The effect of milk yield on some reproductive parameters of the Elsenburg Holstein and Jersey herds

(South African J of Animal Science, 2000, 30, Supplement 1: 34-35

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

Correspondence to Author:

MBC, Total phenolic and flavonoi

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

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

Brief Report: Human Pluripotent Stem Cell Models of Fanconi Anemia Deficiency Reveal an Important Role for Fanconi Anemia Proteins in Cellular Reprogramming and Survival of Hematopoietic Progenitors

Fanconi anemia (FA) is a genomic instability disorder caused by mutations in genes involved in replication-dependant-repair and removal of DNA cross-links. Mouse models with targeted deletions of FA genes have been developed; however, none of these exhibit the human bone marrow aplasia. Human embryonic stem cell (hESC) differentiation recapitulates many steps of embryonic hematopoietic development and is a useful model system to investigate the early events of hematopoietic progenitor specification. It is now possible to derive patient-specific human-induced pluripotent stem cells (hiPSC); however, this approach has been rather difficult to achieve in FA cells due to a requirement for activation of FA pathway during reprogramming process which can be bypassed either by genetic complementation or reprogramming under hypoxic conditions. In this study, we report that FA-C patient-specific hiPSC lines can be derived under normoxic conditions, albeit at much reduced efficiency. These disease-specific hiPSC lines and hESC with stable knockdown of FANCC display all the in vitro hallmarks of pluripotency. Nevertheless, the disease-specific hiPSCs show a much higher frequency of chromosomal abnormalities compared to parent fibroblasts and are unable to generate teratoma composed of all three germ layers in vivo, likely due to increased genomic instability. Both FANCC-deficient hESC and hiPSC lines are capable of undergoing hematopoietic differentiation, but the hematopoietic progenitors display an increased apoptosis in culture and reduced clonogenic potential. Together these data highlight the critical requirement for FA proteins in survival of hematopoietic progenitors, cellular reprogramming, and maintenance of genomic stability

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