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ATM-dependent CHK2 Activation Induced by Anticancer Agent, Irofulven

2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês

10.1074/jbc.m400015200

1083-351X

Jian Wang, Timothy D. Wiltshire, Yutian Wang, Carmenza Mikell, Julian Burks, Cynthia Cunningham, Emily S. Van Laar, Stephen J. Waters, Eddie Reed, Weixin Wang,

Cancer therapeutics and mechanisms

Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is one of a new class of anticancer agents that are semisynthetic derivatives of the mushroom toxin illudin S. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor types. Mechanisms of action studies indicate that irofulven induces DNA damage, MAPK activation, and apoptosis. In this study we found that in ovarian cancer cells, CHK2 kinase is activated by irofulven while CHK1 kinase is not activated even when treated at higher concentrations of the drug. By using GM00847 human fibroblast expressing tetracycline-controlled, FLAG-tagged kinase-dead ATR (ATR.kd), it was demonstrated that ATR kinase does not play a major role in irofulven-induced CHK2 activation. Results from human fibroblasts proficient or deficient in ATM function (GM00637 and GM05849) indicated that CHK2 activation by irofulven is mediated by the upstream ATM kinase. Phosphorylation of ATM on Ser1981, which is critical for kinase activation, was observed in ovarian cancer cell lines treated with irofulven. RNA interference results confirmed that CHK2 activation was inhibited after introducing siRNA for ATM. Finally, experiments done with human colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative; and experiments done by expressing kinase-dead CHK2 in an ovarian cancer cell line demonstrated that CHK2 activation contributes to irofulven-induced S phase arrest. In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner, and p53 phosphorylation on serine 20 is dependent on CHK2 after irofulven treatment. In summary, we found that the anticancer agent, irofulven, activates the ATM-CHK2 DNA damage-signaling pathway, and CHK2 activation contributes to S phase cell cycle arrest induced by irofulven. Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is one of a new class of anticancer agents that are semisynthetic derivatives of the mushroom toxin illudin S. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor types. Mechanisms of action studies indicate that irofulven induces DNA damage, MAPK activation, and apoptosis. In this study we found that in ovarian cancer cells, CHK2 kinase is activated by irofulven while CHK1 kinase is not activated even when treated at higher concentrations of the drug. By using GM00847 human fibroblast expressing tetracycline-controlled, FLAG-tagged kinase-dead ATR (ATR.kd), it was demonstrated that ATR kinase does not play a major role in irofulven-induced CHK2 activation. Results from human fibroblasts proficient or deficient in ATM function (GM00637 and GM05849) indicated that CHK2 activation by irofulven is mediated by the upstream ATM kinase. Phosphorylation of ATM on Ser1981, which is critical for kinase activation, was observed in ovarian cancer cell lines treated with irofulven. RNA interference results confirmed that CHK2 activation was inhibited after introducing siRNA for ATM. Finally, experiments done with human colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative; and experiments done by expressing kinase-dead CHK2 in an ovarian cancer cell line demonstrated that CHK2 activation contributes to irofulven-induced S phase arrest. In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner, and p53 phosphorylation on serine 20 is dependent on CHK2 after irofulven treatment. In summary, we found that the anticancer agent, irofulven, activates the ATM-CHK2 DNA damage-signaling pathway, and CHK2 activation contributes to S phase cell cycle arrest induced by irofulven. Irofulven 1The abbreviations used are: irofulven, 6-hydroxymethylacylfulvene; CHK1, checkpoint kinase 1; CHK2, checkpoint kinase 2; ATM, ataxia telangiectasia-mutated protein; ATR, ATM-RAD3-related; NBS1, Nijmegen breakage syndrome 1; SMC1, structural maintenance of chromosome 1; BRCA1, breast cancer 1; MDM2, murine double minute 2; PBS, phosphate-buffered saline; IR, ionizing radiation; siRNA, small interfering RNA; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; MAPK, mitogen-activated protein kinase; GFP, green fluorescent protein. 1The abbreviations used are: irofulven, 6-hydroxymethylacylfulvene; CHK1, checkpoint kinase 1; CHK2, checkpoint kinase 2; ATM, ataxia telangiectasia-mutated protein; ATR, ATM-RAD3-related; NBS1, Nijmegen breakage syndrome 1; SMC1, structural maintenance of chromosome 1; BRCA1, breast cancer 1; MDM2, murine double minute 2; PBS, phosphate-buffered saline; IR, ionizing radiation; siRNA, small interfering RNA; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; MAPK, mitogen-activated protein kinase; GFP, green fluorescent protein. 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In this study, we found that the anticancer agent, irofulven, activates ATM-CHK2 DNA damage-signaling pathway, and activates NBS1, SMC1, and p53 in an ATM-dependent manner. Irofulven also induces CHK2-dependent p53 phosphorylation on Ser20. This CHK2 activation contributes to irofulven-induced S phase arrest. Cell Culture—All cell lines were maintained in various media supplemented with 10% fetal bovine serum in a 37 °C incubator with 5% CO2 atmosphere. Human ovarian cancer cell lines A2780, A2780/CP70, CAOV3, OVCAR3, and SKOV3 were cultured in RPMI1640. Human colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative HCT116 CHK2–/– (78Jallepalli P.V. Lengauer C. Vogelstein B. Bunz F. J. Biol. Chem. 2003; 278: 20475-20479Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) (generously provided by Prof. Bert Vogelstein, Johns Hopkins University, Baltimore, MD) were cultured in McCoy's 5A medium. The SV40-transformed human normal fibroblast GM00637, the AT (ataxia telangiectasia) fibroblast GM05849 and its parental non-transformed AT fibroblast GM05823 (Coriell Institute, Camden, NJ) were grown in DMEM. The ATM-complemented AT fibroblast (AT22IJE-T-pEBS7-YZ5, generously provided by Prof. Yosef Shiloh, Tel Aviv University, Israel) (80Ziv 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) was grown in Dulbecco's modified Eagle's medium with 100 μg/ml hygromycin (Invitrogen, Carlsbad, CA). GM00847 human fibroblast-expressing tetracycline-controlled, FLAG-tagged kinase-dead ATR (ATR.kd) (81Cliby W.A. Roberts C.J. Cimprich K.A. Stringer C.M. Lamb J.R. Schreiber S.L. Friend S.H. EMBO J. 1998; 17: 159-169Crossref PubMed Scopus (478) Google Scholar) (generously provided by Drs. Stuart L. Schreiber and Shlomo Handeli of the Fred Hutchinson Cancer Research Center, Seattle, WA) was grown in DMEM supplemented with 400 μg/ml of G418 (Invitrogen). For ATR.kd induction, cells were treated with 1.5 μg/ml of doxycycline (Sigma) for 48 h. For UV treatment in control experiments, cells were treated with 50 J/m2 of UV light in Stratalinker 2400 (Stratagene, La Jolla, CA) followed by one additional hour of incubation. To inhibit CDC25A degradation, proteasome inhibitor LLnL (N-Acetyl-Leu-Leu-Norleu-al) (Sigma) (50 μm) were added to cells 30 min before irofulven treatment. Colonogenic Survival Assay—The IC50 concentration of irofulven in ovarian cancer cell lines and the colon cancer cell line HCT116 was determined by colonogenic survival assay. Cells were plated in 6-well plates overnight in complete medium. Cells were treated with different concentrations of irofulven for 1 h. Medium was then replaced with fresh drug-free medium and incubated for 7–10 days. Colonies were stained with PBS containing 0.04% crystal violet and 0.5% paraform-aldehyde for about 10 min. Staining liquid was aspirated and colonies counted. Western Blotting—Western blot was performed as described previously (20Wang W. Waters S.J. MacDonald J.R. Roth C. Shentu S. Freeman J. Von Hoff D.D. Miller A.R. Anticancer Res. 2002; 22: 559-564PubMed Google Scholar). Briefly, cell lysates were prepared in cold immunoprecipitation buffer (10 mm Tris, pH 7.4, 1% Triton X-100, 0.5% Nonidet P-40, 150 mm NaCl, 30 mm sodium fluoride, 40 mm β-glycerophosphate, 20 mm sodium pyrophosphate, 1 mm EDTA, 1 mm EGTA, and 0.2 mm phenylmethylsulfonyl fluoride). Total cellular protein (40 μg) was electrophoresed in SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Western blot protein detection was performed using the ECL kit (Amersham Biosciences) according to the manufacturer's recommendations. Monoclonal antibodies against actin and FLAG were purchased from Sigma. Monoclonal antibody against PARP and polyclonal antibodies against ATR, SMC1, NBS1, CDC25A, p53, and phosphorylated p53 on Ser15 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies against phosphorylated CHK1 on Ser345, phosphorylated CHK2 on Thr68, phosphorylated p53 on Ser20, CHK1 and CHK2 were purchased from Cell Signaling Technology (Beverly, MA). Polyclonal antibody against phosphorylated ATM on Ser1981 was purchased from Rockland Immunochemicals (Gilbertsville, PA). Polyclonal antibody for ATM was purchased from Oncogene Research Products (San Diego, CA). Polyclonal antibodies against phosphorylated NBS1 on Ser343 and phosphorylated SMC1 on Ser957 were purchased from Novus Biologicals (Littleton, CO). BrdU Pulse Labeling and Flow Cytometry—Cells were prelabeled with 10 μm BrdU (Roche Applied Science, Indianapolis, IN) for 1 h before treatment with irofulven for 1 h. After drug removal, cells were incubated in drug-free medium and harvested by trypsinization at different time points. Cells were washed once with cold PBS and fixed in 75% ethanol/PBS. Cells were then washed with 1% BSA/PBS and resuspended in 2 n HCl/0.5% Triton X-100. After incubating for 30 min at room temperature, the cells were washed twice with 1% BSA/PBS and resuspended in 0.2 ml of 1% BSA/PBS. Anti-BrdU antibody (20 μl) (BD PharMingen, San Diego, CA) was added to each cell suspension. After incubating in the dark for 30 min at room temperature, cells were centrifuged and resuspended in PBS containing 10 μg/ml propidium iodide, 20 μg/ml RNase A, 0.1% sodium citrate, and 0.1% Triton X-100. Cells were analyzed by FACSCalibur (BD Biosciences). Cell cycle distributions among BrdU-positive cells were analyzed by ModFit v3.0 software (Verity, Topsham, ME). RNA Interference—The sense and antisense oligonucleotides for small interfering RNA (siRNA) were synthesized and annealed by Dharmacon (Lafayette, CO). The sequences of two double-stranded siRNAs for ATM are CATCTAGATCGGCATTCAG and TGGTGCTATTTACGGAGCT. The siRNA sequence for bacterial green fluorescence protein (GFP) gene is TGGAAGCGTTCAACTAGCA. A2780 cells were transfected twice within a 24-h period starting at 40% confluence with 100 nm final concentrations of siRNAs for ATM (two ATM siRNAs were pooled) and GFP using Oligofectamine (Invitrogen) according to the manufacturer's recommendations. Twenty-four hours after transfection, cells were treated with irofulven for 1 h, washed, and incubated in drug-free media, and harvested 12 h later. Cell lysates were prepared for Western blot as described above. Transfection of Kinase-dead CHK2 and Flow Cytometry—Ovarian cancer cell line CAOV3 was transfected twice within a 24-h period with vector or HA-tagged kinase-dead CHK2 (82Busby E.C. Leistritz D.F. Abraham R.T. Karnitz L.M. Sarkaria J.N. Cancer Res. 2000; 60: 2108-2112PubMed Google Scholar) (generously provided by Dr. Jann Sarkaria of the Mayo Clinic and Foundation, Rochester, MN) using FuGENE 6 (Roche Applied Science, Indianapolis, IN) according to the manufacturer's recommendations. Forty-eight hours after transfection, cells were treated with irofulven for 1 h, washed, and incubated in drug-free media, and harvested 12 h later. Cells were then washed with PBS and fixed in 70% ethanol. After washing twice with PBS, cells were permeabilized with 0.25% Triton X-100 in PBS on ice for 15 min, then washed with 1% BSA/PBS, and the cell pellet was suspended in 100 μl of 1% BSA/PBS containing 1 μg of FITC-conjugated anti-HA antibody (Roche Applied Science) for 3 h at room temperature. Cells were washed three times in PBS and then resuspended in PBS containing 10 μg/ml of propidium iodide and 20 μg/ml of RNase A. 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