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 se nsitivity of mammalian cells to DNA damage by ionizing radiation. We e xamined 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 aga rose were exposed to increasing salt concentrations prior to irradiati on 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 m elanoma, 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 appa rent. While a significant number of histone and non-histone proteins a re 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 mor e strand breaks are required to produce a similar amount of DNA migrat ion in both DU145 and HT144 cells. These results suggest that the asso ciation between proteins and DNA within the nucleus, as probed by extr action with sodium chloride, does not help to explain differences in i ntrinsic radiosensitivity among cells of these diverse tumor cell line s. (C) 1995 by Radiation Research Society