Measurements using the alkaline comet assay predict bladder cancer cell radiosensitivity

Abstract

In the UK, the two main treatments of invasive bladder cancer are radiotherapy or cystectomy. However, B50% of patients undergoing radiotherapy fail to respond. If tumour radiosensitivity could be predicted in advance, it may be possible to improve control rates significantly by selecting for radiotherapy those patients whose tumours are radiosensitive. Additionally, patients who would benefit from surgery would be identified earlier. The alkaline comet assay (ACA) is a sensitive method for the detection of DNA strand break damage in cells. In the present study, using six bladder cancer cell lines of differing radiosensitivities, cell survival was compared to the manifestation of radiogenic DNA damage as assessed by ACA. For all the cell lines, the extent of comet formation strongly correlates with cell killing (R 2 40.96), with a greater response being noted in radiosensitive cells. In repair studies, measures of residual damage correlate with survival fraction at 2 Gy (R 2 40.96), but for only five of the cell lines. Finally, cells from human bladder tumour biopsies reveal a wide range of predicted radiosensitivies as determined by ACA. Overall, these studies demonstrate ACA to be a good predictive measure of bladder cancer cell radiosensitivity at low dose, with potential clinical application.

Potential use of the alkaline comet assay as a predictor of bladder tumour response to radiation

“…Here, we report results in five bladder cancer cell lines using a modified, more sensitive, version of the ACA. Our results are further supported by two independent studies using colorectal tumour cells (Dunne et al, 2003) and bladder tumour cells (Moneef et al, 2003).…”

“…T24 showed a shallow shoulder on the survival curve leading to a radioresistant response at low doses; however, there was a greater rate of change of radiosensitivity above 2 Gy, which lead a radiosensitive response at higher doses. This result has been confirmed by Moneef et al (2003). DNA damage following irradiation was measured in three cell lines using both the Eppendorf and slide variants of the ACA.…”

“…Two studies do not support our findings, however they irradiated the cells prior to embedding thus reducing the sensitivity of their procedure (Muller et al, 1994;Bergqvist et al, 1998). In addition, our study has been confirmed by the concurrently presented studies of bladder tumour cells (Moneef et al, 2003) and colorectal tumour cells (Dunne et al, 2003) suggesting that the method is robust and reproducible in different tumour types and different laboratories. Indeed, it may be that our version of the ACA is capable of exposing differences that are masked in other variants of the same assay.…”

“…In this paper, we present data showing that the ACA can be used as a surrogate measure in the prediction of tumour cell radiosensitivity; the results are supported by two further papers (Dunne et al, 2003;Moneef et al, 2003). Prior to using the ACA, we attempted to measure radiosensitivity in 24 primary bladder tumours using the soft agar clonogenic assay of West et al (1997).…”

“…Whatever the explanation, it seems that some cells clearly have more disruption to their chromatin when irradiated than others. This seems to put a greater strain on the cell's repair capacity since, although the repair rates vary less, the time to return to background levels is longer for cells that have greater initial damage (Table 2; Figure 5; see also Moneef et al (2003).…”