Cells exhibiting strong <i>p16</i> ^<i>INK4a</i> promoter activation in vivo display features of senescence

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 <i>p16</i>^<i>INK4a</i> locus. We used this allele (<i>p16</i>^<i>tdTom</i> ) for the enumeration, isolation, and characterization of individual <i>p16</i>^<i>INK4a</i> -expressing cells (tdTom⁺). The half-life of the knocked-in transcript was shorter than that of the endogenous <i>p16</i>^<i>INK4a</i> mRNA, and therefore reporter expression better correlated with <i>p16</i>^<i>INK4a</i> promoter activation than <i>p16</i>^<i>INK4a</i> transcript abundance. The frequency of tdTom⁺ cells increased with serial passage in cultured murine embryo fibroblasts from <i>p16</i>^{<i>tdTom/+</i>} 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 <i>p16</i>^<i>INK4a</i> 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 <i>p16</i>^<i>INK4a</i> promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence.