Determination of radiation-induced DNA strand breaks in individual cells by non-radioactive labelling of 3' OH ends.

Abstract

To quantify DNA strand breaks generated by ionizing radiation in single cells with preserved morphology, the number of radiation-induced 3' OH ends of nuclear DNA was determined by enzymatic labelling. Confluent CHO-K1 cells were irradiated with doses up to 100 Gy. After fixation and permeabilization of the cell monolayer the nuclear DNA was labelled with Digoxigenin-11-dUTP using terminal transferase. The incorporated nucleotide was detected with an anti-Digoxigenin antibody conjugated with alkaline phosphatase. The phosphatase bound was quantified by a colour reaction and the integrated optical densities of the cell nuclei were measured. For doses ranging from 20 to 100 Gy a linear relationship between dose and labelling signal was obtained. Repair experiments showed a fast component of repair with a half-time of about 14 min, followed by a slower decline to background values, which were reached after 6-8 h. This method allows the measurement of radiation-induced DNA strand breaks in morphologically preserved single cells in a reproducible way, which may be of importance in the prediction of tumour response in radiotherapy.