Features of replicative senescence induced by direct addition of antennapedia‐p16INK4A fusion protein to human diploid fibroblasts

FEBS Letters

Miyazawa, Kazuhiro³  Peters, Gordon¹  Ruas, Marugarida¹  Sakai, Toshiyuki³  Wada, Ikuo²  Kato, Daishiro³  Oka, Takahiro²  Starborg, Maria¹  Hara, Eiji³ 

[1] Imperial Cancer Research Fund Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK;Second Department of Surgery, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-0841, Japan;Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-0841, Japan

关键词: p16INK4A;    Antennapedia;    Cellular senescence;    Cell cycle;    Cdk;    cyclin-dependent kinase;    HDF;    human diploid fibroblasts;    Rb;    retinoblastoma susceptibility gene;    MEF;    mouse embryo fibroblasts;    β-gal;    β-galactosidase;    HTLV-1;    human T-cell leukemia virus type 1;    GST;    glutathione S-transferase;   

DOI  :  10.1016/S0014-5793(98)00426-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The p16^INK4A 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 p16^INK4A 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 p16^INK4A 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 p16^INK4A 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 p16^INK4A 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 p16^INK4A in replicative senescence and raise the possibility of using the antennapedia-tagged protein therapeutically.

【 授权许可】

Unknown