Cells exhibiting strong p16INK4a promoter activation in vivo display features of senescence
Cells exhibiting strong p16INK4a promoter activation in vivo display features of senescence
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DOI：
10.1073/pnas.1818313116
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发表时间：
2019-02-12
影响因子：
11.1
通讯作者：
Sharpless, Norman E.
中科院分区：
文献类型：
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作者：
Liu, Jie-Yu;Souroullas, George P.;Sharpless, Norman E.
在整个生命周期中细胞衰老的激活促进肿瘤抑制，而衰老细胞的持续存在有助于衰老的各个方面。然而，这一理论受到了限制，因为无法在完整的生物体中识别和分离单个衰老细胞。为此，我们通过将荧光染料串联二聚体Tomato（tdTom）“敲入”p16（INK 4a）基因座的外显子1 α来产生小鼠报告菌株。我们使用该等位基因（p16（tdTom））对单个p16（INK 4a）表达细胞（tdTom（+））进行计数、分离和表征。敲入转录本的半衰期短于内源性p16（INK 4a）mRNA的半衰期，因此报告基因表达与p16（INK 4a）启动子激活的相关性优于p16（INK 4a）转录本丰度。在培养的p16（tdTom/+）小鼠胚胎成纤维细胞中，tdTom（+）细胞的频率随着连续传代而增加。在成年小鼠中，tdTom（+）细胞可以在许多组织中以低频率容易地检测到，并且这些细胞的频率随着年龄的增长而增加。使用腹膜炎症的体内模型，我们比较了有或没有p16（INK 4a）激活的细胞表型，发现tdTom（+）巨噬细胞表现出衰老的一些特征，包括增殖减少，衰老相关的β-半乳糖苷酶（SA-β-gal）激活，以及SA分泌表型（SASP）相关转录子编码因子的mRNA表达增加。这些结果表明，携带p16（INK 4a）启动子激活的细胞随着体内老化和炎症而积累，并显示出衰老的特征。
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 1 alpha 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 beta-galactosidase (SA-beta-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.