Radiation research
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In earlier studies using asynchronously growing Chinese hamster cells, we observed substructure in the survival response at low doses. The substructure appeared to result from subpopulations of cells having different, cell cycle phase-dependent radiosensitivity. We have now applied the same flow cytometry and cell sorting technique to accurately measure the responses of cells of eight different asynchronously growing human tumor cell lines, representing a wide range in radiosensitivity. ⋯ A two-population LQ model provides excellent fits to the data for most of the cell lines though, as one might expect with a five-parameter model, the best-fitting value of the various parameters is far from unique, and the values are probably not reliable indicators of the size and radiosensitivity of the different cell subpopulations. At very low dose, below 0.5-1 Gy, another order of substructure is observed: the hypersensitive response; this is described in the accompanying paper (Wouters et al., Radiat. Res. 146, 399-413, 1996).
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It is well known that cells of human tumor cell lines display a wide range of sensitivity to radiation, at least a part of which can be attributed to different capacities to process and repair radiation damage correctly. We have examined the response to very low-dose radiation of cells of five human tumor cell lines that display varying sensitivity to radiation, using an improved assay for measurement of radiation survival. This assay improves on the precision of conventional techniques by accurately determining the numbers of cells at risk, and has allowed us to measure radiation survival to doses as low as 0.05 Gy. ⋯ This hypersensitivity is followed by an increase in radioresistance over the dose range 0.3 to 0.7 Gy, beyond which the response is typical of that seen in most survival curves. Mathematical modeling of the responses suggests that this phenomenon is not due to a small subpopulation of sensitive cells (e.g. mitotic), but rather is a reflection of the induction of resistance in the whole cell population, or at least a significant proportion of the whole cell population. These results suggest that a dose-dependent alteration in the processing of DNA damage over the initial low-dose region of cell survival may contribute to radioresistance in some cell lines.
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Radiation-induced apoptosis detected by gel electrophoresis was measured in cells of three human prostate carcinoma cell lines (TSU, PC-3 and DU-145) and compared to their intrinsic radiosensitivities as measured by clonogenic assays. The intrinsic radiosensitivities of each cell line were defined by their alpha and beta coefficients and their surviving fraction at 2 Gy, derived from complete survival curves. The temporal expression and kinetics of radiation-induced apoptosis for DU-145 cells, the human prostate carcinoma cell line which expressed the highest rate of radiation-induced apoptosis, was characterized further by differential sedimentation and the immunofluorescence assay (Apoptag) which was specific for 3'-OH ends in cellular DNA. ⋯ These may correlate with apoptosis and proliferative cell death, respectively. Of the three prostate cancer cell lines investigated, only DU-145 cells displayed significant levels of radiation-induced DNA fragmentation and rapid cell death, with characteristics of apoptosis. This mechanism of cell death was complete by 24 h after irradiation and was well separated in time from the death of cells by the major mechanisms which occurred after 72 h, and accounted for about 5% of cell inactivation by a single-hit mechanism.
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Apoptosis in HL60 human leukemia cells irradiated in vitro was quantified using a DNA fragmentation assay. Dose-response curves for induction of apoptosis in HL60 cells 6 h after irradiation with 280 kVp X rays in air and hypoxia give an oxygen enhancement ratio (OER) of 2.7. This is similar to the OER of 2.8 obtained from survival curves for HL60 cells using a soft agar clonogenic assay. ⋯ However, if apoptosis is assayed 2-4 days after irradiation, the HL60 cells show greater sensitivity, with 5 Gy in air causing 45-50% apoptosis at 3 days. When apoptosis is measured 3 days after irradiation, the OER is similar to that obtained for survival and for apoptosis at 6 h. Although the HL60 cells exhibit radiation-induced apoptosis if one waits 2-4 days after low doses of radiation, rather than just 6 h, to conduct the assay, the amount of cells undergoing apoptosis is still not sufficient to account for all the loss of clonogenicity seen when HL60 cells are exposed to ionizing radiation.
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Normal tissue toxicity limits radiation therapy and could depend on the extent of damage to the vascular endothelium Aminothiols such as WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] provide radioprotection for normal tissues, but little is known about how the aminothiols specifically affect the endothelium. Bovine aortic endothelial cells in culture were exposed to WR-1065 for 2 h before irradiation (137Cs gamma rays, 1 Gy/min). Alone, WR-1065 demonstrated an antiproliferative effect that was related to dose (0.5-4 mM) and was evident by lowered counts of adherent cells 48 h after exposure. ⋯ WR-1065 pretreatment elevated cellular glutathione (GSH) content more than twofold. Although pretreatment with buthionine sulfoximine inhibited the elevation of GSH, the radioprotective impact of WR-1065 on total DNA strand breaks and colony formation was unaffected. These results suggest that WR-1065 may enable tissue recovery from irradiation by promoting the replication of endothelial cells, possibly by mechanisms independent of GSH.