| Japanese Journal of Clinical Oncology | Pages |
Letter
A Strategy for the Isolation of DNA Fragments Containing Methylated CpG Islands in Human Adenocarcinomas of the Lung
To the Editor:
It was my pleasure to spend three months in the summer of 1999 at the National Cancer Center (NCC) Research Institute. I am indebted to the Foundation for the Promotion of Cancer Research for providing financial support for this period.
My research during this current visit was an extension of the cooperative research initiated in 1996 with Dr Takao Sekiya and Dr Masahiko Shiraishi of the Oncogene Division at the National Cancer Center Research Institute. In this earlier study we investigated the distribution of CpG islands in cloned genomic DNA. In the current research project we turned our attention to the analysis of methylation of CpG islands as a possible means of epigenetic gene silencing in cancer.
Epigenetic gene silencing is associated with many biological phenomena such as X chromosome inactivation and genomic imprinting. In each of these cases one of the alleles is trancriptionally inactive without any apparent alteration of nucleotide sequences. Although the mechanism of epigenetic silencing is far from clear, accumulating evidence strongly suggests that DNA methylation is closely associated with long-term repression of transcription. It has been proposed that binding of the repressor proteins to methylated CpG pairs (mCpG) induces gene silencing, possibly by altering chromatin structure and subsequently basal transcription machinery and RNA polymerase activities.
Cancer develops following the accumulation of genetic and epigenetic changes. Genetic perturbations, in which changes in the specific nucleotide sequence of a gene occur, have been extensively investigated. However, epigenetic changes remain less well understood. In cancer, methylation of CpG islands is considered to be one of the major epigenetic aberrations that cause gene inactivation, although as yet little is known of the genes affected by CpG island methylation. To address this important issue, cooperative research between Dr Masahiko Shiraishi, Dr Takao Sekiya and their laboratory group and myself has been established.
The starting point for the current collaboration was to follow the creation of a library containing methylated CpG island fragments. Initially methylated genomic DNA fragments from a human lung adenocarcinoma were isolated with the use of a methylated DNA binding column. The methylated DNA binding column is an affinity matrix that contains a polypeptide derived from the methyl-CpG binding domain (MBD) of the rat chromosomal protein MeCP2, which preferentially binds DNA at symmetrical mCpG sites. A polypeptide, termed, HMBD, consists of 85 amino acids of the MBD domain and a histidine tag at the N-terminal end, which can then be attached to a solid support. The stoichiometry of binding is 1 HMBD to 1 mCpG, and therefore heavily methylated DNA fragments have a greater affinity with the column than moderately or poorly methylated fragments. This feature allows the selective enrichment of densely methylated DNA fragments.
Following the isolation of methylated DNA fragments, these fragments were subjected to a procedure known as segregation of partly melted molecules (SPM). SPM is a method for the preferential isolation of DNA fragments that contain CpG islands. SPM takes advantage of the reduced rate of strand dissociation of CpG-rich DNA fragments when electrophoresed in a denaturing gradient gel. In an earlier collaboration in which SPM methodology was employed, we demonstrated that DNA fragments containing CpG islands can be detected efficiently from anonymous regions of cloned DNA. As we have reported previously, SPM is a relatively simple procedure and has the advantage over other CpG island detection approaches in that prior knowledge of nucleotide sequences is not required. Following the SPM separation, we now had a population of genomic DNA fragments that were potentially both highly methylated and CpG rich. These fragments were then ligated into the lambda ZapII vector.
Once the library containing mCpG islands derived from a cancer had been established, it was then necessary to identify those genes which possess mCpG islands specifically in cancerous cells but not in their normal counterparts. The screening of the library for such clones is a time-consuming process. It initially requires determination of the nucleic acid sequence of the cloned inserts contained within the lambda ZapII library. Assessments of the sequence of the clones will then determine whether the DNA fragment is derived from a CpG island and also potentially from which gene it is derived (assuming that the gene is already known and the sequence is available in the Genbank database). Having determined that a cloned fragment is derived from a methylated CpG island, a pair of PCR primers corresponding to this region are designed. The next step is to determine the methylation status of the gene from which the CpG clone is derived in DNA from both normal and tumorigenic origins. From these studies one can conclude whether the methylation status of the CpG island associated with a particular gene is altered in tumorigenic and non-tumorigenic tissues.The undertakings of Dr Sekiya, Dr Shiraishi and their laboratory group as well as myself in determining perturbations in CpG methylation, with the intention of comprehensively isolating CpG islands specifically methylated in tumor cells, is an ambitious and long-term project. Although still in an early stage of this process, preliminary results are encouraging and I look forward to hearing from Drs Shiraishi and Sekiya in the months ahead as a fuller picture begins to emerge.
Adam J. Oates
Leeds University Medical School, Leeds, UK
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Copyright© 1999 Foundation for Promotion of Cancer Research.
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