ABSTRACT
Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα(-), PR(-), and Her2(-), named accordingly "Triple-Negative" (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5-12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygro(R)) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50-100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships.
Subject(s)
Cell Culture Techniques/methods , DNA, Complementary/genetics , Estrogen Receptor alpha/genetics , Base Sequence , Blotting, Western , Cell Line, Tumor , Clone Cells/metabolism , Genes, Reporter/genetics , Genetic Vectors/genetics , Humans , Ligands , Luciferases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribosomes/metabolism , Time Factors , Transcription, Genetic , TransfectionABSTRACT
In a multicenter study, we determined the expression profiles of 863 microRNAs by array analysis of 454 blood samples from human individuals with different cancers or noncancer diseases, and validated this 'miRNome' by quantitative real-time PCR. We detected consistently deregulated profiles for all tested diseases; pathway analysis confirmed disease association of the respective microRNAs. We observed significant correlations (P = 0.004) between the genomic location of disease-associated genetic variants and deregulated microRNAs.
Subject(s)
Disease/genetics , MicroRNAs/blood , MicroRNAs/genetics , Gene Expression Profiling , Genetic Variation/genetics , Humans , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL delta (hPOL delta). The hPOL delta enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL delta strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL delta. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL delta.
Subject(s)
DNA Helicases/metabolism , DNA Polymerase III/metabolism , Binding Sites , Cell Line, Transformed , DNA Helicases/analysis , DNA Helicases/chemistry , DNA Polymerase III/analysis , DNA Polymerase III/chemistry , DNA Replication , Humans , Protein Subunits/analysis , Protein Subunits/chemistry , Protein Subunits/metabolism , RecQ HelicasesABSTRACT
AIMS: Postprandial triglyceride-rich lipoproteins (TRL) have a direct effect on vascular smooth muscle cells (SMC) and they increase the risk of atherogenesis. Here, we have tested the hypothesis that the different fatty acid composition of TRL is capable of differentially modifying gene expression in human coronary artery SMC (CASMC). In addition, the effect of TRL on cell proliferation and transcription factor activation was also evaluated. METHODS AND RESULTS: TRL were prepared from plasma of healthy volunteers after the ingestion of meals enriched in refined olive oil (ROO), butter or a mixture of vegetable and fish oils (VEFO). We use cDNA microarrays to determine the genes differentially expressed in TRL-treated CASMC. Correspondence cluster analysis demonstrated that TRL-butter, -ROO and -VEFO provoked different transcriptional profiles in CASMC. Sixty-six genes were regulated by TRL-butter, 55 by -ROO, and 47 by -VEFO. The data revealed that TRL-butter predominantly activated genes involved in the regulation of cell proliferation and inflammation. Likewise, TRL-VEFO induced the expression of genes implicated in inflammation, while TRL-ROO promoted a less atherogenic gene profile. CONCLUSION: The pathophysiological contribution of TRL to the development of atherosclerosis and the stability of atherosclerotic plaques may depend on the fatty acid composition of TRL. Our findings suggest a role for macrophage-inhibiting cytokine-1 (MIC-1) in coronary artery cardiovascular events.
Subject(s)
Coronary Vessels/metabolism , Gene Expression Regulation/physiology , Lipid Peroxidation/physiology , Muscle, Smooth, Vascular/metabolism , Postprandial Period/physiology , Triglycerides/metabolism , Adult , Cell Proliferation/drug effects , Cells, Cultured , Cluster Analysis , Coronary Vessels/drug effects , Cytokines/metabolism , Fatty Acids/metabolism , Gene Expression Profiling , Gene Expression Regulation/drug effects , Growth Differentiation Factor 15 , Humans , Lipid Peroxidation/drug effects , Male , Muscle, Smooth, Vascular/drug effects , NF-kappa B/metabolism , Oligonucleotide Array Sequence Analysis , Oxidation-Reduction , Transcription Factor AP-1/metabolism , Triglycerides/pharmacologyABSTRACT
Hybridization array technology is increasingly being used for the analysis of gene expression in the yeast Saccharomyces cerevisiae. It is a powerful technique in which the relative abundance of all the mRNA molecules transcribed under a particular condition may be simultaneously measured. However, most studies performed using this technique are carried out in batch culture where the growth rate and environment are continuously changing. Often, the experimental condition being studied also impacts on the growth rate of the cells. Changes in growth rate affect the pattern of gene expression. Consequently, the analysis and interpretation of experimental results obtained in this way are inherently problematic due to the difficulty in discriminating between effects due to the experimental condition per se and concomitant growth rate-related effects. Here, we present a method that addresses this problem by exploiting chemostat culture, in which the cells can be grown at a fixed growth rate, in combination with hybridization array technology. We use two experimental examples to illustrate the advantages of using this approach and then describe a specific application of this approach to investigate the effect of carbon and nitrogen limitation at the transcriptome level.
