An Assay for Quantifying DNA Double-Strand Break Repair That Is Suitable for Small Numbers of Unlabeled Cells

Abstract

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 32P-labeled chromosomal DNA. Phosphor screens were exposed to the membranes and scanned on a phosphor imager. The kinetics of induction and repair was determine 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.

Micro-CT for Anatomic Referencing in PET and SPECT: Radiation Dose, Biologic Damage, and Image Quality

“…The sensitivity of the method is demonstrated by the increase in gH2AX staining for the DNA repair-deficient, more radiosensitive BALB/c SCID strain, as is reflected in the slope of both linear curves (0.191 vs. 0.079 for BALB/c SCID and C57BL/6, respectively). More importantly, the results in Figures 1A and 2A clearly illustrate that CT using preset parameters does induce substantial DNA damage that might be sufficient to alter experimental outcome, especially with respect to a radiobiology endpoint (3,15,16). To minimize the biologic consequences of these scans, guidance for lowering the x-ray voltage, flux, and duration settings of the CT acquisition was obtained from the manufacturers.…”

“…Thus, although the biologic effects of radiation were obvious when looking at DNA DSB, they did not translate into macroscopic changes. Nevertheless, care should be taken when performing longitudinal CT studies because exposure to low radiation doses, which might not lead to macroscopic damage, might lead to additional concerns such as radiation hypersensitivity (16) and adaptive response (17).…”

Nuclear clusterin/XIP8, an x-ray-induced Ku70-binding protein that signals cell death

“…Because the 55-kDa nuclear form of CLU͞XIP8 did not increase until 3 days post-IR, it seemed unlikely that this protein played an immediate role in DSB repair, despite its interaction with Ku70. The repair of DSBs occurs within 2 h posttreatment (24,25), although delayed induction and repair of DSBs have been described. Given the proposed role of CLU͞XIP8 as a ''marker of apoptosis'' and the translocation of nuclear CLU͞XIP8 in IRtreated and dying cells (Fig.…”

The Influence of Bromodeoxyuridine on the Induction and Repair of DNA Double-Strand Breaks in Glioblastoma Cells

“…Technical simplicity, short handling times and direct measuring of DNA damage are advantages of these techniques [14]. Studying DSB repair after biologically and clinically relevant radiation doses (< 10 Gy) is difficult, and relatively few data have been published about residual damage after irradiation [22].…”