Wojcik Andrzej, Bochenek Agnieszka, Lankoff Anna, Lisowska Halina, Padjas Anna, Szumiel Irena, von Sonntag Clemens, Obe Günter
Department of Radiobiology and Immunology, Institute of Biology, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce, Poland.
J Photochem Photobiol B. 2006 Jul 3;84(1):15-20. doi: 10.1016/j.jphotobiol.2006.01.008. Epub 2006 Feb 21.
It has been observed previously that 5-bromo-2'-deoxyuridine (BrdU) potentiates the effect of UVC radiation on the level of sister chromatid exchanges. It is not known which type of DNA damage is responsible for this enhancing effect and we have proposed this to be the DNA interstrand crosslink (ICL) which, theoretically, may arise in cells that are labelled with BrdU for one round of replication and exposed to UVC radiation. The aim of the present investigation was to verify if ICLs are indeed formed during this irradiation scenario. CHO-K1 cells were prelabelled with BrdU and exposed to UVC. ICLs were detected by a modified version of the comet assay that relies on the reduction of induced DNA migration in the agarose gel. Carboplatin was used as a positive control. We found that BrdU+UVC treatment indeed results in a reduction of the damage induced by gamma-radiation. Furthermore, we observed that CL-V4B cells exposed to BrdU+UVC, but not to UVC alone, showed a very high level of chromosomal damage. These cells have a deficient Rad51C paralog that renders them extremely sensitive towards ICLs. Interestingly, the cytogenetic results did not correlate with cell survival, where it was found that the CL-V4B cells tolerate BrdU+UVC better than the wild type cells. The possible reasons are discussed. Taken together our results indicate that ICLs are formed in DNA that was prelabelled with BrdU and exposed to UVC radiation.
先前已经观察到，5-溴-2'-脱氧尿苷（BrdU）可增强紫外线C（UVC）辐射对姐妹染色单体交换水平的影响。目前尚不清楚哪种类型的DNA损伤导致了这种增强效应，我们推测这可能是DNA链间交联（ICL），理论上，在经BrdU标记一轮复制并暴露于UVC辐射的细胞中可能会出现这种情况。本研究的目的是验证在这种照射情况下是否确实形成了ICL。将中国仓鼠卵巢细胞K1（CHO-K1）用BrdU预先标记，然后暴露于UVC。通过改良的彗星试验检测ICL，该试验依赖于琼脂糖凝胶中诱导的DNA迁移的减少。卡铂用作阳性对照。我们发现，BrdU+UVC处理确实会导致γ辐射诱导的损伤减少。此外，我们观察到，暴露于BrdU+UVC而非仅暴露于UVC的CL-V4B细胞显示出非常高水平的染色体损伤。这些细胞的Rad51C旁系同源物缺陷，这使它们对ICL极其敏感。有趣的是，细胞遗传学结果与细胞存活率不相关，结果发现CL-V4B细胞比野生型细胞更能耐受BrdU+UVC。文中讨论了可能的原因。综上所述，我们的结果表明，在预先用BrdU标记并暴露于UVC辐射的DNA中形成了ICL。
