RB Regulates the Stability and the Apoptotic Function of p53 via MDM2

Molecular Cell, Vol. 3, 181–193, February, 1999, Copyright 1999 by Cell Press

RB Regulates the Stability and the Apoptotic Function of p53 via MDM2 Jung-Kuang Hsieh,* Florence S. G. Chan,* Daniel J. O’Connor,* Sibylle Mittnacht,† Shan Zhong,* and Xin Lu‡ * Ludwig Institute for Cancer Research Imperial College School of Medicine St. Mary’s Campus, Norfolk Place London, W2 1PG † Institute for Cancer Research, Chester Beatty Fulham Road London SW3 6JB United Kingdom

Summary The binding of RB to MDM2 is shown to be essential for RB to overcome both the antiapoptotic function of MDM2 and the MDM2-dependent degradation of p53. The RB–MDM2 interaction does not prevent MDM2 from inhibiting p53-dependent transcription, but the RB–MDM2 complex still binds to p53. Since RB specifically rescues the apoptotic function but not the transcriptional activity of p53 from negative regulation by MDM2, transactivation by wild-type p53 is not required for the apoptotic function of p53. However, an RB– MDM2–p53 trimeric complex is active in p53-mediated transrepression. These data link directly the function of two tumor suppressor proteins and demonstrate a novel role of RB in regulating the apoptotic function of p53. Introduction Most human tumors have defects in either the p53 or RB pathway. Mutation or inactivation of both tumor suppressors is also found in many different types of human tumors (Weinberg, 1995; Sherr, 1996; Levine, 1997). The concomitant alterations in both p53 and RB often have further negative effects, resulting in increased tumor recurrence and decreased survival of patients, illustrating the cooperative effects of p53 and RB in suppressing human tumor growth (Xu et al., 1995; Cordon-Cardo et al., 1997). Both p53 and RB play important roles in controlling cell cycle progression and apoptosis. The identification of p21WAF1/CIP1 as a p53 target gene demonstrated that p53 can control the phosphorylation status of RB and regulate its activity (Ko and Prives, 1996). However, the ability of both tumor suppressors to interact with the same oncoprotein, MDM2, suggested that there may be another level of regulation between p53 and RB. The rescue of the embryonic lethality of MDM2 null mice in a p53 null background strongly argues that MDM2 is an important cellular inhibitor of p53 (Ko and Prives, 1996). MDM2 can bind to p53 at its transactivation domain and inhibit its transactivation activity. In ‡ To whom correspondence should be addressed (e-mail: x.lu@ ic.ac.uk).

addition, p53 can bind to the promoter region of MDM2 and activate its transcription, forming an autoregulation loop between the expression and function of p53 and MDM2 (Ko and Prives, 1996). Apart from being a transcriptional activator, p53 can also repress many cellular and viral promoters that do not contain p53-binding sites, including c-fos and SV40 large T antigen (Ko and Prives, 1996). Interestingly, the binding between MDM2 and p53 can negatively regulate the transrepression function of p53 (Chen et al., 1995). The MDM2–p53 interaction has also been shown to be able to inhibit p53induced apoptosis in some cells (Ko and Prives, 1996). Recent studies also demonstrated that the MDM2–p53 interaction can target p53 for degradation (Prives, 1998). In contrast to the MDM2–p53 interaction, there is very little known about the biological effect of the MDM2–RB interaction. Stimulation of the transactivation function of E2F1 by MDM2 suggested that the MDM2–RB interaction may promote cell growth (Xiao et al., 1995). However, a direct interaction between MDM2 and E2F1/DP1 has been reported to stimulate the transactivation function of E2F1 in SAOS-2 cells that express an MDM2binding-defective RB (Martin et al., 1995). Therefore, it remains unclear whether the increased transactivation function of E2F1 in the presence of MDM2 is dependent on MDM2–RB binding or independent of it. Since MDM2 is one of the most important regulators of p53 function, we hypothesized that RB may interfere with some aspects of p53 activity through the MDM2–RB interaction. To test this, we investigated whether RB can interfere with the ability of MDM2 to negatively regulate the functions of p53 in transactivation, transrepression, and apoptosis. We also investigated whether RB would have any effect on MDM2-mediated p53 degradation and tested whether p53 and RB compete for MDM2 binding or form a trimeric complex. Results RB Can Overcome the Antiapoptotic Function of MDM2 on p53-Induced Apoptosis To test whether an RB–MDM2 interaction could affect the apoptotic function of p53, we performed experiments in SAOS-2 cells, which are null for p53 and have a C-terminal truncated RB that is defective in binding to E2F1 and MDM2. When a p53 expression plasmid (pCMV-p53) was introduced into SAOS-2 cells, DNA fragmentation typical of apoptotic cells was seen in the cells transfected with the p53 expression plasmid but not with control vector (Figure 1A). Coexpression of MDM2 prevented this apoptotic function of p53 (Figure 1A), but when RB was coexpressed with p53 and MDM2, the antiapoptotic function of MDM2 on p53-mediated apoptosis was diminished (Figure 1A). The amount of fragmented DNA was similar to that in the cells coexpressing RB and p53 (Figure 1A). These results demonstrated that RB can overcome the antiapoptotic function of MDM2 on p53-induced apoptosis. Around 50% of the p53-transfected SAOS-2 cells had a sub-G1 DNA

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Figure 1. RB Can Overcome the Antiapoptotic Function of MDM2 on p53-Induced Apoptosis DNA fragmentation (A) and FACS analysis (B and C) of p53 induced apoptosis in the presence or absence of MDM2 and RB in transfected SAOS-2 cells. The p53 and control plasmid–transfected SAOS-2 cells are labeled as p53 and vector, respectively. For FACS analysis, transfected cells were gated based on the expression of CD20 (data not shown). Apoptosis was measured by the accumulation of cells with a sub-G1 DNA content (labeled M1). The graph in (C) represents the percent of cells with sub-G1 DNA content (apoptotic cells) in transiently transfected SAOS-2 and H1299 cells, respectively, expressing p53 (5 mg/10 cm dish) in the presence or absence of MDM2 and RB as indicated. The mean values were derived from four independent experiments.

content, characteristic of apoptotic cells, in contrast to only 7% of the cells transfected with a control vector (Figures 1B and 1C). Coexpression of MDM2 with p53 reduced the fraction of apoptotic cells from 50% to around 22%, and coexpression of RB, MDM2, and p53 overcame the antiapoptotic function of MDM2 on p53induced apoptosis. As we showed earlier (Hsieh et al., 1997), the ability of RB to overcome the antiapoptotic function of MDM2 on p53-induced apoptosis was not due to any synergistic effect of RB and p53 (Figure 1). In contrast, RB specifically inhibited E2F1-induced apoptosis in SAOS-2 cells (Hsieh et al., 1997). Since the ability of MDM2 to inhibit p53-induced apoptosis is cell type specific (Haupt et al., 1996), we also tested the RB effect on the antiapoptotic function of MDM2 on p53-induced apoptosis in another p53 null cell line, H1299. As for SAOS-2 cells, p53-induced apoptosis was inhibited by coexpression of MDM2 in H1299 cells, and coexpression of RB overcame the antiapoptotic function of MDM2 on p53-mediated apoptosis (Figure 1C). Together, all these results illustrate that RB can overcome the antiapoptotic function of MDM2 on p53mediated apoptosis.

The C Pocket of RB, Residues 792–928, Overcomes the Antiapoptotic Function of MDM2 on p53-Induced Apoptosis RB binds to the transcription factor E2F through its large pocket domain (pocket domains A, B, and C, residues 379–928) and negatively regulates its activity (Weinberg, 1995; Hsieh et al., 1997). It is the C pocket domain of RB that binds to tyrosine kinase c-Abl (Welch and Wang, 1993) and can also interact with MDM2 (Xiao et al., 1995). The three RB mutants we have used (Figure 2A) were pRB(379–928), pRB(379–792), and pRB(792–928), which contain the large pocket domain of RB (pockets A, B, and C), the small pocket domain of RB (A and B), and the C pocket domain of RB, respectively. As shown in Figure 2B, the fraction of apoptotic cells in a p53transfected cell population was reduced by about half (from 30% to 14%) when MDM2 was coexpressed. That reduction was prevented by coexpression of wild-type RB as well as two RB mutants that contain the MDM2binding regions of RB, pRB(379–928), and pRB(792– 928). The central region of RB, pRB(379–792) (pocket A and B), did not affect the antiapoptotic function of MDM2. This was not due to lack of protein expression

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RB Can Prevent MDM2-Mediated p53 Degradation MDM2 can specifically target p53 for degradation through their direct interaction, so the effect of RB and its mutants on MDM2-mediated p53 degradation was also tested. When p53 was coexpressed with MDM2, there was a clear reduction in the level of p53 (Figure 3A), consistent with previous results. Interestingly, when p53, MDM2, and RB were coexpressed, the p53 level remained the same as that seen in the absence of MDM2. Similar levels of MDM2 were detected in all the transfected cells with or without RB, and the level of RB was not affected by the coexpression of MDM2. The ability of RB to prevent MDM2-targeted degradation of p53 was also apparent in H1299 cells (Figure 3B). As coexpression of RB together with MDM2 and p53 can result in a higher level of p53 compared to that coexpressed with just MDM2, we tested whether this activity of RB is dependent or independent of MDM2mediated degradation of p53. As shown in Figure 3C, the coexpression of RB did not alter the expression level of the MDM2-binding-defective p53 mutants, p53(Gln22, Ser23) and p53DI (Ko and Prives, 1996). This was in contrast to the situation with wild-type p53, demonstrating that the effect of RB on the expression level of wildtype p53 is through specifically blocking MDM2-targeted degradation of p53. We then tested whether the ability of the RB mutants to overcome the MDM2 inhibition function on p53induced apoptosis was associated with their ability to protect p53 from MDM2-mediated degradation. When the RB mutants were coexpressed with MDM2 and p53 (Figure 3D), the mutants that protected p53 from MDM2mediated degradation were the same two mutants that overcame the antiapoptotic function of MDM2 on p53induced apoptosis. The mutant pRB(379–792), which only contains the small pocket domain of RB, failed to prevent MDM2-targeted p53 degradation (since similar amounts of protein were expressed by the wild-type RB and RB mutant constructs, Figure 3D). Taken together, these results suggest that the C pocket of RB is essential and sufficient to overcome the antiapoptotic function of MDM2 on p53-induced apoptosis. It can also inhibit MDM2-targeted p53 degradation, indicating an important role for the RB–MDM2 interaction.

Figure 2. C Pocket of RB Is Essential and Sufficient to Overcome the Negative Inhibition of MDM2 on p53-Induced Apoptosis RB mutants used in this study (A) and FACS analysis of the effect of RB mutants on the antiapoptotic function of MDM2 on p53-induced apoptosis (B). The expression of wild-type RB and its mutants used in the study is shown in (C). The graph in (B) represents the percentage of cells with sub-G1 DNA content (apoptotic cells) in transiently transfected SAOS-2 cells expressing p53 (3 mg/10 cm dish) in the presence or absence of MDM2 and RB or RB mutants as indicated. The mean values were derived from three independent experiments.

of the RB mutant, RB(379–792) (Figure 2C). The results indicate that the C pocket domain of RB is essential and sufficient to overcome the antiapoptotic function of MDM2 on p53-mediated apoptosis.

RB Does Not Prevent MDM2 from Inhibiting p53-Mediated Transactivation The effect of RB on the ability of p53 to transactivate various p53-responsive promoters was investigated in the presence or absence of MDM2. Coexpression of MDM2 inhibited the transactivation function of p53 dramatically when the MDM2 promoter was assayed as a p53-responsive promoter in both SAOS-2 (Figure 4A) and H1299 cells (data not shown). Consistent with the observation that RB does not affect the apoptotic function of p53 or its expression, coexpression of RB did not affect the transcriptional activity of p53 significantly. When MDM2, p53, and RB were expressed together, RB was unable to overcome the inhibitory activity of MDM2 on p53-mediated transcription. This was in contrast to all the results shown above where coexpression

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Figure 3. Immunoblotting to Show that RB Can Prevent MDM2 from Targeting p53 for Degradation (A) and (B) show the expression level of RB, p53, MDM2, and GFP in transfected SAOS-2 cells and the expression level of p53 in transfected H1299 cells. The expression levels of RB, MDM2, GFP, and p53 and its MDM2-binding-defective mutants, p53(22Gln, 23Ser) or p53DI, in transfected SAOS-2 cells are shown in (C). In (D), the effect of RB mutants on the expression level of p53 (upper panel), RB and RB mutants (second panel from the top), and MDM2 was measured. Expression of GFP (anti-GFP, Clontech) was used as loading control. RB protein was detected with antibody C-15 from Santa Cruz (3A) and MDM2 with SMP14 (A, C, and D). For 9E10-tagged RB mutants (D), 9E10 antibody cross-linked beads were used to carry out the immunoprecipitation, and 9E10 antibody was used in the immunoblotting. p53-specific monoclonal antibodies used were DO.1 (A and B) or pAb421 (C). The positions of the proteins are indicated by arrows.

of RB overcame the antiapoptotic function of MDM2 on p53-mediated apoptosis. It also contrasted with the effects on the expression level of p53, since RB can prevent MDM2 from targeting p53 degradation, and the amount of p53 expressed in MDM2 and RB cotransfected cells was very similar to that in cells transfected with p53 alone and far greater than that detected in p531MDM2 transfected cells. Further titration of different amounts of RB also did not remove the inhibitory activity of MDM2 on p53 (Figures 4B). To eliminate the possiblity that our results were due to general repression by RB, we tested the ability of the C pocket of RB to release the negative effect of MDM2 on the transactivation function of p53, since this mutant of RB does not repress gene expression (see Figure 5A and data not shown). Interestingly, this RB mutant also failed to remove the inhibitory activity of MDM2 on the transactivation function of p53 in both SAOS-2 and H1299 cells (Figure 4C). Similar results were also obtained with a titration of this C pocket RB mutant (data not shown). Some mutations in p53 can change its ability to transactivate specific p53 target genes such as BAX and IGF-BP3, which are involved in the apoptotic pathway (Levine, 1997). Although coexpression of RB did not prevent MDM2 from inhibiting the transactivation function of p53 on the MDM2 promoter, it might have changed the substrate specificity of p53. This possibility was tested using reporter plasmids containing various p53-responsive promoters (Figure 4A). RB did not alter the inhibitory activity of MDM2 on p53-mediated transactivation on any of the p53-responsive promoters tested. Thus, although RB prevented MDM2-targeted

p53 degradation, the stabilized p53 remained inactive in transactivation of all p53-responsive promoters tested. This is in contrast to the stabilized p53 being competent to induce apoptosis. These results show how coexpression of RB may uncouple the apoptotic function of wildtype p53 from its transactivation function. RB Prevents MDM2 from Releasing p53-Mediated Transrepression To investigate whether coexpression of RB with MDM2 and p53 could prevent the negative effect MDM2 has on the transrepression activity of p53, an SV40-LacZ reporter was used to measure the effect of RB and MDM2 coexpression on the transrepression activity of p53 (p53 represses the early promoter of SV40 [Ko and Prives, 1996]). Wild-type RB and mutant RB(379–928) repressed the transcriptional activity of SV40-LacZ reporter significantly even in the absence of p53 (Figure 5A). This is consistent with RB being a general repressor of transcription, that activity requiring the A and B pocket domains (Weintraub et al., 1995; Chow and Dean, 1996). Expression of the C pocket pRB(792–928) had much less of an effect on the transcriptional activity of SV40-LacZ reporter in H1299 cells in these conditions (Figure 5A), although the pRB(792–928) protein was expressed equally well (Figure 5B). We used pRB(792–928) to investigate the effect RB–MDM2 interaction may have on MDM2-mediated inhibition of p53 transrepression. As shown in Figure 5C, p53 strongly repressed the SV40LacZ reporter activity, and this repression was released by coexpression of MDM2. pRB(792–928) did not have a significant effect on the transrepression activity of p53,

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but when pRB(792–928) was coexpressed with MDM2, it overcame the inhibition function of MDM2 on p53mediated transrepression. The ability of p53 or p531 MDM21RB(792–928) to transrepress the SV40-LacZ reporter activity was observed in both SAOS-2 (Figures 5C) and H1299 cells (data not shown). Since a large percentage of cells expressing wild-type p53 or p531MDM21pRB(792–928) die of apoptosis, we considered whether the reduced transcriptional activity of the SV40-LacZ reporter was a consequence of cell death. We used the protease inhibitor Z-VAD-FMK to block p53-induced apoptosis. As shown in Figure 5D, 100 mM Z-VAD-FMK reduced the percentage of apoptotic cells in wild-type p53-transfected SAOS-2 cells from around 32% to 9%. However, under the same conditions, Z-VAD-FMK did not prevent p53 or p531 MDM21RB(792–928)-mediated transrepression of SV40LacZ reporter activity (Figure 5E). The presence of Z-VADFMK did not affect p53 expression in the transfected cells (data not shown). These results argue that the transrepression function of p53 or p531MDM21RB (792–928) was not due to cell death and indicate that RB–MDM2 interaction may remove the negative regulation of MDM2 on p53-mediated transrepression. The ability of p53 to transrepress gene expression may be the molecular basis of why this stabilized p53 remains active in inducing apoptosis despite the fact that it is incompetent to transactivate many of its responsive promoters, including BAX. The data also show that the apoptotic function of p53 is associated with its transrepression but not its transactivation function. RB Forms a Trimeric Complex with p53 through Binding to MDM2 What is the mechanism that allows RB to specifically regulate individual p53 functions? RB and p53 might compete for MDM2 binding, but this does not explain how coexpression of RB and MDM2 failed to remove the inhibition of MDM2 on p53-mediated transactivation. Alternatively, both p53 and RB might bind to MDM2 simultaneously, and the resulting trimeric complex could prevent MDM2 from its normal function of regulating the activity of p53 in apoptosis, protein degradation, and transcription. In the trimeric model, MDM2 would bind to RB and still occupy the transactivation site of p53, so it could inhibit the transactivation function of p53 regardless of its binding to RB. In addition to masking the transactivation domain of p53, MDM2 could also repress transcription by interfering directly with the basal transcription machinery, independent of p53 (Thut et al., 1997). To test the hypotheses, we initially used in vitro translated p53, MDM2, and RB to investigate the protein–protein interaction between p53 and MDM2 with increasing concentrations of RB. There was no change in the amount of p53 immunoprecipitated with MDM2 in the presence or absence of RB, even when the molar Figure 4. RB and RB(792–928) Do Not Prevent MDM2 from Inhibiting p53-Mediated Transactivation Histograms of p53 transactivation of promoters with or without MDM2 and RB in SAOS-2 cells as indicated (A, B, and C). The luciferase reporter plasmids were MDM2-luc, p21WAF1/CIP1-luc, cyclin G-luc, and BAX-luc. Cells (104 cells/3 cm dish) were transfected with 1 mg of reporter plasmid together with 100 ng of p53 expression plasmid, 200 ng of MDM2 expression plasmid, and 500

ng of RB or RB(379–792) expression plasmid as indicated. Mean values were derived from four independent experiments. The right panel of (B) shows the expression of p53 and RB, which correlate with the data in the left panel in SAOS-2 cells. In (C), MDM2 luciferase reporter plasmid was used in both SAOS-2 and H1299 cells.

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Figure 5. RB and RB(792–928) Can Prevent MDM2 from Inhibiting p53-Mediated Transrepression Cells (104/3 cm dish) were transfected with SV40-LacZ reporter plasmid (1 mg in H1299 cells, [A] and [B], and 5 mg in SAOS-2 cells, [C]) with 1 mg of p53 expression plasmid in the presence or absence of 2 mg of MDM2 expression plasmid. Expression plasmids for RB or RB mutants were titrated as indicated. Mean values were derived from four independent experiments for (C). (B) shows the expression of p53 and RB in the cell lysates used in (A). The ability of Z-VAD-FMK (100 mM) to prevent p53-induced apoptosis in SAOS-2 cells is shown in (D). The transrepresssion activity of p53 in the presence or absence of Z-VAD-FMK is shown in (E) as reporter transcription activity relative to the activity of SV40-LacZ set to a value of 1. Mean values were derived from two independent experiments.

ratio between p53 and the C-terminal RB mutant, pRB(792–928), was 1:20 (Figures 6A and 6B). This result suggests that RB–MDM2 interaction does not displace p53 from interacting with MDM2. To rule out the possibility that RB can directly interact with p53 as well as MDM2, we used an RB-specific antibody, IF8, to immunoprecipitate RB and looked for the coimmunoprecipitation of p53 in the presence and absence of MDM2. In the absence of MDM2, RB failed to coimmunoprecipitate p53, indicating that under these conditions RB does not bind to p53 directly. Consistent with the trimeric protein complex hypothesis, the presence of MDM2 allowed the anti-RB antibody to specifically immunoprecipitate p53 (Figure 6C). Using purified p53, MDM2, and

GST-RB produced in bacteria (Figure 6D, left panel), we demonstrated that the three proteins can form a trimeric complex directly (Figure 6D, right panel). These results show that RB forms a trimeric complex with p53 through its binding to MDM2 in vitro. Similar results were also obtained using a p53 antibody, pAb421, to perform the immunoprecipitation (data not shown). Using various MDM2 mutants, we mapped the RB-binding region on MDM2 to its C-terminal domain (residues 273–321) (Figures 6E and 6F), so the p53- and RB-binding sites are far away from each other on the MDM2 protein sequence. Since MDM2 can bind to dephosphorylated RB (Xiao et al., 1995) and phosphorylation of RB can regulate its activity (Mittnacht, 1998), we investigated the effect of

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Figure 6. RB Forms a Trimeric Complex with p53 through Its Binding to MDM2 In Vitro (A–D) In vitro translated 35S-methionine labeled p53, MDM2, and RB PRB(792–928) proteins are shown in IVT input. Antibody SMP14 was used to immunoprecipitate MDM2 with the coimmunoprecipitation of p53 in the absence or presence of titrated amounts of RB as indicated. The ratio of IVT products of MDM2 and RB used in the experiment was 1:1, 1:2, and 1:5 (A). The ratio between MDM2 and the RB mutant, pRB(792–928), was 1:5, 1:10, and 1:20 (B). The trimeric complex of RB, p53, and MDM2 was detected by an anti-RB antibody IF8, and the in vitro translated proteins used in the immunoprecipitation are shown (C). (D) (left panel) shows the purified p53, MDM2, and GST-RB (Coomassie blue staining). The faster migrating fragments in the lanes labeled as MDM2 and GST-RB were confirmed by immunoblotting as degradation products of MDM2 and GST-RB, respectively (data not shown). (D) (right panel) shows that GST-RB does not bind to p53 directly, and it can only coprecipitate with p53 in the presence of MDM2. GST-RB was pulled down with glutathione beads, and the coprecipitated p53 and MDM2 were detected by anti-p53 and anti-MDM2 antibodies DO.1 and SMP14, respectively. (E and F) RB binds to residues 273–321 of MDM2 protein mapped by a series of MDM2 truncation mutants (6E). In (F), in vitro translated 35Smethionine-labeled MDM2 and its mutants were labeled as IVT MDM2. Proteins that bound to GST-RB(763–928) or GST (negative control) are shown as GST-RB or GST, respectively. (G) MDM2 preferentially binds to hypophosphorylated RB (G). Cyclin E/CDK2 and cyclin D1/CDK4-phosphorylated GST-RB are labeled as phosphorylated GST-RB. In vitro translated 35S-methionine-labeled MDM2, p53, and MDM2 were indicated by arrows. The amount of phosphorylated and nonphosphorylated GST-RB used in the GST pull-down experiment was detected by an anti-GST antibody and is shown in the lower panel.

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Figure 7. RB Can Form a Trimeric Complex with MDM2 and p53 In Vivo, Stabilize the Endogenous p53, and Induce p53-Dependent Apoptosis (A and B) The RB, p53, and MDM2 trimeric complex can be detected in p53 and MDM2 double-knockout mouse fibroblast cells (A) as well as in U20S cells (B). Transfected p53 cells were immunoprecipitated with pAb421, and the expression of p53, RB, and MDM2 (arrowhead) was detected by CM1, C-15, and SMP14, respectively. Cell lysates (4 mg) were immunoprecipitated with RB and p27kip1 antibodies IF8 and SX53G8. The presence of p53 and RB was detected by antibodies CM1 and C-15, respectively. (C–F) Expression of wild-type RB or RB mutants can stabilize endogenous p53 and enhance apoptosis in U2OS and MCF-7 cells by binding to MDM2. 9E10-tagged wild-type RB and RB mutants were detected by the mouse monoclonal antibody 9E10 and visualized by FITCconjugated anti-mouse antibody (C, left panel) or HRP-conjugated anti-mouse antibody (D). Endogenous p53 was detected with antibody CM1 and visualized with a Texas red–conjugated secondary antibody (C, right panel) or HRP-conjugated secondary antibody (D). For (D), the transfected CD20-expressing cell lysates were used to detect the expression level of the endogenous p53 in both U2OS and MCF-7 cells. The expression level of CD20 was used as a loading control. The 9E10 epitope-tagged RB and its mutants were immunoprecipitated with 9E10 cross-linked to beads, and the coimmunoprecipitated p53 was detected with antibody CM1 (E). (F) shows that wild-type RB or MDM2binding mutants of RB can induce p53-dependent apoptosis in U2OS and MCF-7 cells but not in SAOS-2 and H1299 cells. (G and H) Small induction of RB (NPC) was sufficient to induce the endogenous p53 (G) and apoptosis (H). Induction time was labeled as 0, 1, 2, 3, and 4 days. The induced, tagged RB was detected with antibody 9E10 and labeled as RB(induced). Endogenous and induced RB was detected by an antibody C-15 and labeled as RB(total). A very light exposure of the immunoblot was used in this picture. Induced p53 was detected with antibody DO.1, and the expression of PCNA was used as loading control. In (G) (right panel), the induced RB was immunoprecipitated with 9E10 cross-linked to beads, and the immunoprecipitates were analyzed for the presence of RB, MDM2, and p53 as indicated using 9E10, SMP14, and CM1, respectively. In (H), RB was induced for the time shown, and the cells were then stained with annexin V and propidium

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phosphorylation of RB on the interaction between MDM2 and RB. GST-RB was phosphorylated by cyclinE/ CDK2 and cyclinD/CDK4, and its ability to interact with MDM2 and to form the p53–MDM2–RB trimeric complex was compared with unphosphorylated GST-RB. The amount of MDM2 and p53 complexed with phosphorylated GST-RB was lower than that with unphosphorylated GST-RB; note that slightly more phosphorylated GST-RB was used in the experiment (Figure 6G). This result indicated that MDM2 binds preferentially to hypophosphorylated RB. To test whether the RB, MDM2, and p53 trimeric complex exists in cells, we used MDM2 and p53 doubleknockout mouse fibroblast cells (Jones et al., 1996) to show that exogenously expressed p53 cannot complex with endogenous RB in the absence of MDM2. However, introduction of MDM2 allowed the anti-p53 antibody, pAb421, to immunoprecipitate endogenous RB with exogenously expressed p53, presumably through formation of the triple complex (Figure 7A). The RB, p53, and MDM2 trimeric complex was also detected in the cells when all three proteins were produced exogenously; the MDM2-binding-defective mutant p53DI did not form the trimeric complex with either endogenous or exogenous RB when cotransfected with MDM2, although the protein was shown to be expressed (Figure 7A). Furthermore, expression of a p53-binding-defective MDM2 mutant, DMDM2, also failed to mediate the formation of the trimeric complex between p53 and RB. The trimeric complex was detected using anti-p53 (Figure 7A) or antiRB (data not shown) antibodies to perform the immunoprecipitation. These results demonstrated that RB can form a trimeric complex with p53 specifically through its interaction with MDM2 in cells. It also argues that the trimeric complex was formed through the direct interaction of the three proteins, p53, MDM2, and RB. Can such a trimeric complex be detected in cells expressing wild-type p53, RB, and MDM2, and what would be the physiological implications of such a trimeric complex? Both stabilization of p53 and hypophosphorylation of RB can be seen in cells responding to DNA damage. We hypothesized that if the RB, MDM2, and p53 trimeric complex plays an important role in regulating p53-mediated apoptosis, one should be able to detect such a complex in DNA-damaged cells. The osteosarcoma cell line U2OS was used for the experiment since it expresses wild-type p53 and RB. Cells were irradiated with either 10 Gy of g radiation or 10 J/m2 of UV and harvested 14 hr later. An increase in p53 expression was detected in irradiated cells (Figure 7B, left panel). When an antiRB specific antibody, IF8, was used to immunoprecipitate RB, the presence of p53 was clearly detected in the immunoprecipitates from U2OS cells (Figure 7B, right panel). Similar results were obtained in another two cell lines expressing wild-type p53 and RB, human breast carcinoma line MCF-7, and human colorectal carcinoma line RKO (data not shown). The coimmunoprecipitation

of p53 by the RB antibody IF8 was specific because anti-p27 monoclonal antibody SX53G8 (Fredersdorf et al., 1997) did not pull down a detectable amount of p53 under the same conditions. Furthermore, the amount of p53 coimmunoprecipitated with RB was more in the cell lysate irradiated with UV or g radiation than that in the untreated cell lysate, suggesting that the RB, MDM2, and p53 trimeric complex is more abundant in DNAdamaged cells. This may partly be due to the fact that there is more p53 present in irradiated cells, but a higher percentage of RB might form the trimeric complex with p53 through MDM2 binding since there is more hypophosphorylated RB (Mittnacht, 1998).

Expression of RB Stabilizes Endogenous p53 and Enhances Apoptosis by Binding to MDM2 To test whether RB can stabilize endogenous p53 through its ability to bind to MDM2 and form a trimeric complex, we introduced wild-type RB or RB mutants into cell lines MCF-7 and U2OS, which contain wild-type p53. As MDM2 preferentially binds to hypophosphorylated RB, we also included a nonphosphorylatable RB mutant, RB(NPC) (Chew et al., 1998). Expression of wildtype RB and RB mutants competent for MDM2 binding stabilized the endogenous p53 in U2OS and MCF-7 cells (Figures 7C and 7D and data not shown). The unphosphorylatable RB mutant RB(NPC) was at least as active as wild-type RB in stabilizing the endogenous p53. So dephosphorylation did not inhibit the ability of RB to regulate the expression of the endogenous p53; perhaps phosphorylation of RB could affect its ability to counteract the negative regulation of MDM2 on p53, but further studies are required to clarify this. As with exogenously transfected p53, MDM2-bindingdefective RB mutant RB(379–792) failed to stabilize the endogenous p53. This was not due to lack of protein expression, as similar amounts of RB and RB mutants were detected by the antibody 9E10, whose epitope was tagged to all the RB proteins used. The failure of the MDM2-binding-defective RB mutant RB(379–792) to stabilize the endogenous p53 was presumably due to the fact that it did not form a trimeric complex with endogenous p53 in both U2OS (Figure 7E) and MCF-7 cells (data not shown). This was in contrast to wild-type RB and the other MDM2-binding RB mutants, with which a trimeric complex with endogenous p53 was clearly detected. The apoptotic function of the stabilized endogenous p53 in both U2OS and MCF-7 cells was studied using FACS analysis, measuring the percentage of cells in the transfected population containing a sub-G1 DNA content. An approximately 3-fold increase in the percentage of apoptotic cells was detected in both U2OS and MCF-7 cells transfected with wild-type RB or its MDM2-binding mutants. In contrast, the MDM2-binding-defective RB mutant RB(379–792) did not induce

iodide and analyzed by FACS. Cells were gated into regions A, B, C, and D. The x and y axes are the intensity of annexin V and propidium iodide staining, respectively. Healthy proliferating cells are negative for annexin V and propidium iodide staining, and they are located in region A. Apoptotic cells are positive for annexin V staining, and they are located in regions B and C. The number of cells in regions B and C were counted as a proportion of the total number of gated cells and expressed as percent of apoptotic cells in a histogram.

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apoptosis in the transfected cells. The enhanced apoptosis induced by the expression of RB in both U2OS and MCF-7 cells was p53 dependent since wild-type RB and the same RB mutants did not induce apoptosis in the two p53 null cell lines, SAOS-2 and H1299 (Figure 7F). These results indicate that RB can stabilize endogenous p53 by forming a trimeric complex with endogenous MDM2, and the stabilized p53 is able to induce apoptosis. To exclude the possibility that the ability of RB to counteract the inhibitory activity of MDM2 on p53 function depends on very high-level expression, we established a stable U2OS cell line that conditionally expresses the unphosphorylatable RB mutant RB(NPC) in 90% of the cells at a low level (Chew et al., 1998). The RB(NPC) mutant was used because U2OS cells phosphorylate RB very effectively. Expression of the induced RB (tagged with a 9E10 epitope) was detected 1 day after induction; Figure 7G, left panel and labeled as RB(induced). The induced RB was only a small proportion of the endogenous RB since no increase in overall RB expression, labeled as RB(total), was detected with an anti-RB antibody (even with a very short exposure of the immunoblot). Nevertheless, the induction of RB(NPC) was sufficient to cause a significant increase in p53 expression (Figure 7G). The increase in p53 was at the posttranscriptional level since the p53 mRNA level was unchanged (data not shown). The induced RB may stabilize endogenous p53 by forming the trimeric complex through its binding to MDM2 since the monoclonal antibody 9E10 coimmunoprecipitated p53 with the induced RB (Figure 7G, right panel). Furthermore, upon the inducible expression of RB, the increase in p53 protein level was accompanied with an increase in the number of cells stained positive for annexin V as measured by the FACS analysis to indicate the percentage of apoptotic cells (Figure 7H). This result provided further evidence that a small increase in the level of hypophosphorylated RB stabilized p53 and induced apoptosis by forming a trimeric complex with p53 and MDM2. Since the effects seen in these cells are caused by changes in the endogenous p53 and MDM2, RB might be a very important regulator of the apoptotic function of p53 in vivo. Discussion Here we demonstrate, using wild-type proteins expressed at their endogenous levels in cells, that RB can regulate the apoptotic function of p53 through binding to MDM2, thus preventing MDM2 from targeting p53 for degradation. RB can also prevent MDM2 from inhibiting p53-mediated transrepression but not transactivation. RB does so through its ability to form a trimeric complex with p53 via binding to MDM2. The p53 in the trimeric complex was transcriptionally inactive but able to induce apoptosis. We do not yet know how the trimeric complex protects p53 from MDM2-targeted degradation, but the RB binding site on MDM2 (residues 272– 320) is located in the region that is required to mediate p53 degradation. An MDM2 mutant lacking residues 220–437 is incapable of mediating p53 degradation although it can still bind to p53 (Kubbutat et al., 1997).

RB might protect p53 from MDM2-targeted degradation by occupying a site on MDM2 that confers sensitivity to degradation, or it might compete with other degradation proteins binding to a similar site on MDM2 as for E2F1 (Hofmann et al., 1996). The ability of RB to counteract the negative regulation of MDM2 on p53-induced apoptosis would predict that the loss of MDM2 binding would result in an impaired tumor suppression function of RB. Consistent with this hypothesis, patients with a mutant RB lacking 58 amino acids of the C terminus (exons 24 and 25) suffer from retinoblastoma but with only a low penetrance (35% of the eyes at risk) because the deleted RB can still bind to E2F but not MDM2 (Bremner et al., 1997). The low penetrance of the phenotype suggests that a major function of RB is negative regulation of E2F but that the MDM2–RB interaction also has an important tumor suppression function. Uncontrolled E2F1 expression may cause p53-dependent and independent apoptosis (Wu and Levine, 1994; Hsieh et al., 1997), and RB can negatively regulate the apoptosis induced by unrestrained E2F1 expression (Hsieh et al., 1997). RB and E2F1 double-knockout mice demonstrated that the increased apoptosis in RB null mice was mainly caused by the apoptotic function of E2F1 (Tsai et al., 1998). The ability of the E2F1 null background to abolish apoptosis in the lens tissue of RB null mice indicated that previously reported p53-dependent apoptosis (Morgenbesser et al., 1994) could be the result of deregulated apoptotic function of E2F1 (Tsai et al., 1998), consistent with the recent finding that E2F1 is an upstream regulator of p53-mediated apoptosis (Pan et al., 1998). Furthermore, the enhanced apoptosis seen in RB null mice was also reported to be caused by p53dependent and independent pathways (Macleod et al., 1996; Holmberg et al., 1998). All these points suggest that the inhibitory activity of RB on p53-mediated apoptosis seen in RB null mice could be mediated through E2F1. Thus, apart from being a negative regulator of E2F1, RB could also bind to MDM2 and regulate the apoptotic function of p53. Although p53 induces proapoptotic genes such as BAX and IGF-BP3 and studies using p53 mutants suggested a role for p53 transactivation in apoptosis induction (Ko and Prives, 1996), it is now clear that the transactivation function of p53 can be uncoupled from its apoptotic function. This makes a functional distinction between the RB–MDM2–p53 and ARF–MDM2–p53 trimeric complexes (Prives, 1998). There is evidence that the transrepression function of p53 is associated with its ability to induce apoptosis. For example, the p53 mutant p53(175pro) retains transactivation function for some p53-responsive promoters but is defective in both induction of apoptosis and transrepression (Crook et al., 1994; Rowan et al., 1996). Also, BCL-2 and adenovirus E1B 19 kDa protein can inhibit p53-mediated apoptosis; they inhibit p53-mediated transrepression but not transactivation (Ko and Prives, 1996). We used Z-VAD-FMK (Sabbatini et al., 1997) to inhibit p53induced apoptosis, and this did not affect the transrepression function of p53 or the trimeric complex of p53, MDM2, and RB(792–928). Thus, the transrepression by p53 detected in our system was not a consequence of

RB Regulates the Apoptotic Function of p53 via MDM2 191

cell death. The occurrence of apoptosis in response to DNA damage with the same kinetics in thymocytes of BAX knockout mice as in those of wild-type mice (Knudson et al., 1995) also argues that the ability of p53 to transactivate BAX may be uncoupled from its ability to induce apoptosis. Future identification of genes that are transrepressed by p53 in vivo may reveal the precise mechanisms through which p53 induces apoptosis. Increased formation of the RB, p53, and MDM2 trimeric complex in DNA-damaged cells suggests a role in regulating p53-mediated apoptosis in response to DNA damage; there is known to be a discordance between the level of p53 and its transcriptional activity in such cells (Lu et al., 1996), much DNA damage–induced p53 remaining latent for the transactivation function (Hupp and Lane, 1995; Lu et al., 1996), but in the trimeric complex, p53 retains its apoptotic function. It is also clear that the phosphorylation of p53 on Ser-15, which occurs in DNA-damaged cells and prevents its interaction with MDM2 (Prives, 1998), does not happen to all of the p53 in the cell, so p53 is still available to form the trimeric complex. In the context of RB as an upstream regulator of p53-mediated apoptosis, one would anticipate that tumor cells containing wild-type p53 and fulllength RB would respond better to radiotherapy and chemotherapy. Indeed, it has been reported that cancer patients with apparently normal p53 and RB had longer survival times than patients with tumors that were RB negative and had mutated p53 (Xu et al., 1994). Clarification of the interplay between RB, p53, and MDM2 in control of apoptosis may thus be significant for both basic understanding of the control of apoptosis in development and for the application of therapy in cancer patients. Experimental Procedures Cell Culture, Antibodies, and Plasmids Cells were grown in DMEM supplemented with 10% FCS. Monoclonal antibodies to p53 (DO.1 and pAb421), RB (IF8), MDM2 (SMP14), and PCNA (PC10) were used. CM1, C-15, and N-20 are rabbit polyclonal antibodies specific to p53, RB, and BAX, respectively (gifts from Professor David Lane and purchased from Santa Cruz Biotechnology, Inc.). N-20 CD20Leu is an FITC-conjugated monoclonal antibody specific for the cell surface marker CD20 (Becton Dickinson). Anti-GFP monoclonal antibody was purchased from Clontech. All the expression plasmids used in this study were driven by the CMV immediate-early promoter. The luciferase and b-galactosidase reporter plasmids that contain various p53-responsive promoters are MDM2-luc, BAX-luc (both from Dr. Moshe Oren), cyclin G–luc, and SV40-LacZ. The MDM2-binding-defective mutant p53, p53(Gln22, Ser23), and p53DI were gifts from Professor Arnold Levine and Dr. Karen Vousden. Both RB and MDM2 mutants were generated by PCR and cloned into pcDNA-3 expression plasmid (Invitrogen) in-frame with a monoclonal antibody 9E10 tag at the N-terminal end of each fragment. The U2OS cell line expressing inducible 9E10-tagged RB(NPC) has been described (Chew et al., 1998). DNA Transfection, Luciferase, b-Galactosidase, and DNA Fragmentation and FACS Assays DNA was transfected into SAOS-2 cells using calcium phosphate precipitation (O’Connor et al., 1995). For all the transfection experiments, control vector (usually CMV vector) was used to compensate total DNA input. For measuring transcriptional activity, 104 cells in a 3 cm dish were transfected with 100 ng of plasmid DNA expressing p53 and 1 mg of p53 reporter plasmid as indicated. A 5-fold excess

of RB plasmid compared to that of p53 was found to be necessary to give clear detection of RB protein by immunoprecipitation and subsequent immunoblotting (data not shown), and this was the ratio used in the experiments. After transfection, the cells were lysed and assayed as before (Fuchs et al., 1995). The expression level of GFP protein from a cotransfected CMV-GFP plasmid (1 mg) served as a loading control for transfection efficiency. For the transcriptional activity data as well as the FACS data, mean values were derived from duplicate assays replicated in at least two independent experiments. For FACS analysis, 106 cells in a 10 cm dish were transfected with 3–5 mg of p53 plasmid with 6–10 mg MDM2 plasmid and 15–25 mg of RB plasmid. Thirty-six to forty-eight hours after the transfection, both floating and attached cells were used for FACS assay as described (Hsieh et al., 1997). U2OS cells expressing inducible RB were stained with annexin V using the apoptosis detection kit. For the DNA fragmentation assay, 2 3 107 cells were transfected with the indicated plasmid DNA using an amount proportional to that used for the FACS assay. Seventy-two hours after transfection, the cell pellets were used in a DNA fragmentation assay as described (Hsieh et al., 1997). The FACS analysis with Z-VAD-FMK (Enzyme System Products, USA) used cells harvested 24 hr after transfection. For Figure 7D, cells were transfected with CD20 together with RB or RB mutants. Cells expressing CD20 (transfected cells) were stained with FITC-conjugated anti-CD20 antibody. A Biotin conjugated anti-FITC antibody was then added to the cell pellet, and after the incubation, the cells were incubated with straptavidinconjugated magnetic beads to isolate the CD20 expressing cells. Antibody Purification and Cross-Linking of Monoclonal Antibodies to Protein G Beads Anti-myc tag antibody 9E10, anti-RB antibody IF8, anti-p53 antibody pAb421, and anti-p27 antibody SX53G8 were purified from supernatant using ammonium sulphate precipitation and protein G beads. Purified monoclonal antibodies were cross-linked to protein G beads as described (Harlow and Lane, 1988). Production of GST Fusion Proteins and p53 in E. coli and In Vitro Binding Assays p53 protein was produced and purified as described previously (Hupp et al., 1992). The GST-RB(763–928), GST-RB, or GST-MDM2 fusion proteins were produced in E. coli and purified as described (Fredersdorf et al., 1997). The eluted GST-MDM2 was cleaved by incubating in 50 ml of thrombin (1 U/ml PBS, Pharmacia Biotech) and 950 ml PBS for 16 hr at room temperature. Cleaved MDM2 was recovered from the supernatant after further treatment with glutathione beads. MDM2 mutants were in vitro translated with 35S-methionine using the TNT T7 Quick Coupled Transcription/Translation System (Promega). Ten microliters of packed RB-GST or GST prebound glutathione beads were resuspended in 100 ml binding buffer (20 mM Tris–Cl [pH 7.5], 100 mM NaCl, 2 mM EDTA, 0.1% NP-40, 2 mM DTT, 0.05% BSA, 5% glycerol), added to 10 ml of translation mix, and allowed to bind at 48C for 2 hr. Beads were washed once with NET1 1% NP-40, twice with NET (150 mM NaCl, 5 mM EDTA, 50 mM Tris–Cl [pH 7.5]), and then analyzed on an SDS-PAGE gradient gel (10%–15%). In Vitro Translation and Immunoprecipitation RB, MDM2, p53, and the RB mutant pRB(792–928) were in vitro translated with 35S-methionine using the TNT T7 System (Promega). Samples (10 ml) of the in vitro translated protein products were mixed and allowed to interact in binding buffer (NET buffer) in a volume of 200 ml at 48C for 1 hr. Anti-MDM2 antibody SMP14 or anti-RB antibody IF8 immobilized on protein G agarose beads was added to the binding reactions and incubated with mixing at 48C for 1 hr. The beads were then washed extensively with NET containing 0.5% NP-40. The bound proteins were released in SDS gel sample buffer and analyzed by 10% SDS PAGE at pH 9.4. Immunoprecipitation and Immunoblotting Cells were lysed with NET buffer containing 1%NP-40, and 2–4 mg of lysate was incubated with antibodies as indicated. The antibody complexes were isolated using protein G beads, washed three times

Molecular Cell 192

with 1% NP-40/NET buffer, and twice with NET buffer. The immunoprecipitate–protein G beads were boiled in SDS sample buffer, and the supernatant was analyzed on SDS-PAGE gels. For immunoblotting experiments, 20 ml of soluble cellular proteins (5–8 mg/ml of total cellular protein) was loaded on SDS-polyacrylamide gels in SDS sample buffer. After electrophoresis, the proteins were transferred to nitrocellulose paper, and nonspecific binding sites blocked with a 10% solution of reconstituted dried milk powder for 1 hr at room temperature. Primary antibody was added to the blot and incubated for 2–3 hr at room temperature or 48C overnight. Finally, a peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit immunoglobin was incubated with the blot, and bound immunocomplexes detected by the enhanced chemiluminescence (ECL) method, as described by the manufacturer (Amersham). Acknowledgments We would like to thank Dr. Karin Barnouin and Miss Lynn Fallis for their help in purifying and cross-linking some of the antibodies used in this study. We would also like to thank Dr. Stephen Jones for the p53 and MDM2 double-knockout mouse fibroblasts. Our special thanks go to Professor Paul Farrell for the critical reading of the manuscript. This work was supported by Ludwig Institute for Cancer Research and AICR grant. Received May 26, 1998; revised December 14, 1998.

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