1. Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes
- Author
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Michael R. Dohn, John T. Barrett, Patricia V. Schoenlein, Ana M. Sanchez, J. McCoy, D. Y. Hou, and A. Kulharya
- Subjects
Cancer Research ,Genes, myc ,In situ hybridization ,Extrachromosomal circular DNA ,Radiation Tolerance ,Proto-Oncogene Proteins c-myc ,chemistry.chemical_compound ,Extrachromosomal DNA ,Tumor Cells, Cultured ,Medicine ,Double minute ,Humans ,Radiology, Nuclear Medicine and imaging ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Gene ,Tumor Stem Cell Assay ,Cell Line, Transformed ,Radiation ,Micronucleus Tests ,business.industry ,Dose fractionation ,Gene Amplification ,Dose-Response Relationship, Radiation ,Flow Cytometry ,Molecular biology ,Oncology ,chemistry ,Cell culture ,Drug Resistance, Neoplasm ,Dose Fractionation, Radiation ,Genes, MDR ,business ,DNA ,Gene Deletion - Abstract
Purpose: To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines. Methods and materials: The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR 1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy. To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR 1 and MYCC -specific cosmid DNA probes. In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels. Dose–response assays to follow loss of function of the MDR 1 gene from KB-C1 cells were also performed. Results: A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation. Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells. Conclusions: Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype. Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei.
- Published
- 2003