Single‐cell analysis of p16^INK4a and p21^WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Dec 30, 2009Journal of cellular physiology

Single-cell study of two aging markers shows different aging processes in normal and Li-Fraumeni Syndrome skin cells

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Abstract

All fibroblast strains entered replicative senescence at late passages, characterized by growth inhibition and morphological changes.

Cultures exhibited positive staining for senescence-associated beta-galactosidase, indicating the presence of senescence.
Accelerated senescence occurred in early-passage cultures of fibroblasts following exposure to ionizing radiation.
Most normal and ataxia telangiectasia fibroblast strains showed low levels of p16 expression, regardless of culture age.
Sustained nuclear accumulation of p21 was observed during both replicative and accelerated senescence, but did not correlate with p16 levels in p53-proficient fibroblasts.
In p53-deficient Li-Fraumeni syndrome fibroblasts, both types of senescence correlated with p16 expression, but not with p21.
Senescence in Li-Fraumeni syndrome fibroblasts was linked to genomic instability, including polyploidy.

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Herein we used single-cell observation methods to gain insight into the roles of p16(INK4A) and p21(WAF1) (hereafter p16 and p21) in replicative senescence and ionizing radiation-induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li-Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence-associated beta-galactosidase. In addition, proliferating early-passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53-proficient (normal and AT) fibroblasts. In p53-deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation-triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild-type p53 activity. The possibility that one of the tumor-suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results.

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Single‐cell analysis of p16^INK4a and p21^WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Abstract

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