Identification of the motif in versican G3 domain that plays a dominant-negative effect on astrocytoma cell proliferation through inhibiting versican secretion and binding.
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.
Wu
Y
Y
Sunnybrook & Women's College Health Sciences Centre and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M4N 3M5, Canada.
Zhang
Y
Y
Cao
L
L
Chen
L
L
Lee
V
V
Zheng
P S
PS
Kiani
C
C
Adams
M E
ME
Ang
L C
LC
Paiwand
F
F
Yang
B B
BB
D020816:Amino Acid Motifs; D000818:Animals; D001254:Astrocytoma / Q000378:metabolism*; D001667:Binding, Competitive; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism; D019556:COS Cells; D002453:Cell Cycle; D002455:Cell Division; D002466:Cell Nucleolus / Q000378:metabolism; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000737:chemistry* / Q000378:metabolism*; D019938:Cyclin D1 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D017353:Gene Deletion; D005799:Genes, Dominant; D005822:Genetic Vectors / Q000378:metabolism; D005910:Glioma / Q000378:metabolism; D037181:Lectins, C-Type; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured; D053675:Versicans
D011508:Chondroitin Sulfate Proteoglycans; D018076:DNA, Complementary; D037181:Lectins, C-Type; D011509:Proteoglycans; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D053675:Versicans; D019938:Cyclin D1; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases