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Characterisation of the apoptotic response due to low doses of radiation using automated image cytometry Matthews, Jeffrey Blair


Traditionally, cell survival following x-irradiation has been assumed to follow a monotonic dose response, even at very low doses. Recent improvements to the low dose assay have revealed that many cell lines exhibit a complex response whereby cells are hyper-radiosensitive to X-rays at these doses (HRS) followed by an increased radioresistance (IRR) as dose approaches 1 Gy. This hypersensitivity may be eliminated by pre-treatment with small priming doses of x-rays, and there is evidence that the increased radioresistance may be a reflection of an inducible repair mechanism. Because molecular evidence strongly suggests a coupling of DNA repair and apoptosis, or programmed cell death, a hypothesis was put forth that HRS/IRR would be reflected in changes in the levels of apoptotic cell death over this dose region. To test this hypothesis, a very large cell population would be required. To overcome the technical and statistical problems associated with such measurements, an automated image cytometric method of apoptotic cell classification was developed. Image acquisition software was adapted to gather double-stained cell images from slides prepared using cell fixation and staining methods which emphasised apoptotic morphology. Chinese hamster ovary cells were classified individually by discriminant analysis of morphological and nuclear texture features calculated for each image. Discriminant functions were constructed from a manually classified set of over 60,000 cell images categorised as "normal", "apoptotic", "cell doublets" or "debris" and all subsequent cell images collected were classified using these functions. Application of this technique resulted in a 99.8% accuracy in classification of the normal cell population, and 81.7% classification accuracy for apoptotic cells. This method was then applied to study the time course of the apoptotic response of CHO cells following xirradiation. Following irradiation with 5 Gy, no increase above control levels of apoptosis was noted until 18 hours post-irradiation, which corresponded to the release of the G2-block as determined by DNA-content analysis. Apoptotic frequency increased to a peak level of 12.1%±4.6 at 42 hours post-irradiation. CHO cells irradiated with 0.25 or 1.0 Gy also exhibited peak levels at 42 hours, although no cell cycle perturbations were noted following irradiation. A secondary peak in apoptosis was noted 60 hours post-irradiation for these doses. Cells exposed to 0.5 Gy however, showed no distinct peak in apoptosis frequency. Analysis of the cumulative amounts of apoptosis observed at 6 hour time intervals over a 72 hour period following irradiation showed greater levels of apoptosis in the 0.25 Gy irradiated population than in the cells exposed to 0.5 Gy. These results were not statistically significant when subjected to Student's t-test analysis. Experiments using small priming doses of x-rays 6 hours prior to challenge doses failed to show a reduction in apoptotic frequency as would be expected if apoptosis were directly responsible for the HRS/IRR phenomenon. While a direct involvement of apoptosis in HRS/IRR cannot be ruled out, these results do not generally support the original hypothesis. The post-mitotic nature of apoptosis in CHO cells, several generations following low dose irradiation, obscures the relationship of these results to cell survival data. However, there may be some implications for cell survival measurements due to effects on resulting colony size. These studies suggest that the characterisation of the low dose apoptotic response requires further investigation. The automated techniques developed here will aid significantly in this pursuit.

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