pATM and γH2AX are effective radiation biomarkers in assessing the radiosensitivity of 12C6+ in human tumor cells

Abstract

Background: 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 phosphorylated H2AX (γH2AX) and ATM (pATM) in assessing ¹²C⁶⁺ radiosensitivity of tumour cells.

Methods: Human cervical carcinoma HeLa cells, hepatoma HepG2 cells, and mucoepidermoid carcinoma MEC-1 cells were irradiated with different doses of ¹²C⁶⁺. The survival fraction was assayed with clonogenic survival method and the foci of γH2AX and pATM was visualized using immunocytochemical methods. Flow cytometry was used to assay γH2AX, pATM and the cell cycle.

Results: The survival fraction decreased immediately in dose-dependent manner, but in turn, significantly increased during 24 h after ¹²C⁶⁺ irradiation. Both γH2AX and pATM foci accumulated linearly with doses and with a maximum induction at 0.5 h for γH2AX and 0.5 or 4 h for pATM, respectively, and a fraction foci kept for 24 h. The expression of γH2AX and pATM was in relation to cell cycle. The G0/G1 phase cells had the highest expression of γH2AX after 0.5 h irradiation and then decreased to a lower level at 24 h after irradiation. An obvious increase of pATM in G2/M phase was shown after 24 h of 2 and 4 Gy irradiation. The significant G2/M phase arrest was shown. There was a close relationship between the clonogenic survival and γH2AX and pATM expression both in timing and dose in response to ¹²C⁶⁺.

Conclusions: The rate of γH2AX and pATM formation and loss may be an important factor in the response of cells to ¹²C⁶⁺. pATM and γH2AX are effective radiation biomarkers in assessing the radiosensitivity of ¹²C⁶⁺ in human tumor cells.

Keywords: 12C6+; ATM; Human tumor cells; Survival fraction; γH2AX.

Fig. 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 a clonogenic assay was performed. The means and SD are shown for three independent experiments with 3 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 cells; b HepG2 cells; c MEC-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 Gy irradiation

Fig. 2

Digitized images of γH2AX and pATM foci. After exposure to 2 Gy ¹²C⁶⁺ and incubation 0.5 h for γH2AX and 4 h for pATM, cells were grown and irradiated on cover slips. DNA was stained with DAPI and γH2AX and pATM was detected using an Alexa 488-conjugated secondary antibody after staining using anti-phospho-histone H2AX (Ser-139) and anti-phospho-ATM (ser1981) mAb. a Hela-γH2AX; b Hela-pATM; c HepG2-γH2AX; d HepG2- pATM; e MEC-1-γH2AX; f MEC-1-pATM. Scale bar 15 μm

Fig. 3

Foci formation of γH2AX and pATM in Hela, HepG2 and MEC-1 cells observed by immunofluorescent microscopy. The three cell lines are exposured to 0.5, 1, 2 and 4 Gy ¹²C⁶⁺ and subsequently incubated for 0.5, 4 and 24 h for γH2AX and pATM in vitro. a, b, c γH2AX; d, e, f pATM; a, d Hela cells; b, e HepG2 cells; c, f MEC-1 cells. *P < 0.05 vs. 0 Gy irradiation; **P < 0.01 vs. 0 Gy irradiation. 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 the three experiments

Fig. 4

γH2AX and pATM in a cell cycle-dependent manner in Hela, HepG2 and MEC-1 cells. Bivariate (γH2AX and pATM IF vs DNA content) distributions of control and 4 Gy ¹²C⁶⁺ irradiation and subsequent incubation for 0.5 h for γH2AX and 4 h for phosphorylated ATM in vitro. a, b, c, d γH2AX; e, f, g, h pATM; a, e Control (Hela cells); b, f Hela cells; C,G-HepG2 cells; d, h MEC-1 cells

Fig. 5

The expression of γH2AX in a cell cycle-dependent manner in Hela, HepG2 and MEC-1 cells. The three cell lines are exposed to 0.5, 1, 2 and 4 Gy ¹²C⁶⁺ irradiation and then incubated for 0.5, 4 and 24 h in vitro. a, b, c Hela cells; d, e, f HepG2 cells; g, h, i MEC-1cells; a, d G-0.5 h; b, e, h 4 h; c, f, i 24 h. *P < 0.05, **P < 0.01 vs Control. Results are the means and SD for the three experiments

Fig. 6

The expression of pATM in a cell cycle-dependent manner in Hela, HepG2 and MEC-1 cells. The three cell lines are exposed to 0.5, 1, 2 and 4 Gy ¹²C⁶⁺ irradiation then incubated for 0.5, 4 and 24 h in vitro. a, b, c Hela cells; d, e, f HepG2 cells; g, h, i MEC-1cells; a, d G-0.5 h; b, e, h 4 h; c, f, i 24 h. *P < 0.05, **P < 0.01 vs Control. Results are the means and SD for the three experiments

Fig. 7

A Cell cycle distribution of three cell lines. a-Hela, HepG2 and MEC-1 cell are treated with 4 Gy ¹²C⁶⁺ followed incubation for 24 h. a Control (Hela cells), b Hela cells, c HepG2 cells, d MEC-1 cells. B Three cell lines treated with 0.5, 1, 2 and 4 Gy ¹²C⁶⁺ irradiation and subsequently incubated for 0.5, 4 and 24 h. a, b, c 0.5 h; d, e, f 4 h; g, h, i 24 h. a, d, g Hela cells; b, e, h HepG2 cells; c, f, i MEC-1 cells. *P < 0.05, **P < 0.01 vs Control. Results are the means and SD for the three experiments