The Role of p53 in Cell Transformation by BPV-4

Scobie, Linda (1996) The Role of p53 in Cell Transformation by BPV-4. PhD thesis, University of Glasgow.

Abstract

Bovine papillomavirus-type 4 is associated with benign papillomas which can progress to carcinoma in cattle grazing on bracken fern. Bracken is known to contain immunosuppressants and cocarcinogens which can act as cofactors and these are known to be required for progression in vivo. One of the best characterised of these cofactors is the mutagen quercetin. BPV-4 has been shown to be capable of partially transforming primary bovine fibroblasts (PalF) cells in vitro in the presence of an activated ras and when these cells are additionally treated with quercetin they become tumourigenic in nude mice. The transforming functions of BPV-4 are encoded by the E7 and the E8 ORF's. BPV-4 is unusual in that it does not possess an E6 ORF. E6 has been shown to be one of the major transforming oncoproteins of the human papillomaviruses and is known to bind and degrade the tumour suppressor protein p53. This abrogation of the p53 protein is known to contribute to tumour progression by the high risk papillomaviruses such as HPV-16 and 18. As BPV-4 does not encode E6 functions, this would suggest that if p53 dysfunction is important in BPV-4 cell transformation, it may occur by alteration of the cellular gene. In order to look at possible p53 mutations in alimentary cancer of cattle, a 14kb genomic clone containing wild type p53 sequence was isolated from a bovine liver genomic DNA library and sequenced. This clone was used to localise bovine p53 to chromosome 19q15 which is known to be syntenic with human chromosome 17 and mouse chromosome 11 both of which harbour the p53 gene. Immunocytochemical analysis of BPV-4 derived lesions demonstrated that elevated levels of p53 protein were detectable in papillomas but not in carcinomas. BPV-4 E7 was found to be present at all stages of papillomatosis as was BPV-4 viral DNA as detected by in-situ hybridisation. Neither viral DNA nor BPV-4 E7 were detected in normal palatine tissue or in carcinomas which is coincident with the loss of viral DNA previously reported for tumours in vivo. The elevated levels of p53 in some of the papillomas were shown to be a result of the presence of mutations in p53 exon 7 as determined by SSCP-PCR and sequencing. Mutant p53 was also detected in two metastases of upper alimentary canal tumours. The addition of mutant p53 to PalF cells in vitro in the presence of BPV-4, ras and HPV-16E6 caused these cells to become tumourigenic in nude mice. In these tumorigenic PalF cell lines, mRNA expression of the BPV-4 viral oncoprotein E8 was shown to be elevated. Cotransfected mutant p53 mRNA expression was also detectable. This would suggest that p53 mutation plays a role in tumourigenic progression by BPV-4 both in vivo and in vitro. In this study we demonstrate that the lack of an E6 in BPV-4 may be compensated by the presence of a mutated p53 gene in BPV-4 induced carcinomas. These p53 mutations may arise due to cofactors present in bracken fern, and p53 mutations were detected in BPV-4 and ras transfected PalF cells that had been treated with quercetin. However, p53 mutations were detected in transfected PalF cells which had not been exposed to quercetin treatment. We also demonstrate that the additional functions provided by HPV-16 E6 (16E6) in vitro are at least in part, independent of the p53 binding and degradation function. The data suggests that an exogenous 16E6 can supplement in vitro other functions of BPV-4 which are responsible for progression in vivo. HPV-16 E6 was also demonstrated to possess a function confering anchorage independence, which mapped to the cysteines present in the zinc finger motifs of the protein. This study has demonstrated BPV-4 lacks more than one function that can be provided by an exogeneous E6 oncoprotein and that these functions may be independent of p53. In addition, p53 mutations were shown to be present in BPV-4 induced papillomas and carcinomas.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Robert McFarlane
• Keywords: Molecular biology, Virology
• Date of Award: 1996
• Depositing User: Enlighten Team
• Unique ID: glathesis:1996-75306
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 21:16
• Last Modified: 19 Nov 2019 21:16
• URI: https://theses.gla.ac.uk/id/eprint/75306