Both genome instability and replicative senescence stem from the shortest telomere in telomerase-negative cells

Telomeres, the physical ends of linear eukaryotic chromosomes, are bound by proteins that protect chromosome ends from being recognized as DNA double-stranded breaks.1 Telomeres shorten due to incomplete DNA replication and nucleolytic degradation. This shortening is counteracted by a specialized reverse transcriptase called telomerase.2 In the budding yeast Saccharomyces cerevisiae, deletion of any of the genes encoding telomerase components—such as the EST2 gene, which encodes the protein catalytic subunit2—leads to an ever shorter telomere (EST) phenotype and replicative senescence within 60–80 generations.3 Rare cells can escape senescence to become survivors, maintaining their telomeres via DNA recombination.3 There are many genes involved in the regulation of telomere length homeostasis, including the phosphoinositide-3-kinase-related kinases Tel1 and Mec1, yeast orthologs of human ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related), respectively. Yeast cells lacking Tel1 have short but stable telomeres, while mutation of MEC1 confers a mild telomere shortening phenotype.4 Cells lacking both Tel1 and Mec1 have a senescence phenotype similar to that of telomerase-negative strains.4 Telomeric DNA repeats are bound by Rap1, which in turn recruits Rif1 and Rif2 to the telomeres.5 Deletion of RIF1 or RIF2 leads to elongated telomeres, but further deletion of TEL1 largely yields telomeres that mimic those in a tel1Δ single mutant.6 Consistent with these genetic results, Rif1 and Rif2 inhibit the localization of Tel1 to telomeres,7 preventing the further recruitment of telomerase.8 We recently found that telomerasenegative strains lacking Rif1 or Rif2 exhibit accelerated senescence.9 In contrast, deletion of TEL1 delays the senescence of a telomerase-negative strain.4 These observations indicate that Rif1, Rif2 and Tel1 have functions outside the regulation of telomerase. One explanation for these observations is that the Rif proteins prevent accelerated senescence by inhibiting Tel1. To test this hypothesis, we assayed senescence of telomerase-negative rifΔ tel1Δ strains. We sporulated an est2Δ/+ rif1Δ/+ rif2Δ/+ tel1Δ/+ diploid and monitored the growth of the telomerase-negative (i.e., est2Δ) haploid meiotic progeny in liquid culture by dilution of cells to 5 × 104 cells/ml every 24 h (Fig. 1). In this assay, telomerase-negative strains show a progressive loss of viability followed by the appearance of survivors, which restores the growth rate of the cultures.10 We find that in the absence of telomerase, rifΔ mutants senesce after fewer population doublings than RIF cells while tel1Δ mutants senesce after more population doublings than TEL1 strains (Fig. 1), consistent with previous observations.4,9 Surprisingly, the effects of the rifΔ and tel1Δ mutations on the rate of senescence are additive. In the absence of telomerase, a tel1Δ deletion delays senescence by approximately 10 generations, which is also true in a rif1Δ, rif2Δ or double rif1Δ rif2Δ background (Fig. 1). Therefore, although the Rif proteins regulate telomerase-mediated telomere length homeostasis by inhibiting Tel1, the Rif proteins do not prevent accelerated senescence via the inhibition of Tel1. Figure 1 Rif1/2 and Tel1 function in separate pathways during replicative senescence. An est2Δ/+ rif1Δ/+ rif2Δ/+ tel1Δ/+ diploid was sporulated and haploid meiotic progeny were isolated. Spores were allowed to form colonies on YPD ... Interestingly, we find that deletion of MEC1 partially suppresses the accelerated senescence of telomerase-negative rifΔ strains (data not shown). This observation is consistent with previous studies showing that Mec1 is required to induce a cell cycle arrest during senescence.11 Thus, in the absence of telomerase, the Rif proteins prevent accelerated senescence through the inhibition of Mec1, not Tel1. Rif1 and Rif2 inhibit the localization of Tel1 to telomeres.7 The Rif proteins also inhibit 5′ exonucleolytic degradation of telomere ends mediated by the MRX complex.12 MRX-mediated nucleolytic degradation would expose single-stranded DNA (ssDNA) that can become coated with RPA; RPA-ssDNA complexes recruit Mec1/ATR to initiate a DNA damage checkpoint response.1 We propose that while the Rif inhibition of Tel1 localization to telomeres is important to regulate telomerase-mediated telomere length homeostasis, the Rif inhibition of the MRX complex is important to prevent Mec1-mediated accelerated senescence. In telomerase-negative strains lacking Rif1 and Rif2, senescence is accelerated likely because MRX-mediated telomere uncapping occurs earlier after loss of telomerase. Our model suggests that in the rifΔ mutants, the enhanced exonucleolytic degradation triggers a Mec1-dependent DNA damage signal, which helps induce the accelerated senescence. Our findings uncover a new twist in the relationship between the Rif proteins and the Tel1/ATM kinase, but what is the Rif-independent role of Tel1 in delaying senescence? Tel1 is not required for the telomere checkpoint response in senescing cells.11 Insight into the function of Tel1 during senescence would be aided by identifying its relevant substrates. It will also be interesting to compare and contrast the telomeric functions of yeast Tel1 and Mec1 with mammalian ATM and ATR, which are also inhibited at telomeres.1

Saccharomyces cerevisiae, a model organism in telomere biology, has been instrumental in pioneering a comprehensive understanding of the molecular processes that occur in the absence of telomerase across eukaryotes. This exploration spans investigations into telomere dynamics, intracellular signaling cascades, and organelle-mediated responses, elucidating their impact on proliferative capacity, genome stability, and cellular variability. Through the lens of budding yeast, numerous sources of cellular heterogeneity have been identified, dissected, and modeled, shedding light on the risks associated with telomeric state transitions, including the evasion of senescence. Moreover, the unraveling of the intricate interplay between the nucleus and other organelles upon telomerase inactivation has provided insights into eukaryotic evolution and cellular communication networks. These contributions, akin to milestones achieved using budding yeast, such as the discovery of the cell cycle, DNA damage checkpoint mechanisms, and DNA replication and repair processes, have been of paramount significance for the telomere field. Particularly, these insights extend to understanding replicative senescence as an anticancer mechanism in humans and enhancing our understanding of eukaryotes' evolution.

Double strand breaks (DSBs) can be repaired via either Non-Homologous End Joining (NHEJ) or Homology directed Repair (HR). Telomeres, which resemble DSBs, are refractory to repair events in order to prevent chromosome end fusions and genomic instability. In some rare instances telomeres engage in Break-Induced Replication (BIR), a type of HR, in order to maintain telomere length in the absence of the enzyme telomerase. Here we have investigated how the yeast helicase, Mph1, affects DNA repair at both DSBs and telomeres. We have found that overexpressed Mph1 strongly inhibits BIR at internal DSBs however allows it to proceed at telomeres. Furthermore, while overexpressed Mph1 potently inhibits NHEJ at telomeres it has no effect on NHEJ at DSBs within the chromosome. At telomeres Mph1 is able to promote telomere uncapping and the accumulation of ssDNA, which results in premature senescence in the absence of telomerase. We propose that Mph1 is able to direct repair towards HR (thereby inhibiting NHEJ) at telomeres by remodeling them into a nuclease-sensitive structure, which promotes the accumulation of a recombinogenic ssDNA intermediate. We thus put forward that Mph1 is a double-edge sword at the telomere, it prevents NHEJ, but promotes senescence in cells with dysfunctional telomeres by increasing the levels of ssDNA.

The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier to tumor progression in vivo. The p53 signaling pathway plays central roles in the regulation of cellular senescence. Humans, as well as Drosophila and zebrafish, have p53 isoforms; however, their regulation and function are poorly understood. We here examine the expression profiles of two human p53 isoforms, p53β (lacking the C-terminal oligomerization domain due to an alternative mRNA splicing) and Δ133p53 (lacking the N-terminal transactivation and proline-rich domains due to the transcription from an alternative promoter in intron 4), during in vitro and in vivo cellular senescence and their biological activities in regulating cellular senescence. Induced p53β and diminished Δ133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA, the antisense inhibition of which delayed the onset of replicative senescence. The siRNA-mediated knockdown of endogenous Δ133p53 induced cellular senescence, which was attributed to the regulation of p21WAF1 and other p53 transcriptional target genes. In overexpression experiments, while p53β cooperated with full-length p53 to accelerate cellular senescence, Δ133p53 repressed miR-34a expression and extended cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (i.e., elevated p53β and reduced Δ133p53) was observed in vivo in colon adenomas with senescent phenotypes. The decreased p53β and increased Δ133p53 expression found in colon carcinomas might signal an escape from the senescence barrier during the progression from premalignant to malignant tumors in vivo. This study shows that natural p53 isoforms constitute an endogenous regulatory mechanism for p53-mediated replicative senescence and may open up a new p53-based, senescence-mediated strategy to manipulate carcinogenesis and aging. The molecular details of the senescence-associated Δ133p53 repression and p53β induction are currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2915.

The replication of linear chromosome DNA by DNA polymerase leads to the loss of terminal sequences, in the absence of a special mechanism to maintain ends or telomeres. This mechanism is known to consist of short terminal repeats and the enzyme telomerase, which contains RNA complementary to the DNA repeats. There is evidence that telomeric DNA continually decreases in size in the absence of telomerase, and this is followed by cellular senescence. Immortalisation of somatic cells is accompanied, at least in some cases, by acquisition of telomerase activity. The cloning of DNA coding for the RNA component of telomerase has opened up some new experimental approaches, including the study of telomerases with mutant RNA. The telomere theory of cellular senescence appears to provide a molecular basis for the 'Hayflick limit' to human fibroblast growth. However the telomeres and behaviour of primary mouse cells are anomolous, and many immortalised human cell lines lack normal telomerase activity. These exceptions are not easily accommodated in the telomere theory.

Telomeric Recombination in Lymphocytes Implicates ALT, an Alternative Mechanism for Telomere Length Maintenance, in Normal Human Hematopoietic Cells.

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERT flanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, after hTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence.

Repair of telomeric DNA prior to replicative senescence

Most human carcinomas contain genomically unstable cells with multiple genomic errors, and express telomerase activity. Widespread instability can first be observed in vivo at the pre-malignant stage, such as DCIS, and in vitro as finite lifespan cells with critically shortened telomeres approach replicative senescence. We have proposed that telomere-dysfunction induced genomic instability in pre-malignant finite cells may be needed to generate the errors required for telomerase reactivation and immortalization, while also generating many additional “passenger” errors that will be carried forward into resulting carcinomas. Genomic errors that occur prior to immortalization may influence the cancer phenotype and form the basis for ongoing genomic instability in the malignant cells. Further, the prevalence of many genomic errors in primary human carcinomas makes it difficult to identify the driver errors responsible for immortalization using only in vivo tissues. Although immortalization is crucial for human carcinoma development, little is known about the processes that allow telomerase reactivation during malignant progression. This knowledge gap is due, in part, to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. For example, most murine models lack a significant replicative senescence/immortalization barrier; immortalization employing hTERT precludes examination of the errors responsible for telomerase reactivation during carcinogenesis; a reproducible human mammary epithelial cell immortalization model employing agents implicated in in vivo carcinogenesis has been lacking. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the two main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier, enforced by elevated levels of p16INK4A, was bypassed using shRNA to p16. The replicative senescence barrier due to critically shortened telomeres was bypassed in post-stasis HMEC by c-Myc transduction. These results demonstrate that just two pathologically relevant oncogenic agents are sufficient to immortally transform normal human epithelial cells. The non-clonal immortalized lines generated by direct targeting of the senescence barriers exhibited normal karyotypes at early passages, supporting our hypothesis that the genomic instability commonly present in human carcinomas may not be required per se for immortal transformation, but is needed to generate errors that overcome tumor suppressive barriers. This method of achieving efficient HMEC immortalization, in the absence of “passenger” genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression. Additionally, immortalization, associated with telomerase reactivation, is necessary for progression of all subtypes of human beast carcinomas, and could therefore be a valuable therapeutic target. Our reproducible method for immortalization can permit exploration of agents that may prevent or reverse the immortalization process. That transduction of just shRNA to p16 and c-Myc can immortally transform normal HMEC validates our model of the two main tumor-suppressive senescence barriers (Garbe et al, Cancer Res 2009): stasis, a stress-associated arrest independent of telomere length and extent of replication, and replicative senescence due to telomere dysfunction. Citation Format: Martha Stampfer, Lukas Vrba, Laura Fuchs, Arthur Brothman, Mark LaBarge, Bernard Futscher, James Garbe. Efficient immortalization of normal human mammary epithelial cells using two pathologically relevant agents does not require gross genomic alterations. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B008.

Simulated shortening of proliferation-restricting telomeres during clonal proliferation and senescence of human cells.

BackgroundThe Inspiration4 (I4) mission, the first all-civilian orbital flight mission, investigated the physiological effects of short-duration spaceflight through a multi-omic approach. Despite advances, there remains much to learn about human adaptation to spaceflight's unique challenges, including microgravity, immune system perturbations, and radiation exposure.MethodsTo provide a detailed genetics analysis of the mission, we collected dried blood spots pre-, during, and post-flight for DNA extraction. Telomere length was measured by quantitative PCR, while whole genome and cfDNA sequencing provided insight into genomic stability and immune adaptations. A robust bioinformatic pipeline was used for data analysis, including variant calling to assess mutational burden.ResultTelomere elongation occurred during spaceflight and shortened after return to Earth. Cell-free DNA analysis revealed increased immune cell signatures post-flight. No significant clonal hematopoiesis of indeterminate potential (CHIP) or whole-genome instability was observed. The long-term gene expression changes across immune cells suggested cellular adaptations to the space environment persisting months post-flight.ConclusionOur findings provide valuable insights into the physiological consequences of short-duration spaceflight, with telomere dynamics and immune cell gene expression adapting to spaceflight and persisting after return to Earth. CHIP sequencing data will serve as a reference point for studying the early development of CHIP in astronauts, an understudied phenomenon as previous studies have focused on career astronauts. This study will serve as a reference point for future commercial and non-commercial spaceflight, low Earth orbit (LEO) missions, and deep-space exploration.

Cellular senescence is an irreversible and permanent phenomenon of cell cycle arrest. Consequently, cellular senescence as well as apoptosis is recognized as tumor suppressive mechanism. Based on different inducing mechanism, cellular senescence could divide into two groups as replicative senescence and premature senescence. From previous studies, sublethal H2O2 concentration treatment could induce senescence in WI38 cell, and Hdm2 form the nuclear foci in the H2O2-induced senescent cells. In this study, we attempt to investigate whether Hdm2 nuclear foci formation correlate with cellular senescence phenomenon. Besides two established premature senescence systems induced by AOA and H2O2, we set up another two different senescence systems to compare Hdm2 nuclear foci phenomenon between systems. Otherwise, we use various potential senescence-interfering drugs co-treat with H2O2 and observe whether these drugs could disturb cellular senescence and Hdm2 nuclear foci formation corresponsively. we categorize our findings into 3 major groups : First, using Sirt1 activator Resveratrol and Sirt1 inhibitor Nicotinamide, p38 inhibitor SB203580 and Erk inhibitor PD98059 individually co-treat with H2O2 fails to block H2O2-induced senescence and fails to affect Hdm2 nuclear foci formation. Second, using PI3K inhibitor wortmannin could block H2O2-induced senescence, but did not disturb Hdm2 nuclear foci status. Finally, we found HDAC inhibitor TSA co-treat with H2O2 could destroy Hdm2 nuclear foci formation and lower cellular senescence ratio. This finding implies there might be some correlations between Hdm2 nuclear foci formation and cellular senescence phenomenon.

Telomeres, the specific DNA–protein structures found at both ends of each chromosome, protect genome from nucleolytic degradation, unnecessary recombination, repair, and interchromosomal fusion. Telomeres therefore play a vital role in preserving the information in our genome. As a normal cellular process, a small portion of telomeric DNA is lost with each cell division. Telomeres shorten with age. When telomere length reaches a critical limit, the cell undergoes senescence and/or apoptosis. Shorter telomeres have also been implicated in genomic instability and oncogenesis. People older than 60 with short telomeres have three and eight times increased risk to die from heart and infectious diseases, respectively [Starr et al., 2007.] Rate of telomere shortening is therefore critical to an individual’s health and pace of aging. Telomere length may therefore serve as a biological clock to determine the lifespan of a cell. The telomere length limits a cell proliferative ability in cultured human primary cells. Telomere shortens in the absence of telomerase in non-ALT cells. Certain agents associated with specific lifestyles may expedite telomere shortening by inducing damage to DNA in general or more specifically at telomeres and may therefore affect health and lifespan of an individual.

Myelodysplastic syndrome: An inability to appropriately respond to damaged DNA?

Cell cycle restriction of telomere elongation.