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Measurement of Radiation-induced Damage in Human Glioma Cells with Flow Cytometry¹

Brain Tumor Research Center of the Department of Neurological Surgery [J. W., K. L., D. F. D.], Department of Radiation Oncology [D. F. D.], and the Laboratory for Cell Analysis at Department of Laboratory Medicine [W. H.], School of Medicine, University of California, San Francisco, California 94143

Using flow cytometry, we studied DNA supercoiling changes in human glioma cell line SF-126 after irradiation. To release nucleoids (dehistonized DNA in a supercoiled form attached to the nuclear matrix), cells were lysed in a high-salt buffer. Radiation-induced changes in nucleoids were measured by flow cytometry as changes in forward light scatter. Propidium iodide titration curves showed that rewinding of DNA supercoils in irradiated cells was inhibited. To optimize the experimental conditions, we analyzed the effect of lysis time and nucleoid size distribution within the sample. Under optimal conditions, changes in nucleoids were detected after radiation doses as low as 0.5 Gy. The repair of radiation-induced damage in nucleoids followed biphasic kinetics; 50% of the damage was repaired within about 5 min, and the remainder within about 30 min. Interestingly, irradiated S-phase cells showed less damage, as measured by this assay, than irradiated G₁- or G₂-phase cells, which is consistent with the relative radioresistance of S-phase cells as measured with cell survival assays. Our findings show that flow cytometric measurement of supercoiling changes is a sensitive and relatively rapid method for quantitating radiation-induced damage in individual cells.

¹ This study was supported by NIH Grants CA-09215 and CA-13525.

² To whom requests for reprints should be addressed, at Brain Tumor Research Center, Box 0520, Department of Neurological Surgery, University of California, San Francisco, CA 94143-0520. Phone: (415) 476-4590 Fax: (415) 476-9687.

Received 8/11/95. Accepted 10/27/95.