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Previous Article | Next Article 
Molecular and Cellular Biology, April 2004, p. 2808-2819, Vol. 24, No. 7
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.7.2808-2819.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Hai-Su Yang,,
and Philip W. Hinds*
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
Received 1 August 2003/ Returned for modification 5 September 2003/ Accepted 22 December 2003
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ABSTRACT |
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INTRODUCTION |
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In mouse cells, the loss of the p19ARF/p53 pathway is critical for cellular immortalization, while in human cells, the bypass of senescence requires inactivation of the p16INK4a/pRb pathway (22). Indeed, these negative regulators of proliferation accumulate with continued cell division in cultured primary cells, suggesting that they are part of a mechanism that counts the number of cell divisions and thus limits proliferative capacity. The initiating signal of this molecular clock has been attributed to the shortening of telomeres, the progressive erosion of which is thought to eventually trigger growth arrest. In support of this hypothesis, hTERT overexpression and the subsequent restoration of telomerase activity block telomere shortening and ultimately immortalize cells (3). In addition, though hTERT activity is undetectable in normal cells, it is upregulated in tumor cells, further suggesting that dysregulation of hTERT activity is involved in the malignant transformation of cells (20, 25). Significantly, it has been found that immortal cells arising out of hTERT-transduced cells require spontaneous or oncogene-induced disruption of the pRb pathway (10, 19, 30, 34). Thus, pRb likely controls a senescence-instigating pathway that works in synergy with, but is distinct from, that engendered by telomere loss.
The strongest evidence for a telomere-independent senescence pathway is provided by the observation that primary cells will senesce prematurely upon oncogenic stimuli. For instance, the oncogenic, persistently-activated form of the small GTPase Ras, which initially causes proliferation, eventually triggers cell cycle arrest and premature senescence in both mouse embryo fibroblasts and human diploid fibroblasts (HDFs) (38). Ras-induced senescence requires an intact mitogen-activated protein kinase pathway. Indeed, the same senescence effect can be mediated by Ras's downstream effectors, Raf and MEK (21, 38, 47). The senescence induced by Ras is accompanied by an increase in p16INK4a and p19ARF levels and pRb and p53 activation. However, while either p53 or pRb is dispensable in Ras-induced senescence in MEFs, only elimination of pRb function in HDFs by E1A results in senescence inhibition (38). In a conceptually similar but biochemically distinct manner, E2F1 overexpression has been observed to induce a p14ARF-dependent cell cycle arrest and senescence in HDFs (11). However, fibroblasts from patients with constitutive inactivating mutations in p16INK4a are resistant to Ras-induced senescence, although hTERT expression is still required for immortalization (5). Because these cells express functional p14ARF, this work underscores the critical role of the p16INK4a/pRb pathway in premature senescence and its obligate inactivation in the immortalization of human cells.
Significantly, the role of p16INK4a/pRb in the senescence of primary cells can be recapitulated in tumor cells. The reintroduction of pRb or p16INK4a into tumor cells that have lost expression of either protein induces a premature senescence requiring p21CIP1 or, in the absence of an intact p53 pathway, p27KIP1 (1, 8, 9). Intriguingly, cyclin-dependent kinase inhibitors like p14ARF, p21CIP1, and p27KIP1, which are required for senescence, can induce markers of senescence on their own. However, they cannot mediate the senescent shape change, demonstrating that these two processes in senescence are separable (1, 8, 13).
Despite clear evidence for a role for the p16INK4a/pRb pathway in senescence, little is known about the mechanism by which this pathway commits cells to the senescent phenotype characterized by expression of SA-ß-Gal and morphological alteration. Using several model systems of senescence, including long-term passage and acute expression of Ras or pRb, we have found that CDK5, a kinase that hitherto has been considered to be almost solely involved in regulating neuronal activities, plays a central role in the shape change of senescent cells through regulation of Rac1 activity and influences SA-ß-Gal expression in senescent human cells.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunoblotting and immunoprecipitation. Protein expression was detected by immunoblotting. Cells were lysed in 100 to 200 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 mg of aprotinin/ml, 1 µg of leupeptin/ml, 100 µg of phenylmethylsulfonyl fluoride, 4 mM sodium orthovanadate, 2 mM sodium PPi) per 10-cm plate. Protein concentrations in cell lysates were determined by Bio-Rad protein assay. For immunoblotting, 30 to 50 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose by standard procedures. Antibodies used for immunoblotting include: anti-CDK5 DC17 (Santa Cruz), anti-Rac1 (Transduction Laboratories), anti-RhoA (Santa Cruz), anti-Cdc42 (Santa Cruz), anti-Pak1 (Santa Cruz), anti-phospho-Pak1 (Sigma) and anti-pRb Ab-5 (Calbiochem). Proteins were detected by using horseradish peroxidase-conjugated donkey anti-mouse or donkey anti-rabbit antibodies (Jackson Immunosciences). One hundred micrograms of cell lysates was immunoprecipitated with the indicated antibody overnight at 4°C, 30 µl of protein A/G beads was added to the immunoprecipitation mixture for 1 h, and then the beads were washed four times with lysate buffer and separated by SDS-PAGE.
Kinase assay.
To detect CDK5 kinase activity, immunoprecipitation for CDK5 was performed by using 100 µg of cell lysate with anti-CDK5 F8 (Santa Cruz). The immunoprecipitation mixtures were subjected to an in vitro kinase assay essentially as described previously, with slight modifications (1). One microgram of an N-terminal fragment of PSD-95, bacterially expressed, purified His-N-PDZ1-2S (17), was used as a substrate for immunoprecipitated CDK5 (gift of Li-Huei Tsai). For the Pak1 kinase assay, immunoprecipitation with Pak1 antibody was performed as described previously (27). Briefly, the assay was done in the presence of 1 mM ATP, 1 µCi of [32P-
]ATP, 3 µg of histone H4, and kinase buffer (50 mM HEPES [pH 7.5], 10 mM MgCl2) for 30 min at 30°C.
siRNA synthesis. The CDK5 small interfering RNA (siRNA) construct was made as described previously by using the pBS/U6 plasmid (39). The oligonucleotides used to create pBS/U6-siCDK5 were as follows: 1a-GGGAGCTGAAATTGGCTGATA, 1b-AGCTTATCAGCCAATTTCAGCTCCCC, 2a-AGCTTATCAGCCAATTTCAGCTCCCCTTTTTG, and 2b-AATTCAAAAAGGGGAGCT-GAAATTGGCTGATA.
GTPase activity assays. The activity of GTP-Rac, GTP-Rho, and GTP-Cdc42 was determined by using an assay developed by Ren and Schwartz (33). The effectors rhotekin and Pak were used as glutathione S-transferase (GST) fusions to affinity precipitate endogenous, cellular, active Rho, and Rac and Cdc42, respectively. The pEGB-Pak1 and -rhotekin plasmids, kind gifts of J. Settleman, were bacterially expressed and bound to GST beads. The assay was performed as previously described (33).
Immunofluorescence. Transfected cells fixed in methanol for 5 min and acetone for 2 min at the indicated times were immunostained as described previously (1). Antiactin (Sigma) was detected with Cy3-conjugated donkey anti-rabbit secondary antibody (Jackson Immunosciences). Fluorescent images were acquired by using DeltaVision deconvolution microscopy.
SA-ß-Gal assay. The SA-ß-Gal assay was performed as previously described (12). Briefly, cells were washed in phosphate-buffered saline and fixed in 2% formaldehyde-0.2% glutaraldehyde. Then the cells were washed and incubated at 37°C overnight with fresh senescence-associated ß-Gal stain solution (1 mg of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal] per ml, 40 mM citric acid-sodium phosphate [pH 6.0], 150 mM NaCl, 2 mM MgCl2, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide). All visualization and photography of cells were performed on a Leica microscope with Sony digital imaging.
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RESULTS |
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In the osteosarcoma cell line SAOS-2 lacking wild-type pRb and p53 expression, reintroduction of retinoblastoma protein (pRb) results in induction of SA-ß-Gal 7- to 10-days posttransfection (43). Treatment of SAOS-2 cells 48 h after RB transfection with 10 µM roscovitine for the remaining time course of 7 to 10 days completely inhibited SA-ß-Gal induction (Fig. 1A). Similarly, in the pRb/p53 mutant breast carcinoma cell line MDA-MB-468, infection of the cells with adenovirus RB (Ad-RB) can cause senescence after 7 days (43). Treatment of Ad-RB-infected MDA-MB-468 cells with roscovitine 1 through 6 days after infection inhibited SA-ß-Gal induction by approximately 50% (Fig. 1A). The difference in the effect of roscovitine on the two cell lines may be due to our observation that there are higher expression levels of pRb in the MDA-MB-468-infected cells than in SAOS-2-transfected cells (data not shown). Retroviral infection of the normal HDF strain IMR90 with a constitutively active Ras1 mutant, RasV12, causes cell cycle arrest by 5 days after infection and eventual senescence (38). Treatment of RasV12-infected cells with roscovitine 5 days after infection for a subsequent 5 days reduced SA-ß-Gal expression back to the level of the controls (Fig. 1A). Finally, we tested the effects of roscovitine on senescence induced by the continued passage of HDF strains WI38 and IMR90. Treatment of these cells with roscovitine at early passage would cause cell cycle arrest and prevent their continued passage and senescence. Thus, HDFs that had already become senescent were treated with roscovitine for 48 h and showed a reduction in expression of SA-ß-Gal compared to that for dimethyl sulfoxide-treated cells (Fig. 1A). Similarly, we have seen that pRb-expressing SAOS-2 cells treated with roscovitine after the onset of senescence display a diminution of SA-ß-Gal expression (data not shown) but not complete inhibition. Hence, these results suggest that there are differences in the sensitivity of cells to roscovitine's effects on SA-ß-Gal expression dependent upon whether initiation or maintenance of senescence is being assayed. Overall, these results indicate that roscovitine inhibits a phenotypic marker of senescence under a variety of conditions.
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CDK5 activity is required for senescence. The drug roscovitine inhibits a variety of cellular kinases in a dose-dependent manner by competing for their ATP-binding domain. At the concentration employed, 10 µM, roscovitine is a specific inhibitor of CDK2, CDK5, and CDK1. However, both CDK2 (1, 8, 13) and CDK1 (K. Alexander and H. Yang, unpublished data) activities are absent in senescent cells, and overexpressed dnCDK2 or dnCDK1 has no inhibitory effect on pRb-induced senescence. Thus, we decided to investigate a possible role for CDK5, a postmitotic kinase known to regulate neuronal cytoskeletal organization (23), in cells undergoing the process of senescence.
SAOS-2 cells transfected with RB or MDA-MB-468 cells infected with Ad-RB were harvested for cell lysates 2, 4, and 7 days after RB reintroduction. Immunoblot analysis showed an increase in CDK5 protein levels as early as 2 days after reintroduction of pRb in both cell lines (Fig. 2A). Performance of a CDK5 immunoprecipitation and a subsequent in vitro kinase assay using a CDK5 substrate, PSD-95 (M. Morabito, personal communication), showed a concomitant increase in CDK5 activity (Fig. 2A) that was inhibited by roscovitine (data not shown). Interestingly, MDA-MB-468 cells, which had higher expression levels of pRb than SAOS-2, also had higher expression levels of CDK5 and much more CDK5 activity. Similarly, in senescent RasV12-infected IMR90 cells, there was a small but detectable increase in CDK5 levels and a comparable increase in CDK5 activity (Fig. 2A). In senescence induced by the passage of IMR90 HDFs, we found no significant changes in CDK5 levels between early passage (EP), middle passage (MP), and senescent (S) cells (Fig. 2A). However, CDK5 activity in IMR90 cells increased at middle passage and remained high in senescent fibroblasts (Fig. 2A). In addition, in both passaged and RasV12-infected fibroblasts, reduction of pRb phosphorylation correlated with increased CDK5 activity (Fig. 2A).
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To further determine if the ability of roscovitine to block senescence was due to its inhibition of CDK5 function, SAOS-2 cells were cotransfected with pRb and increasing concentrations of a dominant-negative form of CDK5, CDK5N144 (dnCDK5). dnCDK5 was able to block the senescent morphology change in a dose-dependent manner, demonstrating that CDK5 activity is required to attain the senescent phenotype. However, dnCDK5 only partially blocked induction of the senescence marker SA-ß-Gal (Fig. 2C), which might suggest the existence of a CDK5-independent mechanism that participates in SA-ß-Gal induction or, alternatively, that dnCDK5 is not completely effective at blocking CDK5 activity even at higher concentrations.
As dnCDK5 seemed an imperfect mechanism to inhibit CDK5 function in these cells, we made a plasmid-based siRNA construct of CDK5 (siCDK5) to more specifically and effectively inhibit CDK5. Ten days after SAOS-2 cells were transfected with pRb and siCDK5, immunoblot analysis showed that siCDK5 specifically inhibited CDK5 expression levels but had no impact on the homologous kinase CDK2. In addition, in vitro kinase analysis indicated that the pRb-induced elevation of CDK5 activity was inhibited by cointroduction of siCDK5 (Fig. 2D). Most strikingly, siCDK5 inhibited both the senescent morphology change and, to a lesser degree, expression of SA-ß-Gal (Fig. 2E), supporting a role for CDK5 as a regulator of the senescent phenotype.
CDK5 regulates SA-ß-Gal induction through inhibition of Rac1 activity. The data shown above link CDK5 activation to SA-ß-Gal expression and cytoskeletal changes in senescent cells. Because CDK5 has been demonstrated to influence actin polymerization in neurons through interaction with Rac1 (27) and Rac1 has been implicated in controlling cell shape (28, 35), we investigated the role of Rac1 in the senescence phenotype. SAOS-2 cells were cotransfected with pRb and either an activated (RacV12) or dominant-negative (RacN17) form of Rac1. Cells cotransfected with pRb and RacV12 displayed little SA-ß-Gal expression and did not display a flat phenotype, while those cotransfected with pRb and RacN17 had SA-ß-Gal expression levels and morphology comparable to those of senescent cells expressing only pRb (Fig. 3A).
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Rac1 activity decreases in senescing cells. In an effort to test the hypothesis that CDK5 acts to regulate the senescent phenotype through modulation of the activity of small GTPases of the Rho family, GST-Pak1 and GST-rhotekin fusion proteins were used to measure the GTP loading of endogenous Rac1, Cdc42, and RhoA GTPases in senescent cells with and without CDK5 inhibition. To achieve this, cell lysates were incubated with GST beads bound to the effector Pak1 or rhotekin, and the GST-bound proteins were subjected to SDS-PAGE and immunoblotted with Rac1, Cdc42, or RhoA antibody (33). In SAOS-2 cells, there were not detectable levels of Cdc42 and RhoA, although these cells have high expression levels of Rac1. Therefore, SAOS-2 cells transfected with pRb or pRb and CDK5 siRNA were assayed for Rac1 activity as the cells underwent senescence. Rac1 activity decreased in senescing SAOS-2 cells after pRb transfection. However, active Rac1 was accumulated in the presence of CDK5 siRNA, while total Rac1 levels were constant, as determined by direct immunoblotting (Fig. 4A). Similarly, Rac1 activity was reduced in senescent MDA-MB-468 cells infected with Ad-RB, whereas no change in the level of the RhoA- or Cdc42-GTP was detected in the senescent cells compared with that for the parental cells (Fig. 4B). These data suggest that CDK5 activity is required for reduction of activated Rac1 in pRb-induced senescence and that inhibition of CDK5 results in the aberrant accumulation of active Rac1 in senescing cells. Indeed, Rac1 activity also lessened in senescent IMR90 HDF without any change in Rac1 levels, but the activity of RhoA- or Cdc42-GTP showed little change (Fig. 4C). In addition, RasV12-infected senescent fibroblasts also demonstrated a repression of Rac1 activity that was blocked when CDK5 activity was inhibited by roscovitine (Fig. 4D). Overall, these results suggest that Rac1 activity but not that of RhoA or Cdc42 decreases in the process of senescence and that this repression of Rac1 activity is regulated by CDK5.
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Rac1 regulates actin polymerization in senescing cells. CDK5-dependent flattening of senescent cells suggests that extensive changes must occur to the cellular cytoskeleton as cells undergo senescence. Both CDK5 and Rac1 have known roles in regulating cell shape through actin reorganization. With this in mind, we investigated changes to actin structure as cells underwent senescence. We immunostained for actin in cells induced to senesce by pRb (SAOS-2) or passage in culture (IMR90 and WI38). We observed a significant increase in polymerized actin filaments during senescence (Fig. 5A), which was confirmed by staining for F-actin (data not shown). This appearance of polymerized actin structures was accompanied by an overall increase in actin levels (data not shown).
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DISCUSSION |
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Discovery of this role of CDK was unexpected, as CDK5 has been thought to function primarily in developmental signaling in neurons and potentially somatic mesoderm (31). However, given that CDK5 is ubiquitously expressed, it is likely that the kinase plays additional or related roles in other tissues. Indeed, work presented here linking CDK5 to Rac1 activity and cytoskeletal changes in postmitotic, senescent cells is not dissimilar to CDK5's reported biochemical roles in neurite outgrowth. Thus, CDK5, acting in part through Rac1, may function as a general signaling molecule coordinating cellular responses to loss of mitotic capacity in both differentiation and senescence.
Regulation of CDK5 and Rac1 in senescence. The increase in CDK5 activity in senescent cells is observed following a variety of stimuli in human primary and tumor cells, suggesting that a mechanism common to all of these systems may function in CDK5 activation. Indeed, the use of model systems in which senescence is induced by pRb reexpression in tumor cells strongly implicates the pRb pathway in CDK5 upregulation independently of any role for p53. It is not clear at this time whether such a role for pRb is direct, nor is the mechanism of CDK5 activation well understood. There is a clear induction of CDK5 protein in acute systems in which pRb or activated Ras stimulates senescence within days, and this induction appears to be largely independent of transcriptional regulation (K. Alexander, unpublished data). Indeed, pRb-mediated proliferation arrest is dependent on posttranscriptional regulation of p27Kip1 in the SAOS-2 system (1), suggesting that pRb may act directly or indirectly to alter the expression of a number of proteins involved in senescence without associated transcriptional changes.
Regardless of the mechanism of CDK5 activation in senescent cells, our data strongly argue that an important consequence of this activity is downregulation of Rac1 function. Indeed, this function of CDK5 appears to be specific for Rac1, as we observed no change in the levels of active RhoA or Cdc42 in senescent cells. Thus, increased actin polymerization and accompanying cytoskeletal changes may be understood in the context of unmasked RhoA signaling in the relative absence of Rac1 activity in senescent cells. Such a phenotype is consistent with the observed effects of RhoA overexpression in a variety of systems (14, 36). Most importantly, inhibition of CDK5 activity reverses a loss of Rac1 activity that we observe in all senescent cells, and conversely, inhibition of Rac1 by a dominant-negative subunit precludes the need for CDK5 activity for SA-ß-Gal expression and cytoskeletal changes. Taken together, these results imply that CDK5 acts upstream of Rac1 to directly or indirectly inhibit its activity. In addition, our results revealed that CDK5 phosphorylated Pak1 at Thr212 and reduced Pak kinase activity in senescent cells, similar to the reported role of CDK5 in Rac1/Pak1 activity in neurons, where p35/CDK5 kinase associates with Pak1 in a Rac-GTP dependent manner, causing hyperphosphorylation and downregulation of Pak1 kinase activity (27). In theory, the specific inhibition of Rac1 we observed in senescent cells should preclude the need to regulate its effector Pak1. Therefore, we speculate that Pak1 phosphorylation by CDK5 may have additional roles in the senescent phenotype. Indeed, we note that Pak1 has recently been reported to interact with the ERM-related tumor suppressor protein NF2/merlin (18), a protein that our preliminary studies indicate might also play a role in the senescent phenotype (H. Yang, P. Santiago, and P. Hinds, unpublished observations).
Nevertheless, the requirement for CDK5 activity in downregulation of Rac1 activity suggests an additional role for CDK5 beyond regulation of the effector molecule. For example, work in our laboratory has recently identified ezrin as a direct substrate for CDK5 in senescent cells, and the postulated interaction of ezrin with Rho family regulators makes it an attractive candidate to participate in the CDK5/Rac1 pathway in senescence (45). Despite the rudimentary nature of our understanding of Rac1 regulation in senescence, our data indicate that Rac1 activity must somehow be antisenescent, which may be due to the reported proliferative effect of Rac1 in some cells (29, 44) or may imply that established, Rac1-responsive cytoskeletal regulation collaborates with other signals in senescent cells to stimulate SA-ß-Gal expression and, potentially, other aspects of senescence. As discussed below, significant additional studies of the role of cytoskeletal changes in the biochemical and proliferative aspects typical of senescence are needed to fully appreciate the role of Rac1 in these processes.
CDK5 function in senescence and tumor suppression. A prominent role for CDK5 in the senescence process and the likelihood that senescence is tumor suppressive (22, 37) raise the possibility that CDK5, like other senescence regulators, is also a tumor suppressor. In fact, CDK5 has generally been observed to be more highly expressed in many tumor cells than in normal cells, which may result from interrupted senescence signaling in immortal cells akin to elevated p16INK4a expression seen in cells lacking pRb function. Clearly, additional work is warranted to address a general antiproliferative or tumor-suppressive role of CDK5 in postmitotic cells, but our initial indications are that CDK5 at best contributes to, but is not required for, proliferative arrest in senescence. Senescent cells in which CDK5 has been inhibited remain phenotypically senescent in that they do not revert to wild-type shape or size and they do not reenter the cell cycle. This finding supports the idea that cell cycle arrest, likely the most critical characteristic in making senescence irreversible and tumor suppressive, is predominantly mediated by the established functions of pRb and p53, the primary regulators of senescence.
Despite the persistence of growth arrest in senescent cells lacking CDK5 activity, it is quite possible that CDK5 contributes to the irreversibility of senescence. First, the effect of CDK5 inhibition on cell death following senescence reversal in a variety of systems has not been tested (9, 41, 43). Second, we and others have observed that expression of p27Kip1 and p21Cip1 is critical to senescent arrest, and the effect of CDK5 expression on these regulators is unknown. Indeed, the role of CDK5 in cytoskeletal changes and a variety of observations linking cytoskeletal signaling to p27Kip1 expression suggest an intriguing link germane to the establishment of senescence. A third indication that CDK5 may contribute to, but not be required for, phenotypic aspects of senescence contributing to growth arrest derives from the observation that CDK5 repression of Rac1 activity is not the only mechanism to trigger SA-ß-Gal expression in senescent cells, particularly in MEFs. Indeed, we and others have seen that the cell cycle inhibitors p21CIP1 and p27KIP1, which are upregulated during senescence, can on their own induce SA-ß-Gal expression even in the absence of both pRb and p53 (1, 8, 13). Our preliminary data indicate that the CDK5 activity-insensitive SA-ß-Gal expression is likely due to this cell cycle inhibitor induction of SA-ß-Gal, as this pathway seems to remain mostly intact even in the absence of CDK5 activity. However, we have observed that such p27Kip1-induced small senescent cells are less stable than those displaying a morphology characteristic of senescence, providing some evidence for a role for cytoskeletal changes in long-term proliferative arrest (K. Alexander, unpublished).
Our work begins to provide a framework for the molecular events by which the process of senescence occurs downstream of senescence inducers. It seems clear that this will be found to be a complex process involving diverse signaling pathways normally observed to be used in a different context. Still, CDK5 appears to be an important regulator upon which many senescence pathways impinge, which represents a crucial role for CDK5 in normal cells. Recent evidence from mice suggests that tumors that retain an ability to undergo senescence via p16INK4a respond more readily and survive much longer after chemotherapeutic treatment (37). Hence, the fact that the CDK5 pathway to senescence seems to be intact but latent in tumor cells may represent an advantage in cancer treatments in that its activation may perhaps be used as a mechanism to induce aspects of senescence in tumors in vivo and thus impair tumor progression.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant AG20208 to P.W.H.
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FOOTNOTES |
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K.A. and H.-S.Y. contributed equally to this work.
Present address: Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA 02111.
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