A study of the [Ca2+] and the apoptosis of the KB cell lines after 10 Gy irradiation

Moon, Je Woon; Lee, Sam Sun; Heo, Min Suk; Park, Tae Won; You, Dong Soo

Abstract

PURPOSE: Ionizing radiations have been reported as an apoptosis initiating stimulus in various cells and it has established that sustained elevations in [Ca2+] can lead to DNA fragmentation by Ca2+-dependent endonucleases, ultimately resulting in apoptotic cell death. The previous experiments have been reported by using primarily thymocytes and lymphocytes and the change of [Ca2+] was measured only by minutes or hours respectively. We need to evaluate [Ca2+] in both several minutes and hours after irradiation of radiation of radiation therapy and verify the apoptotic cells.
MATERIALS AND METHODS
We have measured [Ca2+] in human gingival epitheloid cancer cell with 10 Gy irradiation, at minutely intervals and hourly intervals using digitized video-intensified fluorescence microscopy and the fluorescent Ca2+ indicator dye, fura-2. In order to find out that the transient rise in [Ca2+] could induced apoptosis, cells were incubated for 1 hour at 37 degrees C with TdT enzyme, rinsed and resuspended containing fluorescence and observed under a confocal fluorescence microscope. MTT assay was done to determine cell activity and LDH assay was done to determine the amount of necrotic cells.
RESULTS
After irradiation, the transient and temporal increasing of [Ca2+] in the KB cells was founded. Though, there was no change in the intracellular [Ca2+] at 30 minutes and 2 hours after irradiation. We could detect of DNA fragmented cells at 4 hours after 10 Gy irradiated cells. There were no significant differences between 4 hour, 1 day, 3 day cells. There were no significant differences in MTT and LDH assay between the irradiated group and the control group after 4 hours and 1 day. Though after 3 days there were differences in MTT and LDH assay between the irradiated group was significantly decreased than the control group, in LDH assay the number of necrotic cell death of the irradiated was higher than the control group.
CONCLUSION
In KB cells there were incipient and temporal increasing of the [Ca2+] with 10 Gy irradiation and the apoptosis was founded from 4 hours later which was earlier than seeing of the change of the amount of the cellular ability and necrosis.