An assay for quantifying DNA double-strand break repair that is suitable for small numbers of unlabeled cells

A system based on pulsed-field gel electrophoresis (PFGE) is described which measures the induction and repair of DNA double-strand breaks (DSBs) in a biologically relevant X-ray dose range (below 10 Gy) using as few as 125 cells per time. This system was used to measure repair in cells of a freshly obtained human glioblastoma multiforme tumor. No prelabeling of the cells is required, and many different cell types can be studied using this system. Under the pulsed-field conditions used, DNA in the range of 2 to 6 Mb enters the PFGE gel and forms an upper compression zone directly under each well. To quantify the DSBs after electrophoresis, the DNA was transferred to nylon membranes and hybridized with P-32-labeled chromosomal DNA. Phosphor screens were exposed to the membranes and scanned on a phosphor imager. The kinetics of induction and repair was determined by measuring the amount of DNA in the compression zones compared to the amount in the wells. EMT-6 cells were used to demonstrate this method. Induction of DSBs by doses of 0-7.5 Gy X rays was assayed using approximately 12,500 cells per dose and was shown to be linear. Double-strand breaks from 1 Gy were detected above background. To determine a lower limit of the number of cells that could be used to measure DSB repair, cells were embedded in agarose at decreasing concentrations per plug, exposed to 7.5 Gy X irradiation and allowed to repair at 37 degrees C for up to 60 min. DNA from approximately 12,500, 1,250 and 125 cells per time was loaded and subjected to PFGE. The average fast-repair half-time was 3 min and the slow-repair half-time was 35 min. The kinetics of DSB repair in glioblastoma multiforme cells was also determined using this system. Agarose plugs were prepared from a cell suspension, irradiated with 7.5 Gy X rays and allowed to repair for up to 90 min. DNA from approximately 1,250 tumor cells was electrophoresed and analyzed as described above for EMT-6 cells. For this particular tumor, approximately 75% of the induced DSBs were repaired after 90 min. Data presented show that this PFGE-based system is an extremely sensitive method for measuring DSB induction and repair after low doses of X rays using very few cells. (C) 1997 by Radiation Research Society