Different doses of radiation on agar colony forming development in C6 glioma cells: Assessment by thymidine labeling index, and bromodeoxyuridine labeling index
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Objective: Gliomas are relatively frequent in adults, and are among the most malignant primary brain tumors. Glioblastoma. multiforme, like many other tumors that exhibit radiation sensitivity in vitro, seems to be very resistant to radiation in vivo, thus suggesting that irradiation may not be a rate-limiting factor for malignant glioma tumor growth. In this study, we aimed to determine the optimal dose of radiation in C6 glioma colony forming assay, which is a valuable tool for antitumor treatment screening. Material and Methods: 10(5) cell/lamella colony forming cells were radiated with 200 cGy, 400 cGy, 800 cGy and 1600 cGy for 10 minutes. Radiosensitivity was measured systematically 24, 48, 72 and 96 hours after the radiation by three methods: soft-agar bilayer assay, thymidinE incorporation, and bromodeoxyuridine (BrdU) incorporation. Results: The soft-agar bilayer assay, which assessed the colony-forming units, showed that the number of colonies in the control group (609, 3 +/- 86.8) were decreased after 200 cGy (8.3 +/- 3.6) and 400 cGy (7.2 +/- 4.3). No colony was detected in 800 cGy and 1600 cGy irradiated cells [3H] Thymidine incorporation was more prominent with higher doses of radiation. BrdU incorporation revealed that even at low doses (200 cGy) of radiation there was a significant decrease of cell proliferation. On higher doses like 1600 cGy it was more prominent. Conclusion: Cell survival, doubling time, and cell phases are parameters of growth kinetics, and the results suggest that C6 glioma cells are radiosensitive and are virtually affected by all radiation doses in our experiment even 200 cGy at 24 hours. Besides, colony forming assay with thymidine labeling index, and BrdU labeling index may be used as new methods for determining radiotherapy doses in clinical applications.