γH2AX foci induced by γ rays and ¹²⁵IdU decay

Abstract

Purpose: γH2AX foci formation was investigated after γ irradiation and after accumulating ¹²⁵IdU decays to study the DNA double strand break (dsb) damage repair response in human breast cancer cells, MCF‐7.

Materials and methods: Confocal laser scanning microscopy (CLSM) was used to detect γH2AX foci formed in response to DNA dsbs induced by 0, 0.5, 1, 2 and 5 Gy γ irradiation and ¹²⁵IdU decays accumulated at −90°C in human breast cancer cells, MCF‐7. ¹²⁵IdU treated cells were labeled with 4 different concentrations of ¹²⁵IdU and then accumulated decays for 6, 19 or 35 days. γH2AX foci formation time for all experiments was 1 hour at 37°C. Visual confirmation of γH2AX foci was achieved by digital imaging (histogram analysis or profile analysis) and by standardizing the scored number of foci. The average numbers of γH2AX foci formed per cell after γ irradiation or accumulated ¹²⁵IdU decays were determined by counting red voxels or counting γH2AX foci in propidium iodide (PI) counterstained nuclei by eye in optically sectioned cells.

Results: Control, unirradiated MCF‐7 cells had an average of 1.7 γH2AX foci per cell and an average of 32 γH2AX foci were scored for cells irradiated with 1 Gy γ rays. The data for doses up to approximately 1 Gy was a good linear fit (r²=0.97) indicating that the assay is sensitive to low doses of γ rays. The average number of γH2AX foci scored in control cells that were frozen and thawed but not irradiated (=2.3) was not statistically significantly different from controls that were not frozen and thawed. The average number of γH2AX foci was linearly related (r²=0.98) to low numbers (<200 decays/cell) of ¹²⁵IdU decays indicating that the assay is also sensitive to low numbers of accumulated ¹²⁵IdU decays. At ¹²⁵I decays greater than 200 decays/cell, the average number of γH2AX foci plateaued. Regression analysis of the data for 0–140 ¹²⁵IdU decays per cell was used to calculate the rate of γH2AX foci formation (=0.26 foci per ¹²⁵I decay).

Conclusions: The γH2AX foci formation assay is sensitive to low doses of γ rays and accumulated ¹²⁵I decays. When ¹²⁵IdU decays were accumulated at −90°C (to overcome confounding DNA damage repair processes that occur during simultaneous ¹²⁵IdU incorporation and decay accumulation at 37°C), 0.26 γH2AX foci were formed per ¹²⁵IdU decay. Methods used to incorporate ¹²⁵I decay may modulate the number of γH2AX foci scored in cells.