RELATIONSHIPS BETWEEN THE CYTOTOXIC EFFECTS OF RESTRICTION ENDONUCLEASES AND RADIATION ON MAMMALIAN-CELLS

Wild-type Chinese hamster ovary (CHO) cells, a radiosensitive mutant C HO line (xrs6), and two human cervical carcinoma cell lines, MS751 and ME180, differ in sensitivity to ionizing radiation with surviving fra ctions at 2 Gy (SF2) of 0.84, 0.06, 0.90 and 0.24 respectively. Restri ction endonucleases (REs) were introduced into the cells by treatment with streptolysin O (SLO) and the effects of this on clonogenic cell s urvival were compared. A comparison was made of REs inducing either bl unt- (AluI) or cohesive-ended (Sau3AI) double-strand breaks. Whilst MS 751 cells were resistant to the effects of both REs, AluI caused signi ficantly greater cell killing than Sau3AI in the other three lines (P < 0.05 for all). Both ME180 and xrs6 were significantly more sensitive to REs than their radioresistant counterparts, MS751 and CHO (P < 0.0 5 for both). In order to investigate the effect of DNA methylation on dsb induction, the isoschizomers MspI and HpaII (cohesive-ended dsb in ducers) were introduced into the cell lines. Both REs recognize the sa me sequence but HpaII: cannot cleave if the internal cytosine is methy lated. MS751 was also resistant to the effects of both of these enzyme s and MspI was more cytotoxic than HpaII in the other three lines (p < 0.03 for all). The differential sensitivity to the two REs was more m arked in the radiosensitive cell lines, suggesting that there may be a greater degree of DNA methylation in radiosensitive cells. The variat ion in sensitivities to REs between the cell lines could not be explai ned in terms of differences in cell poration following SLO treatment b ecause, although MS75 1 was resistant to SLO (25% of cells porated), t he other three lines showed the same level of cell poration (98% of ce lls). With these four cell lines, there was a significant correlation between sensitivity to RE and radiosensitivity for AluI, SauSAI and Ms pI but not for HpaII.