Use of fluorescence in situ hybridization (fish) to study chromosomal damage induced by radiation and bromodeoxyuridine in human colon cancer cells

Purpose:Although the thymidine analog radiation sensitizer bromodeoxyuridine (BrdUrd) increases radiation-induced chromosomal aberrations, it is not known whether these aberrations are uniformly distributed among chromosomes. Using fluorescence in situ hybridization, we carried out a study to test the hypothesis that BrdUrd-induced radiosensitization may be mediated by nonuniform chromosomal damage. Methods and Materials:Log phase HT29 human colon cancer cells were exposed to 10 μM BrdUrd (or media alone) for one cell cycle, and the GI cells were separated by centrifugal elutriation. Half of the control and BrdUrd samples were irradiated with 8 Gy. Cells were then incubated for 24-28 h, and metaphase spreads were prepared. Fluorescence in situhybridization was performed using paint probes for chromosomes 1 and 4. Results:We found that radiation induced 0.20 aberrations per chromosome in chromosome 4. Based on the ratio of the relative lengths of chromosome 1-4 (1.34), it was predicted that chromosome 1 would have ≈ 0.26 aberrations per chromosome. However, we observed 0.39 aberrations per chromosome 1, which was significantly greater than the predicted (p < 0.001by chi-square). Incubation with BrdUrd prior to irradiation significantly increased the aberrations found in chromosome 1 (by a factor of 1.4) and chromosome 4 (by a factor of 1.9) compared to radiation alone (p < 0.001for both chromosome 1 and 4). Conclusion:This study demonstrates that individual chromosomes in human colon cancer cells show significantly different rates of aberration after irradiation. Furthermore, the BrdUrd-mediated increase in radiation-induced chromosomal aberrations may not be uniform among chromosomes.