Respiratory chain protein analysis and gene expression profiles of fibroblast cell lines from 9 patients with SURF1 gene mutations
ABSTRACT: Upregulation of CI, CIII and CV at a protein but not mRNA level in LSCOX cell with COX deficiency due to SURF1 mutations compared to control – evidence for posttranscriptional compensatory mechanism. Two-condition experiment, patients vs. controls cells. Biological replicates: 3 controls and 9 patients, independently grown and harvested.
Project description:Defects of mitochondrial functions lead in humans to vast array of usually multisystemic pathologies and several hundreds of diseases resulting from various defects of mitochondria biogenesis and maintenance, defects of respiratory chain complexes (OXPHOS) or defects of individual mitochondrial proteins are known. We used Agilent Whole Human Genome Microarray for gene expression profiling of genetically heterogeneous group of 13 patients with biochemically proven ATP synthase deficiency. Gene expression data analysis allowed classification of patients into several distinct groups, provided information on subgroup and patient specific gene expression profiles, defined candidate disease causing genes and gave basic information on pathogenic mechanisms associated with ATP synthase deficiency. Keywords: ATP synthase, mitochondrial biogenesis, ROS, gene expression, microarray, human Two-condition experiment, patients vs. controls cells. Biological replicates: 9 control, 13 patients, independently grown and harvested.
Project description:Samples of benign prostate tissue, localized prostate cancer tissue, and metastatic prostate cancer tissue are profiled to study expression changes in diagnosis and progression of prostate cancer. Each tissue sample is also profiled for metabolomics data Keywords: Cancer Progression 41 samples were analyzed (16 benign prostate tissue, 12 local prostate cancer tissue, 13 metastatic prostate cancer tissue)
Project description:Oxaliplatin resistance was induced in 2 colorectal cancer cell lines (LoVo-92, wt-p53 and LoVo-Li, functionally inactive p53) and one ovarian cancer cell line (A2780, wt-p53). Resistance was induced by weekly exposure to oxaliplatin for 4 hrs or 72 hrs with increasing concentrations for a period of 7 months. After RNA isolation, 500ng of RNA of the resistant and parental cell line were linearly amplified and fluorescently labeled with either Cy3-CTP or Cy5-CTP with the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Amstelveen, The Netherlands). Equal amounts (1µg) of Cy3-CTP and Cy5-CTP labeled samples were hybridized to Agilent 4x44K Whole Human Genome arrays (Agilent Technologies) according to the manufacturer. Equal amounts (1µg) of Cy3-CTP and Cy5-CTP labeled samples were hybridized to Agilent 44K Whole Human Genome arrays (Agilent Technologies, Part Number G4112A) according to manufacturer's instructions. Microarrays were scanned using an Agilent DNA Microarray Scanner, and scans were quantified using Agilent Feature Extraction software (version 8.5.1). Raw expression data generated by the Feature Extraction software was imported into R using the LIMMA package in Bioconductor (http://www.bioconductor.org). As overall background levels were very low, no background correction was performed. The intensity distributions within and between arrays were normalized using the quantile scaling algorithm in LIMMA. After normalization, the separate intensity channels were extracted from the ratio measurements and combined to the intensities of the reference. The parental cell line was used as reference.
Project description:ETS gene fusions have been characterized in a majority of prostate cancers, however key molecular alterations in ETS negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier-expression exclusively in a subset of ETS rearrangement negative cancers (~10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier-expression can be detected non-invasively in urine and observed that SPINK1 outlier-expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier-expression model, and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement negative prostate cancers. Keywords: Genetic Modification 22RV1 cells were infected with non-targeting siRNA or siRNA against SPINK1. For reported hybridizations, the reference channel is 22RV1 cells infected with non-targeting siRNA. Duplicate hybridizations were performed with duplicate dye flips, for a total of four arrays. Over and under-expressed signatures were generated by filtering to include only features with significant differential expression (PValueLogRatio < 0.01) in all hybridizations and Cy5/Cy3 ratios > or < 1 in all hybridizations.
Project description:Amplification of a ~2.5 megabase region on chromosome 9p24 is frequent in both primary mediastinal B-cell lymphoma (PMBL) and Hodgkin's lymphoma (HL). To identify the oncogenic genes in this interval, we created a RNA interference library targeting amplicon genes. A genetic screen using this library identified three genes that are essential for the proliferation and survival of PMBL and HL lines with this amplicon, which encode the kinase JAK2, the histone demethylase JMJD2C and a gene of unknown function, RANBP6. Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas and in remodeling their chromatin by globally increasing trimethylation of lysine 9 on histone H3 (H3K9me3) and heterochromatin formation. JAK2 and JMJD2C inhibition silenced MYC and its target genes, which coincided with an increase in H3K9me3 and the heterochromatin protein HP1alpha at the MYC locus. We conclude that amplification of JAK2 and JMJD2C cooperatively reprograms the PMBL and HL epigenome, sustaining their survival and proliferation. Signal from untreated shRNA cells or DMSO control cells (Cy3) was compared to 4 dox-treated JAK2 shRNA cells, 4 dox-treated JMJD2C shRNA cells, or 8 JAK2 inhibitor-treated cells (Cy5).
Project description:LNCaP and its androgen insensitive derivative were profiled in order to identify genes differentially expressed during the conversion to androgen insensitivity. This experiment was performed due to the presence of an ins(7;14) localizing the entire ETV1 locus to chr 14 in LNCaP and C4-2B prostate cancer cells. Keywords: cell type comparison LNCaP and C4-2B were both hybridized to Agilent Whole Human Genome Oligonucleotide Microarrays, with a commercially obtained pool of benign prostate RNA serving as the reference for both cell lines. Dye flips for both cell lines were also performed
Project description:RWPE benign prostatic epithelial cells were infected with a lentivirus expressing ETV1 or GUS (control), and stable clones were isolated by puromycin selection. ETV1 over-expression, recapitulating ETS gene rearrangements observed in vivo, confers invasiveness in the benign prostate cell line RWPE. Keywords: genetic modification Four hybridizations in total were performed using RWPE cells stably expressing ETV1 or GUS (control) on Agilent Whole Human Genome Oligonucleotide Microarrays. RWPE-ETV1 (Cy5) / RWPE-GUS (Cy3) and a dye flip were performed in duplicate
Project description:The goal of this gene expression study was to identify genes whose expression depends on the transription factor IRF4 by knocking down it's expression using shRNA in two IRF4+ myeloma cell lines. Keywords: time series design Two myeloma lines were analyzed over a time course of IRF4-targeted shRNA induction. There are 3 time courses: two using KMS12 (biological replicates) and one with SKMM1. Within each time course there are technical replicates.
Project description:Ataxia-telangiectasia mutated (ATM) kinase plays a central role in maintaining genomic integrity. In both humans and mice, ATM deficiency is associated with an increased incidence of lymphoid cancers that are primarily T cell in origin. We demonstrate here that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early onset IgM+ B cell lymphomas that by histology and gene expression profiling resemble the activated B cell-like (ABC) subset of human diffuse large B cell lymphomas (DLBCL). These ATM-deficient B cell tumors show considerable chromosomal instability and a recurrent genomic amplification of a 4.48 Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC-DLBCL. Further, the amplification of Malt1 in these lymphomas correlates with their dependence on NF-kB, MALT1, and BCR signaling for survival, paralleling human ABC-DLBCL. This study reveals that ATM protects against development of B cell lymphomas that model human ABC-DLBCL and identifies a role for T cells in preventing the emergence of these tumors. We performed gene expression profiling on nine ATMKO.CD3epsilonKO lymphoma cell lines (n=12, 3 technical repeats). We analyzed gene expression of anti-IgM stimulated primary B cells from both ATMKO.CD3epsilonKO (n=7, 5 technical repeats) and ATMWT.CD3epsilonKO mice (n=2), and GC B cells isolated from SRBC-immunized ATMWT mice (n=3, 1 biological repeat and 2 technical repeats). We analyzed gene expression following treatment of two ATMKO.CD3epsilonKO lymphoma cell lines with the BTK inhibitor, PCI-32765, at time points (1, 3, 6, and 24 hours) as compared to time points with vehicle (DMSO) (n=8).
Project description:Pathologic activation of the Toll-like receptor (TLR) pathway underlies various human disorders such as autoimmune diseases, chronic inflammatory diseases and lymphoid malignancies. Current therapy of these diseases relies on immunosuppressive or chemotherapeutic agents, but more effective therapeutics tailored to disease-causing mechanisms are needed. Pivotal to TLR signaling is the IL-1 receptor-associated kinase 4 (IRAK4), which is recruited to TLRs by the adaptor protein MyD88. Recruitment of IRAK kinases to MyD88, triggers the formation of a signaling competent myddosome complex, which underlies the pathogenesis of many immuno-inflammatory disorders, suggesting that IRAK4 inhibitors might be useful in the treatment of these diseases. Gain-of-function MYD88 mutations activate IRAK4 in several mature B cell malignancies, including activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). Development of selective IRAK4 inhibitors has been confounded by the challenging structure of the IRAK4 catalytic domain. Using structure-based drug design methodologies, we identified potent and selective IRAK4 inhibitors. These small molecules suppress LPS-induced TNFalpha production in vitro and in vivo, and are efficacious in mouse models of collagen-induced arthritis and MyD88-dependent inflammatory gout. Human ABC DLBCL cell lines that harbor the activating, oncogenic MyD88 L265P mutation are killed by IRAK4 inhibitors, both in vitro and in mouse xenograft models. IRAK4 inhibitors synergize with the BTK inhibitor ibrutinib, with the Syk inhibitor PRT062607, and with the Bcl-2 inhibitor ABT-199 in killing ABC DLBCL cells, suggesting new therapeutic strategies for this refractory cancer. Four ABC DLBCL cell lines (OCI-Ly10, TMD8, HBL1 and OCI-Ly3), were treated with either ND-2158 or the structurally related negative control compound ND-1659 for 6, 12, 24 or 36 h in culture. Gene expression profiling was performed using two-color human Agilent 4x44K gene expression arrays comparing signal from control compound-treated (ND-1659) control cells (Cy3), to cells treated with ND-2158 for the indicated times (Cy5).