| Summary: | Apoptin, the VP3 protein derived from chicken anaemia virus (CAV), induces tumour cell-specific apoptosis and therefore represents a potential anti-cancer therapeutic agent of the future. In human tumour cells, but not in normal cells, Apoptin is phosphorylated and subsequently translocates to the nucleus, which are essential important steps for its cytotoxic activity. Recently, the β isozyme of protein kinase C (PKCβ) was identified as a kinase phosphorylating Apoptin in multiple myeloma cells. However, the exact mechanism and nature of interaction between PKCβ and Apoptin as well as its importance for Apoptin-induced cell death remain to be characterised. This project aimed to further investigate the physical and functional interaction between PKC isoforms, in particular PKCβ, and Apoptin. Recently, the first human Gyrovirus (HGyV) was isolated which shows significant structural and organisational resemblance to CAV and encodes a homologue of CAV-Apoptin. Using a synthetic HGyV-Apoptin construct the subcellular distribution and apoptotic function of this novel Apoptin protein in several human cancer cell lines was analysed and compared to CAV-Apoptin. The results demonstrated a comparable tumour-specific nuclear translocation and cytotoxic effect between the two proteins. A model of colon carcinoma and normal mucosa cell lines was used to investigate the functional link between PKC and Apoptin in other cancer types. Immunoprecipitation and proximity ligation assay studies confirmed the binding of both CAV- and HGyV-Apoptin to PKCβI in HCT116 colorectal cancer cells and identified the N-terminal region of Apoptin to be important for the interaction with PKC. In contrast to HCT116 cells two normal colon mucosa cell lines tested expressed low levels of PKCβI. This differential expression pattern of PKCβI correlated with reduced Apoptin activation in normal cells, as evident by its localisation to the cytoplasm, decreased phosphorylation and lack of cytotoxic activity. Further studies revealed that PKCβI knockdown in HCT116 cells resulted in reduced Apoptin phosphorylation but did not impair Apoptin cytotoxicity. Additionally, overexpression of PKCβI was not sufficient to activate Apoptin in normal cell lines, indicating that other kinases or processes may contribute to the tumour-specific function of Apoptin. Using the FRET-based PKC activity reporter CKAR and fluorescence lifetime imaging microscopy (FLIM) the effect of Apoptin or other stimuli on PKC activity was analysed. Similar to treatment with the phorbol ester TPA, Apoptin expression in HCT116 cancer cells resulted in a significant increase in PKC activity, correlating with the expression and increased phosphorylation levels of Apoptin. In contrast, normal colon mucosa cell lines with low PKCβI expression showed a delayed and reduced induction of PKC activation in response to Apoptin. Overexpression and knockdown studies in combination with FLIM provided some evidence that Apoptin predominantly activates the PKCβI isoform in HCT116 cells. Another tumour-selective cytotoxic agent, TRAIL, was tested on a panel of head and neck cancer cell lines associated with human papillomavirus (HPV) infection. Addition of the proteasome inhibitor Bortezomib sensitised TRAIL-resistant HPV positive head and neck cancer cells to TRAIL. This seemed to involve activation of caspases, the anti-apoptotic protein XIAP as well as stabilisation of functional p53, but the precise mechanism has so far not yet been established. In conclusion, the results of this study propose an important link between Apoptin and PKCβI in cancer cells, involving their interaction and co-localisation, Apoptin-induced activation of PKC and PKCβI-mediated phosphorylation of Apoptin to promote its nuclear translocation and cytotoxic function. In addition, the novel HGyV-Apoptin was shown to function in a similar manner to CAV-Apoptin, providing a further tumourspecific protein for future studies.
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