Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts.
The p16INK4A cyclin-dependent kinase (Cdk) inhibitor is now recognized as a major tumor suppressor that is inactivated by a variety of mechanisms in a wide range of human cancers. It is also implicated in the mechanisms underlying replicative senescence since p16INK4A RNA and protein accumulate as cells approach their proscribed limit of population doublings in tissue culture. To obtain further evidence of its role in senescence, we have sought ways of overexpressing p16INK4A in primary human diploid fibroblasts (HDF). To circumvent the low transfection efficiency of primary cells we have exploited a recombinant form of the full-length p16INK4A protein fused to a 16 amino acid peptide from the Drosophila antennapedia protein. This peptide has the capacity to cross both cytoplasmic and nuclear membranes allowing the direct introduction of the active protein to primary cells. Here, we show that antennapedia-tagged wild-type p16INK4A protein, but not a functionally compromised tumor-specific variant, causes G1 arrest in early passage HDFs by inhibiting the phosphorylation of the retinoblastoma protein. Significantly, the arrested cells display several phenotypic features that are considered characteristic of senescent cells. These data support a role for p16INK4A in replicative senescence and raise the possibility of using the antennapedia-tagged protein therapeutically.
Kato
D
D
Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Japan.
Miyazawa
K
K
Ruas
M
M
Starborg
M
M
Wada
I
I
Oka
T
T
Sakai
T
T
Peters
G
G
Hara
E
E
D051943:Antennapedia Homeodomain Protein; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004171:Diploidy; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D016193:G1 Phase / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D009687:Nuclear Proteins; D010641:Phenotype; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D014157:Transcription Factors
D051943:Antennapedia Homeodomain Protein; D019941:Cyclin-Dependent Kinase Inhibitor p16; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors