Abstract
Low dose hyper-radiosensitivity (HRS) is observed in some cell lines, e. g. V79, and results in an increase in cell kill, per unit dose, in the low dose region (< 1 Gy). Investigations into this effect are of great interest in radiation protection and cancer treatment using radiotherapy. Cell cycle phase plays a significant role in this effect and with the increase in particle radiotherapy, research into the low dose region, using high LET irradiations, are of particular interest. However, low dose investigations using particles require specific irradiation protocols which are incompatible with gold standard phase synchronisation methods. A protocol for particle irradiation was developed in which the phase of individuals cells could be assessed in situ, prior to irradiation. Phase assessment was made using the fluorescent signal, emitted from previously stained cells, collected using the fluorescent end-station microscope. The protocol was developed to allow the end-station microscope to be used as a cytometry device. Therefore, validation of this technique was carried out by comparing cell data, collected using the end-station microscope and displayed in the form of a fluorescence intensity histogram, to cells analysed using flow cytometry. Both asynchronous and synchronised data were used. Investigations into different fluorescent stains, their toxicity and changes in the radiosensitivity of cells treated with such stains were also investigated. The microscope was characterised and the use of the end-station microscope, as a cytometry device, was shown to be valid. The development of this protocol was complimented by the modification and use of an existing mathematical model, CelCyMUS (Cell Cycle Model University of Surrey) in conjunction with a newly written program FloCytUS (Flow Cytometry University of Surrey). This model allows simulated populations of cells to be grown which can then be passed through either a “virtual” flow cytometer or a “virtual” end-station microscope. These populations can then be fitted to experimental data collected using the corresponding method. Analysis of various data sets allowed cell and instrument parameters to be determined. These parameters were then used to fit simulated to irradiated (using the end-station protocol) data sets. The phase of each simulated cell is recorded by the model thus allowing the fluorescence intensity, relating to a specific phase, to be determined. Using the experimental protocol and mathematical model described, low dose X-irradiation experiments were carried out on V79 cells using facilities at the Royal Surrey County Hospital. The biological end-points investigated were survival, phase delay, apoptosis and DNA damage using the yH2AX assay. It was found the assays used to assess both phase delay and apoptosis were incompatible with the use of the end-station microscope and related software. However, the survival and 7H2AX assays were compatible with this protocol. Data collected were separated into different phases depending on each cell’s fluorescent intensity signal. The intensity ranges associated with the different phases were based on the results of the FloCytUS analysis. It was found that there was a region of slight low dose hypersensitivity with respect to survival for the asynchronous and G1 populations with a more pronounced substructure observed in the G2/M population. With respect to the yH2AX assay there did appear to be an increase in the mean number of foci in the low dose region for the G2/M phase.To summarise, it was found that the end-station microscope could be used as a cytometry device which allowed the cell cycle phase, of an individual cell irradiated, to be determined. FloCytUS modelling complimented this technique and allowed populations of cells to be sorted into separate phases G1, S and G2/M. In doing so, survival and DNA damage foci could be assessed for the different phases of the cell cycle after 250 kVp X-irradiation. Low dose hyper-radiosensitivity was observed in asynchronous, G1 and G2/M populations of cells. The most pronounced hypersensitive effect was observed in the G1 phase and the most pronounced region of increased radiation resistance was observed in the G2/M phase. The γH2AX data showed some low dose substructure indicating that there is also a low dose hyper-radiosensitive response with respect to DNA damage.