| Summary: | 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. === Science, Faculty of === Physics and Astronomy, Department of === Graduate
|
|---|
