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T. J. McFarland, A. L. Dye, B. Ksander, J. Smith, J. T. Stout, B. Appukuttan; Down-Regulation of VEGF by RTEF-1 in Ocular Melanoma Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1685.
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© ARVO (1962-2015); The Authors (2016-present)
Uveal melanoma is the leading ocular cancer observed in U.S. adults today. Treatment options for ocular melanoma vary depending on the tumor size and location within the globe. Often enucleation is the treatment for large tumors. We have previously demonstrated that the 651 isoform of the related transcription enhancer factor-1 (RTEF-1) gene is effective at modulating VEGF gene expression. Due to the importance VEGF plays in tumor growth, it is an ideal target for therapy. The purpose of this study is to evaluate the effect of the 651 RTEF-1 isoform on ocular melanoma cells in culture.
Mel 202 cells were grown in culture until 80% confluent. An equal number of naive cells were plated or transfected with CMV-RTEF-1 651 or GFP plasmid using the Amaxa electroporation device with an optimized protocol. After 24 hours, the media was changed and cells were exposed to a 1% oxygen environment for 4 hours to mimic hypoxia. Media was collected for VEGF ELISA, which was normalized to total media protein, and RNA was isolated for VEGF and RTEF-1 analysis by RT-PCR.
By light microscopy there were more cells with a rounded abnormal morphology in the 651 transfected group compared to the GFP group. There was a 50% reduction in VEGF protein in the 651treated group compared to naive cells (n = 3, p=0.01). Semi-quantitative RT-PCR revealed the presence of the 651 transgene in the transfected samples and a decrease in VEGF levels compared to the control.
As evidenced by these data, RTEF-1 651 was capable of down-regulating VEGF and appears to impact cellular properties of this ocular melanoma cell line. Studies currently underway involve cellular proliferation and determining whether an apoptotic pathway is involved in the morphologic properties observed.
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