract We have applied standardized shotgun and multiple reaction monitoring (MRM) proteomic platforms to detect protein expression changes associated with epidermal growth factor receptor (EGFR) stimulation and inhibition in A431 carcinoma cells. These experiments compared proliferating, stimulated (30 nmol EGF, 4h) with cells co-treated with EGF and either cetuximab (10 μg/ml) or gefitinib (500 nmol). The analyses are done by shotgun proteomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS), using isoelectric focusing of peptide mixtures, followed by reverse phase LC-MS/MS on a Thermo LTQ instrument. Protein inventories (≥2 peptides per protein at 5% peptide FDR) for each set were approximately 3800 proteins at &amp;lt; 3% protein FDR. Differential protein expression was estimated by comparison of spectral count data with a generalized linear modeling (GLM) approach. EGF stimulation induced expression changes in 148 proteins. Effects of the inhibitors on this “stimulation signature” were then determined by GLM comparisons of spectral count data for cetuximab- and gefitinib-treated cell proteomes. These analyses identified 13 proteins whose EGF-induced expression was reversed by both drugs, 15 proteins reversed by cetuximab and 57 proteins reversed by gefitinib. Targeted MRM analysis verified differential expression of the 13 proteins reversed by both drugs, which comprise an “EGF inhibition signature”. These include the transcription factors c-Jun and JunD and the cell cycle inhibitor Cip1, which were upregulated by EGF and the protein CCDC50, which was decreased by EGF. MRM analyses also identified EGF-induced protein expression changes that were selectively reversed by either cetuximab or gefitinib. Serum deprivation, rather than EGF stimulation may account for a subset of the 106 protein changes that were not reversed by inhibitors in EGF treated cells. Our analyses also identified 87 and 149 proteins that were not affected by EGF, yet were significantly altered by cetuximab and gefitinib, respectively. Activation of the EGFR axis is typically measured via the phosphorylation status of EGFR tyrosine kinase substrates and downstream targets, but these measurements are difficult to achieve in tissues due to the low abundance of phosphorylated protein forms and the challenge of preserving phosphorylation status during sample preparation. Protein expression changes directed by EGFR activation may provide more robust measures of EGFR activation and inhibition. Ongoing studies will test the hypothesis that an EGF inhibition signature provides an early index of therapeutic responses to EGFR-targeted cancer therapies. (Supported by NIH Grant U54CA126479) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-331. doi:10.1158/1538-7445.AM2011-LB-331