It was, indeed, a wonderful honor to receive the G. H. A. Clowes Memorial Award and present the Clowes Lecture at the Meeting for the American Association for Cancer Research. I have been fortunate to carry out research in the area of cancer biology at a remarkable time and to have had many truly extraordinary colleagues in this field. When I began my graduate student research in 1961, we did not truly understand the origins of cancer in humans, nor did we have the experimental tools to uncover the mutations or the genes involved in human cancer. Rather, we followed the advice of the exceptional individuals who were beginning the revolution in molecular biology; we chose simple experimental systems with the abiding faith that viruses that cause tumors in hamsters, mice, or chickens would surely teach us something about cancer in humans. For their guidance, advice, friendship, and the example they set for me, I will always be indebted to Harold S. Ginsberg, my thesis advisor, and Robert L. Sinsheimer, my postdoctoral advisor. I learned many lessons about science and life during my stay at the University of Pennsylvania and at the California Institute of Technology. Those were the years that prepared me for science at Princeton University and the exploration of the question: How does SV40 cause tumors in hamsters and transform cells in culture? Princeton in the 1970s was truly fertile ground. Arriving as assistant professors were Bruce Alberts, Mark Kirschner, Uli Laemmli, Harold Weintraub, Abe Worcel, and many others to join a senior faculty with very high standards and well-articulated expectations. It was my good fortune to be in the right place at the right time.

At about this junction, Peter Tegtmeyer had isolated a series of temperature-sensitive mutants of SV40 that blocked infectious virus production at the nonpermissive temperature. One group of these mutants, in complementation group A, failed to transform cells in culture (1), and even if the mutants were used to transform cells at the permissive temperature, they failed to maintain the “transformed phenotype” at the nonpermissive temperature (2, 3). This had been shown previously with polyoma virus, with which tumors only formed in mouse tissues with low temperatures (4). The Tegtmeyer group (5) went on to identify the group A protein as the SV40 large tumor (T) antigen. Animals bearing SV40-induced tumors express the tumor antigen in their cancer cells. The animals recognize this viral protein as foreign and produce antibodies to it. It was these antibodies that Tegtmeyer et al.(5) used to immunoprecipitate the T-antigen and show it had a Mr of ∼100,000 using Laemmli SDS gels.

As we repeated these experiments, it became clear that, in addition to the Mr 100,000 protein immunoprecipitated from SV40-infected or -transformed cells, a number of other proteins were “trapped” in the immunoprecipitate, which we commonly interpreted as proteolytic breakdown products of the viral T-antigen (and some of them were just that). Our first clue that there was more to this observation than that facile explanation came when a graduate student in my laboratory, Dan Linzer, used the antiserum from hamsters bearing SV40-induced tumors (a serum prepared in the laboratory by my technician Angie Teresky) to look for “tumor antigens” in cells not infected or transformed by SV40. Dan Linzer found such an antigen in murine testicular teratocarcinoma cells. A protein of Mr 53,008–54,000 from these cells comigrated in SDS gels with a protein in SV40-transformed cells. Although, I was skeptical and pointed out how many proteins of Mr 53,000 there must be in cells, Dan Linzer used peptide mapping to show that the p53 in SV40-transformed cells and in teratocarcinomas was a cellular protein recognized by SV40 tumor-bearing sera (6). In fact, monoclonal antibodies directed against SV40 large T-antigen, coimmunoprecipitated this p53 protein so it was in a complex with SV40 large T-antigen (6). This result was simultaneously reported by David Lane and Lionel Crawford (7).

By 1982–1984, the first murine and then human cDNA clones of p53 were isolated. In my group, Moshe Oren (then a postdoctoral fellow) and Diane Pennica at Genentech played a central role in obtaining our clones (8, 9). After a 3–4-year confusion about who had wild-type p53 clones or mutant p53 clones, everyone agreed: the mutant p53 cDNA can transform cells in culture, and wild-type p53 cDNA clones actually prevent cellular transformation by a wide variety of oncogenes (10). These hard-won facts surfaced from several years of work in my group by Cathy Finlay and Phil Hinds. Just as Bert Vogelstein’s group (11, 12) reported that p53 mutations were found in both alleles of colon cancer cells, the experiments of Finlay, Hinds, and myself (13) showed that p53 was not an oncogene but a tumor suppressor gene.

From the discovery of p53 in 1979 until 1989, the hope that a simple virus that causes cancer in hamsters could shed light upon the origins of human cancer seemed to become a fair assumption. The years from 1989 to the present have shown just how central the p53 tumor suppressor gene is in human cancers. We now have the hope that we can understand how cancers arise and evolve and, in so doing, understand how to stop the growth of cancer cells.

Since its discovery in 1979 as a cellular target of the transforming large T-antigen of SV40 (6, 7), the importance of the role of the p53 protein as a tumor suppressor has become increasingly clear. Both alleles of the p53 gene are mutated or deleted in a large percentage of a variety of both spontaneous and inherited human tumors (11, 12, 14, 15, 16). In addition, deletion of the p53 gene from mice causes them to be highly predisposed to cancer (17). In cell culture, functional p53 protein activity is often lost during the immortalization process (18), and forced over-expression of p53 causes cells to undergo growth arrest (19, 20, 21) or apoptosis (22, 23, 24, 25, 26). These data amply demonstrate that p53 activity is incompatible with excessive cell growth.

The function of p53 in regulating the growth and division of cells commonly occurs in response to DNA damage (27, 28, 29). The mechanisms by which p53 controls the fate of cells that encounter DNA damage are not fully understood, but several steps of its pathway have been ascertained. First, p53 protein levels rise after DNA damage because p53 is stabilized by a posttranslational mechanism (30). It then functions as a transcription factor, activating the expression of several genes, not all of which have been identified to date. Some of the genes induced by p53 include: p21waf1, which contributes to growth arrest; Bax, which induces apoptosis; GADD45, which functions in DNA repair; and cyclin G, the function of which is not yet known (31, 32, 33, 34). In addition, p53 may have transcription-independent activities, possibly by direct signaling through its proline domain (35), that contribute to its growth arrest and apoptotic activities after DNA damage. By preventing the further division of cells that have encountered genetic insult, p53 checks the uncontrolled growth of cells sustaining oncogenic mutations, thus directly protecting the organism from cancer.

A major step toward understanding the p53 pathway came with the identification of the Mdm2 oncogene (36, 37). It was soon determined not only that Mdm2 is transcriptionally induced by p53 after its stabilization by DNA damage (38, 39, 40, 41) but also that MDM2 binds to p53 and blocks its ability to function as a transcription factor and tumor suppressor (42, 43, 44, 45, 46). MDM2 functions in this capacity by directly blocking p53’s activity as a transcription factor (47, 48, 49) and by targeting it for degradation (50, 51). This inhibition of p53 by MDM2 can clearly be observed in mice. Homologous deletion of Mdm2 is lethal at very early stages of embryogenesis but is completely rescued by the additional deletion of the p53 gene. This result demonstrates that the inhibition of p53 by MDM2 is essential for life at an early developmental stage (52, 53). MDM2 and p53 are, therefore, predicted to form an autoregulatory feedback loop in which p53 limits its own activity through the production of MDM2.

Because the p53 protein becomes stabilized and active as a transcription factor following DNA damage, it is apparent that the inhibition of p53 by MDM2 can be regulated to modulate p53 activity and break the MDM2-p53 autoregulatory feedback loop. This Clowes Memorial Award lecture will discuss recent evidence that supports these ideas: i.e., cell lines that overexpress MDM2 exhibit a significant p53 response after DNA damage, despite the presence of large amounts of its negative regulator, MDM2. Current knowledge of the mechanistic details of MDM2’s inhibition of p53 are then reviewed, to shed light on how MDM2’s inhibition of p53 can be overcome either by the cell in response to DNA damage or by experimental design in an attempt to activate p53 as a potential cancer therapy.

Among tumors that retain two wild-type alleles of p53, many overexpress the MDM2 oncoprotein (54, 55, 56, 57, 58, 59, 60, 61). Through an examination of cell lines derived from such tumors (Table 1), the effect of MDM2 overexpression on the response of p53 to DNA damage can be determined.

Because MDM2 targets p53 for degradation, one prediction is that high levels of MDM2 protein will prevent the stabilization and subsequent increase of p53 that occurs after DNA damage. This predicted effect, however, is not always observed. In the HCT-116 colon carcinoma cell line, the CaCl 7336 melanoma cell line, and the SJSA osteosarcoma cell line, which contain Mdm2 gene amplification or MDM2 overexpression due to enhanced translation (62, 63), p53 protein levels rise in response to DNA-damaging agents. Treatment of these cell lines with 20 J/m2 UV irradiation results in increased p53 levels of at least 2-fold in the HCT-116 cell line, 6-fold in the CaCl 7336 cell lines, and 11-fold in the SJSA cell line, as determined by quantitative Western blot analysis (Fig. 1, top row). No p53 protein was detected in the p53-null H1299 lung carcinoma cell line after UV irradiation as expected (the p53 gene is deleted). After a similar time course of treatment with 10 μm etoposide, the levels of p53 protein increased at least 2-fold, 3-fold, and 8-fold, respectively in the same cell lines (Fig. 1, top row). These results indicate that the 20–100-fold overexpressed MDM2 protein in these cells is unable to degrade p53 and prevent the observed increase in p53 levels after DNA damage.

In addition, it is evident that the levels of p53 and MDM2 protein are inversely proportional in these same cell lines before damage, suggesting that MDM2 plays a role in regulating the low steady-state levels of p53 in the absence of DNA damage. Interestingly, the observed increase in p53 protein levels after DNA damage correlates directly with the level of MDM2 overexpression, such that the cell line with the most MDM2 protein (SJSA) has the greatest fold increase in p53 levels (Fig. 1). Because the different cell lines have approximately equal levels of p53 protein after DNA damage, there appears to be a complete inhibition of MDM2’s ability to degrade p53 after DNA damage, which allows for a substantial increase in p53 levels (Fig. 1).

As well as targeting p53 for degradation, MDM2 also binds to p53 in the nucleus and prevents its ability to activate transcription (45, 49). To determine whether this activity of MDM2 is, like its ability to degrade p53, inhibited after DNA damage, the levels of p53-responsive proteins were observed by Western blot analysis in the same cell lines described above. The level of the MDM2 protein, whose gene is responsive to p53 (38, 39), was observed in the same lysates (Fig. 1, second row). After UV irradiation, MDM2 protein levels fall (in a p53-independent manner), as observed at early time points in Fig. 1 and as has been reported previously (64). At later times after UV irradiation, MDM2 levels increase in each of the cell lines that contain wild-type p53 but not in the p53-null H1299 cell line. A similar, p53-independent (transcriptional) increase in MDM2 levels was observed in these same cells after etoposide treatment. In addition, the levels of p21waf1, the product of another p53-responsive gene (31), rose after both of these DNA-damaging treatments in a p53-dependent manner (Figure 1, third row). The levels of the small GTP-binding protein ran, which is not responsive to p53, was determined from the same lysates as a control, and did not change upon DNA damage (Figure 1, last row). These observations clearly demonstrate that the p53 protein is not only stabilized after DNA damage but is also active as a transcription factor in cell lines that contain high levels of its negative regulator, MDM2. This result demonstrates that the p53-MDM2 autoregulatory feedback loop is modulated in response to DNA damage to allow for the appropriate p53 responses. Some cellular activity, therefore, regulates the p53-MDM2 feedback loop.

To discern how the cell allows for the inhibition of MDM2’s regulation of p53 after DNA damage, an understanding of the details of this regulation is required. In the past several years, a great deal has been learned about the mechanisms by which MDM2 inhibits p53’s activities.

The NH2-terminal domain of MDM2 forms a deep cleft lined with hydrophobic and aromatic amino acids that interact directly with an amphipathic α-helix formed by the NH2 terminus of the p53 protein (42, 65, 66). Through this interaction, MDM2 inhibits the transcriptional activation of p53 both in cotransfection experiments and in an in vitro transcription assay (43, 44, 45, 49). This inhibition of p53 transcriptional activity by MDM2 is thought to result from the blocking of the transactivation domain of p53 (47, 67). MDM2 binds to the same amino acids that p53 uses to contact TAFII31 and TAFII70 and activate transcription (47, 48, 67), suggesting that MDM2 competes with the transcription factor IID transcription factor complex for p53 interaction and, thereby, blocks its ability to activate transcription.

MDM2’s interaction with p53 can also result in the degradation of the p53 protein (50, 51). A recently identified activity of MDM2, that of nuclear-cytoplasmic shuttling, is required for the degradation of p53 by MDM2 and has shed light on the mechanism of this p53 degradation pathway (68, 69). A conserved, leucine-rich sequence in MDM2 resembles a NES3 sequence from several proteins that are exported from the nucleus into the cytoplasm (Refs. 70, 71, 72, 73, 74; Fig. 2,A). MDM2’s ability to shuttle between the nuclear and cytoplasmic compartments of the cell as mediated by its NES and NLS sequences can be observed using heterokaryon assays (68). When human cells are transfected with a human MDM2 expression plasmid and subsequently fused to untransfected murine cells in the presence of cycloheximide, the human MDM2 protein can be detected in both the human and murine nuclei of heterokaryons (Ref. 68; Fig. 2,B, top). This localization pattern occurs as the exogenous MDM2 protein is exported from the human nucleus, in which it is originally located, via its NES to the cytoplasm and then into the murine nucleus using its NLS. The nuclear export of MDM2 is dependent on its NES sequence, as mutations in this sequence (see Fig. 2A) prevent the export of the exogenous MDM2 from the human nucleus in this assay (Ref. 68; Fig. 2 B, bottom).

The nuclear export of MDM2 occurs on a pathway that is used by HIV-rev, as it is blocked by an inhibitor of rev export, which consists of the NLS of SV40 large T antigen fused to NES-containing HTLV1 rex protein (68). HIV-rev, as well as many other proteins that contain homologous NES sequences, are exported from the nucleus via a pathway that has been recently described in some detail (75, 76, 77). CRM1 binds to NES sequences in a RanGTP-dependent fashion, and the protein complex exits through the nuclear pore (75, 76, 77). Hydrolysis of the GTP is thought to trigger the separation of the complex and the release of the NES-containing protein in the cytoplasm. MDM2 is predicted to be exported from the nucleus via this pathway, although direct demonstration of this has not yet been shown.

The impact of MDM2’s ability to shuttle between the nucleus and cytoplasm in the regulation of p53 was subsequently studied. The nuclear export of MDM2 has a rather complicated effect upon the ability of p53 to activate the transcription of a reporter gene during transfection assays. When relatively low levels of p53 were present, MDM2 with a mutated NES (which is retained in the nucleus; Fig. 2) was a better inhibitor of p53 activity than was wild-type MDM2. This observation is likely a result of MDM2’s direct block of p53’s interaction with the transcription machinery in the cell nucleus (68). However, when p53 was present at higher levels, the mutant NES form of MDM2 was a less efficient inhibitor of p53 activity than wild-type MDM2 (68). This result was likely due to: (a) an excess of p53, the transcription activity of which could not be blocked by MDM2 (this requires a 1:1 stoichiometric ratio of p53 to MDM2); and (b) an inability of MDM2 to target p53 for degradation when it lacked nuclear export ability.

To determine whether the nuclear export of MDM2 is, in fact, required for the degradation of p53, p53 protein levels were determined after its expression alone or with either wild-type or the NES-mutant form of MDM2 in the p53-null Saos-2 cells. Equivalent levels of the two forms of MDM2 were expressed during the experiment (Fig. 3). Coexpression of p53 and wild-type MDM2 led to a sharp decrease in the levels of p53 protein compared to the levels of p53 in the absence of MDM2 (Ref. 68; Fig. 3). However, when the mutant-NES form of MDM2 was coexpressed with p53 in these cells, the levels of the protein were unaffected (Fig. 3). The proteasome inhibitor MG132 blocked the reduction in p53 levels observed in the presence of wild-type MDM2. This last result suggests that the decrease in p53 levels by MDM2 is due to proteasomal degradation in the cytoplasm, consistent with previous results (50, 51). Additional support for the assertion that nuclear export of MDM2 is required for the degradation of p53 is that expression of the nuclear export inhibitor fusion protein, NLS-rex, inhibits MDM2’s nuclear export and also blocks the degradation of p53 by MDM2 (68).

Because MDM2’s ability to target p53 for degradation requires its nuclear export ability in the overexpression assays described above, MDM2 likely shuttles p53 to the cytoplasm for subsequent degradation by cytoplasmic proteasomes. If this model is correct, a block of nuclear export should cause an increase in endogenous p53 protein levels in the absence of DNA damage. The drug LMB has been shown to block the export from the nucleus of proteins that contain HIV rev-like NES sequences by blocking the formation of the ternary nuclear export complexes consisting of CRM1, RanGTP, and NES-containing proteins (75, 76, 77). The addition of LMB to cells should therefore inhibit the nuclear export of MDM2, which is predicted to cause p53 protein stabilization.

Consistent with the model in which MDM2 mediates the steady-state levels of p53 by shuttling it from the nucleus for degradation by cytoplasmic proteasomes, the addition of 5 ng/ml LMB to a variety of cell lines containing wild-type (SJSA and the MCF-7 breast carcinoma cell line) or mutant (Sk-mel-2 melanoma cell line) p53 led to a marked increase in the steady-state levels of the p53 protein (Ref. 69; Fig. 4A). Subsequent p53-dependent increases in MDM2 and p21waf1 protein levels were also observed in these cells (Fig. 4 A), suggesting that the p53 produced was functional and was located in the nucleus. This result was confirmed by immunofluorescent studies and substantiates the concepts developed here (69).

The increase in p53 levels by LMB, seen as early as 3 h after the addition of the drug, in fact results from a block of the p53 protein degradation. The addition of LMB to the 12(1) immortalized mouse 3T3 cell line increased the half-life of p53 4-fold, from ∼1 h to almost 4 h (Ref. 69; Fig. 4 B). These results support the validity of the model in which MDM2 and p53 first interact in the nucleus and then shuttle to the cytoplasm, where p53 is then targeted to cytoplasmic proteasomes for degradation.

Thus two mechanisms combine to regulate p53 activity or levels using MDM2: (a) competition for p53 binding to the TAFs and (b) shuttling p53 from the nucleus to the cytoplasm for proteasome degradation. This then provides the cell with several places to block the inhibition of p53 by MDM2 after DNA damage (Fig. 5). For instance, blocking the formation of MDM2-p53 complexes would prevent both of these events. In fact, blocking complex formation by the microinjection of monoclonal antibodies to MDM2 or by transfection of a plasmid expressing a competing, p53-like peptide leads to an increase in p53-dependent transcriptional activity (78, 79). Recent evidence demonstrates that a block in the formation of the MDM2-p53 complex can occur in cells by a phosphorylation event at one or two serine residues located in the NH2 terminus of p53, which is observed after DNA damage (80).

The relative levels of MDM2 and p53 protein in a cell can also control the inhibition of p53 by MDM2. A reduction in MDM2 levels by the transfection of antisense MDM2 constructs increases p53 levels and activity in a variety of cell lines, including those that overexpress MDM2 (81). Cells may, in fact, use a related mechanism after DNA damage to relieve MDM2’s inhibition of p53. MDM2 mRNA and protein levels drop rapidly after UV irradiation of cells in a p53-independent manner, just prior to the increase in p53 protein levels (Ref. 64; Fig. 1).

Finally, nuclear export of MDM2, as a requirement for MDM2’s degradation of p53, presents another potential step for regulation. The addition of the nuclear export inhibitor, LMB, does, in fact, stabilize the p53 protein and increase p53-dependent transcriptional activation (Ref. 69; Fig. 4). Whether or not cells block nuclear export by MDM2 as an additional layer of control over the inhibition of p53 remains to be determined.

The experiments presented and reviewed here have led to the model presented in Fig. 5. In a normal cell, low levels of inactive p53 proteins are maintained by the MDM2 protein, which blocks p53 transcriptional activity in the nucleus and shuttles p53 into the cytoplasm to be degraded by the cytoplasmic proteasome. In response to many types of DNA damage, p53 protein levels rise and p53 activities become functional. DNA damage is detected by a diverse set of repair activities that are specific for the type of DNA damage and that signal to the p53 protein that a specific type of DNA damage has occurred (Table 2). After γ irradiation, the ATM protein kinase appears to phosphorylate the p53 protein at Ser-15 and Ser-31 residues, which, in turn, blocks p53 from binding to MDM2. These events result in increased p53 activities and levels. After UV irradiation, the MDM2 protein is degraded and MDM2 mRNA levels fall (55). In addition, Ser-392 (at the p53 COOH terminus) of p53 is phosphorylated (82), probably by the cyclin H-CDK7 protein kinase, which is part of the nucleotide excision repair complex. Phosphorylation of Ser-392 activates p53 for binding to specific DNA sequences, a prelude to transcriptional activation (83, 84). This same enzyme also phosphorylates the NH2 terminus of p53 (85), blocking p53-MDM2 interactions and stabilizing p53. DNA containing apurinic acid sites are repaired by the apurinic acid sites nuclease, Ref-1, which can regulate the redox potential of p53. This event, in turn, activates p53 for transcription (86). There remain several other possible ways to regulate p53 activities. The histone acetyl-transferases such as the p300 coactivator, acetylate the COOH-terminal lysines of p53 activating it for specific DNA binding (87, 88). A block in the ability of MDM2 to shuttle into the cytoplasm, as shown here, will result in activation of p53 (69). This implies that regulation of the CRM1-RanGTP shuttling pathway could well result in p53 activation (Fig. 5, step B).

Although there is a growing list of potential regulatory events during the activation of p53 by DNA damage (Fig. 5, AFig. 5.,BFig. 5.,CFig. 5. via step B), very little is known about the reversal of this process (Fig. 5, CFig. 5.,BFig. 5.,AFig. 5. via step D). MDM2 levels increase in a p53-dependent fashion forming an autoregulatory loop that acts in a sinusoidal fashion. The interruption of this process favoring different states (A or C) is fertile ground for future research. Indeed, little is known about the regulation of MDM2 protein stability or Mdm2 gene transcription, but clearly, both play a role in p53 activation and inhibition.

In recent years, there have been rather clear suggestions that the MDM2 protein may have p53-independent functions that are related to cell division and the cancer phenotype. Elucidation of these functions will surely open new pathways of understanding cancer biology. The regulation of MDM2 mediated p53 activity by p19Arf (89, 90) has begun to connect the role of p53 back to a broader spectrum of mutations in the cancer cell. This is clearly an area of research that will open new insights into the cellular regulatory pathways that are defective in cancer cells.

Finally, agents that activate p53 in cancer cells all have the potential to selectively kill these cells. Whether this relies upon breaking p53-MDM2 complexes (78, 79), reactivating inactive forms of wild-type p53 in a cell (91), or even introducing a wild-type p53 gene into a cancer cell (92), these approaches are the beginning of a rational basis for therapy. It is clear that p53-mediated apoptosis is modulated by activated oncogene products not found in normal cells (23, 24, 25). It is this fundamental observation that could lead us to drugs or agents with true selective toxicity for tumor cells (expressing activated oncogene products) over normal cells. The generation of such selective drugs must be our clearest goal.

Fig. 1.

p53 responds to DNA damage in cell lines that overexpress MDM2. Cells were treated with 10 μm etoposide or 20 J/m2 UV light at various times before they were harvested, and cell lysates were made. Total protein was separated by SDS-PAGE and transferred to nitrocellulose for Western analysis. The p53 protein was detected with a combination of mAb421 and mAb1801; MDM2 was detected with a combination of 2A9 and 5B10; p21waf1 was detected with polyclonal sera; and ran was detected with a monoclonal antibody from Transduction Laboratories, as described (69). In the wild-type p53-containing SJSA, CaCl 7336, and HCT-116 cell lines, p53 levels rise in response to both types of DNA damage as does MDM2 and p21waf1. H1299 cells lack p53, and the increases in MDM2 and p21waf1 are not observed. ran protein levels are shown as a control.

Fig. 1.

p53 responds to DNA damage in cell lines that overexpress MDM2. Cells were treated with 10 μm etoposide or 20 J/m2 UV light at various times before they were harvested, and cell lysates were made. Total protein was separated by SDS-PAGE and transferred to nitrocellulose for Western analysis. The p53 protein was detected with a combination of mAb421 and mAb1801; MDM2 was detected with a combination of 2A9 and 5B10; p21waf1 was detected with polyclonal sera; and ran was detected with a monoclonal antibody from Transduction Laboratories, as described (69). In the wild-type p53-containing SJSA, CaCl 7336, and HCT-116 cell lines, p53 levels rise in response to both types of DNA damage as does MDM2 and p21waf1. H1299 cells lack p53, and the increases in MDM2 and p21waf1 are not observed. ran protein levels are shown as a control.

Close modal
Fig. 2.

MDM2 is a nuclear-cytoplasmic shuttling protein. A, the leucine-rich NES sequences of several proteins are shown, with conserved hydrophobic residues in boldface type. A similar sequence is conserved in MDM2, shown from four species. The bottom line shows the mutations that were made in this sequence (mtNES); the altered residues are underlined. B, heterokaryon assays were performed as described (68). Briefly, human HeLa cells were transfected with a human-MDM2 expressing plasmid and then fused in the presence of cycloheximide to the murine BALB/c-3T3 cell line. After 45 min, the cells were fixed and stained with 2A9, which is specific for human MDM2 and with an antibody specific for the human nuclear Ku protein. When wild-type MDM2 is transfected (top), it can be exported from the human nucleus and enter the murine nucleus. Mutation of this sequence (bottom), however, prevents the MDM2 protein from leaving the human nucleus, and therefore, no staining is observed in the murine nucleus.

Fig. 2.

MDM2 is a nuclear-cytoplasmic shuttling protein. A, the leucine-rich NES sequences of several proteins are shown, with conserved hydrophobic residues in boldface type. A similar sequence is conserved in MDM2, shown from four species. The bottom line shows the mutations that were made in this sequence (mtNES); the altered residues are underlined. B, heterokaryon assays were performed as described (68). Briefly, human HeLa cells were transfected with a human-MDM2 expressing plasmid and then fused in the presence of cycloheximide to the murine BALB/c-3T3 cell line. After 45 min, the cells were fixed and stained with 2A9, which is specific for human MDM2 and with an antibody specific for the human nuclear Ku protein. When wild-type MDM2 is transfected (top), it can be exported from the human nucleus and enter the murine nucleus. Mutation of this sequence (bottom), however, prevents the MDM2 protein from leaving the human nucleus, and therefore, no staining is observed in the murine nucleus.

Close modal
Fig. 3.

Nuclear export of MDM2 is required for its ability to degrade p53. The p53-null Saos-2 cells were transfected with vectors only (Lane 1, from left to right), plasmids expressing p53 only (Lane 2), plasmids expressing p53 and wild-type MDM2 (Lane 3), or plasmids expressing p53 and the mutant-NES form of MDM2 (Lane 4). The steady-state levels of p53 and MDM2 proteins were then detected as described (68). Wild-type MDM2 led to a dramatic decrease in the levels of p53 (compare Lanes 2 and 3), whereas the mtNES form of MDM2 did not (compare Lanes 2 and 4). Approximately equal amounts of the two forms of MDM2 were expressed. The effect of wild-type MDM2 on p53 levels was blocked by the proteasome inhibitor MG132, indicating that the observed effect is due to p53 degradation by MDM2.

Fig. 3.

Nuclear export of MDM2 is required for its ability to degrade p53. The p53-null Saos-2 cells were transfected with vectors only (Lane 1, from left to right), plasmids expressing p53 only (Lane 2), plasmids expressing p53 and wild-type MDM2 (Lane 3), or plasmids expressing p53 and the mutant-NES form of MDM2 (Lane 4). The steady-state levels of p53 and MDM2 proteins were then detected as described (68). Wild-type MDM2 led to a dramatic decrease in the levels of p53 (compare Lanes 2 and 3), whereas the mtNES form of MDM2 did not (compare Lanes 2 and 4). Approximately equal amounts of the two forms of MDM2 were expressed. The effect of wild-type MDM2 on p53 levels was blocked by the proteasome inhibitor MG132, indicating that the observed effect is due to p53 degradation by MDM2.

Close modal
Fig. 4.

Nuclear export is required for the degradation of endogenous p53. A, the indicated cell lines were treated with the nuclear export inhibitor LMB at various times before they were harvested and cell lysates were made. The levels of endogenous proteins were determined by Western analysis as described (69). In this case, MDM2 was detected by immunoprecitation with polyclonal sera followed by Western analysis with antibodies 2A10 and 4B11. β-Catenin was detected by incubation with an antibody from Transduction Laboratories. The p53 protein levels rose in response to the addition of 5 ng/ml LMB in each cell line that contains p53. MDM2 and p21waf1 levels, however, only increased in the cells that contain wild-type p53 (SJSA and MCF-7) and not in the cell lines with mutant p53 (Sk-mel-2) or no p53 (H1299). ran and β-catenin are included as controls. B, pulse-chase analysis was performed in the 12(1) cell line with and without treatment with 5 ng/ml LMB as described (69). A statistically significant difference was observed in the amount of p53 remaining at each time point. The half-life of p53 increased from ∼1 to ∼4 h with the addition of the drug, indicating that the degradation of p53 depends on nuclear export.

Fig. 4.

Nuclear export is required for the degradation of endogenous p53. A, the indicated cell lines were treated with the nuclear export inhibitor LMB at various times before they were harvested and cell lysates were made. The levels of endogenous proteins were determined by Western analysis as described (69). In this case, MDM2 was detected by immunoprecitation with polyclonal sera followed by Western analysis with antibodies 2A10 and 4B11. β-Catenin was detected by incubation with an antibody from Transduction Laboratories. The p53 protein levels rose in response to the addition of 5 ng/ml LMB in each cell line that contains p53. MDM2 and p21waf1 levels, however, only increased in the cells that contain wild-type p53 (SJSA and MCF-7) and not in the cell lines with mutant p53 (Sk-mel-2) or no p53 (H1299). ran and β-catenin are included as controls. B, pulse-chase analysis was performed in the 12(1) cell line with and without treatment with 5 ng/ml LMB as described (69). A statistically significant difference was observed in the amount of p53 remaining at each time point. The half-life of p53 increased from ∼1 to ∼4 h with the addition of the drug, indicating that the degradation of p53 depends on nuclear export.

Close modal
Fig. 5.

A model of the p53-MDM2 autoregulatory feedback loop and its regulation. A, MDM2 is predicted to interact with p53 in the nucleus, where it prevents p53-activated transcription. MDM2 can also shuttle p53 through the nuclear pore, likely in a CRM1 and RanGTP-dependent manner, for degradation in cytoplasmic proteasomes. B, DNA damage leads to a suppression of these activities by MDM2. Possible mechanisms for this relief of MDM2’s inhibition of p53 are listed. C, without inhibition by MDM2, p53 remains in the nucleus and is active as a transcription factor and tumor suppressor, causing growth arrest or apoptosis. D, after the DNA is repaired, it is predicted that the suppression of MDM2’s inhibition of p53 is lifted, returning the autoregulatory feedback loop to its basal level.

Fig. 5.

A model of the p53-MDM2 autoregulatory feedback loop and its regulation. A, MDM2 is predicted to interact with p53 in the nucleus, where it prevents p53-activated transcription. MDM2 can also shuttle p53 through the nuclear pore, likely in a CRM1 and RanGTP-dependent manner, for degradation in cytoplasmic proteasomes. B, DNA damage leads to a suppression of these activities by MDM2. Possible mechanisms for this relief of MDM2’s inhibition of p53 are listed. C, without inhibition by MDM2, p53 remains in the nucleus and is active as a transcription factor and tumor suppressor, causing growth arrest or apoptosis. D, after the DNA is repaired, it is predicted that the suppression of MDM2’s inhibition of p53 is lifted, returning the autoregulatory feedback loop to its basal level.

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1

Presented by Dr. Arnold J. Levine at the 89th Annual Meeting of the American Association for Cancer Research, March 29, 1998, New Orleans, LA.

3

The abbreviations used are: NES, nuclear export signal; NLS, nuclear localization signal; LMB, leptomycin B.

Table 1

Cell lines with wild-type p53 and MDM2 overexpression

The cell lines listed here have been reported to both have wild-type p53 and overexpress MDM2. The mechanism of MDM2 overexpression is listed, as is the tumor type from which it was derived. The references are also given.
Cell line MDM2 Tumor type (Ref.) 
SJSA (OsA) Amplified Osteosarcoma  62  
U2OS Overexpressed Osteosarcoma  63  
HCT-116 Overexpressed Colon carcinoma  63  
A875 Overexpressed Melanoma  63  
CaCl 7336 Overexpressed Melanoma  63  
WM 8 Overexpressed Melanoma  63  
WM 9 Overexpressed Melanoma  63  
WM 793 Overexpressed Melanoma  63  
WM 115 Overexpressed Melanoma  63  
WM 239 Overexpressed Melanoma  63  
WM 938B Overexpressed Melanoma  63  
JEG3 Overexpressed Choriocarcinoma  60  
JAR Amplified Choriocarcinoma  60  
MCF-7 Overexpressed Breast carcinoma  93  
ZR-75 Overexpressed Breast carcinoma  93  
CCF-STTG1 Amplified Glioblastoma (?)  94  
TP-3G5 Amplified Glioblastoma  94  
LS Amplified Neuroblastoma  95  
NGP Amplified Neuroblastoma  95  
TR14 Amplified Neuroblastoma  95  
RH-18 Amplified Rhabdomyosarcoma  96  
RH-28 Overexpressed Rhabdomyosarcoma  96  
RH-36 Overexpressed Rhabdomyosarcoma  96  
The cell lines listed here have been reported to both have wild-type p53 and overexpress MDM2. The mechanism of MDM2 overexpression is listed, as is the tumor type from which it was derived. The references are also given.
Cell line MDM2 Tumor type (Ref.) 
SJSA (OsA) Amplified Osteosarcoma  62  
U2OS Overexpressed Osteosarcoma  63  
HCT-116 Overexpressed Colon carcinoma  63  
A875 Overexpressed Melanoma  63  
CaCl 7336 Overexpressed Melanoma  63  
WM 8 Overexpressed Melanoma  63  
WM 9 Overexpressed Melanoma  63  
WM 793 Overexpressed Melanoma  63  
WM 115 Overexpressed Melanoma  63  
WM 239 Overexpressed Melanoma  63  
WM 938B Overexpressed Melanoma  63  
JEG3 Overexpressed Choriocarcinoma  60  
JAR Amplified Choriocarcinoma  60  
MCF-7 Overexpressed Breast carcinoma  93  
ZR-75 Overexpressed Breast carcinoma  93  
CCF-STTG1 Amplified Glioblastoma (?)  94  
TP-3G5 Amplified Glioblastoma  94  
LS Amplified Neuroblastoma  95  
NGP Amplified Neuroblastoma  95  
TR14 Amplified Neuroblastoma  95  
RH-18 Amplified Rhabdomyosarcoma  96  
RH-28 Overexpressed Rhabdomyosarcoma  96  
RH-36 Overexpressed Rhabdomyosarcoma  96  
Table 2

Regulation of p53 after DNA damage

The potential signaling molecules that regulate p53 after different types of DNA damage are listed. The changes these molecules make on p53 and the outcomes of the events are listed along with the relevant references.
DNA damage
γ radiationUV radiationApurinic acid
Potential signal molecules ATM;p300/PCAF Cyclin H-CDK7; p300/PCAF Ref-1 
p53 changes Phosphorylation at Ser-15 and -31 Phosphorylation at Ser-392 Redox activation 
  Acetylation at Ser-33 and -37  
Results Acetylation at Ser-33 and -37 Altered p53 conformation Altered conformation of p53 
 MDM2/p53 interaction blocked Enhanced DNA binding  
 Enhanced DNA binding Reduction in MDM2 mRNA  
Refs. 80, 87, and 88 82, 85, 87, and 88  86  
The potential signaling molecules that regulate p53 after different types of DNA damage are listed. The changes these molecules make on p53 and the outcomes of the events are listed along with the relevant references.
DNA damage
γ radiationUV radiationApurinic acid
Potential signal molecules ATM;p300/PCAF Cyclin H-CDK7; p300/PCAF Ref-1 
p53 changes Phosphorylation at Ser-15 and -31 Phosphorylation at Ser-392 Redox activation 
  Acetylation at Ser-33 and -37  
Results Acetylation at Ser-33 and -37 Altered p53 conformation Altered conformation of p53 
 MDM2/p53 interaction blocked Enhanced DNA binding  
 Enhanced DNA binding Reduction in MDM2 mRNA  
Refs. 80, 87, and 88 82, 85, 87, and 88  86  
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