TREATMENT OF CERVICAL CANCER USING RNA-INTERFERENCE AGAINST HUMAN PAPILLOMAVIRUS ONCOGENES

Putral, Lisa (2007). TREATMENT OF CERVICAL CANCER USING RNA-INTERFERENCE AGAINST HUMAN PAPILLOMAVIRUS ONCOGENES PhD Thesis, School of Medicine, University of Queensland.

       
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Author Putral, Lisa
Thesis Title TREATMENT OF CERVICAL CANCER USING RNA-INTERFERENCE AGAINST HUMAN PAPILLOMAVIRUS ONCOGENES
School, Centre or Institute School of Medicine
Institution University of Queensland
Publication date 2007
Thesis type PhD Thesis
Supervisor Dr Nigel McMillan
Abstract/Summary Human papillomavirus (HPV) is the infectious agent responsible for the development of cervical cancer, with type 16 responsible for 50% of all cases. Cervical cancer remains the number one cancer killer of women under 50 worldwide, resulting in more than 270,000 deaths each year. Tumours caused by human HPV infection are an ideal model system for RNA interference-based cancer therapies as the oncogenes which cause cervical cancer, E6 and E7, are only expressed in cervical cancer cells. Furthermore, E6 and E7 are absolutely required for the ongoing proliferation of cervical cancer cells, as it has been demonstrated that upon their suppression, cells undergo apoptosis or senescence. In this study, siRNAs were designed that were directed against the HPV E6 and E7 oncogenes that simultaneously targeted both E6 and E7 with a single siRNA. These siRNAs were shown to reduce E7 protein levels by 80%. Following siRNA treatment, it was observed that there was reactivation of the p53 tumour suppressor pathway. The loss of E6 and E7 in cervical cancer cell lines resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. RNA interference was specific as no effect on HPV-negative cells was observed. It was also shown in this study that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells following a combined treatment, resulting in a four-fold increase in cytotoxicity for the combined treatment as compared to cisplatin alone. Upon co-treatment with cisplatin and shRNA expressed from a plasmid, a decrease in E7 expression was observed with a concurrent increase in p53 protein levels over that seen with either treatment alone. These results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, and that this effect may occur as a result of increased p53 protein levels. This research also investigated the potential for a liposome-based system to facilitate the delivery of siRNA to tumours in vivo. It was demonstrated that liposomes produced by a lyophilisation method entrap nucleic acids with high efficiency (greater than 60%), can fully protect nucleic acids from digestion with nucleases, and are taken up by more than 90% of cells in culture. It was also demonstrated that these liposomes are able to accumulate at tumour sites in vivo in immune-competent mice. Despite excellent cellular uptake, siRNAs entrapped within liposomes were not transfection-competent, suggesting an inability of siRNAs to dissociate from liposomes, localize in the cytoplasm and become available to the silencing machinery. This research lays the groundwork for further development of effective liposomes for the in vivo delivery of siRNA nucleotides.

 
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Created: Fri, 21 Nov 2008, 16:24:17 EST