Methylation profiling of CpG islands in human breast cancer cells

Abstract

CpG island hypermethylation is known to be associated with gene silencing in cancer. This epigenetic event is generally accepted as a stochastic process in tumor cells resulting from aberrant DNA methyltransferase (DNA-MTase) activities. Specific patterns of CpG island methylation could result from clonal selection of cells having growth advantages due to silencing of associated tumor suppressor genes. Alternatively, methylation patterns may be determined by other, as yet unidentified factors. To explore further the underlying mechanisms, we developed a novel array-based method, called differential methylation hybridization (DMH), which allows a genome-wide screening of hypermethylated CpG islands in tumor cells. DMH was used to determine the methylation status of >276 CpG island loci in a group of breast cancer cell lines. Between 5 and 14% of these loci were hypermethylated extensively in these cells relative to a normal control. Pattern analysis of 30 positive loci by Southern hybridization indicated that CpG islands might differ in their susceptibility to hypermethylation. Loci exhibiting pre-existing methylation in normal controls were more susceptible to de novo methylation in these cancer cells than loci without this condition. In addition, these cell lines exhibited different intrinsic abilities to methylate CpG islands not directly associated with methyltransferase activities. Our study provides evidence that, aside from random DNA-MTase action, additional cellular factors exist that govern aberrant methylation in breast cancer cells.

Epigenetics as a mechanism driving polygenic clinical drug resistance

“…Comparison of signal intensities between tumour and normal DNA after autoradiography allows estimation of the number of aberrantly methylated CpG islands in tumours, and individual aberrantly methylated CpG islands can be identified by sequencing. Differential methylation hybridisation (DMH) is an alternative means of examining genome-wide methylation patterns that uses restriction digestion of genomic DNA and ligation to linkers (Huang et al, 1999), followed by digestion with a methylation-sensitive restriction enzyme such as BstUI, PCR amplification and hybridisation to CpG-rich DNA sequences (representing putative CpG islands). Comparison to hybridisation signals obtained from undigested linker-ligated DNA allowed the identification of aberrantly methylated CpG islands.…”

Onecut transcription factor OC2 is a direct target of T-bet in type-1 T-helper cells

“…The CGI inserts were amplified by PCR using the 5 0 -CGGCCGCCTGCAGGTCGACCTTAA-3 0 and 5 0 -AACGCGTTGGGAGCTCTCCCTTAA-3 0 primers. 29 The PCR products were purified using Magnesil (Promega, Madison, WI, USA) and then mixed with dimethyl sulfoxide (DMSO) to a final concentration of 10%. Array Spotter Generation III (Molecular Dynamics, Sunnyvale, CA, USA) was used to assemble the PCR/ dimethyl sulfoxide samples on microarray slides (Type 7 Star; Amersham) as microdots (spot diameter ¼ 250 ml and spot buffer ¼ 30 ml).…”

Aberrant DNA methylation in porcine in vitro‐, parthenogenetic‐, and somatic cell nuclear transfer‐produced blastocysts

“…Differential methylation hybridization analysis was conducted based on a technique developed for global scanning of methylation changes in the human genome (Huang et al, 1999). Porcine CpG island clones from a Porcine CpG Island Library (United Kingdom Human Molecular Reproduction and Development.…”

“…In this study we used a global microarray-based approach, Porcine Differential Methylation Hybridization (PDMH), based on a similar tool that has been developed for humans (Huang et al, 1999), to analyze the CpG methylation in porcine gametes, and embryos. The specific question addressed was to determine if there is a difference in the global methylation profiles in the parthenogenetic-, SCNT-, in vitro, and in vivoproduced blastocysts that explain the developmental differences of these samples.…”