DNA methylator and mismatch repair phenotypes are not mutually exclusive in colorectal cancer cell lines

Pao, Martha M.; Liang, Gangning; Tsai, Yvonne; Xiong, Zhenggang; Laird, Peter W.; Jones, Peter A.

doi:10.1038/sj.onc.1203414

Cited by 16 publications

(13 citation statements)

References 47 publications

(41 reference statements)

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“…It has been postulated previously that, in addition to genetic events (i.e., mutations or allelic losses), epigenetic gene silencing contributes to tumor suppressor gene inactivation. 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.…”

Section: Discussionmentioning

confidence: 99%

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: 99%

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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“…It has also been reported that MSI ϩ colorectal cell lines have a higher de novo methylation activity towards exogenous retroviral sequences than those that are MSI Ϫ (50). However, we did not find an increased frequency of CpG island hypermethylation in MSI ϩ colorectal cell lines (63) or colorectal tumors (86). We have recently suggested that the association between CpG island hypermethylation and mismatch repair deficiency is limited to the association with MLH1 promoter hypermethylation (86).…”

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confidence: 90%

DNA Methyltransferase Deficiency Modifies Cancer Susceptibility in Mice Lacking DNA Mismatch Repair

Trinh¹,

Long²,

Nickel³

et al. 2002

Molecular and Cellular Biology

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We have introduced DNA methyltransferase 1 (Dnmt1) mutations into a mouse strain deficient for the Mlh1 protein to study the interaction between DNA mismatch repair deficiency and DNA methylation. Mice harboring hypomorphic Dnmt1 mutations showed diminished RNA expression and DNA hypomethylation but developed normally and were tumor free. When crossed to Mlh1 ؊/؊ homozygosity, they were less likely to develop the intestinal cancers that normally arise in this tumor-predisposed, mismatch repair-deficient background. However, these same mice developed invasive T-and B-cell lymphomas earlier and at a much higher frequency than their Dnmt1 wild-type littermates. Thus, the reduction of Dnmt1 activity has significant but opposing outcomes in the development of two different tumor types. DNA hypomethylation and mismatch repair deficiency interact to exacerbate lymphomagenesis, while hypomethylation protects against intestinal tumors. The increased lymphomagenesis in Dnmt1 hypomorphic, Mlh1 ؊/؊ mice may be due to a combination of several mechanisms, including elevated mutation rates, increased expression of proviral sequences or proto-oncogenes, and/or enhanced genomic instability. We show that CpG island hypermethylation occurs in the normal intestinal mucosa, is increased in intestinal tumors in Mlh1 ؊/؊ mice, and is reduced in the normal mucosa and tumors of Dnmt1 mutant mice, consistent with a role for Dnmt1-mediated CpG island hypermethylation in intestinal tumorigenesis.

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“…The PCR-amplified region of the promoter was 530 bp, with 28 CpG dinucleotides, including CpGs both upstream of the transcriptional start site and in the first exon. PCR conditions have been previously described (33). Primers for the p16 5Ј region were as follows: 5Ј-GGT GGG GTT TTT ATA ATT AGG AAA GAA TAG TTT TG-3Ј (sense) and 5Ј-TCT AAT AAC CAA CCA ACC CCT CC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Continuous Zebularine Treatment Effectively Sustains Demethylation in Human Bladder Cancer Cells

Cheng

Weisenberger

Gonzales

et al. 2004

Molecular and Cellular Biology

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The abnormal de novo methylation of promoter CpG islands in numerous tumor suppressor and other cancer-related genes has been shown to be associated with their silencing during carcinogenesis (3,18,19,30). This frequent alteration in human cancer cells may represent an alterative mechanism to mutations and chromosomal deletions for gene inactivation during tumorigenesis. The prevalence of aberrant methylation in cancer has encouraged the search for therapeutic agents which can inhibit methylation and which may thus be utilized to reverse this effect by reactivating genes which have become abnormally silenced.5-Azacytidine (5-Aza-CR) and its deoxy analog, 5-aza-2Ј-deoxycytidine (5-Aza-CdR), are two of the most well-known DNA methylation inhibitors (15,20). Both drugs are nucleoside analogs which have been widely used for studying the role of DNA methylation in biological processes as well as for clinical treatment of patients with acute myeloid leukemia and myelodysplastic syndrome (28,34,42). 5-Aza-CR and 5-AzaCdR are potent mechanism-based inhibitors of DNA methyltransferases (DNMTs) and function by substituting for cytosine residues during DNA replication and forming covalent bonds with the DNMT, which ultimately leads to the inhibition of the DNMT's activity (8,12,31,39). Unfortunately, these drugs are both unstable in aqueous solutions and toxic (4, 7, 39), and these characteristics have complicated their clinical use; hence there is the need for an effective, stable, and minimally toxic inhibitor of DNA methylation.Previously, we characterized zebularine [1-(␤-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) as a novel inhibitor of DNA methylation which is stable and minimally toxic both in vitro and in vivo (6). Zebularine is a cytidine analog containing a 2-(1H)-pyrimidinone ring that was originally developed as a cytidine deaminase inhibitor because it lacks an amino group on position 4 of the ring (22,24). Studies with synthetic oligonucleotides containing the 2-(1H)-pyrimidinone ring have demonstrated the formation of a tight complex with bacterial methyltransferases in vitro (17), and this was further corroborated by a recent study demonstrating the crystallization of a bacterial DNA methyltransferase with the 2-(1H)-pyrimidinone ring forming a covalent bond at the active site (43). In a previous study, we have shown that zebularine can be orally administered to cause reactivation and demethylation of a silenced and hypermethylated p16 gene in human bladder tumor cells grown in nude mice (6). Nonetheless, one of the major challenges with the usage and application of nucleoside analogs as inhibitors of DNA methylation is the problem of remethylation of genes that were demethylated after treatment with these agents, which eventually results in their resilencing (5). This phenomenon of remethylation following cessation of drug treatment makes the clinical application of these drugs quite limited.Here we demonstrate that the single treatment of T24 bladder carcinoma cells with zebularine resulted in a rapid i...

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DNA methylator and mismatch repair phenotypes are not mutually exclusive in colorectal cancer cell lines

Cited by 16 publications

References 47 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

DNA Methyltransferase Deficiency Modifies Cancer Susceptibility in Mice Lacking DNA Mismatch Repair

Continuous Zebularine Treatment Effectively Sustains Demethylation in Human Bladder Cancer Cells

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