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Telomerase is an enzyme which resolves the "end replication problem", by adding DNA sequence repeats to 3' ends of newly synthesized DNA strands. The human telomerase consists primarily of hTERT (reverse transcriptase), hTERC(RNA template) and DKC1. As a result of its role in replicative senescence, it has been widely speculated that the presence of an abnormal telomerase can be oncogenic. The aim of this essay is two-fold: to demonstrate that a compelling correlation between abnormal telomerase activity and acute myeloid leukemia (AML) exists, and to illustrate a model whereby short telomeres and elevated telomerase activity assist in oncogenesis in AML. Evidence supporting the link of telomerase activity and AML is firstly drawn from clinical studies of AML patients, where mutations in TERT are observed with statistical certainty despite contrasting evidence from various sources (3)(12)(14)(2). Direct evidence for the role of telomerase in AML is also observed from two sources, where DN-hTERT transduction slows down proliferation of transformed cells (8) and mutated TERT was shown to be essential in the transformation of normal cells into leukemic cells(11). Furthermore, the efficiency of telomerase inhibitors in leukemia treatment is expanded upon as indirect evidence of the role of telomerase in leukemia (5)(6)(7). Lastly, a possible mechanism is proposed whereby the presence of short telomeres (2)(4) result in the chromosomal instability required for transformation, which is coupled to the ability of mutant cells to bypass replicative senescence (4). A method of this verifying this model is then demonstrated, using a mouse model.
One commonality of DNA replication in Eukaryotes is that it is a process involving many key players; proteins which are necessary in order to maintain fidelity in synthesized strands so as to not incur any unwanted mutations onto the daughter cells. One of these proteins is telomerase, which solves the "end replication problem" in the replication of linear DNA, where the fact that synthesis on the lagging strand occurs discontinuously means that the last Okizaki fragment cannot be properly primed (1). Telomerase accommodates for the resultant progressive shortening of DNA strands through the elongation of specific sequences known as telomeres at the ends of DNA (GGGTTA in humans), and is therefore a necessity if DNA replication is to occur for multiple times without loss of information. The telomerase complex in humans consists of many components: hTERT (A reverse transcriptase which is responsible for elongating telomeres), hTERC (containing an RNA template for hTERT), DKC1 (which plays a stabilizing role), and many maintenance proteins such as POT1, TRF1, and TRF2 (9)(2). The involvement of telomerase in DNA replication also makes it seem likely that abnormal activity of telomerase can be partially responsible for oncogenic transformation. In fact, high levels of telomerase activity can result in "immortalization" or uncontrollable cell division, allowing the natural process of replicative senescence resulting from progressive telomere shortening to be bypassed (10). It is therefore unsurprising that substantial links between abnormalities in telomerase have been established with many forms of cancers.
The link between telomerase activity and Acute Myeloid Leukemia (AML) has appeared to be a particularly interesting area of research and discussion. AML is a disease which has caused 9000 deaths along with approximately 12,810 new cases in the United States in 2009 alone, illustrating the importance of research into the mechanisms involved in the development of AML and its subtypes, such as APL (15). At its core, AML is a disease which targets myeloid cells in the bone marrow, resulting in uncontrolled proliferation of leukemic cells and disturbs functions of normal blood cells (16). However, it is often associated with many other harmful symptoms and can quite easily be fatal. In light of the severity of the disease, this article attempts to aggregate the relevant information regarding the link between telomerase and AML, present a possible mechanism by which AML can arise from abnormal telomerase activity, and discuss the possible clinical implications of the research on the topic thus far.
The first step in exploring the link between telomerase and AML is to establish whether such a correlation even exists. Due to the varying circumstances under which clinical experiments occur and the often limiting sample size, many experimenters have reached contrasting conclusions on this topic. For example, Calado et al (3) established that in the AML patients that were tested, mutations in the TERT gene were detected with compelling frequency while mutations were not detected in the control. In a different experiment by Huh et al (11), the authors established that abnormally high levels of hTERT transcripts exist during clinical presentation of the disease. However, the work of Ozgur et al (14) demonstrates that hTERT levels in children appears to have little to no correlation with prognosis factors for AML. In order to solidify the evidence supporting the correlation, two in-vivo experiments are examined where experimenters were not only able to demonstrate that over-expression of hTERT resulted in leukemic transformation, but that the introduction of dominant negative mutations of TERT induced apoptosis of already transformed cells (8) (11). Further proof is provided through the effectiveness of telomerase inhibitors like retinoids/arsenic and BIBR1532 in the treatment of AML (5)(6)(7).Considering the experiments examined, one could conclude that there is certainly a link between hTERT levels and AML.
Secondly, many experiments seem to provide another contradiction: despite high telomerase activity in transformed cells, they appear to have telomeres that are actually shorter than that of controls (2)(4). Hills et al (4) also reached the conclusion that some normal donors had TERT mutations but normal telomere lengths, which seems to suggest that telomere length also plays a role. Experiments by Stewart et al (13) involving the ALT factor confirm the possibility that telomerase serves other functions involved in oncogenesis other than maintaining telomere length. The mechanism proposed by Hills et al (4) about the involvement of telomerase in AML is that telomerase reactivation serves the purpose of immortalizing cancerous cells while short telomeres allow for a greater frequency of genetic mutations. This model appears to fit the experimental results observed from other researchers, and warrants further study. In order to confirm the model, one could attempt transformation of normal hematopoietic cells under specified conditions and test for tumorigenesis. There is certainly a positive correlation between abnormal telomerase activity and shorter telomere lengths with AML, and further research into the subject could pave the way for more effective treatments of the disease.
A Link between telomerase mutations and AML
Perhaps one of the simplest methods to begin establishing a causal link between telomerase abnormalities and AML is through genetic screenings of patients with the disease. In the series of genetic screens done by Calado et al, the authors firstly found that 11 out of 133 of AML patients had some mutations in TERT (8 of which are the A1062T allele), while none were present in healthy controls (3). Furthermore, a subsequent screening for the A1062T allele demonstrated that it appeared with a frequency three times higher in AML patients than in controls (3). It is also worth noting that homozygous mutations were detected in both trials, despite the fact that homozygosity in the TERT gene is normally expected to occur once in 30,000 samples (3). These results demonstrate lucidly that hypomorphic (loss of function) mutations occur with much greater frequency in patients of AML.
Despite the obvious statistical significance presented, a weakness in the experimental protocol is that it only serves to demonstrate correlation and not causality. In other words, the experiment does not demonstrate that loss of function in telomerase actually causes AML. However, the authors did cite evidence that such telomerase mutations are able to cause dyskeratosis congenital (3), which arguably disposes a person for AML. Therefore, some evidence that telomerase abnormalities are linked to AML is seen. Another observation of note is the unusually high occurrence of the A1062T mutation, which occurs in the reverse transcriptase region of the TERT gene (fig. 1). Therefore, further biochemical tests may be warranted to assay the reduced reverse transcriptase activity in the A1062 variant, and any correlation with AML. Yet another implication is that because the most common mutation does not occur at RNA interaction regions (RID), TERC may not play as big a role as TERT in AML (confirmed by the fact that no TERC mutations were detected in either AML patients or controls) (3).
Fig. 1 common TERT mutations observed in AML cases (3).
An alternate method of presenting the link between TERT and AML is through the comparison of TERT expression between AML patients and controls. In the study by Huh et al (12), the authors used real time RT-PCR to detect hTERT mRNA transcripts for AML patients in various stages of the disease (diagnosis, relapse, remission). Real time RT-PCR involves the detection of DNA as it is amplified, and in vitro reverse transcription in order to measure mRNA levels. While hTERT transcripts were not detected in controls, newly diagnosed patients had 73% expression, patients in relapse had 80% expression and patients in remission had 27% expression (fig. 2). Furthermore, the 7 patients that had above average expression of hTERT mRNA did not achieve remission (12).
Fig. 2 hTERT expression levels in  AML patients upon diagnosis,  patients in relapse,  patients in complete remission and  patients in partial remission (12).
The authors in this experiment were able to successfully correlate AML with a reactivation of the TERT gene, as patients with active disease had a much greater frequency of expression than patients without. The result is also confirmed by the fact that higher hTERT expression in patients with the disease is seen to be correlated with a negative prognosis (i.e lower likelihood of remission) (12). Despite the relatively small sample size (21 patients), the experiment clearly confirms that AML is linked to an up-regulation of TERT, and further experimentation with greater sample sizes can only strengthen the conclusion. Probably the most important outcome of the experiments is that hTERT expression levels appears to be a valid diagnostic tool for AML in conjunction with other methods, as healthy controls did not have an appreciable amount of hTERT expression, and expression is correlated with the severity of the disease (i.e patients in remission had lower likelihood of expression). In spite of the clinical implications, the result combined with the study by Calado et al paints a somewhat confusion picture: It would appear that AML patients had higher hTERT expression along with a greater frequency of hypomorphic mutations in the gene. While the simplistic view is that perhaps up-regulation of the gene simply compensates for a lowered enzyme activity as a result of the mutation, the truth behind these observations is clarified in further experiments examining telomere size.
Despite results that generally confirm the link between telomerase and AML, the subject is not without controversy. In the study by Ozgur et al, a similar Real-Time RT-PCR experiment was used to determine whether there was a correlation between hTERT levels and AML prognosis in children (14). The authors were not able to extract a correlation between hTERT levels and remission/relapse frequencies like Huh et al, which suggests that there may be no correlation. In fact, the authors actually found that ALL patients with higher hTERT appeared to have longer survival with the disease (14). While the research does bring to light the controversy on the subject, it primarily examines ALL and not AML as only 9 patients with AML were tested giving the evidence little weight (the fact that only children were tested also makes the evidence weak). Therefore, the study does little to disprove the existence of a correlation between telomerase and AML. In order to provide concrete proof for the existence of the correlation between telomerase and AML, one needs to examine whether direct evidence exists confirming the correlation.
In a study done by Roth et al, the authors used a retrovirus vector to introduce both a functional hTERT gene and a dominant negative mutation into K562 leukemic cells, in the hopes of detecting the effects of telomerase on growth of transformed cells (8). As a result, the DN-hTERT transduced cells had a markedly lower average number of telomeric repeats, lower enzymatic activity (from TRAP assay) along with eitherlower cellular proliferation, or cell death (for two of the clones) (fig. 3). While the decrease in telomeric repeats only serves to confirm that the transduction has taken place, the lower proliferation (20.9Â±4.2% for DN-hTERT compared to 28.9Â±2.6% for the control, and two cell lines transduced with DN-hTERT died off) suggests that hTERT activity is essential in AML development (8). From the results, it is clear that the lowered cellular proliferation in DN-hTERT cells mean that the hTERT activity is integral to the progress of the cancer. In yet another experiment by Warner et al, experimenters were even able to transform normal human blood cells into leukemic cells in a procedure where up-regulation of hTERT is a key step (11). The authors were able to establish that newly transformed cells had elevated hTERT levels upon over-expression of the TLS-ERG oncogene (11). Both of these experiments point to the same conclusion: that elevated telomerase activity is a key step of progressive of the disease, and disruption of hTERT reduces its development. Therefore, these experiments strengthen the evidence tying abnormal telomerase activity to AML.
Fig. 3 Cellular proliferation of leukemic cells upon transduction with hTERT, DN-hTERT, and GFP (control) (8)
A common theme amongst the experiments involving research of telomerase and AML is clinical implications, and the article by Roth et al is no exception. Upon introduction of DN-hTERT into K562 cells, the authors detected that some leukemic cells showed characteristics generally associated with apoptosis (8). While the mechanism for this is unclear at the time of writing, what is clear is that assuming no dangers involved in using the retroviral vector, the introduction of DN-hTERT may become viable gene therapy for AML in the foreseeable future. Furthermore, when the authors performed colony forming assays for DN-hTERT cells, the results were 7.9Ã-102Â±1.8Ã-103CFU/105 cells when compared to 1.1 Ã- 104 Â± 2.7 Ã- 104 CFU/105 cells after 4 weeks for the control (8). This is consistent with the lowered proliferation of DN-hTERT cells, and suggests that telomerase plays a key role in allowing leukemic cells to develop at such great rates. Naturally, further research into exactly how this occurs will allow for a greater understanding of the development of AML and other cancers.
Use of telomerase-specific drugs to target leukemic cells confirm the importance of telomerase in AML
As a result of the causal link between TERT and AML, attempts have been underway to establish telomerase inhibitors as AML treatment. Not only are these trials promising for future treatments, their success also confirms the existence of the already established correlation. In a study by Pendino et al, experimenters attempted to use all-trans retinoic acid (ATRA) along with retinoic acid receptor RARÎ± and RXR agonists (activators) to down-regulate hTERT levels in white blood cell samples from patients with AML and APL (acute promyelocytic leukemia) (5). This is because it has been previously shown that upon ligand binding to RARÎ± and RXR, hTERT expression is repressed (17). After treatment, hTERT expression lowered from 72% to 35% in the test group. Furthermore, despite the difficulty in establishing the ability of the agonists in causing cell death (because most cells die out naturally within 8 days), RARÎ±/RXR were shown to induce cell death in one of the samples (5). In a similar experiment by Tarkanyi et al, the authors attempted to use ATRA in combination with As2O3 to treat APL leukemic cells (6). As a result, the ATRA/As2O3 combination therapy was shown to be far more effective in repressing cellular proliferation (and in some cases, causing cell death) than either treatments alone (fig. 4). An inherent issue with this experiment is that As2O3 alone is able to cause oxidative stress to DNA (and telomeres), meaning that its effect may be unrelated to hTERT inhibition. However, the much greater effect of ATRA/As2O3 combination therapy in reducing hTERT levels over time, and the fact that the two together greatly reduces average telomere size strongly suggests that the 2 act in unison to down-regulate telomerase action (6). Despite their faults, both experiments demonstrate with great certainty that down-regulators of TERT in vivo not only act to suppress TERT, but also reduce cellular proliferation in APL. Therefore, one could conclude that TERT expression is positively linked to the ability of cancerous cells to grow; further evidence that telomerase is a key player in oncogenesis.
Fig. 4 (a) population doublings of leukemic cells upon treatment with ATRA, As2O3, and ATRA/As2O3(circle) and (b) hTERT expression levels (6)
In another experiment by El-Daly et al, the authors tested the use of another telomerase inhibitor (BIBR1532). Not only was BIBR1532 shown to selectively target leukemic cells and not normal hematopoietic cells (this is because telomerase is normally inactivated in normal cells) greater amounts of the inhibitor was shown to have even greater abilities to cause cell death (7). Therefore, the experiment provides proof that telomerase activity differs in leukemic cells, and demonstrate that its inhibition will negatively regulate cellular growth. Both ATRA/As2O3 and BIBR1532 have demonstrated great promise for future APL/AML treatments, and further research into their effects using larger sample sizes can further verify their potency. Research into telomerase activity in AML has proven to be a very important endeavour, as not only does it provide a novel way of early detection (such as through hTERT levels) but also valid forms of treatment. To summarize however, the correlation between telomerase levels and AML has been conclusive proven to be true through screening of hTERT levels in AML patients, through in vivo experiments, and through testing with telomerase inhibitors.
Abnormally short telomeres detected in AML cell samples
As it has been established that AML and other malignancies is often characterized by elevated telomerase activity, the fact that short telomeres are also present is quite surprising. In the study by Swiggers et al, the authors used Q-FISH analysis to quantify telomere lengths in AML cell samples stopped at metaphase (2). Q-FISH is an assay which accurately detects telomere lengths in samples with a resolution of 200 bp, using fluorescence measurements (19). As a result, it was determined that chromosomes containing a loss or gain of chromosomal parts contained the most telomere shortening (average telomere length of 16 a.f.u), while other AML samples had varying degrees of shortening when compared to samples from normal donors (fig 5). In light of the fact that AML with losses or gains of chromosome are associated with telomerase dysfunction while other forms of AML are not (2), it is clear that a statistical correlation is present between short telomeres and telomerase dysfunction in AML.
Fig. 5 Average telomere lengths from Q-Fish analysis for AML samples containing a loss or gain of parts, reciprocal translocations, inversions, no aberrations, and control (2).
What is expected in normal hematopoietic cells is that greater telomerase activity should naturally be associated with longer telomeres. However, in the context of AML, the shorter telomeres make sense not only because AML frequently presents with TERT mutations as previously presented, but also because short telomeres provide the requisite sensitivity to mutations which facilitate oncogenesis. While this may initially be hard to believe, a study carried out by Hills et al (4) confirms the presence of shorter telomeres associated with the previously identified A1062T variant from Calado et al (3). Moreover, Hills et al determined that of the 400 control samples, 7 also had the A1062T mutation but did not have abnormal telomere lengths (3). This seems to suggest that telomere length has a bigger role to play in oncogenesis than TERT mutations, however the evidence is lacking one again due to an insufficient sample size. What is known is that AML is seen to have a very strong correlation with shorter telomere sizes.
In an attempt to explain the presence of such short telomeres in transformed cells, Swiggers et al characterized mRNA levels of POT1, TRF1, and TRF2; 3 proteins which are known to modify telomere length. TRF1 is an inhibitor of telomerase and therefore, its presence would logically lower telomere lengths (2). POT1 and TRF2 are involved in capping of telomeres, and when associated with single stranded DNA, act to regulate telomere length in normal cells(18). The result of the experiments is that while mRNA expression of POT1 and TRF2 are not significantly different in AML caused by telomerase dysfunction, the authors found that TRF1 expression levels had a much greater expression in AML associated with telomerase dysfunction than other forms of AML(approximately 475% against 200%, seen in fig.6). This data allows one to reasonably conclude that the up-regulation of TRF1 in AML associated with telomerase dysfunction resulted in the observed short telomeres. In other to further characterize the degree to which over-expressed TRF1 regulates telomere length in cancerous cells, a Q-FISH analysis experiment can be carried out using similar conditions as the experiment by Swiggers et al, except experimenters can artificially introduce TRF1 transcripts into the system in varying amounts in order to see if the expected linear correlation between TRF1 and shorter telomere size is present. The results from such an experiment will be helpful in elucidating the mechanism behind shorter telomere sizes in AML, and in proving that both TRF1 and hTERT play a role in AML oncogenesis. To summarize, AML is seen to almost universally present with shorter telomeres as a result of the action of the TRF1 negative regulator, further solidifying the link between telomerase and the disease.
Fig. 6 TRF1 mRNA expression levels in AML with gain or losses of chromosomes and without (2)
Possible explanation of the presence of small telomeres in AML
While significant headway has been made regarding the link between telomerase and AML, it is difficult to definitely extract the exact mechanism behind the correlation. What is clear however is that the process of turmorigenesis is a complex one, and that the involvement of telomerase may not be limited to the maintenance of telomere length. This conclusion is drawn from the work of Stewart et al, where the authors attempted to transform GM847, a cell line capable of maintaining telomere length (through an unknown alternative mechanism, or ALT) in the absence of a functional telomerase, by introducing an onco-protein (H-RAS) (13). When compared to a cell line with a functional TERT, GM847 is essential unable to cause tumorigenesis when compared to a cell line with a functional telomerase (table 1). The results therefore suggest that hTERT plays alternative roles in tumorigenesis which are not associated with telomere length. However, an issue with this experiment is that the mechanism for maintaining telomere length by ALT is unclear, and it is possible that ALT is simply not as efficient when compared to hTERT.
Table 1. Ability of GM847 and a telomerase-capable line to carry out tumorigenesis (13)
Thankfully, an explanation already exists which explains the presence of both short telomeres and hTERT mutations. According to Hills et al, short telomere sequences may be responsible for chromosomal instability that is required for transformation, and abnormal telomerase reactivation accounts for immortalization of leukemic cells through bypassing replicative senescence (4). A good method of verifying this hypothesis is through the genetic transduction of normal hematopoietic cells with an oncogene such as TLS-ERG, used by Warner et al (11) under differing conditions. For example in order to test the idea the short telomeres assist in chromosomal instability, one could use Q-Fish to determine telomere lengths, and attempt over-expression of DNA repair genes in vivo upon transformation in order to determine if the link between telomere length and genetic instability is sound. Another example is if upon transformation, an engineered zinc finger nuclease is used to selectively modify hTERT in the cell to determine the extent to which hTERT is responsible. Once all the desired experiments are carried out, the cells can be transplanted into a live mouse model to test the degree to which tumorigenesis occurs. If the initial over-expression of DNA repair genes and use of the selective zinc finger nuclease lower the frequency of tumorigenesis, the hypothesis may be valid.
To summarize, a positive correlation between AML and reactivated telomerase has been established using screening of AML patients, detection of hTERT mRNA, and in vivo attempts to transform normal hematopoietic cells. It has also been established that telomere lengths are significantly shorter in AML cell samples. A hypothesis which fits these two pieces of information is that reactivated telomerase and shorter telomeres are responsible for cellular immortalization and genetic instability respectively, allowing for oncogenesis. This hypothesis can be tested through an in vivo experiment whereby normal blood cells are transformed under certain conditions and tested for tumorigenesis. Research into the various mechanisms by which telomerase and telomeres influence the progress and proliferation of AML cells is a very important area of research, and can provide new methods of early diagnosis and treatment.