Fibach E, Gatt S
Dept of Hematology, Hadassah University Hospital, Jerusalem, Israel.
Leuk Res. 1987;11(11):1019-26. doi: 10.1016/0145-2126(87)90121-4.
Incubation of cells with 12-(1-pyrene) dodecanoic acid (P12), a fatty acid to which a pyrene nucleus has been covalently linked, followed by irradiation with long-wave ultra-violet light (LUV) at 366 nm, resulted in cytotoxicity. Syntheses of macromolecules was significantly decreased after 30 min, while an accumulation of trypan-blue positive, non-viable cells was observed several hours following irradiation. Cloning of the irradiated cells in semi-solid medium showed an exponential dose-response survival curve. Above a threshold dose colony number decreased, although the rate of clonal development and the final size were not affected. The sensitivity of detecting rare surviving cells could be increased by incubating the irradiated cells for several hours in liquid culture followed by concentrating intact cells by gradient sedimentation. Using this procedure, one surviving clonogenic cell could be detected in 10(7) irradiated cells/dish. More than 10 min of irradiation at 773 microV/cm was required to photosensitize the population below detection by this method. The possibility was considered that colonies derived from cells surviving sub-maximal LUV doses represent clones that are resistant to photosensitization, a phenomenon attributed to either inability to take up or metabolize P12, or resistance to the radiation-induced toxicity. Analysis of P12 uptake in the surviving clonal populations showed no significant difference as compared to the parental population. The results suggest that surviving cells reflect a phenotypic heterogeneity caused by variation in the physiological state such as the respective position in the cell cycle and are not genetically resistant mutants.
用12-(1-芘基)十二烷酸(P12，一种芘核已共价连接的脂肪酸)处理细胞，然后用366nm的长波紫外线(LUV)照射，会导致细胞毒性。30分钟后大分子合成显著减少，而照射后数小时观察到锥虫蓝阳性的非存活细胞积聚。在半固体培养基中对照射后的细胞进行克隆，显示出指数剂量反应存活曲线。高于阈值剂量时，集落数减少，尽管克隆发育速率和最终大小不受影响。通过在液体培养基中培养照射后的细胞数小时，然后通过梯度沉降浓缩完整细胞，可以提高检测罕见存活细胞的灵敏度。使用该方法，在10(7)个照射细胞/培养皿中可以检测到一个存活的克隆形成细胞。在773微伏/厘米下照射超过10分钟才能使群体光敏化至该方法无法检测到的程度。有人认为，源自低于最大LUV剂量存活细胞的集落代表对光敏化有抗性的克隆，这种现象归因于无法摄取或代谢P12，或对辐射诱导毒性的抗性。对存活克隆群体中P12摄取的分析表明，与亲代群体相比没有显著差异。结果表明，存活细胞反映了由生理状态变化(如细胞周期中的各自位置)引起的表型异质性，而不是遗传抗性突变体。
