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Endogenously produced urokinase-type plasminogen activator is a major determinant of the basal level of activated ERK/MAP kinase and prevents apoptosis in MDA-MB-231 breast cancer cells

¹ Departments of Pathology,
² Cell Biology², and
³ Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA

View larger version (11K): [in a new window]   Fig. 1. Equilibrium binding of 125I-DIP-uPA to MDA-MB-231 cells. MDA-MB-231 cells were acid-washed and then incubated with increasing concentrations of 125I-DIP-uPA for 4 hours at 4°C. The specific binding isotherm is shown in panel A. Each point represents the mean ± s.e.m. of results from two separate experiments, each with triplicate determinations. The Scatchard transformation of the same data is shown in panel B. B/F, bound/free.

View larger version (18K): [in a new window]   Fig. 2. Accumulation of uPA in MDA-MB-231 cell-conditioned medium. (A) Immunoblot analysis was used to detect uPA in serum-free medium conditioned by MDA-MB-231 cells in the presence or absence of uPA-specific antibody for 24 hours. A standard curve was generated using known amounts of purified uPA (5-50 ng). The low mobility band is assumed to represent uPA-PAI-1 complex, due to the apparent molecular mass. (B) Levels of UPA in CM were quantitated by densitometry, in comparison with the standard curve (n=3).

View larger version (35K): [in a new window]   Fig. 3. ERK phosphorylation in MDA-MB-231 cells treated with uPA- or uPAR-specific antibody. Cells were treated for 24 hours with uPA-specific antibody (panel A), uPAR-specific antibody (panel B) or nonimmune IgG from mouse or rabbit (panel C). Lanes labeled ‘-’ were treated with vehicle. Cell extracts were subjected to SDS-PAGE and electrotransfered to nitrocellulose membranes, which were probed to detect phosphorylated ERK, stripped and then re-probed to detect total ERK. Duplicate determinations from separate cultures are shown in panels A and B.

View larger version (33K): [in a new window]   Fig. 4. Activation of MCF-7 cell ERK by MDA-MB-231 cell CM. MDA-MB-231 cells were allowed to condition serum-free medium for 24 hours. The CM was subjected to centrifugation at 15,000 g for 10 minutes to remove debris and then added to cultures of MCF-7 cells that had been serum-starved for 12 hours. Some samples of CM were pre-incubated with uPA-specific antibody (25 µg/ml) or mouse nonimmune IgG (25 µg/ml) for 20 minutes at 37°C before being added to the MCF-7 cell cultures. ERK phosphorylation was assessed 1 minute after addition of the CM.

View larger version (21K): [in a new window]   Fig. 5. uPA-specific antibody inhibits the growth of MDA-MB-231 cells. (A) The indicated number of MDA-MB-231 cells was plated in 96-well plates, in triplicate, and cultured overnight. The cultures were then transferred into serum-free medium and incubated for 24 hours with uPA-specific antibody (+) or vehicle (-). Cell growth was determined by MTT assay. The number of viable cells in each culture prior to the 24 hour culturing period was determined as a control (labeled ‘C’). The asterisk indicates that antibody significantly altered cell growth compared with vehicle (unpaired t-test, P<0.05, n=3). (B) 4x104 MDA-MB-231 cells were plated in 48-well plates, in triplicate, and cultured at 37°C for 24 hours. The cells were then incubated with increasing amount of uPA-specific antibody, PD098059 (10 µM) or mouse nonimmune IgG (25 µg/ml) for 30 hours. [3H]thymidine incorporation was measured. The asterisk indicates that the value is significantly different from the control (unpaired t-test, P<0.01, n=3).

View larger version (22K): [in a new window]   Fig. 6. uPAR-specific antibody inhibits the growth of MDA-MB-231 cells. (A) 5x103 cells were plated in 96-well plates, in triplicate, and incubated for 24 hours. The cells were subjected to a mild acid wash and incubated with DMSO (0.1% v/v), PD098059 (10 µM), rabbit nonimmune IgG (50 µg/ml), uPAR-specific antibody (50 µg/ml) or PD098059 (10 µM) plus uPAR-specific antibody (50 µg/ml). Cell growth was measured by MTT assay. The asterisk indicates that the treatment is significantly different from control (unpaired t-test, P<0.015, n=3). The dagger indicates that this result is not significantly different than the result obtained with uPAR-specific antibody or PD098059 alone. (B) An identical series of incubations was executed and cell growth was measured by the [3H]thymidine incorporation method. Asterisks/daggers are as for part A.

View larger version (28K): [in a new window]   Fig. 7. uPA-and uPAR-specific antibodies induce apoptosis in MDA-MB-231 cells. (A) MDA-MB-231 cells were cultured in 96-well plates and then incubated in serum-free medium, supplemented with uPA-specific antibody or PD098059 (10 µM) for 24 hours. Cytoplasmic nucleosomes were detected by ELISA. (B) MDA-MB-231 cells were cultured in 6-well plates and treated with DMSO (0.1% v/v), PD098059 (10 µM), mouse nonimmune IgG (25 µg/ml), uPA-specific antibody (25 µg/ml) or uPA-specific antibody (25 µg/ml) plus PD098059 (10 µM) for 24 hours. Caspase-3 activity was then measured. (C) MDA-MB-231 cells were subjected to mild acid wash and then cultured in serum-free medium containing DMSO (0.1% v/v), PD098059 (10 µM), uPAR-specific antibody (50 µg/ml) or uPAR-specific antibody (50 µg/ml) plus PD098059 (10 µM) for 24 hours. Cytoplasmic nucleosomes were detected by ELISA. (D) MDA-MB-231 cells were subjected to a mild acid wash and then cultured for 24 hours in serum-free medium containing DMSO (0.1% v/v), PD098059 (10 µM), rabbit nonimmune IgG (50 µg/ml), uPAR-specific antibody (50 µg/ml) or uPAR-specific antibody (50 µg/ml) plus PD098059 (10 µM). Caspase-3 activity was determined. The asterisk indicates a significant difference compared with control (unpaired t-test, P<0.05, n=3). The dagger indicates that uPA- or uPAR-specific antibody in combination with PD098059 did not significantly alter apoptosis compared with either reagent alone.

View larger version (27K): [in a new window]   Fig. 8. uPA antisense oligodeoxynucleotides inhibit MDA-MB-231 cell growth and promote apoptosis. (A) MDA-MB-231 cells were treated with uPA antisense, sense or nonsense oligodeoxynucleotides for 24 hours, cultured in serum-containing medium for 24 hours and then in serum-free medium for 24 hours. uPA accumulation in serum-free conditioned medium was assessed by immunoblot analysis. (B) uPA levels in CM were quantitated by densitometry and standardized to the level observed in control cultures. (C) MDA-MB-231 cells were treated with oligodeoxynucleotides according to the protocol used to measure uPA levels. Cell growth was determined by MTT assay. The asterisk indicates a significant difference compared with the control (unpaired t-test, P<0.05, n=3). (D) MDA-MB-231 cells were treated with oligodeoxynucleotides. Cytoplasmic nucleosomes were detected by ELISA. The asterisk indicates a significant difference compared with the control (unpaired t-test, P<0.05, n=3). Values in B-D represent mean±s.e.m.

View larger version (41K): [in a new window]   Fig. 9. Expression of uPA and uPAR is regulated by ERK in MDA-MB-231 cells. MDA-MB-231 cells were treated with PD098059 (10 µM or 50 µM) or with vehicle for 24 hours in serum-free medium. (A) Levels of uPA, cell-associated uPAR and soluble uPAR in CM were determined by immunoblot analysis (representative of three separate experiments). (B) PGAD, uPA and uPAR mRNA levels were analyzed in total RNA isolates.