Heterogeneity of DNA Methylation Status Analyzed by Bisulfite-PCR-SSCP and Correlation with Clinico-Pathological Characteristics in Colorectal Cancer

Abstract

Aberrant DNA methylation has been identified as an important mechanism for inactivation of tumor suppressor genes and mismatch repair genes during carcinogenesis. We used bisulfite treatment and the PCR-single strand conformation polymorphism (SSCP) (BiPS) technique to analyze methylation status of the promoter regions of the hMLH1, p16, and HIC1 genes in several cancer cell lines and colorectal cancer tissues. The methylation of the hMLH1, p16 and HIC1 genes was observed in 2, 8, and 13 of 13 cancer cell lines, respectively. The SSCP for p16 and HIC1 in each of the methylation-positive cell lines were similar, indicating relative homogeneity of methylation status and complete methylation in the cell lines. Methylation was observed in 8, 5, and 21 of 25 colorectal cancer tissues for the hMLH1, p16, and HIC1 genes, respectively. The methylated bands revealed by BiPS analysis of the hMLH1 gene were homogeneous, whereas those of the p16 and HIC1 genes were different in each case. The methylation of the promoter region of the HIC1 gene in colorectal cancer was observed most frequently and could serve as a sensitive marker for colorectal cancer. Methylation status of the hMLH1 and p16 gene promoters was correlated with microsatellite instability status, tumor location, and differentiation but not with K-ras mutation or allelic loss of p53.

Methylation of Mitochondrial DNA Is Not a Useful Marker for Cancer Detection

“…The patients were similar to those who participated in our previous studies (4 ). For the patients, both malignant and nonmalignant tissues were examined.…”

Sporadic Colon Cancer: Mismatch Repair Immunohistochemistry and Microsatellite Instability in Omani Subjects

“…Thus, alterations in DNA methylation can silence mismatch repair (MMR) genes such as hMLH1 and hMSH2 [8], and cell cycle regulatory genes such as p16. One study indicated that in CRC there is a distinct pattern of genetic and epigenetic events that are involved in the activation of oncogenes such as K-ras and the inactivation of the tumor suppressor gene p53 [9]. MSI is a hallmark of DNA MMR deficiency that appears to result primarily from inherited and/or acquired alterations in the MMR genes hMLH1 and hMSH2, with the presence of MSI correlating with the absence of either hMLH1 or hMSH2 [10][11][12].…”

Promoter Hypermethylation in Cancer Silences LDHB, Eliminating Lactate Dehydrogenase Isoenzymes 1-4

“…The cancer cell lines were the same as those used in our previous studies (10,11 ). For the patients, both malignant and nonmalignant tissues were examined.…”

“…Each patient consented to the experimental use of specimens and examination of the specimens for pathology. DNA was extracted from the cancer cell lines and the resected tissues by a method described previously (10,11 ).…”