Comments on "Radiation-induced apoptosis in HL60 cells: oxygen effect, relationship between apoptosis and loss of clonogenicity, and dependence of time to apoptosis on radiation dose" by Hopcia et al. (Radiat. Res. 145, 315-323, 1996)

irradiation with doses as high as 20 Gy induced almost no apoptosis within 6 h although irradiation with 5 Gy induced apoptosis in almost half of the cells by 3-4 days. Nevertheless, the authors concluded that the oxygen enhancement ratios (OERs) for apoptosis in HL60 cells at 6 h and 3 days after irradiation were similar to that for the radiationinduced decline in clonogenicity of the cells. The authors deserve a compliment for their effort to determine the OER for apoptosis, which has never been done before. However, we would like to point out that some of the results described in the article differ substantially from the observations reported by other investigators. In the study by Hopcia et al., HL60 cells were very radioresistant so that, in the estimation of the OER for apoptosis at 6 h after irradiation, the oxygenated and hypoxic HL60 cells had to be irradiated with doses as high as 80 Gy and 160 Gy, respectively. We wonder why the HL60 cells used by Hopcia et al. were so resistant to radiation-induced apoptosis while most normal as well as malignant lymphoid cells, including HL60 cells, have been reported to undergo apoptosis within several hours after an exposure to relatively mild external stress including X irradiation, UV irradiation and hyperthermia (2-5). In our previous study we observed that HL60 cells undergo apoptosis in 3 h after heating at 42'C for 60 min (6). The results shown in Figs. 1 and 2 in the paper by Hopcia et al. indicate that the level of apoptosis determined 6 h after irradiation with doses as high as 20 Gy was hardly greater than the apoptosis in the unirradiated control cells. Such a resistance to radiation-induced apoptosis in HL60 cells is, as Hopcia et al. themselves pointed out, distinctly different from the report by Abend et al. (2) that marked apoptosis occurred in HL60 cells within 24 h after an exposure to 2.0-2.5 Gy X rays. As described below, we also observed that significant apoptosis occurs in HL60 cells within several hours after irradiation with doses higher than 2 Gy. Our results on the agarose gel electrophoresis of DNA extracted from HL60 cells 4 h after irradiation are shown in Fig. 1. It can be seen that irradiation with 4 Gy X rays caused a faint but visible ladder composed of DNA fragments, indicating that the cells underwent apoptosis. The intensity of DNA fragmentation increased as the dose was inc eased up to 12 Gy. We repeated the same experiments more than five times and always obtained similar results. Results of our flow cytometry study are shown in Fig. 2. In this particular study, the percentage of apoptotic HL60 cells before irradiation was 4.5% and it increased to 47.3% 4 h