Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody

The human 66 000 mol. wt. plasminogen activator (HPA66; tissue-type plasminogen activator) has been purified from melanoma cells by a one-step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one-polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one-chain form was converted to the two-chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two-chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two-chain form, while the one-chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two-chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase-type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one-chain proenzymes which are converted to active two-chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.     

Plasminogen activating enzyme in cultured glioblastoma cells. An immunofluorescence study with monoclonal antibody

A monoclonal antibody against a 52,000 dalton human plasminogen activating enzyme (HPA52) was used for immunofluorescence staining of cultured glioblastoma cells. The fluorescence was located in the cytoplasm of the cells. A pronounced variation in the staining intensity was observed between the individual cells. The specificity of the fluorescent stain was supported by the findings that 1) no staining was obtained with a monoclonal antibody of the same subclass, but with irrelevant specificity (anti-2,4,6-trinitrophenyl); 2) adsorption with HPA52 purified to homogeneity removed the ability of anti-HPA52 to mediate staining; 3) the glioblastoma cells contained HPA52, as measured by enzymatic assay, while melanoma cells that were not stained did not contain HPA52 activity; 4) dexamethasone reduced both the enzymatically determined HPA52 content and the immunofluorescence in parallel, while progesterone affected none of these parameters; 5) we have previously found that culture fluid conditioned by the glioblastoma cells apart from HPA52 does not contain detectable amounts of any protein that binds to anti-HPA52. Several advantages of immunohistochemical detection of plasminogen activators compared with enzyme histochemical methods are discussed, among these that the immunohistochemical method distinguishes between plasminogen activators of different types.     

Monoclonal antibodies to the angiotensin-converting enzyme from the human lung

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunised with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by ELISA; that of soluble ACE--by immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not effect ACE activity. This antibody is believed useful not only for immunoassay and immunopurification of ACE, but also as a tool for investigation of enzyme distribution in tissue as well as for studying the structure and mechanism of ACE action.     

Monoclonal antibodies to endogenous galactose-specific tumor cell lectins.

A monoclonal antibody, 5D7, was obtained after immunization of syngeneic mice with B16 melanoma cell extracts enriched for endogenous lectin activity and screening for inhibition of lectin-mediated hemagglutination. Binding of this antibody to affinity-purified B16 melanoma galactoside-specific lectin was revealed by solid-phase radioimmunoassay and binding to the surface of viable B16 cells was demonstrated by indirect immunofluorescence. Inhibition of lectin activity and cell surface labeling by 5D7 antibody were also found with several types of cultured human and murine cells including melanoma, sarcoma and carcinoma. This monoclonal antibody should be useful for evaluating the role of tumor cell surface lectins in intercellular interactions and metastasis.     

Monoclonal antibody to human lung angiotensin-converting enzyme

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunized with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by enzyme-linked immunosorbent assay and that of soluble ACE by an immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not affect ACE activity. We believe this antibody will be useful not only for immunoassay and immunopurification of ACE, but also as a tool for the investigation of the tissue distribution of the enzyme as well as for the study of the structure and mechanism of action of ACE.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: A key event in melanoma cell invasiveness in vitro

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on Me Wo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Urokinase-type and tissue-type plasminogen activators are essential for in vitro invasion of human melanoma cells

This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells.     

Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.     

Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor from the conditioned media of a human melanoma cell line

24 established melanoma cell cultures were screened for their secretion of plasminogen activators and plasminogen activator inhibitors into the culture medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by conventional and reverse fibrin autography. Among the cell lines investigated, 22 cell lines predominantly secreting tissue type plasminogen activator (t-PA) and four cell lines additionally secreting urokinase were found. The conditioned media of two cell lines (KRFM and MJZJ) were found to contain plasminogen activator inhibitor (PAI) activity at a Mr position of approximately 50,000. The PAI of one of the two melanoma cell (MJZJ)-conditioned media found to contain PAI activity was purified to apparent homogeneity employing concanavalin A-Sepharose chromatography, gel filtration on Sephadex G-150, chromatography on Affi-Gel blue, and affinity chromatography on a Sepharose 4B immobilized monoclonal anti-t-PA IgG column. The purified melanoma PAI was found to be a single chain protein, acid stable, immunologically related to the endothelial derived PAI. In contrast to endothelial PAI, melanoma PAI presented itself in the conditioned media of the melanoma cells and in the purified preparation to an appreciable extent in its active form.     

Isolation and purification of a substance immunologically cross-reactive with insulin (SICRI) from tumor tissue

1. A substance immunologically cross-reactive with insulin (SICRI) was isolated and purified from murine melanoma B16 and myeloid leukemia. 2. Monospecific anti-insulin immunoglobulin G was coupled with CNBr-activated Sepharose 4B to yield an adsorbent. The immunoaffinity column was used to isolate SICRI from tumor tissue. 3. Purified SICRI yielded a single band with mol. wt 158,000 on SDS-PAGE. After non-denaturing conditions SICRI appeared again in one single peak. 4. Affinity purified SICRI was shown to be a potent growth factor for different human and murine transformed and normal cells. 5. Biochemical and biological data provide evidence that SICRI and insulin are two distinct biologically active agents.     

Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with a lower apparent molecular mass

Purified approximately 54 kDa plasminogen activator inhibitor from human fibrosarcoma cells was converted to an inactive form with slightly higher electrophoretic mobility by incubation with catalytic amounts of urokinase-type or tissue-type plasminogen activator. Serine proteinase inhibitors and a monoclonal antibody against urokinase-type plasminogen activator inhibited the conversion, indicating that it was caused by plasminogen activator-catalyzed proteolysis. These findings represent the first demonstration of a well-defined protein apart from plasminogen, constituting a substrate for plasminogen activators.     

Association between allo-immunoreactive and xeno-immunoreactive subunits of a glycoprotein tumor-associated antigen

Summary Using allogeneic antibody, we previously described a tumor-associated antigen (TAA) in the urine of 68% of melanoma patients. The TAA was purified from urine of a melanoma patient and used as immunogen to develop a murine monoclonal antibody (AD1-40F4) and xenopolyclonal antibodies in a baboon. Sera from melanoma patients treated with whole melanoma cell vaccine were used as the source of human antibody to the glycoprotein antigen. Treatment with 2-mercaptoethanol and separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis resolved the high-molecular-mass glycoprotein TAA into smaller subunits. Immunoblot analysis indicates that the murine monoclonal antibody (AD1-40F4) recognized a 90–100-kDa subunit of the antigen while human anti-TAA antibodies primarily recognized a 65-kDa subunit in addition to the 90–100-kDa subunit. Baboon polyclonal antibodies recognized the same subunits plus a 120-kDa subunit. Blocking studies indicated that the murine monoclonal and baboon polyclonal antibodies recognized the closely related epitopes on the 90–100-kDa subunit, while human antibodies recognized an epitope entirely distinct from that recognized by the mouse antibody. These results demonstrate the epitope complexity associated with the high-molecular-mass glycoprotein TAA.     

湛江沿海潮间带产胞外纤溶活性酶类海洋真菌的生物多样性分析

【目的】研究湛江沿海硇洲岛和徐闻珊瑚礁自然保护区潮间带产胞外纤溶酶样酶和纤溶酶原激活物海洋真菌的生物多样性,为发掘新型溶栓药物奠定基础。【方法】采用马铃薯葡萄糖琼脂(PDA)和酵母膏蛋白胨葡萄糖(YPD)培养基分离培养海洋真菌,采用真菌r DNA转录间隔区1-5.8S r DNA-转录间隔区2(ITS1-5.8S-ITS2)片段的序列分析及其系统进化树构建的方法鉴定分离培养的海洋真菌,采用脱脂牛奶马铃薯葡萄糖琼脂(SM-PDA)培养基培养法筛选产胞外蛋白酶的海洋真菌,采用海水纤维蛋白马铃薯葡萄糖琼脂(FN-PDA)培养基培养法筛选产胞外纤溶酶样酶和/或纤溶酶原激活物的海洋真菌。【结果】从湛江沿海的硇洲岛和徐闻珊瑚礁自然保护区潮间带分离、培养和鉴定了海洋真菌446株,含真菌的98个种,分布于真菌域子囊菌门(Ascomycota)和担子菌门(Basidiomycota)的6个纲、18个目、46个科、65个属;其中产胞外蛋白酶的海洋真菌有265株,61个种,分布于41个属;产胞外纤溶酶样酶的海洋真菌有67株,22个种,分布于14个属;产胞外纤溶酶原激活物的海洋真菌有84株,23种,分布于13个属;优势属为曲霉属(Aspergillus),其次为青霉属(Penicillium)。【结论】湛江沿海潮间带可分离培养的产胞外纤溶酶样酶和纤溶酶原激活物的海洋真菌物种丰富多样,是发掘新型溶栓药的丰富资源。     

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.     

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.     

A colorimetric assay for the simultaneous measurement of plasminogen activators and plasminogen activator inhibitors in serum-free conditioned media from cultured cells

The coupled photometric assay for plasminogen activator reported by Coleman and Green (1981) Methods in Enzymology (Lorand, L., Ed.), Vol. 80, pp. 408-414, Academic Press, San Diego, CA) has been adapted for use with 96-well plates and an automatic microplates spectrophotometer. The assay allows the discrimination between tissue-type and urokinase-type plasminogen activators in cell culture-conditioned media. It provides a level of detection of these enzymes in the range 10(-17) to 10(-13) mol (determined using purified human plasminogen activators), uses no radioisotopes, and is faster and more economical than similar assays using specific peptide substrates for plasminogen activators. Levels of free plasminogen activator inhibitor activity can be simultaneously measured on the same samples by a simple adaptation of the assay. This method allows an easy treatment of the data by interfacing with a computer and should thus be useful when large numbers of samples are assayed.     

Cell binding and tumor inhibiting functions of a new antihuman melanoma murine monoclonal antibody.

Murine, antihuman melanoma cell monoclonal antibody (mAb) 16.C8 was generated by fusing the murine myeloma cell line P3X63/Ag8.653 with splenocytes from a nude mouse bearing a human melanoma xenograft, after reconstitution with splenocytes from syngeneic immunocompetent BALB/c mice. The antibody reacted strongly with fresh human melanoma cells and exhibited preferential reactivity with established human melanoma and neuroectodermal tumor cell lines. Electrophoresis and Western blotting experiments indicated that 16.C8 is directed against a sialoglycoprotein antigen with a molecular weight of 110-120 kDa. mAb 16.C8 mediated lysis of melanoma cells in vitro in antibody-dependent cellular cytotoxicity assays using human mononuclear effector cells isolated from normal volunteers or malignant melanoma patients. In addition, the administration of mAb 16.C8 to nude mice bearing established human melanoma lung and liver metastases resulted in significant inhibition of tumor growth as shown by gross and histologic examination. In contrast, animals treated with Hanks' balanced salt solution or nonspecific immunoglobulin exhibited a large tumor burden. These results suggest that mAb 16.C8 may be of value in treatment of metastatic melanoma in humans.     

Novel properties of human monocyte plasminogen activator

Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing supernatants of human lymphocytes, could be shown to produce and secrete appreciable activities of a 52 000-Mr plasminogen activator. This enzyme was suppressed in control and stimulated cultures by dexamethasone (0.1 microM). Monocyte plasminogen activator could only be assayed under conditions of low ionic strength and had no detectable activity at 0.15 M NaCl. Intracellular enzyme was present as a proenzyme, requiring activation by preincubation with plasminogen containing traces of plasmin, before its activity could be seen on sodium dodecyl sulphate/polyacrylamide gel electrophoresis by a fibrin overlay method. Secreted enzyme was in the active form. Further incubation of lysate or supernatant plasminogen activator with plasminogen did not produce any active enzyme species of Mr 36 000, unlike incubations of urokinase with plasminogen. Moreover, comparisons with other plasminogen activators of Mr 52 000 from transformed cell lines showed that the monocyte activator was unique in its resistance to monocyte minactivin, a specific inactivator of urokinase-type plasminogen activators, and in its sensitivity to human alpha 2-macroglobulin. It was therefore concluded that human monocyte plasminogen activator, although sharing an Mr of 52 000 in common with other such activators, is not identical to the high Mr form of urokinase or the plasminogen activators of transformed cells. On present evidence it is the least likely of these enzymes to be active extracellularly under normal physiological conditions.     

抗t-PA单克隆抗体的制备及初步应用

用猪心t-PA和人黑色素瘤细胞分泌的t-PA作抗原,经腹腔及脾内免疫Bal b/c小鼠。用细胞融合技术制备杂交瘤细胞,细胞融合串达85％,获得4株抗t-PA杂交瘤细胞株,鼠腹水抗体效价达1∶10~5;Western Blot结果表明该单抗所结合的抗原分子量与t-PA相符,证明所获得的单抗为特异的抗t-PA单抗;对t-PA的纤溶活性中和抑制试验结果表明,4株单抗中的1株能完全抑制st-PA的纤溶活性,另外3株表现出程度不等的抑制;其中1株亚类为IgG,另外3株为IgM。杂交瘤细胞株无支原体污染;染色体数目正常。对该抗体进行初步纯化后,将其应用于rt-PA的研究中。