Dahmen Volker, Schmitz Sabine, Kriehuber Ralf
Radiation Biology Unit, Department of Safety and Radiation Protection, Forschungszentrum Jülich, 52425 Jülich, Germany.
Radiation Biology Unit, Department of Safety and Radiation Protection, Forschungszentrum Jülich, 52425 Jülich, Germany.
Mutat Res Genet Toxicol Environ Mutagen. 2017 Nov;823:58-64. doi: 10.1016/j.mrgentox.2017.09.002. Epub 2017 Sep 14.
Triplex-Forming oligonucleotides (TFO) bind sequence-specific to the DNA double helix in-vitro and in-vivo and are a promising tool to manipulate genes or gene regulatory elements. TFO as a carrier molecule for short-range particle emitter such as Auger-Electron-Emitters (AEE) bear the potential to introduce radiation-induced site-specific complex DNA lesions, which are known to induce chromosomal translocations. We studied gene expression, translocation frequency and protein expression in SCL-II cells after transfection with the AEE Iodine-125 (I-125) labeled TFO-BCL2 targeting the human BCL2 gene. The TFO-BCL2 binds to the BCL2 gene in close proximity to a known major-breakage-region (mbr). SCL-II cells were transfected with I-125 labeled TFO and stored for decay accumulation. Monitoring of BCL2 translocations was done with the Fluorescence-In-Situ-Hybridization (FISH) method. The utilized FISH probes were designed to detect a t(14;18) translocation of the BCL2 gene, which is a common translocation leading to an overexpression of BCL2 protein. Analysis of BCL2 gene expression levels was done via quantitative Real-Time PCR. Verification of gene expression on the protein level was analyzed by Western blotting. The relative gene expression of BCL2 in I-125-TFO-BCL2 transfected cells showed a significant up-regulation when compared to controls. Analysis of the BCL2 t(14;18) translocation frequency revealed a significant 1.8- to 2-fold increase when compared to control cells. This 2-fold increase was not reflected on the protein level. We conclude that I-125 decays within the BCL2 gene facilitate the t(14;18) chromosomal translocation in the SCL-II cells and that the increased frequency contributes to the observed overall enhanced BCL2 gene expression.
三链形成寡核苷酸（TFO）在体外和体内均能序列特异性地结合到DNA双螺旋上，是一种用于操纵基因或基因调控元件的有前景的工具。TFO作为短程粒子发射体（如俄歇电子发射体，AEE）的载体分子，有潜力引入辐射诱导的位点特异性复合DNA损伤，已知这种损伤会诱导染色体易位。我们研究了用靶向人BCL2基因的AEE碘 - 125（I - 125）标记的TFO - BCL2转染SCL - II细胞后的基因表达、易位频率和蛋白质表达。TFO - BCL2在靠近已知主要断裂区域（mbr）处与BCL2基因结合。用I - 125标记的TFO转染SCL - II细胞并储存以进行衰变积累。用荧光原位杂交（FISH）方法监测BCL2易位。所使用的FISH探针设计用于检测BCL2基因的t(14;18)易位，这是一种导致BCL2蛋白过表达的常见易位。通过定量实时PCR分析BCL2基因表达水平。通过蛋白质印迹分析蛋白质水平上的基因表达验证。与对照相比，I - 125 - TFO - BCL2转染细胞中BCL2的相对基因表达显示出显著上调。BCL2 t(14;18)易位频率分析显示，与对照细胞相比，显著增加了1.8至2倍。这种2倍的增加在蛋白质水平上未体现出来。我们得出结论，BCL2基因内的I - 125衰变促进了SCL - II细胞中的t(14;18)染色体易位，并且增加的频率导致了观察到的BCL2基因整体表达增强。
