Inhibition of p53-Mediated Growth Arrest by Overexpression of Cyclin ...

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1996, p. 4445–4455 0270-7306/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 16, No. 8

Inhibition of p53-Mediated Growth Arrest by Overexpression of Cyclin-Dependent Kinases KATHRYN M. LATHAM, SCOTT W. EASTMAN, ANDREA WONG,

AND

PHILIP W. HINDS*

Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 Received 22 December 1995/Returned for modification 26 February 1996/Accepted 9 May 1996

Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5&C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdkactivating kinase activity. This is due in part to p53-dependent expression of the p21 cdk inhibitor. Upon shift to 39&C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p21. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p21. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression. tion factors (15, 21, 41, 66). Indeed, many mutant p53 proteins are dominant in their loss of transcription function, preventing this activity of the wild-type protein when both are expressed in the same cell (41, 73). Given the tight association of the abilities of p53 to act as a transcriptional activator and as a growth regulator, one likely candidate for a downstream mediator of its growth-inhibitory effects is the p21 protein encoded by the WAF1/CIP1/SDI1 gene (16, 25, 61). Expression of p21 mRNA and protein is strongly induced by p53 and can itself inhibit cell proliferation upon introduction into cultured cells. This effect of p21 is apparently due to the ability of p53 to associate with and inhibit complexes of cyclins and cyclin-dependent kinases (cdks) (23, 25, 91). Because of this activity, p21 and related proteins have been termed cdk inhibitors (CKIs). In addition to its function as a CKI, p21 may associate independently with the DNA replication-associated protein proliferating-cell nuclear antigen and thus prevent DNA replication directly (5, 84). cdks, in complexes with positive-regulatory cyclin subunits, are key regulators of the cell cycle in all eukaryotes. They form a large family of serine/threonine kinases, whose individual substrate specificities are tailored to the particular cell cycle transition they control. For example, complexes of cdk4 and cdk6 with cyclins D1, D2, and D3 and complexes of cdk2 and cyclin E may each have specific roles in promoting the G1-to-S phase transition (35, 74), in part by collaborating in the phosphorylation and inactivation of the retinoblastoma protein (31, 33, 74). The critical role of cdks in regulating the proper execution and timing of DNA synthesis and mitosis is reflected by the multiple factors that influence their activity. In addition to the periodic synthesis and destruction of cyclin subunits, cells control cdk activity through positive and negative phosphorylation of the cdk itself. Furthermore, cdk activity is subject to inhibition by the induced synthesis and destruction of a variety of CKIs that either compete for cyclin binding or associate with the preformed cyclin-cdk complex (reviewed in references 35 and 58). The ability of the p21 CKI to associate with and inhibit many different cyclin-cdk complexes (25, 26, 91) suggests that p21 has a broad effect on cell cycle progression. Recent experiments have provided evidence for the role of p21 in mediating the ability of p53 to arrest cells in G1. Several groups have found p21 levels to be elevated shortly after p53

Mutation or deletion of the p53 gene is a frequent event in the majority of human tumor types studied. Presumably, this strong selection against wild-type p53 function reflects a critical role ensuring that cell proliferation occurs only under appropriate conditions. The regulatory capacity of p53 as a “checkpoint” is supported by the observation that mice engineered to lack p53 are viable and live several months before they succumb to a variety of cancers (11). Thus, a critical suppressor of tumor formation is lost when p53 genes are absent. The tumor suppressor function of p53 may result from its ability to mediate an important response to DNA damage. When treated with a variety of DNA-damaging agents, normal cells demonstrate a rapid, posttranscriptional induction of p53. Depending on the circumstances, this is accompanied by cell cycle arrest or apoptotic cell death (7, 12, 14, 38, 43, 48, 49). In the absence of p53, cell cycle arrest in G1 or apoptosis is not seen, implicating p53 as a critical regulator of these responses (7, 39, 48, 49). Cell cycle progression or survival in the presence of damaged DNA may then lead to the genomic instability seen in many tumors (46, 94). More importantly, the loss of p53-dependent apoptosis prevents the tumor-suppressive cell death that results from aberrant expression of proliferationpromoting proteins such as myc, E2F-1, and viral oncoproteins (9, 27, 34, 47, 59, 71, 78, 85, 88, 90). Of the various biochemical properties of p53 potentially mediating its growth-regulatory function (reviewed in reference 33), site-specific transcriptional transactivation is most compellingly associated with the tumor-suppressive functions of p53. First, the transactivation function of p53 is inactivated by the viral oncoproteins simian virus 40 (SV40) T antigen and adenovirus E1B (52, 55, 92). Second, the cellular oncoprotein mdm-2 inactivates this function of p53 by binding directly to the region required for interaction with transcriptional machinery (57, 63, 79). The third and most important reason to suspect that transactivation is key to the growth-suppressive function of p53 is that all of the mutant p53 proteins found in human tumors are impaired in their ability to act as transcrip-

* Corresponding author. Phone: (617) 432-2901. Fax: (617) 4320136. Electronic mail address: [email protected]. 4445

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induction by DNA damage, and this correlates with a loss of cdk activity in these cells (12, 14). As expected, this response is lacking in cells without p53 (14); however, p21 can be induced in a p53-independent manner, most often as a response to differentiation signals (13, 24, 36, 37, 54, 65, 77). One model derived from these observations proposes that DNA damage induces p53, which in turn induces high levels of p21, shutting down cdk activity and arresting cells in G1. This model is consistent with a tumor-suppressive effect of p53 mediated at least in part through the ability of p21 to prevent the proliferation of damaged cells and thus the propagation of damaged chromosomes. If p21 is a central mediator of p53-dependent inhibition of cellular proliferation, one prediction is that intragenic or extragenic suppressors of p21 function would themselves be oncogenic, allowing cells an enhanced proliferative capacity in the presence of wild-type p53. In fact, it is apparent from many studies that p53 alterations, although common, do not occur in 100% of tumors originating from a variety of tissues. Nevertheless, deletions or mutations of p21 are rare (21a, 75). Furthermore, elimination of the p21 gene by homologous recombination in mice results in only a partial alteration in p53-mediated suppression of proliferation, suggesting that p21 is not the sole agent through which p53 functions (3, 10). Insensitivity to p53 may require a more pleiotropic mutation, altering p53-dependent functions not limited to inactivation of p21. For example, overexpression of the mdm-2 protein may fulfill such a role in human sarcomas and transfected rat cells by preventing the transactivation function of p53 (19, 62). To begin to explore the extent of the role of p21 in mediating the anti-proliferative functions of p53, we have exploited transformed rat cells that express a temperature-sensitive version of p53 (Ala-1353Val). At 378C, this mutant protein behaves as a typical dominant-negative p53, promoting cellular immortalization and transformation. However, when the temperature is shifted to 32.58C, conformationally and functionally wild-type p53 predominates and cells expressing the protein reversibly arrest in the G1 phase of the cell cycle (22, 50, 53). These cells are thus useful for studying p53-dependent cell cycle arrest in the absence of the pleiotropic effects of DNA-damaging agents. In agreement with the model outlined above, we found that temperature downshift induces p21 and that recovery from the ensuing growth arrest is accompanied by loss of p21 concomitant with kinase reactivation. Intriguingly, overexpression of cdk4 and cdk6, but not cdk2 and several cyclins, not only restores cdk activity but also allows cellular proliferation in the presence of apparently functional p53. These data suggest that cdk4 and cdk6 may act as oncoproteins because of their ability to circumvent a function of p53 in tumor suppression. MATERIALS AND METHODS Cell lines, plasmids, and transfections. The A1-5 rat fibroblast cell line expressing temperature-sensitive p53 and activated ras was kindly provided by A. Levine. The cells were maintained in a humidified 3% CO2 atmosphere at 378C in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 100 U of penicillin G per ml, and 100 mg of streptomycin per ml (all cell culture reagents were from JRH Biosciences). For cell cycle studies, the cells were plated at approximately 500,000 cells per 10-cm-diameter dish, incubated at 378C for 6 to 18 h, and subsequently placed in a 32.58C incubator. Incubation at 32.58C was continued for 36 h to allow the accumulation of wild-type p53 and subsequent growth arrest. Cell cycle reentry was achieved by refeeding the arrested cells with prewarmed (378C) medium prior to placement in a 398C incubator. Transfection of the parental A1-5 cells was performed by the calcium phosphate procedure as described previously (4), and precipitates were allowed to remain on the cells overnight at 378C. Following transfection, the cells were rinsed once with phosphate-buffered saline (PBS), twice with PBS plus 100 mg of calcium chloride and magnesium chloride per liter (PBS1), and once with complete medium and fed with complete medium. At 6 to 10 h later, the cells

MOL. CELL. BIOL. were trypsinized, counted, and plated at 500,000 cells per 10-cm dish. The following morning, the cells were fed with medium containing 6 mg of puromycin per ml and shifted to 32.58C. After 3 weeks of selection, colonies were cloned by trypsinization in glass cloning cylinders (Bellco) and expanded at 32.58C in the presence of puromycin. Plasmids used in transfections included pBabepuro, containing a puromycin resistance cDNA (60); SVgem, carrying the SV40 early region and capable of expressing large T antigen (kind gift of J. Marks); and the vectors Rc/CMV (Invitrogen) and CMVNeoBam3 (2). Rc/CMV-based vectors expressing the D cyclins and cyclin E have been described previously (32), and the cdk2 expression vector was constructed in a similar manner. Vectors expressing cdk4, cdk4NFG, cdk6, and cdk6NFG in CMVNeoBam3 were kindly provided by Sander van den Heuvel (82). Immunoprecipitations and immunoblotting. Cells were lysed in situ with 1 ml of ELB (50 mM N-2-hydroxyethylpiperazine-N9-ethanesulfonic acid [HEPES; pH 7.2], 250 mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40, 1 mM dithiothreitol, 1 mg of aprotinin [Boehringer Mannheim] per ml, 1 mg of leupeptin [Boehringer Mannheim] per ml, 100 mg of Pefabloc SC (AEBSF) [Boehringer Mannheim] per ml, 4 mM sodium orthovanadate, 2 mM sodium PPi) per 10-cm dish either directly or following labeling for 3 h with 500 mCi of Tran35S-Label (ICN) or Expre35S35S (NEN) in 3 ml of methionine-free medium with 10% dialyzed fetal bovine serum (the cells were incubated in methionine-free medium containing 10% dialyzed fetal bovine serum for 30 min before being labeled). Protein concentrations in the cell lysates were determined by the Bio-Rad protein assay. For pRb immunoprecipitations, lysates containing 200 mg of protein were subjected to immunoprecipitation with a cocktail containing 100 ml of tissue culture supernatant from the monoclonal antibody 21C9 raised against residues 248 to 262 of human pRb (87); 7 mg of purified monoclonal antibody AF11, recognizing the “B box” of pRb (kindly provided by Oncogene Science); and 1 mg of monoclonal antibody 245 (Pharmingen). After the mixture had been incubated at 48C for 1 h, rabbit anti-mouse immunoglobulin G (Cappel) and protein A-agarose (Bio-Rad) were added. Incubation was continued for an additional 1 h at 48C with continuous rocking prior to washing of the complexes with ELB. Protein A-bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5% polyacrylamide) and transferred to polyvinylidene difluoride membrane (Millipore) by semidry electroblotting for immunoblotting analysis. The filter was blocked with 5% nonfat dry milk in PBS for 60 to 90 min, rinsed several times with TNET (10 mM Tris [pH 7.5], 2.5 mM EDTA, 50 mM NaCl, 0.1% Tween 20), and subsequently incubated overnight at room temperature with anti-pRb monoclonal antibody 245 diluted to 10 mg/ml in TNET. After three washes with nonfat dry milk-PBS and TNET rinses, bound antibody was detected by a 1-h, room temperature incubation with peroxidaseconjugated donkey anti-mouse antibodies (Jackson Immunoresearch) at approximately 0.3 mg/ml in TNET. After several washes with TNET, peroxidase activity was detected with enhanced chemiluminescence reagents (Amersham). Immunoblotting of other antigens was performed similarly, except that 30 to 50 mg of protein was directly subjected to SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schuell). Purified antibodies were used at 20 to 100 ng/ml in TNET, and whole rabbit sera were diluted 1:1,000 to 1:16,000 in TNET as was determined to be optimal. Rabbit antibodies used in immunoblotting were detected with peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Jackson Immunoresearch) diluted to 0.2 mg/ml in TNET. p21, cyclins, and cdks were immunoprecipitated from unlabeled cell extracts with 200 mg of total protein and 0.5 to 1.5 mg of purified antibody or 1 to 3 ml of whole antiserum. Immunecomplexes were bound to protein A-Sepharose (Pharmacia) and washed four times with ELB plus 0.1% bovine serum albumin (BSA) prior to use in histone H1 kinase assays or SDS-PAGE. For immunoprecipitations of labeled lysates, 0.5 to 1.5 mg of purified antibody or 1 to 3 ml of whole serum was added per ml of lysate (derived from one subconfluent 10-cm dish of A1-5 cells or transfected subclone). Antibody-antigen complexes were collected on protein A-agarose and washed as above prior to separation on SDS-PAGE gels. The gels were fixed in 30% methanol–10% acetic acid, fluorographed by soaking in 15% 2,5-diphenyloxazole (PPO) in acetic acid for 10 min, rinsed in water, and dried. Dried gels were exposed to Kodak XAR5 film for 12 to 72 h at 2808C. Antibodies. For anti-cyclin D1/D2 (C-17 [Santa Cruz], purified anti-C-terminal peptide rabbit polyclonal), original lots of this antibody cross-reacted strongly with cyclin D1, contrary to information supplied by Santa Cruz stating specificity for cyclin D2. Additional immunoprecipitations with cyclin D1-specific monoclonal antibodies HD11 (a gift of E. Lees and E. Harlow) and 72-13G (Santa Cruz) suggest that the predominant D cyclin in these fibroblasts is cyclin D1. For anti-cyclin E, immunoblots, immunoprecipitations, and kinase assays were performed with a polyclonal rabbit-anti-cyclin E raised against the human protein and kindly provided by J. Roberts. For anti-cdk2, whole rabbit antiserum was provided by M. Meyerson and E. Harlow. A purified anti-C-terminal peptide rabbit antiserum, M-2 (Santa Cruz), was also used. For anti-cdk4, whole rabbit antiserum R7 (51) was provided by C. Sherr. The anti-C-terminal peptide antiserum C-22 was from Santa Cruz. For anti-cdk6, anti-C-terminal peptide antiserum C-21 from Santa Cruz was used. For MO-15, a whole rabbit antiserum was kindly provided by T. Ma¨kela ¨. For p21, a whole rabbit antiserum (15431E), raised against full-length human p21, was purchased from Santa Cruz; monoclonal antibody CP36 (96) was kindly provided by B. Dynlacht. For p27kip1, the

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INHIBITION OF p53 FUNCTION BY cdks

anti-mouse p27kip1 monoclonal antibody (K25020) was purchased from Transduction Laboratories. Immunofluorescence. (i) Rb extraction. A1-5 cells plated on glass coverslips were incubated at 32.58C for 36 h and extracted immediately or first refed and shifted to 398C for various times before extraction. To extract pRb, coverslips were rinsed with PBS and incubated in low-salt cracking buffer (10 mM HEPESKOH [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 1 mM dithiothreitol, 1 mg of aprotinin per ml, 1 mg of leupeptin per ml, 100 mg of Pefabloc SC per ml, 4 mM sodium orthovanadate, 2 mM sodium PPi) for 10 min with gentle rocking on ice (56). The coverslips were then directly incubated in 100% methanol at 2208C for 5 min followed by 100% acetone at 2208C for 2 min. After being dried at room temperature, the cells were rehydrated in 0.1% BSA in PBS (BSA-PBS) for 5 min and then incubated at 378C for 60 min with 12.5 mg of anti-pRb monoclonal antibody 245 per ml in BSA-PBS. After three washes in BSA-PBS, the cells were incubated with 1 mg of rabbit anti-mouse IgG per ml in BSA-PBS for 20 min at 378C, washed three times, and incubated at 378C for 30 min with 20 mg of rhodaminated donkey anti-mouse immunoglobulin G (Jackson Immunoresearch) per ml in BSA-PBS containing bisbenzimide as a nuclear counterstain. After three washes in BSA-PBS, the coverslips were briefly rinsed in water and mounted with Fluoromount G (Fisher). The fraction of pRbpositive nuclei was determined by counting the number of red-fluorescing nuclei and dividing by the total number of nuclei counted (at least 100 per coverslip) under UV illumination. (ii) BrdU incorporation. A1-5 cells were plated on coverslips in a 10-cm dish and incubated under cell cycle recovery conditions as described above. Bromodeoxyuridine (BrdU; 20 ml) labeling reagent (Boehringer Mannheim) was added to each plate (containing 10 ml of growth medium) at various times after the temperature shift, and the coverslips were removed after 1 h. The coverslips were rinsed with PBS and fixed at 2208C in 70% ethanol–50 mM glycine (pH 2.0) for 20 to 30 min. Incorporated BrdU was detected with antibody supplied in the 5-Bromo-29-deoxy-uridine labeling and detection kit II (Boehringer Mannheim). After three washes in BSA-PBS, the coverslips were incubated for 45 min at 378C with 20 mg of rhodaminated donkey anti-mouse immunoglobulin G (Jackson Immunoresearch) per ml containing bisbenzimide. The fraction of red-fluorescing, BrdU-positive cells was determined in fields containing at least 100 nuclei. Enzyme assays. (i) Kinase assays. Immunecomplexes were collected overnight at 48C with 30 ml of protein A-agarose from equivalent fractions of ELB cell lysates (50 to 200 mg), as described above. Antigen-antibody complexes were washed four times with ELB (no dithiothreitol or protease inhibitors) and twice with 50 mM Tris (pH 7.4)–10 mM MgCl2. After the final wash, 25 ml of the reaction mix (50 mM Tris [pH 7.4], 10 mM MgCl2, 50 mM ATP, 0.5 mCi of [g-32P]ATP, 1 mM dithiothreitol, and 2 mg of substrate, either histone H1 [Boehringer Mannheim] or GST-cdk2NFG [gift of T. Ma¨kela ¨]) was added to each sample, and this was followed by 20 to 30 min of incubation at 378C. The reactions were stopped by the addition of 25 ml of 23 SDS sample buffer, boiled, and separated by SDS-PAGE (12% polyacrylamide). Prior to autoradiography for 1 to 3 h, the gels were stained with 0.25% Coomassie brilliant blue R-250 in 50% methanol–10% acetic acid for 20 min at 568C, destained in 5% methanol– 7.5% acetic acid, and dried. (ii) b-galactosidase assays. A1-5 cells were transfected with a cytomegalovirus-driven b-galactosidase expression construct and the p53 reporter construct 6FSVCAT (55) at 378C as described above. After being rinsed, one plate was shifted to 32.58C and the other was maintained at 378C. All subcloned, transfection-derived cell lines were transfected directly at 32.58C. Extracts of transfected A1-5 cells and subclones were prepared 48 h after transfection. The cell pellets were resuspended in 0.25 M Tris-HCl (pH 7.6) and lysed by three cycles of alternate freezing (2808C) and thawing (378C). A 30-ml sample of cell extract was incubated with 3 ml of 1003 Mg solution (0.1 M MgCl2, 4.5 M b-mercaptoethanol), 66 ml of 13 ONPG (o-nitrophenyl-b-D-galactopyranoside; 13 is 4 mg of ONPG per ml in 0.1 M sodium phosphate [pH 7.5]), and 201 ml of 0.1 M sodium phosphate (pH 7.5) at 378C for 45 min. The reactions were stopped by adding 0.5 ml of 1 M sodium bicarbonate, and the optical density at 420 nm (OD420) was read. The following is the equation to calculate the b-galactosidase activity: (OD420/0.0045)/[reaction time (minutes) 3 volume (milliliters)]. (iii) Chloramphenicol acetyltransferase (CAT) assays. Transfected cell lysates with equivalent amounts of b-galactosidase activity were incubated with 0.1 mCi of [14C]chloramphenicol (100 mCi/mmol, ICN) and 4 mM acetyl coenzyme A in 1 M Tris-HCl (pH 7.6) at 378C for 1.5 h. The reaction mixtures were extracted with 0.9 ml of ethyl acetate and dried under vacuum at room temperature for 45 min. The samples were then resuspended in 20 ml of ethyl acetate, loaded onto silica gel channeled thin-layer chromatography plates (Baker Si250PA19C; purchased from VWR Scientific), and chromatographed in 5% methanol in chloroform. The plates were air dried and exposed to Kodak XAR5 film. Quantitation of radiolabeled, acetylated chloramphenicol was performed with a Bio-Rad phosphoimager.

RESULTS Cell cycle reentry in temperature-shifted A1-5 cells. The rat fibroblast cell line A1-5 was established by transformation with activated Ha-ras and a temperature-sensitive allele of murine

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p53, val135 (20). These cells are fully transformed at 378C (nonpermissive temperature) and undergo a reversible G1 growth arrest upon incubation at 32.58C (50). To ascertain if this cell cycle regulation retains characteristics of serum-stimulated normal cells and to determine the role of p21 in this process, the phosphorylation state of the retinoblastoma protein, pRb, was monitored during cell cycle recovery of A1-5 cells shifted to 398C from 32.58C. If p53 causes growth arrest in part by preventing G1 cyclin-dependent kinase activity, the retinoblastoma protein should be found in a hypophosphorylated, active state. Consistent with this, immunoblots of immunoprecipitated pRb from arrested cells demonstrate that the majority of pRb migrates as a distinct, fast-migrating band typical of hypophosphorylated pRb. Within 5 h after the temperature shift, slowly migrating, hyperphosphorylated pRb is observed and increases in abundance during cell cycle recovery to become the predominant species 9 to 11 h after the shift (Fig. 1B). These immunoblot data correspond well to the ability of pRb to be extracted by detergent-containing buffers, a measure of the inactivation of pRb by phosphorylation (56). While the majority of p53-arrested A1-5 cells were found to contain unextractable pRb, an increase in pRb extractability was observe 3 h after the temperature shift, and this property of pRb increased during cell cycle recovery (Fig. 1A). In normal human fibroblasts, phosphorylation and inactivation of pRb precedes S-phase entry by several hours (8). By measuring tritiated-thymidine (not shown) and BrdU incorporation (Fig. 1A) into recovering A1-5 cells, it was found that S phase began 2 to 4 h after pRb phosphorylation was first detectable. A1-5 cells thus mimic serum-stimulated, normal fibroblasts in their recovery from p53-mediated growth arrest. Inhibition of pRb phosphorylation and S-phase entry by p53 in A1-5 cells may result from inhibition of cyclin-dependent kinase activity. To directly measure the activity of cdk2, cdk2 immunoprecipitates were incubated with histone H1 and radiolabeled ATP. Subsequent electrophoresis of the histone demonstrated that cdk2 activity was lacking in p53-arrested cells but appeared at 4 h after the temperature shift (Fig. 1C), concomitant with pRb phosphorylation. A temporally similar kinase activity was seen with cyclin E immunoprecipitates (Fig. 1D), consistent with cyclin E being the earliest activator of cdk2. Despite the lack of cyclin E/cdk2 activity in extracts of growth-arrested A1-5 cells, both cyclin E and cdk2 are easily detectable by immunoblot analysis in these extracts and do not increase significantly in abundance as the cells reenter the cell cycle (Fig. 1D). Similarly, cyclin D1 and cdk4 are constitutively expressed in recovering A1-5 cells, as are low levels of cyclins D3 and cdk6 (data not shown; see below). Thus, the function of cyclin E/cdk2 but not the synthesis of the individual components is regulated in a p53-dependent fashion in A1-5 cells. Such a regulation of cdk2 activity could be due to a lack of Thr-160 phosphorylation of cdk2. Phosphorylation of cdk2 at this site by cdk-activating kinase is thought to be a prerequisite to cdk2 activity (58). However, this phosphorylation step is not likely to be limiting in p53-arrested A1-5 cells for two reasons. First, the predominant species of cdk2 identified by immunoblotting migrates at 32 kDa, consistent with the presence of phosphorylation at Thr-160. Upon long exposure of such immunoblots, a minor 34-kDa band appears, and the ratio of these two bands remains unchanged as the cells recover from growth arrest (data not shown). Second, cdk-activating kinase activity on synthetic cdk2 is easily detected in growth-arrested A1-5 cells and does not change with restoration of proliferation (Fig. 1E). Thus, regulation of cdk2 activity in A1-5 cells does not occur at the level of subunit synthesis or cdk-activat-

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FIG. 2. Time course of p21 and p27kip1 CKI expression and association with cdk2 in A1-5 cells released from p53-induced G1 block. A1-5 cells, growtharrested after 36 h of incubation at 32.58C, were harvested at various times following release from G1 block by a temperature shift to 398C. Protein extracts separated by SDS-PAGE were immunoblotted with antibodies to p21 or p27kip1, either directly or following immunoprecipitation with anti-cdk2 antiserum. For reference, the top panel shows anti-cdk2 immunoprecipitates from identical cell lysates that were assayed for associated histone H1 kinase activity as described in Fig. 1.

ing kinase activation and is therefore consistent with the activity of a p53-regulated inhibitor such as p21. To determine if the presence of p21 could account for the timing of cdk2 activity in A1-5 cells, lysates of cells recovering from growth arrest after the temperature shift from 32.5 to 398C were immunoprecipitated with anti-cdk2 antibodies or run directly on SDS-PAGE gels. Analysis of cdk2-associated H1 kinase activity demonstrated activation of cdk2 at 4 to 5 h after the temperature upshift, similar to the result shown in Fig. 1C. Upon direct immunoblotting of lysates with anti-p21 antibodies, p21 was observed in A1-5 cells incubated at 32.58C but not in those incubated at 378C, as expected (Fig. 2). Upon release from growth arrest, the levels of p21 were found to drop with increasing time at 398C, presumably as a result of loss of p53 transcriptional activity and lability of the p21 mRNA and protein. Consistent with activation of cdk2 at 4 to 5 h after temperature upshift, the total amount of p21 and that

FIG. 1. Cell cycle reentry in temperature-shifted A1-5 cells. A1-5 cells were incubated at 32.58C for approximately 36 h to induce the expression of wild-type p53 and G1 growth arrest. The cells were subsequently shifted to 398C to inactivate p53 and allow progression into S phase. The indicated times refer to hours at 398C after temperature upshift. 378C extracts are from asynchronous, proliferative cultures. (A) S-phase entry was monitored by determining the fraction of cells capable of incorporating BrdU. Parallel coverslips were used to monitor phosphorylation of pRb in situ by subjecting the cells to extraction with lowsalt, detergent-containing buffers followed by indirect immunofluorescence with anti-pRb antibodies. Cells lacking pRb fluorescence following this treatment

are those in which pRb has been phosphorylated and inactivated for nuclear tethering. (B) Immunoblot of pRb immunoprecipitated from extracts of cells arrested at 32.58C (328) or shifted to 398C for the indicated times. (C) Extracts were subjected to immunoprecipitation with anti-cdk2 antibodies followed by incubation with histone H1 and radiolabeled ATP. Phosphorylated histone H1 separated by SDS-PAGE is shown. (D) Cell extracts were either subjected to immunoprecipitation with anti-cyclin E antibodies and assayed for histone H1 kinase activity as in panel C or directly subjected to SDS-PAGE followed by immunoblot analysis with anti-cyclin E or anti-cdk2 antibodies. (E) To measure cdk-activating kinase activity, extracts were immunoprecipitated with anti-MO15 antibodies and incubated with GSTcdk2 and radiolabeled ATP.

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TABLE 1. Colony formation in A1-5 cells Mean no. of colonies (fold increase)b in:

plasmida

Vector cdk2 cdk4 cdk4NFG cdk6 cdk6NFG T antigen Cyclin D1 Cyclin D2 Cyclin D3 Cyclin E

Mean fold increase Expt 1

Expt 2

Expt 3

Expt 4

2 (1.0) NDc 15 (7.5) 13 (6.5) 23 (11.5) 12 (6.0) 17 (8.5) 3 (1.5) ND ND ND

4 (1.0) ND 16 (4.0) 10 (2.5) 16 (4.0) 14 (3.5) 4 (1.0) ND ND ND ND

5 (1.0) 5 (1.0) 17 (3.4) 12 (2.4) 25 (5.0) 9 (1.8) 16 (3.2) 8 (1.6) 11 (2.2) 10 (2.0) 3 (0.7)

3 (1.0) 1 (0.3) 10 (3.3) 14 (4.7) 8 (2.7) 14 (4.7) 4 (1.3) 2 (0.7) 3 (1.0) 2 (0.7) ND

1.0 0.7 4.5 4.0 5.8 4.0 3.5 1.3 1.6 1.3 0.7

a

Vectors and expression constructs are described in Materials and Methods. Colonies were stained and counted after puromycin selection at 32.58C as described in Materials and Methods. Numbers represent the mean for three plates per transfection in each experiment. Fold increase 5 mean number of colonies produced by test plasmid/mean number of colonies in vector control. c ND, not determined. b

associated with cdk2 declined significantly in these cells. In contrast, reanalysis of the same filters with antibodies directed against p27kip1, a p21-related CKI (67, 68, 80), demonstrated that this CKI does not decline either in steady-state amount or in association with cdk2 (Fig. 2). These results support a role for loss of p21 but not p27kip1 in cdk2 activation upon recovery from p53-induced G1 growth arrest. Restoration of proliferation of A1-5 cells by introduction of exogenous cdks. The status of pRb, cyclins, and cdks in A1-5 cells incubated at 32.58C suggests that expression of p21 plays a major role in the G1 arrest caused by p53. Because this CKI is thought to act stoichiometrically to inhibit cdk function, we thought it possible that overexpression of cyclins or cdks would restore kinase activity and pRb phosphorylation in A1-5 cells at 32.58C. To test this hypothesis, plasmids encoding various cdks or cyclins were introduced into A1-5 cells by calcium phosphate-mediated transfection. SV40 large T antigen, which can restore proliferation of similar cells (52, 69), was used as a positive control and the empty parent vectors were used as negative controls to score for variants which could proliferate without the introduction of an exogenous gene. In all cases, a puromycin resistance vector (pBabepuro) was cointroduced to allow selection of transfected cells at the p53-permissive temperature of 32.58C. As shown in Table 1, introduction of the parent vectors and pBabepuro resulted in the formation of a small number of potential revertant colonies, most of which remained small, 2 to 3 weeks after transfection. As expected, the introduction of SV40 large T antigen increased the number of proliferating, drug-resistant colonies, and many of these were larger than those produced by the control plasmids. When such large colonies (.5 mm in diameter) were scored, T antigen produced an average number of colonies 3.5-fold above that seen with negative control plasmids. When plasmids encoding cdk2, cyclin E, or D-type cyclins were introduced into A1-5 cells, little effect was seen. However, the introduction of cdk4- or cdk6expressing vectors consistently produced four- to sixfold more large, proliferating colonies than were produced in control transfections, similar to the effect of T antigen. We thought it possible that this effect was due to a pleiotropic effect of cdk4 and cdk6 on a variety of substrates and perhaps on p53 itself. To test if such potential kinase activity was necessary to allow colony formation in A1-5 cells at 32.58C, nonfunctional versions of cdk4 and cdk6, designated cdk4NFG and cdk6NFG (82), respectively, were introduced. As shown in Table 1, these

nonfunctional kinase subunits, while reduced in activity, were also able to restore proliferation of A1-5 cells at the permissive temperature. To demonstrate that the colonies produced by the transfections described above had truly escaped p53-mediated growth arrest, cell lines were established from each type of transfection. Such cell lines were easily expanded from ring-cloned colonies and proliferated with doubling times of 24 to 36 h (data not shown). Immunoblots were performed on lysates of the cells to demonstrate overexpression of the exogenous cdk. As shown in Fig. 3A, cdk4 is easily detectable in parental A1-5 cells at 32.58C. The abundance of cdk4 does not change dramatically at 378C, but a more slowly migrating band appears, perhaps indicating a proliferation-related modification of cdk4

FIG. 3. Immunoblot analysis of cdk4 and cdk6 expression in A1-5 cells and transfected subclones. Lysates of parental A1-5 cells incubated at 37 or 32.58C and representative subclones derived from transfections with SV40 large T antigen, cdk4, cdk4NFG, cdk6, or cdk6NFG were separated by SDS-PAGE and subjected to immunoblot analysis with anti-cdk4 antiserum (A) or anti-cdk6 antiserum (B). (B) Two different chemiluminescent exposures of the same immunoblot for comparison of endogenous cdk6 levels. All subclones were derived and maintained at 32.58C.

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(40). Expression of T antigen in A1-5 cells appears to increase the expression of both cdk4 species somewhat. Consistent with a role for ectopic expression of cdk4 in allowing proliferation of A1-5 cells at 32.58C, each cell line derived from cdk4 or cdk4NFG transfections greatly overexpressed this protein (Fig. 3A). In the case of cdk6, parental A1-5 cells contained low levels of endogenous cdk6, requiring long exposures for detection. T antigen had no obvious effect on this expression, but each cell line derived from a cdk6 or cdk6NFG transfection contained a large amount of the respective protein (Fig. 3B). Thus, cells derived from colonies formed by introduction of cdk4/4NFG or cdk6/6NFG can proliferate continuously at 32.58C, the permissive temperature for p53 function, and display high levels of the relevant exogenous kinase, supporting their role in circumventing p53-mediated growth arrest. Functional state of p53 in cdk4/6-rescued A1-5 cells. The overexpression of cdk4 and cdk6 described above may allow restoration of cell cycle progression in A1-5 cells by interfering with the function of p53, thus preventing p53 from exerting a growth-suppressive effect. Alternatively, such ectopic expression of cdks may leave p53 functionally intact but may interfere with downstream effectors of p53 function, such as p21. To address this issue, we have examined the structure and localization of p53 as well as its function as a transcriptional activator in A1-5 cell subclones overexpressing the various cdks. The structure and localization of murine p53 can be examined by indirect immunofluorescence with the monoclonal antibody PAb246 (93). Many mutant forms of p53 lose reactivity with this antibody (see, for example, references 20 and 30), including the Val-135 mutant expressed at 37 to 398C. However, in A1-5 and similar cells, loss of this epitope is not complete and a fraction of the p53 remains PAb246 reactive (50). Nevertheless, wild-type p53 activity is prevented, probably by association with structurally mutant p53 (41), which in the case of the Val-135 mutant also serves to localize the protein to the cytoplasm (22, 50). Indirect immunofluorescence with PAb246 confirmed that the mutant p53 is cytoplasmic in A1-5 cells at 378C and nuclear at 32.58C, as previously reported. Consistent with retention of conformationally wildtype p53, all T-antigen and rescued subclones also contained PAb246-reactive nuclear p53 (data not shown). Furthermore, immunoblot analysis of p53 from lysates of parental A1-5 cells and cdk-rescued subclones showed no difference in p53 expression when the panspecific antibody PAb241 was used (data not shown). Thus, the conformation and localization of p53 in cdk-rescued A1-5 cells proliferating at 32.58C are indistinguishable from those of p53 in the parental cells arrested at 32.58C. This implies that p53 remains in a functionally wildtype state in these cell clones, although a selection for mutations that do not alter the structure or localization of p53 cannot be ruled out. However, the appearance of such mutations in all rescued cell clones seems unlikely; furthermore, p53 remains transcriptionally active in these cells, as shown below. To demonstrate that p53 can act as a transcriptional activator in cdk-rescued A1-5 cell clones, a trait lost by tumorderived mutant p53 proteins (15, 21, 41, 66), we first tested the activity of a reporter construct dependent on p53 activity for expression of the CAT gene. This construct, 6FSVCAT, contains six copies of the consensus p53-binding site upstream of the CAT cDNA (55). As shown in Fig. 4A, 6FSVCAT gives rise to very little CAT activity upon introduction into A1-5 cells maintained at 378C but produces significant activity when transfected cells are shifted to 32.58C. Similarly, cdk-rescued A1-5 cell clones all showed easily detectable CAT expression at 32.58C. This activity was generally similar to that in the parental cells when normalized to the expression of a cytomeg-

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FIG. 4. p53 function in A1-5 cells and transfected subclones. (A) Parental A1-5 cells maintained at 378C were transfected with 6FSV-CAT (55) containing a synthetic p53-dependent promoter. After transfection, half the cells were maintained at 378C and the other half were shifted to 32.58C. In parallel, A1-5 cell subclones expressing cdk4, cdk4NFG, cdk6, or cdk6NFG were transfected at 32.58C. All transfections contained a cytomegalovirus-driven b-galactosidase construct used as an internal control for transfection efficiency. The ratio of CAT activity to b-galactosidase activity is presented below the autoradiogram with the value for A1-5 cells at 32.58C arbitrarily set to 100. (B) Lysates of asynchronously proliferating WI38 human diploid fibroblasts, A1-5 cells proliferating at 378C or growth arrested at 32.58C, and A1-5 cell subclones expressing cdk4, cdk4NFG, cdk6, or cdk6NFG at 32.58C were subjected to SDS-PAGE. Proteins transferred to nitrocellulose were subsequently subjected to immunoblot analysis with antip21 antibodies. The migrations of human (h) and rat (r) p21 are indicated.

alovirus enhancer-driven b-galactosidase internal control plasmid (Fig. 4A). Although the expression of CAT activity from the p53-dependent construct 6FSVCAT supports the contention that p53 is functionally wild type in cdk-rescued A1-5 cells, it remains possible that the reiteration of consensus p53 DNA binding sites masks a loss of activity on “true,” chromosomal p53dependent promoter elements (66). To demonstrate that a known p53-responsive gene is expressed in cdk-rescued A1-5 cells, the levels of p21 mRNA and protein were examined. Both Northern blot (data not shown) and immunoblot analysis of p21 expression (Fig. 4B) show that each cell line examined expresses p21 mRNA and protein, but such expression is not seen in A1-5 cells proliferating at 378C, as expected from the lack of p53 function. cdk-rescued A1-5 cells therefore retain the properties of wild-type p53 expressed at 32.58C in that the protein is conformationally wild type, localizes to the nucleus, and displays transcriptional transactivation from an exogenous, synthetic promoter as well as an endogenous one. Thus, cdk4 and cdk6 and their nonfunctional counterparts apparently promote proliferation of A1-5 cells not by interfering with p53 function but, rather, by acting downstream of p53 to prevent its growth-suppressive properties. Cell cycle proteins in cdk-rescued A1-5 cells. The observation that cdk-rescued A1-5 cells contain functional p53 and continue to express p21 suggests that such cells are no longer susceptible to the growth-inhibitory function of p21. Because p21 acts stoichiometrically to inhibit cdk activity and because cdk4 and cdk6 can promote A1-5 cell proliferation by an apparently noncatalytic mechanism, we considered it likely that excess cdk subunits could act by titrating p21 away from en-

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dogenous cdks, resulting in their activation. Indeed, such a mechanism has been observed to mediate p27kip1 function in vitro (67) and may play a role in transforming growth factor b regulation of cdk2 (68, 70, 80). In the case of cdk-rescued A1-5 cells, p21 might move from complexes with latent cyclin E-cdk2 and be found preferentially associated with the overexpressed cdk4 or cdk6 subunits. To establish which cyclin-cdk complexes are targeted by p21 in untransfected A1-5 cells, immunoprecipitations were performed with metabolically labeled cell lysates, using cyclin, cdk, and p21-specific antibodies. As shown in Fig. 5A, a band comigrating with rat p21 can be detected in both cdk2 and cyclin D immunoprecipitates of A1-5 cell lysates derived from cells incubated at 32.58C. Similar immunoprecipitations with antibodies specific to each of the D cyclins demonstrated that cyclin D1 is the predominant D-type cyclin in A1-5 cells (data not shown). Surprisingly, coimmunoprecipitation of p21 with a cdk4-specific antibody is difficult to detect in 32.58C A1-5 cell lysates, but a band comigrating with cdk4 coimmunoprecipitates with both cyclin D and p21. Thus, a complex of cyclin D1, cdk4, and p21 is likely to be present in these cells, but p21containing complexes may be poorly recognized by the cdk4 antibody used in these experiments. Consistent with immunoblot data (Fig. 3), immunoprecipitation with cdk6-specific antibodies demonstrates little expression of endogenous cdk6, and no D cyclin or p21 is detected in these immunoprecipitates (Fig. 5A). Thus, the immunoprecipitation experiments shown here support a role for p21 in preventing the function of both cyclin D1-cdk4 and cyclin E-cdk2 in p53-arrested A1-5 cells. To test the hypothesis that cdk4/6 overexpression alters the partitioning of p21, immunoprecipitations similar to those described above were performed with labeled extracts derived from cdk4- or cdk4NFG-rescued cell lines. In these cells, p21 was still found in complex with cdk2 and cyclin D1 (Fig. 5B). Indeed, cdk2 and cyclin D immunoprecipitates are nearly indistinguishable from those derived from parental cell lysates at 32.58C (Fig. 5A). As expected, cdk4 synthesis is greatly elevated in these cells, but as with cdk4 in parental cells, no p21 is observed to coimmunoprecipitate. However, a protein comigrating with cdk4 coimmunoprecipitates with p21, suggesting that a complex of cyclin D1, cdk4, and p21 persists in cells overexpressing cdk4 or cdk4NFG. In contrast to the situation in cells overexpressing cdk4, immunoprecipitation of lysates derived from cells overexpressing cdk6 or cdk6NFG clearly showed the presence of a novel p21-containing complex. Immunoprecipitation with cdk6-specific antisera revealed high levels of the antigen in the transfected cells as expected and also resulted in the coimmunoprecipitation of p21 (Fig. 5C). However, as was the case in cdk4overexpressing cells, both cdk2 and cyclin D immunoprecipitates revealed that p21 remains associated with these proteins in cdk6-rescued cells. Thus, novel complexes of cdk6, p21, and probably cyclin D1 exist in cdk6-rescued cells but do not result in obvious release of p21 from cdk2. Interestingly, immunoprecipitation of cdk6 and cdk6NFG results in the apparent coimmunoprecipitation of other proteins, most notably those with molecular masses of approximately 11 and 15 kDa (Fig. 5C). These proteins are not directly recognized by cdk6 antisera in immunoblot experiments and cannot be reimmunoprecipitated by anti-cdk6 after denaturation as can cdk6 itself, supporting our conclusion that they represent associated cellular proteins (data not shown). Although the identity of these proteins has not been confirmed, their apparent size is consistent with known cdk interactors, such as p13suc1 and members of the p16ink4 CKI family (58). Thus, in addition to p21, cdk overexpression may target other cellular proteins which mod-


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FIG. 5. Expression of cell cycle proteins in growth-arrested and cdk-rescued A1-5 cells. (A) Parental A1-5 cells were incubated at 32.58C for 36 h and subsequently labeled with radioactive methionine for 3 h at 32.58C. Lysates of the labeled cells were subjected to immunoprecipitation with anti-cdk2 (k2), anticdk4 (k4), anti-cyclin D1 and D2 (D), anti-p21 (p21), and anti-cdk6 (k6) antibodies. The immunoprecipitated proteins were separated on 17.5% polyacrylamide gels and visualized by PPO fluorography. The migration of the primary antigen(s) is indicated by an asterisk in each lane. The arrow marks the position of p21, and the migration of molecular mass markers is indicated at the right of the figure in kilodaltons. (B and C) Radiolabeled lysates of representative rescued A1-5 cell subclones expressing cdk4, cdk4NFG, cdk6, or cdk6NFG and proliferating at 32.58C were subjected to immunoprecipitation as described above. The arrowheads indicate the migration of novel 11- and 15-kDa proteins coimmunoprecipitating with cdk6.

ulate cdk activity. The presence of these proteins in such immunoprecipitates emphasizes the potential of overexpressed cdks to alter the stoichiometry of multiple protein complexes with suspected involvement in cell cycle regulation.

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treatment alone is not capable of activating cdk2. Furthermore, whereas p21 immunodepletion removed most cdk2 from control 32.58C lysates, considerable levels of cdk2 remained in lysates derived from cells maintained at 378C and from rescued clones (Fig. 6), suggesting that cdk4/6 overexpression results in the generation of a pool of cdk2 not associated with p21 and containing the majority of cdk2-associated H1 kinase activity. DISCUSSION

FIG. 6. cdk2 activity and association with p21 in cdk4- and cdk6-rescued subclones proliferating at 32.58C. Equivalent amounts of cell extracts derived from parental A1-5 cells incubated at the indicated temperature or from representative subclones expressing cdk4, cdk4NFG, cdk6, or cdk6NFG were assayed directly or following p21 depletion by three sequential rounds of immunoprecipitation with anti-p21 antibody. p21 immunoblots of the lysates are shown in the top two panels, before and after p21 immunodepletion, analyzed either directly or following immunoprecipitations with anti-cdk2 antisera. Shown next are the analagous cdk2 immunoprecipitations assayed for associated histone H1 kinase activity. The last panel shows a cdk2 immunoblot of the direct lysates, also before and after p21 immunodepletion.

Despite the apparently unchanged association of cdk2 with p21 in cdk4/6-rescued A1-5 cell clones, we assumed that such cells must contain cdk2-associated kinase activity, since the function of this protein is necessary for proliferation. To confirm this, extracts derived from rescued cells were subjected to immunoprecipitation with anti-cdk2 antibodies and the precipitates used for histone H1 kinase assays. This experiment clearly demonstrated the presence of active cdk2 in these lysates despite complex formation with p21 (Fig. 6). cdk2 activity in the presence of p21 could result from an inability of associated p21 to inhibit cdk2 or could reflect the presence of at least two populations of cdk2, one of which is bound to p21 and inactive and one of which is a p21-free, active fraction. To distinguish between these possibilities, cell extracts were subjected to immunoprecipitation with a variety of anti-p21 antibodies. While the amount of histone H1 kinase activity present in such immunoprecipitates varied from antibody to antibody, no differences were seen in lysates derived from parental cells incubated at 32.58C with respect to those derived from rescued cells (data not shown). Thus, p21 immunoprecipitation experiments did not support the hypothesis that active cdk2 associated with p21 in the rescued cell clones. To investigate this issue further, cell extracts derived from control A1-5 cells at 32.5 or 378C or from rescued clones were cleared of p21 by three sequential rounds of anti-p21 immunoprecipitation. This treatment cleared most or all p21 from the lysate as judged by direct immunoblotting (Fig. 6). When these lysates were subjected to anti-cdk2 immunoprecipitation and histone H1 kinase assays, most of the cdk2 activity present in the lysates derived from rescued clones and uninhibited parental cells at 378C was retained despite p21 immunodepletion (Fig. 6). Importantly, this treatment did not generate cdk2 activity in the 32.58C cell lysate, suggesting that p21 antibody

A1-5 rat fibroblasts transformed by activated ras and a temperature-sensitive allele of murine p53 (Val-135) undergo growth arrest in G1 at the permissive temperature for p53 function (32.58C) (50). Relief from this growth arrest maintains some of the characteristics of cell cycle progression in normal fibroblasts. This is demonstrated by the fact that phosphorylation of pRb is first observed within 4 h after the shift to the nonpermissive temperature (398C) and is followed by Sphase entry 2 to 3 h later. Interestingly, there is little change in steady-state levels of the G1 cyclins and cdks, but the activity of cdk2 is strongly suppressed at 32.58C, reappearing concomitantly with pRb hyperphosphorylation and inactivation. Consistent with current models of p53 function in growth arrest, synthesis and destruction of p21/WAF1/CIP1 closely parallel the indicators of cell cycle progression described above. Thus, p21 is absent at 398C, when p53 is nonfunctional, but is readily detected at 32.58C. p21 levels diminish upon temperature upshift of growth-arrested A1-5 cells, disappearing from cdk2 and cyclin E immunoprecipitates in a manner consistent with kinase reactivation. These attributes of p21 in A1-5 cells make it an excellent candidate for an important, p53-regulated mediator of cdk activity, pRb phosphorylation, and S-phase entry. The present studies show that overexpression of either cdk4 or cdk6 can restore cdk2 activity in p21-expressing A1-5 cells, allowing them to grow continuously at 32.58C in the presence of functional p53. The ability of nonfunctional counterparts of cdk4 and cdk6 to also override p53 function shows clearly that kinase activity is dispensable for this stimulatory effect. Furthermore, expression of exogenous cdk4 or cdk6 in transfected A1-5 cells generates a p21-free cdk2 fraction that is responsible for most of the cdk2-associated kinase activity in these cell extracts. Indeed, in contrast to observations in other systems (26, 95), there appears to be little active cdk2 associated with p21. The generation of p21-free cdk2 caused by cdk4 or cdk6 could result from a reduction of the total amount of p21 or from sequestration of p21 away from cdk2. Although some cell lines show minor reductions in the steady state level of p21, such a decrease is not seen in all cell lines and may occur as a result, rather than a cause, of cdk4- and cdk6-stimulated proliferation. The most likely mechanism by which overexpressed cdk4 or cdk6 could act to circumvent p21 is by titrating p21 subunits away from cyclin E-cdk2 complexes, thereby reactivating their catalytic activity. Indeed, we observed that all cell clones derived from cdk6 transfections showed complexes between exogenous cdk6 and endogenous p21 (Fig. 5 and data not shown), and we also observed slight increases in the steadystate level of cdk4 bound to p21 in cdk4 transfectants (data not shown). In combination, these results suggest that cdk4 and cdk6 can overcome at least one of the inhibitory effects of p53 by titrating p21 away from cdk2. Interestingly, overexpression of cdk2 or cyclins had little effect in this assay. The failure of certain cdks and cyclins to rescue A1-5 cells could be due to the toxicity of the overexpressed subunits. However, none of the constructs used in

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these experiments caused significant colony reduction upon transfection and drug selection in A1-5 cells proliferating at 378C (data not shown). Alternatively, titration of p21 may require high levels of both a cdk and its cognate cyclin. Thus, while we observed elevated levels of cyclin D1 in A1-5 cells and rescued cell clones maintained at 32.58C (data not shown), possibly from transcriptional upregulation promoted by both activated ras and p53 (1, 6, 76), there may be insufficient cyclin E (or A) present to associate with a significant amount of exogenous cdk2. Furthermore, cdk4- and cdk6-mediated interference with functions of p21 other than cdk inhibition may also promote proliferation in A1-5 cells in a manner that cannot be achieved by elevated cyclin E-cdk2 expression alone. For example, p21 may inhibit replication because of its association with proliferating-cell nuclear antigen (5, 84); this association may also be reduced in cdk4- and cdk6-rescued A1-5 cells. While we have not determined the fraction of p21-associated proliferating cell nuclear antigen in A1-5 cells or cdkoverexpressing subclones, we believe that this effect could not allow cell cycle progression on its own, since cdk2 activity must be present for S-phase entry to occur (64, 81, 82). Finally, the apparent specificity of cdk4 and cdk6 in this assay may be due to a unique ability of these kinases to interfere directly with other components of cdk regulation. Several observations suggest that it is unlikely that the reactivation of cdk2 by p21 titration alone could be sufficient to eliminate the growth-suppressive effect of p53 entirely. For example, murine cells containing homozygous deletion of the p21 gene still respond partially to DNA-damaging treatments (3, 10). The transcriptional activation by p53 of GADD45 and the cyclin G gene or novel targets of p53 may promote a p21-independent G1 arrest in this case. Alternatively, or in addition, nontranscriptional effects of p53 may contribute to cell cycle arrest. An example of such an effect is provided by the ability of p53 to inhibit the translation of cdk4 in mink lung cells following treatment with transforming growth factor b (17, 18). This specific mechanism is unlikely to contribute to growth arrest in A1-5 cells, since cdk4 synthesis remains unchanged in the presence or absence of functional p53 (unpublished observations, but also see Fig. 3 and 5). If p53 is in fact pleiotropic in its ability to cause cell cycle arrest, how might cdk4- and cdk6-overexpressing A1-5 cells lose sensitivity to multiple inhibitory influences? One possibility is that other, unknown alterations exist in these transformed cells that render them insensitive to some of the growth-suppressive effects of p53. Indeed, activated Ha-ras is expressed in these cells and may contribute to p53 insensitivity (45). In keeping with this idea, disruption of the p21 gene in a human colon cancer cell line completely eliminates the radiation-induced G1 block (86), suggesting that these tumor-derived cells are less responsive to p53 than primary murine cells lacking only p21. A second explanation for the ability of cdk4 and cdk6 to circumvent p53 function is that these kinase subunits are themselves pleiotropic, even in a noncatalytic manner. Consistent with this possibility, immunoprecipitation of overexpressed cdk6 in the rescued cell lines results in coimmunoprecipitation of a number of other cellular proteins, including p27kip1 (data not shown) and proteins of 11 and 15 kDa. Preliminary evidence suggests that the smaller of these proteins is the cdk4 and cdk6 inhibitor p15ink4b (89a). This demonstrates that multiple inhibitors can be titrated by cdk6 overexpression but leaves the disposition of p15ink4b in question in cdk4-overexpressing cells, since complexes between cdk4 and p15ink4b have not been observed in these cells with available reagents. In any case, the INK4 family of proteins would not be expected to be targeted by overexpressed cdk2, leading to the possibility


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that p21 titration by cdk2-cyclin E complexes is not sufficient to promote proliferation in the presence of INK4-inactivated cdk4/6. The introduction into A1-5 cells of mutant cdk4/6 proteins that lack the ability to associate with p16ink4 (89), for example, will help determine which functions of cdk4 and cdk6 lead to p53 insensitivity. The results presented here support an oncogenic function of cdk4 and cdk6 that mediates a resistance to the growth-suppressive effects of p53 through titration of (at least) p21, one of several protein inhibitors of cdks. These cell cycle regulators thus join the ranks of known oncogenes such as adenovirus E1A, human papillomavirus E7, and myc that can prevent the growth-suppressive function of p53 even in the absence of physical interaction with p53 (28, 45, 52, 69, 83). Coupled with the ability of ectopic cdk4 expression to render mink lung cells insensitive to transforming growth factor b (18), this function of cdk4 and cdk6 underscores the potential role of cdk4 in human tumor formation. Indeed, such a role for cdk4 in the genesis of human tumors is supported by the observed amplification of cdk4 in sarcomas and other tumors (27, 42, 44, 72). In some cases, cdk4 amplifications are found in cells containing the hdm-2 amplification thought to inactivate p53 (42). In other cases, the hdm-2 gene itself is not overexpressed, leaving cdk4 as a potential target for this oncogenic amplification. Similarly, it is probable that hyperactive cdk4 (or cdk6) would be present in a subset of tumors with p16ink4 deletions (27) and, further, a p16ink4-resistant mutant form of cdk4 has been isolated from a human melanoma (89). Given these observations and our results described above, cdk4 and cdk6 deserve careful consideration as important human oncogenes able to interfere with both the p53 and pRb pathways. ACKNOWLEDGMENTS We thank S. van den Heuvel, B. Dynlacht, E. Harlow, J. Roberts, J. W. Harper, S. Elledge, C. Sherr, and T. Ma¨kela for generously providing reagents and advice. We are grateful to S. Dowdy, L.-H. Tsai, and K. Mu ¨nger for careful reading of the manuscript. This work was supported by grants from The Medical Foundation and the American Cancer Society (Massachusetts Division) to P.W.H. K.M.L. was supported by NIH grants T32-CA09031-18 and F32CA63802-01A1. P.W.H. is a Scholar for The Leukemia Society of America. REFERENCES 1. Albanese, C., J. Johnson, G. Watanabe, N. Eklund, D. Vu, A. Arnold, and R. G. Pestell. 1995. Transforming p21ras mutants and c-ETS-2 activate the cyclin D1 promoter through distinguishable regions. J. Biol. Chem. 270: 23589–23597. 2. Baker, S. J., S. Markowitz, E. R. Fearon, J. K. V. Wilson, and B. Vogelstein. 1990. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 249:912–918. 3. Brugarolas, J., C. Chandrasekaran, J. I. Gordon, D. Beach, T. Jacks, and G. J. Hannon. 1995. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature (London) 377:552–557. 4. Chen, C., and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745–2752. 5. Chen, J., P. K. Jackson, M. W. Kirschner, and A. Dutta. 1995. Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature (London) 374:386–388. 6. Chen, X. B., J. Bargonetti, and C. Prives. 1995. p53, through p21waf1/cip1, induces cyclin D1 synthesis. Cancer Res. 55:4257–4263. 7. Clarke, A. R., C. A. Purdie, D. J. Harrison, R. G. Morris, C. C. Bird, M. L. Hooper, and A. H. Wyllie. 1993. Thymocyte apoptosis induced by p53dependent and independent pathways. Nature (London) 362:849–852. 8. Cobrinik, D., S. F. Dowdy, P. W. Hinds, S. Mittnacht, and R. A. Weinberg. 1992. The retinoblastoma protein and the regulation of cell cycling. Trends Biochem. Sci. 17:312–315. 9. Debbas, M., and E. White. 1993. Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev. 7:546–554. 10. Deng, C., P. Zhang, J. W. Harper, S. J. Elledge, and P. Leder. 1995. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82:675–684.

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