Reagents
The MMP inhibitor CT-1166 was a generous gift from Dr A Docherty, Celltech (Slough, UK). The PAS inhibitor aprotinin, plasminogen and TGFβ were purchased from Sigma. The human mammary epithelial cell (HMEC) line, Mammary Epithelial Basal Medium (MEBM), insulin, hydrocortisone, gentamicin/amphotericin-B and Bovine Pituitary Extract (BPE) were purchased from Clonetics (Walkersville, MD, USA). Human breast tumour cell lines MDA-231, ZR-75-1 and MCF-7 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Gelatinase-A, TIMP-1 and polyclonal sheep antibodies to human MMP-1, -2,- 3, -9 and -13 were gifts from Dr J Reynolds, Strangeways Research Laboratory, Cambridge, UK. Neutralizing mAbs against human uPA or human uPAR were from American Diagnostica, Greenwich, CT, USA.
Cell cultures
Cell lines were cultured in 75-cm2 plastic tissue culture flasks (Costar Corning, Cambridge, MA, USA) as follows: ZR-75-1, MDA-231, MCF-7 and MC-3T3-E1 in α-MEM and HME cells in MEBM medium. All media were supplemented with 10% heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin (Sigma Chemical Co.). Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide and subcultured every third day.
Type I collagen degradation assay
14C-labelled collagen films were prepared as described previously [30]. Breast cells were seeded into collagen coated 16 mm tissue culture wells at a density of 1 × 105 /well. After 4 h incubation at 37°C in MEM supplemented with 10% FCS, the cells were washed twice with phosphate buffered saline (PBS) to remove residual FCS, and incubated for 24 h with 1 ml/well of serum-free MEM with or without the indicated concentrations of plasminogen, TGFβ and the proteinase inhibitors to be tested. At the end of the culture period the media were centrifuged (15 min, 1200 × g) to remove any collagen fibrils, and radioactivity released during collagen degradation quantified by liquid scintillation counting. Residual collagen was digested with bacterial collagenase (50 μg/ml) and assayed for radioactivity. Collagenolysis was expressed as radioactivity released from the films as a percentage of the total ± SEM.
Formation of 3H-labelled, non-mineralized and mineralized bone matrix
The murine calvarial-derived MC3T3-E1 is a well characterized osteoblast culture system providing a suitable model of osteogenesis analogous to in vivo bone formation [15]. The cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide. Culture medium was changed on the first day after seeding and then every 72 h. The cells were split after 7–9 days in culture, before they reached confluence, and plated at a density of 1 × 104 cells/well on collagen-coated 24-well plates (Becton Dickinson, MA, USA). After 4–5 days, when the cultures had reached confluence and the formation of an ECM had started, fresh medium was added containing 1 μCi/ml 3H amino acid mixture for 7 days (Amersham International, Aylesbury, UK) to create a non-mineralized radiolabelled ECM. In order to create a mineralized bone matrix, the medium was supplemented with 10 mM β-glycerol phosphate and the cells cultured for 14 days and the radiolabelled medium changed every 3 days. Unincorporated 3H-radiolabelled amino acids were washed from the remaining ECM using 2 water washes and 75% (v/v) ethanol. The matrices were dried and stored at -20°C until use.
Von Kossa staining for mineralization
Mineralization of matrices was determined by von Kossa staining. The matrices were rinsed with cold PBS and fixed in 10% neutral buffered formalin for 15 min, rinsed with distilled water and left in distilled water for 15 min. Matrices were stained with 2.5 % silver nitrate solution for 30 min at room temperature. The silver nitrate was removed and matrices were rinsed with distilled water before the addition of sodium carbonate formaldehyde for 5 min. After rinsing, the matrices were counterstained with toluidine blue, rinsed with tap water and air dried.
Bone matrix degradation assay
Breast cancer cells (105 /16 mm culture well) were seeded onto the bone matrix as for the type I collagen degradation assay. After 4 h incubation at 37°C in MEM supplemented with 10% FCS, the cells were washed twice with phosphate buffered saline (PBS) to remove residual FCS, and incubated for 24 h with 1 ml/well of serum-free MEM with or without the indicated concentrations of plasminogen, TGFβ and the proteinase inhibitors that were tested. After 24 h incubation, the media were removed and the extent of degraded 3H-radiolabelled matrix released into the medium was determined by liquid scintillation counting. The remaining matrix was degraded as for the type I collagen assay and matrix degradation expressed as above.
PCR and RT-PCR
Confluent breast tumour cells were stimulated with TGFβ (10-10 M) in serum-free medium for 24 h. For PCR analysis, oligonucleotide primers were synthesized based on the published sequences for 14 MMPs (i.e., MMP-1, -2, -3, -7,-8,-9,-10, -11, -12, -13,-14,-15, -16, and -17) [4]. t-PA, u-PA and, u-PAR oligonucleotides were designed using Designer PCR (Research Genetics, AL, USA) and primers purchased from Life Technologies Ltd. The housekeeping gene GAPDH was used as a positive control for the RT-PCR methodology.
Enzymes and buffers for the reverse transcriptase and PCR reactions were obtained from Perkin Elmer (CA, USA). Reverse transcriptase reactions were done according to the manufacturer's protocol using 1 μg of total RNA collected from untreated or TGF-β treated breast cells. PCR reactions were performed in an automated DNA thermal cycler (Perkin Elmer) for 25 cycles of denaturation (95°C, 1 min), annealing (variable time and temperature) and polymerisation (60°C, variable time). Amplification of the house-keeping gene, G3PDH mRNA (35 cycles), provided an internal control for the efficiency of the RT-PCR process. RT-PCR products were analyzed against molecular weight standards (pBR322 HaeIII digest) on a 2.5% agarose gel stained with ethidium bromide, electrophoresed in 0.5× TBE buffer at 100 V for 90 minutes. Gels were examined under ultraviolet light and photographed. The authenticity of the PCR products was verified by sequencing [31].
Western Blot analysis
To identify MMP proteins and uPA present in breast cell conditioned medium, Western blot analysis was performed using their specific antibodies as previously described [32]. Briefly, samples were separated by 8.5% SDS-PAGE, transblotted on to PVDF membranes (Millipore Corp., MA, USA) and immunoblotted with polyclonal sheep antiserum to MMPs or polyclonal goat antiserum to uPA and secondary peroxidase-conjugated anti-sheep / anti-goat antibodies. Labelled proteins were detected with ECL detection solution and exposed to autoradiographic film (Hyperfilm ECL, Amersham International, UK).
Assay of collagenase activity
To measure collagenase activity, TGFβ (10-10 M)-stimulated breast cells were cultured in the presence/absence of 2 ug/ml of human plasminogen for 24 h. Collagenase activity was measured by the degradation of fluorescein isothiocyanate (FITC)-labelled type I collagen using type I activity assay kits. One unit of these activities degrades 1 μg of collagen per min at 37°C.
uPA Assay
Cells were plated in 6-well trays and grown to near confluence, the medium was removed, and the cells were washed and incubated with 2 ml of serum-free medium for 24 hr. The medium was collected, centrifuged, and frozen until assayed. The cells were lysed in 300 ul of 0.1 M Tris (pH 8.1) and 0.1% Triton X-100. The u-PA (10 ul of conditioned medium and 10 μg of cell protein) was assayed as previously described [41] using plasminogen and S2251, the chromogenic substrate for plasmin.
Statistical Analysis
Differences between control and treatment groups were determined by the Mann Whitney U-test.