Expression data from human carcinoma (MCF7) derived cells that have been exposed to insulin analogues
ABSTRACT: Insulin analogues are designed to improve the pharmacokinetic parameters compared to regular human insulin. This provides a sustained control of blood glucose levels in diabetic patients. All novel insulin analogues are tested for their mitogenic side effects, however these assays do not take into account the molecular mode-of-action of different insulin analogues. Insulin analogues can bind the insulin receptor (INSR) and the insulin-like growth factor-1 receptor (IGF1R) with different affinities and consequently will activate different downstream signaling pathways. Here we used a panel of MCF7 human breast cancer cell lines that selectively express either one of the isoforms of the INSR (IRA or IRB) or the IGF1R. We sought to study the role of the different receptors (IRA, IRB and IGF1R) in the mitogenic signaling of insulin-like molecules (including insulin, glargine, X10 (or AspB10) and IGF1). MCF7 IRA, MCF7 IRB or MCF7 IGF1R cells (as described in Arch Toxicol. 2014 Apr;88(4):953-66. doi: 10.1007/s00204-014-1201-2. Epub 2014 Jan 25.) were cultured in RPMI supplemented with 5% (v/v) CDFBS (Hyclone) and used for experiments. Cells have been exposed for 1 or 6 hours to 10 nM of the indicated insulin-like molecule. As a control sample a vehicle stimulation was performed that contained everything except the active compound.
Project description:Despite a high degree of homology, insulin and IGF-1 receptors (IR/IGF1R) mediate distinct cellular and physiological functions. Here, using chimeric and site-mutated receptors, we demonstrate how domain differences between IR and IGF1R contribute the distinct functions of these receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to its higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. We generated mouse brown preadipocytes in which both insulin and IGF-1 receptors (IR and IGF1R) had been genetically inactivated using Cre-lox recombination. These IR and IGF1R DKO cells were then reconstituted with wild-type mouse IR, IGF1R, or one of two chimeric receptors: IR/IGF1R with the IR extracellular domain (ECD) fused to the IGF1R transmembrane and intracellular domains (ICD) and IGF1R/IR with the ECD of IGF1R fused to the ICD domains of IR. Three independent clones for each line were used for the study. For expression analysis, we serum-starved the preadipocytes clones overnight and stimulated cells with 100 nM insulin, IGF-1 or vehicle for 6 h, and subjected the cellular RNA to analysis using Affymetrix Mouse Gene 2.0 ST arrays.
Project description:Dynamic interactions of nuclear lamins with chromatin through so-called lamin-associated domains (LADs) contribute to spatial arrangements of the genome. Here, we provide evidence for pre-patterning of differentiation-driven formation of lamin A/C LADs by domains of histone H2B modified by the nutrient sensor O-linked N-acetylglucosamine (H2BGlcNAc), which we term GADs. We demonstrate a two-step process of lamin A/C LAD formation during in vitro adipogenesis, involving (i) a spreading of lamin A/C-chromatin interactions during the transition from progenitor cell proliferation to cell cycle arrest, and (ii) a genome-scale redistribution these interactions through a process of LAD ‘exchange’ within hours of adipogenic induction. Chromatin state modeling reveals that lamin A/C LADs can be found both in active and repressive chromatin contexts which can be influenced by cell differentiation status. De novo formation of adipogenic lamin A/C LADs occurs non-randomly on GADs, consisting of megabase-size intergenic and repressive chromatin domains. Accordingly, while pre-differentiation lamin A/C LADs are gene-rich, post-differentiation LADs harbor repressive features reminiscent of lamin B1 LADs. Moreover, release of lamin A/C from genes directly involved in glycolysis concurs with their transcriptional upregulation after adipogenic induction, and with concordant downstream elevations in H2BGlcNAc levels and O-GlcNAc cycling. Our results unveil an epigenetic pre-patterning of adipogenic LADs by GADs, suggesting a coupling of developmentally regulated lamin A/C-genome interactions to a metabolically sensitive chromatin modification. Examination of LMNA and H2BGlcNAc binding in ASCs across differentiation
Project description:Adipose stem cells (ASCs) and adipocytes play a crucial role in maintaining energy balance. We aim to examine the temporal relationship between gene expression and histone modification transitions during in vitro differentiation of human ASCs into adipocytes. Here, we examine by RNAseq proliferating ASCs (Day -2 prior to adipogenic induction), confluent ASCs (Day 0, adipogenic induction), pre-adipocytes (Day 3) and maturing adipocytes (Day 9). We find 1060, 5452 and 2216 genes differentially expressed between D-2/D0, D0/D3 and D3/D9 respectively. We identify gene clusters with distinct and dynamic expression patterns. In particular, adipogenic induction is marked by temporal waves of gene induction and downregulation. We report two types of transcriptional waves: (i) those showing transient induction or inactivation at D0, D3 or D9, and involved in sensory perception and immune response functions; and (ii) those showing long-lived induction or repression at these time points. Our data reveal a dynamic network of gene regulation during adipogenesis, involving signaling, immune and developmental processes. We identify 15 unique epigenetic states using Hidden Markov Modeling which reflects an epigenetically highly organised genome showing enhancer states are commonly consecutive. A heatmap for the abundance of epigenetic states for the expression clusters reveals a link between expression and epigenetic marking of the state suggesting an increase in the number of number of chromatin states with increase in expression. Our data point to a model of increased epigenetic complexity associated with gene expression. Examination of expression of profiles of ASCs across differentiation
Project description:To identify the target of miR-212, miR-132 and HIC1, we have employed whole genome microarray expression profiling on the human breast cancer MCF7 cells. To generate miR-212/132 or HIC1 inducible MCF7 cells, doxycycline-dependent miR-212/132 or HIC1 gene expression system was used. Either Tet-ON miR-212/132 MCF7 or Tet-ON HIC1 MCF7 were treated with 1μg/ml of Doxycycline for 36 hours with EMEM containing 0.01 mg/ml bovine insulin and 10% FCS. Two independent experiments were performed.
Project description:A major goal of systems biology is the development of models that accurately predict responses of a cell or organism to perturbation. Constructing such models requires collection of dense measurements of system states, yet transformation of the data into predictive constructs remains a challenge. As a first step towards modeling human immunity, we have analyzed immune parameters in depth both at baseline and in response to perturbation with influenza vaccination. Peripheral blood cell transcriptomes, serum titers, frequencies of 126 cell subpopulations, and B cell responses were assessed before and after vaccination in 63 individuals and used to develop a systematic, computational framework to dissect inter- and intra-individual variation and build predictive models of post-vaccination antibody responses. Strikingly, independent of age and pre-existing antibody titers, accurate models could be constructed using pre-perturbation parameters alone, which were validated using data from independent baseline time-points. Most of the parameters contributing to prediction delineated temporally-stable baseline differences across individuals, raising the prospect of immune responsiveness prediction before intervention. According to CHI protocol 09-H1-0239, PBMC samples from 63 healthy voluntiers were collected 7 days prior to vaccination, immediately before vaccination (day0), and at 3 time points (day1, day7 and day70) post vaccination. The CHI Consortium
Project description:Progesterone and estrogen are important drivers of breast cancer proliferation. Herein, we probed estrogen receptor-α (ER) and progesterone receptor (PR) cross-talk in breast cancer models. Stable expression of PR-B in PR-low/ER+ MCF7 cells increased cellular sensitivity to estradiol and insulin-like growth factor 1 (IGF1), as measured in growth assays performed in the absence of exogenous progestin; similar results were obtained in PR-null/ER+ T47D cells stably expressing PR-B. Genome-wide microarray analyses revealed that unliganded PR-B induced robust expression of a subset of estradiol-responsive ER target genes, including cathepsin-D (CTSD). Estradiol-treated MCF7 cells stably expressing PR-B exhibited enhanced ER Ser167 phosphorylation and recruitment of ER, PR and the proline-, glutamate- and leucine-rich protein 1 (PELP1) to an estrogen response element in the CTSD distal promoter; this complex co-immunoprecipitated with IGF1 receptor (IGFR1) in whole-cell lysates. Importantly, ER/PR/PELP1 complexes were also detected in human breast cancer samples. Inhibition of IGF1R or phosphoinositide 3-kinase blocked PR-B-dependent CTSD mRNA upregulation in response to estradiol. Similarly, inhibition of IGF1R or PR significantly reduced ER recruitment to the CTSD promoter. Stable knockdown of endogenous PR or onapristone treatment of multiple unmodified breast cancer cell lines blocked estradiol-mediated CTSD induction, inhibited growth in soft agar and partially restored tamoxifen sensitivity of resistant cells. Further, combination treatment of breast cancer cells with both onapristone and IGF1R tyrosine kinase inhibitor AEW541 was more effective than either agent alone. In summary, unliganded PR-B enhanced proliferative responses to estradiol and IGF1 via scaffolding of ER-α/PELP1/IGF1R-containing complexes. Our data provide a strong rationale for targeting PR in combination with ER and IGF1R in patients with luminal breast cancer. Overall design: The study contains 4 different sample groups measured in triplicate, for a total of 12 individual samples (12 arrays). From parental MCF7 human breast cancer cell lines, we created two stable clones expressing either (1) an empty vector (pSG5) or (2) the wild type progesterone receptor isoform B (pSG5-PR-B). These two cell lines were treated with either (1) vehicle control (ethanol) or (2) estradiol 10e-7 M (E2) for 6 hours before total RNA harvest. Thus, the experiment contains two cell lines, and two treatments (4 sample groups) treated and analyzed in triplicate (12 microarrays). Standard Illumina HT-12v4 chip controls were used during hybridization.
Project description:IGF1 and IGF1 receptors (IGF1R) are present in the adult heart and have been shown to be essential for myocardial performance. Insulin-like growth factor 1 (IGF1) is produced in numerous tissues particularly by the liver in response to growth hormone stimulation and is an important factor in the regulation of post-natal growth and development. We have generated and characterized transgenic mice over-expressing the IGF1R. We crossed IGF1R transgenic mice with dominant negative (dn)PI3K (p110) and with constitutively active (ca)PI3K(p110) transgenic mice. Expression profiling was performed on the ventricles of IGF1R, IGF1R-caPI3K, IGF1R-dnPI3K, caPI3K, dnPI3K transgenic female mice at 3 months of age. Non-transgenic littermates were used as controls.