Abstract
Epigenetic alterations may contribute to the generation of cancer cells in a multi-step process of tumorigenesis following irradiation of normal body cells. Primary human fibroblasts with intact cell cycle checkpoints were used as a model to test whether X-ray irradiation with 2 and 4 Gray induces direct epigenetic effects (within the first cell cycle) in the exposed cells. ELISA-based fluorometric assays were consistent with slightly reduced global DNA methylation and hydroxymethylation, however the observed between-group differences were usually not significant. Similarly, bisulfite pyrosequencing of interspersed LINE-1 repeats and centromeric α-satellite DNA did not detect significant methylation differences between irradiated and non-irradiated cultures. Methylation of interspersed ALU repeats appeared to be slightly increased (one percentage point; p = 0.01) at 6 h after irradiation with 4 Gy. Single-cell analysis showed comparable variations in repeat methylation among individual cells in both irradiated and control cultures. Radiation-induced changes in global repeat methylation, if any, were much smaller than methylation variation between different fibroblast strains. Interestingly, α-satellite DNA methylation positively correlated with gestational age. Finally, 450K methylation arrays mainly targeting genes and CpG islands were used for global DNA methylation analysis. There were no detectable methylation differences in genic (promoter, 5' UTR, first exon, gene body, 3' UTR) and intergenic regions between irradiated and control fibroblast cultures. Although we cannot exclude minor effects, i.e. on individual CpG sites, collectively our data suggest that global DNA methylation remains rather stable in irradiated normal body cells in the early phase of DNA damage response.
Highlights
Radiation therapy is a highly effective form of cancer treatment
Population doubling time of four different primary human fibroblast strains ranged from 21 h to 27 h with an average of 24.1 ± 2.5 h
The growth curves of these representative strains are shown in S1 Fig. double-strand breaks (DSBs) were visualized by γH2AX foci staining in two fibroblast strains at 6 h and 24 h after irradiation with 2 and 4 Gy, respectively (S2 Fig, upper panel)
Summary
Radiation therapy is a highly effective form of cancer treatment. Ionizing radiation induces DNA damage either directly in exposed cells or indirectly (delayed) in cells several generations after exposure. Accumulating evidence suggests that irradiation induces DNA damage and genome instability and epigenetic alterations, in particular DNA methylation changes [3,4,5,6]. The main focus of epigenetic studies has been DNA methylation alterations several population doublings after irradiation, when radiation-induced genome instability may occur in cells that were not directly exposed [7,8,9]. Our study is focused on direct epigenetic effects in cells within the first cell cycle after irradiation
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