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Extensive DNA Methylation Spanning the Rb Promoter in Retinoblastoma Tumors¹

Kanematsu Laboratories, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050 [C. S., D. S. M., C. L. P., P. M. W., J. H., P. C. V., S. J. C.]; Commonwealth Scientific Industrial Research Organization, Division of Biomolecular Engineering, Sydney Laboratory, P. O. Box 184, North Ryde, NSW 2113 [S. J. C.]; and School of Biological Sciences, A12, University of Sydney, NSW 2006 [M. F.], Australia

The retinoblastoma gene (Rb) is one of the best characterized tumor suppressor genes, and its inactivation is associated with a number of cancers. Previous studies have shown, by restriction enzyme analysis, that the promoter region of the Rb gene is methylated in a significant proportion of primary retinoblastoma tumors. We now report the first detailed methylation sequence analysis of the CpG island spanning the retinoblastoma promoter from hypermethylated retinoblastoma tumors. Our results show methylation is not confined to a specific CpG site, as detected by restriction enzyme studies, but extends to essentially all 27 CpG dinucleotides spanning the retinoblastoma CpG island, including the core promoter. The methylation pattern from each tumor DNA sample is different, ranging from densely to sparsely methylated profiles. Single CpG sites, in particular the E2F transcription factor binding site, as well as blocks of CpGs, were undermethylated in some tumor samples. Possible interference of methylation could be due to the binding of sequence-specific protein factors at these sites in the tumor cells. This study highlights that the dynamics of DNA methylation in cancer cells are clearly different from normal cells and gives an insight into the mechanism of abnormal methylation of CpG islands in cancer cells.

¹ This work has been supported in part by an Anthony Rothe Memorial Grant.

² To whom requests for reprints should be addressed. Phone: (612) 9886-4949; Fax: (612) 9886-4805; E-mail: clark@pelican.dbe.csiro.au.

Received 9/23/96. Accepted 4/ 1/97.

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