MEASUREMENT OF RADIATION-INDUCED DAMAGE IN HUMAN GLIOMA-CELLS WITH FLOW-CYTOMETRY

Using flow cytometry, we studied DNA supercoiling changes in human gli oma cell line SF-126 after irradiation. To release nucleoids (dehiston ized DNA in a supercoiled form attached to the nuclear matrix), cells were lysed in a high-salt buffer. Radiation-induced changes in nucleoi ds were measured by now cytometry as changes in forward light scatter. propidium iodide titration curves showed that rewinding of DNA superc oils in irradiated cells was inhibited. To optimize the experimental c onditions, we analyzed the effect of lysis time and nucleoid size dist ribution within the sample. Under optimal conditions, changes in nucle oids were detected after radiation doses as low as 0.5 Gy. The repair of radiation-induced damage in nucleoids followed biphasic kinetics; 5 0% of the damage was repaired within about 5 min, and the remainder wi thin about 30 min. Interestingly, irradiated S-phase cells showed less damage, as measured by this assay, than irradiated G(1)- or G(2)-phas e cells, which is consistent with the relative radioresistance of S-ph ase cells as measured with cell survival assays. Our findings show tha t flow cytometric measurement of supercoiling changes is a sensitive a nd relatively rapid method for quantitating radiation-induced damage i n individual cells.