A Béliveau1, M Bérubé, P Carrier, C Mercier, S L Guérin. 1. Oncology and Molecular Endocrinology Research Center, Centre Hospitalier Universitaire de Québec and Laval University, Ste-Foy, Québec, Canada.
Abstract
PURPOSE: It has been suggested that the epithelioid morphology and high aggressiveness that is typical of the uveal melanoma cell line TP17 is dependent on the loss of alpha5beta1 integrin expression at the cell surface. The purpose of the current study was to test this hypothesis in the TP17 cell line and investigate the role this integrin may play in the tumorigenicity of the SP6.5 cells, a mixed spindle-epithelioid culture-type human uveal melanoma that shows tumorigenic properties clearly distinct from that of TP17 cells. METHODS: Expression of the alpha5 integrin subunit was restored in the alpha5-TP17 cell line by stably transfecting the cells with a recombinant plasmid encoding the integrin subunit. Flow cytometry and adhesion assays on fibronectin (FN)-coated culture plates were used to monitor alpha5 expression in the cells. The effect of alpha5 expression on both tumorigenicity and cell proliferation was evaluated in vivo in nude mice. In vitro growth properties of the alpha5(+) TP17 cells was evaluated by cell counting and compared with that of the alpha5 parental TP17 cell line. The influence exerted by the alpha5 integrin subunit on the tumorigenic and proliferative properties of the SP6.5 cells was evaluated in vivo in nude mice by exposing the cells to increasing doses of a blocking antibody directed against the alpha5-subunit before subcutaneous injection, and compared with the results obtained with untreated SP6.5 cells. RESULTS: Expression of the alpha5 integrin subunit in the alpha5-TP17 cells was successfully achieved, as evidenced by both flow cytometry and adhesion assays on FN-coated culture plates. Restoring expression of alpha5 in TP17 cells enhanced epithelioid cell morphology and increased the growth properties of this cell line in vivo. The ability of the SP6.5 cells to yield subcutaneous tumors was found to be concentration dependent and was reduced in a dose-dependent manner when the cells were exposed to the anti-alpha5 blocking antibody. CONCLUSIONS: Restoring expression of alpha5 in the alpha5-negative TP17 uveal melanoma cell line influenced the proliferative properties of these cells but did not alter its tumorigenic potential. In contrast, the ability of the SP6.5 cells to yield tumors in vivo in nude mice appeared to be related to expression of this integrin.
PURPOSE: It has been suggested that the epithelioid morphology and high aggressiveness that is typical of the uveal melanoma cell line TP17 is dependent on the loss of alpha5beta1 integrin expression at the cell surface. The purpose of the current study was to test this hypothesis in the TP17 cell line and investigate the role this integrin may play in the tumorigenicity of the SP6.5 cells, a mixed spindle-epithelioid culture-type humanuveal melanoma that shows tumorigenic properties clearly distinct from that of TP17 cells. METHODS: Expression of the alpha5 integrin subunit was restored in the alpha5-TP17 cell line by stably transfecting the cells with a recombinant plasmid encoding the integrin subunit. Flow cytometry and adhesion assays on fibronectin (FN)-coated culture plates were used to monitor alpha5 expression in the cells. The effect of alpha5 expression on both tumorigenicity and cell proliferation was evaluated in vivo in nude mice. In vitro growth properties of the alpha5(+) TP17 cells was evaluated by cell counting and compared with that of the alpha5 parental TP17 cell line. The influence exerted by the alpha5 integrin subunit on the tumorigenic and proliferative properties of the SP6.5 cells was evaluated in vivo in nude mice by exposing the cells to increasing doses of a blocking antibody directed against the alpha5-subunit before subcutaneous injection, and compared with the results obtained with untreated SP6.5 cells. RESULTS: Expression of the alpha5 integrin subunit in the alpha5-TP17 cells was successfully achieved, as evidenced by both flow cytometry and adhesion assays on FN-coated culture plates. Restoring expression of alpha5 in TP17 cells enhanced epithelioid cell morphology and increased the growth properties of this cell line in vivo. The ability of the SP6.5 cells to yield subcutaneous tumors was found to be concentration dependent and was reduced in a dose-dependent manner when the cells were exposed to the anti-alpha5 blocking antibody. CONCLUSIONS: Restoring expression of alpha5 in the alpha5-negative TP17 uveal melanoma cell line influenced the proliferative properties of these cells but did not alter its tumorigenic potential. In contrast, the ability of the SP6.5 cells to yield tumors in vivo in nude mice appeared to be related to expression of this integrin.