The existence of a compensatory mechanism for telomere shortening was first found by Soviet biologist
Alexey Olovnikov in 1973,[4] who also suggested the telomere hypothesis of
aging and the telomere's connections to cancer and perhaps some neurodegenerative diseases.[5]
The role of telomeres and telomerase in
cell aging and
cancer was established by scientists at
biotechnology company
Geron with the cloning of the
RNA and catalytic components of human telomerase[9] and the development of a
polymerase chain reaction (PCR) based assay for telomerase activity called the TRAP assay, which surveys telomerase activity in multiple types of cancer.[10]
The
negative stain electron microscopy (EM) structures of human and Tetrahymena telomerases were characterized in 2013.[11][12] Two years later, the first cryo-electron microscopy (
cryo-EM) structure of telomerase holoenzyme (Tetrahymena) was determined.[13] In 2018, the structure of human telomerase was determined through cryo-EM by UC Berkeley scientists.[14]
Human telomerase structure
The molecular composition of the human telomerase complex was determined by Scott Cohen and his team at the Children's Medical Research Institute (Sydney Australia) and consists of two
molecules each of human
telomerase reverse transcriptase (TERT),
Telomerase RNA Component (TR or TERC), and
dyskerin (DKC1).[15] The genes of telomerase subunits, which include TERT,[16] TERC,[17] DKC1[18] and TEP1,[19] are located on different chromosomes. The human TERT gene (hTERT) is
translated into a
protein of 1132
amino acids.[20] TERT polypeptide
folds with (and carries) TERC, a
non-coding RNA (451
nucleotides long). TERT has a 'mitten' structure that allows it to wrap around the chromosome to add single-stranded telomere repeats.
TERT is a
reverse transcriptase, which is a class of enzymes that creates single-stranded DNA using single-stranded RNA as a template.
The protein consists of four
conserved domains (RNA-Binding Domain (TRBD), fingers, palm and thumb), organized into a "right hand" ring configuration that shares common features with
retroviral reverse transcriptases, viral
RNA replicases and
bacteriophage B-family DNA polymerases.[21][22]
TERT proteins from many eukaryotes have been sequenced.[23]
Mechanism
The
shelterin protein
TPP1 is both necessary and sufficient to recruit the telomerase enzyme to telomeres, and is the only shelterin protein in direct contact with telomerase.[24]
By using TERC, TERT can add a six-nucleotide repeating sequence, 5'-
TTA
GGG (in vertebrates; the sequence differs in other organisms) to the 3' strand of chromosomes. These TTAGGG repeats (with their various protein binding partners) are called telomeres. The template region of TERC is 3'-CAAUCCCAAUC-5'.[25]
Telomerase can bind the first few nucleotides of the template to the last telomere sequence on the chromosome, add a new telomere repeat (5'-GGTTAG-3') sequence, let go, realign the new 3'-end of telomere to the template, and repeat the process. Telomerase reverses
telomere shortening.
Clinical implications
Aging
Telomerase restores short bits of DNA known as
telomeres, which are otherwise shortened after repeated division of a cell via
mitosis.
In normal circumstances, where telomerase is absent, if a cell divides recursively, at some point the progeny reach their
Hayflick limit,[26] which is believed to be between 50 and 70 cell divisions. At the limit the cells become senescent and
cell division stops.[27] Telomerase allows each offspring to replace the lost bit of DNA, allowing the cell line to divide without ever reaching the limit. This same unbounded growth is a feature of
cancerous growth.[28]
A comparative biology study of mammalian telomeres indicated that telomere length of some mammalian species correlates inversely, rather than directly, with lifespan, and concluded that the contribution of telomere length to lifespan is unresolved.[34] Telomere shortening does not occur with age in some
postmitotic tissues, such as in the rat brain.[35] In humans, skeletal muscle telomere lengths remain stable from ages 23 –74.[36] In baboon skeletal muscle, which consists of fully
differentiated postmitotic cells, less than 3% of
myonuclei contain damaged telomeres and this percentage does not increase with age.[37] Thus, telomere shortening does not appear to be a major factor in the aging of the differentiated cells of brain or skeletal muscle. In human liver,
cholangiocytes and
hepatocytes show no age-related telomere shortening.[38] Another study found little evidence that, in humans, telomere length is a significant
biomarker of normal aging with respect to important cognitive and physical abilities.[39]
Some experiments have raised questions on whether telomerase can be used as an
anti-aging therapy, namely, the fact that mice with elevated levels of telomerase have higher cancer incidence and hence do not live longer.[40] On the other hand, one study showed that activating telomerase in cancer-resistant mice by overexpressing its catalytic subunit extended lifespan.[41] A study found that long-lived subjects inherited a hyperactive version of telomerase.[42]
In vitro, when cells approach the
Hayflick limit, the time to senescence can be extended by inactivating the
tumor suppressor proteins
p53 and
Retinoblastoma protein (pRb).[44]Cells that have been so-altered eventually undergo an event termed a "crisis" when the majority of the cells in the culture die. Sometimes, a cell does not stop dividing once it reaches a crisis. In a typical situation, the telomeres are shortened[45] and chromosomal integrity declines with every subsequent cell division. Exposed chromosome ends are interpreted as double-stranded breaks (DSB) in DNA; such damage is usually
repaired by reattaching the broken ends together. When the cell does this due to telomere-shortening, the ends of different chromosomes can be attached to each other. This solves the problem of lacking telomeres, but during cell division
anaphase, the fused chromosomes are randomly ripped apart, causing many
mutations and chromosomal abnormalities. As this process continues, the cell's genome becomes unstable. Eventually, either fatal damage is done to the cell's chromosomes (killing it via
apoptosis), or an additional mutation that activates telomerase occurs.[44]
With telomerase activation some types of cells and their offspring become
immortal (bypass the
Hayflick limit), thus avoiding cell death as long as the conditions for their duplication are met. Many
cancer cells are considered 'immortal' because telomerase activity allows them to live much longer than any other somatic cell, which, combined with uncontrollable cell proliferation[46] is why they can form
tumors. A good example of immortal cancer cells is
HeLa cells, which have been used in laboratories as a model
cell line since 1951.
While this method of modelling human cancer in cell culture is effective and has been used for many years by scientists, it is also very imprecise. The exact changes that allow for the formation of the
tumorigenicclones in the above-described experiment are not clear. Scientists addressed this question by the serial introduction of multiple mutations present in a variety of human cancers. This has led to the identification of mutation combinations that form tumorigenic cells in a variety of cell types. While the combination varies by cell type, the following alterations are required in all cases: TERT activation, loss of p53 pathway function, loss of pRb pathway function, activation of the
Ras or
mycproto-oncogenes, and aberration of the PP2A protein
phosphatase.[47] That is to say, the cell has an activated telomerase, eliminating the process of death by chromosome instability or loss, absence of apoptosis-induction pathways, and continued
mitosis activation.
This model of
cancer in cell culture accurately describes the role of telomerase in actual human tumors. Telomerase activation has been observed in ~90% of all human tumors,[48] suggesting that the immortality conferred by telomerase plays a key role in cancer development. Of the tumors without TERT activation,[49] most employ a separate pathway to maintain telomere length termed
Alternative Lengthening of Telomeres (ALT).[50] The exact mechanism behind telomere maintenance in the ALT pathway is unclear, but likely involves multiple
recombination events at the telomere.
Elizabeth Blackburnet al., identified the upregulation of 70 genes known or suspected in cancer growth and spread through the body, and the activation of
glycolysis, which enables cancer cells to rapidly use sugar to facilitate their programmed growth rate (roughly the growth rate of a fetus).[51]
Approaches to controlling telomerase and telomeres for cancer therapy include
gene therapy,
immunotherapy, small-molecule and signal pathway inhibitors.[52]
Drugs
The ability to maintain functional
telomeres may be one mechanism that allows
cancer cells to grow in vitro for decades.[53] Telomerase activity is necessary to preserve many cancer types and is inactive in
somatic cells, creating the possibility that telomerase inhibition could selectively repress cancer cell growth with minimal side effects.[54] If a drug can inhibit telomerase in cancer cells, the telomeres of successive generations will progressively shorten, limiting tumor growth.[55]
Telomerase is a good
biomarker for cancer detection because most human cancer cells express high levels of it. Telomerase activity can be identified by its catalytic protein domain (
hTERT). This[clarify] is the
rate-limiting step in telomerase activity. It is associated with many cancer types. Various cancer cells and
fibroblasts transformed with hTERT
cDNA have high telomerase activity, while somatic cells do not. Cells testing positive for hTERT have positive nuclear signals. Epithelial stem cell tissue and its early daughter cells are the only noncancerous cells in which hTERT can be detected. Since hTERT expression is dependent only on the number of tumor cells within a sample, the amount of hTERT indicates the severity of cancer.[56]
The lack of telomerase does not affect cell growth until the telomeres are short enough to cause cells to "die or undergo growth arrest". However, inhibiting telomerase alone is not enough to destroy large tumors. It must be combined with surgery,
radiation,
chemotherapy or immunotherapy.[56]
Cells may reduce their telomere length by only 50-252 base pairs per cell division, which can lead to a long
lag phase.[58][59]
A telomerase activator
TA-65 is commercially available and is claimed to delay aging and to provide relief from certain disease conditions.[60][61][62][63][64] This formulation contains a molecule called
cycloastragenol derived from a legume Astragalus membranaceus. Several other compounds have been found to increase telomerase activity:
Centella asiatica extract 8.8-fold,
oleanolic acid 5.9-fold,
astragalus extract 4.3-fold, TA-65 2.2-fold, and
maslinic acid 2-fold.[65]
Immunotherapy
Immunotherapy successfully treats some kinds of cancer, such as
melanoma. This treatment involves manipulating a human's
immune system to destroy cancerous cells. Humans have two major
antigen identifying
lymphocytes:
CD8+
cytotoxic T-lymphocytes (CTL) and
CD4+
helper T-lymphocytes that can destroy cells. Antigen receptors on CTL can bind to a 9-10
amino acid chain that is presented by the
major histocompatibility complex (MHC) as in Figure 4. HTERT is a potential target antigen. Immunotargeting should result in relatively few side effects since hTERT expression is associated only with telomerase and is not essential in almost all somatic cells.[66] GV1001 uses this pathway.[52] Experimental drug and
vaccine therapies targeting active telomerase have been tested in
mouse models, and
clinical trials have begun. One drug,
imetelstat, is being clinically researched as a means of interfering with telomerase in cancer cells.[67] Most of the harmful cancer-related effects of telomerase are dependent on an intact RNA template.
Cancer stem cells that use an alternative method of telomere maintenance are still killed when telomerase's RNA template is blocked or damaged.
Telomerase Vaccines
Two telomerase vaccines have been developed:
GRNVAC1 and
GV1001. GRNVAC1 isolates
dendritic cells and the RNA that codes for the telomerase protein and puts them back into the patient to make
cytotoxic T cells that kill the telomerase-active cells. GV1001 is a peptide from the active site of hTERT and is recognized by the immune system that reacts by killing the telomerase-active cells.[52]
Targeted apoptosis
Another independent approach is to use
oligoadenylated anti-telomerase antisense
oligonucleotides and
ribozymes to target telomerase RNA, reducing dissociation and
apoptosis (Figure 5). The fast induction of apoptosis through antisense binding may be a good alternative to the slower telomere shortening.[58]
Small interfering RNA (siRNA)
siRNAs are small RNA molecules that induce the sequence-specific degradation of other RNAs. siRNA treatment can function similar to traditional
gene therapy by destroying the
mRNA products of particular genes, and therefore preventing the expression of those genes. A 2012 study found that targeting TERC with an siRNA reduced telomerase activity by more than 50% and resulted in decreased viability of immortal cancer cells.[68] Treatment with both the siRNA and radiation caused a greater reduction in tumor size in mice than treatment with radiation alone, suggesting that targeting telomerase could be a way to increase the efficacy of radiation in treating radiation-resistant tumors.
Heart disease, diabetes and quality of life
Blackburn also discovered that mothers caring for very sick children have shorter telomeres when they report that their emotional stress is at a maximum and that telomerase was active at the site of blockages in
coronary artery tissue, possibly accelerating heart attacks.
In 2009, it was shown that the amount of telomerase activity significantly increased following
psychological stress. Across the sample of patients telomerase activity in
peripheral blood mononuclear cells increased by 18% one hour after the end of the stress.[69]
A study in 2010 found that there was "significantly greater" telomerase activity in participants than controls after a three-month meditation retreat.[70]
Mutations in TERT have been implicated in predisposing patients to
aplastic anemia, a disorder in which the
bone marrow fails to produce blood cells, in 2005.[72]
Cri du chat syndrome (CdCS) is a complex disorder involving the loss of the distal portion of the short arm of
chromosome 5. TERT is located in the deleted region, and loss of one copy of TERT has been suggested as a cause or contributing factor of this disease.[73]
Patients with DC have severe bone marrow failure manifesting as abnormal
skin pigmentation,
leucoplakia (a white thickening of the oral mucosa) and
nail dystrophy, as well as a variety of other symptoms. Individuals with either TERC or DKC1 mutations have shorter telomeres and defective telomerase activity in vitro versus other individuals of the same age.[78]
In one family autosomal dominant DC was linked to a
heterozygous TERT mutation.[5] These patients also exhibited an increased rate of telomere-shortening, and
genetic anticipation (i.e., the DC phenotype worsened with each generation).
TERT Splice Variants
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Overview of all the structural information available in the
PDB for
UniProt: O14746 (Human Telomerase reverse transcriptase) at the
PDBe-KB.
Overview of all the structural information available in the
PDB for
UniProt: Q0QHL8 (Tribolium castaneum Telomerase reverse transcriptase) at the
PDBe-KB.