Department of Molecular and Tumor Radiobiology, Frédéric Joliot-Curie National Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary.
Mutat Res. 2011 Nov 1;716(1-2):33-9. doi: 10.1016/j.mrfmmm.2011.07.018. Epub 2011 Aug 5.
One of the key issues of current radiation research is the biological effect of low doses. Unfortunately, low dose science is hampered by the unavailability of easily performable, reliable and sensitive quantitative biomarkers suitable detecting low frequency alterations in irradiated cells. We applied a quantitative real time polymerase chain reaction (qRT-PCR) based protocol detecting common deletions (CD) in the mitochondrial genome to assess direct and non-targeted effects of radiation in human fibroblasts. In directly irradiated (IR) cells CD increased with dose and was higher in radiosensitive cells. Investigating conditioned medium-mediated bystander effects we demonstrated that low and high (0.1 and 2Gy) doses induced similar levels of bystander responses and found individual differences in human fibroblasts. The bystander response was not related to the radiosensitivity of the cells. The importance of signal sending donor and signal receiving target cells was investigated by placing conditioned medium from a bystander response positive cell line (F11-hTERT) to bystander negative cells (S1-hTERT) and vice versa. The data indicated that signal sending cells are more important in the medium-mediated bystander effect than recipients. Finally, we followed long term effects in immortalized radiation sensitive (S1-hTERT) and normal (F11-hTERT) fibroblasts up to 63 days after IR. In F11-hTERT cells CD level was increased until 35 days after IR then reduced back to control level by day 49. In S1-hTERT cells the increased CD level was also normalized by day 42, however a second wave of increased CD incidence appeared by day 49 which was maintained up to day 63 after IR. This second CD wave might be the indication of radiation-induced instability in the mitochondrial genome of S1-hTERT cells. The data demonstrated that measuring CD in mtDNA by qRT-PCR is a reliable and sensitive biomarker to estimate radiation-induced direct and non-targeted effects.
当前辐射研究的一个关键问题是低剂量的生物效应。不幸的是，低剂量科学受到缺乏易于实施、可靠和敏感的定量生物标志物的阻碍，这些标志物适合检测辐照细胞中低频的改变。我们应用了一种基于定量实时聚合酶链反应 (qRT-PCR) 的方案，检测线粒体基因组中的常见缺失 (CD)，以评估人类成纤维细胞中辐射的直接和非靶向效应。在直接辐照 (IR) 的细胞中，CD 随着剂量的增加而增加，并且在放射敏感的细胞中更高。在研究条件培养基介导的旁观者效应时，我们证明了低剂量和高剂量 (0.1 和 2Gy) 诱导了类似水平的旁观者反应，并发现了人类成纤维细胞中的个体差异。旁观者反应与细胞的放射敏感性无关。通过将来自旁观者反应阳性细胞系 (F11-hTERT) 的条件培养基放置在旁观者阴性细胞 (S1-hTERT) 上，并反过来进行，研究了信号发送供体细胞和信号接收靶细胞的重要性。数据表明，在介质介导的旁观者效应中，信号发送细胞比受体细胞更为重要。最后，我们在 IR 后长达 63 天的时间里，对永生敏感 (S1-hTERT) 和正常 (F11-hTERT) 成纤维细胞进行了长期效应的研究。在 F11-hTERT 细胞中，CD 水平一直增加到 IR 后 35 天，然后在第 49 天恢复到对照水平。在 S1-hTERT 细胞中，增加的 CD 水平也在第 42 天正常化，但在第 49 天出现了第二次增加的 CD 发生率，直到 IR 后第 63 天仍保持不变。第二次 CD 波可能是 S1-hTERT 细胞中线粒体基因组辐射诱导不稳定性的迹象。该数据表明，通过 qRT-PCR 测量 mtDNA 中的 CD 是一种可靠和敏感的生物标志物，可用于估计辐射引起的直接和非靶向效应。
