Heterogeneity in Radiation-Induced DNA Damage and Repair in Tumor and Normal Cells Measured Using the "Comet" Assay

Abstract

A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.

The bacterial cytolethal distending toxin (CDT) triggers a G2 cell cycle checkpoint in mammalian cells without preliminary induction of DNA strand breaks

“…The occurrence of early DNA strand breaks in cells exposed to CDT was investigated by using the single cell gel electrophoresis assay, also called the`comet assay'. This test was used in alkaline conditions, a procedure previously reported to detect both single and double strand breaks (Singh et al, 1988;Olive et al, 1990). As expected, etoposide produced DNA strand breaks according to a dose-related response ( Figure 4b).…”

“…This hypothesis was ruled out by the absence of detectable strand breaks in the comet assay (Figure 4) and by the fact that CDT, in contrast to etoposide, did not interfere with the progression of cells through S phase ( Figure 5). The comet assay under alkaline conditions is reported to allow the detection of both single and double strand breaks (Singh et al, 1988;Olive et al, 1990). Compared to other available tests, it allows microscopic individual cell evaluation of DNA damage and, following image analysis, lends itself to quantitative measurement using the tail moment criterion.…”

“…Tail moment accounts for DNA damage. It was de®ned as the product of the percentage of DNA in the comet tail and of the distance between the means of the head and tail DNA distributions (Olive et al, 1990).…”

A p53-independent damage-sensing mechanism that functions as a checkpoint at the G 1 /S transition in Chinese hamster ovary cells

“…Thus, it seems unlikely that every origin in the cell could be directly down-regulated by a cis-acting structural hindrance to initiation. We consider it more likely that this regulatory mechanism(s) involves a trans-acting signal transduction pathway(s), and compelling evidence for this proposal has been obtained (35)(36)(37). In combination with data showing that the repair machinery is activated immediately after DNA damage (29,30), we propose that this G 1 ͞S checkpoint may represent a major control mechanism connecting the replication machinery with damage-sensing and repair pathways.…”

“…One operates at START in mid-G 1 and is apparently mediated by the RAD9 gene product (37,38). A second checkpoint functions near the G 1 ͞S transition and acts between the cell cycle steps effected by the DBF4 and CDC7 gene products (36,37). Interestingly, a defect in the G 1 ͞S checkpoint in yeast dramatically reduces cell survival rates after a radiation challenge (36,37).…”