CpG methylation is maintained in human cancer cells lacking DNMT1

Rhee, Ina; Jair, Kamwing; Yen, Ray-Whay Chiu; Lengauer, Christoph; Herman, James G.; Kinzler, Kenneth W.; Vogelstein, Bert; Baylin, Stephen B.; Schuebel, Kornel E.

doi:10.1038/35010000

Cited by 415 publications

(316 citation statements)

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“…26 For instance, the expression of p16 in the CRC cell line HCT116 is diminished by a mutation on one allele and epigenetic inactivation by promoter hypermethylation of the other allele. 27 In addition, we have shown that PTEN expression in sporadic CRC is influenced by mutations or allelic loss of one allele and promoter hypermethylation of the other allele. 28 Promoter hypermethylation might occur at a very early state.…”

Section: Discussionmentioning

confidence: 95%

APC promoter hypermethylation contributes to the loss of APC expression in colorectal cancers with allelic loss on 5q1

Arnold¹,

Goel²,

Niedzwiecki³

et al. 2004

Cancer Biology & Therapy

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Introduction: Germ-line mutations of the APC gene are associated with familial adenomatous polyposis, and somatic mutations occur frequently in sporadic colorectal cancer. However, to abrogate APC function, both alleles must be inactivated. Recently, it has been demonstrated that epigenetic modification of the APC promoter influences APC silencing. Here we examined the influence of APC methylation on APC expression in tumors with and without LOH at the APC locus.Material and Methods: 137 sporadic colorectal cancer specimens were investigated for LOH at the 5q locus. The methylation status of the APC promoter was determined by methylation-specific PCR. APC expression was performed by immunohistochemistry.Results: expression was reduced or lost in 110 of 137 (80%) tumors and LOH at 5q was found in 13 of 132 (10%) tumors. There was no difference in 5q LOH between tumors with or without intact APC expression. Vice versa, there was no difference in the APC expression in tumors with 5q LOH. Aberrant APC promoter methylation was detected in 33 of 118 (28%) tumors investigated. Of the tumors with 5q LOH for which methylation data were available, 4 of 11 (36%) were methylated versus 28 of 105 (27%) of those without LOH. No difference in methylation was observed in tumors without 5q LOH and normal APC expression and those without 5q LOH and reduced or missing APC expression. Importantly, none of the tumors with 5q LOH and normal APC staining were aberrantly methylated, whereas 50% of the cancers with LOH at 5q and reduced or absent staining were hypermethylated.Conclusions: This report suggests that tumors with 5q LOH and reduced APC expression are more frequently hypermethylated at the APC promoter compared to those tumors with 5q LOH and normal APC expression. The association among APC promoter methylation status, 5q LOH, and reduced or lost APC expression suggests that de novo methylation plays an important role as a "second hit" in silencing APC expression in colorectal neoplasia.

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Section: Discussionmentioning

confidence: 95%

APC promoter hypermethylation contributes to the loss of APC expression in colorectal cancers with allelic loss on 5q1

Arnold¹,

Goel²,

Niedzwiecki³

et al. 2004

Cancer Biology & Therapy

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“…For this task, we utilized a genetic DNMT knockout HCT116 model system (Rhee et al, 2000(Rhee et al, , 2002. We initially confirmed the genotype and phenotype of the DNMT knockout lines by measuring DNMT1 and DNMT3b expression by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively (Supplemental Figure 2a and b), and by analysing global genomic DNA methylation levels using Liquid chromatography-mass spectrometry (LC-MS) (Song et al, 2005) (Supplemental Figure 2c).…”

Section: Activation Of Cg-x Gene Expression By Genetic Knockout Of Dnmentioning

confidence: 99%

“…Recent studies using tumor suppressor genes have provided information concerning the role that specific DNMTs play in mediating gene repression and promoter methylation in human cancer cells (Rhee et al, 2000(Rhee et al, , 2002Leu et al, 2003;Robert et al, 2003;Ting et al, 2004). Some of these studies suggest a critical independent role for DNMT1 (Robert et al, 2003), whereas others implicate cooperativity between DNMT1 and DNMT3b in mediating tumor suppressor gene repression (Rhee et al, 2000(Rhee et al, , 2002Ting et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

“…These genes each contain 5 0 CpG island promoters that encompass their known transcriptional start sites (Figure 1). We examined a number of key epigenetic parameters, including mRNA expression, promoter DNA methylation, and promoter histone code patterns, using an HCT116 model system in which one or both DNMT genes are disrupted by homologous recombination (Rhee et al, 2000(Rhee et al, , 2002. Additionally, we examined CG-X antigen expression and promoter methylation in HCT116 and RKO colon cancer cells following specific targeting of DNMT1 by RNA interference (RNAi).…”

Section: Introductionmentioning

confidence: 99%

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Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

2006

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We examined the function of two key DNA methyltransferase (DNMT) enzymes in epigenetic regulation of Xlinked cancer/germline (CG-X) antigen genes in human cancer cells, using MAGE-A1, NY-ESO-1, and XAGE-1 as models. In HCT116 cells, genetic knockout of DNMT1 caused moderate activation of CG-X genes, DNMT3b knockout had a negligible effect, and double knockout of both enzymes caused robust gene induction. Similarly, dual DNMT knockout caused dramatic hypomethylation of the MAGE-A1 and NY-ESO-1 promoters, DNMT1 knockout showed moderate hypomethylation, and DNMT3b knockout elicited only slight methylation changes. In contrast, both single and double knockout cells showed significant hypomethylation of the XAGE-1 promoter. RNA interference (RNAi) targeting of DNMT1 in HCT116 cells validated the results seen using genetic knockout cells; however, RNAi targeting of DNMT1 in a different colorectal cancer cell line revealed a greater independent role for DNMT1 in mediating CG-X gene repression and promoter methylation in other cell types. Notably, the histone H3 modification pattern at CG-X promoters was altered following DNMT knockout. DNMT1 or DNMT3b knockout reduced dimethylated lysine-9 (diMe-H3K9) levels, but did not significantly affect dimethylated lysine-4 (diMe-H3K4) or acetylated lysine-9 (Ac-H3-K9) levels. In contrast, dual DNMT1/3b knockout reduced the level of diMe-H3K9 and dramatically increased the levels of diMe-H3K4 and Ac-H3K9 at CG-X gene loci. In summary, DNMT1 and DNMT3b were found to perform both redundant and independent functions in epigenetic regulation of CG-X antigen genes in human cancer cells.

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“…A human colon cancer cell line, HCT116 deficient in DNMT1 following targeted recombination, demonstrates demethylation of pericentromeric satellite sequences (Rhee et al, 2000), whereas HCT116 cells lacking both DNMT1 and DNMT3B contain demethylated satellite 2 and Alu repetitive sequences (Rhee et al, 2002) and demonstrate increased chromosomal instability (Karpf and Matsui, 2005). Additional studies of the doubly targeted cells suggest that at least some histone modifications occur before alterations in DNA methylation (Bachman et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins

et al. 2007

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Cancer cells display an altered distribution of DNA methylation relative to normal cells. Certain tumor suppressor gene promoters are hypermethylated and transcriptionally inactivated, whereas repetitive DNA is hypomethylated and transcriptionally active. Little is understood about how the abnormal DNA methylation patterns of cancer cells are established and maintained. Here, we identify over 20 DNMT3B transcripts from many cancer cell lines and primary acute leukemia cells that contain aberrant splicing at the 5 0 end of the gene, encoding truncated proteins lacking the C-terminal catalytic domain. Many of these aberrant transcripts retain intron sequences. Although the aberrant transcripts represent a minority of the DNMT3B transcripts present, Western blot analysis demonstrates truncated DNMT3B isoforms in the nuclear protein extracts of cancer cells. To test if expression of a truncated DNMT3B protein could alter the DNA methylation patterns within cells, we expressed DNMT3B7, the most frequently expressed aberrant transcript, in 293 cells. DNMT3B7-expressing 293 cells have altered gene expression as identified by microarray analysis. Some of these changes in gene expression correlate with altered DNA methylation of corresponding CpG islands. These results suggest that truncated DNMT3B proteins could play a role in the abnormal distribution of DNA methylation found in cancer cells.

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CpG methylation is maintained in human cancer cells lacking DNMT1

Cited by 415 publications

References 24 publications

APC promoter hypermethylation contributes to the loss of APC expression in colorectal cancers with allelic loss on 5q1

APC promoter hypermethylation contributes to the loss of APC expression in colorectal cancers with allelic loss on 5q1

Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins

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