Gene therapy of neuroblastoma
Sammanfattning: Aims: The survival of children with advanced stages of neuroblastoma has not improved significantly during recent years. Therefore additional treatments based on other modalities need to be developed. The objective of this thesis was to investigate the possibility to develop gene therapy of neuroblastoma. It includes studies of the two potential therapeutic genes Apoptosis Signal-regulating Kinase (ASK1) and the suicide gene E. coli Purine Nucleoside Phosphorylase (PNP), neuroblastoma specific therapy, and activation of the immune system.Results: Adenovirus-mediated expression of a constitutively active form of ASK1, promoted apoptosis in two out of four different neuroblastoma cell lines, judged by AnnexinV staining and DNA laddering. ASK1 induced apoptosis by activating the down stream p38MAP kinase signalling pathway which was visualised by western blot analysis. Nevertheless, it was possible to sensitise the unresponsive cell line, NXS2, with the microtubule-interfering agent, paclitaxel. The MASH1-promoter was placed upstream of the luciferase reporter gene or of the E. coli PNP gene in expression vectors. Measuring the luciferase activity, the different sizes of the proximal promoter displayed cell-type specific activity in neuroblastoma cell lines, when compared in non-neuroblastoma cell lines. The shortest MASH1 promoter was combined with the E.coli PNP gene and the toxicity was measured by MTT assay after addition of the prodrug F-araA. The cytotoxicity was 64 to 65 percent in the neuroblastoma cells with low toxicity in the non-neuroblastoma cell lines, relative to the cytotoxicity of the E.coli PNP gene regulated by the strong but non-specific CMV enhancer-promoter. The growth of tumours and the survival of animals challenged with wild type tumour cells were followed in A/J mice, which had been vaccinated with IL-12 and GM-CSF secreting C1300 tumour cells. The survival of animals, subcutaneously injected with the combination of IL-12 and GM-CSF secreting cells was increased compared to vaccination with IL-12 or GM-CSF alone. The immunological responses were analysed by FACS analysis of the splenocytes. The GM-CSF vaccinated animals had a marked increase of activated T helper cells and the GM-CSF and IL-12/GM-CSF groups had an increased fraction of NK1.1 positive cells expressing the NKG2D receptor. The binding of a NKG2D tetramer revealed that the IL-12 and GM-CSF- expressing tumour cells had an upregulated expression of the MHC class I like NKG2D ligands. This is a step forward facilitating detection and eradication of tumours by the NK and NKT cells carrying the NKG2D receptor.Conclusions: Our results show that it is possible to induce apoptosis of neuroblastoma cells in vitro, by ASK1 and E. coli PNP/F-araA, and the MASH1 promoter can be used as a regulatory region for a high and tissue specific expression of the therapeutic gene. Immunotherapy of a syngeneic neuroblastoma mouse model vaccinated with cells secreting IL-12 and GM-CSF increased survival. To further enhance the antitumour effect it would be interesting to combine immunotherapy with apoptosis-inducing gene therapy.
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