Anai H, Maehara Y, Sugimachi K
Cancer Center of Kyushu University Hospital, Faculty of Medicine, Fukuoka, Japan.
Cancer Lett. 1988 May;40(1):33-8. doi: 10.1016/0304-3835(88)90259-5.
DNA strand break in HeLa cells induced by heat was detected using the in situ nick translation method. The cells were incubated at 43 degrees C for various times (15, 30, 45, 60, 90 or 120 min) in Lab-Tek tissue culture chamber/slides and were fixed with ethanol/acetic acid on the slide glass. The break sites in DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and 3H-labeled dTTP. The level of break sites in the DNA was visualized by autoradiographic observation of the grains. The DNA strand break appeared as early as 15 min, increased to 10.3-fold at 45 min of 43 degrees C treatment and this level related reciprocally to clonogenicity of the cell. The nick translation method thus provides a rapid in situ assay for determining heat-induced DNA damage of cultured cells, in a semi-quantitative manner.
采用原位缺口平移法检测热诱导的HeLa细胞DNA链断裂情况。将细胞在Lab-Tek组织培养室/载玻片上于43℃孵育不同时间（15、30、45、60、90或120分钟），然后用乙醇/乙酸固定在载玻片上。在大肠杆菌DNA聚合酶I和3H标记的dTTP存在的情况下，人工翻译DNA中的断裂位点。通过对银粒的放射自显影观察来可视化DNA中断裂位点的水平。DNA链断裂最早在15分钟出现，在43℃处理45分钟时增加到10.3倍，且该水平与细胞的克隆形成能力呈反比。因此，缺口平移法提供了一种快速的原位检测方法，以半定量方式确定培养细胞的热诱导DNA损伤。
