Karen H. Vousden

Degradation of p53 can be targeted by HPV E6 sequences distinct from those required for p53 binding and trans-activation

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.

Clonal p53 mutation in primary cervical cancer: association with human-papillomavirus-negative tumours

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.

Transcriptional activation by p53 correlates with suppression of growth but not transformation

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.

Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.

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)

INTERACTIONS BETWEEN PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSOR GENE PRODUCTS

Publisher Summary Human papillomaviruses (HPVs) have provided unique insight into the mechanisms that regulate the growth and oncogenic progression of human cells. The genital HPV types cause lesions that are not strictly benign; the malignant progression of HPV-infected cells appears to give rise to almost all cervical cancers. The paradigm for tumor suppressor genes has now been joined by a growing number of genes whose loss of function contributes to cancer development; subsequently, many of these genes have been shown to play a role in preventing or delaying progress through the cell cycle. Studies have shown that somatic mutation within the tumor suppressor loci frequently contributes to the development of sporadic cancers. Several cell-encoded proteins have been detected in association with viral proteins E6 and E7, and the identification of some of these as products of tumor suppressor genes has revealed at least some of the mechanisms of viral oncoprotein function. Viral oncoproteins have been identified and the enormous advances in unraveling their mechanism of action have participated in the convergence of many different areas of research. The application of the understanding of the interactions between the viral and host proteins directly to treating such a common human disease may be a fitting culmination to these studies.

Functions of human papillomavirus E6 and E7 oncoproteins

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.

Biological activities of p53 mutants in Burkitt's lymphoma cells

Wild-type human p53 and a series of p53 point mutants isolated from Burkitts 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.

Functional analysis of human papillomavirus type 16 E7 by complementation with adenovirus E1A mutants

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.

HPV E6: ensuring all's well at the end

I mmortality, or our lack of it, must hc one of the most univcrsally tnscinating topics, and the iimitcd life span shown by virtually all normal cells in culture has been interpreted as a reflection of the limited lifispan shown by the whole organism’. Although the concept that cellular senescence is the mechanism through which aging occurs is not fully prover, it is clear that a,1 extended or indefini:e lift span (immortality) is ft aqucntly displayed by tumor cells, indicating that rhc mechanisms that ensure finite life for the cell p!ay :I role in protecting against the devtlopment of cancer. Analysis of Lvents that immortalize cells in culture is therefore likely to illuminate mechanisms ,f tumorigenesis i*r t~~to, hur. alth..sn rodent cells can overcome their normally finite life span with relative ease, immortality is much more difficult to achieve in human cells. Consequently, the ohservation that expression of E6 .md F.7 Rroteins encoded by some genital human papillomavirus types can relatively efficiently immortalize primary human epithelial cells’.” was greeted with interest. Particularly intriguing is the correlation of immortalizing function with oncogenie potentiai because only the virus types that are associated with cervical carcinoma development (high-risk HPVs) clearly show tha activity’. E6 and E7 have been shown to bind and inhibit the function of tumor suppressor proteins (~53 and pRB)‘. The recerrt puhlication by Klingelhutz eta!.’ provides evidence for a new function of E6 in activating telomerase, with a clear potential role in immortalization.