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

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 me 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, me show that antennapedia-tagged mild-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. (C) 1998 Federation of European Biochemical Societies.