Requirement for p27(KIP1) in retinoblastoma protein-mediated senescence

Abstract

In vivo and in vitro evidence indicate that cells do not divide indefinitely but instead stop growing and undergo a process termed cellular proliferative senescence. Very little is known about how senescence occurs, but there are several indications that the retinoblastoma protein (pRb) is involved, the most striking being that reintroduction of RB into RB(-/-) tumor cell lines induces senescence. In investigating the mechanism by which pRb induces senescence, we have found that pRb causes a posttranscriptional accumulation of the cyclin-dependent kinase inhibitor p27(KIP1) that is accompanied by an increase in p27(KIP1) specifically bound to cyclin E and a concomitant decrease in cyclin E-associated kinase activity. In contrast, pRb-related proteins p107 and p130, which also decrease cyclin E-kinase activity, do not cause an accumulation of p27(KIP1) and induce senescence poorly. In addition, the use of pRb proteins mutated in the pocket domain demonstrates that pRb upregulation of p27(KIP1) and senescence induction do not require the interaction of pRb with E2F. Furthermore, ectopic expression of p21(CIP1) or p27(KIP1) induces senescence but not the morphology change associated with pRb-mediated senescence, uncoupling senescence from the morphological transformation. Finally, the ability of pRb to maintain cell cycle arrest and induce senescence is reversibly abrogated by ablation of p27(KIP1) expression. These findings suggest that prolonged cell cycle arrest through the persistent and specific inhibition of cdk2 activity by p27(KIP1) is critical for pRb-induced senescence.

FIG. 1

pRb increases p27^KIP1 levels posttranscriptionally. The SAOS-2 human osteosarcoma cell line was transfected with empty vector (pSVE) or pRb expression plasmids and cell lysates obtained 48 to 72 h posttransfection. (A) Immunoblot of cell cycle proteins. (B) Immunoblot and in vitro kinase assay of cyclin E (left panel) and cyclin A (right panel) immunoprecipitations (IP). Cells labeled for 4 h with ³⁵S-methionine were immunoprecipitated with anti-cyclin E and visualized by SDS-PAGE (middle panel). (C) Immunoblot of cells cotransfected with a p27^KIP1 expression vector. (D) Northern blot of vector and pRb-transfected cells. (E) Transfected cells were treated with cycloheximide for the indicated times, and cell lysates were immunoblotted for p27^KIP1 to determine its half-life. (F) Cells were cotransfected with vector or p27^KIP1 and labeled for 1 h with ³⁵S-methionine, and lysates were obtained and boiled at 100°C and immunoprecipitated with anti-p27^KIP1. (G) Cells cotransfected with p27^KIP1 were labeled for 1.5 h with ³⁵S-methionine and chased with unlabeled methionine. Lysates were then harvested at the indicated timepoints and immunoprecipitated with anti-p27^KIP1.

FIG. 2

pRb-mediated senescence is linked to p27^KIP1 accumulation. SAOS-2 cells were transfected with empty vector (pSVE), RB, HA-p107, or HA-p130 expression plasmids and pBabe-puro to select transfected cells by puromycin drug resistance. (A) Forty-eight hours after transfection cells were placed under puromycin selection. Five days posttransfection cells were labeled with BrdU and immunostained with an anti-BrdU antibody (Roche), and BrdU-positive cells were counted (at least 250 cells). The number of BrdU-positive cells is represented as a decrease in BrdU-positive cells compared to vector-transfected cells and represents the average of at least three experiments. (B) Forty-eight hours posttransfection cells were selected with puromycin and then 10 days after transfection were stained for SA–β-gal activity and flat and SA–β-gal-positive cells were counted. Percent flat and SA–β-gal-positive cells indicates the number of flat cells or SA–β-gal-positive cells divided by the total number of cells counted (at least 100 cells) and represents at least five independent experiments. (C) Immunoblot of cells 2 and 10 days posttransfection. (D) p27^KIP1 expression in senescent cells. Ten days after transfection puromycin-selected cells were immunostained with anti-p27^KIP1.

FIG. 2

pRb-mediated senescence is linked to p27^KIP1 accumulation. SAOS-2 cells were transfected with empty vector (pSVE), RB, HA-p107, or HA-p130 expression plasmids and pBabe-puro to select transfected cells by puromycin drug resistance. (A) Forty-eight hours after transfection cells were placed under puromycin selection. Five days posttransfection cells were labeled with BrdU and immunostained with an anti-BrdU antibody (Roche), and BrdU-positive cells were counted (at least 250 cells). The number of BrdU-positive cells is represented as a decrease in BrdU-positive cells compared to vector-transfected cells and represents the average of at least three experiments. (B) Forty-eight hours posttransfection cells were selected with puromycin and then 10 days after transfection were stained for SA–β-gal activity and flat and SA–β-gal-positive cells were counted. Percent flat and SA–β-gal-positive cells indicates the number of flat cells or SA–β-gal-positive cells divided by the total number of cells counted (at least 100 cells) and represents at least five independent experiments. (C) Immunoblot of cells 2 and 10 days posttransfection. (D) p27^KIP1 expression in senescent cells. Ten days after transfection puromycin-selected cells were immunostained with anti-p27^KIP1.

FIG. 2

pRb-mediated senescence is linked to p27^KIP1 accumulation. SAOS-2 cells were transfected with empty vector (pSVE), RB, HA-p107, or HA-p130 expression plasmids and pBabe-puro to select transfected cells by puromycin drug resistance. (A) Forty-eight hours after transfection cells were placed under puromycin selection. Five days posttransfection cells were labeled with BrdU and immunostained with an anti-BrdU antibody (Roche), and BrdU-positive cells were counted (at least 250 cells). The number of BrdU-positive cells is represented as a decrease in BrdU-positive cells compared to vector-transfected cells and represents the average of at least three experiments. (B) Forty-eight hours posttransfection cells were selected with puromycin and then 10 days after transfection were stained for SA–β-gal activity and flat and SA–β-gal-positive cells were counted. Percent flat and SA–β-gal-positive cells indicates the number of flat cells or SA–β-gal-positive cells divided by the total number of cells counted (at least 100 cells) and represents at least five independent experiments. (C) Immunoblot of cells 2 and 10 days posttransfection. (D) p27^KIP1 expression in senescent cells. Ten days after transfection puromycin-selected cells were immunostained with anti-p27^KIP1.

FIG. 3

E2F repression is not required for pRb upregulation of p27^KIP1. SAOS-2 cells were transiently transfected with empty vector (pSVE), RB, HA-pRbΔ651, HA-pRbΔ657, or HA-pRbΔ663. (A) Cells were cotransfected with an expression vector for the CD20 cell surface protein. Forty-eight hours posttransfection, cells were harvested, fixed, incubated with FITC-conjugated anti-CD20 to identify transfected cells, and stained with PI to determine DNA content. Cell cycle analysis was then performed by FACS, with 10,000 CD20-positive events counted. Results represent the percent increase of cells in the G₁ phase over vector-transfected cells and are the average of at least two independent experiments. (B) Immunoblot of transfected cells 48 h posttransfection. (C) Immunoblot and in vitro kinase assay of lysates immunoprecipitated with anti-cyclin E 48 h after transfection. (D) Immunofluorescence analysis of cyclin E, cdk2, and p27^KIP1 in transfected cells. Cells transfected with vector, RB, or HA-pRbΔ663 were coimmunostained with cyclin E (green), cdk2 (blue), and p27^KIP1 (red) antibodies 48 h posttransfection.

FIG. 3

E2F repression is not required for pRb upregulation of p27^KIP1. SAOS-2 cells were transiently transfected with empty vector (pSVE), RB, HA-pRbΔ651, HA-pRbΔ657, or HA-pRbΔ663. (A) Cells were cotransfected with an expression vector for the CD20 cell surface protein. Forty-eight hours posttransfection, cells were harvested, fixed, incubated with FITC-conjugated anti-CD20 to identify transfected cells, and stained with PI to determine DNA content. Cell cycle analysis was then performed by FACS, with 10,000 CD20-positive events counted. Results represent the percent increase of cells in the G₁ phase over vector-transfected cells and are the average of at least two independent experiments. (B) Immunoblot of transfected cells 48 h posttransfection. (C) Immunoblot and in vitro kinase assay of lysates immunoprecipitated with anti-cyclin E 48 h after transfection. (D) Immunofluorescence analysis of cyclin E, cdk2, and p27^KIP1 in transfected cells. Cells transfected with vector, RB, or HA-pRbΔ663 were coimmunostained with cyclin E (green), cdk2 (blue), and p27^KIP1 (red) antibodies 48 h posttransfection.

FIG. 4

pRb pocket mutants induce senescence and p27^KIP1 accumulation. SAOS-2 cells were transfected with empty vector (pSVE), RB, HA-p107, HA-p130, HA-pRbΔ651, HA-pRbΔ657, or HA-pRbΔ663 and a puromycin resistance plasmid. (A) Cells were puromycin selected 48 h after transfection and at 5 days posttransfection were labeled with BrdU and stained with anti-BrdU, and BrdU-positive cells were counted (at least 250 cells). Results represent the decrease in BrdU-positive cells compared to vector-transfected cells and are the average of at least three experiments. (B) Ten days after transfection puromycin-selected cells were stained for SA–β-gal activity and flat cells (solid bars) and SA–β-gal-positive cells (hatched bars) were counted. Results are the number of flat or SA–β-gal-positive cells divided by the total number of cells counted (at least 100 cells) and represent the average of at least three experiments. (C) Immunoblot at 10 days of lysates from cells transfected with the indicated plasmids and puromycin selected. (D) Ten days posttransfection cyclin E immunoprecipitation and in vitro kinase assay of cells transfected with the indicated plasmids and puromycin selected. (E) Coimmunostaining of vector-, RB-, or HA-pRbΔ663-transfected cells with cyclin E (green), cdk2 (blue), and p27^KIP1 (red) antibodies 10 days posttransfection. Yellow staining in RB-transfected cells indicates colocalization of cyclin E and p27^KIP1.

FIG. 5

p27^KIP1 induces senescence. SAOS-2 cells were transfected with empty vector (pSVE), RB, p16^INK4a, p21^CIP1, p27^KIP1, and dominant-negative Cdk2 (dnCdk2 or dnK2) expression vectors. (A) Cell cycle inhibitors were cotransfected with empty vector (solid bars) or with RB (hatched bars) and with pBabe-puro, selected with puromycin 24 h after transfection, and then stained for SA–β-gal activity 10 days posttransfection. (B) p27^KIP1 immunoblot at 10 days of lysates from cells transfected with the indicated plasmids. (C) Phenotype of pRb and p27^KIP1 SA–β-gal-positive cells 10 days posttransfection. (D) PAI-1 immunoblot of cells transfected with the indicated plasmids 10 days after transfection. (E) Effect of pRb and p27^KIP1 on microtubulin in senescent cells. Ten days posttransfection cells were coimmunostained with p27^KIP1 (green) and α-tubulin (red) antibodies.

FIG. 6

p27^KIP1 is required for pRb-mediated senescence. SAOS-2 cells were transfected with RB and 24 h later were treated with p27^KIP1 mismatch (C) or p27^KIP1 antisense (AS) oligonucleotides. (A) Immunoblot of RB-transfected cell lysates 24 h after p27 antisense treatment. (B) p27^KIP1 immunostaining of RB-transfected cells 6 h (left panel) and 10 h (right panel) after treatment with p27 antisense oligonucleotides. (C) Effect of loss of p27^KIP1 expression on pRb-induced senescence. Starting 24 h posttransfection, cells were treated every 48 h with p27^KIP1 mismatch (pRb C) or p27 antisense (pRb AS) oligonucleotides over a period of 10 days. After 10 days cells were stained for SA–β-gal activity, and flat cells (solid bars) and SA–β-gal-positive cells (hatched bars) were counted as previously described. p27^KIP1 mismatch (top panel) and antisense (bottom panel) oligonucleotide-treated cells were stained for SA–β-gal activity at 10 days. (D) BrdU incorporation of vector (pSVE)- or RB- and HA-pRbΔ651-transfected cells treated every 48 h over a period of 10 days with mismatch or p27 antisense oligonucleotides. Results are the average of at least three experiments. (E) Cells were cotransfected with RB or HA-pRbΔ651 and CD20 expression plasmids and treated 24 h later with p27 mismatch or p27 antisense oligonucleotides, and FACS analysis was performed as previously described 24 h after oligonucleotide treatment. (F) RB-transfected cells were treated with p27 mismatch and antisense oligonucleotides at 2 and 4 days posttransfection, released from oligonucleotide treatment for 1 or 6 days, and then assayed for BrdU incorporation. Results are the average of at least two independent experiments.

FIG. 7

p27^KIP1 partially rescues the ability of pRb pocket mutants to induce flat cells and senescence. SAOS-2 cells were cotransfected with p27^KIP1 and RB, HA-p107, HA-p130, HA-pRbΔ651, HA-pRbΔ657, or HA-pRbΔ663 and a puromycin resistance plasmid. (A) Ten days posttransfection the number of pRb-phenotypic flat cells per total cells counted (at least 100 cells) for vector (CMV) or each protein alone (solid bars) or cotransfected with p27^KIP1 (hatched bars). Results represent the average of three independent experiments. (B) Ten days posttransfection cells were stained for SA–β-gal activity and the SA–β-gal-positive cells per total cells were counted (at least 100 cells) for vector (CMV) or each protein alone (solid bars) or cotransfected with p27^KIP1 (hatched bars). Results are the average of three independent experiments. An asterisk indicates p27 phenotypic senescent cells. (C) p27^KIP1 immunoblot of cotransfected lysates at 10 days posttransfection with vector (pSVE) or the indicated expression plasmid.

FIG. 8

Model for pRb-mediated senescence. pRb represses E2F-mediated transcription of S-phase genes, inducing an acute cell cycle arrest. In a non-E2F-dependent manner, pRb upregulates p27^KIP1 expression, leading to an accumulation of p27^KIP1 levels and a persistent inhibition of cyclin E-cdk2 kinase activity. The specific inhibition of cyclin E kinase activity by the CIP/KIP inhibitors triggers senescence, but it is a unique function of pRb to induce the morphology change associated with senescent cells that appears to strongly correlate with levels of p27^KIP1 expression.