Confirmed Gene Expression Changes Research Articles

Hyponatremia elicits gene expression changes driving osteoclast differentiation and functions

Growing evidence indicates that chronic hyponatremia represents a significant risk for bone loss, osteoporosis, and fractures in our aging population. Our prior studies on a rat model of the syndrome of inappropriate antidiuretic hormone secretion indicated that chronic hyponatremia causes osteoporosis by increasing osteoclastic bone resorption, thereby liberating stored sodium from bone. Moreover, studies in RAW264.7 pre-osteoclastic cells showed increased osteoclast formation and resorptive activity in response to low extracellular fluid sodium ion concentration (low [Na+]). These studies implicated a direct stimulatory effect of low [Na+] rather than the low osmolality on cultured osteoclastic cells. In the present cellular studies, we explored gene expression changes triggered by low [Na+] using RNA sequencing and gene ontology analysis. Results were confirmed by mouse whole genome microarray, and quantitative RT-PCR. Findings confirmed gene expression changes supporting osteoclast growth and differentiation through stimulation of receptor activator of nuclear factor kappa-B ligand (RANKL), and PI3K/Akt pathways, and revealed additional pathways. New findings on low [Na+]-induced upregulation of lysosomal genes, mitochondrial energy production, MMP-9 expression, and osteoclast motility have supported the significance of osteoclast transcriptomic responses. Functional assays demonstrated that RANL and low [Na+] independently enhance osteoclast functions. Understanding the molecular mechanisms of hyponatremia-induced osteoporosis provides the basis for future studies identifying sodium-sensing mechanisms in osteoclasts, and potentially other bone cells, and developing strategies for treatment of bone fragility in the vulnerable aging population most affected by both chronic hyponatremia and osteoporosis. Issue sectionsSignaling Pathways; Parathyroid, Bone, and Mineral Metabolism.

Activating transcription factor-2 (ATF2) is a key determinant of resistance to endocrine treatment in an in vitro model of breast cancer

BackgroundActivating transcription factor-2 (ATF2), a member of the leucine zipper family of DNA binding proteins, has been implicated as a tumour suppressor in breast cancer. However, its exact role in breast cancer endocrine resistance is still unclear. We have previously shown that silencing of ATF2 leads to a loss in the growth-inhibitory effects of tamoxifen in the oestrogen receptor (ER)-positive, tamoxifen-sensitive MCF7 cell line and highlighted that this multi-faceted transcription factor is key to the effects of tamoxifen in an endocrine sensitive model. In this work, we explored further the in vitro role of ATF2 in defining the resistance to endocrine treatment.Materials and methodsWe knocked down ATF2 in TAMR, LCC2 and LCC9 tamoxifen-resistant breast cancer cell lines as well as the parental tamoxifen sensitive MCF7 cell line and investigated the effects on growth, colony formation and cell migration. We also performed a microarray gene expression profiling (Illumina Human HT12_v4) to explore alterations in gene expression between MCF7 and TAMRs after ATF2 silencing and confirmed gene expression changes by quantitative RT-PCR.ResultsBy silencing ATF2, we observed a significant growth reduction of TAMR, LCC2 and LCC9 with no such effect observed with the parental MCF7 cells. ATF2 silencing was also associated with a significant inhibition of TAMR, LCC2 and LCC9 cell migration and colony formation. Interestingly, knockdown of ATF2 enhanced the levels of ER and ER-regulated genes, TFF1, GREB1, NCOA3 and PGR, in TAMR cells both at RNA and protein levels. Microarray gene expression identified a number of genes known to mediate tamoxifen resistance, to be differentially regulated by ATF2 in TAMR in relation to the parental MCF7 cells. Moreover, differential pathway analysis confirmed enhanced ER activity after ATF2 knockdown in TAMR cells.ConclusionThese data demonstrate that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity.

GENE-10. A SIMPLE AND EFFICIENT METHOD FOR GENERATING MULTI-ALLELIC INSERTION-DELETION MUTATIONS AND PRECISE DELETIONS IN PRIMARY HUMAN CELL CULTURES

Abstract CRISPR-Cas9-based technologies have revolutionized experimental manipulation of the human genome by enabling generation of site-specific genetic alterations, such as insertion-deletion (indel) mutations. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary human cells. Here, we present a simple and effective method for fast and penetrant induction of multi-allelic indels and near-precise deletions (ranging from ~50bp to >50kbp) in primary human neural stem/progenitor cell (NPC) and brain tumor-derived stem-like cell cultures using the CRISPR-Cas9 system. We report that mono-, bi-, or multi-allelic (depending on ploidy) indel efficiencies of >90% can be routinely achieved within 3 days, without the need for pre-engineering cells to express Cas9 or isolating clones. As a result, this method enables the quick generation of a series of knockouts at different genomic loci, which we illustrate by successively targeting TP53, CDKN2A, PTEN, and NF1 in human NPC populations, where ³95% loss of protein expression is observed. RNA-seq analysis of these NPC knockout pools confirmed gene expression changes including p53 transcriptional and Rb-axis targets, as well as NF1-dependent repression of major histocompatibility complex class II gene expression. Given its simplicity, this method is readily adaptable to other primary mammalian cell types.

Inflammation potentiates miR-939 expression and packaging into small extracellular vesicles

ABSTRACTExtracellular RNA in circulation mediates intercellular communication in normal and pathological processes. One mode of circulating miRNA transport in bodily fluids is within 30–150 nm small extracellular vesicles (sEVs) or exosomes. Uptake of sEVs can regulate gene expression in recipient cells enabling circulating miRNAs to exert paracrine and systemic effects. Complex regional pain syndrome (CRPS) is a debilitating pain disorder characterized by chronic inflammation. Our previous investigations identified a significant decrease of hsa-miR-939 in whole blood from CRPS patients compared to control; we also observed that overexpression of miR-939 can negatively regulate several proinflammatory genes in vitro. Though downregulated in whole blood, miR-939 was significantly upregulated in sEVs isolated from patient serum. Here we investigated miR-939 packaging into sEVs in vitro under inflammation induced by monocyte chemoattractant protein-1 (MCP-1), a chemokine that is upregulated in CRPS patients. Stimulation of THP-1 monocytes by MCP-1 led to elevated levels of miR-939 in sEVs, which was abrogated using inhibitors of exosome secretion. miRNAs loaded into exosomes largely contain short miRNA sequence motifs called EXOmotifs. Mutation analysis of miR-939 showed that EXOmotif is one of the possible cellular mechanisms responsible for packaging miR-939 into sEVs. We confirmed gene expression changes in recipient cells following the uptake of sEVs enriched in miR-939 using RNA sequencing. Additionally, our data from primary immune cell-derived sEVs of CRPS patients and controls demonstrate that while the relative expression of miR-939 is higher in sEVs derived from B cells, T cells and NK cells relative to monocyte-derived sEVs in controls, only the B cell-derived sEVs showed a significantly higher level of miR-939 in CRPS patients. Differential miRNA sorting into exosomes and its functional impact on recipient cells may contribute to the underlying pathophysiology of CRPS.

Components of the endocannabinoid and dopamine systems are dysregulated in Huntington's disease: analysis of publicly available microarray datasets.

The endocannabinoid system (ECS) and the dopaminergic system (DAS) are two major regulators of basal ganglia function. During Huntington's disease (HD) pathogenesis, the expression of genes in both the ECS and DAS is dysregulated. The purpose of this study was to determine the changes that were consistently observed in the ECS and DAS during HD progression in the central nervous system (CNS) and in the periphery in different models of HD and human HD tissue. To do this, we conducted a meta-analysis of differential gene expression in the ECS and DAS using publicly available microarray data. The consolidated data were summarized as observed changes in gene expression (OCGE) using a weighted sum for each gene. In addition, consolidated data were compared to previously published studies that were not available in the gene expression omnibus (GEO) database. The resulting data confirm gene expression changes observed using different approaches and provide novel insights into the consistency between changes observed in human tissue and various models, as well as disease stage- and tissue-specific transcriptional dysregulation in HD. The major implication of the systems-wide data presented here is that therapeutic strategies targeting the ECS or DAS must consider the dynamic changes in gene expression over time and in different body areas, which occur during HD progression and the interconnectedness of the two systems.

Molecular Profiling of Premalignant Lesions in Lung Squamous Cell Carcinomas Identifies Mechanisms Involved in Stepwise Carcinogenesis

Lung squamous cell carcinoma (SCC) is thought to arise from premalignant lesions in the airway epithelium; therefore, studying these lesions is critical for understanding lung carcinogenesis. Previous microarray and sequencing studies designed to discover early biomarkers and therapeutic targets for lung SCC had limited success identifying key driver events in lung carcinogenesis, mostly due to the cellular heterogeneity of patient samples examined and the interindividual variability associated with difficult to obtain airway premalignant lesions and appropriate normal control samples within the same patient. We performed RNA sequencing on laser-microdissected representative cell populations along the SCC pathologic continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. This is the first gene expression profiling study of airway premalignant lesions with patient-matched SCC tumor samples. Our results provide much needed information about the biology of premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of SCC, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients.

Altered brain gene expression but not steroid biochemistry in a genetic mouse model of neurodevelopmental disorder

BackgroundThe 39,XY*O mouse, which lacks the orthologues of the ADHD and autism candidate genes STS (steroid sulphatase) and ASMT (acetylserotonin O-methyltransferase), exhibits behavioural phenotypes relevant to developmental disorders. The neurobiology underlying these phenotypes is unclear, although there is evidence for serotonergic abnormalities in the striatum and hippocampus.MethodsUsing microarray and quantitative gene expression analyses, and gas chromatography–mass spectrometry, we compared brain gene expression and steroid biochemistry in wildtype (40,XY) and 39,XY*O adult mice to identify non-obvious genetic and endocrine candidates for between-group differences in behaviour and neurochemistry. We also tested whether acute STS inhibition by COUMATE in wildtype (40,XY) adult male mice recapitulated any significant gene expression or biochemical findings from the genetic comparison. Data were analysed by unpaired t-test or Mann Whitney U-test depending on normality, with a single factor of KARYOTYPE.ResultsMicroarray analysis indicated seven robust gene expression differences between the two groups (Vmn2r86, Sfi1, Pisd-ps1, Tagap1, C1qc, Metap1d, Erdr1); Erdr1 and C1qc expression was significantly reduced in the 39,XY*O striatum and hippocampus, whilst the expression of Dhcr7 (encoding 7-dehydrocholesterol reductase, a modulator of serotonin system development), was only reduced in the 39,XY*O hippocampus. None of the confirmed gene expression changes could be recapitulated by COUMATE administration. We detected ten free, and two sulphated steroids in 40,XY and 39,XY*O brain; surprisingly, the concentrations of all of these were equivalent between groups.ConclusionsOur data demonstrate that the mutation in 39,XY*O mice: i) directly disrupts expression of the adjacent Erdr1 gene, ii) induces a remarkably limited suite of downstream gene expression changes developmentally, with several of relevance to associated neurobehavioural phenotypes and iii) does not elicit large changes in brain steroid biochemistry. It is possible that individuals with STS/ASMT deficiency exhibit a similarly specific pattern of gene expression changes to the 39,XY*O mouse, and that these contribute towards their abnormal neurobiology. Future work may focus on whether complement pathway function, mitochondrial metabolism and cholesterol biosynthesis pathways are perturbed in such subjects.

P1-03-02: The Norton-Simon Hypothesis and Cancer Stem Cells: How Cancer Stem Cells May Explain the Effectiveness of Dose-Dense Chemotherapy.

Abstract Background: Systemic therapies are effective initially in controlling and reversing tumor growth; however, residual cancers will invariably re-grow despite this initial response. We have published data from paired human breast cancer samples that standard therapy every three weeks kills dividing daughter cells but not tumor-initiating cells (TICs), so that samples obtained after therapy are enriched for CD44+/CD24−/low putative “tumor-initiating” or “cancer stem” cells, indicating that standard treatment regimens are missing the critical targets, TICs. Interestingly, we have recent data in human breast tumors that indicate that TICs may in fact be chemosensitive initially, with a decrease in TICs observed within two days of chemotherapy, but shortly thereafter, TICS are actually induced by chemotherapy. Materials and Methods: To evaluate TICs response to chemotherapy, mice with human breast tumor xenograft lines BCM-2665a and BCM-2147 were treated with vehicle, 10-, or 33-mg/kg docetaxel, and then tumors were collected for TIC assays and molecular analysis at both 48 and 72h after treatment. Using Affymetrix gene expression microarrays and reverse phase protein array (RPPA) analysis with 119 different validated antibodies, we identified pathways involved in regulation of TICs. Results: According to flow cytometric analysis for TIC markers and mammosphere (MS) formation efficiency, BCM-2665 TICs were reduced by docetaxel treatment compared to vehicle-treated at 48h post 10 mg/kg docetaxel (4-fold decrease) and 48 and 72h post 33 mg/kg docetaxel compared to control (14- and 2-fold decrease, respectively). Although the BCM-2147 TICs did not significantly decrease at any time points or doses tested, they were clearly induced at 72h post-treatment compared to control. Additionally, BCM-2665 TICs were increased within 72h post 10 mg/kg docetaxel, indicating that these time points are ideal for defining the mechanisms responsible for induction of TICs. Ingenuity Pathway Analysis of Affymetrix microarray data for both BCM-2665 and BCM-2147 revealed induction of inflammatory pathways, suggesting leukocyte infiltration associated with induction of TICs. Furthermore, RPPA analysis confirmed gene expression changes from the microarray data, and implicated apoptosis and inflammatory pathways. Sixteen of 28 proteins significantly changed with activation of CSC are involved in development of leukocytes. Discussion: These findings are consistent with the Norton-Simon Hypothesis in that chemotherapy regimens given more frequently may in fact eliminate TICs, thereby explaining the proven increased effectiveness of dose-dense chemotherapy. Based on when TICs became chemoresistant, we are comparing dose-dense treatment (4 mg/kg docetaxel every 3 days) to a traditional single dose of 32 mg/kg, in an effort to eliminate the tumor cells that cause tumor recurrence. Furthermore, our analysis of gene expression at both the RNA and protein level implicated the immune cells as TICs inducers. Since our immunocompromised mice lack T- and B- cells but have active macrophages, macrophages are indicated as inducers of TICs. We are focusing our current efforts at identifying how immune cells activate TICs and thus enhance tumorigenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-02.

Abstract 3915: α-Fetoprotein-producing hepatoma cells have increased Jagged1 gene copy numbers and their growth can be suppressed by gamma secretase inhibitor

Abstract Background: Approximately 70% of patients with hepatocellular carcinoma (HCC) have abnormally high α-fetoprotein (AFP) levels, which increase with tumor progression and angiogenesis. We previously used cDNA microarray comparative genomic hybridization (aCGH) to demonstrate an association between hepatoma AFP-producing status and genomic copy number gain and loss. Although genomic copy number change is reported to be associated with some oncogenic factors, its relationship with molecular pathway aberration has not been elucidated. Here, we examined whether signal pathways are associated with both expression and genomic gain for the purpose of discovering clinically useful molecular targets in hepatoma patients. Method: Changes in DNA copy number of hepatoma cells were analyzed using bacterial artificial chromosome (BAC)-aCGH and compared with gene expression profiles. Candidate genes were identified, and real-time PCR was used to confirm gene expression changes in AFP-producing cells. siRNA was used to inhibit gene expression and the effect of this on hepatoma cell growth was determined. Confirmation of genomic alteration was performed by real-time PCR on clinical HCC samples. Growth of hepatoma cells was examined following the application of an inhibitor of the identified signal transduction-related gene. Results: The BAC clone was significantly amplified in some regions of chromosome 20p from hepatoma cells. This amplified region included the ligand for Notch signal transduction, Jagged1, which was shown to be up-regulated by microarray analysis and real-time PCR in AFP-producing hepatoma cells. Surgically treated AFP-up-regulated HCC samples were used to demonstrate the significant correlation between AFP and Jagged1 gene expression. Interestingly, tissue Jagged1 genomic gain and AFP expression were also correlated. The suppression of Jagged1 using siRNA in an AFP-producing cell line significantly repressed cell growth compared with an AFP-negative cell line. The inhibitor of the Notch pathway, gamma secretase inhibitor (GSI), is currently undergoing clinical trials for the treatment of Alzheimer's disease, and its effectiveness has been shown as a cancer therapy for solid tumors. Here, continuous application of GSI to hepatoma cells resulted in a significant suppression of cell growth. Conclusion: The genomic amplification of Jagged1 contributes to the continuous mRNA expression that activates the Jagged1-Notch signaling pathway in hepatomas. GSI may be a promising candidate for use in the therapy of AFP-producing advanced HCC. 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 3915. doi:10.1158/1538-7445.AM2011-3915

Chronic stress and impaired glutamate function elicit a depressive-like phenotype and common changes in gene expression in the mouse frontal cortex

Major depression might originate from both environmental and genetic risk factors. The environmental chronic mild stress (CMS) model mimics some environmental factors contributing to human depression and induces anhedonia and helplessness. Mice heterozygous for the synaptic vesicle protein (SVP) vesicular glutamate transporter 1 (VGLUT1) have been proposed as a genetic model of deficient glutamate function linked to depressive-like behaviour. Here, we aimed to identify, in these two experimental models, gene expression changes in the frontal cortex, common to stress and impaired glutamate function.Both VGLUT1+/− and CMS mice showed helpless and anhedonic-like behavior. Microarray studies in VGLUT1+/− mice revealed regulation of genes involved in apoptosis, neurogenesis, synaptic transmission, protein metabolic process or learning and memory. In addition, RT-PCR studies confirmed gene expression changes in several glutamate, GABA, dopamine and serotonin neurotransmitter receptors. On the other hand, CMS affected the regulation of 147 transcripts, some of them involved in response to stress and oxidoreductase activity. Interestingly, 52 genes were similarly regulated in both models. Specifically, a dowregulation in genes that promote cell proliferation (Anapc7), cell growth (CsnK1g1), cell survival (Hdac3), and inhibition of apoptosis (Dido1) was observed. Genes linked to cytoskeleton (Hspg2, Invs), psychiatric disorders (Grin1, MapK12) or an antioxidant enzyme (Gpx2) were also downregulated. Moreover, genes that inhibit the MAPK pathways (Dusp14), stimulate oxidative metabolism (Eif4a2) and enhance glutamate transmission (Rab8b) were upregulated.We suggest that these genes could form part of the altered “molecular context” underlying depressive-like behaviour in animal models. The clinical relevance of these findings is discussed.

Abstract 1244: RXR agonist induced changes in gene expression in liver and mammary cancers: Confirmation by RT-PCR on independent samples collected years apart

Abstract Several years ago, our laboratries examined the effects of short-term treatment with the RXR agonist Targretin, a highly effective preventive agent in rodent mammary models, on gene expression. Oligonucleotide microarrays were employed both in normal tissues (liver, lung and mammary gland) and in ER+ mammary cancers (Wang, et al., Molecular Cancer Therapeutics, 2006). It was thought that the gene expression changes observed might serve as potential pharmacodynamic markers, as well as giving mechanistic insights into the efficacy of Targretin. Numerous changes in Targretin treated livers and literally hundreds of changes in Targretin treated mammary cancers were observed by microarray analysis. Pathway alterations in Targretin treated mammary cancers included those for MAPK and TGF-beta signaling. In the present RT-PCR studies, a separate laboratory employed different samples collected approximately four years after the originals. Roughly 20 genes whose expression was modulated in the original liver arrays and a similar number of genes from the original mammary cancer arrays were examined. Of the genes examined in the rat liver, which we expected to be relatively homogeneous, roughly 75% were confirmed by RT-PCR (e.g., cytosolic acyl-CoA thioesterase, CYP 2B2, IGFBP2). More surprisingly, approximately two-thirds of the gene changes observed in the rat mammary cancers (e.g., MMP12, apolipoprotein D, CYP4B1) were confirmed. These results imply that even in different samples collected years apart one can confirm gene expression changes observed in arrays in a relatively homogeneous tissue such as liver. Furthermore, one can confirm gene expression changes observed in mammary cancers, a result potentially more surprising since one might expect substantial heterogeneity in those samples. We are also currently examining expression changes of these same genes following treatment with a series of RXR agonists that are structurally dissimilar to Targretin. Targretin is a cyclic compound, while the analogs have a linear 9-cis-backbone (Muccio et al, Carcinogenesis, 2006). In conclusion, the present data support the use of arrays to identify pharmacodynamic markers that may be utilized with agents of the same class. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1244.

Abstract 1643: Withaferin A analogues up-regulate the MAPK and glutathione metabolic pathways prior to activation of apoptosis in pancreatic cancer cells

Abstract Withaferin A is a natural product known to possess anti-cancer activity. Here we studied the effect of Withaferin A and 11 of its analogs on differentiation state-specific pancreatic cancer cell lines. Three pancreatic cell lines Panc-1 (poorly-differentiated), MiaPaca-2 (moderately differentiated) and BxPC-3 (well differentiated) were treated with 11 analogs and cell proliferation was examined by MTS assays. The IC50 of these 11 analogs were <4.0 µM suggesting they have growth inhibitory effects on pancreatic cancer cell proliferation. Two of these 11 analogs, named KW-120-94-1 and KW-120-95-1 were selected for further study. The IC50s of KW-120-94-1 and KW-120-95-1 were 2.25, 0.56 µM (Panc-1); 1.53, 0.52 µM (MiaPaca-2) and 0.45, 0.29 µM (BxPC-3) respectively. Apoptosis was analyzed by morphology (Acridine Orange and Ethidium Bromide staining), flow cytometry (Annexin V-FITC and Propidium Iodide staining) and PARP cleavage. The data showed that apoptosis was induced both in BXPC-3 and MiaPaca-2 cells in a dose-dependent manner. However, Panc-1 cells were in general resistant to apoptosis. In BxPC-3 cells apoptosis was induced as early as 6 hours after KW-120-95-1 treatment at 1.5µM. To determine the Withaferin A analog dependent ‘tumor profile signature’, gene expression profiling by Affymetrix microarrays were performed in the 3 pancreas cancer cell lines. The cells were treated with KW-120-95-1 for 8 hr at 1.5µM. Differential gene expression was analyzed by ANOVA which identified 33 (MiaPaca-2), 54 (Panc-1) and 48 (BXPC-3) gene changes. The Fisher exact test demonstrated that MAPK signaling and glutathione metabolism pathways were over-expressed within the genes that changed with treatment. Western blotting and real-time RT-PCR were utilized to confirm gene expression changes identified in the MAPK pathway (HSPA1A/HSPA1B, c-Jun) and glutathione metabolism pathway (GCLC, GPX2 and GPX3). Taken together, our data indicate that Withaferin A analogs inhibit pancreatic cancer proliferation and induces apoptosis by activating the MAPK and glutathione (stress) pathways. The molecular target(s) of Withaferin A and analogs are likely to function by activating the MAPK signaling and glutathione metabolism pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1643.

Gene expression changes in the medial prefrontal cortex and nucleus accumbens following abstinence from cocaine self-administration

BackgroundMany studies of cocaine-responsive gene expression have focused on changes occurring during cocaine exposure, but few studies have examined the persistence of these changes with cocaine abstinence. Persistent changes in gene expression, as well as alterations induced during abstinence may underlie long-lasting drug craving and relapse liability.ResultsWhole-genome expression analysis was conducted on a rat cocaine binge-abstinence model that has previously been demonstrated to engender increased drug seeking and taking with abstinence. Gene expression changes in two mesolimbic terminal fields (mPFC and NAc) were identified in a comparison of cocaine-naïve rats with rats after 10 days of cocaine self-administration followed by 1, 10, or 100 days of enforced abstinence (n = 6-11 per group). A total of 1,461 genes in the mPFC and 414 genes in the NAc were altered between at least two time points (ANOVA, p < 0.05; ± 1.4 fold-change). These genes can be subdivided into: 1) changes with cocaine self-administration that do not persist into periods of abstinence, 2) changes with cocaine self-administration that persist with abstinence, 3) and those not changed with cocaine self-administration, but changed during enforced abstinence. qPCR analysis was conducted to confirm gene expression changes observed in the microarray analysis.ConclusionsTogether, these changes help to illuminate processes and networks involved in abstinence-induced behaviors, including synaptic plasticity, MAPK signaling, and TNF signaling.

Coregulation of genes in the mouse brain following treatment with clozapine, haloperidol, or olanzapine implicates altered potassium channel subunit expression in the mechanism of antipsychotic drug action

Antipsychotic drugs are the most effective treatment for the psychotic symptoms of schizophrenia, yet their mechanism of action remains largely unknown. Earlier studies have shown gene expression changes in rodent brains after treatment with antipsychotic drugs. We aimed to further characterize these changes using whole-genome transcript profiling to explore coregulation of genes after multiple antipsychotic drug treatment studies. This study involved transcript profile analysis after 7-day treatment of inbred C57BL/6 mice with conventional (haloperidol) or atypical (clozapine or olanzapine) antipsychotic drugs. Microarray analysis was undertaken using whole-brain mRNA on Affymetrix 430v2 arrays, with quantitative reverse transcriptase-PCR used to confirm gene expression changes. Western blotting was also used to explore translation of gene dysregulation to protein changes and to explore anatomical specificity of such changes. Thirteen genes showed verified regulation by multiple antipsychotic drugs - three genes significantly upregulated and 10 genes significantly downregulated by treatment. These genes encode proteins that function in various biological processes including neurogenesis, cell adhesion, and four genes are involved in voltage-gated ion channels: neural precursor cell developmentally downregulated gene 4 (Nedd4), Kv channel interacting protein 3 (KChip3), potassium voltage-gated channel, shaker-related subfamily, alpha1 (Kcna1) encoding Kv1.1 protein and beta1 (Kcnab1) encoding Kvbeta1 protein. The translation of these gene expression changes to protein dysregulation for Kv1.1, KCHIP3, and NEDD4 was confirmed by western blot, with regional protein analyses undertaken for Kv1.1 and KCHIP3. These results suggest that transcriptional regulation of ion channels, crucial for neurotransmission, may play a role in mediating antipsychotic drug effects.

Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response

BackgroundDespite advances in the understanding of diabetic retinopathy, the nature and time course of molecular changes in the retina with diabetes are incompletely described. This study characterized the functional and molecular phenotype of the retina with increasing durations of diabetes.ResultsUsing the streptozotocin-induced rat model of diabetes, levels of retinal permeability, caspase activity, and gene expression were examined after 1 and 3 months of diabetes. Gene expression changes were identified by whole genome microarray and confirmed by qPCR in the same set of animals as used in the microarray analyses and subsequently validated in independent sets of animals. Increased levels of vascular permeability and caspase-3 activity were observed at 3 months of diabetes, but not 1 month. Significantly more and larger magnitude gene expression changes were observed after 3 months than after 1 month of diabetes. Quantitative PCR validation of selected genes related to inflammation, microvasculature and neuronal function confirmed gene expression changes in multiple independent sets of animals.ConclusionThese changes in permeability, apoptosis, and gene expression provide further evidence of progressive retinal malfunction with increasing duration of diabetes. The specific gene expression changes confirmed in multiple sets of animals indicate that pro-inflammatory, anti-vascular barrier, and neurodegenerative changes occur in tandem with functional increases in apoptosis and vascular permeability. These responses are shared with the clinically documented inflammatory response in diabetic retinopathy suggesting that this model may be used to test anti-inflammatory therapeutics.

Transportation stress alters the circulating steroid environment and neutrophil gene expression in beef bulls

Stress and its association with altered immune function and incidence of respiratory diseases in cattle have lead to concerns over animal health and welfare during truck transportation. Previously, bulls subjected to transportation stress displayed altered expression of candidate neutrophil genes, warranting a broader investigation of the neutrophil transcriptome and possible associations with fluctuations in circulating steroid hormones. In the current study, blood was collected from six Belgian Blue × Friesian bulls at −24, 0, 4.5, 9.75, 14.25, 24, and 48 h relative to initiation of 9 h of truck transportation. Plasma concentrations of cortisol, dehydroepiandrosterone (DHEA), progesterone, and testosterone were measured; cortisol:DHEA ratios were computed. Neutrophil gene expression was monitored by microarray analysis using bovine immunobiology (BOTL-5) microarrays. Eighty-eight genes were identified as being differentially expressed at P < 0.05. Putatively affected genes were grouped into ontological clusters; those of greatest interest for qRT-PCR validation were involved in immune response, apoptosis, wound healing, and several of currently unknown function. Confirmed gene expression changes supported the dramatic effects of transportation stress on the bovine neutrophil transcriptome. Temporal correlations between gene expression profiles and circulating total leukocyte and neutrophil counts were apparent. However, few relationships between gene expression and plasma steroid profiles were detected, possibly due to the biological time-lag between these variables not captured by the blood collection schedule. Further investigation into the factors underlying neutrophil gene expression changes and validations at the protein and cell behavior levels will lead to a better understanding of altered innate immunity in cattle during transportation stress.

Gene expression profiling of initiated epidermal cells with benign or malignant tumor fates.

High-density oligonucleotide array technology was used to search for gene expression changes that may predict or cause sporadic tumorigenesis. To reduce variation among samples, a non-transformed mouse epidermal keratinocyte clone was compared with its carcinoma-producing or papilloma-producing 7,12-dimethylbenz[a]anthracene-initiated cell derivative. The majority of the approximately 12 325 genes or expressed sequence tags (ESTs) analyzed remained unaltered reflective of the clonal nature of the model. Consistent gene expression changes among biological replicates included 69 in malignancy-prone initiated cells, 46 in papilloma-precursor initiated cells and an additional 45 with changes in both initiated cell lineages. The changes covered a broad spectrum of cellular activities, implying that multiple pathways cooperate at the initiation of carcinogenesis. There was a tendency in the malignancy-prone cells for differences in proliferation and apoptosis-related genes. The benign lineage tended toward aberrant expression of genes involved in differentiation and epidermal barrier function. Several independently confirmed gene expression changes revealed plausible effectors to bypass an activated ras protein in proliferation pathways and to compromise the wild-type p53 and other apoptosis pathways at initiation of carcinogenesis. The differential expression patterns of the initiated cells are consistent with the hypothesis that changes in gene expression at initiation may cooperate with subsequent oncogenic changes to determine malignant fate. The cloned epidermal cell lineages allowed detection of putative early cancer genes in a model that is conducive to testing their direct role in epithelial multistep carcinogenesis in vitro and in vivo.

Differential Expression of Critical Cellular Genes in Human Lung Adenocarcinomas and Squamous Cell Carcinomas in Comparison to Normal Lung Tissues

The Atlas human cDNA expression array was used to evaluate gene expression profile changes in the genesis of human lung adenocarcinomas and squamous cell carcinomas. Gene expression changes between adenocarcinomas and squamous cell carcinomas were also analyzed. Of the 588 gene targets, 262 genes were expressed in these tissues and, of these, 45 genes were differentially expressed by at least two-fold in tumor tissues compared to corresponding normal tissues. Semiquantitative reverse-transcriptase polymerase chain reaction was used to confirm gene expression changes. Only those genes that reflected changes in >50% of the analyzed tissues were included in the final analysis. Ultimately, 26 genes were evaluated with 14 genes overexpressed and 12 genes underexpressed compared to matching normal lung tissues. Although similar expression changes were detected in adenocarcinomas and squamous cell carcinomas for most of the genes analyzed, some subtype-specific differences were also found. Genes encoding cell cycle regulators, intracellular signal transducers, cell receptor and adhesion molecules, growth factors, oncogenes, and apoptotic effectors were differentially expressed in this study. These gene expression changes may directly contribute to the initiation or progression of human lung cancer or may be secondary effects of the tumorigenesis process. Regardless, many of these differences may be useful in the diagnosis and/or treatment of this deadly disease.