Role Of Telomere Length Regulation In Cancer Biology Essay

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The telomeres comprise the terminal structure of linear chromosomes. In human cells, they consist of non-coding DNA, specifically of tandem TTAGGG repeats. These G-rich tracts extend many kilobases in length and they include a single-stranded 3' overhang of a few hundred nucleotides, which plays an essential role in their function (1). The telomere area is generally conserved in most organisms (2).

Protection of chromosome ends is accomplished by interaction of telomeric sequences with a certain nucleoprotein complex, termed shelterin in vertebrates. This complex encases the edges of chromosomes and protects them from DNA repair mechanisms, which respond to double strand breaks (DSB) in normal cells. An alternative way of protection is the formation of a T-loop, which is promoted by one of the shelterin complex components, TRF2 (see below): the overhang "invades" in the double-strand sequence and the resulting structure isolates the chromosome edge (2).

Telomeres are very important structures in cell physiology, since they contribute in several procedures, including chromosome stability, transcriptional activity of adjacent genes, chromosomal nuclear localization, segregation during the anaphase, homologous recombination in meiotic cells and repair of DNA double strand breaks (3). In particular, chromosome stability is accomplished by:

preventing the chromosome edges to be recognized as double strand breaks and induce a unnecessary DNA damage response

protecting chromosome edges from enzymatic degradation and

preventing end-to-end fusions between chromosome ends (4).

After every cycle of somatic cell duplication, telomeres lose about 50-200 base pairs, since DNA polymerase fails to complete the synthesis of the terminal ends of the lagging DNA strand. This inevitable process leads to crucial shortening of telomere length, which in turn blocks further cell proliferation. Replicative senescence (as this process is generally known) occurs after about 50 cell divisions and causes a cell cycle arrest in G1 phase, through activation of p53 signaling pathway (3). Specifically, the loss of telomere sequences or the destabilization of the shelterin complex, even in a single telomere, can trigger a DNA damage response and activate the cell's repair mechanisms (2).

2. Telomere binding proteins

Telomeric repeat binding factor 1 (TRF1) and telomeric repeat binding factor 2 (TRF2) are the components of shelterin complex that bind directly to the double-stranded DNA sequences of telomeres. Pot1 is the only other member of the complex that binds directly to DNA; however, it seems to associate with high sequence specificity to the single-stranded 3' overhang. TIN2 (TFR1-Interacting Nuclear Factor 2), TPP1 (TINT1, PIP1, PTOP1) and RAP1 (Repressor Activator Protein 1) complete the shelterin complex and bind indirectly to telomeres, as they interact with TRF1, TRF2 and Pot1 (Fig.1). The shelterin complex regulates length and function of telomeres and protects them from degradation and irregular telomerase activity. It also prevents a double strand break response of repair mechanisms of the cell (3).

Deletion of components of the shelterin complex causes telomere dysfunction, since telomeric sequences are no longer protected. That normally initiates DNA damage responses; these include sensor proteins such as MRE11/Nbs1/Rad50, which detect DNA stimuli and protein kinases such as ATM/ATR/Chk1/Chk2, which mediate the signal transduction pathways. The final steps in such conditions are senescence and/or apoptosis, thus protecting from proliferation genetically unstable cells (4).

Figure 1. Schematic illustration of the three-dimensional structure of telomeres, including formation oh T-loop and protein interactions (3).

3. Maintenance of telomere length

Despite the fact that telomere length can only be shortened in normal somatic cells, in embryonic and stem cells it can be maintained by activation of the telomerase enzyme. Telomerase is a catalytic complex that elongates telomeres at each cell division, but it becomes inactivated during embryonic development (3). The telomerase complex includes TERT protein and the telomerase RNA component (TERC). The addition of telomere repeats takes place through a reverse transcriptase reaction, where TERC serves as a template. TERC includes four conformational domains that define its secondary structure: the core (pseudoknot), CR4-CR5, Box H/ACA and CR7 (Fig. 2).

In most somatic cells, TERC is constitutively expressed. On the contrary, TERT is significantly up-regulated in cancer cells. Regulation of the TERT gene expression is mediated by transcriptional or RNA post-transcriptional modifications. It has been reported that initial down-regulation of the hTERT gene is accomplished through histone deacetylation and the final gene silencing occurs after DNA methylation (6).

Figure 2. Elongation of a telomere by telomerase activity. hTERCT provides the template, while hTERT catalyzes the reverse transcription (3).

4. Mechanism of replicative senescence

The observation that telomere length shortening results in cell cycle arrest or even apoptosis implies that telomeres serve as an intracellular "timer" or "mitotic clock". In other words, telomeres provide a checkpoint which controls cell population and prevents unlimited proliferation. Besides aging (which corresponds in numerous cell divisions), other factors can lead to telomere shortening (UV irradiation, reactive oxygen species) with possibly similar effects in cell physiology (2).

However, in case of inactivation of p53 or pRb pathways, this checkpoint can be bypassed, followed by further cell divisions and extended telomere attrition. The next step of this process is the formation of chromosomal fusions and "breakage-fusion-bridges" cycles, since the chromosomes terminals are no longer protected. This can cause chromosome imbalance, gene amplification, non-reciprocal translocation and differentiated genetic expression. The ultimate consequence of these events is a general cellular response to DNA damage known as crisis, which is pursued by apoptosis in most cases (2, 3).

An open issue in this mechanism is the specific determination of the telomere length or the number of shortened telomeres that are present within a cell, in order to initiate the replicative senescence. Southern blotting techniques demonstrated that a mean length of telomere sequences around 4 kb triggers mitotic senescence. On the other hand, PCR-based techniques revealed that senescent cells may bear very short telomeres. Furthermore, telomere lengths are very heterogeneous within cells but with similar shortening rates. Thus, certain telomeres will become crucially shortened before others and subsequently will be the ones to activate DNA damage repair mechanisms (2).

5. Characteristic points in carcinogenesis

Carcinogenesis is a sequential series of events that follow exposure to carcinogens. In particular, it can be described as the accumulation of genetic and molecular abnormalities that result in formation of immortalized clonal cells. The course from preinvasive histological changes to invasive diseases requires several steps, for instance 10-20 in lung carcinogenesis. The early events of this course (mutations or genetic abnormalities) slowly result in alterations in cell morphology or tissue structure (3).

Cell immortality is one of the main hallmarks of cancer cells and involves the maintenance of telomere length, which is accomplished through telomerase activation, in about 80% cases of human tumors. However, it has not been fully determined which are the processes and alterations that involve telomere length and homeostasis and contribute to the multistep carcinogenic process (3). Another hallmark of cancer cells is their genomic instability and it is generally accepted that a carcinogenetic process can be initiated by genome instability. However, there are many aspects yet to be clarified about the causes and the role of this instability. In cultured human cells, transformation of normal into immortalized cells is carried out through the following course: a. expression of proto-oncogenes and activation of cell proliferation b. inactivation of tumor suppressor genes and c. re-activation or up-regulation of telomerase (5).


1. Shortened telomeres cause genomic instability in vitro

The replicative senescence procedure depends directly from the p53 and Rb signaling pathways. Reports from in vitro studies, where those pathways were disabled, demonstrated that the crucial length of telomeres did not result in cellular growth arrest. On the contrary, cells continued to divide leading to "uncapping" of chromosome ends (as telomeres continued to lose base pairs). In particular, during the primary cell divisions after the crucial telomere shortening (a staged termed pre-crisis), the unprotected chromosomes were targeted by DNA repair mechanisms. This process subsequently led to chromosome fusions, most likely mediated by non-homologous end-joining mechanisms (2).

The consequences of chromosomes fusions become clear at the following division of the cell: during anaphase, the newly-produced dicentric chromosomes will be damaged, since the bridges between them have to be torn apart (segregation procedure). As a result, each daughter cell will inherit a chromosome baring a DSB at one end, which in turn reinitiates the breakage-fusion-bridge (BFB) cycle (Fig. 3). In following cell divisions, the shortened telomeres and the repeated BFB cycles contribute to a growing number of fusions and chromosome damage, ultimately leading to karyotype evolutions and genetic divergence (2).

During the pre-crisis period, cells undergo massive chromosome rearrangements, which are mediated by non-reciprocal translocations. The final results of these conditions are chromosome arm gains and losses as well as limited deletions and amplifications. As the genomic instability increases, cells fail to divide successfully and enter mitotic catastrophe, followed by cell death (crisis) (2).

2. Stabilization of telomeres and immortalization of cells

As mentioned above, a main characteristic of cancer cells is the reactivation of a telomere maintenance mechanism, which provides them an indefinite replication potential. It has also been shown that certain types of phenotypically normal human cells can be immortalized by the forced expression of telomerase (2).

Telomere maintenance is a way of cell survival, when cells have already entered pre-crisis or crisis stages that were previously described. The activation of telomerase stabilizes telomere length and chromosome ends, thus preventing further genomic instability and allowing unconditional cell proliferation. It is has not been clarified yet how the telomere mechanisms are reactivated, however in vitro and in vivo studies showed amplification of the hTERT locus. Other findings include:

Duplication/translocation of the locus have been linked to the reactivation of the enzyme

The promoter of the hTERT gene is a target for many oncogenes, tumor suppressors or other transcriptional factors, which in turn may be have altered expression or activity due to genomic instability (2).

Telomere length can also be maintained by alternative mechanisms (Alternative Lengthening of Telomeres - ALT) that involve homologous recombination between telomeric sequences (5).

It should be noted that even though activation of telomerase inhibits further genetic abnormalities, complete stabilization will probably be observed after several cell divisions and a minimum level of expression of the enzyme. This may result in karyotype divergence in next generations of cells. Another interesting point is that when telomerase is up-regulated, genomic instability is already occurring; a possible result of this situation is that certain chromosome ends emerge from double strand breaks and may not be recognized by telomerase as its substrates. Hence, the ends that will not be recognized may contribute to more BFB cycles, even after telomerase up-regulation (2).

One more question to be answered here is in which point, during the transformation process, is the telomere maintenance mechanism re-activated. Whether this reactivation occurs early or late during genome instability can seriously affect the tumorigenic potential of cells. In the first case, cells will be immortal but there will not have obtained the genetic changes needed for tumor formation. In the second case, the immortalization may be too late for the cells; the massive chromosome damages and rearrangements will be deleterious (2).

3. Genomic instability caused by shortened telomeres may contribute to carcinogenesis

Despite the importance of genome instability in carcinogenesis, the specific roles of genetic abnormalities or the mechanisms responsible for them have not been fully resolved. However, a recent study indicates that telomere shortening (and following telomere dysfunction) may be a basic mechanism, causing chromosome instability, at least in epithelial tissues. Other studies showed that in many types of tumor cells, telomeres appear to be remarkably short and that chromosome instability is consistent with the presence of shortened telomeres (2).

It is reasonable to assume that telomere dysfunction can cause a pre-crisis or crisis situation in early stages of tumor development. If so, telomere shortening initiates BFB cycles, through chromosome fusions. As cells continue to proliferate, genomic instability increases due to further telomere shortening and double strand breaks in more regions. Unfortunately, this process has not been definitely established by in vivo studies on human cells (2).

Summarizing, in vitro results concerning immortalization of cells after a crisis period are quite indicative: their tumorigenic potential is a strong indication that genomic instability caused by telomere dysfunction is compatible to tumor phenotypes and a possible cause of them. Yet, there is still a need for solid in vivo observations. Cells that comprise tissues and organisms reside in a much higher selective environment, compared to cell lines; the karyotypic alterations that arise from genomic instability as described above, may be tolerable in cell cultures but could prove a disadvantage in vivo and preclude tumor formation (2). Another relevant finding is that in vitro, the reactivation of telomerase maintenance mechanism is the necessary genetic alteration to accomplish continuing proliferation. On the contrary, tumor cells in vivo exhibit certain characteristic that involve many more genetic changes.

Studies in mice, in which telomeres were shortened and p53 inactivated, reported chromosome instability in pre-tumor cells, followed by genomic abnormalities that resemble the ones observed in human tumors. The contribution of such rearrangements to tumor development was also suggested by findings in fully developed tumors, where they appeared to affect oncogenic genes. A main difference between this mouse model and original human cancers is the absence of telomerase expression in the first case; as mentioned before, telomerase expression is a basic characteristic of human cancer cells.

4. Telomere shortening correlates to mutator phenotypes

The term "mutator phenotype" refers to the increased mutation rate of cancer cells. Specifically, cancer cells bear mutations in many different genetic loci, apart from those directly associated in carcinogenesis. Through a natural selection mechanism, certain mutations can prove beneficial and become established in vivo. Telomere shortening, through increasing chromosome instability causes widespread mutations and genomic rearrangements. At the same time, cell populations undergo a bottleneck period, since chromosome instability can lead to cell death. Clones with less harmful mutations and a re-activated telomerase length maintenance mechanism will be favored and possibly enter a tumorigenic course (2).

Another interesting finding is that DNA repair mechanisms that respond to double strand breaks or telomere dysfunction are mediated by the same signaling pathways and/or protein factors involved in other types of repair mechanisms. This could possibly suggest that vast DNA repair responses to certain types of damage (such those activated in pre-crisis and crisis stages) have an effect on responses to other types. Since the specific pathways and factors would be "occupied" in double strand breaks repair, other kinds of genetic abnormalities could be facilitated, ultimately leading to mutator phenotypes (2).

5. Telomere length in human cancers

Studies in preinvasive stages of human epithelial carcinogenesis suggest that telomere length abnormalities are present at almost any case. Telomere length shortening was found in most cases of early stage bladder, cervix, colon, oesophagal and oral cavity cancer, as well as in cases of prostate cancer. Studies of metaplastic regions in gallbladder reported telomere modifications in early stages of carcinogenesis. These and other findings strongly suggest that telomere shortening is a basic characteristic of tumor cells, even at the early preinvasive stages of tumor formation (3).

6. Telomere dysfunction due to telomere binding protein defects

TRF2 contributes to protection of chromosome ends by binding to telomere sequences, as part of the shelterin complex. Deletion of TRF2 causes uncapping of chromosomes edges and consequent chromosome fusions, which (as mentioned above) leads to genomic instability and crisis (12). Studies in different types of cancer cells suggested that expression of TRF1 and TRF2 is up-regulated during the carcinogenesis process; maybe as a response to shortened telomeres (3). This enhanced expression of TRF1 and TRF2 could be a compensative mechanism due to telomerase activation, controlling telomere elongation. Other reports include increased levels of TRF2 in preinvasive lesions. This could indicate a basic role of TRF2 in early carcinogenesis by causing telomere dysfunction and/or altering checkpoint controls (3).

Another member of shelterin complex, Pot1, serves as negative regulator of telomerase activity, by preventing the enzyme-substrate interaction. Studies on mouse models showed that deletion of Pot1 gene combined with inactivated p53 pathway lead to chromosome instability and subsequent cellular transformation and tumor formation (2). In yeast pot1+ deletion causes rapid degradation of telomeres (3). A study in gastric carcinoma cells reported that RNA interference which reduced Pot1 expression resulted in loss of 3' overhangs of telomeres, chromosomal instability, apoptosis and senescence. On the contrary, in different cell lines reduction of Pot1 levels led to significant lengthening of telomeres. These data indicate a dual role of Pot1 in telomere length regulation (3).

It has also been reported that defects in TIN2 and TPP1 in p53-inactivated background have similar results (5). Loss of TPP1 function induces a cycle cell arrest mediated by p53 and a DNA damage response mediated by ataxia telangiectasia mutated kinase (ATM kinase). In studies where ATM function was inhibited as well, telomere dysfunction led to cellular transformation and tumor formation in vivo (4, 5).

To sum up, early stages of carcinogenesis are accompanied by shortened telomeres, regardless telomerase activation. In this period, telomere binding proteins could be up- or down-regulated, depending on tissue type and previous genetic alterations of the cells implicated. These proteins contribute to telomere homeostasis by counteracting telomerase activity, through preventing telomerase access to telomere area, maintaining telomere structure and/or inhibition of telomerase enzyme. In cases of down-regulation, cancer cells are subjected to further genomic instability (3).

7. Consequences of telomerase mutations

Studies in Kluyveromyces lactis, showed that certain mutations of the pseudoknot region of TERC effect negatively in telomerase activity while others result in addition of mutant telomeric sequences. The latter leads to cell growth arrest and apoptosis. Similar findings were obtained in Tetrahymena and yeast. Studies in human cancer cells (prostate and breast cancer) demonstrated that even a minor expression of mutant telomerase RNA template can decrease cell viability and enhance apoptosis. Furthermore in human studies, ectopic expression of mutant telomerase RNA templates in immortalized cells has been shown to decrease plating efficiency and proliferation rate and increase the number of senescent cells. Expression of telomerase RNA templates also increased sensitivity of cancer cells to antitumor agents, regardless the initial telomere length of the cells (5).

The above and other findings demonstrate another way of telomere dysfunction that can be considered as a possible initiating step in carcinogenesis. The resulting mutated telomeric sequences added by mutant TERC subunits may affect interaction of telomeres and telomere binding proteins. Specifically, the added "mutant" sequences reduce the affinity of sequence-specific binding proteins and result in ineffective capping of telomere ends. As mentioned above, this initiates a process of chromosome ends fusions and induces genomic instability (5).

8. Potential implications in cancer therapy (5)

The above mentioned findings and conclusions about the contribution of telomere homeostasis in cancer development lead to perspectives of possible treatments for cancer, based on telomere regulation. Indeed, many relevant approaches have been investigated, including gene therapy, immunotherapy, small-molecule and signaling pathway inhibitors.

Gene therapy approaches that target telomere regulation in cancer cells include antisense methods against the hTERT mRNA or the telomerase RNA, suicide gene therapy and oncolytic virus therapy. Antisense methods include antisense oligonucleotides, peptide nucleic acids (PNAs) and chemically modified PNAs. Such methods have been reported to induce inhibition of telomerase activity and cancer cell growth and reduction of telomere length. Suicide gene therapy consists in transfection of telomerase-targeted vectors that bear an enzyme encoding gene, into cancer cells. The next step is the addition of a pro-drug, which results in release of toxins that target only telomerase-positive cells. Finally, in oncolytic virus therapy hTERT promoter controls the replication of an oncolytic virus; the virus replicates specifically in cancer cells, which leads to their selective lysis. An important disadvantage of these methods is that normal stem cells will be targeted as well, since they also express telomerase activity.

Telomerase immunotherapy against cancer cells is based on the fact that hTERT is nearly a universal tumor antigen for CD8+ cells. Cytotoxic T lymphocyte responses that target hTERT induce cell death by apoptosis in a variety of tumor cell types; interestingly, results are observed much sooner, compared to other telomerase inhibitors. An improvement to this approach is the simultaneous activation of CD8+ and CD4+ lymphocytes against hTERT, while its disadvantage is that cells with ALT could resist to therapy.

Small-molecule telomerase inhibitors are natural or synthesized compounds that inhibit or abolish telomerase activity, inducing senescence and/or apoptosis. Most of such inhibitors used in cancer treatment based on telomere regulation are associated with TERT and TERC subunits of telomerase complex. Certain examples of such compounds are BIBR1532 {2-[(E)-3-naphtalen-2-yl-but-2-enoylamino]-benzoic acid}, TNQX (2, 3, 7-trichloro-5-nitroquinoxaline) and pyrimethamine. This compounds act by reversing the mechanism that possibly led to tumor formation: they induce telomere shortening and dysfunction, and subsequently chromosome fusions and genomic instability. That initiates a process resulting in growth arrest and senescence.

Signaling pathways play an essential role in almost every cellular process. Activation of telomerase complex is significantly related to certain signaling pathways, thus the inhibition of those pathways could affect telomerase activation and function. In human cancer cells, the melanoma-associated retinopathy (MAR) signaling pathway has been generally observed and is involved in TERT gene transcription. The Ras/MEK/EPK pathway also contributes to telomerase activation by epidermal growth factor (EGF). Repression of this pathway by many proteins can effect on TERT gene transcription and telomerase function. Protein kinase inhibitors may also contribute to telomerase inhibition, since Protein Kinase C (PKC) and B (Akt) promote telomerase activity by phosphorylating hTERT. Finally, Tumor Necrosis Factor A (TNF-A) is a possible candidate for cancer therapy, since it takes part in translocation of TERT subunit to the nucleus.


Telomere length plays a key role in cell homeostasis, by serving as an intracellular timer and preventing uncontrollable cell replication. When telomere sequences are crucially shortened or ineffectively protected, they are recognized as double strand breaks by the corresponding DNA damage repair mechanisms of the cell. That is the first step for the cells to enter a senescence or apoptosis process. Since it inhibits cell proliferation, this mechanism is considered as a primary anticancer barrier (3). This view is consistent with the presence of shortened of dysfunctional telomeres in early stages of cancer development, as reported by many studies (3).

Telomere length shortening has been strongly associated with occurrence of genomic instability. Telomeres that become critically shortened involve in chromosome fusions that in turn lead to genomic rearrangements. The latter are a great means of introducing mutations and genetic abnormalities, which in cells that escape the normal course of senescence/apoptosis can be the cause or contribute to tumor formation. That can happen by inactivating tumor suppressor genes, up-regulating proto-oncogenes and providing an evolutionary advantage against adjacent cells.

When cells bypass the checkpoint that leads to growth arrest, telomere sequences continue to decrease and induce multiple breakage-fusion-breakage cycles, which promote aberrant chromosome interactions. In most cases, such chromosome abnormalities also lead to cell death, but a few cells can escape that fate by obtaining certain phenotypes that provide genomic stabilization and allow continuing proliferation. A significantly frequent phenotype of this kind is the reactivation or up-regulation of telomerase enzyme complex, which allows telomere stabilization and lengthening. Cells that manage to overcome this crisis have gained non-deleterious genomic rearrangements and an indefinite proliferation potential.

Telomere homeostasis depends on more than a certain length of telomeric nucleotide sequences; it also requires sufficient telomere protection and thus the proper function and amount of telomere binding proteins, as well as a proper regulation of telomerase complex. The destruction of telomere structure can be a result of either gradual or sudden loss of telomere repeats or mutations affecting the telomere-associated proteins. Mutations can also be the cause of reduced affinity of telomere binding proteins, when they occur on the areas encoding the telomerase RNA component; in such cases cells demonstrate genomic instability through end-to-end fusions, non-homologous end joining or homologous recombination.

The current data regarding implications of telomeres in cancer development are quite enlightening, but there is still a great need of further clarification of the processes and mechanisms involved. It has not been completely defined how cells escape senescence or apoptosis fate caused by telomere shortening and it is not fully understood how telomerase activity is regulated in early-staged and established tumors. It is clear that telomere homeostasis depends on many different factors and mechanisms, mainly telomere length, telomere binding proteins and telomerase activity. For this reason, it is necessary to combine analyses and techniques in future studies, in order to obtain a full and detailed molecular description of telomere biology.


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