ABSTRACT
The transcriptional activation of the chicken lysozyme gene (cLys) by lipopolysaccharide (LPS) in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR) triggering eviction of the CCCTC-binding factor (CTCF) from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE). In activated macrophages, a 60 bp region bound by NF-κB, AP1 and C/EBPß controls this CRE, which is strictly dependent on NF-κB binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKKα, known to be recruited by NF-κB to NF-κB-dependent genes is found at the CRE and within the transcribing regions of both cLys and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating cLys transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both cLys transcription and NF-κB binding to the CRE. Finally, comparing cLys with other LPS-inducible genes indicates that IKKα detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3' end of the gene.
Subject(s)
Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Muramidase/genetics , Muramidase/metabolism , NF-kappa B/metabolism , Promoter Regions, Genetic/genetics , Animals , Cell Line , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Macrophages/enzymology , Real-Time Polymerase Chain Reaction , Transcriptional Activation/drug effects , Transcriptional Activation/geneticsABSTRACT
Drug resistance remains a barrier to the effective long term treatment of ovarian cancer. We have established an RNAi-based screen to identify genes which confer resistance to carboplatin or paclitaxel. To validate the screen we showed that siRNA interfering with the apoptosis regulators FLIP and Bcl-X(L) conferred sensitivity to paclitaxel and carboplatin respectively. The expression of 90 genes which have previously been shown to be over-expressed in drug-resistant ovarian cancer was inhibited using siRNA and the impact on sensitivity to carboplatin and paclitaxel was assessed. ENPP2 was identified as a candidate gene causing drug resistance. ENPP2 encodes autotaxin, a phospholipase involved in the synthesis of the survival factor lysophosphatidic acid. siRNA directed to ENPP2 resulted in earlier apoptosis following treatment with carboplatin. 2-carbacyclic phosphatidic acid (ccPA 16:1), a small molecule inhibitor of autotaxin, also accelerated apoptosis induced by carboplatin. Stable ectopic expression of autotaxin in OVCAR-3 cells led to a delay in apoptosis. When serum was withdrawn to remove exogenous LPA, ccPA caused a pronounced potentiation of apoptosis induced by carboplatin in cells expressing autotaxin. These results indicate that autotaxin delays apoptosis induced by carboplatin in ovarian cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Carboplatin/pharmacology , Multienzyme Complexes/physiology , Ovarian Neoplasms/genetics , Phosphodiesterase I/physiology , Pyrophosphatases/physiology , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Female , Humans , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/genetics , Ovarian Neoplasms/pathology , Phosphodiesterase I/antagonists & inhibitors , Phosphodiesterase I/genetics , Phosphoric Diester Hydrolases , Pyrophosphatases/antagonists & inhibitors , Pyrophosphatases/genetics , RNA InterferenceABSTRACT
Transcription of the lysozyme gene is rapidly induced by proinflammatory stimuli such as treatment with bacterial lipopolysaccharide (LPS). Here we show that this induction involves both the relief of repression mediated by the enhancer-blocking protein CTCF that binds to a negative regulatory element at -2.4 kb, and the activation of two flanking enhancer elements. The downstream enhancer has promoter activity, and LPS stimulation initiates the transient synthesis of a noncoding RNA (LINoCR) transcribed through the -2.4 kb element. Expression of LINoCR is correlated with IKKalpha recruitment, histone H3 phosphoacetylation in the transcribed region, the repositioning of a nucleosome over the CTCF binding site, and, eventually, CTCF eviction. Each of these events requires transcription elongation. Our data reveal a transcription-dependent mechanism of chromatin remodeling that switches a cis-regulatory region from a repressive to an active conformation.
Subject(s)
DNA-Binding Proteins/metabolism , Muramidase/genetics , Repressor Proteins/metabolism , Animals , Base Sequence , Binding Sites , CCCTC-Binding Factor , Cell Line , Chickens , DNA Primers/genetics , Enhancer Elements, Genetic , Histones/metabolism , Lipopolysaccharides/pharmacology , Nucleosomes/drug effects , Nucleosomes/metabolism , Phosphorylation , Promoter Regions, Genetic , RNA Interference , RNA Polymerase II/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Untranslated/genetics , Regulatory Elements, Transcriptional , Transcription, Genetic/drug effects , Up-Regulation/drug effectsABSTRACT
Ectopic expression provides an alternative to RNAi to conduct functional genomic screens. We have determined whether transient expression can be used to identify genes which confer drug resistance. We constructed a bigenic vector that allows high throughput cloning and which also encodes a red fluorescent protein to monitor transfection. Assay conditions were optimized to allow detection of changes in sensitivity to carboplatin or paclitaxel. Following transient expression of MDR-1 and MCJ, changes in the sensitivity of Sk-Ov-3 cells to paclitaxel were detected whereas expression of Src, Bcl-2 and Bcl-X(L) decreased the sensitivity of Sk-Ov-3 cells to carboplatin. This approach may potentially be used as an independent screen or as a method to help rank hit identified in screens utilizing methods such as RNAi.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , HSP40 Heat-Shock Proteins/genetics , Oligonucleotide Array Sequence Analysis/methods , Ovarian Neoplasms/genetics , ATP Binding Cassette Transporter, Subfamily B , Animals , Carboplatin/pharmacology , Dose-Response Relationship, Drug , Female , Fluorescent Dyes/metabolism , Genes, MDR , Genes, bcl-2 , Humans , Luminescent Proteins/genetics , Ovarian Neoplasms/drug therapy , Paclitaxel/pharmacology , Transfection , bcl-X Protein/genetics , src-Family Kinases/genetics , Red Fluorescent ProteinABSTRACT
PURPOSE: The effective treatment of ovarian cancer is hampered by the development of drug resistance, which may be mediated by members of the Bcl-2 family of apoptosis regulators. ABT-737 is a recently described inhibitor of members of this family. We investigated whether this compound could sensitize ovarian cancer cells to chemotherapeutic agents. EXPERIMENTAL DESIGN: The sensitivity of ovarian cancer cell lines to ABT-737 in combination with either carboplatin or paclitaxel was tested either in vitro by assessing cell growth/survival and apoptosis or in xenograft studies. RESULTS: As a single agent, ABT-737 inhibited the growth of eight ovarian cancer cell lines, although with relatively poor potency. However, ABT-737, but not a less active enantiomer, increased the sensitivity of several cell lines to carboplatin. The increased sensitivity to carboplatin was accompanied by a decrease in time at which apoptosis was observed when assessed according to the number of attached cells, PARP cleavage, and nucleosome formation. ABT-737 was more effective at sensitizing IGROV-1 cells when ABT-737 was administered after carboplatin. In addition, ABT-737 significantly enhanced the activity of carboplatin in one of three primary cultures derived directly from ascitic tumor cells in patients recently treated with chemotherapy. Small interfering RNA directed to Bcl-X(L) also increased the sensitivity of ovarian cancer cell lines to carboplatin. ABT-737 was also able to augment the inhibition of IGROV-1 tumor xenograft growth beyond that obtained with carboplatin alone. CONCLUSIONS: These data suggest that ABT-737, in combination with carboplatin, may find utility in the treatment of patients with ovarian cancer.
