Telomerase

Encyclopedia of Cancer, 2015


Definition

Telomerase (TE-LУM-ER-ACE) is a ribonucleoprotein enzyme complex (a cellular reverse transcriptase) that maintains chromosome ends and has been referred to as a cellular immortalizing enzyme. Telomerase is composed of both RNA and proteins and uses its internal RNA component (complementary to the telomeric single-stranded overhang) as a template in order to synthesize telomeric DNA (TTAGGG)n directly onto the ends of chromosomes using the catalytic hTERT component. Telomerase is present in most fetal tissues, in normal adult male germ cells, in inflammatory cells, in proliferative cells of renewal tissues, and in most tumor cells. After adding six bases, the enzyme is thought to pause while it repositions (translocates) the template RNA for the synthesis of the next six-base pair repeat. This extension of the 30 DNA template end in turn permits additional replication of the 50 end of the lagging strand, thus compensating for the end replication problem.

Characteristics

Telomeres are the repetitive DNA sequences at the end of all linear chromosomes (Fig. 1). In humans there are 46 chromosomes and thus 92 telomere ends that consist of thousands of repeats of the six nucleotide sequences, TTAGGG. The telomere-telomerase hypothesis of aging and cancer is based on the findings that the cells of most human tumors have telomerase activity, while normal human somatic cells do not. Telomere length is maintained by a balance between processes that lengthen telomeres (telomerase) and processes that shorten telomeres (the end replication problem and oxidative damage). Telomerase is a cellular reverse transcriptase that stabilizes telomere length by adding hexameric (TTAGGG) repeats onto the telomeric ends of the chromosomes, thus compensating for the continued erosion of telomeres that occurs in its absence (Fig. 2). The core catalytic subunit of telomerase, hTERT, is expressed in embryonic cells and in adult male germline cells, but is undetectable in normal somatic cells except for proliferative cells of renewal tissues (e.g., hematopoietic stem cells, activated lymphocytes, basal cells of the epider- mis, proliferative endometrium, and intestinal crypt cells). The hTERT gene maps to chromosome band 5p15.33.

In normal somatic cells, progressive telomere shortening is observed, eventually leading to greatly shortened telomeres and to a limited ability to continue to divide. It has been proposed that telomere shortening may be a molecular clock mechanism that counts the number of times a cell has divided, and when telomeres are short, cellular senescence (growth arrest) occurs. It has been proposed, but not proven, that shortened telomeres in mitotic (dividing) cells may be responsible for some of the changes we associate with normal aging.

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Fig. 1. Telomeres in human chromosomes. Metaphase human chromosomes that have been both stained with DAPI (blue color which stains DNA/chromosomes) and also in situ hybridized with a PNA (peptide nucleic acid) fluorescently labeled telomere probe (red ends of each chromosome)

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Fig. 2. Telomeric sequences are synthesized by telomerase, a ribonucleoprotein enzyme (composed of both RNA and protein). Telomerase contains RNA-dependent DNA polymerase activity which uses its RNA component (complementary to the telomeric single-stranded overhang) as a template in order to synthesize TTAGGG repeats (elongate) directly onto telomeric ends. After adding six bases, the enzyme is thought to pause while it repositions (translocates) the template RNA for the synthesis of the next six-bp repeat. This extension of the 30 DNA template end in turn permits additional replication of the 50 end of the lagging strand, thus compensating for the telomere shortening that occurs in its absence

What are telomeres and what do they do?

Telomeres are repeated DNA sequences that protect the ends of chromosomes from being treated like a broken piece of DNA needing repair. Without telomeres, the ends of the chromosomes would fuse to each other leading to massive genomic instability. Telomeres are also thought to be the “clock” that regulates how many times an individual cell can divide. Telomeric sequences shorten each time the DNA replicates. When at least some of the telomeres reach a critically short length, the cell stops dividing and ages (senesces) which may cause or contribute to some age-related diseases. In cancer, a special cellular reverse transcriptase, telomerase, is reactivated and maintains the length of telomeres, allowing tumor cells to continue to proliferate.

Why do telomeres shorten?

The mechanisms of DNA replication in linear chromosomes are different for each of the two strands (called leading and lagging strands). The lagging strand is made as series of discrete fragments, each requiring a new RNA primer to initiate synthesis. The DNA between the last RNA priming event and the end of the chromosome cannot be replicated because there is no DNA beyond the end to which the next RNA primer can anneal; thus this gap cannot be filled in (this is referred to as the “end replication problem”). Since one strand cannot copy its end, telomere shortening occurs during progressive cell divisions. The shortened telomeres are inherited by daughter cells and the process repeats itself in subsequent divisions. Other factors may lead to telomere loss such as oxidative damage and other end processing events.

What is cellular senescence?

In contrast to tumor cells, which can divide forever (are “immortal”), normal human cells have a limited capacity to proliferate (are “mortal”). In general, cells cultured from a fetus divide more times in culture than those from a child, which in turn divide more times than those from an adult. The length of the telomeres decreases both as a function of donor age and with the number of times a cell has divided in culture. There appear to be two mechanisms responsible for the proliferative failure of normal cells. The first, M1 (mortality stage 1), occurs when there are still at least several thousand base pairs of telomeric sequences left at the end of most of the chromosomes. M1 is induced by a DNA damage signal produced by one or a few of the 92 telomeres that have particularly short telomeres. The M1 mechanism causes growth arrest mediated by the tumor suppressor genes p16, RB1, and p53. If the actions of p53 and p16/pRB are blocked, either by mutation or by binding to viral oncoproteins, then cells can continue to divide and telomeres continue to shorten until the M2 (mortality stage 2) mechanism is induced. M2 represents the physiological result of critically short telomeres when cells are no longer able to protect the ends of the chromosomes, so that end-degradation and end-to-end fusion occurs and causes genomic instability and cell death. In cultured cells, a focus of immortal cells occasionally arises. In most cases, these cells have reactivated the expression of telomerase, which is able to repair and maintain the telomeres (Fig. 3).

If you can stop the shortening of telomeres, will this prevent cellular aging?

While there have been many studies indicating that there is a correlation between telomere shortening and proliferative failure of human cells, the evidence that it is causal has only recently been demonstrated. Introduction of the telomerase catalytic protein component into normal human cells without detectable telomerase results in restoration of telomerase activity. Normal human cells stably expressing transfected telomerase demonstrate extension of lifespan, providing more direct evidence that telomere shortening controls cellular aging. The cells with introduced telomerase maintain a normal chromosome complement and continue to grow in a normal manner. Initial concerns that the introduction of telomerase into normal cells may substantially increase the risk of cancer have not proven true. One way to think about this is that special reproductive tissues maintain high levels of telomerase throughout life, and there is no increased incidence of cancers in these special cells compared with other types of cancer. Thus, the major role of telomerase is to maintain telomere stability and keep the cells dividing. These observations provide the

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Fig. 3. It has been argued that it may not be necessary to exhaust the replicative potential of normal cells in order to form a massive tumor (e.g., after 50 doublings a single cell could generate a tumor of a size greater than 1,000 kg). However, this theoretical argument assumes that all cells survive, which is highly unlikely to be correct. If the frequency of spontaneous mutations is ~10—6, at least a million cells are needed for a second mutation to occur with reasonable probability. Since these mutations must accumulate in the same cell, a series of clonal expansions must occur as is illustrated in this figure. Since it requires 20 cell doublings to generate approximately one million cells, 20 divisions would accompany each mutation. For example, if we assume five mutations are necessary for cancer to arise from a normal cell, more than 100 divisions (doublings) would be required to render a cell malignant. Losses of cells due to apoptosis or inhibition of cell proliferation due to senescence would limit the number of cells in such a tumor to 106–108 cells which is less than a 1 g biomass. Thus, with the possible exception of certain stem cells from the bone marrow, skin, and perhaps the intestine, most normal human cells only divide 50–70 times before their growth arrest. Thus cellular senescence could act as a very effective “brake” on the proliferation of cells that had accumulated a few mutations but not all those prerequisite for malignancy first direct evidence for the hypothesis that telomere length determines the proliferative capacity of human cells.

Can telomerase be used as a product to extend cell lifespan?

The ability to immortalize human cells and retain normal behavior holds promise in several areas of biopharmaceutical research, including drug development, screening, and toxicology testing. The devel- opment of better cellular models of human disease and production of human products are among the immediate applications of this new advance. This technology has the potential to produce unlimited quantities of normal human cells of virtually any tissue type and may have most immediate translational applications in the area of transplantation medicine. In the future it may be possible to take a person’s own cells, manipulate and rejuvenate them without using up their lifespan, and then give them back to the patient. In addition, genetic engineering of telomerase-immortalized cells could lead to the development of cell-based therapies for certain genetic disorders such as muscular dystrophy.

Cell and molecular regulation

Proteins have been identified that directly interact with telomerase, such as p23/hsp90 (molecular chaperones) and TEP1 (telomerase-associated protein 1 with unknown function). In addition, there are likely to be other proteins that help regulate telomerase function that have yet to be identified. The transcriptional regulation of the catalytic subunit of telomerase (hTERT) is clearly complex, but there is evidence that the c-myc gene may be important in some aspects of the transcriptional activation of hTERT. In addition, there is evidence that a gene on chromosome 3 may be involved in the transcriptional repression of hTERT. Since telomerase interacts with the telomeres, there have been a number of proteins identified that directly or indirectly bind to telomeres (TRF1, TRF2, tankyrase, TIN2, hRap1) that are also important in the regulation of telomerase. A single-stranded telomere binding protein is called POT1.

There is also regulation of the level of telomerase activity in specific cell types. Telomerase activity is low in the most primitive stem cells of renewal tissues (e.g., crypts of the intestine, bone marrow cells, resting lymphocytes, basal layer of the epidermis), while telomerase activity is increased in the prolifer- ative descendants of these cells. Thus, there are telomerase competent cells that have low activity when quiescent (not dividing) and increased activity when proliferating (dividing). However, these telomerase competent stem cells do not fully maintain telomere length since such cells obtained from older individuals have shorter telomeres than those derived from younger individuals. Thus, in germline (reproductive) cells and tumor cells, telomerase fully maintains telomere length in contrast to stem cells (with regulated telomerase activity) and most somatic cells (with no detectable telomerase activity) in which telomeres progressively shorten with increased age.

Cellular senescence may have evolved, in part, to protect long-lived organisms, such as humans, against the early development of cancer. Thus, it has been proposed that upregulation or reexpression of telomerase may be a critical event responsible for continuous tumor cell growth. In contrast to normal cells, tumor cells show no net loss of average telomere length with cell division, suggesting that telomere

stability may be required for cells to escape from replicative senescence and proliferate indefinitely. Most, but not necessarily all, malignant tumors may need telomerase to sustain their growth. Immortalization of cells may occur through a mutation of a gene in the telomerase repression pathway. Thus, upregulation or reactivation of telomerase activity may be a rate-limiting step required for the continuing proliferation of advanced cancers. There is experimental evidence from hundreds of independent laboratories that telomerase activity is present in ~90 % of all human tumors, but not in tissues adjacent to the tumors.

Thus, clinical telomerase research is currently focused on the development of methods for the accurate diagnosis of cancer and on novel anti-telomerase cancer therapeutics.

Clinical relevance

There is mounting evidence that cellular senescence acts as a “cancer brake” as it takes many divisions to accumulate all the changes needed to become a cancer cell. In addition to the accumulation of several mutations in oncogenes and tumor suppressor genes, almost all cancer cells are immortal and, thus, have overcome the normal cellular signals that prevent continued division. Young normal cells can divide many times, but these cells are not cancer cells since they have not accumulated all the other changes needed to make a cell malignant. In most instances a cell becomes senescent before it can become a cancer cell. Therefore, aging and cancer are two ends of the same spectrum. The key issue is to find out how to make our cancer cells mortal and our healthy cells immortal, or at least longer lasting. Inhibition of telomerase in cancer cells may be a viable target for anti-cancer therapeutics, while expression of telomerase in normal cells may have important biopharmaceutical and medical applications. In summary, telomerase is both an important target for cancer and for the treatment of age-related disease.

Could telomerase be the “achilles heel” of cancer?

We believe that progressive telomere shortening is halted in cancer cells by the presence of the enzyme telomerase which maintains and stabilizes the telomeres, allowing cells to divide indefinitely. Telomerase activity is detected in almost all human tumors. It is hoped that a therapy can be developed that inhibits telomerase activity and interferes with the growth of many types of cancer. There are several ongoing clinical trials testing these ideas in patients with solid tumors and leukemias.

Will inhibiting telomerase restore the senescence program in cancer cells and if so will this therapy cure cancer?

One research strategy is to inhibit the activity of telomerase, forcing immortal cells into a normal pattern of permanent growth arrest (senescence) or death (apoptosis). Following conventional treatments (surgery, radiotherapy, chemotherapy), anti-telomerase agents would be given to limit the proliferative capacity of the rare surviving tumor cells in the hope that this would prevent cancer recurrence. We believe this treatment would be very selective, in that only cells with an activated telomerase would be affected. As far as we know, that includes only “immortal” tumor cells and germline (reproductive cells) and at lower levels stem cells in renewal tissues.

Will telomerase activity be useful in cancer diagnostics?

Telomerase activity is detected in premalignant specimens (in situ lung and breast cancers), while colon and pancreatic cancers have detectable telomerase activity at later (carcinoma) stages. The ability to use almost any clinical specimen and to demonstrate telomerase may allow the detection of cancers at an earlier stage. For example, telomerase activity is detected in lung cells in cancer patients obtained by bronchial alveolar lavage. In addition, fine needle aspirations (breast, liver, and prostate cancer), washes (bladder and colon), and sedimented cells from urine (bladder and prostate) provide minimally invasive sources of cells to detect telomerase activity and are likely to have immediate diagnostic utility. Telomerase may also be important in the monitoring of minimal residual disease. In an effort to improve the diagnostic value of telomerase determinations, in situ hybridization methods for the demonstration of telomerase on archival paraffin-embedded clinical specimens appear to distinguish cancer from normal cells, correlate well with telomerase activity, and thus may provide added value to telomerase activity assays. In addition, the presence or absence of telomerase may have prognostic value and help risk-stratify patients into those with favorable outcomes (to avoid unnecessary treatments for patients with low or no detectable telomerase) and those with high telomerase activity and with unfavorable outcomes (to help oncologists manage patient treatments more effectively).

Have any telomerase therapeutic agents been identified and what are the potential complications of such strategies?

Since telomerase is expressed in most advanced cancers, methods for telomerase inhibition using small molecules such as modified oligonucleotides may have utility. There are potential risks in the use of such therapy that must be considered, for example, the effects of inhibitors on telomerase-expressing stem cells. However, it is likely that this approach will be less toxic than conventional chemotherapy which affects all proliferating cells, including stem cells. The rate of division of the most primitive stem cells is so much slower than that of most cancer cells that the amount of telomere shortening in the stem cells should be relatively small. Some of the side effects of standard chemotherapy, such as thrombocytopenia, leukopenia, nausea, and hair loss due to the death of the cells in rapidly proliferating tissues, may be reduced by the use of telomerase inhibitors, which are predicted to induce cellular senescence or cell death only after a period of growth. This raises what many consider the most important concern with this proposed treatment regimen, the prolonged time potentially required for a telomerase inhibitor to be effective. Since the mode of action of telomerase inhibitors may require telomeric shortening before inhibition of cell growth or induction of apoptosis, there may be a significant delay in efficacy. Thus methods may have to be devised to increase the rate of telomere shortening when telomerase inhibitors are used therapeutically. Telomerase inhibitors will likely be used together with or following conventional therapies, so that once the bulk of the tumor mass is eliminated, anti-telomerase therapy might prevent the large number of cell divisions required for the regrowth of rare resistant cancer cells. They may also be used in early stage cancer to prevent overgrowth of metastatic cells, as well as in high-risk patients with inherited susceptibility to cancer syndromes to prevent the emergence of telomerase-expressing cells (chemoprevention). Finally, there are ongoing telomerase immunotherapy clinical trials. It appears that tumor cells but not normal stem cells express telomerase epitopes on their cell surface. Scientists have identified these epitopes and have produced synthetic peptides and have injected these into patients with advanced cancer. This approach is showing early signs of efficacy and currently Phase III clinical trials for patients with advanced pancreatic cancer are ongoing.

References

Bodnar AG, Ouellette M, Frolkis M et al (1998) Extension of lifespan by introduction of telomerase in normal human cells. Science 279:349–352

Kim NW, Piatyszek MA, Prowse KR et al (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011–2015

Morales CP, Holt SE, Oullette M et al (1999) Lack of cancer-associated changes in human fibroblasts after immortalization with telomerase. Nat Genet 21:115–118

Shay JW (1998) Telomerase in cancer: diagnostic, prognostic and therapeutic implication. Cancer J Sci Am 4:26–34

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