The potential value of the neutral comet assay and γH2AX foci assay in assessing the radiosensitivity of carbon beam in human tumor cell lines

Abstract

Background: Carbon ions ((12)C(6+)) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The assessment of tumour radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. The aim of the current study was to evaluate the potential value of the neutral comet assay and γH2AX foci assay in assessing (12)C(6+) radiosensitivity of tumour cells.

Materials and methods: The doses of (12)C(6+) and X-rays used in the present study were 2 and 4 Gy. The survival fraction, DNA double-strand breaks (DSB) and repair kinetics of DSB were assayed with clonogenic survival, neutral comet assay and γH2AX foci assay in human cervical carcinoma HeLa cells, hepatoma HepG2 cells, and mucoepidermoid carcinoma MEC-1 cells at the time points of 0.5, 4, 16 and 24 h after (12)C(6+) and X-rays irradiation.

Results: The survival fraction for 12C6+ irradiation was much more inhibited than for X-rays (p < 0.05) in all three tumour cell lines tested. Substantial amounts of residual damage, assessed by the neutral comet assay, were present after irradiation (p < 0.05). The highest residual damage was observed at 0.5 or 4 h, both for (12)C(6+) and X-ray irradiation. However, the residual damage in HeLa and MEC-1 cells was higher for (12)C(6+) than X-rays (p < 0.05). The strongest induction of γH2AX foci was observed after 30 min, for all three tumour cell lines (p < 0.01). The franction of γH2AX foci persisted for at least 24 h after (12)C6+ irradiation; in HeLa cells and MEC-1 was higher than after X-ray irradiation (p < 0.05). The correlation coefficients between the clonogenic survival, neutral comet assay and γH2AX foci assay were not statistically significant, except for some tumour cells at individual irradiation doses and types.

Conclusions: Our study demonstrated that the neutral comet assay and γ-H2AX foci assay could be used to assess the radiosensitivity of (12)C(6+) in human tumour cells.

Keywords: DNA double strand breaks; X-rays; carbon ions; human tumour cells; radiation sensitivity; γH2AX.

FIGURE 1.

A survival curve for the Hela,HepG2 and MEC-1 cell lines, as determined by clonogenic assay. Exponentially growing cells were plated and irradiated, the cells were taken at the indicated time intervals after irradiation of ¹²C⁶⁺ and X-rays and a clonogenic assay was performed. The means and SD are shown for three independent experiments with 4 replicates in each experiment. Untreated cells served as a control. After incubation for two weeks, colonies with cells greater than 50 were counted (A. Hela-2Gy; B. Hela-4Gy; C. HepG2-2Gy; D. HepG2-4Gy; E. MEC-1-2Gy; F. MEC-1-4Gy.) *: P<0.05 vs. 0Gy irradiation; **: P<0.01 vs. 0Gy irradiation; #: P<0.05 vs. the same dose x-rays irradiation; ##: P<0.01 vs. the same dose x-rays irradiation.

FIGURE 2.

Comet images of DSB detected by neutral comet assay 4 h post-irradiation of ¹²C⁶⁺ (A. Hela-Control; B. Hela-4Gy; C. HepG2-Control; D. HepG2-4Gy; E. MEC-1-Control; F. MEC-1-4Gy. Scale bar, 50um).

FIGURE 3.

DNA damage induction and repair profiles measured by the neutral comet assay in Hela, HepG2 and MEC-1 cell lines irradiated with 2 and 4 Gy of ¹²C⁶⁺ and X-rays in vitro. The extent of DNA damage was measured quantitatively by the comet tail moment (TM). Immediately after irradiation, the cell samples were placed at 37° C in a 5% CO2 incubator. The cells were taken at the indicated time intervals after ¹²C⁶⁺ and X-rays exposure and subjected to the comet assay. The means and SD of TM are shown for three independent experiments with 2 replicates in each experiment. Thirty cells were analyzed for each slide (A. Hela-2Gy; B. Hela-4Gy; C. HepG2-2Gy; D. HepG2-4Gy; E. MEC-1-2wGy; F. MEC-1-4Gy.) *: P<0.05 vs. 0Gy irradiation; **: P<0.01 vs. 0Gy irradiation; #: P<0.05 vs. the same dose x-rays irradiation; ##: P<0.01 vs. the same dose x-rays irradiation.

FIGURE 4.

Digitized images of γH2AX foci in Hela, HepG2 and MEC-1 cell lines. After exposure to 4 Gy ¹²C⁶⁺ followed incubated for 30 min, cells were grown and irradiated on cover slips. DNA was stained with DAPI and γH2AX was detected using an Alexa488- conjugated secondary antibody after staining using a monoclonal anti-γH2AX antibody. (A. Hela-Control; B. Hela-4Gy; C. HepG2-Control; D. HepG2-4Gy; E. MEC-1- Control; F. MEC-1-4Gy. Scale bar, 15um)

FIGURE 5.

The percentage of γH2AX foci of Hela, HepG2 and MEC-1 cell lines after exposure to 2 and 4 Gy ¹²C⁶⁺ and X-rays followed incubation for 0.5, 4, 16 and 24 h in vitro. Over 800 randomly selected cells were counted. Cells with three or more foci of any size were classified as positive. Results are the means and SD for three experiments (A. Hela-2Gy; B. Hela-4Gy; C. HepG2-2Gy; D. HepG2-4Gy; E. MEC-1-2Gy; F. MEC-1-4Gy). *: P<0.05 vs. 0Gy irradiation; **: P<0.01 vs. 0Gy irradiation; #: P<0.05 vs. the same dose x-rays irradiation; ##: P<0.01 vs. the same dose x-rays irradiation.