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紫外线（UV）照射技术。具有3μm直径孔的isopore膜过滤器可屏蔽UVC辐射，使其不受培养的人成纤维细胞的影响，导致细胞内的部分辐射，每个细胞核平均约有3个暴露区域。本研究解决的问题，是否在细胞核内的DNA损伤的空间分布是重要的，在触发紫外线诱导的细胞毒性。我们研究了在细胞核中诱导等量的DNA损伤后，部分UV照射的细胞和均匀照射的细胞之间的细胞毒性是否存在差异。我们首先用酶联免疫吸附试验测定了正常人成纤维细胞中DNA损伤的形成。我们发现，每平方米5焦耳的紫外线照射产生的环丁烷嘧啶二聚体和（6-4）光产物的数量与每平方米100焦耳的膜过滤屏相当。在这些剂量下，我们发现两种类型的紫外线照射诱导的细胞毒性水平相似，如MTS测定所评估的。这两种类型的紫外线照射的细胞也有类似的细胞周期分布和细胞凋亡的流式细胞仪测量。此外，两种不同的UV处理之间的修复动力学的任何类型的光损伤没有显着差异。在Cockayne综合征细胞中也得到了类似的结果，这些细胞在转录偶联核苷酸切除修复中有缺陷。目前的结果表明，在研究的光产物范围内，细胞内DNA损伤的空间分布不如触发UV诱导的细胞毒性中的损伤量重要。

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.