It was recently reported that RUNX3 gene expression is significantly downregulated in human gastric cancer cells due to hypermethylation of its promoter region or hemizygous deletion (Cell, 109, 2002). To verify the genetic alterations and methylation status of the RUNX3 gene in colorectal carcinogenesis, we analysed for mutations, loss of heterozygosity (LOH), and RUNX3 gene promoter hypermethylation, in 32 colorectal cancer cell lines. RT–PCR analysis showed undetectable or low RUNX3 expression in 16 cell lines, and no mutations were found in the RUNX3 gene by PCR-SSCP analysis. Of these 16 cell lines, hypermethylation of the RUNX3 promoter was confirmed in 12. The following observations were made: (i) RUNX3 was re-expressed after 5-aza-2′-deoxycytidine treatment, (ii) the RUNX3 promoter was found to be methylated by MS-PCR, and (iii) hypermethylation of the RUNX3 promoter was confirmed by direct sequencing analysis after sodium bisulfite modification in the above 12 cell lines. RUNX3 was neither methylated nor expressed in four cell lines. Of these four, microsatellite instability (MSI) at the RUNX3 locus was found in three, SNU-61 (D1S246), SNU-769A, and SNU-769B (D1S199). This study suggests that transcriptional repression of RUNX3 is caused by promoter hypermethylation of the RUNX3 CpG island in colorectal cancer cell lines, and the results of these experiments may contribute to an understanding of the role of RUNX3 inactivation in the pathogenesis of colorectal cancers.
Aberrant methylation of normally unmethylated CpG islands, located in the 5′ promoter region of genes, is associated with transcriptional inactivation that is as effective as inactivation by gene mutation or deletion. Such inactivation is observed in various human cancers, in genes including RB, p16, VHL, BRCA1, E-cadherin, APC, hMLH1, FHIT, COX2, and CDX1 (reviewed by Melki et al., 2000; Song et al., 2001; Zöchbauer-Müller et al., 2001; Ku et al, 2002; Suh et al., 2002; Yang et al., 2002).
Genes belonging to the RUNX family encode DNA-binding transcription factors that control key events in cell patterning and differentiation. In mammals, the family includes three genes, RUNX1 (PEBP2αB/CBFA2/AML1), RUNX2 (PEBP2αA/CBFA1/AML3), and RUNX3 (PEBP2αC/CBFA3/AML2), and in humans, RUNX1 and RUNX3 are known to be involved in leukemogenesis and gastric carcinogenesis, respectively. RUNX1 is essential for definitive hemopoiesis, and translocations or point mutations involving RUNX1 are frequently found in acute leukemia; haploinsufficiency of RUNX1 results in an increased risk of acute myeloid leukemia. RUNX3 belongs to a small family whose members share a highly conserved region, designated the ‘runt domain’, originally found in the Drosophila runt gene (reviewed by Bangsow et al., 2001; Rini and Calabi, 2001; Lee et al., 2002). Recently it was reported that expression of the human RUNX3 gene, which is located on the short arm of human chromosome 1 at 1p36, was significantly reduced in 45% of human gastric cancer cells due to hemizygous deletions and in 60% due to hypermethylation of the promoter region (Li et al., 2002). It has also been reported that the Runx3 gene is silenced due to hypermethylation of CpG islands in its promoter region in three out of four mouse glandular stomach carcinoma cell lines (Guo et al., 2002). In addition, a runt domain gene is expressed in C. elegans intestinal cells, and double-strand RNA interference targeting of this gene results in malformation of the intestine (Nam et al., 2002).
In the present study, we hypothesized that RUNX3 expression in colorectal cancer cell lines might be, in some cases, silenced by methylation of the CpG islands in its promoter. A total of 32 colorectal cancer cell lines were tested for hypermethylation of the RUNX3 gene promoter and for mutations in the RUNX3 gene. In addition, we have also screened the methylation status of the RUNX3 gene promoter in 87 paired colorectal cancer and normal mucosal tissue samples. We also analysed loss of heterozygosity (LOH) status at chromosome 1p36 in six cell lines, which were available from corresponding normal tissue DNA. We found that the transcriptional inactivation of RUNX3 is closely related with the methylation status of the CpG sites of the RUNX3 gene in human colorectal cancer cell lines.
Expression, mutations and LOH of RUNX3 in colorectal cancer cell lines
Abnormal band shifts were not found in any cell lines using PCR-SSCP for mutational analysis.
RUNX3 expression results were compared with LOH at the RUNX3 locus, determined using microsatellite markers (D1S199, and D1S246). Methylation-specific inactivation (MSI) rather than LOH at 1p36 was found in four cell lines (SNU-61, SNU-769A, SNU-769B, SNU-1047) (Figure 2), and no LOH or MSI in these markers was found in SNU-1040 and SNU-1197. Of these, SNU-769A, SNU-769B, SNU-1040, and SNU-1047 cell lines showed MSI by BAT-26, however, SNU-61 and SNU-1197 cell lines did not show MSI by BAT-26 in our earlier report (Ku et al., 1999).
Of the 87 colorectal carcinomas, 60 (69%) were from the proximal colon (cecum to splenic flexure), and 27 (31%) were from the distal colorectum (splenic flexure to rectum). Of 32 colorectal cancers cell lines, seven originated from the proximal colon and eight originated from the distal colorectum. The origin of the remaining 17 colorectal cancer cell lines was unknown.
Analysis of RUNX-3 methylation by MS-PCR
Using primers for methylated DNA amplification on bisulfite modified DNA, DNA fragments were amplified from 12 cell lines (SNU-81, SNU-1047, SNU-C5, COLO201, COLO205, DLD-1, HCT-8. HCT-15, HT-29, Lovo, NCI-H716, and WiDR) (12 of 32, 37.5%). In four cell lines (SNU-61, SNU-175, SNU-769A, and SNU-769B), which did not express RUNX3 mRNA, bisulfite PCR products were not amplified by methylated DNA-specific PCR. Using primers specific for amplification of unmethylated DNA, PCR products were found in 20 cell lines, including the four cell lines described above, but not in the 12 cell lines that showed amplified methylated DNA by PCR (Figure 3) (Table 1). Using primers that would amplify unmethylated and methylated DNA, PCR products were found all 32 cell lines (data not shown).
In tumor tissues, methylated DNA PCR products were found in 16 out of 87 samples (18.4%). Using primers specific for unmethylated DNA amplification, PCR products were found in all 87 tumor samples, including the 16 cancer samples that amplified the methylated DNA. In normal tissue samples, unmethylated DNA was amplified in all 87 samples and methylated DNA was not (data not shown). Of the 16 tumor tissue samples containing the methylated RUNX3 gene, 12 samples (75%) originated from the proximal colon, and four samples (25%) originated from the distal colorectum.
Methylation profile of the RUNX3 gene CpG island
PCR products amplified using primers specific for unmethylated and methylated DNA were used to investigate the methylation status of 24 CpG sites located between −270 and −121, relative to the transcription initiation site of exon 1 of RUNX3 (Figure 4b). Direct sequencing analysis of the bisulfite-modified PCR products was used to determine if any CpG sites were preferentially methylated in the 12 cell lines that were found to contain methylated DNA. In all 12 cell lines, the 24 individual CpG sites were found to be fully methylated (Figures 4a and b).
Reexpression of RUNX3 after treatment with 5-aza-2′-deoxycytidine and TSA
We investigated whether RUNX3 mRNA was re-expressed after 5-aza-2′-deoxycytidine treatment in 19 cell lines, including the 16 cell lines that did not express RUNX3 mRNA. Cell lines that were cultured in medium without 5-aza-2′-deoxycytidine, the SNU-407 cell line that expressed the RUNX3 gene, and two cell lines, SNU-1 and SNU-5, were included as controls. We found that RUNX3 mRNA was re-expressed in 12 cell lines (SNU-81, SNU-1047, SNU-C5, COLO201, COLO205, DLD-1, HCT-8, HCT-15, HT-29, LOVO, NCI-H716, and WiDR) by RT–PCR analysis. RUNX3 mRNA was not re-expressed in the four cell lines (SNU-61, SNU-175, SNU-769A, and SNU-769B) that did not express RUNX3 mRNA and were not amplified by methylated DNA specific PCR (Figure 5). Furthermore, RUNX3 mRNA expression was not reactivated in these four cell lines by trichostatin-A (TSA) treatment, either alone or when combined with 5-aza-2′-deoxycytidine (data not shown).
Abnormal de novo methylation of CpG islands occurs frequently in human cancers including colorectal cancers; moreover, hypermethylation of CpG islands is known to be associated with transcriptional inactivation (Melki et al., 2000; Trojan et al., 2000; Esteller et al., 2001; Song et al., 2001; Zöchbauer-Müller et al., 2001; Ku et al., 2002; Suh et al., 2002; van Engeland et al., 2002; Yang et al., 2002). It has also been reported that transcriptional inactivation by hypermethylation of the promoter region in some genes, for example, APC, LKB1, p16INK4a, hMLH1, CDX1, RASSF1A, and E-cadherin genes, is involved in colorectal carcinogenesis (reviewed by Trojan et al., 2000; Esteller et al., 2001; Ku et al., 2002; Suh et al., 2002; van Engeland et al., 2002). In this study, we analysed the methylation status of the promoter region and performed mutational analysis on the RUNX3 gene in 32 colorectal cancer cell lines. We first checked whether RUNX3 was expressed in these cell lines by RT–PCR, and found that RUNX3 was expression was downregulated in 16 of the cell lines (50%). This expression ratio of RUNX3 obtained by RT–PCR in colorectal cancer cell lines was similar to that found in gastric cancer cell lines (47%) (Li et al., 2002). Mutations in the RUNX3 gene were not found in the 32 colorectal cancer cell lines, by PCR-SSCP analysis, and mutations of the RUNX3 gene have only been reported in one case of 119 gastric cancers (Li et al., 2002). This result indicates that mutations of the RUNX3 gene are very rare in colorectal cancers as in gastric cancers.
The RUNX3 gene is located on the short arm of human chromosome 1 at 1p36 (Li et al., 2002), a region in which it is suggested that genes playing roles in maintenance of chromosome stability, suppression of tumorigenesis, control of apoptosis, and DNA methylation are located (reviewed by Di Vinci et al., 1998a; 1998b). Indeed, deletions at 1p36 are common in colorectal cancers (Tanaka et al., 1993; Praml et al., 1995; Ogunbiyi et al., 1997; Di Vinci et al., 1998a; 1998b), which suggests that the loss of genes in this region might be implicated in chromosome instability (Di Vinci et al., 1998b). It has also been reported that Runt domain transcription factors are important targets of TGF-β signaling. TGF-β activates Smad2 and Smad3 through TGF-βRII and TGF-βRI receptors, these Smads then associate with Smad4 and enter the nucleus. Each of the receptor regulated-Smads interacts directly with each of the three members of the RUNX class of transcription factors. RUNX3 contacts the MH2 domains of Smad3 and Smad1, present in their C-terminal conserved regions. Given that RUNX3 interacts directly with receptor regulated Smads, inactivation of RUNX3 in cancer fits nicely with the results of earlier studies which found that TGF-β signal transduction pathways are also interrupted in many types of cancers, including those of the gastrointestinal tract (reviewed by Ito and Miyazono, 2003). The gastric mucosa of Runx3 null mice exhibit hyperplasia due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to the growth-inhibitory and apoptosis-inducing action of TGF-β (Li et al., 2002). The TGF-β RII is also frequently altered in colon and gastric cancers, and mutations in Smad2 and Smad4 resulting in their inactivation have been reported in colon cancers. RUNX3 might function as a tumor suppressor in other types of cancers, in addition to gastric cancers, including colon cancers, where mutations or deletions are often found in either TGF-β receptors or Smads (reviewed by Ito and Miyazono, 2003). In the present study, LOH analysis at chromosome 1p36, which includes the RUNX3 gene (Li et al., 2002), showed MSI in the SNU-61 cell line at D1S246 and MSI in SNU-769A, SNU-769B, and SNU-1047 at D1S199. However, no LOH or MSI was found in SNU-1040 or SNU-1197 cells. RUNX3 was not expressed in cell lines showing MSI (i.e. SNU-61, SNU-769A, SNU-769B, and SNU-1047), but RUNX3 mRNA was expressed in SNU-1040 and SNU-1197 cell lines, which lacked LOH and MSI at 1p36. The minimal deleted region in chromosome 1 from gastric cancer cells has been mapped between D1S199 and D1S246, which includes the RUNX3 gene locus (reviewed by Li et al., 2002). It has also been reported that hemizygous deletions of the RUNX3 gene were found in 14 of 46 gastric cancers (30%), and that hemizygous deletions and RUNX3 promoter methylation was found in three of 15 gastric cancer cell lines (Li et al., 2002).
Our experiment suggested that the MSI status rather than LOH at the RUNX3 gene locus, in four cell lines (SNU-61, SNU-769A, SNU-769B, and SNU-1047), might be involved in colorectal carcinogenesis. Moreover, we had already reported that SNU-769A, SNU-769B, and SNU-1047 cell lines showed MSI by BAT-26 (Ku et al., 1999). These cell lines also had frameshift mutations in the targets of MSI within the coding regions of TGF-βRII, BAX, and hMSH3 genes (Ku et al., 1999).
We analysed methylation of the RUNX3 promoter by MS-PCR, after sodium-bisulfite modification, and by direct sequencing analysis. Of the 20 cell lines analysed, methylated DNA was amplified in 12. These cell lines did not express RUNX3 mRNA. Methylation of the 24 CpG sites of the promoter region of the RUNX3 gene was confirmed in these 12 cell lines by direct sequencing analysis of the PCR products, which were amplified with PCR primers specific for methylated and unmethylated DNA. We found that all the C residues of the 24 CpG sites in the promoter region of RUNX3 gene were fully methylated. These results were similar to those obtained in gastric cancer cell lines (Li et al., 2002).
In our experiments the incidence of hypermethylation of the RUNX3 promoter in colorectal cancer cell lines (37.5%) was higher than in colorectal cancer tissues (18.4%). Since DNA was extracted from surgically removed frozen tissue biopsies, the tumor tissues may have been contaminated with normal stromal cells, therefore, masking the true levels of hypermethylation of the RUNX3 promoter in cancer tissues. For further analysis of the methylation status of the RUNX3 promoter in colorectal cancer tissues, expression analysis of RUNX3 could be performed by in situ hybridization (Li et al., 2002) or immunostaining. Laser capture microdissection techniques would allow more precise isolation of cancer cells and normal cells for further analysis of methylation status. Hypermethylation of the RUNX3 gene in corresponding normal tissues was not detected. It has been already reported that cancer cell lines have much higher levels of CpG island hypermethylation than corresponding malignant tissues, which may explain our lower incidence on hypermethylation in tissues versus cell lines. However, cancer cell lines often preserve hypermethylation from the tumors they originate from, so they are indeed useful resources to study hypermethylation (Smiraglia et al., 2001; Paz et al., 2003).
The SNU-1047 cell line showed MSI at D1S199, and the promoter region of its RUNX3 gene was methylated. This result suggests that one allele had been lost by deletion, and the other inactivated by aberrant methylation, and that these events lead to biallelic inactivation and complete lack of RUNX3 gene expression, as found in gastric cancer cell lines (Li et al., 2002). SNU-1040 and SNU-1197, which expressed RUNX3, showed neither methylation of the RUNX3 gene nor LOH at D1S199 and D1S246, and four cell lines (SNU-61, SNU-175, SNU-769A, and SNU-769B) showed neither expression of RUNX3 nor methylation. Of these, SNU-61 showed microsatellite instability at D1S246, and SNU-769A and SNU-769B showed MSI at D1S199. Since both SNU-769A, which originated from a metastasis site in a lymph node, and SNU-769B, which originated from a primary site, were derived from the same patient (Oh et al., 1999) and showed the same MSI results, this suggests that the RUNX3 gene may be inactivated by other mechanisms in these cell lines.
Our results support the importance of promoter methylation in the inactivation of the RUNX3 gene as the RUNX3 gene was re-expressed after treatment with 5-aza-2′-deoxycytidine. This agent reactivates gene expression when hypermethylation of CpG islands is the cause of reduced gene expression.
Our results showed that the RUNX3 mRNA was re-expressed after 5-aza-2′-deoxycytidine treatment in all 12 cell lines that did not express RUNX3 mRNA, and which were hypermethylated at the 24 CpG sites in the promoter region. However, the RUNX3 gene was not re-expressed after treatment with TSA alone, an inhibitor of histone deacetylase, or a mixture of both 5-aza-2′-deoxycytidine and TSA in four cell lines without RUNX3 mRNA or methylation, which suggests the inactivation of RUNX3 expression by another mechanism.
In conclusion, we found hypermethylation in the promoter region of the RUNX3 gene in 12 of 32 colorectal cancer cell lines and 16 of 87 colorectal cancer tissues. This methylation was confirmed by MS-PCR, sequence analysis, and treatment with 5-aza-2′-deoxycytidine. To our knowledge, this is the first demonstration that hypermethylation of the RUNX3 gene in cancers, other than gastric cancer, is associated with the transcriptional inactivation of the RUNX3 gene. Our data demonstrate that hypermethylation of the RUNX3 promoter downregulates gene expression in colorectal carcinoma cell lines, and suggest that this methylation may contribute to the development and progression of human colorectal carcinomas.
Materials and Methods
A total of 32 colorectal cancer cell lines (listed in Table 1) and two gastric cancer cell lines (SNU-1 and SNU-5) were obtained from the KCLB (Korean Cell Line Bank) (Seoul, Korea) or ATCC (American Type Culture Collection). In total, SNU-colorectal cancer cell lines were established and have been reported upon by this laboratory (Park et al., 1987; Oh et al., 1999). SNU-1 and SNU-5 gastric carcinoma cell lines were used as methylation positive (SNU-1) and negative (SNU-5) controls for RUNX3 gene expression (Li et al., 2002). All cell lines were maintained in RPMI1640 supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cultures were maintained in humidified incubators at 37°C in a 5% CO2 and 95% air atmosphere.
Nucleic acid isolation and cDNA synthesis
Genomic DNA and total RNA were isolated from washed-cell pellets and frozen tissue biopsies. Total genomic DNA was extracted using a standard SDS-proteinase K procedure, and total cellular RNA was extracted according to the manufacturer's specifications (Intron Biotechnology, Seoul, Korea). For cDNA synthesis, 2 μg of total RNA was reverse transcribed using random oligo (dT) primer, dNTPs, and 1 μl (200 U) of Superscript™ II reverse transcriptase (Life Technologies, Gaithersburg, MD, USA) in a final volume of 20 μl, for 1 h 15 min at 42°C, after a 10-min denaturation at 70°C. In all, 80 μl of distilled water was then added to the reverse-transcription reaction.
Expression of RUNX3 gene
For RT–PCR analysis, cDNA was amplified using 1 μl of the reverse-transcription reaction, primers, dNTPs, and 0.5 U of Taq DNA polymerase in a final volume of 25 μl. The PCR conditions consisted of 5 min at 94°C for initial denaturation, followed by 35 cycles of 94°C (1 min), 55°C (1 min), and 72°C (1 min), and a final elongation of 7 min at 72°C. PCR amplification was carried out in a programmable thermal cycler (Perkin-Elmer Cetus 9600, Roche Molecular Systems, Inc, NJ, USA). The primers used to amplify the RUNX3 cDNA were as follows: sense, 5′-IndexTermGGCAATGACGAGAACTAC-3′ (located in exon 2); antisense, 5′-IndexTermGGAGAATGGGTTCAGTTC-3′ (located in exon 5). The expected size of the PCR product was 396 bp.
The primer sequences for the RUNX3 isoforms were as follows: For p44 (Accession number: NM_004350.1), sense, 5′-IndexTermCTGTTATGCGTATTCCCGTAG-3′ (nucleotide position, 5–25; start codon, 10–12); antisense, 5′-IndexTermGGAGAATGGGTTCAGTTC-3′ (nucleotide position, 721–738); and for p46 (Accession number: BC013362.2), sense, 5′-IndexTermGGCATCGAACAGCATCTTCG-3′ (nucleotide position, 249–269; start codon, 247–249); antisense, 5′-IndexTermGGAAGGAGCGGTCAAACTG-3′ (nucleotide position, 1027–1045).
Primers for β-actin were used to confirm RNA integrity. Amplified DNA fragments were fractionated in 2% (w/v) agarose gel and stained with ethidium bromide.
Tissue sample collection
In total, 87 paired tumor and normal mucosal tissue samples were obtained from 87 patients with primary colorectal adenocarcinoma. The normal mucosal tissue specimens were collected from the same cancer patients 10 cm, or more, away from the tumor areas. Approximately 2 g of the surgically removed tissues were frozen immediately and stored in liquid nitrogen. The remaining sections of the samples were fixed with formalin and used for further histological examination to confirm the diagnosis postoperatively.
Mutation screening, MS-PCR, and sequence analysis
For MS-PCR, sodium bisulfite modification of genomic DNA was performed as previously reported (Herman et al., 1996). Briefly, 2 μg of genomic DNA was denatured with NaOH and hydroquinone, 3 M sodium bisulfite was added and the mix was incubated at 55°C for 16 h. Following bisulfite modification, the DNA was purified using a Wizard DNA purification system (Promega, Madison, WI, USA), ethanol precipitated, dried, and resuspended in 100 μl distilled water. PCR was performed using PCR primers described previously (Li et al., 2002). Bisulfite-modified DNA was amplified from methylated DNA using primers Rx3-5M and Rx3-3M. For the unmethylated DNA, the Rx3-5U and Rx3-3U PCR primers were used. For amplification and sequencing of bisulfite modified DNA to determine the methylation status, degenerate oligonucleotide primers were designed. The primer sequences were as follows: Rx5-MI, 5′-IndexTermTTAYGAGGGGYGGTYGTAYGYGGG-3′ (Y=C+T) and Rx3-MI, 5′-IndexTermAAAACRACCRACRCRAACRCCTCC-3′ (R=G+A). Amplified DNA fragments were fractionated in a 2% (w/v) agarose gel, stained with ethidium bromide, and visualized under UV light. Amplified bisulfite PCR products were directly sequenced using a Taq dideoxy terminator cycle sequencing kit on an ABI 3100 DNA sequencer (Perkin-Elmer, Foster City, CA, USA).
5-aza-2′-dedoxycytidine and TSA treatment
For 5-aza-2′-deoxycytidine treatment, cells were seeded in two culture flasks at a density of 2 × 105 cells/75-cm2 on day 0. The cells were treated with and without 1–5 μ M of 5-aza-2′-deoxycytidine (Sigma Chemical Co.) for 24 h on days 2 and 5, and the medium was changed 24 h after adding 5-aza-2′-deoxycytidine. Cells were harvested on day 8 for the analysis of RUNX3 expression. DMSO alone was used as a control to exclude nonspecific solvent effects on cells. For TSA treatment, cells were seeded as above, and treated with and without 50–100 ng/ml of TSA (Sigma Chemical Co.) for 36 h on day 2 (Zöchbauer-Müller et al., 2001; Li et al., 2002; Suh et al., 2002). Cells were harvested on day 5 for the analysis of RUNX3 expression.
LOH status was established by PCR using two microsatellite markers flanking chromosome 1p36: D1S199 and D1S246 (Ezaki et al., 1996). All primer sequences were obtained from http://www.gdb.org. Corresponding paraffin-embedded tissue blocks of SNU-61, SNU-769A, SNU-769B, SNU-1040, SNU-1047, and SNU-1197 cell lines were available. Normal epithelial cells and/or lymphocytes of lymph nodes without infiltration of cancer cells were obtained from H&E stained slides. Normal cells were precisely obtained from H&E stained slides using a 30-gauge needle. Approximately 500–1000 dissected normal cells were digested using the proteinase K method as described previously (Hung et al., 1995) and 5 μl of the DNA samples were used directly for each PCR reaction.
Bangsow C, Rubins N, Glusman G, Bernstein Y, Negreanu V, Goldenberg D, Lotem J, Ben-Asher E, Lancet D, Levanon D and Groner Y . (2001). Gene, 279, 221–232.
Di Vinci A, Infusini E, Nigro S, Monaco R and Giaretti W . (1998a). Cancer, 83, 415–422.
Di Vinci A, Infusini E, Peveri C, Sciutto A, Geido E, Risio M, Rossini FP and Giaretti W . (1998b). Int. J. Cancer, 75, 45–50.
Esteller M, Fraga MF, Guo M, Garcia-Foncillas J, Hedenfalk I, Godwin AK, Trojan J, Vaurs-Barriere C, Bignon YJ, Ramus S, Benitez J, Caldes T, Akiyama Y, Yuasa Y, Launonen V, Canal MJ, Rodriguez R, Capella G, Peinado MA, Borg A, Aaltonen LA, Ponder BA, Baylin SB and Herman JG . (2001). Hum. Mol. Genet., 10, 3001–3007.
Ezaki T, Yanagisawa A, Ohta K, Aiso S, Watanabe M, Hibi T, Kato Y, Nakajima T, Ariyama T, Inazawa J, Nakamura Y and Horii A . (1996). Br. J. Cancer, 73, 424–428.
Guo WH, Weng LQ, Ito K, Chen LF, Nakanishi H, Tatematsu M and Ito Y . (2002). Oncogene, 21, 8351–8355.
Herman JG, Graff JR, Myöhänen S, Nelkin BD and Baylin SB . (1996). Proc. Natl. Acad. Sci. USA, 93, 9821–9826.
Hung J, Kishimoto Y, Sugio K, Virmani A, Mclntire DD, Minna JD and Gazdar AF . (1995). J. Am. Med. Assoc., 273, 558–563.
Ito Y and Miyazono K . (2003). Curr. Opin. Genet. Dev., 13, 43–47.
Ku JL, Yoon KA, Kim DY and Park JG . (1999). Eur. J. Cancer, 35, 1724–1729.
Ku JL, Yoon KA, Kim IJ, Kim WH, Jang JY, Suh KS, Kim SW, Park YH, Hwang JH, Yoon YB and Park JG . (2002). Br. J. Cancer, 87, 187–193.
Lee KS, Hong SH and Bae SC . (2002). Oncogene, 21, 7156–7163.
Li QL, Ito K, Sakakura C, Fukamachi H, Inoue KI, Chi XZ, Lee KY, Nomura S, Lee CW, Ban SB, Kim HM, Kim WJ, Yamamoto H, Yamashita N, Yano T, Ikeda T, Itohara S, Inazawa J, Abe T, Hagiwara A, Yamagishi H, Ooe A, Kaneda A, Sugimura T, Ushijima T, Bae SC and Ito Y . (2002). Cell, 109, 113–124.
Melki JR, Vincent PC, Brown RD and Clark SJ . (2000). Blood, 95, 3208–3213.
Nam S, Jin YH, Li QL, Lee KY, Jeong GB, Ito Y, Lee JH and Bae SC . (2002). Mol. Cell. Biol., 22, 547–554.
Ogunbiyi OA, Goodfellow PJ, Gagliardi G, Swanson PE, Birnbaum EH, Fleshman JW, Kodner IJ and Moley JF . (1997). Gastroenterology, 113, 761–766.
Oh JW, Ku JL, Yoon KA, Kwon HJ, Kim WH, Park HS, Yeo KS, Song SY, Chung JK and Park JG . (1999). Int. J. Cancer, 81, 902–910.
Park JG, Oie HK, Sugarbaker PH, Henslee JG, Chen TR, Johnson BE and Gazdar AF . (1987). Cancer Res., 47, 6710–6718.
Paz MF, Fraga MF, Avila S, Guo M, Pollan M, Herman JG and Esteller M . (2003). Cancer Res., 63, 1114–1121.
Praml C, Finke LH, Herfarth C, Schlag P, Schwab M and Amler L . (1995). Oncogene, 11, 1357–1362.
Rini D and Calabi F . (2001). Gene, 273, 13–22.
Smiraglia DJ, Rush LJ, Fruhwald MC, Dai Z, Held WA, Costello JF, Lang JC, Eng C, Li B, Wright FA, Caligiuri MA and Plass C . (2001). Hum. Mol. Genet., 10, 1413–1419.
Song SH, Jong HS, Choi HH, Inoue H, Tanabe T, Kim NK and Bang YJ . (2001). Cancer Res., 61, 4628–4635.
Suh ER, Ha CS, Rankin EB, Toyota M and Traber PG . (2002). J. Biol. Chem., 277, 35759–35800.
Tanaka K, Yanoshita R, Konishi M, Oshimura M, Maeda Y, Mori T and Miyaki M . (1993). Oncogene, 8, 2253–2258.
Trojan J, Brieger A, Raedle J, Esteller M and Zeuzem S . (2000). Gut, 47, 272–276.
van Engeland M, Roemen GM, Brink M, Pachen MM, Weijenberg MP, de Bruine AP, Arends JW, van den Brandt PA, de Goeij AF and Herman JG . (2002). Oncogene, 21, 3792–3795.
Yang Q, Nakamura M, Nakamura Y, Yoshimura G, Suzuma T, Umemura T, Shimizu Y, Mori I, Sakurai T and Kakudo K . (2002). Clin. Cancer Res., 8, 2890–2893.
Zöchbauer-Müller S, Fong KM, Maitra A, Lam S, Geradts J, Ashfaq R, Virmani AK, Milchgrub S, Gazdar AF and Minna JD . (2001). Cancer Res., 61, 3581–3585.
This work was supported in part by the 2002 BK21 Project for Medicine, Dentistry, and Pharmacy and in part by the Basic Research Program (R01-2003-000-10543-0) of the Korea Science & Engineering Foundation.
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Ku, JL., Kang, SB., Shin, YK. et al. Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines. Oncogene 23, 6736–6742 (2004). https://doi.org/10.1038/sj.onc.1207731
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