Hidekazu Ohi

Effects of membrane-associated cathepsin B on the activation of receptor-bound prourokinase and subsequent invasion of reconstituted basement membranes

The present study was undertaken to assess the role of membrane-associated cathepsin B as an activator of receptor-bound single-chain urokinase-type plasminogen activator (pro-uPA) and to determine the importance of receptor-bound uPA activity in the destruction of extracellular matrix by tumor cells with subsequent invasion through basement membranes. Ovarian cancer HOC-I cells express pro-uPA/HMW-uPA and cathepsin B on their surface. uPAs are bound to a specific surface receptor, about 30% of which is saturated. 60% of the receptor-bound uPA is pro-uPA. No reduction in the specific binding of biotinylated DFP-HMW-uPA was observed when cells were cultivated in the presence of E-64, a cysteine proteinase inhibitor, for 24 h. Inhibition of cell-surface cathepsin B activity was associated with a decrease in cell-bound uPA activity to undetectable levels, and > 95% of the membrane-associated uPA was pro-uPA in cells cultivated with E-64. This suggested that receptor-bound pro-uPA cannot be converted to HMW-uPA in the absence of enzymatically-active cathepsin B. The significance of the expression of cell-surface uPA activity regarding invasive potential was examined in an in-vitro Matrigel invasion assay. Decreased cell-surface uPA activity was associated with a decrease in invasive potential. These data support our hypothesis that membrane-associated cathepsin B may be important for the conversion of pro-uPA to HMW-uPA and that receptor-bound uPA activity constitutes an efficient mechanism which contributes to tumor cell invasion. As HOC-I cells produce both uPA and cathepsin B, the implications of tumor-cell-derived pro-uPA activation by cellular proteinase cathepsin B should be considered.

A simple, noninvasive, sensitive method for diagnosis of amniotic fluid embolism by monoclonal antibody TKH-2 that recognizes NeuAcα2-6GalNAc

OBJECTIVE The sialyl Tn structure (NeuAc alpha 2-6GalNAc alpha 1-O-Ser/Thr) recognized by monoclonal antibody TKH-2 is a characteristic component in meconium and amniotic fluid. The purpose of this study was to determine whether amniotic fluid embolism could be detected by quantification of this antigen in maternal serum by means of an assay using antimucin monoclonal antibody TKH-2. STUDY DESIGN Sialyl Tn antigen was measured in the serum of women with meconium-stained amniotic fluid and compared with the level in those with clear amniotic fluid, as well as that in women with a clinical picture suggesting amniotic fluid embolism. The concentration of sialyl Tn antigen was determined by an immunoradiometric competitive inhibition assay. RESULTS Serum sialyl Tn antigen levels in women with meconium-stained amniotic fluid (20.3 +/- 15.4 U/ml) at delivery were slightly higher than those in women with clear amniotic fluid (11.8 +/- 5.6 U/ml). A significantly elevated level of sialyl Tn antigen was observed in serum of patients with amniotic fluid embolism and amniotic fluid embolism-like symptoms (105.6 +/- 59.0 U/ml, p < 0.01). CONCLUSION The method for detecting sialyl Tn antigen in the serum of patients with amniotic fluid embolism is a direct way to demonstrate the release of meconium- or amniotic fluid-derived mucin into the maternal circulation and is a simple, noninvasive, sensitive method for diagnosis of amniotic fluid embolism.

Saturation of tumour cell surface receptors for urokinase-type plasminogen activator by amino-terminal fragment and subsequent effect on reconstituted basement membranes invasion.

Single-chain urokinase-type plasminogen activator (pro-uPA) is bound to a specific surface receptor on ovarian cancer HOC-I cells that is incompletely saturated. Saturation of uncovered receptors by uPA polypeptides with intact amino-terminal fragment (ATF) derived from pro-uPA by limited proteolysis (human leucocyte elastase [HLE] or V8 protease) has been studied. HOC-I cells preferentially invaded reconstituted basement membranes in a time- and plasminogen-dependent manner. This process was inhibitable by preincubation with uPA polypeptides in the medium at levels which suggested that complete saturation of cell surface uPA receptors occurred. This result indicates that occupation of uPA receptors by enzymatically inactive uPA fragments or prevention of rebinding of pro-uPA synthesised by tumour cells to the receptors specifically reduces the invasion of the tumour cells through basement membranes in vitro.

Urinary trypsin inhibitor (UTI) and fragments derived from UTI by limited proteolysis efficiently inhibit tumor cell invasion

We investigated the effects of purified human urinary trypsin inhibitor (UTI) and fragments derived from UTI by proteolysis on the invasive potential of ovarian cancer cells (HOC-I) and gestational choriocarcinoma cells (SMT-ccl) using anin vitro reconstituted basement membrane invasion assay. These cells express cell-associated plasmin and functional uPA receptors that are partially occupied by ligands. SMT-ccl cells, which express threefold higher levels of cell-associated plasmin activity than HOC-I cells, showed approximately twofold increase in their invasive potential. For the invasion assay, HOC-I cells were primed with exogenous plasminogen, but SMT-ccl cells were not. Human leukocyte elastase (HLE)-digested UTI (22 kDa fragment; UTI-22) inhibited plasmin practically with the same strength as native UTI. Trypsindigested UTI (20 kDa fragment; UTI-20), however, did not inhibit plasmin significantly. Treatment of cells with UTI or UTI-22 reduced the incidence of tumor cell invasive capacity, whereas the inhibitory effect of UTI-20 was not remarkable. The inhibitory effect on tumor cell invasion was dose-dependent and non-toxic; moreover, it was not mediated by inhibition of the tumor cell chemotactic response or of cell attachment to matrigel. These results indicate that inhibition of the proteolytic enzyme plasmin specifically reduced the invasive capacity of tumor cellsin vitro.

Increased cell-surface urokinase in advanced ovarian cancer.

Urokinase‐type plasminogen activator (uPA), uPA receptors, and cathepsin B were quantitatcd by using an immunological method, enzyme‐linked immunosorbent assay, and amidolytic activity assays in 15 malignant and 10 benign epithelial ovarian tumors. The levels of uPA and uPA receptors, as well as cathepsin B, were found to be higher in membrane preparations obtained from malignant tumors than in those obtained from benign tumors. Acid‐treated membranes acquired the ability to bind uPA, indicating that uPA is bound to a specific surface receptor that is not completely saturated. Levels of single‐chain uPA (pro‐uPA) and high‐molecular‐weight uPA in membrane preparations were measured by immunoadsorbent‐amidolytic assay. The finding of a significant increase in amidolytic activity following activation of uPAs by plasmin suggested that less than half (30–40%) of all membrane immunoreactive uPAs is present in the enzymatically inactive pro‐uPA form. In the membranes of malignant tumors, levels of uPA receptor and cathepsin B did not vary with stage of disease. On the other hand, we found that the level of receptor‐bound uPA antigen/activity was significantly increased in advanced malignant tumors. Receptor‐bound uPA may play an important role in determining invasive potential of tumor cells. Since ovarian cancer cells produce both pro‐uPA and cathepsin B, the possibility of activation of tumor cell‐derived pro‐uPA by cellular protease cathepsin B must be considered.

CHARACTERIZATION OF CA 125 ANTIGEN IDENTIFIED BY MONOCLONAL ANTIBODIES THAT RECOGNIZE DIFFERENT EPITOPES

The present study was undertaken to characterize the antigenic determinant of the CA 125 macromolecules recognized by newly developed monoclonal antibodies (M 11, 130-22, 145-9, 602-1, and 602-6), examine their relationship among the epitopes recognized by these antibodies, and develop a series of enzyme-linked immunosorbent assays (ELISAs) in which various combinations of antibodies are used in a double-determinant sandwich mode. The antigenic determinants of CA 125 were characterized by several methods including competitive ELISA and immunoblotting. These antibodies, as well as OC 125, reacted with ovarian cancer (HOC-I) cell extract in a dose-dependent manner. Purified CA 125 antigen with a molecular mass of less than 200 kDa (CA 125 < 200 kDa) had a significant inhibitory effect on the reaction between the cancer cell extract and all these antibodies. The reactivity of M 11, 130-22, 145-9, 602-1, and 602-6 to the cancer cell extract was not significantly inhibited by purified CA 125 > or = 200 kDa, whereas the reactivity of OC 125 was completely inhibited by CA 125 > or = 200 kDa. The antigenic determinants of M 11, 145-9, 602-6, 130-22, and 602-1 were closely related to each other, in this order; whereas OC 125 recognized a different epitope on a structurally identical molecule. The use of these monoclonal antibodies in combination with each other may result in the development of a more specific and sensitive assay for CA 125.

Identification and characterization of a Kunitz-type protease inhibitor in ascites fluid from patients with ovarian carcinoma

Urinary trypsin inhibitor (UTI; Mr 40 kDa) is a Kunitz‐type protease inhibitor that efficiently inhibits cell‐associated trypsin and plasmin activities. The aim of this study is to examine the expression pattern of UTI in the human ovarian carcinoma ascites fluid by Western blotting, zymography, immunoprecipitation, immunohistochemistry, biochemical and gene analyses and animal experiments. We have identified and characterized the 40 kDa immunoreactive UTI (UTI40) and 8 kDa degradation fragment (UTI8) in ascites fluid. The levels of UTI40 and UTI8 are elevated in ascites fluid taken from patients with ovarian carcinoma relative to paired plasma samples. The UTI40 and UTI8 were identified immunologically by the reactivity with 2 different anti‐UTI antibodies recognizing different epitopes of the UTI molecule, functionally by its ability to bind trypsin and structurally by its apparent molecular mass with and without deglycosylation treatment. The purified polypeptides have been sequenced and were identical with sequences obtained from UTI and the carboxyl‐terminal domain of UTI, respectively. However, UTI mRNA was not detected in the ovarian carcinoma tissue and ovarian carcinoma cell lines examined. Based on extravasation experiments using intravenously injected biotinylated inter‐α‐trypsin inhibitor (IαI; a precursor of UTI), we conclude that UTI40 and UTI8 found in the ascites fluid may result from (i) the extravasation of plasma proteins such as IαI into the peritoneal cavity via hyperpermeable vessels and (ii) the subsequent degradation of IαI and UTI40 by tumor cell‐associated trypsin‐like enzymes. Int. J. Cancer 87:44–54, 2000.

Characterisation and clinical usefulness of CA130 antigen recognised by monoclonal antibodies, 130-22 and 145-9, in ovarian cancers.

A new cancer-associated antigen CA130, recognised by two monoclonal antibodies (moABs) 130-22 and 145-9, was often found to be present at high levels in the sera of patients with ovarian cancer. There was a strong correlation between CA130 and CA125 values. The epitopes recognised by moABs 130-22 and 145-9 were proved to differ from the CA125 epitope, but to exist on the molecule bearing CA125. Unlike OC125, the majority of 130-22/145-9 activity was associated with a much lower molecular mass (less than 200 kDa), indicating that a lower molecular mass immunoreactive determination may be a unique CA130 antigenic determinant within CA125 molecule. Clinical data demonstrate that, (1) elevated levels of CA130 determinant were found in the sera of 91.3% of women with epithelial ovarian cancer, (2) falling or rising levels of CA130 correlated with regression or progression of ovarian cancer in > 95% of cases, (3) normalisation of serum CA130 levels at response does not imply no microscopic residual disease, but CA130 changes during follow-up support the evaluation of recurrence and can be used as a monitoring marker in an individual patient.

Characterization and clinical evaluation of tumor‐associated antigen CA54/61 identified by monoclonal antibodies MA54 and MA61 in epithelial ovarian cancer

Monoclonal antibodies (moABs) MA54 and MA61, directed toward the O-linked mucin-type glycoprotein, have been established and showed highly specific reactivity with human ovarian cancer. Fetal intestinal and colonic mucosal cells expressed this antigen and meconium staining was also frequently positive. To investigate the characteristic of an epitopic carbohydrate recognized by these moABs, the reactivity of each moAB with meconium extract was monitored by solid-phase enzyme-linked immunosorbent assay with mono-, di-, and oligosaccharides. MA54 and MA61 react with meconium extract and the reactivities of these moABs are neuraminidase sensitive. Ovine submaxillary mucin had a strong inhibitory activity toward the reaction between meconium extract and MA54 as well as MA61, suggesting that these moABs recognize NeuAc 2-6GalNAc epitope in meconium. The second aim of this study is to investigate the possible application of moABs to diagnose ovarian cancer and to compare these levels with those of the CA125 antigen. While serum CA54/61 antigen levels were elevated in 44.4% of ovarian cancer cases and serum CA125 antigen levels were elevated in 86.7% of the same population, the use of both assays indicated a sensitivity of detection of 97.8% (44 of 45 patients) in the population studied.

Characterization and clinical evaluation of tumor-associated antigen CA54/61 identified by monoclonal antibodies MA54 and MA61 in epithelial ovarian cancer

Abstract Monoclonal antibodies (moABs) MA54 and MA61, directed toward the O-linked mucin-type glycoprotein, have been established and showed highly specific reactivity with human ovarian cancer. Fetal intestinal and colonic mucosal cells expressed this antigen and meconium staining was also frequently positive. To investigate the characteristic of an epitopic carbohydrate recognized by these moABs, the reactivity of each moAB with meconium extract was monitored by solid-phase enzyme-linked immunosorbent assay with mono-, di-, and oligosaccharides. MA54 and MA61 react with meconium extract and the reactivities of these moABs are neuraminidase sensitive. Ovine submaxillary mucin had a strong inhibitory activity toward the reaction between meconium extract and MA54 as well as MA61, suggesting that these moABs recognize NeuAc 2–6GalNAc epitope in meconium. The second aim of this study is to investigate the possible application of moABs to diagnose ovarian cancer and to compare these levels with those of the CA125 antigen. While serum CA54/61 antigen levels were elevated in 44.4% of ovarian cancer cases and serum CA125 antigen levels were elevated in 86.7% of the same population, the use of both assays indicated a sensitivity of detection of 97.8% (44 of 45 patients) in the population studied.