Why telomere shortening slowing down cancer?

Why telomere shortening slowing down cancer?

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I am reading Alternative Lengthening of Telomeres in Mammalian Cells, about how telomeres linked to human cancers.

Due to the end-replication problem,5,6 the ends of linear chromosomes shorten with each round of DNA replication.7 In human somatic cells, the progressive telomere shortening that occurs with continued proliferation eventually results in the triggering of a replicative checkpoint. Telomere shortening and the structural changes that it presumably causes, leads to a DNA-damage checkpoint response at the telomere and induction of a permanent p53- and Rb-dependent growth arrest (i.e., replicative senescence).8-10 Because this limits the proliferative capacity of somatic cells, including those that have accumulated oncogenic mutations, telomere shortening and replicative senescence are a potent tumor suppressor mechanism.

The paragraph states a shorter telomere is a potential suppressor to cancers. Why is that? Why exactly a somatic cell proliferate slower with a shorter telomere DNA sequence?

Conversely, why a longer minority leads to human cancers?

Think about the 24 different linear chromosomes in a diploid human XY cell. In the absence of active telomerase enzyme to repair the chromosome ends following one round of cell division, then every time that cell divides each end of each chromosome will get a little bit shorter.

If the cell keeps dividing indefinitely, like a tumor cell, the chromosomes are effectively shrinking. Eventually this chromosome shortening will result in deleting actual genes (at first, 24 chromosomes x 2 chromosome ends = 48 genes). Not all of those genes are essential for cell viability; and the distance, or length, of DNA between the telomere and the first gene on that end will be variable. But as soon as the chromosome shortening removes part of an essential gene, then that cell will quickly die.

It would be like erasing the words at both ends of a sentence, one letter at a time. In the beginning, someone else would still be able to make sense out of your sentence, but eventually they would not be able to fully understand what information you were trying to convey.

Most of the terminally differentiated cells in an adult human are no longer actively dividing, so it is not a problem, they don't need active telomerase. However, cancer is a condition of unregulated cell division, and so all cancer cells have to have some mechanism of repairing the ends of their chromosomes. Reactivation of TERT (Telomerase Reverse Transcriptase, the protein component of the telomerase enzyme) gene expression is extremely common in cancer cells.

TZAP-ing telomeres down to size

The phenomenon of gradual telomere shortening has become a paradigm for how we understand the biology of aging and cancer. Cell proliferation is accompanied by cumulative telomere loss, and the aged cell either senesces, dies or transforms toward cancer. This transformation requires the activation of telomere elongation mechanisms in order to restore telomere length such that cell death or senescence programs are not induced. Most of the time, this occurs through telomerase reactivation. In other rare cases, the Alternative lengthening of telomeres (ALT) pathway hijacks DNA recombination-associated mechanisms to hyperextend telomeres, often to more than 50 kb. Why telomere length is restricted and what sets their maximal length has been a long-standing puzzle in cell biology. Two recent studies published in this issue of EMBO Reports [1] and recently in Science [2] sought to address this important question. Both built on omics approaches that identified ZBTB48 as a potential telomere-associated protein and reveal it to be a critical regulator of telomere length homeostasis by the telomere trimming mechanism. These discoveries provide fundamental insights for our understanding of telomere trimming and how it impacts telomere integrity in stem and cancer cells.

The Same Predictor of Lifespan Is Shared Across Humans and Animals

Measured in creatures like elephants, goats, and humans, one metric shows how long a species will live.

When it comes to selling snake oil, nobody does it quite like the anti-aging industry. But it does get one thing right. Telomeres, the tiny end caps on our chromosomes, seem to protect against aging-related disease, and anti-aging researchers suspect that slowing down how fast telomeres shrink can slow the process of aging. New research published Monday in the Proceedings of the National Academy of Sciences confirms this connection across animal species.

Nobody has successfully lengthened telomeres or slowed down their shrinkage, but previous studies on mice and humans have shown that telomere shortening is linked to aging. The new paper shows that this effect holds true for a wide variety of animal species, including birds and mammals.

The team of Spanish authors showed that the rate of telomere shortening in eight different animals strongly predicted the animals’ average life spans. They showed that the longest-lived animals had telomeres that took the longest time to shorten.

“The results shown here indicate that the telomere shortening rate of a species can be used to predict the life span of that species, at least with the current dataset,” write the study’s authors, led by Kurt Whittemore, Ph.D., a researcher at the Spanish National Cancer Research Center.

Each time our cells divide, the telomeres protecting the DNA in their nuclei shorten. Once they get too short, they can no longer keep chromosomes from unraveling. This means that over time, as telomeres shorten, cells are at greater risk of damage and death. For this reason, some anti-aging therapies, like the unproven approach of the controversial biotech company BioViva, aims to lengthen telomeres.

But as the PNAS study shows, absolute length may not be as important as the rate of shortening.

In the study, regardless of the lengths of animals’ telomeres, it was the rate at which they shortened that was closely correlated with the length of their lifespan.

“We observed that mean telomere length at birth does not correlate with species life span since many short-lived species had very long telomeres, and long-lived species had very short telomeres,” the authors write.

The team used a fluorescence technique to measure telomere lengths in cells from a group of very different animals: lab mouse (Mus musculus), goat (Capra hircus), Audouin’s gull (Larus audouinii), reindeer (Rangifer tarandus), griffon vulture (Gyps fulvus), bottle-nose dolphin (Tursiops truncatus), American flamingo (Phoenicopterus ruber), and Sumatran elephant (Elephas maximus sumatranus).

To create a linear model of how telomeres shorten over time, the team measured samples from different-aged animals of the same species. By comparing these telomere lengths, the team could get a sense of how the telomere lengths of an older individual compare with those of a younger one.

They observed that the mouse, the shortest-lived animal in the study, has an extraordinarily fast rate of telomere shrinking — about 100 times that of a human. The telomere shortening rates of the other animals fell somewhere between those of mice and humans, and all of them correlated to their average lifespans.

The authors admit that future studies on this topic should include a longitudal design, which would follow animals or humans over the course of their entire lives. Since some of the study animals have very long life spans, doing so would not have been possible in the current study.

This study adds support to the idea that the effects of telomere shortening are closely tied to the cell death and damage associated with aging-related diseases. Even though anti-aging researchers have failed to extend human lifespans, this particular hypothesis suggests that they may at least be looking in the right place.

But so far, the best researchers in the field have not been able to slow telomere shortening, even though some have suggested that exercise can do the trick.

“We don’t have any compound that will actually elongate telomeres, despite what you can read on many websites,” Nobel Laureate Carol Greider, Ph.D., previously told Inverse.

“Of course, because we have patients in the hospital dying of these diseases, if there was some sort of a treatment, we would be looking into it. But we have looked into those things that are out there, and it’s basically snake oil.”

Decoding Telomere Biology and Cancer Risk

Sharon Savage leads a team to unravel the secrets of our telomeres.

Dr. Sharon Savage with Nancy, a participant in an NIH Clinical Center study of dyskeratosis congenita, prior to a bone marrow aspiration and biopsy procedure.

Chromosomes have little in common with either shoelaces or bombs, but telomeres—the repeated stretches of DNA that cap chromosome ends—are often compared to components of both.

As plastic tips (aglets) protect shoelaces from fraying, and bomb fuses burn down to imminent danger, telomeres maintain chromosomal stability and protect our genetic data—and when they wear away from aging or environmental stresses, DNA becomes vulnerable to degradation. Each time a cell divides, a process associated with normal aging, its telomeres get shorter, until the cell either undergoes apoptosis (programmed cell death) or senescence (the cell remains alive, but inactive).

Team meetings fuel a variety of research projects with new ideas.

Sharon Savage, M.D., has spent her career studying the association between telomere length and cancer risk, while searching for genetic markers that predict telomere biology disorders. Much of her work focuses on dyskeratosis congenita (DC), a rare and complex inherited cancer predisposition syndrome.

“Although extremely rare,” Dr. Savage explains, “because of its association with cancer and its established link to telomere biology genes, a better understanding of DC could have far-reaching implications for understanding cancer risk and etiology more broadly.”

Human cells contain 46 chromosomes (blue), the ends of which are capped with protective telomeres (white) that vary in length.

When Savage joined the IRP’s Clinical Genetics Branch, mutations in three genes—DKC1, TERT, and TERC—accounted for approximately 40% of DC cases. The remaining 60% of DC patients still lacked an identifiable genetic source of the condition. Working as part of the Inherited Bone Marrow Syndromes (IBMFS) Cohort Study, at NIH’s National Cancer Institute, Savage’s team has since identified four additional genes with mutations associated with telomere maintenance that can cause DC.

Variation in a gene called TINF2 was their first major discovery. In collaboration with researchers at Stanford University, the team next showed that recessive WRAP53 mutations can cause DC—the first study to show that the abnormal location of telomerase, the protein that synthesizes DNA repeats at the ends of telomeres, in the cell is associated with DC. Most recently, her team discovered mutations in a DNA helicase and telomere maintenance gene called RTEL1 in several families, including two of Ashkenazi Jewish ancestry, a clinically severe variant of DC known as Hoyeraal-Hreidarsson syndrome (HH).

Dermatologists Dominique Pichard (left) and Edward Cowen (center) examine Nancy’s son Charlie’s skin and fingernails, two areas of the body frequently affected by DC and other telomere biology disorders.

“From here we collaborated with the Center for Jewish Genetics to define the frequency of RTEL1 mutations in this population and showed that they are common enough to be added to the prenatal testing panel for this population,” Savage says. “This story exemplifies why studying rare diseases is so important. Our RTEL1 discoveries wouldn’t have happened under other circumstances, and the findings will now be applied to help Ashkenazi and other Orthodox Jewish populations manage potential risks associated with this serious disease.”

Treatment for DC is challenging, and current options are limited, so Savage and Suneet Agarwal, M.D., a pediatric oncologist at Dana-Farber Cancer Institute and Boston Children’s Hospital, have formed the Clinical Care Consortium of Telomere Associated Ailments (CCCTAA) to identify new treatments and improve outcomes. First on their list is the urgent need to develop strategies for enrolling greater numbers of DC patients in clinical trials.

Her office can be a good place to catch Dr. Savage for a chat about new ideas.

In parallel, Savage is working with DC Outreach, a support group for affected families that she helped launch, to develop the first clinical guidelines for managing the disease. She also helps DC Outreach in their collaboration with the Genetic Alliance to create a database of DC patients. Thanks to Savage and others, there are now at least 13 known DC genes—all of them related to telomere biology.

Dr. Savage has devoted her career to thinking about ways to help people with telomere biology disorders.

“This work wouldn’t be possible anywhere else,” Savage explains. “First of all, large family studies like ours are important because they provide insight into more common cancers, but they require cross-discipline research collaborations. Second, because of the varied clinical manifestations of DC, our patients need access to a wide range of specialists. The Intramural Research Program combines these unique elements, allowing us to conduct fast-paced, high-risk projects with access to an unparalleled breadth and depth of expertise.”

Such attributes enabled Dr. Savage and her team to create a translational clinical genetics program devoted entirely toward the complex “defusing” of quickly shortening telomeres. Every day, and every patient who visits the clinic, brings them closer to identifying ways of slowing or interrupting that process.

Results and discussion

Chelidonine exhibited dose dependent cytotoxicity

The MTT method was used to assess the cytotoxicity of chelidonine in MCF7 cells. The LD50 value was 8 μM after 48 h treatment (p≤0.05). Chelidonine showed strong cytotoxicity, rapidly reducing viable cell numbers at low concentrations (Fig 1). However, this steep slope in the dose-response curve was subsequently moderated so that 20–30% of cells were still viable at 50 μM. A complete cell death was seen at 100 μM. In the following experiments very low concentrations: 0.01 and 0.05 μM, were used in long term treatments. In telomere length studies treatment with 0.1 μM chelidonine was included too.

Chelidonine increased population doubling time

MCF7 cells were treated with 0.01 or 0.05 μM chelidonine for 48 h after each passage. Chelidonine at 0.01 μM did not change population doublings and doubling time of MCF7 cells significantly no morphological change towards senescence or alteration of growth rates was observed even after continuous treatments of log-phase cultures for almost 1080 h (Fig 2, diamonds). However, a significant reduction of the growth rate occurred in cells treated with 0.05 μM chelidonine in comparison with untreated control (p < 0.005) which is clearly seen after five treatments (Fig 2, squares). At this time point, the treated cells showed approximately 30% less doublings in comparison with the control group. Doubling period in the treated cells was 162.5 ± 0.5 h as compared with 32.6 ± 0.5 h in control cells.

A) Number of population doublings and B) doubling time after long-term treatment with chelidonine (0.01 diamonds or 0.05 μM squares) in comparison with untreated control MCF7 cells (triangles).

Chelidonine strongly reduced telomere length in MCF7

Relative average telomere length can be measured by MMQPCR using primers that hybridize with the telomere hexamer repeats because the number of binding sites for the primers increases as average telomere length increases [37,38]. However, MMQPCR simultaneously counts the copy number of albumin as a single copy gene in each reaction tube using a specific primer pair, so the relative amount of telomere copies (T) to albumin (S) of the untreated control is measured. A representative experiment for the potential effects of chelidonine on MCF7 cells is shown in Fig 3. The T/S value of the treated samples in sequential treatments, 48 h per passage, was compared with that of un-treated cells, which was considered as 1 (triangles). The cells treated with 0.01 μM chelidonine showed only a minor decrease in telomere length after several treatments as the ratio was not stably lower than 1. However, a diminished T/S ratio was observed soon after treatment with 0.05 μM chelidonine (squares). The T/S ratio decrease continued to less than 0.3 after 5 sequential treatments, implying telomere shortening to about 30% of the un-treated control cells. A rapid telomere loss was observed at 0.1 μM chelidonine (circles), so that plating became impossible after only three sequential treatments.

Data represented are the average of two independent experiments each in duplicates ± SD values.

Chelidonine strongly suppresses telomerase activity and hTERT transcription

Quantitative telomerase repeat amplification protocol (qTRAP) measurements showed considerable reduction of telomerase activity in treated MCF7 cells. In other words, chelidonine reduces active telomerase time- and dose-dependently. Fig 4A shows the relative telomerase activity of MCF7 cells after 24, 48 and 72 h treatments with various concentrations of chelidonine. Telomerase activity was reduced ≥40% after 48 h treatment with 0.1 μM chelidonine. The IC50 value for telomerase inhibition in treated cells at this time point was 0.45 ± 0.08 μM (P≤0.05). The method measures the functional amount of the enzyme in equal amounts of total protein. Chelidonine also showed a robust suppression of hTERT transcription which was both time- and concentration-dependent (Fig 4B). The decreased enzyme activity and hTERT mRNA level after 48 h show almost the same pattern, while in a shorter time, 24 h, transcription decrease precedes the loss of enzyme activity.

A) Telomerase activity as measured by q-TRAP assay and B) hTERT transcription levels using quantitative real-time RT-PCR technique in MCF-7 cells after various time of treatment with different concentrations of chelidonine. Mean value ±SEM of three logically independent experiments each containing three samples for each point was presented. (p≤0.01 in Pair-Wise Comparisons via Tukey HSD Test unless marked as + /# /* in the plot evaluating p ≤0.05. “o “represents no significance).

Chelidonine suppresses cell growth reversibly

MCF7 cells that treated once for 48 h with 0.1 or 8 μM chelidonine retained some of their viability/growth rate when compared with un-treated control cells (Fig 5). Also, the cells treated sequentially 2 or 3 times with 0.05 μM chelidonine recovered their growth rate after the compound was removed from the medium. However, after four times treatment, the cell growth was so slow that replating was impossible. As seen in Fig 5 this shows that growth inhibition by chelidonine is reversible cells mostly recovered their growth after removing chelidonine as far was observed.

The mean ± SD values of nine samples were presented.

Chelidonine shifts the splicing pattern of hTERT towards non-enzyme coding variants

Chelidonine reduced the total transcription level of hTERT dose dependently, as seen in Fig 4. The primers used cannot differentiate between the variants. However, telomerase, which is mainly regulated at the transcription level, has non-enzyme coding transcripts too. Transcripts of hTERT undergo a special alternative splicing in MCF-7, which results in at least four different splice variants. Among them the –β variant always figures as the most abundant form while the full length variant is the only active one. Chelidonine induced a clear shift in the splicing pattern of hTERT transcripts, although only at relatively high concentrations (Fig 6). The full-length transcript almost disappeared at the LD50 value, while it is still visible with 2 μM chelidonine treating. This implies that hTERT splicing is not considerably affected by low concentrations of chelidonine. High concentrations, however, suppress both total transcription and full length active variant of hTERT.

PCR products were analysed by gel electrophoresis (3% agarose gels). The white arrows show the location of four splice variants in untreated cells. The upper band is the functional full-length hTERT (FL, 457 bp) which is followed by the three shorter non-enzyme coding variants. Lane 1: negative control, 2: treatment with 5 μM chelidonine, 3: treatment with 2 μM chelidonine, 4: untreated control and 5: 100 bp DNA marker from which 10, 10, 10, 5 and 5 μl was loaded respectively as seen in Fig 4B the total transcription of hTERT was strongly repressed while the major isoform is minus beta. Five microliters of β2-microglobulin PCR products of the related samples have been loaded as control at bottom.

Telomere shortening protects against cancer

As time goes by, the tips of your chromosomes–called telomeres–become shorter. This process has long been viewed as an unwanted side-effect of aging, but a recent study shows it is in fact good for you.

“Telomeres protect the genetic material,” says Titia de Lange, Leon Hess Professor at Rockefeller. “The DNA in telomeres shortens when cells divide, eventually halting cell division when the telomere reserve is depleted.”

New results from de Lange’s lab provide the first evidence that telomere shortening helps prevent cancer in humans, likely because of its power to curtail cell division. Published in eLife, the findings were obtained by analyzing mutations in families with exceptional cancer histories, and they present the answer to a decades-old question about the relationship between telomeres and cancer.

A longstanding controversy

In stem cells, including those that generate eggs and sperm, telomeres are maintained by telomerase, an enzyme that adds telomeric DNA to the ends of chromosomes. Telomerase is not present in normal human cells, however, which is why their telomeres wither away. This telomere shortening program limits the number of divisions of normal human cells to about 50.

The idea that telomere shortening could be part of the body’s defense against cancer was first proposed decades ago. Once an early-stage tumor cell has divided 50 times, scientists imagined, depletion of the telomere reserve would block further cancer development. Only those cancers that manage to activate telomerase would break through this barrier.

Clinical observations seemed to support this hypothesis. “Most clinically detectable cancers have re-activated telomerase, often through mutations,” de Lange says. Moreover, mouse experiments showed that shortening telomeres can indeed protect against cancer. Nonetheless, evidence for the telomere tumor suppressor system remained elusive for the past two decades, and its existence in humans remained controversial.

The solution to a decades-old problem

The telomere tumor suppressor pathway can only work if we are born with telomeres of the right length if the telomeres are too long, the telomere reserve would not run out in time to stop cancer development. Longer telomeres will afford cancer cells additional divisions during which mutations can creep into the genetic code, including mutations that activate telomerase.

For decades, de Lange’s lab has been studying the complex process by which telomeres are regulated. She and others identified a set of proteins that can limit telomere length in cultured human cells, among them a protein called TIN2. When TIN2 is inhibited, telomerase runs wild and over-elongates telomeres. But it was not known whether TIN2 also regulated telomere length at birth.

The stalemate on the telomere tumor suppressor continued until physicians at the Radboud University Medical Center in Holland reached out to de Lange about several cancer-prone families. The doctors found that these families had mutations in TINF2, the gene that encodes the TIN2 protein instrumental to controlling telomere length. That’s when they asked de Lange to step in.

Isabelle Schmutz, a Women&Science postdoctoral fellow in the de Lange lab, used CRISPR gene-editing technology to engineer cells with precisely the same mutations as those seen in the Dutch families and examined the resulting mutant cells. She found that the mutant cells had fully functional telomeres and no genomic instability. They were, for all intents and purposes, normal healthy cells.

But there was one thing wrong with the cells. “Their telomeres became too long, ” de Lange says. Similarly, the patient’s telomeres were unusually long. “These patients have telomeres that are far above the 99th percentile,” de Lange says.

“The data show that if you’re born with long telomeres, you are at greater risk of getting cancer, ” says de Lange. “We are seeing how the loss of the telomere tumor suppressor pathway in these families leads to breast cancer, colorectal cancer, melanoma, and thyroid cancers. These cancers would normally have been blocked by telomere shortening. The broad spectrum of cancers in these families shows the power of the telomere tumor suppressor pathway.”

The study is demonstration of the power of basic science to transform our understanding of medicine. “How telomeres are regulated is a fundamental problem,” de Lange says. “And by working on a fundamental problem, we were eventually able to understand the origins of a human disease.”

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Telomere Shortening and Implications for Cancer and Aging in Humans

Telomeres are highly conserved, specialized nucleotide sequences located at the ends of chromosomes consisting of several hundred tandem six G rich nucleotide repeats in the 5’ to 3’ direction. Telomeres play several important roles in human cells including protection from degradation and homologous recombination. As cells duplicate, the telomere length shortens with each round or replication this leaves chromosomes less protected over time. In humans, telomere shortening rates have been determined to be 20-60 bade pairs per year, depending on the tissue type. Telomeres have a dynamic structure and function, as it changes as cells age. There is a characteristic difference between telomeres found in human somatic cells and those found in cancer cells there are many applications for cells with active telomerase in research and cancer therapies. This review outlines the structure and formation of telomeres and discusses their function as cells age. The role of telomeres in cancer cells will also be reviewed.

Telomeres are located at the ends of linear chromosomes functioning to protect their ends. Telomeres consist of several hundred tandem G rich nucleotide repeats in the 5’ to 3’ direction in humans this sequence is AGGGTT. The length of telomeres is variable based on tissue type [1]. Telomeres can form a telomeric loop (T-loop) at the terminus of linear chromosomes providing more extensive protection [2].

In somatic cells, telomeres are shortened with each round of cell division. As the telomeres shorten, the ends of the chromosomes become more and more vulnerable [3]. Shortening of the telomeres, however, does not occur in most cancer cells. Cancer cells demonstrate telomerase activity throughout their lifespan allowing, the cells to become “immortal” [4]. While all cells contain telomerase, it is typically only active during development, more specifically during the G1 phase [5]. Telomerase will only remain active in cells that also express active human telomerase reverse transcriptase (hTRT) [6]. Better understanding the role of hTRT in cancer cells can lead to more advanced cancer treatments hTRT and other associated proteins can be targeted for drug therapies specifically as it is not active in other somatic cells [7].

Telomere Function and Structure The main known function of telomeres is to protect linear chromosomes from degradation at their terminus. T loop structures form at chromosome ends by looping a 3’ overhang left on the end of the chromosome back and allowing it to become imbedded in the double stranded portion of the chromosome. This process is mediated by the telomeric repeat-binding proteins, TRF1 and TRF2 [8]. TRF1 and TRF2 are included in a complex called the shelterin complex. It consists of TRF1, TRF2, TIN2, Rap1, TPP1 and Pot1 and it functions to protect the ends of chromosomes by forming and maintaining telomeres [9].

TRF2 acts to sequester the single stranded end of telomeres and embed it within a double stranded section of the telomere. This results in a triple stranded structure at the point of insertion which forms a small displacement loop (D loop) [10]. Data shows that TRF2 is localized to the D loop junction and TRF1 is found at the double stranded portion of the telomere. While TRF2 has been classified as a double stranded telomere binding protein, experimental data shows that overexpression of a dominant negative TRF2 cell line has an effect on the singe stranded portion of the telomere. It also led to the activation of the p53 apoptosis pathway [8].

During DNA replication in somatic cells, polymerase is unable to replicate the 3’ end of the lagging strand of DNA (Figure 1. a-c). As a result, with each round of replication your chromosomes are less and less protected [3]. Associations have been made between telomere length and the age of chromosomes [11]. With each round of replication, between 20 and 60 base pairs in the telomere are lost this number is dependent on tissue type [11].

Further functions include the prevention of homologous recombination at the ends of linear chromosomes [3] and the prevention of the activation of premature apoptosis [8]. Work done by Griffith, et al. show that depletion of TRF2 leads to, among other things, activation of a double strand break checkpoint. This checkpoint signals the p53 pathway which often leads to apoptosis. All of this together suggests the necessity of telomeres for maintaining the integrity of chromosomes [8].

T-Loops T-loops were discovered in the Griffith and de Lange laboratories in 1999 changing the way that telomere structure was previously understood. It was previously believed that chromosomes terminated in a 3’ overhang with a GT rich repeat. Klobutcher et al. provided the first indication that there is conservation among various species at chromosome ends they recognized that there is varying length between species but that all contain the same 3’ overhang on the G rich strand [12]. The current understanding of telomere function was discovered in the Griffith lab in 1999. Using electron microscopy, the end structure of chromosomes was visualized as a loop. Griffith et al. describe a three stranded displacement loop (D-loop) which folds over allowing the single stranded overhang to tuck into the double stranded structure. This process is accomplished with sequestering proteins, TRF1 and TRF2 [8].

TRF1 and TRF2 sequester the single stranded 3’ end of the telomere and loops it back to form a T-loop. While TRF1 is acting to align double stranded sequences of the T loop, TRF2 closes the loop by inserting the single strand into a section of the double stranded section of the telomere and forming a D loop structure [8].

The Discovery of Telomerase Telomerase was first discovered by Carol Greider and Elizabeth Blackburn in 1985. Greider and Blackburn first described telomerase as telomere terminal transferase and were later able to characterize it as an RNA ribonucleoprotein [10] [13]. The discovery of telomerase opened the field for new research not long after the initial discovery in Tetrahymena thermophile, Jack Szostak discovered telomerase activity in human HeLa cells. HeLa cells were isolated from the ovarian cancer patient, Henrietta Lacks, several years prior and coined the immortal cells. Until this time, it was unknown what made these cells immortal. This era was the beginning of a new understanding of cell aging [4].

In 2009, the Nobel Prize in Physiology or Medicine went to Elizabeth Blackburn, Carol Greider and Jack Szostak for their work on telomere function and the mechanism of telomerase [14].

Regulation of telomerase Telomerase is a ribonucleoprotein complex containing a telomeric RNA subunit (TR), a catalytic core, and telomerase reverse transcriptase (hTRT). It is also surrounded by several accessory proteins [14].The TR component of telomerase is an RNA sequence complimentary to the repeated sequences found in the telomere in humans this sequence is 3′-CAAUCCCAAUC-5′ [15]. Interestingly, only 6 nucleotides in this sequence are repeated in telomeres, the others act to orient the telomerase on the DNA. The RNA template sequence functions as a primer on the 3’ overhang of the lagging strand during DNA replication (Figure 1. d). The telomerase moves step wise in the 5’-3’ direction, continually adding repeats of 6 nucleotides long (Figure 1. e). Once this step is complete, DNA polymerase α is able to complete lagging strand elongation (Figure 1. F) [1].

The catalytic subunit of telomerase in Homo sapiens is Human telomerase reverse transcriptase (hTRT) [6]. Nakayama et al. characterized hTRT by measuring telomerase activity in human fibroblast cells overexpressing hTRT. They were able to see that when hTRT is overexpressed in telomerase negative cells, telomerase activity is induced. In cells containing hTRT mutations, telomerase activity iss not induced thus characterizing the relations ship between hTRT and telomerase activity. This led them to measure activity in cancerous liver cells and non-cancerous liver cells where they saw consistent results. They came to the conclusion that hTRT is the catalytic protein responsible for activating telomerase and that it plays an important role in the immortality of cancer cells [6].

Telomeres and aging

The p53 tumor suppressor protein is responsible for imposing apoptosis and cell cycle arrest. As telomeres shorten critically to unsafe lengths, p53 is activated and senescence is promoted. Senescence is when cells no longer replicate, eventually leading to apoptosis [16]. This mechanism allows cells the ability to maintain the integrity of their chromosomes. Those with hyper shortened telomeres lose their protection and are subject to translocations, deletions, homologous recombination, and other damage [7]. This kind of damage can lead to tissue aging and degenerative disorders [16].

Some aging phenotypes in humans can be contributed to shortened telomere lengths, as shown in telomerase knockout mice. Telomerase knockout mice in late generations show signs of aging including graying fur and the inability to respond to physical stressors such as wound repair. Many of the phenotypes seen correlated with skin internal organs including kidneys, brain, and cardiovascular system do not show advanced aging with shortened telomeres [18]. While this information shows promise in the field of telomeres and aging, it cannot be directly applied to humans. Mouse telomeres range from 50-150 kbp while in humans, they are about 15 kbp in length. This, combined with the short lifespan of mice in comparison to humans [17], suggests that the instability of chromosomes with shortened telomeres is likely more dependent on the vulnerability than their actual shortened length. Less telomeric protection at exposed chromosome ends can lead to recombination and cancer.

Aged chromosomes with shortened telomeres have a correlation with abnormalities such as Robertsonian fusions (end to end fusions). They are also correlated with spontaneous tumor generation. Cancer generation was observed in telomerase knockout mice there was a significant correlation in tumor generation in tissues that have been proven telomerase dependent such as the skin and testes [18].

Telomeres and Cancer

Telomerase activity in cancer cells The majority of cancer cell lines have activated telomerase activity giving them an extended, and sometimes immortal, life [4]. An estimated 80% of tumors contain active telomerase throughout their lifespan. It was recently shown that these telomerase positive tumors also express contain long interspersed nuclear elements-1 (LINE-1) which are responsible for telomere maintenance in pathological cells [19]. cMyc and Krüppel-like factor-4 (KLF-4) are potential oncogenes cMyc codes for a transcription factor while KLF-4 is a transcription factor itself. When Aschacher et al performed LINE-1 knockdown, they observe increased telomere dysfunction and decreased levels of cMyc and KLF-4 were also knocked down. This suggests that LINE-1 acts as a regulatory element by means of cMyc and KLF-4 [19].

Cancer cells as research tools The research done by Aschacher et al. proposes LINE-1 as a rational target for inhibiting telomerase activity in cancer cells. Their experimentation shows that G2 cell cycle arrest is observed in LINE-1 depleted cell lines. This suggests that inhibition of the effects of LINE-1, either by directly targeting it or through cMya and KLF-4 , could be an effective way of repression tumor growth as LINE-1 is only found in active tumor cells [19].

Another proposed mark for cancer drugs is TRF2. TRF2 has shown to be essential in the formation of telomeres [8]. TRF2 disruption can impair telomere maintenance and elicit a DNA damage response. A chemical inhibitor is being studied to bind to the TRF2 domain to switch off its signaling [20].

Figure 1: Mechanism of telomerase. (a.) Lagging and leading strands are primed by primase to prepare for elongation by polymerases α and δ (b.). The result is a 3’ overhang on the lagging strand caused by nucleolytic degradation of the 5’ end [23](c.) leaving the chromosome vulnerable to damage and shortening after each round of replication without the action of telomerase (d). Telomerase containing telomeric template attaches to the overhang and elongates (e.) the 3’ overhang with a series of tandem 6 nucleotide repeats. (f). Primase with polymerases α and δ are then able to complete double strand synthesis. Conclusion

The future of research regarding telomeres and cancer research lies in using associated proteins as drug targets. As telomerase activity is noted in 80% of tumor cells, telomerase and its associated proteins are a good place to start. It is a specific target as most adult somatic cells do not have active telomerase [21]. The downfall of this method is that existing cancer cells with elongated telomeres will have to go through significant numbers of cell division until senescence is reached [22].

Stress speeds up aging through telomere shortening

Aging and stress in the workplace: accelerated telomere shortening

Stress in the workplace occurs when there is no balance between what a person perceives the constraints are and how well they feel they can deal with them. Although stress is not a disease, prolonged exposure to it has negative effects on health. It is called chronic stress [5].

Many studies have shown a link between chronic stress in the workplace and a degradation in health. The risk to develop cardiovascular diseases increases as the capacity of the immune system decreases [6]. Although the missing link between stress and health (and aging) has not yet been singled out, we know that it tampers with cell function. However, cell environment plays an important role when regulating telomere length and telomerase activity. Researchers studied healthy women under different levels of chronic stress to determine if it had any impact on telomere length or influence on their physiological age [6].

The subjects under a more important stress had shorter telomeres. On average, there is a 550bp difference in telomere sequence length, no matter the chronological age of the subject, between the subjects undergoing high levels of stress and those with low levels of stress in the workplace [6]. The difference is linked to a 10 years increase of the biological age [6].

In the high-level stress group, telomerase activity is 48% lower than in subjects in the group with lower levels of stress. When this decrease of telomerase activity becomes chronic, it contributes to accelerated telomere shortening [6].

This proves the influence of extracellular factors, such as stress in the workplace, on telomere shortening. It would then be strongly linked to an increase in oxidative stress, a decrease of telomerase activity and an acceleration of telomere shortening. All of these would result in early cell senescence [4], which impacts the lifespan of the cells as well as the physiological age.

Telomeres are repeated DNA sequences that form the end caps of chromosomes. A little of their length is lost with each cell division, and cells self-destruct or become senescent and cease replication when telomeres become too short. This is a part of the Hayflick limit on cell replication: near all cells in the body can only divide a limited number of times. Stem cells are the first exception, using telomerase to extend telomeres. Cancer cells are the second exception, employing either telomerase or the alternative lengthening of telomeres (ALT) mechanisms that do not operate in normal cells. Telomere lengthening is a universal mechanism in cancer, and thus there is considerable interest in producing a single class of treatment, based on interference in telomere lengthening, that can potentially shut down all cancers.

The original vision for whole-body interdiction of telomere lengthening, a part of the SENS rejuvenation research agenda, was to turn off the processes that lengthen telomeres throughout the body. Perhaps temporarily, or, in a more futuristic option, perhaps permanently when deployed in conjunction with periodic replacement of stem cell populations. Since the original proposition was put forward, research into ALT hasn't made all that much progress, perhaps because only 10% of cancers exhibit this behavior. Research into interfering with telomerase-based telomere lengthening has progressed, however, and diversified into a number of interesting lines of work. All of these seem likely to be targeted to cancer cells, either as an inherent result of the mechanism, or by combining the therapy with a selective delivery system.

One recent example of many is the work of Maia Biotechnology, building on an approach that sabotages telomerase-based telomere lengthening in a subtle way that has the outcome of killing cells. Today's research materials are another example of a program at an earlier stage of exploration, more focused on an indirect approach to reducing telomerase activity, one that can involve signaling applied outside the cell. This makes it an attractive basis for the development of therapies.

Researchers have in the past provided evidence to suggest that shelterins, proteins that wrap around telomeres and act as a protective shield, might be therapeutic targets for cancer treatment. Subsequently, they found that eliminating one of these shelterins, TRF1, blocks the initiation and progression of lung cancer and glioblastoma in mouse models and prevents glioblastoma stem cells from forming secondary tumours. Now researchers go one step further and describe for the first time how telomeres can be regulated by signals outside the cell that induce cell proliferation and have been implicated in cancer. The finding opens the door to new therapeutic possibilities targeting telomeres to help treat cancer.

Researchers have outlined a link between TRF1 and the PI3K/AKT signalling pathway. This metabolic pathway, which also encompasses mTOR, is one of the pathways most frequently affected in numerous tumorigenic processes. However, it was not known whether preventing TRF1 regulation by this pathway can have an impact on telomere length and its ability to form tumours. AKT acts as a transmitter of extracellular signals triggered by, among others, nutrients, growth factors, and immune regulators, to the interior of cells.

Researchers modified the TRF1 protein in cells to make it unresponsive to AKT, using the gene-editing tool CRISPR/Cas9. This way, TRF1 and the telomeres became invisible to any extracellular signals transmitted by AKT. Telomeres in these cells shortened and accumulated more damage most importantly, the cells were no longer able to form tumours, indicating that telomeres are important targets of AKT and its role in cancer development.

The paper shows that telomeres are among the most important intracellular targets of the AKT pathway to form tumours, since, although neither the function of AKT nor of any of the thousands of proteins that are regulated by it was altered, only blocking AKT's ability to modify telomeres was sufficient to slow tumour growth. The next step will be to generate genetically modified mice with telomeres that are invisible to AKT. The authors anticipate that these mice will be more resistant to cancer.

The telomere-bound shelterin complex is essential for chromosome-end protection and genomic stability. Little is known on the regulation of shelterin components by extracellular signals including developmental and environmental cues. Here, we show that human TRF1 is subjected to AKT-dependent regulation. To study the importance of this modification in vivo, we generate knock-in human cell lines carrying non-phosphorylatable mutants of the AKT-dependent TRF1 phosphorylation sites by CRISPR-Cas9.

We find that TRF1 mutant cells show decreased TRF1 binding to telomeres and increased global and telomeric DNA damage. Human cells carrying non-phosphorylatable mutant TRF1 alleles show accelerated telomere shortening, demonstrating that AKT-dependent TRF1 phosphorylation regulates telomere maintenance in vivo. TRF1 mutant cells show an impaired response to proliferative extracellular signals as well as a decreased tumorigenesis potential. These findings indicate that telomere protection and telomere length can be regulated by extracellular signals upstream of PI3K/AKT activation, such as growth factors, nutrients, or immune regulators, and this has an impact on tumorigenesis potential.

Telomere lengthening is much safer method of avoiding SASP caused by replicative senescence then any senolytic ever would be. Dual inhibitor/activator substances like sylibinin and baicalin (but more potent) would be the best - inhibiting telomerase in cancer cells and activating telomerase in healthy cells at the same time.

the body has powerful method to turn off telomerase - its TGFbeta (part of the SASP) but switching telomerase off doesn't kill cancer - it kills the host.

Oncosens is the part of SENS with the slowest progress. It needs a very good gene editing technology and a very good stem cell replacement technology. Despite the big money put in both, it's not clear at all to me whether they will arrive in time for me or any other middle-aged person.

By hook or by crook we need to defeat cancer comprehensively, or everything else will at best buy us time until the inevitable strikes us all.

Aubrey said, I think, last year, that anti cancer immune therapy is progressing so well that we might not need resort to whole body telomerase..

I know what he said, but immune therapy will never be a comprehensive solution, and anyway I don't see it progressing so fast in the clinic.

WILT is a non-starter to begin with

It's clearly the one part of SENS Aubrey knew he was creating an option which had no merit / potential for human use, about but had to create the 7th bucket

It's the equivalent of using chemotherapy for weight loss

Turning off or getting rid of all telomerase, and then radically improving senolytics, seems like a good technique for enhanced longevity.


Work in the laboratory of the author is supported by National Institutes of Health Grant AI-29524, Canadian Institutes of Health Research Grant MOP38075, and the National Cancer Institute of Canada (with support from the Terry Fox Run).

We thank Irma Vulto for assistance in the preparation of Figure 6.

Address for reprint requests and other correspondence: P. M. Lansdorp, Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada (e-mail: [email protected] ).