Sándor Nikolett, Schilling-Tóth Boglárka, Kis Enikő, Benedek Anett, Lumniczky Katalin, Sáfrány Géza, Hegyesi Hargita
Division of Molecular Radiobiology and Biodosimetry, F. Joliot-Curie National Research Institute for Radiobiology and Radiohygiene, Anna 5., Budapest, Hungary.
Division of Cellular and Immune-radiobiology, F. Joliot-Curie National Research Institute for Radiobiology and Radiohygiene, Anna 5., Budapest, Hungary.
Mutat Res Genet Toxicol Environ Mutagen. 2015 Nov;793:142-9. doi: 10.1016/j.mrgentox.2015.06.009. Epub 2015 Jun 17.
We have investigated the importance of GDF-15 (secreted cytokine belonging to the TGF-β superfamily) in low and high dose radiation-induced cellular responses. A telomerase immortalized human fibroblast cell line (F11hT) was used in the experiments. A lentiviral system encoding small hairpin RNAs (shRNA) was used to establish GDF-15 silenced cells. Secreted GDF-15 levels were measured in culture medium by ELISA. Cell cycle analysis was performed by flow cytometry. The experiments demonstrated that in irradiated human fibroblasts GDF-15 expression increased with dose starting from 100mGy. Elevated GDF-15 expression was not detected in bystander cells. The potential role of GDF-15 in radiation response was investigated by silencing GDF-15 in immortalized human fibroblasts with five different shRNA encoded in lentiviral vectors. Cell lines with considerably reduced GDF-15 levels presented increased radiation sensitivity, while a cell line with elevated GDF-15 was more radiation resistant than wild type cells. We have investigated how the reduced GDF-15 levels alter the response of several known radiation inducible genes. In F11hT-shGDF-15 cells the basal expression level of CDKN1A was unaltered relative to F11hT cells, while GADD45A and TGF-β1 mRNA levels were slightly higher, and TP53INP1 was considerably reduced. The radiation-induced expression of TP53INP1 was lower in the silenced than in wild type fibroblast cells. Cell cycle analysis indicated that radiation-induced early G2/M arrest was abrogated in GDF-15 silenced cells. Moreover, radiation-induced bystander effect was less pronounced in GDF-15 silenced fibroblasts. In conclusion, the results suggest that GDF-15 works as a radiation inducible radiation resistance increasing factor in normal human fibroblast cells, acts by regulating the radiation-induced transcription of several genes and might serve as a radiation-induced early biomarker in exposed cells.
我们研究了生长分化因子15（GDF - 15，属于转化生长因子-β超家族的分泌型细胞因子）在低剂量和高剂量辐射诱导的细胞反应中的重要性。实验使用了一种端粒酶永生化的人成纤维细胞系（F11hT）。采用编码小发夹RNA（shRNA）的慢病毒系统建立GDF - 15沉默细胞。通过酶联免疫吸附测定法（ELISA）测量培养基中分泌的GDF - 15水平。通过流式细胞术进行细胞周期分析。实验表明，在受辐照的人成纤维细胞中，GDF - 15表达从100毫戈瑞开始随剂量增加。在旁观者细胞中未检测到GDF - 15表达升高。通过用慢病毒载体编码的五种不同shRNA使永生化人成纤维细胞中的GDF - 15沉默，研究了GDF - 15在辐射反应中的潜在作用。GDF - 15水平显著降低的细胞系表现出辐射敏感性增加，而GDF - 15水平升高的细胞系比野生型细胞更具辐射抗性。我们研究了降低的GDF - 15水平如何改变几种已知辐射诱导基因的反应。在F11hT - shGDF - 15细胞中，相对于F11hT细胞，细胞周期蛋白依赖性激酶抑制因子1A（CDKN1A）的基础表达水平未改变，而生长停滞和DNA损伤诱导蛋白45A（GADD45A）和转化生长因子-β1（TGF - β1）的mRNA水平略高，而TP53诱导核蛋白1（TP53INP1）则显著降低。在沉默的成纤维细胞中，TP53INP1的辐射诱导表达低于野生型细胞。细胞周期分析表明，在GDF - 15沉默细胞中，辐射诱导的早期G2/M期阻滞被消除。此外，在GDF - 15沉默的成纤维细胞中，辐射诱导的旁观者效应不太明显。总之，结果表明GDF - 15在正常人成纤维细胞中作为一种辐射诱导的辐射抗性增加因子起作用，通过调节几种基因的辐射诱导转录发挥作用，并且可能作为暴露细胞中辐射诱导的早期生物标志物。
