Radiation-induced DNA double-strand breaks produced in histone-depleted tumor cell nuclei measured using the neutral comet assay.

Removal of histones and other nuclear proteins greatly enhances the sensitivity of mammalian cells to DNA damage by ionizing radiation. We examined the possibility that the ease of dissociation of histones, or the association of other nuclear proteins with DNA, may differ between radioresistant and sensitive human tumor cells. Cells embedded in agarose were exposed to increasing salt concentrations prior to irradiation and examination using a microscopic gel electrophoresis method, the neutral comet assay. Induction of double-strand breaks increased by a factor of about 20 when cells of four human tumor cell lines, HT144 melanoma, HT29 adenocarcinoma, DU145 prostate carcinoma and U87 glioma, were exposed to 2 M NaCl prior to irradiation. Subtle differences in sensitivity to induction of double-strand breaks by radiation between cells of the four cell lines were also observed after extraction with 0.7-1.1 M NaCl; however, no correlation with radiosensitivity was apparent. While a significant number of histone and non-histone proteins are present after extraction with 1.2 M NaCl, these proteins apparently have only a minor influence on radiosensitivity. However, if they are allowed to remain with DNA during electrophoresis, about 15 times more strand breaks are required to produce a similar amount of DNA migration in both DU145 and HT144 cells. These results suggest that the association between proteins and DNA within the nucleus, as probed by extraction with sodium chloride, does not help to explain differences in intrinsic radiosensitivity among cells of these diverse tumor cell lines.