Evaluation of telomerase control elements and radiation-inducible Waf1 promoter for the enhancement of targeted radiotherapy in neuroblastoma cells

Cosimo, Emilio (2007) Evaluation of telomerase control elements and radiation-inducible Waf1 promoter for the enhancement of targeted radiotherapy in neuroblastoma cells. PhD thesis, University of Glasgow.

Abstract

Introduction: Neuroblastoma has a long-term survival rate of only 15%. While patients with early stage disease can be treated with surgical excision of the tumour, those with inoperable disease require intensive treatment. However, little progress has been made in the survival rates of patients with advanced neuroblastoma. Targeted radiotherapy using [1311]meta-iodobenzylguanidine ([131l]MIBG has induced favourable remissions in some neuroblastoma patients when used as a single agent. However, uptake of the radiopharmaceutical in malignant sites is heterogeneous and approximately 15% of neuroblastoma patients are MIBG negative by scintigraphy and therefore do not progress to [131l]MIBG therapy. Therefore, the full potential of this therapy may only be realised by improving the drug accumulation capacity of neuroblastoma cells. One way to achieve this is by the introduction of cDNA of the noradrenaline transporter (NAT) into neuroblastoma cells. NAT is responsible for the intracellular accumulation of [131l]MIBG. In this strategy, the NAT transgene will be under the control of tumour specific promoter sequences such as the telomerase promoters or the radiation- inducible WAF1 promoter. Furthermore, this gene therapy approach could be improved by the use of immunoliposomes as a non-viral DNA delivery system. Aims: The aims of this study were to determine the potency of the telomerase promoters with respect to the NAT transgene expression, and to assess whether the radiopharmaceuticals [131l]MIBG and [211At]MABG could induce the activity of the WAF1 promoter. Finally, the capacity of GD2-targeted coated cationic immunoliposomes to transfer plasmid DNA specifically to GD2-positive neuroblastoma cells was evaluated. Results: Both telomerase promoters (hTR and hTERT) were able to drive the expression of the NAT transgene in neuroblastoma cells. Furthermore, this resulted in enhanced toxicity of the [131l]MIBG and [211At]MABG to the transfected cells, compared to that of untransfected cells. The hTERT promoter displayed the greatest activity for both [131l]MIBG and [211At]MABG treatments. The WAF1 promoter activity was inducible not only by external beam gamma-rays but also by the beta-emitter radionuclide 1311 in the form of [131l]MIBG or by the alpha-emitter radionuclide 211At conjugated to benzylguanidine ([211At]MABG). In vitro estimation of the equivalent radiation dose of both radiopharmaceuticals was performed. This demonstrated that levels of WAF1 promoter activation caused by [1311]MIBG or [211At]MABG were comparable to that by gamma-radiation. Preliminary toxicity experiments showed that, after irradiation, toxicity of [131I]MIBG improved in neuroblastoma cells transfected with the construct containing the NAT cDNA downstream of the WAF1 promoter sequence. The preparation of GD2-targeted coated cationic immunoliposomes, used in this study, successfully encapsulated plasmid DNA, and were specifically bound to and internalised by GD2-positive neuroblastoma cells. Unfortunately, low transfection efficiency indicated limited usefulness of this methodology. Conclusion: Increase in [1311]M1BG or [211At]MABG toxicity was achieved in neuroblastoma cells transfected with the NAT transgene under the control of the hTR or hTERT promoter. If the overexpression of the NAT transgene and the improved toxicity of radiolabelled drugs are confirmed in pre-clinical models, there is potential for therapeutic gain. The WAF1 promoter was activated by both radiopharmaceuticals, and preliminary experiments suggest that pre-exposure to ionizing radiation could increase the cytotoxicity of [1311]M1BG, via WAF1 promoter-controlled overexpression of the NAT transgene. These results together indicate potential for immediate applications in neuroblastoma patients, such as bone marrow purging. The technology of GD2-targeted, coated cationic immunoliposomes has great potential for its target-specificity and internalisation capacity. The low transfection effectiveness observed in this study may be improved with advances in the current methodology. These results suggest that further advances in promoter control and immunoliposomal technology could enable the application of NAT gene transfer in combination with [131I]MIBG targeted radiotherapy.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Nuclear physics and radiation, Oncology
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Mairs, Dr. Robert and Boyd, Dr. Marie
• Date of Award: 2007
• Depositing User: Enlighten Team
• Unique ID: glathesis:2007-71014
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 09 May 2019 14:28
• Last Modified: 21 May 2021 09:32
• URI: https://theses.gla.ac.uk/id/eprint/71014