Genetic and epigenetic changes in clonal descendants of irradiated human fibroblasts

Abstract

To study delayed genetic and epigenetic radiation effects, which may trigger radiation-induced carcinogenesis, we have established single-cell clones from irradiated and non-irradiated primary human fibroblasts. Stable clones were endowed with the same karyotype in all analyzed metaphases after 20 population doublings (PDs), whereas unstable clones displayed mosaics of normal and abnormal karyotypes. To account for variation in radiation sensitivity, all experiments were performed with two different fibroblast strains. After a single X-ray dose of 2 Gy more than half of the irradiated clones exhibited radiation-induced genome instability (RIGI). Irradiated clones displayed an increased rate of loss of chromosome Y (LOY) and copy number variations (CNVs), compared to controls. CNV breakpoints clustered in specific chromosome regions, in particular 3p14.2 and 7q11.21, coinciding with common fragile sites. CNVs affecting the FHIT gene in FRA3B were observed in independent unstable clones and may drive RIGI. Bisulfite pyrosequencing of control clones and the respective primary culture revealed global hypomethylation of ALU, LINE-1, and alpha-satellite repeats as well as rDNA hypermethylation during in vitro ageing. Irradiated clones showed further reduced ALU and alpha-satellite methylation and increased rDNA methylation, compared to controls. Methylation arrays identified several hundred differentially methylated genes and several enriched pathways associated with in vitro ageing. Methylation changes in 259 genes and the MAP kinase signaling pathway were associated with delayed radiation effects (after 20 PDs). Collectively, our results suggest that both genetic (LOY and CNVs) and epigenetic changes occur in the progeny of exposed cells that were not damaged directly by irradiation, likely contributing to radiation-induced carcinogenesis. We did not observe epigenetic differences between stable and unstable irradiated clones. The fact that the DNA methylation (DNAm) age of clones derived from the same primary culture varied greatly suggests that DNAm age of a single cell (represented by a clone) can be quite different from the DNAm age of a tissue. We propose that DNAm age reflects the emergent property of a large number of individual cells whose respective DNAm ages can be highly variable.

Epigenetic signatures of Werner syndrome occur early in life and are distinct from normal epigenetic aging processes

“…Aging is associated with hypo‐ and hypermethylation events at specific regions of the genome (Unnikrishnan et al, ). Recently, we showed that the methylation of various repeat families decreased, whereas that of rDNA increased during in vitro aging of fibroblast clones (Flunkert et al, ). Here, we used the same bisulfite pyrosequencing assays to quantify mean methylation of ALU, LINE1, and α‐satellite repeats in WRN ‐, LMNA ‐, and POLD1 ‐mutant patients and controls (Table ).…”

“…Perturbations of this highly organized process cause nucleolar stress that is involved in the pathogenesis of many human diseases including cancer, and metabolic and cardiovascular disorders (Wang et al, ). Impaired ribosome biogenesis (Rattan, ) and rDNA hypermethylation (Flunkert et al, ) have also been linked to aging. During in vitro aging of fibroblasts, rDNA hypermethylation was reported to be more pronounced in two WS patients, compared with four controls (Machwe, Orren, & Bohr, ).…”

Common fragile site instability in normal cells: Lessons and perspectives

“…84 A good example of the valuable contribution of studies using normal cells is coming from a recent study carried out using normal fibroblasts. 85 Clonal cell populations generated from cells treated with low doses of IR displayed clusters of CNVs at FRA3B and FRA7J. 85 Interestingly, three CNVs, affecting directly or indirectly the FHIT gene, were detected in clones showing karyotypic instability.…”

“…85 Clonal cell populations generated from cells treated with low doses of IR displayed clusters of CNVs at FRA3B and FRA7J. 85 Interestingly, three CNVs, affecting directly or indirectly the FHIT gene, were detected in clones showing karyotypic instability. At the same time, the majority of cell clones derived from irradiated cells did not exhibit genomic alterations at FHIT, 85 although FRA3B is a well-known hot spot for radiation-induced fragility.…”

“…85 Interestingly, three CNVs, affecting directly or indirectly the FHIT gene, were detected in clones showing karyotypic instability. At the same time, the majority of cell clones derived from irradiated cells did not exhibit genomic alterations at FHIT, 85 although FRA3B is a well-known hot spot for radiation-induced fragility. 86,87 These results can be interpreted in light of the physiological protective function of FHIT against IR-induced DNA damage and its role of genome stability caretaker.…”