p16INK4 mediates contact-inhibition of growth - Nature

Oncogene (1999) 18, 277 ± 281 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc

SHORT REPORT

p16INK4 mediates contact-inhibition of growth Raimund J Wieser, Dagmar Faust, Cornelia Dietrich and Franz Oesch Institut fuÈr Toxikologie, Obere Zahlbacherstrasse 67, D-55131 Mainz, Germany

Growth of non-transformed cells in vitro is regulated by density-dependent mechanisms via cell-cell contacts, leading to arrest in late G1-phase at con¯uency (contact-inhibition of growth). In the present study it is shown that this results from p16INK4-mediated dissociation of the complex cdk4-cyclin D1, which is responsible for the inactivation of the gate keeper of G1-S transition, the retinoblastoma protein pRb. As a consequence of the inactivation of cdk4, downstream the activation of cdk2 and hyperphosphorylation and thus inactivation of pRb was impaired. Direct evidence for the central role of p16INK4 in growth control comes from the observation that a competitive inhibitor of p16INK4 repressed contact inhibition of growth. These ®ndings provide an explanation for the high incidence of mutation or loss of INK4 in human tumours. Keywords: contact-inhibition; cdk4; p16INK4 The cell cycle is regulated by an ordered cascade of phosphorylation reactions by a speci®c family of serine-threonine kinases, the cyclin-dependent kinases (cdks) (Lees, 1995; Sherr, 1996), and speci®c check points ensure that the cell's proper cycle is tightly controlled. In order to determine at which point of the G1-phase cells are arrested by contact-dependent inhibition of growth, time course studies were performed. In FH109 cells, human embryonal diploid lung ®broblasts (Wieser et al., 1985), contactdependent inhibition of growth is exclusively mediated by the interaction of two cell membrane proteins on adjacent cells, i.e. by the glycoprotein, contactinhibin (Wieser et al., 1990), which binds to its receptor referred to as contactinhibin receptor (Gradl et al., 1995). Cell-cell contacts were imitated by the addition of glutaraldehyde-®xed cells (in which contactinhibin remains active, as its growth inhibitory activity is mediated exclusively by its N-glycans not a€ected by the ®xation process) to sparsely seeded ®broblasts in the presence of serum. In previous studies we have demonstrated that this system mimics physiological conditions concerning cell proliferation and differentiation which occur in con¯uent cultures (Wieser et al., 1985; Wieser and Oesch, 1986), thus enabling the study of early e€ects of cell-cell contact-dependent signals. FH109 cells were sparsely seeded and cultured for 24 h in DMEM/0.5% FCS to achieve partial synchronization. Control cells were stimulated with 10% FCS in DMEM whereas contact-inhibited cells were generated

Correspondence: RJ Wieser Received 13 January 1998; revised 6 July 1998; accepted 7 July 1998

by the addition of ®xed cells in DMEM/10% FCS prepared from con¯uent cultures (Wieser et al., 1985). These experiments revealed that when ®xed cells were added within the ®rst 8 h after serum-stimulation, proliferation was inhibited by 70 ± 80% as determined by [3H]-thymidine incorporation, which correlates with the reduction of growth rate achieved by seeding ®broblasts to con¯uency (Figure 1). The addition of ®xed cells 10 h after serum stimulation no longer resulted in any growth inhibition, indicating that contact-dependent inhibition of growth is limited to a window in the cell cycle corresponding to mid G1. Since cdk4-cyclin D1 is suggested to act in mid to late G1, regulation of cdk4-activity was examined. We ®rst studied protein levels of cdk4 and cyclin D1 by Western blot analysis. Neither cdk4 nor cyclin D protein levels were a€ected by treatment with ®xed cells (Figure 2a) indicating that reduced synthesis or increased degradation of either protein was not the reason for growth arrest. These results point to an important di€erence between growth-arrest achieved by contact-inhibition and serum-deprivation (Dietrich et al., 1997). In the latter case, synthesis of D-type cyclins is repressed, and consequently, levels of these labile proteins decline rapidly leading to a loss of cdk4activity.

Figure 1 Time course of inhibition of proliferation by ®xed cells of serum-stimulated FH109 cells. Cell culture Human diploid lung ®broblasts (FH109; Wieser et al., 1985) were cultured in Dulbecco's modi®ed Eagle's medium (DMEM) with 10% fetal calf serum (FCS; Roth) at 10% CO2. Proliferation assay 56103 cells were cultured in microtiter plates for 24 h in 100 ml of DMEM/0.5%FCS. Then 100 ml of DMEM/10% FCS were added and after the times indicated, ®xed cells were added. Fixation of cells with glutardialdehyde Con¯uent monolayers of FH109 cells were treated with trypsin, washed three times with phosphatebu€ered saline (PBS) and ®xed by dropwise adding of glutardialdehyde as described by Oesch et al. (1987). After a total of 24 h, cell proliferation was determined by measurement of [3H]thymidine-incorporation into DNA after a 30 min pulse with 0.25 mCi of [3H]thymidine (Rinderknecht and Weiler, 1983). (7)=cells which were cultured for 48 h in DMEM/0.5% FCS. Controls (=100%) received DMEM/10% FCS without ®xed cells

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The activity of cdk4 is strongly dependent on its association with cyclin D1. To check the association of cdk4 with its regulatory subunit cyclin D1, cell extracts were immunoprecipitated with anti-cyclinD1-antibodies followed by SDS ± PAGE and Western blotting with anti-cdk4-antibodies. It is shown (Figure 2b) that 12 h after the addition of ®xed cells cdk4 was not detectable in D1-immunoprecipitates. Accordingly, kinase activity of cyclin D1-immunoprecipitates measured with pRb fusion protein as substrate was reduced by 70% in immunoprecipitates from treated cells compared with untreated proliferating cells (Figure 2c). One protein with key regulatory function of cdk4activity is the speci®c inhibitor p16INK4 which leads to a

dissociation of cyclin D1 from the complex. Antip16INK4-immunoprecipitates of extracts prepared from cells cultured in the presence of ®xed cells probed by Western blotting with anti-cdk4-antibodies revealed a strong signal of cdk4. In contrast, in immunoprecipitates of proliferating cells only a faint signal was observed (Figure 2d). These results indicate that contact-inhibition of growth triggers association of p16INK4 with cdk4 thereby releasing cyclin D1 which results in inactivation of cdk4. Since protein levels of p16 were only increased about twofold in cell extracts (Figure 2d), association of p16INK4 with cdk4 is supposed to be regulated by further mechanisms, possibly by the regulation of an assembly factor as it c

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Figure 2 Analysis of cdk4 activation in FH109 cells cultured in the presence or absence of ®xed cells. (a) Protein levels of cdk4 and cyclin D1. Cells were cultured after the addition of ®xed cells in DMEM/10% FCS (+) or DMEM/10% FCS alone (7) for the times indicated and harvested for SDS ± PAGE (Laemmli, 1970), (7.5% acrylamide) followed by Western blot analysis with anticdk4 antibodies (0.1 mg/ml, Santa Cruz) and anti-cyclin D1 antibodies (0.1 mg/ml, Santa Cruz). Cell extracts Cells were lysed for 20 min at 48C in RIPA-bu€er (50 mM Tris/HCl pH 7.5, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, protease and phosphatase inhibitors 10 mM NaF, 1 mM Na3VO4, and the protease inhibitors aprotinin, soyabean trypsin inhibitor, PMSF, iodoacetamide, pepstatin, leupeptin). Proteins of the supernatant were precipitated (Wessel and FluÈgge, 1984) and solubilized in SDS-sample bu€er. Protein determination was performed according to (Smith et al., 1985). (b) Analysis of the association of cyclin D1 with cdk4. Cells were cultured after the addition of ®xed cells in DMEM/10% FCS (+) or DMEM/10% FCS alone (7) for the times indicated and harvested for immunoprecipitation with anti-cyclin D1 antibodies. Immmunoprecipitates were analysed by Western blot for the presence of cdk4. Immunoprecipitation FH109 cells were lysed in RIPA-bu€er as described above and immunoprecipitated for 2 ± 4 h at 48C with anti-cyclin D1 antibodies (10 mg, Santa Cruz) covalently coupled to protein A-Sepharose (12.5 mg, Sigma). The immunoprecipitate was washed three times with RIPA-bu€er and proteins eluted with 100 ml of diethylamine (100 mM, pH 11.5) for 10 min at room temperature. The supernatant was neutralized with HCl, the proteins precipitated and solubilized in SDS-sample bu€er. (c) Analysis of cdk4 activity. FH109 cells were cultured in the presence (+) or absence (7) of ®xed cells for 14 h and harvested for immunoprecipitation with anti-cyclin D1 antibodies. Kinase activity was measured after ®ve washing steps of the immunoprecipitates with kinase bu€er, using GST-pRb fusion-protein (1 mg, Santa Cruz) as substrate, ATP (30 mM), and [g-33P]ATP (25 mCi) in 50 ml of kinase bu€er (50 mM Tris/HCl, pH 7.4). The reaction mixture was incubated for 30 min at 378C and stopped by the addition of 50 ml of 26Laemmli sample bu€er. Proteins were precipitated, separated by SDS ± PAGE (12.5%) and phosphorylation of GST-pRB visualized by ¯uorography. pRbpp=phosphorylated GST-fusion protein. (d) Analysis of the association of p16INK4 with cdk4 (upper part) and the p16INK4 protein level (lower part) in FH109 cells cultured in the presence or absence of ®xed cells. Cells were cultured after the addition of ®xed cells in DMEM/10% FCS (+) or DMEM/10% FCS alone (7) for the times indicated and harvested for immunoprecipitation with anti-p16INK4 antibodies. Immunoprecipitates were analysed by Western blot for the presence of cdk4. Extracts from cells cultured with or without ®xed cells were analysed by Western blotting for the expression of p16INK4

p16INK4-mediated contact-inhibition of growth RJ Wieser et al

has been described vice versa for the association of cyclin D with cdk4 after growth factor stimulation (Matsushime et al., 1992). It is well known that cdk4-cyclinD is responsible for the hyperphosphorylation of the gate keeper of the G1S-phase transition, the retinoblastoma gene product pRb (Bartek et al., 1996). In its hypophosphorylated state it binds to a familiy of transcription factors, the E2Fs, hence inhibiting transcription. Hyperphosphorylation leads to its functional inactivation resulting in the loss of binding to E2F thus permitting entry into Sphase (DeCaprio et al., 1989; Mittnacht and Weinberg, 1991). From mid to late G1-phase, pRb is phosphorylated by the cyclin D-cdk4 complex resulting in induction of cyclin E expression which in association with cdk2 also contributes to pRb phosphorylation at G1/S transition (Sherr, 1994; Weinberg, 1995; Botz et al., 1996). Therefore, the activity of cdk2 and the degree of phosphorylation of pRb was studied. Figure 3a shows that 14 ± 16 h after the addition of ®xed cells the faster migrating form of cdk2 decreased. Since the faster

migrating form represents activated cdk2, phosphorylated at Thr 160 (Dulic et al., 1992; Gu et al., 1992), the results indicate that treatment with ®xed cells leads to inhibition of cdk2. This was con®rmed by demonstrating markedly reduced kinase activity of anti-cyclin E-immunoprecipitates, compared with the activity found in immunoprecipitates of untreated cells (Figure 3b). Impaired cdk2-activity in treated cells was also re¯ected by the phosphorylation state of pRb (Figure 3c). Since phosphorylation of pRb results in reduced anity for the nuclear compartment we fractionated cell extracts into nuclear and cytosolic compartments according to (Mittnacht and Weinberg, 1991). Compared with proliferating cells, in contactinhibited cells the slower migrating, hyperphosphorylated species representing inactivated pRb was strongly reduced in the cytosol, indicating that contactinhibition of growth by a€ecting both cdk4 and cdk2 kinase activities impairs hyperphosphorylation and thus inactivation of pRb. One of the factors which are known to regulate cdk2-activity is the small inhibitory protein p27KIP1.

a

b

c

Figure 3 Analysis of cdk2-activation and pRb-phosphorylation in FH109 cells cultured in the presence or absence of ®xed cells. (a) Protein levels of cdk2. Cells were cultured after the addition of ®xed cells (+) in DMEM/10% FCS or DMEM/10% FCS alone (7) for the times indicated and harvested for Western blot analysis with anti-cdk2 antibodies (0.1 mg/ml, Santa Cruz). cdk2*=cdk2 activated by phosphorylation at Thr 160. (b) Analysis of cdk2 activity. FH109 cells were cultured in the presence (+) or absence (7) of ®xed cells for 14 h and harvested for immunoprecipitation with anti-cyclin E-antibodies. Kinase activity was measured as described in Figure 2c, with histone H1 (1 mg, Sigma) as substrate. (c) Analysis of phosphorylation status of pRb in FH109 cells. FH109 cells were cultured in the presence (+) or absence (7) of ®xed cells for 14 h. For pRb-detection (Mittnacht and Weinberg, 1991) cells were extracted with TM-bu€er (10 mM Tris/HCl pH 7.4, 2 mM MgCl2, 5 mM KCl, 1 mM DTT, 1 mM NaH2P2O7, 10 mM NaF, 1 mM Na3VO4, and the protease inhibitors aprotinin, soybean trypsin inhibitor, PMSF, iodoacetamide, pepstatin, leupeptin) containing 0.1% Triton X-100. After separation of nuclei from cytosol by centrifugation at 400 g and SDS ± PAGE (7.5% acrylamide) separation, the cytosolic fractions were probed by Western blot for pRb. pRb=hypophosphorylated pRb, pRbppp=hyperphosphorylated pRb

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The inhibitor blocks cdk2-activity directly by binding to the complex and indirectly by inhibiting cdkactivating kinase which is responsible for the activating phosphorylation at Thr 160 (Polyak et al., 1994). p27 is present in cdk4-cyclinD complexes without a€ecting the activity of cdk4. When this complex dissociates, p27 is freed and now available for interacting with and thereby inhibiting cdk2. In agreement with ®ndings by others (Polyak et al., 1994; Ko€ et al., 1993; Hengst and Reed, 1996) we have previously shown that contact-inhibition resulted in accumulation of p27KIP1, leading to reduced kinase activity of cdk2 and therefore to decreased phosphorylation of pRb, indicating that the observed inhibition of cdk2 in this study resulted from the action of p27 (Dietrich et al., 1997). If p16INK4 was the critical molecule transmitting the growth-negative signals generated by cell-cell contacts, then impairing the function of p16 should lead to repression of contact-dependent inhibition of growth. Starting from the observation that: (i) a 20mer peptide of p16INK4 contained the entire information to bind cdk4, to inhibit pRb phosphorylation and hence to block entry into S-phase (Fahraeus et al., 1996), and that (ii) a mutated form of this peptide was still able to bind cdk4 while only slightly inhibiting cdk4-kinase activity (Yang et al., 1996), we used this mutated peptide to competitively inhibit interaction of endogenous p16INK4 with cdk4. It was shown that in cultures loaded with the mutated p16INK4 peptide nearly twofold higher cell numbers (Figure 4) as in control cultures were found. Concomitantly, criss-crossed growth and the emergence of many foci, both being characteristic phenotypes of cultures of transformed cells, were observed. A twofold increase in cell density is the maximal value which can be achieved in con¯uent cultures of FH109 ®broblasts by impairing contactinhibition of growth, as shown in previous studies (Wieser et al., 1990; Gradl et al., 1995). Although we have not tested the hypothesis, according to the morphology of the cells in these cultures and in agreement with similar observations described in literature, it is suggested that in overcrowded cultures apoptosis takes place (Yasaka et al., 1996). Our results are in agreement with several other observations: (i) Deletions and mutations involving INK4a occurs in many di€erent human tumours (Hall and Peters, 1996; Elledge et al., 1996; Sherr and Roberts, 1995); (ii) p16INK4-null mice develop spontaneously many di€erent tumours at an early age and are highly susceptible to treatment with carcinogens (Serrano et al., 1996); (iii) Fibroblasts derived from p16INK4-null mice show enhanced saturation-density (up to threefold over controls) (Serrano et al., 1996) indicating loss of contact-dependent inhibition of

Figure 4 E€ect of a competitive p16INK4-peptide on contactdependent inhibition of growth of FH109 cells. The competitive p16INK4 peptide 84DAAREGFLDTLVVLHRAWARC104 (consisting of aa 84 ± 103 of p16INK4 containing the mutation (bold letter) at position 101 with an exchange of W for G, plus a terminal cysteine residue) of p16INK4 was synthesized by the Proteinchemisches Labor of the University Mainz and introduced into FH109 cells by electroporation. Brie¯y, cells were trypsinized and resuspended at 2.56106 cells/ml in phosphate bu€ered saline containing 500 mM of the competitive p16INK4 peptide or control peptide (antennapedia peptide `penetratin', Appligene). Electroporation was performed with EPI 2500 (Fa Dr Fischer, Heidelberg, Germany) for 2 msec at 450 volt. Electroporated cells were transferred into DMEM/10%FCS and seeded at 56105 into 35 mm dishes. After 3 days in culture, cells were trypsinized and counted using a hemocytometer. Shown are the results of two independent experiments and represent the mean cell number of two dishes per experiment. +comp. peptide: +, cells loaded with the competitive peptide, 7, cells loaded with the control peptide

growth; (iv) By expression of exogenous p16INK4 in glioblastoma cells lacking endogenous p16INK4 contact inhibition of growth is restored (Higashi et al., 1997). In contrast, in cells derived from p27KIP1-null mice, contact-dependent inhibition of growth was not a€ected although p27KIP1 is upregulated in contactinhibited cells and blocks cdk2-activity (Nakayama et al., 1996). In the present study we show for the ®rst time that interaction of p16INK4 with cdk4 plays a crucial role in contact-dependent inhibition of growth, which might explain the frequent inactivation of this cdk-inhibitor in human tumours.

Acknowledgements This work was supported by grants of the Deutsche Forschungsgemeinschaft SFB 519/A6 and Thyssen Stiftung and is part of the PhD. thesis of FD.

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