Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes.
OBJECTIVE
To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines.


METHODS
The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 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 MDR1 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 MDR1 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.
Schoenlein
Patricia V
PV
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA. pschoel@mail.mcg.edu
Barrett
John T
JT
Kulharya
A
A
Dohn
Michael R
MR
Sanchez
Ana
A
Hou
D-Y
DY
McCoy
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D002461:Cell Line, Transformed / Q000187:drug effects / Q000528:radiation effects; D019583:Dose Fractionation, Radiation; D004307:Dose-Response Relationship, Radiation; D019008:Drug Resistance, Neoplasm / Q000235:genetics / Q000528:radiation effects*; D005434:Flow Cytometry; D005784:Gene Amplification; D017353:Gene Deletion; D019450:Genes, MDR / Q000187:drug effects / Q000528:radiation effects*; D016259:Genes, myc / Q000187:drug effects / Q000528:radiation effects*; D006801:Humans; D015162:Micronucleus Tests; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis*; D011836:Radiation Tolerance / Q000187:drug effects / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000528:radiation effects; D014410:Tumor Stem Cell Assay
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016271:Proto-Oncogene Proteins c-myc