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Title: Identification of the Apoptin tumour specific kinase/s
Author: Cole, Daryl
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2013
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A chicken anaemia virus (CAV) protein, Apoptin, has been shown to induce specific killing targeted at human transformed cells, leaving non-transformed cells unaffected. Investigation has revealed that Apoptin is phosphorylated on a threonine residue in transformed cells, suggesting a role for kinase activation in Apoptin cytotoxicity. The aim of this study was to identify the kinase or kinases involved in Apoptin cytotoxicity using a serine/threonine kinase siRNA library. In addition, we have recently identified that Apoptin interacts and is phosphorylated by Protein Kinase C (PKC). Also, a potential role for cyclic Guanosine Monophosphate dependent protein kinase (PKG) in the regulation of Apoptin has been investigated. PKC/PKG expression was investigated in cell lines in relation to Apoptin sensitivity. Subsequent modification of PKC/PKG kinase expression was investigated and its effects on Apoptin sensitivity. Further to these studies, a recently discovered human derived gyrovirus producing a protein with homology to CAV Apoptin was investigated. The results indicated that in HCT116 colon cancer cells Apoptin cytotoxicity was not related to the kinase activity of the siRNA library targets. PKCĪ²1 was confirmed to be important in sensitivity to Apoptin in multiple myeloma cell lines, however, this importance in Apoptin-induced cell death in head and neck cancer is currently unclear. PKG expression was found to be higher in normal cells compared to transformed cells. In contrast, PKC expression was shown to be higher in transformed cells when compared to normal cells. Apoptin phosphorylation experiments proved to be inconclusive with regards to kinase expression. Physical interaction between Apoptin, PKC and PKG was detected by immunoprecipitation, however, evidence of functional effects based solely on these interactions was not found. The results indicate that PKG and PKC expression is potentially linked to Apoptin function in the cell lines tested. In addition, a newly discovered Human gyrovirus (HGyV) Apoptin was shown to have a similar cellular localisation to CAV Apoptin, and was also found to be toxic to transformed cells, though more indiscriminately than CAV Apoptin. In conclusion, the results presented here in this study propose a novel role for PKG and PKC in the tumour specific activation of Apoptin in normal and transformed cells. In the absence of PKG, transformed cells activate Apoptin through PKC leading to nuclear localisation and apoptosis. PKG expression in normal cells appears to block this process, resulting in cytoplasmic localisation of Apoptin and abrogation of Apoptin induced cell death. Additionally, the novel viral protein human gyrovirus Apoptin has been shown to behave in a similar manner to CAV Apoptin, providing a further tumour specific protein for future study. The information obtained during this study will therefore be important in the development of therapeutic strategies for not only determining sensitivity to Apoptin of certain tumours but also for the sensitisation of tumour cells to Apoptin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available