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The stability of the p53 tumor suppressor protein is regulated by interaction with Mdm2, the product of a p53-inducible gene. Mdm2-targeted degradation of p53 depends on the interaction between the two proteins and is mediated by the proteasome. We show here that in addition to the N-terminal Mdm2 binding domain, the C terminus of p53 participates in the ability of p53 to be degraded by Mdm2. In contrast, alterations in the central DNA binding domain of p53, which change the conformation of the p53 protein, do not abrogate the sensitivity of the protein to Mdm2-mediated degradation. The importance of the C-terminal oligomerization domain to Mdm2-targeted degradation of p53 is likely to reflect the importance of oligomerization of the full-length p53 protein for interaction with Mdm2, as previously shown in vitro. Interestingly, the extreme C-terminal region of p53, outside the oligomerization domain, was also shown to be necessary for efficient degradation, and deletion of this region stabilized the protein without abrogating its ability to bind to Mdm2. Mdm2-resistant p53 mutants were not further stabilized following DNA damage, supporting a role for Mdm2 as the principal regulator of p53 stability in cells. The extreme C terminus of the p53 protein has previously been shown to contain several regulatory elements, raising the possibility that either allosteric regulation of p53 by this domain or interaction between this region and a third protein plays a role in determining the sensitivity of p53 to Mdm2-directed degradation.

The two distinct proteins encoded by the CDKN2A locus are specified by translating the common second exon in alternative reading frames. The product of the alpha transcript, p16(INK4a), is a recognized tumour suppressor that induces a G1 cell cycle arrest by inhibiting the phosphorylation of the retinoblastoma protein by the cyclin-dependent kinases, CDK4 and CDK6. In contrast, the product of the human CDKN2A beta transcript, p14(ARF), activates a p53 response manifest in elevated levels of MDM2 and p21(CIP1) and cell cycle arrest in both G1 and G2/M. As a consequence, p14(ARF)-induced cell cycle arrest is p53 dependent and can be abrogated by the co-expression of human papilloma virus E6 protein. p14(ARF) acts by binding directly to MDM2, resulting in the stabilization of both p53 and MDM2. Conversely, p53 negatively regulates p14(ARF) expression and there is an inverse correlation between p14(ARF) expression and p53 function in human tumour cell lines. However, p14(ARF) expression is not involved in the response to DNA damage. These results place p14(ARF) in an independent pathway upstream of p53 and imply that CDKN2A encodes two proteins that are involved in tumour suppression.

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest, functions which have been shown to be separable activities of p53. We have characterized a series of p53 mutants with amino acid substitutions at residue 175 and show that these mutants fall into one of three classes: class I, which is essentially wild type for apoptotic and cell cycle arrest functions; class II, which retains cell cycle arrest activity but is impaired in the induction of apoptosis; and class III, which is defective in both activities. Several residue 175 mutants which retain cell cycle arrest function have been detected in cancers, and we show that these have lost apoptotic function. Furthermore, several class II mutants have been found to be temperature sensitive for apoptotic activity while showing constitutive cell cycle arrest function. Taken together, these mutants comprise an excellent system with which to investigate the biochemical nature of p53-mediated apoptosis, the function of principal importance in tumor suppression. All of the mutants that showed loss of apoptotic function also showed defects in the activation of promoters from the potential apoptotic targets Bax and the insulin-like growth factor-binding protein 3 gene (IGF-BP3), and a correlation between full apoptotic activity and activation of both of these promoters was also seen with the temperature-sensitive mutants. However, a role for additional apoptotic activities of p53 was suggested by the observation that some mutants retained significant apoptotic function despite being impaired in the activation of Bax- and IGF-BP3-derived promoters. In contrast to the case of transcriptional activation, a perfect correlation between transcriptional repression of the c-fos promoter and the ability to induce apoptosis was seen, although the observation that Bax expression induced a similar repression of transcription from this promoter suggests that this may be a consequence, rather than a cause, of apoptotic death.

The p53 tumor suppressor protein can adopt both latent, non-DNA binding and active, DNA binding forms, and p53 activity is thought to be regulated in cells, at least in part, through a conformational shift which leads to sequence specific DNA binding. In vitro, this allosteric regulation of DNA binding by p53 has been shown to be mediated through the C-terminus of the protein. We show here that although deletion of the C-terminal 16 amino acids of p53 did not activate DNA binding, deletion of a further eight amino acids resulted in constitutive activation of DNA binding activity. Simultaneous mutation of the three lysine residues within these eight amino acids also resulted in constitutive DNA binding activity, although this was reduced when only two of these lysines were altered. The deletion or point mutants of p53 showing constitutive DNA binding activity did not display clear evidence of DNA binding site specificity, although some binding site preference was seen with the point mutants. Each of the constitutively active p53 mutants retained transcriptional activity and induced both cell cycle arrest and apoptosis in transiently transfected cells at rates comparable with the wild type protein.

The tumor suppressor protein p53 plays a pivotal role in protection against the development of cancer and is inactivated in many human malignancies. p53 is thought to prevent accumulation of genomic alterations by hindering cell proliferation in response to genotoxic stress, and two of the principal functions of p53 are the induction of cell-cycle arrest and the activation of apoptotic cell death. Because p53 is an extremely efficient inhibitor of cell growth, keeping p53 function under control in normal cells is critical. One of the principal mechanisms by which cells achieve this is by regulating the p53 protein level, although the ability of the protein to adopt active and latent forms and its cellular localization also contribute to the regulation of its function. Here, we summarize recently identified mechanisms that regulate the stability of the p53 protein and discuss the potentially immense clinical relevance of these observations in developing therapeutical approaches that aim to restore p53 function in human tumors.

AP-1 transactivation appears to be required for mouse JB6 cell neoplastic transformation induced by the tumor promoter TPA or epidermal growth factor (EGF). Exposure to AP-1 transrepressing retinoids and glucocorticoids and expression of a dominant negative c-jun (TAM67) blocked tumor promoter-induced AP-1 transactivation and neoplastic transformation. The aim of the present study was to extend the inquiry of the role of AP-1 and other transcription factors to human neoplastic progression. Expression of human papillomavirus (HPV) 16 or 18 E6 and E7 immortalizes human keratinocytes and inhibits serum/calcium-stimulated differentiation. Further transformation by v-fos co-expression renders these keratinocytes tumorigenic in nude mice. We have analysed two series of E6/E7 immortalized human keratinocyte cell lines that show progressing phenotypes ranging from differentiation sensitive to anchorage-independent to tumorigenic in nude mice. We analysed the activities of AP-1 and NF-kappaB which may 'cross-talk'. Both DNA binding and transactivation of AP-1 and NF-kappaB transcription factors showed elevation in the anchorage-independent (16RH) and tumorigenic (18 v-fos) keratinocyte lines compared to the less progressed but immortalized cell lines. HPV E7 was expressed at a constant level shown by quantitative RT-PCR in both the more and the less progressed lines, indicating that E7 is not the factor limiting this progression. Blocked shift/supershift analysis indicates that Fos family member proteins especially Fra-1 and Fra-2 are related to progression and no changes found in the Jun family member proteins although they are present in the AP-1/DNA binding complex. When a dominant negative mutant c-jun driven by a human keratin 14 promoter was co-transfected with AP-1 or NF-kappaB reporters, both AP-1 and NF-kappaB activities were suppressed in the more progressed cell lines 16RH and 18 v-fos but not in the less progressed 16RL or 18 cell lines. Overexpression of the same dominant negative c-jun did not inhibit p53 dependent reporter transactivation, indicating the specificity of inhibition of AP-1 and NF-kappaB transactivation in the HPV-immortalized cells. Stable transfectants of this mutant c-jun in the two more progressed cell lines 16RH and 18 v-fos showed reduced AP-1 and NF-kappaB activation and reduced anchorage-independent growth. Together, these results indicate that activation of AP-1, NF-kappaB or both may contribute to neoplastic progression in HPV immortalized human keratinocytes and that specific targeting of the elevated levels seen in benign or malignant tumors might be effective for prevention or treatment of human cancer.

Certain types of human papillomavirus (HPV), such as types 16 and 18, are etiological agents for carcinogenesis of the uterine cervix. These HPVs have two oncogenes, E6 and E7, that have transforming activities in established murine cells. Tumorigenicity and decreased serum requirement for cell growth are conferred by the E6 gene, whereas anchorage-independent growth is mainly governed by the E7 gene. To understand the mechanism of cellular transformation by the HPV16 E6 gene, we examined three mutant E6 proteins defective for p53 binding, p53 degradation, or transactivation of the adenovirus E2 promoter for the ability to induce tumorigenicity and decreased serum requirement. The results showed that tumorigenicity and decreased serum requirement were associated with the ability of E6 to bind to p53, although the subsequent degradation of p53 was not required for these functions.

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 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.

The tumour-suppressor p53 is a short-lived protein that is maintained at low, often undetectable, levels in normal cells. Stabilization of the protein in response to an activating signal, such as DNA damage, results in a rapid rise in p53 levels and subsequent inhibition of cell growth. Tight regulation of p53 function is critical for normal cell growth and development, and one mechanism by which p53 function is controlled is through interaction with the Mdm2 protein. Mdm2 inhibits p53 cell-cycle arrest and apoptic functions and we show here that interaction with Mdm2 can also result in a large reduction in p53 protein levels through enhanced proteasome-dependent degradation. Endogenous levels of Mdm2 are sufficient to regulate p53 stability, and overexpression of Mdm2 can reduce the amount of endogenous p53. Because mdm2 is transcriptionally activated by p53, this degradative pathway may contribute to the maintenance of low p53 concentrations in normal cells. Furthermore, mechanisms regulating the Mdm2-induced degradation of p53 may play a role in controlling the extent and duration of the p53 response.

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 E7 protein encoded by human papillomavirus type 16 shows transforming and immortalizing activities which are mediated, in part, through the interaction of the viral oncoprotein with the pRB protein family. This interaction is not solely responsible for E7 function, however, and other properties of E7, such as the interaction with basal transcription factors such as TBP, are likely to be of importance. We show here that three regions of the viral protein contribute to the interaction between E7 and TBP; the pRB-binding domain, the casein kinase II phosphorylation region and the C-terminal dimerization domain. Mutations within each region reduced the interaction of E7 with TBP in vitro, and simultaneous alterations within each of these regions completely abrogated binding. Unlike the pRB interaction, the association of E7 with TBP was enhanced following phosphorylation of E7 by casein kinase II, demonstrating a functional significance for phosphorylation of the viral protein.

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 human papillomavirus type 16 (HPV-16), the type most often associated with cervical cancer, immortalizes primary keratinocytes and inhibits serum/calcium-stimulated differentiation in culture. In this study, we have used a model of keratinocyte immortalization based upon HPV-16 to analyze perturbation of function and expression of E-cadherin, a Ca(2+)-dependent cell-cell adhesion molecule expressed by normal keratinocytes, and its associated proteins. An immortalized keratinocyte cell line generated by cotransfection with HPV-16 E6 and E7 showed decreased membrane E-cadherin expression and redistribution of alpha-, beta-, and gamma-catenin from the undercoat membrane to the cytoplasm. No changes in the level of expression were seen. Selection of the immortalized keratinocyte cell line for resistance to differentiation generated a more transformed cell line with an invasive phenotype, down-regulated E-cadherin and alpha-catenin, and up-regulated the epidermal growth factor receptor (EGFr). Transfection of an E-cadherin expression construct into the differentiation-resistant cell line restored membrane-bound E-cadherin and catenin expression, down-regulated the EGFr, and reversed the invasive phenotype. These results indicate that overexpression of the EGFr correlates with perturbation of the E-cadherin/catenin complex seen in the HPV-16 E6- and E7-transfected keratinocytes and may underlie a functional interaction between growth-regulatory factors and adhesion molecules (E-cadherin/catenin).

Several genes have been identified as targets for transcriptional activation by the p53 tumour suppressor protein. Rodent cyclin G was previously identified as a p53 responsive gene and in order to assess the role played by cyclin G as a mediator of p53 function in humans cells we have isolated full length human cyclin G1 and identified a related gene designated cyclin G2. Both human G-cyclins are induced by the DNA damaging agent actinomycin-D and although the induction of cyclin G1 is clearly p53 dependent, activation of cyclin G2 expression was observed in the absence of p53. Based on sequence similarity, the G-cyclins and the recently identified cyclin I form a distinct sub-group within the larger cyclin family, possibly reflecting some degree of functional similarity.

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 activity of the p53 tumor suppressor protein is regulated, at least in part, through the stability of the protein. p53 degradation in normal cells is controlled by ubiquitin-dependent proteolysis, and activation of p53 following DNA damage is associated with an increase in the stability of the protein. The human papillomavirus-encoded E6 protein abrogates p53 function by targeting it for rapid degradation, also through the ubiquitin pathway. Although the p53 protein is ubiquitinated following interaction with E6, we show here that none of the lysine residues within p53 are specifically required for E6-targeted degradation. Mutation of lysine residues within the C-terminus of p53 resulted in resistance to E6-mediated degradation in vitro, although the ability of the two proteins to form a complex was not affected. The same mutant was efficiently targeted for degradation in cells, however, illustrating a lack of correlation between the in vitro and the in vivo assays.

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 cell cycle arrest and apoptotic functions of p53 both contribute to the role of this tumour suppressor protein in preventing replication of cells suffering DNA damage. Although the ability of p53 to function as a sequence-specific transcription factor appears to be directly and causally linked to the implementation of an arrest at the G1 stage of the cell cycle, the contribution of transcriptional activation to the apoptotic response is less clear. It seems likely that several p53 activities, both transcriptionally dependent and transcriptionally independent, can play a role in mediating cell death. The requirement for each of these functions appears to depend on the cell type, the cell environment and other genetic alterations already sustained by the cell in which p53 function is activated.

The p53 tumor suppressor protein is a transcriptional activator, which can mediate apoptotic cell death in a variety of cell types. To determine whether sequence-specific trans-activation is a prerequisite for the induction of apoptosis by p53, the apoptotic effects of various p53 deletion mutants were monitored in an assay based on the transient transfection of HeLa cells. A truncated protein (p53dl214), containing only the first 214 amino-terminal residues of murine p53, induced extensive apoptosis, albeit at a slower rate than trans-activation-competent wild-type p53. p53dl214 also suppressed the transformation of rat fibroblasts by several oncogene combinations and particularly by myc plus ras and HPV E7 plus ras. p53dl214 lacks a major portion of the DNA-binding domain and cannot activate p53-responsive promoters. Moreover, a human p53 protein carrying mutations in residues 22 and 23 also triggered HeLa cell apoptosis, despite failing to induce significant activation of relevant p53 target promoters. These data suggest the existence of two p53-dependent apoptotic pathways--one requiring activation of specific target genes, and the other independent of sequence-specific trans-activation. The latter pathway may actually be totally uncoupled from the binding of p53 to its consensus DNA sites. The relative contribution of trans-activation-independent apoptosis to tumor suppression by p53 may be dictated by the specific genetic lesions present in the particular tumor.

The tumour suppressor protein p53 normally functions as a tetramer in a defined conformational state. Mutations within p53 which contribute to cancer development frequently induce a conformational shift in the protein which correlates with loss of wild type growth suppressor functions. Both the cell encoded mdm2 protein and the human papillomavirus oncoprotein E6 can regulate p53 function and we have examined the interaction of these proteins with p53. The E6/p53 association is sensitive to conformational alterations in the p53 protein, although oligomerisation is not necessary for this interaction to occur. Analysis of C-terminal p53 truncations has indicated that the region between residues 327 and 347 may play a role in E6 binding. Since monomeric forms of p53 retain transcriptional and transformation suppressor activities, our results indicate that E6 targets p53 proteins which retain these wild type functions. Conversely, the interaction of p53 with mdm2 is not dependent on the conformation of the p53 protein but is significantly impaired by loss of quaternary structure. It is possible that mdm2 plays a role in mediating activities of p53 which, unlike transcriptional activation, depend on oligomerisation.

Human papillomavirus (HPVs) adenovirus and simian virus 40 (SV40) are small DNA viruses which can show oncogenic activity. Although not otherwise related, all three have adopted very similar strategies to deregulate cell growth; each virus encoding oncoproteins which interact with the same cellular targets. Of particular interest are the interactions with the cell encoded pRB and p53 proteins, products of tumour suppressor genes. Somatic mutation results in the loss of the pRB and p53 function in many cancers and the contribution of the viruses to tumour development appears to reflect their ability to inactivate these cellular proteins. Both pRB and p53 negatively regulate progress through the cell cycle and the action of the viral proteins has highlighted the central importance of these tumour suppressor proteins in maintaining normal cell growth.

The tumor suppressor protein p53 shows growth and transformation suppression functions that are frequently lost by mutant proteins detected in cancers. Using a large series of p53 mutants, we have demonstrated an excellent correlation between transcriptional activation and growth suppression in p53-null human cells. Not all transcriptionally active mutants retain the ability to suppress transformation in primary rodent cells, however, and two tumor-derived point mutants displayed some evidence of both transforming and transactivating activity. Transformation by these mutants was not mediated by transdominant repression of endogenous p53 transactivating function, and cell lines expressing these p53 proteins showed elevated p53 transcriptional activity. Our results suggest that activation of transcriptional regulation by p53 will not necessarily result in tumor suppression.

Using a new series of p53 mutants targeting the conserved regions we have analysed the relationship of various activities of the protein. Mdm-2 and human papillomavirus (HPV) E6, two proteins which interact with and abrogate p53 function, were shown to bind independently. Deletion of the conserved regions of the protein in which most of the naturally occurring mutations are found (boxes II-V) abrogated transcriptional activity and the ability to interact with E6, supporting the importance of this DNA binding domain to these activities. Nevertheless, these mutants retained the ability to interact with mdm2. One mutant, deleted of all the C-terminal sequences, showed loss of mdm2 binding, E6 binding and transcriptional activity. More subtle mutations within the C-terminus of the protein, including alterations of the cdc2 and CKII phosphorylation sites, had no effect on the transcriptional trans-activation, mdm-2 or E6 binding functions, indicating that phosphorylation of these sites is not essential for these activities. Deletion of conserved box I sequences abolished the interaction with mdm-2 without loss of transcriptional activation or transformation suppressor activity, suggesting that mdm-2 is not a downstream effector of p53 function.

Stabilisation and activation of p53 contributes to the G1 arrest exhibited by many cells in response to DNA damage. One function of p53 is the transcriptional activation of an inhibitor of cyclin dependent kinases; enzymes which phosphorylate and inactivate the growth inhibitory function of the pRB tumour suppressor protein. In this study we show that expression of either of the human papillomavirus encoded E6 and E7 oncoproteins allows cell cycle progression following DNA damage. This suggests that both viral proteins can function in the same pathway; E6 by directly targeting p53 for degradation and E7 through the interaction with pRB, one of the potential downstream effectors of p53.

Epstein-Barr virus (EBV), hepatitis B virus (HBV), HTLV-I and some human papillomaviruses (HPVs) appear to contribute to the development of a large proportion of certain human cancers. Although the epidemiological evidence linking infection with these viruses to malignancies is generally convincing, only for the HPVs have experimental systems indicated a clear role for some of the HPV encoded genes in tumour cell growth. In these cases, molecular analysis is revealing the mechanisms by which the virally encoded oncogenes function to perturb the normal regulation of cell growth. For HTLV-I, EBV and HBV the mechanism by which the viruses contribute to tumour cell growth is obscure, even though much has been learned about cell transformation in culture by these viruses. A detailed understanding of the mechanism of oncogenesis will be required to design therapeutic drugs for the treatment of these cancers. Prophylactic vaccination resulting in prevention of infection may be an effective approach to reduce the incidence of some of these common cancers.

Aims: To determine the sensitivity of the hybrid capture method for human papillomavirus (HPV) detection and potential clinical uses as a screening method for the identification of cervical intraepithelial neoplasia., Methods: The presence of oncogenic types of HPV was tested for in samples taken from the cervix at colposcopy, and compared with detection by polymerase chain reaction (PCR) in 60 patients. Both sets of results were corrected with the pathology determined by biopsy and smear cytology., Results: Hybrid capture detection showed 86% agreement with PCR. Eighty three percent of CIN 3 lesions, 62% of CIN 2, 59% of CIN 1 and 21% of normal controls were positive for oncogenic HPV types., Conclusion: The hybrid capture detection method is reliable, sensitive, and easy to use. The addition of HPV testing to cytological screening would detect a greater proportion of cervical dysplasia with a higher false positive rate.

The identification of certain human papillomaviruses as human tumour viruses has been paralleled by our understanding of how the viral oncoproteins function. The virally encoded E6 and E7 proteins act, in part, to abrogate the activities of the tumour suppressor proteins p53 and pRB. The interaction between these viral proteins and regulators of cell growth offers targets for future therapeutic developments.

The E6 protein encoded by human papillomavirus type 16 (HPV16), a genital virus with oncogenic potential, can target cellular p53 for rapid degradation following the formation of a complex including the two proteins. Some studies suggest that the E6 proteins encoded by HPV6 and 11, viral types which are normally limited to benign lesions, may also interact with p53, although the association is weaker than that seen with HPV16 E6. The present study demonstrates that E6 proteins from HPV16 and HPV6 can modulate the transcriptional regulatory functions of p53 in several cell types. A series of E6 mutants was used to show that association between E6 and p53 is necessary for this activity and that E6 proteins which retain the ability to associate with p53 but show no detectable degradation activity in vitro can, to some extent, abrogate p53 mediated transcriptional trans-regulation. This activity is augmented, however, by the ability of the E6 protein to target bound p53 for rapid degradation. These results suggest that some degree of modulation of p53 function is necessary in the normal viral life cycle but also demonstrate a correlation between the efficiency of this activity and oncogenic potential of the virus.

HPV16 is a human tumour virus encoding two principal oncoproteins, E6 and E7. Expression of E7 can induce DNA synthesis in quiescent cells and this property coincides with its ability to bind to the cell proteins pRb and p107. As these cell proteins are regulators of the transcription factor E2F, we have investigated whether the interaction with E7 could result in induction of cell cycle regulated genes. We show that B-myb, whose induction at the G1/S boundary is regulated by release from E2F mediated transcriptional repression, is a target for transcriptional activation by E7 and is the first E7 responsive cell gene to be identified. E7 transactivation leads to both inappropriate transcription of B-myb during G1 and constitutive over-expression in cycling cells. B-Myb plays an essential role in cell cycle progression, and activation by E7 is likely to contribute to the mitogenic activity of the viral oncoprotein. Regulation of the B-myb promoter in NIH3T3 cells correlates with binding of distinct p107-containing complexes at the E2F binding site, and analysis of E7 mutants confirms that B-myb transcription in these cells is regulated through interactions with p107 rather than pRb. These results provide the first example of a potentially specific role for p107 in the regulation of the cell cycle.

Rat embryo fibroblasts immortalized with a temperature-sensitive p53 mutant and ras rapidly stop proliferating at 33 degrees C. Expression of the viral oncoproteins human papillomavirus type 16 (HPV-16) E7 or adenovirus E1A efficiently overcame this growth arrest, although cells rescued by E7 or E1A displayed no detectable changes in p53 expression. Co-transfection of HPV-16 E6, which binds and directs the degradation of human p53, affected neither growth of the cells at 33 degrees C nor the amount or conformation of the p53 protein, possibly reflecting an inability of E6 to interact with mouse p53 in rodent cells. Our results suggest either that the temperature-sensitive p53 failed to regain full wild-type suppressor activity at 33 degrees C or that E7 and E1A can modulate wild-type p53 growth-suppressor activity without altering the conformation or stability of the protein.

Wild-type human p53 and a series of p53 point mutants isolated from Burkitt's lymphoma (BL) cell lines were tested for their ability to inhibit DNA synthesis in a p53-negative BL cell line and to bind and be degraded by the human papillomavirus type 16 E6 protein. All the mutants lost the wild-type ability to inhibit DNA synthesis, demonstrating that they are all functionally altered. Binding to E6 and consequent degradation of the p53 mutants frequently correlated with changed suppressor properties in BL cells.

We have produced human papillomavirus type 16 E7 protein in a bacterial expression system and examined the mitogenic activity of this protein in Swiss 3T3 cells after scrape loading. The ability of E7 to induce cellular DNA synthesis in quiescent mouse fibroblasts is strongly enhanced by the presence of a single growth factor such as insulin. Although only weakly mitogenic, introduction of E7 alone resulted in the rapid induction of the transcriptionally active form of E2F, which was not enhanced further by the addition of insulin. Mutant E7 proteins defective for RB binding failed to induce the active form of E2F or act synergistically with insulin to stimulate DNA synthesis. The ability of E7 to regulate E2F may therefore be necessary, but is not sufficient, for full induction of DNA synthesis.

Primary human papillomavirus (HPV) positive anogenital cancers normally develop without somatic mutation within the p53 gene. In this study, however, we have identified p53 point mutations in metastases arising from HPV positive cervical carcinomas, suggesting that acquisition of p53 mutation may play a role in the progression of some HPV associated primary cancers. p53 mutants identified in anogenital cancers exhibit a dominant transforming phenotype and increased resistance to HPV16 E6 directed degradation. The association of p53 mutation with metastases may explain the poor prognosis reported for HPV negative primary cancers, many of which already contain mutant p53. A high proportion of p53 mutations detected in both primary and metastatic cancers are GC-->TA transversions, strongly suggesting a role for external carcinogens in the development of these cancers.

Functional analysis of human papillomavirus type 16 E7 protein by complementation with adenovirus E1A mutants in baby rat kidney cells has shown that the retinoblastoma gene product (RB)-binding region of E7 can substitute in trans for that of E1A. An N-terminal E7 mutant was unable to complement an E1A mutant unable to bind p300, indicating that the two mutants were defective for functionally equivalent activities. E7 proteins with mutations within the RB-binding region were also unable to complement either the non-p300-binding E1A mutant or the N-terminal E7 mutant, suggesting that these mutations affect more than just RB binding.

The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Analyses of cancer cell lines and of anal cancers suggest an inverse correlation between infection with human papillomavirus (HPV) and somatic mutation of the p53 tumour-suppressor gene. We have investigated this association in primary cervical tumours. Tumour-tissue samples from 28 women with primary cancer of the cervix were analysed for presence of HPV sequences and for somatic mutations of the p53 gene. Southern blot analysis and the polymerase chain reaction (PCR) showed that 25 of the tumours contained HPV sequences; 20 were HPV16 positive and 5 HPV18 positive. 17 tumours subjected to restriction fragment length polymorphism analysis for the short arm of chromosome 17 showed no evidence of allelic deletion. Sequencing of the entire coding region of the p53 gene by asymmetric PCR detected heterozygous point mutations in only 3 HPV-negative tumours. By contrast, in 21 HPV-positive cancers the p53 sequence was wild-type throughout. Our data indicate that loss of wild-type p53 function is important in the pathology of cervical cancer and that in the absence of an HPV-encoded gene product that mediates loss of p53 function, somatic mutation of the gene is required. This pattern of p53 mutation may partly explain the apparently worse prognosis of HPV-negative cervical cancers.

Human papillomavirus (HPV) research dates back to the turn of the century, when Ciuffo demonstrated that human warts are caused by an infectious agent, only later identified as a virus (1). More recently, the HPVs have generated particular interest, since they include some of the few viruses clearly involved in the development of certain human cancers (2). Although most papillomaviruses give rise to benign, self-limiting proliferations, a number of human and animal papillomaviruses have also been shown to have oncogenic potential. This link between papillomaviruses and malignant disease was first recognized more than 50 years ago, when it was shown that benign lesions induced by the cottontail rabbit papillomavirus could progress to invasive carcinoma (3). The change from benign lesions to malignancy and the identification of factors that contribute to this conversion has been extremely useful as a model for tumor progression. However, following the initial studies that so elegantly identified the oncogenic potential of papillomaviruses, research into these viruses slowed considerably for many years. Further investigation has been hampered by the failure to develop an in vitro culture system for viral propagation. In general, papillomaviruses are limited to a single host and show strong tropism with respect to the cell type that they infect. Most papillomaviruses infect only cutaneous or mucosal epithelial cells at specific anatomical sites. Viral DNA replication and transcription of some viral sequences occurs in cells throughout the epithelium.

Human papillomavirus (HPV) types 16 and 18 appear to play a role in the development of ano-genital malignancies, whereas HPV 6 and 11 are usually associated with benign lesions. One HPV16 oncoprotein, E6, complexes with and promotes degradation of the cellular tumor suppressor p53. Here we show that E6 proteins of both oncogenic and benign HPV types associate in vitro with p53, but binding by E6 proteins of benign HPV types cannot target p53 for degradation. A C-terminal region of E6 conserved among all HPV types is important for p53 binding. However, N-terminal sequences of E6 conserved only between oncogenic HPV types are necessary to direct p53 degradation. p53 binding by E6 appears necessary but not sufficient for this activity. All E6 proteins tested showed comparable transcriptional trans-activating activity, a property that does not require the ability to bind or direct degradation of p53.

A panel of 12 Burkitt's lymphoma cell lines and four other B cell lines were tested for the presence of mutations in p53. Protein analysis using a mutant-specific antibody and sequencing of both cDNA and genomic DNA revealed changes relative to the standard p53 protein sequence in 12 of the 16 lines studied, including 10 of the BL lines. Mutation of p53 in the BL lines was usually accompanied by loss of the other allele of p53. Testing of the mutated p53 cDNAs for gain of transforming activity or loss of growth suppression activity showed that several of the BL mutants were functionally altered from wild-type p53.

We have examined a series of squamous cell carcinomas (SCC) of the anus, anal intraepithelial neoplasia grade III (AINII) lesions and haemorrhoids for the presence of sequences from transforming human papillomavirus (HPV) types by polymerase chain reaction (PCR)/Southern blotting. In addition, the same DNAs have been analysed for abnormalities in the c-myc, p53 and retinoblastoma (Rb-1) gene loci by Southern blotting. HPV16 sequences were detected in a total of 38 of 50 (76%) and HPV18 sequences in 4 of the 50 cancers (8%). Of 12 haemorrhoids examined, none contained HPV16 or HPV18 sequences. Amplification of c-myc was demonstrated in 15 of the 50 cancers (30%), of which 13 were HPV16 positive, and one also positive for HPV18. Amplification of c-myc was not observed in the 5 AINIII or any of the 41 haemorrhoid DNAs analysed. Rearrangement of c-myc was not seen in any of the DNAs. Gross rearrangement, or loss of p53 or Rb-1 loci was not observed in any normal or tumor tissue. However, in preliminary analysis of p53 sequence, three tumours negative for HPV were heterozygous for p53 point mutation whereas six HPV positive tumours and two haemorrhoids were wild-type sequence throughout exons four to ten.

Clinical and experimental evidence is consistent with a key role for transforming human papilloma viruses (HPVs) in the aetiology of anogenital carcinoma. Cervical carcinoma does, however, occasionally occur in the absence of HPV sequences (Riou et al., 1990). We have used a direct cDNA/PCR sequencing protocol to analyse the sequence of p53 mRNA expressed by HPV positive and negative cervical carcinoma cell lines. Six cell lines which contain HPV sequences express p53 mRNA which has wild-type sequence throughout conserved boxes 2, 3, 4 and 5. The two HPV negative cell lines (C33a and HT3) express mutant p53 mRNA. In each case the mutation occurs in an evolutionarily conserved amino acid. Our data suggest that loss of wild-type p53 function is important in development of cervical carcinoma, and that this might be achieved either by mutation within the p53 gene or the presence of a virally encoded p53 binding protein.

Mutations within the tumor suppressor genes Rb-1 and p53 are commonly found in many human malignancies, and loss of wild-type function of both p53 and RB appear to be important events in the development of these malignancies. Interference with normal RB and p53 function in the cell has apparently also been exploited by the oncogenic genital human papillomaviruses (HPVs), which encode transforming proteins capable of binding cellular RB and p53 proteins. We have investigated the expression of RB and p53 in a series of eight cervical carcinoma cell lines, six of which contain HPV sequences and two of which have arisen apparently independently of HPV infection. In the six HPV-positive lines, no evidence of abnormal RB or p53 protein could be detected. However, there was evidence for abnormal RB and p53 in the two HPV-negative lines. These data are consistent with the hypothesis that loss of wild-type RB and p53 function is necessary for tumor development and that such loss can occur either by mutation within the cellular gene or by expression of viral proteins capable of complexing wild-type cellular proteins.

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.

Human papillomaviruses (HPVs) appear to play a role in the etiology of the vast majority of virus-associated human malignancies. Studies of viral gene expression in carcinomas suggest the importance of two HPV encoded proteins, E6 and E7, in malignant development and these proteins have been shown to encode transforming and immortalising activities. The two proteins show some functional resemblance to the transforming proteins of other small DNA tumour viruses such as adenovirus and SV40. Recent evidence suggests that one important function of these virus-encoded proteins is binding the products of the cellular tumour suppressor genes RB and p53, revealing an exciting link between oncogenes and anti-oncogenes.