Cellular radiosensitivity correlates with residual dna damage in primary normal human fibroblasts

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Radiation Oncology, Biology, Physics Volume 27, Supplement 1

human colon adenocarcinoma cells vhen alloved to repair after irradiated to 15 Gy. Fig.6-Excess angle of dispersion is plotted against the time allowed for repair. Conclusion: Both assays can be used to demonstrate a good radiation dose response for various human tumor

cells. Eovever, acridine orange flov cvtometrv assay is less user-deoendent. time-consuming procedure vhich-allovs both the-radiation dose-responsh study concomitantly using the same human tumor cell suspension.

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67 CELLULAR

RADIOSENSITIVITY

CORRELATES WITH RESIDUAL DNA DAMAGE IN PRIMARY NORMAL HUMAN FIBROBLASTS

Reinhard Wurm, Neil Burnet, Nisha Duggal, John Yamold, John Peacock Institute of Cancer Research & Royal Marsden Hospital, Sutton, Surrey, U.K. Purpose: Following the identification of a link between intrinsic cellular radiosensitivity and normal tissue radioresponsiveness we are evaluating the relationship between radiation-induced cell death and DNA damage in primary normal human tibroblasts. The aim is to analyse the underlying mechanisms which in addition might provide the basis to use DNA damage assays as rapid and reliable tests to predict normal tissue response to radiation. Materials and Methods: Fibroblasts from skin biopsies were grown as monolayer. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose-rate (HDR) l-2 Gy/min and low dose-rate (LDR) 0.01 Gy/min. SF, (survival at 2 Gy), and D,,et (radiation dose to reduce surviving fraction to 0.01) were taken for comparison with DNA damage. DNA damage was measured in “C-thymidine labelled cells in plateau phase expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage cells were embedded in agarose plugs and irradiated at HDR on ice. Repair was studied alter HDR irradiation on ice by incubating cells embedded in agarose plugs at 37°C for various times. Residual DNA damage was measured in monolayer by allowing a four hour repair period after HDR irradiation and immediately after LDR irradiation at 0.02 Gy/min. Results: Following highdose rate (HDR) irradiation almost linear survival curves were obtained and cell survival varied by a factor of 1.5 (SF, 0.15 to 0.23). This range increased after LDR irradiation (SF, 0.14 to 0.39) and revealed differences in dose-rate sparing. Initial DNA damage demonstrated a similar dose response for all strains with a linear induction up to 30 Gy. The repair was characterised by a fast initial component and reached a plateau at = 60% between 90 and 120 min. After that no further repair was observed for at least three hours. There was no difference in tbe kinetics of repair between the tibroblast strains. Residual damage was similar following a four hour repair period after HDR and immediately after LDR irradiation. No damage was detectable up to 40 Gy. Above this threshold it increased linearly with dose and the slope of the dose response curve of the different strains showed a close correlation with cell survival. Conclusion: We conclude that residual damage as measured by PFGE in primary normal human tibroblasts is the major determinant of the initial slope of the cell survival curve (i.e. LDR response). The observation that initial damage was similar in all cell lines implies that cellular differences in sensitivity result from their inherent ability to process DNA damage. If assays of DNA damage are to be used as predictors of normal tissue response to radiation levels of residual DNA damage provide the most likely correlation with cell kill.