Longo J A, Nevaldine B, Longo S L, Winfield J A, Hahn P J
Department of Radiation Oncology, State University of New York Health Science Center, Syracuse 13210, USA.
Radiat Res. 1997 Jan;147(1):35-40.
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.
本文描述了一种基于脉冲场凝胶电泳（PFGE）的系统，该系统可在生物学相关的X射线剂量范围（低于10 Gy）内，每次仅使用125个细胞来测量DNA双链断裂（DSB）的诱导和修复情况。该系统用于测量新鲜获取的多形性胶质母细胞瘤肿瘤细胞中的修复情况。无需对细胞进行预标记，并且使用该系统可以研究许多不同的细胞类型。在所使用的脉冲场条件下，2至6 Mb范围内的DNA进入PFGE凝胶，并在每个孔的正下方形成一个上部压缩区。为了在电泳后对DSB进行定量，将DNA转移到尼龙膜上，并用32P标记的染色体DNA进行杂交。磷屏与膜接触并在磷成像仪上进行扫描。通过测量压缩区中的DNA量与孔中的DNA量之比来确定诱导和修复的动力学。使用EMT-6细胞来演示此方法。使用每剂量约12,500个细胞测定0至7.5 Gy X射线剂量诱导的DSB，结果显示为线性关系。1 Gy剂量产生的双链断裂在背景之上可被检测到。为了确定可用于测量DSB修复的细胞数量下限，将细胞以每块琼脂糖塞中逐渐降低的浓度包埋，暴露于7.5 Gy X射线照射下，并在37℃下修复长达60分钟。每次加载来自约12,500、1,250和125个细胞的DNA，并进行PFGE分析。快速修复的平均半衰期为3分钟，缓慢修复的平均半衰期为35分钟。还使用该系统确定了多形性胶质母细胞瘤细胞中DSB修复的动力学。从细胞悬液制备琼脂糖塞，用7.5 Gy X射线照射，并允许修复长达90分钟。对来自约1,250个肿瘤细胞的DNA进行电泳，并如上所述对EMT-6细胞进行分析。对于这种特定的肿瘤，90分钟后约75％的诱导DSB得到修复。所呈现的数据表明，这种基于PFGE的系统是一种极其灵敏的方法，可使用极少的细胞来测量低剂量X射线照射后的DSB诱导和修复情况。
