Evaluation of CRPV based cell systems for the therapeutic studies of papilloma virus associated head and neck cancer

This study focused on establishing an in vitro expression system for studying papillomavirus (cottontail rabbit papilloma virus, CRPV) associated rabbit squamous cell carcinomas. This system was chosen since the CRPV associated VX2 carcinoma of the New Zealand White rabbit is an established animal m...

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Bibliographic Details
Main Author: Ambreen, Ghazala
Contributors: Bakowsky, Udo (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2021
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Summary:This study focused on establishing an in vitro expression system for studying papillomavirus (cottontail rabbit papilloma virus, CRPV) associated rabbit squamous cell carcinomas. This system was chosen since the CRPV associated VX2 carcinoma of the New Zealand White rabbit is an established animal model for human head and neck squamous cell carcinoma (HNSCC). Literature research revealed several reports regarding VX2 carcinoma derived cell lines. Unfortunately, these cell lines were either discontinued or not readily available. This situation prompted us to generate our own VX2 cell line. Developing a VX2 carcinoma derived cell line would allow to perform in vitro studies thereby helping to reduce the number of animal experiments. The 2nd chapter of the thesis therefore deals with the development and characterization of a VX2 carcinoma derived cell line. It was presumed that this VX2 carcinoma cell line would help to conduct various experiments before performing tests in the VX2 rabbit animal model. VX2 cells were isolated from a VX2 tumor that was excised from the rabbit ear. Cells were allowed to grow and proliferate under standard laboratory conditions. The VX2 cell line survived for about 150 passages which is much longer than expected for cultures of normal cells, however, permanent cell growth could not be achieved. Immunostaining of VX2 cells at initial passages demonstrated the progressive loss of tumor associated fibroblasts giving rise to a VX2 cell line with a pronounced proliferative capacity. Flow cytometry results illustrated the presence of two VX2 cell populations, a small and a large one. Cell sorting, with subsequent cultivation for 5 days followed by western blot analysis favored the hypothesis that the larger VX2 cell population had the highest proliferative potential. Real time PCR was performed to analyze and confirm the presence of CRPV E6 and E7 oncogene transcripts in the VX2 cells using primers designed specifically to detect CRPV E6 and E7 transcripts. Expression of various proliferation markers, apoptosis related genes, EMT (epithelial mesenchymal transition) markers and CRPV E6 & E7 transcripts were consistently found in the VX2 carcinoma generated cell line as well as in the original VX2 tumor. Another objective, presented in the 3rd chapter of the thesis, was to evaluate therapeutic strategies in the VX2 carcinoma derived cell line. For this purpose, photodynamic therapy (PDT) was selected as a non-invasive treatment to investigate its biological effects on the VX2 carcinoma derived cells. Liposomal encapsulated curcumin was used as a photosensitizer. VX2 cells were treated with curcumin liposomes alone, PDT alone or a combination of curcumin liposomes and PDT. Cytotoxicity studies such as the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that addition of PDT could reduce the IC50 values of curcumin liposomes. Lysosomal disruption, live & dead staining and apoptosis assays were discussed in detail. A significant reduction in colony formation and cell migration was observed in cells exposed to both curcumin liposomes and PDT. In addition to the CRPV associated rabbit VX2 cell line, human papilloma virus (HPV) positive cell lines such as HeLa (HPV-18 positive cervical cancer cell line) and UD-SCC-2 (HPV-16 positive head and neck cancer cell line) were also included during this study to observe the therapeutic effects of curcumin loaded liposomes and PDT therapy. After assessing different parameters using PDT in papilloma virus associated cell lines, it could be concluded that a combination of curcumin liposomes along with PDT was significantly more effective when compared to a treatment with curcumin liposomes or PDT alone. The 4th chapter described an alternative approach for the in vitro study of CRPV oncogenes. Recombinant mammalian expression vectors containing CRPV E6 and E7 oncogenes with and without GFP and RFP reporter genes were successfully generated by PCR cloning using VX2 carcinoma derived RNA as a source. Recombinant clones were validated by restriction enzyme digestion and sequence analysis. Theses clones were transiently transfected in COS-7 & VX2 cells using polyethylenimine (PEI) based lipopolyplexes. The expression of CRPV E6 and E7 recombinant clones was assessed using different techniques. Microscopic results revealed successful expression of CRPV E6 & E7 genes by monitoring GFP and RFP reporter genes. Quantitative PCR demonstrated significant expression of E6 & E7 mRNA in transfected cell lines while western blot analysis demonstrated the expression of E7 recombinant proteins but not of E6 presumably due to failure of recombinant E6 proteins to dissolve sufficiently in the lysis buffer. The results presented in the 4th chapter provide an alternative platform for the study of anti-papillomavirus E6 and E7 therapeutic approaches. With this system, the consequences of targeted antiviral therapies could be easily evaluated by visual examination using fluorescence microscopy or quantitatively monitored in multiplate capable fluorescence detectors. The effect of experimental therapies on viral mRNA and protein expression levels could be measured via PCR and western blot analysis, e.g. by using anti-GFP or anti-RFP antibodies as no antibodies directed against CRPV E6 or E7 proteins are currently commercially available. In conclusion, this CRPV expression system could provide an in vitro platform for the evaluation of antiviral therapeutic approached prior to in vivo testing of e.g. promising therapeutic formulations in the CRPV positive rabbit VX2 carcinoma which serves as an animal model for human HNSCC.
Physical Description:133 Pages