Tulley, Paul; (2000) The Role of Corrective Gene Therapy Targeting the C-myc Oncogene in Melanoma. Doctoral thesis (M.D), UCL (University College London).

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Abstract

C-myc oncogene expression is known to be a highly significant prognostic factor in melanoma. Corrective gene therapy targeting this gene has been demonstrated to inhibit the growth of melanoma in-vitro and in-vivo. This project has investigated the role of the c-myc oncogene in interferon resistance, the efficacy of combining c-myc antisense with interferon and chemotherapeutic drugs and has also designed and tested a ribozyme targeting the c-myc oncogene. The efficacy of interferon as adjuvant therapy for melanoma has recently been questioned. Overexpression of the c-myc oncogene has been shown to be associated with interferon resistance in cell lines. This project investigated the relationship between c-myc gene expression and interferon sensitivity in a series of 45 uveal melanomas using flow cytometry and the ATP chemosensitivity assay. High c-myc expression was found to correlate with interferon resistance as measured by the maximum percentage of inhibition (p=0.05) and the interferon sensitivity index (p=0.07). These results demonstrate that tumours with high c-myc expression and poor prognosis are also associated with interferon resistance. To assess whether corrective gene therapy targeting the c-myc gene could be used to improve the efficacy of interferon, 4 melanoma cell lines were treated with c-myc antisense and interferon, individually or in combination, and the effects on cell growth measured using the ATP-chemosensitivity assay. Combination of c-myc antisense with interferon was found to significantly increase the growth inhibitory effects of interferon in the cell lines expressing the highest levels of the c-myc gene. Super-additive effect was demonstrated in the A375m cell line. This suggests that corrective gene therapy could be used to improve the efficacy of interferon as adjuvant therapy. The prognosis for patients with advanced melanoma is very poor as standard chemotherapy drugs are generally ineffective. Tumour c-myc overexpression is known to be associated with chemo-resistance. C-myc antisense was therefore combined with a number of chemotherapeutic drugs and the effects on the growth of the A375m melanoma cell line assessed with the ATP-chemosensitivity assay. Combination of c-myc antisense with cis-platinum resulted in synergistic effect on cell growth inhibition, suggesting that this strategy could be used to overcome chemo-resistance in advanced melanoma. As an initial step in the development of c-myc antisense for use in patients a ribozyme targeting exon 2 of c-myc mRNA was designed, synthesised and subsequently tested in-vitro using the MTS assay. Although inhibition of melanoma cell growth was demonstrated to occur, this was found to be a non-specific transfection-related effect and not accompanied by down-regulation of c-myc expression. A second ribozyme, targeting a different part of exon 2 of the c-myc gene, has subsequently been designed which is known to be effective in down-regulating the c-myc oncogene in other cell lines.

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