Characterization of RTEF-1 in Choroidal Melanoma Cells Under Hypoxic Conditions

Abstract

Purpose: : We have identified 3 novel ocular specific related transcriptional enhancer factor-1 (RTEF-1) isoforms; 936, 447, and 651 within ocular endothelial cells. RTEF-1 isoforms differ in their ability to regulate VEGF gene expression and appear to act independently of the hypoxia response element (HRE) mediated pathway. Hypoxia and VEGF play a role in the stimulation of angiogenesis during tumor development. We aim to determine if RTEF-1 is present in human ocular melanoma (OM) cells and its role in VEGF regulation in OM cells under hypoxic conditions.

Methods: : Choroidal melanoma cells (OCM3, MEL 270, MEL 202, OMM1.3) were cultured under normoxic or hypoxic conditions. Total RNA and protein was purified. Semi-quantitative RT-PCR and Western blot analysis were used to compare RTEF-1 RNA and protein from hypoxic and normoxic conditions (n=3). Sequencing determined isoform exon structure. Isoform modulation of the human VEGF promoter was determined by pSEAP reporter assays. Plasmid electroporation was optimized for each cell line with the Amaxa Nucleofection machine. One of each RTEF-1 isoform (447, 651 and 936) or full length (1305) were co-electroporated with VEGF reporter constructs into OM cells. Reporter protein levels were compared.

Results: : Optimal nucleofection programs were identified and noted to be different between OM cell types. Protein data indicated that multiple isoforms exist, ranging in size from 20 to 50 kDa. Isoforms differed between cell lines. The full length 1305 RTEF-1 (~50 kDa) is the primary product expressed. Densitometry of Western blot data showed the 50 kDa isoform to be more abundant in OCM3 cells than other OM cell lines. The 50 kDa product is increased in hypoxic OCM3 cells. In contrast, another RTEF-1 isoform (~38 kDa) with a size corresponding to the 651 isoform, appears to be decreased under hypoxic conditions in Mel 202 cells. RT-PCR showed the presence of RTEF-1 isoforms in OM cells. Isoforms differed between hypoxic and normoxic conditions. In addition, a new 750bp isoform of RTEF-1 was discovered in Mel 202 cells under hypoxic conditions.

Conclusions: : Alternative isoforms of RTEF-1 exist within OM cells. These isoforms may differentially regulate the activity of the VEGF gene within OM cells. This observation has potential implication for the treatment of ocular tumors and other VEGF-dependent diseases in the human eye.

Keywords: melanoma • transcription factors • vascular endothelial growth factor