In normal human cells, telomerase is repressed, leading to telomere shortening that triggers replicative senescence. However, in most tumors, telomerase is up-regulated and is essential for telomere maintenance and tumor cell growth. Although long considered a viable target for tumor therapy, successful inhibition of telomerase in cancer therapy remains to be described.
It has long been known that telomeres are sensitive to damage by reactive oxygen species ROS , but the impact of oxidation on telomerase function in living cells was not known. Using gene knockouts in colon cancer cells, the investigators demonstrate that the antioxidant enzyme peroxiredoxin 1 PRDX1 and the nudix phosphohydrolase superfamily enzyme MTH1 cooperate to retain, upon oxidative stress, telomeres in a telomerase-extendible state.
Considering that cancer cells are more vulnerable to ROS than noncancer cells, this work may open new avenues targeting telomeres and telomerase in tumor cells.
Telomerase counteracts the telomere shortening that results from the end replication problem and nucleolytic processing. Telomerase is active during embryogenesis but, with the exception of some stem cell compartments, repressed in the human soma, and, as a result, telomeres shorten. This shortening limits the replication potential of somatic cells, thereby serving a powerful tumor suppressor function.
Most cancers overcome this barrier by activation of telomerase and ultimately rely on telomerase-mediated telomere lengthening for survival. Reactive oxygen species ROS are generated by cell-intrinsic factors and environmental exposures. Free nucleotide pools are highly susceptible to oxidation, and insertion of oxidized nucleotides into the genome during replication is both mutagenic and toxic. ROS-mediated damage of nucleotides and nucleic acids is countered by cellular antioxidant defenses that repair and prevent oxidative damage.
The base excision repair pathway can remove this base modification; however, this pathway requires a complementary DNA strand. Recent studies indicate specific enzyme pathways that play a role in protection of telomeres from ROS. The Lingner group Aeby et al. The Opresko laboratory Fouquerel et al. The impact of ROS on telomere length maintenance by telomerase is not well understood. In vitro studies predicted that ROS could either promote or repress telomerase-mediated lengthening.
Moreover, it was shown that 8-oxo dGTP is used by telomerase as a substrate but, once incorporated, can function as a chain terminator Aeby et al.
On the other hand, internal 8-oxo G suppresses G-quadruplex formation, thereby increasing telomerase accessibility and loading Fouquerel et al. They showed that knockout of each gene led to increased expression of the other, suggesting that cells respond to disruption of one antioxidant system by increasing another.
The most frequent DNA damage induced by oxidative stress is base modification, and the principal product is 8-oxoG [42]. For this reason, we performed the standard comet assay together with its FPG-modified version, which is more sensitive to the presence of oxidised bases. Genomic damage decreased 15 hrs after treatment, although it persisted significantly higher than the control value. At 15 hrs, Tail DNA values obtained from the FPG-modified assay are not statistically different from those produced by the standard version of the comet assay for both doses used.
A complete repair of genomic damage was observed 24 hrs after treatment both with the standard and FPG-modified comet assay, as evidenced by the similar values yielded by the treated samples and the controls. Standard and FPG-modified versions of the comet assay have been used to evaluate the fold increase in genomic damage induced by hydrogen peroxide with respect to the control value.
The net cleavage sites generated by FPG activity were calculated subtracting the value of total DNA damage yielded by the samples not treated with the enzyme from the DNA damage value obtained from samples treated with the enzyme.
All values are normalised to the control values. DNA damage increases in a dose dependent manner after 1 hr treatment. We observed a time-dependent decrease of genomic damage to the control value 15 and 24 hrs after treatment. Error bars denote the standard error.
The high incidence of guanine residues in telomeric sequences makes these more susceptible to oxidative damage, especially to the accumulation of oxidised bases such as 8-oxoG. A new technique that uses quantitative PCR qPCR with telomere-specific primers is an extremely sensitive method to measure the amount of oxidised residues by analysing the abundance of telomeric FPG-sensitive sites within telomeric DNA [33].
Quantitative PCR was used to evaluate the amount of FPG-sensitive sites within telomeric sequences after 1 hr treatment with hydrogen peroxide.
At subsequent recovery times 1, 15 and 24 hrs , we observed a time-dependent decrease of telomeric damage that persists at a value significantly higher than the control value. Statistical analysis was performed between treated and control samples. We performed telomere length analysis Fig. The kinetics of telomere length are shown in Fig.
Normal telomere length was restored 72 hrs after treatment and did not change for up to 96 hrs. Conversely, values at other times were no different than the control. Error bars were calculated using standard error propagation rules. The results demonstrated no telomerase activity for either dose and for all times post treatment, confirming that H 2 O 2 did not induce telomerase activation.
The graph displays data on telomerase activity. The error bars were calculated using standard error propagation rules. To demonstrate that the telomere restoration observed at 72 and 96 hrs post-treatment in human primary fibroblasts is not due to the activation of the ALT pathway, we performed CO-FISH analysis.
The results revealed no statistical differences in T-SCE frequency at these times after oxidative stress Fig. The T-SCE ratio was calculated as the ratio of the frequency from treated samples to the frequency from control samples. The results revealed no significant differences between treated and control samples.
To evaluate if telomere length restoration could be attributed to a cellular selection system that favours cells with longer telomeres, we performed a cell growth curve Fig. Cell viability was not different between treated and untreated cells Fig. As previously demonstrated by Baglole et al. After treatment, cells were seeded t 0 and harvested at different times. Total cells were counted using an electronic haemocytometer a , and the percentage of viable cells was determined by propidium iodide exclusion by flow cytometry analysis b.
The main goal of this work was to assess the role of telomeres on chromosome instability CIN. Representative images of Bi-nucleate BN cells with different abnormal nuclear morphologies. We observed a significant increase of NPBs 48 hrs after treatment and a decrease at subsequent times. We again observed a significant increase of NPBs 48 hrs after treatment and a decrease with time until 96 hrs. Error bars denote standard error.
The results revealed a dose-related increase of ANMs 48 hrs after treatment for both Fig. NPBs have been shown to correlate with telomere defects [24] , [25]. Given that our work has demonstrated that oxidative stress induces telomere shortening and ANM, we decided to study the specific correlation between telomere length and NPBs by comparing results obtained from Q-FISH Fig.
The results are shown in Fig. At the longer times, we observed the restoration of telomere length and a decrease in the frequency of NPBs. Telomeres are the nucleoprotein structures that protect the ends of linear chromosomes, preserving genome and chromosome stability [1].
Oxidative stress damages DNA, especially telomere structure [45] , [46]. Oxidative stress that produces ROS has been shown to accelerate telomere shortening in replicating fibroblasts in vitro [47].
Other studies have shown accelerated telomere shortening in cells from patients with mutations in mitochondrial DNA characterised by an increased production of reactive oxygen species [48]. The evidence causally linking reactive oxygen species with telomeres is derived from experiments in which oxidative stress induced by arsenic yields telomere attrition, chromosome instability and apoptosis [49]. However, it is unclear how oxidative DNA damage compromises telomere length and integrity. It was hypothesised that telomere sequences, rich in guanine residues, are more susceptible to oxidative stress, mainly by the formation of 8-oxoG [18].
Furthermore, these base modifications could lead to single strand breaks, leading to the loss of the distal fragments of telomeric DNA following replication [50] and, thus, telomere shortening [51] , [52]. Starting from these assumptions, our interest was focused on the analysis of new biomarkers of chromosome instability, recently considered a new valuable tool to measure chromosome rearrangement and DNA damage [53] especially after telomere damage [25].
To our knowledge, no study demonstrated a direct relationship between telomere oxidation and the development of gross nuclear abnormalities.
Therefore, the main aim of this work was to demonstrate a direct link between the two processes, i. To assess the DNA damage induced by the acute oxidative stress treatment as well as to compare genomic and telomeric damage, we treated MRC-5 cells with two doses of hydrogen peroxide and we performed two different analyses — one on the whole genome by the FPG-modified alkaline comet assay and the other on telomeric sequences by the FPG-sensitive base lesions within telomeric DNA.
The analysis of the entire genome immediately time 0 and 1 hr after treatment revealed an increase in damage that was completely rescued 24 hrs later. These results could be interpreted to signify that, as expected, the genomic damage was repaired within this time. In contrast, the analysis of telomeric sequences indicated an increase in DNA damage 1 hr after treatment that decreased over time but persisted at a significant level 24 hrs after treatment for both doses of hydrogen peroxide.
The differences between the two techniques can be explained by considering that the comet assay highlights damage in the entire genome and not in the damage site specifically e. The persistence of damaged bases in telomeric versus non-telomeric G-rich DNA suggests not only that the telomere structure is susceptible to oxidative stress but also that DNA damage repair at telomeres is less effective than in non-telomeric regions [54].
These results are in line with a previous study in which the authors demonstrated that acute oxidant exposure causes a high incidence of FPG-sensitive sites in telomeric DNA fragments [55]. These lesions persist at a higher level in telomeres after 6 hrs of recovery time, suggesting that the repair of oxidative DNA damage may be less effective in telomeres in vivo, most likely because the sequence context of telomere repeats and certain telomere configurations may contribute to the vulnerability of telomeres to the processing of oxidative DNA damage.
Furthermore, unrepaired oxidative telomere damage can have undesired consequences during replication if not readily repaired.
With these assumptions, we focused our attention on telomeres to test whether persistent base damage has an impact on telomere length. Our data revealed no telomere length modulation 24 hrs after treatment, while a significant telomere shortening was observed after 48 hrs.
One likely explanation is that the persistent telomeric damage observed 24 hrs after treatment due to the 8-oxoG is not repaired and is responsible for the telomere shortening observed at 48 hrs, indicating that this phenomenon is dependent on replication. At the subsequent times, 72 and 96 hrs, we observed a restoration of telomere length, suggesting that telomere shortening is a transient consequence of acute oxidative stress. Telomere shortening is a well-accepted cause of chromosome instability.
For this reason, after demonstrating a change in telomere length, the next step was to investigate the relationship between the telomere shortening observed and chromosome instability through the analysis of biomarkers strictly related to telomere dysfunction such us MN, NBUDs and NPBs [25].
Especially for NPBs, we observed a dose-dependent increase 48 hrs after treatment. This result relates very well with the telomere shorting observed in the same time frame, allowing us to confirm the relationship between telomere shortening and the observed markers of chromosome instability.
Because telomere shortening generated fusions of broken chromosome ends [15] , [25] , we believe that such fusions could induce an increase in observed chromosome bridges. Additionally, our analysis showed a decrease of NPBs at later times, which could be associated with the restoration of telomere length.
The strict correlation between NPBs and telomere length indicates that this biomarker represents a good readout for telomere defects and any consequent chromosome segregation errors. We hypothesised that telomere shortening leads to an increase in the rate of chromosome bridges; when telomere length is recovered, a decrease in the frequency of chromosome bridges is observed Fig.
To understand the mechanism leading to restored telomere length, we performed a telomerase activity assay to evaluate the possible involvement of this enzyme in the observed telomere modulation, and our results indicated no contribution. In our opinion, after acute oxidative stress, telomere restoration could be the result of a cellular selection system that promotes cells with longer telomeres due to their higher growth rate.
Indeed, our results on cell viability and cell growth demonstrated that doubling time increased in treated cells, indicating that hydrogen peroxide treatment significantly reduced the MRC-5 proliferation rate. The idea is that cells with shorter telomeres could be responsible for the decreased growth rate, which is particularly noticeable in the log phase of the curve between 48 hrs, when telomeres are shortened, and 72 hrs, when telomere length is rescued. The results on cell viability did not indicate any differences between treated and control samples, allowing us to exclude an effect on cell viability in treated samples for our analysis times.
This hypothesis could also explain the brief chromosome instability, which is restored in a short time. In this work, we demonstrated a link between the oxidation process and abnormal nuclear morphologies. Oxidative stress has been shown to induce 8-oxoG at telomeric sequences.
In addition, telomeres are repaired less efficiently than the whole genome [14] , and the presence of base damage could interfere with the replication fork at the telomere, extending the portion of unreplicated ends [12] and disrupting the binding of shelterin protein [19]. The presence of unrepaired 8-oxoG can lead to GC to TA transversions after two rounds of replication [16]. Finally, processing of oxidative lesions may lead to changes in telomere repeat number. These possibilities are explored in more detail below, along with evidence from studies that support these models.
Consequences of replication fork stalling and blocks at telomeres. The schematic shows a model for how telomere fragility or telomere losses arise from DNA lesions that stall or block replication fork progression, respectively.
DNA replication fork encounters with single strand breaks SSBs can cause the fork to collapse into a double strand break. Fragile telomeres manifest as multi-telomeric foci at a chromatid end, and are proposed to result from uncondensed regions arising from accumulated unreplicated ssDNA.
Telomeres losses manifest as chromatid ends lacking sufficient telomeric DNA for detection with a telomeric probe. Telomerase can suppress telomere losses by extending a pre-maturely truncated telomere. Is oxidative damage to telomeric DNA responsible for accelerated telomere shortening under oxidative stress?
These lesions include damaged pyrimidines and purines, as well as SSBs and abasic sites. Guanine is the most susceptible of the natural bases to oxidation, commonly generating 8-oxoguanine 8-oxoG , which is even more sensitive to oxidation, ultimately giving rise to hydantoin lesions Fleming and Burrows, ; Luo et al.
Consistent with this, several studies reported more SSBs or 8-oxoG lesions in telomeres compared to microsatellite repeats and bulk genomic DNA, after cellular exposures to oxidizing agents Coluzzi et al. Indeed, 8-oxoG cannot be repaired in the context of folded telomeric G-quadruplex structures Zhou et al.
More work is required to determine how efficiently oxidative base damage is repaired at telomeres, compared to elsewhere in the genome. Most oxidative lesions are repaired by BER, which is essential for genome stability and for preserving telomeres.
These lesions can be cytotoxic or mutagenic, and thereby promote carcinogenesis for review see Wallace et al. While these core steps are conserved, several variations in BER sub-pathways exist, assisted by additional proteins. BER intermediates can be cytotoxic and therefore, efficient hand-off to each downstream processing enzyme is essential Sobol et al. Given that 8-oxoG is one of the most common oxidative lesions, multiple pathways exist to deal with this form of damage.
When 8-oxoG forms opposite C it is recognized by OGG1 glycosylase, which removes the lesion, generating an abasic site Figure 3ii. OGG1 can further process this site with its AP-lyase function, however OGG1 has high affinity for abasic sites, and becomes trapped by its own product Hill et al.
Remarkably, an unbiased screen in yeast for genes that alter telomere length revealed that ogg1 deletion strains had longer telomeres than wild type Askree et al. An independent study confirmed this result and reported that the lengthening was partly telomerase dependent Lu and Liu, This may represent a hormesis situation, in which low levels of 8-oxoG at telomeres promotes lengthening, whereas high amounts cause telomere losses and aberrations.
Mechanistically, we showed that a single 8-oxoG in telomeric ssDNA disrupts the folded G-quadruplex structures that impede telomerase loading, thereby promoting telomere elongation Fouquerel et al. The increased 8-oxoG at telomeres may lead to shelterin disruption Lu and Liu, ; Opresko et al.
Processing of 8-oxoG. When 8-oxoG forms opposite C by direct oxidation i , it is recognized by OGG1 ii which removes the modified base. Proteins in parentheses can stimulate OGG1 activity. This is because 8-oxoG miscodes for A. Changes in the telomeric sequence would disrupt shelterin binding. This suggests that either 8-oxoG is efficiently repaired at telomeres prior to replication, or that MUTYH efficiently removes A opposite 8-oxoG at telomeres to prevent mutations.
Sequencing telomeres from oxidative stress conditions in OGG1 and MUTYH singly and doubly deficient cells, is needed to fully elucidate the mutagenic potential of 8-oxoG at telomeres, and the roles for these glycosylases in preserving telomeric repeats.
Models for 8-oxoG induced mutations at telomeres. A The schematic shows three possible scenarios of 8-oxodGTP insertion opposite A during telomere replication and the resulting change in telomeric repeat sequence. Interestingly, these are the most common variant repeats reported from telomere sequencing studies Lee et al. Lee et al. However, 8-oxodGTP is a telomerase chain terminator in vitro Aeby et al. Consistent with this, acute MTH1 depletion increases telomere loss and cell death in telomerase positive cancer cells harboring critically short telomeres, but not in cancer cell with longer telomere reserves Fouquerel et al.
This suggests that 8-oxodGTP, and potentially oxidized versions of dATP, which are normally removed by MTH1, inhibit telomerase restoration of critically short telomeres. Some cancer cell lines are more sensitive to MTH1 inhibition, compared to normal cells, which may be due differences in the reliance on telomerase activity for short-term survival Gad et al.
Whether MTH1 inhibition leads to mutagenesis at telomeres remains to be determined. Whether a similar mechanism exists to assist telomerase extension from a terminal 8-oxoG remains unknown. While 8-oxoG is primarily mutagenic, other oxidized purines are more cytotoxic due to their ability to block DNA replication and transcription.
Since 8-oxoG has a lower redox potential than the normal unmodified bases, it can be further oxidized to other lesions including spiroiminodihydantoin Sp and guanidinohydantoin Gh Luo et al. These distorting hydantoin lesions impede DNA replication and transcription Henderson et al.
How BER proceeds in the absence of a templating base is unclear, but the processing of lesions in telomeric ssDNA and G-quadruplexes has important implications for telomere integrity.
The offending lesion s responsible for telomere loss is difficult to define because NEIL glycosylases remove several lesion types including ring opened 2,6-diaminohydroxyformamidopyrimidine FapyG and oxidized pyrimidines see below. Roles for NEIL1 at telomeres have not been examined. Analyses using more sensitive measurements such as qFISH could be more revealing. Collectively, these studies suggest that NEIL glycoslyases have important roles in protecting telomeres against ROS-induced base damage, particularly to preserve DNA replication and transcription at telomeres.
Pyrimidine bases are also susceptible to damage by free radicals, giving rise to various lesions including thymine glycol Tg , 5-hydroxycytosine, and 5-hydroxyuracil. Tg is the most common oxidized thymine lesion and is cytotoxic because it can block DNA replication McNulty et al. However, only Neil3 can excise Tg from a telomeric G-quadruplex and shows a strong preference for excising Tg from telomeric versus non-telomeric duplex DNA Zhou et al.
Biochemical and biophysical studies indicate that unlike 8-oxoG, the presence of a single Tg only slightly disrupts telomeric G-quadruplexes Lee et al. However, Tg alters the structural conformations and dynamics of the telomeric G-quadruplex in a manner that favors telomerase binding Lee et al.
These studies reveal that Tg likely causes telomere defects through interference with telomere replication rather than telomerase inhibition. While the members of the BER pathway are well known, studies show proteins from other repair pathways also promote the repair of oxidative lesions.
For example, Nek7 kinase is recruited to telomeres after the localized induction of superoxide anion, and stabilizes shelterin TRF1 Tan et al. Whether these proteins protect telomeres by stimulating BER, or by removing oxidative lesions through NER, remains to be determined. Mismatch repair also contributes to oxidative damage repair in human cells.
This effect was reduced when MTH1 was overexpressed, indicating MMR processes 8-oxoG misincorporated during replication or repair events. These studies also raise the possibility that MMR proteins are also important for protecting telomeres from oxidative base damage.
Processing of oxidative DNA damage can be more detrimental than the initial damage, if toxic intermediates arise in the repair pathway that are not properly resolved. However, roles in preserving telomeres under oxidative stress remains controversial. However, these cells displayed a significant increase in chromosome end-to-end fusions after 26 population doublings.
Failures in repair of SSBs leads to numerous neurological disorders reviewed in Rulten and Caldecott, The impact of SSBs or processing of oxidative base damage in telomeres in neural cells remains to be examined. The repair of closely spaced damaged bases i. Importantly, the efficiency and accuracy of DSB repair at the telomeres may be comprised or altered by the presence of shelterin proteins and the highly repetitive nature of the sequence reviewed in Doksani and de Lange, This raises the possibility that BER processing of 8-oxoG in telomeric repeats may also lead to changes in telomeric repeat number.
G-quadruplexes that could impact repair processing reviewed in Fouquerel et al. Accumulating evidence indicates that oxidative stress correlates with accelerated telomere shortening and dysfunction in studies from human tissues, mouse models, cell culture and biochemical experiments.
As discussed in this review, several mechanisms have been proposed to explain how elevated ROS alters telomere length homeostasis; the most prominent being oxidative DNA damage. However, many questions remain. Given that ROS can damage numerous cellular components, it is difficult to determine whether changes in telomere length and integrity under oxidative stress conditions are due to indirect factors, or direct damage to the telomeres.
Tools to selectively induce oxidative base damage at telomeres will be useful for elucidating how the formation and processing of oxidative lesions impacts telomere maintenance, cellular function, as well as organism health and aging. Answering the question of whether telomeres are more susceptible to oxidative base damage in cells requires more accurate tools and methods to measure and quantify various types of oxidative lesions at telomeres. This is particularly challenging due to the highly repetitive telomeric sequence, their location at chromosome ends, and their low abundance.
The ability to measure different lesion types will be important since cytotoxic lesions, mutagenic lesions, single strand breaks, and repair intermediates can lead to different telomere outcomes.
The same features that make damage detection in telomeres difficult also make sequencing telomeres extremely challenging. Advances in next generation sequencing tools and protocols should help overcome these barriers, and will be required to determine whether oxidative base damage leads to mutations in telomeric DNA and accumulation of variant repeats.
Future studies are also required to address how oxidative base damage, and the processing of damage by repair enzymes, impact non-proliferating cells including neurons.
Activation of DNA repair at the telomeres has the potential to dramatically alter telomere lengths even in the absence of replication. Understanding how the formation and processing of oxidative damage alters telomere length homeostasis and integrity will be valuable for developing intervention strategies that protect telomeres in the face of oxidative stress, and promote healthy aging.
Numerous diseases associated with oxidative stress are also associated with shortened telomeres. Studies in human tissues, mouse models and cell culture provide evidence that oxidative stress is associated with accelerate telomere shortening. Telomeres are highly sensitive to oxidative DNA damage, which can induce telomere losses and dysfunction. We apologize to those investigators whose work was not cited in the interest of preparing a concise review.
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Barnes RP 1 ,. Search articles by 'Elise Fouquerel'. Fouquerel E 1 ,. Opresko PL 1. Affiliations 3 authors 1. Share this article Share with email Share with twitter Share with linkedin Share with facebook. Abstract Telomeres are dynamic nucleoprotein-DNA structures that cap and protect linear chromosome ends. Free full text.
Mech Ageing Dev. Author manuscript; available in PMC Jan 1. PMID: Ryan P. Barnes , Elise Fouquerel , and Patricia L. Author information Copyright and License information Disclaimer. Copyright notice. The publisher's final edited version of this article is available at Mech Ageing Dev. See other articles in PMC that cite the published article.
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