DNA Mismatch Repair-dependent Activation of c-Abl/p73α/GADD45α-mediated Apoptosis
2008; Elsevier BV; Volume: 283; Issue: 31 Linguagem: Inglês
10.1074/jbc.m709954200
ISSN1083-351X
AutoresLong Shan Li, Julio C. Morales, Arlene Hwang, Mark W. Wagner, David A. Boothman,
Tópico(s)Cholangiocarcinoma and Gallbladder Cancer Studies
ResumoCells with functional DNA mismatch repair (MMR) stimulate G2 cell cycle checkpoint arrest and apoptosis in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G2 arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G2 arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G2 arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73α and GADD45α protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73α/GADD45α signaling in cell death responses. STI571 (Gleevec™, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73α, and GADD45α knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73α knockdown blocked MMR-dependent apoptosis, but not G2 arrest, thereby uncoupling G2 arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73α/GADD45α retrograde signaling. Cells with functional DNA mismatch repair (MMR) stimulate G2 cell cycle checkpoint arrest and apoptosis in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G2 arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G2 arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G2 arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73α and GADD45α protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73α/GADD45α signaling in cell death responses. STI571 (Gleevec™, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73α, and GADD45α knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73α knockdown blocked MMR-dependent apoptosis, but not G2 arrest, thereby uncoupling G2 arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73α/GADD45α retrograde signaling. Loss of DNA mismatch repair (MMR) 3The abbreviations used are: MMR, DNA mismatch repair; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; h, human; 6-TG, 6-thioguanine; shRNA, short hairpin RNA; shSCR, scrambled shRNA; shp53, p53 shRNA; shABL, c-Abl shRNA; shp73, p73α shRNA; shGADD45α, GADD45α shRNA; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; TUNEL, terminal deoxynucleotidyl-transferase dUTP nick end labeling; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 3The abbreviations used are: MMR, DNA mismatch repair; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; h, human; 6-TG, 6-thioguanine; shRNA, short hairpin RNA; shSCR, scrambled shRNA; shp53, p53 shRNA; shABL, c-Abl shRNA; shp73, p73α shRNA; shGADD45α, GADD45α shRNA; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; TUNEL, terminal deoxynucleotidyl-transferase dUTP nick end labeling; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. leads to a mutator phenotype (1Jiricny J. Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (953) Google Scholar). MMR-deficient cells lack G2 arrest responses (2Hawn M.T. Umar A. Carethers J.M. Marra G. Kunkel T.A. Boland C.R. Koi M. Cancer Res. 1995; 55: 3721-3725PubMed Google Scholar) and are resistant (3Kat A. Thilly W.G. Fang W. Longley M.J. Li G. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6424-6428Crossref PubMed Scopus (426) Google Scholar), in terms of cell death and long-term survival, to alkylating agents. MMR-proficient cells respond to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) by inducing G2 arrest and cell death, typically through apoptosis (4Meyers M. Hwang A. Wagner M.W. Boothman D.A. Environ. Mol. Mutagen. 2004; 44: 249-264Crossref PubMed Scopus (53) Google Scholar). Thus, along with correcting DNA mismatches and 1–2-bp loops, MMR also acts to arrest cells in G2, presumably preventing mutation "fixation" through cell division. Furthermore, MMR acts to eliminate damaged cells by signaling apoptosis. The signaling responses leading from MMR-dependent detection of DNA damage to G2 arrest, as well as to downstream alterations in extrinsic or intrinsic apoptotic signals resulting in mitochondrial dysfunction, remain ill defined. Hereditary nonpolyposis colorectal cancers are a result of genetic deletion of MMR genes, typically human (h) MSH2 (mutS homolog-2) or MLH1 (mutL homolog-1) (5Fishel R. Lescoe M.K. Rao M.R.S. Copeland N.G. Jenkins N.A. Garber J. Kane M. Kolodner R. Cell. 1993; 75: 1027-1038Abstract Full Text PDF PubMed Scopus (2598) Google Scholar, 6Bronner C.E. Baker S.M. Morrison P.T. Warren G. Smith L.G. Lescoe M.K. Kane M. Earabino C. Lipford J. Lindblom A. Tannergard P. Bollag R.J. Godwin A.R. Ward D.C. Nordenskjld M. Fishel R. Kolodner R. Liskay R.M. Nature. 1994; 368: 258-261Crossref PubMed Scopus (1923) Google Scholar). Notably, many sporadic human colon cancers also have defective MMR as a result of epigenetic changes such as hMLH1 promoter hypermethylation and loss of expression (7Kim H. Kim Y.H. Kim S.E. Kim N.-G. Noh S.H. Kim H. J. Pathol. 2003; 200: 23-31Crossref PubMed Scopus (71) Google Scholar). These cancers have hallmark microsatellite instability (8Li L.S. Kim N.-G. Kim S.H. Park C. Kim H. Kang H.J. Koh K.H. Kim S.N. Kim W.H. Kim N.K. Kim H. Am J. Pathol. 2003; 163: 1429-1436Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and are resistant (sometimes ≥100-fold) to alkylating agents (9Karran P. Carcinogenesis. 2001; 22: 1931-1937Crossref PubMed Scopus (196) Google Scholar). Apoptosis is a distinct cell death process that occurs under various physiological and pathological situations. Initiation of apoptosis can occur by extrinsic or intrinsic pathways. Both pathways typically culminate in caspase activation. Caspases are cysteine protease zymogens that cleave specific cellular substrates and dismantle the cell, leading to specific morphologic features of apoptosis, including nuclear blebbing, DNA fragmentation, mitochondrial dysfunction, and specific proteolyses of DNA repair proteins. Intrinsic apoptosis is initiated by release of apoptogenic factors such as cytochrome c, apoptosis-inducing factor, and Smac/DIABLO. Once released into the cytosol from mitochondria, cytochrome c interacts with Apaf-1 and activates caspase-9 (10Bao Q. Shi Y. Cell Death Differ. 2007; 14: 56-65Crossref PubMed Scopus (321) Google Scholar). Caspase-9 then activates other effector caspases (e.g. caspase-3, -6, and -7) that dismantle and "clean up" the cell. Extrinsic apoptosis is initiated by stimulation of death receptors of the tumor necrosis factor receptor superfamily, such as CD95 (APO-1/Fas) and TRAIL receptors. In contrast to intrinsic apoptosis that activates caspase-9 from mitochondrial dysfunctional processes (10Bao Q. Shi Y. Cell Death Differ. 2007; 14: 56-65Crossref PubMed Scopus (321) Google Scholar), stimulation of the extrinsic apoptotic pathway initially activates caspase-8, which, in turn, propagates the apoptotic signal by direct cleavage of downstream effector caspases such as caspase-9 and caspase-3. Although activated caspase-8 is fairly specific to the extrinsic apoptotic pathway, once activated, it eventually triggers intrinsic apoptotic processes, causing cytochrome c release and caspase-9 activation (11Wajant H. Science. 2002; 296: 1635-1636Crossref PubMed Scopus (721) Google Scholar). Reciprocally, once caspase-9 is activated, it can stimulate caspase-8 activation. Thus, the temporal kinetics of initial caspase-9 versus caspase-8 activation can be used to differentiate intrinsic versus extrinsic apoptotic signaling pathways, respectively. Alkylating agents are commonly used cancer chemotherapeutic agents that can kill cells by apoptotic processes (12Patenaude A. Deschesnes R.G. Rousseau J.L.C. Petitclerc E. Lacroix J. Cote M.-F. C.-Gaudreault R. Cancer Res. 2007; 67: 2306-2316Crossref PubMed Scopus (23) Google Scholar). Monofunctional alkylating agents can be divided into two major groups, Sn1 and Sn2. Sn2-type alkylating agents include methyl methanesulfonate and dimethyl sulfate. Sn1-type alkylating agents include temozolomide, MNNG, and N-methyl-N′-nitrosourea and are cytotoxic to cancer cells at lower concentrations than Sn2-type agents. Sn1-type compounds have therefore been used more often as antitumor agents with limited success. Treatment of cancer cells with Sn1-type agents such as MNNG and temozolomide gives rise to N7-methylguanine, N3-methyladenine, O4-methylthymine, and O6-methylguanine DNA lesions. N7-Methylguanine and N3-methyladenine DNA lesions are removed by base excision repair. O6-Methylguanine can be repaired by O6-methylguanine-DNA methyltransferase, which removes the methyl group from guanine in a single step and transfers it to an internal cysteine residue (Cys145) on O6-methylguanine-DNA methyltransferase (as reviewed in Ref. 13Gerson S.L. J. Clin. Oncol. 2002; 20: 2388-2399Crossref PubMed Scopus (372) Google Scholar). This reaction inactivates O6-methylguanine-DNA methyltransferase, but directly restores guanine in DNA without DNA incision, synthesis, or ligation steps. Cells that do not express detectable O6-methylguanine-DNA methyltransferase levels or are treated with O6-benzylguanine (a selective O6-methylguanine-DNA methyltransferase inhibitor) are hypersensitive to O6-methylguanine-inducing agents (13Gerson S.L. J. Clin. Oncol. 2002; 20: 2388-2399Crossref PubMed Scopus (372) Google Scholar). Other DNA repair pathways such as nucleotide excision repair and MMR may also process O6-methylguanine lesions (4Meyers M. Hwang A. Wagner M.W. Boothman D.A. Environ. Mol. Mutagen. 2004; 44: 249-264Crossref PubMed Scopus (53) Google Scholar). A hallmark of MMR-mediated response to these DNA lesions is prolonged G2 arrest and lethality. In contrast, MMR-deficient cells are highlighted by "damage tolerance" with heightened mutation rates (14Davis T.W. Patten C. W.-V Meyers M. Kunugi K.A. Cuthill S. Reznikoff C. Garces C. Boland C.R. Kinsella T.J. Fishel R. Boothman D.A. Cancer Res. 1998; 58: 767-778PubMed Google Scholar). Defining the exact retrograde signaling events from MMR-specific DNA lesion detection to G2 arrest and apoptosis remains important in understanding the efficiency of mutational avoidance of the MMR system. Numerous studies have attempted to elucidate the signaling events that occur between MMR detection of DNA lesions in the nucleus and those involved in programmed cell death. Alkylation-induced signaling and apoptotic responses require MutSα (the hMSH2-hMSH6 heterodimer) and MutLα (the hMLH1-hPMS2 heterodimer) protein complexes (9Karran P. Carcinogenesis. 2001; 22: 1931-1937Crossref PubMed Scopus (196) Google Scholar, 15Stojic L. Mojas N. Cejka P. de Pietro M. Ferrari S. Marra G. Jiricny J. Genes Dev. 2004; 18: 1331-1344Crossref PubMed Scopus (197) Google Scholar) and human exonuclease I (16Schaetzlein S. Kodandaramireddy N.R. Ju Z. Lechel A. Stepczynska A. Lilli D.R. Clark A.B. Rudolph C. Kuhnel F. Wei K. Schlegelberger B. Schirmacher P. Kunkel T.A. Greenberg R.A. Edelmann W. Rudolph K.L. Cell. 2007; 130: 863-877Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). A significant number of signaling molecules, including p53, Chk1, Chk2, ATR, ATM, and p38α mitogen-activated protein kinase (MAPK), were reported to regulate MMR-dependent signaling cascades, culminating in G2 arrest and apoptosis (15Stojic L. Mojas N. Cejka P. de Pietro M. Ferrari S. Marra G. Jiricny J. Genes Dev. 2004; 18: 1331-1344Crossref PubMed Scopus (197) Google Scholar, 17Duckett D.R. Bronstein S.M. Taya Y. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12384-12388Crossref PubMed Scopus (157) Google Scholar, 18Hirose Y. Katayama M. Stokoe D. Haas-Kogan D.A. Berger M.S. Pieper R.O. Mol. Cell. Biol. 2003; 23: 8306-8315Crossref PubMed Scopus (124) Google Scholar). A recent study suggested that ATR, and not ATM, is activated in the presence of MutSα and MutLα protein complexes (19Yoshioka K.-i. Yoshioka Y. Hsieh P. Mol. Cell. 2006; 22: 501-510Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), although this evidence was derived from cell-free extracts. In addition to the above-mentioned signaling pathways, a role for p53 in MMR-dependent apoptosis was also suggested. Our laboratory was the first to demonstrate a differential MMR-dependent p53 stabilization after 6-thioguanine (6-TG) or IR exposure (14Davis T.W. Patten C. W.-V Meyers M. Kunugi K.A. Cuthill S. Reznikoff C. Garces C. Boland C.R. Kinsella T.J. Fishel R. Boothman D.A. Cancer Res. 1998; 58: 767-778PubMed Google Scholar). Results from other laboratories suggested that MMR-dependent apoptosis in human colon cancer cells after MNNG or N-methyl-N′-nitrosourea treatment is dependent on p53 and hMLH1, although isogenic cells were not used (20Yanamadala S. Ljungman M. Mol. Cancer Res. 2003; 1: 747-754PubMed Google Scholar). Furthermore, temozolomide-induced apoptotic responses were reported to be dependent on p53 (21Roos W.P. Kaina B. Trends Mol. Med. 2006; 12: 440-450Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar), although these responses were not examined for MMR dependence. In contrast, MNNG- or temozolomide-induced apoptosis was observed in TK6 cells that express viral E6 protein, making cells functionally null for p53 (22Hickman M.J. Samson L.D. Mol. Cell. 2004; 14: 105-116Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Other studies suggested that p73, a homolog of p53, is activated in response to cisplatin in an MMR-specific manner (23Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (504) Google Scholar, 24Gong J. Costanzo A. Yang H.-Q. Melino G. Kaelin W.G. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (833) Google Scholar, 25Yuan Z.-M. Shioya H. Ishiko T. Sun X. Gu J. Huang Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (539) Google Scholar). Indeed, hPMS2 (a binding partner of hMLH1 and component of the hMutLα complex) may directly interact with stabilized p73α after cisplatin exposure (26Shimodaira H. Yoshioka-Yamashita A. Kolodner R.D. Wang J.Y.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2420-2425Crossref PubMed Scopus (135) Google Scholar). A role for p73α in MMR-dependent apoptosis was not, however, mechanistically explored. Thus, to date, an in-depth study of MMR-dependent retrograde (nucleus-to-cytoplasm) signaling that culminates in cell death (apoptosis) has not been performed. Using isogenic hMLH1-deficient versus hMLH1-corrected human colon cancer cells, we elucidated the hMLH1/c-Abl/p73α/GADD45α (growth arrest- and DNA damage-inducible-45α) signaling pathway that controls apoptosis in response to MNNG treatment. We demonstrate functional roles for c-Abl and GADD45α in MMR-dependent G2 arrest, apoptosis, and lethality. We also demonstrate a specific role for c-Abl-induced p73α stabilization in apoptotic responses and survival after MNNG treatment, although this p53 homolog plays no role in G2 arrest responses. Thus, we provide the first evidence that MMR-dependent G2 arrest responses can be uncoupled from apoptosis initiation. G2 arrest may therefore be required for mutational avoidance, but not for apoptosis or lethality. Finally, we show that although p53 is differentially stabilized in an MMR-dependent manner, this signaling response plays no apparent role(s) in G2 arrest responses, apoptosis, or lethality after MNNG treatment. Clinically, our data strongly suggest that inhibitors of c-Abl such as Gleevec™ are ill suited for combination therapy with temozolomide and cisplatin, which depend on functional MMR responses for efficacy. Reagents and Chemicals—MNNG, staurosporine, propidium iodide, puromycin, and RNase A were purchased from the Sigma. MNNG was dissolved in Me2SO at 100 mm and stored at -20 °C. STI571 (Gleevec™, Novartis, East Hanover, NJ) was dissolved in water at 5 mm and stored at -20 °C. Stock concentrations were determined by spectrophotometric analyses. Plasmids and Short Hairpin RNA (shRNA)—Full-length hMLH1 cDNA was generously provided by Drs. A. Buermeyer and R. M. Liskay (Oregon Health Sciences University, Portland, OR). Human DD1 transcriptionally inactive, dominant-negative p53 mutant cDNA was a kind gift from Dr. George Stark (Cleveland Clinic, Cleveland, OH). Human p73α and p73β isoforms were kind gifts from Dr. Meredith Irwin (Hospital for Sick Children Research Institute, Ontario, Canada). Human papillomavirus E6 cDNA was obtained from Dr. C. Reznick (University of Wisconsin Medical School, Madison, WI), cloned into a mammalian cytomegalovirus expression vector, and used to transfect RKO or HCT116 cells as indicated (14Davis T.W. Patten C. W.-V Meyers M. Kunugi K.A. Cuthill S. Reznikoff C. Garces C. Boland C.R. Kinsella T.J. Fishel R. Boothman D.A. Cancer Res. 1998; 58: 767-778PubMed Google Scholar). Human scrambled shRNA (shSCR) sequence and p53 shRNA (shp53) were obtained from Dr. M. Jackson (Case Western Reserve University, Cleveland) (27Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3963) Google Scholar). Cell Culture, Transfections, Treatments, and Colony-forming Ability Assays—HCT116 and HCT116 3-6 cells were generously provided by Dr. C. R. Boland (Baylor College of Medicine, Dallas, TX). MMR-deficient RKO6 and MMR-proficient RKO7 clones were generated by transfecting cytomegalovirus-driven vector-only plasmid DNA or cytomegalovirus-directed plasmid expressing full-length hMLH1 cDNA using Lipofectamine 2000™ (Invitrogen) according to the manufacturer's protocol. Stable clones were selected with G418 by limiting dilution. The human SK-N-AS neuroblastoma cell line was purchased from American Type Culture Collection (Manassas, VA). Mouse MEF1-1 (Gadd45α+/+) and MEF11-1 (Gadd45α-/-) cells were provided by Dr. A. J. Fornace (Georgetown University, Washington, D. C.) and grown as described (28Hollander M.C. Sheikh M.S. Bulavin D.V. Lundgren K. Augeri-Henmueller L. Shehee R. Molinaro T.A. Kim K.E. Tolosa E. Ashwell J.D. Rosenberg M.P. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.-X. Fornace A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar). All other human cells were maintained in Dulbecco's modified Eagle's medium (Cambrex Bio Science, Walkersville, MD) containing 10% fetal bovine serum (HyClone, Logan, UT) supplemented with penicillin (10 units/ml) and streptomycin (10 units/ml). For stable shp53, c-Abl shRNA (shABL), p73α shRNA (shp73), and GADD45α shRNA (shGADD45α) knockdown clones, RKO6 and RKO7 cells were infected with Polybrene-supplemented medium obtained from Phoenix and 293T packaging cells transfected with lentiviral vector pSUPER-p53, retroviral vector pShag2-c-Abl, lentiviral vector pLKO2-p73α, and lentiviral vector pLKO2-GADD45α as described in the accompanying article (46Wagner M.W. Li L.S. Morales J.C. Galindo C.L. Garner H.R. Bornmann W.G. Boothman D.A. J. Biol. Chem. 2008; 283: 21382-21393Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). All drug treatments were performed in medium lacking antibiotics or selective agents. For alkylation exposure, cells were incubated with MNNG (2–10 μm, 1 h). Where indicated, cells were pretreated with 25 μm STI571 for 2 h prior to MNNG exposure. Cells were then washed and scraped, and whole cell extracts were harvested at 24–96 h as indicated. Cells were also pretreated as indicated with the caspase inhibitor Z-VAD-fmk (caspase inhibitor I, EMD Biosciences, La Jolla, CA) or Z-DEVD-fmk (caspase-3 inhibitor II, EMD Biosciences) at 20 or 50 μm, respectively. Colony-forming assays were then performed (14Davis T.W. Patten C. W.-V Meyers M. Kunugi K.A. Cuthill S. Reznikoff C. Garces C. Boland C.R. Kinsella T.J. Fishel R. Boothman D.A. Cancer Res. 1998; 58: 767-778PubMed Google Scholar) three or more times in triplicate, and statistics were completed as described below. Flow Cytometry—For cell cycle distribution assays, adherent and floating cells were treated with 50 μg/ml propidium iodide and 100 μg/ml RNase A at 4 °C and analyzed for DNA content and apoptotic populations using TUNEL assays (29Meyers M. Wagner M.W. Hwang H.-S. Kinsella T.J. Boothman D.A. Cancer Res. 2001; 61: 5193-5201PubMed Google Scholar). Cells were then analyzed using a FACSCalibur flow cytometer (BD Biosciences), and 10,000 events were plotted using CellQuest software. Immunoblot Analyses—Whole cell extracts and Western blots were prepared as described (30Choi Y.R. Kim H. Kang H.J. Kim N.-G. Kim J.J. Park K. -S. Paik Y.-K. Kim H.O. Kim H. Cancer Res. 2003; 63: 2188-2193PubMed Google Scholar). For Western blots, primary antibodies against the following proteins were used at the indicated dilutions: caspase-8 (1C12; Cell Signaling, Beverly, MA), 1:1000; cleaved caspase-9 (Asp315; Cell Signaling), 1:1000; PARP-1 (Cell Signaling), 1:1000; Bax (Cell Signaling), 1:1000; hMLH1 (Ab-2; Oncogene Research Products, Boston, MA), 1:1000; hMSH2 (Ab-1; Oncogene Research Products), 1:500; hPMS2 (Pharmingen), 1:1000; c-Abl (Ab-3; Oncogene Research Products), 1:1000; p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA), 1:5000; GADD45α (H-165; Santa Cruz Biotechnology), 1:1000; glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 6C5; EMD Chemicals, Inc., San Diego, CA), 1:50,000; p21 (Ab-1; EMD Chemicals, Inc.), 1:1000; p73α (5B429; Imgenex Corp., San Diego, CA), 1:2000; and phospho-specific p53 (Ser15; Cell Signaling), 1:1,000. Detection was performed using appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology) and SuperSignal chemiluminescence substrate (Pierce) on Fuji RX medical x-ray film. Statistics—All studies were performed in triplicate at a minimum. The Western blots presented are data from experiments performed three times with similar results. Quantification of protein levels was performed by scanning x-ray films and analyzing the scans using NIH Image J software. Statistical significance was determined using paired Student's t tests. Lethality Correlates with Apoptosis in MNNG-treated MMR-corrected RKO Cells—Human RKO cells are MMR-deficient due to hMLH1 promoter hypermethylation and are incapable of forming the hMutLα complex (i.e. hMLH1-hPMS2 heterodimer), rendering them deficient in both hMLH1 and hPMS2 protein expression. A prior study reported that hMLH1 overexpression is toxic (31Zhang H. Richards B. Wilson T. Lloyd M. Cranston A. Thorburn A. Fishel R. Meuth M. Cancer Res. 1999; 59: 3021-3027PubMed Google Scholar); however, we selected RKO clones that express hMLH1 at levels comparable to endogenous protein amounts in wild-type cells. Selection of clones with hMLH1 levels comparable to endogenous levels in wild-type cells was critical for long-term stability. Various RKO clones were established, and all showed increased sensitivity to MNNG or 6-TG (Fig. 1) (46Wagner M.W. Li L.S. Morales J.C. Galindo C.L. Garner H.R. Bornmann W.G. Boothman D.A. J. Biol. Chem. 2008; 283: 21382-21393Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). However, no clone demonstrated polyploidy in 3 or more years in culture, unlike previous reports of RKO clones in which hMLH1 expression was driven to abnormally high levels (32Yan T. Berry S.E. Desai A.B. Kinsella T.J. Clin. Cancer Res. 2003; 9: 2327-2334PubMed Google Scholar). MMR+ clone RKO7 was chosen to further explore the role of hMLH1 expression in apoptotic responses after MNNG treatment. A dramatic but delayed MMR-dependent apoptotic response in RKO7 cells after a single dose of MNNG (10 μm, 1 h) was observed, with peak levels appearing at 72–96 h post-exposure (Fig. 1A). Apoptosis gradually declined in RKO7 cells at times >96 h, until few cells remained. MMR+ RKO7 cells showed a strong correlation between lethality (i.e. loss of colony-forming ability) and apoptosis induction at 96 h (Fig. 1B). In contrast, MMR- RKO6 cells were not responsive to MNNG in terms of apoptosis (Fig. 1B), even at doses ≥10 μm, where loss of survival was noted. In fact, RKO cells exposed to 20 μm MNNG did not elicit appreciable apoptotic responses, but did show an enlarged and "flattened out" morphology. Cell death in MMR- RKO6 cells after high dose MNNG exposure was not caused by apoptosis. To determine that this MMR-dependent apoptosis response was unique to MNNG treatment, we treated MMR-proficient and MMR-deficient cells with cisplatin. Using TUNEL as an output for apoptosis, we found that there was no appreciable difference in the amount of apoptosis measured in MMR-proficient and MMR-deficient cells (supplemental Fig. 1). MMR-dependent Apoptosis Induced by MNNG Occurs via the Intrinsic Pathway—To determine whether MMR-dependent apoptosis in response to MNNG (10 μm, 1 h) occurred through intrinsic or extrinsic apoptotic pathways, we monitored the kinetics of caspase-8 and caspase-9 as well as PARP-1 proteolyses in RKO6 (MMR-) versus RKO7 (MMR+) cells. Significant caspase-9 activation cleavage (35 kDa) occurred at 48 h post-treatment in RKO7 cells at a time concomitant with significant levels of apoptosis (Fig. 2A, arrow). Notably, caspase-9 cleavage was noted at 48 h post-treatment, nearly 20 h prior to the activation cleavage of caspase-8 (43 kDa; arrow) observed at 72 h. Activation of caspase-9 prior to caspase-8 strongly suggested that the intrinsic apoptotic pathway was activated by MMR in response to MNNG. Pretreatment of MNNG-treated RKO7 cells with the caspase inhibitor Z-VAD-fmk or Z-DEVD-fmk prevented PARP-1 cleavage and abrogated apoptosis (Fig. 2B), suggesting that MMR-mediated apoptosis is caspase-dependent. In contrast, RKO6 (MMR-) cells did not undergo apoptosis in response to MNNG. Exposure of MMR+ and MMR- cells to staurosporine caused apoptosis equally in both cells independent of MMR function (Fig. 2A). Thus, RKO6 (MMR-) cells are functionally capable of inducing apoptosis in response to staurosporine and do not lack the capacity to die by this mechanism. Abrogating p53 Function Does Not Affect MMR-dependent Apoptosis—Our laboratory was the first to report differential MMR-dependent stabilization of p53 in cells after 6-TG or IR treatment, and we hypothesized that MMR-dependent cell death and lethality may be p53-dependent (14Davis T.W. Patten C. W.-V Meyers M. Kunugi K.A. Cuthill S. Reznikoff C. Garces C. Boland C.R. Kinsella T.J. Fishel R. Boothman D.A. Cancer Res. 1998; 58: 767-778PubMed Google Scholar). We confirmed differential p53 stabilization and phosphorylation (Ser15) in RKO7 (MMR+) versus RKO6 (MMR-) cells in response to MNNG (Fig. 3A), a signaling pathway that would be consistent with the activation of ATM, ATR, or DNA-dependent protein kinase (33Burma S. Kurimasa A. Xie G. Taya Y. Araki R. Abe M. Crissman H.A. Ouyang H. Li G.C. Chen D.J. J. Biol. Chem. 1999; 274: 17139-17143Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 34Canman 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 (1704) Google Scholar, 35Kim W.-J. Beardsley D.I. Adamson A.W. Brown K.D. Toxicol. Appl. Pharmacol. 2005; 202: 84-98Crossref PubMed Scopus (24) Google Scholar, 36Tibbetts R.S. Brumbaugh K.M. Williams J.M. Sarkaria J.N. Cliby W.A. Shieh S.-Y. Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; 13: 152-157Crossref PubMed Scopus (863) Google Scholar). Furthermore, significant increases in Bax and p21 expression, known downstream p53 target genes, were observed in RKO7 cells in a prolonged manner compared with transient responses in RKO6 cells (Fig. 3A). As with G2 arrest responses (see Fig. 2 in the accompanying article) (46Wagner M.W. Li L.S. Morales J.C. Galindo C.L. Garner H.R. Bornmann W.G. Boothman D.A. J. Biol. Chem. 2008; 283: 21382-21393Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), RKO6 cells responded initially to MNNG by stabilizing and phosphorylating p53 (Ser15) from 4–12 h, with accompanying minor increases in p21 and Bax; however, the responses rapidly declined as noted (14Davis T.W. Patten C. W.-V Meyers M. Kunugi K.A. Cuthill S. Reznikoff C. Garces C. Boland C.R. Kinsella T.J. Fishel R. Boothman D.A. Cancer Res. 1998; 58: 767-778PubMed Google Scholar). Thus, elevated transcriptionally functional p53 levels may be involved in MMR-dependent apoptosis after MNNG exposure. To examine its role in MMR signaling, we functionally inactivated p53 using several different methods in MMR- versus MMR+ RKO cells. p53 was functionally inactivated in RKO cells by E6 expression or stable shRNA-mediated p53 knockdown. E6-expressing RKO7 clones (i.e. B2, B4, B5, and B9) were isolated and expressed significantly less p53 protein compare with vector alone RKO7 (MMR+) cells (Fig. 3B). RKO7 cells that expressed E6 and were functionally depleted of p53 protein levels showed similar levels of apoptosis as vector alone containing RKO7 cells in response to MNNG (Fig. 3B). In contrast, RKO6 cells expressing or lacking E6 did not elicit apoptosis after MNNG exposure. Expression of E6 in HCT116 3-6 cells did not affect MNNG-induced apoptosis (Fig. 2A and supplemental Fig. 2). To further confirm that p53 was not required for MMR-mediated apoptosis, we generated stable knocked down p53 using shp53 (Fig. 3C). Stable clones showed no detectable p53 protein levels in either RKO6 or RKO7 cells after stable infection
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