Altered regulation of G1 cyclins in senescent human diploid fibroblasts: accumulation of inactive cyclin E-Cdk2 and cyclin D1-Cdk2 complexes.

Abstract

Senescent human diploid fibroblasts are unable to enter S phase in response to mitogenic stimulation. One of the key deficiencies in mitogen-stimulated senescent ceUls is their failure to phosphorylate the retinoblastoma protein, which acts as an inhibitor of entry into S phase in 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 to the G1/S boundary. Surprisingly, we found 10-to 15-fold higher constitutive amounts of both cyclin E and cyclin Dl in senescent cells compared to quiescent early-passage ceUs. Nevertheless, cyclin E-associated kinase activity in senescent cells was very low and did not increase signficantly upon mitogenic stimulation even though cyclin E-Cdk2 complexes were abundant. In contrast to earlypassage cells in late G, phase, senescent cells contained mainly underphosphorylated cyclin E and proportionally more unphosphorylated and inactive Cdk2, perhaps accounting for the low kinase activity. We also show that a majority of the Cdk2 in senescent cells, but not in early-passage cells, was complexed with cyclin Dl. Cyclin Dl-Cdk2 complexes, severalfold enriched in senescent cells, contained exclusively unphosphorylated Cdk2. Amounts of cyclin A, which ordinarily accumulates in S and G2 phases, were extremely low in stimulated senescent ceUls. We suggest that the failure to activate cyclin E-Cdk2 kinase activity in senescent cells may account for the inability ofthese cells to phosphorylate the retinoblastoma protein in late

Active Cyclin A-CDK2 Complex, a Possible Critical Factor for Cell Proliferation in Human Primary Lung Carcinomas

“…27,37 Alternatively, as demonstrated in the present study, CDK2-cyclin E complexes accumulate in these well differentiated cells without catalyzing significant kinase activity, suggested in a previous report using senescent human diploid fibroblasts. 38 If this is the case, the lack of kinase activity could be due to the action of CDK-cyclin inhibitor proteins p21, p27, p57, or others, and/or to changes in the phosphorylation state of CDK2 that modulate its activity. 5,38 -41 Although these hypotheses seemed applicable to most of the cases in the present study, we do not have reasonable explanation for a case of SmCC (case 7) and a cell line Lu135 both of which manifested higher cyclin E-associated kinase activity.…”

Senescence from G2 arrest, revisited

“…It is initiated by inactivation of cyclin D1-Cdk4/6 and cyclin E1-Cdk2 complexes that positively regulate G1/S phase progression. 27,28 The best-characterized role of cyclin D1-Cdk4/6 complexes is in triggering pRb phosphorylation, 29,30 while cyclin E1-Cdk2 complexes control the onset of DNA replication by directly phosphorylating a number of replication proteins (reviewed in ref. 31 ).…”

“…pRb phosphorylation, 40 together with the presence of G1 cyclins (cyclins D1 and E1) 27 and the lack of mitotic cyclins (cyclin B1), 41 is an early-recognized hallmark of senescence. Very soon after its discovery, p16 Ink4a (p16), another pRb regulator that specifically inhibits the cyclin D1-associated kinases Cdk4 and Cdk6, was also implicated in senescence 28,[42][43][44] (Fig.…”

Mitogen Stimulation Cooperates with Telomere Shortening To Activate DNA Damage Responses and Senescence Signaling

“…Cellular senescence induced by telomere shortening is characterized by typical changes in cell morphology (flattened cells with enlarged cytoplasm) (5,23) and senescence-associated ␤-galactosidase (SA-␤-Gal) activity at pH 6 (10). At the molecular level, growth arrest at the senescence stage is induced by an attenuation of gene expression and an accumulation of cells in the late G 1 phase of the cell cycle (13,35,37,40) which fail to phosphorylate pRb after mitogen stimulation (50). Phosphorylation of pRb during G 1 phase is carried out by cyclin D-Cdk4/6 and cyclin E-Cdk2 complexes.…”