Radiosensibility of melanoma cell by beta chronic irradiation at very low-dose-rate

MICHELIN S.; GALLEGOS C.E.; DUBNER D.

Congreso; 15th International Congress of Radiation Research; 2015

INTRODUCTIONThe effects of ionizingradiation are partially known below certain levels, and the LNT model is used forpublic protection. This model is controversial among scientists and the pasttwo decades of research have indicated that low dose effects cannot be exactly predictedby the extrapolation of effects from high doses. The objective of this work is todetermine the biological effect of beta irradiation at low dose and very lowdose rate on melanoma cells. MATERIALS AND METHODS: Beta irradiation system: The system is composed by two identical tissueculture superposed flasks. The bottom flask (irradiation flask) was loaded by adding3 ml of water containing 150 µCi of 32P orthophosphate solution. Theabsorbed dose in the upper culture flask and the isodose curves were calculatedapplying MCNPX 2.5f Monte Carlo code coupled to photon and electron crosssections from ENDF/B-VI library, and validated by Gafchromic EBT2 filmdosimetry. The irradiation and cell culture system was kept in a cell incubatorat 37°C with a 5% CO2 atmosphere 24, 48, 72 and 144 h, until the desiredfinal dose (0.2, 0.5, 1 or 2 Gy) was reached. The initial dose rate was 12-15mGy/h.Clonogenic survival: The humanmelanoma cell line M8 was used. Cells were seeded on 25 cm2 tissueculture flasks 5 h before the start of theirradiation. When the cultures reached the desired dose, the number of cell colonieswas determined by May-Grunwald stain.Cell cycle analysis of M8 cells: Samples were collected after 24, 48, 72 and 144 h ofbeta irradiation and fixed in ethanol 70% (v/v). The cells were resuspended inPBS 1X containing 100 μg/ml RNAse and 40 μg/ml propidium iodide and assessedfor cell cycle distribution by flow cytometry.Apoptosis detection: The cells were collected after 24, 48, 72 and 144 h of irradiation(equivalent to 0.2, 0.5, 1 and 2 Gy respectively), stained with AnnexinV-FITC/Propidium iodide and analyzed by flow cytometry for the determination ofapoptosis levels. RESULTS The clonogenic survival at 0.2Gy was lower than the survival at 0.5 Gy, but higher than at 1 and 2 Gy At 2 Gythe survival was near 50% of the control.Beta irradiation causedarrest of M8 cells in the G2/M phase of the cell cycle. The level of G2/M arrestincreased together with the increment of the irradiation dose. Apoptosis levels becamestatistically significant after 48h of irradiation. CONCLUSIONSWe determined theeffectiveness of low doses of beta irradiation at a very low dose rate (12-15mGy/h) in producing significant cell death of the M8 melanoma cell line.In addition, we could demonstratethe progressive cell cycle arrest in the G2/M phase of irradiated M8 cells and asignificant increase in apoptosis after 0.5 Gy. Further studies are in progressin order to elucidate the mechanisms that influence cell killing at low doserate, particularly at the lowest dose used.This irradiation system issimple, economic, and offers the possibility of irradiation with different doserates, by just changing the 32P activity. We consider our resultswould be a useful contribution for the optimization of radiotherapy protocols.