INTRODUCTION

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Instead of having an infinite capacity to proliferate, eukaryotic cells are thought to have a limited replicative lifespan. They undergo a terminal arrest of cellular division and take on an altered cellular state, termed senescence (6, 21). Cellular senescence was first observed by Hayflick and Moorhead in human fibroblasts passaged in culture and has since been seen to occur upon passage of many cell types from various species and upon oncogenic stimuli (25, 38, 45, 48, 52, 69). Importantly, senescence is believed to be tumor suppressive, as bypass of senescence leads to immortalization and, potentially, oncogenic transformation.

Senescent cells share several basic characteristics. One of these is stringency in human cellswhile rodent cells often spontaneously immortalize, human cells do not (6, 21). Also, though senescent cells continue to be metabolically active, they undergo cell cycle arrest in the G₁ phase that is irreversible in that they can not be mitogen stimulated to reenter S phase (6, 21). In addition, cells that have senesced exhibit changes in the expression of proteins that regulate cell cycle and take on an altered cellular morphology (5, 21, 52). Recently, the triggering of senescence has been associated with telomere shortening, and senescent cells, in contrast to immortal cells, exhibit decreased telomerase activity (31, 63, 66, 67). In fact, in transformed cells telomerase activation was found to be sufficient to allow cells to escape from senescent crisis (16, 23). However, the mechanism by which senescence occurs is relatively unclear, though evidence suggests that the retinoblastoma protein (pRb), a potent tumor suppressor, may play a role (32).

pRb, the product of the RB1 gene, is a 105-kDa phosphoprotein that regulates cell cycle progression from the G₁ to S phase by reversibly inhibiting E2F-mediated transcription of genes required for S-phase entry (19, 47, 58, 65). pRb releases E2F when the former is phosphorylated and inactivated by the cyclin D-cdk4-cdk6 kinase in G₁ and the cyclin E-cdk2 kinase at the G₁-S boundary (4, 28, 33). The activity of these kinase complexes is negatively regulated by cyclin-dependent kinase inhibitors. Members of the INK4 family (p15, p16, p18, and p19) inhibit D-type cyclins, while CIP/KIP family members (p21, p27, and p57) inhibit E- and A-type cyclins (36, 51). In almost all human cancers, either RB or components of its regulatory pathway are mutated, suggesting that loss of pRb function is critical for oncogenesis.

In addition, the p53 gene, another potent tumor suppressor, is also found to be mutated or deleted in most human tumors (29). The primary anti-oncogenic function of p53 may be its rapid upregulation and subsequent induction of cell cycle arrest and apoptosis upon detection of DNA damage signals (20, 34, 50). An important mediator of p53-induced cell cycle arrest is its transcriptional target, the cyclin-dependent kinase inhibitor p21^CIP1 (20). Many oncogenic, proliferation-promoting events have been demonstrated to induce p53-dependent apoptosis, suggesting that in cancer cells, selective loss of p53 protects them from programmed cell death (55).

Ample evidence implicates a major role for tumor suppressors in cellular senescence (6, 21). However, recent findings indicate that pRb may be a crucial regulator of certain forms of senescent cell cycle exit in human cells, while p53 may be less critical. p53 and p21 levels are often seen to increase in senescent human diploid fibroblasts (2, 3, 38, 48, 69). Nevertheless, it has been observed that bypass of replicative senescence by human diploid fibroblasts did not require p53 inactivation, though this immortalization did occur with the introduction of the pRb-inactivating viral oncoprotein E7 in combination with increased telomerase activity (32). Similarly, in human cells p53 was found to be dispensable in oncogenic Ras-induced senescence, while E1Awhich inactivates and sequesters pRbblocked the senescence triggered by oncogenic Ras (48). Also, the reestablishment of the pRb pathway by the readdition of p16^INK4a in cells mutated for p16^INK4a led to senescence (15). Finally, reintroduction of pRb into RB^/ tumor cell lines induced senescence, even in cells that did not contain wild-type p53 (67). Characteristic of senescence, the state induced by pRb was found to be irreversiblethe inactivation of pRb in senescent cells led to the abortive reentry of these cells into S phase and their subsequent apoptotic death (58, 67).

With pRb clearly implicated in senescence, we wanted to determine its unique role in the senescence process and establish the mechanism by which pRb-mediated senescence occurs. We have found that, even in the absence of an intact p53 pathway, pRb is able to induce senescence, causing an accumulation of p27^KIP1 in senescent cells. The posttranscriptional upregulation of p27^KIP1 by pRb is critical to the ability of pRb to maintain cellular arrest but is not sufficient for the singular function of pRb to promote the morphological change associated with senescent cells.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cell culture, plasmids, and transfection. The human osteosarcoma cell line SAOS-2, subclone 2.4, was used for these studies (28). The cells were maintained in Dulbecco's modified Eagle's medium (Gibco/BRL) supplemented with 15% heat-inactivated fetal bovine serum and penicillin-streptomycin in a 5% CO₂ incubator at 37°C. The pRb expression plasmid was constructed in the pSVE vector (28, 57). pCMVHA-p107 and pCMVHA-p130 were gifts from Jim DeCaprio and have been described previously (61, 71). Made in the pSG5L vector (Stratagene), pRb651, pRb657, and pRb663 were constructed as described previously and were generous gifts of William Kaelin (46). pCMVp16 has been described before (27). pCMVCdk2NFG was a gift from Sander Van der Heuvel (1993). For puromycin selection, the vector pBabepuro was used (41).

Immunoblotting and immunoprecipitation. Expression of transfected and endogenous proteins was detected by immunoblotting and metabolic labeling. Cells were lysed in 100 µl of ELB (50 mM HEPES [pH 7.2], 250 mM NaCl, 2 mM EDTA, 0.1% NP-40, 1 mM dithiothreitol) plus protease and phosphatase inhibitors (1 µg of aprotinin per ml, 1 µg of leupeptin per ml, 100 µg of Pefabloc, 4 mM sodium orthovanadate, 2 mM sodium PP_i) per 10-cm plate. For metabolic labeling, cells were incubated in methionine-free media with 15% dialyzed fetal bovine serum for 45 min, labeled for 4 h (cyclin E immunoprecipitation), 1.5 h (pulse-chase-p27 immunoprecipitation), or 1 h (p27 immunoprecipitation) with 500 µCi of Expre-³⁵S³⁵S (NEN) in 2 ml of methionine-free media plus 15% dialyzed fetal bovine serum, and then lysed in 1 ml of ELB plus inhibitors. For the p27 immunoprecipitation, harvested lysates were first boiled at 100°C for 5 min. Protein concentrations in cell lysates were determined by Bio-Rad protein assay.

Cell cycle analysis. Determination of the percentage of cells in G₁ phase for the indicated plasmids was done using fluorescence-activated cell sorting (FACS) as described previously (71). For 10-cm or 6-well plates, 5 or 0.5 µg, respectively, of the expression plasmid for the B-cell surface marker CD20, pCMVCD20 (62), was cotransfected with 10 or 0.5 µg, respectively, of the indicated plasmids. Forty-eight hours posttransfection the cells were rinsed off the plates with phosphate-buffered saline (PBS) plus 0.1% EDTA, pelleted, and stained with 20 µl of fluorescein isothiocyanate (FITC)-conjugated CD20 (Pharmingen). Cells were fixed with 90% ethanol and left at 4°C for 1 h to several days. Prior to FACS analysis cells were stained with 20 µg of propidium iodide (PI) per ml and treated with 200 µg of RNase A per ml for 30 min at room temperature in the dark. Flow cytometric analysis was performed on a Becton Dickinson machine, with the intensity of PI and FITC measured by CellQuest and the cell cycle analysis done using ModFit (Becton Dickinson).

Immunofluorescence. On 6-well plates, SAOS-2 cells at 80% confluency were transfected with the indicated plasmids using the Fugene 6 reagent. Cells were split to coverslips 24 h after transfection and at the indicated time were fixed for 5 min in methanol followed by 2 min in acetone at 20°C. Coverslips were then washed three times in PBS plus 0.1% BSA, incubated in primary antibody for 1 h at 37°C, washed in PBS plus 0.1% BSA again, and then incubated in secondary antibody for 30 min at 37°C. Primary antibodies used for staining were monoclonal anti-p27 K25050 (1:100 dilution) (Transduction Laboratories), goat polyclonal anti-cyclin E C-19 (1:50 dilution) (Santa Cruz), rabbit polyclonal anti-cdk2 M2 (1:50 dilution) (Santa Cruz), and monoclonal anti--tubulin (1:1,000 dilution) (Sigma). Secondary antibodies consisted of Oregon green-conjugated anti-rabbit (1:1,000 dilution) (Molecular Probes), Cy5-conjugated goat anti-rabbit (1:250 dilution) (Amersham Pharmacia Biotech), Cy3-conjugated goat anti-mouse (1:100 dilution), and FITC-conjugated donkey anti-goat (1:50 dilution) (Jackson Immunosciences). Since both the p27^KIP1 and -tubulin antibodies were from the same host, the antibody staining was done serially. First cells were incubated with anti-p27^KIP1 antibody, then with rabbit anti-mouse immunoglobidin G antibody (1:100), and then Oregon green-conjugated anti-rabbit antibody. Following p27^KIP1 staining, -tubulin staining was performed as described above. After staining, coverslips were mounted using Fluoromount G and visualized using a Leica microscope with Sony digital imaging (p27 single staining) or a Delta Vision deconvolution microscope (coimmunostaining).

SA--gal assay. For the senescence-associated -galactosidase (SA--gal) assay, SAOS-2 cells were cotransfected with the indicated plasmids and pBabepuro and selected 24 h posttransfection. The cells were maintained in selective media for 10 days, and the SA--gal assay was performed as described previously (17). All photography of SA--gal-positive cells was done on a Leica microscope with Sony digital imaging.

Antisense oligonucleotide treatment. SAOS-2 cells at 80% confluency were transfected on 6-well plates using the Fugene 6 reagent with 0.5 to 1 µg of the indicated plasmids. Twenty-four hours after transfection the cells were split to another 6-well plate and coverslips in a 24-well plate. Twenty-four hours after the cells were split, they were transfected using the Fugene 6 transfection reagent with p27 antisense (p27AS) or p27 mismatch (p27C) oligonucleotides. Six-well plates were transfected with 1 µg of the oligonucleotides (to 0.5 µg of DNA), and 24-well plates were transfected with 0.3 µg of the oligonucleotides (to 0.1 µg of DNA). The oligonucleotides were constructed to base pairs 306 to 320 of murine KIP1 as follows: p27AS, UGG CUC UCC UGC GCC, and p27C, UCC CUU UGG CGC GCC (13; Biosource International-Keystone). For immunoblotting, immunofluorescence, and FACS, 10 h after oligonucleotide treatment cells were harvested and extracts were prepared for immunoblotting or harvested cells were fixed and PI stained for FACS as described previously. For BrdU and SA--gal assays, cells were transfected every 48 h with p27C and p27AS oligonucleotides for the indicated time. We found that the cells did not take up the oligonucleotides as well in selective medium, so the selective medium was replaced with nonselective medium immediately prior to transfection of the oligonucleotides. Cells were then placed under selection again 10 to 18 h after oligonucleotide transfection. After the indicated incubation time, the SA--gal or BrdU assay was performed as previously described.

Northern blotting. Total RNA was isolated from subconfluent cultures using Trizol (Gibco/BRL). Ten micrograms of RNA was resolved by electrophoresis, transferred to Hybond-N⁺ membranes, and probed according to standard procedures. The 500-bp DNA probe was constructed to the human p27^KIP1 gene and labeled with [³²]dCTP using the High Prime DNA labeling kit (Roche Molecular Biochemicals).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

pRb acutely increases p27^KIP1 to inhibit cyclin E-associated kinase activity. To determine the distinct role of pRb in senescence, we chose to employ the osteosarcoma tumor cell line SAOS-2, which is mutated for both p53 and RB. Past work has indicated that pRb causes an acute G₁-S cell cycle arrest in SAOS-2 cells and senescence (22, 28, 47, 58, 67); hence, prolonged cellular arrest by pRb may be important in the maintenance of the senescent phenotype. In order to further understand the nature of the G₁ arrest imposed by pRb, we investigated changes to cell cycle proteins upon reintroduction of RB into SAOS-2 cells.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   pRb increases p27KIP1 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 p27KIP1 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 p27KIP1 to determine its half-life. (F) Cells were cotransfected with vector or p27KIP1 and labeled for 1 h with 35S-methionine, and lysates were obtained and boiled at 100°C and immunoprecipitated with anti-p27KIP1. (G) Cells cotransfected with p27KIP1 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-p27KIP1.

pRb increases p27^KIP1 levels posttranscriptionally. With the increase in p27^KIP1 levels implicated in pRb-mediated cell cycle arrest, we wanted to investigate how pRb was regulating p27^KIP1 expression. Interestingly, cotransfection of RB with p27^KIP1 resulted in increased expression of the exogenously introduced p27^KIP1, suggesting that pRb regulation of p27^KIP1 may be posttranscriptional (Fig. 1C). We confirmed this by performing a Northern blotting of cells transfected with RB either with or without p27^KIP1, demonstrating that pRb did not increase either endogenous (Fig. 1D) or exogenous (data not shown) p27^KIP1 levels transcriptionally.

pRb, but not p107 or p130, induces senescence and p27^KIP1 accumulation. The pRb-like proteins p107 and p130 are thought to have functions in the cell that are both overlapping with and distinct from those of pRb. Others have shown that p107 and p130 can acutely arrest SAOS-2 cells in G₁ phase in a manner comparable to that of pRb but do not comparably induce the pRb-mediated morphology change, termed flat-cell formation, after prolonged expression (28, 46). In addition, both p107 and p130 can directly inhibit cdk2 in a manner similar to that of p27^KIP1 (8, 13, 70). Hence, we wanted to determine whether senescence was a unique function of pRb and if the increase in p27^KIP1 levels played a role in maintaining this terminally arrested phenotype. To do this we investigated the ability of ectopically expressed p107 and p130 to induce senescence in SAOS-2 cells.

View larger version (86K): [in this window] [in a new window]   FIG. 2.   pRb-mediated senescence is linked to p27KIP1 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--gal activity and flat and SA--gal-positive cells were counted. Percent flat and SA--gal-positive cells indicates the number of flat cells or SA--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) p27KIP1 expression in senescent cells. Ten days after transfection puromycin-selected cells were immunostained with anti-p27KIP1.

pRb upregulation of p27^KIP1 is not E2F-dependent. pRb is thought to enforce cell cycle arrest through its interaction with E2F by repressing E2F-mediated transcription of target genes whose expression is required for S-phase entry. In agreement with this, it was shown that mutation of the gene encoding pRb in the pocket domain abolished its ability to stably bind E2F, repress transcription, and induce an acute G₁ growth arrest (46). To determine if the pRb-E2F interaction was involved in pRb's regulation of p27^KIP1, we employed these pRb pocket mutants. With a flexible linker sequence, NAAIRS, inserted at the first amino acid indicated by the mutant name, HA-pRb651 and HA-pRb657 bind E2F in solution but not on DNA, while HA-pRb663 does not bind E2F either in solution or on DNA (46). By FACS analysis we confirmed that transiently at 48 h pRb induced a significant G₁ arrest, while the non-E2F-regulating pRb mutants, pRb651, pRb657, and pRb663, did not (Fig. 3A) (46).

View larger version (86K): [in this window] [in a new window]   FIG. 3.   E2F repression is not required for pRb upregulation of p27KIP1. SAOS-2 cells were transiently transfected with empty vector (pSVE), RB, HA-pRb651, HA-pRb657, or HA-pRb663. (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 p27KIP1 in transfected cells. Cells transfected with vector, RB, or HA-pRb663 were coimmunostained with cyclin E (green), cdk2 (blue), and p27KIP1 (red) antibodies 48 h posttransfection.

pRb mutants deficient in E2F regulation induce a delayed cell cycle arrest and senescence that correlates with p27^KIP1 accumulation. It has been reported that some of the non-E2F binding pRb mutants could cause the morphology change in SAOS-2 cells that has recently been linked to senescence and differentiation (46, 64). We wanted to determine if the flat cells produced by those mutants, pRb651 and pRb663, were senescent, clarifying if pRb regulation of E2F was required for pRb-mediated senescence. We determined the abilities of cells transfected with HA-pRb651, HA-pRb657, and HA-pRb663 to incorporate BrdU compared to those of the other pocket proteins, pRb, p107, and p130. We found that by 5 days, pRb651 and pRb663 had achieved an arrested cellular state, decreasing the number of cells in S phase comparably to that of p107 (Fig. 4A). pRb657, however, was not as successful at halting cellular proliferation. When we assayed the ability of the pRb pocket domain mutants to induce senescence, pRb651 and pRb663, mutants that had established cell cycle arrest by 5 days, retained the ability to induce flat cells that stained positively for SA--gal activity (Fig. 4B).

View larger version (46K): [in this window] [in a new window]   FIG. 4.   pRb pocket mutants induce senescence and p27KIP1 accumulation. SAOS-2 cells were transfected with empty vector (pSVE), RB, HA-p107, HA-p130, HA-pRb651, HA-pRb657, or HA-pRb663 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--gal activity and flat cells (solid bars) and SA--gal-positive cells (hatched bars) were counted. Results are the number of flat or SA--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-pRb663-transfected cells with cyclin E (green), cdk2 (blue), and p27KIP1 (red) antibodies 10 days posttransfection. Yellow staining in RB-transfected cells indicates colocalization of cyclin E and p27KIP1.

CIP1/KIP1 inhibitors induce senescence but not flat cells. The observation that the cell cycle arrest produced by a nonphosphorylatable pRb mutant can still be bypassed by cyclin E overexpression indicates that cyclin E must have a pRb-independent role in cell cycle progression (10, 37). This, in fact, underscores the requirement for a mechanism to inhibit cyclin E activity independent of the pRb-E2F interaction in senescent cells. With a strong correlation between p27^KIP1 accumulation and senescence, we wanted to determine if p27^KIP1 played a causative role in senescence downstream of pRb.

View larger version (43K): [in this window] [in a new window]   FIG. 5.   p27KIP1 induces senescence. SAOS-2 cells were transfected with empty vector (pSVE), RB, p16INK4a, p21CIP1, p27KIP1, 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--gal activity 10 days posttransfection. (B) p27KIP1 immunoblot at 10 days of lysates from cells transfected with the indicated plasmids. (C) Phenotype of pRb and p27KIP1 SA--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 p27KIP1 on microtubulin in senescent cells. Ten days posttransfection cells were coimmunostained with p27KIP1 (green) and -tubulin (red) antibodies.

p27^KIP1 is required for pRb-mediated senescence. With p27^KIP1 implicated in a causative role in senescence, we wanted to determine if its accumulation was necessary for pRb-mediated senescence. To eliminate p27^KIP1 expression from SAOS-2 cells, we chose to use p27^KIP1 antisense oligonucleotides previously described (12). The efficacy of the p27 antisense oligonucleotides in reducing pRb upregulation of p27^KIP1 was assessed by immunoblot and immunofluorescence. p27 antisense oligonucleotides significantly reduced the pRb-induced increase in p27^KIP1 levels, while the control oligonucleotide had no effect (Fig. 6A). Similarly, staining of oligonucleotide-treated cells for p27^KIP1 demonstrated a sharp decrease in p27^KIP1 expression in pRb-transfected cells incubated with the p27 antisense oligonucleotides, confirming their specific effect in inhibiting p27^KIP1 expression (Fig. 6B).

View larger version (32K): [in this window] [in a new window]   FIG. 6.   p27KIP1 is required for pRb-mediated senescence. SAOS-2 cells were transfected with RB and 24 h later were treated with p27KIP1 mismatch (C) or p27KIP1 antisense (AS) oligonucleotides. (A) Immunoblot of RB-transfected cell lysates 24 h after p27 antisense treatment. (B) p27KIP1 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 p27KIP1 expression on pRb-induced senescence. Starting 24 h posttransfection, cells were treated every 48 h with p27KIP1 mismatch (pRb C) or p27 antisense (pRb AS) oligonucleotides over a period of 10 days. After 10 days cells were stained for SA--gal activity, and flat cells (solid bars) and SA--gal-positive cells (hatched bars) were counted as previously described. p27KIP1 mismatch (top panel) and antisense (bottom panel) oligonucleotide-treated cells were stained for SA--gal activity at 10 days. (D) BrdU incorporation of vector (pSVE)- or RB- and HA-pRb651-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-pRb651 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.

p27^KIP1 partially rescues the ability of pRb pocket mutants to induce senescence. With the indication that p27^KIP1 was both necessary and sufficient for senescence, one prediction would be that supplying exogenous p27^KIP1 to cells transfected with pocket proteins that have diminished or no ability to induce senescence would rescue their ability to do so. To test this hypothesis we cotransfected p27^KIP1 with HA-p107, HA-p130, RB, and the pRb pocket domain mutants. Exogenous expression of p27^KIP1 had no effect on the ability of p107 or p130 to form flat cells but slightly augmented the incidence of p107 senescent cells (Fig. 7A and B). This result again demonstrates that p27^KIP1 has no ability on its own to induce the flat-cell phenotype, but instead its expression is linked to senescence. In addition, there was an increase in senescent cells when p27^KIP1 was overexpressed with p130, but these senescent cells were phenotypically identical to the senescent cells induced by p27^KIP1 alone, not pRb-mediated flat cells (Fig. 7B). Thus, p130, which is unable to induce flat cells, showed no benefit from p27^KIP1 overexpression and instead inhibited the ability of p27^KIP1 to induce senescence itself (Fig. 7B). Interestingly, these results seemed to correlate with the expression levels of cotransfected p27^KIP1p107 did not induce p27^KIP1 levels above that of cells transfected with p27^KIP1 alone while, similar to its effect on endogenous p27^KIP1 at 10 days, p130 appeared to repress exogenous p27^KIP1 expression (Fig. 7C).

View larger version (30K): [in this window] [in a new window]   FIG. 7.   p27KIP1 partially rescues the ability of pRb pocket mutants to induce flat cells and senescence. SAOS-2 cells were cotransfected with p27KIP1 and RB, HA-p107, HA-p130, HA-pRb651, HA-pRb657, or HA-pRb663 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 p27KIP1 (hatched bars). Results represent the average of three independent experiments. (B) Ten days posttransfection cells were stained for SA--gal activity and the SA--gal-positive cells per total cells were counted (at least 100 cells) for vector (CMV) or each protein alone (solid bars) or cotransfected with p27KIP1 (hatched bars). Results are the average of three independent experiments. An asterisk indicates p27 phenotypic senescent cells. (C) p27KIP1 immunoblot of cotransfected lysates at 10 days posttransfection with vector (pSVE) or the indicated expression plasmid.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Reintroduction or reactivation of pRb in human tumor cell lines that lack functional pRb often results in senescence. In this study we have investigated the contribution of pRb to senescence by reintroducing RB into an osteosarcoma tumor cell line mutated for both RB and p53. In doing so we examined the transient and prolonged effects of pRb on cell cycle protein levels and activities, cellular proliferation, and cellular morphology and the importance of these changes in cellular function to senescence. We found that soon after pRb expression, p27^KIP1 synthesis increased in an E2F-independent manner, cyclin E-cdk2 kinase activity decreased, and the cells arrested in the G₁ phase. These properties persisted upon prolonged pRb expression and progression into the senescent state, suggesting that they are important in the senescence process.

Most significantly, we found that only pRb and not p107 or p130 could induce sustained p27^KIP1 synthesis and senescence, despite the fact that p107 and p130 can cause cell cycle arrest through E2F repression and cdk2 inhibition (11, 53, 71). Indeed, recent evidence points to p107 and p130 being the primary regulators of cellular proliferation through E2F-dependent mechanisms. p130 was seen to be the predominant pocket protein bound to E2F target gene promoters in G₀ and early G₁, while p107 dominated at late G₁ and S phase (30, 56). Further, mouse embryo fibroblasts (MEFs) from RB^/ mice exhibit normal cell cycle regulation and few E2F target gene alterations, suggesting that E2F regulatory functions may be adequately performed by p107 and p130 in RB^/ MEFs (30). Given this ability of p107 and p130 to control cell cycle through E2F association at least as well as pRb, it is interesting that p107 and p130 induce senescence poorly or not at all in comparison to pRb. This strongly indicates that pocket protein-mediated E2F repression and subsequent cell cycle arrest are not enough to initiate a senescent phenotype; rather, a specific function of pRb is required.

Despite the importance of E2F regulation in pocket protein-mediated cell cycle arrest, we found senescence induction to best correlate with p27^KIP1 induction. Wild-type pRb and senescence-competent mutants all induced persistent upregulation of p27^KIP1, whether or not they were able to block the cell cycle through interaction with E2F. Further, ectopic expression of p27^KIP1 could induce aspects of senescence on its own, and ablation of p27^KIP1 expression prevented senescence induction by wild-type pRb. Thus, even in the absence of pRb and p53, p27^KIP1 can cause SAOS-2 cells to enter the senescence pathway. Indeed, p27^KIP1- or p21^CIP1-mediated inhibition of cdk2 appears to be specifically required for senescence induction. Loss of cdk2 activity at the hands of p130 does not lead to senescence, and instead we observed an inhibition of both p27^KIP1 expression and senescence in the presence of p130. Together, these data argue that p27^KIP1 (or p27^KIP1-cdk2 complexes) may play an active role in the senescence program rather than passively allowing cell cycle exit to occur as a consequence of lack of kinase activity. This E2F-independent induction of p27^KIP1 and senescence by pRb may in part explain the prevalence of RB mutation in cancer. Perhaps tumor cells selectively inactivate pRb to prevent its initiation of a senescence program upon oncogenic stimuli or cellular exhaustion of proliferative capacity.

Although the evidence outlined above demonstrates mechanistic differences in p27^KIP1 induction and E2F regulation by pRb, it is important to note that these functions likely collaborate in cell cycle arrest. For example, higher levels of cdk2 were found after expression of senescence-competent, E2F nonbinding pRb mutants, suggesting that the level of cyclin E-cdk2 complex might be regulated by E2F and thus affect the ability of p27^KIP1 to effect cell cycle arrest. Further, wild-type pRb-mediated arrest was attenuated by inhibition of p27^KIP1 expression despite the retention of an E2F binding domain, suggesting that E2F regulation and cdk2 inhibition must both occur to achieve cell cycle arrest. Indeed, the fact that an active cyclin E-cdk2 kinase complex can clearly bypass pRb-mediated cell cycle arrest potentially explains the requirement for the blocking of both E2F and cyclin E proliferative pathways for complete cell cycle arrest (10, 37). Exactly how pRb leads to increased p27^KIP1 synthesis is under study but may be related to a recently described mechanism of p27^KIP1 translational control (40). This translational regulation of p27^KIP1 expression is mediated by a 5' U-rich element which could explain how pRb regulates endogenous p27^KIP1 levels (40). However, this element does not appear to be in the p27^KIP1 construct used in these studies, suggesting that another regulatory mechanism may be in place. Still, the lack of this particular U-rich element does not preclude the possibility that a related sequence exists in the construct that would result in translational regulation of p27^KIP1 in a manner similar to the mechanism previously described.

The two collaborative functions of pRb described above are apparently augmented by a third function that results in the unique morphological alteration elicited by pRb. Although the flat-cell phenotype is similar to that seen in many senescent cell systems, pRb's induction of this phenotype is now clearly shown to be a consequence neither of senescence induction nor of combined E2F repression and cdk2 inhibition. First, despite a slight change in morphology, CIP/KIP inhibition of cdk2 did not lead to the formation of the enlarged, flattened cells seen in pRb senescent cells, although two markers of senescence, SA--gal activity and induction of plasminogen activator inhibitor, were efficiently induced. Second, p130 was found to be completely unable to induce flat cells despite its block of E2F and cdk2 activity. These observations are reminiscent of those reported recently that unlink phenotypic senescence and cell cycle arrest in human diploid fibroblasts (18). However, these data do not preclude the possibility that p27^KIP1 expression is necessary but not sufficient for the flat-cell phenotype, nor do they argue against a role for the cytoskeletal alterations in augmenting p27^KIP1 expression. Indeed, we found a tight correlation between p27^KIP1 induction and flat-cell formation by pRb mutants, and the observation that the small number of flat cells induced by p107 express the highest levels of p27^KIP1 suggests that these responses to pocket protein expression are related. How and why cells undergo this pRb-dependent, extensive morphology change involving, as we found, at least microtubule reorganization is unknown but currently being studied and is likely to contribute significantly to pRb's ability to induce permanent cell cycle exit.

Altogether our data suggest the model of pRb-induced senescence shown in Fig. 8. With the proper senescence-promoting stimulus, pRb represses E2F transcriptional activity to initiate an acute cell cycle arrest. In a non-E2F-dependent manner, pRb increases p27^KIP1 levels posttranscriptionally, leading to an accumulation of p27^KIP1, and it is in this way that pRb pocket domain mutants unable to stably interact with E2F induce growth arrest and senescence. The increased levels of p27^KIP1 inhibit cyclin E-cdk2 kinase activity, triggering a prolonged G₁ arresta function that exogenous expression of p21^CIP1 and p27^KIP1 (or dominant-negative cdk2) duplicates. It is this persistent inhibition of cyclin E kinase activity specifically by p27^KIP1 that results in the formation of small, round senescent cells. However, it is a unique function of pRb to induce an extensive cellular morphology alteration, producing enlarged, flattened cells that are senescent. Changes in cytoskeletal signaling in these flat cells may in fact maintain the irreversible cell cycle exit and high levels of p27^KIP1 expression.

View larger version (27K): [in this window] [in a new window]   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 p27KIP1 expression, leading to an accumulation of p27KIP1 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 p27KIP1 expression.

This model reinforces the idea that the p53/p21 pathway is not crucial for cellular senescence in human cells and that pRb can achieve this antioncogenic cellular state at least in SAOS-2 cells through p27^KIP1 induction. However, the observation of an increase in p53 activity and a requirement for p21^CIP1 in senescent primary human cells (3, 18) and in p53-positive human tumor cells (15) suggests that the pRb-p27^KIP1 pathway may be subordinated by the existence of an intact p53 pathway or may be specific to SAOS-2 cells or certain cell types. We believe that the pRb-p27^KIP1 pathway is a general response to senescence stimuli in the absence of p53 for two reasons. First, we have observed that the simultaneous blocking of p53 function and reestablishment of the pRb pathway by the reintroduction of p16^INK4a into U20S cells, an osteosarcoma cell line that is wild type for pRb and p53, result in the upregulation of p27^KIP1 instead of p21^CIP1 in these senescent cells (data not shown). Second, our preliminary data indicate that this pRb-p27^KIP1 senescence-inducing mechanism occurs in other cell types shown to be susceptible to pRb-mediated senescence (67). Additionally, evidence is accumulating for p27^KIP1-dependent, p53-independent mechanisms of senescence in normal cells. For example, recent work with MEFs has shown that inhibition of phosphoinositide 3-kinase leads to a senescence that is associated with an elevation of p27^KIP1 levels but not p53, p19^ARF, p16^INK4a, or p21^CIP1 levels (14). Thus, certain physiological stimuli, tissue contexts, or intracellular environments may modulate the pathways by which cells enter senescence in an effort to avoid tumorigenesis. The prevalence of disruption of the pRb pathway in tumors is likely to partially result from pRb's critical role in cell cycle exit programs such as the one described here. The mechanism through which pRb augments p27^KIP1 synthesis and the specific role of p27^KIP1-mediated cdk2 inhibition in senescence remain to be elucidated but promise to yield significant clues to an important tumor-suppressive process.