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• W1966373529 abstract "DNA damage and replication stress activate the Chk1 signaling pathway, which blocks S phase progression, stabilizes stalled replication forks, and participates in G2 arrest. In this study, we show that Chk1 interacts with Hsp90, a molecular chaperone that participates in the folding, assembly, maturation, and stabilization of specific proteins known as clients. Consistent with Chk1 being an Hsp90 client, we also found that Chk1 but not Chk2 is destabilized in cells treated with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG-mediated Chk1 loss blocked the ability of Chk1 to target Cdc25A for proteolytic destruction, demonstrating that the Chk1 signaling pathway was disrupted in the 17-AAG-treated cells. Finally, 17-AAG-mediated disruption of Chk1 activation dramatically sensitized various tumor cells to gemcitabine, an S phase-active chemotherapeutic agent. Collectively, our studies identify Chk1 as a novel Hsp90 client and suggest that pharmacologic inhibition of Hsp90 may sensitize tumor cells to chemotherapeutic agents by disrupting Chk1 function during replication stress. DNA damage and replication stress activate the Chk1 signaling pathway, which blocks S phase progression, stabilizes stalled replication forks, and participates in G2 arrest. In this study, we show that Chk1 interacts with Hsp90, a molecular chaperone that participates in the folding, assembly, maturation, and stabilization of specific proteins known as clients. Consistent with Chk1 being an Hsp90 client, we also found that Chk1 but not Chk2 is destabilized in cells treated with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG-mediated Chk1 loss blocked the ability of Chk1 to target Cdc25A for proteolytic destruction, demonstrating that the Chk1 signaling pathway was disrupted in the 17-AAG-treated cells. Finally, 17-AAG-mediated disruption of Chk1 activation dramatically sensitized various tumor cells to gemcitabine, an S phase-active chemotherapeutic agent. Collectively, our studies identify Chk1 as a novel Hsp90 client and suggest that pharmacologic inhibition of Hsp90 may sensitize tumor cells to chemotherapeutic agents by disrupting Chk1 function during replication stress. Hsp90 is a ubiquitously expressed and abundant molecular chaperone that participates in the folding, assembly, maturation, and stabilization of specific proteins (known as clients). Hsp90 carries out these functions as an integral component of a multiprotein chaperone complex that contains additional chaperones and co-chaperones (reviewed in Refs. 1Pearl L.H. Prodromou C. Adv. Protein Chem. 2001; 59: 157-186Crossref PubMed Scopus (178) Google Scholar, 2Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 3Pratt W. Toft D. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1276) Google Scholar). Hsp90 regulates client function by cycling between two physiologically important states. In its ATP-bound state, Hsp90 interacts with co-chaperones Cdc37, p23, and an assortment of immunophilin-like proteins, forming a complex that stabilizes and protects client proteins from proteasomal degradation (1Pearl L.H. Prodromou C. Adv. Protein Chem. 2001; 59: 157-186Crossref PubMed Scopus (178) Google Scholar, 2Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 3Pratt W. Toft D. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1276) Google Scholar). In its ADP-bound form, Hsp90 recruits Hsp70 and p60/Hop, forming a complex that targets clients for proteasomal degradation. Many new Hsp90 clients have been discovered recently with the aid of the Hsp90 inhibitors geldanamycin, radicicol, and their derivatives. These agents occupy the Hsp90 ATP-binding site, mimicking the ADP-bound state and targeting clients to the proteasome for degradation (4Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1125) Google Scholar, 5Grenert J.P. Sullivan W.P. Fadden P. Haystead T.A. Clark J. Mimnaugh E. Krutzsch H. Ochel H.J. Schulte T.W. Sausville E. Neckers L.M. Toft D.O. J. Biol. Chem. 1997; 272: 23843-23850Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 6Roe S.M. Prodromou C. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. J. Med. Chem. 1999; 42: 260-266Crossref PubMed Scopus (896) Google Scholar, 7Schulte T.W. Akinaga S. Soga S. Sullivan W. Stensgard B. Toft D. Neckers L.M. Cell Stress Chaperones. 1998; 3: 100-108Crossref PubMed Scopus (365) Google Scholar). Several of these clients, such as Akt, Her2/Neu, Bcr-Abl, and Raf-1, are important participants in signaling pathways that drive tumor cell proliferation and survival (8Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5171) Google Scholar, 9Neve R.M. Lane H.A. Hynes N.E. Ann. Oncol. 2001; 12: S9-S13Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 10Raitano A.B. Whang Y.E. Sawyers C.L. Biochim. Biophys. Acta. 1997; 1333: F201-F216PubMed Google Scholar, 11Naumann U. Eisenmann-Tappe I. Rapp U.R. Recent Res. Cancer Res. 1997; 143: 237-244Crossref PubMed Scopus (41) Google Scholar). In addition to their role in proliferation, the constitutive activation of these signaling proteins also enhances the survival of tumor cells following DNA damage. Thus, Hsp90-directed therapy has been viewed as a mechanism to simultaneously target numerous oncogenic signaling pathways and sensitize cells to chemotherapeutic agents (2Neckers L. Trends Mol. Med. 2002; 8: S55-S61Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 12Blagosklonny M.V. Leukemia. 2002; 16: 455-462Crossref PubMed Scopus (229) Google Scholar). Although the oncogenic signaling pathways enhance survival following DNA damage, DNA damage also activates checkpoint signaling pathways that play pivotal roles in the survival of genotoxin-treated cells. The DNA damage-activated checkpoint pathways are evolutionarily conserved signaling pathways that regulate cell cycle progression, programmed cell death, and DNA repair (reviewed in Refs. 13Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1676) Google Scholar and 14Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2645) Google Scholar). One checkpoint signaling pathway that has emerged as a key regulator of cellular responses is the Chk1 signaling pathway, which is activated by replication stress and various types of DNA damage. Following replication fork stalling, single-stranded regions of DNA accumulate that bind the single-stranded DNA-binding protein RPA (15Walter J. Newport J. Mol. Cell. 2000; 5: 617-627Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 16Lupardus P.J. Byun T. Yee M.C. Hekmat-Nejad M. Cimprich K.A. Genes Dev. 2002; 16: 2327-2332Crossref PubMed Scopus (144) Google Scholar, 17You Z. Kong L. Newport J. J. Biol. Chem. 2002; 277: 27088-27093Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2059) Google Scholar). RPA binding is then followed by the chromatin recruitment of the phosphatidylinositol 3-kinase-related kinase ATR and its binding partner ATRIP (18Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2059) Google Scholar). The stalled fork also recruits DNA polymerase α (16Lupardus P.J. Byun T. Yee M.C. Hekmat-Nejad M. Cimprich K.A. Genes Dev. 2002; 16: 2327-2332Crossref PubMed Scopus (144) Google Scholar, 17You Z. Kong L. Newport J. J. Biol. Chem. 2002; 277: 27088-27093Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), which then participates in the Rad17-dependent recruitment of the PCNA-like Rad9-Hus1-Rad1 (9-1-1) clamp complex to chromatin (17You Z. Kong L. Newport J. J. Biol. Chem. 2002; 277: 27088-27093Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 19Zou L. Cortez D. Elledge S.J. Genes Dev. 2002; 16: 198-208Crossref PubMed Scopus (437) Google Scholar). Once bound to chromatin, the 9-1-1 complex facilitates the ATR-mediated phosphorylation and activation of Chk1 (reviewed in Ref. 20Melo J. Toczyski D. Curr. Opin. Cell Biol. 2002; 14: 237-245Crossref PubMed Scopus (399) Google Scholar). Activated Chk1 performs several functions that promote cell survival. First, Chk1 increases the time available for DNA repair by arresting cells in G2. This arrest occurs when Chk1 phosphorylates Cdc25C and Cdc25A, two cell cycle phosphatases that activate the Cdk1-cyclin B complex (reviewed in Ref. 21Rhind N. Russell P. J. Cell Sci. 2000; 113: 3889-3896Crossref PubMed Google Scholar). Second, activated Chk1 slows progression through S phase by blocking the firing of unfired origins of the replication (22Guo N. Faller D.V. Vaziri C. Cell Growth Differ. 2002; 13: 77-86PubMed Google Scholar, 23Heffernan T.P. Simpson D.A. Frank A.R. Heinloth A.N. Paules R.S. Cordeiro-Stone M. Kaufmann W.K. Mol. Cell Biol. 2002; 22: 8552-8561Crossref PubMed Scopus (208) Google Scholar, 24Zhao H. Watkins J.L. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14795-14800Crossref PubMed Scopus (422) Google Scholar, 25Zachos G. Rainey M.D. Gillespie D.A. EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (223) Google Scholar). In this checkpoint, Chk1 phosphorylates Cdc25A, leading to Cdc25A degradation (24Zhao H. Watkins J.L. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14795-14800Crossref PubMed Scopus (422) Google Scholar, 26Shimuta K. Nakajo N. Uto K. Hayano Y. Okazaki K. Sagata N. EMBO J. 2002; 21: 3694-3703Crossref PubMed Scopus (111) Google Scholar, 27Mailand N. Falck J. Lukas C. Syljuasen R.G. Welcker M. Bartek J. Lukas J. Science. 2000; 288: 1425-1429Crossref PubMed Scopus (656) Google Scholar). Because Cdc25A is required for activation of Cdk2 complexes, which then control the firing of origins of replication, activation of this pathway blocks S phase progression (28Sexl V. Diehl J.A. Sherr C.J. Ashmun R. Beach D. Roussel M.F. Oncogene. 1999; 18: 573-582Crossref PubMed Scopus (87) Google Scholar, 29Blomberg I. Hoffman I. Mol. Cell Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (255) Google Scholar, 30Hoffmann I. Draetta G. Karsenti E. EMBO J. 1994; 13: 4302-4310Crossref PubMed Scopus (424) Google Scholar, 31Jinno S. Suto K. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (400) Google Scholar). Third, Chk1 stabilizes stalled replication forks. Although the relevant substrate is not known, in the absence of Chk1 function, the stalled forks irreversibly collapse. Correspondingly, disruption of the Chk1 signaling pathway is associated with increased sensitivity to genotoxins. For example, cells lacking the upstream regulators Hus1 and Rad9 are highly sensitive to ultraviolet radiation and the replication inhibitor hydroxyurea (32Weiss R.S. Enoch T. Leder P. Genes Dev. 2000; 14: 1886-1898PubMed Google Scholar, 33Roos-Mattjus P. Hopkins K.M. Oestreich A.J. Vroman B.T. Johnson K.L. Naylor S. Lieberman H.B. Karnitz L.M. J. Biol. Chem. 2003; 278: 24428-24437Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Additionally, cells in which Chk1 has been deleted by gene targeting are sensitive to the replication inhibitor aphidicolin and ionizing radiation (25Zachos G. Rainey M.D. Gillespie D.A. EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (223) Google Scholar). Consistent with this finding, UCN-01, a pharmacologic Chk1 inhibitor (34Graves P.R. Yu L. Schwarz J.K. Gales J. Sausville E.A. O'Connor P.M. Piwnica-Worms H. J. Biol. Chem. 2000; 275: 5600-5605Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 35Busby E.C. Leistritz D.F. Abraham R.T. Karnitz L.M. Sarkaria J.N. Cancer Res. 2000; 60: 2108-2112PubMed Google Scholar), also sensitizes cells to genotoxins (36Luo Y. Rockow-Magnone S.K. Joseph M.D. Bradner J. Butler C.C. Tahir S.K. Han E.K. Ng S.C. Severin J.M. Gubbins E.J. Reilly R.M. Rueter A. Simmer R.L. Holzman T.F. Giranda V.L. Anticancer Res. 2001; 21: 23-28PubMed Google Scholar, 37Playle L.C. Hicks D.J. Qualtrough D. Paraskeva C. Br. J. Cancer. 2002; 87: 352-358Crossref PubMed Scopus (31) Google Scholar, 38Xiao H.H. Makeyev Y. Butler J. Vikram B. Franklin W.A. Radiat. Res. 2002; 158: 84-93Crossref PubMed Scopus (20) Google Scholar, 39Byrd J.C. Shinn C. Willis C.R. Flinn I.W. Lehman T. Sausville E. Lucas D. Grever M.R. Exp. Hematol. 2001; 29: 703-708Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 40Sampath D. Shi Z. Plunkett W. Mol. Pharmacol. 2002; 62: 680-688Crossref PubMed Scopus (57) Google Scholar, 41Shi Z. Azuma A. Sampath D. Li Y.X. Huang P. Plunkett W. Cancer Res. 2001; 61: 1065-1072PubMed Google Scholar). Taken together, these observations have raised the possibility that inhibitors of the Chk1 signaling pathway may be useful clinical agents to sensitize tumor cells to genotoxic chemotherapy agents; however, no clinically viable Chk1 inhibitor has yet emerged. Although a variety of previous studies have implicated Hsp90 function in signaling pathways regulating cell growth, survival, and apoptosis, the role of Hsp90 in checkpoint signaling has not been explored. Moreover, it remains unclear how Hsp90 inhibitors sensitize tumor cells to genotoxic chemotherapeutics. In the present study, we investigated whether Hsp90 regulates genotoxin-induced checkpoint signaling pathways. We report that Chk1 is an Hsp90 client that is lost from cells treated with 17-allylamino-17-demethoxygeldanamycin (17-AAG), 1The abbreviations used are: 17-AAG17-allylamino-17-demethoxygeldanamycinGSTglutathione S-transferaseHAhemagglutininMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethony phenol)-2-(4-sulfophenyl)-2H-tetrazolium. a geldanamycin derivative that is used in clinical trials for the treatment of tumors. Collectively, these studies suggest a novel mechanism by which Hsp90 inhibitors sensitize cells to anti-tumor chemotherapeutic agents. 17-allylamino-17-demethoxygeldanamycin glutathione S-transferase hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethony phenol)-2-(4-sulfophenyl)-2H-tetrazolium. Cell Culture, Cell Cycle Analysis, and Cell Transfection—HeLa, OVCAR3, and ML-1 cells were grown in RPMI 1640 (BioWhittaker) supplemented with 10% fetal bovine serum. The cell cycle was analyzed by incubating trypsinized cells in 0.1% sodium citrate, 0.1% Triton X-100, 50 μg/ml propidium iodide, and 1 μg/ml RNase A for 30 min at room temperature. Cell cycle profiles were obtained by flow cytometry and analyzed with CellQuest software (Becton Dickinson). For HeLa cell transfections, the cells were trypsinized (0.5–1 × 107 cells/transfection), resuspended in RPMI 1640 containing 10% fetal calf serum, and resuspend in 0.35 ml of the same medium. Plasmid DNA (40 μg/transfection) dissolved in RPMI 1640 was then added to the cells and incubated for 5 min. The cell-DNA mix was transferred to a 0.4-cm electroporation cuvette and electroporated with a 10-mS, 350-V pulse in a BTX T820 square wave electroporator. The cells were then replated and cultured for 20–24 h. Reagents and Antibodies—17-AAG was obtained from R. Schultz (Developmental Therapeutics Program, National Cancer Institute) and from T. Mueller (Kosan Biosciences). Purified GST-Cdc25C (amino acids 200–256) was prepared as described (42Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar). [γ-32P]ATP (4500 Ci/mmol) was purchased from ICN Radiochemicals. Polyclonal anti-Chk1 and anti-Chk2 and monoclonal anti-Chk2 antibodies were provided by Junjie Chen (Mayo Foundation) (43Ward I.M. Wu X. Chen J. J. Biol. Chem. 2001; 276: 47755-47758Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Monoclonal antibody to Hsp90 (H9010) has been previously described (44Barent R.L. Nair S.C. Carr D.C. Ruan Y. Rimerman R.A. Fulton J. Zhang Y. Smith D.F. Mol. Endocrinol. 1998; 12: 342-354Crossref PubMed Google Scholar). HA-conjugated agarose (sc-7392AC) and polyclonal Chk1 (sc-7898) antibodies were purchased from Santa Cruz Biotechnology and used according to the manufacturer's instructions. Antibodies recognizing human Rad9, Rad1, Hus1, and Rad17 were raised as previously described (45Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 46Burtelow M.A. Kaufmann S.H. Karnitz L.M. J. Biol. Chem. 2000; 275: 26343-26348Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Anti-phospho-Chk1 (Ser345) was purchased from Cell Signaling Technology. Biotinylated anti-HA monoclonal antibody 3F10 was purchased from Roche Applied Science. Streptavidin and protein A-conjugated horseradish peroxidase was from Amersham Biosciences. The anti-Cdc25A antibody (Ab3) was purchased from Neomarkers. HA-tagged expression vectors for Hus1, Chk1, and c-Raf have been described previously (35Busby E.C. Leistritz D.F. Abraham R.T. Karnitz L.M. Sarkaria J.N. Cancer Res. 2000; 60: 2108-2112PubMed Google Scholar, 45Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 47Sutor S.L. Vroman B.T. Armstrong E.A. Abraham R.T. Karnitz L.M. J. Biol. Chem. 1999; 274: 7002-7010Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Chk1 Kinase Assays—HeLa cells (1 × 107) were transfected with 2 μg of pEF-BOS-Chk1-HA2 and 38 μg of empty vector as described above. Following overnight incubation, the cells were pretreated with Me2SO or 17-AAG for 1 h prior to treatment with gemcitabine. Following an additional 1-h incubation, the cells were lysed, and HA-tagged Chk1 was immunoprecipitated using HA-conjugated agarose following a previously described Chk1 assay procedure (48Zhao 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 (436) Google Scholar). The immunopurified Chk1 was then incubated with [γ-32P]ATP and the Chk1 substrate GST-Cdc25C (encoding amino acids 200–256 of Cdc25C) at 30 °C for 30 min. The reactions were terminated by adding 4× SDS-PAGE sample buffer. The kinase reactions were resolved on a 12.5% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. Radiolabeled proteins were visualized using an Amersham Biosciences storm 840 Phosphorimager. Immunoprecipitations—HeLa cells were plated in 10-cm dishes and grown to 90% confluence prior to cell lysis. Approximately 3 × 107 cells were lysed for 10 min on ice in a buffer containing 10 mm HEPES, pH 7.4, 150 mm KCl, 10 mm MgCl2, 0.1% Nonidet P-40, 20 mm β-glycerophosphate, 1 mm sodium orthovanadate, 20 μg/ml aprotinin, 10 μg/ml pepstatin, 20 nm microcystin-LR, and 20 μg/ml leupeptin. To identify the Chk1-Hsp90 interaction, the buffer described above was supplemented with 20 mm sodium molybdate. The lysates were centrifuged for 10 min at 4 °C at 15,000 × g. The clarified lysates were then immunoprecipitated with the indicated antibodies. Washed immunoprecipitates were resolved on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were probed with antibodies as described above to detect associated proteins. Immunoblotting—The cell lysates were prepared as described for immunoprecipitations. The cell lysates were mixed with an equal volume of 2× SDS-PAGE sample buffer, boiled for 5 min, and resolved on 10% SDS-polyacrylamide gels. Anti-phospho-Chk1 (Ser345) immunoblotting was performed according to the manufacturer's directions (Cell Signaling Technology). To detect HA-tagged proteins that co-immunoprecipitated with Hsp90, the immunoprecipitates were immunoblotted with biotinylated anti-HA antibody. Biotinylated anti-HA antibody was detected with streptavidin-conjugated horseradish peroxidase. All other immunoblots were performed as previously described (45Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 46Burtelow M.A. Kaufmann S.H. Karnitz L.M. J. Biol. Chem. 2000; 275: 26343-26348Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Cell Viability—The cells were treated with varying concentrations of gemcitabine and 17-AAG for 24–48 h and stained with trypan blue. Viable (trypan blue-excluding) and nonviable (trypan blue-stained) cells were then counted using a hemocytometer. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays were performed by plating 15,000 cells/well in 96-well plates, treating with drugs as described, and processing according to the manufacturer's directions (Promega). The Hsp90 Inhibitor 17-AAG Selectively Destabilizes Chk1— Many key signaling proteins in mammalian cells are Hsp90 clients. One way to identify potential Hsp90 clients is to examine whether a given protein is destabilized when cells are treated with an Hsp90 inhibitor. To examine whether any of the proteins in the Chk1 signaling pathway are potential Hsp90 clients, we treated HeLa cells with 1 μm 17-AAG, a concentration that maximally disrupts Hsp90 function (49Basso A.D. Solit D.B. Munster P.N. Rosen N. Oncogene. 2002; 21: 1159-1166Crossref PubMed Scopus (251) Google Scholar, 50Munster P.N. Srethapakdi M. Moasser M.M. Rosen N. Cancer Res. 2001; 61: 2945-2952PubMed Google Scholar). The cellular levels of Rad9, Hus1, Rad1, Rad17, ATR, and Chk1 were then examined by immunoblotting at various time points after 17-AAG addition (Fig. 1A). The levels of Rad9, Hus1, Rad1, Rad17, and ATR were not affected by 17-AAG treatment. In contrast, Chk1 levels were reduced by an 8-h 17-AAG treatment and were maximally suppressed after 24 h of exposure (Fig. 1 and data not shown). Radicicol, a structurally unrelated Hsp90 inhibitor also depleted Chk1 (data not shown). We also examined whether Chk1 disappeared in OVCAR3 cells (Fig. 1B), an ovarian tumor line, and ML-1 cells (Fig. 1C), a myeloid leukemia cell line. In both cell lines, 17-AAG promoted Chk1 loss, demonstrating that the effect of 17-AAG on Chk1 levels is not limited to a single cell line. To determine whether another checkpoint protein kinase was affected by Hsp90 inhibition, we also examined Chk2 levels in all three 17-AAG-treated cell lines (Fig. 1). Like Chk1, Chk2 is also activated by DNA damage but responds primarily to double-stranded DNA breaks in an ATM-dependent manner. Unlike Chk1, Chk2 levels did not decrease with 17-AAG treatment. Taken together, these results indicated that Chk1 is selectively lost when Hsp90 is inhibited. Chk1 levels are low in cells in G1/G0, (51Kaneko Y.S. Watanabe N. Morisaki H. Akita H. Fujimoto A. Tominaga K. Terasawa M. Tachibana A. Ikeda K. Nakanishi M. Kaneko Y. Oncogene. 1999; 18: 3673-3681Crossref PubMed Scopus (132) Google Scholar). The fact that geldanamycin and 17-AAG induce G1 and G2/M arrest (50Munster P.N. Srethapakdi M. Moasser M.M. Rosen N. Cancer Res. 2001; 61: 2945-2952PubMed Google Scholar, 52Nimmanapalli R. O'Bryan E. Bhalla K. Cancer Res. 2001; 61: 1799-1804PubMed Google Scholar, 53Srethapakdi M. Liu F. Tavorath R. Rosen N. Cancer Res. 2000; 60: 3940-3946PubMed Google Scholar, 54Hostein I. Robertson D. DiStefano F. Workman P. Clarke P.A. Cancer Res. 2001; 61: 4003-4009PubMed Google Scholar) raised the possibility that 17-AAG-induced Chk1 loss was merely the result of cell cycle redistribution caused by 17-AAG. To address this question, we devised a strategy to arrest cells in S phase, where Chk1 levels are high (51Kaneko Y.S. Watanabe N. Morisaki H. Akita H. Fujimoto A. Tominaga K. Terasawa M. Tachibana A. Ikeda K. Nakanishi M. Kaneko Y. Oncogene. 1999; 18: 3673-3681Crossref PubMed Scopus (132) Google Scholar). HeLa cells were blocked in S phase by treating them with a low concentration of gemcitabine, a nucleoside analog that is incorporated by DNA polymerase into replicating DNA, blocking further chain elongation (55Kaye S.B. Br. J. Cancer. 1998; 78: 1-7Crossref PubMed Scopus (45) Google Scholar, 56Noble S. Goa K.L. Drugs. 1997; 54: 447-472Crossref PubMed Scopus (222) Google Scholar). Gemcitabine-treated HeLa cells accumulated in S phase (Fig. 2A) and activated the Chk1 signaling pathway as evidenced by Chk1 phosphorylation on Ser345 (Fig. 2B), a site that is phosphorylated by ATR and is essential for Chk1 activation (57Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (199) Google Scholar, 58Zhao H. Piwnica-Worms H. Mol. Cell Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). As previously reported, treatment with 17-AAG alone resulted in an accumulation of cells in G1 and G2/M (Fig. 2A). In contrast, when the cells were pretreated with gemcitabine, which slowed their progression through S phase, and then treated with 17-AAG, they remained in S phase. Under these conditions, Chk1 levels were also dramatically reduced (Fig. 2B, lane 4), and, correspondingly, Chk1 phosphorylation on Ser345 was not detected. This demonstration that Chk1 is lost in S phase-arrested cells indicates that Chk1 loss is not due to cell cycle arrest in G1. Chk1 Interacts with Hsp90 —Because many proteins that are destabilized by 17-AAG treatment are bona fide Hsp90 clients, the results in Figs. 1 and 2 raised the possibility that Chk1 may also be an Hsp90 client. An additional characteristic of Hsp90 clients is that they interact with Hsp90. To evaluate a potential Chk1-Hsp90 interaction, we immunoprecipitated endogenous Chk1 and the previously identified Hsp90 client Akt (59Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (846) Google Scholar, 60Basso A.D. Solit D.B. Chiosis G. Giri B. Tsichlis P. Rosen N. J. Biol. Chem. 2002; 277: 39858-39866Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar) from HeLa cell lysates (Fig. 3A). No Hsp90 was found in the preimmune serum control, whereas both Akt and Chk1 co-immunoprecipitated with Hsp90. We also performed the reciprocal co-immunoprecipitation experiment. However, because Chk1 co-migrates with immunoglobulin heavy chain, we always detected the co-migrating immunoglobulin heavy chain when we immunoblotted for Chk1 (data not shown). To circumvent this problem, we transiently transfected HeLa cells with HA-tagged Chk1 and immunoblotted the immunoprecipitates with biotinylated anti-HA monoclonal antibody, which allowed detection of Chk1 in the Hsp90 immunoprecipitates without interference from the heavy chain (Fig. 3B). As has been reported previously for other Hsp90 clients (61Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 62Grammatikakis N. Lin J.H. Grammatikakis A. Tsichlis P.N. Cochran B.H. Mol. Cell Biol. 1999; 19: 1661-1672Crossref PubMed Scopus (229) Google Scholar, 63Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1337) Google Scholar), Hsp90 inhibition decreased the interaction between Hsp90 and Chk1. As controls, we also examined the interaction between Hsp90 and two additional HA-tagged proteins: Hus1, which is not an Hsp90 client, and c-Raf, a bona fide Hsp90 client. As expected, Hus1 did not co-precipitate with Hsp90, whereas c-Raf associated strongly with Hsp90. Like the Hsp90-Chk1 interaction, the Hsp90-c-Raf interaction was also disrupted by 17-AAG. These results, in conjunction with the results in Fig. 1, strongly suggest that Chk1 is indeed an Hsp90 client. The Chk1 Signaling Pathway Is Not Rapidly Inhibited in 17-AAG-treated Cells—The results presented above demonstrated that Chk1 is an Hsp90 client and that Chk1 is slowly lost from 17-AAG-treated cells. However, Hsp90 inhibition occurs quickly following 17-AAG treatment, and some clients, such as the progesterone receptor, rapidly lose their ability to respond to stimuli when Hsp90 is inhibited (64Smith D.F. Whitesell L. Nair S.C. Chen S. Prapapanich V. Rimerman R.A. Mol. Cell Biol. 1995; 15: 6804-6812Crossref PubMed Scopus (272) Google Scholar). To determine whether Hsp90 inhibition rapidly affects the ability of Chk1 to respond to upstream signals and to be activated by genotoxic stress, we pretreated cells with 17-AAG for 1 h and asked whether Chk1 was still phosphorylated on Ser345 by its upstream activating kinase ATR. As shown in Fig. 4A, 17-AAG did not block Chk1 phosphorylation, indicating that even when Hsp90 is inhibited, this client still responds to upstream signals. Moreover, these results also indicate that short term Hsp90 inhibition does not block any of the upstream events required for Chk1 phosphorylation. To assess the effect of Hsp90 inhibition on Chk1 catalytic activity, we developed a Chk1 assay using transiently expressed HA-tagged Chk1 in HeLa cells. (We were unable to analyze endogenous Chk1 activity in any cell line we assayed (data not shown).) Treatment of HeLa cells with gemcitabine activated the catalytic activity of Chk1 (Fig. 4B), and this activation was not blocked by a 1-h pretreatment with 17-AAG. Taken together, these results suggest that Hsp90 is not required to continuously maintain Chk1 in a state that is able to receive and respond to upstream activating signals. 17-AAG-mediated Chk1 Loss Blocks Cdc25A Loss—Chk1-mediated Cdc25A phosphorylation leads to Cdc25A proteolytic destruction, thereby blocking cell cycle progression (24Zhao H. Watkins J.L. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14795-148" @default.
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• W1966373529 title "Hsp90 Inhibition Depletes Chk1 and Sensitizes Tumor Cells to Replication Stress" @default.
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