Okayasu R, Iliakis G
Thomas Jefferson University, Department of Radiation Oncology and Nuclear Medicine, Philadelphia, PA 19107.
Int J Radiat Biol. 1989 Apr;55(4):569-81. doi: 10.1080/09553008914550621.
The effect of detergent type, pH and temperature during lysis on the DNA elution dose response was studied under non-winding conditions in exponentially growing, plateau-phase and synchronous S-phase CHO cells. Lysis with sodium-N-laurylsarcosine (NLS) increased the DNA elution rate and resulted in higher DNA elution for the same absorbed radiation dose than lysis with sodium dodecyl sulphate (SDS). This increase in elution caused a reduction in the shoulder width of the DNA elution dose-response curve, but did not significantly affect the final slope. One hour incubation at elevated temperatures (60 degrees C) during lysis either with NLS or SDS further increased DNA elution. Under these conditions DNA elution dose-response curves with a small or zero shoulder were obtained with exponentially growing, plateau-phase or synchronized S-phase cells. DNA elution was reduced to about 50 per cent of the controls when the pH of the SDS lysis solution was adjusted from 9.6 to 7.6. This effect was observed in cells that were lysed at room temperature, as well as in cells lysed at 60 degrees C. When NLS was used for lysis, a similar reduction in pH did not alter the DNA elution dose-response curve at either lysis temperature. Based on these results it is suggested that the shoulder observed in the DNA elution dose-response curve reflects partial separation of DNA from associated proteins. A direct and unconditional correlation of the DNA filter elution behaviour, as observed under non-unwinding conditions, with the induction of DNA dsb may thus not always be justified. Caution is required when elution results are used to establish correlations between the level of induction of DNA dsb and cell killing.
在指数生长期、平台期和同步化 S 期的中国仓鼠卵巢（CHO）细胞中，在非缠绕条件下研究了裂解过程中去污剂类型、pH 和温度对 DNA 洗脱剂量反应的影响。与十二烷基硫酸钠（SDS）裂解相比，用 N-月桂酰肌氨酸钠（NLS）裂解可提高 DNA 洗脱速率，并且在相同吸收辐射剂量下导致更高的 DNA 洗脱量。洗脱量的这种增加导致 DNA 洗脱剂量反应曲线的肩部宽度减小，但对最终斜率没有显著影响。用 NLS 或 SDS 裂解时，在高温（60℃）下孵育 1 小时可进一步增加 DNA 洗脱。在这些条件下，指数生长期、平台期或同步化 S 期细胞获得的 DNA 洗脱剂量反应曲线的肩部较小或为零。当 SDS 裂解溶液的 pH 从 9.6 调节至 7.6 时，DNA 洗脱量降低至对照的约 50%。在室温下裂解的细胞以及在 60℃下裂解的细胞中均观察到这种效应。当使用 NLS 进行裂解时，类似的 pH 降低在任何一种裂解温度下均未改变 DNA 洗脱剂量反应曲线。基于这些结果，表明在 DNA 洗脱剂量反应曲线中观察到的肩部反映了 DNA 与相关蛋白质的部分分离。因此，在非解旋条件下观察到的 DNA 滤膜洗脱行为与 DNA 双链断裂（dsb）诱导之间的直接和无条件相关性可能并不总是合理的。当使用洗脱结果来建立 DNA dsb 诱导水平与细胞杀伤之间的相关性时需要谨慎。
