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RL Shearer, PR Van Ginkel, AS Polans; Over-expression Of The Receptor Tyrosine Kinase Axl Promotes Ocular Melanoma Cell Survival . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1125.
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Purpose: The aim of this project is to determine whether the over-expression of Axl and the stimulation by its ligand, Growth Arrest Specific Protein 6 (Gas6), enhances the survival of uveal melanoma cells in vitro. Secondarily, alterations in gene expression as a function of Gas6 stimulation will help define the cellular events by which Axl provides selective advantage to tumor cells. Methods: Axl expression was compared between normal uveal melanocytes and established cell lines of uveal melanoma by RNase protection assay and by Western analysis. Gas6 stimulation of Axl and the subsequent degree of mitogenesis and cell survival were assessed by bromodeoxy uridine incorporation and trypan blue exclusion, respectively. Cell lines transfected with an Axl anti-sense construct were used to demonstrate the specificity of the Axl and Gas6 effects. Gene expression was assessed using gene chip arrays. Results: Axl is expressed at higher levels in the epithelioid cell line Mel-290 than in normal uveal melanocytes and the spindle type uveal melanoma cell line Mel-270. Axl transcripts also were detected in fresh biopsies and archival specimens of primary uveal melanoma. Gas6, the ligand for Axl, mediates mitogenesis and cell survival in serum starved Mel-290 cells in culture. The effect of Gas6 was specific for the Axl pathway, since transfected cells expressing lower levels of Axl demonstrated diminished synthesis of DNA and eventually died from serum starvation. Basic fibroblastic growth factor (bFGF) could not substitute for Gas6 in these experiments. Fluorescence In Situ Hybridization (FISH) indicates a possible amplification of the Axl gene on chromosome 19 in Mel-290 cells, providing a structural basis for the elevated expression of Axl. Conclusion: The over-expression of Axl may provide selective advantage during the growth and metastasis of uveal melanoma, especially for long-lived micrometastases associated with the disease.
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