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1 Hamon Center for Therapeutic Oncology Research, and 2 Departments of Pathology, 3 Internal Medicine, and 4 Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, and 5 Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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At present three active DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and one candidate gene, DNMT2, have been identified in mammals (8)
. DNMT1, the first DNA methyltransferase to be cloned, is responsible for maintaining DNA methylation patterns during DNA replication (9)
. Fournel et al. (10)
showed recently that ablation of DNMT1 expression with antisense oligonucleotides resulted in loss of promoter methylation, re-expression of p16ink4A, and inhibition of cell proliferation in the bladder cancer cell line, T24. In contrast, Rhee et al. (11)
demonstrated that targeted deletion of DNMT1 by homologous recombination in the colon cancer cell line HCT116 was not sufficient to cause promoter demethylation and gene re-expression. In these experiments, DNMT1 deletion resulted in only a small decrease (
20%) in overall genomic methylation, and imprinted genes were not re-expressed. Rhee et al. (12)
additionally showed that deletion of both DNMT1 and DNMT3B reduced overall genomic methylation by >95% as well as promoter methylation of specific genes, and caused the re-expression of multiple genes (p16ink4A and TIMP-3), resulting in substantial growth suppression of HCT116 cells. Paradoxically, a more recent publication by Robert et al. (13)
showed that DNMT1 depletion using either antisense or small interfering RNA (siRNA) techniques led to demethylation of p16ink4A and MLH1 promoters, and re-expression of p16ink4A in the same HCT116 cells. Therefore, it is still unclear how the different DNMT genes act alone or in concert to maintain or establish DNA methylation patterns in individual types of human cancers.
To address this issue, we used RNA interference (RNAi) technology to knock down DNMT1 protein expression in the non-small cell lung cancer cell line, NCI-H1299, and the breast cancer cell line, HCC1954. Using quantitative assays for DNA methylation and mRNA expression, we found that DNMT1 knockdown led to a dramatic loss of methylation (>80%) compared with nontreated controls at the promoters of RASSF1A, p16ink4A, CDH1, and HPP1, and re-expression of RASSF1A, p16ink4A, CDH1, HPP1, and SEMA3B in lung and breast cancer cells. These findings provide quantitative evidence of the role of DNMT1 activity in both lung and breast cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Preparation and Transfection of siRNAs.
siRNAs targeting DNMT1 were designed and prepared as described previously (16)
. The two siRNA sequences against DNMT1 were 5'-CGGUGCUCAUGCUUACAACTT-3' (sense) and 5'-GUUGUAAGCAUGAGCACCGTT-3' (antisense), and 5'-CGAGUUGCUAGACCGCUUCTT-3' (sense) and 5'-GAAGCGGUCUAGCAACUCGTT-3' (antisense). The siRNA sequences against the human T-cell leukemia virus gene (Tax) and Lamin A/C were as reported previously (16
, 17) . The siRNA target sequences were tested in a basic local alignment search tool search of GenBank (National Center for Biotechnology Information database) to ensure that only the corresponding gene is the target. RNA oligonucleotides were obtained from the core facility in University of Texas Southwestern Medical Center (see website for details).6
The sense and antisense oligonucleotides were annealed to make siRNA (18)
and stored at –20°C before use. One day before transfection, cells were seeded such that they were 30–50% confluent the next day. Cells were transfected with 100 nM of siRNA using Oligofectamine transfection reagent (Invitrogen) in Opti-MEM I reduced serum medium (Invitrogen) at 37°C in a 5% CO2 atmosphere for 6 h. The medium was removed and replaced with fresh RPMI 1640 supplemented with 5% fetal bovine serum. Control cells were treated with Oligofectamine alone or with Tax and Lamin A/C siRNA. Transfection was repeated at 2, 4, and 6 days for a total of 4 treatments. Cells were grown and harvested at 3, 5, 7, 9, 14, and 23 days after the initial transfection for additional analysis.
Western Blot Analysis.
Cells were grown and harvested at 80–90% confluency, and cellular proteins were extracted with lysis buffer [40 mM HEPES-NaOH (pH 7.4), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and 150 mM NaCl] containing Complete Mini, a mixture of protease inhibitors (Roche, Indianapolis, IN). Total protein was electrophoresed on SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). After blocking with 5% nonfat dry milk and 0.1% Tween 20 in Tris-buffered saline, membranes were incubated with the mouse monoclonal anti-DNMT1 (Imgenex, San Diego, CA), the rabbit polyclonal anti-DNMT3B (a kind gift from Dr. A. Robert MacLeod, MethylGene Inc., Montreal, Quebec, Canada), the rabbit monoclonal anti-p16ink4A (Santa Cruz Biotechnology, Santa Cruz, CA), or the mouse monoclonal anti-actin (Sigma) antibodies. The membranes then were developed with peroxidase-labeled antibodies (Amersham Pharmacia, Piscataway, NJ) by Super Signal chemiluminescence substrate (Pierce, Rockford, IL). Actin protein levels were used as a control for equal protein loading.
Quantitative Methylation-Specific PCR (MSP) Assay.
Genomic DNA was obtained from cell lines by digestion with proteinase K (Life Technologies, Inc.), followed by phenol:chloroform (1:1) extraction. One µg of genomic DNA was denatured with 2 N NaOH and modified with sodium bisulfite, as described previously (19)
. The modified DNA was purified using the Wizard DNA purification kit (Promega, Madison, WI), treated with 3 N NaOH, precipitated with ethanol, and resuspended in water. Sodium bisulfite-treated genomic DNA was amplified by fluorescence-based real-time MSP (Perkin-Elmer Corp., Foster City, CA) as described previously (20)
. For the internal reference gene, MYOD1, the primers and probe were designed to avoid CpG nucleotides. The methylation ratio is defined as the ratio of the fluorescence emission intensity values for the target PCR products to those of the MYOD1 PCR products, multiplied by 100. The ratio is then divided by the ratio of the nontreated sample and multiplied by 100 to yield a percentage. The sequences of the primers and probes are shown in Table 1
. Quantitative real-time MSP assays were performed in a reaction volume of 25 µl by using components supplied in a TaqMan PCR Core Reagent kit (Perkin-Elmer Corp.). Each assay was performed in triplicate. The final reaction mixtures contained the forward and reverse primers at 300 nM each; the probe at 100 nM; 200 µM each of dATP, dGTP, dCTP, and dTTP; 5.5 mM MgCl2; 1x TaqMan Buffer A; 1 unit of HotStarTaq DNA polymerase (Qiagen Inc., Valencia, CA); and 2 µl bisulfite-converted genomic DNA. PCR was performed under the following conditions, 95°C for 12 min, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. We performed quantitative real-time MSP with the Gene Amp 5700 Sequence Detection System (Perkin-Elmer Corp.). DNA from lymphocytes of a healthy volunteer treated with SssI methyltransferase (New England BioLabs, Beverly, MA) was used as a positive control. The same untreated, unmethylated DNA was used as a negative control for methylated alleles. Water blanks were included with each assay.
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Soft Agar-Growth Assay.
Cells were transfected with siRNAs for a total of four treatments, and 7 days after the initial transfection, cells were replated for soft agar-growth assay. Briefly, 300 viable cells were suspended and plated in 0.33% agar in RPMI 1640 (Life Technologies, Inc.) supplemented with 20% fetal bovine serum and layered over a 0.50% agar base medium in 12-well plates. After 2 weeks, the number of colonies >100 cells were counted in triplicate plates.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. Both siRNAs targeted to DNMT1 mRNA led to substantial down-regulation of DNMT1 expression 3 days after the initial transfection in the NCI-H1299 cell line and HCC1954. These effects continued until at least day 9 (Fig. 1)
. We routinely observe targeted gene silencing in 90% of the NCI-H1299 cells transfected with siRNA as detected by immunofluorescent staining of individual cells (data not shown). DNMT1 down-regulation was specific as evidenced by a consistent level of actin protein (Fig. 1)
and DNMT3B protein (Fig. 3D)
in the context of siRNA targeted to DNMT1, whereas DNMT1 protein expression was not affected by siRNA targeted to Lamin A/C (data not shown).
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To compare the methylation levels of each gene before and after treatment with siRNA, we converted the mean ratio of promoter methylation to a percentage. RNAi-mediated down-regulation of DNMT1 protein expression resulted in a significant decrease in methylation levels at the RASSF1A, p16ink4A, and CDH1 promoters in NCI-H1299 (Fig. 2A)
, and similar effects were observed for RASSF1A, p16ink4A, and HPP1 in HCC1954 (Fig. 2B
; P < 0.001; all genes examined, repeated measures ANOVA).
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; Fig. 2, A and B
). The level of methylation for all genes was reduced on day 3, more so on day 5, and reached a nadir on day 7, whereupon maximal demethylation seems to have been reached (Fig. 2, A and B)
. As the data indicate, there does not appear to be a significant decrease in methylation levels between day 7 and 9. The methylation level of all of the genes tested was reduced by up to 80% when compared with promoter methylation levels in untreated cells. Reduction of promoter methylation was greatest in the HPP1 promoter in HCC1954 (Fig. 2B)
and p16ink4A in NCI-H1299 (Fig. 2A)
, yet in neither case was methylation completely lost. It is known that siRNA can be used to specifically knock down target genes, but RNAi never completely eliminates the targeted gene products (26) . Thus, the presence of basal amounts of promoter methylation we observed, even with extended siRNA treatment, may result from residual DNMT1 protein or other DNMTs. Other methyltransferases such as DNMT3B or methyl-DNA binding proteins may affect methylation levels in the promoters of TSGs. Rhee et al. (12) demonstrated that genetic disruption of DNMT1 by homologous recombination did not lead to promoter demethylation and re-expression of p16ink4A in the colon cancer cell line HCT116, whereas p16ink4A was demethylated and re-expressed in HCT116 cells, in which both DNMT1 and DNMT3B were disrupted. Therefore, knockdown of both DNMT1 and DNMT3B or other factors may be required to achieve complete demethylation of genes involved in cancer pathogenesis.
5-Aza-CdR treatment results in global demethylation of genomic DNA in many cancer cell lines. Upon removal of 5-Aza-CdR and continued culture, remethylation occurs slowly and in a sequence-specific manner (27) . The propensity of particular regions of DNA to become remethylated may result from selective pressure, such as TSG function, or some cryptic sequence information within loci that are preferentially remethylated. Recent research using 5-Aza-CdR indicates that de novo methylation of CpG sites in the p16ink4A promoter is not stochastic. Thus, the kinetics of selective CpG island remethyl-ation in the promoters of genes may reflect differences in the contribution individual CpG sites have to gene repression. However, due to the nonspecificity and cytotoxicity of 5-Aza-CdR, it is unclear which DNMT is responsible for the apparent nascent methylation, or whether remethylation is really the result of the expansion of a resistant subclone within the treated population of cells (27) .
To address these issues and to determine how persistent loss of promoter methylation was in the context of the specific down-regulation of DNMT1 protein, we maintained the treated cell lines in the absence of any additional siRNA treatment. We then reexamined the methyl-ation level of all genes at day 14 and day 23 after initial treatment. The kinetics of remethylation varied between genes in both cell lines; however, remethylation (returning to 40–80% of starting levels) and loss of gene expression was observed in all cases by day 23 (Fig. 2, A and B)
. These results indicate that the appearance of de novo methylated CpG sites within multiple gene promoters occurs in tandem with the re-expression of DNMT1 protein. This finding clarifies the results from the 5-Aza-CdR experiments described above, because it suggests that DNMT1, as opposed to DNMT3A and DNMT3B, has important in vivo, de novo DNA methyltransferase activity. A previous report has demonstrated that DNMT1 has de novo methylase activity, but only in vitro (28)
. The variation in remethylation kinetics between the two cell lines may result from differences in their doubling times (NCI-H1299 have a doubling time of 25 h, whereas HCC1954 double every 31 h), because de novo methylation has been shown to be dependent on cell division (27)
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Demethylation Induced by RNAi-Mediated DNMT1 Knockdown Restored the Expression of Several Tumor Suppressor Genes in Lung and Breast Cancer Cell Lines.
To establish whether loss of promoter methylation mediated by DNMT1 siRNA resulted in the quantitative re-expression of genes, we analyzed the expression status of RASSF1A and SEMA3B genes in NCI-H1299, and RASSF1A and HPP1 genes in HCC1954 line by real-time RT-PCR (Fig. 3, A and B)
. RNAi-mediated DNMT1 knockdown induced the expression of all genes examined (P < 0.001, repeated measures ANOVA). The expression levels of all genes in DNMT1 siRNA-treated cells were 2–8-fold higher than that of untreated cells. We examined the expression status of SEMA3B because it is silenced by tumor-associated promoter methylation in NCI-H1299, and is located on 3p21, a known tumor suppressor locus as reported by us and others (29
, 30)
. The expression level of p16ink4A and CDH1 genes in NCI-H1299 and p16ink4A in HCC1954 were examined by 37-cycle end point RT-PCR. NCI-H1299 cells treated with DNMT1 siRNA expressed p16ink4A mRNA from day 5 to day 23 and expressed CDH1 from day 3 to day 23 (Fig. 3C)
. HCC1954 cells treated with DNMT1 siRNA expressed p16ink4A from day 3 to day 23 (Fig. 3C)
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Because the p16ink4A gene locus has a complicated structure, it was not possible to design an isoform-specific TaqMan probe. Thus, we sought to verify gene induction by Western blot. Both of two different siRNAs targeted to DNMT1 restored p16ink4A protein expression (Fig. 3D)
. Thus, there is a clear inverse relationship between the presence of methyl-CpGs in the promoter of p16ink4A, and the expression of p16ink4A mRNA and protein. We additionally compared the effect of the DNMT1 siRNA (DNMT1-2) on the restoration of gene expression with that of 5-Aza-CdR treatment in these cell lines. siRNA inhibitors of DNMT1 protein expression are at least as effective at restoring mRNA expression as 5-Aza-CdR treatment (Table 2)
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We assessed the effect of the DNMT1 knockdown on in vitro growth of NCI-H1299 cells by soft agar growth assay. Surprisingly, there was no significant difference in colony number between treatment of DNMT1 siRNAs (DNMT1-1 and DNMT1-2) and that of Tax siRNA (means ± SD of colony number by treatments with DNMT1-1, DNMT1-2, and Tax siRNAs were 115 ± 13, 100 ± 14, and 108 ± 9, respectively). Thus, we could not demonstrate an obvious effect of the loss of DNMT1 expression on the in vitro tumor growth of NCI-H1299 cells. Whereas this was unexpected, there are several possible explanations for this result. The first is that the genes we monitored (e.g., RASSF1A or SEMA3B) really do not function as TSGs. The study of SEMA3B as a TSG is early, there are multiple methylation and functional studies of the role of p16ink4A and RASSF1A strongly implicating them as TSGs in lung and other cancers (29, 30, 31, 32, 33) . The tumor cells were plated after 7 days and 4 RNAi treatments the colonies were not scored until 14 days later, it is possible that transient re-expression of the tumor suppressor genes by DNMT1 siRNA was not sufficient to inhibit colony formation due to the short-term inhibition of DNMT1 expression. In fact, a recent study showed that prolonged knockdown of DNMT1 by a tetracycline-inducible vector-based siRNA induced growth arrest, whereas growth resumed 1–2 days after the siRNA knockdown was relaxed (34) . It is also possible that the tumor cells have developed other ways to bypass these growth regulatory molecules. For example, the p53 null status of H1299 cells (they are homozygously deleted for p53) prevents transient re-expression of the proteins from inducing apoptosis. In fact, a previous study showed that adenovirus-mediated exogenous p16 expression alone did not induce apoptosis in H1299 cells, but only exhibited apoptosis after the addition of exogenous p53 expression (35) . Finally, it is possible that DNMT1 knockdown led to the expression of proteins (e.g., those involved in the differentiated state), which either made the cells resistant to tumor suppressor function or caused growth arrest, preventing subsequent induction of apoptosis by other re-expressed proteins. All of these mechanisms will require future study. However, the lack of a dramatic effect on growth of H1299 cells by DNMT1 knockdown indicates to us that the use of agents that block methylation may have to be combined with other approaches before being clinically active. Additional studies of single cells and clones after knockdown will be needed to verify that individual cells can undergo DNMT1 knockdown, loss of promoter methylation, and re-expression of genes followed by later promoter remethylation and gene silencing. In this regard, additional investigations using RNAi vectors that can stably suppress the expression of other DNMTs and/or methyl-DNA binding proteins will elucidate how DNA methylation contributes to cancer pathogenesis, and enable us to systematically analyze the DNA methylation machinery as a target for therapeutic intervention of cancer.
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FOOTNOTES |
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Note: M. Suzuki and N. Sunaga contributed equally to this work.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: John D. Minna, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center; 6000 Harry Hines Boulevard, Dallas, TX 75390-8593. Phone: (214) 648-4921; Fax: (214) 648-4940; E-mail: john.minna{at}utsouthwestern.edu
6 Internet address: http://cbi.swmed.edu/pages/oligonet_index.htm. 
Received 9/26/03. Revised 2/23/04. Accepted 2/27/04.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, Oligofectamine; 
, Tax; 
, Lamin A/C;
, DNMT1-1;
, DNMT1-2.
), DNMT1-1 (
), or DNMT1-2 (
) small interfering RNA four times (on days 0, 2, 4, and 6). Cells were harvested at 0, 3, 5, 7, 9, 14, and 23 days, and DNA was extracted and treated with sodium bisulfite. Real-time methylation-specific PCR was performed as described ("Materials and Methods"). Each ratio was normalized to MYOD1 and converted to a percentage based on the same ratio in untreated cells. Each point represents averages from three independent experiments; bars, ±SE. A, NCI-H1299. B, HCC1954.