Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts.
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.
Alcorta
D A
DA
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Xiong
Y
Y
Phelps
D
D
Hannon
G
G
Beach
D
D
Barrett
J C
JC
D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002455:Cell Division; D002460:Cell Line; D002478:Cells, Cultured; D016922:Cellular Senescence; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050760:Cyclin-Dependent Kinase Inhibitor p27; D050761:Cyclin-Dependent Kinase Inhibitor p57; D018844:Cyclin-Dependent Kinases / Q000302:isolation & purification / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012867:Skin / Q000166:cytology* / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
C493964:CDKN1C protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050761:Cyclin-Dependent Kinase Inhibitor p57; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases