Computational analysis of the number, area and density of gamma-H2AX foci in breast cancer cells exposed to (111)In-DTPA-hEGF or gamma-rays using Image-J software.

PURPOSE: To develop a simple method for the quantification of gamma-H2AX focus number, density and size.

METHODS

MDA-MB-468 human breast cancer cells were treated overnight with (111)In-diethylenetriaminepentaacetic acid human epidermal growth factor ((111)In-DTPA-hEGF, 0-142 kBq/pmol) or exposed to gamma-radiation to induce DNA double strand breaks (DSB). DNA DSB formation was evaluated by detection of phosphorylated histone H2AX on serine 139 (gamma-H2AX) using immunofluorescence. Confocal microscopy was used to capture images of gamma-H2AX foci and cell nuclei. Image-J software with customized macros was used to quantify gamma-H2AX foci.

RESULTS

The number of gamma-H2AX foci per nucleus scored using Image-J correlated strongly with the number scored using direct visual confirmation (coefficient of determination, R(2) = 0.950; 60 nuclei scored). The mean density (grayscale values per pixel), area and integrated density (IntDen) of individual foci increased linearly as the specific radioactivity (SR) increased up to 67 kBq/pmol (R(2) values of 0.826, 0.964, 0.978, respectively). The mean number of foci per nucleus, the combined area of gamma-H2AX foci per nucleus and the IntDen per nucleus also increased linearly with SR, giving R(2) values of 0.926, 0.974 and 0.983, respectively. Similar linear relationships were observed with the gamma-ray absorbed dose up to 3.0 Gy.

CONCLUSIONS

The density, area and IntDen of individual foci, as well as the number of gamma-H2AX foci, total focus area and IntDen per nucleus were successfully quantified using Image-J with customized macros.