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.     

Functional analysis of the cellular receptor for urokinase in plasminogen activation. Receptor binding has no influence on the zymogenic nature of pro-urokinase

Plasminogen activation catalyzed by the urokinase-type plasminogen activator (uPA) constitutes a reciprocal zymogen activation system, as plasmin can efficiently activate pro-uPA, the single-chain zymogenic form of the protease. We have previously shown that the overall efficiency of this plasminogen activation system is greatly enhanced by its assembly on the cell surface, involving binding of pro-uPA to its cellular binding site uPAR, and the concurrent cellular binding of plasminogen. We have now studied the effect of a recombinant soluble form of uPAR (residues 1-277) on the proteolytic reactions of this system. In contrast to the increased efficiencies of plasminogen activation and pro-uPA activation observed with cell-surface uPAR, soluble uPAR had an inhibitory effect on both of these individual reactions. Soluble uPAR also caused no increase in the low, but discernible, intrinsic activity of pro-uPA. Consistent with the observations on the isolated reactions, the overall activity of the pro-uPA-mediated plasminogen activation system was significantly inhibited. These observations confirm the previous interpretation of the observations made with cell-surface uPAR that the mechanism of the enhanced plasmin generation is due to the catalytically favorable interaction of uPAR-bound uPA/pro-uPA with cell-bound plasminogen/plasmin, rather than direct effects on the properties of uPA or pro-uPA on binding to uPAR.     

Expression of the verotoxin receptor glycolipid, globotriaosylceramide, in ovarian hyperplasias

The presence of cell surface receptor glycolipid, globotriaosylceramide (Gb3), is essential to confer susceptibility to the E. coli-derived verotoxin (VT). Our earlier studies showed that Gb3 is expressed in ovarian carcinoma cell lines. The Gb3 content of normal ovary, benign and malignant primary ovarian tumors, and their metastases have now been compared by verotoxin thin-layer chromatogram (TLC) overlay of the glycolipid tissue extracts. FITC-labeled VT1 B subunit binding to frozen tumor sections was also monitored histochemically. Low to undetectable levels of Gb3 were found in "normal" ovarian tissue. Gb3 was markedly increased in both benign and malignant tumors, suggesting that increased Gb3 may be related to proliferation, rather than malignancy per se. Mucinous tumors showed the least Gb3 elevation; serous tumors were variable, showing higher levels of Gb3 in less differentiated malignant tumors. By far the highest Gb3 content was observed for secondary ovarian metastases and tumors refractory to chemotherapy. Frozen sections of neoplastic ovarian tissue overlaid with fluorescein-conjugated VT1 B subunit show extensive binding to tumor cells, particularly in poorly differentiated samples and blood vessels adjacent to, and within, the tumor mass. Tumor foci were stained but stromal tissue was consistently negative both in primary tumors and metastases. VT staining of well-differentiated primary ovarian tumor sections was weak, corresponding to their low Gb3 content, but strong staining was observed in sections from a highly differentiated primary tumor from a patient who was unexpectedly refractory to clinical chemotherapy. These studies suggest that verotoxin/Gb3 targeting may provide the basis for new treatments for ovarian cancer.     

Plasminogen activation by pro-urokinase in complex with its receptor--dependence on a tripeptide (Spectrozyme plasmin)

The intrinsic activity of single-chain pro-urinary-type plasminogen activator (pro-uPA) and whether its receptor (uPAR) potentiates this activity remains controversial. In this report, the pro-uPA/uPAR-(1-281)-peptide complex in solution is shown to have equivalent plasminogen-activator activity to that of active two-chain uPA (tc-uPA). However, the activity of the complex was dependent on a synthetic tripeptide, Spectrozyme plasmin (Spl, H-D-2-aminohexanoic acid(Ahx)-hexatyrosyl-lysine-p-nitroanilide), which can also be used as a chromogenic substrate for plasmin. Furthermore, this activity could be completely suppressed by commonly used carrier proteins and detergents. The pro-uPA/uPAR-(1-281)-peptide complex at 1 nM displayed similar activity to that of tc-uPA for either [Glu1]plasminogen or [Lys77]plasminogen in chromogenic assays with Spl present as the plasmin substrate. When assayed with another plasmin substrate, S2251, the pro-uPA/uPAR-(1-281)-peptide complex was unable to activate plasminogen. The pro-uPA/uPAR-(1-281)-peptide complex and tc-uPA also showed a similar extent of plasminogen activation as measured by SDS/PAGE, when incubated with plasminogen and Spl in the presence of 100 micro M aprotinin, and plasminogen activation by pro-uPA alone was also stimulated in the presence of Spl in this assay. Activation of plasminogen by the pro-uPA/uPAR-(1-281)-peptide strictly required the presence of Spl, and pro-uPA remained in single-chain form during these assays. This activity of the pro-uPA/uPAR-(1-281)-peptide complex but not that of tc-uPA was completely inhibited by human serum albumin, bovine serum albumin, Tween-80, Triton X-100, and Pluronic-F68. Taken together, the data indicates that uPAR-(1-281)-peptide itself is not sufficient to augment pro-uPA activity and the presence of an effector molecule (e.g. Spl) is required to elicit the full plasminogen-activator activity of the pro-uPA/uPAR-(1-281)-peptide complex. It remains to be seen whether there is a physiological counterpart to this phenomenon.     

Telomerase activity in gynecological tumors

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends using an RNA component as a template. Extension of telomeric repeats by telomerase prevents telomere shortening with cell divisions and contributes to chromosomal stability, possibly leading to immortalization of the cells. In the present study, we determined the telomerase activity of gynecological tumors and cell lines using a newly developed non-radioisotope telomeric repeat amplification protocol. A total of 21 cell lines derived from cervical cancer, endometrial cancer, ovarian cancer, and choriocarcinoma was examined, and all lines were found to be positive for telomerase activity, although the activity varied among cell types. A total of 50 gynecological malignant tumors was also examined, and 10 of 12 (83%) cervical cancers, 12 of 13 (92%) endometrial cancers, 18 of 21 (86%) ovarian cancers, 2 of 2 tubal cancers, and 1 of 1 vulvar cancer were found to be positive for telomerase activity. A total of 88% of gynecological tumors tested was thus found to be telomerase positive. However, no significant correlation was observed between telomerase activity and clinical features for any tumor type, although ovarian tumors expressing high telomerase activity tended to be more invasive. In contrast to that in malignant tumors, telomerase expression was weak and less common in premalignant lesions, with 5 of 7 cervical intraepithelial lesions and 4 of 6 borderline ovarian tumors exhibiting faint activity. Nine benign uterine lesions were also examined, and all were negative for telomerase activity except 1 uterine myoma, which had a weak signal. Three benign ovarian cysts examined had weak telomerase activity. These findings suggest that telomerase activation is common in gynecological malignant tumors and may be a critical step in their pathogenesis. However, premalignant lesions and some types of benign tumors also express weak telomerase activity.     

Recall and attitudes in patients with prostate cancer

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are important in the regulation of tumor tissue progenesis, cell differentiation, tumor cell motility, and tumor cell invasiveness. We have recently reported that the levels of uPA and uPAR were higher in malignant astrocytomas than in low-grade gliomas. In the present study, we measured the levels of uPA and uPAR during the growth of glioblastomas in nude mice. Using fibrin zymography, densitometry, and an enzyme-linked immunosorbent assay, we found that the enzyme activity and content of uPA were increased 4- to 10-fold during tumor formation. Using a receptor assay and an enzyme linked immunosorbent assay, we found the numbers and content of uPAR were increased 5- to 15-fold during tumor formation. In addition, immunohistochemical staining for uPA and uPAR revealed strong immunoreactivity in tumor cells with the staining more intense on day 28 than on day 14. These results suggest that the upregulation of uPA and uPAR plays a major role in the formation of gliomas.     

Enhancement of the expression of urokinase-type plasminogen activator from PC-3 human prostate cancer cells by thrombin

The presence of procoagulants and fibrin deposition have been demonstrated in malignant tumors. Although thrombin, a key enzyme in coagulation, has other various biological functions, the significance of its presence in tumors is not known. We studied the effects of thrombin on the expression of urokinase-type plasminogen activator (uPA) which is known to play a role in tumor invasion, using a human prostate cancer cell line PC-3. Human alpha-thrombin added to cultures of PC-3 produced a dose-dependent and time-dependent increased secretion of uPA that was greatest at 3-6 h after exposure to thrombin. Increase in uPA antigen paralleled the increase in mRNA level, which reached a maximum at 4 h. Thrombin showed the maximum effect on uPA expression at a concentration 1-2 units/ml. Zymography showed that transient exposure to thrombin induced an increase in fibrinolytic activity which could be quenched by anti-uPA antibody. The thrombin receptor-activating peptide also caused an increase in uPA protein and mRNA level, indicating the presence of the same thrombin specific receptor on PC-3 cells as on platelets and endothelial cells. Thrombin did not affect the expression of other components of the plasminogen activation system, tissue-type plasminogen activator and type-1 plasminogen activator inhibitor, and uPA receptor. These results indicate that thrombin increases uPA expression selectively by the stimulation of a functional thrombin receptor on PC-3 cells. Since uPA is known to play a role in pericellular proteolysis of extracellular matrix, thrombin may be involved in the regulation of tumor invasion and metastasis.     

Evaluation of an assay for detecting telomerase activity in neoplastic tissues of dogs

OBJECTIVE: To determine feasibility of using the telomere repeat amplification protocol (TRAP) assay to detect telomerase activity in tumors of dogs. SAMPLE POPULATION: Samples of tumor or normal tissue were obtained from client-owned dogs that underwent surgical biopsy during the period of January 1996 through December 1997. PROCEDURE: The TRAP assay was used to detect telomerase activity in malignant or benign tumors of dogs. Telomerase status (positive or negative) was compared with results of histologic examination for each sample to estimate specificity and sensitivity of this assay for the diagnosis of malignancy. RESULTS: Of 26 malignant tumors, 24 were telomerase positive on TRAP assay, whereas 3 of 4 benign tumors and 3 of 3 normal tissues were telomerase negative. Analysis of these results indicated an estimated sensitivity of 92% and specificity of 86% for tumor analysis, using the TRAP assay. CONCLUSION: The TRAP assay can be used to measure telomerase activity in malignant tumors of dogs. CLINICAL RELEVANCE: Because telomerase activation may be required for indefinite longevity of cells, it may also serve as a tumor marker and therapeutic target. The TRAP assay can be used to detect telomerase in samples of fluid as well as tissues obtained from solid tumors. Therefore, it may have considerable clinical value in rapid and noninvasive diagnosis of neoplasia in dogs. Additional studies must be completed to more accurately determine sensitivity and specificity of the assay.     

Synergistic effects of prostaglandin F2alpha and tumor necrosis factor to induce luteolysis in the pig

Telomerase activity was detected in germ cells, stem cells and cancer cells. In tumors of the ovary, an organ that contains germ cells, the authors examined availability to detect telomerase activity. Telomerase activity of malignant tumors was extremely high compared with that of normal ovaries and benign tumors. Strength and frequency of telomerase activity in malignant tumors was significant different from that in benign tumors. Telomere length tended to be smaller for malignant tumors of advanced stage, but no significant relationship between telomere length and telomerase activity and tumor stage could be recognized. Telomerase activity may be a useful marker for the diagnosis of ovarian tumors.     

In vitro regulation of pericellular proteolysis in prostatic tumor cells treated with bombesin

Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen-insensitive prostatic tumor cells. This study examines the bombesin-mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo-invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell-surface uptake of uPA and MMP-9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p-aminobenzamidine. Moreover, HMW-uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p-aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP-9 and TIMP-1 production. MMP-9 activation can be also obtained by HMW-uPA treatment, suggesting that plasma-membrane-bound uPA can start a proteolytic cascade involving MMP-9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the "non-random" degradation of the extracellular matrix in which membrane uPA-uPAreceptor complexes could start bombesin-induced directional protein degradation during metastatic spread.     

Coupling of gonadotropin-releasing hormone receptor to Gi protein in human reproductive tract tumors

The signaling pathway by which GnRH acts in peripheral tumors is distinct from that in the anterior pituitary. We attempted to identify the guanosine triphosphate (GTP)-binding protein (G protein) subtypes linked to GnRH receptor in the genital tract tumor membranes. Surgically removed ovarian carcinomas and uterine leiomyosarcomas were screened for GnRH receptor expression before plasma membrane isolation. The G alpha i was detected by immunoblotting of membrane extracts with specific antibody and pertussis toxincatalyzed ADP-ribosylation from nicotinamide adenine dinucleotide. Membrane phosphotyrosine phosphatase activity was determined as a GnRH-sensitive membrane event using synthetic substrate p-nitrophenyl in a spectrophotometric assay. Pertussis toxin, but not cholera toxin, brought about ADP-ribosylation of an immunodetected G alpha i of 41 kDa in the GnRH receptor-positive tumor membrane. Incubation with a GnRH analog and GTP decreased the ADP-ribosylation activity in a dose-dependent manner; a half-maximal effect occurred with 30 nmol/L buserelin (P < 0.01). The apparent inhibition by GnRH of the ADP-ribosylation demonstrated that GnRH resolved the alpha-subunit of the Gi to GTP-bound form in the membranes. The action of GnRH was neutralized by a competitive antagonist, antide. Pretreatment of the membrane with the pertussis toxin completely inhibited GnRH-sensitive phosphotyrosine phosphatase activity (P < 0.01). These data demonstrate the coupling of GnRH receptor to Gi protein subfamily. The Gi which couples GnRH receptor to the effector may define the difference of responses by peripheral tumor and the anterior pituitary.     

Differential expression of estrogen receptor-alpha and -beta messenger RNAs as a potential marker of ovarian carcinogenesis

Although estrogen receptor (ER)-alpha is expressed in both benign and malignant ovarian tumors, the role of ER in ovarian carcinogenesis of epithelial tumors is still unknown. In view of the recent characterization of ER-beta, a second form of ER that seems to be highly expressed in ovaries, we reexamined this issue by studying the relative expression of ER-alpha and -beta in human ovarian tumor progression. We developed a competitive PCR assay based on coamplification of the two ERs in target nucleotide sequences displaying a high homology (exons 3 and 4). Coamplification experiments with varying amounts of plasmids containing ER-alpha and -beta cDNAs showed that this assay was reliable for discriminating as little as a 2-fold difference in the initial ER-alpha:ER-beta cDNA ratio. The relative expression of ER-alpha compared with ER-beta mRNAs was studied in human ovarian cancer cell lines (n = 5) and in normal ovaries (n = 6), then in human benign and malignant tumor samples including ovarian cysts (n = 24), borderline tumors (n = 3), and cancers (n = 10). In normal ovaries, ER-beta mRNA was the predominant ER form, whereas in ovarian cancer cell lines ER-alpha mRNA was markedly increased as compared with ER-beta. In benign and borderline tumors, ER-beta mRNA was detected in 78% of tumors, whereas ER-alpha mRNA was detected in 29%. In ovarian carcinomas, both ER-alpha and -beta mRNAs were expressed in 80% of tumors. The ER-alpha:ER-beta mRNA ratio was >1 in only one cyst sample (4%). In contrast, the ER-alpha:ER-beta mRNA ratio was markedly increased in ovarian cancers because 60% showed an ER-alpha:ER-beta mRNA >1. In situ hybridization experiments showed overlapping tissular distribution of ER-beta and -alpha expression in cancers and cysts, with a main localization in the epithelium and only a low level of expression in stromal cells. In summary, we found an increase in the ER-alpha:ER-beta mRNA ratio in ovarian carcinomas as compared with normal ovaries and cysts. These data suggest that overexpression of ER-alpha relative to ER-beta mRNA may be a marker of ovarian carcinogenesis.     

Activation of the ras-mitogen-activated protein kinase pathway and phosphorylation of ets-2 at position threonine 72 in human ovarian cancer cell lines

The activation status of the ras pathway was studied in eight ovarian tumor cell lines. Three biochemical parameters indicative of ras activation were tested: (a) the ratio of the ras-GTP:ras-GDP complex; (b) the activity of mitogen-activated protein kinases p42/p44; and (c) ets-2 phosphorylation at position threonine 72, a mitogen-activated protein kinase phosphorylation site in vivo. Four of the ovarian tumor cell lines had an activated ras pathway by these three parameters, whereas only one of these contained a mutated ras gene. In addition, ras/ets-2 responsive genes such as the urokinase plasminogen activator (uPA) were activated in these four cell lines. Transient transfection assays indicated that the compound ets-AP1 oncogene responsive enhancer present in the uPA gene was the target of ras signaling in ovarian tumor cells and that the combination of activated ras and ets-2 could superactivate the uPA enhancer element. Coexpression of the dominant-negative ras-Asn17 cDNA gene abrogated activity of this uPA element in ovarian tumor cells. These data indicate that ets-2 is a nuclear target of ras action in ovarian tumor cell lines and that ras signaling pathways may be activated in ovarian cancer by mechanisms independent of direct genetic damage to ras genes.     

A competitive chromogenic assay to study the functional interaction of urokinase-type plasminogen activator with its receptor

Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various extracellular matrix components. uPA is focused to the cell surface via binding to a specific receptor (uPAR, also termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeling and tumor invasion. We have developed a competitive microtiter plate-based chromogenic assay which allows the analysis of uPA/uPAR interaction. The plates are coated with recombinant uPAR expressed in Chinese hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is added to the microtiter plate-attached uPAR. The amount of receptor-bound uPA is then determined indirectly via addition of plasminogen, which is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reduction of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respectively, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed in CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides derived from the sequence of the uPAR-binding region of uPA, and iv) antibodies directed against uPAR. This assay may be helpful in identifying uPA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.     

Pharmacokinetic characteristics and tolerability of a novel intravenous immunoglobulin preparation

Cathepsin D content and activity were determined in matched paired sets of colorectal tumor tissue and normal mucosa and correlated with a number of biological and clinical parameters. Significantly higher cathepsin D activity was measured in tumor cytosol compared to paired normal mucosa (p < 0.02), in Dukes' stage A tumors compared to Dukes' B and C (p < 0.05), in tumors < 5 cm compared to those > 5 cm, or in tumors with a low proliferation rate compared to those with a high proliferation rate (p < 0.05). Moreover, significant differences in enzyme activity between tumor tissue and paired normal mucosa were observed in node-positive and G2 tumors (p < 0.05). No significant correlation between cathepsin D activity and other biological parameters was found. Further, no differences in cathepsin D content between tumor tissue and paired normal mucosa were observed except in Dukes' stage A tumors (p < 0.02). A significantly increased cathepsin D content was also observed in tumors > 5 cm compared to tumors < 5 cm (p < 0.01). No relationship between tumor cathepsin D content and clinical stage was detected. However, a significant correlation (p < 0.05) was observed between the tumor-specific content of this enzyme and tumor grade. Finally, there was no relationship between tumor-specific cathepsin D activity and content (r = -0.27, p = 0.23). These data suggest that cathepsin D activity rather than content correlates with the malignant progression of colorectal cancer. This phenomenon should be taken into consideration when clinical studies are undertaken to assess the potential prognostic value of proteolytic enzymes involved in tumor progression.     

Markers of tumor angiogenesis and proteolysis independently define high- and low-risk subsets of node-negative breast cancer patients

PURPOSE: To compare the prognostic impact of tumor angiogenesis factors (vascular endothelial growth factor [VEGF], angiogenin, and basic fibroblast growth factor [bFGF]), tumor proteolysis factors (urokinase-type plasminogen activator [uPA] and plasminogen activator inhibitor-1 [PAI-1]), and conventional tumor markers (stage, grade, and steroid receptors) in early breast cancer. PATIENTS AND METHODS: In the primary clinical study, tumor angiogenesis and other factors were detected in frozen biopsies from 305 primary breast tumors. VEGF expression was assessed by chemiluminescence immunosorbent assay (ICMA); angiogenin, bFGF, uPA, and PAI-1 by enzyme-linked immunosorbent assay (ELISA); and steroid receptors (estrogen receptor [ER] and progesterone receptor [PgR]) by enzyme immunoassay (EIA). In the validating clinical study, another set of 190 node-negative primary breast tumor samples were collected at a separate institution. RESULTS: Univariate analysis of the primary study showed that VEGF levels were positively correlated with recurrence (P < .001). Angiogenin levels were positively correlated with disease relapse (P < .005) for the overall collective group, but not within the node-negative subset. No significant correlations were found between tumor bFGF levels and patient survival. In multivariate regression analysis, the only independent predictors of relapse-free survival (RFS) were VEGF, uPA, and lymph node status. In the validation set, the distribution of VEGF and uPA values were similar to those in the primary study; low expression of both VEGF and uPA identified patients with a < or = 20% likelihood of recurrence within 7 years. CONCLUSION: Separate primary and validating clinical studies concur that tumor VEGF level is the most important prognostic parameter among several markers of tumor angiogenesis and proteolysis.     

The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria

The urokinase plasminogen activator (uPA) is a proteolytic enzyme which converts the proenzyme plasminogen to the active serine protease plasmin. A cell surface receptor for uPA (uPAR) is attached to the cell membrane by a glycosyl-phosphatidylinositol anchor. Binding of uPA to uPAR leads to an enhanced plasmin formation and thereby an amplification of pericellular proteolysis. We have shown previously that uPAR is expressed on normal blood monocytes and granulocytes, but is deficient on affected blood monocytes and granulocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH), and that uPAR is present in plasma from these patients. In this study a newly established sensitive enzyme-linked immunosorbent assay (ELISA) has been applied for quantitation of uPAR in plasma. Unexpectedly, we found that uPAR is not only present in PNH plasma but also in plasma from healthy individuals. In 39 healthy individuals the mean plasma-uPAR value +/- SD was 31 +/- 15 pM, median 28 (range 11-108), and the corresponding value for six PNH patients was 116 +/- 67 pM, median 90 (range 61-228). The elevated uPAR-level in PNH patients was highly significant (Mann-Whitney test; P < 0.0001), and may possibly contribute to the propensity for thrombosis in PNH by inhibition of the fibrinolytic system. Binding of pro-uPA by uPAR in plasma may interfere with the appropriate binding of pro-uPA to cell-bound uPAR and therefore inhibit cell-associated plasmin generation and fibrinolysis. It is likely that the uPAR in normal plasma reflects the overall level of activity of the uPAR-mediated cell surface proteolysis. The present ELISA may be used for studies of uPAR levels in plasma from patients with conditions in which this activity might be increased, such as cancer and inflammatory disorders. Future studies will determine if uPAR in plasma is a parameter of clinical importance in these diseases.     

Failure of the polyethylene after bipolar hemiarthroplasty of the hip. A report of five cases

Due to the difficulties in separating malignant and benign ovarian cysts by transvaginal ultrasound and other techniques, there is a need for biochemical markers in serum or cyst fluids. In the present study we have evaluated the levels of the chemokine interleukin-8 (IL-8) in ovarian cysts. IL-8 is known to be expressed in the normal ovary and to influence proliferation and angiogenesis of several nonovarian types of tumors. Cyst fluids from benign (n = 15) and malignant (n = 13) ovarian tumors were analyzed. The levels of IL-8 were found to be significantly (13-fold) higher in cyst fluids from malignant tumors (18.1 +/- 7.5 ng/ml; mean +/- SE) compared to benign cysts (1.3 +/- 0.7 ng/ml). The plasma levels of IL-8 were considerably lower (2.9 and 0.3% of levels in benign and malignant cyst fluids, respectively) than in cyst fluids. No difference in the plasma levels of patients with benign or malignant tumor could be detected. In contrast, the levels of CA 125 were significantly higher in plasma of patients with malignant disease with the inverse relation in cyst fluids. In conclusion, the levels of IL-8 are markedly elevated in cyst fluid from malignant tumors compared to benign. This specific increase indicates a role for this cytokine in ovarian tumor biology.     

Interaction of 5-HT1B/D ligands with recombinant h 5-HT1A receptors: intrinsic activity and modulation by G-protein activation state

Many 5-HT1B/D receptor ligands have affinity for 5-HT1A receptors. In the present study, the intrinsic activity of a series of 5-HT1B/D ligands was investigated at human 5-HT1A (h 5-HT1A) receptors by measuring G-protein activation in recombinant C6-glial and HeLa membranes, using agonist-stimulated [35S]GTPgammaS binding. In these two membrane preparations, the density of h 5-HT1A receptors (i.e., 246 to 320 fmol mg(-1) protein) and of their G-proteins, and the receptor: G-protein density ratio (0.08 to 0.18) appeared to be similar. It was found that: (i) the maximal [35S]GTPgammaS binding responses induced by the 5-HT1B/D receptor ligands in the HeLa preparation at 30 microM GDP were comparable to that of the native agonist 5-HT; (ii) as compared to 5-HT (1.00), similar potencies but lower maximal responses were observed in the C6-glial preparation at 0.3 microM GDP for zolmitriptan (0.89), dihydroergotamine (0.81), rizatriptan (0.71), CP122638 (0.69), naratriptan (0.60) and sumatriptan (0.53); and that (iii) maximal [35S]GTPgammaS binding responses induced by 5-HT1B/D ligands in the C6-glial preparation were either unaffected or significantly enhanced by increasing the GDP concentration from 0.3 to 30 microM and higher concentrations. These features differ from those observed with 5-HT1A receptor agonists; the latter display the same rank order of potency and efficacy in both membrane preparations, and increasing the amount of GDP with C6-glial membranes results in an attenuation of both the agonist's maximal effect and the apparent potency of partial agonists. The differential regulation of 5-HT1A and 5-HT1B/D agonist responses by GDP suggests that different G-protein subtypes are involved upon 5-HT1A receptor activation by 5-HT1A and 5-HT1B/D agonists.