Identification of the Motif in Versican G3 Domain That Plays a Dominant-negative Effect on Astrocytoma Cell Proliferation through Inhibiting Versican Secretion and Binding

Yaojiong Wu,Yaou Zhang,Liu Cao,Liwen Chen,Vivian Lee,Peng-Sheng Zheng,Chris Kiani,Mark E Adams,Lee Cyn Ang,Frouz Paiwand,Burton B Yang

doi:10.1074/jbc.m100618200

Yaojiong Wu, Yaou Zhang + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m100618200

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Journal: The Journal of biological chemistry	Publication Date: Apr 1, 2001
Citations: 53	License type: cc-by

Affiliation: University of Toronto

Abstract

This study was designed to investigate the mechanisms by which mutant versican constructs play a dominant-negative effect on astrocytoma cell proliferation. Although a mini-versican or a versican G3 construct promoted growth of U87 astrocytoma cells, a mini-versican lacking epidermal growth factor (EGF) motifs (versicanDeltaEGF) and a G3 mutant (G3DeltaEGF) exerted a dominant-negative effect on cell proliferation. G3DeltaEGF-transfected cells formed smaller colonies, arrested cell cycle at G(1) phase, inhibited expression of cell cycle proteins cdk4 and cyclin D1, and contained multiple nucleoli. In cell surface binding assays, G3 products expressed in COS-7 cells and bacteria bound to U87 cell surface. G3DeltaEGF products exhibited decreased binding activity, but higher levels of G3DeltaEGF products were able to inhibit the binding of G3 to the cell surface. G3DeltaEGF expression inhibited secretion of endogenous versican in astrocytoma cells and also inhibited the secretion of mini-versican in COS-7 cells co-transfected with the mini-versican and G3DeltaEGF constructs. The effect seems to depend on the expression efficiency of G3DeltaEGF, and it occurred via the carbohydrate recognition domain.

Highlights

Versican, a member of the large aggregating chondroitin sulfate proteoglycan family, was initially detected in the limb bud of chick embryo [1] and later cloned in human fibroblasts and chick embryo [2,3,4,5]
Proliferation Assays—Growth media from COS-7 cells transfected with different recombinant constructs were mixed in a 1:1 ratio with native culture medium (DMEM supplemented with 2.5% fetal bovine serum (FBS)), and the mixture was introduced into glioma cells cultured in 96-well tissue culture plates at a density of 2 ϫ 103 cells/well
The mini-versican gene was expressed in COS-7 cells, and its expression and secretion were confirmed by Western blot

Summary

EXPERIMENTAL PROCEDURES

Materials and Cell Cultures—Lipofectin, Geneticin (G418), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Hank’s balanced salt solution, trypsin/EDTA were from Life Technolo-. Proliferation Assays—Growth media from COS-7 cells transfected with different recombinant constructs were mixed in a 1:1 ratio with native culture medium (DMEM supplemented with 2.5% FBS), and the mixture was introduced into glioma cells cultured in 96-well tissue culture plates at a density of 2 ϫ 103 cells/well. Cell lysate and culture medium were harvested for analysis of gene expression on Western blot. Cell proliferation was tested in glioma cell lines stably transfected with versican⌬EGF construct and the control vector. To test whether one product could compete with another for binding to glioma cells, 50 ␮l of peptides purified from bacteria was mixed with different amounts of competing medium (from G3- or G3⌬EGF-transfected COS-7 cells). Equal amounts of proteins from each treatment were analyzed on Western blot to estimate the binding of the above products to glioma cells.

Versican Enhances Glioma Proliferation

RESULTS

DISCUSSION