Telomerase reverse transcriptase
Telomerase reverse transcriptase is a catalytic subunit of the enzyme telomerase, which, together with the telomerase RNA component, comprises the most important unit of the telomerase complex.
Telomerases are part of a distinct subgroup of RNA-dependent polymerases. Telomerase lengthens telomeres in DNA strands, thereby allowing senescent cells that would otherwise become postmitotic and undergo apoptosis to exceed the Hayflick limit and become potentially immortal, as is often the case with cancerous cells. To be specific, TERT is responsible for catalyzing the addition of nucleotides in a TTAGGG sequence to the ends of a chromosome's telomeres. This addition of repetitive DNA sequences prevents degradation of the chromosomal ends following multiple rounds of replication.
hTERT absence is associated with the disorder Cri du chat.
Function
is a ribonucleoprotein polymerase that maintains telomere ends by addition of the telomere repeat TTAGGG. The enzyme consists of a protein component with reverse transcriptase activity, encoded by this gene, and an RNA component that serves as a template for the telomere repeat. Telomerase expression plays a role in cellular senescence, as it is normally repressed in postnatal somatic cells, resulting in progressive shortening of telomeres. Studies in mice suggest that telomerase also participates in chromosomal repair, since de novo synthesis of telomere repeats may occur at double-stranded breaks. Alternatively spliced variants encoding different isoforms of telomerase reverse transcriptase have been identified; the full-length sequence of some variants has not been determined. Alternative splicing at this locus is thought to be one mechanism of regulation of telomerase activity.Regulation
The hTERT gene, located on chromosome 5, consists of 16 exons and 15 introns spanning 35 kb. The core promoter of hTERT includes 330 base pairs upstream of the translation start site, as well as 37 base pairs of exon 2 of the hTERT gene. The hTERT promoter is GC-rich and lacks TATA and CAAT boxes but contains many sites for several transcription factors giving indication of a high level of regulation by multiple factors in many cellular contexts. Transcription factors that can activate hTERT include many oncogenes such as c-Myc, Sp1, HIF-1, AP2, and many more, while many cancer suppressing genes such as p53, WT1, and Menin produce factors that suppress hTERT activity. Another form of up-regulation is through demethylation of histones proximal to the promoter region, imitating the low density of trimethylated histones seen in embryonic stem cells. This allows for the recruitment of histone acetyltransferase to unwind the sequence allowing for transcription of the gene.Telomere deficiency is often linked to aging, cancers and the conditions dyskeratosis congenita and Cri du chat. Meanwhile, over-expression of hTERT is often associated with cancers and tumor formation. The regulation of hTERT is extremely important to the maintenance of stem and cancer cells and can be used in multiple ways in the field of regenerative medicine.
Stem cells
hTERT is often up-regulated in cells that divide rapidly, including both embryonic stem cells and adult stem cells. It elongates the telomeres of stem cells, which, as a consequence, increases the lifespan of the stem cells by allowing for indefinite division without shortening of telomeres. Therefore, it is responsible for the self-renewal properties of stem cells. Telomerase are found specifically to target shorter telomere over longer telomere, due to various regulatory mechanisms inside the cells that reduce the affinity of telomerase to longer telomeres. This preferential affinity maintains a balance within the cell such that the telomeres are of sufficient length for their function and yet, at the same time, not contribute to aberrant telomere elongation.High expression of hTERT is also often used as a landmark for pluripotency and multipotency state of embryonic and adult stem cells. Over-expression of hTERT was found to immortalize certain cell types as well as impart different interesting properties to different stem cells.
Immortalization
hTERT immortalizes various normal cells in culture, thereby endowing the self-renewal properties of stem cells to non-stem cell cultures. There are multiple ways in which immortalization of non-stem cells can be achieved, one of which being via the introduction of hTERT into the cells. Differentiated cells often express hTERC and TP1, a telomerase-associated protein that helps form the telomerase assembly, but does not express hTERT. Hence, hTERT acts as the limiting factor for telomerase activity in differentiated cells. However, with hTERT over-expression, active telomerase can be formed in differentiated cells. This method has been used to immortalize prostate epithelial and stromal-derived cells, which are typically difficult to culture in vitro. hTERT introduction allows in vitro culture of these cells and available for possible future research. The introduction of hTERT has an advantage over the use of viral protein for immortalization in that it does not involve the inactivation of tumor suppressor gene, which might lead to cancer formation.Enhancement
Over-expression of hTERT in stem cells changes the properties of the cells. hTERT over-expression increases the stem cell properties of human mesenchymal stem cells. The expression profile of mesenchymal stem cells converges towards embryonic stem cells, suggesting that these cells may have embryonic stem cell-like properties. However, it has been observed that mesenchymal stem cells undergo decreased levels of spontaneous differentiation. This suggests that the differentiation capacity of adult stem cells may be dependent on telomerase activities. Therefore, over-expression of hTERT, which is akin to increasing telomerase activities, may create adult stem cells with a larger capacity for differentiation and hence, a larger capacity for treatment.Increasing the telomerase activities in stem cells gives different effects depending on the intrinsic nature of the different types of stem cells. Hence, not all stem cells will have increased stem-cell properties. For example, research has shown that telomerase can be upregulated in CD34+ Umbilical Cord Blood Cells through hTERT over-expression. The survival of these stem cells was enhanced, although there was no increase in the amount of population doubling.
Clinical significance
Deregulation of telomerase expression in somatic cells may be involved in oncogenesis.Genome-wide association studies suggest TERT is a susceptibility gene for development of many cancers, including lung cancer.
Role in cancer
activity is associated with the number of times a cell can divide playing an important role in the immortality of cell lines, such as cancer cells. The enzyme complex acts through the addition of telomeric repeats to the ends of chromosomal DNA. This generates immortal cancer cells. In fact, there is a strong correlation between telomerase activity and malignant tumors or cancerous cell lines. Not all types of human cancer have increased telomerase activity. 90% of cancers are characterized by increased telomerase activity. Lung cancer is the most well characterized type of cancer associated with telomerase. There is a lack of substantial telomerase activity in some cell types such as primary human fibroblasts, which become senescent after about 30–50 population doublings. There is also evidence that telomerase activity is increased in tissues, such as germ cell lines, that are self-renewing. Normal somatic cells, on the other hand, do not have detectable telomerase activity. Since the catalytic component of telomerase is its reverse transcriptase, hTERT, and the RNA component hTERC, hTERT is an important gene to investigate in terms of cancer and tumorigenesis.The hTERT gene has been examined for mutations and their association with the risk of contracting cancer. Over two hundred combinations of hTERT polymorphisms and cancer development have been found. There were several different types of cancer involved, and the strength of the correlation between the polymorphism and developing cancer varied from weak to strong. The regulation of hTERT has also been researched to determine possible mechanisms of telomerase activation in cancer cells. Importantly, mutations in the hTERT promoter were first identified in melanoma and have subsequently been shown to be the most common noncoding mutations in cancer. Glycogen synthase kinase 3 seems to be over-expressed in most cancer cells. GSK3 is involved in promoter activation through controlling a network of transcription factors. Leptin is also involved in increasing mRNA expression of hTERT via signal transducer and activation of transcription 3, proposing a mechanism for increased cancer incidence in obese individuals. There are several other regulatory mechanisms that are altered or aberrant in cancer cells, including the Ras signaling pathway and other transcriptional regulators. Phosphorylation is also a key process of post-transcriptional modification that regulates mRNA expression and cellular localization. Clearly, there are many regulatory mechanisms of activation and repression of hTERT and telomerase activity in the cell, providing methods of immortalization in cancer cells.
Therapeutic potential
If increased telomerase activity is associated with malignancy, then possible cancer treatments could involve inhibiting its catalytic component, hTERT, to reduce the enzyme's activity and cause cell death. Since normal somatic cells do not express TERT, telomerase inhibition in cancer cells can cause senescence and apoptosis without affecting normal human cells. It has been found that dominant-negative mutants of hTERT could reduce telomerase activity within the cell. This led to apoptosis and cell death in cells with short telomere lengths, a promising result for cancer treatment. Although cells with long telomeres did not experience apoptosis, they developed mortal characteristics and underwent telomere shortening. Telomerase activity has also been found to be inhibited by phytochemicals such as isoprenoids, genistein, curcumin, etc. These chemicals play a role in inhibiting the mTOR pathway via down-regulation of phosphorylation. The mTOR pathway is very important in regulating protein synthesis and it interacts with telomerase to increase its expression. Several other chemicals have been found to inhibit telomerase activity and are currently being tested as potential clinical treatment options such as nucleoside analogues, retinoic acid derivatives, quinolone antibiotics, and catechin derivatives. There are also other molecular genetic-based methods of inhibiting telomerase, such as antisense therapy and RNA interference.hTERT peptide fragments have been shown to induce a cytotoxic T-cell reaction against telomerase-positive tumor cells in vitro. The response is mediated by dendritic cells, which can display hTERT-associated antigens on MHC class I and II receptors following adenoviral transduction of an hTERT plasmid into dendritic cells, which mediate T-cell responses. Dendritic cells are then able to present telomerase-associated antigens even with undetectable amounts of telomerase activity, as long as the hTERT plasmid is present. Immunotherapy against telomerase-positive tumor cells is a promising field in cancer research that has been shown to be effective in in vitro and mouse model studies.