Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide analysis of parental and wild-type SK-N-Be(2)C neuroblastoma cell lines as well as those co-treated with the histone deacetylase inhibitor SAHA

ABSTRACT: Analysis of varied biologic pathways in neuroblastoma SK-N-Be(2)C cells grown in doxorubicin and/or SAHA. We hypothesized that SK-N-Be(2)C cells undergo a mesenchymal change through mesenchymal change resulting in a more invasive as well as drug resistant phenotype, and that the mechanism of SAHAs effect on drug resistance may be revealed through this analysis. Total RNA was isolated from wild type, SAHA treated wild type cells, doxorubicin resistant, and SAHA treated doxorubicin resistant SK-N-Be(2)C cell lines in triplicate.

ORGANISM(S): Homo sapiens

SUBMITTER: Chunfa Jie

PROVIDER: E-GEOD-47669 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Objective: To assess the role of aldoketoreductases and other doxorubicin pharmacokinetic or pharmacogenomic genes in doxorubicin cytotoxicity, resistance, DNA binding activity, and subcellular localization, Methods: We conducted a whole genome microarray study to identify differences in between doxorubicin-sensitive MCF-7cc cells and doxorubicin-resistant MCF-7Dox2-12 cells in terms of their expression of genes related to doxorubicin pharmacokinetics or pharmacodynamics. Targets were then validated by pharmacologic inhibition in conjunction with drug metabolite profiling, drug localization, drug cytotoxicity, and drug DNA binding studies. Results: 2063 differentially expressed transcripts were identified, including 17% and 43% of genes or gene families associated with doxorubicin pharmacokinetics or pharmacodynamics (p values of significance of 0.05 and <0.0001, respectively). The largest changes in the expression of genes associated with doxorubicin pharmacokinetics and pharmacodynamics were chiefly among the aldo-keto reductases (AKRs) Akr1c2, Akr1c3 and Akr1b10 which convert doxorubicin to doxorubicinol. We observed that doxorubicinol exhibits dramatically reduced drug toxicity, reduced drug DNA-binding activity, and altered drug subcellular localization to lysosomes. Pharmacologic inhibition of these AKRs in MCF-7Dox2-12 cells restored drug cytotoxicity, and drug localization to the nucleus. Conclusion: These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledgebases to identify highly relevant genes associated with doxorubicin resistance. The products of one or more of these genes could effectively be shown to alter the drug’s properties, while inhibiting them restored drug DNA binding, cytotoxicity, and subcellular localization. Doxorubicin resistant cell lines of breast MCF-7 cells were generated for gene expression profilling. Two colour microarray of Agilent whole human genome nucleotide arrays was conducted with four labelling replicates of both forward and reverse labellings plus another set of 8 arrays with forward labelling. Sixteen arrays were used for this experiments. The co-cultured control cells MCF-7cc12 was generated by parallel selection process in the absence of drug.

Project description:Recently, more than a thousand large intergenic non-coding RNAs (lincRNAs) have been reported. These RNAs are evolutionarily conserved in mammalian genomes and thus presumably function in diverse biological processes. Here, we report the identification of lincRNAs that are regulated by p53. One of these lincRNAs (lincRNA-p21) serves as a repressor in p53-dependent transcriptional responses. Inhibition of lincRNA-p21 affects the expression of hundreds of gene targets enriched for genes normally repressed by p53. The observed transcriptional repression by lincRNA-p21 is mediated through the physical association with hnRNP-K. This interaction is required for proper genomic localization of hnRNP-K at repressed genes and regulation of p53 mediated apoptosis. We propose a model whereby transcription factors activate lincRNAs that serve as key repressors by physically associating with repressive complexes and modulating their localization to sets of previously active genes. Gene expression profiles of different p53 inducible mouse embryonic fibroblasts (p53LSL/LSL MEFs) across different time points after DNA damage. A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 transcriptional response. [1] MEF profiling: p53 LSL/LSL MEFs were treated with adenoCRE for p53 restoration or adenoGFP as control for 24h and treated with 500nM doxorubicin for the indicated times. [2] hnRNPKKD profiling: p53 LSL/LSL MEFs were treated with adenoCRE for p53 restoration for 24h, transfected with siRNA oligos targeting different transcripts for 24h and then treated with 100nM doxorubicin for 13h. [3] lincP21KD profiling: p53 LSL/LSL MEFs were treated with adenoCRE for p53 restoration for 24h, transfected with siRNA oligos targeting different transcripts for 24h and then treated with 100nM doxorubicin for 13h. [4] RAS profiling: For p53 restoration, cultured tumor cell lines were incubated with 500nM 4-hydroxytamoxifen for the indicated times.

Dataset Information

Genome-wide analysis of parental and wild-type SK-N-Be(2)C neuroblastoma cell lines as well as those co-treated with the histone deacetylase inhibitor SAHA

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