Genomics

Dataset Information

25

Genome Position and Gene Amplification


ABSTRACT: Amplifications, regions of focal high level copy number change, lead to overexpression of oncogenes or drug resistance genes in tumors. Their presence is often associated with poor prognosis; however the use of amplification as a mechanism for overexpression of a particular gene in tumors varies. To investigate the influence of genome position on propensity to amplify, 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. These studies suggest that genome context together with the particular challenges to genome stability experienced during the progression to cancer contribute to the propensity to amplify a specific oncogene or drug resistance gene, whereas the overall functional response to drug (or other) challenge may be independent of the genomic location of an oncogene. Keywords: Gene amplification, array CGH, chromosomal fragile sites Overall design: We introduced a mutant form of DHFR (L22F), which confers greater resistance to methotrexate than the wild type (endogenous) gene into HCT116+chr3 cells. We 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 (82 methotrexate resistant colonies).

INSTRUMENT(S): UCSF HumArray 3.1

SUBMITTER: Donna G Albertson  

PROVIDER: GSE6262 | GEO | 2007-10-08

SECONDARY ACCESSION(S): PRJNA100547

REPOSITORIES: GEO

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Publications


Amplifications, regions of focal high-level copy number change, lead to overexpression of oncogenes or drug resistance genes in tumors. Their presence is often associated with poor prognosis; however, the use of amplification as a mechanism for overexpression of a particular gene in tumors varies. To investigate the influence of genome position on propensity to amplify, we integrated a mutant form of the gene encoding dihydrofolate reductase into different positions in the human genome, challeng  ...[more]

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