Gelsolin functions as a metasatsis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect

Abstract

Gelsolin, an actin-binding protein, is implicated as a critical regulator in cell motility. In addition, we have reported that cellular levels of gelsolin are decreased in various tumor cells, and overexpression of gelsolin by gene transfer suppresses tumorigenicity. We sought to assess the effects of gelsolin overexpression on metastasis and to determine the importance of a carboxyl-terminus that confers Ca 2؉ dependency on gelsolin for effects of its overexpression. Expression vectors with cDNA encoding either full-length wild-type or His321 mutant form, isolated from a flat revertant of Rastransformed cells and a carboxyl-terminal truncate, C-del of gelsolin, were transfected into a highly metastatic murine melanoma cell line, B16-BL6. Expression of introduced cDNA in transfectants was confirmed using Western blotting, 2-dimensional gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR). We characterized phenotypes of transfectants, such as growth rate, colony formation in soft agar, cell motility and metastasis formation in vivo. Transfectants expressing the wild-type, His321 mutant and C-del gelsolin exhibited reduced growth ability in soft agar. Although expression of integrin ␤1 or ␣4 on the cell surface of transfectants was not changed, wild-type and His321 mutant gelsolin, except for C-del gelsolin, exhibited retardation of cell spreading, reduced chemotatic migration to fibronectin and suppressed lung colonization in spontaneous metastasis assay. Gelsolin may function as a metastasis suppressor as well as a tumor suppressor gene. The carboxylterminus of gelsolin is important for retardation of cell spreading, reduced chemotasis and metastasis suppression. © 2001 Wiley-Liss, Inc. Key words: gelsolin; metastasis; cell spreading; cell motilityMetastasis, the spread of malignant tumor cells from a primary site to distant sites, is the most life-threatening complication of cancer and a major problem of cancer treatment. 1,2 The metastatic process consists of multiple steps: (i) dissociation of tumor cell(s) from the primary site with a concomitant loss of cell-cell and cell-extracellular matrix (ECM) adhesions; (ii) tumor-cell adhesion to and subsequent local digestion of basement membrane; (iii) retraction of endothelial cells and subsequent intravasation; (iv) survival within the vasculature; (v) extravasation from vasculature at a distant site and (vi) growth in a "foreign" or ectopic organ environment. 3,4 Much attention has been paid to the interaction of cell-cell or cell-ECM adhesion [5][6][7] and to proteolysis by a variety of classes of degradative enzymes and their inhibitors, 8,9 even though tumor cell migration is also an essential step for establishment of cancer metastasis. It is well known that the actin cytoskeleton is a major component of the cell motility machinery. 10,11 Evidence is accumulating that changes in actin cytoskeletal organization, adhesiveness and motility are important not only for tumor development and progression but also may be critical for deter...

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