Transcriptional recapitulation and subversion of embryonic colon development by mouse colon tumor models and human colon cancer.

BACKGROUND: The expression of carcino-embryonic antigen by colorectal cancer is an example of oncogenic activation of embryonic gene expression. Hypothesizing that oncogenesis-recapitulating-ontogenesis may represent a broad programmatic commitment, we compared gene expression patterns of human colorectal cancers (CRCs) and mouse colon tumor models to those of mouse colon development embryonic days 13.5-18.5. RESULTS: We report here that 39 colon tumors from four independent mouse models and 100 human CRCs encompassing all clinical stages shared a striking recapitulation of embryonic colon gene expression. Compared to normal adult colon, all mouse and human tumors over-expressed a large cluster of genes highly enriched for functional association to the control of cell cycle progression, proliferation, and migration, including those encoding MYC, AKT2, PLK1 and SPARC. Mouse tumors positive for nuclear beta-catenin shifted the shared embryonic pattern to that of early development. Human and mouse tumors differed from normal embryonic colon by their loss of expression modules enriched for tumor suppressors (EDNRB, HSPE, KIT and LSP1). Human CRC adenocarcinomas lost an additional suppressor module (IGFBP4, MAP4K1, PDGFRA, STAB1 and WNT4). Many human tumor samples also gained expression of a coordinately regulated module associated with advanced malignancy (ABCC1, FOXO3A, LIF, PIK3R1, PRNP, TNC, TIMP3 and VEGF). CONCLUSION: Cross-species, developmental, and multi-model gene expression patterning comparisons provide an integrated and versatile framework for definition of transcriptional programs associated with oncogenesis. This approach also provides a general method for identifying pattern-specific biomarkers and therapeutic targets. This delineation and categorization of developmental and non-developmental activator and suppressor gene modules can thus facilitate the formulation of sophisticated hypotheses to evaluate potential synergistic effects of targeting within- and between-modules for next-generation combinatorial therapeutics and improved mouse models.

GenomeTrafac: a whole genome resource for the detection of transcription factor binding site clusters associated with conventional and microRNA encoding genes conserved between mouse and human gene orthologs.

Transcriptional cis-regulatory control regions frequently are found within non-coding DNA segments conserved across multi-species gene orthologs. Adopting a systematic gene-centric pipeline approach, we report here the development of a web-accessible database resource--GenomeTraFac (http://genometrafac.cchmc.org)--that allows genome-wide detection and characterization of compositionally similar cis-clusters that occur in gene orthologs between any two genomes for both microRNA genes as well as conventional RNA-encoding genes. Each ortholog gene pair can be scanned to visualize overall conserved sequence regions, and within these, the relative density of conserved cis-element motif clusters form graph peak structures. The results of these analyses can be mined en masse to identify most frequently represented cis-motifs in a list of genes. The system also provides a method for rapid evaluation and visualization of gene model-consistency between orthologs, and facilitates consideration of the potential impact of sequence variation in conserved non-coding regions to impact complex cis-element structures. Using the mouse and human genomes via the NCBI Reference Sequence database and the Sanger Institute miRBase, the system demonstrated the ability to identify validated transcription factor targets within promoter and distal genomic regulatory regions of both conventional and microRNA genes.

Transcriptional response to persistent beta2-adrenergic receptor signaling reveals regulation of phospholamban, which alters airway contractility.

Beta(2)-Adrenergic receptors (beta(2)AR) are expressed on airway smooth muscle cells and act to relax the airway on activation by beta-agonists. These agents are utilized for treating asthma but are associated with adverse outcomes. To ascertain the effects of persistent beta(2)AR activation on gene expression, cultured airway smooth muscle cells derived from wild-type (WT) and transgenic mice overexpressing beta(2)AR were subjected to DNA microarray analysis; 319 genes were increased and 164 were decreased. Differential expression was observed in genes from 22 Gene Ontology Slim categories, including those associated with ion transport and calcium ion binding. A 60% decrease (P = 0.008) in phospholamban (PLN), an intracellular Ca(2+) concentration ([Ca(2+)]i)-handling protein that is at a signaling nodal point in cardiomyocytes, was observed in beta(2)AR-overexpressing cells and confirmed at the protein level. To isolate the physiological effect of decreased PLN in airway smooth muscle, airway contraction and relaxation responses were studied in WT and PLN(-/-) mice. PLN(-/-) mice had a markedly reduced constrictive response to methacholine. In contrast, the bronchodilatory effect of beta-agonist was not different between WT and PLN(-/-) mice. These results revealed an unanticipated therapeutic effect of beta-agonists, PLN downregulation, which acts to decrease airway hyperreactivity. Thus agents that inhibit PLN may act synergistically with the bronchodilating action of beta-agonists. A number of other genes related to [Ca(2+)]i are also differentially regulated by beta(2)AR activity, some of which may act to oppose, or augment, the efficacy of chronic beta-agonists. These genes or pathways may also represent additional targets in the treatment of asthma and related obstructive lung diseases.

Large-scale molecular comparison of human schwann cells to malignant peripheral nerve sheath tumor cell lines and tissues.

Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14(Arf), and p16(INK4a) proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100beta protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool.

Gene expression profiles of Mst1r-deficient mice during nickel-induced acute lung injury.

Previous studies have shown that mice deficient in the tyrosine kinase domain (TK-/-) of the receptor Mst1r have an increased susceptibility to nickel (Ni)-induced acute lung injury (ALI). Mst1r TK-/- mice have decreased survival times, alterations in cytokine and nitric oxide regulation, and an earlier onset of pulmonary pathology compared with control mice, suggesting that Mst1r signaling, in part, may regulate the response to ALI. To examine the role of Mst1r in ALI in more detail, we compared the gene expression profiles of murine lung mRNA from control and Mst1r TK-/- mice at baseline and after 24 h of particulate Ni sulfate exposure. Microarray analyses showed a total of 343 transcripts that were significantly changed, either by Ni treatment, or between genotypes. Genes responsible for inflammation, edema, and lymphocyte function were altered in the Mst1r TK-/- mice. Interestingly, the genes for several granzymes were increased in Mst1r TK-/- mice before Ni exposure, compared with controls. In addition, the Mst1r TK-/- lungs showed clusters of cells near the vascular endothelium and airways. Immunohistochemistry indicates these clusters are composed of macrophages, T cells, and neutrophils, and that the clusters display granzyme protein production. These results suggest that Mst1r signaling may be involved in the regulation of macrophage and T-lymphocyte activation in vivo during injury. This assessment of gene expression indicates the importance of genetic factors in contributing to lung injury, and points to strategies for intervention in the progression of inflammatory diseases.

Gene expression microarray data analysis of decidual and placental cell differentiation.

Gene expression analysis using DNA microarray approaches have provided new insights into the physiology and pathophysiology of many biological processes. These include identification of genetic programs and pathways that underlie cell and tissue differentiation and gene expression programs responsive to genetic perturbations, drugs, toxins, and infectious agents. In this chapter, we present methods for the analysis of microarray data using earlier investigations from our laboratory as examples of how gene expression patterns for cellular differentiation may be detected and analyzed for biological significance and how regulated genes may be classified into functional categories and pathways.

Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system.

BACKGROUND: In this study we have built and mined a gene expression database composed of 65 diverse mouse tissues for genes preferentially expressed in immune tissues and cell types. Using expression pattern criteria, we identified 360 genes with preferential expression in thymus, spleen, peripheral blood mononuclear cells, lymph nodes (unstimulated or stimulated), or in vitro activated T-cells. RESULTS: Gene clusters, formed based on similarity of expression-pattern across either all tissues or the immune tissues only, had highly significant associations both with immunological processes such as chemokine-mediated response, antigen processing, receptor-related signal transduction, and transcriptional regulation, and also with more general processes such as replication and cell cycle control. Within-cluster gene correlations implicated known associations of known genes, as well as immune process-related roles for poorly described genes. To characterize regulatory mechanisms and cis-elements of genes with similar patterns of expression, we used a new version of a comparative genomics-based cis-element analysis tool to identify clusters of cis-elements with compositional similarity among multiple genes. Several clusters contained genes that shared 5-6 cis-elements that included ETS and zinc-finger binding sites. cis-Elements AP2 EGRF ETSF MAZF SP1F ZF5F and AREB ETSF MZF1 PAX5 STAT were shared in a thymus-expressed set; AP4R E2FF EBOX ETSF MAZF SP1F ZF5F and CREB E2FF MAZF PCAT SP1F STAT cis-clusters occurred in activated T-cells; CEBP CREB NFKB SORY and GATA NKXH OCT1 RBIT occurred in stimulated lymph nodes. CONCLUSION: This study demonstrates a series of analytic approaches that have allowed the implication of genes and regulatory elements that participate in the differentiation, maintenance, and function of the immune system. Polymorphism or mutation of these could adversely impact immune system functions.

Critical role for transcription factor AP-2alpha in human trophoblast differentiation.

To examine whether AP-2alpha is a critical component of the genetic program that directs human trophoblast differentiation, we used DNA microarray analyses to characterize the effects of a dominant-negative form of the AP-2 protein upon in vitro differentiating cytotrophoblast cells. Human cytotrophoblast cells (>95% pure) were cultured for 3 days in the presence of control medium or medium containing an adenovirus that expresses a dominant-negative mutant of AP-2 (Ad2.AP-2D/N) or an adenovirus lacking the AP-2 mutant gene (Ad.WT). DNA microarray analyses using Affymetrix human U95Av2 GeneChips were performed on RNA extracted from the three groups of cells immediately prior to and after 3 days of cell culture. Cells infected with Ad2.AP-2D/N or Ad2.WT underwent morphological differentiation similar to that of uninfected cells, with greater than 90% of the cells in each group fusing to form multinucleated syncytiotrophoblast cells. However, Ad2.AP-2D/N markedly inhibited the induction or repression of many genes that were regulated in the noninfected and Ad2.WT-infected cells during differentiation. Eighteen of the 25 most induced genes and 17 of the 20 most repressed genes during differentiation were AP-2 dependent, with the majority of these related to extracellular organization, cellular communication, and signal transduction. Taken together, these findings strongly suggest that AP-2 plays a critical role for both the induction and repression of genes that comprise postsyncytialization gene expression programs of trophoblast differentiation and maturation. AP-2, however, is not required for the fusion of cytotrophoblast cells to form a syncytium or the expression of syncytin.

Differential regulation of activator protein-1 and heat shock factor-1 in myocardial ischemia and reperfusion injury: role of poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme activated in response to DNA strand breaks, has been implicated in cell dysfunction in myocardial reperfusion injury. PARP-1 has also been shown to participate in transcription and regulation of gene expression. In this study, we investigated the role of PARP-1 on the signal transduction pathway of activator protein-1 (AP-1) and heat shock factor-1 (HSF-1) in myocardial reperfusion injury. Mice genetically deficient of PARP-1 (PARP-1(-/-) mice) exhibited a significant reduction of myocardial damage after occlusion and reperfusion of the left anterior descending branch of the coronary artery compared with their wild-type littermates. This cardioprotection was associated with a reduction of the phosphorylative activity of JNK and, subsequently, reduction of the DNA binding of the signal transduction factor AP-1. On the contrary, in PARP-1(-/-) mice, DNA binding of HSF-1 was enhanced and was associated with a significant increase of the cardioprotective heat shock protein (HSP)70 compared with wild-type mice. Microarray analysis revealed that expression of several AP-1-dependent genes of proinflammatory mediators and HSPs was altered in PARP-1(-/-) mice. The data indicate that PARP-1 may exert a pathological role in reperfusion injury by functioning as an enhancing factor of AP-1 activation and as a repressing factor of HSF-1 activation and HSP70 expression.

Absence of poly(ADP-ribose)polymerase-1 alters nuclear factor-kappa B activation and gene expression of apoptosis regulators after reperfusion injury.

Poly(ADP-ribose) polymerase-1 (PARP-1) is activated in response to DNA injury in eukaryotic cells and has been implicated in cell dysfunction in reperfusion injury. In this study we investigated the role of PARP-1 on apoptosis in early myocardial reperfusion injury. Mice genetically deficient of PARP-1 (PARP-1-/-) and wild-type littermates were subjected to myocardial ischemia and reperfusion. Myocardial injury was assessed by measuring the serum levels of creatine phosphokinase and oligonucleosomal DNA fragments in the infarcted area. Expression of the anti-apoptotic protein, Bcl-2, and the pro-apoptotic protein, Bax, was analyzed by Western blot. Activation of caspases, important executioners of apoptosis, and activation of the nuclear factor kappa B (NF-kappa B) pathway were evaluated. Gene expression profiles for apoptotic regulators between PARP-1-/- and wild-type mice also were compared. Myocardial damage in PARP-1-/- mice was reduced significantly, as indicated by lower serum creatine phosphokinase levels and reduction of apoptosis, as compared with wild-type mice. Western blot analyses showed increased expression of Bcl-2, which was associated with reduction of caspase-1 and caspase-3 activation. This cardioprotection was associated with significant reduction of the activation of I kappa B kinase complex and NF-kappa B DNA binding. Microarray analysis demonstrated that the expression of 29 known genes of apoptotic regulators was significantly altered in PARP-1-/- mice compared with wild-type mice, whereas 6 known genes were similarly expressed in both genotypes. The data indicate that during reperfusion absence of PARP-1 leads to reduction of myocardial apoptosis, which is associated with reduced NF-kappa B activation and altered gene expression profiles.

Microarray-based discovery of highly expressed olfactory mucosal genes: potential roles in the various functions of the olfactory system.

We sought to gain a global view of tissue-specific gene expression in the olfactory mucosa (OM), the major site of neurogenesis and neuroregeneration in adult vertebrates, by examination of its overexpressed genes relative to that in 81 other developing and adult mouse tissues. We used a combination of statistical and fold-difference criteria to identify the top 269 cloned cDNAs from an array of 8,734 mouse cDNA elements on the Incyte Mouse GEM1 array. These clones, representing known and poorly characterized gene transcripts, were grouped according to their relative expression patterns across the other tissues and then further examined with respect to gene ontology categories. Approximately one-third of the 269 genes were also highly expressed in developing and/or adult central nervous system tissues. Several of these have been suggested or demonstrated to play roles in neurogenesis, neuronal differentiation, and/or neuronal migration, further suggesting that many of the unknown genes that share this expression pattern may play similar roles. Highly OM-specific genes included a palate, lung, and nasal epithelium carcinoma-associated gene (Plunc); sphingosine phosphate lyase (Sgpl1), and paraoxonase 1 (Pon1). Cell-type-specific expression within OM was established using in situ hybridization for several representative expression pattern clusters. Using the ENSEMBL-assembled mouse genome and comparative genomics analyses to the human genome, we assigned many of the unknown expressed sequence tags (ESTs) and poorly characterized genes to either novel or known gene products and provided predictive classification. Further exploration of this database will provide additional insights into genes and pathways critical for olfactory neurogenesis, neuronal differentiation, olfaction, and mucosal defense.