Malignant mouse melanoma cells do not form tumors when mixed with cells of a non-malignant subclone: relationships between plasminogen activator expression by the tumor cells and the host's immune response.

Mouse melanoma clones B559 and B78 are highly tumorigenic when injected into C57BL/6J mice. Tmor formation by these cells is suppressed when they are mixed with nonmalignant bromodeoxyuridine-grown clone C3471 before injection. C3471 cells suppress tumor formation only in immunocompetent hosts; mixtures of B559 and C3471 cells or C3471 cells alone form tumors in antithymocyte serum (ATS)-treated mice. Explants of C3471 tumors grown in ATS-treated mice form tumors in immunocompetent mice, most of which regress. Inability of C3471 or mixtures of C3471 with malignant cells to grow in normal mice, as contrasted with ability to grow in immunosuppressed mice, indicates that host response is involved. Both tumorigenic clones have high plasminogen activator activity, whereas nontumorigenic clone C3471 has none. Mixture of either tumorigenic clone with C3471 cells decreases plasminogen activator in vitro. C3471 tumor explants from ATS-treated mice initially express plasminogen activator, but lose the capacity to express this activity upon prolonged cultivation in vitro. Explants from B559 tumors retain plasminogen activator in long term culture. Close physical contact between C3471 and B559 cells appears essential both for inhibiton of plasminogen activator expression by B559 cells in vitro, and for tumor suppression in vivo. These findings suggest that production of plasminogen activators by tumor cells may play an important role in suppressing the host's immune response locally to an inoculum of syngeneic tumor cells.     

Human tissue-type plasminogen activator synthesized by using a baculovirus vector in insect cells compared with human plasminogen activator produced in mouse cells

A cDNA fragment encoding the human tissue-type plasminogen activator was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream from the polyhedrin promoter. The induction kinetics of t-PA was followed, after infection of Spodoptera frugiperda cells, at both mRNA and protein levels. Fibrinolytically active plasminogen activator accumulated in the culture medium and reached 2.5 micrograms/ml after 120 h. The protein was compared with recombinant plasminogen activator produced in mouse cells and was found to be slightly smaller. This difference in size was found to be caused by N-linked oligosaccharides which are shorter in the recombinant activator obtained from insect cells. The molecules produced in such cells contain at least two different types of N-linked glycans, since only one out of three oligosaccharides is sensitive to endoglycosidase H. However, all glycan structures bind strongly to concanavalin A-Sepharose.     

Modulation of cell-associated plasminogen activator activity by cocultivation of a stem cell and its tumorigenic descendant.

The effect of the presence of one cell type on the plasminogen activator activity of another cell type was studied. The cell types, AC and D, were isolated from a rat neuroblastoma (I. Imada and N. Sueoka, Dev. Biol. 66:97-108, 1978). AC cells are stem cells capable of multipotential differentiation in vitro and have little or no cell-associated plasminogen activator activity. D cells are tumorigenic and have high levels of cell-associated plasminogen activator activity. When AC cells were cocultivated with D cells, the plasminogen activator activity of the D cells was dramatically inhibited. The presence of as few as 1,250 AC cells inhibited 70% of the plasminogen activator activity of 20,000 D cells, as determined by a highly quantitative assay. The amount of inhibition by AC cells was proportional to the number of AC cells present. At increasing numbers of AC cells and a constant number of D cells, the Vmax for the activation of plasminogen proportionately decreased and the Km remained constant, implying that AC cells did not alter the structure or concentration of plasminogen. Inhibition was not mediated by a soluble inhibitor secreted by AC cells. Rather, attachment of AC cells adjacent to D cells, i.e., cell-to-cell contact, seemed to be required for inhibition. The substratum-attached material of AC cells, that which remained on the microwell surface after removal of AC cells with EDTA, inhibited D cell plasminogen activator activity. If plasminogen activator activity is involved in metastasis, then regulation of the plasminogen activator activity of one cell type by another cell type may be involved in determining which cells in a tumor can metastasize and where secondary tumors can arise.     

Inhibition of a plasminogen activator from oncogenic virus-transformed mouse cells by rabbit antibodies against the enzyme

Antisera were raised in rabbits against an electrophoretically pure 48 000 dalton plasminogen activator from mouse cells transformed by an oncogenic virus. The IgG fraction of the antisera inhibited 48 000 dalton mouse plasminogen activators from a variety of sources (neoplastic and nonneoplastic), a 29 00) dalton plasminogen activator from mouse urine and a 48 000 dalton plasminogen activator from rat urine. No inhibition was observed of a 75 000 dalton plasminogen activator extracted from mouse lung, of mouse plasmin or of plasminogen activators from human urine and from oncogenic-virus transformed chicken cells. The IgG antibodies were stronger and more specific inhibitors of the 48 000 dalton mouse plasminogen activator than any previously tested compounds.     

Proteolytic activation of tissue-type plasminogen activator by the culture media of mouse cancer cells

Human single-chain tissue-type plasminogen activator (tPA) was activated by the culture media of mouse B16 melanoma and Lewis lung carcinoma cells. This activation was due to the proteolytic conversion of single-chain tPA to two-chain tPA. Typical serine proteinase inhibitors, such as diisopropylfluorophosphate and aprotinin, strongly inhibited the proteolytic activation, suggesting that the enyzme responsible for this activation is a serine proteinase. Through a process of gel filtration, the molecular weight of the putative tPA-activating enzyme was estimated to be approximately 35 kDa. Expression of the tPA mRNA was demonstrated by Northern blot analysis both for the melanoma and carcinoma cells. Zymographic experiments showed that the culture medium of the melanoma cells contained active two-chain tPA. These results indicate that a common serine proteinase may be involved in the proteolytic activation of single-chain tPA in these cancer cells.     

TGF-beta inhibits human cutaneous melanoma cell migration and invasion through regulation of the plasminogen activator system

Over the past decades, the incidence of cutaneous melanoma in developed countries has increased faster than any other cancer. Although most patients have localized disease at the time of diagnosis and are cured by surgical excision of the primary tumor, melanoma can be highly malignant and the survival dramatically decreases for advanced stage melanomas. It is thus necessary to understand the progression of this disease. Cell migration and invasion promote tumor metastasis, the major cause of melanoma cancer morbidity and death. In this study, we investigated the role of the TGFβ/Smad signaling pathway in melanoma tumor progression and found TGFβ to potently inhibit both cell migration and invasion in human melanoma cell lines, established from different patients. Furthermore, we elucidated the molecular mechanisms by which TGFβ exerts its effects and found the plasminogen activation system (PAS) to play a central role in the regulation of these effects. We found TGFβ to strongly up-regulate the Plasminogen Activator Inhibitor-1 (PAI-1) in melanoma cells, leading to reduced plasmin generation and activity and, in turn to inhibition of cell migration and invasion. Together, our results define TGFβ as a potent suppressor of tumor progression in cutaneous melanoma, inhibiting both cell migration and invasion.     

Responsiveness of tumorigenic and non-tumorigenic CHEF18 Chinese hamster cells to 1-beta-D-arabinofuranosylcytosine treatment.

In cultured mammalian cells, sister chromatid exchanges are easily induced by agents that perturb the scheduled timing of DNA replication. In this work a blockage of DNA synthesis induced by 1-beta-D-arabinofuranosylcytosine was applied to non-tumorigenic and tumorigenic CHEF18 Chinese hamster cells, and their responsiveness was compared. The data show that both the induction of sister chromatid exchanges and the reduction of the colony-forming ability were less extensive in non-tumorigenic than in tumorigenic CHEF18 cells. The results suggest that a tight control of the scheduled timing of DNA replication is present in non-tumorigenic CHEF18 cells and perhaps this feature avoids the generation of those chromosomal structures that are responsible for the abnormal induction of sister chromatid exchanges and for the elevated cytotoxicity seen in tumorigenic cells.     

Polarized secretion of urokinase-type plasminogen activator by epithelial cells.

Numerous epithelial cell types produce and secrete plasminogen activators (PAs) and/or PA inhibitors (PAIs). When epithelial cells were grown on polycarbonate filters and their apical and basolateral secretion products analyzed, PA activity accumulated in a highly polarized fashion; depending upon the cell line, the compartment of PA accumulation was either apical (MDCK I cells and HBL-100 cells) or basolateral (LLC-PK1, CaCo-2, and HeLa cells). By contrast, PAI-1 was recovered in roughly equal amounts in both compartments. Basolateral accumulation of urokinase-type plasminogen activator (uPA), but not its apical targeting, required an acidic compartment and the integrity of the cytoskeleton. Polarity of uPA accumulation did not result from removal of the free enzyme from the opposite compartment through its binding to the cell surface. Transfection with wild-type or mutated murine uPA demonstrated that neither the "growth factor" domain nor the kringle domain is required for the appropriate sorting of the protein. We propose that polarized secretion of PAs is one mechanism whereby cells spatially control extracellular proteolysis.     

Modulation of urokinase plasminogen activator gene expression during the transition from quiescent to proliferative state in normal mouse cells.

We have investigated the regulation of urokinase (u-PA) mRNA in quiescent mouse fibroblasts and keratinocytes stimulated to divide by the addition of serum or epidermal growth factor (EGF), respectively. Serum stimulation of quiescent fibroblasts (BALB/c 3T3 or Swiss 3T3) results in an early and transient increase of u-PA mRNA level, which precedes by several hours the onset of DNA synthesis. A similar response is elicited by EGF stimulation of quiescent keratinocytes. The increase of u-PA mRNA parallels that of c-myc mRNA, does not require protein synthesis and is at least in part due to increase in template activity of the u-PA gene. Induction of terminal differentiation of mouse keratinocytes results in a decrease of u-PA mRNA which parallels the decrease of thymidine incorporation. In conclusion, variation in the level of u-PA mRNA is seen during G0/G1 transition and correlates with the proliferative state of these normal mouse cells.     

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.     

Inhibition of urokinase-type plasminogen activator receptor induces apoptosis in melanoma cells by activation of p53

The urokinase-type plasminogen activator receptor (uPAR) is involved in several biological processes, including proteolysis, adhesion, migration and inflammation. Increased expression of uPAR is associated with metastasis in several tumor types. We studied the biological role of uPAR in melanoma and found that inhibition of uPAR via RNA interference induced massive death in three different metastatic cell lines. Annexin-V staining and caspase activation analysis revealed induction of the mitochondrial apoptotic pathway. The expression of members of the Bcl-2 family (Bax, Bcl-2, Bak and Bcl-x(L)) was changed in a pro-apoptotic manner. uPAR inhibition induced the expression of the tumor suppressor p53 and of its downstream target gene p21. Inhibition of p53 rescued cells from apoptosis indicating that p53 was critical for apoptosis induction. Apoptosis was observed in melanoma cells carrying activating BRAF mutations and occurred in the presence of extracellular signal-regulated kinase (ERK) phosphorylation. uPAR can activate focal adhesion kinase (FAK), which is implicated in adhesion-dependent tumor cell survival. However, inhibition of FAK did not induce apoptosis. Our data suggest a new function of uPAR acting as a survival factor for melanoma by downregulating p53. Inhibition of uPAR induces a pro-apoptotic signalling pathway via p53 that is independent of ERK or FAK signalling. These findings may offer new treatment strategies for metastatic melanoma.     

High level expression of human tissue-type plasminogen activator gene in mouse C127 cell.

We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.     

Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.     

Production of plasminogen activator by migrating cephalic neural crest cells.

Neural crest cells migrate extensively during embryonic development and differentiate into a wide variety of cell types. Our working hypothesis is that during migration, embryonic cells secrete proteases which modify local microenvironments, thereby facilitating directed cellular movements. In this communication, we report studies on the migration of cephalic neural crest cells in the avian embryo. We demonstrate that these cells produce high levels of the serine protease, plasminogen activator (PA), at the time of their initial migration from the neural tube and during their migration to and colonization of the developing head and neck.     

Tumorigenicity of herpesvirus-transformed cells correlates with production of plasminogen activator.

Our studies first demonstrated that established hamster cell lines transformed in vitro by herpesviruses activate plasminogen more effectively than normal hamster fibroblasts. This ability is probably due to increased levels of the enzyme plasminogen activator (PA). In the studies described here, the 333-8-9 cell line, originally transformed by herpes simplex virus type 2 strain 333, was used to derive subclonal lines that maintained stable PA phenotypes over the course of long in vitro passage. We were interested in correlating tumor formation by the subclones with their fibrinolytic capacity. Cells were, therefore, single-cell subcloned twice, and resulting cultures were tested for ability to activate plasminogen in vitro. PA activity was then quantitated by [125I]fibrin lysis assay, and high- and low-activity subclones were isolated; these retained high- or low-activity phenotypes. Syngeneic newborn hamsters were inoculated with these subclones and observed for the appearance of palpable tumors. A strong correlation between enzyme activity and tumor formation was observed in four separate trials; animals receiving high-PA subclones developed tumors more rapidly than those inoculated with the parental cell line. Tumors were also excised from test animals, and the cell lines established from the tumors were tested in vitro at different passages for their ability to activate plasminogen. These tumor cells were then reinoculated into syngeneic animals to confirm the tumorigenicity of cell lines with high fibrinolytic activity. In these experiments, the positive correlation between PA production and tumorigenicity was confirmed.