The soluble form of urokinase receptor promotes angiogenesis through its Ser88-Arg-Ser-Arg-Tyr9² chemotactic sequence.

BACKGROUND: The urokinase plasminogen activator receptor (u-PAR) focuses the proteolytic activity of the urokinase plasminogen activator (u-PA) on the endothelial cell surface, thus promoting angiogenesis in a protease-dependent manner. The u-PAR may exist in a glycophosphatidylinositol-anchored and in a soluble form (soluble u-PAR [Su-PAR]), both including the chemotactic Ser88 -Arg-Ser-Arg-Tyr9² internal sequence. OBJECTIVE: To investigate whether Su-PAR may trigger endothelial cell signaling leading to new vessel formation through its chemotactic Ser88 -Arg-Ser-Arg-Tyr9² sequence. METHODS AND RESULTS: In this study, the formation of vascular-like structures by human umbilical vein endothelial cells was assessed by using a matrigel basement membrane preparation. First, we found that Su-PAR protein promotes the formation of cord-like structures, and that this ability is retained by the isolated Ser(88) -Arg-Ser-Arg-Tyr9² chemotactic sequence, the maximal effect being reached at 10 nmol L⁻¹ SRSRY peptide (SRSRY). This effect is mediated by the α(v) ß3 vitronectin receptor, is independent of u-PA proteolytic activity, and involves the internalization of the G-protein-coupled formyl-peptide receptor in endothelial cells. Furthermore, exposure of human saphenous vein rings to Su-PAR or SRSRY leads to a remarkable degree of sprouting. Finally, we show that Su-PAR and SRSRY promote a marked response in angioreactors implanted into the dorsal flank of nude mice, retaining 91% and 66%, respectively, of the angiogenic response generated by a mixture of vascular endothelial growth factor and fibroblast growth factor type 2. CONCLUSIONS: Our results show a new protease-independent activity of Su-PAR that stimulates in vivo angiogenesis through its Ser88 -Arg-Ser-Arg-Tyr9² chemotactic sequence.

A conditioned medium from a human liposarcoma-derived cell line induces p53-dependent apoptosis in several tumor cell lines.

A novel cell line, named LSA, has been obtained, stabilized, and characterized from a human liposarcoma. These cells have morphological and biochemical features strongly resembling the adipocytes and were able to grow in the Ham's F12 medium, in presence or absence of FCS. A conditioned medium (LSA-CM) was obtained by growing the LSA cells in the F12 medium in the absence of FCS. LSA-CM had cytostatic and cytotoxic effects (apoptosis and necrosis) associated with down-regulation of c-myc and upregulation of p53 in several human cell lines (breast, lung, glioblastoma, etc. ). The MCF-7 and glioblastoma cells were killed by LSA-CM in 5-6 days, whereas the same cells were killed by LSA-CM co-incubated with low doses of cisplatin in 30 h. LSA-CM peri-tumoral injections for 15 days in Balb-c-fc3H mice affected by mammary tumors, resulted in the rapid disruption of tumors and absence of metastases. In contrast, in the untreated animals the tumor masses were 4 times larger than initial lesions, and numerous metastases were found in the lungs. The toxicity analysis of LSA-CM, performed on three different animal species, showed that LSA-CM is absolutely free of acute, subacute, and subchronic toxicity. The possible use of LSA-CM/cisplatin for cancer treatment is discussed.

Establishment and growth regulation of a novel ovarian cancer cell line from a poorly-differentiated adenocarcinoma: proposal for a new treatment.

A continuously growing cultured cell line has been obtained in vitro, starting from a specimen of ascites fluid obtained from a patient with ovarian cancer, in whom a poorly-differentiated adenocarcinoma was diagnosed. This cell line, named OC-A1, is routinely grown in standard, serum-supplemented culture medium and has been fully stabilized to long-term growth and characterized for both cultural and genetic parameters. OC-A1 cells express a set of characteristics, as determined in vitro which, when compared with the in vivo primary tumor, confirm the high malignity of this cancer. In addition, karyotype analysis showed a translocation of chromosome 8 which is correlated with the amplification of c-myc oncogene. However, the expression of this oncogene was found to be significantly inhibited by a new regulatory activity, recently found to be present in a liposarcoma cell line. Conditioned medium from these cells was indeed able to inhibit the growth of OC-A1 cells, arresting their cell cycle in the G1 phase and inducing them to apoptosis. Finally, the cell programmed death appeared to be related to the expression of antioncogene p53.

In vivo paracrine interaction between urokinase and its receptor: effect on tumor cell invasion.

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.

Complementation between urokinase-producing and receptor-producing cells in extracellular matrix degradation.

The respective roles of urokinase plasminogen activator (u-PA) and the u-PA receptor in extracellular matrix degradation was investigated. Human pro-u-PA and the human u-PA receptor were expressed independently by two different mouse LB6 cell lines. The matrix degradation capacity of these cell lines individually or in coculture was studied. Although pro-u-PA-producing cells alone degrade the matrix in the presence of plasminogen, u-PA-receptor producing cells do not. Cocultivation of a small fraction of pro-u-PA-producing cells with the receptor-producing cells increases the rate of matrix degradation at least threefold. By immunoprecipitation it was shown that cocultivation of the two cell lines increases the conversion of the inactive pro-u-PA to the active two chain u-PA. The enhancement of matrix degradation and of pro-u-PA activation requires actual binding of pro-u-PA to its receptor because it is inhibited by u-PA-receptor antagonists. The u-PA receptor must be cell associated, as binding of pro-u-PA to a receptor solubilized from the cell surface with phosphatidyl-inositol specific phospholipase C did not enhance the activation of pro-u-PA in the presence of plasminogen. The finding that activity of u-PA is enhanced when it is bound to its receptor, even when the receptor is produced by a different cell, might have important implications for the mechanisms of u-PA-induced extracellular proteolysis in vivo.

A soluble, ligand binding mutant of the human urokinase plasminogen activator receptor.

A truncated version of the human urokinase plasminogen activator receptor has been obtained by in vitro mutagenesis by insertion of a premature nonsense codon in the urokinase plasminogen activator receptor cDNA. This results in a protein truncated immediately upstream of the region which appears to be required for membrane attachment of the receptor via a glycolipid anchor. The modified receptor cDNA inserted into an expression vector has been transfected into mouse LB6 cells. Transfectants produce a urokinase plasminogen activator (u-PA)-binding protein that is secreted into the medium. It can be cross-linked to iodinated ATF (amino-terminal fragment of u-PA) and can also inhibit binding of iodinated ATF to mouse LB6 cells that express the wild type human receptor. The soluble u-PA receptor will be used in a variety of experiments aimed at identifying the role and mechanism of u-PA in physiological and pathological invasive processes, as well as in therapeutical attempts to block or decrease cancer cell invasion and in general u-PA-mediated tissue destruction.

Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis.

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.

Purification to homogeneity of the major "4S" PAH binding protein from "non responsive" DBA/2N mouse liver by affinity chromatography.

DBA/2N is a genetically non responsive inbred strain of mice in which administration of polycyclic aromatic hydrocarbons (PAHs) does not induce microsomal monooxygenase activity. DBA/2N mouse liver cytosol contains a polycyclic aromatic hydrocarbon-binding protein that sediments, in a sucrose gradient, at 4S ("4S" PAH-BP). Its binding kinetic and physicochemical properties indicate that this protein is practically indistinguishable from the "4S" PAH-BP identified and characterized in liver cytosol of rats and other PAH responsive rodents including C57 B1/6J mice. "4S" PAH-BP was purified to homogeneity from DBA/2N mouse liver by ammonium sulfate fractionation of the cytosol, followed by Sephadex G-200 chromatography and, finally, affinity chromatography using 1-aminopyrene-Sepharose 6B. This procedure yielded about 50 micrograms of protein from 50-60 g of mouse liver, with a recovery of 18%. "4S" PAH-BP as a complex with 3H-(benzo-a-pyrene) was more than 99% pure. A single band was seen on polyacrylamide gel electrophoresis under non denaturing conditions. H-BaP comigrated with the protein band. 3H-BaP bound to the protein was displaced by PAHs with a specificity identical to that obtained using crude cytosol. On electrophoresis in SDS gels, the purified protein migrated as a single protein band with an apparent molecular weight of 40,000.

"4S" polycyclic aromatic hydrocarbon binding protein. Its role as a benzo(a) pyrene cytosolic carrier to the microsomes of DBA/2N mouse liver.

Rodent liver cytosol and other biological systems contain two proteins that bind polycyclic aromatic hydrocarbons (PAH) in a non covalent manner and that sediment at a different rate when centrifuged on sucrose gradient. The role of the smaller protein ("4S" PAH-BP) was studied. When DBA/2N mouse liver homogenate was incubated with 3H-BaP, most of the radioactivity was found in the microsomal subcellular fraction. The cytosol binding activity apparently decreased but reincubation of the cytosol with the radioactive ligand completely restored "4S" PAH-BP activity. The microsomal uptake of 3H-BaP can be studied in a reconstituted system in which microsomes are incubated with radioactive benzo(a)pyrene in the presence of crude cytosol. In these conditions the microsomal uptake rate of 3H-BaP increased with the temperature and at 37 degrees C ten minutes were required to reach the plateau. When cytosol was substituted by HEDG buffer, the amount of radioactivity found in the microsomes decreased drastically. 0.2 microM was the benzo(a)pyrene concentration required to saturate the microsomes. When microsomes were incubated with ammonium sulfate cytosolic fractions or with homogeneously purified "4S" PAH-BP, the 3H-BaP uptake was restored and reached the maximum with 3 micrograms/ml of purified protein. The radioactive benzo(a)pyrene bound to microsomes was oxidated in the presence of NADPH regenerating system. The oxidated products were discharged from microsomes only when "4S" PAH-BP was either present during the incubation or added at its end. Thus, this protein is able to transfer benzo(a)pyrene to the microsomal metabolization sites and to facilitate the release of oxidized products and, presumably, bind them.

"4 S" polycyclic aromatic hydrocarbon binding protein: further characterization and kinetic properties.

A protein that binds polycyclic aromatic hydrocarbons (PAHs) with high affinity and sediments in a sucrose gradient at 4 S has been described in rat liver cytosol. This "4 S" PAH binding protein precipitates at a 40-60% ammonium sulfate saturation. This partial purification procedure allows assay of this protein by using purified 3H-benzo(a)pyrene (3H-BaP) as radioactive ligand and dextran-coated charcoal as adsorbent for unreacted 3H-BaP. The 3H-BaP binding activity measured as a function of pH shows its maximum activity between pH 7.3 and 10.5. The "4 S" PAH binding protein is stable up to 42 degrees C even in the absence of the ligand. At 65 degrees C the binding sites for 3H-BaP are destroyed. The binding activity assayed as a function of protein concentration is linear between 0.4 and 2 mg/ml at 0 degrees C, whereas at 37 degrees C higher protein concentrations (4 mg/ml) can be reached. Exposure to guanidine X HCl (3 M) and urea (5 M) for 20 min at 4 degrees C inhibits the PAH binding completely to the "4 S" protein. Quick dilution or dialysis does not restore the binding activity. The dissociation rate of the "4 S" PAH binding protein measured in the presence of an excess of unlabeled ligand at 0 degrees C is biphasic and shows a two-step, first-order kinetic pattern. At 37 degrees C the dissociation rate is linear and faster, and is complete after 5 min of incubation. The association rate shows the same behavior: the binding is complete after 10 min at 0 degrees C, whereas at 37 degrees C the reaction is 10 times as fast. The dissociation equilibrium constants at 0 degrees C and 37 degrees C are respectively 2.45 X 10(-9) M and 1.09 X 10(-9) M. The high rates of association and dissociation of BaP to "4 S" PAH binding protein were used to set up an assay to exchange radioactive 3H-BaP with cold BaP.

Polycyclic aromatic hydrocarbon binding macromolecules. Identification, characterization and temperature activation of a 4.5 S binding nucleoprotein.

A macromolecule binding 3H-methylcholanthrene (3H-MCA) and 3H-benzo(a)pyrene (3H-BaP) and sedimenting in the 4-5 S region of sucrose gradient (4.5 S) was identified in rat liver cytosol. The binding was displaced by 100-fold molar excess unlabeled ligands whereas 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was ineffective. The dissociation constant for both polycyclic aromatic hydrocarbons (PAHs) was of the order of 10(-8) M or lower. Both 3H-MCA and 3H-BaP bound to 4.5 S in a non covalent manner, since 92% of the bound radioactivity was extractable with ethyl ether. Furthermore the binding was strongly reduced by urea 8 M and by guanidine. HCl 4 M (99 and 70% respectively). Thin layer chromatography of the ethyl ether-solubilized radioactivity showed a peak comigrating with PAHs used as standards. When chromatographed on Sephadex G-200, 4.5 S was eluted as a sharp peak with an apparent molecular weight of 50-60,000 daltons. Enzyme treatment of liver cytosol showed that the 4.5 S binding sites were destroyed by micrococcal nuclease (92% of inhibition). Papain and phosphodiesterase I and II reduced the binding to 50%, whereas DNase I, DNase II, RNase, phospholipase A2 and C and trypsin were ineffective. These data suggest that the PAHs binding macromolecule of rat liver cytosol is a protein associated with a polynucleotide. The binding of both PAHs was enhanced by increasing the incubation temperature, the maximum being reached after 20-30 min at 37 degrees C. After 2.5 min at 65 degrees C, binding sites were completely destroyed. The same temperature-induced "activation" was obtained also by prewarming the cytosol at 37 degrees C in the absence of ligands.

Interaction of rat liver glucocorticoid receptor with heparin.

When rat liver cytosol containing [3H]dexamethasone-glucocorticoid receptor complex is exposed to immobilized heparin (Sepharose-heparin; Seph-hep) the steroid receptor complex binds to the substituted Sepharose avidly [Kd = 3.5 (+/- 1.7) X 10(-10) M], and 80-90% of the receptor present is adsorbed to the solid phase after 40 min at 0 degree C. The binding is enhanced by Mn2+ (10 mM) and Mg2+, whereas Ca2+ and Sr2+ are ineffective. Sodium molybdate (10 mM) does not influence the reaction but enhances receptor stability. Moreover, binding of the receptor to Seph-hep is dependent on the ionic strength of the medium, because binding is totally reversed by 300 mM KCl. The bound [3H]dexamethasone-receptor complex can be recovered from Seph-hep with solutions (4 mg/mL) of heparin (95% release), dextran sulfate (88%), and chondroitin sulfate (63%); total calf liver RNA is less effective (9%), whereas dextran, D-glucosamine, N-acetyl-D-glucosamine, D-glucuronic acid, and sheared calf thymus DNA are totally ineffective (less than 3%). Both "native" and temperature "transformed" forms of the glucocorticoid receptor interact with immobilized heparin. These results strongly suggest that the receptor site that binds heparin is distinct from that binding DNA. An immediate application of this newly found ability of the glucocorticoid receptor to interact with heparin is the use of Seph-hep for affinity chromatography purification of the glucocorticoid receptor. A purification of 10-fold, with a recovery of 55-65%, can be achieved by using either 4 mg/mL heparin or 300 mM KCl to elute [3H]dexamethasone-receptor bound to the resin.

Estradiol and progesterone receptors in malignant gastrointestinal tumors.

Estradiol and progesterone receptors were assayed in tumors from 79 patients with primary colorectal and 56 patients with stomach adenocarcinomas. Eighteen of 79 colorectal cancers contained estradiol receptor, while 34 specimens were positive for progesterone receptor. In stomach cancer, the positive samples were 8 for estradiol and 14 for progesterone receptors. In both types of tumors, the Kd was in the range of 10(-10) M for estradiol and 10(-9) M for progesterone receptor, respectively. In colorectal adenocarcinomas, the presence of progesterone receptor seems to be partially correlated to the presence of estradiol receptor while, in stomach tumors, this correlation is lost. The positivity of at least one receptor in colorectal cancers is higher in the female sex. The contrary occurs for stomach cancer. Sucrose gradient centrifugation showed that cytoplasmic estradiol receptor of stomach cancer sedimented at 8S or 4 to 5S at low ionic strength. The isoelectric point of stomach cancer estradiol receptor is 6.5.