Real-time imaging of transcriptional activation in live cells reveals rapid up-regulation of the cyclin-dependent kinase inhibitor gene CDKN1A in replicative cellular senescence.
Cellular replicative senescence is a permanent growth arrest state that can be triggered by telomere shortening. The cyclin-dependent kinase (Cdk) inhibitor p21(CIP1/WAF1) (p21), encoded by the CDKN1A gene, is a critical cell cycle regulator whose expression increases as cells approach senescence. Although the pathways responsible for its up-regulation are not well understood, compelling evidence indicates that the upstream triggering event is telomere dysfunction. Studies of replicative senescence have been complicated by the asynchrony of its onset, which is caused by the continuous and stochastic variability in individual cell lifespans. In fact, the actual entry into senescence has never been observed in a single unperturbed cell. We report here a new in vitro human model system that allows entry into senescence to be monitored in real-time in individual viable cells. We used homologous recombination to generate non-immortalized fibroblast cells with the enhanced yellow fluorescence protein (EYFP) gene knocked into one CDKN1A gene copy, allowing promoter activity to be visualized as fluorescence intensity. Gamma irradiation, DNA-damaging drugs, expression of p14(ARF) or oncogenic Ras, and replicative exhaustion all resulted in elevated EYFP expression, demonstrating its proper control by physiological signalling circuits. Analysis by time-lapse microscopy of cultures approaching replicative senescence revealed that p21 levels rise abruptly in individual aging cells and remain elevated for extended periods of time.
Herbig
Utz
U
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
Wei
Wenyi
W
Dutriaux
Annie
A
Jobling
Wendy A
WA
Sedivy
John M
JM
D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics* / Q000528:radiation effects; D003001:Cloning, Molecular; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D005720:Gamma Rays; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics* / Q000528:radiation effects; D025803:Tumor Suppressor Protein p14ARF / Q000096:biosynthesis; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008164:Luminescent Proteins; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins