Kapiszewska M, Reddy N M, Lange C S
Institute of Molecular Biology, Jagiellonian University, Krakow, Poland.
Int J Radiat Biol. 1991 Oct;60(4):635-46. doi: 10.1080/09553009114552461.
Trypsin is the enzyme commonly used to prepare single cell suspensions from monolayer and spheroid cultures, both to determine survival and to assay DNA damage. Trypsin induces rounding, dissociation and radiosensitization of anchorage-dependent cells. Radiosensitivity and chromatin structure were compared between trypsin-treated (0.05%) round V79 cells from monolayers and spheroids vs. untreated spread monolayer cells in situ. The fluorescent halo technique was used to measure the changes in DNA supercoiling in nucleoids isolated from control and irradiated round and spread cells. Maximal halo diameters, the amount of initial and residual radiation-induced DNA damage (estimated from nucleoid halo diameter changes), and the radiosensitivity were higher in round cells than in spread monolayer V79 cells. The effects on cellular radiosensitivity and maximal halo diameter of other agents which also round and dissociate cells, e.g. 0.25% trypsin, pronase E and a non-enzymatic cell-dissociation solution, were similar to those of 0.05% trypsin. In LY-S cells, which are anchorage-independent, DNA loop size, the initial amount of DNA damage and radiosensitivity were not affected by trypsin. We suggest that the higher radiosensitivity of anchorage-dependent cells under immediate trypsinization and plating conditions, compared to cells with postirradiation in situ repair incubation, is due to correlated changes in cell shape and chromatin structure.
胰蛋白酶是常用于从单层培养物和球体培养物中制备单细胞悬液的酶，既用于确定细胞存活率，也用于检测DNA损伤。胰蛋白酶可诱导贴壁依赖性细胞变圆、解离并产生放射增敏作用。比较了用胰蛋白酶处理（0.05%）的单层和球体来源的圆形V79细胞与未经处理的原位铺展单层细胞之间的放射敏感性和染色质结构。采用荧光晕圈技术测量从对照及照射后的圆形和铺展细胞中分离出的核小体中DNA超螺旋的变化。圆形细胞的最大晕圈直径、初始和残留辐射诱导的DNA损伤量（根据核小体晕圈直径变化估算）以及放射敏感性均高于铺展的单层V79细胞。其他能使细胞变圆和解离的试剂，如0.25%胰蛋白酶、链霉蛋白酶E和一种非酶促细胞解离溶液，对细胞放射敏感性和最大晕圈直径的影响与0.05%胰蛋白酶相似。在不依赖贴壁的LY-S细胞中，DNA环大小、DNA损伤初始量和放射敏感性不受胰蛋白酶影响。我们认为，与经照射后原位修复孵育的细胞相比，贴壁依赖性细胞在立即用胰蛋白酶处理和铺板条件下具有更高的放射敏感性，这是由于细胞形状和染色质结构的相关变化所致。
