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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.

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 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 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).

The presence and consistent expression of the genes encoding the human papillomavirus (HPV) E6 and E7 proteins in the great majority of cervical tumours presents the opportunity for an immunotherapeutic approach for control of the disease. This report describes the construction and characterisation of a recombinant vaccinia virus designed to express modified forms of the E6 and E7 proteins from HPV16 and HPV18, the viruses most commonly associated with cervical cancer. The recombinant virus (designated TA-HPV) was based on the Wyeth vaccine strain of vaccinia, and was shown to express the desired gene products. Studies in mice indicated that the recombinant virus was less neurovirulent than the parental virus and was capable of inducing an HPV-specific CTL response. This pre-clinical evaluation has provided a basis for the initiation of human trials in cervical cancer patients.

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.

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.

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.

Human papillomaviruses (HPVs) contribute to the development of almost all cervical cancers, a common and often fatal human disease. The mechanisms by which the HPVs function in malignant progression appear to be related to the activity of the two viral oncoproteins, E6 and E7, which form complexes with several cell proteins normally involved in controlling cell growth. Of particular interest has been the association of E6 with p53 and E7 with Rb, both products of tumor suppressor genes. Expression of E6 and E7 is likely to overcome the regulation of cell proliferation normally mediated by proteins like p53 and Rb, allowing uncontrolled growth and providing the potential for malignant transformation. These activities of E6 and E7 support the importance of the tumor suppressor proteins in the maintenance of normal cell proliferation and provide novel approaches to understanding the mechanisms by which they function.

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.

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.

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)

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.