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Expression data from hormone-starved MCF7/BUS cell culture

ABSTRACT: "To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics, and employed it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed 3~5 times larger numbers of genes than it induced, whereas at higher concentrations (30-100 pM) it induced 1.5~2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, TGF-? and SDF-1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, dependent on the E2 concentration especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dose-dependent aspects of E2 actions. (paper abstract) Keywords: Dose Response, Time Course MCF7/BUS cells were exposed to varying concentrations of 17beta-estradiol (E2) for 48 hours. Experiments were repeated five times independently. In another experiment, MCF7/BUS cells were subjected to hormone starvation. At day 0, 1, and 2, RNA samples were isolated from cell culture. Experiments were repeated three times independently."

ORGANISM(S): Homo sapiens

SUBMITTER: Coser KR

PROVIDER: S-ECPF-GEOD-4668 | biostudies-other | 2003 Nov

REPOSITORIES: biostudies-other

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Project description:To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics, and employed it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed 3~5 times larger numbers of genes than it induced, whereas at higher concentrations (30-100 pM) it induced 1.5~2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, TGF-? and SDF-1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, dependent on the E2 concentration especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dose-dependent aspects of E2 actions. (paper abstract) Keywords: Dose Response, Time Course MCF7/BUS cells were exposed to varying concentrations of 17beta-estradiol (E2) for 48 hours. Experiments were repeated five times independently. In another experiment, MCF7/BUS cells were subjected to hormone starvation. At day 0, 1, and 2, RNA samples were isolated from cell culture. Experiments were repeated three times independently.

Project description:We propose the hypothesis that loss of estrogen receptor function which leads to endocrine resistance in breast cancer, also results in de-differentiation from an epithelial to a mesenchymal phenotype that is responsible for increased aggressiveness and metastatic propensity. siRNA mediated silencing of the estrogen receptor in MCF7 breast cancer cells resulted in estrogen/tamoxifen resistant cells (pII) with altered morphology, increased motility with rearrangement and switch from an actin to a vimentin based cytoskeleton, and ability to invade simulated components of the extracellular matrix. Phenotypic profiling using an Affymetrix Human Genome U133 plus 2.0 GeneChip indicated fold changes ≥ 3 in approximately 2500 identifiable unique sequences, with about 1270 of these being up-regulated in pII cells. Changes were associated with genes whose products are involved in cell motility, loss of cellular adhesion and interaction with the extracellular matrix. Selective analysis of the data also showed a shift from luminal to basal cell markers and increased expression of a wide spectrum of genes normally associated with mesenchymal characteristics, with consequent loss of epithelial specific markers. Over-expression of several peptide growth factors and their receptors are indicative of an increased contribution to the higher proliferative rates of pII cells as well as aiding their potential for metastatic activity. Signalling molecules that have been identified as key transcriptional drivers of epithelial to mesenchymal transition were also found to be elevated in pII cells. We suggest that these data support our hypothesis that induced loss of estrogen receptor in previously antiestrogen sensitive cells is a trigger for the concomitant loss of endocrine dependence and onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type. Inhibition of this transition through targeting of specific mediators may be a useful supplementary strategy to circumvent the effects of loss of endocrine sensitivity. We used microarrays to detail the global programme of gene expression underlying Epithelial to mesenchymal transition and identified distinct classes of regulated genes during this process. MCF7 cell line was selected before and after (PII) silencing of Estrogen receptor for RNA extraction and hybridization on Affymetrix expression microarrays.

Project description:Multiple gene expression studies have demonstrated that breast cancer biological diversity is associated with distinct transcriptional programs. Transcription factors, because of their unique ability to coordinate the expression of multiple genes, are speculated to play a role in generating phenotypic plasticity associated with cancer progression including acquired drug resistance. Combinatorial libraries of artificial zinc-finger transcription factors (ZF-TFs) provide a robust means for inducing and understanding various functional components of the cancer phenotype. Herein, we utilized combinatorial ZF-TF library technology to better understand how breast cancer cells acquire resistance to a fulvestrant, a clinically important anti-endocrine therapeutic agent. We isolated six ZF-TF library members capable of inducing stable, long-term anti-endocrine drug-resistance in two independent estrogen receptor positive breast cancer cell lines. Comparative gene expression profile analysis of the ZF-TF-transduced breast cancer cell lines revealed a 72-gene cluster that constituted a common signature for the fulvestrant-resistance phenotype. Pathway enrichment-analysis of gene expression data revealed that the ZF-TF-induced fulvestrant resistance is associated with an estrogen receptor negative-like gene set and four unique myb-regulated gene sets. Furthermore, we identified a set of genes strongly expressed in the ZF-TF-induced fulvestrant-resistant cells that was correlated with a lower probability of distant metastasis-free or death-from-relapse-free survival of breast cancer patients. MCF7-R73 cells, a monoclonal MCF7 subline that is highly sensitive to fulvestrant-induced cytocidal activity, underwent retroviral transduction with the zinc finger transcription factor (ZF-TF) activator library or with a control plasmid encoding only the NF-kB p65 activation domain. Both populations of cells were enriched for transduced cells by selecting for growth in puromycin and for fulvestrant-resistant cells by selecting with 100 nM fulvestrant. After 6 weeks of continuous treatment with fulvestrant, hundreds of drug-resistant colonies emerged from the population of cells infected with the ZF-TF activator library. By contrast, as expected, the control MCF7cell line transduced by NF-kB p65-only underwent massive cell death resulting in the complete absence of resistant colonies. DNA encoding the zinc-finger arrays was rescued by PCR from genomic DNA of pooled fulvestrant-resistant cells. The sequences of the ZF-TFs were determined and 46 unique ZF-TF clones identified. These 46 unique ZF-TFs were re-cloned into the retroviral vector and converted into clonal virus stocks that were used to transduce MCF7-R73 cells. These 46 retrovirally transduced cell populations were then challenged with fulvestrant. As compared with the control MCF-238 cells, MCF7-R73 cells transduced with six unique ZF-TFs demonstrated survival and growth in the presence of 100nM fulvestrant.

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Expression data from hormone-starved MCF7/BUS cell culture

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