Limoli C L, Ward J F
Department of Radiology, University of California at San Diego, La Jolla 92093-0610.
Radiat Res. 1994 Jun;138(3):312-9.
Previous work has established that DNA double-strand breaks (DSBs) are formed when Chinese hamster cells are substituted with 5-bromo-2'-deoxyuridine (BrdU) and exposed to UVA light in the presence of Hoechst dye #33258. Double-strand breaks produced by this treatment (5.1 x 10(-6) DSBs/BrdU residue/kJ m-2) were found to depend linearly on the level of BrdU substitution, Hoechst dye and fluence of UVA light. To examine the biological consequences of these novel DSBs, clonogenic assays were used to score cell survival, and elution assays were used to measure strand break levels at various times after photolysis. Using this system, marked cell killing was observed; photosensitivity could be increased by four orders of magnitude compared to cells without BrdU and dye. Decreases in the F0 value and the shoulder of survival curves followed increasing levels of BrdU substitution. In addition, the results indicate that DSBs produced by this photolysis protocol are two to three times more effective in causing cell killing than the DSBs produced by the action of ionizing radiation. To investigate the cause of the toxicity, repair of DSBs after photolysis was measured. Unexpectedly, DSB levels increased two- to threefold over 1 h at 37 degrees C, then decreased to initial damage levels over the next 2 h. The implications of this break induction are discussed in terms of mechanism and cell killing.
先前的研究已经证实，当用5-溴-2'-脱氧尿苷（BrdU）替代中国仓鼠细胞，并在存在Hoechst染料#33258的情况下暴露于UVA光时，会形成DNA双链断裂（DSB）。发现这种处理产生的双链断裂（5.1×10⁻⁶个DSB/BrdU残基/kJ m⁻²）线性依赖于BrdU替代水平、Hoechst染料和UVA光的通量。为了研究这些新型DSB的生物学后果，使用克隆形成试验来评估细胞存活率，并使用洗脱试验来测量光解后不同时间的链断裂水平。使用该系统，观察到明显的细胞杀伤；与没有BrdU和染料的细胞相比，光敏性可提高四个数量级。随着BrdU替代水平的增加，存活曲线的F0值和肩部降低。此外，结果表明，这种光解方案产生的DSB在导致细胞杀伤方面比电离辐射作用产生的DSB有效两到三倍。为了研究毒性的原因，测量了光解后DSB的修复情况。出乎意料的是，在37℃下，DSB水平在1小时内增加了两到三倍，然后在接下来的2小时内降至初始损伤水平。从机制和细胞杀伤方面讨论了这种断裂诱导的意义。
