Risk of developing ovarian cancer among women with ovarian endometrioma: a cohort study in Shizuoka, Japan.

Although some studies have indicated that endometriosis may increase the risk of developing ovarian cancer, there are no data from epidemiologic studies in Japan. We prospectively analyzed all cases of ovarian endometrioma enrolled in the prefecture-wide Shizuoka Cohort Study on Endometriosis and Ovarian Cancer Programme, which was initiated in 1985. To evaluate the risk of ovarian cancer by time periods subsequent to ovarian endometrioma diagnosis, a cohort of 6,398 women with a clinically documented ovarian endometrioma in Shizuoka between 1985 and 1995 was identified from the Shizuoka Cancer Registry (SCR), with follow-up through 2002. Ovarian cancer incidence among cohort members was ascertained by linkage to the SCR using a unique person-identification number. Standardized incidence ratios (SIR) and their 95% confidence intervals (CI) were computed by a use of prefecture-wide rates of ovarian cancer, adjusted for age and calendar year. During follow-up of up to 17 years of the ovarian endometrioma cohort, 46 incident ovarian cancers were identified, yielding that the ovarian cancer risk was elevated significantly among patients with ovarian endometrioma (SIR = 8.95, 95% CI = 4.12-15.3). The SIR did not increase with increasing follow-up duration. The risk increased with increasing age at ovarian endometrioma diagnosis, with a SIR equal to 13.2 (95% CI = 6.90-20.9) in women above 50 years of age. Our findings for the first time support the hypothesis that ovarian endometrioma increases the subsequent risk of developing ovarian cancer in Shizuoka, Japan.

Mobile intracardiac calcinosis: a new risk of thromboembolism in patients with haemodialysed end stage renal disease.

Cardiac calcinosis is a common complication of end stage renal disease. A newly observed risk of thromboembolism is reported in four patients with mobile cardiac calcinosis, treated with long term dialysis. Rapidly growing mobile calcification was confirmed by echocardiography. Each patient had an imbalance in serum calcium x inorganic phosphate (Ca x P product >/= 50); this imbalance could not be treated due to the sudden death of the patient or the need for surgical resection to prevent recurrent cerebral thromboembolism. Histological examination revealed intracardiac calcinosis in three cases, and each case showed haemodialysis hypoparathyroidism (intact PTH < 160 pg/ml). Thromboembolism in such cases is rare, however it indicates a need for cautious echocardiographic monitoring in end stage renal disease in patients with an uncontrolled Ca x P product.

Relationship of urokinase type plasminogen activator, plasminogen activator inhibitor type 1 and activated protein C in fibrinolysis of human placenta.

Suppression of the fibrinolytic activity plays an important role in the prevention of hemorrhage during pregnancy and labor. A hypofibrinolytic and hypercoagulable state may be established in the placenta during pregnancy. However, little infraction is present in the normal placenta. This evidence shows that placenta maintains the fibrinolytic activity in spite of hypercoagulable state. As there is a high amount of APC in the placenta, APC is thought to be involved in fibrinolysis of placenta. Thus, we studied the role of APC on fibrinolysis in placenta. (1) uPA activity of cell membrane reappears after incubation with uPA/PAI-1 complex and a large amount of APC by flow cytometry, (2) APC was made PAI-1/APC complex after incubation of uPA/PAI-1 complex with APC. Our results suggest that APC is the important substance for fibrinolysis in the placenta by decreasing of PAI activity.

Urokinase-type plasminogen activator (uPA) and its receptor (CD87): a new target in tumor invasion and metastasis.

Extravasation and intravasation of tumor cells in solid malignant tumors is controlled by 3 steps: 1) attachment to and interaction of tumor cells with components of the basement membrane and the extracellular matrix, 2) local proteolysis, and 3) tumor cell migration. Evidence has accumulated that different types of tumor-associated proteases, their inhibitors and receptors are involved in tumor invasion and metastasis. Four different classes of proteases are known to be correlated with the malignant phenotype: 1) Matrix metalloproteases; including collagenases, gelatinases and stromelysins. 2) Cysteine proteases; including cathepsins B and L. 3) Aspartyl protease cathepsin D. 4) Serine proteases; including plasmin and tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). A strong independent prognostic value (relapse-free and/or overall survival) has especially been demonstrated for uPA and its inhibitor PAI-1 in patients with cancer of the breast, ovary, stomach, esophagus, colon, lung, and kidney thus predicting the course of the cancer disease. The strong correlation between elevated uPA and/or PAI-1 values in primary cancer tissues and the malignant phenotype of cancer cells has prompted to explore new tumor biology-oriented concepts in order to suppress uPA or uPA receptor (CD87) expression or to abrogate interaction of uPA with CD87. Various very different approaches to interfere with the expression or reactivity of uPA or CD87 at the gene or protein level were successfully tested including antisense oligonucleotides, antibodies, inhibitors and recombinant or synthetic uPA and CD87 analogues.

Model system for isolation of competent ovarian-carcinoma cells from fresh tumor-tissue by a magnetic separation technique (macs).

Flow cytometric analysis of tumor cells in carcinomas is hampered by the presence of a variety of different cells in the tumor tissue and the surrounding stroma. To obtain single competent tumor cells, we have established a model system which can be applied to separate living cells from fresh ovarian carcinoma tissue. Due to the lack of tumor-cell surface specific antibodies, we isolated tumor cells by a procedure called 'negative tumor cell selection'. For this purpose, fresh ovarian carcinoma tissue, immediately after surgery, was subjected to mechanical disintegration using an automated mincing device to obtain a single-cell suspension (approximately 10(7) cells/g). Collagenase D (0.005%) was added to prevent further aggregation. Cells other than tumor cells were then labeled with a set of monoclonal antibodies directed to cell surface antigens: CD3 (T-cells), CD14 (monocytes), CD15 (granulocytes), CD45R (T-/B-cells) and 5B5 (fibroblasts). Anti-isotype antibodies coupled to ferrit microbeads were then reacted with the cell suspension and those cells reacting with the microbeads retained on a steel wool matrix in a magnetic field (1). Tumor cells not reacting with the microbeads were recovered by a simple wash of the steel wool matrix. All incubation steps were at 4 degrees C. This procedure, which takes about 2 hours, enables fast and simple isolation of single, living competent tumor cells from fresh tumor tissue and also from ascitic or pleuritic effusions. In a model system with cultured ovarian carcinoma cells and human leukocytes, tumor cell purity was about 93% and about 97% when re-subjected to the same procedure (respective recovery rates 75% and 50%). The still unlabeled tumor cells can subsequently be analyzed by flow cytometry or by central laser scanning microscopy for the presence of various surface antigens including receptors for proteases or growth factors. Moreover, after detergent treatment and fixation, flow cytometric multiparameter analysis such as simultaneous labeling of intracellular and surface antigens as well as nuclear DNA staining for ploidy and S-phase determination becomes possible.

Inhibition of metastasis of Lewis lung carcinoma by a synthetic peptide within growth factor-like domain of urokinase in the experimental and spontaneous metastasis model.

Four synthetic peptides (residues 20-30 and 17-34) within the growth factor-like domain (GFD) of murine and human urokinase-type plasminogen activator (uPA) were examined to determine whether they inhibit production of experimental and spontaneous lung metastasis by murine Lewis lung carcinoma (3LL) cells. In an in vivo experimental metastasis assay, which determines mainly the later steps of the metastatic migration process (extravasation from the bloodstream and then growth into pulmonary tumor), none of the peptides introduced by i.v. single co-injection into syngeneic C57B1/6 mice inhibited pulmonary metastasis, when 3LL cells were pre-incubated with the peptides followed by i.v. co-injection of the peptide and cells. In addition, none of the peptides, when injected i.p. daily for 7 days after i.v. tumor cell inoculation, reduced the number of lung tumor colonies. In a second in vivo assay that measures metastasis from a primary tumor (spontaneous metastasis model), multiple i.p. injections of the mouse peptide 17-34 for 7 days after s.c. tumor cell inoculation significantly inhibited metastatic lung tumor colonization in a dose-dependent manner, whereas human peptide 17-34 had no effect. Mouse and human peptide 20-30 had no effect either. The inhibition of lung metastasis was not due to direct antitumor effects of mouse peptide 17-34. Our results indicate that occupation of uPA receptors on 3LL cells by the enzymatically inactive mouse peptide 17-34 or prevention of rebinding of uPA synthesized by tumor cells to their receptor specifically reduced tumor cell invasion and formation of metastasis and that uPA may regulate more efficiently the mechanism involved in the entry of tumor cells into vascular circulation than extravasation during the metastatic process.

Inhibition of the metastasis of Lewis lung carcinoma by antibody against urokinase-type plasminogen activator in the experimental and spontaneous metastasis model.

A selective inhibitory antibody, raised against human high molecular weight urokinase-type plasminogen activator (HMW-uPA), was examined to determine whether it would inhibit production of experimental and spontaneous lung metastasis by murine Lewis lung carcinoma (3LL) cells. Polyclonal antibody to human uPA cross-reacts with the murine uPA and inhibits murine uPA activity. When examined with an in vitro assay system using a modified Boyden chamber, the anti-catalytic IgG to uPA suppressed the invasion of tumor cells through Matrigel. Anti-uPA IgG inhibited neither the cell proliferation nor the binding of tumor cells to Matrigel, and showed no significant suppression of chemotactic migration of tumor cells to fibronectin. In an in vivo spontaneous metastasis assay, multiple subcutaneous (s.c.) injections of anti-uPA IgG (up to a concentration of 200 micrograms [= 500 inhibitory unit/mouse/day] for 7 days immediately after s. c. tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner. The inhibition of lung metastasis was not due to direct antitumor effects of anti-uPA IgG. In an in vivo experimental metastasis assay, multiple s. c. injections of anti-uPA IgG for 7 days after intravenous (i. v.) tumor cell inoculation did not reduce the number of lung tumor colonies. These results suggest that uPA more efficiently regulates the mechanism involved in the entry into vascular circulation of tumor cells (intravasation) than in extravasation, during the metastatic process.

Role of activated protein C in facilitating basement membrane invasion by tumor cells.

The present study was undertaken to investigate the role of plasminogen activator inhibitor type 1 (PAI-1) and activated protein C (APC) in the regulation of tumor cell invasion. PAI-1 was purified in active form from conditioned medium of human umbilical vein endothelial cells under denaturing conditions (4 M guanidine-HCl). The purified inhibitor reacts with urokinase-type plasminogen activator (uPA) and APC. Two selected human lines, HOC-I (ovarian cancer cells) and SMT-ccl (choriocarcinoma cells), preferentially invaded through reconstituted basement membranes in an in vitro invasion assay using a modified Boyden chamber. The present study determined the efficacy of these two agents (PAI-1 and APC) used alone or in combination in inhibiting or facilitating tumor cell invasion. Active PAI-1 inhibited the tumor cell surface receptor-bound uPA activity. In an in vitro invasion assay, active PAI-1 reduced tumor cell invasive potential in a dose-dependent manner. When SMT-ccl cells saturated with uPA-PAI-1 complexes were treated with a 50-fold molar excess of APC, PAI-1-APC complex was demonstrated in conditioned medium, indicating that PAI-1 was dissociated from receptor-bound uPA on tumor cells and that tumor cell-associated uPA restored its enzymatic activity. Although APC alone had no effect on tumor cell invasion, the addition of APC to the cells saturated with uPA-PAI-1 complexes showed regeneration of tumor cell surface receptor-bound uPA activity and produced substantial and efficient invading effects. These data suggest that PAI-1 activity may be neutralized by APC or that APC may promote tumor cell invasion via inactivation of PAI-1 by formation of a stable PAI-1-APC complex. These observations suggest that APC may play a critical role in the initiation of a hematogenous metastatic process (extravasation step).

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.

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.

Increased cell-surface urokinase in advanced ovarian cancer.

Urokinase-type plasminogen activator (uPA), uPA receptors, and cathepsin B were quantitated 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.

Tumor-associated urokinase-type plasminogen activator: biological and clinical significance.

Evidence has accumulated that invasion and metastasis in solid tumors require the action of tumor-associated proteases, which promote the dissolution of the surrounding tumor matrix and the basement membranes. Receptor-bound urokinase-type plasminogen activator (uPA) appears to play a key role in these events. uPA converts plasminogen into plasmin and thus mediates pericellular proteolysis during cell migration and tissue remodeling under physiological and pathophysiological conditions. uPA is secreted as an enzymatically inactive proenzyme (pro-uPA) by tumor cells and stroma cells. uPA exerts its proteolytic function on normal cells and tumor cells as an ectoenzyme after having bound to a high-affinity cell surface receptor. After binding, pro-uPA is activated by serine proteases (e.g. plasmin, trypsin or plasma kallikrein) and by the cysteine proteases cathepsin B or L, resp. Receptor-bound enzymatically active uPA converts plasminogen to plasmin which is bound to a different low-affinity receptor on tumor cells. Plasmin then degrades components of the tumor stroma (e.g. fibrin, fibronectin, proteoglycans, laminin) and may activate procollagenase type IV which degrades collagen type IV, a major part of the basement membrane. Hence receptor-bound uPA will promote plasminogen activation and thus the dissolution of the tumor matrix and the basement membrane which is a prerequisite for invasion and metastasis. Tissues of primary cancer and/or metastases of the breast, ovary, prostate, cervix uteri, bladder, lung and of the gastrointestinal tract contain elevated levels of uPA compared to benign tissues. In breast cancer uPA and PAI-1 antigen in tumor tissue extracts are independent prognostic factors for relapse-free and overall survival.