Hyper-phosphorylated retinoblastoma protein suppresses telomere elongation

Immortalized cell lines maintain telomeres by the expression of telomerase or by a mechanism designated alternative lengthening of telomeres (ALT). Although DNA polymerase alpha (pol-alpha) is reported to be required for telomere maintenance, the critical role of pol-alpha in telomere maintenance has not been firmly determined. We examined the role of retinoblastoma protein (pRb) and pol-alpha in the regulation of telomere length, using telomere-fiber FISH. Telomere length varied dependent on the intracellular abundance of pol-alpha or pRb in HeLa cells. A proportion of hyper-phosphorylated pRb (ppRb) molecules localized to sites of telomeric DNA replication in HeLa cells. Pol-alpha might thus contribute to telomere maintenance, and might be regulated by ppRb.     

ATR contributes to telomere maintenance in human cells

Telomere maintenance is essential to preserve genomic stability and involves several telomere-specific proteins as well as DNA replication and repair proteins. The kinase ATR, which has a crucial function in maintaining genome integrity from yeast to human, has been shown to be involved in telomere maintenance in several eukaryotic organisms, including yeast, Arabidopsis and Drosophila. However, its role in telomere maintenance in mammals remains poorly explored. Here, we report by using telomere-fluorescence in situ hybridization (Telo-FISH) on metaphase chromosomes that ATR deficiency causes telomere instability both in primary human fibroblasts from Seckel syndrome patients and in HeLa cells. The telomere aberrations resulting from ATR deficiency (i.e. sister telomere fusions and chromatid-type telomere aberrations) are mainly generated during and/or after telomere replication, and involve both leading and lagging strand telomeres as shown by chromosome orientation-FISH (CO-FISH). Moreover, we show that ATR deficiency strongly sensitizes cells to the G-quadruplex ligand 360A, enhancing sister telomere fusions and chromatid-type telomere aberrations involving specifically the lagging strand telomeres. Altogether, these data reveal that ATR plays a critical role in telomere maintenance during and/or after telomere replication in human cells.     

Telomerase can inhibit the recombination-based pathway of telomere maintenance in human cells

Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.     

Telomere instability in a human cancer cell line.

Telomere maintenance is essential in immortal cancer cells to compensate for DNA lost from the ends of chromosomes, to prevent chromosome fusion, and to facilitate chromosome segregation. However, the high rate of fusion of chromosomes near telomeres, termed telomere association, in many cancer cell lines has led to the proposal that some cancer cells may not efficiently perform telomere maintenance. Deficient telomere maintenance could play an important role in cancer because telomere associations and nondisjunction have been demonstrated to be mechanisms for genomic instability. To investigate this possibility, we have analyzed the telomeres of the human squamous cell carcinoma cell line SQ-9G, which has telomere associations in approximately 75% of the cells in the population. The absence of detectable telomeric repeat sequences at the sites of these telomere associations suggests that they result from telomere loss. The analysis of telomere length by quantitative in situ hybridization demonstrated that, compared to the human squamous cell carcinoma cell line SCC-61 which has few telomere associations, SQ-9G has more extensive heterogeneity in telomere length and more telomeres without detectable telomeric repeat sequences. The dynamics of the changes in telomere length also demonstrated a higher rate of fluctuation in telomere length, both on individual telomeres and coordinately on all telomeres. These results demonstrate that telomere maintenance can play a role in the genomic instability seen in cancer cells.     

Telomerase enzymatic component hTERT shortens long telomeres in human cells

Telomere lengths are tightly regulated within a narrow range in normal human cells. Previous studies have extensively focused on how short telomeres are extended and have demonstrated that telomerase plays a central role in elongating short telomeres. However, much about the molecular mechanisms of regulating excessively long telomeres is unknown. In this report, we demonstrated that the telomerase enzymatic component, hTERT, plays a dual role in the regulation of telomere length. It shortens excessively long telomeres and elongates short telomeres simultaneously in one cell, maintaining the optimal telomere length at each chromosomal end for efficient protection. This novel hTERT-mediated telomere-shortening mechanism not only exists in cancer cells, but also in primary human cells. The hTERT-mediated telomere shortening requires hTERT’s enzymatic activity, but the telomerase RNA component, hTR, is not involved in that process. We found that expression of hTERT increases telomeric circular DNA formation, suggesting that telomere homologous recombination is involved in the telomere-shortening process. We further demonstrated that shelterin protein TPP1 interacts with hTERT and recruits hTERT onto the telomeres, suggesting that TPP1 might be involved in regulation of telomere shortening. This study reveals a novel function of hTERT in telomere length regulation and adds a new element to the current molecular model of telomere length maintenance.     

RTEL1 contributes to DNA replication and repair and telomere maintenance

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.     

Telomeres: hallmarks of radiosensitivity

Telomeres are the very ends of the chromosomes. They can be seen as natural double-strand breaks (DSB), specialized structures which prevent DSB repair and activation of DNA damage checkpoints. In somatic cells, attrition of telomeres occurs after each cell division until replicative senescence. In the absence of telomerase, telomeres shorten due to incomplete replication of the lagging strand at the very end of chromosome termini. Moreover, oxidative stress and accumulating reactive oxygen species (ROS) lead to an increased telomere shortening due to a less efficient repair of SSB in telomeres. The specialized structures at telomeres include proteins involved in both telomere maintenance and DNA repair. However when a telomere is damaged and has to be repaired, those proteins might fail to perform an accurate repair of the damage. This is the starting point of this article in which we first summarize the well-established relationships between DNA repair processes and maintenance of functional telomeres. We then examine how damaged telomeres would be processed, and show that irradiation alters telomere maintenance leading to possibly dramatic consequences. Our point is to suggest that those consequences are not restricted to the short term effects such as increased radiation-induced cell death. On the contrary, we postulate that the major impact of the loss of telomere integrity might occur in the long term, during multistep carcinogenesis. Its major role would be to act as an amplificator event unmasking in one single step recessive radiation-induced mutations among thousands of genes and providing cellular proliferative advantage. Moreover, the chromosomal instability generated by damaged telomeres will favour each step of the transformation from normal to fully transformed cells.     

The Role of Telomeres in Etoposide Induced Tumour Cell Death

Etoposide, a topoisomerase II poison is used in the treatment of a number of solidtumours. Contradictory data exist on the role of the telomere/telomerase complex inetoposide induced apoptosis. Therefore we examined the effects of etoposidetreatment in the neuroblastoma cell line SHSY5Y, with very short telomeres and theacute lymphoblastic T cell line 1301, which displays extremely long telomeres. Bothshort-term and continuous exposure to the drug was examined. Etoposide inducedwidespread DNA damage followed by DNA damage foci formation and ultimatelygrowth arrest and apoptosis in a concentration-dependent manner. However, length oftelomeres and of single stranded telomeric G rich overhangs did not changesignificantly under the treatments in any cell line. There was no significant inductionof single-strand breaks in the G- rich strand of telomeres. Telomerase activity wastransiently upregulated under low concentrations of etoposide, while highconcentrations resulted in decreased telomerase activity only after onset of apoptosis.Telomerase overexpression protected against etoposide induced apoptosis infibroblasts. The data suggest that telomeres are not major signal transducers towardsgrowth arrest or apoptosis after etoposide treatment. However, upregulation oftelomerase might be part of an attempted adaptative response, which protects cells bya mechanism that might be independent of telomere length maintenance.     

Telomerase: Structure,functions, and activity regulation

Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells proliferation potential is strictly limited and senescence follows approximately 50–70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. No doubt, DNA polymerase is not capable to completely copy DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis, and cell death. However, in tumor cells the system of telomere length maintenance is activated. Besides catalytic telomere elongation, independent telomerase functions can be also involved in cell cycle regulation. Inhibition of the telomerase catalytic function and resulting cessation of telomere length maintenance will help in restriction of tumor cell replication potential. On the other hand, formation of temporarily active enzyme via its intracellular activation or due to stimulation of expression of telomerase components will result in telomerase activation and telomere elongation that can be used for correction of degenerative changes. Data on telomerase structure and function are summarized in this review, and they are compared for evolutionarily remote organisms. Problems of telomerase activity measurement and modulation by enzyme inhibitors or activators are considered as well.     

Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination

Mammalian telomeres have heterochromatic features, including trimethylated histone H3 at lysine 9 (H3K9me3) and trimethylated histone H4 at lysine 20 (H4K20me3). In addition, subtelomeric DNA is hypermethylated. The enzymatic activities responsible for these modifications at telomeres are beginning to be characterized. In particular, H4K20me3 at telomeres could be catalyzed by the novel Suv4-20h1 and Suv4-20h2 histone methyltransferases (HMTases). In this study, we demonstrate that the Suv4-20h enzymes are responsible for this histone modification at telomeres. Cells deficient for Suv4-20h2 or for both Suv4-20h1 and Suv4-20h2 show decreased levels of H4K20me3 at telomeres and subtelomeres in the absence of changes in H3K9me3. These epigenetic alterations are accompanied by telomere elongation, indicating a role for Suv4-20h HMTases in telomere length control. Finally, cells lacking either the Suv4-20h or Suv39h HMTases show increased frequencies of telomere recombination in the absence of changes in subtelomeric DNA methylation. These results demonstrate the importance of chromatin architecture in the maintenance of telomere length homeostasis and reveal a novel role for histone lysine methylation in controlling telomere recombination.     

Cell cycle-dependent recruitment of telomerase RNA and Cajal bodies to human telomeres

Telomerase is a ribonucleoprotein enzyme that counteracts replicative telomere erosion by adding telomeric sequence repeats onto chromosome ends. Despite its well-established role in telomere synthesis, telomerase has not yet been detected at telomeres. The RNA component of human telomerase (hTR) resides in the nucleoplasmic Cajal bodies (CBs) of interphase cancer cells. Here, in situ hybridization demonstrates that in human HeLa and Hep2 S phase cells, besides accumulating in CBs, hTR specifically concentrates at a few telomeres that also accumulate the TRF1 and TRF2 telomere marker proteins. Surprisingly, telomeres accumulating hTR exhibit a great accessibility for in situ oligonucleotide hybridization without chromatin denaturation, suggesting that they represent a structurally distinct, minor subset of HeLa telomeres. Moreover, we demonstrate that more than 25% of telomeres accumulating hTR colocalize with CBs. Time-lapse fluorescence microscopy demonstrates that CBs moving in the nucleoplasm of S phase cells transiently associate for 10-40 min with telomeres. Our data raise the intriguing possibility that CBs may deliver hTR to telomeres and/or may function in other aspects of telomere maintenance.     

Targeted DNA damage at individual telomeres disrupts their integrity and triggers cell death

Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising <1% of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence in HeLa cells and cell death in HeLa, U2OS and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including chromatid telomere loss and telomere associations, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage.     

Pharmacological intervention strategies for affecting telomerase activity: future prospects to treat cancer and degenerative disease

Telomerase enzyme is a ribonucleoprotein maintaining the length of the telomeres by adding G-rich repeats to the end of the eukaryotic chromosomes. Normal human somatic cells, cultured in vitro, have a strictly limited proliferative potential undergoing senescence after about 50-70 population doublings. In contrast, most of the tumor cells have unlimited replicative potential. Although the mechanisms of immortalization are not understood completely at a genetic level, the key role of the telomere/telomerase system in the process is clear. The DNA replication machinery is not able to replicate fully the DNA at the very end of the chromosomes; therefore, about 50-200 nucleotides are lost during each of the replication cycles resulting in a gradual decrease of telomere length. Critically short telomere induces senescence, subsequent crisis and cell death. In tumor cells, however, the telomerase enzyme prevents the formation of critically short telomeres, adding GGTTAG repeats to the 3' end of the chromosomes immortalizing the cells. Immortality is one of the hallmarks of cancer. Besides the catalytic activity dependent telomere maintenance, catalytic activity-independent effects of telomerase may also be involved in the regulation of cell cycle. The telomere/telomerase system offers two possibilities to intervene the proliferative activity of the cell: (1) inhibition the telomere maintenance by inhibiting the telomerase activity; (2) activating the residual telomerase enzyme or inducing telomerase expression. Whilst the former approach could abolish the limitless replicative potential of malignant cells, the activation of telomerase might be utilized for treating degenerative diseases. Here, we review the current status of telomerase therapeutics, summarizing the activities of those pharmacological agents which either inhibit or activate the enzyme. We also discuss the future opportunities and challenges of research on pharmacological intervention of telomerase activity.     

Rapid shortening of telomere length in response to ceramide involves the inhibition of telomere binding activity of nuclear glyceraldehyde-3-phosphate dehydrogenase

Ceramide has been demonstrated as one of the upstream regulators of telomerase activity. However, the role for ceramide in the control of telomere length remains unknown. It is shown here that treatment of the A549 human lung adenocarcinoma cells with C(6)-ceramide results in rapid shortening of telomere length. During the examination of ceramide-regulated telomere-binding proteins, nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified to associate with both single- and double-stranded telomeric DNA with high specificity in vitro. The association of nuclear GAPDH with telomeres in interphase nuclei was also demonstrated by co-fluorescence in situ hybridization and chromatin immunoprecipitation analysis. Further data demonstrated that the nuclear localization of GAPDH is regulated by ceramide in a cell cycle-dependent manner parallel with the inhibition of its telomere binding activity in response to ceramide. In addition, the results revealed that nuclear GAPDH is distinct from its cytoplasmic isoform and that telomere binding function of nuclear GAPDH is strikingly higher than the cytoplasmic isoform. More importantly, the functional role for nuclear GAPDH in the maintenance and/or protection of telomeric DNA was identified by partial inhibition of the expression of GAPDH using small interfering RNA, which resulted in rapid shortening of telomeres. In contrast, overexpression of nuclear GAPDH resulted in the protection of telomeric DNA in response to exogenous ceramide as well as in response to anticancer drugs, which have been shown to induce endogenous ceramide levels. Therefore, these results demonstrate a novel function for nuclear GAPDH in the maintenance and/or protection of telomeres and also show that mechanisms of the rapid degradation of telomeres in response to ceramide involve the inhibition of the telomere binding activity of nuclear GAPDH.     

细胞DNA损伤检控系统在维持端粒稳定中的功能

真核生物的DNA损伤检控系统是维持细胞基因组稳定的一个重要机制,该系统能检测细胞在生命活动过程中出现的DNA损伤并引发细胞周期阻滞,对DNA损伤进行修复,以维持细胞遗传的稳定性。端粒是位于真核细胞染色体末端由重复DNA序列和蛋白质组成的复合物,具有保护染色体、介导染色体复制、引导减数分裂时的同源染色体配对和调节细胞衰老等作用。虽然端粒与DNA双链断裂都具有作为线性染色体末端的共同特点,但正常端粒并不像DNA双链断裂那样激活DNA损伤检控系统。另一方面,端粒又与DNA损伤相似,因为多种DNA损伤检控蛋白在端粒长度稳定中起重要作用。因此DNA损伤检控系统既参与了维持正常端粒的完整性,又可对端粒损伤作出应答。现就DNA损伤检控系统在维持端粒稳定中的作用及其对功能缺陷端粒的应答作一简要综述。     

DNA-PKcs Inhibition Sensitizes Cancer Cells to Carbon-Ion Irradiation via Telomere Capping Disruption

Heavy-ion irradiation induces a higher frequency of DNA double strand breaks (DSBs) which must be properly repaired. Critical shortening of telomeres can trigger DNA damage responses such as DSBs. Telomeres are very sensitive to oxidative stress such as ionizing radiation. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the central component in the non-homologous end joining (NHEJ) repair complex and participates in telomere maintenance. Therefore, it is expected to enhance the cell killing effect of heavy-ion irradiation via DNA-PKcs inhibition. To test this hypothesis, cellular radiosensitivity was measured by the clonal genetic assay. DNA damage repair was relatively quantified by long PCR. Apoptosis was quantified by flow-cytometric analysis of annexin V/PI double staining, and senescence was analyzed by galactosidase activity. Telomere length was semi-quantified by real-time PCR. P53 and p21 expression was determined by western blotting. Our data demonstrated that MCF-7 and HeLa cells with DNA-PKcs inhibition were more susceptible to carbon-ion irradiation than Those without DNA-PKcs inhibition. Even though NHEJ was inhibited by the DNA-PKcs specific inhibitor, NU7026, most DNA damage induced by carbon-ion irradiation was repaired within 24 hours after irradiation in both cell lines. However, potential lethal damage repair (PLDR) could not restore cellular inactivation in DNA-PKcs inhibited cells. MCF-7 cells showed extensive senescence and accelerated telomere length reduction, while HeLa cells underwent significant apoptosis after irradiation with NU7026 incubation. In addition, both cell lines with shorter telomere were more susceptible to carbon-ion radiation. Our current data suggested that DNA-PKcs inhibition could enhance cellular sensitivity to carbon-ion radiation via disturbing its functional role in telomere end protection. The combination of DNA-PKcs inhibition and carbon-ion irradiation may be an efficient method of heavy-ion therapy.     

Telomere dynamics in a human cancer cell line

Telomere maintenance is thought to be essential for immortalization of human cancer cells to compensate for the loss of DNA from the ends of chromosomes and to prevent chromosome fusion. We have investigated telomere dynamics in the telomerase-positive squamous cell carcinoma cell line SCC-61 by marking the ends of chromosomes with integrated plasmid sequences so that changes in the length of individual telomeres could be monitored. Despite having very short telomeres, SCC-61 has a relatively stable genome and few telomere associations. The marked telomeres in different SCC-61 clones have similar mean lengths which show little change with increasing time in culture. Thus, each marked telomere is maintained at a specific length, which we term the equilibrium mean length (EML). The Gaussian distribution in the length of the marked telomeres demonstrates that telomeres continuously fluctuate in length. Consistent with this observation, the mean lengths of the marked telomere in subclones of these cell lines initially differ, but then gradually return to the EML of the original clone with increasing time in culture. The analysis of a clone with two marked telomeres demonstrated that changes in telomere length can occur on each marked telomere independently or coordinately on both telomeres. These results suggest that the short telomeres in many tumor cell lines do not result from an inability to properly maintain telomeres at a specific length.     

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length.     

Telomere recombination in normal mammalian cells

Two mechanisms of telomere length maintenance are known to date. The first includes the use of a special enzymatic telomerase complex to solve the problems that arise during the replication of linear DNA in a normal diploid and part of tumor cells. Alternative lengthening of telomeres (ALT), which is based on the homologous recombination of telomere DNA, represents the second mechanism. Until recently, ALT was assumed to be expressed only in 15–20% of tumors lacking active telomerase and,, together with telomerase reactivation represented one of two possibilities to overcome the replicative senescence observed in somatic mammalian cells due to aging or during cell culturing in vitro. Previously described sporadic cases of combinations of the two mechanisms of telomere length maintenance in several cell lines in vitro were attributed to the experimental design rather than to a real biological phenomenon, since active cellular division without active telomerase was considered to be the “gold standard” of ALT. The present review describes the morphological and functional reorganizations of mammalian telomeres observed with ALT activation, as well as recently observed and well-documented cases of combinations between ALT-like and telomerase-dependent mechanisms in mammalian cells. The possible role of telomere recombination in telomerase-dependent cells is discussed.