Cells exhibiting strong p16 INK4a promoter activation in vivo display features of senescence

Proc Natl Acad Sci U S A. 2019 Feb 12;116(7):2603-2611. doi: 10.1073/pnas.1818313116. Epub 2019 Jan 25.

Abstract

The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by "knocking-in" a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the p16 INK4a locus. We used this allele (p16 tdTom ) for the enumeration, isolation, and characterization of individual p16 INK4a -expressing cells (tdTom+). The half-life of the knocked-in transcript was shorter than that of the endogenous p16 INK4a mRNA, and therefore reporter expression better correlated with p16 INK4a promoter activation than p16 INK4a transcript abundance. The frequency of tdTom+ cells increased with serial passage in cultured murine embryo fibroblasts from p16 tdTom/+ mice. In adult mice, tdTom+ cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging. Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16 INK4a and found that tdTom+ macrophages exhibited some features of senescence, including reduced proliferation, senescence-associated β-galactosidase (SA-β-gal) activation, and increased mRNA expression of a subset of transcripts encoding factors involved in SA-secretory phenotype (SASP). These results indicate that cells harboring activation of the p16 INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence.

Keywords: aging; cdkn2a; senescence.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Proliferation
  • Cellular Senescence / genetics*
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics*
  • Enzyme Activation
  • Fibroblasts / metabolism
  • Half-Life
  • Humans
  • Mice
  • Phenotype
  • Promoter Regions, Genetic*
  • RNA, Messenger / genetics
  • beta-Galactosidase / metabolism

Substances

  • Cyclin-Dependent Kinase Inhibitor p16
  • RNA, Messenger
  • beta-Galactosidase