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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01097

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Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells

David J. Kurz^1,3,*, Stephanie Decary^1,*, Ying Hong^1,2, Elisabeth Trivier¹, Alexander Akhmedov³ and Jorge D. Erusalimsky^1,2,

¹ Department of Medicine, University College London, 5 University Street, London, WC1E 6JF, UK
² The Wolfson Institute for Biomedical Research, University College London, The Cruciform Building, Gower Street, London, WC1E 6BT, UK
³ Cardiovascular Research, Institute of Physiology, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland

View larger version (23K): [in a new window]   Fig. 1. Generation of oxidative stress by experimental interference with the metabolism of glutathione in human endothelial cells. Scheme depicting the central position of the glutathione redox-cycle in the intracellular detoxification of peroxides and the sites of pharmacological intervention used in this study (details in text). The proposed mechanism by which these alterations of redox homeostasis affect telomere integrity are shown. GSH, reduced glutathione; GSSG, oxidised glutathione; SOD, superoxide dismutase.

View larger version (27K): [in a new window]   Fig. 2. Increase of the intracellular oxidant status by low concentrations of t-BHP or BSO without impairment of cell-cycle progression. (A) Intracellular pro-oxidant status was determined by fluorimetric detection of DCF. Values represent the increase in fluorescence recorded 30 minutes after addition of 0.1 µM t-BHP or 10 µM BSO, relative to the values recorded in control cells. Data are the mean (± s.d.) of triplicate wells from two experiments. (B) Cells were treated with 0.1 µM t-BHP (empty circles), 10 µM BSO (filled squares) or vehicle (filled circles) 48 hours after passage and simultaneously incubated with 10 µg/ml BrdU. Labelled cells were identified by immunocytochemistry at the indicated time points after treatment. Results represent the mean (± s.d.) of three replicate dishes from the third passage. (C) Whole cell lysates were prepared from cultures grown under control or pro-oxidant conditions during the fourth and fifth passage. Oxidised proteins were quantified as described in Materials and Methods. Results are the mean (± s.d.) of three replicate cultures. *P<0.05. (D) Intracellular pro-oxidant status was determined by flow cytometry in late passage cultures (>25 CPD) before (baseline) and 30 minutes after addition of 200 µM H2O2. Values are the mean of three replicate dishes.

View larger version (33K): [in a new window]   Fig. 3. Premature onset of senescence under chronic oxidative stress. (A) Long term growth curves of control and stressed cultures. (B) Cell density at confluence of control and stressed cultures as a function of CPD at each passage. (C) Brightfield photomicrographs showing senescence-associated ß-galactosidase staining of control and stressed HUVECs at similar CPD levels.

View larger version (32K): [in a new window]   Fig. 4. Accelerated telomere attrition and appearance of long TRFs under chronic oxidative stress. (A,C) Southern blot hybridisation of a telomeric probe to genomic DNA from HUVEC of different replicative ages. A shows blots from cells were grown under normal conditions or chronically treated with 0.1 µM t-BHP during successive passages; C shows blots from cells treated with 10 µM BSO. Note in both cases the appearance of long TRF species (*) in the stressed cells at later passages. (B) The mean TRF lengths corresponding to the blots shown in A are plotted as a function of the replicative age.

View larger version (30K): [in a new window]   Fig. 5. Increased heterogeneity of TRF length under chronic oxidative stress. (A) FISH of a (CCCTAA)3 telomeric probe to metaphase chromosomes from HUVEC of the indicated replicative ages after long term culture under pro-oxidant (t-BHP) or normal conditions. Photographs were taken using a 100x objective and identical exposure times. Note the bright telomeres (arrows) and the greater heterogeneity of signals in the t-BHP treated sample. (B) Box and whiskers plots for the relative telomere fluorescence intensities of ten representative metaphase spreads each of control and t-BHP-treated HUVEC cultures at 20-25 CPD. Boxes represent the spread of telomere signals between the 25th and 75th percentile, while bars show the range between the weakest and strongest telomere spot fluorescence. The horizontal line within the box represents the median. (C) Cumulative frequency distributions of all telomere signals from the metaphases shown in B.

View larger version (46K): [in a new window]   Fig. 6. Lack of evidence for chromosomal end-to-end fusions as the source of the long TRFs. (A) Time course of Bal31 exonuclease digestion of genomic DNA from HUVEC of the indicated replicative ages and culture conditions. Southern blot analysis for mean TRF length determination was performed as described in Fig. 4. The three lanes for each treatment are from samples digested with Bal31 for 30, 60 and 120 minutes, respectively. (B) The mean TRF length is plotted against the time of Bal31 digestion.

View larger version (34K): [in a new window]   Fig. 7. Rapid decrease in telomerase activity following exposure to oxidative stress. (A,B) Comparison between telomerase activity levels in HUVEC of the indicated replicative ages cultured under pro-oxidant (BSO) or normal conditions. (A) TRAP products (50 base pairs); IC, internal polymerase chain reaction control in the TRAP assay. (B) Relative telomerase activity levels corresponding to the blots shown in A. (C) Time course of telomerase activity in young HUVEC (6 CPD) following a single exposure to 10 µM BSO. Values represent the mean (± s.d.) of duplicate cultures from representative experiments. (D) Immunoblots showing levels of hTERT in nuclear extracts at the indicated time points after the initial oxidant treatment at passage 2 and 3.

View larger version (58K): [in a new window]   Fig. 8. In search of evidence for the existence of ALT in HUVEC subjected to chronic oxidative stress. Indirect immunofluorescence staining of TRF1 (green) and PML protein (red) in late passage HUVEC grown under pro-oxidant conditions (BSO) and in U2OS cells. Co-localisation of TRF1 and PML appears yellow (arrows). DNA was counterstained with DAPI (blue).

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