Single-cell analysis of p16(INK4a) and p21(WAF1) expression suggests distinct mechanisms of senescence in normal human and Li-Fraumeni Syndrome fibroblasts.
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.
Mirzayans
Razmik
R
Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada. razmikm@cancerboard.ab.ca
Andrais
Bonnie
B
Scott
April
A
Paterson
Malcolm C
MC
Murray
David
D
D001260:Ataxia Telangiectasia / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation / Q000528:radiation effects; D048430:Cell Shape; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics / Q000528:radiation effects; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology / Q000528:radiation effects; D005434:Flow Cytometry; D042822:Genomic Instability; D006801:Humans; D016864:Li-Fraumeni Syndrome / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008856:Microscopy, Fluorescence; D011123:Polyploidy; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D001616:beta-Galactosidase