NHEK cells were plated at a density of 8 x 10 000/cm2 and the cell cultures were grown for 24 hours before addition of 2 mM N-Acetyl-L-Cystein. RNA obtained from cultures grown for 1, 12 and 24 hrs after NAC treatment were compared to RNA from untreated cells at the corresponding time points. I.e 1 hour NAC treated vs 1 hour untreated cells etc. Each EXTRACT represents an individual mRNA extraction and subsequent cDNA synthesis from a batch of totalRNA originating from one cellculture dish.
Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro.
Specimen part, Subject, Compound, Time
View SamplesCaco-2 human colon carcinoma cells were seeded at a density of 9 x 10 000 cells/cm2 and the cell cultures were grown for 24 hours before addition of 10 mM N-Acetyl-L-Cystein. RNA obtained from cultures grown for 1, 12 and 24 hrs after NAC treatment were compared to RNA from untreated cells at the corresponding time points. I.e 1 hour NAC treated vs 1 hour untreated cells etc. Each "SAMPLE" represents a biological replicate (i.e. separate cellcultures treated similarily) although I have given identical SAMPLE numbers in pairs.
Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro.
Specimen part, Cell line, Subject, Compound, Time
View SamplesThe cellular response to DNA damage is vital for maintaining genomic stability and preventing undue cell death or cancer formation. The DNA damage response (DDR), most robustly mobilized by double-strand breaks (DSBs), rapidly activates an extensive signaling network that affects numerous cellular systems, leading to cell survival or programmed cell death. A major component of the DDR is the widespread modulation of gene expression. We analyzed transcriptional responses to ionizing radiation (IR) in 5 human cell lines to elucidate the scope of this response and identify its gene targets. According to the mRNA expression profiles most of the responses were cell line-specific. Data analysis identified significant enrichment for p53 target genes and cell cycle-related pathways among groups of up-regulated and down-regulated genes, respectively.
Transcriptional modulation induced by ionizing radiation: p53 remains a central player.
Cell line, Time
View SamplesScreens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here an approach to screening for agents with epithelial CSC-specific toxicity. We implemented this method in a chemical screen and discovered compounds showing selective toxicity for breast CSCs. One compound, salinomycin, reduces the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates the ability to identify agents with specific toxicity for epithelial CSCs
Identification of selective inhibitors of cancer stem cells by high-throughput screening.
Specimen part
View SamplesAdipose stroma in the mouse mammary gland undergoes remodeling throughout the 5 stages of development. These include nulliparous (virgin;never been pregnant), pregnant, lactating, involuting and regressed.
Pregnancy-associated breast cancers are driven by differences in adipose stromal cells present during lactation.
Specimen part
View SamplesMicroarrays were used to determine relative global gene expression changes in WT and BRCA1-mutation carrier breast epithelium as well as tumors created from WT and BRCA1-mutation carrier breast epithelial cells.
Genetic predisposition directs breast cancer phenotype by dictating progenitor cell fate.
Specimen part, Disease, Disease stage
View SamplesTris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a high-production volume organophosphate flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) results in genome-wide alterations in methylation during cleavage (2 hpf) as well as epiboly delay or arrest (at higher concentrations) during late-blastula and early-gastrula (4-6 hpf). To determine whether these TDCIPP-induced effects were associated with impacts on the transcriptome, embryos were exposed to vehicle (0.1% DMSO) or 2 M TDCIPP from 0.75 hpf to 6 hpf, and total RNA was extracted from triplicate embryo pools per treatment and hybridized onto duplicate Affymetrix Zebrafish Gene 1.0 ST Arrays per RNA sample. Based on transcriptome-wide profiling, TDCIPP resulted in a significant impact on biological pathways involved in dorsoventral patterning and bone morphogenetic protein (BMP) signaling. Consistent with pathway-level responses, TDCIPP exposure also resulted in strongly dorsalized embryos by 24 hpf a phenotype that mimicked the effects of dorsomorphin, a potent and selective BMP inhibitor. Moreover, the majority of dorsalized embryos were preceded by epiboly arrest at 6 hpf. Our microarray data also revealed that the expression of sizzled (szl) a gene encoding a secreted Frizzled-related protein that limits BMP signaling was significantly decreased by nearly 4-fold at 6 hpf. Therefore, we used a splice-blocking morpholino to test the hypothesis that knockdown of szl phenocopies TDCIPP-induced delays in epiboly progression. Interestingly, contrary to our hypothesis, injection of szl MOs did not affect epiboly progression but, similar to chordin (chd) morphants, resulted in mildly ventralized embryos by 24 hpf. Overall, our findings suggest that TDCIPP-induced epiboly delay may be independent of szl expression and function, and that TDCIPP-induced dorsalization may similar to dorsomorphin be due to interference with BMP signaling during early zebrafish.
Tris(1,3-dichloro-2-propyl) phosphate disrupts dorsoventral patterning in zebrafish embryos.
Specimen part, Time
View SamplesA tissue like buccal mucosa (from cheek swabs) would be an ideal sample material for rapid, easy collection for testing of biomarkers as an alternative to blood. A limited number of studies, primarily in the smoker/oral cancer literature, address this tissue's efficacy for quantitative PCR or microarray gene expression analysis. In this study both qPCR and microarray analyses were used to evaluate gene expression in buccal cells. An initial study comparing blood and buccal cells from the same individuals looked at relative amounts of four genes. The RNA isolated from buccal cells was degraded but was of sufficient quality to be used with RT-qPCR to detect expression of specific genes. Second, buccal cell RNA was used for microarray-based differential gene expression studies by comparing gene expression between smokers and nonsmokers. The isolation and amplification protocol allowed use of 150-fold less buccal cell RNA than had been reported previously with human microarrays. We report here the finding of a small number of significant gene expression differences between smokers and nonsmokers, using buccal cells as target material. Additionally, Gene Set Enrichment Analysis confirmed that these genes were changing expression in the same pattern as seen in an earlier buccal cell study performed by another group. Our results suggest that in spite of a high degree of RNA degradation, buccal cells from cheek mucosa could be used to detect differential gene expression between smokers and nonsmokers. However the RNA degradation, increase in sample variability and microarray failure rate show that buccal samples should be used with caution as source material in expression studies.
Examining smoking-induced differential gene expression changes in buccal mucosa.
Specimen part
View SamplesGlucocorticoids (GC) and 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) are steroid hormones with anti-inflammatory properties with enhanced effects when combined. We previously showed that transcriptional response to GCs was correlated with inter-individual and inter-ethnic cellular response. Here, we profiled cellular and transcriptional responses to 1,25(OH)2 D3 from the same donors. We studied cellular response to combined treatment with GCs and 1,25(OH)2 D3 in a subset of individuals least responsive to GCs. We found that combination treatment had significantly greater inhibition of proliferation than with either steroid hormone alone. Overlapping differentially expressed (DE) genes between the two hormones were enriched for adaptive and innate immune processes. Non-overlapping differentially expressed genes with 1,25(OH)2 D3 treatment were enriched for pathways involving the electron transport chain, while with GC treatment, non-overlapping genes were enriched for RNA-related processes. These results suggest that 1,25(OH)2 D3 enhances GC anti-inflammatory properties through a number of shared and non-shared transcriptionally-mediated pathways.
Comparison of cellular and transcriptional responses to 1,25-dihydroxyvitamin d3 and glucocorticoids in peripheral blood mononuclear cells.
Sex, Age, Specimen part, Treatment
View SamplesAs part of the civil aviation safety program to define the adverse effects of ethanol on flying performance, we present results of our DNA microarray analysis of samples from a timecourse study of individuals given ethanol orally, and then evaluated by breathalyzer to monitor blood alcohol content (BAC). At five blood alcohol levels, T1-T5, blood was drawn such that the samples represented 0%, 0.04%, 0.08% BAC, and return to 0.04%, and 0.02% BAC. Microarray analysis showed that changes in gene expression could be detected across the time-course. We verified these expression changes by quantitative polymerase chain reaction (qPCR). Candidate target genes identified from the microarray analysis were clustered by expression change pattern, examined for shared functions and functional network membership. Five coordinately expressed groups were revealed and functional analysis showed shared transcription factor binding sites and functions for members of the clusters. These functions include protein synthesis and modification, expected for changes in gene expression, hematological and immune functions, expected for a blood sample, and pancreatic and hepatic function, expected as response to ethanol. The results provide a first look at changing gene expression patterns in blood during acute increase of ethanol concentration and its depletion due to metabolism or excretion and demonstrate that it is possible to detect significant changes in gene expression using total RNA isolated from whole blood. The analysis approach for this study can be utilized as part of a workflow to identify target genes by timecourse changes in gene expression that may affect pilot performance.
Microarray characterization of gene expression changes in blood during acute ethanol exposure.
Sex, Specimen part
View Samples