Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal, NBS and AT fibroblasts.

PURPOSE

To study the relationship between cellular radiosensitivity and DNA damage measured by the comet assay.

MATERIALS AND METHODS

Experiments were performed with nine human fibroblast lines (six normal, one NBS, and two AT). Cellular radiosensitivity was determined by colony assay and DNA damage was assessed by the comet assay.

RESULTS

The cellular radiosensitivity of the fibroblast lines used covered a broad range with SF2 values varying between 1.3% and 53%. The comets analysed immediately after irradiation with doses up to 5 Gy showed marked differences among the cell lines; the relative initial tail moment at a dose of 5 Gy, ITM5, varied from 2.7+/-0.2 to 5.0+/-0.3. This variation was considered not to result from different numbers of DNA strand breaks induced but from differences in chromatin structure. There was an inverse correlation between SF2 and ITM5, i.e. radiosensitive cell lines exhibited a higher initial tail moment than radioresistant cell lines. In contrast, the repair kinetics measured with the comet assay for a dose of 2Gy followed by an incubation of up to 2h showed little variation and were found not to correlate with SF2. Repair kinetics as well as the amount of residual damage measured by this version of the comet assay were fairly similar to those measured by the alkaline unwinding technique and unlike that measured by neutral gel electrophoresis, indicating that this comet assay detects primarily single-strand breaks and alkali-labile sites, not double-strand breaks.

CONCLUSIONS

The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.