Expression of matrix metalloproteinases-1, -2, and -9; tissue inhibitors of matrix metalloproteinases-1 and -2; cathepsin B; urokinase plasminogen activator; and plasminogen activator inhibitor, type I in skull base chordoma

Little is known about proteinase expression in skull base chordoma, a rare bone tumor exhibiting local invasiveness. Using immunohistochemical techniques, we investigated the expression of matrix metalloproteinases (MMPs)-1, -2, and -9; tissue inhibitors of matrix metalloproteinases (TIMPs)-1 and -2; cathepsin B (CatB); urokinase plasminogen activator (uPA); and plasminogen activator inhibitor, type I (PAI1), in 45 patients with skull base chordoma (45 primary and 25 autologous recurrent lesions). We compared these data with clinicopathologic parameters and the expression of cell differentiation markers. MMP-1, MMP-2, TIMP-1, CatB, uPA, and PAI1 were frequently expressed, and there was a significant correlation in the expression of some proteinases. Immunoreactivity for MMP-1, MMP-2, CatB, and uPA was significantly higher in lesions exhibiting tumor infiltration of host bone than in those without such components. Expression of MMP-1, TIMP-1, CatB, and uPA was associated with that of low-molecular-weight cytokeratin (CAM5.2). There were no differences in proteinase expression in 25 pairs of primary and their recurrent lesions, and proteinase expression did not predict local recurrences. However, patients with higher expression of both MMP-1 and uPA showed worse prognosis compared with the others. In conclusion, expression of some proteinases correlated with CAM5.2 expression and seemed to play an important role in a synergistic manner in the invasion process in skull base chordoma. The authors believe that elevated expression of MMP-1 and uPA can be used to identify patients with a worse prognosis in skull base chordoma.     

Promigratory Effect of Plasminogen Activator Inhibitor-1 on Invasive Breast Cancer Cell Populations

The urokinase-type plasminogen activator (uPA) system is a dynamic complex in which the membrane receptor uPAR binds uPA that binds the plasminogen activator inhibitor (PAI)-1 localized in the extracellular matrix, resulting in endocytosis of the whole complex by the low-density lipoprotein receptor-related protein (LRP). High expression of PAI-1 is paradoxically associated with marked tumor spreading and poor prognosis. We previously reported a nonproteolytic role of the [uPAR:uPA:PAI-1:LRP] complex operative in cell migration. Here we explored whether matrix PAI-1 could be used as a migration support by human breast cancer cells. We showed that the uPA system and LRP are localized at filopodia of invasive cells, and that formation/internalization of the [uPAR:uPA:PAI-1:LRP] complex is required for attachment and migration of cancer cells on plastic and on a PAI-1 coat. PAI-1 increased both filopodia formation and migration of cancer cells suggesting a chemokine-like activity. Migration velocity, expression of the uPA system, use of the [uPAR:uPA:PAI-1:LRP] complex to migrate, and promigratory effects of PAI-1 paralleled cancer cell invasiveness. Phenotyping and functional analysis of invasive cancer cell subclones indicated that different cell subpopulations may use different strategies to migrate depending on both the environment and their expression of the uPA system, some of them taking advantage of abundant available PAI-1.     

Immunohistochemical expression of uPA, uPAR, and PAI-1 in breast carcinoma. Fibroblastic expression has strong associations with tumor pathology

The urokinase-type plasminogen activator (uPA) system has been implicated in tumor spread. We have used immunohistochemistry to examine three components of this system, ie, uPA, uPA receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1), in a pilot study on 142 cases of breast carcinoma. We wished to determine whether there were any relationships between expression of the proteins in either tumor cells or fibroblasts and clinical and pathological features. Strong uPA expression in each cell type was significantly related to high tumor grade (P = 0.013 and 0.008, respectively), and was more common in invasive than in in situ carcinomas (P < 0.0001). Fibroblastic expression of uPAR was only related to the presence of invasion (P < 0.0001). Strong PAI-1 expression in both cell types was seen in high-grade tumors (tumor cells, P = 0.012; fibroblasts, P < 0.001), but only fibroblastic expression was related to the presence of invasion (P = 0.042). Fibroblastic expression of both uPA and uPAR were positively correlated with tumor size. Although patients with strong fibroblastic expression of uPA showed a tendency toward a shorter time to relapse, none of the plasminogen activator proteins were significantly associated with relapse-free survival. These results suggest that strong expression of uPA, uPAR, and PAI-1 in fibroblasts rather than in tumor cells have the most impact on the clinical behavior of breast cancer. Larger prospective studies are needed to confirm these findings.     

Co-stimulation of human breast cancer cells with transforming growth factor-beta and tenascin-C enhances matrix metalloproteinase-9 expression and cancer cell invasion

Transforming growth factor-beta (TGF-beta), tenascin-C (TN-C) and matrix metalloproteinases (MMPs) have been demonstrated independently to be associated with disease progression and poor prognosis in patients with breast cancer. The present study explored effects of TGF-beta and TN-C on MMP-9 expression and cancer invasion. An experimental study was designed to analyse MDA-MB-231 breast cancer cells, known for their high invasiveness, after stimulation with TGF-beta1 and/or TN-C. TGF-beta1 stimulated TN-C expression in the cells. Co-stimulation of MDA-MB-231 cells with TN-C and TGF-beta increased MMP-9 expression at both the gene (28-fold) and the protein levels. The in vitro invasion also increased (4-fold). GM6001 inhibited the invasion induced by the co-stimulation. The combined effect of TN-C and TGF-beta resulted in enhanced MMP-9 expression and cancer invasion in vitro.     

Regulation of tissue-degrading factors and in vitro invasiveness in progression of breast cancer cells

Hormone-independent growth and invasiveness represent phenotypic properties acquired during early progression of breast cancer. We compared human mammary adenocarcinoma cells, MCF-7, which are estrogen-dependent and poorly metastatic, with the estrogen-independent and highly metastatic subline, MCF7/LCC1, with regard to expression of tissue-degrading factors of the matrix metalloproteinase (MMP)-and urokinase (uPA)-dependent degradative pathways, as well as for their in vitro invasive properties. Both cell lines showed low constitutive mRNA expression of the MMP inhibitor TIMP-1. Baseline expression of TIMP-2 mRNA was also very low in MCF-7 cells, whereas the MCF7/LCC1 level was much higher (~10- fold). Furthermore, both cell lines revealed low constitutive capacity to migrate in an in vitro invasion assay. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nM) induced the mRNAs for TIMP-1 as well as for MMP-1, MMP-9, the uPA receptor, and the uPA inhibitor PAI-1, am ongst which only the responses of MMP-9 and PAI-1 were cell-specific. The mRNA levels of MMP-9 and PAI-1 were ~10-fold and ~15-fold higher in MCF7/LCC1 cells compared to MCF-7 cells. The secretion of immuno-reactive PAI-1 was considerably elevated (. 20-fold) in TPA-treated MCF7/LCC1 cells, whereas the TPA-dependent level of 92-kDa MMP-9 was only ~2-fold higher in MCF7/LCC1 cells than in MCF-7 cells. In both cell lines treatment with TPA was associated with an increase (~10-fold) in in vitro migration, which in the MCF7/LCC1 cells was significantly attenuated by a reconstituted basement membrane extract (Matrigel). These data suggest that TPA-responsive in vitro invasive properties that are probably associ-ated with PAI-1 expression may co-vary with progression from hormone-dependent to -independent breast cancer. © Rapid Science 1998     

A novel 3-dimensional culture system as an in vitro model for studying oral cancer cell invasion

Tissue microenvironment plays a critical role in tumour growth and invasion. This study established a novel 3-dimensional (3-D) cell invasion model for direct microscopic observation of oral cancer cell invasion into the underlying basement membrane and connective tissue stroma. A multilayer cell construct was developed using the OptiCell chamber, consisting of a lower layer of oral mucosa fibroblasts embedded in collagen gel and an overlaying upper layer of oral cancer cells. The two layers are separated by a basement membrane composed of reconstituted extracellular matrix. To verify the applicability of the cell invasion model, multilayer cell constructs of oral squamous cell carcinoma and oral mucosal fibroblasts were exposed to extrinsic urokinase-type plasminogen activator (uPA) or plasminogen activator inhibitor (PAI-1), which are known effectors of cell migration. In addition, the constructs were exposed to both normoxic and hypoxic culture conditions. Microscopic study showed that the presence of uPA enhanced cell invasion, while PAI-1 inhibited cell migration. Western blot and zymographic analysis demonstrated that hypoxia up-regulated uPA and matrix metalloproteinases (MMPs) expression and activity; conversely, PAI-1 level was down-regulated in response to hypoxic challenge as compared to normoxic condition. Our results indicated that the novel 3-D invasion model could serve as an excellent in vitro model to study cancer cell invasion and to test conditions or mediators of cellular migration.     

Plasminogen activator system modulates invasivecapacity and proliferation in prostatic tumor cells

The malignant phenotype of prostatic tumor cells correlates with the expression of both uPA and itscell-membrane receptor (uPAR); however, there is little information concerning the role of cell-bound uPAin matrix degradation and invasion. Our results suggest that cell-associated uPA plays a key role in regulat-ingthe amount of plasmin present at the surface of prostatic carcinoma (PRCA) cells and show that differ-entialproduction of uPA corresponds with the capacity to bind and activate plasminogen. In addition, weprovide direct evidence that both uPA secretion and the presence of uPA-uPAR complexes characterize theinvasive phenotype of PRCA cells and suggest the existence of several pathways by which tumor cells acquireplasmin activity. LNCaP cells (which do not produce uPA but express uPAR) may activate plasmin throughexogenous uPA. In vivo, the source of uPA may be infiltrating macrophages and/or fibroblasts as observedin several other systems. PAI-1 accumulation in the conditioned medium (CM) limits plasmin action to thepericellular microenvironment. Our results indicate that MMP-9 and MMP-2 are also activated by plasmingenerated by cell-bound but not by soluble, extracellular uPA. Plasmin activation and triggering of the pro-teolyticcascade involved in Matrigel invasion is blocked by antibodies against uPA (especially by anti- A-chainof uPA which interacts with uPAR) and by PA inhibitors such as p-aminobenzamidine which mayregulate levels of cell-bound uPA. uPA may also regulate growth in PRCA cells. Indeed, antibodies againstuPA A-chain (and also p-aminobenzamidine treatment) interfere with the ATF domain and inhibit cell growthin uPA-producing PC3 and DU145 prostate cancer cell lines, whereas exogenous uPA (HMW-uPA with ATF)induces growth of LNCaP prostate tumor cell line. These data support the hypothesis that in prostatic can-cerpatients at risk of progression, uPA/plasmin blockade may be of therapeutic value by blocking both growthof the primary tumor and dissemination of metastatic cells. ©Kluwer Academic Publishers     

Expression and activation of proteases in co-cultures

The present study concerned the expression and activation of metalloproteinase-2 (MMP-2), metalloproteinase-9 (MMP-9) and the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system in co-cultures of human colon carcinoma cell spheroids (HT29, LS180, SW948) with human normal colon epithelium (CCD 841 CoTr), myofibroblasts (CCD-18Co) and endothelial cells (HUVEC). Additionally, the influence of monensin on the production and function of the proteases was tested.Tumor cells expressed small amounts of MMP-2, MMP-9 and uPA. Normal cells generally produced proportionally higher concentrations of these proteases (especially MMP-2, compared with significantly smaller yields of MMP-9 and significantly lower amounts of uPAR than tumors. In co-cultures of tumor spheroids with normal cell monolayers, the concentration of the proteases was equal to the sum of the enzymes produced in monocultures of both types of cells. The highest activity of uPA, measured as the reduction of the chromogenic substrate (S-2444), was detected in supernatants and lysates of endothelial cells. Interestingly, in normal cells, the higher expression of proteases, mainly uPA, measured as the level of protein concentration, was closely linked with their lower activity and inversely, in tumor cells, the low level of the expression of the enzymes correlated with their high enzymatic activity. In zymography analysis, mainly pro-MMPs were detected both in culture supernatants and cell lysates. The highest amounts of active forms of the MMPs were detected in tumor spheroids co-cultured with endothelial cells. Monensin inhibited MMPs and uPA secretion but significantly increased uPAR release, mainly from normal cells.In conclusion, during direct interactions of tumor cells with normal cells, MMPs and the uPA/uPAR system play an important role in the degradation of ECM and tumor development, but as we found, there is a reverse relationship between the concentration and the enzymatic activity of MMPs and uPA/uPAR in co-culture models.     

Signalling networks associated with urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in breast cancer tissues: new insights from protein microarray analysis

The urokinase-type plasminogen activator (uPA) and the main uPA inhibitor PAI-1 play important roles in cell migration and invasion in both physiological and pathological contexts. Both factors are clinically applicable predictive markers in node-negative breast cancer patients that are used to stratify patients for adjuvant chemotherapy. In addition to their classical functions in plasmin regulation, both factors are key components in cancer-related cell signalling. Such signalling cascades are well described in cell culture systems, but a better understanding of uPA- and PAI-1-associated signalling networks in clinical tissues is needed. We examined the expression of uPA, PAI-1, and 21 signalling molecules in 201 primary breast cancer tissues using protein microarrays. Expression of uPA was significantly correlated with the expression of ERK and Stat3, while expression of PAI-1 was correlated with the uPA receptor and Akt activation, presumably via integrin and HER-receptor signalling. Analysis of uPA expression did not reveal any significant correlation with staging, grading or age of the patients. The PAI-1 expression was correlated with nodal stage. Network monitoring for uPA and PAI-1 in breast cancer reveals interactions with main signalling cascades and extends the findings from cell culture experiments. Our results reveal possible mechanisms underlying cancer development.     

反义转化生长因子β1基因转染大鼠肾系膜细胞及其对纤溶？…

目的 通过对大鼠肾小球系膜细胞（ＭｓＣ）转染反义转化生长因子β１（ＴＧＦ－β１）基因,观察该细胞克隆基质金属蛋白酶家族（ＭＭＰｓ）和纤溶酶原激活物抑制剂１（ＰＡＩ－１）表达的改变,方法 采用脂质体进行基因转染,并用Ｗｅｓｔｅｒｎ ｂｌｏｔ法加以鉴定,后分别应用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ、Ｗｅｓｔｅｒｎ ｂｌｏｔ和酶谱分析法检测该细胞克隆ＭＭＰｓ和ＰＡＩ－１表达的变化。结果 成功地建立低表达ＴＧＦ－     

Increased expression of matrix metalloproteinases-21 and -26 and TIMP-4 in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma is known for early aggressive local invasion, high metastatic potential, and a low 5-year survival rate. Matrix metalloproteinases (MMPs) play important roles in tumor growth and invasion. Earlier studies on pancreatic cancer have found increased expression of certain MMPs to correlate with poorer prognosis, short survival time or presence of metastases. We studied the expression of MMP-21, -26, and tissue inhibitor of matrix metalloproteinases (TIMP)-4 in 50 tissue samples, including 25 adenocarcinomas, seven other malignant pancreatic tumors, and 18 control samples of non-neoplastic pancreatic tissue with immunohistochemistry. The regulation of MMP-21, -26, and TIMP-4 mRNAs by cytokines was studied with RT-PCR in pancreatic cancer cell lines PANC-1, BxPC-3, and AsPC-1. MMP-21, -26, and TIMP-4 were detected in cancer cells in 64, 40, and 60% of tumors, respectively. MMP-21 expressed in well-differentiated cancer cells and occasional fibroblasts, like TIMP-4, tended to diminish in intensity from grade I to grade III tumors. Patients with metastatic lymph nodes had increased expression of MMP-26 in actual tumor samples. All cultured cancer cell lines expressed MMP-21 basally at low levels, and presence of the protein was confirmed immunohistochemically in cultured cells. MMP-21 expression was induced by epidermal growth factor (EGF) in PANC-1 cells. MMP-26 was neither expressed basally nor induced by tumor necrosis factor alpha, transforming growth factor beta-1 (TGFbeta1), EGF, or interferon gamma. Basal TIMP-4 expression was lowest in the poorly differentiated cancer cell line PANC-1 compared to better-differentiated BxPC-3 and AsPC-1 cells. TIMP-4 expression was induced by TGFbeta1 in PANC-1 cells and by EGF in BxPC-3 cells. Our findings suggest that MMP-21 is not a marker of invasiveness, but rather of differentiation, in pancreatic cancer and it may be upregulated by EGF. The putative role of MMP-26 as a marker of metastases warrants further studies. Unlike other TIMPs, TIMP-4 was not upregulated in relation to aggressiveness of pancreatic cancer.     

Regulation of the plasminogen activator inhibitor-2 (PAI-2) gene in murine macrophages. Demonstration of a novel pattern of responsiveness to bacterial endotoxin.

We investigate the regulation of plasminogen activator inhibitor-2 (PAI-2) in murine macrophages. PAI-2 mRNA was inducible by bacterial lipopolysaccharide (LPS) in primary cells and macrophage-like cell lines. Evidence is presented for a role for autocrine factors, including cyclooxygenase products but not the cytokines tumor necrosis factor alpha or interferon-beta (IFN-beta). PAI-2 mRNA levels generally varied inversely from those of its target, urokinase-type plasminogen activator (uPA), and the macrophage growth factor CSF-1, which induces uPA, inhibited PAI-2 expression in cells treated subsequently with LPS. Expression of PAI-2 was distinct from that of other LPS-inducible genes in terms of induction time course, LPS dose response, and sensitivity to co-stimulation with IFN-gamma. Induction of PAI-2 mRNA in subclones of the cell line RAW 264 was not uniform, reflecting heterogeneous expression in the parent line. The expression pattern of PAI-2 is discussed in terms of a possible role in LPS-induced pathology such as septicemia.     

Mature breast adipocytes promote breast cancer cell motility

Adipocytes express substances involved in both normal physiology and pathological processes. One such adipocyte protein is the Serpin (serine protease inhibitor) plasminogen activator inhibitor-1 (PAI-1). PAI-1 functions to inhibit urokinase type plasminogen activator (uPA) though PAI-1 itself is also implicated in breast cancer progression. While the role of adipocytes in breast cancer development is not fully understood, obesity is a known risk factor associated with breast cancer. Thus, we characterized adipocytes from breast and omental tissues for PAI-1 and uPA, and the influence of adipocytes on breast cancer cell motility. Using preadipocyte cells from breast and omental adipose tissue, we differentiated each site into mature adipocytes. PAI-1 protein was found in breast adipocytes>omental preadipocytes>omental adipocytes>breast preadipocytes. Interestingly, uPA protein was not detected in any of these cell types. We then incubated breast adipocyte conditioned media (Adip-CM) and preadipocyte conditioned media (PreAdip-CM) on both normal (MCF-10A) and malignant (MCF-10CA1) breast epithelial cell lines. Adip-CM, but not PreAdip-CM, (a) increased cell motility in both MCF-10A and MCF-10CA1 cells; (b) increased cell-associated uPA activity in both cell lines; (c) increased phosphorylated-Akt levels in MCF-10CA1 cells; and (d) gene array profiles show altered expression of several genes associated with cancer adhesion, metastasis and signaling. Our results suggest that mature breast adipocytes are capable of altering the epithelial cell phenotype, producing a more motile cell type and further provide a potential link between obesity and risk of breast cancer.     

Thrombospondin-1 (TSP-1) up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human tumor cells: Exploring the functional significance in tumor cell invasion

Thrombospondin-1 (TSP-1), a matrix-bound adhesive glycoprotein, has been shown to modulate tumor progression. We previously demonstrated that TSP-1 up-regulates matrix metalloproteinases MMP-2 and MMP-9. Our studies suggested that the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is a key determinant in tumor cell invasion. We now report that TSP-1 up-regulates TIMP-1 expression in both human breast and prostate cancer cell lines. The effect of TSP-1 on TIMP-1 expression was examined in human breast adenocarcinoma cell lines (MDA-MB-231) and human prostate cancer cell lines (PC3-NI and PC3-ML) treated with exogenous TSP-1. TIMP-1 expression was also examined in TSP-1 stably transfected breast cancer cell line (MDA-MB-435). Northern and western blot analysis revealed TIMP-1 mRNA and TIMP-1 protein expression increased with increasing concentrations of TSP-1. This effect was inhibited by antibodies against the type I repeat domain of TSP-1 further suggesting that TSP-1 mediates TIMP-1 secretion. Inhibition of TSP-1 induced TIMP-1 levels increased tumor cell invasion. We conclude that TSP-1 is involved in influencing the critical balance between MMPs and their inhibitors, maintaining the controlled degradation of the extracellular matrix needed to support metastasis and our results may provide an explanation for the divergent activities reported for TSP-1 in tumor progression.     

Influence of preoperative core biopsies on uPA/PAI-1 expression in breast cancer tissue

The urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1) are involved in tumor invasion and metastasis as well as wound healing and inflammation. We investigated the impact of tissue injury by preoperative sampling on the level of uPA/PAI-1 in paraffin tissue of 55 breast cancer cases by immunohistochemistry. The tumor area surrounding the biopsy channel was compared with distant intact tumor tissue. uPA and PAI-1 were constantly expressed by the tumor cells. Fibroblastic expression was higher in the tumor area surrounding the biopsy channel than in intact tissue with 47 vs 29 positive cases for uPA (p<0.001) and 35 vs 25 positive cases for PAI-1 (p=0.055). A decrease in fibroblastic enzyme expression from the biopsy area to the intact tumor tissue was seen in 21 cases for uPA and 16 cases for PAI-1. This difference was most evident in cases with an interval of 6 days and longer between biopsy and surgery. In conclusion, fibroblastic inflammatory reaction around the biopsy channel affects stromal uPA and PAI-1 expression, which possibly leads to falsely increased enzyme levels in ELISA. Tissue specimen for ELISA analysis should not be taken from the tumor area around the biopsy channel in the resection specimen.     

Kinetics of matrix metalloproteinases and their regulatory factorsin mercuric chloride-induced tubulointerstitial fibrosisin Brown Norway rats

We investigated the kinetics of matrix metalloproteinases (MMPs) and their regulatory factors mRNAs in the kidneys of mercuric chloride-treated Brown Norway rats. The expression of MMP-1 mRNA remained at lower levels than control, while other MMPs mRNAs were upregulated. The expression of tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA showed significant upregulation. On the other hand, the expressions of TIMP-2 and TIMP-3 mRNAs were not significantly changed. In the plasmin-dependent pathway, the expression of plasminogen activator inhibitor (PAI-1) mRNA was continuously increased, while the expression of urokinase-type plasminogen activator (uPA) mRNA was not increased. The signals of TIMP-1 and PAI-1 mRNAs examined by in situ hybridization, were localized in the regenerative epithelial cells of the proximal tubules. In conclusion, these findings suggest that the activity of MMPs may bealtered by MMP-1 downregulation and inhibition of MMP activity by PAl-1 and TIMP-1 generated from tubular epithelial cells.     

Invasion by esophageal cancer cells: functional contribution of the urokinase plasminogen activation system, and inhibition by antisense oligonucleotides to urokinase or urokinase receptor

Early metastasis contributes to the very poor prognosis of esophageal carcinoma. The recent immunohistochemical finding that invasive esophageal carcinomas express elevated levels of urokinase (uPA) and urokinase receptor (uPA-R) in vivo suggest that the plasminogen activation system may contribute to metastasis in esophageal cancer. The aim of our study was to functionally investigate, at the molecular level, the relative contribution of uPA and uPA-R to the invasiveness of esophageal cancer cells in vitro. The three esophageal cancer cell lines, OC1-3, generated in our laboratory, were analyzed for uPA and uPA-R expression by RT-PCR, immunoenzymatic staining, and quantitative ELISA. Invasiveness of all cell lines was quantified as percentage cellular invasiveness in a standardized Matrigel in vitro assay. OC1 and OC3, which were found to coexpress both uPA and uPA-R, displayed stronger invasiveness (44% and 32.5% respectively) relative to OC2 (19%) which expressed uPA-R but was negative for uPA. Transfection of OC2 cells with the uPA cDNA resulted in two variants, OC2.uPA1 and OC2.uPA2, stably expressing functional uPA. Both transfectants exhibited enhanced invasiveness (60% and 50% respectively) relative to the parent uPA-negative OC2 cells (19%). Antisense oligonucleotide inhibition of either uPA or uPA-R expression resulted in a similar, marked reduction in invasiveness of esophageal tumor cells which normally coexpress both molecules (OC1, OC3 and the uPA-expressing OC2-transfectant clones). Neither antisense treatment altered the basal invasiveness of OC2, which expresses uPA-R but not uPA. In conclusion, coexpression of uPA with its receptor, uPA-R, is required for functional involvement of the urokinase system in invasion by esophageal carcinoma cells. Our results suggest that these synergistic mediators of invasiveness are quantitatively major contributors to the invasiveness of esophageal carcinoma.