Mitochondrial localisation of hTERT protects against nuclear DNA damage and mitochondrial ROS production after endogenous and exogenous stress

PhD ThesisUnder oxidative stress condition, telomerase catalytic subunit can shuttle from the nucleus and localises within mitochondria. hTERT can improve mitochondrial functions and contribute to a decreased oxidative stress suggesting an entirely new function of telomerase in protecting mitochondria and cells under stress. However, there are still many questions about the mechanism and what factors influence the protective function of telomerase. In this study we investigated the kinetic exclusion of hTERT, the catalytic subunit of telomerase, in various cell lines under different oxidative stress conditions. We also used organelle specific hTERT localisation vectors to model hTERT localisation and investigated a correlation between hTERT location, nuclear DNA damage and ROS production. We found that cells excluded endogenous hTERT from the nucleus in a heterogeneous fashion independently of the cell types. Importantly, nuclear DNA damage showed a significant correlation with the localisation of hTERT. Cells where hTERT remained in the nucleus displayed high DNA damage while cells which excluded hTERT from the nucleus displayed no or very low DNA damage. Our results from specific hTERT localisation vectors specified that mitochondrial localisation of hTERT protects the nucleus from DNA damage and did not showed any sign of apoptosis induction while nuclear localisation of hTERT correlated with higher amounts of DNA damage and apoptosis. Moreover, mitochondrial localisation of hTERT decreased mitochondrial ROS generation levels directly after both endogenous and exogenous stress which we interpret as the reason for the prevention of nuclear DNA damage. Additionally, we analysed whether p53 status might influence the protective function of telomerase. Our results in an isogenic cell pair of glioblastoma cells showed that p53 status does not prominently influence the protective function of mitochondrial hTERT under low stress condition. However, nuclear hTERT of cells which contained inactive p53 displayed a significantly higher nuclear DNA damage than cells which contained an active p53 and this became more pronounced when stress levels were increased. We hypothesise that telomerase localisation might possibly interact with p53 when a cancer cell is under stress condition. However, the molecular mechanism for that is unknown. Our results demonstrate a novel link between mitochondrial localisation of hTERT, decrease of mitochondrial ROS generation and the protective capacity of hTERT to nuclear DNA from damage after stress treatments.King Mongkut’s University of Technology North Bangkok and Ministry of Science and Technology scholarship, Thailand

EVALUATION OF ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF VERNONIA AMYGDALINA LEAF EXTRACTS AS AN AUXILIARY IN NATURAL HAIR SHAMPOO

Objective: This study evaluated the phytochemical compounds, antioxidant, and antimicrobial activities of Vernonia amygdalina (VA) leaf extract and used it as an auxiliary herbal constituent in natural hair shampoo. Methods: VA leaf was macerated in various solvents. All crude extracts were phytochemical compounds screened and determined the antioxidant and antimicrobial activities before formulating a natural shampoo formula. Natural shampoos were evaluated with physic-chemical properties and sensory satisfaction. Results: About 95% ethanolic extract was the most suitable substrate for product development. It reveals the inhibition zone from 7.00±0.00 to 15.00±1.00 mm and possesses a broad spectrum of antibacterial agents against both Gram-positive and Gram-negative bacteria. The MICs and minimum bactericidal concentrations value ranged from 6.250 to 100 mg/ml and 25 to >200 mg/ml, respectively. About 95% ethanolic extract also revealed high antioxidant activity. The IC50 value of DPPH of 95% ethanolic extract was 1.88±0.02 μg/ml (% inhibition of 85.73±0.01) and the ferric reducing ability power was 23.00±0.50 mg AAE/100 gDW. These high biological activities may be due to a broad range of phytochemical compounds, including saponins, tannins, flavonoids, terpenoids, steroids, cardiac glycosides, and alkaloids. Conclusions: The VA leaf was suitable to use as an ingredient in natural shampoos with a low detrimental effect on normal skin flora. The shampoo with 0.00188% of 95% ethanolic extract was the most suitable formula in terms of characteristics and stability. This formula also obtained the highest satisfaction level under our sensory evaluation

Aletris aurea

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. However, 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. Here we show that endogenous telomerase (TERT protein) shuttles from the nucleus into mitochondria upon oxidative stress in cancer cells and analyzed the nuclear exclusion patterns of endogenous telomerase after treatment with hydrogen peroxide in different cell lines. Cell populations excluded TERT from the nucleus upon oxidative stress in a heterogeneous fashion. We found a significant correlation between nuclear localization of telomerase and high DNA damage, while cells which excluded telomerase from the nucleus displayed no or very low DNA damage. We modeled nuclear and mitochondrial telomerase using organelle specific localization vectors and confirmed that mitochondrial localization of telomerase protects the nucleus from inflicted DNA damage and apoptosis while, in contrast, nuclear localization of telomerase correlated with higher amounts of DNA damage and apoptosis. It is known that nuclear DNA damage can be caused by mitochondrially generated reactive oxygen species (ROS). We demonstrate here that mitochondrial localization of telomerase specifically prevents nuclear DNA damage by decreasing levels of mitochondrial ROS. We suggest that this decrease of oxidative stress might be a possible cause for high stress resistance of cancer cells and could be especially important for cancer stem cells

Mitochondrially located TERT reduces nuclear DNA damage after

<p>H₂O₂<b>treatment in comparison to nuclear TERT localization in 4 different cell lines. A</b>: Organelle specific TERT vectors transfected into HeLa cells. Upper panel: representative images of cells transfected with mitochondrial and nuclear TERT shooter vectors with and without treatment with 200 µM 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>B</b>: Organelle specific TERT vectors transfected into MCF7 cells. Panels as described for A. <b>C–F</b>: Quantification of cells with high levels of DNA damage foci for transfected and un-transfected cells with and without x-irradiation. <b>C</b>: MCF7 after 20 Gy X- irradiation. <b>D:</b> U87 after 20 Gy X-irradiation. <b>E</b>: MRC-5/SV40 after 10 Gy X-irradiation. Bars are mean ± SE from 3 independent experiments. * P<0.05.</p

Mitochondrially localized TERT protects against mitochondrial ROS generation after H₂O₂ treatment and irradiation in 4 different cell lines. A:

<p>Upper panel: 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 h 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>B:</b> MCF7 cells, panels as described for A. <b>C:</b> Quantification of ROS in U87 cells after 3 h of 100 µM H₂O₂ treatment. <b>D–F:</b> Quantification of ROS levels after X-irradiation. <b>D:</b> MCF7 after 20 Gy X-irradiation. <b>E:</b> U87 after 20 Gy X-irradiation <b>F:</b> MRC-5/SV40 after 10 Gy X-irradiation. Bars represent mean ± SE from 3 independent experiments. * P<0.05.</p

Mitochondrial TERT protects from apoptosis induction after H₂O₂ treatment and X-irradiation compared to cells transfected with nuclear TERT.

<p>Representative images of activated caspase 3 (shown in red) in <b>A</b>: Hela, <b>B</b>: MRC/SV40, <b>C</b>: U87 cells transfected with mito TERT and nuclear TERT (myc-tag, shown in green) after 400 µM H₂O₂ treatment for 3 h or irradiation with 20 Gy. <b>D</b>: Quantification of the percentage of apoptotic cells of the 3 cell lines after H₂O₂ treatment, E: 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.</p

TERT shuttles from nucleus to mitochondria upon H₂O₂ treatment in cancer cells. A:

<p>Example rendered 3D volume projections of deconvolved confocal images from HeLa and MCF7 cells untreated (control, left panel) or treated with 400 µM H₂O₂ for 3 h (right panel). Green represents mitotracker green fluorescence, red anti-TERT immuno-fluorescence and blue nuclear DNA (DAPI). Marked colocalization between mitotracker green and TERT is displayed by red-green mixing being displayed as yellow. <b>B–D:</b> TERT localization kinetics in 3 cell line populations after treatment with 400 µM H₂O₂ over 5 days. <b>B:</b> HeLa <b>C:</b> MCF7 <b>D:</b> MRC-5/hTERT. Black bars: nuclear TERT, red bars: cytoplasmic TERT. Bars are means ± SE from at least 30 cells per time point and cell line from 3 independent experiments.</p

Nuclear TERT localization correlates with high DNA damage levels after treatment with H₂O₂

<p><b>while mitochondrial telomerase prevents it. A–C:</b> Representative images of TERT localization (green), and γH2A.X staining (red). Blue: DAPI nuclear counterstain <b>A:</b> HeLa <b>B:</b> MCF7 <b>C:</b> MRC-5/hTERT cells. Cells were treated for 3 h with 400 µM H₂O₂. TERT localization was determined as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052989#pone-0052989-g001" target="_blank">Figure 1B</a> 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. <b>D:</b> Correlation between subcellular TERT localization and nuclear DNA damage levels (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 h 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.</p

Quantification and significances of z-stacks for determination of correlation coefficient for co-localization of hTERT to mitochondria in three cell lines.

<p>Mean correlation coefficients were determined from between 8–10 cells per cell line/treatment. Pearson correlation coefficients were determined per cell for deconvolved, 3D rendered images, subtracting any background for each channel, using Huygens Colocalization analyzer plugin (Huygens, SVI, Netherlands). All datasets passed Normality tests and P values are from Student's T tests comparing untreated and treated cells per cell line.</p