Hsp90 Inhibition Depletes Chk1 and Sensitizes Tumor Cells to Replication Stress

• DOI: 10.1074/jbc.m309054200
• Publication Date: 2003-12-19T21:32:55Z
• Authors: Sonnet J.H. Arlander; Alex K. Eapen; Benjamin T. Vroman; Robert J. McDonald; David O. Toft; Larry M. Karnitz
• Keywords: CHEK1; CDC25A; Hsp90 inhibitor

DNA damage and replication stress activate the Chk1 signaling pathway, which blocks S phase progression, stabilizes stalled forks, participates in G2 arrest. In this study, we show that interacts with Hsp90, a molecular chaperone folding, assembly, maturation, stabilization of specific proteins known as clients. Consistent being an Hsp90 client, also found but not Chk2 is destabilized cells treated inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG-mediated loss blocked ability to target Cdc25A for proteolytic destruction, demonstrating pathway was disrupted 17-AAG-treated cells. Finally, disruption activation dramatically sensitized various tumor gemcitabine, phase-active chemotherapeutic agent. Collectively, our studies identify novel client suggest pharmacologic inhibition may sensitize agents by disrupting function during stress. ubiquitously expressed abundant (known clients). carries out these functions integral component multiprotein complex contains additional chaperones co-chaperones (reviewed 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 PDF (622) 3Pratt W. Toft D. Exp. Biol. 2003; 228: 111-133Crossref (1276) Scholar). regulates cycling between two physiologically important states. its ATP-bound state, Cdc37, p23, assortment immunophilin-like proteins, forming protects from proteasomal degradation (1Pearl ADP-bound form, recruits Hsp70 p60/Hop, targets clients degradation. Many new have been discovered recently aid inhibitors geldanamycin, radicicol, their derivatives. 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We report lost (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. geldanamycin derivative trials treatment tumors. anti-tumor agents. glutathione S-transferase hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethony Culture, Cycle Analysis, Transfection—HeLa, OVCAR3, ML-1 were grown RPMI 1640 (BioWhittaker) supplemented 10% fetal bovine serum. analyzed incubating trypsinized 0.1% sodium citrate, Triton X-100, 50 μg/ml propidium iodide, 1 RNase A 30 min at room temperature. profiles obtained flow cytometry CellQuest software (Becton Dickinson). HeLa transfections, (0.5–1 × 107 cells/transfection), resuspended containing calf serum, resuspend 0.35 ml same medium. Plasmid (40 μg/transfection) dissolved added incubated 5 min. cell-DNA mix transferred 0.4-cm electroporation cuvette electroporated 10-mS, 350-V pulse BTX T820 square wave electroporator. replated cultured 20–24 h. Reagents Antibodies—17-AAG Schultz (Developmental Therapeutics Program, National Institute) Mueller (Kosan Biosciences). Purified GST-Cdc25C (amino acids 200–256) prepared described (42Sarkaria Busby Tibbetts Roos Taya 4375-4382PubMed [γ-32P]ATP (4500 Ci/mmol) purchased ICN Radiochemicals. Polyclonal anti-Chk1 anti-Chk2 monoclonal antibodies provided Junjie Chen (Mayo Foundation) (43Ward I.M. Wu X. 276: 47755-47758Abstract (89) Monoclonal antibody (H9010) previously (44Barent Nair Carr D.C. Ruan Rimerman R.A. Fulton Zhang Smith Endocrinol. 342-354Crossref HA-conjugated agarose (sc-7392AC) polyclonal (sc-7898) Santa Cruz Biotechnology according manufacturer's instructions. Antibodies recognizing human Rad9, Rad1, Hus1, Rad17 (45Volkmer 274: 567-570Abstract (176) 46Burtelow M.A. S.H. 26343-26348Abstract (102) Anti-phospho-Chk1 (Ser345) Signaling Technology. Biotinylated anti-HA 3F10 Roche Applied Streptavidin A-conjugated horseradish peroxidase Amersham Biosciences. anti-Cdc25A (Ab3) Neomarkers. HA-tagged expression vectors Chk1, c-Raf (35Busby 45Volkmer 47Sutor S.L. Armstrong 7002-7010Abstract (65) Kinase Assays—HeLa (1 107) transfected 2 μg pEF-BOS-Chk1-HA2 38 empty vector above. overnight incubation, pretreated Me2SO or 17-AAG h prior gemcitabine. 1-h lysed, immunoprecipitated using assay procedure (48Zhao Weng Y.C. Yuan S.S. Lin Y.T. Hsu H.C. Gerbino Song M.H. Zdzienicka M.Z. Gatti Shay J.W. Ziv Shiloh Lee E.Y. 405: 473-477Crossref (436) immunopurified (encoding amino 200–256 Cdc25C) °C reactions terminated adding 4× SDS-PAGE sample buffer. resolved on 12.5% SDS-polyacrylamide gel onto polyvinylidene difluoride membrane. Radiolabeled visualized Biosciences storm 840 Phosphorimager. Immunoprecipitations—HeLa plated 10-cm dishes 90% confluence lysis. Approximately 3 lysed 10 ice buffer mm HEPES, pH 7.4, 150 KCl, MgCl2, Nonidet P-40, 20 β-glycerophosphate, orthovanadate, aprotinin, pepstatin, nm microcystin-LR, leupeptin. To Chk1-Hsp90 interaction, above molybdate. lysates centrifuged 4 15,000 g. clarified indicated antibodies. Washed immunoprecipitates gels membranes. membranes probed detect proteins. Immunoblotting—The immunoprecipitations. mixed equal volume 2× buffer, boiled min, gels. immunoblotting performed directions (Cell Technology). co-immunoprecipitated immunoblotted biotinylated antibody. detected streptavidin-conjugated peroxidase. All other immunoblots Viability—The varying concentrations gemcitabine 24–48 stained trypan blue. Viable (trypan blue-excluding) nonviable blue-stained) counted hemocytometer. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays plating cells/well 96-well plates, treating drugs described, processing (Promega). Inhibitor Selectively Destabilizes Chk1— mammalian way potential examine given inhibitor. any μm 17-AAG, concentration maximally disrupts (49Basso A.D. Solit D.B. Munster P.N. Rosen 1159-1166Crossref (251) 50Munster Srethapakdi Moasser M.M. 2945-2952PubMed levels Rad17, ATR, examined points after (Fig. 1A). affected treatment. contrast, reduced 8-h suppressed 24 exposure data shown). Radicicol, structurally unrelated depleted (data disappeared OVCAR3 1B), ovarian line, 1C), myeloid leukemia line. both lines, promoted loss, effect limited single determine another inhibition, all three lines 1). Like responds primarily double-stranded breaks ATM-dependent manner. Unlike did decrease results selectively inhibited. low G1/G0, (51Kaneko Y.S. Watanabe Morisaki Akita Fujimoto Tominaga Terasawa Tachibana Ikeda Nakanishi Kaneko 3673-3681Crossref (132) fact induce G1 G2/M (50Munster 52Nimmanapalli O'Bryan Bhalla 1799-1804PubMed 53Srethapakdi Liu F. Tavorath 3940-3946PubMed 54Hostein Robertson DiStefano Workman Clarke P.A. 4003-4009PubMed Scholar) 17-AAG-induced merely result redistribution caused 17-AAG. address question, devised strategy phase, where high them nucleoside analog incorporated into replicating DNA, further chain elongation (55Kaye S.B. 78: 1-7Crossref (45) 56Noble Goa Drugs. 54: 447-472Crossref (222) Gemcitabine-treated accumulated 2A) evidenced Ser345 2B), site phosphorylated essential (57Liu Q. Guntuku Cui X.S. Matsuoka Tamai Luo Carattini-Rivera DeMayo Bradley Donehower L.A. 1448-1459Crossref (199) 58Zhao 4129-4139Crossref (871) As reported, alone resulted accumulation 2A). slowed they remained phase. Under conditions, 2B, lane 4), and, correspondingly, detected. demonstration phase-arrested indicates due G1. Interacts —Because many bona fide Figs. client. An characteristic interact Hsp90. evaluate endogenous identified Akt (59Sato Fujita Tsuruo 97: 10832-10837Crossref (846) 60Basso Chiosis Giri Tsichlis 39858-39866Abstract (546) 3A). No preimmune serum control, whereas reciprocal co-immunoprecipitation experiment. However, because co-migrates immunoglobulin heavy chain, always co-migrating circumvent problem, transiently antibody, allowed detection without interference 3B). reported (61Fujita Sato Ishida 10346-10353Abstract (185) 62Grammatikakis J.H. Grammatikakis Cochran B.H. 1661-1672Crossref 63Whitesell E.G. De Costa Myers C.E. 91: 8324-8328Crossref (1337) decreased interaction Chk1. controls, proteins: c-Raf, expected, co-precipitate strongly Hsp90-Chk1 Hsp90-c-Raf results, conjunction Fig. 1, indeed Pathway Is Not Rapidly Inhibited Cells—The presented demonstrated slowly quickly treatment, some progesterone receptor, rapidly lose respond stimuli inhibited (64Smith Whitesell Prapapanich 1995; 6804-6812Crossref (272) affects signals stress, asked still activating ATR. shown 4A, block phosphorylation, indicating even inhibited, signals. indicate short term does events phosphorylation. assess catalytic activity, developed (We unable analyze activity line assayed shown).) Treatment 4B), pretreatment continuously maintain able receive Loss Blocks Loss—Chk1-mediated leads thereby 14795-148