Inhibition of intraperitoneal tumor growth of human ovarian cancer cells by bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Several proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (N-hTIMP-1-chCysWT, N-hTIMP-1-chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.     

A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction. Cystatin with a urokinase receptor binding site

Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19-31). The inhibition constants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K(i), papain (pm), 1.9-2.4; K(i), cathepsin B (nm), 1.0-1.7; K(i), cathepsin L (pm), 0.12-0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. The measured K(D) value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 17 nm (K(D) value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors.     

Inhibition of NF-kappa B-Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1.

The essential role of urokinase-type plasminogen activator (uPA) in tumor invasion and metastasis stresses the necessity of a fine-tuned cellular control over its expression. It has been shown that changes in uPA directly correlate with changes in cell invasiveness. We examined the role of Rel-related proteins in uPA synthesis by human ovarian cancer cells by inhibiting their expression using the antisense (AS) oligodeoxynucleotide (ODN) technology. Exposure of OV-MZ-6 cells to 10 microM phosphorothioate (PS)-derivatized AS-ODN directed to Rel A led to a maximal 50% decrease of uPA antigen in cell lysates and a 70% reduction in cell cultures supernatants accompanied by a significant transient decline in uPA mRNA levels. Antisense-PS-ODN directed to NF-kappa B1 (p50) or c-rel had no effect on uPA protein expression. AS-PS-ODN directed to Rel A also affected the proteolytic capacity of OV-MZ-6 cells reflected by an approximately 70% decrease in the fibrinolytic capacity of the cells within 24 h compared to untreated controls. AS-PS-ODN directed to I kappa B alpha expression increased uPA in cell culture supernatants up to 50%. uPA receptor (uPAR) production and synthesis of plasminogen activator inhibitor type-1 (PAI-1) were not altered by either AS-PS-ODN applied. Western blot and gel retardation analyses revealed constitutive expression of Rel-related proteins in nuclear protein extracts of OV-MZ-6 cells. Thus these proteins seem to be implicated in uPA regulation and may thereby contribute to tumor spread and metastasis.     

High level synthesis of recombinant soluble urokinase receptor (CD87) by ovarian cancer cells reduces intraperitoneal tumor growth and spread in nude mice

Focussing of the serine protease urokinase (uPA) to the tumor cell surface via interaction with its receptor (uPAR) is an important step in tumor invasion and metastasis. The human ovarian cancer cell line OV-MZ-6#8 was stably transfected with expression plasmids either encoding cell-associated uPAR (GPI-uPAR) or a soluble form of uPAR (suPAR) lacking its glycan lipid anchor. In vitro, high level synthesis of functionally active recombinant suPAR inhibited cell proliferation and led to reduced cell-associated fibrin matrix degradation, whereas fibrinolytic activity was increased in OV-MZ-6#8 cells overexpressing GPI-uPAR. Both OV-MZ-6#8-derived clones were inoculated into the peritoneum of nude mice and tested for tumor growth and spread. High level synthesis of recombinant suPAR (without altering the physiological expression levels of GPI-uPAR and uPA in these cells) resulted in a significant reduction of tumor burden (up to 86%) in the xenogeneic mouse model. In contrast, overexpression of GPI-uPAR in tumor cells did not affect tumor growth. Our results demonstrate that high levels of suPAR in the ovarian cancer cell vicinity can act as a potent scavenger for uPA, thereby significantly reducing tumor cell growth and cancer progression in vivo.     

Inhibition of cytotrophoblastic (JEG-3) cell invasion by interleukin 12 involves an interferon gamma-mediated pathway

Trophoblast invasion, like tumor invasion, shares common biochemical mechanisms. However, in contrast to tumor invasion of a host tissue, trophoblastic invasion during implantation is strictly regulated, temporospatially. Factors responsible for these important regulatory processes are presently unknown; however, studies indicate that cytokines and growth factors represent in the peri-implantation uterine milieu as the possible candidates. In this study we investigated the role of interleukin (IL) 12 in regulating trophoblast invasion and the expression of trophoblast proteases (matrix metalloprotease (MMP)-2, MMP-9, and urokinase-type plasminogen activators) and their inhibitors (tissue inhibitors of metalloprotease (TIMP) 1, TIMP-2, and plasminogen activator inhibitor (PAI)-1) using an in vitro tissue culture system of human choriocarcinoma cell line JEG-3. Our major findings show an anti-invasive role of IL-12, associated with an inhibitory effect on the proteases but with an opposite up-regulating influence on the protease inhibitor, TIMP-1, whereas TIMP-2 and plasminogen activator inhibitor 1 remained unaltered. Stimulation of JEG-3 cells with IL-12 also induced interferon (IFN)-gamma production, which when neutralized using a monoclonal anti-IFN-gamma antibody, F12, abrogates its ability to down-regulate the MMPs. IL-12 also mediates an IFN-gamma-dependent up-regulation of E-cadherin, thereby implying that alteration in cell-cell adhesion besides regulating the proteases and the inhibitors possibly contributes to the observed anti-invasive role of this cytokine. TIMP-1, although stimulated by IL-12, was found to be unaltered by antibody F12, thereby implying a possibility of an IL-12-dependent-IFN-gamma independent regulation. These findings thereby suggest an important role of IL-12 in modulation of trophoblast proteases and their inhibitors besides regulating cell-cell interactions and invasion during implantation, with far reaching possibilities for understanding the mechanism(s) and regulations of invasion and metastasis.     

Effect of compressive force on the expression of MMPs, PAs, and their inhibitors in osteoblastic Saos-2 cells

Bone matrix turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor type-1 (PAI-1). We previously demonstrated that 1.0g/cm(2) of compressive force was an optimal condition for inducing bone formation by osteoblastic Saos-2 cells. Here, we examined the effect of mechanical stress on the expression of MMPs, TIMPs, tPA, uPA, and PAI-1 in Saos-2 cells. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and with or without continuously compressive force (0.5-3.0g/cm(2)) for up to 24h. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 gene expression were estimated by determining the mRNA levels using real-time PCR, and the protein levels were determined using ELISA. The expression levels of MMP-1, MMP-2, MMP-14, and TIMP-1 markedly exceeded the control levels at 1.0g/cm(2) of compressive force, whereas the expression levels of MMP-3, MMP-13, TIMP-2, TIMP-3, TIMP-4, tPA, uPA, and PAI-1 markedly exceeded the control levels at 3.0g/cm(2). These results suggest that mechanical stress stimulates bone matrix turnover by increasing these proteinases and inhibitors, and that the mechanism for the proteolytic degradation of bone matrix proteins differs with the strength of the mechanical stress.     

Urokinase directly activates matrix metalloproteinases-9: a potential role in glioblastoma invasion

Previous reports showed that urokinase plasminogen activator (uPA) converts plasminogen to plasmin which then activates matrix metalloproteinases (MMPs). Here, we report that uPA directly cleaved pro-MMP-9 in a time-dependent manner at both C- and N-terminus and generated two gelatinolytic bands. uPA-activated-MMP-9 efficiently degraded fibronectin and blocked by uPA inhibitor B428 and recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1). B428 inhibited basal and PMA-induced active MMP-9 in glioblastomas (GBM) U1242 cell media as well as cell invasion in vitro. A combination of MMP-9 and uPA antibodies more significantly inhibited U1242 cell invasion than uPA or MMP-9 antibody alone. Both uPA and MMP-9 were highly expressed in U1242 cell and GBM patient specimens. Furthermore, two active MMP-9 fragments with identical molecular weights to the uPA-activated MMP-9 products were detected in GBM patient specimens. These results suggest that uPA-mediated direct activation of MMP-9 may promote GBM cell invasion.     

Matrix metalloproteinases and their tissue inhibitors in the developing neonatal mouse uterus

Postnatal development of the mouse uterus involves differentiation and development of the endometrial glands as well as the myometrium. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in extracellular matrix breakdown and morphogenesis of many epitheliomesenchymal organs. As a first step to understanding their roles in postnatal mouse uterine development, MMPs and TIMPs found to be expressed in the neonatal mouse uterus by microarray analysis were localized by in situ hybridization. The MMP-2 mRNA was detected only in the uterine stroma, whereas the MMP-10 mRNA was present only in the uterine epithelium from Postnatal Day (PND) 3 to PND 9. All other MMPs (MMP-11, MMP-14, and MMP-23) as well as TIMP-1, TIMP-2, and TIMP-3 were detected in both epithelial and stromal cells of the endometrium, but not in the myometrium. Uterine extracts were then analyzed by gelatin and casein gel zymography to detect active gelatinases and stromelysins, respectively. Five major gelatinase bands of activity were detected and inhibited by the MMP inhibitors, EDTA or 1,10-phenanthroline, but not by PMSF, a serine protease inhibitor. Western blot analysis confirmed the presence of MMP-2 and MMP-9 proteins in the uterus. Immunoreactive MMP-9 protein was detected only in the endometrial stroma, whereas immunoreactive MMP-2 protein was detected in both the stroma and epithelium of the uterus. Casein zymography detected three major bands of activity ( approximately 54, 63, and 80 kDa) that were inhibited by the serine protease inhibitor, PMSF, but not by the MMP inhibitors, EDTA or 1,10-phenanthroline, suggesting that they were serine proteases. These results support the hypothesis that MMPs and TIMPs regulate postnatal development of the mouse uterus.     

Flavonoids inhibit VEGF/bFGF-induced angiogenesis in vitro by inhibiting the matrix-degrading proteases

Flavonoids have been proposed to act as chemopreventive agents in numerous epidemiological studies and have been shown to inhibit angiogenesis and proliferation of tumor cells and endothelial cells in vitro. Angiogenesis requires tightly controlled extracellular matrix degradation mediated by extracellular proteolytic enzymes including matrix metalloproteinases (MMPs) and serine proteases, in particular, the urokinase-type plasminogen activator (uPA)-plasmin system. In this study, we have investigated the antiangiogenic mechanism of the flavonoids, genistein, apigenin, and 3-hydroxyflavone in a human umbilical vein endothelial cell (HUVEC) model. The stimulation of serum-starved HUVECs with vascular endothelial growth factor/basic fibroblast growth factor (VEGF/bFGF) caused marked increase in MMP-1 production and induced the pro-MMP-2 activation accompanied by the increase in MT1-MMP expression. However, pretreatment with flavonoids before VEGF/bFGF stimulation completely abolished the VEGF/bFGF-stimulated increase in MMP-1 and MT1-MMP expression and pro-MMP-2 activation. Genistein blocked VEGF/bFGF-stimulated increase in TIMP-1 expression and decrease in TIMP-2 expression. Apigenin and 3-hydroxyflavone further decreased TIMP-1 expression below basal level and completely abolished TIMP-2 expression. VEGF and bFGF stimulation also significantly induced uPA expression, most strikingly the level of 33 kDa uPA, and increased the expression of PA inhibitor (PAI)-1. Genistein, apigenin, and 3-hydroxyflavone effectively blocked the generation of 33 kDa uPA, and further decreased the activity of the 55 kDa uPA and the expression of PAI-1 below the basal level. In conclusion, these data suggest that genistein, apigenin, and 3-hydroxyflavone inhibit in vitro angiogenesis, in part via preventing VEGF/bFGF-induced MMP-1 and uPA expression and the activation of pro-MMP-2, and via modulating their inhibitors, TIMP-1 and -2, and PAI-1.     

Curcumin, demethoxycurcumin and bisdemethoxycurcumin differentially inhibit cancer cell invasion through the down-regulation of MMPs and uPA

Curcumin (Cur), a component of turmeric (Curcuma longa), has been reported to exhibit antimetastatic activities, but the mechanisms remain unclear. Other curcuminoids present in turmeric, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) have not been investigated whether they exhibit antimetastatic activity to the same extent as curcumin. The regulation of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) play important role in cancer cell invasion by cleavage of extracellular matrix (ECM). In this line, we comparatively examined the influence of Cur, DMC and BDMC on the expressions of uPA, MMP-2, MMP-9, membrane Type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinases (TIMP-2), and in vitro invasiveness of human fibrosarcoma cells. The results indicate that the differential potency for inhibition of cancer cell invasion was BDMC> or =DMC>Cur, whereas the cell migration was not affected. Zymography analysis exhibited that curcumin, DMC and BDMC significantly decreased uPA, active-MMP-2 and MMP-9 but not pro-MMP-2 secretion from the cells in a dose-dependent manner, in which BDMC and DMC show higher potency than curcumin. The suppression of active MMP-2 level correlated with inhibition of MT1-MMP and TIMP-2 protein levels involved in pro-MMP-2 activation. Importantly, BDMC and DMC at 10 microM reduced MT1-MMP and TIMP-2 protein expression, but curcumin slightly reduced only MT1-MMP but not TIMP-2. In addition, three forms of curcuminoids significantly inhibited collagenase, MMP-2, and MMP-9 but not uPA activity. In summary, these data demonstrated that DMC and BDMC show higher antimetastasis potency than curcumin by the differentially down-regulation of ECM degradation enzymes.     

Gelatinase A activity directly modulates melanoma cell adhesion and spreading.

Interaction of cells with the extracellular matrix (ECM) plays an important role in the regulation of cell behavior. Formation of adhesive contacts leads to transduction of signals into the cell and results in altered gene expression and modulation of the cellular phenotype. Specific adhesive interactions of the fibronectin and vitronectin receptors with their ligands in the matrix modulates expression of ECM-degrading metalloproteases. These proteases are involved in the acquisition of the invasive phenotype by a number of cell types. The activity of matrix metalloproteases (MMPs) is reduced by endogenous inhibitors referred to as tissue inhibitors of metalloproteases (TIMPs). Alterations in the balance between the activity of MMPs and TIMPs alters cellular invasion through effects on matrix degradation. In this study we demonstrate that inhibition of endogenous gelatinase A activity in A2058 human melanoma cells results in enhanced cellular adhesion. To further explore this phenomenon, we have used retroviral infection vectors to control the amount of the MMP inhibitor TIMP-2 in human melanoma A2058 cells. Altering the production of TIMP-2 modulates not only proteolysis of the extracellular matrix, but also the adhesive and spreading properties of the cells and results in altered cell morphology. These effects of TIMP-2 appear to be mediated by inhibition of gelatinase A activity. We conclude that gelatinase A, in addition to contributing to proteolysis of ECM components, also functions to proteolyse cell surface components that mediate attachment of A2058 cells to the ECM. Thus, gelatinase A may function to modulate cell attachment and facilitate cell migration and invasion.     

Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells

Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP-1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP-mediated activation of proMMP-2. © 1996 Wiley-Liss, Inc.     

Matrix metalloproteinases and lysosomal cysteine proteases in osteoclasts contribute to bone resorption through distinct modes of action

The effects of inhibitors of matrix metalloproteinases (MMPs) and lysosomal cysteine proteases on osteoclastic pit formation in dentine slices were investigated. A nonspecific cysteine protease inhibitor, E-64, inhibited pit formation on naked slices in a concentration-dependent manner, and at 10 microM E-64 reduced the pit volume by 70%. However, up to 10 microM of the MMP inhibitor, BB-94, did not show any inhibition of pit formation. On the other hand, on slices coated with reconstituted basement membrane, both BB-94 and E-64 at 10 microM showed a marked decrease in pit volume by 73% and 68%, respectively. By a combination of treatment with both BB-94 and E-64, pit formation could be completely suppressed. These results suggest that MMPs are necessary for the migration of precursor and/or immature osteoclasts to bone surface through basement membranes, while cysteine proteases are essential for the osteoclastic degradation of bone collagen.     

Characterization of matrix metalloproteinase expressed by human embryonic kidney cells

There is little information available on the proteases expressed by human embryonic kidney (HEK) cells, which are often used for expression of recombinant proteins and production of adenovirus vector. The expression profile of proteases in HEK cell line was investigated using zymography, mRNA analysis, western blotting and protein array. The major protease was gelatinase A [or matrix metalloproteinase (MMP)-2]. Beside, other MMPs, such as MMP-1, -2, -3, -8, -9, -10, -13 and membrane type (MT) 1- and 3−MMP, as well as tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, were also expressed by HEK cells. Characterization of MMP and TIMP profiles expressed by HEK cells provides the basis for degradation control of recombinant protein and adenovirus vector during culture and purification processes.     

Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases

Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31,-H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs.     

Expression level and glycan dynamics determine the net effects of TIMP-1 on cancer progression

Tissue inhibitor of metalloproteinases (TIMPs; TIMP-1, -2, -3 and -4) are endogenous inhibitor for matrix metalloproteinases (MMPs) that are responsible for remodeling the extracellular matrix (ECM) and involved in migration, invasion and metastasis of tumor cells. Unlike under normal conditions, the imbalance between MMPs and TIMPs is associated with various diseased states. Among TIMPs, TIMP-1, a 184-residue protein, is the only N-linked glycoprotein with glycosylation sites at N30 and N78. The structural analysis of the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1 suggests new possibilities of the role of TIMP-1 glycan moieties as a tuner for the proteolytic activities by MMPs. Because the TIMP-1 glycosylation participate in the interaction, aberrant glycosylation of TIMP-1 presumably affects the interaction, thereby leading to pathogenic dysfunction in cancer cells. TIMP-1 has not only the cell proliferation activities but also anti-oncogenic properties. Cancer cells appear to utilize these bilateral aspects of TIMP-1 for cancer progression; an elevated TIMP-1 level exerts to cancer development via MMP-independent pathway during the early phase of tumor formation, whereas it is the aberrant glycosylation of TIMP-1 that overcome the high anti-proteolytic burden. The aberrant glycosylation of TIMP-1 can thus be used as staging and/or prognostic biomarker in colon cancer. [BMB Reports 2012; 45(11): 623-628]     

Tumor-associated urokinase-type plasminogen activator: biological and clinical significance.

Evidence has accumulated that invasion and metastasis in solid tumors require the action of tumor-associated proteases, which promote the dissolution of the surrounding tumor matrix and the basement membranes. Receptor-bound urokinase-type plasminogen activator (uPA) appears to play a key role in these events. uPA converts plasminogen into plasmin and thus mediates pericellular proteolysis during cell migration and tissue remodeling under physiological and pathophysiological conditions. uPA is secreted as an enzymatically inactive proenzyme (pro-uPA) by tumor cells and stroma cells. uPA exerts its proteolytic function on normal cells and tumor cells as an ectoenzyme after having bound to a high-affinity cell surface receptor. After binding, pro-uPA is activated by serine proteases (e.g. plasmin, trypsin or plasma kallikrein) and by the cysteine proteases cathepsin B or L, resp. Receptor-bound enzymatically active uPA converts plasminogen to plasmin which is bound to a different low-affinity receptor on tumor cells. Plasmin then degrades components of the tumor stroma (e.g. fibrin, fibronectin, proteoglycans, laminin) and may activate procollagenase type IV which degrades collagen type IV, a major part of the basement membrane. Hence receptor-bound uPA will promote plasminogen activation and thus the dissolution of the tumor matrix and the basement membrane which is a prerequisite for invasion and metastasis. Tissues of primary cancer and/or metastases of the breast, ovary, prostate, cervix uteri, bladder, lung and of the gastrointestinal tract contain elevated levels of uPA compared to benign tissues. In breast cancer uPA and PAI-1 antigen in tumor tissue extracts are independent prognostic factors for relapse-free and overall survival.     

Protease activity in protein-free NSO myeloma cell cultures

Summary Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that this cell line produced and released/secreted several proteases. Two caseinolytic activities at 45–50 and 90 kDa were identified as aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular masses of 30–35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using an enzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid and cysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product or affecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, but addition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibody product was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5–4, the aspartic acid proteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM, one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotinin significantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminal amino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitor and cystatin C.