In vitro inhibition of human glioblastoma cell line invasiveness by antisense uPA receptor

Abstract

The cell surface urokinase-type plasminogen activator receptor (uPAR) has been shown to be a key molecule in regulating plasminogen-mediated extracellular proteolysis. To investigate the role of uPAR in invasion of brain tumors, human glioblastoma cell line SNB19 was stably transfected with a vector capable of expressing an antisense transcript complementary to the 300 base pair of the 5' end of the uPAR mRNA. Parental and stably transfected (vector, sense, and antisense) cell lines were analysed for uPAR mRNA transcript by Northern blot analysis, and receptor protein levels were measured by radioreceptor assays and Western blotting. Signi®-cant reduction of uPAR sites was observed in the antisense transfected cell lines. The levels of uPAR mRNA were signi®cantly decreased in antisense clones compared to control, vector and sense clones. The invasive potential of the cell lines in vitro was measured by Matrigel invasion assay and migration of cells from spheroids to monolayers. The antisense transfected cells showed a markedly lower level of invasion and migration than the controls. The antisense clones were more adhesive to the ECM components compared to parental, vector and sense clones. All transfected (vector, sense and antisense) clones and parental cells produced similar levels of uPA activity without any signi®cant dierence however, MMP-2 activity was decreased in antisense clones compared to controls. These results demonstrate that uPAR expression is critical for the invasiveness of human gliomas and down regulation of uPAR expression may be a feasible approach to decrease invasiveness.

Adenovirus-mediated p16/CDKN2 gene transfer suppresses glioma invasion in vitro

“…An established human glioma cell line SNB19 (kindly provided by Dr Richard Morrison, MD Anderson Cancer Center, Houston, TX) (Go et al, 1996;Mohanam et al, 1997) was used in the current study. The cells were maintained in Dulbecco's modi®ed Eagle's medium/F12 medium (1:1, v/v) supplemented with 10% fetal calf serum in a humidi®ed atmosphere containing 5% CO 2 at 378C.…”

Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells

“…The cells were collected by centrifugation, homogenized in RIPA bu er (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mM EDTA, and 50 mM Tris Ph 7.4) with protease inhibitors and centrifuged at 18 0006g for 15 min. The supernatant was boiled in SDS sample bu er under reducing conditions and applied on a 12.5% polyacrylamide gel prepared as described earlier (Mohanam et al, 1997). Separated proteins were electroblotted onto a nitrocellulose membrane, and nonspeci®c binding was blocked by incubation with 5% milk powder in washing bu er.…”

“…Cells were trypsinized, and 200 ml of cell suspension (3610 5 cells per ml) were added in triplicate wells. After 24 h incubation, the cells that passed through the ®lter into lower wells were quantitated as described earlier (Mohanam et al, 1997) and expressed as a percentage of the sum of the cells in the upper and lower wells. Cells on the lower side of the membrane were ®xed, stained with Heme-3 and photographed.…”

“…Total cellular RNA was extracted from con¯uent cultures as described earlier (Mohanam et al, 1997). Aliquots of 10 mg of RNA was separated by electrophoresis on 1% agarose formaldehyde gels, capillary transferred to a nylon membrane overnight and the ®lters were hybridized at 658C with 32 Pradiolabelled cathepsin B cDNA probe (Sivaparvathi et al, 1995).…”

“…Cells were seeded in 35 mm culture dishes grown overnight, transfected with 5 mg DNA using Lipofectin (Mohanam et al, 1997) and then cultured for 48 h in complete medium before transfer to petridishes containing complete medium and 800 mg/ml G418. After 3 ± 4 weeks in culture, G418-resistant colonies were isolated by means of cloning cylinders and characterized for the reduction in production of cathepsin B protein.…”