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New approaches to gene therapy and prognostic markers in melanoma

Ramsden, A; (2005) New approaches to gene therapy and prognostic markers in melanoma. Doctoral thesis , UCL (University College London). Green open access

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Abstract

The c-myc proto-oncogene plays a key role in cell proliferation, malignant transformation and apoptosis. Previous work at RAFT demonstrated that c-myc expression levels are an accurate prognostic marker in melanoma. Ribozymes are catalytic RNA molecules that cleave specific mRNA sequences to prevent gene expression. A ribozyme was constructed targeting the c-myc translation initiation site. A375M melanoma cells were transfected using liposomes and growth was assessed using a MTS growth assay, c-myc expression was measured with immuno-staining and flow-cytometry. Ribozyme treatment of A375M melanoma cells reduced c-myc expression when compared to non-specific controls (p<0.001). Ribozyme treatment also significantly reduced growth of A375M cells in-vitro compared to untreated controls (p<0.001). Ribozymes were investigated in combination with interferon and cis- platinum. A prospective investigation of c-myc expression in 117 melanomas was undertaken using immunostaining and flow cytometric analysis. Clinico-pathological details were also studied. Analysis revealed that high c-myc expression was associated with a worse prognosis (p=0.043). Multivariate analysis demonstrated c-myc as an independent prognostic marker when measured against other routine parameters including Breslow thickness. To facilitate rapid screening for molecular alterations in melanoma, a tissue micro-array was created using historical paraffin embedded specimens. 126 tumours were included in the array block representing all stages of disease progression. Analysis was performed on a variety of genes associated with c-myc examining prognostic significance. High expression of p27 and p53 were significantly associated with improved survival (p<0.05). Cyclins A, E and D1 correlated with disease progression (p<0.05). Conclusions. Ribozymes can effectively block c-myc gene expression in vitro and may have a role in the treatment of melanoma. Flow cytometric analysis of c-myc expression provides a new important independent prognostic marker for melanoma. Tissue array technology is a simple and powerful tool in the search for new prognostic markers in melanoma.

Type: Thesis (Doctoral)
Title: New approaches to gene therapy and prognostic markers in melanoma
Identifier: PQ ETD:593254
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest. Third party copyright material has been removed from the ethesis. Images identifying individuals have been redacted or partially redacted to protect their identity.
URI: http://discovery.ucl.ac.uk/id/eprint/1445930
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