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. 1999 Mar;19(3):2109-17.
doi: 10.1128/MCB.19.3.2109.

Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts

G H Stein  1 , L F Drullinger, A Soulard, V Dulić
Affiliations

Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts

G H Stein et al. Mol Cell Biol. 1999 Mar.

Abstract

The irreversible G1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G1 cyclin-cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21(Sdi1,Cip1,Waf1), which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16(Ink4a), suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E- and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by </=50% compared to young cells. We also provide new evidence that p21 may play a role in inactivation of the DNA replication factor proliferating cell nuclear antigen during early senescence. Finally, because p16 accumulates in parallel with the increases in senescence-associated beta-Gal activity and cell volume that characterize the senescent phenotype, we suggest that p16 upregulation may be part of a differentiation program that is turned on in senescent cells. Since p21 decreases after senescence is achieved, this upregulation of p16 may be essential for maintenance of the senescent-cell-cycle arrest.

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Figures

FIG. 1
FIG. 1
Age-related changes in CKIs and putative markers of differentiation in IMR90. HDF were analyzed at 14 days after each subcultivation. (A) Cumulative population doublings (PD) and the percentage of [3H]thymidine-labeled nuclei (LN) before and after serum stimulation. (B) Cell density at quiescence and the amount of p21 and p27 determined by immunoblotting and densitometry, such that each CKI is quantitated relative to its own maximum in this experiment. (C) Cell volume and p16 relative to the maximum achieved for each of these parameters and the percentage of cells that were SA–β-Gal positive. Abbreviations: EOL, end of lifespan, no further population doublings; SEN, senescent, %LN-Stim of <5%; late SEN, elevated p16.
FIG. 2
FIG. 2
All in vivo p21-bound cyclin-Cdk2 complexes are inactive. Extracts were prepared from young quiescent IMR90 (0 h) and serum-stimulated young and senescent IMR90 (16 h). (A) Western blot analysis of cyclin E, Cdk2, p27, and p21, in cyclin E immunoprecipitates (I.P.) from total cell extracts. Arrows indicate isoforms of Cdk2. (B and C) Protocol and results, respectively. Cell extracts were immunodepleted of p21 by incubation with saturating amounts of p21-specific antibodies (+) or mock-treated with protein A-Sepharose (−). Supernatants, mock-treated and depleted of p21, were immunoblotted for p21, cyclin E, and cyclin A and used for the preparation of cyclin E and cyclin A immunoprecipitates, which were analyzed for their Cdk2 content and histone H1 kinase activity. (D) Histone H1 kinase activity of cyclin A and p21 immunocomplexes. Nonspecific antisera, cyclin A, and p21 immunoprecipitates were assayed for both histone H1 kinase activity and cyclin A, Cdk2, and p21 content as described in Materials and Methods. The arrow labeled “P” denotes the T160-phosphorylated (activated) Cdk2 isoform. Note that cyclin E binds to both forms of Cdk2, whereas cyclin A binds only the activated form of Cdk2.
FIG. 3
FIG. 3
Cyclin D1-Cdk complexes are inactive in senescent HDF. Young, senescent, and late senescent (LS) IMR90 were harvested before (0 h) and/or after serum stimulation (16 h). A Western blot analysis of total cell lysates, probed for cyclin D1-dependent pRb phosphorylation at Ser-780 (30), is shown. The total protein demonstrates equal loading, and an analysis of pRb, cyclin D1, p27, p21, and p16 confirms the young, senescent, and late senescent status of the cells.
FIG. 4
FIG. 4
Persistence of cyclin D1-Cdk4/6 complexes in senescent HDF. (A) Western blot analysis of cyclin D1, Cdk2, Cdk4, Cdk6, p21, and/or p16 in cyclin D1 and p16 immunoprecipitates from young, senescent, and late senescent IMR90 harvested before (0 h) and/or after serum stimulation (16 h). (B) Differential association between various CKI, Cdk, and G1 cyclins in young and late senescent HDF at 16 h after serum stimulation. Western blot analysis of seven proteins (cyclin D1, Cdk6, Cdk4, Cdk2, p27, p21, and p16) in p21, p16, Cdk4, Cdk6, cyclin D1, cyclin E, and nonspecific (P.I.) complexes immunoprecipitated from equal amounts (150 mg) of cell extracts prepared from serum-stimulated young (Y) and late senescent (LS) IMR90 fibroblasts. Except in the case of Cdk4 (75% removal) and cyclin E (60% removal), the remaining supernatants were virtually depleted for the indicated protein. Prolonged ECL exposure was necessary to detect the presence of Cdk6 in p21 and cyclin D1 immunocomplexes (S.E., short exposure; L.E., long exposure). Because the Western blots were probed sequentially with different antibodies, the arrows indicate residual Cdk2 and cyclin D1 signals that remained after their “stripping.” Note that cyclin D1 binds only the inactive, unphosphorylated form of Cdk2, whereas cyclin E binds both forms of Cdk2 (parallel arrows).
FIG. 5
FIG. 5
Increased association of p21 with cyclin D1-Cdk4/6 complexes in senescent HDF. (A) Western blot analysis of cyclin D1, Cdk2, Cdk4, p27, and p21 in cyclin D1 immunoprecipitates. (B) Western blot analysis of cyclin D1, Cdk2, Cdk4, Cdk6, and p21 in cyclin D1-immunoprecipitates after p21 immunodepletion as described in Fig. 2B.
FIG. 6
FIG. 6
Accumulation of PCNA-p21-cyclin D1-Cdk complexes in early senescent HDF. Young, senescent, and late senescent (LS) IMR90 were harvested before (0 h) and/or after serum stimulation (16 h). (A) PCNA levels in total cell extracts. (B and C) Western blot analysis of p21 and p16 immunoprecipitates (I.P.) (B) and cyclin D1 immunoprecipitates (C). In both p21 and cyclin D1 immunoprecipitates, the strong PCNA accumulation correlates with increased Cdk2. (D) p21-dependent association of PCNA with cyclin D1-Cdk2 complexes, as assessed by Western blot analysis of cyclin D1 immunoprecipitates prior to (−) and after (+) p21 immunodepletion (see Fig. 2B). Note that the senescent cells used in this figure show no increase in p16, indicating that they were harvested at a very early senescent stage, where cyclin D1 is usually at its highest level. The combination of high cyclin D1, high p21 (to stabilize cyclin D1-Cdk complexes), and low p16 may account for the high amounts of cyclin D1-associated Cdks in the cyclin D1 immunoprecipitates.
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