Nuta Otilia, Rothkamm Kai, Darroudi Firouz
Nazarbayev University, School of Sciences and Humanities, Department of Biology, Nur-Sultan, 010000, Kazakhstan.
Department of Radiotherapy and Radiation Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Radiat Res. 2020 May;193(5):451-459. doi: 10.1667/RR15495.1. Epub 2020 Mar 9.
Findings from previous studies have suggested that the telomerase system is involved in radiation-induced genomic instability. In this study, we investigated the involvement of telomerase in the development and processing of chromosomal damage at different cell cycle stages after irradiation of human fibroblasts. Several response criteria were investigated, including cell survival, chromosomal damage (using the micronucleus assay), G-induced chromatid aberrations (using the conventional G assay as well as a chemically-induced premature chromosome condensation assay) and DNA double-strand breaks (DSBs; using γ-H2AX, 53BP1 and Rad51) in an isogenic pair of cell lines: BJ human foreskin fibroblasts and BJ1-hTERT, a telomerase-immortalized BJ cell line. To distinguish among G, S and G phase, cells were co-immunostained for CENP-F and cyclin A, which are tightly regulated proteins in the cell cycle. After X-ray irradiation at doses in the range of 0.1-6 Gy, the results showed that for cell survival and micronuclei induction, where the overall effect is dominated by the cells in G and S phase, no difference was observed between the two cell types; in contrast, when radiation sensitivity at the G stage of the cell cycle was analyzed, a significantly higher number of chromatid-type aberrations (breaks and exchanges), and higher levels of γ-H2AX and of Rad51 foci were observed for the BJ cells compared to the BJ1-hTERT cells. Therefore, it can be concluded that telomerase appears to be involved in DNA DSB repair processes, mainly in the G phase. These data, taken overall, reinforce the notion that hTERT or other elements of the telomere/telomerase system may defend chromosome integrity in human fibroblasts by promoting repair in G phase of the cell cycle.
先前研究的结果表明，端粒酶系统参与辐射诱导的基因组不稳定。在本研究中，我们调查了端粒酶在人成纤维细胞受辐照后不同细胞周期阶段染色体损伤的发生和处理过程中的作用。研究了几个反应标准，包括细胞存活、染色体损伤（使用微核试验）、G诱导的染色单体畸变（使用传统的G试验以及化学诱导的早熟染色体凝聚试验）和DNA双链断裂（DSBs；使用γ-H2AX、53BP1和Rad51），采用一对同基因细胞系：BJ人包皮成纤维细胞和BJ1-hTERT，一种端粒酶永生化的BJ细胞系。为了区分G、S和G期，细胞用CENP-F和细胞周期蛋白A进行共免疫染色，这两种蛋白在细胞周期中受到严格调控。在0.1-6 Gy范围内的剂量进行X射线辐照后，结果表明，对于细胞存活和微核诱导，总体效应主要由G和S期的细胞决定，两种细胞类型之间未观察到差异；相反，当分析细胞周期G期的辐射敏感性时，与BJ1-hTERT细胞相比，BJ细胞观察到明显更多的染色单体型畸变（断裂和交换）以及更高水平的γ-H2AX和Rad51灶。因此，可以得出结论，端粒酶似乎参与DNA双链断裂修复过程，主要在G期。总体而言，这些数据强化了这样一种观念，即hTERT或端粒/端粒酶系统的其他元件可能通过促进细胞周期G期的修复来保护人成纤维细胞中的染色体完整性。
