Radiation response in vitro of fibroblasts from a fanconi anemia patient with marked clinical radiosensitivity
Radiation response in vitro of fibroblasts from a fanconi anemia patient with marked clinical radiosensitivity
复制标题
DOI：
10.1007/s00066-004-1250-1
复制
发表时间：
2004-12-01
影响因子：
3.1
通讯作者：
Plowman, PN
中科院分区：
文献类型：
--
作者：
Djuzenova, C;Flentje, M;Plowman, PN
背景：范可尼贫血（Fanconi anemia， FA）是一种常染色体隐性染色体不稳定性疾病，以进行性全血细胞减少和癌症易感性为特征。放射治疗对FA患者合并癌症的风险仍有待研究。最近，Marcou等（2001）报道了一例女性FA合并扁桃体鳞状细胞癌经放疗后出现严重临床放射敏感性的病例。相比之下，她的体外辐照皮肤成纤维细胞显示出几乎正常的放射敏感性，通过菌落生存试验确定。材料和方法：鉴于这种差异，本研究采用碱性单细胞凝胶电泳（Comet）和细胞松弛素阻断微核（MN）试验进一步分析了这种特殊FA成纤维细胞菌株（命名为425BR）的辐射响应。此外，采用Western blot和局灶免疫荧光染色检测DNA修复蛋白hMrell、Rad50和Rad51的表达水平。结果：彗星实验显示，辐照FA细胞的初始DNA片段比对照（1BR3）成纤维细胞高2倍，DNA重新连接过程慢3倍。此外，尽管FA细胞中MN的基线水平低于对照组，但在2-4 Gy照射下，FA成纤维细胞比对照细胞更容易（约两倍）产生MN。DNA修复蛋白（hMrell、Rad50和Rad51）的Western blot分析未发现放射前后FA患者的成纤维细胞中蛋白表达水平或迁移模式有任何异常。与此同时，FA患者体外辐照后的细胞与对照细胞相比，具有局灶性DNA修复Rad51蛋白的细胞核数量明显减少。结论：受辐照FA成纤维细胞DNA损伤和MN诱导增加，含有Rad5l的DNA修复灶形成减少，这可能与先前报道的FA患者的临床放射敏感性有关。研究中对这一特殊FA细胞株的发现指出了仅基于菌落存活试验来预测细胞系和肿瘤的辐射反应的困难。
Background: Fanconi anemia (FA) is an autosomal recessive chromosome instability disorder characterized by progressive pancytopenia and cancer susceptibility. The risks of radiation therapy in FA patients who have cancer remain to be investigated. Recently, Marcou et al. (2001) reported a case of severe clinical radiosensitivity in a female FA patient with a tonsillar squamous cell carcinoma treated by radiotherapy. By contrast, her in vitro irradiated skin fibroblasts revealed nearly normal radiosensitivity as determined by the colony survival assay.Material and Methods: In view of this discrepancy, the radiation response of this particular FA fibroblast strain (designated 425BR) was further analyzed in the present study by means of the alkaline single-cell gel electrophoresis (Comet) assay, and also by the cytochalasin-blocked micronuclei (MN) test. In addition, the expression levels of DNA repair proteins, hMrell, Rad50, and Rad51, were investigated using Western blot and foci immunofluorescence staining.Results: The Comet assay revealed that the initial DNA fragmentation in irradiated FA cells was two times higher and the DNA rejoining process was three times slower than that in control (1BR3) fibroblasts. Moreover, although the baseline Level of MNs was Lower in FA cells than in controls, the FA fibroblasts were more prone (about two times) to MN production than control cells when irradiated with 2-4 Gy. Western blot analysis of the DNA repair proteins (hMrell, Rad50, and Rad51) did not reveal any abnormalities in protein expression Levels or their migration patterns in the fibroblasts derived from an FA patient either before or after irradiation. At the same time, in vitro irradiated cells from the FA patient exhibited a significantly reduced number of nuclei with focally concentrated DNA repair Rad51 protein than in control cells.Conclusion: The increased DNA damage and MN induction in irradiated FA fibroblasts, and the reduction of the formation of DNA repair foci containing Rad5l suggest a possible Link to the profound clinical radiosensitivity reported earlier for this FA patient. The findings on this particular FA cell strain presented in the study point toward the difficulties involved in the prediction of the radiation response of cell Lines and tumors based solely on the colony survival test.