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Studies on the effect of radiation on 3T3 cell motility Thurston, Gavin O.


The ability of mammalian cells to locomote is important in a variety of normal and pathological processes. Previous work has suggested that low doses of x-irradiation may perturb cell motility, a finding that may have important consequences in embryogenesis, cancer metastasis, and immune response. This thesis has sought to study in more detail the effect of radiation on mammalian cell motility. Work performed in other laboratories used the colloidal gold assay and time lapse cinemicroscopy to study x-irradiation induced changes to 3T3 fibroblast motility in tissue culture. These studies were repeated here, with qualitative results similar to those reported earlier. However, these methods were not amenable to a detailed quantitative analysis. For this, spatial and temporal information on the motility and dynamic morphology of a large number of cells is required. Such a task would be impossible to perform manually, thus an automated microscope system was developed that used a computer-driven precision stage and a solid state optical sensor to track individual cells in tissue culture. Information on motility and morphology was concurrently extracted from many cells. As part of the thesis, several techniques were developed to analyze and display these data, and to correlate motility and morphology observations. These techniques were directed at preserving the actual process of 3T3 cell motility, and parameters were measured to quantify the short term persistence of cell movement (on a time scale of 0.5 to 2 hours), and the long term persistence of cells in maintaining certain characteristic behaviour (on a time scale of 3 to 12 hours). The response of 3T3 fibroblasts to x-irradiation was characterized by a number of parameters. The population average cell speed was measured following treatment, and a dose response and time response was determined in the range of 1.5 Gy. Other motility parameters indicate that the normal process of cell motility, evidenced by a series of motile segments, was disrupted by x-rays. This was thought to reflect perturbation to the control mechanisms of cell motility. The morphology of 3T3 cells stained with Coomassie blue was examined in an effort to correlate the observed motility changes with changes in the fixed cell morphology. This stain is a general structural protein stain with higher affinity toward microfilaments. High doses of x-rays were required to produce significant perturbation to cell morphology, and in the dose regime of interest, the morphology of irradiated cells was not identifiably different from control. Of note is that it was the well spread, quiescent cells that seemed least perturbed by large doses of irradiation. In summary, x-rays apparently disrupt the normal process of cell motility. Several lines of evidence suggest that actively migrating cells are the most perturbed by irradiation. This work has developed techniques to quantify cell motility in a meaningful way, and to characterize the x-ray induced perturbations.

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