Study on the anti-cancer mechanisms of low dose-rate γ irradiation

To investigate appropriate dose range in low-dose-rate γ irradiation and to establish a useful method for the research of radioactive biological effect produced by low does-rate γ irradiation, we have studied apoptosis of Hela carcinoma cells induced by different low dose rates of γ irradiation in vitro. The cultured cells were exposed to defined radiation doses from a 60Co therapy source. The radiation source delivered a dose rate of 1Gy/min and gave total dose of 1, 2, 5, 10 and 15 Gy respectively. Using Annexin V labeled by fluorescein and PI, followed by flow cytometry and DNA ladder analysis and confocal microscope, apoptotic cells were detected and quantified. The re- sults are as follows: (1) Determination of apoptosis by flow cytometry indicated that percentage of apoptotic cells was increasing with dose and time. When cells were cultured for 168 h after irradiation, the highest percentage of apop- tosis was yielded in all groups. When the cells were irradiated with 1—2 Gy, no significant increase in the number of apoptotic cells could be seen. After a total dose of 5 Gy, however, a significant increase in the ratio of apop- totic/nonapoptotic cells was observed, and apoptosis reached a peak level of (72.57±2.04)% (P 0.001). (2) The typi- cal appearance of Hela cells stained by annexin V-Fluos for detection of apoptosis exhibited a plasma mem-brane-localized fluorescence that is an early sign of apoptosis under laser confocal microscope. PI entered into the cells slowly while in later apoptosis, with special laser which were red in center and green around, however, necrotic cell only emitted red laser. (3)The presence of apoptotic cells were confirmed by the DNA ladder formation assay, with typical formation of apoptotic fragments. DNA laddering reached the greatest after a single radiation dose of 5 Gy. (4) Over the dose interval from 2 to 15 Gy with a dose rate of 1 Gy/min, the survival of the cells decreased from (58.95±0.36)% to (1.67±0.35)% (P 0.001) by clonogenic assay. It was concluded that the low-dose, low-dose-rate γ irradiation delivered by 60Co sources to Hela cells can induce a significant apoptosis rate, and a maximum level of apoptosis as high as (72.57±2.04)% for an irradiation dose of 5Gy can be reached .