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First published online 11 March 2008
doi: 10.1242/jcs.019372

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Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress

¹ Crucible Laboratory, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, NE4 6BE, UK
² Henry Wellcome Biogerontology Laboratory and Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, NE4 6BE, UK
³ School of Clinical and Laboratory Sciences, Newcastle University, Newcastle upon Tyne, NE4 6BE, UK
⁴ Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, NE4 6BE, UK

View larger version (36K): [in this window] [in a new window]   Fig. 1. TERT overexpression does not maintain telomere length under hyperoxia. (A) Telomere restriction fragment length in MRC5-TERT cells grown for the indicated times (in days) under normoxia (left) or under 40% hyperoxia (right). Population doublings are indicated (PD). M indicates the positions of a HINDIII size marker. (B) Telomere shortening rates per PD in MRC5 (black bars) and MRC5-TERT (white bars) under normoxia (left) and hyperoxia (right). Data are mean±s.e.m. from four experiments measured in quadruplicate. The asterisk indicates a significant difference between parental and TERT-overexpressing cells with P<0.05 (ANOVA). (C) Metaphase spreads from MRC5 (left) and MRC5-TERT (right) cells grown under hyperoxia for the indicated times (in days) hybridised with a telomeric PNA probe (red). Chromosomes are stained with DAPI (blue). (D) Frequency distributions of telomere fluorescence signal intensities in MRC5-TERT (top) and MRC5 (bottom) cells grown under hyperoxia for the indicated times. Red bars indicate the median telomere length. (E) Frequencies of chromosomal end-to-end fusions per metaphase in MRC5 (black) and MRC5-TERT (white, broken lines) under hyperoxia. Data are mean±s.e.m. from at least 25 metaphases per condition.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Growth arrest of MRC5-TERT cells under hyperoxia is telomere dependent. (A,B) ImmunoFISH of MRC5 cells (A) and MRC5-TERT cells (B) under hyperoxia (49 days and 42 days, respectively) showing telomeres (red) and -H2A.X-containing foci (green). Boxed areas are shown at higher magnification on the right. Significant colocalisation of telomeres and -H2A.X foci is indicated in white (arrowheads). (C) Average foci-telomere Pearson correlation coefficients for MRC5 fibroblasts in senescence under normoxia (MRC5 SEN), MRC5 cells after 7 weeks hyperoxia (MRC5 HYP), MRC5-TERT cells after 6 weeks hyperoxia (TERT HYP) and MRC5 cells 48 hours after 20Gy -irradiation (MRC5 IR) as negative control. All experimental conditions are significantly different from the negative control, but not from each other. Data are mean±s.e.m., with the number of cells evaluated given for each condition.

View larger version (71K): [in this window] [in a new window]   Fig. 3. Telomerase is excluded from the nucleus under oxidative stress and colocalises with mitochondria. (A) Percentage of total TRAP activity in the extranuclear fraction of MRC5-TERT after H2O2. (B) Percentage of total TRAP activity in the extranuclear fraction following hyperoxic treatment for the indicated times. Data are mean±s.e.m. from three independent experiments. (C) Change in mitochondrial (white) and nuclear (black) TRAP activity after 3 hours of 500 mM H2O2 exposure or 52 days of hyperoxia. Data are mean±s.e.m. from six replicate measurements. All values are significantly different from controls (100%). (D-G) TERT immunofluorescence (red) in MRC5-TERT under normoxia (D, left), following treatment with H2O2 at the indicated concentrations (D, middle and right), under hyperoxia for the indicated times (E), in cells grown under hyperoxia for 50 days followed by shift back to normoxia for the indicated times (F), and co-stained after 70 days hyperoxia with Mitotracker green (G). Colocalisation of mitochondria and TERT-containing foci appears in yellow.

View larger version (30K): [in this window] [in a new window]   Fig. 4. TERT overexpression protects mitochondrial DNA integrity and mitochondrial function under oxidative stress. Frequency of lesions in mtDNA as measured by the relative amplification efficiency of an 11 kb mtDNA fragment in MRC5 (black circles) and MRC5-TERT (white circles) following treatment with hydrogen peroxide in the indicated concentrations (A) and under long-term hyperoxia (B). Data are mean±s.e.m. from quadruplicate measurements. (C) Mitochondrial superoxide generation as measured by MitoSOX fluorescence intensity per cell. (D) Cellular peroxide levels as measured by DHR123 fluorescence. (E) MMP as measured by JC1 fluorescence ratio. (F) UCP2 expression as measured by duplex RT-PCR with Gapdh as control. All data in C to F were measured in MRC5 (black bars) and MRC5-TERT (white bars) under normoxia (left) and after 1 week of hyperoxia (right), and are mean±s.e.m. from three experiments. Differences between parental and TERT-overexpressing cells marked by an asterisk are significant with P<0.05 (ANOVA). (G) Expression of endogeneous TERT was measured by semi-quantitative TERT RT-PCR 2 days after transfection with the indicated siRNAs. GAPDH was measured as control. (H) MitoSOX and DHR fluorescence intensity in HUVECs at 2 days after transfection with the indicated siRNAs. Data are mean±s.e.m. from quadruplicate measurements. Asterisks indicate significant differences to cells treated with control siRNA with P<0.05 (ANOVA).

View larger version (34K): [in this window] [in a new window]   Fig. 5. TERT overexpression decreases mitochondrial mass and mtDNA content, reverses expression of candidate genes for retrograde response and improves resistance against apoptosis. (A) Relative mitochondrial mass per cell (relative to that in MRC5 cells under normoxia) as measured by NAO fluorescence in flow cytometry. (B) Relative MtDNA copy number as measured by real-time PCR. All data are mean±s.e.m. from at least three experiments. Asterisks indicate significant differences between parental and TERT-overexpressing cells with P<0.05. (C-F) Relative mRNA expression levels of candidate marker genes for retrograde response (Passos et al., 2007) in senescent MRC-5 (SEN), young MRC-5 (YOU) and MRC5-TERT cells (TERT). Expression colour code is indicated at the bottom. (C) Ca2+-related signalling, (D) metabolism, (E) mitochondrial function and (F) stress response. (G) Caspase 3/7 activity (in arbitrary units) in MRC5 and MRC5-TERT cells treated for 2 hours with H2O2 at the indicated concentrations and assayed after 24 hours. Data are mean±s.e.m. from three experiments. (H) Caspase 3/7 activity (in arbitrary units) in MRC5 and MRC5-TERT cells treated for 48 hours with etoposide at the indicated concentrations and assayed after 3 days. Data are mean±s.d. from two experiments.

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