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Jon R Backstrom, Stephanie M Evans, Ashwath Jayagopal; Melanoma Inhibitor of Apoptosis (ML-IAP) - Driven Expression of the Pro-Apoptotic Protein BAD in Uveal Melanoma Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1285.
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© ARVO (1962-2015); The Authors (2016-present)
The paucity of effective treatments and low survival rate of patients with metastatic uveal melanoma warrant new targeting strategies. Our goal is to develop adeno-associated viruses that selectively express cargo genes (such as the pro-apoptotic gene, BAD) in uveal melanoma cells that have metastasized to the liver. Expression of ML-IAP (birc7/livin) is restricted to melanomas and RPE. Therefore, we evaluated the promoter activity of ML-IAP in uveal melanoma and RPE cell lines.
The ML-IAP promoter and BAD cDNA were cloned from the human melanoma cell line SK-MEL-28. Site-directed mutagenesis was used to mutate the ML-IAP promoter at consensus transcription factor binding sites and BAD cDNA at previously characterized phosphorylation sites. Plasmids with ML-IAP/BAD upstream of either CMV/Renilla luciferase or CMV/mCherry were introduced into primary and metastatic uveal melanoma cell lines derived from the same patient. A CMV/Renilla plasmid without ML-IAP/BAD was used in separate wells as a negative control for BAD activity since ML-IAP-driven expression of BAD results in cell death and concomitant lower luciferase activity. Immunocytochemistry with antibodies against cleaved caspase-3 was used to monitor apoptosis of cells transiently expressing BAD-FLAG and mCherry.
Higher levels of endogenous ML-IAP protein were detected on immunoblots prepared from primary melanoma cells than from metastatic cells. The effectiveness of introduced ML-IAP/BAD was abrogated (highest luciferase activity) with a single serine-to-aspartic acid mutation at the Bcl2/Bcl-XL interaction site of BAD and increased (lowest luciferase activity) with serine-to-alanine mutations at three phosphorylation sites (BAD-3SA). Therefore, BAD-3SA was utilized for subsequent analyses of the ML-IAP promoter. The ML-IAP/BAD-3SA construct was most effective in primary melanoma cells and least effective in the RPE cell line ARPE-19. Mutations at all of the E-box sites in the ML-IAP promoter (MITF, USF, and AP4) prevented BAD-3SA activity in primary melanoma cells, but not metastatic cells.
The ML-IAP promoter is capable of driving expression of BAD at levels that promote apoptosis of uveal melanoma cells. Furthermore, the data raise the intriguing possibility that transcription factor interactions at the ML-IAP promoter change as cells transition from primary to metastatic.
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