Abstract
One main function of telomeres is to maintain chromosome and genome stability. The rate of telomere shortening can be accelerated significantly by chemical and physical environmental agents. Reactive oxygen species are a source of oxidative stress and can produce modified bases (mainly 8-oxoG) and single strand breaks anywhere in the genome. The high incidence of guanine residues in telomeric DNA sequences makes the telomere a preferred target for oxidative damage. Our aim in this work is to evaluate whether chromosome instability induced by oxidative stress is related specifically to telomeric damage. We treated human primary fibroblasts (MRC-5) in vitro with hydrogen peroxide (100 and 200 µM) for 1 hr and collected data at several time points. To evaluate the persistence of oxidative stress-induced DNA damage up to 24 hrs after treatment, we analysed telomeric and genomic oxidative damage by qPCR and a modified comet assay, respectively. The results demonstrate that the genomic damage is completely repaired, while the telomeric oxidative damage persists. The analysis of telomere length reveals a significant telomere shortening 48 hrs after treatment, leading us to hypothesise that residual telomere damage could be responsible for the telomere shortening observed. Considering the influence of telomere length modulation on genomic stability, we quantified abnormal nuclear morphologies (Nucleoplasmic Bridges, Nuclear Buds and Micronuclei) and observed an increase of chromosome instability in the same time frame as telomere shortening. At subsequent times (72 and 96 hrs), we observed a restoration of telomere length and a reduction of chromosome instability, leaving us to conjecture a correlation between telomere shortening/dysfunction and chromosome instability. We can conclude that oxidative base damage leads to abnormal nuclear morphologies and that telomere dysfunction is an important contributor to this effect.
Highlights
Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes
At 15 hrs, Tail DNA values obtained from the FPGmodified assay are not statistically different from those produced by the standard version of the comet assay for both doses used
Given that our work has demonstrated that oxidative stress induces telomere shortening and Abnormal nuclear morphology (ANM), we decided to study the specific correlation between telomere length and nucleoplasmic bridges (NPBs) by comparing results obtained from Quantitative-Fluorescence ‘‘in situ’’ Hybridization Analysis (Q-FISH) (Fig. 3b) and NPBs analysis (Fig. 8) for each dose and for all recovery times
Summary
Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes. Their primary role is to maintain chromosome and genome stability by preventing chromosomal ends from being recognised as double-strand breaks and by protecting them from end-to-end fusion and degradation [1].In humans, telomeric DNA is typically 10–15 Kb and is composed of tandem (TTAGGG)n hexanucleotide repeats [1].In addition, telomeres interact with a number of proteins that can influence chromosome-end integrity and dynamics [2]. Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes. Their primary role is to maintain chromosome and genome stability by preventing chromosomal ends from being recognised as double-strand breaks and by protecting them from end-to-end fusion and degradation [1]. Telomeres interact with a number of proteins that can influence chromosome-end integrity and dynamics [2]. Among them is telomerase, which is a ribonucleoprotein complex that regulates telomere-length maintenance by adding telomeric repeats to the chromosome 39-end using an RNA template [3]. Alternative Lengthening of Telomeres (ALT) is a separate mechanism of telomere length maintenance based on recombination that is active in the remaining 15% of cases [4,5]
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