Telomere Length and Telomerase Activity in Cancer

Telomere Length and Telomerase Activity in Cancer Conducted on the mean telomere length and the activity of telomerase in bone marrow samples to measure the survival and prognosis of B cell chronic lymphocytic leukemia and found that the patients with a mean telomere length under 6Kb correlated with high telomerase activity similarly the telomere length over 6Kb had a significant correlation with lower telomerase activity (Bechter and Eisterer et al, 1998). This study portrays that telomere length alone is not sufficient enough information to provide prognostic characteristic in cancer therapy but together with the activity of telomerase it is more useful in clinical medicine. Table 2: Mean telomere length and telomerase activity measured and detected in different types of cancer The telomere length for most cancers range from very low to higher than normal somatic cell telomere length. Telomerase activity was also detected in most cancers except in pediatric bone where certain types of bone cancers were did not show detectable telomerase activity (Remes and Norrback et al, 2000, Hiyama and Gollahon et al, 1996, Tasumoto and Hiyama et al, 2000, Sotillo-Pineiro and Sierrasesumaga et al, 2004, Sommerfeid and Meeker et al, 1996, Albanell and Engalhardt et al, 1997). However there are some problems with this technique that makes this topic debatable like for example each type of cancer is unique which make is harder to pick a biological marker that is universally reliable hence the telomerase activity for each type of cancer must be studied individually. Another issue is that this method used highly sensitive assays to obtain telomere length and telomerase activity which in return means that it is more susceptible to false positive results or false negative results. A false positive result can arise due to contaminated lymphocytes as a result of inflammation in the surrounding issues that have no malignant cancer spread may also show telomerase activity. To overcome this issue Kinoshita, et al suggested, in a study evaluating telomerase activity in bladder cancer, that telomerase assays should be performed after antibiotic treatment. False negative results on the other hand may arise from PCR taq polymerase inhibitors in the sample such as bile s alt, heparin and hemoglobin. A highly sensitive telomerase activity assay has been proposed by Yaku and Murashima, et al (2013) that includes the use of magnetic beads which enables the washing process of PCR products to remove any inhibiting contaminants from the samples and hereby limiting the detection of false negative results. What are the current methods of telomere detection? Table 3: Different technique used to measure telomere length and their advantages and disadvantages. There are many different assays that have been developed to measure the activity of telomerase though the most common method used is the telomeric repeat amplification protocol also known as the TRAP/PCR assay. Although this technique is more sensitive and rapid compared to the conventional assays TRAP assay is not suitable for diagnostic purposes in large clinical samples due to its high technicality, obtains time consuming stages and cannot provide reliable quantitative data however many modification of the TRAP assay has been constructed to achieve a better suitable and more efficient way to detect telomerase activity some of which include bioluminescent TRAP-ELIPA (Zu and He et al, 2002), real time quantitative TRAP or (RTQ-TRAP) (Hou and Xu et al, 2001) and the fluorescent TRAP assay (Table 3). On the other hand, several studies have demonstrated enzymatic amplification rather than PCR based amplification to determine telomerase activity such as the DNAzyme assay (Wang and Donov on et al, 2013) however although this assay showed higher sensitivity than the TRAP assay, the enzymes used in the assay are also prone to inhibitions by other biological molecules in the sample. A more indirect approach in detecting telomerase activity has also been carried out by measuring the components of telomerase such as the hTERT mRNA using RTQ-PCR. As the hTR component of telomerase is found in all types of cells whereas hTERT mRNA is only found in cells that express telomerase therefore due to the strong correlation between hTERT mRNA levels and telomerase activity it becomes the next ideal target for detecting telomerase activity and hence cancer. This method was evaluated in peritoneal disseminated cells of gastrointestinal cancers by Botchkina and Rivadeneira et al, (2008) and the study concluded that this method had 100% sensitivity and 100% negative predictive value hence have an important diagnostic value. Furthermore additional longitudinal studies on larger clinical samples are required to fully benefit from its clinical cancer diagnostic values nonetheless telomerase activity can be combined with present cytological diagnosis methods to make a more accurate diagnosis. Telomerase activity can also be used to detect poor prognosis and help identify those that have a more advantage of benefiting from adjuvant treatments and due to its biological marker properties it can also be useful in the development of anti-cancer therapies. Can telomere length be used to predict life expectancy? A study conducted on domestic dog breeds show that peripheral blood mononuclear cell telomere length is a strong predictor of average life span and the breeds with shorter telomere lengths were found to have an increase probability of deaths caused by cardiovascular diseases. The correlation of telomere length in domestic dogs shows similar telomere biology to humans including telomere length, attrition and absence of somatic cell telomerase activity in comparison to studies conducted in mice models (Fick and Fick et al, 2012). Mice have rodent telomeres which have a significantly different dynamics in contrast to human telomeres and are much longer with shorter life span, making it difficult to address whether or not telomere length can be used to predict life expectancy directly. However studies that have been conducted on mice also show that the rate of increasing short telomere length predicts longevity in mammals providing more evidence for the association between telomere lengt h and life expectancy (Vera and Jesus et al, 2012). As telomere shortening is inversely correlated with age which in return is very likely to correlate with age associated issues including lifespan. One of the largest family studies on telomere length in humans also reported a correlation between telomere length, aging and life span as well as a significant indication that telomere length is also influenced by genetics specifically from the paternal inheritance (Njajau and Cawthon et al, 2007). This study suggests the possibility that the genes that are controlling telomere length may also affect lifespan (Figure 5). Another more recent study conducted by Heidinger and Blount et al, (2011) on Zebra finches, measuring telomere lengths from nesting stage to various points of its natural lifespan producing the strongest available for the relationship between telomere length and life span. This study suggests that telomere length at early stages in life is a strong indicator of lifespan however as humans retain a higher life expectancy co mpared to zebra finches it’s harder to compare these findings to humans. Therefore similar studies should be carried out to evaluate the importance of early life telomere length and lifespan in humans. Figure 5 The mean telomere length in wild type( WT) mice and transgenic telomerase reverse transcriptase (TgTERT) mice both decreased with age whereas the amount of short telomeres measured increased with age (5A). This shows that telomere length is significatly negatively correlated with life span in mice similarly to the strong negative association between age and telomere length in humans supported by results obtained by Njajou et al (5B). There is a distinct market in the industry that includes various privet companies and clinics such as Life length, RepeatD and TeloMe, which are providing the public the ability to determine an individual’s biological age through measuring the length telomeres via blood tests. Most of these companies use flow-PCR, RTQ-PCR or Q-FISH to estimate telomere length however to be able to estimate an individual’s life expectancy is a debateable topic however there are various benefits that comes with obtaining information about how short or long ones telomere is like for example the biological age is an indicator of overall health status and essentially help people proceed a healthier life style with better understanding of how life style actions are effect aging process. It is important to take in to consideration that the telomere length tests that are currently available are not able to predict life span as it is effected by a wide range of factors than telomeres alone. More over these tests can be used to see if there in an improvement in the rate of biological aging and can even be used to monitor the prognosis of current treatments and help disease prevention however the uses of these tests in a clinical environment still requires further longitudinal research and understanding in more depth of telomeres and its involvement in different diseases than what is currently known. This includes more studies of how telomere length is affected during early stages of life and how the inherited and environmental factors affect telomere length. Is it possible to alter telomere length using modern technology or in the near future? Table 4: The population doublings and telomerase activity detected in pBabe, pBabest2 and pBabest2-AS infected BJ cells. The reconstructions of telomerase activity in normal neonatal human fibroblast cell strain (BJ) which do not poses telomerase activity but however express the RNA subunit of telomerase complex (hTR) have been reported to display elongation of telomeres and extend replicative life span. The cells were infected with cDNA coding for hTERT sub-cloned in the retroviral vector pBabe, pBabest-2 and pBabest-2-As. The PBabest-2 under the control promotor of the Maloney Murine leukaemia long terminal repeat sense strand exceeded the normal estimated life span of BJ cells of 87-90 PDs and hence showing evidence for forced expression of telomerase activity results in extended life span (Vaziri and Benchimol et al, 1998). This study reveals that pBabest-2 cells can potentially replace genetically unstable cell lines through the expression of telomerase in gene therapy in order to treat age related diseases and cancer. Consequently other studies have shown that the inhibition of telomerase leads to telomere shortening and cell death and therefore can be used as anticancer drugs. Telomerase inhibiting drugs are currently under phase 2 of clinical trials and its effects on telomere shortening is reversible however the process of telomere shortening with inhibition requires a long period of time before a significant change in cell growth is seen due to the presence of the cells alternative telomere lengthening mechanisms (ATL) for maintaining telomeres. Figure 6 The HME50-5E cells underwent apoptosis against number of days after transfected with complementary 2’-O-MeRNA oligomer telomerase inhibitors. More than 50% of cells went under apoptosis after 100 days after the initial transfection (Herbert et al, 1999). Herbert and Pitts et al, 1999, reported that peptide nucleic acid (PNA) and 2-O-MeRNA oligomers inhibit the activity of hTR telomerase in immortal human cell lines HME50-5E and DU145 hence can be used as a potential chemotherapeutic drug (figure 6). The use of targeting telomerase in direct anticancer therapies also has its drawbacks as telomerase activity is also found in some somatic human cells that may also be targeted during treatment. Additionally a small minority of cancers have been reported to exhibit no significant telomerase activity therefore the treatment may not be as effective in certain cancers compared to others due to drug resistance. Another issue with this approach is that chemically related molecules may carry the risk if being unintentionally inhibited which could result in harmful side effects. Based on the research that that has been carried out on telomeres it is well established that telomere length can be easily manipulated however like in most biological pathways when the natural process are disrupted there are always positive and negative consequence. In this case involuntarily altering telomere length requires the manipulation of telomerase activity (Lee and Hills et al, 2013). The increase in telomerase activity has been associated with multiple different cancers which could therefore potentially increase the risk of developing cancer while reducing the risk of developing CHD and other diseases that are associated with telomere shortening. Furthermore additional studies are required to develop a more advanced technique that can alter telomere length and at the same time prevent or repress cancer. Alternatively a follow up study reported that a series of lifestyle changes such as plant based diet, moderate exercise, stress management and increased social support provided to men with low risk for prostate cancer, revealed an increase in relative telomere length in the intervention group compared to the controls after 5 years. This study shows a significant correlation between telomere length and lifestyle changes, indicating that telomere length can also be altered naturally by reducing the environmental risk factors associated with telomere shortening without having to interfere with telomerase activity (Ornish and Lin et al, 2013). Conclusion The discovery of telomeres and telomerase is without a doubt a major revolutionary step in the scientific field which has led to the understanding of biological age and its association with low mortality medical conditions such as CHD and Cancer. However there are still some structural aspects of telomeres and telomerase that are not fully understood, like for example telomere binding factors TRF1 and TRF2 have only a few studies which suggest that they are part of a telomere maintaining control mechanism (Smogorzewska and Steensel et al, 2000). Therefore these negative regulators can also be possibly targeted in therapies in the future as they have been found to be associated with telomerase activity and hence telomere shortening and eventually lead to programed death of cancerous cells through apoptosis. Although most telomerase inhibition drugs have failed due to long lag phase period before effective shortening of telomeres occur, it can still be used in combination with present chemotherapeutic drugs and provide a more efficiently induced cell death (Cerone and Londono-Vallejo et al, 2006). Moreover telomere length is an independent biomarker for assessing cardiovascular diseases therefore telomere length testing can be used in clinical medicine as a risk factor for early diagnosis faster than current biomarkers such as cholesterol levels. The future will undoubtedly point to new discoveries of how telomere and age related diseases are associated and help prevent them.