Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human colon cancer C85 cells upon prolonged exposure to methotrexate

ABSTRACT: The aim of the experiment was to identify the genes whose expression was upregulated at the maintenance phase of premature senescence attained after exposure of human colon cancer C85 cells (or CCCL-1) to 1 micromolar methotrexate for 5 days. Gene expression pattern of C85 cells at the senescence initiation phase attained after exposure to 1 micromolar methotrexate for 48 h, was used as a referral system.

ORGANISM(S): Homo sapiens

SUBMITTER: Magdalena Dabrowska

PROVIDER: E-MTAB-7205 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We investigated the influence of genome position on propensity to amplify. First, we integrated a mutant form of DHFR into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells. We observed site specific differences in methotrexate sensitivity, organization of amplicons and amplification frequency. One site was uniquely associated with a significantly enhanced propensity to amplify and recurrent amplicon boundaries, possibly implicating a rare folate sensitive fragile site in initiating amplification. Hierarchical clustering of gene expression patterns and subsequent gene enrichment analysis revealed two clusters differing significantly in expression of MYC target genes independent of integration site. We introduced a mutant form of DHFR (L22F), which confers greater resistance to methotrexate than the wild type (endogenous) gene into HCT116+chr3 cells and isolated independent clones containing DHFR* at different positions in the genome and identified genome sequences flanking the integration site of DHFR* using inverse PCR. For further analysis, we selected only clones, which were considered to have a single insertion of DHFR* by inverse PCR (13 independent insertion sites). The individual insertion site clones were further characterized with respect to genome copy number profiles. To select methotrexate resistant colonies, we exposed cells to a concentration of methotrexate that was three to four times the IC-50 for each integration site. Genomic copy number profiles were obtained for isolated resistant colonies (GSE6262) by using UCSF HumArray platform (GPL4421). Twelve methotrexate resistant colonies (four different integration sites) were selected for microarray analysis of gene expression at the mRNA level. The untreated integration site clone was used as the reference (Cy5 labeled cDNA) for each of the hybridizations with its respective resistant colonies (Cy3 labeled cDNA). Hybridizations were carried out on arrays of printed long oligonucleotides (70mers) containing 21,000 elements (Operon V2.0, printed in J. David Gladstone Institutes, Genomics Core Laboratory).

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Transcription profiling of human colon cancer C85 cells upon prolonged exposure to methotrexate

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