We know that a cell have a nucleus and in that nucleus are chromosomes. Chromosomes have a protective cap at the end of its arms called a telomere, which have a repeating DNA sequence. In vertebrates the sequence is 5'-TTAGGG-3', but the sequence differs in other organisms. As healthy cells divide, we know that overtime the telomeres shorten until senescence. This is due to the repression of telomerase.
Additionally, telomerase is a ribonucleoprotein polymerase that is RNA dependent. It is an enzyme best known for its role in telomere maintenance. It is found to be expressed at high levels in most cancer, germ, and embryonic & adult stem cells, contributing to pluripotency and immortality. Expression also plays a role in cellular senescence, as it is normally repressed in somatic cells. In order to maintain telomeres, telomerase (the enzyme) needs its catalytic subunit (abbreviated TERT/hTERT in humans) as well as the RNA component (abbreviated TERC or TR) which contains the template for telomere synthesis, and its ribonucleoprotein.
Here we can see the complex and how TERC is used by TERT to add the six nucleotide sequence. The addition of repetitive DNA sequences prevents degradation of the chromosomal ends following multiple rounds of replication. Specifically: TERC, TERT, and the ribonucleoprotein (in human Telomerase it is Dyskerin) compose the Telomerase Complex.
Out of the telomerase complex, TERT has become a point of interest. At the time of publication, in recent years, evidence accumulated that telomerase, particularly TERT, was involved in various non-telomere-related functions such as regulation of gene expression, growth factors, and cell proliferation.
Additionally, it's been shown from multiple sources that TERT shuttles from the nucleus and translocates to mitochondria upon exogenous (or outside) stress. This study shows that there is a protective role of telomerase within mitochondria and that the Inability of telomerase shuttling leads to: cellular stress, prevention of immortalization and increase in sensitivity against genotoxic stress.
Cancer cells express high levels of telomerase, an important prerequisite for indefinite proliferation and immortality. Additionally, telomerase contributes to tumorigenesis via non-telomere dependent mechanisms which have not been yet throughly understood. Knowing these things, telomerase has been suggested to be an important anti-cancer target and the first clinical trials of telomerase inhibitors (imetelstat) have successfully been initiated.
Cancer cell survival after therapeutic treatments can be heterogeneous; meaning some cells respond to the treatment while others seem to be resistant which contributes to tumor cell survival. Since telomerase is regulated at multiple levels, including subcellular localization, having better insight into the biological consequences of different subcellular localizations of TERT specifically, might lead to the development of more effective anti-cancer treatments.
Modeling the different subcellular localizations of telomerase was possible through the utilization of organelle-targeted "shooter" vectors. Doing so allows for it to be demonstrated that mitochondrial telomerase prevents nuclear DNA damage, as well as the induction of apoptosis after treatment with H2O2 and irradiation.
Essentially, reduced generation of mitochondrial TERT prevents nuclear DNA damage. Thus, exclusion of telomerase from the nucleus after stress, such as anti-cancer therapeutic treatment, could be a protective mechanism that decreases nuclear DNA damage and apoptosis by reducing oxidative stress within mitochondria. This might contribute to increased resistance of those cancer cells against various anti-cancer treatments.
Lipofectamine is a common transfection reagent, produced by Invitrogen. It increases the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection.
Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer.
MitoSOX™ Red reagent is a novel fluorogenic dye specifically targeted to mitochondria in live cells. Oxidation of MitoSOX™ Red reagent by superoxide produces red fluorescence.
The production of superoxide by mitochondria can be visualized in fluorescence microscopy using the MitoSOX™ Red reagent.
Reactive Oxygen Species (ROS) are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis. During times of environmental stress (e.g., UV or heat exposure), ROS levels can increase dramatically. This may result in significant damage to cell structures.
Cumulatively, this is known as oxidative stress. ROS are also generated by exogenous sources such as ionizing radiation.
γH2AX, is the phosphorylated form of H2AX, and is involved in the steps leading to chromatin decondensation after DNA double-strand breaks.
γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of ionizing irradiation, RNF8 protein can be detected in association with γH2AX.
An assay for γH2AX generally reflects the presence of double-strand breaks in DNA, though the assay may indicate other minor phenomena as well.
What exactly is irradiation?
Irradiation is the process by which an object is exposed to radiation. The exposure can originate from various sources, including natural sources. It excludes the exposure to non-ionizing radiation, such as infrared, visible light, microwaves from cellular phones or electromagnetic waves emitted by radio and TV receivers and power supplies.
Gamma rays, X-rays, and the higher ultraviolet part of the electromagnetic spectrum are all forms of ionizing radiation.
One gray is the absorption of one joule of energy, in the form of ionizing radiation, per kilogram of matter.
Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis
Published online 2013 Jan 9