Background: Although subclinical persistent infections with the human polyomaviruses are ubiquitous worldwide, they are known to vary in relation to geographical location, diseases present and may associate with different human tumours, especially in immunocompromised patients. The current study hypothesised that there may be co-operativity between HPV and polyomaviruses, particularly in HIV positive women, that could influence the rate of progression to invasive cervical carcinoma. Patients and Methods: Novel PCR methods were developed for the detection of SV40, MCV, JCV and BKV polyomavirus DNA. These were used to test DNAs extracted from 220 cervical smears and 77 invasive cervical carcinomas (ICCs) from HIV positive and negative Kenyan women of known HPV status. An expression plasmid was constructed containing JCV Large T (LT) antigen and this, in addition to empty vector control, used to stably transfect HPV16 E6/E7 immortalised human keratinocytes. Expression of LT was analysed in transfected cell lines by PCR, immunocytology and Western blotting. These cells were then used to test for changes in Cell contact growth inhibition; Growth rate and Epithelial to Mesenchymal Transition (EMT). Screening of full transcriptome microarrays was carried out on vector and LT transfected cells and their sensitivity to the drug mefloquine tested by comparison of growth rates and live/dead cell assays. Results: PCR accurately detected ~18 copies of SV40, MCV, JCV and BKV DNA in addition to simultaneous detection of JCV and BKV. None of the clinical samples tested were positive for SV40, MCV, or BKV DNA. However, JCV DNA was detected in 24/297 (8%) of cervical specimens. Comparison of the incidence of JCV in cervical smears and ICCs showed a ~3-fold increase in samples from HIV positive women with ICC (P=0.025) whereas no significant difference was found between smears and ICCs from HIV negative women (P=0.553). Analysis of the consequences of ectopic expression of JCV LT in E6/E7 immortalised human keratinocytes showed no difference in either growth rates or contact inhibition and changes in the EMT marker vimentin were found to be related to cellular clonality. Microarray analysis showed LT related alterations in gene expression which could have bearing on its carcinogenic potential in addition to changes related to clonality. JCV LT expressing monoclonal cell were the most sensitive to mefloquine treatment. Conclusion: The simultaneous JCV/BKV detection method, described herein, is unique and has been evaluated by the WHO for this purpose. The results indicate the prevalence JCV and BKV with respect to the African geographical location and suggest that JCV may combine with high-risk HPV in a sub-set of HIV positive women to influence the rate of progression to invasive cervical carcinoma. In vitro JCV LT was found not to be an overt oncogene in the cell system used although cell cloning procedures clearly affected the assays. LT induced changes in total gene expression were consistent with neoplastic progression although a high proportion of genes with unknown function were dsyregulated with respect to clonality. The anti JCV drug mefloquine showed some selectivity for LT expressing cells and further investigation of this indication is warranted.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:686755 |
| Date | January 2015 |
| Creators | Alosaimi, Bandar |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/studies-on-potential-cooperativity-between-different-types-of-tumour-virus(5dea86d5-418d-46a0-943c-da7d53653423).html |
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