ABSTRACT: Profile of RNA expression in a C-33A cell line derived from an HPV negative cervical carcinoma in the presence or absence of HPV1 E2 expression. mRNA profiles of C-33A cells in presence or absence of HPV1 E2 expression were generated by deep sequencing using Illumina GAIIx. Two samples (no replicates). One control and one experimental.
Project description:Profile of RNA expression in a C-33A cell line derived from an HPV negative cervical carcinoma in the presence or absence of HPV1 E2 expression. Overall design: mRNA profiles of C-33A cells in presence or absence of HPV1 E2 expression were generated by deep sequencing using Illumina GAIIx. Two samples (no replicates). One control and one experimental.
Project description:The emergence of anti-estrogen resistance in breast cancer is an important clinical phenomenon affecting long-term survival in this disease. Identifying factors that convey cell survival in this setting may guide improvements in treatment. Estrogen (E2) can induce apoptosis in breast cancer cells that have been selected for survival after E2 deprivation for long periods (MCF-7:5C cells), but the mechanisms underlying E2-induced stress in this setting have not been elucidated. Here, we report that the c-Src kinase functions as a key adapter protein for the estrogen receptor (ER, ESR1) in its activation of stress responses induced by E2 in MCF-7:5C cells. E2 elevated phosphorylation of c-Src, which was blocked by 4-hydroxytamoxifen (4-OHT), suggesting that E2 activated c-Src through the ER. We found that E2 activated the sensors of the unfolded protein response (UPR), IRE1α (ERN1) and PERK kinase (EIF2AK3), the latter of which phosphorylates eukaryotic translation initiation factor-2α (eIF2α). E2 also dramatically increased reactive oxygen species production and upregulated expression of heme oxygenase HO-1 (HMOX1), an indicator of oxidative stress, along with the central energy sensor kinase AMPK (PRKAA2). Pharmacologic or RNA interference-mediated inhibition of c-Src abolished the phosphorylation of eIF2α and AMPK, blocked E2-induced ROS production, and inhibited E2-induced apoptosis. Together, our results establish that c-Src kinase mediates stresses generated by E2 in long-term E2-deprived cells that trigger apoptosis. This work offers a mechanistic rationale for a new approach in the treatment of endocrine-resistant breast cancer. MCF-7:5C cells were treated with vehicle (0.1% EtOH) as control, E2 (10-9mol/L), 4-OHT (10-6mol/L), E2 (10-9mol/L) plus 4-OHT (10-6mol/L), PP2 (5x10-6mol/L), and E2 (10-9mol/L) plus PP2 (5x10-6mol/L) respectively for 72 hours.
Project description:Profiling of bone marrow-derived erythroid progenitor cells at E1 (CFU-e), E2 (proerythroblasts), and E3 (maturing erythroblast) stages of development under stress erythropoiesis conditions and in response to EPO challenge. MACS-isolated E1, E2, and E3 stage EPCs were cultured in SP34ex media for 6 hrs in the absence of hematopoietic growth factors and the presence of insulin (to enforce survival and anti-apoptotic effects) and then exposed to rhEPO for 90 minutes. Gene expression analysis was performed using Affymetrix Mouse Genome 430 2.0 arrays; microarray data were analyzed using Bioconductor for R.
Project description:Estrogen receptor-α (ERα) is an important driver of breast cancer and is the target for hormonal therapies, anti-estrogens and drugs that limit estrogen biosynthesis (aromatase inhibitors). Mutations in the ESR1 gene identified in metastatic breast cancer provide a potential mechanism for acquired resistance to hormone therapies. We have used CRISPR-Cas9 mediated genome editing in the MCF-7 breast cancer cell line, generating MCF-7-Y537S. MCF-7-Y537S cells encode a wild-type (tyrosine 537) and a mutant (serine 537) allele. Growth of the line is estrogen-independent and expression of ERα target genes is elevated in the absence of estrogen. ER ChIP-seq was carried out to map global ERα binding sites in the presence and absence of estrogen. RNA-seq following estrogen treatment was used for gene expression analysis. We show that expression of ER target genes and ER recruitment to ER binding regions is similar in MCF-7 and MCF-7-Y537S cells, except that ER recruitment to DNA and expression of ER target genes is frequently elevated in the absence of estrogen. Hormone depleted MCF7 Luc or Y537S cells were treated with 10nM E2 or ethanol, as vehicle control, for 8 hours, with 3 replicates (2 replicates for Y537S + E2). RNA-seq was carried out using Illumina Hiseq 2500.
Project description:MCF-7 is an estrogen receptor-positive breast cancer cell line. This experiment is designed to study (1) the effect of estradiol (E2) exposure and (2) lysine methyltransferase 2B (KMT2B) knockdown in MCF-7 cells. Cells were grown for 72 hours prior to treatment with vehicle or 10 nM E2 for 4 and 24 hours. Additionally, to assess the effect of KMT2B knockdown, MCF-7 cells were transfected with KMT2B targeting siRNA or scrambled control siRNA in the absence or presence of E2. RNA were isolated using Trizol and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 array.
Project description:To explore the global mechanisms of estrogen-regulated transcription, we used chromatin immunoprecipitation coupled with DNA microarrays to determine the localization of RNA polymerase II (Pol II), estrogen receptor alpha (ERalpha), steroid receptor coactivator proteins (SRC), and acetylated histones H3/H4 (AcH) at estrogen-regulated promoters in MCF-7 cells with or without estradiol (E2) treatment. In addition, we correlated factor occupancy with gene expression and the presence of transcription factor binding elements. Using this integrative approach, we defined a set of 58 direct E2 target genes based on E2-regulated Pol II occupancy and classified their promoters based on factor binding, histone modification, and transcriptional output. Many of these direct E2 target genes exhibit interesting modes of regulation and biological activities, some of which may be relevant to the onset and proliferation of breast cancers. Our studies indicate that about one-third of these direct E2 target genes contain promoter-proximal ERalpha-binding sites, which is considerably more than previous estimates. Some of these genes represent possible novel targets for regulation through the ERalpha/AP-1 tethering pathway. Our studies have also revealed several previously uncharacterized global features of E2-regulated gene expression, including strong positive correlations between Pol II occupancy and AcH levels, as well as between the E2-dependent recruitment of ERalpha and SRC at the promoters of E2-stimulated genes. Furthermore, our studies have revealed new mechanistic insights into E2-regulated gene expression, including the absence of SRC binding at E2-repressed genes and the presence of constitutively bound, promoter-proximally paused Pol IIs at some E2-regulated promoters. These mechanistic insights are likely to be relevant for understanding gene regulation by a wide variety of nuclear receptors. Keywords: MCF7 cells, E2, Estrogen, RNA, ER, RNA Polymerase II, SRC, Acetylated histones, ChIP-chip Overall design: See Materials and Methods in the associated publication for more details
Project description:Estrogens(E2) are important steroid hormones that regulate differentiation, proliferation, and apoptosis in hormone-dependent breast cancer.In order to detect the E2-dependent transcription program associated with the observed cell cycle response, we analyzed the effect of H2ac knockdown on MCF-7 gene expression using microarray. Interestingly, we noticed that 51% of the E2-upregulated genes are down-regulated by depletion of H2ac. The data also show that H2ac regulated E2-dependent genes through E2-induction signaling pathway. MCF-7 cell line was transfected with scrambled or H2ac siRNA in the absence or persence of E2.