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INTRODUCTION The current study examined whether and how severe injustice such as a school attack threatens the belief in a just world (BJW). METHOD We collected longitudinal data on the BJW from adolescents in China who witnessed random school attacks on the news (N = 227). RESULTS Change analyses provided evidence that the BJW increased after witnessing severe injustice. Furthermore, we tested for moderating effects of buffer variables, such as life satisfaction and perceived social support, on change in the BJW. Findings showed that these variables buffered the threat to the BJW after observing unfairness. DISCUSSION We discuss these results in the context of justice motive theory and suggest implications for future research and practical implications.

Although moral courage is a highly desirable behavior whose determinants need to be understood, research has largely neglected the emotions involved in moral courage. Does anger about the norm violation or (anticipated) guilt enhance such interventions even if general mood does not? As previous studies have often failed to overcome the limitations of self-reported emotions and the use of behavior intention measures, we used a multimethod emotion measurement while observing real behavior. By realizing a real theft scenario in the laboratory (N = 68), we found that anger but neither guilt nor general mood predicted intervention behavior. Our findings complement and expand previous studies by showing that people who experience and express anger more strongly are able to overcome the psychological barrier of potential negative (social) consequences in a situation in which a fast and immediate intervention is needed, whereas others stand and watch. Copyright © 2014 John Wiley & Sons, Ltd.

Chemotherapeutic drugs kill cancer cells, but it is unclear why this happens in responding patients but not in non-responders. Proteomic profiles of patients with oesophageal adenocarcinoma may be helpful in predicting response and selecting more effective treatment strategies. In this study, pretherapeutic oesophageal adenocarcinoma biopsies were analysed for proteomic changes associated with response to chemotherapy by MALDI imaging mass spectrometry. Resulting candidate proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated for functional relevance in vitro. Clinical impact was validated in pretherapeutic biopsies from an independent patient cohort. Studies on the incidence of these defects in other solid tumours were included. We discovered that clinical response to cisplatin correlated with pre-existing defects in the mitochondrial respiratory chain complexes of cancer cells, caused by loss of specific cytochrome c oxidase (COX) subunits. Knockdown of a COX protein altered chemosensitivity in vitro, increasing the propensity of cancer cells to undergo cell death following cisplatin treatment. In an independent validation, patients with reduced COX protein expression prior to treatment exhibited favourable clinical outcomes to chemotherapy, whereas tumours with unchanged COX expression were chemoresistant. In conclusion, previously undiscovered pre-existing defects in mitochondrial respiratory complexes cause cancer cells to become chemosensitive: mitochondrial defects lower the cells' threshold for undergoing cell death in response to cisplatin. By contrast, cancer cells with intact mitochondrial respiratory complexes are chemoresistant and have a high threshold for cisplatin-induced cell death. This connection between mitochondrial respiration and chemosensitivity is relevant to anticancer therapeutics that target the mitochondrial electron transport chain.

Intergroup contact and group relative deprivation have both been shown to play a key role in the understanding of intergroup relations. Nevertheless, we know little about their causal relationship. In order to shed some light on the directionality and causality of the relationship between intergroup contact and group relative deprivation, we analysed responses by East and West Germans from k= 97 different cities, collected 6 (N(T)(1) = 1,001), 8 (N(T)(2) = 747), and 10 years (N(T)(3) = 565) after reunification. Multi-level cross-lagged analyses showed that group relative deprivation at T1 led to more (rather than less) intergroup contact between East and West Germans 2 years as well as 4 years later. We found no evidence for the reverse causal relationship, or moderation by group membership. Furthermore, admiration mediated the positive effect of relative deprivation on intergroup contact for both East and West Germans. This intriguing finding suggests that intergroup contact may be used as a proactive identity management strategy by members of both minority and majority groups.

Epigenetic drugs are promising add-ons to cancer treatment; still, adverse effects concerning tumour promotion have been reported occasionally. In this in vitro study, we investigated the effect of combination treatment of decitabine with anthracycline-based chemotherapy [5-fluorouracil plus epirubicine plus cyclophosphamide (FEC)] on viability and metastatic activity of breast cancer cell lines, MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). The effect of decitabine and its combined treatment with FEC on viability of both cancer cell lines was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide and adenosine triphosphate (ATP) cell survival assays. DNA methylation specific real-time polymerase chain reaction (PCR) (Methylight®) was employed to document the methylation status of the metastasis-relevant urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-I (PAI-1) genes. Additionally, protein expression levels of uPA and PAI-1 were determined using enzyme-linked immunosorbent assays. Invasion capacity of cells was assayed using Matrigel® invasion assay. Decitabine lowered the viability of MCF-7 cells, although MDA-MB-231 cells were not affected. Decitabine did not augment FEC-mediated cytotoxicity in both cell lines. In MCF-7 cells, methylation of the uPA and PAI-1 gene promoter was significantly reduced by decitabine or decitabine plus FEC. Protein levels of uPA and PAI-1 were induced by all treatments. Decitabine significantly induced the invasion capacity of MCF-7 cells, whereas all of the drugs resulted in decreased invasion capacity of MDA-MB-231. Our results suggest differential effects of single-dose decitabine and its combination with FEC on the metastatic capacity and survival of breast cancer cell lines endowed with different metastatic behaviour.

The present paper aims to elucidate under what conditions victims of injustice who seek revenge feel satisfied and perceive that everybody got what he or she deserved. Two hypotheses are discussed: The comparative suffering hypothesis states that seeing the offender suffer from fate is sufficient for evoking satisfaction and perceptions of deservingness among victims. The understanding hypothesis states that revenge can only be satisfactory when the offender understands it as a response to his or her prior behavior. These hypotheses were tested in three experimental studies. The comparative suffering hypothesis received only weak support. The understanding hypothesis, on the other hand, received much stronger support: When the offender understood revenge as punishment, revenge led to satisfaction and deservingness among victims. These findings are discussed with regard to the question why people take revenge. Copyright © 2010 John Wiley & Sons, Ltd.

According to the social justice literature, fraternal relative deprivation causes protest, but has little impact on well-being. We consider this view incomplete and predict that fraternal relative deprivation can impair well-being if it is enduring and difficult to ameliorate. As part of a longitudinal study of the German unification process, measures of egoistic relative deprivation, fraternal relative deprivation, life satisfaction, mental health, and protest were obtained on three occasions of measurement (1996, 1998, 2000) from a demographically heterogeneous sample of 1276 East German citizens. Model tests and parameter estimation were performed with LISREL. In line with our predictions, unique longitudinal effects of fraternal relative deprivation on well-being were identified. No longitudinal effect of fraternal relative deprivation on protest was identified.

We previously showed that integrin alphavbeta3 overexpression and engagement by its ligand vitronectin increased adhesion, motility, and proliferation of human ovarian cancer cells. In search of differentially regulated genes involved in these tumor biological events, we previously identified the integrin-linked kinase (ILK) to be under control of alphavbeta3. In the present investigation we demonstrated significantly upregulated ILK protein as a function of alphavbeta3 in two ovarian cancer cell lines, OV-MZ-6 and OVCAR-3, and proved co-localization at the surface of alphavbeta3-overexpressing cells adherent to vitronectin. Increase of ILK protein was reflected by enhanced ILK promoter activity, an effect, which we further characterized with regard to transcriptional response elements involved. Abrogation of NF-kappaB/c-rel or p53 binding augmented ILK promoter activity and preserved induction by alphavbeta3. The AP1-mutant exhibited decreased promoter activity but was also still inducible by alphavbeta3. Disruption of the two DNA consensus motifs for Ets proteins led to divergent observations: mutation of the Ets motif at promoter position -462 bp did not significantly alter promoter activity but still allowed response to alphavbeta3. In contrast, disruption of the second Ets motif at position -85 bp did not only lead to slightly diminished promoter activity but also, in that case, abrogated ILK promoter induction by alphavbeta3. Subsequent co-transfection studies with ets-1 in the presence of the second Ets motif led to additional induction of ILK promoter activity. Taken together, these data suggest that ets-1 binding to the second Ets DNA motif strongly contributes to alphavbeta3-mediated ILK upregulation. By increasing ILK as an important integrin-proximal kinase, alphavbeta3 may promote its intracellular signaling and tumor biological processes arising thereof in favor of ovarian cancer metastasis.

Urokinase-type plasminogen activator (uPA) binds with high affinity to its specific cell surface receptor (uPAR) (CD87) via a well-defined sequence within the N-terminal region of uPA (uPA(19-31)). Since this uPA/uPAR-interaction plays a significant role in tumor cell invasion and metastasis, it has become an attractive therapeutic target. Two small peptidic cyclic competitive antagonists of uPA/uPAR-interaction have been developed, based on the uPAR binding site in uPA: WX-360 (cyclo(21,29)[D-Cys21]-uPA(21-30)[S21C;H29C]) and its norleucine (Nle) derivative WX-360-Nle (cyclo(21,29)[D-Cys21]-uPA(21-30)[S21C;K23Nle;H29C]). These peptides display an only five to 10-fold lower affinity to uPAR as compared to the naturally occurring uPAR-ligand uPA. In this study, WX-360 and WX-360-Nle were tested in nude mice for their potency to inhibit tumor growth and intraperitoneal spread of lacZ-tagged human ovarian cancer cells. Intraperitoneal administration of either cyclic peptide (20 mg peptide/kg; 1x daily for 37 days) into the tumor-bearing nude mice resulted in a significant reduction of tumor weight and spread within the peritoneum as compared to the untreated control group. This is the first report demonstrating effective reduction of tumor growth and spread of human ovarian cancer cells in vivo by small synthetic uPA-derived cyclic peptides competitively interfering with uPA/uPAR-interaction. Thus, both WX-360 and WX-360-Nle are promising novel compounds to reduce dissemination of human ovarian carcinoma.

In a longitudinal questionnaire field study on psychological consequences of German unification, the intergroup situation between East and West Germans was investigated. Data were collected in 1996 and 1998. The sample consisted of 585 East Germans and 387 West Germans who had never lived in the other part of Germany. It was assumed that East Germans' social identity is threatened due to their fraternal deprivation in comparison with West Germans. It was predicted that East Germans would employ ingroup bias as an identity management strategy in order to protect their emotional well-being against harmful consequences of fraternal deprivation. In line with this prediction, it was found that (a) East Germans feel fraternally deprived compared to West Germans on important quality of life dimensions, (b) they display ingroup bias vis-a-vis West Germans, (c) ingroup bias increases with increasing East German identity, (d) ingroup bias is determined longitudinally by relative deprivation, and (e) ingroup bias buffers the effect of relative deprivation on mental health over time. As expected, ingroup bias and the effects of ingroup bias were larger for the dimension of personal integrity than for the dimensions of sympathy and competence. Ingroup bias is interpreted as compensatory self-enhancement. West Germans feel fraternally privileged compared to East Germans and consider their advantages to be undeserved. Unexpectedly, West Germans display outgroup bias on the stereotype dimensions of integrity. This bias is interpreted as an effort to appease the moral outrage of East Germans and to silence their own guilty conscience due to undeserved advantages. Copyright © 2002 John Wiley & Sons, Ltd.

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays an important role in physiological processes such as thrombolysis and fibrinolysis, as well as pathophysiological processes such as thrombosis, tumor invasion and metastasis. In addition to inhibiting serine proteases, mainly tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, PAI-1 interacts with different components of the extracellular matrix, i.e. fibrin, heparin (Hep) and vitronectin (Vn). PAI-1 binding to Vn facilitates migration and invasion of tumor cells. The most important determinants of the Vn-binding site of PAI-1 appear to reside between amino acids 110-147, which includes alpha helix E (hE, amino acids 109-118). Ten different PAI-1 variants (mostly harboring modifications in hE) as well as wild-type PAI-1, the previously described PAI-1 mutant Q123K, and another serpin, PAI-2, were recombinantly produced in Escherichia coli containing a His(6) tag and purified by affinity chromatography. As shown in microtiter plate-based binding assays, surface plasmon resonance and thrombin inhibition experiments, all of the newly generated mutants which retained inhibitory activity against uPA still bound to Vn. Mutant A114-118, in which all amino-acids at positions 114-118 of PAI-1 were exchanged for alanine, displayed a reduced affinity to Vn as compared to wild-type PAI-1. Mutants lacking inhibitory activity towards uPA did not bind to Vn. Q123K, which inhibits uPA but does not bind to Vn, served as a control. In contrast to other active PAI-1 mutants, the inhibitory properties of A114-118 towards thrombin as well as uPA were significantly reduced in the presence of Hep. Our results demonstrate that the wild-type sequence of the region around hE in PAI-1 is not a prerequisite for binding to Vn.

Intrinsic or acquired resistance to chemotherapy is responsible for failure of current treatment regimens in breast cancer patients. The Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug-resistant tumor phenotype. In human breast cancer, overexpression and nuclear localization of YB-1 is associated with upregulation of P-glycoprotein. In our pilot study, we analyzed the clinical relevance of YB-1 expression in breast cancer (n = 83) after a median follow-up of 61 months and compared it with tumor-biologic factors already used for clinical risk-group discrimination, i.e., HER2, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1). High YB-1 expression in tumor tissue and surrounding benign breast epithelial cells was significantly associated with poor patient outcome. In patients who received postoperative chemotherapy, the 5-year relapse rate was 66% in patients with high YB-1 expression. In contrast, in patients with low YB-1 expressions, no relapse has been observed so far. YB-1 expression thus indicates clinical drug resistance in breast cancer. Moreover, YB-1 correlates with breast cancer aggressiveness: in patients not treated with postoperative chemotherapy, those with low YB-1 expression are still free of disease, whereas the 5-year relapse rate in those with high YB-1 was 30%. There was no significant correlation between YB-1 expression and either HER2 expression or uPA and PAI-1 levels. Risk-group assessment achieved by YB-1 differed significantly from that by HER2 or uPA/PAI-1. In conclusion, YB-1 demonstrated prognostic and predictive significance in breast cancer by identifying high-risk patients in both the presence and absence of postoperative chemotherapy, independent of tumor-biologic factors currently available for clinical decision making. © 2001 Wiley-Liss, Inc.

The glycosylphosphatidylinositol (GPI)-anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane-anchored and soluble isoforms (s-uPAR) in vitro and in vivo. Recent evidence indicates that s-uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s-uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI-specific phospholipase D (GPI-PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI-PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI-PLD activity by GPI-PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI-PLD also regulated uPAR expression, possibly by releasing a GPI-anchored growth factor. Our data suggest that cellular GPI-PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI-anchored proteins by generating functionally distinct, soluble counterparts.

In a field study with 295 factory employees, three hypotheses were tested: (1) Procedural injustice at work is correlated negatively with job satisfaction and psychosomatic well-being. (2) The perception of procedural injustice depends on the person's chronic justice sensitivity. (3) Justice sensitivity moderates the correlation of procedural injustice with satisfaction and well-being, the correlation becoming larger with increasing justice sensitivity. Procedural injustice was defined as the discrepancy between desired (ought) and perceived (is) procedures. Justice sensitivity and procedural fairness according to Leventhal's criteria (consistency, nonpartiality, accuracy, correctability, representativeness) and one additional criterion (open information) were measured via questionnaire. Job satisfaction, number of sick days during the last six months and number of days a person felt sick at work during the last six months served as indicators of psychosomatic well-being. The first and second hypotheses were supported by the data. Partial support was also obtained for the third hypothesis: Justice sensitivity moderated the correlation of procedural unfairness with (a) the number of days the person felt sick at work and (b) the sum of this variable with the number of sick days. Copyright © 1999 John Wiley & Sons, Ltd.

Elevated levels of the urokinase-type plasminogen activator (uPA) in tumor cells are conductive to tumor cell spread and metastasis. In a previous study we observed that suppression of RelA dramatically reduced endogenous uPA synthesis in the human ovarian cancer cell line OV-MZ-6. Because the uPA promoter contains three potential Rel-like protein binding motifs (RRBE, 5'-NF-kappaB, and 3'-NF-kappaB) we conducted the first thorough systematic uPA promoter analysis to examine the direct impact of Rel proteins on uPA gene transcription. Disruption of RRBE resulted in a approximately 40% decrease in uPA promoter activity, mutation of the 5'-NF-kappaB motif led to an additional 20% decrease. The 3'-NF-kappaB motif was not active. Overexpression of RelA significantly enhanced uPA promoter activity, whereas IkappaB-alpha overexpression reduced uPA promoter activity by 40%. These data were supported by the finding that endogenous uPA was also increased sixfold by overexpression of RelA and decreased by 30% upon overexpression of IkappaB-alpha. Transfection of OV-MZ-6 cells with antisense deoxynucleotides directed to RelA expression reduced uPA promoter activity by at least 40%. Our data clearly suggest that by binding to uPA promoter elements, Rel transcripton factors contribute directly to elevated uPA gene expression in human ovarian cancer cells, thereby promoting the multiple functions of uPA during tumor growth and metastasis.

Evidence has accumulated that urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1) and receptor (uPAR) are involved in tumor invasion and metastasis. We analyzed the DNA sequences encoding these factors to see if they are altered in the ovarian cancer cell lines OV-MZ-6, OV-MZ-19, and OVCAR-3. In the uPA-encoding cDNA derived from OV-MZ-6 cells (but not in the uPA-cDNA from OVCAR-3 and OV-MZ-19), a so-far unknown mutation was identified in codon 121, resulting in a proline to leucine exchange. This exchange creates an AluI restriction site making restriction fragment length polymorphism (RFLP) analyses possible. Previously published PAI-1 sequences pointed to a variation of amino acid 15 of the PAI-1 signal sequence representing either threonine or alanine, which was confirmed in the present study. The uPAR cDNAs of all three cell lines encoded the published wild-type sequence. In order to elucidate the possible role of the Pro121Leu exchange in uPA and the Ala/Thr variants in the signal sequence of PAI-1 in the development and/or progression of human ovarian cancer, we studied the presence of these mutants or variants in a series of 22 ovarian cancer tissues. In addition to the wild-type sequence, the Pro121Leu exchange in the uPA sequence was detected in 10 out of 22 tumor tissues; 11 tumors carried exclusively the Pro121 allele; in one case exclusively the Leu121 allele was detected. In 18/22 tumors, triplet 15 in the signal sequence of PAI-1 encoded alanine, four DNAs contained both the Ala and the Thr allele. Furthermore, we analyzed another known common single-base-pair insertion/deletion polymorphism (ins/del allele) found in the promoter region of the PAI-1 gene and thought to be of functional importance in regulating PAI-1 gene expression. The PAI-1 ins-allele was found in 3/22, the del-allele in 6/22 and both alleles in 13/22 ovarian cancer tissues. In genomic DNA isolated from peripheral blood of 23 healthy donors, we observed similar allele frequencies of the three polymorphisms as found in the 22 ovarian carcinomas. Taken together, these results suggest that the polymorphisms observed in the uPA and PAI-1 genes may not be linked to ovarian cancer.

The amino-terminal fragment of human uPA (ATF; amino acids 1-135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae. Recombinant yeast ATF, modified and extended by an amino-terminal in-frame insertion of a His6 tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell-surface-associated uPAR. The ATF expression plasmid served as a template for the construction of a series of site-directed mutants in order to define those amino acids that are important for binding to uPAR. All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate. Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20-30) were individually substituted with alanine. Lys23, Tyr24, Phe25, IIe28, and Trp30 were important determinants for uPAR binding. A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14-32 of ATF. Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPAR-binding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thr18 by alanine or Asn32 by serine had hardly any effect, replacement of Asn22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR. Deletion of either Val20, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutants. Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR-binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR-binding activity. The results presented for the ATF mutants and uPA-derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as potential therapeutic agents to block tumor cell-associated uPA/uPAR interaction.

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.

The receptor for urokinase-type plasminogen activator (uPAR) may contribute to the invasive and metastatic capacity of tumor cells by focusing the serine protease urokinase-type plasminogen activator (uPA) to the cell surface. uPA activates plasminogen to plasmin which in turn degrades extracellular matrix proteins or activates other proteases. Mature uPAR is a heavily glycosylated protein of about 284 amino acids attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor. A set of different polyclonal uPAR antibodies has been generated in order to investigate the role of uPAR in tumor spreading in more detail. For this purpose, uPAR (lacking the GPI anchor) was expressed in E. coli and Chinese hamster ovary (CHO) cells. Recombinant uPAR from E. coli (corresponding to amino acids 1-284 of human uPAR) was expressed with an N-terminal histidine-tag insertion and purified by nickel chelate affinity chromatography. Soluble uPAR, synthesized by CHO cells (corresponding to amino acids 1-277 of human uPAR), was isolated by ligand (uPA) affinity chromatography. Expression in E. coli led to a nonglycosylated form of uPAR, whereas uPAR produced by CHO cells seemed to be glycosylated to a similar extent as the naturally occurring human form of uPAR (as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Prior to immunization the N-termini of the recombinant uPAR variants were determined by amino acid sequence analysis. Polyclonal antibodies were generated in chickens and purified from egg yolk. The reaction patterns of these antibodies were analyzed by Western blot analyses and flow cytofluorometry.

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase-type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single-chain proenzyme (pro-uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro-uPA. Enzymatic assays, SDS-PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro-uPA. As determined by N-terminal amino acid sequence analysis, activation of pro-uPA by cathepsin L is achieved by cleavage or the Lys158-lle159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147-5152) cleavage of pro-uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro-uPA of 1:2,000). Nevertheless, even at pH 7.0, pro-uPA was activated by cathepsin L, although a 10-fold higher concentration of cathepsin L was required. As tumor cells may produce both pro-uPA and cathepsin L, implications for the activation of tumor cell-derived pro-uPA by cathepsin L may be considered. Different pathways activation of pro-uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro-uPA; (ii) activation by serine proteases (plasmin, kallikrein. Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).

Carcinogenesis in the human colon is associated with a marked increase in the tissue content of the urokinase-type plasmmogen activator (u-PA). This study was performed to determine the type of cells responsible for the u-PA increase in carcinomas of the colon and in their precursor lesions, the adenomas, by immunohistological evaluation applying monoclonal antibody 3689 directed to the β-chain of u-PA. Normal intestinal mucosa (n= 17) showed hardly any staining of u-PA, but some lamina propria cells were faintly positive. Carcinomas (n= 17) and adenomas (n= 16) showed a considerable and comparable staining intensity of u-PA in neoplastic columnar epithelial cells, and this staining was found to be diffuse and cytoplasmic. In a majority of the neoplastic tissues the u-PA staining was found to be patchy and not related to known risk markers of malignancy such as dysplasia in the adenomas, or to prognostic determinants such as Dukes' classification or differentiation in the carcinomas. The observation of strong u-PA positive lamina propria cells in adenomas but infrequently observed in normal mucosa and carcinomas was noteworthy. u-PA staining intensity of the tissue sections was found to correlate well with the u-PA antigen level in the tissue extracts determined by ELISA (r= 0.52, P= 0.0001) but poorly with the u-PA activity determined enzymatically (r= 0.28, P= 0.05). In conclusion, the u-PA increase in neoplasia of the human colon can be attributed to an increased diffuse cytoplasmic content of u-PA in neoplastic columnar epithelial cells.

Proteolytic enzymes released from granulocytes upon stimulation with the chemotactic N-formyl peptide FNLPNTL (in the presence of cytochalasin B) prevented activation of tumor cell single-chain urokinase-type plasminogen activator (pro-uPA) by plasmin. Elastase was identified by the use of eglin C (elastase inhibitor) and a monoclonal antibody to elastase as the functional proteolytic enzyme in granulocyte supernatants. Action of purified granulocyte elastase on pro-uPA generated enzymatically inactive two-chain uPA linked by disulfide bridges which was indistinguishable by SDS-PAGE from plasmin-generated HMW-uPA. The major elastase cleavage site in pro-uPA was located between Ile159 and Ile160; a minor one between Thr165 and Thr166. Elastase cannot substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. However, when pro-uPA was first activated by plasmin to form enzymatically active HMW-uPA, this enzymatic activity was not impaired by subsequent elastase treatment.

Quantitative analysis of ligand-occupied receptor interactions with elements of the cytoskeleton and with intracellular compartments requires a sensitive and simple method of identifying the receptor-ligand complex in living cells. Toward this goal, we have prepared a photoactivatable arylazide derivative of the chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, which can be radiolabeled to high specific activity with 125I. This derivative was biologically active as judged by its ability to elicit superoxide anion production by human PMNL at nanomolar concentrations (ED50 approximately 0.7 nM). When incubated at 0 degree C with whole PMNL, radioactive ligand became specifically and saturably associated with a 60-70,000-dalton species (as assessed by SDS-PAGE) after exposure to UV light. Addition of 10-100-fold excess of unlabeled parent or unlabeled azidopeptide derivative completely blocked uptake into this species. Approximately 20-40% of the available surface receptor-binding sites were covalently labeled under these conditions. Subcellular fractionation of the labeled cells on sucrose gradients after homogenization showed that the labeled species was primarily associated with plasma membrane-rich fractions. The labeled receptor could be completely solubilized with Triton X-100 in a form which eluted as a single species with a Stoke's radius of less than 50 A on Sepharose 4B columns. In addition, the solubilized receptor-ligand complex bound specifically to wheat germ agglutinin, indicating that it is probably a glycoprotein. The ability to label the receptor in living PMNL with a high efficiency should facilitate the study of receptor dynamics and receptor physiochemical properties in this system.

Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4 degrees C or incubated at 37 degrees C for 2 min or less. Fractionation of cells incubated at 37 degrees C for longer times revealed that the radioactivity sedimented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37 degrees C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% +/- 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction cosedimenting with dense granules.