The Total Amount of DNA Damage Determines Ultraviolet-Radiation-Induced Cytotoxicity after Uniform- or Localized Irradiation of Human Cells

我们最近开发了一种微孔紫外线（UV）照射技术。孔径为3μm的等孔滤膜可屏蔽体外培养的人成纤维细胞的UVC辐射，导致细胞内平均每个细胞核约3个暴露区域的局部照射。目前的研究解决了细胞核内DNA损伤的空间分布在触发紫外线诱导的细胞毒性中是否重要的问题。我们研究了部分紫外线照射细胞和均匀紫外线照射细胞在细胞核中诱导等量DNA损伤后是否存在细胞毒性差异。我们首先用酶联免疫吸附法测定了正常人成纤维细胞中DNA损伤的形成。我们发现，5 J / m^2的紫外线照射产生的环丁烷嘧啶二聚体和（6-4）光产物相当于100 J / m^2的膜过滤屏蔽。在这些剂量下，我们发现两种类型的紫外线照射诱导相似水平的细胞毒性，通过MTS试验评估。两种类型的紫外线照射细胞也有相似的细胞周期分布和凋亡的流式细胞术测量。此外，在两种不同的紫外线处理中，两种类型的光斑的修复动力学没有显著差异。在转录偶联核苷酸切除修复缺陷的Cockayne综合征细胞中也获得了类似的结果。目前的结果表明，在所研究的光产物范围内，在触发紫外线诱导的细胞毒性时，DNA损伤的空间分布不如损伤的数量重要。

We have recently developed a micropore ultraviolet (UV) irradiation technique. An isopore membrane filter with 3μm diameter pores shields UVC radiation from cultured human fibroblasts, leading to partial irradiation within the cells with an average of about 3 exposed areas per nucleus. The present study addressed the question of whether the spatial distribution of DNA damage within a cell nucleus is important in triggering UV-induce cytotoxicity. We have examined whether there are differences incytotoxicity between partially UV-irradiated cells and uniformly irradiated cells after equal amounts of DNA damage were induced in the cell nuclei. We first determined DNA damage formation in normal human fibroblasts using anenzyme-linked immunosorbent assay. We found that 5 J per m^2 UV irradiation produced an equivalent amount of cyclobutane pyrimidine dimers and (6-4) photoproducts per cell as 100 J per m^2 with the membrane filter shield. At those doses, we found that both types of UV irradiation induced similar levels of cytotoxicity as assessed by an MTS assay. Both types of UV irradiated cells also had similar cell-cycle distribution and apoptosis as measured by flowcytometry. Moreover, no significant differences in repair kinetics for either type of photolesion were observed between the two different UV treatments. Similar results were obtained in Cockayne syndrome cells that are defective in transcription-coupled nucleotide excision repair Present results indicate that in the range of photoproducts studied, the spatial distribution of DNA damage within a cell is less important than the amount of damage in triggering UV-induced cytotoxicity.