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An assay for the autoubiquitination activity of the E3 ligaseHDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used tomeasure the activity of other E3s andmay be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.

Overexpression of ectopic mutant p53 represses wild-type p53-stimulated transcription, known as a dominant negative effect. On the other hand, overexpression of wild-type p53 can repress transcription stimulated by several transcription factors, including hypoxia-inducible factor-1 (HIF-1). Using a panel of well-characterized Arg175 p53 mutants we found that only mutants (Tyr175, Trp175, Asp175 and Phe175) which have completely lost their ability to transactivate repress wild-type p53-stimulated Bax, p21 and PG13 promoter constructs. In contrast, Asn175, Gln175, Leu175 and Pro175 mutants which partially retained transactivating functions did not exert dominant negative effects against PG13 and p21 promoter constructs. However, these latter mutants failed to activate Bax and, instead, exerted a dominant negative effect on a Bax-Luc promoter construct. We conclude that a dominant negative effect is promoter selective as a consequence of selective loss of transactivating function. Albeit less potent than wild-type p53, all Arg175 p53 mutants retained partial ability to repress HIF-1-stimulated transcription. We propose that transrepression and the dominant negative effect have similar mechanisms and may involve competition with transcription factors (wild-type p53, HIF-1, etc.) for cofactors such as p300. Thus, a p53(22/23) mutant, which is deficient in p300 binding, did not exert dominant negative effects. Like transrepression, the dominant negative effect required overexpression of mutant p53 and, therefore, is not dominant. In the presence of a wild-type p53 allele, levels of endogenous mutant p53 protein were low in heterozygous cells. Endogenous mutant p53 became overexpressed only after loss of the second p53 allele. Therefore, endogenous mutant p53s are unable to display a dominant negative effect. This explains why loss of the second p53 allele is required to eliminate p53 functions in cancer cells.

Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.

The loss of p53-mediated apoptosis (programmed cell death) has been implicated as an important event in tumour progression in a number of systems. p53 can induce or potentiate apoptosis through several mechanisms, both by regulating the expression of genes which can participate in the apoptotic response and through transcriptionally independent means. There appears to be cell type variability in both the response to p53 expression and in the requirement for p53 transcriptional transactivation for the induction of apoptosis. It seems clear, however, that the induction of p53 in untransformed cells is more likely to result in cell-cycle arrest, whereas the expression of p53 in their transformed counterparts is more likely to result in the induction of apoptosis, and this may, in part, reflect the deregulated expression of E2F-1 in tumour cells. The synergistic action of p53 and E2F-1 in the induction of apoptosis has raised the possibility that the reactivation of p53 in transformed cells can be an effective tumour therapy.

The p53 tumor‐suppressor gene product is frequently inactivated in malignancies by point mutation. Although most tumor‐derived p53 mutants show loss of sequence specific transcriptional activation, some mutants have been identified which retain this activity. One such mutant, p53175P, is defective for the suppression of transformation in rodent cells, despite retaining the ability to suppress the growth of p53‐null human cells. We now demonstrate that p53175P can induce a cell‐cycle arrest in appropriate cell types but shows loss of apoptotic function. Our results therefore support a direct role of p53 transcriptional activation in mediating a cell‐cycle arrest and demonstrate that such activity is not sufficient for the full apoptotic response. These data suggest that either p53 can induce apoptosis through a transcriptionally independent mechanism, a function lost by p53175P, or that this mutant has specifically lost the ability to activate genes which contribute to cell death, despite activation of genes responsible for the G1 arrest. This dissociation of the cell‐cycle arrest and apoptotic activities of p53 indicates that inactivation of p53 apoptotic function without concomitant loss of growth inhibition can suffice to relieve p53‐dependent tumor‐suppression in vivo and thereby contribute to tumor development.

The p53 tumor suppressor protein can induce both cell cycle arrest and apoptosis in DNA-damaged cells. In human carcinoma cell lines expressing wild-type p53, expression of E7 allowed the continuation of full cell cycle progression following DNA damage, indicating that E7 can overcome both G1 and G2 blocks imposed by p53. E7 does not interfere with the initial steps of the p53 response, however, and E7 expressing cells showed enhanced expression of p21(waf1/cip1) and reductions in cyclin E- and A-associated kinase activities following DNA damage. One function of cyclin-dependent kinases is to phosphorylate pRB and activate E2F, thus allowing entry into DNA synthesis. Although E7 may substitute for this activity during cell division by directly targeting pRB, continued cell cycle progression in E7-expressing cells was associated with phosphorylation of pRB, suggesting that E7 permits the retention of some cyclin-dependent kinase activity. One source of this activity may be the E7-associated kinase, which was not inhibited following DNA damage. Despite allowing cell cycle progression, E7 was unable to protect cells from p53-induced apoptosis, and the elevated apoptotic response seen in these cells correlated with the reduction of cyclin A-associated kinase activity. It is possible that inefficient cyclin A-dependent inactivation of E2F at the end of DNA synthesis contributes to the enhanced apoptosis displayed by E7-expressing cells.

The effect of a potent mammary carcinogen, anti benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide, on the progress of human mammary carcinoma MCF-7 cells through the cell cycle was investigated. While these cells, which express wild-type p53, were arrested in G1 after treatment with actinomycin D (a positive control), treatment with the mammary carcinogen did not cause G1 arrest but instead delayed the cells in the DNA synthesis phase. In concert with the absence of a G1 arrest, it was found that though both chemical treatments led to increased levels of p53, only the p53 induced by actinomycin D was transcriptionally active and increased the levels of the cyclin dependent kinase inhibitor, p21(waf1/cip1). Since treatment of the cells with the mammary carcinogen did not abrogate the G1 arrest induced by actinomycin D, the lack of p21(waf1/cip1) and of G1 arrest, resulting from treatment with the mammary carcinogen alone, was not due to some general inhibition of transcription or translation. An analogous difference between these two chemicals was demonstrated also in other human cell systems. The stealth-like property of the mammary carcinogen that allows it to damage DNA without turning on the cells' 'guardian of the genome' defense mechanism presumably increases the likelihood of malignant change because DNA replication continues on a damaged template. It is suggested that this stealth characteristic may be a major contributor to the high carcinogenic potency of this mammary carcinogen and possibly to that of other highly potent carcinogens.

The p53 tumor suppressor protein is a sequence-specific transcriptional activator, a function which contributes to cell cycle arrest and apoptosis induced by p53 in appropriate cell types. Analysis of a series of p53 point mutants has revealed the potential for selective loss of the ability to transactivate some, but not all, cellular p53-responsive promoters. p53 175P and p53 181L are tumor-derived p53 point mutants which were previously characterized as transcriptionally active. Both mutants retained the ability to activate expression of the cyclin-dependent kinase inhibitor p2lcip1/waf1, and this activity correlated with the ability to induce a G1 cell cycle arrest. However, an extension of this survey to include other p53 targets showed that p53 175P was defective in the activation of p53-responsive sequences derived from the bax promoter and the insulin-like growth factor-binding protein 3 gene (IGF-BP3) promoter, while p53 181L showed loss of the ability to activate a promoter containing IGF-BP3 box B sequences. Failure to activate transcription was also reflected in the reduced ability of the mutants to bind the p53-responsive DNA sequences present in these promoters. These specific defects in transcriptional activation correlated with the impaired apoptotic function displayed by these mutants, and the results suggest that activation of cell cycle arrest genes by p53 can be separated from activation of genes with a role in mediating the p53 apoptotic response. The cellular response to p53 activation may therefore depend, at least in part, on which group of p53-responsive genes become transcriptionally activated.

The transforming function of human papillomavirus type 16 (HPV16) E7 has been shown to depend on activities additional to the ability to bind RB. In this paper we describe two further properties of E7 which may also contribute to transformation, an association with a histone H1 kinase at the G2/M phase of the cell cycle and an ability to bind the RB-related protein p107. The region of E7 identified previously as important for RB binding was found to be involved in the association with the kinase and complex formation with p107, although analysis of E7 point mutants within this region revealed a difference in the precise sequence requirement for RB and p107 binding. Association with the kinase activity correlated with the ability to bind RB, but the restriction of the kinase association to the G2/M phase of the cell cycle implies that this activity might not be directly mediated by RB binding. Since kinase-binding-deficient E7 mutants are also transformation defective, this may represent an independent function of E7 which plays a role in the G2/M phase of the cell cycle.

The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E2F-1, suggesting that it may be mediated through alleviation of E2F-dependent transcriptional repression. These results indicate that E2F-1 can show independent cell cycle progression and apoptotic functions, consistent with its putative role as a tumor suppressor.

In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7, t-8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (anti-BPDE) generated a transforming oncogene when the modified DNA was transfected into NIH 3T3 cells. The protooncogene was also activated by heating the plasmid at 70 degrees C, pH 4, to generate apurinic/apyrimidinic sites in the DNA. DNA isolated from transformed foci was analyzed by hybridization with 20-mer oligonucleotides designed to detect single point mutations within two regions of the gene commonly found to be mutated in tumor DNA. Of 23 transformants studied, 7 contained a mutation in the region of the 12th codon, whereas the remaining 16 were mutated in the 61st codon. Of the codon-61 mutants, 6 were mutated at the first base position (C X G), 5 at the second (A X T), and 5 at the third (G X C). The point mutations induced by anti-BPDE were predominantly G X C----T X A and A X T----T X A base substitutions, whereas four N-acetoxy-2-acetylaminofluorene-induced mutations were all G X C----T X A, and a single depurination-induced activation that was analyzed contained an A X T----T X A transversion. Together, these methods provide a useful means of determining point mutations produced by DNA-damaging agents in mammalian cells.

The E7 open reading frame of human papillomavirus type 16 (HPV16) has been shown to be selectively retained in cervical tumors and to encode both transforming and trans-activating functions in murine cells, supporting the notion that expression of E7 contributes towards the progression of premalignant cervical lesions. A comparison among E7 sequences of different HPV types reveals some homology at the amino acid level. Of particular interest are two regions, one which contains significant homology to a region of adenovirus E1a and simian virus 40 large T (LT), and a second region which contains two conserved Cys-X-X-Cys motifs. To determine the importance of these domains to the function of the E7 protein, a series of mutants carrying substitutions at amino acids in the region of E1a-LT homology and at the Cys-X-X-Cys motifs were constructed. The mutated E7 sequences were placed under the control of a strong heterologous promoter (Moloney long terminal repeat), and the activity of the mutants was assayed in NIH 3T3 cells, a cell line in which both the transforming function and the trans-activating function of E7 could be determined. A single amino acid substitution analogous to a mutation in E1a which destroys the transforming ability of this protein abolished both transformation and trans-activation by E7. Mutations at the Cys-X-X-Cys motifs demonstrated that this region contributes to the transforming potential of E7, although proteins in which both motifs were interrupted retained a low level of transforming activity. Mutations in the region of E1a-LT homology which occur within a recognition sequence for casein kinase II did not markedly affect transforming activity of E7 but severely reduced trans-activating ability. This indicates that efficient trans-activation is not required for transformation by HPV16 E7 in these cells.

We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low-molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not.

The p53 tumor suppressor protein is activated in cells in response to DNA damage and prevents the replication of cells sustaining genetic damage by inducing a cell cycle arrest or apoptosis. Activation of p53 is accompanied by stabilization of the protein, resulting in accumulation to high levels within the cell. p53 is normally degraded through the proteasome following ubiquitination, although the mechanisms which regulate this proteolysis in normal cells and how the p53 protein becomes stabilized following DNA damage are not well understood. We show here that p53 can also be a substrate for cleavage by the calcium-activated neutral protease, calpain, and that a preferential site for calpain cleavage exists within the N terminus of the p53 protein. Treatment of cells expressing wild-type p53 with an inhibitor of calpain resulted in the stabilization of the p53 protein. By contrast, in vitro or in vivo degradation mediated by human papillomavirus E6 protein was unaffected by the calpain inhibitor, indicating that the stabilization did not result from inhibition of the proteasome. These results suggest that calpain cleavage plays a role in regulating p53 stability.

The E7 protein is one of the principle transforming proteins encoded by human papillomavirus type 16 (HPV16), a virus strongly associated with the development of cervical carcinoma. In the present study we show that cotransfection of wild-type human or murine p53 sequences with E7 and ras markedly reduces transformation in baby rat kidney cells, although no effect of p53 is seen on the ability of E7 to transform an established mouse line to anchorage independence. In contrast, expression of mutant p53 strongly potentiates the transforming function of E7 and confers marked growth factor independence to cells cotransformed by E7 and ras. These data suggest that E7 and p53 function in separate yet complementary biochemical pathways.

The bovine papillomavirus E6 gene can independently transform mouse C127 cells. To characterize E6 in greater detail, we created 16 site-directed mutations in E6, including substitution mutations in the cysteine codons of the four Cys-X-X-Cys motifs that are conserved in all papillomavirus E6 proteins. Proteins mutated in six of the seven cysteines tested, as well as those lacking the nonconserved C-terminus, were stable in transfected cells but were unable to induce morphological transformation, indicating that these amino acids play an important role in the function of E6.