Transcription profiling of wild_type and IGF-I receptor over-expressing mouse lung carcinoma cells identifies a type I insulin like growth factor receptor regulated gene expression profile associated with an altered site-specificity of metastasis.
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ABSTRACT: Identification of a type I insulin like growth factor receptor regulated gene expression profile associated with an altered site-specificity of metastasis.
Project description:The liver is frequently challenged by surgery-induced metabolic overload, viruses, or toxins, which induce the formation of reactive oxygen species. To determine the effect of oxidative stress on liver regeneration and to identify the underlying signalling pathways, we studied liver repair in mice lacking the Nrf2 transcription factor. In these animals, expression of several cytoprotective enzymes was reduced in hepatocytes, resulting in oxidative stress. As a consequence, tissue damage was aggravated, and liver regeneration after partial hepatectomy was delayed. Using in vitro and in vivo studies we identified oxidative stress-induced insulin/insulin-like growth factor resistance as the underlying mechanism. This deficiency impaired the activation of p38 mitogen-activated kinase, Akt kinase, and downstream targets after hepatectomy, resulting in enhanced death and delayed proliferation of hepatocytes. Our results reveal novel roles of Nrf2 in the regulation of growth factor signalling and in tissue repair. In addition, they provide new insight into the mechanisms underlying oxidative stress-induced defects in liver regeneration and thus offer new avenues to improve regeneration in patients with acute or chronic liver damage. Experiment Overall Design: Livers from Nrf2 k.o. and wt mice; 3 hybridizations per genoype: RNA samples were pooled from 3 individual animals
Project description:In order to identify the genes that are regulated by TGF-beta in glioma, we serum starved two glioma cell lines, U373MG and U87MG, for 16h and we treated them with vehicle,100pM TGF-beta, 2uM inhibitor of the TGF-beta Receptor I(TbRI)(LY2109761), or both 100pM TGF-beta plus 2uM TbRI for 3h. Then, cell were collected and total RNA was extracted.
Project description:Mononuclear phagocytes play an important role in the removal of apoptotic cells by expressing cell surface receptors that recognize and remove apoptotic cells. Based on the knowledge that cigarette smoking is associated with increased lung cell turnover, we hypothesized that alveolar macrophages (AM) of normal cigarette smokers may exhibit enhanced expression of apoptotic cell removal receptor genes. AM obtained by bronchoalveolar lavage of normal non-smokers (n=11) and phenotypic normal smokers (n=13, 36 +/- 6 pack per year) were screened for mRNA expression of all known apoptotic cell removal receptors using Affymetrix HG-U133 Plus 2.0 chips with TaqMan RT-PCR confirmation. Of the 14 known apoptotic receptors expressed, only MER Tyrosine Kinase (MERTK), a transmembrane tyrosine kinase receptor, was significantly up-regulated in smokers. MERTK expression was then assessed in AM of smokers vs nonsmokers by TaqMan RT-PCR, immunohistochemistry, Western and flow analysis. Smoker AM had up-regulation of MERTK mRNA levels (smoker vs non-smoker, 3.6-fold by microarray, p<0.003; 9.5-fold by TaqMan RT-PCR, p<0.02). Immunohistochemistry demonstrated a qualitative increase in MERTK protein expression on AM of smokers. Increased protein expression of MERTK on AM of smokers was confirmed by Western and flow analyses (p< 0.007 and p< 0.0002, respectively). MERTK, a cell surface receptor that recognizes apoptotic cells, is expressed on human AM, and its expression is up-regulated in AM of cigarette smokers. This may reflect an increased demand for removal of apoptotic cells in smokers, an observation with implications for the development of chronic obstructive pulmonary disease (COPD), a disorder associated with dysregulated apoptosis of lung parenchymal cells. Experiment Overall Design: Alveolar macrophages were obtained from bronchoalveolar lavage.
Project description:Processing of Immunoglobulin heavy chain (IgH) mRNA is a paradigm for competition between splicing and polyadenylation. In plasma cells pre-mRNA is polyadenylated mainly at the promoter-proximal secretory site while B-cells utilize a cryptic 5â?? splice site in the last secretory-specific exon; these are mutually exclusive events for all IgH pre-mRNAs. Transcription elongation factor ELL2, more abundant in plasma cells relative to B-cells, was down-modulated by overexpression of heterogenous ribonucleoprotein F, a condition which reduced production of secretory IgH mRNA. Transfection of B-cells with ELL2 and the IgH reporter showed an accelerated use of the secretory poly(A) site, positioned in competition with the splice to M1; a small interfering RNA to ELL2 reduced expression of IgH secretory mRNA. Co-transcription factors ELL1 and PC4 were ineffective at driving secretory-poly(A) site use. ELL2 had little effect on poly(A) site choice with reporters containing tandem-linked poly(A) sites. Shorter forms of ELL2 protein result from both internal initiation at M186 and protein processing. An alternative splicing reporter driven by IgH or non-Ig promoters revealed that ELL2 and its M186 initiated form were able to accelerate exon skipping. Therefore, ELL2 influences IgH pre-mRNA processing through facilitating skipping of the alternative splice to the membrane form. Experiment Overall Design: AxJ plasma cells were stably transfected to overexpress hnRNP-F, -H or empty vector. Clones showing high overexpression levels of F or H by western blot were selected. The IgH sec to mb ratios of these clones were determined. A global gene expression analysis was performed on mRNA from two clones from hnRNP-F, which demonstrated a lower sec:mb ratio, and one from each of the controls: overexpression of hnRNP-H, or transfection with empty vector, or A20 B-cells, using Affymetrix gene micro array technology.
Project description:Gene expression in eukaryotes is tightly linked to the methylation state of specific lysine residues within the N-terminal region of the core histone proteins. While the mechanisms connecting histone lysine methylation to effector protein recruitment and control of gene activity are increasingly well understood, it remains unknown whether non-histone chromatin proteins are targets for similar modification-recognition systems. Here we show that histone H3 and the H3 methyltransferase G9a share a conserved methylation motif that is both necessary and sufficient to mediate in vivo interaction with the potent epigenetic regulator Heterochromatin Protein 1 (HP1). As with H3, G9a-HP1 interaction is dependent on lysine methylation and can be reversed by adjacent phosphorylation. NMR analysis demonstrates that the HP1 chromodomain recognizes methyl-G9a through a binding mode similar to that used in recognition of methyl-H3, and that adjacent phosphorylation directly antagonizes G9a-HP1 interaction. In addition to uncovering the chromodomain as a generalized methyl-lysine binding module, these data identify histone-like modification cassettes (or âhistone mimicsâ) as an entirely new class of non-histone methylation targets, and directly demonstrate the relevance of the principles underlying the histone code to the regulation of non-histone proteins. Experiment Overall Design: Two independent Affymetrix gene expression microarray analyses were performed on samples from G9a-deleted MEFs reconstituted with empty vector (delta), wild type FLAG-G9a (WT), FLAG-G9a K165A (K165A) or FLAG-G9a H1093K catalytic mutant (H1093K).
Project description:The vertebrate nuclear hormone receptor steroidogenic factor 1 (SF1; NR5A1) controls reproductive development and regulates the transcription of steroid-modifying cytochrome P450 genes. We find that the SF1-related Drosophila nuclear hormone receptor HR39 is also essential for sexual development. In Hr39 mutant females, the sperm-storing spermathecae and glandular parovaria are absent or defective, causing sterility. Our results indicate that spermathecae and parovaria secrete reproductive tract proteins required for sperm maturation and function, like the mammalian epididymis and female reproductive tract. Hr39 controls the expression of specific cytochrome P450 genes and is required in females both to activate spermathecal secretion and repress male-specific courtship genes such as takeout. Thus, a pathway that, in vertebrates, controls sex-specific steroid hormone production, also mediates reproductive functions in an invertebrate. Our findings suggest that Drosophila can be used to model more aspects of mammalian reproductive biology than previously believed. Experiment Overall Design: Wild type and Hr39(04443) Spermathecae, Wild type and Hr39(04443) Reproductive Tract
Project description:Different mutations in the gene encoding humans IGF-I cause intrauterine growth retardation, postnatal growth failure, microcephaly, mental retardation, bilateral sensorineural deafness and multiple dysmorphic features. Insight into the role of IGFs in inner ear cochlear ganglion neurogenesis has come from the study of genetically modified mice. Postnatal cochlear development is severely impaired in mice Igf1-/-, which develop smaller cochlea and cochlear ganglia, an immature tectorial membrane and they display a significant decrease in the number and size of auditory neurons. We used microarrays to define the genetic signatures of Igf-1 +/+ and Igf-1-/- mouse cochea and identify the differentially expressed genes. Experiment Overall Design: Cochleae from two E18.5 were isolated from both Igf-1+/+ wild type and Igf-1-/- null mice and pooled to obtain RNA. Heterozygous male and female with a genetic background C57BL/6J were mated to obtain embryos 18.5 days post coitus (E18.5). Three independent pools were used. Cochlear tissues included the otic capsule but not vestibular tissues.