Abstract 2975: Widespread cell cycle inhibition and features of premature aging in mice with conditional expression of p16Ink4a

Abstract

Abstract Diverse approaches have shown that interphase Cyclin dependent kinases 2, 4, and 6 drive phosphorylation of retinoblastoma family proteins, transcription of E2F-dependent genes, and entry into the cell cycle in cultured mammalian cells. In contrast, targeted gene disruption of interphase Cdks and activating E2Fs in mice, individually and in combination, is surprisingly well tolerated. For example, absence of all activating E2Fs did not reduce proliferation of intestinal epithelial cells, the most highly proliferative cells in mammals. These observations have raised doubt about the need for Cdk2/4/6 and E2F activity for most normal development and tissue homeostasis. However, targeted gene disruption carries the important caveat of protein under-expression. Absence of interphase Cdks creates voids filled by Cdk1, which displays compensatory increased binding to interphase cyclins. In an alternative approach to dissect the role of Cdk2/4/6 in normal tissues, we generated transgenic mice with conditional expression of p16Ink4a from a doxycycline (Dox)-inducible promoter (TetO-p16). p16 is a major tumor suppressor and specific Cdk2/4/6 inhibitor. p16 accumulates and contributes to cell cycle arrest in settings of replicative senescence, neoplasia, pre-neoplasia, and certain aging progenitors. Nonetheless, the ability of p16 to inhibit cell cycle entry in most normal tissues remains unclear. TetO-p16 mice are healthy and fertile but a few demonstrated spontaneous patches of alopecia, suggesting that leaky p16 expression may cause deficits in hair regeneration. To achieve broad p16 induction, we mated TetO-p16 mice to mice that express the reverse tetracycline transactivator from a cytomegalovirus promoter (CMV-rtTA). Exogenous p16 induction was readily detected by immunoblotting, immunohistochemistry, and immunofluorescence. Consistent with known properties of the CMV-rtTA transgene, Dox treatment resulted in widespread but mosaic p16 induction. In intestinal epithelial cells, exogenous p16 bound efficiently to Cdk4. p16 expression in crypt transit-amplifying cells inhibited bromodeoxyuridine (BrdU) incorporation by 75%. Expression in stem cells marked by the Lgr5-lacZ transgene inhibited BrdU incorporation by 85%. Mice treated with Dox from day 20 to 40 developed proliferative deficits in all rapidly renewing tissues and features of premature aging, including intestinal shortening, hair loss and hypopigmentation, weight loss, reduced fat stores, and anemia. These results suggest that Cdk2/4/6 activity is required for normal tissue maturation and homeostasis, that p16 is an effector of aging, and that inhibition of cell proliferation is sufficient to elicit signature features of aging. Initial results suggest that at least some p16-imposed aging features are reversible. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2975. doi:10.1158/1538-7445.AM2011-2975