Determination of radiation-induced DNA strand breaks in individual cells by non-radioactive labelling of 3' OH ends.
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.
Kodym
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R
Clinic for Radiotherapy and Radiobiology, University of Vienna, Austria.
Hörth
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D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004249:DNA Damage / Q000528:radiation effects*; D004253:DNA Nucleotidylexotransferase; D004260:DNA Repair; D004278:DNA, Superhelical / Q000737:chemistry; D006861:Hydrogen Peroxide; D066298:In Vitro Techniques; D011839:Radiation, Ionizing; D013997:Time Factors
D004278:DNA, Superhelical; D006861:Hydrogen Peroxide; D004253:DNA Nucleotidylexotransferase