Linda S Yasui1. 1. Northern Illinois University, Department of Biological Sciences, DeKalb, IL 60115, USA. lyasui@niu.edu
Abstract
PURPOSE: GammaH2AX foci formation was investigated after gamma 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 yH2AX foci formed in response to DNA dsbs induced by 0, 0.5, 1, 2 and 5 Gy gamma irradiation and 125IdU decays accumulated at -90 degrees 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. gammaH2AX foci formation time for all experiments was 1 hour at 37 degrees C. Visual confirmation of gammaH2AX foci was achieved by digital imaging (histogram analysis or profile analysis) and by standardizing the scored number of foci. The average numbers of gammaH2AX foci formed per cell after gamma irradiation or accumulated (125)IdU decays were determined by counting red voxels or counting gammaH2AX 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 gammaH2AX foci per cell and an average of 32 yH2AX foci were scored for cells irradiated with 1 Gy gamma 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 gamma rays. The average number of gammaH2AX 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 yH2AX 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 yH2AX foci plateaued. Regression analysis of the data for 0-140 125IdU decays per cell was used to calculate the rate of yH2AX foci formation (=0.26 foci per 125I decay). CONCLUSIONS: The gammaH2AX foci formation assay is sensitive to low doses of gamma rays and accumulated 125I decays. When 125IdU decays were accumulated at -90 degrees C (to overcome confounding DNA damage repair processes that occur during simultaneous 125IdU incorporation and decay accumulation at 37 degrees C), 0.26 gammaH2AX foci were formed per 125IdU decay. Methods used to incorporate 125I decay may modulate the number of gammaH2AX foci scored in cells.
PURPOSE:GammaH2AX foci formation was investigated after gamma irradiation and after accumulating 125IdU decays to study the DNA double strand break (dsb) damage repair response in humanbreast cancer cells, MCF-7. MATERIALS AND METHODS: Confocal laser scanning microscopy (CLSM) was used to detect yH2AX foci formed in response to DNA dsbs induced by 0, 0.5, 1, 2 and 5 Gy gamma irradiation and 125IdU decays accumulated at -90 degrees C in humanbreast 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. gammaH2AX foci formation time for all experiments was 1 hour at 37 degrees C. Visual confirmation of gammaH2AX foci was achieved by digital imaging (histogram analysis or profile analysis) and by standardizing the scored number of foci. The average numbers of gammaH2AX foci formed per cell after gamma irradiation or accumulated (125)IdU decays were determined by counting red voxels or counting gammaH2AX 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 gammaH2AX foci per cell and an average of 32 yH2AX foci were scored for cells irradiated with 1 Gy gamma 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 gamma rays. The average number of gammaH2AX 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 yH2AX 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 yH2AX foci plateaued. Regression analysis of the data for 0-140 125IdU decays per cell was used to calculate the rate of yH2AX foci formation (=0.26 foci per 125I decay). CONCLUSIONS: The gammaH2AX foci formation assay is sensitive to low doses of gamma rays and accumulated 125I decays. When 125IdU decays were accumulated at -90 degrees C (to overcome confounding DNA damage repair processes that occur during simultaneous 125IdU incorporation and decay accumulation at 37 degrees C), 0.26 gammaH2AX foci were formed per 125IdU decay. Methods used to incorporate 125I decay may modulate the number of gammaH2AX foci scored in cells.