Differences in gene expression in A2780C20 vs. A2780
ABSTRACT: Differences in gene expression in A2780C20 vs. A2780 and A2780 treated with temsirolimus at 24-hr and 48-hr. The A2780 are human ovarian carcinoma cellls were purchased to the European Collection of Cell Cultures (ECACC). The A2780CP20 are cisplatin resisatnt cells derived of the A2780 cells. A2780CP20 cells were generated by Dr. Robert Ozols (J. Natl. Cancer Inst. 84, 264-267 (1992) by treatment of A2780 cells with incresing concentrations of cisplatin. The goal of this study is to determine whether the cisplatin and temsirolimus resistance are co-regulated. The objectives of this study are to identify additional genes differentially expressed in cisplatin sensitive and cisplatin resistant ovarain cancer cells and the changes in gene expreession upon temsirolimus treatment of A2780 cells. RNA was isolated from A2780, A2780CP20, A2780 plus temsirolimus 24-hr, and A2780 plus temsirolimus 48-hr. Labelled samples were hybridized to Affymetrix GeneChip Gene 1.0 ST Human Arrays.
Project description:A2780 ovarian cancer cells and a cisplatin resistant derivate of A2780 cells, obtained from ECACC, UK, No. 93112519 [A2780] and No. 93112517 [A2780 cis] were seeded out in T-75 flasks at a density of 2x106 for A2780sens and 3x106 for A2780cis cells in 15 ml of medium and preincubated overnight. Medium was removed and 15 ml fresh medium (37°C) with different concentrations of cisplatin, liposomal cisplatin or empty liposomes were added and incubated for 72 h at 37°C in a 5% CO2 incubator. In case of A2780sens cells, 1.72 µM cisplatin (IC50 concentration) and in case of A2780cis cells 8.94 µM cisplatin (IC50 concentration) were added. Liposomal formulations contained equal cisplatin concentrations. Empty liposomes were added in the same concentration as the liposomal cisplatin, to analyze the impact of liposomal lipids (A2780sens: 0.80µmol lipid, A2780cis: 4.15 µmol lipid). After incubation, medium was removed and cells were washed thrice with 10 ml PBS. 1 ml RLT-buffer was added and cells lysates were stored at -80°C until RNA extraction.
Project description:Differences in gene expression in A2780C20 vs. A2780 and A2780 treated with temsirolimus at 24-hr and 48-hr. The A2780 are human ovarian carcinoma cellls were purchased to the European Collection of Cell Cultures (ECACC). The A2780CP20 are cisplatin resisatnt cells derived of the A2780 cells. A2780CP20 cells were generated by Dr. Robert Ozols (J. Natl. Cancer Inst. 84, 264-267 (1992) by treatment of A2780 cells with incresing concentrations of cisplatin. The goal of this study is to determine whether the cisplatin and temsirolimus resistance are co-regulated. The objectives of this study are to identify additional genes differentially expressed in cisplatin sensitive and cisplatin resistant ovarain cancer cells and the changes in gene expreession upon temsirolimus treatment of A2780 cells. Overall design: RNA was isolated from A2780, A2780CP20, A2780 plus temsirolimus 24-hr, and A2780 plus temsirolimus 48-hr. Labelled samples were hybridized to Affymetrix GeneChip Gene 1.0 ST Human Arrays.
Project description:Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, subsequently resulting in a poor long-term prognosis. To model the onset of drug resistance, and investigate the DNA methylation alterations associated with cisplatin resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation microarray analyses. We treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation analyses by differential methylation hybridization (DMH) using a customed 44K promoter CGI microarray.
Project description:Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared to A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes PKM, GPI, Aldolase, LDH, and PGK were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, while vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step towards a comprehensive understanding of drug resistance in ovarian cancer.
Project description:This SuperSeries is composed of the following subset Series: GSE28646: Gene expression profiling in A2780, CP70 and CP70 following Decitabine and/or PXD101 treatment GSE28647: Genome-wide methylation profiling identifies candidate DNA methylation drivers of acquired cisplatin resistance in ovarian cancer. Refer to individual Series
Project description:Cisplatin and other platinum-based drugs are widely used in the treatment of ovarian cancer, but most patients acquire the drug resistance that greatly compromises the efficacy of drugs. Understanding the mechanism of drug resistance is important for finding new therapeutic approaches. In the present study, we found that the expression of vimentin was down-regulated in drug-resistance ovarian cancer A2780-DR and SKOV-3/DDP cells compared to the drug sensitive A2780 and SKOV-3 cells. Overexpression of vimentin in A2780-DR cells markedly increased their sensitivity to cisplatin, whereas knockdown of vimentin in A2780 cells increased the resistance to cisplatin, demonstrating that vimentin plays an important role in cisplatin resistance. Quantitative proteomic analysis identified 95 differentially expressed proteins between A2780-VIM-KN and control cells, which involved in many cellular processes. Down-regulation of endocytic proteins and the up-regulation of exocytic proteins were proposed to contribute the decreased cisplatin accumulation in A2780-VIM-KN cells. Cancer stem cell markers were found to be up-regulated in A2780-VIM-KN cells which were more facile to form spheroids as compared to control cells. Our results also showed that down-regulation of vimentin increased the 14-3-3 mediated retention of Cdc25C in the cytoplasm, leading to inactivation of Cdk1 and the prolonged G2 arrest that allows the longer period of time for cells to repair cisplatin-damaged DNA. Taken together, the down-regulation of vimentin enhances cells’ resistance to cisplatin via mediating multiple cellular processes, suggesting that vimentin is a potential target for treatment of drug resistant ovarian cancer.
Project description:UCHL1 is related to apoptosis and proliferation in cancers and we sought to identify genes regulated by UCHL1 UCHL1 was knocked down in A2780 (A2780-shUCHL1). A2780-shUCHL1 and A2780-control were cultured in RPMI and total RNA was extracted and hybridizated on Affymetrix.GeneChip.HG-U133_Plus_2
Project description:Time-course and concentration-effect experiments with multiple time points and drug concentrations provide far more valuable information than experiments with just two design-points (treated vs. control), as commonly performed in most microarray studies. Analysis of the data from such complex experiments, however, remains a challenge. Here we present a semi-automated method for fitting time profiles and concentration-effect patterns, simultaneously, to gene expression data. The submodels for time-course included exponential increase and decrease models with parameters such as initial expression level, maximum effect, and rate-constant (or half-time). The submodel for concentration-effect was a 4-parameter Hill model. The method was applied to an Affymetrix HG-U95Av2 dataset consisting of 51 arrays. The specific study focused on the effects of two platinum drugs, cisplatin and oxaliplatin, on A2780 human ovarian carcinoma cells. Replicates were available at most time points and concentrations. Eighteen genes were selected; time-course and concentration-effect were modeled simultaneously. Comparisons of model parameters helped distinguish genes with different expression patterns between the two drug treatments. This overall paradigm can help in understanding the molecular mechanisms of the agents, and the timing of their actions. Experiment Overall Design: For the time-course experiments, samples were exposed to IC90 concentrations of oxaliplatin (32 μM) or cisplatin (25 μM) for 2 hours, and then allowed to grow in drug free medium. Cells were harvested and processed at the following times: before treatment, immediately after treatment (0 h), and at 2 h, 6 h, 16 h and 24 h after treatment. Control experiments were done following a similar design, but with exposure to drug-free medium. For the concentration-effect experiments, samples were treated with specific growth-inhibitory concentrations of cisplatin or oxaliplatin for 2 hours. The previously-estimated IC10, IC25, IC50, IC75 and IC90 concentrations (for 2 hr drug exposure; 72 hr total growth) for cisplatin and oxaliplatin growth inhibition of A2780 were 2.6, 4.0, 6.4, 12 and 25 μM and 2.8, 4.8, 8.5, 17 and 32 μM respectively. Cells were harvested and processed at 16 h after completion of treatment
Project description:Transcriptional profiling of human ovarian cancer cells comparing control vehicle-treated A2780 and OVCAR-3 (Human ovarian cancer cell lines) cells with A2780 and OVCAR-3 cells treated with 5μM LS-98 for 24 hours.Goal was to determine the effects of LS-98 compound on the global A2780 and OVCAR-3 cells gene expression. One-condition experiment, control vehicle-treated A2780 and OVCAR-3 vs. LS098 treated-A2780 and OVCAR-3 cells. Biological replicates: 2 replicates.
Project description:The taxanes are widely used in the treatment of breast and other cancers. While their cytotoxicity has been attributed to the induction of cell cycle arrest in mitosis through the stabilization of microtubules, we found that docetaxel promotes soluble tumor necrosis factor alpha (TNF-alpha production in MCF-7 breast tumor cells. TNF-alpha induces apoptotic cell death in a variety of cell types by binding to one of its receptors (TNFR1) which promotes death-inducing signaling complex (DISC) formation. Consistent with this view, we also report that selection of MCF-7 cells for survival in increasing concentrations of paclitaxel or docetaxel results in selection of drug-resistant variants that are resistant to TNF-alpha cytotoxicity. MCF-7 cells selected to 3-5 nM docetaxel produced >30-fold more TNF-alpha than control MCF-7CC cells but had strongly reduced levels of TNFR1. In contrast, expression of TNFR2 was unchanged, resulting in enhanced cell survival through the activation of the NF-kappaB p50 and p65 subunits. Gene expression profiling of docetaxel resistant MCF-7 cells compared to parental MCF-7 cells was performed for the changes of TNF related genes, and also confirmed in ovarian cell line A2780. Two docetaxel resistant cell lines of breast MCF-7 and ovarian A2780 were generated for gene expression profilling. Two colour microarray of Agilent whole human genome nucleotide arrays was conducted with two replicate of both forward and reverse labellings for MCF-7. Four arrays were used for this experiments. And it was four replicate of both forward and reverse labellings for A2780 cells. Eight arrays were used for this experiments