Establishment and Validation of Real-Time Polymerase Chain Reaction Method for CDH1 Promoter Methylation

Abstract

Aberrant methylation of the promoter region has emerged as the major mechanism for silencing tumor suppressor genes. However, for some genes, such as E-cadherin (CDH1), methylation and protein expression demonstrate considerable heterogeneity, making correlations difficult. We compared methylation and protein expression status of CDH1 in 56 primary breast carcinomas using semiquantitative assays. Aberrant CDH1 methylation was studied by methylation-specific polymerase chain reaction (MSP) and semiquantitative real-time MSP assays. The Cdh1 expression was investigated by immunostaining on archival formalin-fixed sections from 34 primary carcinomas and their accompanying normal epithelium and preinvasive and metastatic lesions. Membrane-specific Cdh1 expression in the neoplastic cells was quantified by image analysis using an automated cellular imaging system and a continuous score. Aberrant promoter methylation of the CDH1 was present in 24 of 56 (43%) breast carcinomas by MSP assay. There was excellent concordance between the standard MSP assay and the real-time assay (91%, P < 0.0001). The concordance between loss of Cdh1 expression and CDH1 methylation by standard MSP was 71% (P ‫؍‬ 0.02). Furthermore, there was a strong correlation between the semiquantitative assays for methylation and protein expression (r ‫؍‬ 0.47, P ‫؍‬ 0.005). We conclude that promoter methylation of CDH1 significantly correlated with the Cdh1 expression level, demonstrating that epigenetic silencing is a valid pathway for silencing of tumor suppressor genes in primary breast carcinomas.

Constitutive Expression Exposes Functional Redundancy between the Arabidopsis Histone H2A Gene HTA1 and Other H2A Gene Family Members

“…Real-time PCR has been used by several research groups to estimate gene copy number and for quantification of transcript levels (Bustin, 2000;Kandasamy et al, 2002;Milligan et al, 2002;Toyooka et al, 2002). Because we used Arabidopsis roots to test for Agrobacterium-mediated transformation competence, we used this tissue for our RNA analysis and subsequently compared these results with those using RNA extracted from whole plants.…”

Detection of Promoter Hypermethylation of Multiple Genes in the Tumor and Bronchoalveolar Lavage of Patients with Lung Cancer

“…Thus far, genes such as APC, CDKN2A, CDH1, RAR-␤2, and RASSF1A have been found to harbor hypermethylated promoters in over 30% of lung tumors (10,11). The development of the real-time methylationspecific PCR, which is more sensitive than conventional methylation-specific polymerase chain reaction (MSP) by 10-fold, has simplified the study of the genes inactivated by promoter hypermethylation in human cancer and has the advantage of increasing specificity attributable to the use of an internally binding fluorogenic hybridization probe for each gene (12,13). Recent publications have demonstrated the presence of promoter hypermethylation of various genes in bodily fluids, including bronchoalveolar lavage (BAL) DNA of lung cancer patients (14 -18).…”

RNA Interference-Mediated Knockdown of DNA Methyltransferase 1 Leads to Promoter Demethylation and Gene Re-Expression in Human Lung and Breast Cancer Cells

“…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 bisulfitetreated 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.…”