Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells

Abstract

The intrinsic radiation sensitivity of normal and tumour tissue is a major determinant of the outcome of radiotherapy. There is currently no established test that can be used routinely to measure the radiosensitivity of the cells in an individual patient's cancer in a manner that can inform treatment planning. The purpose of this study was to evaluate, in four human colorectal adenocarcinoma cell lines, two possible end points as surrogate markers of radiation response--apoptosis and induction of DNA single-strand breaks--and to compare the results with those of a conventional clonogenic assay. Cell lines (SW707 SW480, SW48 and HT29) known to differ in radiosensitivity were exposed to single doses of X-rays ranging from 0.5 to 5 Gy and cell survival was measured using the clonogenic assay. Apoptosis was determined on the basis of morphology under fluorescent microscopy and DNA damage/repair was measured, as tail moment, using an adaptation of the alkaline comet assay. The relationship between surviving fraction at 2 Gy (SF2) and the percentage of apoptotic cells 24 h after the same dose was complex, but apoptosis accurately predicted the order of radiosensitivities as measured by SF2. Initial damage measured after 2 Gy using the alkaline comet assay gave a close correlation with SF2 (r2=0.95), whereas there was no correlation between initial DNA damage repair rate and SF2.

Figure 1

Radiation cell survival curves for four colon adenocarcinoma cell lines. Error bars represent ±1 s.e.m.

Figure 2

Apoptotic frequency for four colon adenocarcinoma cell lines at different times after a range of X-ray doses: 0.5 Gy (•), 1.0 Gy (▴), 2.0 Gy (▪) and 5 Gy (♦). Error bars represent±1 s.e.m.

Figure 3

DNA damage measured immediately after irradiation by the alkaline comet assay, expressed as mean tail moment as a function of radiation dose (0.5, 2 and 5 Gy) in four colon adenocarcinoma cell lines. Error bars represent ±1 s.e.m.

Figure 4

DNA damage expressed as mean tail moment, measured by the alkaline comet assay, as a function of time after a radiation dose of 5 Gy in four colon adenocarcinoma cell lines. Error bars represent±1 s.e.m.

Figure 5

Correlation between surviving fraction after 2 Gy X-rays and DNA damage expressed as mean tail moment, measured immediately after 2 Gy by the alkaline comet assay. Error bars represent±1 s.e.m.

Figure 6

Relationship between surviving fraction after 2 Gy X-rays and the percentage of apoptotic cells. Error bars represent ±1 s.e.m.