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Restriction Landmark Genomic Scanning (RLGS-M)-based Genome-wide Scanning of Mouse Liver Tumors for Alterations in DNA Methylation Status¹

Genome Science Laboratory, The Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305 Japan [T. O. A., Y. O., M. I., H. O., H. K., M. M., Y. H.] and Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263 [C. P., W. A. H.]

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect, and subsequently clone, genomic regions with alterations in DNA methylation associated with tumorigenesis. Use of a methylation-sensitive enzyme for the landmark cleavage allows analysis of changes in methylation patterns. In this study, we used RLGS-M to analyze SV40 T antigen-induced mouse liver tumors derived from interspecific F₁ hybrids between Mus spretus (S) and C57BL/6 (B6). Because 575 S- and B6-specific RLGS loci/spots have been mapped, tumor-related alterations in the RLGS profile could be immediately localized to specific chromosomal regions. We previously found that the loss of contiguous loci/spots could be attributed primarily to DNA loss, whereas loss of solitary loci/spots could be attributed primarily to DNA methylation. In this study, we examined 30 mouse liver tumor samples for loss of the 507 mapped loci/spots. Fourteen solitary loci/spots found to be absent or reduced in more than 75% of tumor samples were cloned and subjected to DNA sequence analyses. Two loci were identified as 4 integrin and p16/CDKN2, genes reported to be involved in tumorigenesis. Thus, RLGS-M can detect alterations in the methylation status of known tumor suppressor genes and provide a method for detecting and subsequently cloning novel genomic regions that undergo alterations in methylation during tumorigenesis.

¹ This study was supported by special coordination funds and a research grant for the Genome Exploration Research Project from the Science and Technology Agency of the Japanese government; a grant-in-aid for scientific research on priority areas and the Human Genome Program from the Ministry of Education, Science, Sports and Culture, Japan; a grant for research on aging and health; a grant-in-aid for a second term comprehensive 10-year strategy for cancer control from the Ministry of Health and Welfare of the Japanese government (to Y. H.); and a grant from Core Research of Evolution Science and Technology (CREST) from Japan Science and Technology Corporation (JST). This work was also supported by Grant RO1-CA686612 from the National Cancer Institute (to W. A. H.).

² To whom requests for reprints should be addressed.

Received 3/ 4/97. Accepted 6/ 2/97.

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