Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Herman, James G.; Graff, Jeremy R.; Myöhänen, Sanna; Nelkin, Barry D.; Baylin, Stephen B.

doi:10.1073/pnas.93.18.9821

Cited by 5,068 publications

(4,169 citation statements)

References 28 publications

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“…Methylation-associated silencing of HSulf-1 in ovarian cell lines and primary ovarian tumors Primers encompassing 12 CpG sites within the CpG-rich region in Exon 1A of HSulf-1 (Cross et al, 1994;Lai et al, 2003) were designed to amplify and sequence the bisulfite-modified DNA as described previously (Herman et al, 1996;Shridhar et al, 2001). Overall methylation was determined for a subset of cell lines and primary ovarian tumors (fresh frozen) with or without HSulf-1 expression.…”

Section: Resultsmentioning

confidence: 99%

Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance

et al. 2007

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To investigate the mechanism by which HSulf-1 expression is downregulated in ovarian cancer, DNA methylation and histone acetylation of HSulf-1 was analysed in ovarian cancer cell lines and primary tumors. Treatment of OV207 and SKOV3 by 5-aza-2 0 -deoxycytidine resulted in increased transcription of HSulf-1. Sequence analysis of bisulfite-modified genomic DNA from ovarian cell lines and primary tumors without HSulf-1 expression revealed an increase in the frequency of methylation of 12 CpG sites in exon 1A. Chromatin immunoprecipitation assays showed an increase in histone H3 methylation in cell lines without HSulf-1 expression. To assess the significance of HSulf-1 downregulation in ovarian cancer, OV167 and OV202 cells were transfected with HSulf-1 siRNA. Downregulation of HSulf-1 expression in OV167 and OV202 cells lead to an attenuation of cisplatin-induced cytotoxicity. Moreover, patients with ovarian tumors expressing higher levels of HSulf-1 showed a 90% response rate (27/30) to chemotherapy compared to a response rate of 63% (19/30) in those with weak or moderate levels (P ¼ 0.0146, v 2 test). Collectively, these data indicate that HSulf-1 is epigenetically silenced in ovarian cancer and that epigenetic therapy targeting HSulf-1 might sensitize ovarian tumors to conventional first-line therapies.

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Section: Resultsmentioning

confidence: 99%

Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance

et al. 2007

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“…Methylation-specific PCR Methylation-specific PCR was done as previously described with slight modifications (Herman et al, 1996). EZ DNA methylation kit (ZymoResearch, Orange, CA, USA) was used for the bisulfite treatment of DNA as per the manufacturer's instructions.…”

Section: Protein Extraction and Immunoblottingmentioning

confidence: 99%

p21WAF1/CIP1 induction by 5-azacytosine nucleosides requires DNA damage

et al. 2008

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Decitabine (DAC) and 5-azacitidine have recently been approved for the treatment of myelodysplastic syndrome. The pharmacodynamic effects of DAC and 5-azacitidine outside their known activity as inhibitors of DNA methyltransferases (DNMTs) require further investigation. The purpose of this study was to investigate the effect of DAC on the expression of p21 WAF1/CIP1 , a gene with a putative CpG island surrounding its promoter region. Promoter methylation analysis of p21 WAF1/CIP1 in leukemia cells revealed the absence of CpG methylation. However, DAC upregulated p21 WAF1/CIP1 expression in a dosedependent manner (ED 50 ¼ 103.34 nM) and induced G2/M cell cycle arrest in leukemia cells. Sequential application of DAC followed by different histone deacetylase inhibitors induced expression of p21 WAF1/CIP1 synergistically. Upregulation of p21 WAF1/CIP1 paralleled DAC-induced apoptosis (ED 50 ¼ 153 nM). Low doses of DAC induced c-H2AX expression (ED 50 ¼ 16.5 nM) and upregulated p21 WAF1/CIP1 in congenic HCT 116 colon cancer cells in a DNMT-independent and p53-dependent fashion. Inhibition of p53 transactivation by pifithrin-a or the kinase activity of ATM by either the specific ATM inhibitor KU-5593 or caffeine abrogated p21 WAF1/CIP1 upregulation, indicating that DAC upregulation of p21 WAF1/CIP1 was p53-and ATM-dependent in leukemia cells. In conclusion, DAC upregulates p21 WAF1/CIP1 in DNMT-independent manner via the DNA damage/ATM/ p53 axis.

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“…Subsequently, the DNA promoter methylation status of the CDH13 and CDH1 gene was investigated by MSP assay as described previously. 23 Both specific primers for methylated and unmethylated promoters and annealing temperature applied were described in previous reports. 3,4,23 The primers for the methylated CDH13 were (sense) 5 0 -TCGCGGGGTTCGTT TTTCGC-3 0 and (antisense) 5 0 -GACGTTTTCATTCA TACACGCG-3 0 (annealing temperature: 571C, 243 bp product) and for the unmethylated form were (sense) 5 0 -TTGTGGGGTTTGTTTTTTGT-3 0 and (antisense) 5 0 -AACTTTTCATTCATACACACA-3 0 (annealing temperature: 531C, 242 bp product).…”

Section: Bisulfite Modification and Methylation-specific Pcrmentioning

confidence: 99%

Tumor-specific downregulation and methylation of the CDH13 (H-cadherin) and CDH1 (E-cadherin) genes correlate with aggressiveness of human pituitary adenomas

et al. 2007

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The gene products of CDH13 and CDH1, H-cadherin and E-cadherin, respectively, play a key role in cell-cell adhesion. Inactivation of the cadherin-mediated cell adhesion system caused by aberrant methylation is a common finding in human cancers, indicating that the CDH13 and CDH1 function as tumor suppressor and invasion suppressor genes. In this study, we analyzed the expression of H-cadherin mRNA and E-cadherin protein in 5 normal pituitary tissues and 69 primary pituitary adenomas including all major types by quantitative real-time RT-PCR (qRT-PCR) and immunohistochemistry, respectively. Reduced expression of H-cadherin was detected in 54% (28/52) of pituitary tumors and was significantly associated with tumor aggressiveness (Po0.05). E-cadherin expression was lost in 30% (21 of 69) and significantly reduced in 32% (22 of 69) of tumors. E-cadherin expression was significantly lower in grade II, III, and IV than in grade I adenomas (P ¼ 0.015, P ¼ 0.029, and P ¼ 0.01, respectively). Using methylation-specific PCR (MSP), promoter hypermethylation of CDH13 and CDH1 was detected in 30 and 36% of 69 adenomas, respectively, but not in 5 normal pituitary tissues. Methylation of CDH13 was observed more frequently in invasive adenomas (42%) than in non-invasive adenomas (19%) (Po0.05) and methylation of CDH1 was more frequent in grade IV adenomas compared with grade I adenomas (Po0.05). Methylation of either CDH13 or CDH1 was identified in 35 cases (51%) and was more frequent in grade IV invasive adenomas than in grade I non-invasive adenomas (Po0.05 and Po0.05, respectively). Downregulation of expression was correlated with promoter hypermethylation in CDH13 and CDH1. In conclusion, the tumor-specific downregulation of expression and methylation of CDH13 and CDH1, alone or in combination, may be involved in the development and invasive growth of pituitary adenomas.

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Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Cited by 5,068 publications

References 28 publications

Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance

Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance

p21WAF1/CIP1 induction by 5-azacytosine nucleosides requires DNA damage

Tumor-specific downregulation and methylation of the CDH13 (H-cadherin) and CDH1 (E-cadherin) genes correlate with aggressiveness of human pituitary adenomas

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