5-ASA Affects Cell Cycle Progression in Colorectal Cells by Reversibly Activating a Replication Checkpoint

Background & Aims: Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. Methods: CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116p53−/−, HCT116+chr3, and LoVo were treated with 5-ASA for 2–96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. Results: We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Conclusions: Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis. Background & Aims: Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. Methods: CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116p53−/−, HCT116+chr3, and LoVo were treated with 5-ASA for 2–96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. Results: We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Conclusions: Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis. Patients with inflammatory bowel disease (IBD) of the colon have increased risk of developing colorectal cancer (CRC).1Itzkowitz S.H. Yio X. Inflammation and cancer IV Colorectal cancer in inflammatory bowel disease: the role of inflammation.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G7-G17Crossref PubMed Scopus (1076) Google Scholar Early age at diagnosis, extent of disease, severity of inflammation, presence of primary sclerosing cholangitis, and family history of cancer have been established as independent risk factors for the development of CRC in ulcerative colitis (UC).1Itzkowitz S.H. Yio X. Inflammation and cancer IV Colorectal cancer in inflammatory bowel disease: the role of inflammation.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G7-G17Crossref PubMed Scopus (1076) Google Scholar, 2Ekbom A. Helmick C. Zack M. Adami H.O. 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The chemopreventive efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) against intestinal tumors has been well established, but, because NSAIDs may aggravate the symptoms of colitis, their sustained use for the purpose of CRC chemoprevention has been contraindicated in IBD. A promising candidate drug for chemoprevention in IBD patients is mesalazine, (the active compound of sulfasalazine) or 5-aminosalicylic acid (5-ASA). In fact, the discontinuation of 5-ASA therapy was associated with a higher CRC risk.5Eaden J. Abrams K. Ekbom A. Jackson E. Mayberry J. Colorectal cancer prevention in ulcerative colitis: a case-control study.Aliment Pharmacol Ther. 2000; 14: 145-153Crossref PubMed Scopus (529) Google Scholar Successful prevention of colonic dysplasia and cancer further supports the potential role of 5-ASA as a chemopreventive agent in IBD,6Rubin D.T.D.A. Huo D.Z. Yadron N. Hanauer S.B. Use of 5-ASA is associated with decreased risk of dysplasia and colon cancer in ulcerative colitis.Gastroenterology. 2003; 124: A36Abstract Full Text PDF Google Scholar but the mechanism underlying these effects remains unknown.7Croog V.J. Ullman T.A. Itzkowitz S.H. Chemoprevention of colorectal cancer in ulcerative colitis.Int J Colorectal Dis. 2003; 18: 392-400Crossref PubMed Scopus (42) Google Scholar It is assumed that 5-ASA has similar genetic and molecular targets as NSAIDs, which is further supported by its structural similarity with aspirin. It was reported that 5-ASA had antiproliferative effects on colon cancer cell lines,8Allgayer H. Review article: mechanisms of action of mesalazine in preventing colorectal carcinoma in inflammatory bowel disease.Aliment Pharmacol Ther. 2003; 18: 10-14Crossref PubMed Scopus (100) Google Scholar but other studies have failed to show a significant antiproliferative effect of 5-ASA against several colon cancer cells in culture9MacGregor D.J. Kim Y.S. Sleisenger M.H. Johnson L.K. Chemoprevention of colon cancer carcinogenesis by balsalazide: inhibition of azoxymethane-induced aberrant crypt formation in the rat colon and intestinal tumor formation in the B6-Min/+ mouse.Int J Oncol. 2000; 17: 173-179PubMed Google Scholar or against colon tumors in a rat model.10Millar A.D. Rampton D.S. Chander C.L. Claxson A.W. Blades S. Coumbe A. Panetta J. Morris C.J. Blake D.R. Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis.Gut. 1996; 39: 407-415Crossref PubMed Scopus (263) Google Scholar Several groups found that 5-ASA significantly induced apoptosis and decreased proliferation in colorectal mucosa in patients.11Reinacher-Schick A. Seidensticker F. Petrasch S. Reiser M. Philippou S. Theegarten D. Freitag G. Schmiegel W. Mesalazine changes apoptosis and proliferation in normal mucosa of patients with sporadic polyps of the large bowel.Endoscopy. 2000; 32: 245-254Crossref PubMed Scopus (80) Google Scholar, 12Bus P.J. Nagtegaal I.D. Verspaget H.W. Lamers C.B. Geldof H. Van Krieken J.H. Griffioen G. Mesalazine-induced apoptosis of colorectal cancer: on the verge of a new chemopreventive era?.Aliment Pharmacol Ther. 1999; 13: 1397-1402Crossref PubMed Scopus (105) Google Scholar Most studies have focused on 5-ASA's anti-inflammatory properties. A few reports suggest that 5-ASA might act by blocking the transcription factor nuclear factor (NF)-κB and/or stabilizing the inhibitor IκB both in vivo13Bantel H. Berg C. Vieth M. Stolte M. Kruis W. Schulze-Osthoff K. Mesalazine inhibits activation of transcription factor NF-κB in inflamed mucosa of patients with ulcerative colitis.Am J Gastroenterol. 2000; 95: 3452-3457Crossref PubMed Google Scholar and in mouse models.14Kaiser G.C. Yan F. Polk D.B. Mesalamine blocks tumor necrosis factor growth inhibition and nuclear factor κB activation in mouse colonocytes.Gastroenterology. 1999; 116: 602-609Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar It has been recently reported that 5-ASA affects the Wnt/β-catenin pathway in adenomatous polyposis coli (APC) mutated cells, by inhibiting the activity of PP2A phosphatase and therefore stimulating β-catenin degradation.15Bos C.L. Diks S.H. Hardwick J.C. Walburg K.V. Peppelenbosch M.P. Richel D.J. Protein phosphatase 2A is required for mesalazine-dependent inhibition of Wnt/{β}-catenin pathway activity.Carcinogenesis. 2006; 27: 2371-2382Crossref PubMed Scopus (68) Google Scholar 5-ASA has also been shown to interact with the Wnt/β-catenin target peroxisome proliferator-activated receptor (PPAR)-γ, which is involved in inflammation-driven colon carcinogenesis.16Su C.G. Wen X. Bailey S.T. Jiang W. Rangwala S.M. Keilbaugh S.A. Flanigan A. Murthy S. Lazar M.A. Wu G.D. A novel therapy for colitis utilizing PPAR-γ ligands to inhibit the epithelial inflammatory response.J Clin Invest. 1999; 104: 383-389Crossref PubMed Scopus (738) Google Scholar A recent study showed that 5-ASA treatments had a beneficial effect on colitis induced in wild-type mice but not in heterozygous PPAR-γ (+/−) mice.17Rousseaux C. Lefebvre B. Dubuquoy L. Lefebvre P. Romano O. Auwerx J. Metzger D. Wahli W. Desvergne B. Naccari G.C. Chavatte P. Farce A. Bulois P. Cortot A. Colombel J.F. Desreumaux P. Intestinal anti-inflammatory effect of 5-aminosalicylic acid is dependent on peroxisome proliferator-activated receptor-gamma.J Exp Med. 2005; 201: 1205-1215Crossref PubMed Scopus (417) Google Scholar In epithelial cells, 5-ASA increased PPAR-γ expression and its activation and promoted its translocation into the nucleus, all effects independent from its weak cyclooxygenase (COX) inhibitory properties. Finally, Monteleone et al18Monteleone G. Franchi L. Fina D. Caruso R. Vavassori P. Monteleone I. Calabrese E. Naccari G.C. Bellinvia S. Testi R. Pallone F. Silencing of SH-PTP2 defines a crucial role in the inactivation of epidermal growth factor receptor by 5-aminosalicylic acid in colon cancer cells.Cell Death Differ. 2006; 13: 202-211Crossref PubMed Scopus (38) Google Scholar recently showed that 5-ASA disrupts epidermal growth factor receptor (EGFR) signaling and inhibits EGFR activation by enhancing the activity of the SH-PTP1 and SH-PTP2 phosphatases both in CRC cell lines and in ex vivo organ cultures of CRC explants. One mechanism by which genome stability is maintained and cellular proliferation is regulated is that of activating checkpoint responses.19Hartwell L.H. Weinert T.A. Checkpoints: controls that ensure the order of cell cycle events.Science. 1989; 246: 629-634Crossref PubMed Scopus (2568) Google Scholar Cell cycle checkpoints are activated by DNA damage and incomplete DNA replication.20Zhou B.B. Elledge S.J. The DNA damage response: putting checkpoints in perspective.Nature. 2000; 408: 433-439Crossref PubMed Scopus (2679) Google Scholar If DNA synthesis is inhibited or disturbed, an S-phase checkpoint stabilizes replication components and slows down DNA synthesis.21Dimitrova D.S. Gilbert D.M. Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis.Nat Cell Biol. 2000; 2: 686-694Crossref PubMed Scopus (135) Google Scholar Recent lines of research suggested that 5-ASA alters cell cycle progression22Reinacher-Schick A. Schoeneck A. Graeven U. Schwarte-Waldhoff I. Schmiegel W. Mesalazine causes a mitotic arrest and induces caspase-dependent apoptosis in colon carcinoma cells.Carcinogenesis. 2003; 24: 443-451Crossref PubMed Scopus (98) Google Scholar: when colon cells were exposed to high doses of 5-ASA, an increase of aberrant mitotic cells and mitotic arrest was observed, followed by cellular death. Our group has recently determined the influence of 5-ASA on the rate of mutation in a repetitive DNA sequence in cultured colorectal cells; this compound reduces the rate of spontaneous mutations, independently of its anti-inflammatory properties.23Gasche C. Goel A. Natarajan L. Boland C.R. Mesalazine improves replication fidelity in cultured colorectal cells.Cancer Res. 2005; 65: 3993-3997Crossref PubMed Scopus (52) Google Scholar We hypothesized that one key mechanism by which 5-ASA reduces the mutation rate is by interacting with cellular machineries involved in progression through the cell cycle. This would result in slowing down processes such as DNA replication (S phase) and cell division (mitosis) through the onset of cell cycle checkpoints, which would give the cell the opportunity to either repair the damage that the DNA may have encountered or undergo apoptosis. In both cases, this would prevent accumulation of mutated or damaged cells and would lead to maintenance of DNA integrity. In this study, we therefore investigated the effects of 5-ASA cell cycle progression with the aim of determining the underlying cellular mechanisms. Here, we show that 5-ASA inhibits the proliferation of colon cancer cells in a dose- and time-dependent manner and leads to the accumulation of cells in S phase independently of both p53 and an active mismatch repair (MMR) complex. High concentrations of 5-ASA cause apoptosis and induce the appearance of abnormal mitotic cells. HT29 (mutant p53R273H), LoVo (hMSH2 mutant), and HCT116 (hMLH1 mutant) colorectal cancer cells were obtained from the ATCC. HCT116p53−/− cells (p53 null) were originated in Prof. Burt Vogelstein's laboratory as described.24Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Requirement for p53 and p21 to sustain G2 arrest after DNA damage.Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2592) Google Scholar HCT116+chr3 (hMLH1 wild-type) were a kind gift from Dr. Boland's laboratory.25Koi M. Umar A. Chauhan D.P. Cherian S.P. Carethers J.M. Kunkel T.A. Boland C.R. Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N'-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation.Cancer Res. 1994; 54: 4308-4312PubMed Google Scholar Cells were grown in monolayers in Iscove's modified Dulbecco's medium (IMDM; GIBCO/Invitrogen, Lofer, Austria) containing 2 nmol/L glutamine and 10% fetal bovine serum (FBS) at 5% CO2. The medium for HCT116+chr3 contained 400 μg/mL of G418 (GIBCO/Invitrogen). 5-ASA (Sigma-Aldrich Handels GmbH, Vienna, Austria) was dissolved in culture medium at 40 mmol/L and pH adjusted to 7.2 with NaOH. Aphidicolin (Sigma), Nocodazole (Sigma), and bromodeoxyuridine (BrdU) (Sigma) were dissolved in dimethyl sulfoxide (DMSO) and sterile filtered. Caffeine (Sigma) was dissolved in H2O and used at 5 mmol/L. Cells were treated at subconfluent densities. All experiments were performed in triplicate. Total cell lysates were obtained by harvesting pellets of treated cells in lysis buffer (20 mmol/L Hepes, pH 7.5, 100 mmol/L NaCl, 0.2% NP-40, 10% glycerol, 1 mmol/L EDTA, 1 mmol/L DTT) in the presence of protease inhibitors (1 mmol/L Pefabloc [Promega GmbH, Mannheim, Germany], 1 mmol/L aprotinin, and Complete Protease Inhibitor mix [Roche]) and phosphatase inhibitors (50 mmol/L NaF, 50 mmol/L β-glycerophosphate, and 1 mmol/L Na3VO4 [Sigma]). Protein lysates were clarified by centrifugation at 16,000g for 15 minutes at 4°C. To obtain the DNA-bound protein fraction, after the first lyses, the nuclear pellets were resuspended in urea-based buffer (6 mol/L urea; 150 mmol/L NaCl; 0.5% NP-40), the DNA was fragmented by sonication, and all cellular debris was precipitated by centrifugation (16,000 rpm/20 minutes at 4°C). Cell lysates and DNA-bound protein fractions were snap frozen in liquid N2 and kept at −80°C until use. Western blots were performed according to standard procedures. Briefly, equal amounts of proteins (50–150 μg) were denatured in SDS-sample buffer and resolved on NuPAGE Tris-Acetate 4%–12% gradient gels (Invitrogen). Proteins were transferred onto a Hybond-P (Amersham Pharmacia Biotech, Uppsala, Sweden) nitrocellulose membrane in Tris-Glycine buffer and 20% methanol for 3 hours. Membranes were blocked in phosphate-buffered saline (PBS) and 5% nonfat milk for 1 hour, incubated with the appropriate primary antibody according to the manufacturer's protocol, followed by the specific horseradish peroxidase (HRP)-conjugated secondary antibody. Chemoluminescent signals were detected using ECL reagents (Amersham). All antibodies for immunoblotting were diluted in PBS, 3% BSA, and 0.03% NaN3. Antibodies used were as follows: mouse monoclonal antibody (mAb) anti-p53 DO7 (Calbiochem); rabbit polyclonal antibody (pAb) antiphospho-p53 Ser15 (Cell Signaling Technology, Danvers, MA); mAb anti-p21waf1/cip1 (Cell Signaling); pAb for hMSH2 and hMLH1 (Pharmingen/Becton-Dickinson, Cowley, Oxford, UK); pAb antitubulin (Abcam, Cambridge, UK); pAb anti-ATR; mAb anti-MCM2; pAb anti-RPA; pAb anti-Cdc45; pAb anti-γH2AX and pAb anti-phospho-γH2AX Ser39; pAb anti-Claspin; pAb anti-Chk1 and pAb anti-phospho-Chk1 Ser317 and Ser345 (Cell Signaling); and pAb anti-Rad17 and pAb anti-phospho-Rad17 Ser645 (Cell Signaling). Yeast DNA polymerase δ26Burgers P.M. Gerik K.J. Structure and processivity of two forms of Saccharomyces cerevisiae DNA polymerase δ.J Biol Chem. 1998; 273: 19756-19762Crossref PubMed Scopus (165) Google Scholar was kindly provided by P. M. Burgers. Cells were plated in 96-well microtiter plates at a density of 5000 cells/well in medium. Twenty-four hours later, cells were treated with 5-ASA (0–20 mmol/L). After treatment, cells were incubated for 3 hours at 37°C with a solution of MTT (Sigma) at a concentration of 50 mg/100 mL. The cells were lysed in 10% SDS and 0.01 N HCl for 12 hours. For each sample, the absorbance of the reduced intracellular formazan product was read at 570 nm in a microtiter plate reader (Bio-Rad Laboratories, Hercules, CA). Each assay was performed in triplicate. Cells were harvested, washed in PBS, and fixed in 70% ethanol for 30 minutes at room temperature; subsequently, they were centrifuged for 5 minutes at 1500 rpm, resuspended in PBS-containing propidium iodide (20 mg/mL, Sigma) and RNAse (60 mg/mL, Sigma), and stained at room temperature in the dark for 15 minutes. DNA content was measured by flow cytometry, and the cell cycle distribution was analyzed using CellQuest Software (BD Technology, Mountain View, CA). For the analysis of the S-phase population, subconfluent adherent cells were pulsed with 10 μmol/L BrdU (Sigma) for 60 minutes prior to harvesting and fixation in 70% ethanol. Cells were subsequently denatured in 2 mol/L HCl and stained with anti-BrdU monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by FITC-conjugated secondary anti-mouse IgG (Molecular Probes/Invitrogen, Lofer, Austria). After staining with propidium iodide solution, cells were analyzed by flow cytometry as described. Cells were seeded in 8-well chambers (Nalgene; Nalge Ltd, Hereford, UK) at 37°C to subconfluent density. Twenty-four hours later, cells were treated with 5-ASA for 24–48 hours. After treatment, cells were fixed in freshly prepared 4% paraformaldehyde (pH 7.4). Cells were permeabilized in 0.5% Triton/PBS for 10 minutes and then blocked with 3% BSA/PBS for 1 hour. After blocking, cells were incubated with mAb antitubulin (Santa Cruz) for 2 hours, to stain the mitotic spindle or the interphasic filaments, followed by FITC-conjugated secondary anti-mouse IgG (Molecular Probes) for 45 minutes. Cells were mounted onto slides in the presence of the DAPI-containing mounting medium (Vectashield, Burlingame, CA) and visualized using a Carl Zeiss Axioplan 2 fluorescence microscope. Images were captured with a Photometrics CoolSNAP fx digital camera (Roper Scientific, Tuscon, AZ) and processed using MetaMorph (Molecular Devices, Downingtown, PA) and Canvas X (ACD Systems, British Columbia, Canada) software. The fidelity of polymerase (Pol) δ was measured using an in vitro DNA synthesis assay with an M13mp2-derived gapped substrate that contained the lacZα gene in the gapped region, as previously described.27Bebenek K. Kunkel T.A. Analyzing fidelity of DNA polymerases.Methods Enzymol. 1995; 262: 217-232Crossref PubMed Scopus (193) Google Scholar The template contained a run of 7 template T residues in the +1 reading frame that yields colorless M23 plaques. Single-base deletion errors by Pol δ restore the correct reading frame and yield dark blue M13 plaques. To fill the gap, Pol δ was incubated with gapped M13mp2 molecules in a reaction mixture containing 40 mmol/L Tris-HCl, pH 7.8, and 8 mmol/L magnesium acetate and each of the 4 dNTPs at 250 μmol/L. Reactions also contained either 0.1 mmol/L, 1 mmol/L, or 10 mmol/L 5-ASA or solvent alone (4% DMSO) as a control. Reaction mixtures were incubated at 30°C for 60 minutes. Gap filling was confirmed by gel electrophoresis,27Bebenek K. Kunkel T.A. Analyzing fidelity of DNA polymerases.Methods Enzymol. 1995; 262: 217-232Crossref PubMed Scopus (193) Google Scholar and aliquots of reaction products were introduced into the MC1061 mutS strain of Escherichia coli by electroporation, and samples were plated along with the CSH50 complementation strain on minimal media containing IPTG and X-Gal.27Bebenek K. Kunkel T.A. Analyzing fidelity of DNA polymerases.Methods Enzymol. 1995; 262: 217-232Crossref PubMed Scopus (193) Google Scholar The reversion frequency was calculated from the ratio of revertant (blue) plaques to the total number of plaques scored. Changes in the cell cycle distribution were measured by parent t test. A P value of <.001 was regarded as significant. Experiments were repeated at least 3 times. The effect of 5-ASA on cellular viability and cellular growth was evaluated by an assay based on the activity of mitochondrial dehydrogenase, which cleaves a soluble yellow tetrazolium salt (MTT) to blue formazan crystals, a conversion that occurs only in living cells. HCT116, LoVo, and HT29 cells were treated with 5-ASA (0–20 mmol/L) for 48 hours. 5-ASA treatment induced a significant concentration-dependent reduction in the proliferation of all cell lines (Figure 1). We aimed to establish whether the reduction in growth in CRC cells was due to 5-ASA-induced changes in cell cycle progression. HT29, HCT116, and LoVo cells were cultured in the presence of 0–20 mmol/L ASA for 48 hours, and the cell cycle distribution was analyzed by flow cytometry (Figure 2A). Upon treatment with 10–20 mmol/L 5-ASA, the cell cycle profile of all cell lines changed within 48 hours, with an apparent increase in the number of cells in S phase. The best effect on cell proliferation arrest was observed at 20 mmol/L 5-ASA. Higher 5-ASA concentrations (above 20 mmol/L) affected cell survival (data not shown).Figure 2Cell cycle distribution in 5-ASA-treated cells. (A) HT29, HCT116, and LoVo cell lines were treated with 5, 10, or 20 mmol/L 5-ASA for 48 hours. After staining with propidium iodide, the cellular DNA content was visualized by flow cytometry, and the cell cycle distribution was analyzed using CellQuest flow cytometry software (BD Technology). All cell lines showed changes in the cell cycle profile following 5-ASA exposure within 48 hours of treatment. In further experiments, HCT116 cells (B) and HT29 cells (C) were exposed to 5, 10, or 20 mmol/L 5-ASA for 48 hours. Following treatment, 5-ASA was removed, and the cells were pulsed with 10 μmol/L BrdU for 1 hour. After permeabilization and DNA-antigen unmasking, samples were stained with anti-BrdU antibody to visualize replicating cells and PI to visualize the total DNA content. Cells were analyzed by flow cytometry using CellQuest flow cytometry software (BD Technology). Both cell lines showed an increase in the percentage of cells in S phase following treatment with 5-ASA. (D) HCT116 cells were treated with low doses of 5-ASA (1.25–5 mmol/L) for 120 hours (upper panels) or 168 hours. After pulsing with 10 μmol/L BrdU and staining with anti-BrdU antibody and PI, cells were analyzed by flow cytometry. At later time points, an increase in the S-phase arrested population is visible at 1.25 mmol/L 5-ASA. In the flow cytometry dot blot, the upper region (R2) contains cells in S phase, the lower left region (R3) represents cells in G1, and the lower right region (R4) represents cells in G2/M. The flow cytometry profiles are representative of at least 3 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The cell cycle distribution that we observed in colon cells upon treatment with 5-ASA would be consistent with an increase in the proportion of replicating cells (S phase), suggesting that DNA replication is slowed down in these cells. Fluorescent detection of BrdU incorporation into newly synthesized DNA provides an exact measure of the number of cells in S phase. To verify our observation, we pulsed 5-ASA-treated cells with BrdU for 1 hour and measured the number of actively replicating cells by flow cytometry analysis. After 48 hours exposure to 5-ASA, both HCT116 (Figure 2B) and HT29 (Figure 2C) showed a significant increase in the number of cells in S phase when compared with the nontreated population (percentage S phase-HCT116: 0 mmol/L: 47.8% ± 2.9%; 10 mmol/L: 62.6% ± 2.8%; HT29: 0 mmol/L: 42.2% ± 3.6%; 10 mmol/L: 59.1% ± 3.8%, P < .0001). Moreover, prolonged treatment with low doses of 5-ASA (1.25–2.5 mmol/L) for 120 hours (Figure 2D) caused a similar S-phase arrest (120 hours: percentage S-phase-HCT116: 0 mmol/L: 46.9% ± 1.8%; 2.5 mmol/L: 61.1% ± 0.3%, P < .0001), suggesting that the increase in the replicating population upon 5-ASA-treatment is dose and time dependent. The DNA MMR system repairs base mismatches after DNA replication, inhibits recombination between nonidentical DNA sequences, and provokes both checkpoint and apoptotic responses following certain types of DNA damage. It was recently demonstrated that aspirin alters expression of MMR proteins and induces apoptosis through COX- and p53-independent mechanisms.28Goel A. Chang D.K. Ricciardiello L. Gasche C. Boland C.R. A novel mechanism for aspirin-mediated growth inhibition of human colon cancer cells.Clin Cancer Res. 2003; 9: 383-390PubMed Google Scholar MMR-deficient cells are resistant to many chemotherapeutic agents and are capable of bypassing the S and G2/M checkpoint in vitro.29Yan T. Schupp J.E. Hwang H.S. Wagner M.W. Berry S.E. Strickfaden S. Veigl M.L. Sedwick W.D. Boothman D.A. Kinsella T.J. Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation.Cancer Res. 2001; 61: 8290-8297PubMed Google Scholar We therefore investigated whether MMR-deficient and -proficient colonic cells respond differently to 5-ASA-treatment: hMLH1-mutant HCT116 cells and the nearly isogenic HCT116+chr3 cells30Chauhan D.P. Yang Q. Carethers J.M. Marra G. Chang C.L. Chamberlain S.M. Boland C.R. Antisense inhibition of hMLH1 is not sufficient for loss of DNA mismatch repair function in the HCT116+chromosome 3 cell line.Clin Cancer Res. 2000; 6: 3827-3831PubMed Google Scholar (in which an additional copy of chromosome 3, bearing the hMLH1 wild-type gene, has been reintroduced; Figure 3A) were exposed to 5-ASA for 48 hours (Figure 3C, upper and middle panels, respectively). Both cell lines showed a significant increase in the S-phase population, with no considerable difference between the MLH1-expressing and the MLH1-mutant cell line (percentage S phase-HCT116+chr3: 0 mmol/L: 50.8% ± 1.1%; 10 mmol/L: 63.8% ± 2.6%, P < .0001). This suggests that hMHL1 does not have a significant role in tuning the response to 5-ASA. DNA damage leads primarily to G1 arrest in p53 wild-type cells, whereas p53-defective tumor cells preferentially arrest in S phase or G2/M.31Yao S.L. Akhtar A.J. McKenna K.A. Bedi G.C. Sidransky D. Mabry M. Ravi R. Collector M.I. Jones R.J. Sharkis S.J. Fuchs E.J. Bedi A. Selective radiosensitization of p53-deficient cells by caffeine-mediated activation of p34cdc2 kinase.Nat Med. 1996; 2: 1140-1143Crossref PubMed Scopus (159) Google Scholar In our study, colon cells expressing either wild-type p53 (HCT116) or a mutant form of the tumor suppressor protein (HT29) responded similarly to 5-ASA treatment by arresting in S phase. It has been reported that the presence of mutations in the p53 gene does not affect DNA replication fidelity or DNA repair.3