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Loss of p16^INK4 Expression by Methylation Is Associated with Lifespan Extension of Human Mammary Epithelial Cells¹

Children's Medical Research Institute, Westmead, Sydney, NSW 2145, Australia [L. I. H., J. R. N., A. A. N., E. L. M., R. R. R.]; Department of Surgery, Westmead Hospital, Westmead, NSW 2145, Australia [P. B.]; and Kanematsu Laboratories, CSIRO, Division of Molecular Science, North Ryde, NSW 2113, Australia [J. R. M., S. J. C.]

Inactivation of p16^INK4 tumor suppressor gene function is frequently observed in breast cancer. We examined p16^INK4 expression in human mammary epithelial cell (HMEC) cultures established from four normal donors. Normal HMECs divide a limited number of times before proliferation ceases in a state referred to as selection (or M₀). The cell subpopulation that emerges spontaneously from selection undergoes a further limited period of proliferation before senescence. By immunofluorescence and Western blot analysis of four independent cultures, we have shown loss of p16^INK4 expression in postselection HMECs. In contrast, p16^INK4 was present in both early and late passage fibroblasts from the same individuals. Bisulfite genomic sequencing revealed extensive methylation of the p16^INK4 CpG island in post- but not preselection cells. Thus, the extended period of growth observed in postselection HMECs is associated with hypermethylation of the p16^INK4 CpG island and loss of p16^INK4 expression. Although postselection HMECs are widely considered to be normal, these data indicate that they have sustained an epigenetic alteration.

¹ Supported by the Kathleen Cuningham Foundation for Breast Cancer Research and the Carcinogenesis Fellowship of the New South Wales Cancer Council.

² To whom requests for reprints should be addressed, at Children's Medical Research Institute, 214 Hawkesbury Road, Westmead, Sydney, NSW 2145, Australia. Phone: 61-2-9687-2800; Fax: 61-2-9687-2120; E-mail: rreddel@mail.usyd.edu.au.

Received 4/27/98. Accepted 7/ 1/98.

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