) The objective of this proposal is to develop efficient strategies for screening cancer cell genomes for hypermethylated promoter region CpG islands which may be associated with gene inactivation. Such CpG regions, on autosomal chromosomes in normal cells, are generally free of methylation as part of the process that maintains genes in an active, or ready, transcription state. It has become apparent over the past several years, that, in tumors of all types, a large number of important genes may harbor aberrant methylation in promoter region CpG islands. This change is the mark for transcriptional silencing events which provide for an alternative to coding region mutations for the inactivation of tumor suppressor genes such as Rb, VHL, p16, pl5, E-cad, and hMLH1. Thus, identification of random hypermethylated CpG islands in each of the types of human cancer could provide an important contribution to the search for: a) major tumor suppressor genes and b) genes which, when inactivated in tumors, lead to loss of cell cycle control, regulation of differentiation, entry into apoptosis, and other events which contribute to tumor predisposition and/or progression. The strategies under construction depend upon initial restriction of tumor and normal cell DNA with methylation sensitive enzymes for sites which occur far more frequently in CpG rich areas than in the majority of the genome, and which will not cut when cytosines in the recognition sequence are methylated. Subsequent PCR steps then amplify methylated CpG rich regions preferentially and final subtraction steps, or gel resolution procedures, are utilized to enrich for and identify CpG islands selectively methylated in tumor DNA. Methods to most efficiently identify the best candidate sequences for full characterization, including gene identification, are also being developed as are construction of the best strategies for entry of all data into existing and/or new databases and automation of the major steps in the screening schema. Preliminary data indicate that the techniques constructed to date will be extremely successful and several CpG rich regions selectively hypermethylated in tumor DNA have already been isolated. The final technology established should provide an important addition to approaches for screening the human genome for disease related genes.
| Baylin, S B; Herman, J G (2000) DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 16:168-74 |
| Toyota, M; Ho, C; Ohe-Toyota, M et al. (1999) Inactivation of CACNA1G, a T-type calcium channel gene, by aberrant methylation of its 5' CpG island in human tumors. Cancer Res 59:4535-41 |
| Toyota, M; Ho, C; Ahuja, N et al. (1999) Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. Cancer Res 59:2307-12 |
| Toyota, M; Ahuja, N; Suzuki, H et al. (1999) Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype. Cancer Res 59:5438-42 |
