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Response of chinese hamster spheroids to mulifraction irradiation Brown, Ruth Caro


The response of mammalian cells to ionizing radiation has been extensively studied with single cells exposed to acute doses. Little information is, however, available for cells growing in tissues, especially for cells subjected to multiple exposures. Our aim in this thesis was therefore to use a more complex in vitro system, three dimensional spheroids grown from V79-171b Chinese hamster lung cells, to determine the role of repair, redistribution and repopulation during multifraction irradiation. Repair and redistribution effects were isolated by using spheroids under normal culture conditions of 37°C, or at 22°C where repair occurs but cell proliferation is markedly inhibited. As expected, we found that cells surviving an initial 8 Gy dose showed cell cycle dependent fluctuations in radiosensitivity when allowed to progress at 37°C before exposure to a second 8 Gy dose. Sublethal radiation damage was repaired more rapidly at 37°C than at 22°C, and was also affected by proliferation. Due, however, to the small proliferating population in the spheroid system, a large initial dose was required to produce a population with enough synchrony for the expected split dose survival fluctuations throughout the cell cycle to be observed. When two doses of 6 Gy separated by 4 hours were administered to spheroids, the subsequent cellular radiosensitivity to a third dose remained quite constant for at least 10 hours, indicating a more extended mitotic delay than observed in the two dose experiments. Mitotic delay consequently was not linear with dose, a result apparently dependent upon the fractionation scheme used, and the complexity of the multicell system. In multifraction schedules where doses of 6 Gy or 8 Gy were administered daily for 6 days, we found, as expected, that repair, redistribution and repopulation all affected cell viability. However, each effect dominated at different times throughout the experiments. The overall cytotoxicity for each 6 Gy fraction decreased with increasing fraction number, while the 8 Gy fraction survival remained fairly constant. A novel feature of our experimental design, administering each 6 Gy or 8 Gy fraction in 1-2 Gy increments, also allowed evaluation of successive responses to the clinically relevant dose of 2 Gy. Cell survival at that level fluctuated greatly due to a decreasing repair capacity, 'and an increasing effect of repopulation with fraction number. Using two radioactive Iridium sources of different activities, high dose rate fractionated exposure was compared to continuous low dose rate irradiation. Also, the linear quadratic model was used to predict the equivalent doses. We found that the model did not provide a good prediction; more repair of radiation induced damage was observed at the lower dose rates than the higher dose rates, an effect which could not be incorporated into this theoretical model. We conclude that, with fractionated radiation exposures to the spheroid system, repair, redistribution, repopulation and cell killing all contribute to the multifraction responses. Each has varying significance on each fraction. An equal effect per fraction, often implicit in radiotherapy regimens, is therefore only achieved in the fortuitous situation where repair, redistribution, repopulation and cell killing combine in different proportions to result in the same overall survival.

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