In
tumor specimens such as those from
neuroblastoma, ovarian, and lung
carcinoma patients, the prevalence of extrachromosomal
circular DNA molecules harboring amplified genes has been well established. In some cases, the amplified genes have been identified as oncogenes, and their increased expression appears to contribute to the maintenance and progression of the
malignancy. The aim of this study was to investigate the effect of fractionated
radiation treatment, given in daily doses similar to those administered clinically, on the stability of extrachromosomal
circular DNA molecules in
cancer cells. Our studies were conducted with multidrug-resistant KB cells, which harbor extrachromosomal copies of the multidrug resistance gene (MDR1) almost exclusively on
circular DNA molecules of approximately 750 and 1500 kb pairs. This size range is representative of extrachromosomal
circular DNA molecules that have been shown to harbor amplified oncogenes in vivo. Exponentially growing MDR KB cells were exposed to 1400 and 2800 cGy ionizing radiation administered in 7 and 14 fractions, respectively, at 200 cGy per fraction/day. A statistically significant decrease in MDR1 extrachromosomal gene copy number was reproducibly detected in the irradiated cells compared with unirradiated cells passaged for the duration of the experiment in the absence of
radiation treatment. This decrease was accompanied by a reduction in multidrug resistance and in
P-glycoprotein levels, as determined by clonogenic dose-response assays and Western analyses, respectively.
P-glycoprotein is a multidrug transporter encoded by the MDR1 gene. Fluorescence in situ hybridization studies further determined that extrachromosomal
circular DNA loss correlated to the entrapment of these
DNA molecules in radiation-induced micronuclei. These results indicate that radiation-induced loss of extrachromosomally amplified genes from
tumor cells via their entrapment in micronuclei contributes to the improved therapeutic response observed for some
cancers.