A tissue inhibitor of metalloproteinases (T1MP)-1 was isolated from human polymorphonuclear leukocytes (PMNL) in a complex with latent 95-kDa gelatinase (matrixmetalloproteinase, . It was separated from the enzyme by gel fitration in the presence of SDS. Using a competitive ELISA procedure, we determined that 10% of the isolated gelatinase was complexed with TIMP-1. The presence of the inhibitor in isolated PMNL could also be demonstrated by indirect immunofluorescence using a specific antibody against TIMP-1. Cellular mRNA was isolated from PMNL, which were highly purified by separation via formylMet-Leu-Pro-stimulated chemotactic migration in a Boyden chamber. Using reversetranscription PCR and Nothern blotting, TIMP-1 mRNA was shown to be present in PMNL, suggesting that these cells are also capable of synthesizing TIMP-1.Keywords. Tissue inhibitor of metalloproteinases (TIMP-1) ; neutrophils ; metalloproteinase.Human polymorphonuclear leukocytes (PMNL) are phagocytic cells with a short life span, estimated to be 24-48 h after release from the bone marrow. They are the primary effector cells in sites of acute inflammatory processes, in which they move along a chemotactic gradient from the bloodstream to the site of inflammation. To penetrate the vessel walls and tissue barrier, they secrete specific proteinases which are stored in their granules. Matrix metalloproteinase (MMP)-9, also called gelatinase B, is assumed to be the required proteinase for leukodiapedesis Bakowski et al., 1992). Gelatinase B is a member of the matrix metalloproteinase family and, amongst other substrates, cleaves type-IV collagen, the main component of basement membranes.The matrix metalloproteinases are secreted as zymogens which undergo extracellular activation. Specific inhibitors exert control over the active enzymes in the extracellular matrix. Two tissue inhibitors of metalloproteinases (TIMPs) are so far known. TIMP-1 is an 28.5-kDa glycoprotein secreted by many cell types, including fibroblasts and macrophages. TIMP-2 is an unglycosylated 21 -kDa protein with significant sequence similarity to TIMP-1. These inhibitors form stoichiometric 1 : 1 complexes with active metalloproteinases (Docherty et al., 1992).In addition to these complexes of the active enzyme with its inhibitor, complexes between the latent 72-kDa gelatinase (MMP-2) and the latent 95-kDa gelatinase (MMP-9) with TIMP-2 and TIMP-1, respectively, have been isolated, which can still be activated by mercurial compounds, as can the noncomplexed latent enzymes. The physiological role and significance of these latent enzyme-inhibitor complexes in vivo is unknown. In human blood cells, TIMP-1 was only detected in platelets and megakaryocytes (Cooper et al., 1985) and macrophages and monocytes (Opdenakker et al., 1991).Here, we report the isolation of TIMP-1 in the free form and in complex with 95-kDa gelatinase from human PMN leukocytes. The detection of TIMP-1 transcripts in highly purified PMNL provided evidence that these cells are capable of regulating metalloprot...

Significant evidence has accumulated that DNA-methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene can serve as a prognostic and predictive biomarker in breast cancer. PITX2 DNA-methylation data have been obtained so far from microarray and polymerase chain reaction (PCR)-based research tests. The availability of an analytically validated in vitro methylation-specific real-time PCR assay format (therascreen PITX2 RGQ PCR assay) intended for the determination of the percent methylation ratio (PMR) in the (PITX2) promoter 2 prompted us to investigate whether the clinical performance of these different assay systems generate comparable clinical outcome data. Mathematically converted microarray data of a previous breast cancer study (n = 204) into PMR values leads to a PITX2 cut-off value at PMR 14.73. Recalculation of the data to experimentally equivalent PMRs with the PCR PITX2 assay leads to a cut-off value at PMR 12 with the highest statistical significance. This cut-off predicts outcome of high-risk breast cancer patients to adjuvant anthracycline-based chemotherapy (n = 204; Hazard Ratio 2.48; p < 0.001) comparable to microarray generated results (n = 204; Hazard ratio 2.32; p < 0.0001). The therascreen PITX2 RGQ PCR assay is an analytically validated test with high reliability and robustness and predicts outcome of high-risk breast cancer patients to anthracycline-based chemotherapy.

Arg-Gly-Asp (RGD); confocal laser scanning microscopy (CLSM); epidermal growth factor receptor (EGF-R); Epithelial ovarian cancer (EOC); extracellular matrix (ECM); Fédération Internationale de Gynécologie et d'Obstétrique (FIGO); focal adhesion kinase (FAK); glyceraldehyde 3-phosphate dehydrogenase (GAPDH); glycophorin A (GpA); integrin mediated death (IMD); mitogen activated protein kinases (MAPK); propidium iodide (PI); transmembrane domain (TMD)

A tissue inhibitor of metalloproteinases (T1MP)-1 was isolated from human polymorphonuclear leukocytes (PMNL) in a complex with latent 95-kDa gelatinase (matrixmetalloproteinase, . It was separated from the enzyme by gel fitration in the presence of SDS. Using a competitive ELISA procedure, we determined that 10% of the isolated gelatinase was complexed with TIMP-1. The presence of the inhibitor in isolated PMNL could also be demonstrated by indirect immunofluorescence using a specific antibody against TIMP-1. Cellular mRNA was isolated from PMNL, which were highly purified by separation via formylMet-Leu-Pro-stimulated chemotactic migration in a Boyden chamber. Using reversetranscription PCR and Nothern blotting, TIMP-1 mRNA was shown to be present in PMNL, suggesting that these cells are also capable of synthesizing TIMP-1.

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