Power, Olive Mary; (1998) Cellular and molecular mechanisms affecting tumour radiosensitivity : An in vitro study. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The response of tumour cells in vitro to ionising radiation can, to a certain extent, predict the response of tumours to various radiotherapy treatment modalities. This thesis considers some of the factors known to be involved in the radiation response of human tumour cells in vitro. These parameters include radiation-induced cell-cycle perturbations, apoptosis and DNA damage repair. A panel of eight human tumour cell lines with markedly differing radiosensitivities were assessed in order to determine the key factors governing their radiation response. A wide range of doses spanning both the low dose region (0-2 Gy and 0-5 Gy) and the clinically relevant region (1-4 Gy) were used to determine whether differences in responses could distinguish cells which were radiosensitive or resistant. Ionising radiation produced a cell cycle delay in all cell lines in one or both of the cellular checkpoints. A Gl/S delay was detected in those cell lines that expressed wild-type p53, and the duration of this delay appeared to be directly related to the level of constitutive protein. p53 protein stabilisation was observed after 4 h, even at doses of 0-2 Gy, although a Gl/S delay was only detectable at higher doses. There was no direct relationship between p53 status and survival although wild-type p53 expression was more prevalent in the radiosensitive cell lines (3/4 sensitives are wild-type versus 2/4 resistants). A G2/M delay could only be detected at doses of > 1 Gy. This delay appeared to be dose independent in the resistant cell lines, suggesting a threshold dose of IGy, above which no further effect is observed. A radiation-induced reduction of cyclin B1 protein was observed in all cell lines implicating this protein in the induction of a G2/M delay. The duration of G2/M delay was significantly longer in the radiosensitive cell lines at 4 Gy (7-20 h versus 4-6 h at 4 Gy). The proportion of cells that exited the G2/M block and re-entered GO/G1 phase was also significantly less in the radiosensitive cell lines at this dose and was directly related to the increased level of apoptosis observed in these cell lines. It is speculated that the induction of apoptosis occurs after cells have attempted to repair damage before the onset of mitosis, eliminating those cells with irreparable damage. Apoptosis at low doses were not significantly different to constitutive levels, suggesting a limited role of apoptosis in the low dose hypersensitivity response previously observed in some of the cell lines in this study. Radiosensitive cell lines also demonstrated reduced initial DNA damage, measured by CHEF, (1.3-1.7 versus 1.8-2.2 excluding mutant radiosensitive phenotypes) and DSB break repair (20-30% versus 15-20%). While the responses to radiation were expectedly varied it is speculated that radioresistance is associated with increased repair while radiosensitivity is associated with increased apoptosis.

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