Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts

Alcorta, D. A., Xiong, Y., Phelps, D., Hannon, G., Beach, D., Barrett, J. C. (November 1996) Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proceedings of the National Academy of Sciences of the United States of America, 93 (24). pp. 13742-13747. ISSN 00278424 (ISSN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/8943005
DOI: 10.1073/pnas.93.24.13742

Abstract

Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs. Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs. During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly 40-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 is the major CDK inhibitor for both CDK4 and CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors. Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16. p16 up-regulation is a key event in the terminal stages of growth arrest in senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.

Item Type: Paper
Uncontrolled Keywords: cyclin dependent kinase enzyme inhibitor gene product messenger RNA retinoblastoma protein cell aging cell immortalization cell proliferation conference paper diploidy embryo enzyme inhibition fibroblast gene expression human human cell messenger RNA synthesis newborn priority journal protein phosphorylation protein synthesis senescence Carrier Proteins Cell Cycle Proteins Cell Division Cell Line Cells, Cultured Cyclin-Dependent Kinase 4 Cyclin-Dependent Kinase 6 Cyclin-Dependent Kinase Inhibitor p16 Cyclin-Dependent Kinase Inhibitor p27 Cyclin-Dependent Kinase Inhibitor p57 Cyclin-Dependent Kinases Enzyme Inhibitors Fibroblasts Humans Infant, Newborn Male Microtubule-Associated Proteins Nuclear Proteins Protein Binding Protein-Serine-Threonine Kinases Proto-Oncogene Proteins Recombinant Fusion Proteins Skin Tumor Suppressor Proteins
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > fibroblasts
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > fibroblasts
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > fibroblasts

bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > kinase
organs, tissues, organelles, cell types and functions > cell types and functions > cell functions > senescence
CSHL Authors:
Communities: CSHL labs > Beach lab
CSHL labs > Hannon lab
Depositing User: Matt Covey
Date: 26 November 1996
Date Deposited: 21 May 2014 19:52
Last Modified: 21 May 2014 19:52
PMCID: PMC19411
Related URLs:
URI: http://repository.cshl.edu/id/eprint/30173

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