{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T19:18:06Z","timestamp":1791314286810,"version":"4.3.1"},"reference-count":44,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5406","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1999,2,26]]},"abstract":"<jats:p>Although broken chromosomes can induce apoptosis, natural chromosome ends (telomeres) do not trigger this response. It is shown that this suppression of apoptosis involves the telomeric-repeat binding factor 2 (TRF2). Inhibition of TRF2 resulted in apoptosis in a subset of mammalian cell types. The response was mediated by p53 and the ATM (ataxia telangiectasia mutated) kinase, consistent with activation of a DNA damage checkpoint. Apoptosis was not due to rupture of dicentric chromosomes formed by end-to-end fusion, indicating that telomeres lacking TRF2 directly signal apoptosis, possibly because they resemble damaged DNA. Thus, in some cells, telomere shortening may signal cell death rather than senescence.<\/jats:p>","DOI":"10.1126\/science.283.5406.1321","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:40:50Z","timestamp":1027762850000},"page":"1321-1325","source":"Crossref","is-referenced-by-count":898,"title":["p53- and ATM-Dependent Apoptosis Induced by Telomeres Lacking TRF2"],"prefix":"10.1126","volume":"283","author":[{"given":"Jan","family":"Karlseder","sequence":"first","affiliation":[{"name":"Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10021, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dominique","family":"Broccoli","sequence":"additional","affiliation":[{"name":"Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10021, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yumin","family":"Dai","sequence":"additional","affiliation":[{"name":"Cell Genesys, Foster City, CA 94405, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen","family":"Hardy","sequence":"additional","affiliation":[{"name":"Cell Genesys, Foster City, CA 94405, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Titia","family":"de Lange","sequence":"additional","affiliation":[{"name":"Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10021, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.270.5242.1663"},{"key":"e_1_3_1_2_3","doi-asserted-by":"publisher","DOI":"10.1038\/ng1097-231"},{"key":"e_1_3_1_2_4","unstructured":"; T. Bilaud et al. ibid. p. 236."},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/385740a0"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80932-0"},{"key":"e_1_3_1_5_2","unstructured":"A. Smogorzewska and T. de Lange unpublished observations."},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.71.3.1842-1849.1997"},{"key":"e_1_3_1_7_2","unstructured":"Details of the cloning of the adenoviral constructs and the expression of the TRF alleles are given at the Science Web site (www.sciencemag.org\/feature\/data\/986472.shl). Plaque-purified viruses were expanded on 293 cells purified by CsCl banding and stored at \u201320\u00b0C in 50% glycerol. For infections 2 \u00d7 10 5 cells were mixed with virus in 1.4 ml of media and plated into six-well dishes. HeLa Saos-2 SW 626 and HT-1080 were infected with 12 plaque-forming units (pfu) per cell; IMR-90 WI-38 HS68 and MRC-5 with 36 pfu\/cell; MEFs with 100 pfu\/cell; and B and T cells with 360 pfu\/cell. We have found that long-term (>4 days) adenovirus infection inhibits cell growth and alters the expression of cell-cycle markers. For this reason data were collected within 96 hours after infection. The adenoviral growth inhibition prevented the analysis of a possible senescence response to TRF proteins."},{"key":"e_1_3_1_8_2","unstructured":"We used the M2 monoclonal antibody (Kodak) to detect the FLAG epitope the 9E10 monoclonal antibody to detect the myc tag antibody #371 to detect TRF1 and antibody #508 to detect TRF2 by indirect immunofluorescence (IF) (2 3). Rabbit antibodies (#371 and #508) were detected with tetramethyl rhodamine isothiocyanate (TRITC)\u2013conjugated donkey antibodies to rabbit immunoglobulin (IgG) and mouse antibodies (M2 9E10) were detected with fluorescein isothiocyanate (FITC)\u2013conjugated donkey antibodies to mouse IgG (Jackson). The secondary antibodies did not cross-react. DNA was stained with 4 6-diamino-2-phenylindole (DAPI) (0.2 \u03bcg\/ml)."},{"key":"e_1_3_1_9_2","doi-asserted-by":"publisher","DOI":"10.1093\/emboj\/16.7.1785"},{"key":"e_1_3_1_10_2","unstructured":"TRF1 gel-shift assays (8) were performed with 2 \u03bcg cellular protein extracted in buffer containing 0.1% NP-40 and 0.4 M KCl (2) at 48 hours after infection. A 90% drop in TRF1 binding activity was observed in extracts of cells expressing AdTRF1 \u0394M as compared to noninfected control cells."},{"key":"e_1_3_1_11_2","unstructured":"TUNEL labeling was performed using the Oncor ApopTaq Direct In Situ Apoptosis Detection Kit. Annexin-V labeling was performed using a Clontech ApoAlert Annexin-V-FITC Apoptosis kit. For FACS analysis 2 \u00d7 10 5 cells were fixed in 70% ethanol stained with propidium iodide and analyzed using a Becton-Dickinson FacsScan and CellQuest software."},{"key":"e_1_3_1_12_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.94.25.13618"},{"key":"e_1_3_1_12_3","unstructured":". The modest G 2 accumulation observed in HeLa cells infected with AdTRF1 and AdTRF2 appears to be a HeLa-specific phenotype because it does not occur in HT-1080 (2) WI-38 and IMR-90."},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1101\/gad.8.21.2540"},{"key":"e_1_3_1_13_3","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(95)00083-2"},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","unstructured":"X. Chen L. J. Ko L. Jayaraman C. Prives Genes Dev. 10 2438 (1996);","DOI":"10.1101\/gad.10.19.2438"},{"key":"e_1_3_1_14_3","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.93.24.14094"},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(90)90409-8"},{"key":"e_1_3_1_16_2","unstructured":"Protein (30 \u03bcg) extracted in 50 mM Hepes-KaOH (pH 7.5) 150 mM NaCl 1 mM EDTA 2.5 mM EGTA and 0.5% NP-40 was fractionated on SDS\u2013polyacrylamide gels transferred to nitrocellulose membranes blocked in phosphate-buffered saline (PBS) containing 10% milk and 0.1% Tween 20 and incubated in PBS with 0.1% milk and 0.1% Tween 20 with the following antibodies: anti-p53 (Santa Cruz DO1) anti-Cyclin D1 (Santa Cruz R124) anti-Bax (Santa Cruz B-9) anti-p21 (Santa Cruz F-5) anti-FLAG (Kodak M2) or anti-myc 9E10. Blots were washed (three times) in PBS with 0.1% milk and 0.1% Tween 20 and incubated with horseradish peroxidase\u2013conjugated donkey anti-rabbit or sheep anti-mouse (Amersham). Bound antibodies were detected using the ECL kit (Amersham)."},{"key":"e_1_3_1_17_2","unstructured":"MEFs (passage 6 or less) were grown in Dulbecco's modification of Eagle's medium and 15% fetal bovine serum. Although MEFs show diminished infectability with adenoviruses satisfactory infection levels (90%) can be achieved using a threefold higher multiplicity of infection compared to primary human fibroblasts (100 pfu\/cell). Although MEFs normally do not undergo apoptosis in response to DNA damage when infected with adenovirus they display a strong apoptotic response 72 hours after \u03b3-irradiation (20 gray). Thus the apoptotic response of MEFs to AdTRF2 \u0394B\u0394M is partly due to adenoviral infection. However adenoviral infection alone does not initiate an apoptotic response because MEFs infected with AdTRF2 or AdTRF2 \u0394B did not show more TUNEL-positive cells 96 hours after infection than did uninfected controls."},{"key":"e_1_3_1_18_2","unstructured":"To document the dominant negative activity of AdTRF2 \u0394B\u0394M in MEFs we stained cells with anti-hTRF2 #508 (3) which cross-reacts with mouse Trf2 generating a punctate nuclear pattern reflecting Trf2 on mouse telomeres. Upon infection of MEFs with AdTRF2 \u0394B\u0394M this punctate pattern was lost or reduced (3). The dominant negative activity of AdTRF2 \u0394B\u0394M was also evident from the induction of anaphase bridges and apoptosis."},{"key":"e_1_3_1_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(95)90039-X"},{"key":"e_1_3_1_20_2","first-page":"27","volume":"85","author":"Serrano M.","year":"1996","unstructured":"Serrano M., et al., ibid. 85, 27 (1996).","journal-title":"ibid."},{"key":"e_1_3_1_21_2","doi-asserted-by":"publisher","DOI":"10.1038\/359288a0"},{"key":"e_1_3_1_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(00)00002-6"},{"key":"e_1_3_1_23_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.281.5383.1674"},{"key":"e_1_3_1_23_3","unstructured":"; C. E. Canman et al. ibid. p. 1677."},{"key":"e_1_3_1_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(92)90593-2"},{"key":"e_1_3_1_24_3","doi-asserted-by":"publisher","DOI":"10.1101\/gad.10.19.2401"},{"key":"e_1_3_1_24_4","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(93)90496-D"},{"key":"e_1_3_1_25_2","unstructured":"CD4 + T cells were isolated using a MiniMACS column and \u03b1CD4 beads as described by the supplier (Milteny Biotech). Alternatively primary human T cells were purified by a modification (www.sciencemag.org\/feature\/data\/986472.shl) of a previously described method ["},{"key":"e_1_3_1_25_3","doi-asserted-by":"publisher","DOI":"10.1084\/jem.180.3.1159"},{"key":"e_1_3_1_25_4","unstructured":"]. Resting T cells (99%) were enriched using depletion with antibodies to HLA-DR [American Type Culture Collection (ATCC)] and CD69 (Pharmingen) adsorbed onto Dynabeads (Dynal). T cells were stimulated with anti-CD3 (OKT-3; ATCC) and anti-CD28 (Pharmingen) in RPMI medium containing recombinant human interleukin-2 (200 U\/ml) and 1 \u03bcM BrdU. After 12 hours cells were infected with adenoviruses in fresh medium containing 1 \u03bcM BrdU. Cells were collected onto cover slips at 24 hours after infection fixed for 1 hour at \u201320\u00b0C in 70% ethanol and 50 mM glycine (pH 2.5) and processed for TUNEL labeling. Subsequently BrdU incorporation was visualized with a Boehringer BrdU Labeling and Detection Kit #1 with a TRITC-coupled secondary antibody (Jackson). Based on the BrdU incorporation infection with AdTRF2 and AdTRF2 \u0394B\u0394M resulted in 55% and 83% inhibition of S phase respectively."},{"key":"e_1_3_1_26_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.15.1.177"},{"key":"e_1_3_1_27_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.279.5349.349"},{"key":"e_1_3_1_27_3","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(98)70109-5"},{"key":"e_1_3_1_27_4","doi-asserted-by":"publisher","DOI":"10.1101\/gad.12.12.1769"},{"key":"e_1_3_1_27_5","doi-asserted-by":"publisher","DOI":"10.1126\/science.279.5349.334"},{"key":"e_1_3_1_27_6","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(98)70105-8"},{"key":"e_1_3_1_28_2","doi-asserted-by":"publisher","DOI":"10.1038\/33345"},{"key":"e_1_3_1_29_2","first-page":"518","volume":"10","author":"de Lange T.","year":"1990","unstructured":"de Lange T., et al., Mol. Cell. Biol. 10, 518 (1990);","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_29_3","doi-asserted-by":"publisher","DOI":"10.1038\/346866a0"},{"key":"e_1_3_1_30_2","unstructured":"We thank T. Jacks and R. DePinho for MEFs and helpful discussion; N. Dyson C. Sherr C. Prives and K. Kuchler for antibodies; D. Unutmaz and M. Alberts for primary human T cells; T. Jenuwein for CMV-myc TRF1; F. Isdell for expert assistance in the FACS analysis; and C. Price and members of the de Lange lab for their insights. Supported by the Human Frontiers Science Program (J.K.) Merck (D.B.) and grants from the New York Community Trust Rita Allen Foundation and NIH (GM49046) (T.d.L.)."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.283.5406.1321","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T03:16:44Z","timestamp":1705115804000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.283.5406.1321"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1999,2,26]]},"references-count":44,"journal-issue":{"issue":"5406","published-print":{"date-parts":[[1999,2,26]]}},"alternative-id":["10.1126\/science.283.5406.1321"],"URL":"https:\/\/doi.org\/10.1126\/science.283.5406.1321","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1999,2,26]]}}}