ALTERED REGULATION OF G(1)-CYCLINS IN SENESCENT HUMAN-DIPLOID FIBROBLASTS - ACCUMULATION OF INACTIVE CYCLIN-E-CDK2 AND CYCLIN-D1-CDK2 COMPLEXES

Senescent human diploid fibroblasts are unable lo enter S phase in res ponse to mitogenic stimulation. One of the key deficiencies in mitogen -stimulated senescent cells is their failure to phosphorylate the reti noblastoma protein, which acts as an inhibitor of entry into S phase i n its unphosphorylated form. Recent data suggest that cyclin-dependent kinases (Cdks) regulated by G1 cyclins (D type and E) are responsible for the primary phosphorylation of the retinoblastoma protein prior t o the G1/S boundary. Surprisingly, we found 10- to 15-fold higher cons titutive amounts of both cyclin E and cyclin DI in senescent cells com pared to quiescent early-passage cells. Nevertheless, cyclin E-associa ted kinase activity in senescent cells was very low and did not increa se significantly upon mitogenic stimulation even though cyclin E-Cdk2 complexes were abundant. In contrast to early-passage cells in late G1 phase, senescent cells contained mainly underphosphorylated cyclin E and proportionally more unphosphorylated and inactive Cdk2, perhaps ac counting for the low kinase activity. We also show that a majority of the Cdk2 in senescent cells, but not in early-passage cells, was compl exed with cyclin Dl. Cyclin D1-Cdk2 complexes, severalfold enriched in senescent cells, contained exclusively unphosphorylated Cdk2. Amounts of cyclin A, which ordinarily accumulates in S and G2 phases, were ex tremely low in stimulated senescent cells. We suggest that the failure to activate cyclin E-Cdk2 kinase activity in senescent cells may acco unt for the inability of these cells to phosphorylate the retinoblasto ma protein in late G1 phase, which in turn may block the expression of late G1 genes such as cyclin A that are required for entry into S pha se.