Epigenomic Stress Response
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.m213219200
ISSN1083-351X
AutoresSnezana Milutinovic, Qianli Zhuang, Alain Niveleau, Moshe Szyf,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2′-deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novosynthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such asNF-κB, JunB, ATF-3, and GADD45β (growth arrestDNA damage 45β gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information. The DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2′-deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novosynthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such asNF-κB, JunB, ATF-3, and GADD45β (growth arrestDNA damage 45β gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information. 5-aza-2′-deoxycytidine phosphate-buffered saline bovine serum albumin bromodeoxyuridine BCL2-interacting killer antibody reverse transcriptase Proper epigenomic regulation of gene expression is essential for the integrity of cell function. One critical component of the epigenome is the pattern of distribution of methylated cytosines in CG dinucleotide sequences in the genome (1Razin A. EMBO J. 1998; 17: 4905-4908Crossref PubMed Scopus (662) Google Scholar). Methylation of CGs marks genes for inactivation by either interfering with the binding of methylated DNA-sensitive transcription factors (2Prendergast G.C. Ziff E.B. Science. 1991; 251: 186-189Crossref PubMed Scopus (432) Google Scholar) or by recruiting methylated DNA-binding proteins such as MeCP2, which in turn recruit corepressor complexes and histone deacetylases to the chromatin associated with the gene (3Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2802) Google Scholar). The methylation pattern can thus determine the chromatin structure and state of activity of genes. Disruption in the proper maintenance of the DNA methylation pattern results in aberrant gene expression, as is observed in tumor suppressor genes that are hypermethylated in cancer (4Merlo A. Herman J.G. Mao L. Lee D.J. Gabrielson E. Burger P.C. Baylin S.B. Sidransky D. Nat. Med. 1995; 1: 686-692Crossref PubMed Scopus (1880) Google Scholar). Aberrant hypomethylation can also result in improper activation of genes (5Jackson-Grusby L. Beard C. Possemato R. Tudor M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. Lander E. Jaenisch R. Nat. Genet. 2001; 27: 31-39Crossref PubMed Scopus (573) Google Scholar). The main enzyme responsible for replicating the DNA methylation pattern is DNMT1 (DNA methyltransferase1). This enzyme shows preference for hemimethylated DNA and is therefore believed to faithfully copy the DNA methylation pattern (6Gruenbaum Y. Cedar H. Razin A. Nature. 1982; 295: 620-622Crossref PubMed Scopus (310) Google Scholar). Multiple mechanisms have been proposed to coordinate the inheritance of DNA methylation patterns with DNA replication. First, DNMT1 expression is regulated with the cell cycle (7Detich N. Ramchandani S. Szyf M. J. Biol. Chem. 2001; 276: 24881-24890Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 8Szyf M. Bozovic V. Tanigawa G. J. Biol. Chem. 1991; 266: 10027-10030Abstract Full Text PDF PubMed Google Scholar), and it is up-regulated by proto-oncogenes Ras and Jun (9MacLeod A.R. Rouleau J. Szyf M. J. Biol. Chem. 1995; 270: 11327-11337Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 10Rouleau J. MacLeod A.R. Szyf M. J. Biol. Chem. 1995; 270: 1595-1601Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 11Bigey P. Ramchandani S. Theberge J. Araujo F.D. Szyf M. Gene (Amst.). 2000; 242: 407-418Crossref PubMed Scopus (108) Google Scholar), Fos (12Bakin A.V. Curran T. Science. 1999; 283: 387-390Crossref PubMed Scopus (216) Google Scholar), and T antigen (13Slack A. Cervoni N. Pinard M. Szyf M. J. Biol. Chem. 1999; 274: 10105-10112Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Second, DNMT1 is localized to the replication fork (14Leonhardt H. Page A.W. Weier H.U. Bestor T.H. Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (832) Google Scholar) and is associated with the replication protein proliferating cell nuclear antigen (15Chuang L.S. Ian H.I. Koh T.W. Ng H.H. Xu G. Li B.F. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (787) Google Scholar). Third, DNA methylation occurs concurrently with DNA replication (16Araujo F.D. Knox J.D. Szyf M. Price G.B. Zannis-Hadjopoulos M. Mol. Cell. Biol. 1998; 18: 3475-3482Crossref PubMed Scopus (74) Google Scholar). This temporal and physical association of DNMT1 with DNA replication is believed to have evolved to guarantee concordant replication of DNA and its methylation pattern. Previous studies have shown that inhibition of DNMT1 can lead to inhibition of initiation of DNA replication (17Knox J.D. Araujo F.D. Bigey P. Slack A.D. Price G.B. Zannis-Hadjopoulos M. Szyf M. J. Biol. Chem. 2000; 275: 17986-17990Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), but it is not clear whether this response is a consequence of induction of tumor suppressor genes such as p21 (18Milutinovic S. Knox J.D. Szyf M. J. Biol. Chem. 2000; 275: 6353-6359Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) or p16 (19Fournel M. Sapieha P. Beaulieu N. Besterman J.M. MacLeod A.R. J. Biol. Chem. 1999; 274: 24250-24256Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), leading to retreat from the cell cycle. A conditional knockout of murine dnmt1 gene was also shown to reduce the rate of cell division (5Jackson-Grusby L. Beard C. Possemato R. Tudor M. Fambrough D. Csankovszki G. Dausman J. Lee P. Wilson C. Lander E. Jaenisch R. Nat. Genet. 2001; 27: 31-39Crossref PubMed Scopus (573) Google Scholar), but it is still unclear whether inhibition of DNMT1 leads to a change in cell cycle kinetics similar to DNA damage response checkpoints. Multiple mechanisms have been established to guard the integrity of the genome in response to DNA damage. For example, two parallel, cooperating mechanisms, both regulated by ATM, jointly contribute to the rapid and transient inhibition of firing of origins of DNA replication in response to ionizing radiation (20Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar, 21Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (873) Google Scholar, 22Maser R.S. Mirzoeva O.K. Wells J. Olivares H. Williams B.R. Zinkel R.A. Farnham P.J. Petrini J.H. Mol. Cell. Biol. 2001; 21: 6006-6016Crossref PubMed Scopus (184) Google Scholar). This stalling of DNA synthesis is required to prevent genetic instability by coordinating replication and repair. We reasoned that similar mechanisms guard the integrity of epigenomic information in response to a disruption in the DNA methylation machinery. In this paper, we test this hypothesis by determining the response of human cell lines to a knockdown of DNMT1 mRNA, encoding the enzyme responsible for the replication of the DNA methylation pattern. Our data suggest that cells respond to this epigenomic stress by an intra-S-phase arrest of DNA synthesis as well as by inducing a large number of stress response genes. The slow down in DNA synthesis during S-phase protects the DNA from a global loss of the methylation pattern. This mechanism is not triggered by 5-aza-2′-deoxycytidine (5-aza-CdR),1 which causes an extensive loss of DNA methylation. Both A549, a human non-small cell lung carcinoma cell line, and T24, a human bladder transitional carcinoma-derived cell line, were obtained from the ATCC (Manassas, VA). A549 cells were grown in Dulbecco's modified Eagle's medium (low glucose) supplemented with 10% fetal calf serum and 2 mm glutamine. T24 cells were maintained in McCoy's medium supplemented with 10% fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. 18–24 h prior to treatment, cells were plated at a concentration of 3 × 105 cells/100-mm tissue culture dish or 5 × 104 cells/well in a six-well plate in the absence of antibiotics. The phosphorothioate oligodeoxynucleotides used in this study were MG88 (human DNMT1 antisense oligonucleotide) and its mismatch control MG208, which has a 6-base pair difference from MG88 (19Fournel M. Sapieha P. Beaulieu N. Besterman J.M. MacLeod A.R. J. Biol. Chem. 1999; 274: 24250-24256Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Oligonucleotides were transfected into cells with 6.25 μg/ml Lipofectin (Invitrogen) in serum-free Opti-MEM (Invitrogen). The oligonucleotide-containing Opti-MEM medium was removed from the cells and replaced with regular growth medium after 4 h. The treatment was repeated every 24 h. The cells were harvested 24, 48, 72, and 96 h following the first transfection. For 5-aza-CdR treatment, cells were grown in regular culture medium in the presence of 10−6m 5-aza-CdR (Sigma) dissolved in Me2SO. The 5-aza-CdR-containing medium was freshly replaced every 24 h. To determine the level of cellular DNA methyltransferase activity, nuclear extracts were prepared, and DNA methyltransferase activity was assayed as described previously (8Szyf M. Bozovic V. Tanigawa G. J. Biol. Chem. 1991; 266: 10027-10030Abstract Full Text PDF PubMed Google Scholar). For Western blot analysis of DNMT1, 50 μg of nuclear protein was fractionated on a 5% SDS-polyacrylamide gel, transferred to polyvinylidene difluoride membrane, and reacted with the polyclonal anti-DNMT1 antibody (New England Biolabs) at a dilution of 1:2000 in the presence of 0.05% Tween and 5% milk, and it was then reacted with anti-rabbit IgG (Sigma) at a dilution of 1:5000. The amount of total protein per lane was determined by Amido Black staining (23Ramchandani S. MacLeod A.R. Pinard M. von H.E. Szyf M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 684-689Crossref PubMed Scopus (168) Google Scholar). The intensity of DNMT1 and total protein signal was measured by scanning densitometry, and the ratio of DNMT1/total nuclear protein was calculated. Total RNA was extracted using the standard guanidium isothiocyanate method (24Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63222) Google Scholar). cDNA was synthesized in a 20-μl reaction volume containing 2 μg of total RNA, 40 units of Moloney murine leukemia virus reverse transcriptase (MBI), 5 μmrandom primer (Roche Molecular Biochemicals), a 1 mmconcentration of each of the four deoxynucleotide triphosphates, and 40 units of RNase inhibitor (Roche Molecular Biochemicals). mRNA was denatured for 5 min at 70 °C, the random primers were annealed for 10 min at 25 °C, and mRNA was reverse transcribed for 1 h at 37 °C. The reverse transcriptase was heat-inactivated for 10 min at 70 °C, and the products were stored at −20 °C until use. PCR was performed in a 50-μl reaction mixture containing 3 μl of synthesized cDNA product, 5 μl of 10× PCR buffer, 1.5–2.0 mm MgCl2, 0.2 mm dNTP, 1 unit ofTaq polymerase (all from MBI) and 0.5 μm of each primer. The primer sequences that were used for the different mRNAs were GADD45β (growth arrestDNA damage 45β gene) (sense, 5′-GTGTACGAGTCGGCCAAGTT-3′; antisense, 5′-AGGAGACAATGCAGGTCTCG-3′); ATF-3 (sense, 5′-AAGAGCTGAGGTTTGCCATC-3′; antisense, 5′-GACAGCTCTCCAATGGCTTC-3′); JunB (sense, 5′-TGGAACAGCCCTTCTACCAC-3′; antisense, 5′-GGAGTAGCTGCTGAGGTTGGT-3′); β-actin (sense, 5′-GTTGCTAGCCAGGCTGTGCT-3′; antisense, 5′-CGGATGTCCACGTCACACTT-3′); MAGEB2 (sense, 5′-AGCGAGTGTAGGGGGTGCG-3′; antisense, 5′-TGAGGCCCTCAGAGGCTTTC-3′); BCL2-interacting killer (BIK) (sense, 5′-GGCCTGCTGCTGTTATCTTT-3′; antisense, 5′-CCAGTAGATTCTTTGCCGAG-3′); SSX2 (sense, 5′-CAGAGTACGCACGGTCTGAT-3′; antisense, 5′-GATTCCCACGGTTAGGGTCA-3′). Amplifications were performed in a Biometra T3 thermocycler (Biomedizinische Analytik GmbH) using the following programs: for GADD45β, first cycle 94 °C for 3 min, 58 °C for 1 min, and 72 °C for 1 min, second cycle 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min followed by 37 cycles of 94 °C for 1 min, 54 °C for 1 min, and 72 °C for 1 min; for ATF-3 and JunB, an initial cycle of 94 °C for 3 min 60 °C for 1 min and 72 °C for 1 min, followed by 34 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min; for β-actin, first cycle 94 °C for 3 min, 64 °C for 1 min, and 72 °C for 1 min, second cycle 94 °C for 1 min, 62 °C for 1 min, and 72 °C for 1 min, followed by 25 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min; for MAGEB2, BIK, and SSX2, first cycle 94 °C for 30 s, 62 °C for 30 s, 72 °C for 30 s, second cycle 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and 30 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s. The numbers of cycles were selected and tested so that the PCR amplification remained in the linear phase. 10 μl of the PCR products were applied on a 1.2% agarose gel and visualized by ethidium bromide staining. Densitometric analysis was performed using Scion Imaging Software (Scion Inc., Frederick, MD). Total RNA (2 μg) was reverse transcribed as described above in the presence of 12.5 μCi of 35S-labeled dCTP (1250 Ci/mmol) (ICN) to quantify the efficiency of reverse transcription. Equal amounts of reverse transcribed cDNA (70,000 cpm as determined by the incorporation of 35S-labeled dCTP) were subjected to PCR amplification in the presence of increasing concentrations of a competitor DNA fragment that amplifies with the same set of primers but yields a product that is shorter by 48 base pairs. The following primers were used: 5′-ACCGCTTCTACTTCCTCGAGGCCTA-3′ (DNMT1 sense), 5′-GTTGCAGTCCTCTGTGAACACTGTGG-3′(DNMT1 antisense), and 5′-CGTCGAGGCCTAGAAACAAAGGGAAGGGCAAG (primer used to generate the competitor). PCR conditions were as follows: 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min (33 cycles). Genomic DNA was extracted with DNA extraction buffer (1% SDS, 5 mm EDTA, 150 mmNaCl) followed by proteinase K digestion, phenol/chloroform extractions and ethanol precipitations. Bisulfate treatment was performed as described previously (25Clark S.J. Harrison J. Paul C.L. Frommer M. Nucleic Acids Res. 1994; 22: 2990-2997Crossref PubMed Scopus (1625) Google Scholar). The methylation status of the p16gene was determined by methylation-specific PCR (26Herman J.G. Graff J.R. Myohanen S. Nelkin B.D. Baylin S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9821-9826Crossref PubMed Scopus (5235) Google Scholar) as modified by Palmisano et al. (27Palmisano W.A. Divine K.K. Saccomanno G. Gilliland F.D. Baylin S.B. Herman J.G. Belinsky S.A. Cancer Res. 2000; 60: 5954-5958PubMed Google Scholar). Cells were plated in a six-well plate (5 × 104/well). For the final 4 h of incubation, 1 μCi/ml [methyl-3-3H]thymidine (PerkinElmer Life Sciences) was added to the medium. After washing twice with PBS, the cells were incubated in 10% trichloroacetic acid for 30 min at 4 °C, washed twice with cold 10% trichloroacetic acid, and then lysed with 1 n NaOH and 1% SDS. [3H]thymidine incorporation was measured using a liquid scintillation counter (LKB Wallac). Global DNA methylation was evaluated by staining the cells with specific monoclonal antibody against 5-methylcytidine using the protocol described previously (28Habib M. Fares F. Bourgeois C.A. Bella C. Bernardino J. Hernandez-Blazquez F. de C.A. Niveleau A. Exp. Cell Res. 1999; 249: 46-53Crossref PubMed Scopus (61) Google Scholar) with slight modifications. Briefly, cells were washed with phosphate-buffered saline (PBS) supplemented with 0.1% Tween 20 and 1% bovine serum albumin (PBST-BSA), fixed with 0.25% paraformaldehyde at 37 °C for 10 min and 88% methanol at −20 °C for at least 30 min. After two washes with PBST-BSA, the cells were treated with 2 n HCl at 37 °C for 30 min and were then neutralized with 0.1 m sodium borate (pH 8.5). The cells were blocked with 10% donkey serum in PBST-BSA for 20 min at 37 °C, incubated with anti-5-methylcytidine antibody (1 μg/ml) for 45 min at 37 °C, followed by staining with donkey anti-mouse IgG conjugated with Rhodamine Red-X (Jackson ImmunoResearch Laboratories). Finally, the cells were washed with PBS three times and were resuspended in PBS for flow cytometry analysis. A549 cells were transfected with 200 nm MG208 or MG88 or were treated with 1 μm5-aza-CdR or Me2SO, for 48 h. Total RNA was extracted with RNAeasy (Qiagen). Micoarray analysis was performed as previously described (29Golub T.R. Slonim D.K. Tamayo P. Huard C. Gaasenbeek M. Mesirov J.P. Coller H. Loh M.L. Downing J.R. Caligiuri M.A. Bloomfield C.D. Lander E.S. Science. 1999; 286: 531-537Crossref PubMed Scopus (9252) Google Scholar). Briefly, 20 μg of RNA was used for cDNA synthesis, followed by in vitro transcription with a T7 promoter primer having a poly(T) tail. The resulting product was hybridized and processed with the GeneChip system (Affymetrix) to a HuGeneFL DNA microarray containing oligonucleotides specific for ∼12,000 human transcripts. Data analysis, average difference, and expression for each feature on the chip were computed using Affymetrix GeneChip Analysis Suite version 3.3 with default parameters. The gene expression analysis was performed by the Montreal Genome Center. Cells were incubated with 10 μm BrdUrd (Sigma) for the last 2 h before harvesting. Incorporated BrdUrd was stained with anti-BrdUrd antibody conjugated with fluorescein isothiocyanate (Roche Molecular Biochemicals) following the manufacturer's protocol (30Vanderlaan M. Thomas C.B. Cytometry. 1985; 6: 501-505Crossref PubMed Scopus (101) Google Scholar). After the last washing, the cells were resuspended in PBS containing 50 μg/ml propidium iodide and 10 μg/ml RNase A for 30 min at room temperature and then analyzed with FACScan (BD Bioscience) for both fluorescein isothiocyanate and propidium iodide fluorescence. DNMT1 activity is physically and temporally associated with the DNA replication machinery. The absence of DNMT1 from the replication fork could potentially lead to an epigenomic catastrophe. We have previously proposed that the coordination of DNMT1 expression and DNA replication evolved as a mechanism to protect the coordinate inheritance of genetic and epigenetic information (31Szyf M. Trends Pharmacol. Sci. 2001; 22: 350-354Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 32Szyf M. Front. Biosci. 2001; 6: D599-D609Crossref PubMed Google Scholar). To test this hypothesis, we determined the cellular response to a knockdown of DNMT1 protein. We took advantage of a previously described antisense oligonucleotide, which specifically knocks down DNMT1mRNA (19Fournel M. Sapieha P. Beaulieu N. Besterman J.M. MacLeod A.R. J. Biol. Chem. 1999; 274: 24250-24256Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) MG88 and its mismatch control MG208 (see Fig.1A for sequence and alignment with human and mouse DNMT1 mRNA). We first optimized the time and concentration at which MG88 specifically knocks down DNMT1activity in A549 cells in comparison with MG208. The results presented in Fig. 1B show that MG88 reduces DNA methyltransferase activity in a dose- and time-dependent manner relative to MG208. Inhibition of DNA methyltransferase activity approximates 80% after 48 h of MG88 treatment, whereas no inhibition is observed following MG208 treatment. Therefore, for our further analysis we chose to treat the cells with a 200 nm concentration of either MG88 or MG208 for 48 h. We confirmed the antisense mechanism of action of MG88 by demonstrating that DNMT1mRNA levels are knocked down following a 48-h treatment with this oligonucleotide in comparison with MG208 treatment using a competitive RT-PCR assay for DNMT1 (Fig. 1, C andD). To confirm that DNMT1 inhibition results in reduction of DNMT1 protein, nuclear extracts prepared from either MG208- or MG88-treated cells (200 nm for 48 h) were subjected to a Western blot analysis and reacted with anti-DNMT1 antibody (Fig.1E). Quantification of the signal by densitometry reveals 85% reduction in protein levels. Inhibition of DNMT1mRNA by MG88 was also confirmed in a gene array expression analysis presented in Table II.Table IIStress-responsive genes up-regulated by MG88 after 48 h of treatmentRatioGenBank™ accession no.NameAbbreviated name17.6AF078077Growth arrest and DNA damage-inducible, βGADD45β15.1M59465Tumor necrosis factor, α-induced protein 3TNFAIP312.1S76638Nuclear factor of κ light polypeptide gene enhancer in B-cells 2 (p49/p100)NFKB211.6L19871Activating transcription factor 3ATF-39.1J04111v-jun avian sarcoma virus 17 oncogene homologJUN9.1U19261Tumor necrosis factor receptor-associated factor 1TRAF17.4U72206Rho/Rac guanine nucleotide exchange factor 2ARHGEF27.1J04111v-jun avian sarcoma virus 17 oncogene homologJUN6.9U10550GTP-binding protein overexpressed in skeletal muscleGEM6.9V01512v-fos FBJ murine osteosarcoma viral oncogene homologFOS6.2M58603Nuclear factor of κ light polypeptide gene enhancer in B-cells 1 (p105)NFKB16.0L49169FBJ murine osteosarcoma viral oncogene homolog BFOSB5.8M59287CDC-like kinase1CLK15.6M60974Growth arrest and DNA-damage-inducible, alphaGADD45α5.5L36463Ras inhibitorRIN15.3M69043Nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, αNFKB1A5.2X51345junB proto-oncogeneJUNB4.8D87116Mitogen-activated protein kinase kinase 3MAP2K34.5AF059617Serum-inducible kinaseSNK4.1S82240ras homolog gene family, member EARHE3.8AB014551Rho/Rac guanine nucleotide exchange factor 2ARHGEF23.7X61498Nuclear factor of κ light polypeptide gene enhancer in B-cells 2 (p49/p100)NFKB23.6U83981Growth arrest and DNA damage-inducible 34GADD343.6AF050110TGFB-inducible early growth responseTIEG3.2AB011093Rho-specific guanine nucleotide exchange factor p114P114-RHO-GEF3.1S81439TGFB-inducible early growth responseTIEG3.1AI304854Cyclin-dependent kinase inhibitor 1B (p27, Kip1)CDKN1B2.9M62831Immediate early proteinETR1012.9U03106Cyclin-dependent kinase inhibitor 1A (p21, Cip1)CDKN1A2.9L08246Myeloid cell leukemia sequence 1 (BCL2-related)MCL12.8X52560CCAAT/enhancer-binding protein, βCEBPB2.8M36820GRO2oncogeneGRO22.8AF093265Homer, neuronal immediate early gene, 3HOMER-32.7AI038821v-Ha-ras Harvey rat sarcoma viral oncogene homologHRAS2.7AF016266Tumor necrosis factor receptor superfamily, member 10bTNFRSF10B2.6X82260Ran GTPase-activating protein 1RANGAP12.6D87953N-mycdownstream-regulatedNDRG12.5X54489GRO1 oncogene (melanoma growth stimulating activity, α)GRO12.5L20320Cyclin-dependent kinase 7 (homolog of Xenopus MO15 Cdk-activating kinase)CDK7−3.4X63692DNA (cytosine-5-)-methyltransferase 1DNMT1A549 cells were transfected with 200 nm of either MG208 or MG88 for 48 h. Total RNA was subjected to a differential expression microarray analysis using HuGeneFL DNA microarrays containing oligonucleotides specific for approximately 12,000 human transcripts as described under "Materials and Methods." The first column indicates the -fold difference of the normalized expression of the indicated genes in MG88- versus MG208-treated A549 cells. The second column lists the accession numbers of the genes. The third column lists the names of the genes, and the last column provides their abbreviated names. Open table in a new tab A549 cells were transfected with 200 nm of either MG208 or MG88 for 48 h. Total RNA was subjected to a differential expression microarray analysis using HuGeneFL DNA microarrays containing oligonucleotides specific for approximately 12,000 human transcripts as described under "Materials and Methods." The first column indicates the -fold difference of the normalized expression of the indicated genes in MG88- versus MG208-treated A549 cells. The second column lists the accession numbers of the genes. The third column lists the names of the genes, and the last column provides their abbreviated names. Several studies have previously demonstrated that inhibition of DNMT1 results in inhibition of cell growth (33Bigey P. Knox J.D. Croteau S. Bhattacharya S.K. Theberge J. Szyf M. J. Biol. Chem. 1999; 274: 4594-4606Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). One possible explanation for the reduced cell growth is that knockdown of DNMT1 results in inhibition of firing of DNA replication origins (17Knox J.D. Araujo F.D. Bigey P. Slack A.D. Price G.B. Zannis-Hadjopoulos M. Szyf M. J. Biol. Chem. 2000; 275: 17986-17990Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). We therefore addressed the question of whether this inhibition of DNA replication reflects a distinct alteration in cell cycle kinetics, similar to the DNA damage checkpoints that trigger arrest at distinct phases of the cell cycle (34Bartek J. Lukas J. Curr. Opin. Cell Biol. 2001; 13: 738-747Crossref PubMed Scopus (469) Google Scholar). A549 cells were treated with a 200 nm concentration of either MG88 or MG208 for 24–96 h as described under "Materials and Methods." As observed in Fig. 2,DNMT1 knockdown results in a significant decrease in overall DNA synthetic capacity of A549 cells. This is illustrated by the reduced incorporation of [3H]thymidine into DNA 24 h after the initiation of treatment, as has been previously reported (17Knox J.D. Araujo F.D. Bigey P. Slack A.D. Price G.B. Zannis-Hadjopoulos M. Szyf M. J. Biol. Chem. 2000; 275: 17986-17990Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To exclude the possibility that inhibition of DNA synthesis by MG88 is independent of DNMT1 expression and is a toxic side effect of the sequence, we took advantage of the species specificity of MG88. As shown in Fig. 1A, there is a 6-base pair mismatch between MG88 and the mouse dnmt1 mRNA. We therefore determined whether MG88 would inhibit DNA synthesis in a mouse adrenal carcinoma cell line, Y1, which was previously shown by us to be responsive to a mouse dnmt1 antisense oligonucleotide (23Ramchandani S. MacLeod A.R. Pinard M. von H.E. Szyf M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 684-689Crossref PubMed Scopus (168) Google Scholar). The results shown in Fig. 2B demonstrate that 48-h treatment with 200 nm MG88 had no significant impact on the DNA synthetic capacity of Y1 cells in comparison with MG208, supporting the hypothesis that MG88 inhibition of DNA synthesis is DNMT1-dependent. We then addressed the question of whether this inhibition in DNA synthesis represents a slowdown in the rate of DNA synthesis or a reduction in the fraction of cells that are in the synthetic phase, which would indicate a change in cell cycle phase kinetics. We pulsed MG88- and MG208-treated cells with BrdUrd 48 h after the initiation of treatment and sorted the cells that incorporated BrdUrd using fluorescence-activated cell sorting as described under "Materials and Methods." As illustrated in Fig. 2C, DNMT1 knockdown reduces the fraction of cells that incorporate DNA (the M1 population). However, the reduction in the fraction of cells that synthesize DNA (up to 42%) does not account for the overall reduction in DNA replication shown by the [3H]thymidine incorporation assay, which is >95%. Although there is no significant cell death that can account for this disparity, this difference might reflect the fact that cell number following MG88 treatment is reduced. The reduction in the fraction of cells that incorporate DNA could be a consequence of a phase specific cell cycle arrest. However, preliminary results using flow cytometry sorting of propidium iodide-stained cells failed to
Referência(s)