Angiostatin Expression in Human and Murine Melanoma Cell Lines

Abstract

Abstract: : Purpose: To investigate angiostatin activity in human uveal and murine melanoma cell lines. Methods: Human uveal melanoma (MEL270, MEL285, MEL290), murine melanoma (B16LS9, B16F10, Queens), and bovine vascular endothelial (EJG) cells were grown in culture. EJG (0.1ml, 1X10₅/ml) and 0.4 ml MEM were added to lightly gelatin coated 24-well-plates. The plates were incubated overnight, and the human uveal and murine melanoma cell lines (0.3ml, 1X10₆/ml) were added to individual wells (6 wells/specimen). EJG supernatant was the control. Human glu-plasminogen (50µL, 1mg/ml) was added to each of 3 wells for each supernatant tested and controlled. Serum-free RPMI was added to each well, totaling a volume of 500µL in each well. ₃H was diluted to 1:10 in serum-free RPMI and 10µL was added to each well. The plates were incubated for 24h at 37°C. 10%SDS(100µL) was added to each well and the plate remained at room temperature for 20 minutes. 0.25mlPBS was added to each well. The contents of each well were transferred to a scintillation vial, and the radioactivity of each vial was counted for 1 min in a liquid scintillation counter. Data were presented as %angiostatin activity=[%cell proliferation in absence of plasminogen]-[%cell proliferation in presence of plasminogen]. Results: Angiostatin activities of human uveal melanoma MEL270, MEL285 and MEL290 were 0.73%, 0.43% and 1.53% and murine melanoma B16LS9, B16F10 and Queens were 1.27%, 0.57% and -1.07% compared with -2.63% for EJG cells. Conclusions: Angiostatin activities of the melanoma cell lines tested varied according to the cell line. These results suggest that angiostatin expression of primary melanoma may suppress micrometastases, depending on the cell line.

Keywords: 464 melanoma • 610 tumors • 506 pathology: experimental