Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis
Abstract​​​​​​​
Most cancer cells express high levels of telomerase and proliferate indefinitely. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction. The molecular mechanisms for this protective function remain elusive and it is unclear whether it is connected to telomere maintenance or is rather a non-telomeric function of the telomerase protein, TERT. It was shown recently that the protein subunit of telomerase can shuttle from the nucleus to the mitochondria upon oxidative stress where it protects mitochondrial function and decreases intracellular oxidative stress. 

Introduction
What exactly is "Telomerase?" Telomerase is an enzyme best known for its role in telomere maintenance. Cells with low or no telomerase expression lose telomere repeats during cell division, eventually resulting in cellular senescence. Most cancer cells, germ cells and embryonic stem cells express high levels of telomerase, thus contributing to pluripotency and immortality. In order to maintain telomeres the enzyme (telomerase) needs its catalytic subunit (TERT) as well as the RNA component (TERC or TR) which contains the template for telomere synthesis


We know that a cell has 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 replicationSpecifically: 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. ​

METHODS AND MATERIALS 
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.​
RESULTS AND DISCUSSION
Figure 1​
{A} ​
Confirmation of shuttling endogenous telomerase was made after H2O2 treatment in HeLa and MCF7 cells from the nucleus to mitochondria and the exclusion was quantified and compared to MRC-5/hTERT cells {A}​
Further evaluation of shuttling kinetics of TERT analyzed 3 additional cell lines (2 cancer, and MRC-5) and followed over 5 days.​
In the left panel HeLa and MCF7 cells are untreated and in the right panel they were treated with 400 µM H2O2 for 3 hours (right panel). ​
The Green represents mitotracker green fluorescence, red, the anti-TERT immuno-fluorescence and blue nuclear DNA (DAPI). Marked co-localization between mitotracker green and TERT is displayed by red-green mixing being displayed as yellow. ​
{B - D} ​
In all 3 cell lines nuclear TERT exclusion started around 45 min after onset of H2O2 treatment. In hTERT over-expressing fibroblasts the exclusion reached its maximum of 60% after 3 hours while it took both cancer cell lines up to a day to reach an exclusion level of 50–60%​
Intriguingly, the nuclear exclusion level of 50–60% persisted in all 3 cell lines up to 5 days, the longest time points we analyzed​
This data leads to the conclusion that nuclear TERT exclusion is a rather persistent process that can last up to several days after a single bolus dose of 400 µM H2O2. ​


In B - D: TERT localization kinetics of the 3 cell line populations after treatment with 400 µM H2O2 over 5 days. {B: HeLa C: MCF7 D: MRC-5/hTERT.}​
The black bars are nuclear TERT, red bars are cytoplasmic TERT. Bars are standard error/standard deviation of the means was compiled from at least 30 cells per time point and cell line from 3 independent experiments. Meaning at every single indicated time frame (0 min, 15 min etc) 30 cells were compared from all 3 cell lines in 3 independent experiments. 
Figure 2

Nuclear TERT localization correlates with high DNA damage levels after treatment with H2O2while mitochondrial telomerase prevents it​.

{A–C} ​
Green is representative of TERT localization​
Red is the γH2A.X staining. ​
Blue: is a DAPI nuclear counterstain ​
A: HeLa ​
B: MCF7 ​
C: MRC-5/hTERT cells. ​
Cells were treated for 3 hours with 400 µM H2O2. TERT localization was determined as described for Figure 1B and grouped into 3 categories: nuclear TERT (N) TERT (C) and intermediary TERT (I) localization. ​
Examples for the 3 different localizations are indicated with arrows. ​
D: Here we can find a correlation between subcellular TERT localization and nuclear DNA damage levels counting the (number of γH2A.X foci). Cytoplasmic TERT localization correlates with low nuclear DNA damage in all 3 cell lines while nuclear TERT localization results in high nuclear damage after 3 hours of treatment with 400 µM H2O2. Intermediary TERT localization results in intermediate DNA damage levels. ​
Black bars: HeLa, red bars: MCF7, green bars: MRC-5/hTERT. ​
Bars are mean ± SE from at least 40–100 cells per cell line in repeated experiments. * P<0.05.​
If more than 75% of total TERT was in the nucleus, or cytoplasmic/mitochondrial if more than 75% of TERT was outside the nucleus with the remaining cells being classified as an intermediate phenotype.​
 ​
A clear heterogeneity for nuclear TERT exclusion between cells within a population  
Figure 3

(Mitochondrially located TERT reduces nuclear DNA damage after)​
Here we compare H2O2 treatment to nuclear TERT localization in 4 different cell lines.​
A: Organelle specific TERT vectors transfected into HeLa cells.  representative images of cells transfected with mitochondrial and nuclear TERT shooter vectors with and without treatment with 200 µM of H2O2 for 3 hours. TERT staining (using myc-tag) fused to TERT protein (green) and γH2A.X staining (red) for DNA damage foci. ​
Arrows show transfected cells. ​
Lower panel: Quantification of cells with high levels of DNA damage foci for transfected and un-transfected cells with and without H2O2 treatment. Bars are mean ± SE from 3 independent experiments, *P<0.05. B: Organelle specific TERT vectors transfected into MCF7 cells. Panels as described for A. ​
​​​​​​​
Next, the different TERT localizations were modeled separately by over-expressing nuclear and mitochondrial organelle specific vectors expressing the catalytic telomerase subunit TERT fused to a myc-tag in 3 cancer cell lines: HeLa, MCF7, and U87 glioblastoma ​
 ​
Figure {3A an B; upper panels} ​
Localization for mitochondrial (mito TERT) and nuclear TERT shown ​
Cells were transiently transfected and treated with H2O2 or irradiation and analyzed for γH2A.X DNA damage foci and TERT localization using the fused myc-tag. 
Figure 3.2​
At the top are the Lower panels of A and B: which show Quantification of cells with high levels of DNA damage foci for transfected and un-transfected cells with and without H2O2 treatment​
While, 
C–F: show Quantification of cells with high levels of DNA damage foci for transfected and un-transfected cells with and without x-irradiation. ​
C: has MCF7 after 20 Gy X- irradiation. D: has U87 after 20 Gy X-irradiation. E: has MRC-5/SV40 after 10 Gy X-irradiation.​
More in-depth with E:
To exclude endogenous telomerase interaction with over-expressed shooter TERT protein, the experiment was repeated in an SV40 immortalized MRC-5 cell line that maintains its telomeres via an alternative lengthening mechanism. Post irradiation, the same protective effect of mitochondrial telomerase was found. 
Figure 4

Mitochondrial TERT protects from apoptosis induction after H2O2 treatment and X-irradiation compared to cells transfected with nuclear TERT.​
Activated caspase 3 (shown in red) in A: Hela, B: MRC/SV40, C: U87 cells were transfected with mito TERT and nuclear TERT (myc-tag, shown in green) after 400 µM H2O2 treatment for 3 hours or irradiation with 20 Gy. ​
D: shows the Quantification of the percentage of apoptotic cells of the 3 cell lines after H2O2 treatment.
E: shows the Quantification of the percentage of apoptotic cells of the 3 cell lines after X-irradiation. Bars present mean and standard error from around 45 transfected cells per condition and cell line. * p<0.05.​
Since high amounts of nuclear DNA damage are thought to decrease survival of cells, it was analyzed whether the used stress treatments would also compromise cell survival and induce apoptosis. ​Three (3) cell lines transfected with both TERT shooter vectors with H2O2 and X-irradiation and determined apoptosis induction using an antibody against activated caspase 3. ​
Caspase-3 is activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways, where it is responsible for chromatin condensation and DNA fragmentation.​
Strikingly, not a single cell transfected with mitochondrial TERT showed any sign of apoptosis while around 20% of un-transfected cells and between 40-60 of cells expressing the nuclear shooter were apoptotic. 
Figure. 5 
(Mitochondrially localized TERT protects against mitochondrial ROS generation after H2O2 treatment and irradiation in 4 different cell lines.)​
{A} Hela ​
{B}: MCF7 cells​
Upper panel {for both}: Representative images of ROS staining (red, mitosox) and TERT localization (myc-tag, green) after organelle specific TERT transfection and 100 µM H2O2 treatment for 3 hours in HeLa cells. ​
Upper row: mito- TERT, lower row: nuclear TERT. Arrows indicate transfected cells. ​
Lower panel: Quantification of ROS levels measured as percentage of mitosox positive area from whole cytoplasm using ImageJ in transfected and un-transfected cells. B: MCF7 cells, panels as described for A.​
Figure 5 A - E​
The result confirm that the induced DNA damage found in cells with nuclear TERT localization impacts directly on cell survival versus mitochondrial TERT efficiently protecting against apoptosis. ​
To elucidate, or make clear, the mechanism by which mitochondrial TERT might protect cancer cells from nuclear DNA damage, the amount of mitochondrial superoxide generation in addition to irradiation using mitosox staining as a measure of mitochondrial superoxide generation in addition to myc-TERT staining for nuclear and mitochondrial "shooter" vectors in the same 3 cancer cell lines. ​
Figure 5 - F​
Again, MRC-5/SV50 cells were used without endogenous telomerase to confirm the results from the 3 cancer cell lines and found the same protective effect of mitochondrially localized TERT on ROS levels. ​
It has been demonstrated previously that exogenous ROS generation by irradiation in fibroblasts damages mitochondria and accelerates nuclear DNA damage creating a positive feedback loop. It is  suggested that such a functional interaction between mitochondria and the nucleus also exists in cancer cells and other telomerase positive cells, where telomerase enters mitochondria in order to decrease ROS which are induced by chemotherapeutic drugs and irradiation. Thus, it seems that anti-cancer treatments can induce a novel, hitherto unknown mechanism of telomerase shuttling that prevents nuclear DNA damage by decreasing mitochondrial ROS generation via induction of telomerase shuttling. Due to its heterogeneous pattern it could also explain the resistance of some, but not all, cancer cells against therapeutic treatments.

Results demonstrate mitochondrial telomerase localization specifically decreases mitochondrial ROS generation and cellular oxidative stress after induction of exogenous stress generated H2O2  by or irradiation in cancer cells, preventing damage to nuclear DNA. Shuttling of telomerase from the nucleus to mitochondria seems to promote cellular survival versus cells where telomerase is not able to leave the nucleus and DNA damage accumulation is observed. 


References

Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis
Published online 2013 Jan 9

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