Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes.

The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration.

Beta 1 integrin is essential for teratoma growth and angiogenesis.

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of beta 1 integrin during teratoma formation, we compared teratomas induced by normal and beta1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, beta 1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of beta 1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in beta1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface. Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell-derived endothelial cells. In contrast, beta 1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although beta 1- deficient endothelial cells were absent in teratomas, beta 1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in beta1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in beta 1-null embryoid bodies.

The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies.

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.

Control of cell growth by c-Myc in the absence of cell division.

The c-Myc protein (Myc) is a transcription factor, and deregulated expression of the c-myc gene (myc) is frequently found in tumours. In Burkitt's lymphoma (BL), myc is transcriptionally activated by chromosomal translocation. We have used a B-cell line called P493-6 that carries a conditional myc allele to elucidate the role of Myc in the proliferation of BL cells. Regulation of proliferation involves the coordination of cell growth (accumulation of cell mass) and cell division [1] [2] [3]. Here, we show that division of P493-6 cells was strictly dependent on the expression of the conditional myc allele and the presence of foetal calf serum (FCS). More importantly, cell growth was regulated by Myc without FCS: Myc alone induced an increase in cell size and positively regulated protein synthesis. An increase in protein synthesis is thought to be one of the causes of cell mass increase. Furthermore, Myc stimulated metabolic activities, as indicated by the acidification of culture medium and the activation of mitochondrial enzymes. Our results confirm the model that Myc is involved in the regulation of cell growth [4] and provide, for the first time, direct evidence that Myc induces cell growth, that is, an increase in cell size, uncoupled from cell division.

The transcriptional program of a human B cell line in response to Myc.

The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

CTp11, a novel member of the family of human cancer/testis antigens.

To identify new genes that may contribute to the metastatic pathway of neoplastic cells, we compared mRNA expression of the parental human melanoma cell line 1F6 and its metastatic variant 1F6m using mRNA differential display. We isolated a cDNA clone that was exclusively expressed in 1F6m. Northern blot analysis on a broader panel of human melanoma cell lines with different metastatic capacity following s.c. inoculation into nude mice demonstrated that the gene was expressed only in the most aggressive, highly metastatic cell lines, giving a band of 0.5 kb. The isolated full length cDNA clone showed an open reading frame of 97 amino acids. To study the subcellular localization of the gene product, COS-1 cells were transfected with cDNA of the gene fused to eGFP. We found the fusion protein to be exclusively present in the nucleus. A computer search showed strong homology with human genomic clones all localized on chromosome X (Xq26.3-Xq27.1) and with several expressed sequence tags, all from testis. Localization of the gene on chromosome X was confirmed by genomic PCR on a panel of human chromosome-specific rodent/human hybrid cell lines. Northern blotting and reverse transcription-PCR on 17 different normal human tissue samples showed that the gene was only expressed in normal testis. Reverse transcription-PCR on a great number of different human tumor cell lines showed expression in 25-30% of the melanoma and bladder carcinoma cell lines. Only 2 of 29 other tumor cell lines were positive. Nested PCR analysis of a series of fresh human melanocytic tumors demonstrated expression in 7 of 10 melanomas tested. No expression was seen in benign melanocytic tumors. In addition to melanoma, some malignant tumors from other histological types were also found to be positive. Based on these data, we conclude that the described gene, CTp11 (cancer/testis-associated protein of 11 kDa), is a novel member of the family of cancer/testis antigens.

Decreased expression of both the low-density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor and its receptor-associated protein in late stages of cutaneous melanocytic tumor progression.

We recently found that the proteins of the proteolytic system of plasminogen activation emerge in late stages of melanocytic tumor progression. A large body of evidence suggests a role for two proteins, the low-density lipoprotein receptor-related protein (LRP)/alpha(2)-macroglobulin receptor and its receptor-associated protein (RAP), in the internalization of components of the plasminogen activation system. Here, we present data on the presence of these two proteins in human melanoma cell lines which differ in metastatic capacity, their corresponding xenografts, and in cutaneous melanocytic lesions. With flow cytometry, we found surface expression of LRP to be restricted to urokinase plasminogen activator, producing highly metastatic cell lines. These cell lines also produce higher levels of LRP mRNA, whereas RAP mRNA and protein are expressed at equal levels in all cell lines and not expressed at the cell surface. Xenografts of cell lines producing high levels of LRP remarkably contain only a small fraction of LRP-positive tumor cells. Using immunohistochemistry on frozen sections of 107 human melanocytic lesions comprising the various stages of melanocytic tumor progression, we found that expression of both LRP and RAP decreased in tumor progression. Furthermore, we noted that LRP and RAP are coexpressed within the same lesion. Using immunofluorescence double staining, we found that LRP and RAP colocalize in the same cells in the lesions studied and in the same cell structures in the cell lines studied. In conclusion, our results indicate that LRP and RAP are coordinately expressed in a decreased fashion in melanocytic tumor progression. Based on the staining results in xenografts and in human melanocytic lesions, we conclude that a strong correlation between expression of LRP and urokinase-type plasminogen activator seems not to exist in in vivo melanomas.

Beta1 integrin promotes but is not essential for metastasis of ras-myc transformed fibroblasts.

To investigate the role of beta1 integrin during tumor metastasis, we established a ras-myc transformed fibroblastoid cell line with a disrupted beta1 integrin gene on both alleles (GERM 11). Stable transfection of this cell line with an expression vector encoding beta1A integrin resulted in beta1A integrin-expressing sublines. Tumors were induced by subcutaneous injection of GERM 11 cells and 3 independent beta1 integrin expressing sublines (GERM 116, 1A10, 2F2) into syngeneic mice. After 10 days tumors were surgically removed. While average weights of GERM 11 and GERM 116 tumors were similar, tumors induced by the high expressing clones 1A10 and 2F2 were markedly smaller, suggesting an inverse correlation of tumor growth and beta1 integrin expression. The metastasis potential of all three beta1 integrin-expressing GERM 11 sublines tested was significantly higher than that of the beta1-deficient GERM 11 cells. GERM 116 tumors led in all animals to severe metastasis in lung and liver, while GERM 11 tumors induced only a few metastatic foci in the lung. Stroma of both tumors contained nidogen and high amounts of tenascin C, but only a few very low levels of fibronectin, laminin-1, and collagen type I. Beta1 integrin, therefore, increases but is not essential for metastasis of ras-myc transformed fibroblasts.

Isolation of DICE1: a gene frequently affected by LOH and downregulated in lung carcinomas.

In the development and progression of sporadic tumors multiple tumor suppressor genes are inactivated that may be distinct from predisposing cancer genes. Previously, a tumor suppressor locus on human chromosome 13q14 that is distinct from the retinoblastoma predisposing gene 1 (RB1) has been identified in lung, head and neck, breast, ovarian and prostate tumors. By an approach that combines genomic difference cloning and positional cloning we isolated the cDNA of a novel gene (DICE1) located at 13q14.12-14.2. The DICE1 gene is highly conserved in evolution and its mRNA is expressed in a wide variety of fetal and adult tissues. The DICE1 cDNA encodes a predicted protein of 887 amino acids corresponding to an 100 kD protein that shows 92.9% identity to the carboxy-terminal half of the mouse EGF repeat transmembrane protein DBI-1. The DBI-1 protein interferes with the mitogenic response to insulin-like growth factor 1 (IGF-I) and is presumably involved in anchorage-dependent growth. When compared to normal lung tissue expression of the DICE1 mRNA was reduced or undetectable in the majority of non-small cell lung carcinomas analysed. The location of the DICE1 gene in the region of allelic loss, its high evolutionary conservation and the downregulation of expression in carcinoma cells suggests that DICE1 is a candidate tumor suppressor gene in non-small cell lung carcinomas and possibly in other sporadic carcinomas.

Chimeric interleukin 2 receptor alpha chain antibody derivatives with fused mu and gamma chains permit improved recruitment of effector functions.

In order to investigate the feasibility of shuffling effector functions of monoclonal antibodies, we constructed chimeric antibodies with fused heavy chains. The derivatives studied are based on a monoclonal antibody directed against the alpha chain of the human Il2-R. Derivatives studied were the IgG1 and IgM isotypes; IgM delta, lacking the ability of multimerization due to a deletion; IgMc gamma 1 and IgGlc mu, with fused mu and gamma 1 chains and vice versa. IgG1, IgM delta and IgMc gamma 1 were secreted as monomers, IgM and IgG1c mu as polymers. The Ki values for competition with radio-iodinated Il2 with respect to binding to the Il2-R were markedly lower for polymeric than for monomeric derivatives (300-400 pM versus 2500-6500 pM). Recruitment of complement mediated by the deposition of C3 fragments, either of heterologous (rabbit) or homologous (human) origin, was mediated only by the polymeric derivatives IgM and IgG1c mu. ADCC was mediated by monomeric IgG1 and polymeric IgG1c mu, the latter derivative being active at concentrations 100-fold lower than the former. Together, the results demonstrate that both CDC and ADCC effector functions can be combined on a polymeric antibody derivative with fused gamma 1 and mu chains. In addition, such a derivative, due to its polymeric nature, has a high binding affinity. These properties may be important for the elimination of target cells in vivo.

Increased expression of bone sialoprotein in bone metastases compared with visceral metastases in human breast and prostate cancers.

The recent demonstration that bone sialoprotein (BSP) is expressed in osteotropic cancers suggests that this bone matrix protein might be implicated in the preferential seed and growth of metastatic cells in bone. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. The exact mechanisms by which BSP may favor bone metastases formation are not clearly established yet. Although BSP expression has been detected in breast, prostate, lung, thyroid, and neuroblastoma primary tumors, no information regarding its expression in metastases is available to date. In this study, we have examined BSP expression in 15 bone and 39 visceral metastatic lesions harvested from 8 breast cancer patients and 7 prostate cancer patients who died of disseminated disease. We were able to retrieve the primary lesions from 5 of the 8 breast cancer patients as well as from all 7 prostate cancer patients. All the primary breast tumor patients and 5 of the 7 primary prostate cancer patients expressed a detectable level of BSP. Bone metastases from all 8 breast cancer patients and from 5 out of 7 prostate cancer patients exhibited detectable levels of the protein. Metastatic cells in close contact with bone trabeculae usually were highly positive for BSP. BSP also was detected in secondary lesions developed at visceral sites including liver, thyroid, lung, and adrenal glands. However, BSP expression was significantly lower in visceral metastases than in skeletal ones (Mann-Whitney test, p < 0.05). Our data represent the first demonstration of an increased expression of BSP in bone metastases compared with nonskeletal metastases in human breast and prostate cancers and add weight to the body of evidence attributing a significant role to this protein in the genesis of bone metastases.

Tumorigenic transformation of established murine fibroblasts by transfection with amplification promoting DNA elements.

Deregulated replication of the cellular genome is assumed to result in tumorigenic transformation of the cell. We tested this hypothesis by using mouse DNA elements that promote amplification of transfected DNA in mammalian cells when linked to a selectable marker gene that is driven by a truncated promoter (Holst et al., Cell 52, 355-365 (1988); Wegner et al., Nucl. Acids Res. 17, 9909-9932 (1989)). Here, the DNA elements muNTS-1, e-1, e-2, e-4, e-5, and e-12 were inserted into the plasmid p-hyg, which contains the gene for resistance to hygromycin B driven by a constitutive promoter. After transfer into established rat fibroblasts (208F), transfected DNA constructs persist in low copy numbers (1-10 copies per cell) integrated into high molecular weight DNA. We observed a neoplastically transformed phenotype in 40% to 70% of hygromycin-resistant colonies of 208F cells which is dependent on the DNA element transfected. 208F cells transfected with vector DNA exhibit a "normal" phenotype and are not tumorigenic. The transformed cells, on the other hand, induced malignant tumors after injection into immunodeficient NMRI nu/nu mice. In contrast to established cells, primary rat embryo fibroblasts (REF) with limited life span are neither neoplastically transformed nor immortalized after transfection of e-4 DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of stable mouse myeloma cells constitutively secreting human tissue-type plasminogen activator.

Expression plasmids for human tissue-type plasminogen activator (t-pA) were introduced into mouse myeloma cells and stable cell lines constitutively secreting t-pA established by selection with mycophenolic acid. Expression of t-pA is driven either by the simian virus 40 early promoter or by immunoglobulin regulatory elements of either light or heavy chains of the mouse. The availability of myeloma cells secreting a heterologous protein is of importance for biotechnological applications, because large-scale fermentation of myeloma cells is well established.

Kringle 1 domain of human tissue-type plasminogen activator is a functional module mediating fibrinogen-stimulated plasminogenolytic activity.

The functions of the finger and kringle-2 (K2) domains of human tissue-type plasminogen activator (t-PA) in mediating fibrin-stimulated plasminogenolytic activity are well documented. Contradictory results have been reported for the kringle-1 (K1) domain with respect to this property. To clarify this issue we have deleted the finger and the K2 domains of t-PA according to the exon-intron organization of the gene by site-directed mutagenesis. The resulting derivative (GK1L) was constitutively expressed in permanent clones of Chinese hamster ovary cells. The secreted proteins have been partially purified and characterized by Western blotting. Since the plasminogenolytic activity of GK1L is stimulated by fibrin, the K1 domain of t-PA must be a functional domain in this context.

Expression of heterobispecific antibodies by genes transfected into producer hybridoma cells.

We report the expression of heterobispecific antibodies (Ab) by transferred genes. The kappa and gamma 1 genes of a mouse anti-idiotypic Ab (IgG1) were transfected into a mouse hybridoma cell line secreting Ab (IgG1), directed against an isoenzyme of human creatine kinase. Stable cell lines secreting the parental Ab derived from the introduced genes and a mixture of hybrid Ab were established. The transfected Ab specificity was expressed at similar levels as in a nonproducer background (50 ng-1 microgram/ml), heterobispecific Ab was expressed in microgram quantities (1-4 micrograms/ml) in all cell lines examined. As shown by isoelectric focusing analysis, hybrid Ab (heterobifunctional and other species) are expressed to a similar extent in the transfected cell lines as the Ab in the parental Ab-producing cells.

Genes encoding a mouse monoclonal antibody are expressed in transgenic mice, rabbits and pigs.

To study the expression pattern of immunoglobulin-encoding genes in transgenic animals, we have introduced the genes for the light and heavy chain of a mouse monoclonal antibody (mAb) into the germ-line of mice (control), rabbits and pigs. The transgenes were detected in the mouse lines, two rabbit lines and pigs. Titers of 100-200 micrograms mAb/ml (rabbits) and up to 1000 micrograms mAb/ml (pig) were measured in the sera of the transgenic animals. Isoelectric focusing experiments with serum followed by immunofixation revealed that in the transgenic pig only a minority of the bands were identical to those of the purified mouse mAb. In transgenic rabbits we found no coincidence of bands at all. The results can be explained by assuming tissue- and cell-type-specific glycosylation, modification and possible heterologous chain associations. Expression of Ab in the serum of animals could help to protect against diseases (e.g., influenza in pigs).

Expression of antibody cDNA in murine myeloma cells: possible involvement of additional regulatory elements in transcription of immunoglobulin genes.

Expression vectors for cDNA of the kappa and gamma 1 chains of a monoclonal antibody directed against creatine kinase were introduced into murine myeloma cells. Kappa and gamma 1 cDNA were either under the control of the SV40 early promoter or of the cognate promoters and enhancers of the light- and heavy-chain genes. Secretion of immuno-reactive kappa and gamma 1 chains into the culture medium was demonstrated with the SV40 promoter as well as with the cognate promoters. Expression of gamma 1 cDNA with the SV40 early promoter was about twice as high as with the heavy-chain promoter and enhancer. Expression of kappa cDNA under the control of the SV40 early promoter was about 17 times higher than with the light-chain promoter and enhancer. These expression levels were compared to those of a genomic immunoglobulin (Ig) kappa determinant, including introns. Such an entire kappa gene led to expression of the light chain at levels double those with the kappa cDNA construction using the SV40 promoter and about 35 times as high when using kappa cDNA and the cognate promoter and enhancer. This result might indicate that, besides the cognate promoter and enhancer elements, other intragenic elements are involved in the regulation of Ig expression. However, the SV40 early promoter seems to be able to compensate for the absence of these postulated regulatory elements probably located in the introns.

Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective pressure.

By linking an expression cassette for human tissue-type plasminogen activator (t-PA) to an amplifiable marker gene, its introduction into Chinese hamster ovary dhfr- cells and subsequent amplification with methotrexate, we have generated cell lines that overproduce the heterologous protein and contain 300-1100 copies of the expression constructs integrated into the hamster genome. We present a detailed investigation of the fate of amplified sequences in the presence and absence of selective pressure by parallel examination of three producer cell lines with respect to relevant parameters. These include the determination of t-PA production upon continuous propagation in culture, the genomic organization of the integrated expression constructs by Southern blotting, and the localization of homogeneously staining regions by in-situ hybridization with biotinylated probes and visualization by interference reflection microscopy. We conclude that in the three cell lines examined, the decrease in production of t-PA in the absence of methotrexate selection is accompanied by decreases in the number of integrated expression constructs and the size of the amplified regions, whereas all these parameters are stable when selective pressure is maintained. The instability is probably due to the head-to-tail mode of integration of the expression constructs in the hamster genome, which increases the frequency of homologous recombination between the integrated plasmids in recombination-proficient cells in the absence of selective pressure.

A new expression system for mammalian cells based on putative replicator sequences of the mouse and a truncated thymidine kinase gene.

We have constructed a new expression vector for mammalian cells. The vector contains a truncated tk gene for amplification under selective conditions, a sequence putatively supporting the replication of plasmid DNA in eukaryotic cells (murine autonomously replicating sequence) and an expression cassette for the cDNA to be studied. As a model cDNA we have used that of human tissue-type plasminogen activator (t-PA). Analysis of Hirt supernatants and chromosomal DNA from L cells, prepared six weeks after isolation of the clones indicated a 50- to 500-fold amplification of the expression construct in the cells. Concomitantly, the expression of t-PA was dramatically increased. Our data are consistent with episomal persistence of the expression construct, with a head-to-tail mode of integration into the mouse genome and with coexistence of both episomal plasmids and head-to-tail integrates. In tk-deficient cell lines other then L-cells, such as mouse mastocytoma or rat hepatoma cells, a strong selection against the persistence of the expression construct was noted. After long-term propagation of the L-cells under selective conditions the expression of the indicator gene continually decreases, but finally a constant plateau level of expression is established. Expression could be restored to the original level by blocking more efficiently the de novo synthesis of nucleosides.

A protease-hypersensitive deletion derivative of human tissue-type plasminogen activator.

We have constructed amplified Chinese hamster ovary cell lines constitutively synthesizing human tissue-type plasminogen activator (t-PA) or a derivative in which the domains homologous to epidermal growth factor and kringle 1 have been removed [delta(G + K1)]. The properties of the secreted proteins were investigated when synthesized in the presence or absence of the serine protease inhibitor aprotinin in the medium. t-PA in the culture supernatants was either single-chain or two-chain protein. The protease activity of both forms was stimulated by fibrin. The biochemical properties of delta(G + K1) were significantly different when harvested from cells grown under different culturing conditions. Protease activity of delta(G + K1) was stimulated ten- to 20-fold by fibrin when harvested from medium with aprotinin, but was stimulated only two- to three-fold when aprotinin was absent from the serum. Characterization of the secreted proteins revealed that the heavy-chain equivalent of delta(G + K1) is degraded when serine protease inhibitor is absent in the culture medium. These results indicate that the functional and biochemical properties of restructured versions of t-PA may depend on the presence of protease(s) in the culture supernatants.