ABSTRACT
To elucidate the physiological role of human stromelysin-3 (hST-3) in tumor progression and/or wound healing, insulin-like growth factor-binding protein-1 (IGFBP-1) was analyzed as a potential physiological substrate. hST-3 proteolysis generates two fragments of 16 and 9 kDa that react with IGFBP-1 monoclonal antibody, although they do not bind insulin-like growth factor-I (IGF-I) in ligand blot. N-terminal sequencing shows that hST-3 cleaves IGFBP-1 at the His140-Val141 bond located in the IGFBP-1 midregion. We show that IGFBP-1 inhibits IGF-I-induced survival and proliferation of BAF/3 cells, as well as IGF-I-mediated activation of phosphatidylinositol 3-kinase (PI 3-K). Co-incubation of the IGF-I. IGFBP-1 complex with hST-3 restores IGF-I-induced proliferation and PI 3-K kinase activity in these cells. BAF/3 proliferation is significantly increased with the hST-3-treated IGF-I.IGFBP-1 complex compared with that obtained using IGF-I alone. To produce this enhanced proliferation, IGF-I must bind to IGFBP-1 before hST-3 proteolysis, demonstrated using an IGF-I variant that does not bind IGFBP. IGFBP-1 also inhibits IGF-I-induced proliferation of the MCF-7 breast adenocarcinoma, and this inhibition was not seen in hST-3-transfected MCF-7 cells. Such proteolysis may thus play a role in in vivo tumor progression. These results indicate that hST-3 may regulate IGF-I bioavailability by proteolyzing IGFBP, thus favoring cell survival and proliferation.
Subject(s)
Insulin-Like Growth Factor Binding Protein 1/metabolism , Metalloendopeptidases/metabolism , Humans , Hydrolysis , Insulin-Like Growth Factor I/metabolism , Matrix Metalloproteinase 11 , Metalloendopeptidases/genetics , Molecular Weight , Peptide Mapping , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Transfection , Tumor Cells, CulturedABSTRACT
To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) by zymogram analysis and fluorescent substrate degradation assays. FITC-casein and the fluorogenic peptide Dnp-Pro-beta-cyclo-hexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH 2 were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tested except MMP-9. MMP-2 was not significantly able to degrade the fluorogenic peptide. Gelatin zymography was the most sensitive method to detect the activity of both gelatinases but quantitation problems compromise its use. The degradation of fluorogenic substrates by MMPs could be inhibited by the chelating agent EDTA and by the tissue inhibitor of metalloproteinases 2 (TIMP-2), an MMP-specific inhibitor. Fluorometric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.
Subject(s)
Collagenases/analysis , Electrophoresis, Polyacrylamide Gel , Fluorometry , Gelatinases/analysis , Matrix Metalloproteinase 3/analysis , Metalloendopeptidases/analysis , Recombinant Fusion Proteins/analysis , Calcium/physiology , Caseins/metabolism , Chelating Agents/pharmacology , Collagenases/metabolism , Edetic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Gelatinases/antagonists & inhibitors , Gelatinases/metabolism , Humans , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 7 , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , Proteins/pharmacology , Recombinant Fusion Proteins/antagonists & inhibitors , Tissue Inhibitor of Metalloproteinase-2ABSTRACT
The baculovirus expression system was used to produce recombinant human matrilysin. Expression of promatrilysin reached a peak at 72 h post-infection. Most of the recombinant protein remained in the intracellular fraction in an insoluble form, which after renaturation was purified by S-Sepharose and Green A Dyematrex chromatography in order to remove host proteases. Active recombinant matrilysin degraded casein, type I and type IV collagens and fibronectin. Expression of recombinant human matrilysin using the baculovirus system represents a useful tool for obtaining large amounts of this metalloproteinase in order to carry out further biochemical studies and to screen for inhibitors.
Subject(s)
Baculoviridae/genetics , Gene Expression , Metalloendopeptidases/genetics , Metalloendopeptidases/isolation & purification , Animals , Base Sequence , Cell Line , Colonic Neoplasms , DNA, Complementary/chemistry , Humans , Matrix Metalloproteinase 7 , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/metabolism , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Spodoptera , Transfection , Tumor Cells, CulturedSubject(s)
Cell Movement , Fluoresceins/metabolism , Animals , Inflammation/pathology , Mice , Spectrometry, Fluorescence , Tumor Cells, CulturedABSTRACT
Two methods based on the reversion of adriamycin-resistance o the increase of Rhodamine 123 accumulation in a multidrug resistant (MDR) cell line have been simplified and adapted for the screening of MDR reversal agents. Both methods are carried out in microtiter plates, are highly sensitive and can be easily automated. In both assays verapamil and the cyclosporine derivative PSC 833 could be detected at concentrations lower than 1 and 0.05 microM, respectively. Depending on the MDR cell line used, drugs exhibiting the collateral sensitivity phenomenon can be selected in the cytotoxicity assay, while interferences due to sample toxicity are easily avoided in the dye accumulation assay.
