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• W2078465024 abstract "Cells derived from patients with the human genetic disorder ataxia-telangiectasia (A-T) display many abnormalities, including telomere shortening, premature senescence, and defects in the activation of S phase and G2/M checkpoints in response to double-strand DNA breaks induced by ionizing radiation. We have previously demonstrated that one of the ATM substrates is Pin2/TRF1, a telomeric protein that binds the potent telomerase inhibitor PinX1, negatively regulates telomere elongation, and specifically affects mitotic progression. Following DNA damage, ATM phosphorylates Pin2/TRF1 and suppresses its ability to induce abortive mitosis and apoptosis (Kishi, S., Zhou, X. Z., Nakamura, N., Ziv, Y., Khoo, C., Hill, D. E., Shiloh, Y., and Lu, K. P. (2001)J. Biol. Chem. 276, 29282–29291). However, the functional importance of Pin2/TRF1 in mediating ATM-dependent regulation remains to be established. To address this question, we directly inhibited the function of endogenous Pin2/TRF1 in A-T cells by stable expression of two different dominant-negative Pin2/TRF1 mutants and then examined their effects on telomere length and DNA damage response. Both the Pin2/TRF1 mutants increased telomere length in A-T cells, as shown in other cells. Surprisingly, both the Pin2/TRF1 mutants reduced radiosensitivity and complemented the G2/M checkpoint defect without inhibiting Cdc2 activity in A-T cells. In contrast, neither of the Pin2/TRF1 mutants corrected the S phase checkpoint defect in the same cells. These results indicate that inhibition of Pin2/TRF1 in A-T cells is able to bypass the requirement for ATM in specifically restoring telomere shortening, the G2/M checkpoint defect, and radiosensitivity and demonstrate a critical role for Pin2/TRF1 in the ATM-dependent regulation of telomeres and DNA damage response. Cells derived from patients with the human genetic disorder ataxia-telangiectasia (A-T) display many abnormalities, including telomere shortening, premature senescence, and defects in the activation of S phase and G2/M checkpoints in response to double-strand DNA breaks induced by ionizing radiation. We have previously demonstrated that one of the ATM substrates is Pin2/TRF1, a telomeric protein that binds the potent telomerase inhibitor PinX1, negatively regulates telomere elongation, and specifically affects mitotic progression. Following DNA damage, ATM phosphorylates Pin2/TRF1 and suppresses its ability to induce abortive mitosis and apoptosis (Kishi, S., Zhou, X. Z., Nakamura, N., Ziv, Y., Khoo, C., Hill, D. E., Shiloh, Y., and Lu, K. P. (2001)J. Biol. Chem. 276, 29282–29291). However, the functional importance of Pin2/TRF1 in mediating ATM-dependent regulation remains to be established. To address this question, we directly inhibited the function of endogenous Pin2/TRF1 in A-T cells by stable expression of two different dominant-negative Pin2/TRF1 mutants and then examined their effects on telomere length and DNA damage response. Both the Pin2/TRF1 mutants increased telomere length in A-T cells, as shown in other cells. Surprisingly, both the Pin2/TRF1 mutants reduced radiosensitivity and complemented the G2/M checkpoint defect without inhibiting Cdc2 activity in A-T cells. In contrast, neither of the Pin2/TRF1 mutants corrected the S phase checkpoint defect in the same cells. These results indicate that inhibition of Pin2/TRF1 in A-T cells is able to bypass the requirement for ATM in specifically restoring telomere shortening, the G2/M checkpoint defect, and radiosensitivity and demonstrate a critical role for Pin2/TRF1 in the ATM-dependent regulation of telomeres and DNA damage response. Mutations in theATM 1ATMataxia-telangiectasia-mutatedA-Tataxia-telangiectasia cellsPBSphosphate-buffered salineBrdUrddeoxybromouridineSA-β-Galsenescence-associated β-galactosidaseFITCfluorescein isothiocyanateGFPgreen fluorescent proteinx-gal5-bromo-4-chloro-3-indolyl-b-d-galactopyranosideFISHfluorescentin situ hybridizationDAPI4′,6-diamidino-2-phenylindole 1ATMataxia-telangiectasia-mutatedA-Tataxia-telangiectasia cellsPBSphosphate-buffered salineBrdUrddeoxybromouridineSA-β-Galsenescence-associated β-galactosidaseFITCfluorescein isothiocyanateGFPgreen fluorescent proteinx-gal5-bromo-4-chloro-3-indolyl-b-d-galactopyranosideFISHfluorescentin situ hybridizationDAPI4′,6-diamidino-2-phenylindole gene are responsible for the genetic disease ataxia telangiectasia (A-T) (1Savitsky K. Bar S.A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanijali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jasper N.G.J. Taylor A.M.R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collin F.C. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2345) Google Scholar). Cells derived from A-T patients display many abnormalities, including telomere shortening, premature senescence, and hypersensitivity to ionizing radiation (2Lavin M.F. Shiloh Y. Annu. Rev. Immunol. 1997; 15: 177-202Crossref PubMed Scopus (536) Google Scholar, 3Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1648) Google Scholar). Exposure of normal mammalian cells to ionizing radiation leads to a delay in progression of the cell from G1 to S phase, inhibition of DNA synthesis, and a delay in progression from G2 phase into mitosis (4Leeper D.B. Schneiderman M.H. Dewey W.C. Radiat. Res. 1972; 50: 401-417Crossref PubMed Scopus (55) Google Scholar, 5Konig K. Baisch H. Radiat. Environ. Biophys. 1980; 18: 257-266Crossref PubMed Scopus (19) Google Scholar). Similar mechanisms are also presented in yeast cells (6Weinert T.A. Hartwell L.H. Mol. Cell. Biol. 1990; 10: 6554-6564Crossref PubMed Scopus (182) Google Scholar, 7Li R. Murray A.W. Cell. 1991; 66: 519-531Abstract Full Text PDF PubMed Scopus (924) Google Scholar). These cell cycle checkpoints allow cells to repair damaged DNA and to maintain genomic stability (3Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1648) Google Scholar, 8Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2292) Google Scholar). However, A-T cells are defective in activating checkpoints at the G1/S, during S phase, and at the G2/M in response to ionizing radiation exposure (2Lavin M.F. Shiloh Y. Annu. Rev. Immunol. 1997; 15: 177-202Crossref PubMed Scopus (536) Google Scholar). ATM is a protein kinase that is activated by ionizing DNA damage and is critical for genome stability, telomere maintenance, and induction of cell cycle checkpoints (2Lavin M.F. Shiloh Y. Annu. Rev. Immunol. 1997; 15: 177-202Crossref PubMed Scopus (536) Google Scholar, 3Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1648) Google Scholar). ATM has been shown to phosphorylate and regulate many key regulators, including p53, β-adaptin, c-Abl, Chk1–2, Brca1, and Nijmegen breakage syndrome protein (9Shafman T. Khanna K.K. Kedar P. Spring K. Kozlov S. Yen T. Hobson K. Gatei M. Zhang N. Watters D. Egerton M. Shiloh Y. Kharbanda S. Kufe D. Lavin M.F. Nature. 1997; 387: 520-523Crossref PubMed Scopus (418) Google Scholar, 10Baskaran R. Wood L.D. Whitaker L.L. Canman C.E. Morgan S.E., Xu, Y. Barlow C. Baltimore D. Wynshaw-Boris A. Kastan M.B. Wang J.Y. Nature. 1997; 387: 516-519Crossref PubMed Scopus (485) Google Scholar, 11Banin S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1688) Google Scholar, 12Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1684) Google Scholar, 13Sarkaria J.N. Tibbetts R.S. Busby E.C. Kennedy A.P. Hill D.E. Abraham R.T. Cancer Res. 1998; 58: 4375-4382PubMed Google Scholar, 14Khanna K.K. Keating K.E. Kozlov S. Scott S. Gatei M. Hobson K. Taya Y. Gabrielli B. Chan D. Lees-Miller S.P. Lavin M.F. Nat. Genet. 1998; 20: 398-400Crossref PubMed Scopus (401) Google Scholar, 15Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar, 16Cortez D. Wang Y. Qin J. Elledge S.J. Science. 1999; 286: 1162-1166Crossref PubMed Scopus (864) Google Scholar, 17Kim S.T. Lim D.S. Canman C.E. Kastan M.B. J. Biol. Chem. 1999; 274: 37538-37543Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 18Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (371) Google Scholar, 19Zhao S. Weng Y.C. Yuan S.S. Lin Y.T. Hsu H.C. Lin S.C. Gerbino E. Song M.H. Zdzienicka M.Z. Gatti R.A. Shay J.W. Ziv Y. Shiloh Y. Lee E.Y. Nature. 2000; 405: 473-477Crossref PubMed Scopus (433) Google Scholar). For example, ATM phosphorylates p53 and thereby increases transcription of the Cdk inhibitor p21 and the Cdc2 sequester 14-3-3ς. Furthermore, ATM also phosphorylates Chks, which eventually leads to inhibition of Cdc2 activation. These multiple and redundant pathways have been shown to be involved in cell cycle checkpoint regulation (2Lavin M.F. Shiloh Y. Annu. Rev. Immunol. 1997; 15: 177-202Crossref PubMed Scopus (536) Google Scholar, 3Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1648) Google Scholar). ataxia-telangiectasia-mutated ataxia-telangiectasia cells phosphate-buffered saline deoxybromouridine senescence-associated β-galactosidase fluorescein isothiocyanate green fluorescent protein 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside fluorescentin situ hybridization 4′,6-diamidino-2-phenylindole ataxia-telangiectasia-mutated ataxia-telangiectasia cells phosphate-buffered saline deoxybromouridine senescence-associated β-galactosidase fluorescein isothiocyanate green fluorescent protein 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside fluorescentin situ hybridization 4′,6-diamidino-2-phenylindole An increasing body of evidence supports an important role for ATM in regulating telomere metabolism. Cells derived from humans and mice with a defective ATM gene show prominent defects related to telomere dysfunction (1Savitsky K. Bar S.A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanijali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jasper N.G.J. Taylor A.M.R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collin F.C. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2345) Google Scholar, 20Barlow C. Hirotsune S. Paylor R. Liyanage M. Eckhaus M. Collins F. Shiloh Y. Crawley J.N. Ried T. Tagle D. Wynshaw B.A. Cell. 1996; 86: 159-171Abstract Full Text Full Text PDF PubMed Scopus (1249) Google Scholar, 21Xu Y. Ashley T. Brainerd E.E. Bronson R.T. Meyn M.S. Baltimore D. Gene Dev. 1996; 10: 2411-2422Crossref PubMed Scopus (736) Google Scholar, 22Xu Y. Baltimore D. Gene Dev. 1996; 10: 2401-2410Crossref PubMed Scopus (347) Google Scholar, 23Elson A. Wang Y. Daugherty C.J. Morton C.C. Zhou F. Campos-Torres J. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13084-13089Crossref PubMed Scopus (449) Google Scholar). Both primary and transformed A-T cells have been found to have unusually short telomeres and chromosome end-to-end associations, and primary A-T cells show premature aging/senescence phenotype (24Rudolph N.S. Latt S.A. Mutat. Res. 1989; 211: 31-41Crossref PubMed Scopus (59) Google Scholar, 25Beamish, H., Khanna, K. K., and Lavin, M. F. (1994)Radiat. Res. S130–133Google Scholar, 26Pandita T.K. Pathak S. Geard C.R. Cytogenet. Cell Genet. 1995; 71: 86-93Crossref PubMed Scopus (164) Google Scholar, 27Xia S.J. Shammas M.A. Shmookler R.J. Mutat. Res. 1996; 364: 1-11Crossref PubMed Scopus (56) Google Scholar, 28Metcalfe J.A. Parkhill J. Campbell L. Stacey M. Biggs P. Byrd P.J. Taylor A.M. Nat. Genet. 1996; 13: 350-353Crossref PubMed Scopus (295) Google Scholar, 29Smilenov L.B. Morgan S.E. Mellado W. Sawant S.G. Kastan M.B. Pandita T.K. Oncogene. 1997; 15: 2659-2665Crossref PubMed Scopus (91) Google Scholar). Furthermore, expression of a dominant-negative ATM fragment in normal cells results in a decrease in average telomere repeat length (29Smilenov L.B. Morgan S.E. Mellado W. Sawant S.G. Kastan M.B. Pandita T.K. Oncogene. 1997; 15: 2659-2665Crossref PubMed Scopus (91) Google Scholar, 30Morgan S.E. Lovly C. Pandita T.K. Shiloh Y. Kastan M.B. Mol. Cell. Biol. 1997; 17: 2020-2029Crossref PubMed Scopus (152) Google Scholar). Moreover, ATM has been implicated in regulation of chromosome end associations and telomere nuclear matrix interactions (31Smilenov L.B. Dhar S. Pandita T.K. Mol. Cell. Biol. 1999; 19: 6963-6971Crossref PubMed Scopus (68) Google Scholar). In yeast, deletion of ATM homologous genes TEL1 and MEC1 also leads to accelerated telomere shortening, premature aging, and the G2/M checkpoint defect (32Lustig A.J. Petes T.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1398-1402Crossref PubMed Scopus (201) Google Scholar, 33Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-665Crossref PubMed Scopus (665) Google Scholar, 34Greenwell P.W. Kronmal S.L. Porter S.E. Gassenhuber J. Obermaier B. Petes T.D. Cell. 1995; 82: 823-829Abstract Full Text PDF PubMed Scopus (347) Google Scholar, 35Ritchie K.B. Mallory J.C. Petes T.D. Mol. Cell. Biol. 1999; 19: 6065-6075Crossref PubMed Scopus (222) Google Scholar). Interestingly, yeast TEL1 partially substitutes for human ATM in suppressing ionizing radiation-induced apoptosis and telomere shortening in A-T cells (36Fritz E. Friedl A.A. Zwacka R.M. Eckardt-Schupp F. Meyn M.S. Mol. Biol. Cell. 2000; 11: 2605-2616Crossref PubMed Scopus (25) Google Scholar), and overexpression of telomerase elongates telomeres and extends the life span of A-T cells (37Wood L.D. Halvorsen T.L. Dhar S. Baur J.A. Pandita R.K. Wright W.E. Hande M.P. Calaf G. Hei T.K. Levine F. Shay J.W. Wang J.J. Pandita T.K. Oncogene. 2001; 20: 278-288Crossref PubMed Scopus (84) Google Scholar). These results indicate that ATM plays a crucial role in regulation of telomere maintenance and the G2/M checkpoint. However, overexpression of telomerase does not rescue radiosensitivity, telomere fusion, or cell cycle checkpoint defects in A-T cells (37Wood L.D. Halvorsen T.L. Dhar S. Baur J.A. Pandita R.K. Wright W.E. Hande M.P. Calaf G. Hei T.K. Levine F. Shay J.W. Wang J.J. Pandita T.K. Oncogene. 2001; 20: 278-288Crossref PubMed Scopus (84) Google Scholar). In addition, deletion of both TEL1 and MEC1 in yeast does not affect telomerase activity and still allows telomerase to act when the telomere structure is disrupted (38Chan S.W. Chang J. Prescott J. Blackburn E.H. Curr. Biol. 2001; 11: 1240-1250Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). These results indicate that the primary function of ATM in telomere maintenance is not to regulate telomerase activity but rather to act on telomeres or telomere proteins (38Chan S.W. Chang J. Prescott J. Blackburn E.H. Curr. Biol. 2001; 11: 1240-1250Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Our previous studies indicate that one of the ATM substrates in the regulation of telomeres and mitotic progression is the telomere protein Pin2/TRF1 (39Kishi S. Zhou X.Z. Nakamura N. Ziv Y. Khoo C. Hill D.E. Shiloh Y. Lu K.P. J. Biol. Chem. 2001; 276: 29282-29291Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Pin2/TRF1 was originally identified by its ability to bind telomeric DNA repeats (TRF1) (40Chong L. van Steensel B. Broccoli D. Erdjument B.H. Hanish J. Tempst P. de Lange T. Science. 1995; 270: 1663-1667Crossref PubMed Scopus (615) Google Scholar) or to bind the mitotic kinase NIMA and suppress its ability to induce mitotic catastrophe (Pin2) (41Lu K.P. Hanes S.D. Hunter T. Nature. 1996; 380: 544-547Crossref PubMed Scopus (782) Google Scholar,42Lu K.P. Prog. Cell Cycle Res. 2000; 4: 83-96Crossref PubMed Scopus (73) Google Scholar). Pin2 is identical to TRF1 with the exception of a 20-amino acid internal deletion but is 5–10-fold more abundant than TRF1 in the cell (43Shen M. Haggblom C. Vogt M. Hunter T. Lu K.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13618-13623Crossref PubMed Scopus (80) Google Scholar); they are likely generated from the same genePIN2/TRF1 (44Young A.C. Chavez M. Giambernardi T.A. Mattern V. McGill J.R. Harris J.M. Sarosdy M.F. Patel P. Sakaguchi A.Y. Somat. Cell Mol. Genet. 1997; 23: 275-286Crossref PubMed Scopus (13) Google Scholar). For clarity, we will use Pin2 for the 20-amino acid deletion isoform and TRF1 for the 20-amino acid-containing isoform, as they were originally identified (40Chong L. van Steensel B. Broccoli D. Erdjument B.H. Hanish J. Tempst P. de Lange T. Science. 1995; 270: 1663-1667Crossref PubMed Scopus (615) Google Scholar, 41Lu K.P. Hanes S.D. Hunter T. Nature. 1996; 380: 544-547Crossref PubMed Scopus (782) Google Scholar,43Shen M. Haggblom C. Vogt M. Hunter T. Lu K.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13618-13623Crossref PubMed Scopus (80) Google Scholar), and use Pin2/TRF1 for endogenous proteins. Overexpression of Pin2/TRF1 accelerates telomere loss, whereas dominant-negative Pin2/TRF1 increases telomere length, indicating that Pin2/TRF1 is a negative regulator of telomere elongation (45van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1045) Google Scholar). Furthermore, Pin2/TRF1 interacts with tankyrase and Tin2, which modulate telomere metabolism (46Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (891) Google Scholar, 47Kim S.H. Kaminker P. Campisi J. Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar). Although Pin2/TRF1, tankyrase, and Tin2 do not directly inhibit telomerase activity (45van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1045) Google Scholar, 46Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (891) Google Scholar, 47Kim S.H. Kaminker P. Campisi J. Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), we have recently demonstrated that Pin2/TRF1 binds a potent telomerase inhibitor, PinX1, which directly inhibits telomerase, shortens telomeres, and induces cells into crisis (48Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). These results indicate that Pin2/TRF1 plays a key role in the regulation of telomere maintenance. In addition, we have shown that both the protein level and subcellular localization of Pin2/TRF1 are tightly regulated during the cell cycle. Pin2/TRF1 levels are increased in the G2/M (43Shen M. Haggblom C. Vogt M. Hunter T. Lu K.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13618-13623Crossref PubMed Scopus (80) Google Scholar). Furthermore, Pin2/TRF1 specifically localizes to the mitotic spindle during mitosis and affects microtubule assembly (49Nakamura M. Zhou X.Z. Lu K.P. Curr. Biol. 2001; 11: 1062-1067Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 50Nakamura M. Zhou X.Z. Kishi S. Kosugi I. Tsutsui Y. Lu K.P. Curr. Biol. 2001; 11: 1512-1516Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Moreover, overexpression of Pin2/TRF1 induces abortive mitosis and apoptosis in cells containing short telomeres but not in those containing long telomeres (51Kishi S. Wulf G. Nakamura M. Lu K.P. Oncogene. 2001; 20: 1497-1508Crossref PubMed Scopus (56) Google Scholar). These results indicate that Pin2/TRF1 also plays an important role in mitotic progression. This is consistent with other studies linking telomere regulation to mitotic progression. For example, elimination or mutation of telomeres causes a Rad9p-mediated cell cycle arrest in G2 in budding yeast (52Sandell L.L. Zakian V.A. Cell. 1993; 75: 729-739Abstract Full Text PDF PubMed Scopus (683) Google Scholar), triggers abortive mitosis and apoptosis in Drosophila (53Ahmad K. Golic K.G. Genetics. 1999; 151: 1041-1051Crossref PubMed Google Scholar), or causes a severe delay or block in anaphase, displaying an anaphase bridge inTetrahymena (54Kirk K.E. Harmon B.P. Reichardt I.K. Sedat J.W. Blackburn E.H. Science. 1997; 275: 1478-1481Crossref PubMed Scopus (189) Google Scholar). Interestingly, we have also demonstrated that Pin2/TRF1 binds with ATMin vitro and in vivo (39Kishi S. Zhou X.Z. Nakamura N. Ziv Y. Khoo C. Hill D.E. Shiloh Y. Lu K.P. J. Biol. Chem. 2001; 276: 29282-29291Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Furthermore, ATM activated by DNA damage directly phosphorylates Pin2/TRF1 preferentially on serine 219 and also suppresses its ability to induce abortive mitosis and apoptosis (39Kishi S. Zhou X.Z. Nakamura N. Ziv Y. Khoo C. Hill D.E. Shiloh Y. Lu K.P. J. Biol. Chem. 2001; 276: 29282-29291Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Moreover, point mutations in Pin2/TRF1 mimicking ATM phosphorylation completely abolished its ability to induce apoptosis, whereas replacing the ATM phosphorylation site with a nonphosphorylatable residue rendered Pin2 resistant to suppression by ATM (39Kishi S. Zhou X.Z. Nakamura N. Ziv Y. Khoo C. Hill D.E. Shiloh Y. Lu K.P. J. Biol. Chem. 2001; 276: 29282-29291Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In addition, overexpression of Pin2/TRF1 results in phenotypes similar to those of ATM mutations, including accelerated telomere shortening (45van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1045) Google Scholar, 46Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (891) Google Scholar, 47Kim S.H. Kaminker P. Campisi J. Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar, 48Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), abortive mitosis, and apoptosis (51Kishi S. Wulf G. Nakamura M. Lu K.P. Oncogene. 2001; 20: 1497-1508Crossref PubMed Scopus (56) Google Scholar). These results suggest that ATM may inhibit the function of Pin2/TRF1 during DNA damage response. However, the physiological importance of Pin2/TRF1 in mediating ATM-dependent regulation remains to be determined. To address this question, we here inhibited the function of endogenous Pin2/TRF1 in A-T cells by stable expression of two different dominant-negative Pin2/TRF1 mutants. Both the mutants increased telomere length in A-T cells. More importantly, both the mutants reduced radiosensitivity and restored the G2/M checkpoint defect without inhibiting Cdc2 activation in A-T cells. In contrast, neither of the mutants affected the S phase checkpoint defect in same cells. These results indicate that inhibition of Pin2/TRF1 can specifically suppress telomere shortening, the G2/M checkpoint defect, and radiosensitivity in A-T cells and demonstrate a critical role for Pin2/TRF1 in mediating some aspects of phenotypes associated with ATM mutations. For stable expression of ATM, pEBS7 vector encoding full-length ATM tagged with FLAG or the control vector were stably transfected into parental A-T22IJE-T cells as described (39Kishi S. Zhou X.Z. Nakamura N. Ziv Y. Khoo C. Hill D.E. Shiloh Y. Lu K.P. J. Biol. Chem. 2001; 276: 29282-29291Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 55Ziv Y. Bar-Shira A. Pecker I. Russell P. Jorgensen T.J. Tsarfati I. Shiloh Y. Oncogene. 1997; 15: 159-167Crossref PubMed Scopus (223) Google Scholar). After selection with hygromycin B (200 μg/ml) and limited dilution, multiple clones were isolated and checked for ATM expression by immunoblotting analysis with anti-ATM antibody (Ab-3) and anti-FLAG antibody (M5). For stable expression of Pin2 mutants, cDNA encoding Pin2-(1–372) and Pin2-(1–316) were cloned into the pEGFP-C1 vector and stably transfected into A-T22IJE-T cells. After selection with G418 (1 mg/ml), GFP-expressed cells were picked up under a fluorescence microscope. Multiple stable clones were obtained with similar properties. To detect expression of GFP fusion proteins in the cells, trypsinized cells were resuspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, washed in phosphate-buffered saline (PBS), and then immediately analyzed by flow cytometry (BD PharMingen) for detection of the GFP fluorescence intensity of individual cells with nontransfected cells as a negative control or by immunoblotting analysis with anti-GFP antibodies. Telomere restriction fragment length was determined as described previously (48Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Briefly, genomic DNA was isolated and digested with restriction enzymes HinfI and RsaI (New England Biolabs), separated on 0.7% agarose gels (2 μg of DNA per lane). The gels were dried, but not completely, and then hybridized in-gel with a 500-bp telomeric DNA fragment labeled with [α-32P]dCTP by standard protocols, followed by autoradiography. Telomere FISH was carried out as described previously (39Kishi S. Zhou X.Z. Nakamura N. Ziv Y. Khoo C. Hill D.E. Shiloh Y. Lu K.P. J. Biol. Chem. 2001; 276: 29282-29291Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Briefly, cells grown on coverslips were washed once in Tris-buffered saline (TBS) and incubated in 3.7% formaldehyde in TBS for 10 min at room temperature. These prepared cells were then denatured in a hybridization mixture containing 70% deionized formamide, 20 mm Tris pH 7.0, 1% bovine serum albumin, and 10 nm Cy3-labeled PNA telomere repeat probe (PerSeptive Biosystems, Framingham, MA) for 10 min at 80 °C. A hybridization was performed for 12 h at room temperature. Finally, DNA was counterstained with 0.5 mg/ml DAPI, and preparations were mounted in antifade solution (Vectashield, Vector Labs). Samples were observed using a fluorescence microscope, and digital images were recorded with a CCD camera. Quantitative fluorescence intensity of individual cells was evaluated by NIH Image software. To stain for senescence-associated β-galactosidase (SA-β-Gal), cells grown in dishes or on coverslips were washed with PBS and fixed in 0.5% glutaraldehyde. The cells were then incubated with staining solution (1 mg/ml 5-bromo-4-chloro-3-indolyl-galactoside, 5 mm potassium ferrocyanide, 5 mm potassium ferricyanide, and 1 mm MgCl2 in PBS at pH 6.0) for ∼12 h at 37 °C, as reported previously (48Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 56Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5558) Google Scholar). Cells were rinsed in PBS, and the staining and cell morphology were determined under a microscope. Experiments were carried out according to previously published protocols (55Ziv Y. Bar-Shira A. Pecker I. Russell P. Jorgensen T.J. Tsarfati I. Shiloh Y. Oncogene. 1997; 15: 159-167Crossref PubMed Scopus (223) Google Scholar, 57Ziv Y. Bar-Shira A. Jorgensen T.J. Russell P.S. Sartiel A. Shows T.B. Eddy R.L. Buchwald M. Legerski R. Schimke R.T. et al.Somatatic Cell Mol. Genet. 1995; 21: 99-111Crossref PubMed Scopus (16) Google Scholar). Briefly, 50% confluent monolayer cultures at logarithmic stage were irradiated, and after 14–21 days the resultant colonies were fixed and stained with 2% crystal violet in 50% ethanol and counted under a dissecting microscope. For cell cycle analysis, cells were harvested by trypsinization, resuspended in Dulbecco's modified Eagle's medium supplemented with 10% serum, washed in PBS, and then fixed in 70% ethanol. After washing cells once with PBS containing 1% bovine serum albumin, DNA was stained with propidium iodide (10 μg/ml) containing 250 μg/ml of ribonuclease A, followed by flow cytometry analysis (BD PharMingen) as described (42Lu K.P. Prog. Cell Cycle Res. 2000; 4: 83-96Crossref PubMed Scopus (73) Google Scholar, 51Kishi S. Wulf G. Nakamura M. Lu K.P. Oncogene. 2001; 20: 1497-1508Crossref PubMed Scopus (56) Google Scholar). DNA synthesis was assayed by incubation with 10 μm BrdUrd for 60 min, and incorporation of BrdUrd into cells was determined by staining cells with PE-conjugated anti-BrdUrd monoclonal antibody, followed by flow cytometry according to the manufacturer's protocol (BD PharMingen) as described (48Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Tyr-15 phosphorylation status and histone H1 kinase activity of Cdc2 were assayed as described previously (42Lu K.P. Prog. Cell Cycle Res. 2000; 4: 83-96Crossref PubMed Scopus (73) Google Scholar, 58Lu K.P. Hunter T. Cell. 1995; 81: 413-424Abstract Full Text PDF PubMed Scopus (141) Google Scholar). Overexpression of Pin2/TRF1 induces telomere shortening, abortive mitosis, and apoptosis (45van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1045) Google Scholar, 51Kishi S. Wulf G. Nakamura M. Lu K.P. Oncogene. 2001; 20: 1497-1508Crossref PubMed Scopus (56) Google Scholar). Furthermore, ATM phosphorylates Pin2/TRF1 and suppresses its ability to induce abortive mitosis and apoptosis (39Kishi S. Zhou X.Z. Nakamura N. Ziv Y. Khoo C. Hill D.E. Shiloh Y. Lu K.P. J. Biol. Chem. 2001; 276: 29282-29291Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Interestingly, A-T cells contain shortened telomeres and enter abortive mitosis and apoptosis upon ionizing radiation (25Beamish, H., Khanna, K. K., and Lavin, M. F. (1994)Radiat. Res. S130–133Google Scholar, 26Pandita T.K. Pathak S. Geard C.R. Cytogenet. Cell Genet. 1995; 71: 86-93Crossref PubMed Scopus (164" @default.
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• W2078465024 title "A Critical Role for Pin2/TRF1 in ATM-dependent Regulation" @default.
• W2078465024 cites W1543500873 @default.
• W2078465024 cites W1618806168 @default.
• W2078465024 cites W1633875338 @default.
• W2078465024 cites W1842203567 @default.
• W2078465024 cites W1965461792 @default.
• W2078465024 cites W1968069432 @default.
• W2078465024 cites W1971958721 @default.
• W2078465024 cites W1972303536 @default.
• W2078465024 cites W1973085375 @default.
• W2078465024 cites W1973916918 @default.
• W2078465024 cites W197969011 @default.
• W2078465024 cites W1980725176 @default.
• W2078465024 cites W1988876917 @default.
• W2078465024 cites W1989821481 @default.
• W2078465024 cites W1994163195 @default.
• W2078465024 cites W2005466445 @default.
• W2078465024 cites W2011788692 @default.
• W2078465024 cites W2029531565 @default.
• W2078465024 cites W2031863792 @default.
• W2078465024 cites W2033200608 @default.
• W2078465024 cites W2035154953 @default.
• W2078465024 cites W2036911210 @default.
• W2078465024 cites W2037728125 @default.
• W2078465024 cites W2039839254 @default.
• W2078465024 cites W2046589688 @default.
• W2078465024 cites W2046994769 @default.
• W2078465024 cites W2050674095 @default.
• W2078465024 cites W2054837582 @default.
• W2078465024 cites W2057949133 @default.
• W2078465024 cites W2058792468 @default.
• W2078465024 cites W2062232773 @default.
• W2078465024 cites W2063681671 @default.
• W2078465024 cites W2064727462 @default.
• W2078465024 cites W2067191626 @default.
• W2078465024 cites W2067539799 @default.
• W2078465024 cites W2074615071 @default.
• W2078465024 cites W2075906592 @default.
• W2078465024 cites W2078242581 @default.
• W2078465024 cites W2079357292 @default.
• W2078465024 cites W2081106264 @default.
• W2078465024 cites W2084147994 @default.
• W2078465024 cites W2085305202 @default.
• W2078465024 cites W2086992094 @default.
• W2078465024 cites W2089380061 @default.
• W2078465024 cites W2089574530 @default.
• W2078465024 cites W2091494325 @default.
• W2078465024 cites W2092446580 @default.
• W2078465024 cites W2093606537 @default.
• W2078465024 cites W2094957076 @default.
• W2078465024 cites W2098212826 @default.
• W2078465024 cites W2104908670 @default.
• W2078465024 cites W2106087857 @default.
• W2078465024 cites W2112671059 @default.
• W2078465024 cites W2127440297 @default.
• W2078465024 cites W2128252882 @default.
• W2078465024 cites W2129031151 @default.
• W2078465024 cites W2132380666 @default.
• W2078465024 cites W2140832807 @default.
• W2078465024 cites W2148555433 @default.
• W2078465024 cites W2153923420 @default.
• W2078465024 cites W2159168998 @default.
• W2078465024 cites W2159258181 @default.
• W2078465024 cites W2160840611 @default.
• W2078465024 cites W2161680599 @default.
• W2078465024 cites W2172192445 @default.
• W2078465024 cites W2313767788 @default.
• W2078465024 cites W2315612846 @default.
• W2078465024 cites W2325498452 @default.
• W2078465024 cites W4212962560 @default.
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