Vectors and methods for recombinant production of uPA-binding fragments of the human urokinase-type plasminogen receptor (uPAR)

Activation of plasminogen to plasma is inhibited by preventing the binding of a receptor binding form of urokinase-type plasminogen activator to a urokinase-type plasminogen activator receptor in a mammal, thereby preventing the urokinase-type plasminogen activator from converting plasminogen into plasmin. DNA fragments which encode for soluble, active fragments of the urokinase-type plasminogen activator are provided.

FIELD OF INVENTION 
The present invention relates to a method for preventing or counteracting 
localized proteolytic activity in a mammal, in particular a human, the 
method comprising inhibiting the activation of plasminogen to plasmin by 
preventing the binding of a receptor binding form of urokinase-type 
plasminogen activator (in the following termed u-PA) to a u-PA receptor in 
the mammal and thereby preventing the u-PA from converting plasminogen 
into plasmin; the invention also relates to a pure u-PA receptor (in the 
following termed u-), to DNA coding for the u-, to the production of 
u- or parts thereof for use as a therapeutic or diagnostic component, 
to u- antibodies and the production of u-PA receptor binding u-PA 
molecules for use as a therapeutic or diagnostic component. In a further 
aspect, the invention relates to the regulation of the activity of a 
receptor binding form of u-PA, the activation of pro-u-PA to u-PA by 
plasmin and the regulation of the number of u-s on the cell and the 
binding affinity of the u-/u-PA binding as well as the therapeutic 
aspects of these findings. In yet a further aspect, the invention relates 
to the detection of u- by labelled u-PA. 
GENERAL BACKGROUND 
According to the literature, urokinase-type plasminogen activator (u-PA) 
has been found in all mammalian species so far investigated. Several 
findings relate u-PA to tissue degradation and/or cell migration, 
presumably through a breakdown of the extracellular matrix, caused by 
plasmin together with other proteolytic enzymes. This relation has been 
most extensively studied in postlactational involution of the mammary 
gland and the early phase of trophoblast invasion after implantation of 
the fertilized egg in the uterus. The hypothesis of a role of u-PA in 
tissue degradation and cell migration is further supported by the more 
exact localization made possible by the immunocytochemical findings of 
u-PA in epithelial cells of involuting mammary glands, in areas with 
tissue degradation in psoriasis, in association with the release of 
spermatocytes during spermatogenesis, and in keratinocytes of the 
epithelial outgrowth during wound healing (see Dan.o slashed. et al., 
1988, Gr.o slashed.ndal-Hansen et al., 1988). 
It is also conceivable that u-PA plays a role in the degradative phase of 
inflammation, and there have also been reports that u-PA interferes with 
the lymphocyte-mediated cytotoxicity against a variety of cells, and a 
direct role of u-PA in the cytotoxic effect of natural killer cells has 
been proposed. A role of u-PA has been proposed in angiogenesis and in 
endothelial cell migration, a process important in tumor growth. 
u-PA is produced by many cultured cell types of neoplastic origin. It has 
been found that explants of tumor tissue released more u-PA than the 
corresponding normal tissue. u-PA has been identified in extracts from 
human lung, colon, endometrial, breast, prostate and renal carcinomas, 
human melanomas, murine mammary tumors, the murine Lewis lung tumor, and 
in ascites from human peritoneal carcinomatosis. An immunohistochemical 
study of invasively growing and metastasing Lewis lung carcinomas in mice 
consistently showed the presence of u-PA, but also a pronounced 
heterogenecity in the content of u-PA in different parts of the individual 
tumors. A high u-PA content was found in areas with invasive growth and 
degradation of surrounding normal tissue, while other areas were devoid of 
detectable u-PA. The u-PA was located in the cytoplasm of the tumor cells 
and extracellularly surrounding the tumor cells. 
Degradation of the surrounding normal tissue is a central feature of 
invasiveness of malignant tumors. The constant finding of u-PA in 
malignant tumors and the findings indicating that u-PA plays a role in 
tissue degradation in normal physiological events have led to the 
assumption that u-PA plays a similar role in cancer development. The 
hypothesis of u-PA playing a role in tissue destruction involves the 
assumption that plasmin, together with other proteolytic enzymes, degrades 
the extracellular matrix. It is noteworthy in this context that most 
components of the extracellular matrix can be degraded by plasmin. These 
include laminin, fibronectin, proteoglycans, and possibly some types of 
collagen, but not all. In addition, as originally reported by Vaes and 
collaborators, plasmin can activate latent collagenases which in turn can 
degrade the other types of collagen (see Dan.o slashed. et al., 1988). 
The majority of the cancer patients in the treatment failure group succumb 
to the direct effects of the metastases or to complications associated 
with the treatment of metastases. Therefore, much research has been 
focused on identifying specific biochemical factors which can be the basis 
for diagnostic or therapeutic strategies. The extracellular matrix is 
composed of glycoproteins such as fibronectin and laminin, collagen and 
proteoglycans. Extracellular matrix becomes focally permeable to cell 
movement only during tissue healing and remodelling, inflammation, and 
neoplasia. Liotta (1986) has proposed a three-step hypothesis: The first 
step is tumor cell attachment via cell surface receptors. The anchored 
tumor cell next secretes hydrolytic enzymes (or induces host cells to 
secrete enzymes) which can degrade the matrix locally (including 
degradation of the attachment components). Matrix lysis most probably 
takes place in a highly localized region close to the tumor cell surface. 
The third step is tumor cell locomotion into the region of the matrix 
modified by proteolysis. Thus, invasion of the matrix is not merely due to 
passive growth pressure but requires active biochemical mechanisms. 
Many research groups have proposed that invasive tumor cells secrete 
matrix-degrading proteinases. A cascade of proteases including serine 
proteases and thiol proteases all contribute to facilitating tumor 
invasion. One of the crucial cascades is the plasminogen activation 
system. Regulation of the proteolysis can take place at many levels 
including tumor cell-host cell interactions and protease inhibitors 
produced by the host or by the tumor cells themselves. Expression of 
matrix-degrading enzymes is not tumor cell specific. The actively invading 
tumor cells may merely respond to different regulatory signals compared to 
their non-invasive counterparts (Liotta, 1986). 
The assumption that the plasminogen activation system, through a breakdown 
of extracellular matrix proteins, plays a role in invasiveness and 
destruction of normal tissue during growth of malignant tumors is 
supported by a variety of findings. These include a close correlation 
between transformation of cells with oncogenic viruses and synthesis of 
u-PA, the finding that u-PA is involved in tissue destruction in many 
non-malignant conditions, and the immunohistochemical localization of u-PA 
in invading areas of tumors (see Dan.o slashed. et al., 1985, Saksela, 
1985, for reviews). 
Further support for this hypothesis has come from studies with 
anticatalytic antibodies to u-PA in model systems for invasion and 
metastasis. Such antibodies were found to decrease metastasis to the lung 
from a human u-PA producing tumor, HEp-3, transplanted onto the 
chorioallantoic membrane of chicken embryos (Ossowski and Reich, 1983, 
Ossowski 1988), penetration of amniotic membranes by B16 melanoma cells 
(Mignatti et al., 1986), basement membrane invasion by several human and 
murine cell lines of neoplastic origin (Reich et al., 1988), and formation 
of lung metastasis after intravenous injection of B16 melanoma cells in 
mice (Hearing et al., 1988). In some of these studies (Mignatti et al., 
1986, Reich et al., 1988), a plasmin-catalyzed activation of 
procollagenases (see Tryggvason et al., 1987) appeared to be a crucial 
part of the effect of plasminogen activation. 
A requirement for the regulation of a proteolytic cascade system in 
extracellular processes is the precise localization of its initiation and 
progression. For example, in the complement and coagulation systems, 
cellular receptors for various components are known and serve to localize 
reactions that either promote or terminate the reaction sequence 
(Muller-Eberhard, 1988, Mann et al., 1988). In the plasminogen activation 
system, the role of fibrin in the localization of plasminogen activation 
catalyzed by the tissue-type plasminogen activator (t-PA) is well known 
(Thorsen et al., 1972, Hoylaerts et al., 1982). 
Immunocytochemical studies have suggested that in the invasive areas of 
tumors, u-PA is located at the membrane of the tumor cells (Skriver et 
al., 1984), and recent findings indicate that at cell surfaces, u-PA is 
generally bound to a specific receptor and that this localization may be 
crucial for the regulation of u-PA catalyzed plasminogen activation in 
time and space (see Blasi et al., 1987). Preliminary reports suggest that 
also t-PA may bind to cell surface receptors and retain its enzymatic 
activity (Beebe, 1987, Barnathan et al., 1988, Hajjar and Nachmann, 1988, 
Kuiper et al., 1988). This phenomenon, however, awaits further 
clarification concerning the nature of the binding sites. 
Surface receptor for u-PA 
The cellular receptor for u-PA (u-) was originally identified in blood 
monocytes and in the monocyte-like U937 cell line (Vassalli et al., 1985), 
and its presence has been demonstrated on a variety of cultured cells, 
including several types of malignant cells (Stoppelli et al., 1985, 
Vassalli et al., 1985, Plow et al., 1986, Boyd et al., 1988a, Nielsen et 
al., 1988), human fibroblasts (Bajpai and Baker, 1985), and also in human 
breast carcinoma tissue (Needham et al., 1987). The receptor binds active 
54 kD u-PA, its one-polypeptide chain proenzyme, pro-u-PA (see below), as 
well as 54 kD u-PA inhibited by the active site reagent DFP, but shows no 
binding of the low molecular weight (33 kD) form of active u-PA (Vassalli 
et al., 1985; Cubellis et al., 1986). Thus, binding to the receptor does 
not require the catalytic site of u-PA, and in agreement with these 
findings, the binding determinant of u-PA has been identified in the 
amino-terminal part of the enzyme, in a region which in the primary 
structure is remote from the catalytic site. The receptor binding domain 
is located in the 15 kD amino-terminal fragment (ATF, residues 1-135) of 
the u-PA molecule, more precisely within the cysteine-rich region termed 
the growth factor region as this region shows homologies to the part of 
epidermal growth factor (EGF) which is responsible for binding to the EGF 
receptor. The amino acid residues which appear to be critical for binding 
are located within the sequence 12-32 (Appella et al., 1987). Synthetic 
peptides have been constructed that inhibit the binding of very low (100 
nM) concentrations. The lack of cross-reactivity between the murine and 
the human peptides indicates that the binding between u-PA and u- is 
strongly species specific. 
Binding of u-PA to u- is specific in the sense that as yet no other 
protein has been found to compete for binding to the receptor, though 
several proteins structurally related to u-PA, including t-PA and 
plasminogen, have been tested (Stoppelli et al., 1985, Vassalli et al., 
1985, Nielsen et al., 1988). Fragments of u-PA containing only the 
receptor binding domain, e.g. ATF, ensure specificity of the binding to 
the receptor, since other molecules that might bind u-PA (protease nexin 
and the specific plasminogen activator inhibitors PAI-1 and PAI-2) 
recognize the catalytically active region (Stoppelli et al., 1985; Nielsen 
et al., 1988). PAI-1 is able to form a covalent complex with u-PA but not 
with pro-u-PA (Andreasen et al., 1986). 
The number of receptors reported varies strongly among the cell types 
studied, from a few thousand molecules per cell on normal monocytes (Miles 
and Plow, 1987) up to 3.times.10.sup.5 on some colon carcinoma cell lines 
(Boyd et al., 1988a), and some variation apparently also occurs in the 
binding affinity, which is in the 0.1-10 nM range (for a review, see Blasi 
1988). Further, on certain cell lines the number of receptors can be 
regulated by the addition of various agents such as phorbol myristate 
acetate (PMA) in U937 cells (Stoppelli et al., 1985, Nielsen et al., 
1988), epidermal growth factor in A431 cells (Blasi et al., 1986) and HeLa 
cells (Estreicher et al., 1989) and dimethylformamide in colon carcinoma 
cells (Boyd et al., 1988b). In the first-mentioned case, a large decrease 
in affinity for the ligand occurs concomitantly with an increase in the 
number of receptors (Nielsen et al., 1988, Picone et al., 1989). 
Preliminary molecular studies on the u-PA receptor have been carried out. A 
u-PA receptor assay has been developed and an approximately 2200-fold 
purification has been accomplished, using metabolically labelled material 
and affinity chromatography with immobilized pro-u-PA (Nielsen et al., 
1988). Characterization of the partly purified protein has shown that the 
receptor is a 55-60 kD glycoprotein, the molecular weight of which is 
unchanged after cleavage of disulfide bonds, suggesting that it consists 
of a single polypeptide chain. Until the present invention, nothing was 
known about the structural properties of the receptor, responsible for 
binding to the ligand. In the study of Nielsen et al., the purified u- 
preparation shows essentially one radiolabelled band after SDS-PAGE 
followed by autoradiography. This analysis, however, does not show the 
purity of the preparation as it does not detect unlabelled proteins that 
may be present in an amount that may be higher than that of the u-. 
Similar considerations hold true for a recent study by Estreicher et al. 
(1989), in which attempts at purifying u- were done from cells that had 
been surface-labelled with .sup.125 I. By detergent separation followed by 
incubation with diisopropylfluorophosphate labelled u-PA (DFP-u-PA) and 
affinity chromatography with immobilized antibodies to u-PA, a labelled 
band of approximately 45,000 kD was obtained after SDS-PAGE and 
autoradiography. It is not clear whether this band represents u-. No 
cross-linking studies have been performed on the purified preparation, and 
its apparent molecular weight is distinctly lower than than of u- as 
reported by Nielsen et al. (1988) and found in the present study (see 
Example 1). In addition, it cannot be evaluated whether contaminating 
non-labelled proteins are present, and as only a part of the lane in the 
SDS-PAGE is shown, even an evaluation of whether contaminating labelled 
proteins are present is impossible. 
Preparation of antibodies to u- has hitherto not been described. 
Proenzyme to u-PA (pro-u-PA) 
Several studies have indicated that u-PA is released from many types of 
cultured cells as a single-chain proenzyme with little or no plasminogen 
activating capacity (Nielsen et al., 1982, Skriver et al., 1982, Eaton et 
al., 1984, Kasai et al., 1985, Pannell and Gurewich 1987). By limited 
proteolysis with catalytic amounts of plasmin, this proenzyme can be 
converted to its active two-chain counterpart. The proenzyme nature of 
single-chain u-PA is also reflected in the finding that it has essentially 
no amidolytic activity with synthetic substrates (Wun et al., 1982, Eaton 
et al., 1984, Lijnen et al., 1986, Stump et al., 1986a, 1986b, Nelles et 
al., 1987, Pannell and Gurewich 1987), and that it has little or no 
reactivity with macromolecular inhibitors (Eaton et al., 1984, Vassalli et 
al., 1985, Andreasen et al., 1986, Stephens et al., 1987) and synthetic 
inhibitors (Nielsen et al., 1982, Skriver et al., 1982, Wun et al., 1982, 
Gurewich et al., 1984, Kasai et al., 1985). 
This picture of single-chain u-PA as an essentially inactive proenzyme is 
in contrast to the interpretation reached by some other investigators 
(Collen et al., 1986, Lijnen et al., 1986, Stump et al., 1986a, 1986b). 
They concluded that single-chain u-PA from several sources had 
considerable plasminogen activating capability, and that recombinant 
single-chain u-PA had an activity that was even higher than that of 
two-chain u-PA. For these studies, a coupled plasminogen activation assay 
was used in which the activity of generated plasmin was measured with a 
chromogenic substrate. Such assays for pro-u-PA are self-activating and 
are strongly influenced by small amounts of contaminating or generated 
two-chain u-PA or plasmin. As discussed in detail elsewhere (Petersen et 
al., 1988), it is therefore possible that the high activity of one-chain 
u-PA found in these studies was apparent and not due to intrinsic activity 
of single-chain u-PA. Consistent with this interpretation is a report on a 
variant of recombinant single-chain u-PA which by site-directed 
mutagenesis was made partly resistant to plasmin cleavage. This variant of 
single-chain u-PA had an activity that in coupled assays was 200-fold 
lower than that of two-chain u-PA (Nelles et al., 1987). 
Recent kinetic studies, which included measures to prevent self-activation 
in the assays for pro-u-PA, have confirmed the low intrinsic activity of 
pro-u-PA (Ellis et al., 1987, Petersen et al., 1988, Urano et al., 1988). 
In one study with a highly purified preparation of pro-u-PA from HT-1080 
fibrosarcoma cells, it was shown that the pro-u-PA had a capacity for 
plasminogen activation that was lower than that of a 250-fold lower 
concentration of two-chain u-PA. It was not possible to decide whether 
this low activity was intrinsic or due to contamination (Petersen et al., 
1988). 
In the intact organism, pro-u-PA is the predominant form of u-PA in 
intracellular stores, and it also constitutes a sizable fraction of the 
u-PA in extracellular fluids (Skriver et al., 1984, Kielberg et al., 
1985). Extracellular activation of pro-u-PA may therefore be a crucial 
step in the physiological regulation of the u-PA pathway of plasminogen 
activation. The plasmin-catalyzed activation of pro-u-PA provides a 
positive feedback mechanism that accelerates and amplifies the effect of 
activation of a small amount of pro-u-PA. The initiation of the u-PA 
pathway of plasminogen activation under physiological conditions, however, 
involves triggering factors that activate pro-u-PA as described herein. 
Mutants of human single-chain pro-u-PA in which lysine 158 is changed to 
another amino acid (e.g. Glu or Gly) are not, or are only to a small 
extent, converted to active two-chain u-PA (Nelles et al., 1987). 
u-PA at focal contact sites 
At the surface of HT-1080 fibrosarcoma cells and human fibroblasts, u-PA 
has been found to be unevenly distributed, distinctly located at cell-cell 
contact sites and at focal contacts that are the sites of closest 
apposition between the cells and the substratum (Pollanen et al., 1987, 
1988, Hebert and Baker 1988). u-PA was not detected in the two other types 
of cell-substratum contact, i.e. close contacts and fibronexuses, making 
it an intrinsic component at focal contact sites (Pollanen et al., 1988). 
u-PA at the focal contact sites is receptor-bound (Hebert and Baker, 
1988). The focal contact sites are located at the termini of 
actin-containing microfilament bundles, the so-called stress fibers or 
actin cables (Burridge, 1986). These sites contain several structural 
components (actin, talin) and regulatory factors (the tyrosine kinase 
protooncogene products P60.sup.src, P120.sup.gag-abl, P90.sup.gag-yes, 
P80.sup.gag-yes), that are all located on the cytoplasmic side (see 
Burridge, 1986). 
Plasminogen binding sites on cell surfaces 
Plasminogen, as well as plasmin, binds to many types of cultured cells, 
including thrombocytes, endothelial cells and several cell types of 
neoplastic origin (Miles and Plow, 1985, Hajjar et al., 1986, Plow et al., 
1986, Miles and Plow 1987, Burtin and Fondaneche, 1988). The binding is 
saturable with a rather low affinity for plasminogen (K.sub.D 1 .mu.M). At 
least in some cell types, binding of plasmin appears to utilize the same 
site as plasminogen, but the binding parameters for plasmin indicate that 
more than one type of binding site for plasminogen and plasmin may exist. 
Thus, on some cell types, plasmin and plasminogen bind with almost equal 
affinity (Plow et al., 1986), while on others plasmin apparently binds 
with a higher affinity (K.sub.D 50 nM) than plasminogen (Burtin and 
Fondaneche, 1988). The binding is inhibited by low amounts of lysine and 
lysine analogues and appears to involve the kringle structure of the heavy 
chains of plasminogen and plasmin (Miles et al., 1988). 
The binding capacity varies between cell types and in many cell types is 
quite high (10.sup.5 -10.sup.7 binding sites per cell). The chemical 
nature of the binding sites are not known. A membrane protein, GPIIb/IIIa, 
seems to be involved in the binding of plasminogen to thrombocytes (Miles 
et al., 1986) and, particularly on thrombin-stimulated thrombocytes, also 
fibrin may be involved in plasminogen binding (Miles et al., 1986). In its 
purified form, the thrombocyte protein thrombospondin forms complexes 
(K.sub.D 35 nM) with plasminogen (Silverstein et al., 1984). Also 
immobilized laminin (Salonen et al., 1984) and fibronectin (Salonen et 
al., 1985) bind plasminogen (K.sub.D 3 nM and 90 nM, respectively) 
Surface plasminogen activation 
Some cell types bind both u-PA and plasminogen (Plow et al., 1986, Miles 
and Plow, 1987, Burtin and Fondaneche, 1988, Ellis et al., 1988). 
Receptor-bound pro-u-PA can be activated by plasmin (Cubellis et al., 
1986) and, at least in part, receptor-bound two-chain u-PA retains its 
ability to activate plasminogen (Vassalli et al., 1985). 
Addition of u-PA and plasminogen to cells holding binding sites for both 
molecules leads to the occurrence of cell-bound plasmin (Plow et al., 
1986, Burtin and Fondaneche, 1988). These studies did not allow a rigorous 
discrimination between an activation process occurring in solution or 
between surface-bound reactants. 
An interaction between binding sites for u-PA and plasminogen is suggested 
by the finding that u-PA binding in two cell lines led to an increased 
binding capacity for plasminogen. Binding of plasminogen in these studies 
had no effect on the binding capacity for u-PA (Plow et al., 1986). An 
enhancement of u-PA binding caused by plasminogen was also found by Burtin 
and Fondaneche (1988) in a cell line of neoplastic origin, even though the 
plasminogen binding sites demonstrated in the two studies were apparently 
not identical (see above). 
Recently, Ossowski (1988) published findings that the invasive ability of 
human tumor cells (into modified chick embryo chorioallantoic membranes in 
an in vivo assay) which have surface u-PA receptors, but which do not 
produce u-PA, could be augmented by saturating their receptors with 
exogenous u-PA. This finding, however, is only suggestive (as stated by 
the author) and it does not demonstrate that binding to the receptor per 
se is necessary. It is possible that the u-PA added to the cells was 
carried to the site of invasiveness because of receptor binding, but 
released from the receptor before exerting its activation. In addition, 
this study was carried out with two-chain u-PA and therefore does not 
simulate endogenous u-PA of the single-chain form. In the study of 
Ossowski, it was also found that an increased production of mouse u-PA in 
human cells transfected with mouse u-PA cDNA under the control of a human 
heat shock promoter did not increase invasiveness. Mouse u-PA does not 
bind to human u-, but the published data cannot be taken as a proof 
that this lack of effect of mouse u-PA is due to this lack of receptor 
binding because several other explanations are possible, e.g. 1) that the 
mouse u-PA does not activate chicken plasminogen as efficiently as human 
u-PA, 2) that in this system there are lacking mechanisms of converting 
one-chain mouse u-PA to the two-chain form, 3) that the heat shock in 
itself decreases the ability of the cells to invade, 4) that the heat 
shock treatment does not increase the production of mouse u-PA when it is 
followed by implantation that changes the microenvironments of the cells. 
No attempts were made in this study to investigate the effect on invasion 
of displacement of u-PA from its receptor. 
Ellis et al. (1989) recently published evidence indicating that the 
reactions leading to plasminogen activation can take place when 
single-chain u-PA and plasminogen are added to U937 cells, and that they 
occur more efficiently when both plasminogen and pro-u-PA are bound to the 
surface. This experiment, however, was performed in the absence of serum, 
i.e. under conditions where the plasminogen activation with the 
preparations used by Ellis et al. will also take place in solution (cf. 
Ellis et al., 1987), and these studies do not exclude the possibility that 
one or more of the processes involved (e.g. the plasminogen activation 
catalyzed by two-chain u-PA) actually occurred when the u-PA was not 
receptor-bound. Moreover, these studies used a purified preparation of 
single-chain u-PA that has a catalytic activity considerably higher than 
that found for single-chain u-PA by other groups (Pannell and Gurewich, 
1987; Urano et al., 1988; Petersen et al., 1988). Ellis' preparation may 
therefore be contaminated with two-chain u-PA and thus be distinctly 
different from the endogenous single-chain u-PA produced by cells in situ. 
In the experiments according to Ellis et al., 1989, binding of the added 
single-chain u-PA to the receptor was prevented by preincubation of the 
cells with the amino-terminal fragment of u-PA. These experiments do not, 
therefore, as do the following examples, demonstrate displacement of 
endogenously produced u-PA, a prerequisite for any therapeutic use of this 
approach. 
SUMMARY OF THE INVENTION 
The present invention is based upon the discovery that under conditions 
similar to those present extracellularly in the intact organism (i.e. in 
the presence of serum containing inhibitors of plasmin and of plasminogen 
activators), plasminogen activation initiated by endogenous u-PA occurs 
only when the u-PA is receptor-bound, upon the provision of pure u-PA 
receptor, and upon the provision of the possibility of producing the u-PA 
receptor or characteristic and valuable sequences thereof or analogues to 
sequences thereof by recombinant DNA technology. On the basis of these 
findings and developments, new and potentially extremely valuable 
therapeutic, prophylactic and diagnostic methods and products, together 
with associated basic methods and products, are provided by the present 
invention. 
Plasminogen binds to cell surfaces, and surprisingly it was found that a 
large part, if not all, of the cell surface plasminogen activation is 
catalyzed by surface-bound u-PA, and that binding of plasmin to the 
surface is necessary for the activation of pro-u-PA. In the absence of 
plasminogen, most of the cell surface u-PA is present in its single-chain 
proenzyme form (pro-u-PA), while addition of plasminogen leads to the 
formation of receptor-bound two-chain u-PA. The latter reaction is 
catalyzed by cell-bound plasmin. Receptor-bound u-PA is accessible to 
inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound 
plasmin is inaccessible to serum inhibitors. 
A model for cell-surface plasminogen activation can be made in which 
plasminogen binding to cells is followed by plasminogen activation by 
trace amounts of bound active u-PA, to form bound plasmin, which in turn 
serves to produce more active u-PA from bound pro-u-PA. This exponential 
process is subject to regulation by endogenous PAI-1, and limited to the 
pericellular space. 
The new findings include the requirement, in the presence of serum, for 
binding of plasminogen, the ability of bound u-PA under these conditions 
to activate plasminogen, the presence of pro-u-PA on the cells, the 
ability of bound plasmin to activate pro-u-PA, and the ability of 
endogenous plasminogen activator inhibitor PAI-1, as well as added 
plasminogen activator inhibitor PAI-2, to regulate the surface plasminogen 
activation. By these means tumor cells can acquire the broad-spectrum 
proteolytic activity of plasmin, bound to their surface in such a way that 
it is protected from inactivation by serum protease inhibitors, and 
ideally situated to be employed in the degradation of the pericellular 
matrix. 
Binding of u-PA to its receptor localizes u-PA not only to the cell 
surface, but focalizes it to distinct parts of the surface that at least 
in some cell types are the cell-cell and cell-substrate contact. The 
location of pro-u-PA at the focal contact sites suggests that u-PA 
catalyzed plasminogen activation is involved in the breakdown of the 
contacts, e.g. during cell movement. A selective activation of pro-u-PA at 
these sites provides a means of obtaining a directional pericellular 
proteolysis. pro-u-PA activation might be intracellularly initiated and 
mediated by a transmembrane signal through the u-PA receptor. 
Human tumor cells are very commonly found to secrete plasminogen activator 
of the urokinase type (u-PA). By this means they are able to recruit the 
proteolytic potential available in the high concentration of plasminogen 
in plasma and other body fluids. The invasive properties of tumor cells 
may be at least partly dependent on their proteolytic capability mediated 
through the broad spectrum of activity of plasmin and including its 
indirect actions in activating other latent proteases, such as 
collagenases. The expression of protease activity by tumor cells 
facilitates their penetration of basement membranes, capillary walls and 
interstitial connective tissues, allowing spread to other sites and 
establishment of metastases. 
A stepwise pathway of pericellular proteolysis geared to cell migration can 
be envisaged: binding of u-PA and plasminogen to the cell surface will 
lead to extracellular proteolysis and to the local severing of cell-cell 
and cell-substrate connections. This region of the cell is therefore free 
to move and this will transpose u-PA to a region in which PAI-1 is 
present. PAI-1 will inactivate u-PA and in the absence of local 
proteolytic activity, the cell will form new connections with the matrix, 
a process required for further migration. 
The expression of the u-PA gene is finely regulated by a variety of agents 
that affect cell growth; however, until recently very little was known of 
the regulation of the u- function and synthesis. It is known that the 
affinity of the u-PA receptor can be modified by e.g. the tumor promoter 
PMA. This indicates that the cells are endowed with mechanisms that 
modulate the u-PA:u- interaction. While this interaction appears to act 
at the level of the receptor itself, the effect of the plasminogen 
activator inhibitors demonstrates a second level of modulation, i.e. at 
the level of the active ligand itself. It is possible that the two levels 
of regulation might actually be interconnected, i.e. that the binding of 
the inhibitor to surface-bound u-PA influences the affinity of the 
receptor. 
The change in affinity is a regulatory mechanism capable of modifying the 
ratio between soluble and surface-bound u-PA, i.e. regulating the location 
of u-PA. It is possible that the effects on synthesis and affinity of 
u- normally take place either in different cells or in the same cells, 
but in response to different stimuli. The physiological signal for the 
affinity-regulating mechanism may be connected with the level and possibly 
the fine localization of the u-PA activity on the cell surface. 
Formation of a PAI-1/u-PA complex on the u-PA receptor is followed by 
internalization and degradation of at least the u-PA part of the complex, 
thus representing a novel way of eliminating u-PA activity from the cell 
surface. The results of Example 8 show that blocking binding to the u-PA 
receptor, or to PAI-1 and/or PAI-2 inhibitors, should result in an 
increase in the half life of therapeutically administered pro-u-PA and 
u-PA, thus allowing a decrease of the therapeutically efficient dosage. 
Further characterization of the interaction of u-PA and u- required the 
purification of the u-. The number of u- produced by the 
monocyte-like cell U937 can be increased several fold by phorbol esters 
like PMA. This fact was used to produce sufficient quantities of the 
receptor for purification. In Example 1, a complete purification of the 
u-PA receptor is described, involving temperature-induced phase separation 
of a detergent extract from cells, and affinity chromatography with 
immobilized DFP-inactivated u-PA. This resulted in a preparation that 
shows one band at approximately 55-60 kD after SDS-PAGE and silver 
staining, with a load of approximately 1 .mu.g of the receptor. 
The purified protein could be chemically cross-linked with u-PA. Its amino 
acid composition and N-terminal sequence were determined (30 residues, 
some of which with some uncertainty). It was found to be heavily 
N-glycosylated, deglycosylation resulting in a protein with an apparent 
molecular weight of about 30-35 kD. The apparent molecular weight of u- 
from different cell lines and from PMA-stimulated and non-stimulated U937 
cells varied somewhat. This heterogeneity disappeared after 
deglycosylation and was thus due to differences in glycosylation of u- 
from the various sources. 
The presence of several variants of the same receptor appears to be rather 
common in mammalian cells. The modulation of the u- molecules 
demonstrated in Example 1 may represent an important feature in the 
regulation of extracellular proteolysis and thus in the degradation of the 
extracellular matrix and basement membrane components, processes that are 
at the core of cell migration and invasiveness. In cases where different 
cell types have different kinds of receptors where the protein part of 
u- is glycosylated in different ways, it is possible to distinguish 
between the cell types for which a prevention of the localized proteolytic 
activity is needed, which is of a particular value when cancer cells 
produce a u- which is glycosylated in a way sufficiently different from 
the glycosylating of the u- of normal cells to permit distinguishing by 
means of e.g. u- antibodies. 
In Example 2, isolation of a ligand binding domain of u- is identified 
and characterized. This provides potentially therapeutically valuable 
information on peptides that may inhibit the ligand binding. 
Characterization of the primary structure of the complete u- molecule 
was obtained by cloning of the cDNA copy of the mRNA of the u-PA receptor 
as explained in detail in Example 3. 
The deduced amino acid sequence indicated that u- is produced as a 313 
residues long protein with a 282 residues long hydrophilic N terminal part 
(probably extracellular) followed by 21 rather hydrophobic amino acids 
(probably a trans-membrane domain). The potential extracellular part is 
organised in 3 repeats with striking homologies, particularly with respect 
to the pattern of cysteines. This may indicate the presence of distinct 
domains that may bind different ligands. 
The receptor purification and cDNA cloning allowed to recognize that the 
u- is at least in some cases terminally processed and anchored to the 
cell surface via a glycolipid anchor, and that the surface location can be 
regulated by the phospholipase PI-PLC, but not by the phospholipases 
A.sub.2 and D (Example 4). Furthermore, it was found that also harvest 
fluid from cells that were not treated contain some free u-, indicating 
release from the cells that may be mediated by an endogenous 
phospholipase. This may be a physiological mechanism and it is possible 
that measurement of free receptor, e.g. in serum, may be a diagnostically 
valuable indicator of some pathological processes. 
The u- cDNA was used to show that u- mRNA could be regulated in some 
cell types by substances such as PMA, dexamethasone, mEGF and TGF-.beta.-1 
(Example 5). The findings indicate that these and similar substances may 
be therapeutically useful in regulating u- synthesis. 
Furthermore, u- cDNA was used to produce fragments of u- antisense 
mRNA which proved useful for the detection of u- mRNA in tissue 
sections by in situ hybridization. Particularly interesting is the finding 
that u- mRNA is consistently present in human colon carcinomas and is 
located in cells at the invasive front of the tumors, thus indicating the 
production of u- by these cells and a role of u- in localizing u-PA 
at these sites. 
In Example 7 it is demonstrated that after incubation of monolayer cultures 
of human HT-1080 fibrosarcoma cells with purified native human plasminogen 
in serum containing medium, bound plasmin activity can be eluted from the 
cells with tranexamic acid, an analogue of lysine. The bound plasmin is 
the result of plasminogen activation on the cell surface; plasmin activity 
is not taken up onto cells after deliberate addition of plasmin to the 
serum containing medium. The cell surface plasmin formation is inhibited 
by an anti-catalytic monoclonal antibody to u-PA, indicating that this 
enzyme is responsible for the activation. 
The vast majority of u-PA secreted by the fibrosarcoma cells, and present 
on the cell surface was in the single-chain, proenzyme form. After 
addition of native plasminogen, bound u-PA was found to be in the 
two-chain form, a reaction known to be catalysed by plasmin. However, 
under serum culture conditions, in the presence of a large excess of 
plasmin inhibitors, the binding of plasminogen and its activation product 
(plasmin) to the cell surface was a prerequisite for the formation of the 
two-chain u-PA. It is likely that the activation of pro-u-PA occurs when 
it is actually surface bound. However, it is conceivable that cell-bound 
plasmin activates pro-u-PA in the immediate environment of the cells and 
that the u-PA formed could subsequently exchange with bound pro-u-PA. 
The binding and subsequent protection of plasmin was abolished by low 
concentrations of the lysine analogue, tranexamic acid. It is therefore 
likely that plasmin binding involves the lysine affinity sites situated in 
the heavy-chain kringles of plasmin. Plasmin released from the cells was 
partially inactivated in the serum medium. As long as the plasmin remained 
bound, it was protected from serum inhibitors but could be inhibited by 
aprotinin or an anti-catalytic monoclonal antibody. 
This result provides a possible explanation for the effectiveness of 
aprotinin in certain therapeutic applications, such as the promotion of 
healing of corneal ulcers. Plasmin has been shown to be produced in this 
condition, yet one would expect that it would be inactivated by serum 
inhibitors. If a significant fraction is bound to cells, however, this may 
escape inhibiton and retard development of healing tissue, until an 
effective inhibitor is applied externally. 
The experiments with plasmin uptake and release in serum medium clearly 
established the existence of a one-way movement of plasmin activity from 
the cells into the medium, and not vice versa. Plasmin formation was not 
detected when cells were grown in serum medium without addition of a 
native preparation of plasminogen. 
In Example 7 it is shown that preincubation of the cells with DFP-incubated 
u-PA led to a decrease in surface-bound plasmin, indicating that a large 
part, if not all, of the cell surface plasminogen activation was catalyzed 
by surface-bound u-PA. 
In some cells, e.g. U937 cells, plasminogen activator activity is largely 
dependent on addition of exogenous u-PA. In Example 8, u-PA is 
administered in a binding step followed by washing of the cells and assay. 
The activity can be competed by receptor binding u-PA antagonists, e.g. 
synthetic peptides and DFP-treated u-PA, and can be inhibited by the 
addition of PAI-1. Thus PAI-1 also binds to, and inhibits the activity of 
receptor-bound u-PA in U937 cells. 
Example 8 also shows that PAI-1/u-PA complexes bind the receptor of U937 
cells with the same specificity and affinity as free u-PA. PAI-1 is able 
to interact also with u-PA pre-bound on the receptor on the U937 cells. 
This results in the formation of a typical covalent PAI-1/u-PA complex 
that is not detectably internalized, and in the inhibiton of the u-PA 
activity. The affinity of complexed u-PA is slightly decreased compared to 
free u-PA. Possibly the presence of the bulky PAI-1 molecule may pose a 
problem of steric hindrance. 
In Example 8, it is further shown that when u-PA/PAI-1 complexes are bound 
to the U937 cells, they are subsequently degraded and internalized. 
Example 9 shows that PAI-1 and PAI-2 rather rapidly inhibit receptor-bound 
u-PA, although the respective association rate constants are lower than 
those for inhibition of u-PA in solution. 
It is furthermore demonstrated that binding of u-PA to solubilized (Example 
10) or purified (Example 13) u- inhibits the ability of u-PA to 
activate plasminogen in solution, in contrast to the stimulation of the 
activity which is observed when u-PA is bound to u- on a cell surface 
concomitantly with surface binding of plasminogen. Purified u- also 
inhibits plasmin catalyzed pro-u-PA activation in solution (Example 13). 
Apart from pointing to an important regulatory biological mechanism for 
limiting u-PA catalyzed plasminogen activation to the receptor binding 
sites at the cell surface, these findings indicate that solubilized u- 
or derivatives thereof may prove to be valuable therapeutical reagents for 
inhibition of u-PA activity. 
Polyclonal antibodies were developed by immunization of mice with purified 
u- (Example 11). These antibodies precipitated .sup.125 I-labelled 
purified u- in a dose-dependent manner, with a significant 
precipitation being obtained by the antiserum in a dilution of 1:7500. In 
a reverse-phase radioimmunoassay, the antiserum was found to immunocapture 
radiolabelled u-, and in an ELISA immobilized u- in an amount of 1 
ng was detected with the immune serum diluted 1:8000. By Western blotting, 
the antibodies detected both purified u- and u- in the crude 
detergent phase of extracts of PMA-treated U937 cells. In the latter case, 
no reaction with proteins with electrophoretic mobility different from 
u- was detected, indicating a high degree of specificity of the 
antibodies. The Example also includes a description of methods for 
development of monoclonal antibodies using the above-mentioned methods for 
screening of hybridomas for antibody production. 
In addition, Example 11 describes that antibodies to u- can be used to 
specifically prevent ligand binding. It is furthermore shown (Example 10) 
that u- antibodies inhibit u-PA catalyzed cell surface plasminogen 
activation. These or similar antibodies may also be used to specifically 
target bacterial or vegetable toxins with the purpose of destroying 
potentially metastatic tumor cells. 
In Example 12, a method for the visualization of the u- on cells and in 
tissue sections is described, using biotinylated DFP-treated u-PA followed 
by incubation with streptavidin-fluorescin isothiocyanate. The method is 
very sensitive, and its specificity can readily be tested by competition 
experiments (e.g. with the amino-terminal fragment of u-PA (ATF), t-PA, 
EGF, etc.). 
Based upon the present findings, the present invention provides inhibition 
of receptor binding of u-PA as a means of inhibiting some of its 
physiological functions in relationship to therapeutic prevention of 
localized proteolytic activity, e.g. invasion and metastasis of cancer 
cells, inflammatory bowel disease, premalignant colonic adenomas, septic 
arthritis, osteoarthritis, rheumatoid arthritis (for which a direct 
involvement of excess u-PA production has been demonstrated), 
osteoporosis, cholesteatoma, and a number of skin and corneal diseases for 
which an excess plasminogen activation has been shown to be the 
pathogenetic cause, such as corneal ulcers, keratitis, epidermolysis 
bullosa, psoriasis, and pemphigus. Since u-PA receptors are present in 
several blood and endothelial cells, their regulation might also 
significantly affect intravascular fibrinolytic activity in physiological, 
pathological and pharmacological conditions. The above-mentioned diseases 
would be the first obvious targets for a therapy based on administration 
of substances that block or decrease cell surface plasminogen activation. 
Because of a role of u-PA in implantation of the fertilized egg, a 
contraceptive effect is expected of measures that inhibit receptor 
binding. The therapy and prophylaxis will involve systemic or topical 
treatment with agents that block or reduce receptor bound plasminogen 
activator activity, such as will be explained below. 
The present invention also provides valuable reagents and methods for 
diagnostic or research purposes, such as the u-PA receptor (u-), the 
u- cDNA, anti u- antibodies, and u-PA antagonists like modified u-PA 
and pro-u-PA molecules, obtained by chemical, biological, or synthetic 
means, such as explained below. 
While the present specification and claims relate predominantly to the 
urokinase type plasminogen activator (u-PA), it is obvious that the same 
approach can and should be used for tissue-type plasminogen activator 
(t-PA). Both u-PA and t-PA are widely used in thromboembolic therapy. The 
identification of the u-PA/t-PA receptors and of agents that prevent 
binding and/or internalization and degradation of u-PA/t-PA to these 
receptors can be exploited to increase the half life of the administered 
substance by administering the substance together with an agent which will 
prevent or reduce binding of the substance to its receptor in the 
cardiovascular system and therefore to reduce the doses and their side 
effects. 
DETAILED DISCLOSURE OF THE INVENTION 
In one aspect, the invention relates to a method for preventing or 
counteracting localized proteolytic activity in a mammal, in particular a 
human, comprising inhibiting the activation of plasminogen to plasmin by 
preventing the binding or inducing the specific degradation of a receptor 
binding form of u-PA to a u-PA receptor (u-) in the mammal and thereby 
preventing u-PA from converting plasminogen into plasmin. 
In the present specification and claims, the term "localized proteolytic 
activity" is intended to designate a proteolytic activity which is located 
at one or several distinct regions in a human body, or at distinct cells, 
as opposed to an overall proteolytic activity exerting itself 
substantially anywhere in the body. The localized proteolytic activity can 
be inhibited generally in a mammal, in particular a human, or locally. The 
term "preventing or counteracting" is intended to designate a situation 
where the binding of u-PA to u- is completely inhibited, or a situation 
where the binding is sufficiently inhibited so as to inhibit the undesired 
effect of the plasminogen activator. The term "inducing the specific 
degradation" is intended to designate a process by which the 
receptor-binding form of u-PA is degraded in a specific manner, e.g. 
internalized such as described in Example 8 in which the specific 
degradation is induced by adding PAI-1. 
In the present context, the term "a receptor binding form of u-PA" is 
intended to mean any form of u-PA possessing a site that binds to a site 
at a u-, that is to say that the u-PA contains the u- binding site. 
The receptor binding form of u-PA can thus be pro-u-PA, u-PA, an 
amino-terminal fragment of u-PA, a u-PA that is irreversibly inhibited by 
e.g. diisopropyl fluorophosphate (DFP), p-nitrophenyl-p'-guanidinobenzoate 
(NPGB), or any other inhibitor or any other modification of u-PA that can 
bind to a u-. 
The usage of the term "a u-" indicates that even though the polypeptide 
part of u- in a species might be the same for all u-s, there is a 
plurality of u-s as for example the carbohydrate part or the mechanism 
of surface attachment of the u- can be different. It may even be so 
that some cells, e.g. cancer cells, have substantially different u-s 
which might have important therapeutic significance as it might be 
possible to block the binding of u-PA to u-s residing on a cancer cell 
without affecting the binding of u-PA to u-s on non-pathological cells 
or of specifically killing cancer cells that express u-. 
The enzyme urokinase-type plasminogen activator (u-PA) has only one 
well-defined macromolecular substrate, namely plasminogen. By cleavage at 
Arg.sup.560, plasminogen is activated to the broad spectrum protease 
plasmin. By the term "preventing u-PA from converting plasminogen into 
plasmin" is therefore meant that this activation by u-PA is substantially 
inhibited or a situation where the activation is sufficiently inhibited so 
as to inhibit or reduce the undesired effect of the plasmin. 
The prevention of the binding of a receptor binding form of u-PA to a u- 
is, e.g. suitably performed by blocking the u- by administration, to 
the mammal, of a substance binding to the u- so as to occupy a site of 
the receptor to which a receptor binding form of u-PA is normally bound, 
the substance being administered in an amount effective to reduce the 
binding of the receptor binding form of u-PA to the receptor. In the 
present context the term "blocking the u-" means that a substance that 
is not able to activate plasminogen to plasmin is bound to u-, 
preferably by a substantially irreversible binding, thereby preventing a 
receptor binding form of u-PA of catalyzing the conversion of plasminogen 
into plasmin. The term "binding to a u- so as to occupy a site of the 
receptor to which a receptor binding form of u-PA is normally bound" is 
intended to mean that the substance binds to the u- so that a receptor 
binding form of u-PA can not be bound to the u-. 
The prevention of the binding of a receptor binding form of u-PA to a u- 
may be performed by administering a modification of u-PA which has 
retained its capability of binding to the u-, but which is not capable 
of converting plasminogen to plasmin, to the mammal. An important example 
of such a modification of u-PA is u-PA inhibited at its catalytically 
active site with a substantially irreversible inhibitor, i.e. a substance 
which binds to the catalytically active site by a substantially 
irreversible bond. A number of such inhibitors are known, one example of 
an irreversible inhibitor being diisopropyl fluorophosphate (u-PA 
inhibited by this inhibitor is termed DFP-u-PA). 
Particularly interesting is the administration of complexes between u-PA 
and PAI-1 for inhibition of receptor binding of u-PA. These complexes not 
only bind to u- (Example 8), but also cause internalization of the 
complexes (and presumably also of u-). Thus, their inactivation of the 
u-PA binding capacity of u- has the character of being irreversible. 
The findings described in Examples 8 and 9 make it justified to 
contemplate that u-PA-PAI-2 complexes will have a similar effect and 
utility. 
The modification of u-PA may also be obtained by binding an antibody to its 
catalytically active site region. The antibody may be either polyclonal 
antibodies or monoclonal antibodies and may be prepared as described in 
greater detail below. 
Another useful modification of u-PA is an amino-terminal fragment of u-PA 
(ATF-u-PA) (cf. Stoppelli et al., 1985). 
The prevention of the binding of a receptor binding form of u-PA to u- 
may also be performed by administering a substance comprising a sequence 
which is identical or substantially identical to a site of u-PA which 
binds to the u-; such substance may for example be a molecule 
comprising a sequence which is identical or substantially identical to a 
u- binding site of u-PA amino residues 12-32, which are known to be 
involved in the receptor binding of u-PA. 
Another way of preventing the binding of a receptor binding form of u-PA to 
a u- is to administer a u- or a u-PA-binding modification thereof to 
the mammal so as to occupy the cell receptor-binding site of u-PA, thereby 
preventing the receptor binding form of u-PA from binding to the 
cell-bound receptor. Normally, it will not be preferred to administer a 
complete u-, but rather a water-soluble form thereof, in other words a 
part thereof comprising a u-PA binding sequence. Such a part will often 
have been made by truncation of a larger sequence by removing part of the 
cDNA sequence of a plasmid vector containing the human u- cDNA, cf. 
Example 3. Particularly interesting is the finding that u- can be 
solubilized by removal of the glycerol-phosphoinositol anchor, e.g. with a 
lipase as shown in Example 4. The soluble forms of u-, such as the 
above-mentioned truncated forms, are also useful in that they may be 
coupled by chemical methods or recombinant DNA methods to plasminogen 
activator inhibitors such as plasminogen activator inhibitor Type 1 
(PAI-1) or Type 2 (PAI-2), whereby an interesting double effect comprising 
either receptor blocking or ligand blocking, or both, may be obtained. 
The prevention of the binding of a receptor binding form of u-PA to a u- 
may also be obtained by administering a modification of pro-u-PA which has 
retained its capability of binding to the u-, but which is not capable 
of being converted into u-PA. Typically, such a modification of pro-u-PA 
is one in which the sequence of u-PA normally cleavable by plasmin has 
been changed so that the u-PA is not cleaved by plasmin. An example of 
this is pro-u-PA in which Lys.sup.158 has been substituted with Glu or Gly 
by site-directed mutagenesis. 
A very interesting method of preventing the binding of a receptor binding 
form of u-PA to a u- and thereby preventing the cell surface 
plasminogen activation is the use of antibodies against u- such as 
demonstrated in Example 11 (prevention of binding) and Example 10 
(prevention of cell surface plasminogen activation). The antibodies may be 
polyclonal antibodies, preferably of high specificity such as the 
antibodies illustrated in Example 11, or a monoclonal antibody. The 
antibody may be an antibody that is reactive with non-carbohydrate 
moieties of the u-, or it may be an antibody that is reactive with 
carbohydrate moieties of the u-, the latter permitting a valuable 
distinction between target cells where cells expressing distinct variants 
of u- are the cells involved in the undesired proteolysis. The 
antibodies may be administered in various ways as described below. 
Another strategy for preventing or counteracting localized proteolytic 
activity in a mammal, in particular a human, comprises inhibiting the 
activation of plasminogen to plasmin by substantially reducing the 
activity of a receptor-bound form of u-PA by administering, to the mammal, 
a plasminogen activator inhibitor in a sufficient amount to inhibit the 
activation of plasminogen. The plasminogen activator inhibitor may be 
PAI-1 or PAI-2 which, according to the present invention, have been found 
to inhibit u-PA, also when it is receptor-bound. 
Another strategy for preventing or counteracting localized proteolytic 
activity in a mammal, in particular a human, comprises inducing the 
selective internalization of the receptor-bound u-PA by, for example, 
blocking its activity by administering the specific inhibitor PAI-1 or 
increasing PAI-1 synthesis with hormones (estrogens, glucocorticoids, 
polypeptide hormones), cytokines (interleukins, interferons, TNF) or 
growth factors (EGF, IGF-1, IGF-2, PDGF, FGF, TGF.alpha., TGF.beta.) and 
any other factor that induces PAI-1 synthesis, thereby causing u-PA 
degradation and internalization. 
Another strategy for inducing intracellular u-PA degradation consists in 
administering compounds that would induce dimerization of receptors, such 
as PAI-1 dimers, as in other receptors dimerization appears to be involved 
in internalization. 
Another strategy for preventing or counteracting localized proteolytic 
activity is removal of u- from cell surfaces by treatment with an agent 
which destroys the glycerol-phosphoinositol anchor, e.g. a phosholipase, 
such as PI-PLC as described in Example 4. The agent will preferably be 
administered locally. 
Yet another strategy for preventing or counteracting localized proteolytic 
activity in a mammal, in particular a human, comprises inhibiting the 
activation of plasminogen to plasmin by altering the binding affinity of 
the u-PA/u- by modifying u- in the mammal (e.g. by treatment with 
the phorbol ester PMA or with EGF) and thereby preventing u-PA from 
converting plasminogen into plasmin. 
The most effective alteration of the binding activity is believed to be a 
reduction of the binding affinity because, at a given concentration of 
u-PA in the pericellular fluid, a reduced affinity will lead to a reduced 
number of bound u-PA molecules and thereby a reduced proteolytic activity. 
A reduction of the binding affinity may be obtained by administering a 
substance selected from the group consisting of hormones, growth factors 
(such as epidermal growth factor EGF!) or cytokines. 
The PAI-1 induced internalization of receptor-bound u-PA can also be 
exploited to selectively kill tumor cells by administering a PAI-1 
derivative which is covalently bound (by chemical or genetic methods) to a 
bacterial or plant toxin. Upon internalization of the u-PA-PAI-1-toxin 
complex, the cells can be selectively killed by the action of the toxin. 
It is likely that some disorders are related to a reduced amount or an 
impaired function of u-. These may include some cases of impaired wound 
healing and also some cases of thromboembolic disorders. A role of u-PA 
(and therefore probably also of u-) in thrombolysis under some 
conditions is suggested by the finding by inventors of the present 
invention of u-PA being present in endothelial cells during acute 
inflammation and in cancer. Under normal conditions, the endothelial cells 
contain t-PA, but no u-PA. It is furthermore interesting that the disease 
paroxysmal nocturnal hemoglobinuria is associated with an impaired ability 
to form glycerol-phosphoinositol anchors and that this disease is often 
associated with thromboembolic disorders (See: Selvaraj et al., 1988, and 
references therein). According to the present invention, it is therefore 
contemplated that a therapeutic effect may be obtained by administering 
u- or a derivative thereof having u- function or by conferring to 
the cells of the patient the ability to produce functionally intact u- 
or a derivative thereof having u- function by transfection with the 
u- cDNA or a variant thereof. Alternatively, the synthesis of u- may 
be increased by administration of various hormones, growth factors, or 
cytokines, e.g. dexamethazone, mEGF, and TGF-.beta.-1 as indicated by the 
findings described in Example 5. 
A completely different, and potentially therapeutically very valuable 
application of u-, solubilized u- and variants thereof is as an 
inhibitor of u-PA-catalyzed plasminogen activation in solution. The 
hitherto known specific inhibitors of plasminogen activators, that is, 
PAI-1 and PAI-2, inhibit both u-PA and t-PA. Purified u- as well as 
u- solubilized by removal of the glycerol-phosphoinositol anchor 
inhibits u-PA in solution as demonstrated in Examples 10 and 13. u- 
does not bind t-PA. Thus, u- is contemplated to be more advantageous 
than PAI-1 and PAI-2 in cases where specific inhibition of u-PA is needed. 
Potentially very valuable is also the therapeutic use of u-, 
solubilized u- and variants thereof for inhibiting the activation of 
the virtually inactive single-chain pro-u-PA molecule to active two chain 
u-PA. 
The finding that the extracellular part of u- consists of three repeats 
with considerable mutual homologies (Example 3) renders it probable that 
it can bind different ligands, that is, that it can bind other ligands 
apart from the proven binding of u-PA. It would be justified to assume 
that some of these may involve yet unknown plasminogen receptors or 
plasminogen binding sites because of the strong enhancing effect obtained 
by concomitant binding of u-PA and plasminogen to the cell surface as 
described in Example 7. Potential alternative ligands for u- may also 
be proteins located at cell-cell and focal cell-substratum contact sites 
because of the preferential location of receptor-bound u-PA at these sites 
in some cell types. Variants or parts of u- that inhibit binding of 
such ligands may be valuable in inhibition of cell surface plasminogen 
activation, and prevention of binding of u- to such ligands may be 
functionally important and therapeutically valuable in a broad spectrum of 
diseases. 
u- exists in various forms, such as the glycosylation variants described 
in Example 1, the variants with different sensitivity to the lipase PI-PLC 
suggested by the findings described in Example 4, and the variants in 
ability to stimulate cell surface plasminogen activation found on 
PMA-stimulated and PMA-non-stimulated U937 cell, respectively, as 
described in Example 10. In some diseases that involve increased or 
decreased u- function, some of these forms may be preferentially 
changed. Therapy directed against correcting some distinct forms may 
therefore be particularly therapeutically valuable in such diseases. 
The administration of the various above-mentioned principles to a mammal, 
preferably a human being, may be performed by any administration method 
which is suitable for administering proteins or peptides or antibodies. 
Typical administration routes are parenteral, oral, nasal, topical or 
rectal administration. In each case, the active ingredient to be 
administered should be formulated in a manner which will protect the 
active ingredient against degradation, in particular by enzymes. In many 
cases, the parenteral administration is the safest way of administering 
proteins and peptides. The parenteral administration route should be 
selected dependent on where the active ingredient is to be released, e.g. 
intravenously, intramuscularly or subcutaneously, etc. It is also 
important to consider the necessity of "packing" the active ingredient in 
a suitable manner in order to 
1) obtain a sufficient therapeutic concentration level for a suitable time, 
2) avoid first-pass metabolism, 
3) avoid allergic and immunological reactions, and 
4) avoid undesired side effects by 
5) obtaining transport of the active ingredient to the site of action. 
When the active ingredient is administered perorally, suitable measures 
should be taken to protect the active ingredient from enzymatic 
degradation in the gastrointestinal tract, e.g. by packing the active 
ingredient in such a way that it will not be released from the formulation 
(i.e. the pharmaceutical composition) until it has reached the site where 
either the active ingredient is to exert its activity locally (i.e. in the 
gastrointestinal tract) or from where the absorption may take place (e.g. 
M-cells in the colon). 
When rectal administration is performed, it is often desirable to use the 
so-called enhancers which are capable of making active ingredients of the 
peptide type pass the rectal mucosa and thereby become absorbed. 
Nasal administration is an administration form which is presently 
intensively investigated in order to provide absorption of substances of 
the peptide type from the nasal cavity. In principle, this may take place 
in two ways, firstly by using enhancers, and secondly by using the 
bioadhesion principle in which the active ingredient may be maintained for 
a long period of time at a suitable domain in the nose. 
Topical administration may be performed by formulating the active 
ingredient in a salve, an ointment, a lotion, a creme, etc. 
The pharmaceutical compositions of the invention may for example include 
pharmaceutically acceptable excipients adapted to the character of the 
active ingredients in accordance with the above discussion. Suitable 
excipients may include liposomes and/or microspheres. The preparation of 
the pharmaceutical compositions may be performed in accordance with 
methods described in the literature for compositions of the types 
described herein. 
Based upon the findings concerning the dose-related effect of DFP-u-PA in 
Example 7, it is contemplated that a suitable pericellular (extracellular) 
concentration thereof is in the range of from 1 .mu.g/ml to well above 10 
.mu.g/ml, such as, e.g., 100 .mu.g/ml, but it is also noted from FIG. 11 
that even concentrations smaller than 1 .mu.g/ml do have a significant 
effect for which reason a practical lower limit could be set at 0.1 
.mu.g/ml. This would correspond to a unit dosage of between about 1.4 mg 
and 1.4 g, preferably in the range of about 50 to 300 mg such as about 150 
mg for an average adult person. The same considerations apply with respect 
to NPGB-u-PA, the amino-terminal fragment of u-PA, and pro-u-PA that is 
modified so that it cannot be cleaved by plasmin. Evidently, the higher 
the affinity between the modified form binding to a receptor, the lower is 
the dosage required. Based on the above data, a person skilled in the art 
will be able to determine suitable dosage ranges from preliminary in vitro 
and in vivo experiments. 
The treatments will normally be continued for weeks or often months and are 
suitably combined with treatment with other medicaments against the 
conditions in question. 
Another strategy of treating the conditions and diseases mentioned above is 
to target a cell that contains a u- on the surface by a medicament, 
comprising administering the medicament bound to a substance that binds to 
a u-. The substance may be a receptor binding form of u-PA, or it may 
be an antibody against u- such as a polyclonal or a monoclonal 
antibody, e.g. an antibody particularly directed to a variant of u- 
present in a cancer cell type. 
The medicament may typically be an anti-cancer agent such an alkylating 
agent, e.g. melphalan, chlorambucil, busulfan, cisplatin, thiotepa, an 
antimetabolite, such as methotrexate, fluracil, azathioprin, an 
antimitoticum, typically vincristine, vinblastine, or an antibiotic such 
as doxorubicin, daunorubicin or bleomycin. The medicament may also 
comprise bacterial or other toxins. 
Another important aspect of the present invention is a method of targeting 
a cell that contains a u- on the surface by a diagnostic, comprising 
administering the diagnostic bound to a receptor binding form of u-PA. The 
diagnostic may be a radioactive substance which is physiologically 
tolerable such as, e.g., technetium. 
An important field of the present invention is a number of diagnostic 
methods which methods, or the importance thereof, are based upon the 
findings according to the invention. One important aspect thereof is a 
method of detecting a u- in a tissue section comprising treating the 
tissue section with a substance that binds to a u-, and visualizing the 
presence of the bound substance. The substance may in principle be any of 
the above substances which bind to u-, but it is especially preferred 
that the substance is an antibody, in particular a labelled antibody or an 
unlabelled antibody which is subsequently detected by an immunostaining 
method. The antibody may be a polyclonal or monoclonal antibody, and 
particularly interesting antibodies are antibodies that distinguish 
between various forms of u-. A detailed description of diagnostic kits, 
materials and methods based on antibodies is given further below. 
The substance which binds to u- may also be a form of u-PA, either 
labelled, for example biotinylated DFP-treated u-PA that subsequently is 
detected by streptavidin-fluorescin isothiocyanate, or an unlabelled form 
which is subsequently detected by immunostaining. 
Another field of the invention is the use of antibodies against a u- for 
the quantification of the u- in biological material using antibodies 
aganist the u-. While this method may be performed using either 
polyclonal or monoclonal antibodies, an interesting embodiment uses a 
combination of polyclonal and monoclonal antibodies, the monoclonal 
antibodies being more specific and the polyclonal antibodies generally 
having a higher binding affinity. 
The quantification method may be of the ELISA type or may be a 
radioimmunoassay. These assays may be produced by methods known per se. 
One aspect of the invention relates to a method of producing pure u-, 
the method comprising subjecting a u--containing material to affinity 
chromatography with immobilized antibodies to u- and eluting the u-, 
e.g. under acidic conditions. 
The present invention also relates to pure u-. As mentioned above, pure 
u- has been made for the first time in accordance with the present 
invention. Pure u- in glycosylated form shows, in an SDS-PAGE at a load 
of approximately 1 .mu.g, substantially one and only one silver stained 
band having an apparent molecular weight in the range of about 55-60 kD. 
The presence of substantially one and only one silver stained band in this 
SDS-PAGE is a proof of the purity of the u-. Another proof of the 
purity of the u- is the presence of a single amino-terminal amino acid 
sequence in purified u- preparations. While it has been found that 
different cells may produce u-s having different glycosylation, the 
glycosylated u-s, upon deglycosylation, were all found to have an 
identical electrophoretic mobility (corresponding to substantially one and 
only one band at about 30-35 kD in an SDS-PAGE), indicating that the 
peptide part of the molecule is identical in all cases. 
As appears from the Examples, pure u- in glycosylated form may be 
prepared from a biological material containing u- by 
temperature-induced phase separation of detergent extracts followed by 
affinity chromatography purification with immobilized DFP-u-PA. The 
detergent is preferably a non-ionic detergent such as a polyethylene 
glycol ether, e.g. Triton X-114. The temperature was found to be 
relatively critical in the range of 34.degree.-40.degree. C., such as 
about 37.degree. C., for 10 minutes. 
The pure u- in unglycosylated form may be prepared by deglycosylation 
with, e.g., peptide/N-glycosidase F. 
The present invention also relates to a novel method of purification of 
u-, exploiting the ability of phospholipase C to release the receptor 
in the medium, thereby providing a direct method of preparing and 
purifying an extracellular, soluble form of u- which is able to bind 
u-PA and which can be used as a u-PA scavenger. 
On the basis of the amino-terminal amino acid sequence of pure u-, a 
24-mer nucleotide probe was synthesized and used to screen a library to 
identify and isolate recombinant clones carrying the cDNA for u-. The 
identity of the cDNA clones was confirmed by comparing the nucleotide 
sequence of this cDNA clone with the amino terminal sequence of the 
purified u-, and by expressing said cDNA in mouse L cells and assaying 
their u-PA-binding properties. 
The abbreviations of the amino acids used herein are the following: 
______________________________________ 
Three-letter 
One-letter 
Amino acid abbreviation 
symbol 
______________________________________ 
Alanine Ala A 
Arginine Arg R 
Asparagine Asn N 
Aspartic acid Asp D 
Asparagine or aspartic acid 
Asx B 
Cysteine Cys C 
Glutamine Gln Q 
Glutamic acid Glu E 
Glutamine or glutamic acid 
Glx Z 
Glycine Gly G 
Histidine His H 
Isoleucine Ile I 
Leucine Leu L 
Lysine Lys K 
Methionine Met M 
Phenylalanine Phe F 
Proline Pro P 
Serine Ser S 
Threonine Thr T 
Tryptophan Trp W 
Tyrosine Tyr Y 
Valine Val V 
______________________________________ 
One aspect of the invention relates to a polypeptide comprising a 
characteristic amino acid sequence derived from a u- which polypeptide 
comprises at least 5 amino acids and up to the complete sequence of u- 
as shown below as the DNA sequence and the deduced amino acid sequence of 
the clone p-u-1 (hereinafter Sequence A. The signal peptide is 
underlined and the first 30 amino acids, the sequence of which has been 
determined on the purified protein with an Applied Biosystems gas phase 
sequencer (see Example 1), are overlined. The putative transmembrane 
domain is doubly underlined. The star symbols indicate the potential 
N-linked glycosylation sites. 
##STR1## 
or an analogue thereof. 
The invention relates to any polypeptide comprising at least 5 amino acids 
and up to the complete sequence of u- from amino acid 1 to 313, and any 
analogue to such a polypeptide. 
The polypeptide may be coupled to a carbohydrate or lipid moiety. It may 
typically be glycosylated as mentioned above. 
In the present context, the term "characteristic amino acid sequence 
derived from the u-" is intended to mean an amino acid sequence, such 
as an epitope, which comprises amino acids constituting a substantially 
consecutive stretch (in terms of linear or spatial conformation) in u-, 
or amino acids found in a more or less non-consecutive conformation in 
u-, which amino acids constitute a secondary or tertiary conformation 
having interesting and useful properties, e.g. as therapeutics or 
diagnostics. Thus, amino acids present at different positions in u- but 
held together e.g. by chemical or physical bonds, e.g. by disulphide 
bridges, and thereby forming interesting tertiary configurations are to be 
understood as "characteristic amino acid sequences". The characteristic 
amino acid sequence may comprise a consecutive subsequence of the amino 
acid sequence of u- of greater or smaller length or a combination of 
two or more parts of such subsequences which may be separated by one or 
more amino acid sequences not related to u-. Alternatively, the 
characteristic amino acid sequences may be directly bonded to each other. 
In the present context, the term "epitope" refers to a sequence or 
subsequence of the polypeptides of the invention or a derivative or an 
analogue thereof capable of stimulating or interacting with 
immunocompetent cells, especially epitopes against which antibodies 
showing desirable properties in regard to diagnosis can be raised. 
The term "analogue" is used in the present context to indicate a protein or 
polypeptide of a similar amino acid composition or sequence as the 
characteristic amino acid sequence derived from the u-, allowing for 
minor variations which do not have an adverse effect on the immunogenicity 
of the analogue. The analogous polypeptide or protein may be derived from 
mammals or may be partially or completely of synthetic origin. 
The present invention also relates to a substantially pure polypeptide 
which is recognized by an antibody raised against or reactive with a 
polypeptide comprising the amino acid sequence defined above. 
In the present context, the term "substantially pure" is understood to mean 
that the polypeptide in question is substantially free from other 
components, e.g. other polypeptides or carbohydrates, which may result 
from the production and/or recovery of the polypeptide or otherwise be 
found together with the polypeptide. The high purity of the polypeptide of 
the invention is advantageous when the polypeptide is to be used for, 
e.g., the production of antibodies. Also due to its high purity, the 
substantially pure polypeptide may be used in a lower amount than a 
polypeptide of a conventional lower purity for most purposes. The 
purification of the polypeptide of the invention may be performed by 
methods known to a person skilled in the art, but particularly the low 
concentrations of u- in biological material and the strongly 
hydrophobic nature of the receptor has hitherto hampered its purification. 
Now, however, the combination of temperature-induced phase separation of 
detergent extracts of cells and affinity chromatography with immobilized 
DFP-treated u-PA has led to its successful purification in amounts high 
enough (100-200 .mu.g) to have enabled a partial amino acid sequencing and 
further characterization. 
In another aspect, the invention relates to a DNA fragment comprising a 
nucleotide sequence encoding the u- described above. The DNA fragment 
may be used in a method of preparing the u- or parts thereof by 
recombinant DNA techniques or as a diagnostic agent (i.e. a DNA probe). 
The use of the DNA fragment of the invention in the production of a 
recombinant polypeptide (e.g. by inserting the fragment in a suitable 
vector, transforming the vector into a suitable host organism 
(microorganism or cultured animal cell), cultivating the organism so as to 
produce the polypeptide and subsequently recovering the polypeptide from 
the organisms) includes a number of advantages. It is possible to provide 
large amounts of u- or any fragment thereof and the u- polypeptide 
produced may be isolated in a substantially pure form, free from 
contaminating substances. The DNA fragment of the invention may also be 
used as a diagnostic agent for the detection of mRNA encoding u- or 
parts thereof in a sample, which diagnostic agent comprises a labelled DNA 
sequence which is homologous with a DNA sequence coding for at least part 
of u-. 
The pure u- (natural or recombinant) of the invention may be used in the 
preparation of polyclonal or monoclonal antibodies. The antibodies may be 
used for the identification and/or quantification of at least part of the 
above described polypeptide present in a sample thus making it possible to 
diagnose diseases related to presence of abnormal numbers of the u- on 
the surface of mammalian cells. The sample may be any part of the human 
organism, e.g. be a body fluid or tissue part containing the polypeptide, 
e.g. a tissue sample such as a biopsy, e.g. a bone marrow tissue sample, a 
blood sample, a urine sample, a sample of cerebrospinal fluid, serum, 
plasma or any product prepared from blood or lymph, secretions or any 
sample obtained from a human cavity containing cells with a u-PA receptor. 
The polypetide of the invention may be coupled to a carbohydrate, a lipid 
moiety or modified in other ways, e.g. phosphorylated or hydroxylated. In 
particular the polypeptide may be glycosylated, the coupled carbohydrate 
moiety having molecular weights of 20-30 kD in the natural molecule. 
Coupling of the polypeptide to one or more moieties may for instance be 
due to a posttranslational modification of the polypeptide performed by an 
organism producing the polypeptide or a modification resulting from 
chemical synthesis. 
The polypeptide of the invention may also be a fusion protein in which 
characteristic amino acid sequences) from u- is/are fused to another 
polypeptide sequence. The polypeptide to which the characteristic amino 
acid sequence(s) from u- is/are fused may be one which results in an 
increased expression of the protein when expressed in an organism, or 
facilitates or improves the purification and recovery of the fusion 
protein from said organism in terms of a more easy and economical 
recovery, or confers to the u- the property of inhibiting u-PA (as it 
would be in the case of a u--PAI-1 fusion). 
In some cases, it may be advantageous to cleave the fusion protein so as to 
obtain a polypeptide which substantially solely comprises characteristic 
amino acid sequence(s) from u-. In these cases, the characteristic 
amino acid sequence(s) from u- is/are preferably fused to a polypeptide 
sequence which may be specifically recognized by a cleaving agent, e.g. a 
chemical such as cyanogen bromide, hydroxylamine and 
2-nitro-5-thiocyanobenzoate, or an enzyme, e.g. a peptidase, proteinase or 
protease, e.g. trypsin, chlostripain, and staphylococcal protease or 
factor Xa. 
As mentioned above, one aspect of the present invention relates to a DNA 
fragment encoding the polypeptide of the invention. In particular, the 
invention relates to a DNA fragment comprising substantially the 
nucleotide sequence (1), or a subsequence thereof coding for a subsequence 
of the polypeptide of the invention. 
Each of the nucleotides of the above sequence is represented by the 
abbreviations generally used, i.e. 
A represents adenine 
T represents thymidine 
G represents guanine 
C represents cytosine. 
This nucleotide sequence encodes the entire protein part of u-. The DNA 
sequence shown above has been established as described in Example 3. 
The cDNA of the u- represents a rather rare clone, based on the fact 
that it is expressed at the most at 800,000 molecules/cell. It has in fact 
been found with a frequency of less than 6.times.10.sup.-6. The cDNA is 
about 1.4 kb long based on its restriction map (FIG. 4B), has a 5' 
untranslated sequence of about 40 residues and an about 40 nucleotides 
long poly-A stretch at the 3' end. 
In order to examine the relatedness of DNA not related to u- and the 
gene encoding at least part of u-, DNA hybridization is a useful 
method. Hybridization may be performed as follows: Pure DNA comprising the 
gene encoding u- from the plasmid p-u-1 is prepared using the large 
scale method described in Maniatis et al. (1982), pages 86-96. More 
specifically, the u- gene may be excised from the plasmid by digestion 
of the plasmid DNA with suitable restriction enzymes. The insert is then 
separated from the plasmid DNA by use of agarose gel electrophoresis. The 
insert is labelled by any labelling principle, such as the ones disclosed 
herein. The foreign DNA to be examined is coupled to a matrix, e.g. a 
nitrocellulose filter. The filter is subjected to a suitable treatment 
suited to the kind of matrix employed so as to couple the DNA to the 
matrix, in the case of a nitrocellulose filter e.g. by baking the filter 
at a temperature of 80.degree. C. for 2 hours. The membrane is exposed to 
a prehybridization solution of a composition, at a temperature and for a 
period of time recommended suited to the membrane in question. The 
membrane is then placed in the hybridization solution containing the 
labelled denatured DNA probe obtained from the p-u-1 plasmid (the u- 
gene). Hybridization is preferably carried out overnight at a suitable 
temperature. The membrane is then washed and incubated with a volume of 50 
ml 2.times. SSC at 65.degree. C. for 30 minutes. The procedure is repeated 
once. The membrane is then incubated in 15 ml 2.times. SSC containing 0.1% 
SDS. Incubation is performed at 65.degree. C. for 30 minutes. All 
incubations including prehybridization and washings are performed with 
gentle agitation. The filter is air-dried and wrapped in a suitable 
plastic wrap (e.g. Saran Wrap), the filter is then applied to an x-ray 
film so as to obtain an auto-radiographic image. Exposition is preferably 
carried out at -70.degree. C. with intensifying screens for a period of 
time which is determined by the positive control used. Any hybridization 
of the foreign DNA and the u- gene is an indication of similarity of 
the two DNA probes, i.e. that the foreign DNA is a DNA fragment of the 
invention. Another approach of determining similarity between DNA 
sequences is by determining the nucleotide sequence of the DNA sequence to 
be compared with the DNA sequence of the invention by conventional DNA 
sequencing analysis, and comparing the degree of homology with the DNA 
sequence of the invention. Preferably, a degree of homology of at least 
about 70%, e.g. at least about 80% such as at least about 95% is obtained. 
The DNA fragment of the invention may comprise a nucleotide sequence 
encoding a polypeptide fused in frame to the nucleotide sequence encoding 
the characteristic amino acid sequence with the purpose of producing a 
fused polypeptide. When using recombinant DNA technology, the fused 
sequence may be inserted into an appropriate vector which is transformed 
into a suitable host organism. Alternatively, the DNA fragment of the 
invention may be inserted in the vector in frame with a gene carried by 
the vector, which gene encodes a suitable polypeptide. The host organism, 
which might be of eukaryotic or prokaryotic origin, for instance a yeast 
or a mammalian cell line, is grown under conditions ensuring expression of 
the fused sequence after which the fused polypeptide may be recovered from 
the culture by physico-chemical procedures, and the fused polypeptide may 
be subjected to gel filtration and affinity chromatography using an 
antibody directed against the antigenic part(s) of the fused polypeptide. 
After purification, the polypeptide of the invention and the polypeptide 
to which it is fused may be separated, for instance by suitable 
proteolytic cleavage, and the polypeptide of the invention may be 
recovered, e.g. by affinity purification or another suitable method. 
The DNA fragment may also comprise a suitable nucleotide sequence 
controlling the expression of the DNA fragment. The regulatory nucleotide 
sequence is conveniently a part of the expression vector used for the 
production of the polypeptides, when such a vector is employed. 
The DNA fragment described above may be obtained directly from genomic DNA 
or by isolating mRNA and transferring it into the corresponding DNA 
sequence by using reverse transcriptase producing cDNA. When obtaining the 
DNA fragment from genomic DNA, it is derived directly by screening for 
genomic sequences, hybridizing to a DNA probe prepared on the basis of the 
full or partial amino acid sequence of u-. When the DNA is of 
complementary DNA (cDNA) origin, it may be obtained by preparing a cDNA 
library on the basis of mRNA from cells containing a u- or parts 
thereof. Hybridization experiments may then be carried out using synthetic 
oligonucleotides as probes to identify the cDNA sequence encoding the 
u- or part thereof. cDNA differs from genomic DNA in, e.g. that it 
lacks certain transcriptional control elements and introns which are 
non-coding sequences within the coding DNA sequence. These elements and 
introns are normally contained in the genomic DNA. The DNA fragment may 
also be of synthetic origin, i.e. prepared by conventional DNA 
synthesizing method, e.g. by using a nucleotide synthesizer. The DNA 
fragment may also be produced using a combination of these methods. 
Also interesting is a DNA or RNA fragment comprising the sequence 
complementary to the above DNA sequence or a part thereof or the mRNA 
corresponding to said DNA sequence. It is contemplated that a DNA or RNA 
fragment complementary to at least part of the mRNA corresponding to the 
polypeptide of the invention is effective in arresting the translation of 
the polypeptide in the human cells, and thereby inhibiting the synthesis 
of u- polypeptides. In other systems, it has been shown that DNA or RNA 
fragments complementary to the mRNA encoding a given protein is capable of 
arresting the translation of the protein. Thus, the insertion of an 
antisense-oncogene in a human cell line (as described by Holt, J. T. et 
al., Proc. Natl. Acad. Sci. USA, 1986, 83, 4794-4798) and a plant enzyme 
in a transgenic plant (as described by Kroll, Nature, 1988, 333, 866) have 
been found to have this effect. Furthermore in some cases, antisense DNA 
or RNA complementary to virus mRNA has been shown to be able to inhibit 
the infection rate of the SP-phage in an E. coli strain (as described in 
Hirashima, A. et al., Proc. Natl. Acad. Sci. USA, 1986, 83, 7726-7730), or 
of the HIV-virus in an infected human CD4 cell line (Reitz, M. et al., 
Proc. Natl. Acad. sci., 1987, 84, 7706-7710). 
The above DNA or RNA fragment should comprise a number of nucleotides which 
is sufficient for obtaining the desired specificity and hybridization of 
the DNA or RNA fragment to the mRNA corresponding to the polypeptide of 
the invention. Preferably, the DNA or RNA fragment is of a size which 
allows the safe transport of the fragment through the cell membrane, i.e. 
without any substantial disruption of the fragment transported over the 
membrane. To obtain a sufficient specificity and/or hybridization in terms 
of linear or spatial structure of the fragment, it is contemplated that 
the DNA or RNA fragment should have a size of at least about 5 
nucleotides, preferably at least about 8 nucleotides. To ensure a safe 
transport of the DNA or RNA fragment through the cell membrane, it is 
contemplated that the DNA or RNA fragment should have a size of at the 
most about 100 nucleotides, preferably at the most about 80 nucleotides. 
Thus, it is contemplated that the DNA or RNA fragment having a size of 
about 10-60 nucleotides, such as about 12-50 nucleotides, e.g. about 14-40 
nucleotides, preferably about 15-25 or 15-22 nucleotides is useful. The 
DNA or RNA fragment may be complementary to any part of the mRNA, e.g. to 
a part of the mRNA comprising the ribosomal binding site or part thereof, 
or the start codon for the gene encoding the polypeptide of the invention, 
or to a sequence which is repeated one or more times. 
The DNA or RNA fragment may comprise multiple phosphate-modified 
oligodeoxyribonucleotides such as oligo-alkyl phosphotriesters, 
oligomethylphosphonates or oligophosphorothioates to improve the 
resistance against nucleases or the transport across cell membranes. The 
DNA or RNA fragment may be prepared by conventional methods, e.g. the 
methods outlined above. 
In a further aspect, the invention relates to an expression vector which is 
capable of replicating in a host organism and which carries a DNA fragment 
as described above. The vector may be any vector which conveniently can be 
subjected to recombinant DNA procedures, the choice of vector often 
depending on the host cell into which it is to be introduced. Thus, the 
vector may either be one which is capable of autonomous replication, i.e. 
a vector which exists as an extrachromosomal entity, the replication of 
which is independent of chromosomal replication, such as a plasmid, or a 
vector which is replicated together with the host chromosome, such as a 
bacteriophage. 
When a microorganism or a mammalian cell line is used as the host organism, 
examples of useful vectors are plasmids such as natural or synthetic 
plasmids, eg. plasmids related to pBR322 such as pEX 1-3, the pRIT-family, 
the pUC-family and the like, and viruses such as adenovirus, vaccinia 
virus, retrovirus, Baculo virus, Epstein-Barr-virus, SV40-related virus 
and bovine papilloma virus. Examples of suitable bacteriophages include 
M13 and lambda. 
The invention also relates to an organism which carries and is capable of 
expressing a DNA fragment as defined above and which not in its native 
form expresses said DNA fragment. The DNA fragment may be carried on a 
vector as described above or may be integrated in the genome of the 
organism. Examples of suitable organisms include microorganisms such as 
bacteria, yeasts, fungi and higher eucaryotic organisms or cells including 
plant and mammalian cells. However, also higher organisms such as animals, 
e.g. sheep, cattle, goats, pigs, etc. is contemplated to be useful as host 
organisms for the production of the polypeptide of the invention. 
The present invention also relates to a method of producing the 
polypeptides described above. Suitably, the polypeptides are prepared 
using recombinant DNA-technology e.g. the methods disclosed in Maniatis et 
al. op. cit. More specifically, the polypeptides may be produced by a 
method which comprises cultivating or breeding an organism carrying a 
DNA-fragment encoding a characteristic amino acid sequence from an u-, 
e.g. the above described DNA fragment, under conditions leading to 
expression of said DNA fragment, and subsequently recovering the 
polypeptide from the organism. 
As described above, the organism which is used for the production of the 
polypeptide may be a higher organism, e.g. an animal, or a lower organism, 
e.g. a microorganism. Irrespective of the type of organism employed for 
the production of the polypeptide, the DNA fragment encoding the 
characteristic amino acid sequence from an u- should be introduced in 
the organism. Conveniently, the DNA fragment is inserted in an expression 
vector, e.g. a vector as defined above, which is subsequently introduced 
into the host organism. The DNA fragment may also be directly inserted in 
the genome of the host organism. The insertion of the DNA fragment in the 
genome may be accomplished by use of a DNA fragment as such or cloned in 
bacteria, phage lambda or other vectors, carrying the DNA fragment and 
being capable of mediating the insertion into the host organism genome. 
The insertion of the DNA fragment into an expression vector or into the 
genome of the host organism may be accomplished as described e.g. by 
Colbere-Garapin F. et al., J. Molec. Biol., 150; 1-14 (1981): A New 
Dominant Hybrid Selective Marker for Higher Eucaryotic Cells. 
Also a higher organism, e.g. an animal, may be employed for the production 
of the polypeptides of the invention. In such cases, transgenic techniques 
known in the art may be employed for the production of the polypeptide. 
Examples of suitable animals are sheep, cattle, pigs, etc. When transgenic 
techniques are employed, the DNA encoding the polypeptide of the invention 
is suitably inserted into the genome of the animal in such a position that 
the polypeptide of the invention is expressed together with a polypeptide 
which inherently is expressed by the animal, preferably a polypeptide 
which is easily recovered from the animal, e.g. a polypeptide which is 
secreted by the animal, e.g. a milk protein, or the like. Suitably, the 
DNA fragment of the invention is inserted in the genome of the animal in 
frame with the DNA fragment encoding the polypeptide inherent to the 
animal so as to obtain expression of a fusion protein comprising on the 
one hand the polypeptide of the invention and on the other hand the 
polypeptide related to the host organism, e.g. the animal. The resulting 
fusion protein may then be subjected to posttranslational modification so 
as to obtain the polypeptide of the invention. 
Similarly, when using an expression vector for the production of the 
polypeptide of the invention, the DNA fragment may be inserted in frame 
with a second DNA fragment encoding another polypeptide so as to obtain an 
expression of fusion protein. 
When the polypeptide of the invention comprises one or more distinct parts, 
e.g. being a fusion protein comprising on the one hand characteristic 
amino acid sequence(s) from u- and on the other hand amino acid 
sequence(s) constituting a polypeptide which is not related to u-, the 
DNA fragments encoding each of these polypeptides may be inserted in the 
genome or expression vector separately or may be coupled before insertion 
into the genome or expression vector by use of conventional DNA techniques 
such as described in Maniatis et al. op. cit. 
The conditions under which the organism producing the polypeptide of the 
invention is cultured or breeded should of course be adapted to the 
organism employed. Conventional cultivation and breeding techniques may be 
employed. In the case of microorganism, the cultivation is e.g. carried 
out in a culture medium conventionally used for fermentation purposes, 
e.g. Luria Broth medium, and under conditions with respect to pH, 
temperature, aeration, etc. suited to the type of microorganism in 
question, e.g. as disclosed in Maniatis et al. op. cit. 
Subsequent to the expression of the polypeptide in the host organism, the 
polypeptide is recovered or isolated from the organism. The polypeptide 
may be isolated or recovered from the culture by a method comprising one 
or more affinity chromatography and/or size chromatography steps, and 
optionally employing a step using an antibody reactive with and/or being 
raised against said polypeptide. Of course, the procedure used for 
recovering of the polypeptide depends on the kind of host organism used as 
well as the polypeptide produced. 
In the case of using transgenic techniques for the production of the 
polypeptide, the polypeptide may e.g. be recovered from the animal 
material, e.g. the milk, in which it is produced by extraction, 
centrifugation, affinity chromatography, ion exchange chromatography, gel 
filtration, or other conventionally used polypeptide isolation and 
purification techniques. 
When the polypeptide of the invention is produced using microorganisms as a 
host organism, the recovery and isolation of the polypeptide will also of 
course depend on the kind of microorganism employed. Suitably, the 
recovering of the polypeptide from the microorganism comprises treatment 
of the microorganism so as to release the polypeptide, e.g. by rupturing 
the microorganism, i.e. partly or totally, and subsequently recovering the 
polypeptide by well-known methods such as precipitation, gel filtration, 
ion exchange chromatography, or HPLC reverse phase chromatography or 
immuno affinity chromatography or the like. 
More specifically, the polypeptide of the invention may be isolated from a 
biological material containing the polypeptide, e.g. a suspension of cells 
producing the polypeptide, by use of a method comprising adsorbing the 
biological material to a matrix comprising an immobilized monoclonal or 
polyclonal antibody as described herein, eluting the polypeptide from the 
matrix, and recovering the polypeptide from the eluate. Examples of 
procedures for isolating the polypeptide are: 
a) A procedure employing antibodies reactive with u- compounds or with 
u--reactive compounds (e.g. u-PA itself or derivatives thereof) which 
is suited for the obtainment of a u- containing fraction with high 
yield and purity. The procedure may be performed by immobilizing the 
specific antibodies, preferably monoclonal antibodies, to a matrix, 
contacting said matrix with the preparation containing the released u- 
compounds, washing, and finally treating the antigenantibody complex fixed 
to the matrix so as to release the u- compounds in a purified form. A 
preferred way is to isolate the u- compounds by means of column 
affinity chromatography involving antibodies fixed to the column matrix. 
b) Procedures involving various forms of affinity chromatography, gel 
filtration, ion exchange or high performance liquid chromatography (HPLC). 
c) Preparative electrophoresis procedures; for instance the following 
procedure: A supernatant from a centrifuged enzyme treated cell or cell 
line preparation is subjected to a gel electrophoresis, such as a sodium 
dodecyl sulphate-polyacrylamidgel electrophoresis (SDS-PAGE) (cf. Laemmli, 
U.K. Nature, 227:680-685; 1970), or an agarose gel electrophoresis. 
Subsequently, labelled antibodies, such as monoclonal antibodies, reactive 
with u-, are used to identify bands primarily constituted by the 
isolated u- compounds. For instance, the antibodies may be used in any 
conventional immunoblotting technique. The markers may be isotopes or 
fluorescein labels detectable by means of relevant sensitive films. After 
identification, the u- containing bands of the gel may be subjected to 
a treatment resulting in the release of the u- compounds from the gels, 
such as procedures involving slicing up the gel and subsequent elution of 
u- compounds. Optionally, the amino acid sequence of the u- proteins 
obtained may be determined. 
d) Procedures involving solubilization of u- from expressing cells using 
phosphatase C and/or D, and the use of the above-mentioned procedures for 
purification. 
Prior to cultivation of the microorganism, the DNA fragment encoding the 
polypeptide of the invention may be subjected to modification, before or 
after the DNA fragment has been inserted in the vector. The polypeptide 
produced may also be subjected to modification. The modification may 
comprise substitution, addition, insertion, deletion or rearrangement of 
one or more nucleotides and amino acids in the DNA fragment and the 
polypeptide, respectively, or a combination of these modifications. The 
term "substitution" is intended to mean the replacement of any one or more 
amino acids or nucleotides in the full amino acid or nucleotide sequence 
with one or more others, "addition" is understood to mean the addition of 
one or more amino acids or nucleotides at either end of the full amino 
acid or nucleotide sequence, "insertion" is intended to mean the 
introduction of one or more amino acids or nucleotides within the full 
amino acid or nucleotide sequence, and "deletion" is intended to indicate 
that one or more amino acids or nucleotides have been deleted from the 
full amino acid or nucleotide sequence whether at either end of the 
sequence or at any suitable point within it. "Rearrangement" is intended 
to indicate that one or more amino acids or nucleotides or the sequence 
has been exchanged with each other. The DNA fragment may, however, also be 
modified by subjecting the organism carrying the DNA fragment to 
mutagenization, preferably site directed mutagenization so as to 
mutagenize said fragment. When the organism is a microorganism, the 
mutagenization may be performed by using conventional mutagenization means 
such as ultraviolet radiation, ionizing radiation or a chemical mutagen 
such as mitomycin C, 5-bromouracil, methylmethane sulphonate, nitrogen 
mustard or a nitrofuran or mutagens known in the art, e.g. mutagens of the 
type disclosed in Miller, J. H., Molecular genetics, Unit III, Cold Spring 
Harbor Laboratory 1972. 
Examples of suitable modifications of the DNA sequence are nucleotide 
substitutions which do not give rise to another amino acid sequence of the 
protein, but which, e.g., correspond to the codon usage of the specific 
organism in which the sequence is inserted; nucleotide substitutions which 
give rise to a different amino acid sequence and therefore, possibly, a 
different protein structure without, however, impairing the critical 
properties of the polypeptide encoded by the DNA sequence; a subsequence 
of the DNA sequence shown above encoding a polypeptide which has retained 
the receptor properties of the native u-; or a DNA sequence hybridizing 
to at least part of a DNA prepared on the basis of the DNA sequence shown 
above, provided that it encodes a polypeptide which has the biological 
property of u-. 
The polypeptide produced as described above may be subjected to 
posttranslational modifications such as for instance thermal treatment, 
treatment with a chemical such as formaldehyde, glutar aldehyde or a 
suitable proteolytic enzyme, e.g. a peptidase or proteinase, such as 
trypsin, phospholipases, glycopeptidases. 
It is well-known that use of recombinant DNA-techniques, including 
transgenic techniques, may be associated with another kind of processing 
of the polypeptide than the processing of the polypeptide when produced in 
its natural environment. Thus, when a bacterium such as E. coli is used 
for the production of the polypeptide of the invention, the amino acid 
residues of the polypeptide are not glycosylated, whereas the polypeptide 
may be glycosylated when produced in another microorganism or organism. 
However, it may be advantageous to remove or alter the processing 
characteristics caused by the host organism in question, and 
post-translational modification of the polypeptide as well as of the DNA 
sequence may serve this purpose. 
The term "truncated polypeptide" refers to a polypeptide deleted for one or 
more amino acid residues eventually resulting in changing of the 
properties of the polypeptide, e.g. solubility. In a further meaning, the 
term "truncated polypeptide" refers to a mixture of polypeptides all 
derived from one polypeptide or expressed from the gene encoding said 
polypeptide. Such truncated polypeptides might arise for instance in 
vector/host cell systems in which part of the cDNA has been deleted by 
restriction enzyme digestion or other suitable methods, resulting in the 
expression of a protein not normally produced in that system. 
Also, the polypeptide of the invention may be prepared by the well-known 
methods of liquid or solid phase peptide synthesis utilizing the 
successive coupling of the individual amino acids of the polypeptide 
sequence or the coupling of individual amino acids forming fragments of 
the polypeptide sequence which fragments subsequently are coupled so as to 
result in the desired polypeptide. The solid phase peptide synthesis may 
e.g. be performed as described by R. B. Merrifield, J. Am. Chem. Soc. 85, 
1963, p. 2149. In solid phase synthesis, the amino acid sequence is 
constructed by coupling an initial amino acid to a solid support and then 
sequentially adding the other amino acids in the sequence by peptide 
bonding until the desired length has been obtained. In this embodiment, 
the solid support may also serve as the carrier for the polypeptide of the 
invention in a vaccine preparation as described below. The preparation of 
synthetic peptides may be carried out essentially as described in 
Shinnick, Ann. Rev. Microbiol. 37, 1983, pp. 425-446. 
Another aspect of the invention is a monoclonal or polyclonal antibody 
reactive with u- compounds, and a method for the preparation thereof. 
The term "antibody" refers to a substance which is produced by a 
vertebrate or more precisely a cell of vertebrate origin belonging to the 
immune system as a response to exposure to the polypeptides of the 
invention. 
The variant domain of an antibody is composed of variable and constant 
sequences. The variant part of the domain is called the idiotype of the 
antibody. This part of the antibody is responsible for the interaction 
with the antigen, the antigen binding. 
The idiotypic structure is antigenic and can thus give rise to specific 
antibodies directed against the idiotypic structure. This has been done in 
mice. The antibodies raised against the idiotype, the anti-idiotypic 
antibodies, may mimic the structure of the original antigen and therefore 
may function as the original antigen to raise antibodies reactive with the 
original antigen. This approach may be advantageous as it circumvents the 
problem associated with the characterization and synthesis of the 
important immunogenic parts of the protein in question. This is most 
important in the case of conformational epitopes, which might otherwise be 
difficult to identify. It has been shown for a number of organisms that 
protective immunity can be induced in this way (e.g. Trypanosoma druzel, 
Trypanosoma brucei, Hepatitis B virus, and Plasmodium knowlesii). 
The antibodies of the present invention may be produced by a method which 
comprises administering in an immunogenic form at least a natural or 
synthetic part of the polypeptide of the invention to obtain cells 
producing antibodies reactive with said polypeptide and isolating the 
antibody containing material from the organism or the cells. The methods 
of producing antibodies of the invention will be explained further below. 
The antibody is preferably a monospecific antibody. The monospecific 
antibody may be prepared by injecting a suitable animal with a 
substantially pure preparation of the polypeptide of the invention 
followed by one or more booster injections at suitable intervals (e.g. one 
or two weeks to a month) up to four or five months before the first 
bleeding. The established immunization schedule is continued, and the 
animals are bled about one week after each booster immunization, and 
antibody is isolated from the serum in a suitable manner (cf. e.g. Harboe 
and Ingild, Scand. J. Immun. 2 (Suppl. 1), 1973, pp. 161-164.) 
For purposes not requiring a high assay specificity, the antibody may be a 
polyclonal antibody. Polyclonal antibodies may be obtained, e.g. as 
described in Harboe and Ingild, see above. More specifically, when 
polyclonal antibodies are to be obtained, the u- compound preparation 
is, preferably after addition of a suitable adjuvant, such as Freund's 
incomplete or complete adjuvant, injected into an animal. When the 
immunogens are human u- compounds, the animals may be rabbits. The 
animals are bled regularly, for instance at weekly intervals, and the 
blood obtained is separated into an antibody containing serum fraction, 
and optionally said fraction is subjected to further conventional 
procedures for antibody purification, and/or procedures involving use of 
purified u- compounds. 
In another preferred embodiment, monoclonal antibodies are obtained. The 
monoclonal antibody may be raised against or directed substantially 
against an essential component of u- compounds, i.e. an epitope. The 
monoclonal antibody may be produced by conventional techniques (e.g. as 
described by Kohler and Milstein, Nature 256, 1975, p. 495) e.g. by use of 
a hybridoma cell line, or by clones or subclones thereof or by cells 
carrying genetic information from the hybridoma cell line coding for said 
monoclonal antibody. The monoclonal antibody may be produced by fusing 
cells producing the monoclonal antibody with cells of a suitable cell 
line, and selecting and cloning the resulting hybridoma cells producing 
said monoclonal antibody. Alternatively, the monoclonal antibody may be 
produced by immortalizing an unfused cell line producing said monoclonal 
antibody, subsequently growing the cells in a suitable medium to produce 
said antibody, and harvesting the monoclonal antibody from the growth 
medium. 
The immunized animal used for the preparation of antibodies of the 
invention is preferably selected from the group consisting of rabbit, 
monkey, sheep, goat, mouse, rat, pig, horse and guinea pigs. The cells 
producing the antibodies of the invention may be spleen cells or lymph 
cells, e.g. peripheric lymphocytes. 
When hybridoma cells are used in the production of antibodies of the 
invention, these may be grown in vitro or in a body cavity of an animal. 
The antibody-producing cell is injected into an animal such as a mouse 
resulting in the formation of an ascites tumor which releases high 
concentrations of the antibody in the ascites of the animal. Although the 
animals will also produce normal antibodies, these will only amount to a 
minor percentage of the monoclonal antibodies which may be purified from 
ascites by standard purification procedures such as centrifugation, 
filtration, precipitation, chromatography or a combination thereof. 
An example of a suitable manner in which the monoclonal antibody may be 
produced is as a result of fusing spleen cells from immunized mice (such 
as Balb/c mice) with myeloma cells using conventional techniques (e.g. as 
described by R. Dalchau, J. Kirkley, J. W. Fabre, "Monoclonal antibody to 
a human leukocyte-specific membrane glycoprotein probably homologous to 
the leukocyte-common (L-C) antigen of the rat", Eur. J. Immunol. 10, 1980, 
pp. 737-744). The fusions obtained are screened by conventional techniques 
such as binding assays employing u- compounds isolated by the 
above-described methods. 
In a further aspect, the invention relates to a diagnostic agent capable of 
detecting and/or quantitating u- or a derivative thereof in a sample. 
In accordance with the above discussion, such diagnostic agent may be 
valuable in diagnosis of cancer and other disorders involving tissue 
invasion and tissue remodelling, considering the involvement of u- in 
these processes. The finding that u- mRNA is consistently found in the 
invasion front in colon carcinoma as shown in Example 6 herein strongly 
supports this notion. In this connection, it is also interesting that 
serum from breast cancer patients has an increased concentration of u-PA 
compared with normal individuals (Gr.o slashed.ndahl-Hansen et al., 1988) 
and that the u-PA content in breast cancer tissue has been shown to be a 
valuable prognostic marker in this disease such as has been published in 
the priority year of the present application (Janicke et. al., 1989, 
1990). The fact that the presence of u- is a prerequisite to u-PA 
function makes it likely that u-PAE content in cancer tissue is an even 
better diagnostic and prognostic marker. A new aspect of the potential 
diagnostic and prognostic use of u- determinations is the release of 
u- from cultured cells (described in Example 4) that occurs even in the 
absence of exogeneously added phospholipase. This finding raises the 
possibility that u- is also released into body fluids under some 
physiological and pathophysiological conditions and particularly in 
cancer. Determination of concentrations of u- or degradation products 
thereof in body fluids, such as serum, urine, and ascites fluid may 
therefore prove to be diagnostically and/or prognostically valuable. 
The diagnostic agent, may, e.g, be an antibody as defined above. 
Alternatively, the diagnostic agent may be in the form of a test kit 
comprising in a container a polypeptide comprising a characteristic amino 
acid sequence of u-, e.g. a sequence including or included in the 
sequence (1). The diagnostic agent may be used in the diagnosis of 
diseases related to abnormal numbers of u-s residing on the cell. 
The diagnostic agent may be one which is suited for use in an agglutination 
assay in which the solid particles to which the antibody is coupled 
agglutinate in the presence of a polypeptide of the invention in the 
sample subjected to testing. In this type of testing, no labelling of the 
antibody is necessary. For most uses it is, however, preferred that the 
antibody is provided with a label for the detection of bound antibody or, 
alternatively (such as in a double antibody assay), a combination of 
labelled and unlabelled antibody may be employed. The substance used as 
label may be selected from any substance which is in itself detectable or 
which may be reacted with another substance to produce a detectable 
product. Thus, the label may be selected from radioactive isotopes, 
enzymes, chromophores, fluorescent or chemiluminescent substances, and 
complexing agents. 
Examples of enzymes useful as labels are .beta.-galactosidase, urease, 
glucose oxidase, carbonic anhydrase, peroxidases (e.g. horseradish 
peroxidase), phosphatases (e.g. alkaline or acid phosphatase), 
glucose-6-phosphate dehydrogenase and ribonuclease. 
Enzymes are not in themselves detectable, but must be combined with a 
substrate to catalyze a reaction the end product of which is detectable. 
Thus, a substrate may be added to the reaction mixture resulting in a 
coloured, fluorescent or chemiluminescent product or in a colour change or 
in a change in the intensity of the colour, fluorescence or 
chemiluminescence. Examples of substrates which are useful in the present 
method as substrates for the enzymes mentioned above are H.sub.2 O.sub.2, 
p-nitrophenylphosphate, lactose, urea, .beta.-D-glucose, CO.sub.2, RNA, 
starch, or malate. The substrate may be combined with, e.g. a chromophore 
which is either a donor or acceptor. 
Fluorescent substances which may be used as labels for the detection of the 
components as used according to the of invention may be 
4-methylumbelliferyl-phosphate, 4-methylumbelliferyl-D-galactopyranoside, 
and 3-(p-hydroxyphenyl) propionic acid. These substances may be detected 
by means of a fluorescence spectrophotometer. Chemiluminescent substances 
which may be peroxidase/eosin/EDTA, isoluminol/EDTA/H.sub.2 O.sub.2 and a 
substrate therefor. 
Chromophores may be o-phenylenediamine or similar compounds. These 
substances may be detected by means of a spectrophotometer. Radioactive 
isotopes may be any detectable and in a laboratory acceptable isotope, 
e.g. .sup.125 I, .sup.131 I, .sup.3 H, .sup.35 P, .sup.35 S or .sup.14 C. 
The radioactivity may be measured in a .gamma.-counter or a scintillation 
counter or by radioautography followed by densitometry. 
Complexing agents may be Protein A, Protein G (which form a complex with 
immunoglobulins), biotin (which forms a complex with avidin and 
streptavidin), and lectin (which forms a complex with carbohydrate 
determinants, e.g. receptors). In this case, the complex is not in itself 
directly detectable, necessitating labelling of the substance with which 
the complexing agent forms a complex. The marking may be performed with 
any of the labelling substances described above. 
In an embodiment of the invention an antibody or a polypeptide of the 
invention may be coupled to a bridging compound coupled to a solid 
support. The bridging compound, which is designed to link the solid 
support and the antibody may be hydrazide, Protein A, glutaraldehyde, 
carbodiimide, or lysine. 
The solid support employed is e.g. a polymer or it may be a matrix coated 
with a polymer. The matrix may be of any suitable solid material, e.g. 
glass, paper or plastic. The polymer may be a plastic, cellulose such as 
specially treated paper. nitrocellulose paper or cyanogenbromide-activated 
paper. Examples of suitable plastics are latex, a polystyrene, 
polyvinylchloride, polyurethane, polyacrylamide, polyvinylacetate and any 
suitable copolymer thereof. Examples of silicone polymers include 
siloxane. 
The solid support may be in the form of a tray, a plate such as a 
mitrotiter plate, e.g. a thin layer or, preferably, strip, film, threads, 
solid particles such as beads, including Protein A-coated bacteria, or 
paper. 
The polypeptide and antibody of the invention may be used in an assay for 
the identification and/or quantification of at least a form and/or a part 
of said polypeptide present in a sample. The identification and/or 
quantification performed by the use according to the present invention may 
be any identification and/or quantification involving u- compounds or a 
form of u- compounds. Thus, both a qualitative and a quantitative 
determination of u- compounds may be obtained according to the use of 
the present invention. The identification and/or quantification may be 
performed for both a scientific, a clinical and an industrial purpose. As 
will be further described below, it is especially important in clinical 
routine to identify or quantify u- compounds. 
The sample may be a specimen obtained from a living organism such as a 
human or an animal. The specimen may be blood, e.g. an erythrocyte 
enriched fraction, or a tissue sample e.g. comprising liver cells. In a 
very interesting embodiment of the present invention, the specimen is 
urine. 
In one preferred embodiment of the invention it is preferred that the 
antibody used in the method of the invention is a monoclonal antibody as 
this generally provides a higher precision and accuracy of the assay, at 
the same time possibly requiring less time to perform. Furthermore, a 
mixture of two or more different monoclonal antibodies may be employed as 
this may increase the detection limit and sensitivity of the test. The 
monoclonal antibody may be obtained by the method described below. 
Antibodies possessing high avidity may be selected for catching 
techniques. 
The antibody used in the present method is preferably in substantially pure 
form (purified according to suitable techniques or by the methods of the 
invention, see below) in order to improve the precision and/or accuracy of 
the assays of the invention. 
The determination of antibodies reactive with the polypeptide of the 
invention and being present in a sample, e.g. as defined above, may be 
carried out by use of a method comprising contacting the sample with the 
polypeptide of the invention and detecting the presence of bound antibody 
resulting from said contacting and correlating the result with a reference 
value. 
When the polypeptide of the invention is to be employed in an assay for 
determining the presence of u- compounds in a sample, it may be in the 
form of a diagnostic reagent or a diagnostic agent. As will be apparent to 
a person skilled in the art several techniques may be applied in 
connection with such diagnostic reagents. 
When, according to the invention, any part of said polypeptide is coupled 
to a solid support, an antibody against the component may then be added to 
the solid support. Alternatively, the antibody is coupled to a solid 
support. 
As a further alternative, any u- compounds present in the sample is 
coupled to a solid support. It may then be incubated with the polypeptide 
component by addition of the component to the solid support followed by 
adding an antibody labelled with a detectable marker. 
The use of a DNA fragment for the detection of the presence of modified, 
rearranged DNA sequences related to u- in tumor or other diseases may 
advantageously be carried out utilizing the principles of the polymerase 
chain reaction as described by Randall et al., Science, 1985, 230: 
1350-1354, Randall et al., Science, 1988, 239: 487-491, and Stoflet et 
al., Science, 1988, 239: 491-494. The polymerase chain reaction (PCR) is a 
procedure used for the amplification of DNA present in a sample. The 
procedure involves the use of two oligonucleotide primers which flank the 
DNA fragment to be amplified. The oligonucleotide primers may e.g. be 10- 
to 20-mers and comprise the flanking regions of the u- gene or be part 
of the u- gene. The oligonucleotide primers are constructed so as to 
enable hybridization of one primer to the plus strand 5' of the target 
DNA, and of another primer to the minus strand 5' of the Target DNA. The 
preferred distance between the two primers is 500-2000 base pairs for 
diagnostic purposes, whereas longer distances could be accepted for 
preparative purposes. The primers are hybridized with the opposite DNA 
strands to be amplified and are extended by using DNA polymerase, e.g. the 
Klenow fragment of E. coli DNA polymerase I or another useful DNA 
polymerase such as the Taq DNA polymerase, so as to synthesize a DNA 
sequence which is complementary to the DNA sequence to which the primers 
are annealed. Subsequent to the synthesis of these complementary 
sequences, the DNA synthesized is denatured, e.g. by heating, from the 
"parent DNA strings", and the parent strings as well as the newly 
synthesized DNA strings are subjected to a new PCR amplification cycle. In 
this manner, it is possible to obtain a substantial amplification of 
specific DNA sequences which are present in a sample. By use of the PCR 
amplification method, it may be possible to amplify and thus detect the 
presence of originally very small and undetectable amounts of DNA 
sequences present in a sample, and thereby e.g. identifying a cancer cell.

EXAMPLE 1 
Purification and Characterization of u- 
MATERIAL AND METHOD 
SDS-PAGE. When not stated otherwise, SDS-PAGE was performed according to 
Laemmli, U.K., "Cleavage of structural proteins during the assembly of the 
head of bacteriophage T4", Nature 227: 680-682, 1970, using 6-16% gradient 
slab gels. Pretreatment of samples under nonreducing conditions was 
performed without boiling. When reducing conditions were used, the samples 
were boiled for 5 minutes in the presence of 20 mM DTT. 
Phast-gel SDS-PAGE was performed on a Phast gel apparatus (Pharmacia), 
using ready-made 10-15% gradient gels. Electrophoresis was performed 
according to the recommendations of the manufacturer. Silver staining was 
performed according to Heukeshoven and Dernick, 1988. 
Tricine-SDS-PAGE of samples to be electroblotted for amino acid analysis or 
NH.sub.2 -terminal amino-acid sequencing was performed in a Mini Protean 
II apparatus (BioRad) according to Schagger and von Jagow, 1987, on a 0.75 
mm homogeneous 7.7% T, 3% C gel. The gel was pre-electrophoresed for 3 
hours at 15 mA in the gel buffer with 12 mM 3-mercaptopropanoic acid added 
as a scavenger. The freeze-dried sample was dissolved directly in 50 .mu.l 
of the sample buffer with 40 mM dithioerythritol as the reducing agent, 
and boiled for 2 minutes. The gel buffer used for pre-electrophoresis was 
replaced with electrophoresis buffer, after which electrophoresis was 
performed for 4 hours at 60 V. 
Electroblotting of samples for amino acid analysis or NH.sub.2 -terminal 
amino acid sequencing. After electrophoresis, the 
Tricine-SDS-polyacrylamide gel was electroblotted onto a polyvinylidene 
difluoride (PVDF) membrane (Millipore), using a semi-dry electroblotting 
apparatus (JKA Instruments, Denmark). Electroblotting took place at pH 
11.0 in 10 mM CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), including 
0.4 mM dithioerythritol and 10% methanol, and was performed at 0.8 
mA/cm.sup.2 for 2 hours. The protein was localized by staining with 
Coomassie R250 for 2 minutes and brief destaining, followed by wash in 
water (Matsudaira, 1987). 
Alkylation of electroblotted protein and amino acid sequencing. The 
Coomassie-stained protein band was cut out from the PVDF-membrane and 
treated with 25 mM iodoacetamide in 50 mM sodium borate, pH 8.0, for 1 
hour in the dark at room temperature. After the reaction, it was washed 
extensively with water and dried under argon. The protein on the dried 
filter was sequenced on an Applied Biosystems protein sequencer, model 
477A. The on-line HPLC identification system for the PTH amino acid 
derivatives included the derivative of carboxymethylcysteine (produced by 
deamidation of the amidomethyl derivative during conversion). The correct 
identification of this derivative was assured by a test-sequencing of 
chicken lysozyme (with cysteine at residue no. 6) after parallel 
preparative electrophoresis, electroblotting and alkylation. 
Determination of amino acid composition and amino sugars. For hydrolysis of 
electroblotted u-, areas of PVDF membranes containing Coomassie-stained 
and in situ alkylated protein were treated with 6M HCl containing 0.05% 
phenol for 20 h in vacuo at 110.degree. C. Amino acid analysis was 
performed on a Waters amino acid analyzer equipped with a post-column 
o-phthaldialdehyde identification system, as described (Barkholt and 
Jensen, 1989). 
Cell culture for analytical studies. The following human cell lines were 
obtained from the indicated sources: the histiocytic lymphoma cell line 
U937 (here designated as U937a) (E. K. O. Kruithof, University Hospital 
Center, Lausanne, Switzerland), a variant of this cell line, designated 
U937b (A. Fattorossi, Research Lab of Aeronautica Militare, Rome, Italy), 
the promyeloid leukemic cell line HL-60 (American Type Culture Collection 
(ATCC)), the bladder carcinoma cell line 5637 (ATCC), the larynx 
epidermoid carcinoma cell line HEp-2 (ATCC), the epidermoid carcinoma cell 
line A-431 (E. Helseth, University of Trondheim, Norway), the cervix 
carcinoma cell line HeLa (ATCC), the colon carcinoma cell line HCT 116 
(ATCC), the conjunctiva cell line Chang (ATCC), the choriocarcinoma cell 
line JEG-3 (A. Vaheri, University of Helsinki, Finland), the amnion cell 
line AV3 (ATCC), and the fibrosarcoma cell line HT-1080 (A. Vaheri). The 
U937a and b and HL-60 cells were grown in suspension, while all the other 
cell lines were grown as monolayers. The HT-1080 and A-431 cells were 
grown in Dulbecco's modified Eagle's medium with 10% heat-inactivated 
fetal calf serum. All other cell lines were propagated in RPMI 1640 medium 
with 5% heat-inactivated fetal calf serum and 2 mM L-glutamine. All media 
were supplemented with 200 units/ml penicillin, 25 .mu.g/ml streptomycin. 
All cells were cultured at 37.degree. C. in a humid atmosphere with 5% 
CO.sub.2. Adherent cells were harvested with a rubber scraper. PMA 
induction of U937b cells was performed at a density of 
0.5-1.times.10.sup.6 cells/ml with 150 nM PMA. A 4-day treatment was used 
whereby the cells adhere to the plastic surface. The PMA-induced adherent 
U937b cells were harvested with a rubber scraper. 
Large-scale production of U937a cells. The U937a cells were grown in 
1-liter spinner flasks to reach a density of 1.0-1.5.times.10.sup.6 
cells/ml in RPMI 1640 medium supplemented with 2 mM L-glutamine, 5% fetal 
calf serum (heat inactivated), 200 units/ml penicillin, 25 .mu.g/ml 
streptomycin (or without antibiotics). Each flask contained 500 ml cell 
culture. 
Phorbol 12-myristate 13-acetate (PMA) induction and harvest of U937a cells. 
The 500 ml cell suspension of one spinner flask was added to 1 liter of 
fresh medium without serum. 150 .mu.l of PMA stock solution in 
dimethylsulfoxide (1 mg PMA/ml) was added, to reach a final concentration 
of 150 nM PMA. The culture was transferred to a 10-layer cell factory 
(Nunc, Denmark) and grown for 3.5 days in the factory. Upon addition of 
the PMA solution, the cells stop dividing and attach to the surface. 
The 1.5 liter supernatant, still containing a large number of less adherent 
cells, was harvested. The more strongly adherent cells were harvested by 
washing the factory with 500 ml of PBS (without Ca.sup.++ and Mg.sup.++) 
containing 0.1% EDTA, and vigorous shaking. The two cell suspensions were 
pooled to yield a total 2-liter harvest. The cells were collected by 
centrifugation. 
Cell lysis and detergent phase separation. PMA-stimulated U937a cells were 
washed and acid-treated as described by Nielsen et al., 1988. 20 ml lysis 
buffer (0.1M Tris/HCl, pH 8.1, 1% Triton X114, 10 mm EDTA, 10 .mu.g/ml 
Aprotinin) and 0.2 ml 100 mM phenylmethylsulfonylfluoride in 
dimethylsulfoxide were added to 10.sup.9 acid-treated cells at 0.degree. 
C. The suspension was mixed thoroughly, left on ice for 5 minutes, mixed 
again and left at 0.degree. C. for another 5 minutes, after which it was 
clarified by centrifugation at 4.degree. C., 16,000.times.g for 10 
minutes. 
The clarified lysate was subjected to temperature-induced phase separation 
(Bordier, 1981) by incubation at 37.degree. C. for 10 minutes, after which 
the detergent phase was collected by centrifugation for 10 minutes at 
20.degree. C., 1,800.times.g. The upper phase was discarded. The lower 
phase (approximately 2 ml) was washed by addition of 18 ml 0.1M Tris/HCl, 
pH 8.1, at 0.degree. C., followed by complete mixing to restore a clear, 
one-phase solution, and repeated phase separation by warming and 
centrifugation, as above. 
After removal of the new upper phase, the lower phase was made up to 20 ml 
by addition of 0.1M Tris/HCl, pH 8.1. In order to avoid renewed phase 
separation during subsequent handling and purification, 500 .mu.l 10% w/v 
3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) was 
added to yield a clear, single-phase detergent fraction. Minor amounts of 
non-dissolved material were removed from this solution by centrifugation 
for 15 minutes at 4.degree. C., 3,300.times.g. 
Lysates and detergent phases from other cell types (as indicated) were 
prepared in the same manner, except that smaller amounts of cell material 
were used. The amounts of all reagents were reduced proportionally. In one 
experiment, 0.5% CHAPS was used as the lysis detergent instead of 1% 
Triton X114. In that experiment, no phase separation was performed. 
Preparation of affinity matrix. 2.5.times.10.sup.6 IU (approximately 25 mg) 
of u-PA (Serono) was dissolved in 25 ml 0.1M Tris/HCl, pH 8.1, 0.1% Tween 
80. The enzyme was inactivated by addition of 250 .mu.l of a fresh 500 mM 
stock solution of diisopropylfluorophosphate (DFP) in isopropanol and 
incubation for 4 hours at 37.degree. C., with a further addition of the 
same amount of DFP after the first 2 hours. 
The reaction was stopped by extensive dialysis at 0.degree. C. against 
0.25M NaHCO.sub.3, 0.5M NaCl, 0.1% Triton X-100, pH 8.5. 
In a a total volume of 50 ml, the dialyzed material was coupled to 12.5 ml 
of CNBr-activated Sepharose (Pharmacia) that had been freshly equilibrated 
with 0.25M NaHCO.sub.3, 0.5M NaCl, pH 8.5 (coupling buffer). The reaction 
proceeded overnight at 4.degree. C. and was stopped by equilibration of 
the matrix with 1M ethanolamine/HCl, pH 8.0 and incubation for 24 hours at 
4.degree. C. The matrix (DFP-u-PA-Sepharose) was washed with the coupling 
buffer and pre-eluted with the appropriate elution buffer (see below) 
before use. 
Affinity purification. The clarified detergent fraction obtained from 
6.times.10.sup.9 U937a cells was diluted with 1 vol washing buffer-1 (10 
mM sodium phosphate, 140 mM sodium chloride, 0.1% CHAPS, pH 7.4) and 
chromatographed on a column containing 8 ml DFP-u-PA-Sepharose, 
equilibrated with the same buffer. After application of the sample, the 
column was washed with washing buffer-1, followed by washing buffer-2 (10 
mM sodium phosphate, 1M sodium chloride, 0.1% CHAPS, pH 7.4). The column 
was eluted from below with elution buffer (0.1M acetic acid, 0.5M sodium 
chloride, 0.1% CHAPS, pH 2.5). Elution fractions were immediately titrated 
to pH 7.5 by addition of the appropriate volume of 0.1M sodium phosphate, 
1.0M sodium carbonate, pH 9.0. u--containing fractions were identified 
by chemical cross-linking to the .sup.125 I-labelled amino terminal (ATF) 
fragment of urokinase, followed by SDS-PAGE and autoradiography. Purified 
u- samples for amino acid analysis or NH.sub.2 -terminal amino acid 
sequencing were dialyzed against 0.1% acetic acid and lyophilized. 
Protein labelling with .sup.125 I. .sup.125 I-labelling of ATF was 
performed as described previously (Nielsen et al., 1988), except that 0.1% 
Triton X100 was replaced by 0.01% Tween 80. Purified u-, concentrated 
by freeze-drying after dialysis against 0.1% acetic acid, was iodinated in 
the same manner, except that 1.5 .mu.g protein was treated with 250 .mu.Ci 
.sup.125 I in a volume of 25 .mu.l. 
Chemical cross-linking assay. Cross-linking of u- in complex mixtures or 
purified fractions to .sup.125 I-labelled ATF was performed as described 
for solubilized receptor (Nielsen et al., 1988), except that 2 mM 
disuccinimidylsuberate (DSS) was used for cross-linking. Cross-linking of 
purified u- to DFP-treated u-PA for analysis by SDS-PAGE and 
silver-staining was performed in the same manner, except that non-labelled 
DFP-treated u-PA was used as the ligand. 
Enzymatic deglycosylation. For the deglycosylation studies on u- in cell 
lysates and detergent fractions, the receptor was selectively labelled 
before the degradation by chemical cross-linking to .sup.125 I-labelled 
ATF. 
Lyophilized, purified u- was radioiodinated directly. 
For complete removal of N-bound carbohydrate, the samples were denatured 
under mildly reducing conditions by the addition of SDS and dithiothreitol 
to final concentrations of 0.5% and 1.6 mM, respectively, and boiling for 
3 minutes. Aliquots of the denatured samples (10 .mu.l) were adjusted to 
include 200 mM sodium phosphate, pH 8.6, 1.5% Triton X-100, 10 mM 1,10 
phenanthroline (added from a methanol stock solution) and either 1 unit of 
peptide:N-glycosidase F (N-glycanase, Genzyme), or no enzyme, in a total 
volume 30 .mu.l. Deglycosylation was performed at 37.degree. C. for 20 
hours. During studies on non-fractionated cell lysates obtained after 
lysis with CHAPS, 100 mM .beta.-mercaptoethanol was used for reduction 
instead of dithiothreitol, and 10 mM EDTA was included during 
deglycosylation instead of 1,10 phenanthroline. 
For desialylation, 70 .mu.l lysate samples labelled by cross-linking to 
.sup.125 I-ATF, were made up to 200 .mu.l with 0.05M sodium acetate, pH 
5.0. 90 .mu.l aliquots of the mixture received either 14 .mu.l of 33 
ng/.mu.l neuraminidase (Boehringer-Mannheim) or no enzyme. Desialylation 
was performed overnight at 37.degree. C. 
RESULTS 
Purification. PMA-stimulated U937a cells were acid-treated to remove any 
surface-bound u-PA and lysed in a Triton X114 containing buffer. The 
detergent extract was subjected to temperature-induced phase separation, 
and the isolated detergent phase was used as the raw material for affinity 
chromatography. The acid eluates were neutralized and analyzed, either 
directly or after concentration by dialysis against 0.1% acetic acid and 
lyophilization. The electrophoretic appearance of the purified material is 
shown in FIGS. 1A-C. 
After SDS-PAGE and silver staining (FIG. 1A), the eluted protein migrated 
as one broad band, covering the range from approximately 55 to 60 kDa. 
Outside this range, no protein material was detected. A single band with 
the same apparent molecular mass was also found when SDS-PAGE was 
performed under nonreducing conditions. (FIG. 1C, lane 5). 
Analysis for binding activity toward the ATF of urokinase was performed by 
chemical cross-linking to .sup.125 I-labelled ATF followed by SDS-PAGE and 
autoradiography. ATF-binding activity co-eluted with silver-stainable 
protein. The conjugate formed between ATF and the purified protein 
migrated as a 70-75 kDa component during electrophoresis (FIG. 1B, lane 
2). As demonstrated previously for partially purified u- (Nielsen et 
al., 1988), the formed conjugate was indistinguishable from the 
cross-linked product formed with ATF on intact, PMA-stimulated U937 cells 
(not shown), as well as in non-purified detergent extracts from the same 
cells. Binding and cross-linking to .sup.125 I-labelled ATF was specific 
and saturable. Thus, it could be competed for by an excess of unlabelled 
ATF, active u-PA or DFP-treated u-PA, while no competition was obtained 
with unrelated proteins such as, for example, bovine serum albumin, or 
with related proteins, such as t-PA, plasminogen or epidermal growth 
factor (FIG. 1B). 
To study the functional integrity and the purity of the purified protein, a 
cross-linking experiment was performed with non-labelled components (FIG. 
1C). In this experiment, DFP-treated u-PA was chosen as the u--specific 
ligand instead of ATF, since, because of the higher molecular weight, this 
ligand would lead to a conjugate clearly separable from the purified 
protein itself by SDS-PAGE. It is seen that all protein material present 
in the purified preparation was able to bind to the nonlabelled ligand 
(compare lanes 4 and 3), thus confirming the identity to u- (Nielsen et 
al., 1988) and the purity of the purified protein. The binding capability 
was indeed a property of the only protein detectable in the preparation by 
silver staining. 
Quantification by amino acid analysis indicated a purification yield of 6-9 
.mu.g polypeptide (corresponding to about 10-15 .mu.g u- glycoprotein; 
see below) from 6.times.10.sup.9 cells. 
Amino acid composition and NH.sub.2 -terminal amino acid sequences. The 
amino acid composition of the purified protein after preparative 
electrophoresis, electroblotting and alkylation with iodoacetamide is 
shown in table 1. This composition includes a strikingly high content of 
cysteine residues. Further, it is noted that rather few lysine residues 
are present. The analysis system employed allows the quantification of 
glucosamine and galactosamine in addition to the amino acids. Glucosamine 
was detected in an amount corresponding to approximately 30 mol of 
N-acetylglucosamine per mol protein, correcting for loss during 
hydrolysis. In contrast, no galactosamine was identified. 
The high number of glucosamine residues detectable after acid hydrolysis, 
as well as the large decrease in apparent molecular mass following 
treatment with peptide:N-glycosidase F (see below), indicate that large 
side chains of N-linked carbohydrate are present in the protein. The 
failure to detect any galactosamine indicates that this type of O-linked 
carbohydrate is absent in u-. However, the presence of other O-linked 
oligosaccharides that escape detection by amino acid analysis cannot be 
excluded. 
Two amino acid sequencing experiments were performed. In the first 
sequencing experiment, direct NH.sub.2 -terminal sequencing of 
affinity-purified u- was performed after dialysis and lyophilization. A 
partial sequence (Table 2A) was obtained, and it was demonstrated that 
only one sequence was present in the purified material. 
In the second sequencing experiment, dialyzed and lyophilized, purified 
u- was subjected to Tricine-SDS-PAGE, electroblotted onto a 
PVDF-membrane, Coomassie-stained, alkylated, and excised as described 
above, and then subjected to NH.sub.2 -terminal sequencing. This sequence 
is shown in Table 2B. 
As seen in Table 2, all amino acid residues identified proved identical 
when comparing the two sequences. Furthermore, positions 3, 6 and 12, 
which were identified only in the second experiment, all proved to be 
cysteines. Thus, the lack of any identification at these positions in the 
first experiment was to be ascribed to the lack of alkylation. It was 
clear that the only detectable NH.sub.2 -terminal sequence in the 
preparation was associated with the electrophoretic mobility of u-. 
Consequently, no additional sequences were hidden in the form of, for 
example, low molecular weight peptide components associated with the major 
polypeptide chain. 
A search in the Georgetown University protein data base did not reveal any 
identity, nor even pronounced homology, of the u- NH.sub.2 -terminal 
amino acid sequence to any known protein. 
The amino terminus, like the amino acid composition of the entire protein, 
is rich in cysteine residues. 
Data for probe construction (Example 2) were derived from the sequencing 
shown in Table 2A. For this construction, position 6 of the amino acid 
sequence was tentatively assigned Asn; see footnote a of Table 2A. 
Glycosylation. Purified .sup.125 I-labelled u-PA receptor was treated with 
Peptide:N-glycosidase F. This enzyme is capable of removing all kinds of 
N-bound carbohydrate, the cleavage site being between the asparagine side 
chain and the innermost N-acetyl glucosamine residue (Tarentino et al., 
1985). FIG. 2 shows the electrophoretic appearance of the deglycosylated 
protein. The electrophoretic band observed after autoradiography of the 
.sup.125 I-labelled protein was always slightly broader than that seen 
after direct protein staining. However, the reaction turned the 
heterogeneous 55-60 kDa receptor (lane 1) into a deglycosylated protein of 
only 35 kDa that migrated as a much sharper band (lane 2), thus further 
confirming that the initially heterogeneous material all represented 
variants of the same protein. 
Glycosylation heterogeneity and variation among cell lines. In another 
series of experiments, unpurified detergent fractions from cell lysates, 
or non-fractionated lysates, containing the receptor were subjected to 
treatment with the same enzyme as used above. In these experiments, a 
selective labelling of u- was performed before the deglycosylation 
reaction by chemical cross-linking to .sup.125 I-labelled amino terminal 
fragment (ATF) of urokinase (Nielsen et al., 1988). 
It is seen (FIG. 3) that the cell lysates from which the receptor was 
purified gave rise to a 70-75 kDa u--ATF conjugate (lane 1) that could 
be deglycosylated to yield an approximately 50 kDa product (lane 3). ATF 
is known not to contain N-bound carbohydrate. Thus, as the change in 
apparent molecular weight was the same as that seen for the purified 
protein above, this experiment provided independent evidence that the 
heavy glycosylation found is indeed a property of the only significant ATF 
binding component in the detergent lysates of these cells. 
When cross-linking was performed on nonstimulated U937a cell extracts (FIG. 
3, lane 2), the conjugate formed reproducibly migrated with a slightly 
higher electrophoretic mobility than that found after PMA stimulation, the 
apparent molecular mass being 70 kDa. After deglycosylation, however, the 
conjugates from the PMA-treated and the non-treated cells became 
indistinguishable (compare lanes 3 and 4). The receptor purified from 
PMA-stimulated U937a cells, therefore, is a glycosylation variant of that 
present in nonstimulated cells. 
When detergent lysates obtained from other cell lines were analyzed by 
chemical cross-linking to ATF, variations in the electrophoretic migration 
of the radiolabelled product were observed in certain cases. In these 
analyses, for comparison, individual adjustment of dilution factors was 
necessary in order to correct for the large variation in u- content 
among various cell types (Nielsen et al., 1988). In separate experiments, 
however, it was assured that the dilution had no effect on the migration 
of the individual conjugates. 
Including the patterns described above, a total of 4 distinguishable 
electrophoretic patterns were found. As reported previously (Nielsen et 
al., 1988), the majority of cell lines yielded a single conjugate band of 
70 kDa, as was the case for e.g. U937a cells not treated with PMA (FIG. 3, 
lane 2). Thus, this pattern was found for e.g. A-431 epidermoid carcinoma 
cells, HeLa cervix carcinoma cells, 5637 bladder carcinoma cells, HCT 116 
colon carcinoma cells, AV3 amnion cells, JEG-3 choriocarcinoma cells, and 
Chang conjunctiva cells. 
The fibrosarcoma cell line HT-1080 contained a third u- variant, giving 
rise to a single conjugate band of a slightly lower molecular weight 
(approximately 6 5 kDa; not shown). 
The fourth pattern was found during studies on a strain of U937 cells 
different from the strain used as raw material for purification. When not 
treated with PMA, this strain (here designated U937b) showed the same 
conjugate band as did the above U937a cells. However, the response to PMA 
treatment was reproducibly different. Thus, PMA-treated U937b cells gave 
rise to two conjugate bands. The uppermost band seemed identical to that 
found in PMA-treated U937a. The lower band appeared sharp and migrated as 
a 55 kDa component (not shown). The latter band was found only after 
cross-linking in solubilized material. When cross-linking was performed on 
intact cells (Nielsen et al., 1988), only the uppermost band was present 
(not shown), suggesting that the lower band could represent an 
intracellular precursor or degradation product of the receptor. 
However, when samples representing the 4 patterns above were subjected to 
enzymatic deglycosylation after the cross-linking to .sup.125 I-ATF, the 
molecular weight variation was abolished. The resulting conjugate band was 
sharp, and migrated as a 50 kDa component, irrespective of the identity of 
the parent cell line (not shown). 
Thus, N-bound glycosylation was responsible, not only for molecular u- 
heterogeneity within the PMA-stimulated U937 line and occurrence of two 
bands in the PMA-stimulated U937b line, but also for the electrophoretic 
difference between u-s from non-stimulated and PMA-stimulated U937 
cells and for the variation among different cell lines (i.e. HT-1080 
fibrosarcoma cells compared to the other cell lines tested). 
Removal of sialic acids. The above cross-linking labelling system for u- 
in unpurified detergent fractions was employed for the study of enzymatic 
desialylation (not shown). Neuraminidase treatment of cross-linked 
detergent fractions from PMA-stimulated U937a cells led to an 
approximately 5 kDa reduction in the apparent molecular weight of the 
ATF-u- conjugate. Thus, the glycosylation includes several sialic acid 
residues. The change in molecular weight, though undoubtedly present, 
appeared somewhat smaller when U937a cells without PMA-stimulation were 
used in the desialylation experiment. However, a preliminary comparison 
suggested that sialylation could not account for the whole difference 
between the u-s in non-stimulated and PMA-stimulated cells. 
TABLE 1 
______________________________________ 
Amino acid composition of affinity purified u-, 
determined after Tricine-SDS-PAGE, electroblotting 
onto a PVDF membrane, and alkylation 
______________________________________ 
Asp/Asn 33.2 
Thr.sup.a 21.4 
Ser.sup.b 26.3 
Glu/Gln.sup.c 
43.2 
Pro 11.4 
Gly 28.2 
Ala 8.4 
Cys (as Cys(Cm)) 
28.4 
Val 11.9 
Met.sup.d 7.7 
Ile 6.7 
Leu 26.5 
Tyr 8.0 
Phe 5.7 
His 12.8 
Lys 11.1 
Arg 20.0 
Glucosamine.sup.e 
30.8 
______________________________________ 
.sup.a Corrected for a 5% loss during hydrolysis. 
.sup.b Corrected for a 10% loss during hydrolysis. 
.sup.c Slight overestimation possible, due to formation of pyroglutamic 
acid in amino acid standard mixture. 
.sup.d Corrected for a 30% loss normally observed during electrophoresis 
and blotting (35). 
.sup.e Corrected for a 50% loss during hydrolysis. 
Hydrolysis of 70 pmol of protein was performed for 20 hours directly on the 
PVDF membrane. The number of residues is calculated assuming a total of 
310 residues. Correction for losses during electrophoresis and blotting 
(Met) and during hydrolysis (Thr, Ser, glucosamine) has been performed 
according to correction factors found for standard proteins analyzed under 
the same conditions. 
TABLE 2 
__________________________________________________________________________ 
N-terminal amino acid sequence of u-. Parentheses indicate an 
identification classified as tentative. Question mark indicates no 
identification. Where footnotes are present, they indicate the best 
guess. 
A. Direct sequencing of affinity purified u- after dialysis 
against 0.1M acetic acid and lyophilization. The initial yield was 
70 pmol PTH-Leu at step 1. Note that direct sequencing does not allow 
the identification of cysteine residues. 
Res. no. 
1 2 3 4 5 6 7 8 9 10 11 12 
13 14 15 16 
__________________________________________________________________________ 
Amino acid 
Leu 
? ? Met 
Gln 
?.sup.a 
Lys 
Thr 
Asn 
Gly 
Asp 
? Arg 
Val 
(Glu) 
Glu (SEQ ID NO. 1) 
residue 
__________________________________________________________________________ 
B. Sequence obtained after Tricine-SDS-PAGE, electroblotting and 
alkylation. The PVDF membrane contained 35 pmol u-, as estimated 
from a parallel amino acid analysis experiment (Table 1). The initial 
yield was 19.5 pmol PTH-Leu at step 1. The repetitive yield, based on 
Leu 1, Leu 19 and Leu 23, was 96%. Cys indicates the identification 
of the PTH derivative of carboxymethyl cysteine in the alkylated 
protein. 
Res. no. 
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 
__________________________________________________________________________ 
Amino acid 
Leu 
? Cys 
Met 
Gln 
Cys 
Lys 
Thr 
Asn 
Gly 
Asp 
Cys 
(Arg) 
Val 
Glu 
residue 
__________________________________________________________________________ 
Res. no. 
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 
__________________________________________________________________________ 
Amino acid 
Glu 
(His) 
Ala 
Leu 
Gly 
Gln 
?.sup.b 
Leu 
?.sup.c 
(Arg) 
Thr 
(Thr) 
Ile 
Val 
?.sup.d (SEQ ID NO. 2) 
residue 
__________________________________________________________________________ 
.sup.a Asn 
.sup.b Asp 
.sup.c Arg/Cys 
.sup.d Arg/Thr 
EXAMPLE 2 
Isolation and Identification of the Ligand Binding Domain of u- 
METHODS 
Enzymatic degradation: Affinity purified u- was dialyzed against 0.1% 
acetic acid and lyophilized as described in Example 1. The freeze-dried 
material was redissolved in incubation buffer (0.05M Tris/HCl, 0.05% 
CHAPS, pH 8.1) to yield a protein concentration of approx 25 .mu.g/ml. 9 
.mu.l samples of this u- solution were treated with chymotrypsin 
(Worthington; final concentrations ranging from 8-200 ng/ml), by addition 
of 1 .mu.l of the appropriate stock solution of the enzyme, dissolved in 
incubation buffer. The samples were incubated for 16 h at 37.degree. C. 
after which the degradation was stopped by addition of 0.5 .mu.l of 20 mM 
phenylmethylsulfonylfluoride, dissolved in dimethylsulfoxide. The samples 
were stored at -80.degree. C. until analysis. 
Analysis: Direct electrophoretic analysis was performed by Tricine SDS-PAGE 
(see example 1) on a 10% T, 3% C gel after reducing sample treatment. The 
reagent system of Henkeshoven and Dernick (1988) was used for silver 
staining. 
Samples to be analyzed by chemical cross-linking to .sup.125 I-ATF were 
50-fold diluted in 0.1M Tris/HCl, 1% Triton X-114, pH 8.1. The diluted 
samples were either clarified by addition of 0.25% w/v CHAPS (final 
concentration) or subjected to a single round of temperature induced phase 
separation (see Example 1). After the phase separation of 1 vol. of 
diluted sample, each phase (i.e., the detergent and buffer fraction, 
respectively) was made up to 1 vol. by addition of 0.1M Tris/HCl, pH 8.1, 
and clarified by addition of 0.25% CHAPS (final concentration). 
Deglycosylation of samples, cross-linked to .sup.125 I-ATF 
Enzymatic deglycosylation with N-Glycanase (Genzyme) was performed 
according to example 1, except that the actual concentrations during the 
deglycosylation step were the following: 0.08% SDS; 0.26 mM 
dithiothreitol; 0.11M sodium phosphate; 0.9% Triton X-100; 5.3 mm 1,10 
phenanthroline; 33.3 units/ml N-glycanase. 
Identification of the binding domain fragment, generated by chymotrypsin 
Direct confirmation of the identity of the 16 kD chymotryptic fragment of 
u- (see "Results" below) to the binding domain of the receptor requires 
a cross-linking experiment using non-labelled DFP-u-PA or ATF as the 
ligand and analysis by SDS-PAGE and silver staining, using the metods 
already adopted (see Example 1). For further analyses, the fragment will 
be generated on a preparative scale (i.e., using purified protein in the 
range of 20-50 .mu.g as the starting material). The N-terminal amino acid 
sequence of the fragment will be obtained by the methods described in 
Example 1 (i.e., Tricine SDS-PAGE, electroblotting and amino acid 
sequencing). Identification of the fragment will subsequently be done by 
comparison to the amino acid sequence derived from u- cDNA. For a 
closer identification of the binding determinant, synthetic peptides 
covering the chymotryptic fragment will be constructed. The peptides will 
be assayed for their potential inhibitory activity against the binding 
reaction between u- and the ligand, as studied by cell binding assays 
(Nielsen et al., 1988; Appella et al., 1987) or by chemical cross-linking 
assay. 
Methods not specified above were as described in Example 1. 
Results 
Samples of purified u- were subjected to degradation with chymotrypsin 
and subsequently analysed by Tricine-SDS-PAGE (FIG. 4). Treatment with the 
enzyme in the concentration range of 8-200 ng/ml (lanes 1-3) led to the 
appearance of a 16 kD degradation product that migrated as a sharp band, 
and a broad band covering the range from 45-65 kD. (Note that the sharp 
bands at 67 kD were due to the reducing sample treatment (Hashimoto et 
al., 1983) and not related to u-; these bands were present also in 
samples devoid of added protein (not shown)). No further products were 
detected. The non degraded u- samples showed one broad band, covering 
the range from 60-70 kD in this electrophoretic system (lanes 4 and 5); no 
additional components were observed. 
In parallel, the samples were analyzed in the chemical cross-linking assay, 
using .sup.125 I-ATF as the ligand (FIG. 5). While the non-degraded 
samples (lanes 4 and 5) showed the 70-75 kD conjugate band which is 
characteristic for the intact u- (see Example 1), the intensity of this 
band was much reduced in the degraded samples (lanes 1-3). In contrast, 
the degraded samples showed an approx. 30 kD cross-linked conjugate; i.e. 
the size to be expected for a conjugate formed between the above 
mentioned, 16 kD u- degradation product and the 15 kD ATF. The presence 
of a minor binding activity corresponding to intact u- was ascribed to 
the cleavage being slightly incomplete; compare to the molecular weight 
pattern of FIG. 4. When analysis was preceeded by phase separation in the 
Triton X-114 system, it came out that th 30 kD conjugate was formed by a 
product preferentially present in the buffer phase, whereas the binding 
activity corresponding to intact u- partitioned into the detergent 
phase (not shown). 
If cross-linked samples were subjected to enzymatic deglycosylation before 
electrophoretic analysis, the molecular weight of the formed conjugate was 
reduced (FIG. 6). Thus, the approx. 30 kD conjugate of the chymotrypsin 
treated samples (lanes 1 and 2), was turned into an approx. 22 kD product 
after treatment with N-glycanase (lanes 4 and 5), while deglycosylation of 
the non proteolyzed samples (lanes 3 and 6) led to a result consistent 
with Example 1. 
In conclusion, the only detectable u- fragment in the lower molecular 
weight (i.e., below 40 kD) region, formed by chymotrypsin in the 
concentration range tested, was a 16 kD product, consistent with the 
expected size for the fragment with binding activity observed after 
cross-linking to .sup.125 I-ATF. Unlike the intact u-, the ligand 
binding fragment proved hydrophilic in the Triton X-114 system, suggesting 
that this fragment does not include the diacylglycerol part of the protein 
(see Example 4). The deglycosylation experiment showed that the ligand 
binding fragment is glycosylated and suggested that the polypeptide part 
of the fragment comprised only 6-10 kD, corresponding to approx. 50-90 
amino acid residues. 
EXAMPLE 3 
Cloning of u- 
cDNA Libraries Used 
A human cDNA library was used made from SV 40 transformed human GM637 
fibroblasts in a plasmid vector based on pBR322 (carrying an ampicillin 
resistance gene) (Okayama H, Berg P, "High-efficiency cloning of 
full-length cDNA", Mol. Cell. Biol. 2: 161-170, 1982). The library was 
kindly donated by Dr. Okayama. This library was selected on the basis of 
the known high number of u- in GM637 cells (Blasi, unpublished). 
The plasmid vector (FIG. 8) uses the SV 40 promoter and has high expression 
in various eukaryotic cells, but very low or no expression in prokaryotes. 
Screening Procedures 
The library was screened with synthetic oligonucleotide probes made on the 
basis of amino acid sequence data from purified receptor protein (Tables 
4-5). The melting temperatures were calculated from Lathe, J. Mol. Biol. 
183: 1-12, 1985. The equation used was modified from: 
EQU t.sub.m =16.6 log M+0.41(% G+C)+81.5 
in which M is the monovalent cation concentration (molarity) in 5.times. 
SSC, 16.6 log M has a value of -2), and % G+C is the base composition. The 
melting temperature calculated from the equation applies to an infinitely 
long stretch of DNA. To account for probe length and degree of homology, 
the following formula was applied: 
EQU t.sub.w =t.sub.m -(810/l)-1.2 (100-h) 
in which l is the length of the DNA (number of bases), and h is the percent 
homology. 
The hybridization conditions were then further tested in pilot experiments 
to maximize the signal to noise ratio. Briefly, nitrocellulose filters 
containing DNA from the plasmid library were hybridized to the 
end-labelled oligonucleotide probe at various temperatures and salt 
concentrations (all within the range calculated from Lathe, supra). The 
filters were produced according to Grunstein and Hogness ("Colony 
hybridization: A method for the isolation of cloned DNAs that contain a 
specific gene", Proc. Natl. Acad. Sci. USA 72: 3961, 1975). The 
hybridization conditions to be used for the screening were chosen as the 
ones giving the minimum amount of background hybridization. In Table 3, 
the amino acid sequence derived frm a preliminary amino-terminal 
sequencing of purified u- (see Example 1) and the derived 
oligonucleotide sequence are presented. 
TABLE 3 
______________________________________ 
The amino acid sequence of the N-terminal peptide 
and the derived synthetic oligonucleotide. 
______________________________________ 
##STR2## 
##STR3## 
##STR4## 
The hybridization conditions used for this probe were 5x SSC and 
50.degree. C. 
______________________________________ 
Outline of Screening Strategy 
Initially, the plasmid library was screened with the N-terminal probe using 
the procedure of Grunstein and Hogness (supra). The detailed procedure is 
described below. Several positive clones were found but after the third 
rescreening, only one remained. The purity of the clone was checked and 
DNA was prepared from it (see large scale DNA preparation below). The DNA 
was digested with several different restrictions enzymes, and a map of the 
restriction sites found in the clone was constructed (see procedure in 
Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring 
Harbor Laboratory, 1982). The insert was further analysed by DNA 
sequencing (see procedure below). The clone was able to code for 7 out of 
the 8 amino acids in the N-terminal peptide used to construct the 24-mer 
probe. The sequence in the probe starts with an A whereas the clone had a 
T in this position, resulting in the substitution of Cys for Met. The 
clone was thus isolated by a specific hybridization but could not code for 
the correct peptide. 
Further DNA sequence analyses showed stop codons in all reading frames, and 
the clone was definitively eliminated as the gene for the u-PA receptor. 
To eliminate the problem of finding this clone again, an oligonucleotide 
was constructed (TGGTGATATGAAGGAGAGAA) from an internal sequence of the 
clone and used as a probe to test the clones isolated in subsequent 
screenings (see below). 
The library was then rescreened using chloramphenicol amplification of 
plasmids (Maniatis et al., supra) to increase the signal intensity. This 
procedure resulted in a total of 7 positive clones (see Table 4) of which 
two were eliminated on the basis of hybridization to the probe made from 
the original false positive clone. 
Outline of DNA Sequencing Strategy 
Sequencing on Plasmid DNA 
SV 40 primer: 
EQU 5' CAGTGGATGTTGCCTTTAC 3' 
This primer was made in the inventors' laboratory on an Applied Biosystems 
391 A DNA Synthesizer. Primers for the pEMBL18 vector were purchased from 
Biolabs. 
Sequencing Procedure 
The procedure used for sequencing followed Hattori et al., 1985 (NAR 
13:7813) for double-stranded sequencing. 
Large-scale Preparation of Plasmid DNA 
Large-scale plasmid DNA preparations were used for restriction enzyme 
analyses of the isolated clones and for the isolation of fragments for 
further sequence analyses. The cloning vector used (pEMBL 18, Biolabs 
Inc.) was also produced in this way. Plasmid DNA was prepared according to 
described procedures (Maniatis, supra). 
Radioactive Labelling of DNA Probes 
The synthetic oligonucleotides were end-labelled using T4 polynucleotide 
kinase and .gamma.-.sup.32 P-ATP. Gel purified DNA fragments were 
nick-translated using a BRL nick translation kit and .alpha.-.sup.32 
P-ATP. The probes were purified on NENSORB 20 columns (NEN) following the 
manufacturer's specifications. Nick-translated probes were denatured for 
10 minutes at 100.degree. C. before use. 
Results 
The results of the screening of the chloramphenicol amplified Okayama-Berg 
cDNA library are presented in Table 4 below. 
TABLE 4 
______________________________________ 
Results of screening the Okayama-Berg plasmid library 
Numbers Numbers positive 
screened First screen 
Second screen 
Third screen 
______________________________________ 
10.sup.6 27 14 7 
______________________________________ 
Large-scale DNA preparations were made from the five plasmid clones 
remaining after elimination of the two false positives (see above). The 
clones were mapped using restriction enzymes and the 5' ends were 
sequenced using an SV 40 primer which hybridizes to the vector. All the 
clones contained an insert of about 1400 bp, and on the basis of the maps 
and the sequences, the clones were determined to be identical. One clone 
was fully sequenced. On the basis of DNA sequence (1), this clone, named 
p-u-1, was found to be able to code for the u-PA receptor. 
This clone has been deposited in plasmid form in the Deutsche Sammlung von 
Mikroorganismen, Mascheroder Weg 1b, D-3300 Braunschweig, Federal Republic 
of Germany, on 5 Apr., 1989 in accordance with the provisions of the 
Budapest Treaty for the International Recognition of the Deposit of 
Microorganisms for the Purposes of Patent Procedure, and has received the 
accession No. DSM 5277. 
Complete sequence of p-u--1 cDNA 
The complete sequence of one of the isolated clones (p-u--1) was 
obtained on double-stranded DNA in both orientations using commercial 
primers for pEMBL18 (M13 primers) and internal synthetic primers (see 
above). The sequence is shown in Sequence (1) in the Detailed Description 
of the invention. The restriction map and the sequencing strategy are 
illustrated in FIGS. 7A-C. The cDNA clone is 1364 nucleotides long from 
the 5' end to the beginning of the polyA stretch. At the 5' end, 46 
nucleotides precede the first ATG codon which is followed by a 1005 
nucleotides sequence with an open reading frame, ending with a 
nonanucleotide containing two in frame stop codons. 312 nucleotides of 3' 
untranslated sequence separate the first stop (TAA) codon from the polyA 
sequence. The assignment of the ATG at nucleotide 47 as the translation 
start site agrees with the consensus for initiating regions (Kozak, 1987) 
as discussed above. The translated sequence starts with a hydrophobic 
sequence which conforms to the rules for the signal peptide (von Heijne, 
1986) (see above). The putative signal peptide is followed by 313 amino 
acid residues. The sequence shown in Sequence (1) was compared with the 
initial amino terminal sequence (FIG. 7A), and it was observed that in 
fact the original sequence contained an error at position 6 (Asn instead 
of Cys) which, however, did not prevent the isolation of the right cDNA 
clone. This is in fact proven by the 25/26 matches of the sequence derived 
from the cDNA with the definitive N-terminal protein sequence (see Example 
1) determined in the course of this study after carboxymethylation and 
electroblotting of the purified protein the region of homology is 
underlined in sequence (1)!. The calculated amino acid content agrees well 
with the one measured on the U937 protein (see Example 1). Also the 
calculated molecular weight (34,633) agrees well with the migration of the 
deglycosylated protein (see Example 1). 
The human u- is a relatively small protein of 313 amino acid residues. 
The amino acid sequence contains five potential N-linked glycosylation 
sites, in agreement with the high level of glycosylation of the protein 
(see Example 1). Starting at amino acid position 282, a sequence of 21 
hydrophobic amino acids flanked by arginine residues may represent a 
membrane spanning domain of the u- (FIG. 7C). At the C-terminal 
(possibly intracellular) side of the presumptive membrane-spanning 
segment, the arginine is followed by 9 additional hydrophobic amino acids 
ending with a carboxy-terminal threonine. Because of the high 
hydrophobicity of the ten carboxy-terminal residues, u- may contain no 
intracytoplasmic domain at all, i.e. also the carboxy-terminal 10 residues 
may be buried in the membrane. The sequence of the carboxy-terminal about 
30 amino acid residues would also be compatible with a signal peptide for 
glycolipid-anchored, phospholipase C-sensitive membrane attachment 
(Ferguson and Williams, 1988). The u- is a slightly acidic protein (6 
net acid charges), is very rich in cysteine, rich in glycine and leucine, 
and poor in lysine. The u- is also rich in serine and threonine 
residues, which might indicate O-linked glycosylation (Russell et al., 
1984). However, deglycosylation and sugar composition studies indicate 
that the receptor contains only N-linked carbohydrates (see Example 1). 
The u- sequence is not similar to any known protein: a search in the 
Georgetown University data bank did not yield any extended homology. In 
particular, it bears no resemblance to the tissue factor, a receptor for 
factor VII of the coagulation pathway, which in common with u- has the 
low molecular weight and the unusually large extent of glycosylation 
(Morrissey et al., 1987). The very high proportion of cysteine residues, 
however, is common to many extracellular portions of receptors, like the 
epidermal growth factor receptor (Yarden & Ullrich, 1988), the epidermal 
growth factor precursor (Bell et al., 1986), and many others (Appella et 
al., 1988). However, there does not appear to be a common pattern of 
cysteine spacings in these proteins. 
Further studies of the u- amino acid sequence revealed that the entire 
extracellular portion of the molecule is organized into three homologous 
cysteine rich domains (1-92, 93-191, and 192-281) as follows: 
##STR5## 
(Amino acid residues that are identical in at least two of the repeats are 
indicated through underlining and italics while conservative substitutions 
are indicated with italics only). 
The second and third repeats are the most closely related (about 25 percent 
identity). Significantly, the pattern of cysteines is strikingly similar 
in these two repeats. These findings may indicate that the extracellular 
part of u- has three distinct domains that have a similar secondary 
structure (e.g. reflecting that they are binding sites for ligands) but 
still being different (e.g. reflecting that they bind different ligands). 
Transfection of p-u--1 cDNA in mouse LB6 cells 
The functionality of p-u--1 clone was tested by transfecting it into 
mouse LB6 cells and testing transfectants by the caseinolytic plaque 
assay. This assay is based on the ability of plasmin to degrade casein 
which gives rise to clear plaques in an opaque background. Since LB6 cells 
produce no plasminogen activator, plasmin cannot be produced. In the 
presence of u-PA receptors, however, cells can bind u-PA and hence acquire 
the ability to degrade casein (Vassalli et al., 1985). The murine LB6 
cells produce no plasminogen activator (unpublished observation) but have 
u-PA receptors. However, because binding is strictly species-specific 
(Belin & Vassalli, personal communication, 1985; Appella et al., 1987; 
Estreicher et al., 1989), LB6 cells cannot bind human u-PA. Expression of 
human u- cDNA by LB6 cells should provide these cells with the ability 
to bind human u-PA which can be visualized by the formation of clear 
plaques in the caseinolytic plaque assay. The vector used in the cDNA 
library is an expression vector that contains the SV40 promoter at the 5' 
end and polyadenylation and splice sites at the 3' end (Okayama & Berg, 
1983). Expression of human u-PA receptors in transfected cells will, 
therefore, prove that the p-u--1 clone encodes a complete cDNA 
sequence. 
MATERIALS AND METHOD 
Cell culture and reagents 
Mouse LB6 cells (Corsaro and Pearson, 1981) were cultured in Dulbecco's 
modified minimal essential medium (DMEM) supplemented with 10% fetal calf 
serum, 2 mM glutamine and 10 IU/ml of penicillin and streptomycin. Human 
high molecular weight urokinase and prourokinase were provided by Lepetit 
SpA (Nolli et al., 1989). The amino terminal fragment of human u-PA, ATF, 
was a gift from Abbott Laboratories. The synthetic peptides human 
u-2-32(ala19)! and mouse u-3-33(ala20)! have been described before 
(Appella et al., 1987). Plasminogen was from Sigma Chemical Co. 
Transfection and caseinolytic plaque assay 
2.times.10.sup.5 LB6 cells were transfected either with 9 .mu.g of 
p-u--1 DNA plus 1 .mu.g of pRSVneo DNA, or with 9 .mu.g of pRSVCAT 
plous 1 .mu.g of pRSVneo DNA using a modification of the calcium phosphate 
coprecipitation technique (Pozzatti et al., 1986). Cells were plated in 
0.8 mg/ml G418-containing DMEM, 10% foetal calf serum, and colonies were 
isolated after about 13 days. The pools of transfected clones were tested 
(in the case of p-u--1 DNA) by the caseinolytic plaque assay (Vassalli 
et al., 1977) and positive clones were picked. After one subcloning, 
several clones from each transfection were tested for human u-PA binding 
using the same technique. Cells (plated one day before at 100,000/dish) 
were washed with PBS, incubated in the presence of 0.2 nM human u-PA for 1 
hour at 37.degree. C., washed extensively and covered with a thin agar 
layer containing 1.3% casein and 17 .mu.g/ml plasminogen. The plates were 
incubated at 37.degree. C. for 3 hours, stained with Coomassie brilliant 
blue R and photographed. In some experiments, specific competitors were 
used during the binding step. 
RESULTS 
Expression of p-u--1 in mouse cells 
As described in Materials and methods, p-u--1 and pRSVneo DNA was 
cotransfected into mouse LB6 cells and a pool of G418 resistant clones was 
isolated and analysed for human u-PA binding (0.2 nM) by the caseinolytic 
plaque technique. Control experiments showed that all cells were negative 
in this assay in the absence of added u-PA or plasminogen. After 
incubation with human u-PA and in the presence of plasminogen, the pool of 
G418 resistant cells that had received p-u--1 DNA gave a high number of 
caseinolytic plaques; control cells (transfected with pRSVCAT and pRSVneo 
DNA) were negative (data not shown). Transfected cells were subcloned and 
single colonies from each transfection tested. The results obtained with 
one such clone are shown in FIGS. 9A-F. LB6 cells transfected with 
p-u--1 DNA formed caseinolytic plaques upon binding human u-PA (see 
FIGS. 9A vs. 9B), whereas those transfected with pRSVCAT DNA did not (see 
FIG. 9B). Specificity is shown by the ability of the amino-terminal 
fragment of u-PA (ATF), i.e. a truncated u-PA molecule maintaining the 
binding capacity but deprived of the catalytic activity (Stoppelli et al., 
1985) (FIG. 9, panel D), and by the synthetic peptide human 
u-2-32(ala19)! (FIG. 9E) to compete with human u-PA. On the contrary, 
the mouse u-3-33(ala20)! does not compete for the binding (FIG. 9F). 
These are the results predicted on the basis of the species specificity of 
u-PA binding (Stoppelli et al., 1985; Appella et al., 1987; Estreicher et 
al., 1989). 
Assessment of p-u--1 cDNA expression in mouse LB6 cells 
The expression of the human u- by mouse LB6 cells transfected with 
p-u--1 was further analysed by binding competition experiments using 
unlabelled and iodinated ATF. The molecular properties of the u- 
expressed by the transfected cells were analysed by SDS-PAGE and 
radiography of material from these cells cross-linked to iodinated ATF. 
MATERIALS AND METHOD 
Cell culture and reagents 
Mouse LB6 cells were grown in DMEM as described in this Example. Iodination 
of ATF has been described previously by Stoppelli et al. (1985). The 
cross-linking reagent disuccinimidyl suberate was from Pierce Chemical Co. 
Binding of .sup.125 I-ATF 
About 300,000 LB6/RSVCAT or LB6/p-u--1 cells in a 30 mm dish were washed 
with PBS containing 1 mg/ml bovine serum albumin, incubated in serum-free 
medium for 1 hour at 37.degree. C., and then incubated with 47,000 cpm 
.sup.125 I-ATF (1500 cpm/fmole) at 37.degree. C. for 60 minutes in the 
presence of different concentrations of unlabelled ATF. The experiment was 
carried out in duplicate. At the end of the incubation, the cells were 
washed with PBS-bovine serum albumin, incubated for 15 minutes at 
37.degree. C. in 0.5N NaOH, and the cell lysate was collected and counted 
(Stoppelli et al., 1985). Specific binding was calculated by subtracting 
the radioactivity not competed by 100 nM ATF. 
Cross-linking of .sup.125 I-ATF to the u- 
Cross-linking of LB6/p-u--1 cells with .sup.125 I-ATF was carried out 
using disuccinimidyl suberate (DSS) as previously described (Picone et 
al., 1989). Duplicate dishes of 2.6.times.10.sup.5 cells were washed with 
PBS-bovine serum albumin (1 mg/ml), incubated with 60,000 cpm .sup.125 
I-ATF (1500 cpm/fmole) in serum-free DMEM supplemented with 25 mM Hepes, 
pH 7.4 for 60 minutes at 37.degree. C., washed four times with PBS-bovine 
serum albumin solution, and cross-linked with 1 mM DSS for 15 minutes at 
room temperature. Cross-linking was stopped with 10 mM (final 
concentration) ammonium acetate and incubated for 10 minutes at room 
temperature. Cells were scraped with PBS containing 1 mM EDTA, 1 mM PMSF, 
collected by centrifugation, resuspended in 25 .mu.l of distilled water, 
and counted. The cells were then lysed directly in Laemmli buffer 
containing 5% .beta.-mercaptoethanol (Laemmli, 1970). In control samples, 
100 nM unlabelled ATF was present during the binding step. The cell 
extract was analysed by SDS-polyacrylamide (12.5%) gel electrophoresis 
under reducing conditions (Laemmli, 1970), along with molecular weight 
markers (Rainbow, Amersham) (myosin; phosphorylase b; bovine serum 
albumin; ovalbumin; carbonic anhydrase; trypsin inhibitor; lysozyme). The 
gel was dried and exposed to X-ray film. 
RESULTS 
Expression of p-u--1 DNA in LB6 cells is supported by quantitative 
binding data with .sup.125 I-ATF. FIG. 10A shows a binding-competition 
plot in which control LB6 cells (LBS/RSVCAT) do not bind .sup.125 I-ATF, 
whereas LB6 cells transfected with p-u--1 DNA do. The binding is 
specifically competed by unlabelled ATF. Scatchard plot of the data gave a 
Ka of about 10.sup.8 moles.sup.-1 and about 25,000 receptors/cell. 
In order to verify that the p-u--1 expressed in the transfected LB6 
cells has the correct molecular properties, cross-linking studies were 
performed with the LB6/p-u--1 cells. Cells were incubated with human 
.sup.125 I-labelled ATF, bound ATF cross-linked with disuccinimidyl 
suberate, the cells lysed and analysed by SDS-polyacrylamide gel 
electrophoresis. The results are shown in FIG. 10B. Whereas the ligand 
migrates with a molecular of about 17,000 daltons, migration of the 
cross-linked ligand corresponds to a molecular weight of slightly less 
than 69,000 identical to that obtained with human GM637 cells (from which 
the cDNA clone is derived). This is the molecular weight expected for the 
intact ATF-u- complex (Nielsen et al., 1988). Considering the possible 
cell-dependent difference in glycosylation, and the fact that PMA-treated 
cells possess a u- of a slightly higher molecular weight because of 
their higher extent of glycosylation, the data presented in FIG. 10B are 
in perfect agreement with those obtained with purified u- (Nielsen et 
al., 1988). 
This Example then shows expression of the human u- gene in mouse LB6 
cells by the following findings: p-u--l DNA transfected LB6 cells bind 
labelled human ATF and unlabelled human u-PA as shown by direct binding 
assay (FIG. 10A) and the caseinolytic plaque assay (FIG. 9). The binding 
is specific as shown by the ability of human ATF, human synthetic peptide 
u-2-32(ala19)!, but not mouse synthetic peptide u-3-33(ala20)! to 
compete for binding (FIGS. 9A-F 10A). The ATF-u- complex has the 
correct molecular weight (FIG. 10B). 
Production of a Soluble Receptor Protein Containing the Binding Site for 
Urokinase 
Receptors are anchored at the plasma membrane by a stretch of hydrophobic 
amino acids (the trans-membrane domain) or through a glycolipid anchor. 
Most integral membrane proteins have a single trans-membrane domain, 
although cases have been described of multiple trans-membrane domains. In 
many cases, the trans-membrane domain is present in the middle of the 
protein sequence, i.e. between the carboxy terminal portion (generally 
intracellular) and the amino terminus (generally extracellular, containing 
the binding site for the ligand in the case of most receptors). A 
carboxy-terminal hydrophobic region is also a signal for glycolipid-anchor 
processing. 
The available information on the structure of the U- indicates that it 
is a protein of about 35,000 daltons, i.e. about 330 amino acids. 
An amino acid sequence compatible with both a trans-membrane domain and a 
glycolipid anchor signal is present at the carboxy terminus. 
In order to produce a soluble receptor, it is necessary to modify the 
protein in such a way as to eliminate the hydrophobic, membrane-spanning 
domain or the glycolipid anchor signal, while retaining both the signal 
sequence for secretion and the extracellular, ligand-binding portion of 
the u-. To this end, two constructions have been made. In one of these, 
the carboxy-terminal 8 last amino acids have been eliminated by inserting 
a stop codon at the unique PFLM-1 site of the u- cDNA. The following 
sequence depicts the carboxy-terminal region of the normal u-: 
EQU GCC AGA CTG TGG GGA GGC ACT CTC CTC TGG ACC TAA (SEQ ID NO.6) 
EQU Ala Arg Leu Trp Gly Gly Thr Leu Leu Trp Thr Stop (SEQ ID NO.7) 
The sequence cut by the restriction endonuclease PFLM-1 is: 
EQU CCANNNNNTGG (SEQ ID NO.8), 
and the bases substituting the N's in the u- sequence are underlined in 
the sequence shown above. Cutting p-u--1 DNA with PFLM-1 results in the 
following ends: 
##STR6## 
p-u--1 DNA was cut with PFLM-1, the ends filled with T4 DNA polymerase 
to produce 
##STR7## 
and the following linker (CTAGTCTAGACTAG) (SEQ ID NO.13) containing 
non-sense codons in all frames was inserted to obtain: 
EQU AGA CTC TAG TCT AGA CTA GAC TGT (SEQ ID NO.14), 
which codes for a u- molecule ending with Arg Leu and thus missing the 
last 8 amino acids (mutant p-u--PFLM-1). This clone has been deposited 
as plasmid DNA in the Deutsche Sammlung von Mikroorganismen, Mascheroder 
Weg 1b, D-3300 Braunschweig, Federal Republic of Germany, on 27 Mar. 1990, 
in accordance with the provisions of the Budapest Treaty for the 
International Recognition of the Deposit of Microorganisms for the 
Purposes of Patent Procedure, and has received Accession No. DSM 5865. 
p-u--PFLM-1 clone has been transfected into LB6 cells as described above 
and its expression compared with that of wildtype p-u--1 cDNA. As shown 
in FIG. 11, this mutant expresses a u- molecule that is partly 
recovered in the medium and partly retained in the cells. In fact, 
cross-linking to iodinated ATF shows a single band in the medium and two 
bands in the Triton X-114 extract (prepared as described in Example 1). 
The lower molecular weight band corresponds to a molecular weight of the 
non-glycosylated u-. Only the high molecular weight band is present on 
the cell surface (see below). The data presented in FIG. 11 indicate that 
approximately 10 times as much protein is present in the medium with 
respect to what is retained in the cell. 
A second mutant has been prepared in which the carboxy-terminal 36 amino 
acids have been deleted from the u-PA receptor, thus leaving a protein 
with no trans-membrane and no glycolipid anchor domain. To obtain this 
mutant, oligonucleotide-directed mutagenesis was employed, using the 
system commercially available from Amersham, to insert a single EcoRV 
site. To this end, the following oligonucleotide was used which hybridizes 
to the nucleotides 935-952 of the u- cDNA sequence: 
EQU GACCTGGATATCCAGTA (SEQ ID NO.15) 
(the underlined sequence indicates the EcoRV site, the bold nucleotide 
indicates the site of the mutation, T to C). This mutation 
(p-u--Ile278) as such results in a Val to Ile substitution in position 
278. The p-u-Ile278 DNA was cut with EcoRV and the same linker 
containing stop codons in all frames was inserted. This results in a 
receptor protein of only 278 molecules, lacking both the trans-membrane 
domain and the glycolipid anchor domain. This mutant (p-u-278stop) is 
expected to be unable to attach to the cell surface, to be secreted in the 
medium, and to bind pro-u-PA, ATF, DFP-u-PA and active u-PA, in general 
the same molecules bound by the normal u-PA receptor. It should therefore 
be useful as a u-PA or pro-u-PA scavenger in all the cases where a 
reduction of u-PA activity is desired. 
EXAMPLE 4 
u- Has a Glycosyl-Phosphotidylinositol Anchor and is C-Terminally 
Processed 
MATERIALS AND METHODS 
Materials 
PVDF membranes (Immobilon-P) were from Millipore. n.sup.w,N.sup.'w 
-dimethyl-Arg was from Sigma, N.sup.w -monomethyl-Arg from Calbiochem, 
whereas N.sup.w,N.sup.w -dimethyl-Arg was a kind gift from Dr. T. Ogawa 
(University of Tokushima, Japan). Ethanolamine was from Merck. Na.sup.125 
I, 9,10(n)-.sup.3 H!-myristic acid (53 Ci/mmol), myo-2-.sup.3 H!inositol 
(18.3 Ci/mmol) and 1-.sup.3 H!ethanolamine hydrochloride (19 Ci/mmol) 
were from Amersham. 
Proteins 
Acetylcholinesterases from human and bovine erythrocytes, phospholipase 
A.sub.2 from bee venom and myelin basic protein from bovine brain were 
from Sigma. Phospholipase D from cabbage and phosphatidylinositol-specific 
phospholipase C from Bacillus cereus (PI-PLC) were from Boehringer 
Mannheim. u- was purified from PMA-stimulated U937 cells as in Example 
1. Active human u-PA was purchaged from Serono and was DFP-inactivated as 
described (Nielsen et al., 1988); the amino terminal fragment (ATF) of 
u-PA was a kind gift from Dr. G. Cassani (LePetit, Italy). ATF, u- and 
DFP-inhibited u-PA were radiolabelled as described (Nielsen et al., 1988) 
except that 0.1% (v/v) Triton X-100 was replaced by 0.1% (w/v) CHAPS in 
the case of u- and by 0,01% (v/v) Tween 80 in the case of ATF and 
DFP-u-PA. Preparation of polyclonal rabbit antibodies against human u- 
was carried out as described in Example 11. 
Phospholipase treatment of intact U937 cells 
Adherent, PMA-stimulated U937 cells (approx. 2.times.10.sup.7 /dish) were 
initially washed with serum-free RPMI 1640 medium including 25 mM HEPES, 
pH 7.4 (Buffer A). The cells were subsequently acid treated for 3 min at 
room temperature in 50 mM glycin/HCl, 0.1M NaCl (pH 3.0) to dissociate any 
endogenously produced u-PA, bound to its receptor in an autocrine fashion. 
The supernatants were discharged immediately after neutralization with 0.2 
vol of 0.5M HEPES, 0.1M NaCl (pH 7.5) and the cells were washed twice with 
buffer A. In some experiments exogenously added .sup.125 I-labelled 
DFP-uPA (1 nM) were allowed to rebind to the unoccupied u- by 
incubation for 2 hours at 4.degree. C. in buffer A followed by 
3.times.wash in the same buffer without added ligand. Incubation of these 
adherent U937 cells with the various phospholipases were performed in 
buffer A at 37.degree. C. on a shaking table. 
In vivo labelling 
Cell culture was performed as described in Example 1. Prior to metabolic 
labelling human U937 cells (5.times.10.sup.7 cells/dish) were 
PMA-stimulated (150 nM) for 5 hours in order to increase expression of 
u-. For labelling with .sup.3 H!ethanolamine and .sup.3 H!myristic 
acid the cells were cultured in RPMI 1640 medium, while labelling with 
myo-.sup.3 H!inositol was performed in Eagle's minimum essential medium. 
Both media were supplemented with: 2 mM L-glutamine, 5 mM Na-pyruvate, 200 
units/ml penicillin, 25 .mu.g/ml streptomycin, 25 mM HEPES (pH 7.4), 0.5 
mg/ml defatted BSA and 4.times.normal concentration of non-essential amino 
acids. All tracers were added from stock solutions in 25 mg/ml defatted 
BSA, 0.1M HEPES (pH 7.4) to a final concentration of 0.1 mCi/ml in 10 ml 
media and metabolic labelling was allowed to proceed for 15 hours at 
37.degree. C. Subsequently, the adherent cells were acid treated, washed 
and lyzed with 5 ml ice-cold 1% precondensed Triton X-114, 0.1M Tris (pH 
8.1), 10 .mu.g/ml Trasylol, 1 mM PMSF and 0,2 mM ZnCl.sub.2. Finally, 
detergent-phase separation was performed as described in Example 1. 
Immunoprecipitation of biosynthetically labelled u- 
To each aliqout of 2 ml clarified detergent phase was added 12 .mu.g 
preimmune rabbit IgG and the mixture was incubated for 2 hours at 
4.degree. C. After addition of 100 .mu.l of a 50% (v/v) suspension of 
Protein A Sepharose (Pharmacia) in 0.1M Tris (pH 8.1), 0.1% CHAPS and 0.1% 
defatted BSA, incubation at 4.degree. C. was continued for 2 hours with 
concomitant mixing. The supernatant was recovered by centrifugation (5 
minutes at 5,000.times.g) and incubation was proceeded overnight at 
4.degree. C. after addition of 12 .mu.g of polyclonal anti-u- rabbit 
IgG and finally for an additional 3 hours with a new aliquote of Protein A 
Sepharose as above. The immobilized immunocomplexes were then extensively 
washed in 0.1M Tris (pH 8.1)/0.1% CHAPS including either 0.1% (w/v) 
defatted BSA (once), 0.1% defatted BSA/1M NaCl (once) or without further 
additions (twice). The Protein A Sepharose thus washed was collected by 
centrifugation and finally suspended in 50 .mu.l of 0.1M Tris (pH 6.8) 
containing 2% (w/v) SDS and boiled for 5 minutes before analysis by 
SDS-PAGE. 
Tricine-SDS-PAGE and amino acid analysis 
Tricine-SDS-polyacrylamide gels were prepared according to Schagger and von 
Jagow, 1987 in a Bio-Rad Mini-Protean II apparatus (8 cm.times.7 
cm.times.0.75 mm). The homogenous gel (7.5% T and 3% C) was cast 1 day in 
advance and subjected to pre-electrophoresis at pH 8.45 with 0.5M Tris, 
0.1% (w/v) SDS and 12 mM 3-mercaptopropionic acid (added as scavenger) for 
4 hours at 15 mA/gel. Purified, lyophilized u- was reduced by boiling 
for 2 minutes in 4% (w/v) SDS, 12% (w/v) glycerol, 50 mM Tris and 40 mM 
dithiotreitol at pH 6.8. The gel buffer used for pre-electrophoresis was 
replaced with the original electrophoresis buffer (Schagger and von Janow, 
1987) except that 1 mM 3-mercaptopropionic acid was included in the catode 
buffer. Electrophoresis was performed at 60 V for 4 hours. Electrotransfer 
onto a 0.45 .mu.m PVDF-membrane was performed at pH 11 in 10 mM 
3-(cyclohexylamino)-1-propane sulfonic acid, 10% v/v methanol and 0.4 mM 
dithiotreitol by the semi-dry approach at 0.8 mA/cm.sup.2 for 2 hours as 
previously described (Ploug et al., 1989). 
The Coomassie stained u- was prepared for amino acid analysis by acid 
hydrolysis directly on the excised PVDF-membrane at 110.degree. C. in 100 
.mu.l of redistilled 6M HCl including 0.05% (w/v) phenol and 5 .mu.l of 1% 
(w/v) DTDPA in 2M NaOH as published (Ploug et al., 1989). Amino acid 
analysis was performed on a Waters amino acid analyzer, equipped with 
o-phtaldialdehyde derivatization essentially as described (Barkholt and 
Jensen, 1989). However, the chromatographic system was modified slightly 
to increase resolution of basic amino acids. Elution was still performed 
by a pH-gradient resulting from mixing two non-halide buffers A and B (for 
composition see Barkholt and Jensen, 1989), but the gradient consisted of 
the following linear segments: initial eluant 100% A, 88% A and 12% B at 
15 min, 60% A and 40% B at 24 min, 55% A and 45% B at 26 min, 50% A and 
50% B at 36 min, 30% A and 70 B at 40 min, 25% A and 75% B at 64 min, 100% 
A at 65 min and 100% A from 65 to 70 min. 
Miscellaneous analyses 
SDS-PAGE, chemical cross-linking with disuccinimyl suberate (DSS) and an 
analytical detergent phase separation was performed with Triton X-114 as 
described in Example 1. 
Direct autoradiography (.sup.125 I) and fluorography (.sup.3 H) were 
performed with an X-ray film (Kodak X-Omat) at -80.degree. C. using 
intensifying screens (Cronex). In the case of fluorograms the X-ray film 
was pre-exposed (0,2-0,3 A) and the polyacrylamide gels were impregnated 
with Amplify according to the manufacturer's instructions (Amersham). 
RESULTS 
Amino acid analysis of purified u- 
Amino acid analysis of the purified u- (see Example 1) revealed the 
presence of an unidentified compound in the acid hydrolysate that reacted 
with o-phtaldialdehyde and eluted just after ammonia during cation-exhange 
chromatography (FIG. 12). A similar peak was observed when u- was 
purified from non-stimulated U937 cells (2.times.10.sup.10 cells), but 
otherwise treated identically (data not shown). This unknown compound 
behaved as a covalent constituent of u-, as it persisted within the 
purified protein despite boiling it in 2% SDS followed by Tricine-SDS-PAGE 
and electroblotting onto a 0.45 .mu.m polyvinylidene diflouride (PVDF) 
membrane in the presence of 10% (v/v) MeOH. Furthermore, the compound was 
a specific constituent of the Coomassie stained u-, as it was absent, 
when appropriate pieces of PVDF-membranes just above and below the protein 
stained area were excised and prepared for amino acid analysis by the same 
procedure (FIG. 12 insert). In addition, several stained proteins and 
peptides previously analyzed by this approach did not reveal the presence 
of this particular component (Ploug et al., 1989). 
For amino acid analysis in this study, a special gradient was designed for 
the cation-exchange chromatography that allowed an increased resolution of 
common as well as various uncommon, basic amino acids without impairing 
reproducibility of their retention times (see Materials and Methods 
section). By this method the unidentified compound in u- reproducibly 
eluted after 55.3 min, between ammonia (53.5 min) and arginine (60.8 min). 
As various physiological occurring arginine derivatives are expected to 
possess approx. similar retention times, several methylated arginine 
derivatives were tested, including: N.sup.w,N.sup.w -dimethylarginine 
(53.8 min), N.sup.w,N.sup.w -dimethylarginine (54.4 min) and N.sup.w 
-monomethylarginine (58.6 min). None of these retention times were in 
agreement with the one observed for the unidentified compound in u-. 
However, when authentic ethanolamine was tested, it showed exactly the 
same retention time as that for the unidentified compound. Furthermore, 
upon hydrolysis of both human and bovine erythrocyte 
acetylcholinesterases, a compound with this retention time was also 
observed, whereas it was absent in the hydrolysate from e.g. myelin basic 
protein. Acetylcholinesterases isolated from erythrocytes contain 
ethanolamine as a covalent constituent in a glycolipid membrane anchor, 
while myelin basic protein posseses a partly methylated arginine residue. 
It is therefore concluded that u- does contain ethanolamine, covalently 
linked to the protein by acid labile bonds (e.g. ester or amide bonds). 
Quantitative analysis of the data in FIG. 12 shows that each u- 
molecule contains 2-3 ethanolamine residues (see also Table 5). 
Release of u- from cell surfaces by PI-PLC treatment 
The presence of ethanolamine in purified u- suggests that this cellular 
receptor may be anchored to the plasma membrane by 
glycosylphosphatidylinositol (GPI). The majority of such GPI-anchored 
proteins are susceptible to bacterial phosphatidylinositol-specific 
phospholipase C (PI-PLC), which release the proteins into the medium by 
removing the diacylglycerol portion of the glycolipid (Low, 1989). We 
therefore investigated whether PI-PLC could release .sup.125 I-labelled 
DFP-treated u-PA, initially bound to the cell surface of PMA-stimulated 
U937 cells. As shown in FIG. 13, approx 50% of the cell associated 
radioactivity was released within the first 15 min by PI-PLC. Furthermore, 
the rate of release was only slightly decreased when PI-PLC concentration 
was reduced to only 50 ng/ml (data not shown). In contrast, neither 
phospholipase A.sub.2 (FIG. 13) nor phospholipase D (not shown) was able 
to induce any enhanced liberation of 125I-labelled DFP-u-PA from the cell 
surface as compared to the blind sample, although these phospholipases 
were present in rather high concentrations (&gt;5 .mu.g/ml, FIG. 13). 
Trypsin, on the other hand, efficiently released all cell surface 
associated radioactivity (not shown), thus demonstrating the physical 
accessibility of the receptor bound u-PA. 
As shown in FIG. 14A, u-PA released to the medium by PI-PLC was essentially 
non-degraded and consisted primarily of intact two-chain u-PA (Mr 50,000) 
along with a smaller amount of its amino terminal fragment (ATF, Mr 
17,000). The receptor-binding domain of u-PA resides in both of these 
components (Appella et al., 1987). Accordingly, these two molecular 
species did bind to the cell surface during preincubation with .sup.125 
I-labelled DFP-u-PA. In contrast, the low molecular weight form of u-PA 
(Mr 33,000), devoid of the receptor-binding domain, was eliminated by the 
washing procedures. These data indicate that u-PA and ATF were released 
from the cell surface by PI-PLC, while they were specifically associated 
to u-. 
When cross-linking analysis was performed concomitantly with sampling in 
this experiment by addition of 1 mM disuccinimidyl suberate (DSS) to the 
withdrawn supernatants, soluble u-PA containing complexes were detected 
only in the media from the PI-PLC treated cells (FIG. 14B). The 
electrophoretic mobility of this conjugate in SDS-PAGE (Mr 110,000) was 
identical to that of a u-PA/u- complex (Nielsen et al., 1988). The mock 
treated sample showed only free u-PA in the medium, reflecting a slow, 
spontaneous dissociation of u-PA from the u-. This experiment further 
supports the interpretation that u-PA released by PI-PLC is in complex 
with u-. Finally, it was demonstrated directly that a specific release 
of the u- protein itself by PI-PLC was the real cause for the observed 
release of the .sup.125 I-labelled ligands. In this experiment, 
PMA-stimulated U937 cells were initially acid treated to remove endogenous 
u-PA and then incubated with PI-PLC. Subsequently, the presence of any 
u-PA binding components released into the media was assayed by 
cross-linking to .sup.125 I-labelled DFP-u-PA. This experiment revealed 
that PI-PLC induced a fast conversion of the unoccupied u- from a 
membrane-anchored form into a soluble protein (Mr 60,000) that still 
expressed high affinity towards .sup.125 I-labelled DFP-u-PA (FIG. 14C) as 
well as .sup.125 I-labelled ATF (data not shown). Furthermore, by SDS-PAGE 
and immunoblotting, a protein with similar Mr was detected in the 
serum-free medium after PI-PLC treatment of PMA-stimulated U937 cells, 
using a polyclonal mouse antiserum raised against purified human u- 
(data not shown). Hence, this soluble protein resembles cell-associated 
u- in both functional (binding specificity) and structural terms (Mr 
and antigenicity). Analysis of non-stimulated U937 cells in suspension 
revealed a similar PI-PLC dependent release of u- (not shown). 
A slow, endogenous release of u- could, however, be detected after 
prolonged incubation in serum-free media without PI-PLC treatment (FIG. 
14C); this finding may indicate that the cells either produce and secrete 
a soluble u- or more likely, that they produce a GPI-specific 
phospholipase. 
Altered hydrophobicity of purified u- after PI-PLC treatment 
When purified u- was subjected to detergent-phase separation by Triton 
X-114, it almost quantitatively partitioned into the detergent phase, as 
assessed by cross-linking to .sup.125 I-labelled ATF (FIG. 15A), thus 
demonstrating the very hydrophobic properties of the receptor. Incubation 
with PI-PLC altered the hydrophobicity of the u-PA binding protein 
substantially, as more than 50% of the ATF-binding activity was now 
recovered in the aqueous phase (FIG. 15B). It proved impossible to achieve 
a higher level of this conversion in the purified u- preparation by 
increasing the concentration of PI-PLC. These data are in accordance with 
the fraction of cell associated u-PA which had been released in the 
previous experiment by PI-PLC treatment of intact PMA-stimulated U937 
cells (FIG. 13). This finding may indicate that a partial resistance 
(approx. 50%) against bacterial PI-PLC is a genuine feature of the u- 
population In vivo. Other phospholipases (PLD and PLA.sub.2) did not 
induce any significant change in the hydrophobic properties of the 
purified u- (FIG. 15C). 
A similar behaviour was seen when samples of .sup.125 I-labelled u- were 
analyzed by charge-shift electrophoresis after enzymatic treatment with 
various phospholipases. Only PI-PLC was able to transform a significant 
portion of the labelled u- (again approx. 50%) into a hydrophilic form 
that migrated independently of the composition of detergents in the 
polyacrylamide gel (data not shown). This experiment shows that the PI-PLC 
induced change in phase-partitioning of the ATF binding activity is 
totally accounted for by an identical change in the hydrophobicity of the 
u- protein itself. 
In vivo labelling 
Biosynthetic labelling of a component (Mr 50-60,000), capable of binding to 
DFP-u-PA, was obtained after incubation of PMA-stimulated U937 cells with 
either .sup.3 H!-ethanolanine, myo-.sup.3 H!-inositol or .sup.3 
H!-myristic acid (data not shown). This protein was isolated from the 
detergent lysates of U937 cells by immunoprecipitation with specific 
polyclonal antibodies against u- and analysed by SDS-PAGE and 
fluorography (see Materials and Methods). 
Post-translational processing of the carboxyl terminus 
Apart from demonstrating the presence of approx. 2 mol ethanolamine/mol 
u- (FIG. 12 and Table 5), amino acid analysis revealed additional 
information about potential post-translational processing of this membrane 
receptor. When the calculated amino acid composition for the purified 
u- was compared with that predicted for the nascent protein from cDNA 
sequence, several reproducible and significant discrepancies arose (Table 
5). In particular, the actual determinations of Ala and Leu were too low, 
whereas those of Tyr and Phe were too high (Table 5). Interestingly, 
however, it was possible to bring the calculated and the predicted amino 
acid compositions into perfect agreement provided that the last 29-31 
COOH-terminal residues were removed during some posttranslational event 
(Table 5). Thus, on the basis of the determined amino acid composition and 
the accuracy/precision normally obtained for this equipment, it is assumed 
that there exists a COOH-terminal processing site in u-. According to 
this model, processing is expected to occur at one of the residues 
Ser.sub.282, Gly.sub.283 or Ala.sub.284 --as indicated in FIG. 16. 
TABLE 5 
______________________________________ 
Amino acid composition of purified u- compared with that 
deduced from its cDNA before and after the proposed COOH-terminal 
processing.sup.a 
Entire u- sequence (Leu.sub.1 -Thr.sub.313) 
A) Predicted Determined after 
Amino acid 
from cDNA acid hydrolysis 
SD 
______________________________________ 
Asp + Asn 
29 32.7 0.5 
Thr.sup.b 
25 21.9 0.5 
Ser.sup.b 
25 25.8 0.5 
Glu + Gln.sup.c 
37 41.8 1.3 
Pro 12 11.1 0.3 
Gly 29 29.4 1.1 
Ala 11 8.3 0.1 
Cys.sup.d 
28 28.8 1.0 
Val 12 12.1 0.2 
Met 7 6.0 0.6 
Ile 8 6.7 0.1 
Leu 31 26.9 0.7 
Tyr 7 7.8 0.2 
Phe 5 5.7 0.1 
His 13 12.8 0.1 
Lys 10 10.8 0.2 
Arg 20 20.3 0.2 
Trp 4 nd nd 
Ethanolamine 
-- 2.6 0.4 
______________________________________ 
Assumed u- sequence after processing (Leu.sub.1 -Ala.sub.284) 
B) Predicted Determined after 
Amino acid 
from cDNA acid hydrolysis 
SD 
______________________________________ 
Asp + Asn 
29 29.8 0.4 
Thr.sup.b 
20 20.0 0.5 
Ser.sup.b 
24 23.6 0.4 
Glu + Gln.sup.c 
36 38.1 1.2 
Pro 9 10.2 0.3 
Gly 26 26.8 1.0 
Ala 8 7.6 0.1 
Cys.sup.d 
28 26.3 0.9 
Val 12 11.0 0.2 
Met 6 5.5 0.5 
Ile 7 6.1 0.1 
Leu 24 24.5 0.6 
Tyr 7 7.1 0.1 
Phe 5 5.2 0.1 
His 12 11.6 0.1 
Lys 10 9.9 0.2 
Arg 19 18.6 0.2 
Trp 2 nd nd 
Ethanolamine 
-- 2.4 0.4 
______________________________________ 
Footnotes to Table 5 
.sup.a. Purified u was prepared for amino acid analysis as described i 
the legend to FIG. 12 A-B. The presented values represent the average of 
independent determinations. The data were normalized relative to all amin 
acids, except tryptophan, assuming a total number of 309 residues for the 
nascent u and 282 for the fully processed protein (omitting 4 and 2 
tryptophan residues, respectively). Amino acid numbering was based upon 
the cDNA sequence for u without the signal sequence (Example 3). 
.sup.b. The values for these hydroxyamino acids were corrected for 
decomposition during hydrolysis Ser (5%) and Thr (10%). 
.sup.c. A slight overestimation is expected due to the formation of 
pyroglutamic acid in the amino acid standard mixture. 
.sup.d. In one sample cysteine was derivatized before hydrolysis by in 
situ alkylation using iodoacetamide and subsequently quantified as 
Scarboxymethylcysteine after acid hydrolysis. In general, the yield of 
this alkylation procedure is 95% (Ploug, 1989). Otherwise, cysteine was 
derivatized during hydrolysis in the presence of 3,3dithiodipropionic aci 
(DTDPA) and quantified as the mixed disulfide compound (Cysx) formed 
between cysteine and DTDPA. 
.sup.e. nd = not determined. 
.sup.f. SD = standard deviation (absolute number of residues). 
The results in this Example unequivocally demonstrate that u- has a 
glycosyl-phosphotidylinositol anchor and is C-terminally processed. 
EXAMPLE 5 
Regulation of u- and u- mRNA Levels 
MATERIALS AND METHODS 
Materials 
Phorbol 12-myristate 13-acetate (PMA), dexamethasone and dibutyl cyclic AMP 
were obtained from Sigma. Deoxycytidine 5'-.alpha.-.sup.32 P! 
triphosphate (specific activity 3000 Ci/mmol), and Rainbow .sup.14 C! 
protein molecular weight markers were purchased from The Radiochemical 
Centre, Amersham, U.K. A kit for random primed labelling reaction and 
murine epidermal growth factor (mEGF) were purchased from Boehringer 
Mannheim, BRG. Porcine transforming growth factor .beta.-type 1 
(TGF-.beta.1) was obtained from R and D Systems, Minneapolis, Minn., USA. 
Cell culture 
The human histiocytic lymphoma cell line U937 (American Type Culture 
Collection (ATCC), CRL 1593) was obtained from Dr. A. Fattorssi (Research 
Lab. of Aeronautica Militare, Rome, Italy) and cultured in RPMI 1640 
medium with 10% heat inactivated fetal calf serum and 2 mM L-glutamine at 
a density of 0,5.times.10.sup.6 cells/ml at the onset of the experiment. 
The medium was supplemented with 100 units/ml of penicillin and 25 ug/ml 
streptomycin. The human rhabdomyosarcoma (RD) and adenocarcinoma (A549) 
cell lines (ATCC CCL 136 and ATCC CCl 185, respectively) were obtained 
from Flow laboratories, Irvine, U.K., and kept in Dulbecco's modified 
Eagle's medium supplemented with 10% fetal calf serum until confluency, as 
described earlier (Lund et al., 1988). The cell lines were tested for and 
found free from Mycoplasma infection. PMA and the other compounds were 
present during different time periods and in varying concentrations, as 
indicated for each experiment. The adherent cells were released by a 
rubber policeman, and harvested for RNA analysis as described (Mayer et 
al., 1988). 
RNA analysis 
Total cellular RNA was isolated from the cells as described by Chomczynsky 
and Sacchi (1987). The RNA was analyzed by hybridizing Northern blots as 
described (Lund et al., 1987), except that random primed labelled plasmid 
probes were used. The plasmid used as a probe for u- mRNA (p-u--1) 
carries cDNA covering the entire coding region and the 3'- and the 
5'-untranslated regions (Example 3). 
Chemical cross-linking assay was performed as described in Example 1. 
RESULTS 
Effect of PMA on u- mRNA levels 
Total RNA was extracted from control U937 cells and from U937 cells treated 
with PMA at different time periods. The size and relative concentration of 
mRNA specific for u- was analyzed by Northern blot filters, which were 
hybridized with a plasmid containing a full length cDNA coding for u- 
(FIGS. 17A-B). 
In the Northern blot the signal for u- is extremely weak for the control 
cells but visible after longer exposure (result not shown). After 3 hours 
of PMA treatment, a visible signal for u- mRNA is seen with a maximal 
effect after 24 hours of PMA treatment. 
As a control for equal loading of RNA, the Northern filter was stripped and 
rehybridized with a human .beta.-actin cDNA probe (Ponte et al., 1983). No 
or only a little effect on the level of hybridization with the 
.beta.-actin cDNA was seen after PMA treatment. 
Effect of PMA treatment on the u- protein level 
The effect of PMA on production of u- protein was studied by 
cross-linking experiment. .sup.125 I-labelled aminoterminal fragment (ATF) 
of the urokinase were chemically cross linked to the detergent phase of 
phase-separated Triton X-114 extracts prepared from U937 cells treated 
with PMA for different time periods. FIG. 18 shows a weak signal of 
.sup.125 I-ATF cross-linked to the u- in control U937 cells. After 
increasing time of PMA treatment both an increase in the strength of 
signal and a change to a lower electrophoretic mobility was seen. 
Effect of PMA, dexamethasone, mEGF and TGF-.beta.-1 on u- mRNA levels in 
A549 and RD cells 
FIG. 19 shows that u- mRNA levels are increased after 48 hours of 
stimulation with PHA (150 nM), mEGF (20 ng/ml and TGF-.beta.1 7,5 ng/ml) 
in both A549 and RD cells. Dexamethasone treatment (10.sup.-6 M) for 48 
hours increased the u- mRNA level only in RD cells. 
Effect of dibuturyl cAMP treatment on the u- protein level in U937 cells 
The effect of dibuturyl cAMP on production of u- protein was studied by 
the cross linking assay as described. FIG. 20 shows a weak signal of 
.sup.125 I-ATF cross-linked to the u- in control U937 cells. After 
increasing time of dibuturyl cAMP treatment both an increase in the 
strength of signal and a change to a lower electrophoretic mobility was 
seen. 
EXAMPLE 6 
In Situ Hybridization for u-Par mRNA 
MATERIALS AND METHODS 
Materials. The following materials were obtained from the indicated 
sources: T7 and T3 polymerase, pBluescriptKS(+) plasmid vector 
(Stratagene; California, USA); RNasin and DNase I (Promega, Wisconsin, 
USA); 35!S-UTP (1300 Ci/mmol) (NEN Dupont, Massachusetts, USA); 
Dithiothretiol and restriction endonucleases (Boehringer Mannheim, 
Mannheim, FRG); K5 autoradiographic emulsion (Ilford, Cheshire, England); 
Formamide (Fluka, Buchs, Switzerland); Salmon Sperm DNA (Type III, Sigma, 
Missouri, USA). All other materials were as described previously 
(Kristensen et al., 1984; Kristensen et al., 1990), or of the best 
commercially available grade. 
Tissue preparation. Following surgery, tissue specimens from 13 patients 
with adenocarcinoma of the colon were dissected and placed in 4% or 10% 
(wt/vol) formalin--0.9% NaCl solution for 24-48 hours before embedding in 
paraffin wax. 
Preparation of RNA probes. Fragments of the complete human u- cDNA (see 
Example 3) were subcloned using standard techniques (Maniatis et al., 
1982), and two subclones were prepared: pHUR04: PstI(184)-PstI(451) 
fragment and pHUR06: BamHI(497)-BamHI(1081) fragment in pBluescriptKS(+), 
base pair numbers corresponding to sequence as listed in Example 3. Pure 
plasmid preparations were prepared by banding in CsCl gradients and the 
plasmids were linearized for transcription using Smal restriction 
endonuclease (pHUR04) or SpeI and EcoRI (pHUR06). 5 .mu.g of the 
linearized plasmid was extracted with phenol and with 
chloroform/isoamylalcohol (25:1), precipitated with ethanol and 
redissolved in water. Each transcription reaction contained linearized DNA 
template (1 .mu.g), RNasin (40 U), 40 mM Tris-Cl, pH 7.6, 6 mM MgCl2, 10 
mM NaCl, 2 mM Spermidine, 10 mM DTT, 1 mM GTP, 1 mM ATP, 1 mM CTP, 4 .mu.M 
35!S UTP and the relevant polymerase (T3 or T7, 40 U). The pHUR04 
template was transcribed with the T3 polymerase and the pHUR06 template 
linearized with EcoRI was transcribed with T7 polymerase, yielding 
antisense transcripts. The pHUR06 template linearized by digestion with 
SpeI was transcribed with the T3 polymerase yielding sense transcripts. 
After transcription performed for 120 min at 37.degree. C., the template 
DNA was removed by addition of RNase-free DNase I (1 U), yeast t-RNA (20 
.mu.g), RNasin (20 U) and incubation at 37.degree. C. for 15 min. After 
extraction with phenol and chloroform/isoamylalcohol (25:1) RNA was 
precipitated by ethanol by centrifugation at 15000.times.g, 4.degree. C., 
for 10 10 minutes after addition of ammonium acetate (final concentration 
2M), and redissolved in 10 mM DTT. The RNA was hydrolyzed in 0.1M sodium 
carbonate buffer, pH 10.2, containing 10 mM DTT to an average size of 100 
bp. Hydrolysis time was calculated as described (Cox et al., 1984). After 
hydrolysis, the reaction was neutralized by addition of an equal amount of 
0.2M sodium acetate buffer, pH 6.2, containing 10 mM DTT and the RNA was 
precipitated twice with ethanol, as above. The RNA probe was redissolved 
in 10 mM DTT and radioactivity measured using scintillation counting. 
Probe preparations always contained more than 4.times.10.sup.6 cpm/.mu.l, 
and the amount of TCA precipitable material was usually above 90%. The two 
corresponding RNA probes transcribed from the opposite strands of the 
pHUR06 plasmid template were adjusted to the same radioactivity 
concentration by addition of 10 mM DTT, and deionized formamide was added 
to a final concentration of 50%. Probes were stored at -20.degree. C. 
until use. 
In situ hybridization. In situ hybridization was performed using a method 
adapted from a number of published procedures (e.g. Cox et al., 1984; 
Angerer et al., 1987). Slides were dipped in 0.5% gelatin, 0.5% 
chrome-alum, dried at room temperature, baked at 180.degree. C. for 3 
hours and stored dust-free at room temperature. Paraffin sections were 
cut, placed on slides, heated to 60.degree. C. for 30 minutes, 
deparaffinized in xylen and rehydrated through graded alcohols to PBS 
(0.01M sodium phosphate buffer pH 7.4, containing 0.14M NaCl). The slides 
were then washed twice in PBS, acid treated in 0.2M HCl for 20 minutes and 
washed for 5 minutes in PBS. This was followed by incubation in 5 .mu.g/ml 
Proteinase K in 50 mM Tris-Cl, pH 8.0, with 5 mM EDTA for 7.5 min, washing 
twice in PBS (2 min) and fixation in 4% (wt/vol) paraformaldehyde in PBS 
for 20 min. Fixative was removed by washing with PBS and slides were 
immersed in 100 mM triethanolamine in a beaker on a magnetic stirrer. As 
the solution was being stirred, acetic acid anhydride was added (final 
concentration 0.2% (vol/vol)) and the addition was repeated after 5 min. 
Finally, the slides were washed in PBS (5 min), dehydrated in graded 
ethanols and airdried at room temperature. The probe was heated to 
80.degree. C. for 3 min and allowed to cool before addition to the 
hybridization mix. The final hybridization solution contained RNA probe 
(80 .mu.g/.mu.l), deionized formamide (50%), dextran sulphate (10%), t-RNA 
(1 .mu.g/.mu.l), Ficoll 400 (0.02% (wt/vol), polyvinylpyrrolidone (0.02% 
(wt/vol)), BSA Fraction V (0.02% (wt/vol)), 10 mM DTT, 0.3M NaCl, 0.5 mM 
EDTA, 10 mM Tris-Cl and 10 mM NaPO4 (pH 6.8). The hybridization solution 
was applied to the slides (approx. 20 .mu.l pr. section) and sections 
covered by alcohol washed, autoclaved coverslips. Sections were hybridized 
at 47.degree. C. overnight (16-18 hours) in a chamber humidified with 10 
ml of a mixture similar to the hybridization solution, except for probe, 
dextran sulphate, DTT and t-RNA (washing mix). After hybridization, the 
position of air bubbles occasionally formed over the section was marked, 
and coverslips were removed by incubation in washing mix for 1 hour at 
50.degree. C. The washing mix was changed, and washing continued for 1 
hour at 50.degree. C. Sections were washed in 0.5M NaCl, 1 mM EDTA, 10 mM 
Tris-Cl (pH 7.2, NTE) with 10 mM DTT at 37.degree. C. for 15 min, and 
treated with RNase A (20 .mu.g/ml) in NTE at 37.degree. C. for 30 min. 
This was followed by washing in NTE at 37.degree. C. (2.times.30 min), and 
washing in 2 liters of 15 mM sodium chloride, 1.5 mM sodium citrate, pH 
7.0 with 1 mM DTT for 30 min at room temperature with stirring. Sections 
were then dehydrated in grading solutions of ethanol, all containing 300 
mM ammonium acetate until 99% ethanol, and air-dried. Finally, 
autoradiographic emulsion was applied following the manufacturer's 
recommendations, and sections were stored in black airtight boxes with 
dessicant at 4.degree. C. until developed after 1-2 weeks of exposure. 
RESULTS 
Tissues were analyzed with antisense transcripts from the two 
non-overlapping clones pHUR04 and pHUR06 and with sense transcripts from 
pHUR06. 
Areas of normally appearing mucosa were in all cases devoid of 
hybridization signal (not shown). 
At invasive foci of carcinoma, hybridization signal was consistently seen 
when using pHUR06 antisense transcripts. A particularly prominent 
hybridization signal was found above cells at the leading edge of 
disrupted tumor glands in areas with clear signs of inflammation and 
degradation of surrounding mesenchymal tissue (FIGS. 21A-B). In other 
areas of infiltrating carcinoma where tumor glands show a more organized 
structure, hybridization signal was located above cells closely associated 
with coherent strands of tumor cells (FIG. 21D) or above cells integrated 
at the serosal surface of the neoplastic epithelium itself (FIG. 21C). It 
was not possible from the sections to identify with certainty the cell 
type(s) in question, nor could the identity of some cells in areas of 
neovascularization that showed hybridization signal be firmly established 
(FIG. 21E). After intensive photographing at high magnification 
(400-1000.times.) of selected areas of the tumor, silver bromide crystals 
were removed by immersion in periodic acid for 5 min and the slides were 
reexamined. By this technique, cells showing hybridization signal can be 
studied in greater detail and this technique is at present being pursued 
for a final assesment of cell type(s). 
The hybridization signals obtained with pHUR06 antisense transcripts were 
confirmed on adjacent sections using antisense transcripts from pHUR04 
(not shown). Unspecific binding of radioactive probe was demonstrated 
using sense transcripts from pHUR06 and in all tumors analyzed gave rise 
to a signal uniformly distributed above tissue sections and with an 
intensity comparable to that obtained with pHUR06 antisense transcripts in 
areas of no hybridization (e.g. normally appearing mucosa) (not shown). 
EXAMPLE 7 
Role of u- in Cell Surface Plasminogen Activation 
MATERIALS AND METHODS 
Cell cultures 
Human fibrosarcoma cells (HT-1080, CCL 121) were obtained from the American 
Type Culture Collection, Rockville, Md. Confluent cell layers were grown 
in plastic Linbro wells (2 cm.sup.2 ; Flow Laboratories) in Eagle's 
minimal essential medium (MEM) supplemented with 10% heat-inactivated 
(56.degree. C. for 60 minutes) fetal calf serum (Gibco), 100 IU/ml 
penicillin and 50 .mu.g/ml streptomycin. After reaching confluence, the 
cells were rinsed three times with MEM containing 0.2% bovine serum 
albumin (BSA), then changed to either serum-free medium (0.5 ml) or medium 
containing 10% heat-inactivated and plasminogen-depleted (i.e. absorbed 
with lysine-Sepharose; Pharmacia, Uppsala, Sweden) fetal calf serum as 
indicated in the Examples. 
In the Examples concerning plasmin binding to cells from medium, human 
plasmin (approximately 18 CU/mg; Kabi Diagnostica, Stockholm, Sweden) was 
added to the cultures at final concentrations of 0-5 .mu.g/ml. The cells 
were incubated for 3 hours at 37.degree. C. before assay of cell-bound and 
supernatant plasmin (see below). For plasmin release experiments, cells 
were loaded for 1 hour at 37.degree. C. with 0-5 .mu.g/ml plasmin in 
serum-free medium, then rinsed three times with MEM. 
Human plasminogen (with glutamic acid N-terminal) was prepared by affinity 
chromatography on lysine-Sepharose (Deutsch, D. G., and E. T. Mertz, 
"Plasminogen: Purification from human plasma by affinity chromatography", 
Science 170: 1095-1097, 1970) from freshly separated, unfrozen human 
plasma pretreated with 10 .mu.M p-nitrophenyl guanidinobenzoate, 1 mM 
phenylmethylsulfonylfluoride and 0.1 .mu.g/ml of an anti-catalytic murine 
monoclonal IgG antibody to human t-PA (ESP-2; see MacGregor, I. R. et al., 
"Characterization of epitopes on human tissue plasminogen activator 
recognised by a group of monoclonal antibodies", Thromb. Haem. 53: 45-50, 
1985); American Diagnostica, Greenwich, Conn.). 
Inhibition studies made use of the following reagents added to cell 
cultures: an anti-catalytic murine monoclonal IgG antibody to human 
plasmin (anti-plg 1, 20 .mu.g/ml; see Sim, P-S. et al., "Monoclonal 
antibodies inhibitory to human plasmin: definitive demonstration of a role 
for plasmin in activating the proenzyme of urokinase-type plasminogen 
activator", Eur. J. Biochem. 158: 537-542, 1986); aprotinin (Trasylol, 
Bayer, Leverkusen, FRG; 200 KIU/ml); tranexamic acid (Cyclokapron, Kabi 
Vitrum, Stockholm; 10 .mu.M and 100 .mu.M); human type-2 plasminogen 
activator inhibitor minactivin (see Golder, J. P. et al., "Minactivin: A 
human monocyte product which specifically inactivates urokinase-type 
plasminogen activators", Eur. J. Biochem. 136: 517-522, 1983), PAI-2 
purified from cultures of human U-937 histiocytic lymphoma cells (see 
Leung, K-C. et al., "The resistance of fibrin-stimulated tissue 
plasminogen activator to inactivation by a class PAI-2 inhibitor 
(minactivin)", Thromb. Res. 46: 755-766, 1987) titration equivalent of 3.6 
IU u-PA/ml; an anti-catalytic murine monoclonal IgG antibody to human u-PA 
(clone 2 (10 .mu.g/ml) in Nielsen, L. S. et al., "Enzyme-linked 
immunosorbent assay for human urokinase-type plasminogen activators and 
its proenzyme using a combination of monoclonal and polyclonal 
antibodies", J. Immunoassay 7: 209-228, 1986); the anti-catalytic 
monoclonal antibody to human t-PA (10 .mu.g/ml); a neutralising murine 
monoclonal IgG antibody to human PAI-1 (Nielsen, L. S. et al., "Monoclonal 
antibodies to human 54,000 molecular weight plasminogen activator 
inhibitor from fibrosarcoma cells--inhibitor neutralization and one-step 
affinity purification", Thromb. Haem. 55: 206-212, 1986) (10 .mu.g/ml) and 
diisopropyl fluorophosphate (DFP)-inactivated u-PA (0-10 .mu.g/ml). 
DFP-inactivated u-PA for competition studies 
Active two-chain u-PA (Ukidan, Serono) was dissolved in 0.1M Tris-HCl, pH 
8.1, 0.1% Tween 80 (Tris/Tween). A freshly prepared solution of 500 mM DFP 
(Sigma) in isopropanol was added to yield a final DFP concentration of 5 
mM. After thorough mixing, the sample was incubated for 2 hours at 
37.degree. C., after which period addition of DFP was repeated as above. 
After renewed incubation for 2 hours at 37.degree. C., the reaction was 
terminated by thorough dialysis at 0.degree. C. against Tris/Tween. No 
residual DFP inhibitor could be detected when the preparation was tested 
in an activity assay of soluble urokinase. 
Metabolic labelling of cell-bound u-PA 
Confluent layers of HT-1080 cells were rinsed three times with 
methionine-free MEM medium containing 0.2% BSA, then prelabelled for 5 
hours at 37.degree. C. with 170 .mu.Ci/ml (.sup.35 S)methionine (800 
Ci/mmol, Amersham). Human plasminogen (50 .mu.g/ml) and the neutralising 
monoclonal antibody to human PAI-1 (10 .mu.g/ml) were added to one of two 
cultures, and the incubations continued for another 3 hours. Aprotinin 
(200 KIU/ml) was added to both cultures before the medium was removed, 
after which the cells were rinsed three times with Dulbecco's medium 
containing 0.2% BSA. The cell-bound u-PA was then eluted with 50 mM 
glycine/HCl (pH 3.0) containing 0.1M NaCl for 3 minutes at 23.degree. C. 
(Stoppelli et al., 1986). The acid eluate was neutralised with 0.5M 
Tris-HCl (pH 7.8) before immunoprecipitation for 2 hours at 23.degree. C. 
with 3 .mu.g/ml of goat IgG antibodies to human u-PA (American 
Diagnostica) or 3 .mu.g/ml goat IgG antibodies to human t-PA (American 
Diagnostica) as control. Immune complexes were collected by adsorption to 
protein A-Sepharose in an end-over mixer for 1 hour. Immunoprecipitates 
were washed several times with immunoprecipitation buffer 10 mM Tris-HCl 
(pH 7.5), 50 mM NaCl, 0.5% sodium deoxycholate, 0.5% NP-40, 0.1% sodium 
dodecyl sulfate (SDS)! containing 100 KIU/ml aprotinin, twice with PBS and 
finally with 20 mM Tris-HCl (pH 7.5). Immunocomplexes were solubilised by 
boiling in Laemmli's sample buffer (cf. Laemmli, supra) under reducing 
conditions (10% .beta.-mercaptoethanol), and electrophoresed in 10% 
SDS-polyacrylamide gels. Fixed gels were treated with Amplify.RTM. 
(Amersham) and exposed to Kodak XAR-5 film at -70.degree. C. 
u-PA assays 
Cell culture supernatants were assayed for pro-u-PA and active u-PA by the 
following modification of an immunocapture method (Stephens et al., 1988; 
Stephens et al., 1987). Microtitre wells of polystyrene immunoplates (type 
269620, A/S Nunc, Roskilde, Denmark) were coated overnight at 37.degree. 
C. with 50 .mu.l of a solution of goat IgG antibodies to human u-PA (cat. 
#398, American Diagnostica). The coating solution contained 2.5 .mu.g of 
IgG per ml of 0.1M sodium carbonate (pH 9.8). After rinsing, the wells 
were treated with conditioned medium (50 ml) for 2 hours at 23.degree. C., 
then rinsed again. Half the wells were then treated with 50 .mu.l of 
freshly prepared 2 .mu.M p-nitrophenyl guanidinobenzoate (NPGB, Sigma) 
(Dan.o slashed., K., and E. Reich, "Plasminogen activator from cells 
transformed by an oncogenic virus--Inhibitors of the activator reaction", 
Biochim. Biophys. Acta 566: 138-151, 1979) for 20 minutes at 37.degree. C. 
The other half (controls) received 50 .mu.l of rinsing buffer (0.05% Tween 
20 in PBS). After rinsing, u-PA was assayed in all the wells by addition 
of 40 .mu.l of plasminogen solution (100 .mu.g/ml in assay buffer 
consisting of 50 mM sodium glycinate (pH 7.8), 0.1% Triton X-100, 0.1% 
gelatin and 10 mM 6-aminocaproic acid which also contained a very low 
concentration of plasmin (10 ng/ml)), and incubation took place for 30 
minutes at 37.degree. C. This concentration of plasmin in the plasminogen 
incubation was sufficient to enable full realization of the potential 
activity of pro-u-PA (cf. Petersen et al., 1988). The plasmin produced by 
this incubation was assayed by its thioesterase activity (Green, G. D. G., 
and E. Shaw, "Thiobenzyl benzyloxycarbonyl-L-lysinate, substrate for a 
sensitive calorimetric assay for trypsin-like enzymes", Anal. Biochem. 93: 
223-226, 1979) by the addition of 200 .mu.l of a solution containing 200 
mM potassium phosphate (pH 7.5), 200 mM KCl, 0.1% Triton X-100, 220 .mu.M 
Z-lysine thiobenzyl ester (Peninsula Laboratories, Belmont, Calif.) and 
220 .mu.M 5,5'-dithiobis(2-nitrobenzoic acid) (Sigma). This mixture was 
incubated for 30 minutes at 37.degree. C., and the absorbancies of the 
wells were read at 405 nm. Active u-PA (60,000 IU/mg) was purchased from 
Calbiochem-Behring (La Jolla, Calif.) and pro-u-PA (potential activity 
90,000 IU/mg) was obtained from American Diagnostica. 
Pro-u-PA and active u-PA bound to the cell layer were recovered for 
immunocapture assays by the same method as was used in the metabolic 
labelling (see above). Each culture well (2 cm.sup.2) was eluted with 150 
.mu.l of acid glycine at pH 3 (Stoppelli et al., 1986). For conditioned 
medium and cell-bound u-PA, the u-PA activity assayed after NPGB treatment 
was expressed as a percentage of the total activity obtained without NPGB 
treatment, and this percentage used as an index of pro-u-PA content 
(pro-u-PA index). The conditions used for the NPGB treatment were 
previously established (Stephens et al., 1988) to allow selective 
inactivation of active u-PA, while leaving the pro-u-PA unchanged and 
still able to be activated by the added plasmin to the same extent as 
untreated pro-u-PA. 
Plasmin assays 
The plasmin activity of culture supernatant samples (50 .mu.l) was assayed 
directly by incubation with the thioester substrate solution above (200 
.mu.l) for 30 minutes (serum-free supernatants) or 3 hours 
(serum-containing supernatants) at 37.degree. C. An estimate of the amount 
of active plasmin present was made from calibration curves using human 
plasmin dilutions in serum-free medium covering the appropriate ranges of 
activity. 
Plasmin bound to the cell layer was recovered and assayed as follows. After 
harvest of culture medium, the cells were rinsed three times with PBS 
(plasmin assays of further rinses were negative); then the bound plasmin 
was specifically eluted (Miles, L. A., and E. F. Plow, "Binding and 
activation of plasminogen on the platelet surface", J. Biol. Chem. 260: 
4303-4311, 1985) with a solution of 1 mM tranexamic acid in the same 
rinsing solution (150 .mu.l/well). Plasmin activity was assayed in eluate 
samples (50 .mu.l) as above with an incubation time of 3 hours at 
37.degree. C. Tranexamic acid at 1 mM had no effect on the thioesterase 
activity of plasmin in these assays. 
RESULTS 
Plasminogen is activated on the cell surface 
After addition of purified preparations of human plasminogen to cultures of 
human fibrosarcoma cells (HT-1080) growing in a medium with 10% 
plasminogen-depleted fetal calf serum, plasmin activity could be recovered 
as a bound fraction from the cell layer. Upon varying the concentration of 
added plasminogen, the bound plasmin activity increased in a 
dose-dependent manner (FIG. 22). The binding was specific so that after 
rinsing of the cells with isotonic buffer, the plasmin could be released 
by 1 mM tranexamic acid. This agent disrupts interactions with plasminogen 
or plasmin which involve the lysine affinity sites of the heavy-chain 
kringles (Miles, supra). The plasmin released from HT-1080 cell surfaces 
was conveniently measured by its thioesterase activity, a method which was 
unaffected by the presence of tranexamic acid. Some plasmin activity was 
also detected in the medium. At a concentration of 40 .mu.g/ml human 
plasminogen added to 0.5 ml of medium above a confluent 2 cm.sup.2 cell 
layer, activity corresponding to 28 ng of plasmin could be recovered from 
the cell layer with tranexamic acid, while 10 ng was measurable in the 
medium after 3 hours of incubation at 37.degree. C. This concentration of 
plasminogen is well below the 200 .mu.g/ml present in normal human plasma. 
To test whether the cell surface plasmin might have been derived from 
either preformed plasmin (added as a trace contaminant with the 
plasminogen preparation) or from plasmin formed in the medium and 
subsequently bound to the cells, plasmin was added to the culture medium 
of HT-1080 cells. As shown in FIG. 23, virtually no plasmin activity was 
detected on the cell surface when the medium contained 10% fetal calf 
serum, while there was a considerable dose-dependent plasmin binding in 
the absence of serum. 
These findings indicated that the cell-bound plasmin activity found in the 
experiment shown in FIG. 22 was formed by activation of plasminogen on the 
surface of the cells. 
Incubation of cells carrying plasmin with fresh serum-free medium showed 
that approximately 40% of the activity remained bound after 2 hours at 
37.degree. C. (FIGS. 24A and 24B). When the cells were incubated in 10% 
serum-containing medium, the same fraction (40%) of this activity could be 
recovered from the cells; the bound plasmin was not inactivated by the 
serum. However, only about 11% (compared to 60% for serum-free medium) 
could be detected in the serum-containing medium (FIG. 24B). When 1 mM 
tranexamic acid was added to the serum-containing medium, no plasmin 
activity could be recovered from the cells (FIG. 24A). 
Cell surface plasminogen activation is catalyzed by cell-bound u-PA HT-1080 
cells are prolific producers of u-PA (Saksela, O., et al., "Plasminogen 
activators, activation inhibitors and alpha-2-macroglobulin produced by 
cultured normal and malignant human cells", Int. J. Cancer 33: 609-616, 
1984), but although they synthesize some t-PA, this does not appear to be 
secreted (R. Stephens, unpublished observations). To test which of the 
activators was responsible for the cell-surface plasminogen activation, 
the cells were incubated with plasminogen in the presence of monoclonal 
antibodies that inhibit each of the activators. The results in Table 5 
show that inhibition of the enzymatic activity of u-PA resulted in 
virtually no plasmin activity being detected on the cell surface while 
inhibition of t-PA did not decrease the amount of plasmin activity, 
indicating that the cell surface plasminogen activation was catalyzed by 
u-PA. Bound plasmin activity was also reduced in cultures containing PAI-2 
(Golder, supra), aprotinin or an anti-catalytic monoclonal antibody to 
human plasmin (Sim, supra). 
In HT-1080 cell cultures, u-PA is present both in the medium and bound to 
the cell surface (Nielsen et al., 1988). To test whether the surface-bound 
u-PA was involved in the cell-surface plasminogen activation in serum 
cultures, the cells were preincubated with either the anti-catalytic u-PA 
antibody or PAI-2, and the cells were then thoroughly washed before being 
incubated with plasminogen in serum medium. Both inhibitors caused a 
significant decrease in the cell-bound plasmin activity, while no 
inhibition of the u-PA activity in the medium was detected (Table 6). 
An alternative method of studying the role of cell-bound versus free u-PA 
is illustrated in FIG. 25. u-PA is bound to its receptor, u-, at the 
surface of HT-1080 cells (Nielsen et al., 1988). This binding 30 does not 
involve the active site of u-PA (Blasi, F., 1988), and u- therefore 
also binds u-PA that has been treated with the irreversible active-site 
titrant, DFP (Nielsen et al., 1988). To decrease the amount of 
receptor-bound catalytically active u-PA, the HT-1080 cells were 
preincubated for 18 hours with DFP-inactivated u-PA which, when present in 
a large molar excess, resulted in a decrease of approximately 70% in 
surface-bound u-PA that was released by acid treatment (Stoppelli et al., 
1986). Concomitantly, there was a comparable decrease in the amount of 
plasmin generated on the cell surface (FIG. 25). 
These results indicate that a large part, if not all, of the cell surface 
plasminogen activation in serum cultures was catalyzed by the 
surface-bound u-PA. 
Surface-bound plasmin activates pro-u-PA 
u-PA is released into the medium of HT-1080 cells as a single-chain 
proenzyme, pro-u-PA, that can be converted to two-chain active u-PA by 
plasmin (Nielsen et al., 1982; Sim, supra). The enzymatic activity of the 
proenzyme is at least 250-fold lower than that of the two-chain u-PA 
(Petersen et al., 1988), and it does not react with PAI-1 (Andreasen et 
al., 1986) or PAI-2 (Stephens et al., 1987; Wun et al., 1987). With the 
use of metabolic labelling, recovery of receptor-bound u-PA by acid 
treatment, immunoprecipitation, SDS-PAGE under reducing conditions and 
fluorography, it was found (FIG. 26) that the receptor-bound u-PA was 
almost exclusively present in the single-chain form when the cells were 
incubated in serum medium without added plasminogen. By contrast, 
virtually all was in the two-chain form when the cells were incubated with 
50 .mu.g/ml human plasminogen in serum medium for 3 hours. 
As an alternative way of distinguishing between pro-u-PA and active u-PA, 
the fact that low molecular weight active site reagents for u-PA do not 
bind to pro-u-PA (Nielsen et al., 1982) was utilized. One of these, NPGB, 
was used in a convenient test to distinguish between the two u-PA forms in 
an immunocapture assay (Stephens et al., 1988; Stephens et al., 1987). The 
u-PA present in samples was first absorbed to u-PA antibodies bound to 
microtitre wells. Half the wells for each sample were treated with NPGB, 
the other half were control treated. The total u-PA activity 
(pro-u-PA+active u-PA) in the untreated wells and the pro-u-PA in the 
treated wells were then measured by a coupled plasminogen activator assay 
in the presence of an initial concentration of 10 ng/ml plasmin, and the 
results were expressed as a pro-u-PA index. FIG. 27A shows that the 
proportion of the total surface-bound u-PA that was present as pro-u-PA 
decreased dramatically when the cells were incubated with human 
plasminogen in serum medium in comparison with plasminogen-free cultures 
(from approximately 90% to approximately 10%). 
These findings suggest that conversion of the surface-bound pro-u-PA to 
active u-PA by plasmin plays a role in cell surface plasminogen 
activation. 
In the experiment described in FIG. 27, there was a markedly lower amount 
of total u-PA activity on the cells incubated with plasminogen (FIG. 27B). 
It was found that this difference was nearly abolished when a monoclonal 
antibody that neutralizes human PAI-1 was added during the incubation 
(FIG. 27B). HT-1080 cells release large amounts of PAI-1 (Nielsen et al., 
supra) that binds to active u-PA, but not to pro-u-PA (Andreasen et al., 
1986). The apparent decrease in total u-PA after incubation with added 
plasminogen (FIG. 27B) can therefore be attributed to PAI-1 binding to 
active u-PA on the cell surface and inhibiting its activity in the 
subsequent assay. 
To prevent the interference of PAI-1, the neutralizing PAI-1 antibody was 
therefore included in the next experiment in which the effect of the 
plasmin inhibitor aprotinin and the effect of an anti-catalytic monoclonal 
antibody to human plasmin on the conversion of pro-u-PA to active u-PA 
were studied. As shown in Table 7, both these inhibitors increased the 
relative amount of pro-u-PA, thus demonstrating that the activation of 
cell-bound pro-u-PA was catalyzed by plasmin. To study whether this was an 
effect of cell-bound plasmin, the effect of tranexamic acid in a 
concentration of 100 .mu.M was also tested, which concentration completely 
inhibits binding of plasmin to the cells, but does not affect the ability 
of plasmin to activate pro-u-PA in solution (R. Stephens, unpublished 
results). This treatment markedly decreased the relative amount of active 
u-PA, indicating that the activation of the cell surface pro-u-PA is 
catalyzed by the surface-bound plasmin. 
TABLE 6 
______________________________________ 
Effect of inhibitors of plasmin and u-PA on the formation 
of bound plasmin in HT-1080 cells in serum culture 
Bound plasmin activity 
u-PA activity in medium 
(%) (%) 
Incubation 3 h 17 h 3 h 17 h 
______________________________________ 
Control 3.5 7.1 100 100 
Plg 100 100 111 83 
Plg + anti-u-PA 
6.2 11 9.9 29 
Plg + anti-t-PA 
101 123 102 81 
Plg + PAI-2 48 19 89 77 
Plg + aprotinin 
13.2 7.8 93 99 
Plg + anti-Plg-1 
2.8 6.4 90 88 
Plg + TA (10 .mu.M) 
9.7 34 116 87 
Plg + TA (100 .mu.M) 
2.2 13 92 95 
______________________________________ 
The following additions were made to cell layers growing in MEM medium (0.5 
ml) containing 10% heat-inactivated and plasminogen-depleted fetal calf 
serum: native human plasminogen (Plg, 40 .mu.g/ml); anti-catalytic 
monoclonal antibody to human u-PA (10 .mu.g/ml); anti-catalytic monoclonal 
antibody to human t-PA (10 .mu.g/ml); PAI-2 (titration equivalent of 3.6 
UI u-PA/ml); anti-catalytic monoclonal antibody to human plasmin (20 
.mu.g/ml); aprotinin (200 KIU/ml); tranexamic acid (TA, as shown). The 
cultures were incubated for the times shown before assay of cell-bound 
plasmin. The incubation with plasminogen was used as the 100% control for 
bound plasmin. 
TABLE 7 
______________________________________ 
Effect of pretreatment of HT-1080 cells with u-PA 
inhibitors on subsequent ability to produce 
bound plasmin in serum culture 
Bound plasmin 
u-PA activity 
Preincubation 
Plg activity (%) 
in medium (%) 
______________________________________ 
Control - 2.2 100 
Control + 100 86 
Anti-u-PA + 32 96 
PAI-2 + 54 86 
______________________________________ 
Confluent cell layers in serum medium (0.5 ml) were preincubated for 1 hour 
at 37.degree. C. with an anti-catalytic monoclonal antibody to human u-PA 
(10 .mu.g/ml) or PAI-2 (titration equivalent of 3.6 IU u-PA/ml). The cells 
were then rinsed three times with serum-free MEM medium before incubation 
for 3 hours at 37.degree. C. with MEM medium containing 10% 
heat-inactivated and plasminogen-depleted fetal calf serum and native 
human plasminogen (Plg, 40 .mu.g/ml). The incubation with plasminogen was 
used as the 100% control for bound plasmin while the control for u-PA was 
the incubation without plasminogen. 
TABLE 8 
______________________________________ 
Effectors of pro-u-PA activation and plasmin 
on the surface of HT-1080 cells in serum medium 
Pro-u-PA index 
Bound plasmin 
Incubation (%) activity (ng) 
______________________________________ 
Control 85 0 
Plg 50 12 
Plg + anti-PAI-1 21 33 
Plg + anti-PAI-1 + aprotinin 
93 3.3 
Plg + anti-PAI-1 + anti-Plg-1 
72 2.1 
Plg + anti-PAI-1 + TA (100 .mu.M) 
88 0 
______________________________________ 
Confluent cell layers were incubated for 2 hours at 37.degree. C. with MEM 
medium (0.5 ml) containing 10% heat-inactivated and plasminogen-depleted 
fetal calf serum with the following additions: native human plasminogen 
(Plg, 40 .mu.g/ml); neutralizing monoclonal antibody to human PAI-1 (10 
.mu.g/ml); aprotinin (200 KIU/ml); anti-catalytic monoclonal antibody to 
human plasmin (20 .mu.g/ml); and tranexamic acid (TA, as shown). Half the 
wells were then treated with aprotinin (200 KIU/ml) and used for assay of 
bound u-PA and its pro-u-PA index. The other half were used for elution 
and assay of bound plasmin. 
EXAMPLE 8 
Accessibility of Receptor Bound u-PA to PAI-1 and Internalization of the 
u-PA/PAI-1 Complexes 
MATERIALS AND METHODS 
Reagents. PAI-1 was purified as described previously (Nielsen et al., 
1986). Pro-u-PA was purified from human A431 epidermoid carcinoma cells 
(Fabricant et al., Proc. Natl. Acad. Sci. USA 74, 565-569, 30 1977) as 
described by Corti et al. in Peptides of Biological Fluids (H. H. Peeters, 
Ed.), 33, 623-626, 1985, and was a kind gift from E. Sarubbi and A. 
Soffientini. Two-chain u-PA and u-PA amino-terminal fragment (ATF) 
purification (Stoppelli et al., 1985) and DFP-treated u-PA preparation 
(Andreasen et al., 1986) have previously been described. Human plasmin (4 
units/mg), human plasminogen (6 U/mg), aprotinin (15 TIU/mg) and 
benzamidine-Sepharose were purchased from Sigma. The synthetic peptide 
human u-2-32(ala20)! has been described by Appella et al., 1987. 
Cells and cell culture. Human monocyte-like U937 cells derived from a 
histiocytic lymphoma (Sundstrom C. and Nilsson, K., Int. J. Cancer 17: 
565-577, 1976) were grown in RPMI 1640 medium supplemented with 10% fetal 
calf serum. 
Preparation of u-PA/PAI-1 complex. PAI-1 was activated before use by 
treatment with 4M guanidine-HCl for 1 hour at 37.degree. C. (Hekman, C. M. 
and Loskutoff, D. J., J. Biol. Chem. 260, 11581-11587, 1985). Guanidine 
was removed by centrifugation through Centricon 10 (Amicon, Danvers, 
Massachusetts). The u-PA/PAI-1 complex was formed after incubation of the 
proteins at the ratios indicated in the results section for 1 hour at room 
temperature. 
Iodinations. 1 .mu.g portions of protein (ATF, u-PA or pro-u-PA) in 30 mM 
sodium phosphate buffer (pH 7.4) were iodinated with 1 mCi of Na.sup.125 I 
(Amersham Ltd., Amersham, UK) and 5 .mu.g of Iodogen (Pierce Chemical Co., 
Rockford, Ill.) for 4 minutes at 4.degree. C., and the reaction was 
stopped with excess N-acetyltyrosine. Iodinated proteins were separated 
from unincorporated radioactivity by gel filtration on Sephadex G-25. The 
specific activity obtained ranged within 80-150 .mu.Ci/.mu.g protein. 
Iodinated u-PA was further purified by chromatography on 
benzamidine-Sepharose (Holmberg et al., 1976) to isolate molecules still 
retaining enzymatic activity. 
Binding assay. Before binding, U937 cells were incubated for 1 hour at 
4.degree. C. in RPMI 1640 medium supplemented with 0.1% bovine serum 
albumin and 50 mM Hepes (pH 7.4). The cells were then acid-treated in 50 
mM glycine-HCl, 100 mM NaCl (pH 3) for 3 minutes at 4.degree. C. and 
quickly neutralized with half a volume of 0.5M Hepes, 100 mM NaCl (pH 
7.4). One million cells were then resuspended in 0.2 ml of binding buffer 
(phosphate buffered saline supplemented with 0.1% bovine serum albumin) 
containing iodinated ligands (about 50,000 cpm corresponding to 0.1 nM for 
ATF and 0.05 nM for pro-u-PA and u-PA) and incubated for the indicated 
time at 4.degree. C. After binding, the cells were centrifugated and 
washed with cold phosphate buffered saline--0.1% bovine serum albumin. 
Non-specific binding was determined in the presence of 100 nM unlabelled 
u-PA. 
Plasmin cleavage of pro-u-PA. .sup.125 I-pro-u-PA was allowed to bind to 
cells as described above. After washing, the cells were incubated in the 
presence of plasmin (10 .mu.g/ml) at room temperature for 10 minutes. 
lodinated pro-u-PA in solution was activated under the same conditions. 
The reaction was stopped by the addition of aprotinin to a final 
concentration of 125 .mu.g/ml. 
Amidolytic assay. u-PA activity was assayed by incubating 100 .mu.l 
aliquots of binding mixtures or supernatants of binding assays in 0.05M 
Tris-HCl (pH 7.5), 40 mM NaCl, 0.01% Tween 80, with 1 mM of the 
plasmin-specific substrate S2390 (Kabi Vitrum, Sweden) and 0.5 .mu.M 
plasminogen in a final volume of 0.3 ml. The time dependence of the colour 
development was measured following the absorbance at 405 nm (Petersen et 
al., 1988). 
Gel electrophoresis. SDS polyacrylamide gel electrophoresis was carried out 
in 7.5-15% polyacrylamide gradient gels. Samples were applied in Laemmli 
buffer (Laemmli, supra) without previous reduction and heat denaturation. 
Gels were dried and exposed to Kodak XAR-5 films. .sup.14 C-labelled 
molecular weight standards (rainbow mixture, Amersham Ltd., UK) were run 
alongside. 
Zymography and caseinolytic plaque assay. Plasminogen activator activity in 
electrophoretic gels was revealed by zymography (Granelli-Piperno, A. and 
E. Reich, J. Exp. Med. 148: 223-234 (1978)), layering the polyacrylamide 
gel over an agarose gel (1%) containing casein (2% non-fat dry milk) and 
plasminogen (40 .mu.g/ml), in 255 mM Tris-HCl (pH 7.5). 
The caseinolytic plaque assay was carried out essentially as described by 
Goldberg, A. R. (Cell 2: 95-102, 1974); briefly, U937 cells were 
resuspended in RPMI 1640 medium containing 0.8% agar, 1.3% non-fat dry 
milk, and 13 .mu.g/ml plasminogen, and layered into a 30 mm plastic dish. 
After incubation for 3 hours at 37.degree. C., the plates were visually 
scored and photographed. The whole plate was then dried and stained in 70% 
methanol, 10% acetic acid and 0.2% Coomassie blue. 
Immunoaffinity chromatography. For the demonstration of PAI-1/u-PA 
complexes in acid washes of cells, these washes were diluted five fold 
with 0.1M Tris-HCl buffer (pH 8.1)--0.1M NaCl and passed twice over a 1 ml 
polyclonal anti-PAI-1 IgG-Sepharose 4B column equilibrated with the same 
buffer. The columns were washed with 10 volumes of buffer and eluted with 
1M NaCl in 0.1M acetic acid (pH 2.7), dialysed against 0.03% SDS, 
lyophilized and subjected to SDS-gel electrophoresis (see above). 
RESULTS 
The interaction of preformed PAI-1/u-PA complexes with u-PA receptors was 
studied as was that of PAI-1 with receptor-bound u-PA to assess if u-PA 
could, at the same time, interact with its inhibitor and its receptor, and 
if the two moieties of u-PA, the receptor-binding amino terminus and the 
inhibitor-binding, catalytically active carboxy terminus (Stoppelli et 
al., 1985), are completely independent. The ability of PAI-1 to bind and 
inhibit receptor-bound u-PA would also demonstrate that the latter can be 
regulated mainly as the soluble u-PA and would strongly suggest that bound 
u-PA is catalytically active. 
Effect of u-PA and u-PA/PAI-1 complex on binding of .sup.125 I-ATF to the 
u-PA receptor 
In order to study the interaction between PAI-1 and receptor-bound u-PA, it 
was first tested whether purified PAI-1 competes with ATF for binding to 
the receptor on U937 cells, and it was found that it does not, event at a 
1000:1 excess (data not shown). Then, the ability of unlabelled u-PA and 
preformed u-PA/PAI-1 complex to compete with .sup.125 I-ATF for receptor 
binding was compared. FIGS. 29A-B shows the dependence of the inhibition 
of .sup.125 I-ATF binding to U937 cells on the concentration of unlabelled 
u-PA or u-PA/PAI-1 complex. Since PAI-1 forms stoichiometric covalent 
complexes with u-PA (Hekman et al., supra), a constant 50-fold excess of 
PAI-1 was used throughout. In all cases, complete inhibition of u-PA 
activity was observed (not shown) and, as shown in FIG. 29 (insert), all 
u-PA in the competing binding mixtures migrates as a PAI-1/u-PA complex in 
zymography. The data presented in FIG. 29 indicate that complexing of u-PA 
by PAI-1 does not dramatically alter its ability to compete with ATF for 
receptor binding. The slight difference in the shape of the competition 
curves, suggesting that u-PA is a 2-3 fold better ligand for the u- 
than the u-PA/PAI-1 complex, has been observed consistently and may 
reflect a real difference in dissociation constants. 
Binding of u-PA/PAI-1 complex to u- 
To directly ascertain that PAI-1/u-PA complexes bind to the u-PA receptor, 
preformed .sup.125 I-u-PA/PAI-1 complexes were incubated with U937 cells 
for 1 hour at 4.degree. C. After the binding step, the cells were washed 
and lysed, and the cell-associated radioactivity was analyzed by SDS-PAGE 
under non-reducing conditions. As shown in FIG. 30, in the absence of 
PAI-1 and of any unlabelled competitors, cell-bound radioactivity migrates 
mostly as a 50 kD band. However, with preformed u-PA/PAI-1 complexes, a 
cell-bound 90 kD band appears, corresponding to the migration of the 
u-PA/PAI-1 complex. This band represents receptor-bound u-PA/PAI-1 complex 
as it is competed for by unlabelled 85 nM ATF or u-PA. 
Further analysis of FIG. 30 shows that .sup.125 I-u-PA used for this 
experiment was in fact contaminated with 33 kD low molecular weight u-PA. 
In the presence of a 50 or 150 fold excess of PAI-1, much of the u-PA of 
the binding mixture is complexed to give 75 kD and 90 kD PAI-1 complexes, 
the former representing that with low molecular weight u-PA. However, 
although present in the binding mixture and in the supernatants of the 
binding incubations, the 75 kD band is not found associated to U937 cells, 
which is in keeping with the notion that u-PA binds its receptor via the 
amino-terminal domain, which is missing in the low molecular weight u-PA. 
Surprisingly, in the absence of PAI-1 in the binding mixture, two weaker 
bands with molecular -weights of about 69 and 90 kD are detected. This 
background was dependent on the presence of the cells and could not be 
eliminated by different pretreatment of the cells. These bands were not 
retained on Sepharose 4B columns coupled with anti-PAI-1 IgG. This is in 
contrast to the complexes found on cells after incubation with preformed 
PAI-1/u-PA complexes which, as expected, could be isolated from the acid 
washes of cells by immunoaffinity chromatography (data not shown). This is 
in agreement with the very low levels of PAI-1 in U937 cells (Lund et al., 
1988). The nature of the two contaminating bands, therefore, remains 
unknown and will require further investigation. They may represent 
complexes of receptor-bound u-PA with PAI-2 (Genton et al., 1987) or with 
protease nexin-1 (Baker et al., Cell 21: 37-47, 1980). 
The specificity of the binding of the u-PA/PAI-1 complex to the u-PA 
receptor was further investigated. u-PA binds the receptor through its 
amino-terminal extremity, and the binding is competed equally well by ATF 
or u-PA (Stoppelli et al., 1985). Accordingly, it was found that the 
binding of the u-PA/PAI-1 complex can be competed to the same extent by 
ATF and u-PA, with 50% competition reached around 1-2 nM (data not shown). 
Thus, even when complexed to its inhibitor, u-PA still binds specifically 
to its receptor. 
Binding of PAI-1 to receptor-bound u-PA 
The above experiments show that a u-PA/PAI-1 complex can bind specifically 
to the u-PA receptor. It was then tested whether PAI-1 can bind to 
pre-bound u-PA. With the aim of reducing-complex formation in the absence 
of exogenous added PAI-, single-chain pro-u-PA was bound to U937 cells (1 
hour at 4.degree. C.) and then, the receptor-bound pro-u-PA was converted 
into two-chain u-PA with plasmin (Cubellis et al., 1986). Then the plasmin 
inhibitor trasylol and PAI-1 were added; in addition, excess 
receptor-binding synthetic peptide was present to prevent reassociation of 
previously dissociated pro-u-PA or u-PA (Cubellis et al., 1986). Finally, 
the cells were lysed and the state of labelled u-PA analyzed by SDS-PAGE 
under non-reducing conditions. The results are shown in FIG. 31. 
Cells to which no plasmin and no PAI-1 had been added shown only a single 
50 kD band (pro-u-PA). Plasmin activation of pro-u-PA coincides with the 
appearance of the 90 kD band, the intensity of which is proportional to 
the amount of added PAI-1. Also in this case, however, although weak, the 
cell-associated 90 kD band is observed in the absence of exogenous PAI-1. 
Since it only appears after pro-u-PA activation, it most likely represents 
a complex of u-PA with a plasminogen activator inhibitor. The extent of 
activation of pro-u-PA to two-chain u-PA was analyzed in parallel by 
SDS-PAGE under reducing conditions and, in all cases, essentially all of 
the bound pro-u-PA was shown to be converted to the two-chain form (data 
not shown). Comparison of activation of pro-u-PA and binding to PAI-1 in 
the presence and absence of cells (compare lanes "cell-bound" vs. "in 
solution", FIG. 31) did not reveal any dramatic difference. In conclusion, 
this experiment shows that PAI-1 can interact with two-chain u-PA even 
when it is receptor-bound. 
Effect of PAI-1 on cell-bound u-PA activity of U937 cells 
u-PA/PAI-1 complex formation inhibits receptor-bound u-PA activity. To 
study this, the caseinolytic plaque assay was employed in which cells are 
plated in agar in the presence of plasminogen and casein. The presence of 
a plasminogen activator activity is visualized by the appearance of a 
clear plaque, due to the digestion of casein by plasmin. In FIGS. 32A-G, 
caseinolytic plaques observed around individual U937 cells are shown, 
which are representative of the entire cell population. In all instances, 
plaque formation was plasminogen-dependent (not shown). Since U937 cells 
produce a very small amount of u-PA, some very small plaques are observed 
in the absence of exogenous enzyme (FIG. 32A), and the entire plate 
actually scores as negative (not shown). If U937 cells are incubated for 
60 minutes with 10 nM u-PA, washed and then plated, they now clearly score 
as positive (FIG. 32B). DFP-treated u-PA obviously does not confer 
activity to U937 cells (FIG. 32C), and preincubation of u-PA with a 75 
fold excess of PAI-1 completely blocks the activity (FIG. 32D). Thus, 
u-PA/PAI-1 complex is inactive also when cell-associated. It was then 
tested whether the activity that is seen in these assays is indeed 
receptor-bound. U937 cells were first incubated with 10 nM DFP-u-PA for 60 
minutes, washed and reincubated with active u-PA. Under these conditions, 
no activity can be detected (FIG. 32E). Thus, the previously measured 
activity (FIG. 32B) can be competed by a u-PA analogue with a blocked 
active site. In the reverse experiment, first incubation with u-PA 
followed by a second incubation with DFP-u-PA, the latter does not prevent 
plaque formation (FIG. 32G). Thus, very little dissociation of 
receptor-bound u-PA occurs during the time of the experiment. Finally, 
when U937 cells are first incubated with u-PA and subsequently with excess 
PAI-1 in the presence of DFP-u-PA, the activity can be completely blocked 
(FIG. 32F). Thus, PAI-1 is indeed capable of inhibiting the activity of 
receptor-bound u-PA, in suspension-growing U937 cells. 
Fate of receptor-bound u-PA/PAI-1 complexes 
It has been shown that receptor-bound u-PA remains associated with the cell 
surface, is not detectably internalized or degraded, and can be 
dissociated from the surface by a mild acidic treatment (Stoppelli et al., 
1985; Stoppelli et al., 1986; Vassalli et al., 1985). It was investigated 
whether receptor-bound u-PA/PAI-1 complex has a similar fate. 
The basic experiment was carried out in two steps: first, labelled ligands 
were incubated with acid-washed cells at 4.degree. C. for 90 minutes (step 
1). In all cases, more than 90% of the binding occurring during step 1 was 
inhibited by u-PA or ATF while no inhibition was obtained with low 
molecular weight u-PA, demonstrating the specificity of the interaction 
(data not shown). In step 2, the cells were incubated at 37.degree. C. for 
3 hours in binding buffer containing no ligands. The amount and the state 
of the ligand were assayed by quantitation of the radioactivity (and the 
extent of TCA precipitability) at different times during step 2. To this 
end, both the radioactivity recovered in the cell-associated form and in 
the supernatant were measured. The former was distinguished in 
radioactivity extracted by an acid wash (see Methods) representing 
receptor-bound ligand, and radioactivity resistant to the acid wash, 
representing cell-trapped internalized ligand (Haigler, 1980; Stoppelli et 
al., 1986). 
Four different iodinated ligands were tested: two-chain u-PA (u-PA), 
DFP-inactivated u-PA (DFP-u-PA), the amino-terminal fragment of u-PA (ATF) 
and the preformed u-PA:PAI-1 complex. The amount of receptor-bound ligand 
(cell-associated, acid-extracted radioactivity), of cell-trapped ligand 
(cell-associated, acid-resistant radioactivity) and of degraded (i.e. not 
precipitable by 10% TCA) ligand in the supernatant were measured at 
different times during step 2 incubation. In all cases, the acid-extracted 
and cell-associated radioactivity was more than 90% TCA-precipitable at 
all times with all ligands, and their migration in SDS gel electrophoretic 
analysis indicated intact ligands (not shown, but see below). FIG. 33 
shows the fate of the ligand during step 2 incubation at 37.degree. C. In 
the case of ATF and DFP-u-PA, the receptor-bound fraction decreases slowly 
in agreement with previous data (Stoppelli et al., 1985); for u-PA, the 
decrease is somewhat faster. In the case of u-PA:PAI-1, the initial sharp 
loss of receptor-bound ligand continues throughout the incubation and 
after 2-3 hours at 37.degree. C., very little complex is found still to be 
surface-bound. The non-degraded, internalized ligand constitutes a small 
fraction in the case of ATF, but is clearly higher in all other cases. In 
particular in the case of the u-PA:PAI-1 complex, it increases rapidly 
reaching about 40% of the total radioactivity around 30 minutes, and 
decreasing thereafter. While very little ligand is degraded in the case of 
ATF and DFP-u-PA, a larger fraction is degraded in the case of u-PA (20% 
after 3 hours) and much more in the case of the u-PA:PAI-1 complex (65% 
after 3 hours). In the latter case, the time course suggests a 
precursor-product relationship between the cell-trapped and the degraded 
ligand. Possibly, therefore, the u-PA:PAI-1 complex, but not ATF and 
DFP-u-PA, is internalized and then degraded. In the experiment shown in 
FIGS. 33A-D, u-PA might represent an intermediate case (low-level 
internalization and degradation) (see below). The low-level degradation of 
u-PA might also reflect internalization of covalent u-PA:PAI-2 complexes 
since U937 cells produce this inhibitor. To test this hypothesis, the 
experiments were repeated including 50 nM low molecular weight u-PA during 
steps 1, 2 and in all washing buffers with the aim of titrating endogenous 
u-PA-binding PAI-like proteins. Quantitative data obtained under these 
conditions (FIG. 34) show a time-dependent decrease of the surface-bound 
ligand, no accumulation in the pellet, and complete recovery of the 
radioactivity in the supernatants of step 2 in TCA-precipitable form. 
Thus, the low-level degradation observed with u-PA (FIGS. 33A-D) seems to 
be due to the formation of covalent complexes with endogenous, low 
molecular weight u-PA-titratable inhibitors. It is therefore concluded 
that under the experimental conditions, ligand degradation only occurs 
when u-PA is in complex with exogenous PAI-1 and possibly with endogenous 
PAI-2. 
To test the role of lysosomes in u-PA:PAI-1 degradation, chloroquine was 
employed, which is a drug inhibiting lysosomal protein degradation (De 
Duve et al., 1974; Carpenter and Cohen, 1976; McKanna et al., 1979). In 
the absence of chloroquine (FIG. 35A), a typical precursor-product 
relationship is observed in step 2 between the rate of accumulation of 
ligand in the pellet in a non-acid-extractable form and the rate of 
release of degraded ligand in the supernatant (in a TCA-soluble form). The 
latter reaches 60% of the total bound ligand after 3 hours. While the 
presence of 0.5 mM chloroquine does not affect the ability of the 
u-PA:PAI-1 complex to bind to the U937 cells (not shown) during step 2 
incubation (FIG. 35B), a slight decrease was observed in the rate of loss 
of receptor-bound ligand and of its accumulation in the pellet; most 
prominently, however, degradation of the ligand is strongly inhibited 
(from 60 to 20% after 3 hours). These data suggest that degradation of the 
u-PA:-PAI-1 complex occurs intracellularly in the lysosomes. 
CONCLUSIONS 
The results unequivocally show that while ATF, DFP-treated u-PA and free 
active u-PA (in particular when excess low molecular weight u-PA is 
present to titrate endogenous inhibitors) are not internalized nor 
degraded, the u-PA:PAI-1 complex is internalized and degraded, most likely 
in the lysosomes. 
Previous data have shown the absence of internalization of receptor-bound 
ATF, u-PA and pro-u-PA (Vassali et al., 1985; Stoppelli et al., 1985; 
Bajpai and Baker, 1985a; Stoppelli et al., 1986). These data are fully 
confirmed in the present study. 
Also, free u-PA is apparently internalized and degraded by U937 cells, 
although at a slower rate (FIGS. 33A-D). This is due to internalization of 
complexes formed between u-PA and endogenous proteins which interact with 
the u-PA active site, possibly the inhibitor PAI-2 (Vassalli et al., 1984; 
Genton et al., 1987). 
Unlike the internalization of the nexin-protease complexes which are formed 
in solution and subsequently bind to the cells and are internalized via so 
far uncharacterized receptors (Baker et al., 1980), the u-PA:PAI-1 complex 
is bound to the receptor itself (see Example 8) and subsequently undergoes 
internalization and degradation. This receptor, therefore, must alternate 
between two possible configurations: one in which it binds active u-PA and 
in which it dictates plasminogen activation on the cell surface; and 
another in which it binds the inhibited enzyme and in which it favours 
internalization and degradation of the ligand. This property could be 
exploited for internalizing toxins and thus specifically kill the cells 
that express the u-PA receptor, or by forcing the state of the receptor 
from one state (i.e. exposed) to another, through PAI-1 or PAI-1 
analogues. 
EXAMPLE 9 
Inhibition of Receptor Bound u-PA by PAI-1 and PAI-2 
MATERIALS AND METHODS 
Plasminogen was purified from fresh human plasma as previously described 
(Dan.o slashed. and Reich, 1979), and was further separated into its two 
isoforms by elution from lysine-Sepharose with a linear gradient of 
6-amino-hexanoic acid. Plasminogen isoform 2 was used in all experiments 
described here. u-PA (M.sub.r 55,000) was obtained either by plasmin 
activation of pro-uPA (Ellis et al., 1987) or as Ukidan (Serono). Both 
preparations were greater than 95% high molecular weight u-PA by 
SDS-polyacrylamide gel electrophoresis. The concentration of active u-PA 
in these preparations was determined by active-site titration with 
p-nitrophenyl-p-guanidinobenzoate (Sigma Chem. Co.). DFP-inactivated u-PA 
was prepared as described in Example 1. The murine monoclonal antibody to 
u-PA was clone 2 from Nielsen et al., 1986. Active PAI-1 was purified from 
the serum-free conditioned medium of Hep G2 cells by affinity 
chromatography on immobilized anhydro-urokinase (Wun et al., 1989). PAI-2 
was purified from U937 cell lysates by chromatofocusing as described 
(Kruithof et al., 1986). The concentrations of active inhibitor in the 
various PAI preparations were determined by titration against u-PA 
immediately before use in the kinetic experiments. PAI-1 or PAI-2 at 
varying concentrations between 1 nM and 100 nM were incubated with 
active-site titrated u-PA (20 nM) for 1 hour at 37.degree. C. in 0,05M 
Tris, 0,1M NaCl pH 7,4 containing 0,2% bovine serum albumin. Residual u-PA 
activity was then measured by hydrolysis of 0,2 mM Glu-Gly-Arg-AMC 
(Bachem, Switzerland). 
U937 cells were grown in suspension in RPMI 1640 medium supplemented with 
5% heat-inactivated fetal calf serum. PMA-stimulation of U937 cells was 
performed at a cell density of 0.5.times.10.sup.6 cells/ml with 150 nM PA 
for 4 days. The adherent cell population was harvested with a rubber 
scraper and resuspended in PBS. 
Prior to their use in the kinetic experiments cells were washed 3 times in 
PBS and resuspended in PBS containing 2 mg/ml fatty-acid free bovine serum 
albumin (Sigma Chem. Co.). In experiments where cells were pre-incubated 
with u-PA this was performed at a u-PA concentration of 1.4 nM and at 
2.times.10.sup.7 cells/ml for 20 minutes at 37.degree. C., followed by 3 
washes in PBS containing 2 mg/ml fatty-acid free bovine serum albumin. The 
cells were then incubated at a final concentration of 1.times.10.sup.6 
cells/ml in 0.05M Tris-HCl, 0.1M NaCl with plasminogen 2 (0.175 .mu.M) and 
0.2 mM of the plasmin specific fluorogenic peptide substrate 
H-D-Val-Leu-Lys-AMC (Bachem, Switzerland). These incubations were made in 
10-mm plastic fluorimeter cuvettes which were maintained at 37.degree. C. 
and gently stirred in a Perkin-Elmer LS-5 spectrofluorimeter equipped with 
a micro magnetic stirrer. The fluorescence was measured at 1 minute 
intervals at an excitation wavelength of 380 nm and an emission wavelength 
of 480 nm, with both slits set to 5 nm. These data were converted to 
plasmin concentrations by calculating the rate of change in fluorescence 
between each time point and comparison with a calibration curve 
constructed using active-site titrated plasmin. 
The effect of PAI's on the activity of cell-bound u-PA was determined by 
the addition of varying concentrations of PAI-1 (0.18-18.4 nM) or PAI-2 
(1.13-56.7 nM) to the incubations at the same time as the addition of 
plasminogen. Curves were then constructed of plasmin concentration against 
time. 
The concentrations of inhibitors used in these studies were at least 
100-fold higher than the concentration of cell-bound u-PA, meaning that 
the incubations were performed under pseudo-first order reaction 
conditions. The following general equation describes the progressive 
inhibition curve for reactions performed under such conditions: 
EQU pln!.sub.t =pln!.sub..infin. (1-e.sup.k app.sup.t) equation.1 
where k.sub.app is the apparent pseudo-first order rate constant and 
pln!.sub.t and pln!.sub..infin., respectively, are the plasmin 
concentrations at time, t and at infinite time when u-PA is completely 
inhibited. 
pln!.sub..infin. was calculated from the relationship: 
EQU pln!.sub..infin. =1/k.sub.app (.DELTA.pln!.sub.0) equation.2 
where .DELTA.pln!.sub.0 is the initial rate of plasmin generation which 
was determined in control incubations in the absence of inhibitors. The 
experimentally obtained plasmin generation curves were fitted to equation 
1 by non-linear regression. 
Association rate constants (second-order rate constants) were calculated 
from the slope of the line of double reciprocal plots of k.sub.app against 
inhibitor concentration. 
RESULTS 
Plasminogen activation by cell-bound u-PA 
In order to study the interaction of cell receptor bound u-PA with PAI-1 
and PAI-2 it was first necessary to determine the activity of receptor 
bound u-PA against its physiological substrate plasminogen. The U937 cells 
used in this study secrete low concentrations of u-PA, which is found to 
occupy some of the u-PA receptors on the cell surface. This endogenously 
bound u-PA was demonstrated to activate plasminogen, giving a linear rate 
of plasmin generation (FIG. 36). This suggests that the u-PA is in the 
active two-chain form, consistent with other observations (Stephens et 
al., 1988). The identity of the bound enzyme was confirmed as u-PA, rather 
than tPA or unidentified activator, by its complete inhibition by an 
anticatalytic monoclonal antibody to u-PA (FIG. 36). Incubation of the 
cells with exogenously added u-PA resulted in an increased rate of 
plasminogen activation (FIG. 36), due to saturation of the previously 
unoccupied receptors. Alternatively, the endogenously-bound u-PA could be 
eluted from the cells by brief acid treatment (Stoppelli et al., 1986) and 
the cells then saturated with exogenously added u-PA, which gave rise to 
an approximately 50% higher rate of plasmin generation (FIG. 36). The 
binding of u-PA to the cells could be competed by preincubation of the 
acid-washed cells with a 100-fold molar excess of DFP-inactivated u-PA 
(87% inhibition of plasminogen activation), demonstrating that the binding 
was via specific interaction with the u-PA receptor. This was further 
demonstrated by preincubation of the cells with 25 ug/ml of a polyclonal 
antibody raised against the purified human u-PA receptor, which resulted 
in 82% inhibition of plasminogen activation as detailed in Example 10. 
Pre-immune IgG from the same animal in which this antibody was raised had 
no effect on u-PA binding. In control experiments cells prepared by each 
of the above methods were found to be indistinguishable with respect to 
their inhibition by PAI's, therefore the studies subsequently described 
were performed using cells with endogenously-bound u-PA. 
Inhibition of u-bound u-PA by PAI-1 
The PAI-1 used for these studies was purified from the conditioned medium 
of Hep G2 cells, and in contrast to PAI-1 preparations purified from other 
cell-types does not require pre-treatment with denaturants for inhibitory 
activity. This preparation contains the NH.sub.2 -terminal fragment(s) of 
vitronectin which may be reponsible for the stabilization of the PAI-1 
activity. The effect of this PAI-1 preparation on plasminogen activation 
by cell-bound u-PA is shown in FIG. 37. It can be seen that PAI-1 inhibits 
u-PA catalyzed plasminogen activation on the cell-surface in a time and 
concentration dependent manner and also that at the higher concentrations 
of PAI-1 used there is complete inhibition of u-PA activity within the 
time-course of the experiments. These data give an association rate 
constant for the inhibition of cell-bound u-PA by PAI-1 of 
4.5.times.10.sup.6 M.sup.-1 s.sup.-1 (FIG. 39). The inhibition by PAI-1 of 
plasminogen activation by u-PA in solution was determined as 
7.9.times.10.sup.6 M.sup.-1 s.sup.-1 (FIG. 39), or as 7.6.times.10.sup.6 
M.sup.-1 s.sup.-1 by measuring the inhibition of u-PA directly using a 
u-PA specific fluorogenic peptide substrate (data not shown). u-PA bound 
to its cellular receptor appears therefore to be inhibited very 
efficiently by PAI-1, at a rate approximately 60% that of u-PA in 
solution. 
Vitronectin is able to interact with cells through an Arg-Gly-Asp adhesion 
sequence and thereby promote cell attachment and spreading. This sequence 
is apparently still available in PAI-1/vitronectin complexes (Salonen et 
al., 1989) and may therefore be playing a role in the inhibition of 
cell-bound u-PA. To determine whether this occurs U937 cells were 
pre-incubated with the peptide Gly-Arg-Gly-Asp-Ser (0.5 mg/ml) for 30 
minutes prior to incubation with the PAI-1 preparation (0.9 or 4.5 nM). 
The rates of inhibition of cell-bound u-PA were found to be the same in 
the presence or absence of the peptide. 
Inhibition of u-bound u-PA by PAI-2 
The PAI-2 used in these studies was purified from U937 cell lysates and 
therefore consists of the intracellular, mainly non-glycosylated form of 
PAI-2. Previous studies have shown that the glycosylated and 
non-glycosylated forms of PAI-2 are functionally identical (Wohlwend et 
al., 1987). FIG. 38 shows the inhibition of cell-bound u-PA by varying 
concentrations of PAI-2. There is once again a concentration and time 
dependent inhibition of plasminogen activation, and complete inhibition of 
plasminogen activation was observed within the time-course of the 
experiment at the higher concentrations of PAI-2. These concentrations 
were approximately 10-fold higher than those used with PAI-1, consistent 
with the association-rate constant which was determined as 
3.3.times.10.sup.5 M.sup.-1 s.sup.-1 for PAI-2 (FIG. 39), compared to 
4.5.times.10.sup.6 M.sup.-1 s.sup.-1 for PAI-1. The rate of inhibition of 
u-PA in solution by PAI-2 was determined as 5.3.times.10.sup.5 M.sup.-1 
s.sup.-1 by plasminogen activation (FIG. 39) and 5.2.times.10.sup.5 
M.sup.-1 s.sup.-1 by direct assay (data not shown). This data demonstrates 
that PAI-2 inhibits cell-bound u-PA with an association-rate constant that 
is approximately 60% of that obtained for u-PA in solution, which is very 
similar to the effect observed with PAI-1. Therefore, similarly to PAI-1, 
PAI-2 is virtually as efficient an inhibitor of cell-bound u-PA as it is 
of u-PA in solution. 
Inhibition of u-PA bound to u- on PMA-stimulated U937 cells 
Stimulation of U937 cells with PMA has been shown to be accompanied by an 
increase in the number of u-PA receptors per cell, and a concomitant 
reduction in the affinity of these receptors for u-PA, which may be 
related to the increased glycosylation of the receptor observed under 
these conditions. Therefore, as the u-PA receptor on U937 cells appears to 
acquire somewhat different properties upon PMA-stimulation, we determined 
whether this form of the receptor caused any alteration in the inhibition 
of bound u-PA. PAI-1 was found to inhibit cell-bound u-PA with a lower 
association-rate constant than on unstimulated cells, 1.7.times.10.sup.6 
M.sup.-1 s.sup.-1 compared to 4.5.times.10.sup.6 M.sup.-1 s.sup.-1 (Table 
9), which represents approximately 20% of the rate of inhibition of u-PA 
in solution. The inhibition of cell-bound u-PA by PAI-2 was also reduced 
to a similar extent upon PMA stimulation of the cells, from 
3.3.times.10.sup.5 M.sup.-1 s.sup.-1 to 1.1.times.10.sup.5 M.sup.-1 
s.sup.-1 (Table 9). 
TABLE 9 
______________________________________ 
Association rate constants for the inhibition of free and u-bound 
u-PA by PAI's. 
Situation of Association rate constant, M.sup.-1 s.sup.-1 
u-PA PAI-1 PAI-2 
______________________________________ 
In solution 7.9 .times. 10.sup.6 
5.3 .times. 10.sup.5 
U937 cells 4.5 .times. 10.sup.6 
3.3 .times. 10.sup.5 
PMA-stimulated U937 cells 
1.7 .times. 10.sup.6 
1.1 .times. 10.sup.5 
______________________________________ 
EXAMPLE 10 
Inhibition of Cell Surface Plasminogen Activation by u- Antibodies and 
Inhibition of u-PA Catalyzed Plasminogen Activation in Solution by 
Solubilized u- 
MATERIALS AND METHODS 
Plasminogen activation by u--bound u-PA on U937 cells was determined as 
described in detail in Example 9. Briefly, varying concentrations of 
plasminogen (0.09 uM and 2.26 .mu.M) were incubated with U937 cells 
(pre-incubated with active u-PA and subsequently washed) in the presence 
of the plasmin specific fluorogenic substrate Val-Leu-Lys-AMC (Bachem, 
Switzerland). Plasmin generation was determined from the rate of change of 
the increase in fluorescence due to substrate hydrolysis, measured at 
excitation and emission wavelengths of 380 nm and 480 nm, respectively. 
These plasmin generation rates were subsequently plotted against the 
plasminogen concentration in a double-reciprocal manner to determine the 
individual kinetic constants, K.sub.m and V.sub.max, for the reaction. 
V.sub.max, the maximum reaction velocity, was converted to k.sub.cat, the 
catalytic rate constant, by division of V.sub.max by the concentration of 
u-PA bound to u-. 
The concentration of u-PA bound to u- on U937 cells was determined using 
.sup.125 I-u-PA (prepared as described in Example 8) which was incubated 
with the cells in parallel incubations to the kinetic experiments and 
treated identically. .sup.125 I-u-PA bound to u- was then quantitated 
using standard gamma-counting techniques. 
In experiments where the effect of u- released from cells by treatment 
with PI-PLC (phosphatidylinositol phospholipase C, Boehringer Mannheim 
Biochemica) on the enzymatic activity of u-PA was studied, the following 
procedure was used. U937 cells were treated with 150 nM PMA (phorbol 
myristate acetate) for 4 days. The cell layer was washed 3 times in PBS 
and 2.times.10.sup.7 cells incubated with 3.3 .mu.g of PI-PLC in 5 ml of 
PBS. Aliquots were then removed at various time points. The presence of 
u- in these supernatants was demonstrated by cross-linking to .sup.125 
I-ATF using DSS as described in Example 1. The effect of this soluble form 
of u- on u-PA enzymatic activity was determined by incubation of 
varying concentrations of supernatant with 60 .mu.M u-PA in 0,05M Tris pH 
7,4, 0,1M NaCl, 0,2% bovine serum albumin. In some experiments the 
supernatants were first pre-incubated for 30 minutes with 5 ug/ml of a 
monoclonal antibody against PAI-2 (MAI-21; Biopool, Umea, Sweden). After 
30 minutes of incubation between u-PA and soluble u- in the 
supernatant, residual u-PA activity was determined by the addition of an 
equal volume of a solution containing 0,2 Mg/ml Glu-plasminogen (Kabi, 
Stockholm, Sweden) and0,2 mM Val-Leu-Lys-AMC in 0,05M Tris pH 7,4, 0,1M 
NaCl, 2 mM trans-4-(aminomethyl)-cyclohexane-carboxylic acid (Sigma Chem. 
Co.). Hydrolysis of the fluorogenic substrate was monitored continuously 
in a Perkin-Elmer LS5 spectrofluorimeter with the excitation and emission 
wavelengths set at 380 nm and 480 nm, respectively. Residual u-PA 
concentrations were calculated from these data by reference to standard 
curves constructed using u-PA of known concentration. 
RESULTS 
Kinetics of plasminogen activation by u-PA bound to u- on U937 cells. 
u-PA bound to u- on U937 cells was found to activate its natural 
substrate plasminogen with different kinetic characteristics from those 
displayed in the absence of u-. The activation of Glu-plasminogen by 
u--bound u-PA followed an apparently Michaelis-Menton type kinetic 
mechanism. This was characterized by a K.sub.m of 0.67 .mu.M and a 
k.sub.cat of 5.6 min (FIG. 40). Both of these constants were different 
from those obtained with u-PA in solution, i.e. the absence of U937 cells. 
In this situation, the K.sub.m was much higher at 25 .mu.M (equivalent to 
an approximately 40-fold lower affinity for plasminogen in the absence of 
cell-associated u-) and the k.sub.cat higher at 44 min.sup.-1 
(equivalent to an approximately 8-fold higher catalytic rate in the 
absence of cell-associated u-). Therefore, u-PA binding to u- on 
U937 cells causes plasminogen activation to be saturated at lower 
plasminogen concentrations than in solution, but this is accompanied by a 
reduction in the catalytic rate. However, the overall effect is a 5-fold 
increase in the catalytic efficiency (k.sub.cat /K.sub.m) of u-PA when 
bound to u- on U937 cells (Table 10). As plasminogen (and plasmin) is 
known to bind to U937 cells, as well as a wide variety of other cells 
(Ellis et al., 1989; Plow et al., 1986), these constants measure 
plasminogen activation taking place at the surface of cells possessing 
u-, i.e. cell-surface plasminogen activation. 
Table 10 also shows similar data for plasminogen activation by u-PA bound 
to u- on PMA-stimulated U937 cells. The K.sub.m for plasminogen 
activation is now 1.43 .mu.M, still much lower than for the reaction in 
solution. However, the k.sub.cat also falls from 5.6 min.sup.-1 to 1.23 
min.sup.-1, resulting in an overall reduction in plasminogen activation 
(k.sub.cat /K.sub.m) of approximately 10-fold when compared to 
unstimulated cells. 
TABLE 10 
______________________________________ 
Kinetic constants for Glu-plasminogen activation in the presence of 
U937 associated u- 
K.sub.m 
k.sub.cat k.sub.cat /K.sub.m 
______________________________________ 
u-PA in solution 
25 .mu.M 44 min.sup.-1 
1.76 .mu.M.sup.-1 min.sup.-1 
u-PA - u- 0.67 5.6 8.36 
on U937 cells 
u-PA - u- 1.43 1.23 0.86 
on PMA-U937 
______________________________________ 
Inhibition of cell-surface plasminogen activation by a polyclonal antibody 
to u- 
The polyclonal rabbit antibody raised against purified u- (see Example 
11) was used to demonstrate that the cell-surface plasminogen activating 
activity of u-PA demonstrated in the previous section was indeed due to 
u-PA binding to u-, and also to demonstrate that this antibody did 
block binding of u-PA to u- in solution. 
Firstly, the effect of this antibody on u-PA activity in solution was 
determined. In 4 experiments anti-u- (100 .mu.g/ml for 30 min) gave a 
residual u-PA activity of 90.1+9.3%, compared to 88.6+12.3% for pre-immune 
IgG from the same animal. Therefore the anti-u- antibody gave no 
specific inhibition of u-PA activity. 
When pre-incubated with U937 cells at a concentration of 25 .mu.g/ml for 30 
minutes, the anti-u- antibody resulted in a decrease in the subsequent 
plasminogen activating activity of 76% (mean of three experiments, range 
66%-82%). In contrast the preimmune IgG gave &lt;1% inhibition, whilst 
DFP-u-PA gave 90% inhibition (range 74%-100% in three experiments). 
Therefore the anti-u- polyclonal antibody effectively inhibits the 
cell-surface plasminogen activation. 
Effect of u- released from cells by PI-PLC treatment on u-PA activity. 
Supernatants from PI-PLC-treated PMA-stimulated U937 cells contain a 
soluble form of u-, as determined by DSS crosslinking to .sup.125 
1-ATF. When these supernatants were incubated with u-PA, there was a 
concentration-dependent decrease in u-PA activity (FIG. 41) which was much 
larger than the decrease in u-PA activity caused by control supernatants, 
i.e. not treated with PI-PLC and not containing significant amounts of 
soluble u-. A proportion of the inhibitory activity of both 
supernatants was due to PAI-2 secreted from the cells, and this inhibitory 
activity could be neutralized with antibodies to PAI-2 (FIG. 41). After 
this treatment, the inhibition of u-PA by the Pl-PLC-treated supernatant 
was still apparent. To demonstrate that this inhibitory activity was, in 
fact, due to u- liberated from the cells, the supernatants were also 
pre-incubated for 30 min with either DFP-inactivated u-PA (100-fold excess 
over u-PA) or 25 .mu.g/ml polyclonal antibody to u- (see Example 11). 
The results are shown in Table 11. It can be seen that preincubation with 
either of these reagents, which will abolish binding of u-PA to u-, 
also decreases the inhibitory activity of the Pl-PLC-treated supernatants 
by approximately 40%. There is also a minor effect observed with the 
control supernatants, which is due to the small amounts of u- observed 
in the sample by .sup.125 I-ATF cross-linking. 
These findings clearly demonstrate that u- which has been solubilized by 
removal of the glycosyl-phosphatidylinositol anchor inhibits the ability 
of u-PA to activate plasminogen in solution. 
TABLE 11 
______________________________________ 
Residual u-PA activity 
-PI-PLG 
+Pl.PLC 
______________________________________ 
-- 88% 34% 
+DFP-u-PA 100% 75% 
+anti-u- 100% 72% 
antibody 
______________________________________ 
PMA-stimulated U937 cells were treated with PI-PLC for 120 minutes. 
Supernatants from both treated and control cells were incubated with 
monoclonal antibodies to PAI-2. 20 .mu.l of supernatant was incubated with 
u-PA in a final volume of 100 .mu.l. 
EXAMPLE 11 
Production of Antibodies to u- 
Immunization of mice 
Mice of the BALB/c strain were immunized with u- purified on a 
diisopropylfluoride urokinase-type plasminogen activator (DFP-u-PA) ligand 
affinity column. The mice were given three intraperitoneal injections with 
5 .mu.g of u- with 3 week intervals. 8-10 days after the last 
injection, serum was tested in both ELISA and Western blotting for 
reactivity against u-. When positive reaction was detected, a final 
booster injection of 10-15 .mu.g of u- was given intraperitoneally. 
Production of monoclonal mouse antibody 
Standard protocols for fusion were followed and are briefly outlined below: 
a) The isolated spleen from BALB/c mice was mechanically disrupted, and a 
homogeneous cell suspension was prepared in serum-free medium. 
b) Myeloma cells and X63-Ag 8.653 cells (Kearney, J. Immunol. 123: 
1548-1550, 1979) in logarithmic phase of growth were isolated for fusion 
with BALB/c spleen lymphocytes. The myeloma cells were resuspended in 
serum-free medium. 
c) The spleen lymphocytes and myeloma cells were mixed in a ratio of 1:1. 
d) Cells were fused by dropwise addition of 1 ml of 50% (wt/vol) 
polyethylene glycol 4000 (PEG) at 37.degree. C. (1 ml/10.sup.8 cells). 
e) Fusion was stopped by gentle addition of serum-free medium. 
f) After centrifugation, the supernatant was removed and the cells were 
washed once in serum containing medium. Then the cells were carefully 
resuspended in hypoxanthine-aminopterin-thymidine (HAT)-containing medium. 
g) The fused cells at a concentration of approximately 5.times.10.sup.5 
cells/well were distributed in 50 .mu.l aliquots to wells of flat-bottomed 
microtiter plates containing 2.5.times.10.sup.4 macrophages in 150 .mu.l 
selection medium. 
h) The cells were incubated at 37.degree. C. in 5% CO.sub.2 in a humid 
incubator. 
i) The selection medium was renewed after a week or when needed. 
j) The wells were inspected for hybridoma growth. When vigorous growth and 
change of colour to yellow were observed, supernatants were removed for 
screening of antibody activity. 
k) 10-14 days after fusion, HAT medium was replaced by HT medium and later, 
e.g. after 10 days, by regular medium. 
l) Positive wells were transferred into cups of 24-well plates and then to 
small (25 cm.sup.2) culture flasks. 
m) Hybrid cells secreting the desired antibodies were frozen in liquid 
N.sub.2 as early as possible. 
n) Positive hybridoma clones were cloned by limited dilution, retested, 
recloned and retested until a hybridoma secreting only one type of 
monoclonal antibody was established. 
Screening procedures for production of monoclonal u- antibodies 
Radioimmunoprecipitation assay (RIPA). This assay as well as the reverse 
solid phase radioimmune assay were developed because the amount of 
purified antigen was limited. 
Materials 
1) .sup.125 I-iodinated purified u- (Iodogen method). 
2) Reaction buffer: 0.1% bovine serum albumin+0.1% Triton X-100 in PBS 
(0.1% BSA, 0.1% Triton X-100/PBS). 
3) Washing buffer: reaction buffer+0.5M NaCl. 
4) Protein A Sepharose CL 4B swollen and diluted 1:1 in reaction buffer 
(Prot. A Seph. solution). 
5) Eppendorf plastic tubes. 
Procedure 
1) Add 100 .mu.l of radiolabelled u- diluted in reaction buffer (about 
3-5.times.10.sup.5 cpm/ml) into Eppendorf tubes. 
2) Add 100 .mu.l of immune serum/non-immune serum serial diluted in 
reaction buffer and include relevant controls. 
3) Incubate for 1 hour at 4.degree. C. without shaking. 
4) Add 50 .mu.l of Prot. A. Seph. solution. 
5) Incubate for 1 hour at 4.degree. C. on an end-over-end rotor. 
6) After the last incubation, add 1 ml of reaction buffer to the test tubes 
and let the Prot. A Seph. solution settle. Remove supernatant. 
7) Replace reaction buffer with 1 ml of washing buffer and repeat step 6. 
8) Repeat step 6. 
9) Cut the lid of the test tubes and count. 
Reverse solid phase radioimmunoassay 
Materials 
1) 96-well plates (Costar). 
2) Coating buffer: 0.1M Na.sub.2 CO.sub.3, pH 9.8. 
3) Rabbit anti-mouse Ig (RaM Ig 11.6 mg/ml) (Dako Z109). 
4) Blocking buffer: 25% fetal calf serum in PBS (25% FCS/PBS). 
5) Dilution buffer: PBS, pH 7.4 (PBS). 
6) Washing buffer: PBS+0.1% Tween 20, pH 7.4 (PBS/Tween 20). 
7) .sup.125 I-lodinated purified u-. 
Procedure 
1) Coat the wells with 100 .mu.l of RaM Ig diluted in 0.1M Na.sub.2 
CO.sub.3, pH 9.8, to a concentration of 20 .mu.g/ml. 
2) Incubate overnight at 4.degree. C. on a shaker. 
3) Next day, wash the wells 4.times. in PBS/Tween 20. 
4) Block the remaining active sites in the wells with 25% FCS/PBS, 
200 .mu.l/well, for 1/2 hour at room temperature (RT). Gentle shaking. 
5) Wash 4.times. in PBS/Tween 20. 
6) Add 100 .mu.l/well of immune/non-immune sera serial diluted in PBS or 1% 
BMP/PBS and include relevant controls. 
7) Incubate for 1 hour at 37.degree. C. with gentle shaking. 
8) Wash 4.times. in PBS/Tween 20. 
9) Add 100 .mu.l/well of radiolabelled u- diluted in PBS 
(3-5.times.10.sup.5 cpm/ml) or 1% BMP/PBS. 
10) Incubate for 1 hour at 37.degree. C. with gentle shaking. 
11) Wash 4.times. in PBS/Tween 20. 
12) Count the wells. 
Enzyme-linked Immunosorbent Assay (ELISA) 
Materials 
1) 96-well plates (U-form high binding capacity, Nunc). 
2) u- purified (10 .mu.g/ml). 
3) Horseradish peroxidase-conjugated rabbit anti-mouse Ig (HRP-REM Ig). 
4) PBS buffer, pH 7.4 (PBS). 
5) PBS+0.1% Tween 20, pH 7.4 (PBS/Tween 20). 
6) Blocking buffer: 25% fetal calf serum in PBS (25% FCS/PBS) or 1% skimmed 
milk powder (SMP) in PBS. 
7) Citrate buffer: 0.1M citrate, pH 5.0. 
8) Substrate solution: 1,2-Phenylenediamine dihydrochloride (OPD) tablets 
in citrate buffer, e.g. 3 OPD tab. in 15 ml of citrate buffer+5 .mu.l of 
H.sub.2 O.sub.2 (30%). 
9) Stop buffer: 1 M H.sub.2 SO.sub.4. 
Procedure 
1) Coat the wells with 100 .mu.l of purified u- diluted in 0.1M Na.sub.2 
CO.sub.3, pH 9.8, to a concentration of 10 ng/ml. 
2) Incubate overnight at 4.degree. C. 
3) Next day, wash the wells 4.times. in PBS/Tween 20. 
4) Block the remaining active sites in the wells with 25% FCS/PBS or 1% 
SMP/PBS, 200 .mu.l/well, for 1/2 hour at RT. Gentle shaking. 
5) Wash as step 3. 
6) Add 100 .mu.l/well of immune/non-immune sera serial diluted in PBS and 
include relevant controls. 
7) Incubate for 1 hour at 37.degree. C. with gentle shaking. 
8) Wash as step 3. 
9) Add 100 .mu.l/well of secondary antibody HRP-RaM Ig diluted 1:500 in 
PBS. 
10) Incubate as step 7. 
11) Wash as step 3. 
12) Wash 1.times. in 0.1M citrate buffer, pH 5.0. 
13) Add 100 .mu.l/well of substrate solution. 
14) Stop the reaction with 150 .mu.l/well of 1M H.sub.2 SO.sub.4 when 
bright yellow colour appears, 15-30 minutes. 
15) Read on an ELISA-reader with a 490 nm filter. 
Preparation of polyclonal rabbit antibodies against u- 
Samples of purified human u-PA receptor (Example 1) were subjected to 
SDS-polyacrylamide gel electrophoresis under non-reducing conditions on a 
6-16% gradient gel. By the use of fluorescent molecular weight markers run 
in neighbouring lanes, the electrophoretic region corresponding to the 
antigen was excised. The gel piece was lyophilized and subsequently 
macerated in a Mikro-Dismembrator II apparatus (B. Braun AG, Federal 
Republic of Germany). The polyacrylamide powder was reconstituted in 
Tris-buffered saline, mixed with Freund's incomplete adjuvant and used for 
injection of a New Zealand white rabbit. The animal received 5 injections, 
each containing approximately 3 .mu.g of the antigen, over a 10 week 
period, followed by a single 8 .mu.g injection after an additional 7 
weeks. Serum was drawn 1 week after the last injection, and IgG was 
prepared by Protein A-Sepharose chromatography. In order to remove 
antibodies against trace impurities in the injected antigen, the antibody 
was absorbed by consecutive passages through columns containing 
immobilized human u-PA and the protein mixture constituting the Triton 
X-114 detergent phase from PMA-stimulated U937 cells (see Example 1), 
respectively. The antibody preparation obtained did not inhibit the 
amidolytic or plasminogen activator activity of u-PA in solution. 
Specificity of u- antibodies evaluated by Western blotting 
Electrophoresis. SDS-PAGE was carried out in slab gels with a linear 6-16% 
polyacrylamide concentration gradient according to Laemmli (supra). 
Samples were run under reducing conditions. The samples were reduced 
immediately before electrophoresis in Laemmli buffer except that 
2-mercaptoethanol was replaced with dithiothreitol for 3 minutes at 
100.degree. C. The following molecular weight markers were used: 
phosphorylase b (molecular weight about 94,000), bovine serum albumin 
(molecular weight about 67,000), ovalbumin (molecular weight about 
43,000), carbonic anhydrase (molecular weight about 30,000), soybean 
trypsin inhibitor (molecular weight about 20,100), and .alpha.-lactalbumin 
(molecular weight about 14,400). 
Western Blotting--Samples of affinity purified u- or detergent phase 
from Triton X-114 extracts of PMA-stimulated U937 cells were subjected to 
SDS-PAGE under reducing conditions on 6-16% gradient gels. The gels were 
electroblotted onto nitrocellulose sheets. The sheets were rinsed and 
blocked with 30% fetal calf serum in Tris-buffered saline, pH 7.4. The 
sheets were incubated with mouse anti-u- serum or control serum (i.e. 
mouse antiserum against porcine mucins), diluted in fetal calf serum in 
Tris-buffered saline. The sheets were rinsed, incubated with secondary 
antibody (alkaline phosphatase-conjugated rabbit anti-mouse Ig (Dakopatts, 
Copenhagen)), and developed with nitro blue 
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate/Levamisol. 
Western blotting analysis of rabbit u- antibody was performed in the 
same manner, except for the following modifications: SDS-PAGE was 
performed under non-reducing conditions. Newborn calf serum was used 
instead of fetal calf serum. Only 10% serum was included in the primary 
antibody incubation step. Alkaline phosphatase conjugated swine 
anti-rabbit Ig (Dakopatts code 306), 100-fold dilution, was used as the 
secondary antibody. 
Assay for Inhibition of Cellular ATF Binding--U937 cells were washed and 
acid-treated, as described (Nielsen et al., 1988). The cells were 
resuspended in 100 .mu.l of PBS, 0.1% bovine serum albumin, and 100 .mu.l 
of prediluted anti-u- serum was added. Control samples received 100 
.mu.l of prediluted control serum (i.e. mouse antiserum raised against 
porcine mucins). The samples were incubated for 1 hour at 4.degree. C. 
with gentle stirring. After the incubation, 100 .mu.l of .sup.125 I-ATF 
was added and incubation was continued for another hour. In the 300-.mu.l 
reaction volume, the final concentration of .sup.125 I-ATF was 2.2 nM, and 
the final dilutions of anti-u- serum/control serum ranged from 1:300 to 
1:153,600. The cells were then washed 3 times with 1 ml of PBS-bovine 
serum albumin, and the bound radioactivity was measured in a gamma 
counter. Under these conditions, 12% of the radioactivity became 
cell-bound when no antiserum had been added. 90% of the bound 
radioactivity was displaced when the cells were preincubated with 700 nM 
non-labelled u-PA. 
RESULTS 
As shown in FIG. 42, serum from immunized mice precipitated .sup.125 
I-labelled purified u-. The anti-u- serum diluted 1:75, 1:750, 
1:7500 and 1:75000 gave a 25%, 18%, 5% and 1% precipitation, respectively. 
The non-immune serum at the same dilutions and the other controls gave 
precipitations in the range of 0.5-1%. 
Using a reverse solid phase radioimmunoassay, the antiserum was used to 
immunocapture .sup.125 I-labelled purified u- (FIG. 43). A 2-fold 
serial dilution of the anti-u- serum 1:500-1:32000 showed that the same 
amount of .sup.125 I-u- (about 2% of total) was captured at a serum 
dilution up to 1:4000 and dropped to half the amount at 1:32000. The same 
serial dilution of non-immune serum and the other controls resulted in a 
capture of .sup.125 I-u- of about 0.5% of total. 
The reaction of immune versus non-immune serum in an ELISA is shown in FIG. 
44. 1 ng of purified u- coated per well was sufficient to be detected 
with the immune serum diluted 1:8000. Both the non-immune serum at all 
dilutions and other controls gave reaction values at background level. 
The mouse antiserum against human u- was used in a competition 
experiment in which U937 cells were preincubated with the antiserum 
followed by addition of .sup.125 I-ATF. As shown in FIG. 45, the 
anti-u- serum was able to completely inhibit the specific binding of 
.sup.125 I-ATF to the cells. 50% inhibition was obtained at a 1:2400 
dilution. Under the same conditions, a control serum showed only slight 
inhibition, i.e. about 20% at the highest concentration used (a 1:300 
dilution). In Western blotting, the u- contained within the detergent 
phase from PMA-treated U937 cells, as well as the purified u-, were 
detected by the anti-u- serum (FIG. 45, inset, lanes 1 and 2). The 
control immune serum gave no reaction with the same preparations (lanes 3 
and 4). 
Rabbit polyclonal antibodies were prepared by immunizing a rabbit with 
polyacrylamide gel material containing affinity-purified u- that had 
subsequently been subjected to preparative SDS-PAGE. The IgG fraction was 
isolated from the obtained antiserum and absorbed by passage through 
columns with immobilized human u-PA and immobilized membrane-protein 
mixture derived from PHA-stimulated U937 cells, respectively. The antibody 
recognized u- in the Triton X-114 detergent phase from PMA-stimulated 
U937 cells (FIG. 46A). Thus, a protein in the 50-65 kD range was 
recognized (lanes 1 and 2) which could be identified as being u- by the 
ability to form a 100-110 kD conjugate with DFP-treated u-PA after the 
performance of chemical cross-linking (see Example 1 for methods) (lane 
3). No staining was obtained with DFP-treated u-PA alone (lanes 5 and 6), 
and the cross-linking procedure did not alter the electrophoretic 
appearance of u- when no DFP-treated u-PA was added (lane 2). In none 
of the samples was any band stained with the pre-immune IgG from the same 
rabbit, prepared in the same manner (FIG. 46B). 
The effect of the rabbit antibody on the ligand binding capability of u- 
was studied in a different experiment (not shown) in which a purified 
sample of u- (Example 1; approximately 20 ng/ml) was preincubated with 
the purified and absorbed IgG from the rabbit anti-u- serum (final IgG 
concentration 90 .mu.g/ml during preincubation). This treatment completely 
hindered the subsequent formation of crosslinked conjugates with .sup.125 
I-ATF. The IgG from the pre-immune serum had no effect on the 
cross-linking assay at the same concentration. 
EXAMPLE 12 
Visualization of the u-PA Receptor 
Method: u-PA was purified by affinity chromatography on monoclonal 
antibodies and activated by treatment with plasmin (Nielsen et al., 1982). 
Alternatively, two-chain u-PA was obtained commercially (Serono u-PA). 
u-PA was DFP-inactivated as described for the preparation of columns for 
purification of the receptor. 
u-PA was dialyzed overnight against 0.1M Na.sub.2 HCO.sub.3 with 0.1% 
Triton X-100. N-biotin-hydroxysuccinimide was dissolved in 
N,N-dimethylformamide (5 mM). To the u-PA preparation was added 0,1 .mu.l 
of this solution per .mu. of u-PA, and the reaction was allowed to run for 
1 hour at room temperature. Excess labelling compound was removed by 
dialysis overnight against 0.1M NaHPO.sub.4, pH 8.0, with 0.5M NaCl and 
0.1% Triton X-100. 
Cultured cells (PMA-treated U937) or cryostat sections of freshly frozen 
human chorion were treated for 3 minutes at room temperature with 0.05M 
glycine, pH 3.0 with 0.1M NaCl, neutralized with 0.5M 30 HEPES, pH 7.5 
with 0.1M NaCl and incubated at 4.degree. C. with 200 nM of biotinylated 
DFP-treated u-PA dissolved in PBS with 0.1% BSA (PBS-BSA). Competition 
experiments were performed by simultaneous incubation with biotinylated 
DFP-treated u-PA (200 nM) and purified unlabelled u-PA (2 .mu.M). 
After incubation, slides were washed for 2.times.5 minutes in PBS-BSA, and 
fixed with 4% paraformaldehyde for 5 minutes. After washing for 3.times.5 
minutes in PBS-BSA, unspecific binding sites were blocked by 5 minutes of 
incubation with 25% newborn calf serum in TBS. Sections were briefly 
washed with TBS and incubated with streptavidin-fluorescin isothiocyanate 
(Amersham) diluted 1:100 in TBS-BSA for 30 minutes. After a brief rinse 
and washing for 2.times.10 minutes in TBS, slides were incubated with 
biotinylated anti-avidin (Vector) (5 .mu.g/ml) in TBS-BSA for 30 minutes. 
After rinsing and washing for 2.times.10 minutes in TBS, the incubation 
with streptavidin-fluorescin isothiocyanate was repeated. Finally, the 
sections were rinsed and washed for 2.times.10 minutes in TBS, 
contrast-stained for 2 minutes in Meyer's hematoxylin (standard method) 
and, in the case of U937 cells, also in Eriochrome Black (cf. Schenk E A 
and Churukin C J, "Immunofluorescence counterstains", Cytochem. 22: 
962-966, 1974; Johnson G D et al., "Fading of immunofluorescence during 
microscopy: a study of the phenomenon and its remedy", J. Immunol. Methods 
50: 231-242, 1982). Sections were mounted using DABCO-glycerol and viewed 
using a LEITZ epifluorescence microscope. 
RESULTS 
The u-PA receptor could be visualized on the surface of U937 cells, as 
shown in FIG. 47A. The signal was particularly prominent over cellular 
extensions. Incubation with a surplus of unlabelled u-PA showed 
competition of the signal (FIG. 47B). 
Cryostat sections of human chorion showed a diffuse signal over the 
cellular layer (FIG. 48A). Competition with purified u-PA inhibited this 
binding (FIG. 48B). 
EXAMPLE 13 
Effect of Purified u- on u-PA Catalyzed Plasminogen Activation and 
Plasmin Catalyzed PRO-u-PA Activation 
Methods for the study of the effect of added, purified u- on assays for 
u-PA mediated plasminogen activation and plasmin mediated pro-u-PA 
activation 
Both assays were carried out in microtiter plates, using chromogenic 
substrates (see below), the cleavage of which was followed by measuring 
the absorbance at 405 nm in an ELISA reader. Proteolysis buffer (0.1M 
Tris/HCl, pH 8.1, 0.1% Triton X-100) was used as the reaction buffer and 
for the dilution of all samples. Affinity purified u- (see Example 1) 
was added as indicated or substituted by a protein devoid sample of the 
same buffer compositon. Materials and methods not specified below were 
those described by Petersen et al. (1988). All samples were analysed in 
triplicate. 
Assays for plasminogen activation 
Human 54 kDa two-chain u-PA (Ukidan, Serono) was preincubated with u- or 
buffer at the concentrations indicated for 15 min at room temperature. 
Plasminogen (10 .mu.g/ml final concentration) and 
H-D-Valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride (Kabi product 
S-2251), termed substrate S1 below (final concentration 400 .mu.M) were 
added in a final reaction volume of 250 .mu.l, and cleavage of the 
substrate was followed during incubation at 37.degree. C. Standard curves 
were drawn from assays of the following final concentrations of u-PA: 8, 
16, 32, 64, 128 and 256 pg/ml. 
Assay for pro-u-PA activation 
Human pro-u-PA was preincubated with u- or buffer for 10 min at room 
temperature. Plasmin (10 ng/ml final concentration) was added 30 and the 
samples were incubated at 37.degree. C. Aliquots were taken after the 
following periods of incubation: 1, 2, 5, 10, 20, 30 and 60 min. After the 
periods indicated, plasmin activity within each sample was stopped by the 
addition of Trasylol (10 .mu.g/ml final concentration). Each aliquot was 
assayed for u-PA amidolytic activity by addition of 400 .mu.M (final 
concentration) of L-Pyroglutamyl-glycyl-L-arginine-p-nitronanilide 
hydrochloride (Kabi product (S-2444; termed substrate S2 below) in a final 
reaction volume of 200 .mu.l, followed by incubation at 37.degree. C. and 
absorbance measurement. The absorbance values were compared to a standard 
curve obtained with known concentrations of 54 kDa two-chain u-PA (Ukidan, 
Serono) in the same assay of amidolytic activity, performed simultaneously 
and using the same buffer composition. 
RESULTS 
Effect of purified u- on u-PA plasminogen activation activity. 
Samples of 54 kDa, two chain u-PA (final concentrations rangning from 8-256 
pg/ml) were preincubated in the presence or absence of affinity purified 
u- (approx. 1 nM final concentration). After the preincubation step, 
plasminogen (final concentration 10 .mu.g/ml) was added and plasmin 
generation was measured spectrophotometrically after addition of the 
chromogenic plasmin substrate S1 (see "Methods"; 400 .mu.M final 
concentration) and incubation for 2 h at 37.degree. C. 
In this assay, the u- concentration used led to an apparent 50% 
inhibition of u-PA activity in the dynamic u-PA concentration range of 
32-256 .mu.g/ml. Thus, the standard curves obtained in the presence and 
absence of u- were superimposable according to a model in which the 
activity of any u-PA concentration in the presence of u- was equivalent 
to the activity of 50% of the same concentration in the absence of u-. 
Effect of purified u- on plasmin mediated pro-u-PA activation 
Samples of pro-u-PA (63 ng/ml final concentration) were preincubated in the 
presence or absence of affinity purified u- (approx. 2 nM 30 final 
concentration), followed by addition of plasmin (final concentration 10 
ng/ml). The samples were incubated at 37.degree. C. At various time 
intervals aliquots were taken and mixed with Trasylol for the termination 
of pro-u-PA activation. 
The generated u-PA activity in each aliquot was measured 
spectrophotometrically after addition of the chromogenic u-PA substrate 
(S2) (see Methods; 400 .mu.M final concentration) and incubation for 19 h 
at 37.degree. C. The activity was expressed as the equivalent 
concentration of commercial two-chain u-PA (see Methods), as read from a 
standard curve drawn from a simulteneous and parallel experiment. 
The curves of u-PA activity vs. time of plasmin treatment were linear in 
the range from 2-20 min. In the absence of u-, u-PA activity was 
generated at a velocity of 0.60 ng/ml equivalent two-chain u-PA per min. 
In the presence of u- at the concentration used, the activation 
velocity was reduced to 0.18 ng/ml equivalent two-chain u-PA per min. This 
reduction was due to a real inhibition of pro-u-PA activation since the 
presence of u- had no effect on the activity of two-chain u-PA against 
the substrate S2. 
Independence of kinetic results on hydrophobic properties of u- (see 
Example 4 for principles and methods). 
Samples of purified u- were treated with PI-PLC (500-fold final dilution 
of the Boehringer Mannheim preparation) for 30 min at 37.degree. C. This 
treatment led to an approx. 50% delipidation of u- as judged by the 
shift of the ATF cross-linking activity towards the buffer phase in the 
Triton X114 phase separation system (see Example 1). 
The above mentioned assays for u-PA plasminogen activator activity and for 
pro-u-PA activation, respectively, were reproduced in the presence of 50% 
the delipidized u- preparation. The results were identical to those 
obtained with the intact u- which were reproduced in parallel. 
These results demonstrate that pure u- also after removal of the 
glycerol-phosphoinositol anchor inhibits the activity of u-PA in solution, 
in perfect agreement with the conclusion obtained in Example 10. 
REFERENCES 
Andreasen PA, Nielsen L S, Kristensen P, Gr.o slashed.ndahl-Hansen J, 
Skriver L, Dan.o slashed. K (1986) Plasminogen activator inhibitor from 
human fibrosarcoma cells binds urokinase-type plasminogen activator, but 
not its proenzyme. J Biol Chem 261: 7644-7651 
Angerer L M, Stoler M H, Angerer R C (1987) In Situ Hybridization with RNA 
probes: An annotated Recipe. In In situ hybridization. Applications to 
Neurobiology. Oxford University Press, Oxford, pp. 71-96. 
Appella E, Robinson E A, Ullrich S J, Stoppelli M P, Corti A, Cassani C, 
Blasi F (1987) The receptor-binding sequence of urokinase. A biological 
function for the growth-factor module of proteases. J Biol Chem 262: 
4437-4440 
Appella E, Weber I T, Blasi F (1988) Structure and function of epidermal 
growth factor-like regions in proteins. FEBS L. 231: 1-4 
Bajpai A, Baker J B (1985) Cryptic urokinase binding sites on human 
foreskin fibroblasts. Biochem Biophys Res Commun 133: 475-482 
Bajpai, A, Baker J B (1985a) Biochem Biophys Res Commun 133: 994-1000 
Baker J B, Low D A, Simmer R L, Cunningham D D (1980) Cell 21: 37-45 
Barkholt V, Jensen A L (1989) Amino acid analysis: Determination of 
cysteine plus half-cysteine in proteins after hydrochloric acid hydrolysis 
with a disulfide compound as additive. Anal Biochem 177: 318-322 
Barnathan E S, Cines D B, Barone K, Kuo A, Larsen G R (1988) Differential 
binding of recombinant wild type and variant t-PA to human endothelial 
cells. Fibrinolysis 2, Suppl 1: 28 
Beebe D P (1987) Binding of tissue plasminogen activator to human umbilical 
vein endothelial cells. Thromb Res. 46: 241-254 
Bell G I, Fong N M, Stempien M M, Wormsted M A, Caput D, Ku L, Urdea M S, 
Rall S B, Sanchez-Pescador L (1986) Human epidermal growth factor 
precursor: cDNA sequence, expression in vitro and gene organization. Nucl. 
Ac. Res. 14: 8427-8446 
Blasi F (1988) Surface receptors for urokinase plasminogen activator. 
Fibrinolysis 2: 73-84 
Blasi F, Stoppelli M P, Cubellis M V (1986) The receptor for 
urokinase-plasminogen activator. J Cell Biochem 32: 179-186 
Blasi F, Vassalli J-D, Dan.o slashed. K (1987) Urokinase-type plasminogen 
activator: proenzyme, receptor, and inhibitors. J Cell Biol 104: 801-804 
Bordier C (1981) Phase Separation of integral membrane proteins in Triton 
X-114 solution. J Biol Chem 256: 1604-1607 
Boyd D, Florent G, Kim P, Brattain M (1988a) Determination of the levels of 
urokinase and its receptor in human colon carcinoma cell lines. Cancer Res 
48: 3112-3116 
Boyd D, Florent G, Murano G, Brattain M (1988b) Modulation of the urokinase 
receptor in human colon cell lines by N,N-dimethylformamide. Biochim 
Biophys Acta 947: 96-100 
de Bruin P A F, Crama-Bohbouth G, Werspaget H W, Verheijen J H, Dooijewaard 
G, Weterman I T, LLamers C B H W (1988) Thrombosis and Haemostasis 60: 2; 
262-266 
Burridge K (1986) Substrate adhesions in normal and transformed 
fibroblasts: Organization and regulation of cytoskeletal, membrane and 
extracellular matrix components at focal contacts. Cancer Rev 4: 18-78 
Burtin P, Fondaneche M-C (1988) Receptor for plasmin on human carcinoma 
cells. J Natl Cancer Inst 80: 762-765 
Carpenter G, Cohen S (1976) J Cell BIol 71: 159-171 
Chomczynski P, Sacchi, N (1987) Single-step method of RNA isolation by acid 
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 
156-159. 
Collen D, Zamarron C, Lijnen H R, Hoylaerts M (1986) Activation of 
plasminogen by pro-urokinase. II. Kinetics. J Biol Chem 261: 1259-1266 
Corsaro C M, Pearson M L (1981) Enhancing the efficiency of DNA-mediated 
gene transfer in mammalian cells. Somat. Cell Gen. 7: 603-616 
Cox, K H, DeLeon D V, Angerer L M, Angerer R C (1984) Detection of mRNAs in 
Sea Urchin Embryos by in Situ Hybridization Using Asymmetric RNA Probes. 
Develop Biol 101: 485-502 
Cubellis M V, Nolli M L, Cassani G, Blasi F (1986) Binding of single-chain 
pro-urokinase to the urokinase receptor of human U937 cells. J Biol Chem 
261: 15819-15822 
Cubellis M V, Andreasen P A, Ragno P, Mayer M, Dan.o slashed. K, Blasi F 
(1989) Proc Natl Acad Sci USA 86: 4828-4830 
Dan.o slashed. K, Andreasen P A, Gr.o slashed.ndahl-Hansen J, Kristensen P, 
Nielsen L S, Skriver L (1985) Plasminogen activators, tissue degradation 
and cancer. Adv Cancer Res 44: 139-266 
Dan.o slashed. K, Nielsen L S, Pyke C and Kellermann, G M (1988) 
Plasminogen activators and neoplasia. In: Tissue-Type Plasminogen 
Activator (t-PA): Physiological and Clinical Aspects. C. Kluft, ed., CRC 
Press, Boca Raton. 1988, pp. 19-46 
de Duve C, du Barsy T, Poole B, Trouet A, Tulkens P, Van Hoof F (1974) 
Biochem Pharmacol 23: 2495-2531 
Eaton D L, Scott R W, Baker J B (1984) Purification of human fibroblast 
urokinase proenzyme and analysis of its regulation by proteases and 
protease nexin. J Biol Chem 259: 6241-6247 
Ellis V, Scully M F, Kakkar V V (1987) Plasminogen activation by 
single-chain urokinase in functional isolation. J Biol Chem 262: 
14998-15003 
Ellis V, Scully M F, Kakkar V V (1988) Role of human U937 monocytes in 
controlling single-chain urokinase-initiated plasminogen activation. 
Fibrinolysis 2: supp. 1, 112 
Ellis V, Scully M F, Kakkar V V (1989) Plasminogen aktivation initiated by 
single-chain urokinase-type plasminogen activator. J Biol Chem 264, 
2185-88 
Estreicher A, Wohlwend A, Belin D, Schleuning W-D, Vassalli J-D (1989) 
Characterization of the cellular binding site for the urokinase-type 
plasminogen activator. J Biol Chem 264: 1180-1189 
Ferguson and Williams (1988) Cell surface anchoring of proteins via 
glycosyl-phosphatidyl inositol structures. Ann Rev Biochem 57: 285-320 
Genton C, Kruithof E K O, Schleuning W-D (1987) J Cell Biol 104: 705-712 
Gr.o slashed.ndahl-Hansen J, Agerlin N, Munkholm-Larsen P, Bach F, Nielsen 
L S, Dombernowsky P, Dan.o slashed. K (1988) Sensitive and specific 
enzyme-linked immunosorbent assay for urokinase-type plasminogen activator 
and its application to plasma from patients with breast cancer. J Lab Clin 
Med 111: 42-51 
Gr.o slashed.ndahl-Hansen J, Lund L R, Ralfk.ae butted.r E, Ottevanger V, 
Dan.o slashed. K (1988) Urokinase- and tissue-type plasminogen activators 
in keratinocytes during wound reepithelialization in vivo. The Journal of 
Investigative Dermatology 90: 790-795 
Gurewich V, Pannell R, Louie S, Kelley P, Suddith R L, Greenlee R (1984) 
Effective and fibrin-specific clot lysis by a zymogen precursor form of 
urokinase (pro-urokinase). A study In Vitro and in two animal species. J 
Clin Invest 73: 1731-1739 
Haigler H T, Maxfield F R, Willingham M C, Pastan I (1980) J Biol Chem 255: 
1239-1241 
Hajjar K A, Harpel P C, Jaffe E A, Nachman R L (1986) Binding of 
plasminogen to cultured human endothelial cells. J Biol Chem 261: 
11656-11662 
Hajjar K A, Nachmann R L (1988) Assembly of the fibrinolytic system on 
cultured endothelial cells. Fibrinolysis 2, Suppl 1: 118 
Hashimoto F, Horigome T, Kanbayashi M, Yoshida K, Sugano H (1983) An 
improved method for separation of low-molecular-weight polypeptides by 
electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem 
129: 192-199 
Hearing V J, Law L W, Corti A, Appella E, Blasi F (1988) Modulation of 
metastatic potential by cell surface urokinase of murine melanoma cells. 
Cancer Res 48: 1270-1278 
Hebert C A, Baker J B (1988) Linkage of extracellular plasminogen activator 
to the fibroblast cytoskeleton: Colocalization of cell surface urokinase 
with vinculin. J Cell Biol 106: 1241-1247 
Heukeshoven J, Dernick R (1988) Improved silver staining procedure for fast 
staining in Phast System Development Unit. Electrophoresis 9: 28-32 
Hopp T P, Woods K R (1981) Prodiction of protein antigenic determinants 
from amino acid sequences. Proc. Natl. Acad. Sci. USA 78: 3824-3828 
Hoylaerts M, Rijken D C, Lijnen H R, Collen D (1982) Kinetics of the 
activation of plasminogen by human tissue plasminogen activator. Role of 
fibrin. J Biol Chem 257: 2912-2919 
Janicke F, Schmitt M, Hafter A, Hollrieder A, Babic R, Ulm K, Gossner W, 
Graeff H (1990) Urokinase-type plasminogen activator (u-PA) antigen is a 
predictor of early relapse in breast cancer. Fibrinolysis 1-10 
Janicke F, Schmitt M, Ulm K, Gossner W, Graeff H (1989) Urokinase-type 
plasminogen activator antigen and early relapse in breast cancer. The 
Lancet, 1049 
Kasai S, Arimura H, Nishida M, Suyama T (1985) Proteolytic cleavage of 
single-chain pro-urokinase induces conformational change which follows 
activation of the zymogen and reduction of its high affinity for fibrin. J 
Biol Chem 260: 12377-12381 
Kielberg V, Andreasen P A, Gr.o slashed.ndahl-Hansen J, Nielsen L S, 
Skriver L, Dan.o slashed. K (1985) Proenzyme to urokinase-type plasminogen 
activator in the mouse In vivo. FEBS Lett 182: 441-445 
Kristensen P, Larsson L-I, Nielsen L S, Grondahl-Hansen J, Andreasen P A, 
Dan.o slashed. K (1984) Human endothelial cells contain one type of 
plasminogen activator. FEBS Lett 168: 33-37 
Kristensen P, Pyke C, Lund L R, Andreasen P A, Dan.o slashed. L (1990) 
Plasminogen activator inhibitor type 1 in Lewis lung carcinoma. 
Histochemistry. In press. 
Kozak M (1987) An analysis of 5'-noncoding sequences from 699 vertebrate 
messenger RNAs. Nucl. Ac. Res. 15: 8125-8132 
Kuiper J, Otter M, Rijken D C, van Berkel T J C (1988) In vivo interaction 
of tissue-type plasminogen activator with rat liver cells. Fibrinolysis 2, 
Suppl 1: 28 
Kyte J, Doolittle R F (1982) A simple method for displaying the hydropathic 
character of a protein. J. Mol. Biol 157: 105-132 
Laemmli U K (1970) Cleavage of the structural proteins during the assembly 
of the head of bacteriophage T4. Nature 227: 680-685 
Lijnen H R, Zamarron C, Blaber M, Winkler H E, Collen D (1986) Activation 
of plasminogen by pro-urokinase. I. Mechanism. J Biol Chem 261: 1253-1258 
Liotta L A (1986) Tumor invasion and metastases--role of the extracellular 
matrix: Rhoads Memorial Award Lecture. Cancer Res. 46: 1-7 
Low M G (1989) The glycosyl-phosphatidylinositol anchor of membrane 
proteins. Biochim Biophys Acta 988: 427-454 
Lund L R, Riccio A, Andreasen P A, Nielsen L S, Kristensen P, Laiho M, 
Saksela 0, Blasi F, Dan.o slashed. K (1987) Transforming growth 
factor-.beta. is a strong and fast acting positive regulator of the level 
of type-1 plasminogen activator inhitor mRNA in WI-38 human lung 
fibroblasts. EMBO J 6: 1281-1286 
Lund L R, Georg B, Nielsen L S, Mayer M, Dan.o slashed. K, Andreasen P 
(1988) Mol Cell Endocrinol 60: 43-53 
Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring 
Harbor Laboratory 
Mann K G, Jenny R J, Krishnaswamy S (1988) Cofactor proteins in the 
assembly and expression of blood clotting enzyme complexes. Ann Rev 
Biochem 57: 915-956 
Matsudaira P (1987) Sequence from picomole quantities of proteins 
electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262: 
10035-10038 
Matsuo O, Tanaka S, Kikuchi H (1988) Effect of urinary trypsin inhibitor on 
osteoarthritis. Trombosis Research 52: 237-245 
Mayer M, Lund L R, Riccio A, Skouv J, Nielsen L S, Stacey S N, Dan.o 
slashed. K, Andreasen P A (1988) Plasminogen activator inhibitor type-1 
protein, mRNA and gene transcription are increased by phorbol esters in 
human rhabdomyosarcoma cells. J Biol Chem 263: 15688-15693 
Mignatti P, Robbins E, Rifkin D B (1986) Tumor invasion through the human 
amniotic membrane: Requirement for a proteinase cascade. Cell 47: 487-498 
Miles L A, Dahlberg C M, Plow E F (1988) The cell-binding domains of 
plasminogen and their function in plasma. J Biol Chem 263: 11928-11934 
Miles L A, Ginsberg M H, White J G, Plow E F (1986) Plasminogen interacts 
with human platelets through two distinct mechanisms. J Clin Invest 77: 
2001-2009 
Miles L A, Plow E F (1985) Binding and activation of plasminogen on the 
platelet surface. J Biol Chem 260: 4303-4311 
Miles L A, Plow E F (1986) Topography of the high-affinity lysine bindding 
site of plasminogen as defined with a specific antibody probe. 
Biochemistry 25: 6926-6933 
Miles L A, Plow E F (1987) Receptor mediated binding of the fibrinolytic 
components, plasminogen and urokinase, to peripheral blood cells. Thromb 
Haemostas 58: 936-942 
Morrissey J H, Falhrai H, Edgington T S (1987) Molecular cloning of the 
cDNA for tissue factor, the cellular receptor for the initiation of the 
coagulation protease cascade. Cell 50: 129-135 
Muller-Eberhard H J (1988) Molecular organization and function of the 
complement system. Ann Rev Biochem 57: 321-347 
Needham G K, Sherbet G V, Farndon J R, Harris A L (1987) Binding of 
urokinase to specific receptor sites on human breast cancer membranes. Eur 
J Cancer 55: 13-16 
Nelles L, Lijnen H R, Collen D, Holmes W E (1987) Characterization of 
recombinant human single chain urokinase-type plasminogen activator 
mutants produced by site-specific mutagenesis of lysine 158. J Biol Chem 
262: 5682-5689 
Nielsen L S, Hansen J G, Skriver L, Wilson E L, Kaltoft K, Zeuthen J, Dan.o 
slashed. K (1982) Purification of zymogen to plasminogen activator from 
human glioblastoma cells by affinity chromatography with monoclonal 
antibody. Biochemistry 24: 6410-6415 
Nielsen L S, Kellerman G M, Behrendt N, Picone R, Dan.o slashed. K, Blasi F 
(1988) A 55,000-60,000 M.sub.r receptor protein for urokinase-type 
plasminogen activator. J Biol Chem 263: 2358-2363 
Nielsen L S, Andreasen P A, Gr.o slashed.ndahl-Hansen J, Huang J-Y, 
Kristensen P, Dan.o slashed. K (1986) Thromb. Haemost. 55: 206-212 
Nolli M L, Sarubbi E, Corti A, Robbiati F, Soffientini A, Blasi F, Parenti 
F, Cassani C (1989) Production and characterization of human recombinant 
single chain urokinase-type plasminogen activator from mouse cells. 
Fibrinolysis 3: 101-106 
Okayama H, Berg P (1983) A cDNA cloning vector that permits expression of 
cDNA inserts in mammalian cells. Mol Cell Biol 3: 280-289 
Ossowski L (1988) Plasminogen activator dependent pathways in the 
dissemination of human tumor cells in the chick embryo. Cell 52: 321-328 
Ossowski L, Reich E (1983) Antibodies to plasminogen activator inhibit 
human tumor metastasis. Cell 35: 611-619 
Pannell R, Gurewich V (1987) Activation of plasminogen by single-chain 
urokinase or by two-chain urokinase--a demonstration that single-chain 
urokinase has a low catalytic activity (pro-urokinase). Blood 69: 22-26 
Petersen L C, Lund L R, Nielsen L S, Dan.o slashed. K, Skriver L (1988) 
One-chain urokinase-type plasminogen activator from human sarcoma cells is 
a proenzyme with little or no intrinsic activity. J Biol Chem 263: 
11189-11195 
Picone R, Kajtaniak E L, Nielsen L S, Behrendt N, Mastronicola M R, 
Cubellis M V, Stoppelli M P, Pedersen S, Dan.o slashed. K, Blasi F (1989) 
Regulation of urokinase receptors in monocyte-like U937 cells by phorbol 
ester PMA. J Cell Biol 108: 693-702 
Ploug M, Jensen A L, Barkholt V (1989) Determination of amino acid 
compositions and NH2-terminal sequences of peptides electroblotted onto 
PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel 
electrophoresis: Application to peptide mapping of human complement 
component C3. Anal Biochem 181: 33-39. 
Plow E F, Freaney D E, Plescia J, Miles L A (1986) The plasminogen system 
and cell surfaces: evidence for plasminogen and urokinase receptors on the 
same cell type. J Cell Biol 103: 2411-2420 
Pollanen J, Hedman K, Nielsen L S, Dan.o slashed. K, Vaheri A (1988) 
Ultrastructural localization of plasma membrane-associated urokinase-type 
plasminogen activator at focal contacts. J Cell Biol 106: 87-95 
Pollanen J, Saksela O, Salonen E-M, Andreasen P, Nielsen L S, Dan.o 
slashed. K, Vaheri A (1987) Distinct localizations of urokinase-type 
plasminogen activator and its type-1 inhibitor under cultured human 
fibroblasts and sarcoma cells. J Cell Biol 104: 1085-1096 
Ponte P, Gunning P, Blau H, Kedes L (1983) Human actin genes are single 
copy for a-cardiac actin, but multicopy for .beta.- and 
.alpha.-cytoskeletal genes: 3'-untranslated regions are isotype specific 
but are conserved in evolution. Mol Cell Biol 3: 1783-1791 
Pozzatti R, Muscel R, Williams S J, Padmanabhan R, Howard B, Liotta L, 
Khoury G (1986) Primary rat embryo cells transformed by one or two 
oncogenes show different metastatic potential. Science 232: 223-227 
Reich R, Thompson E, Iwamoto Y, Martin G R, Deason J R, Fuller G C, Miskin 
R (1988) Inhibition of plasminogen activator, serine proteinases and 
collagenase IV prevents the invasion of basement membranes by metastatic 
cells. In press 
Russell D W, Schneider W J, Yamamoto T, Luskey K L, Brown M S, Goldstein J 
L (1984) Domain map of the LDL receptor: sequence homology with the 
epidermal growth factor precursor. Cell 37: 577-585 
Saksela O (1985) Plasminogen activation and regulation of pericellular 
proteolysis. Biochim Biophys Acta 823: 35-65 
Salonen E-M, Saksela O, Vartio T, Vaheri A, Nielsen L S, Zeuthen J (1985) 
Plasminogen and tissue-type plasminogen activator bind to immobilized 
fibronectin. J Biol Chem 260: 12302-12307 
Salonen E-M, Zitting A, Vaheri A (1984) Laminin interacts with plasminogen 
and its tissue-type activator. FEBS Lett 172: 29-32 
Schagger H, von Jagow G (1987) Tricine-sodium dodecyl 
sulfate-polyacrylamide gel electrophoresis for the separation of proteins 
in the range from 1-100 kDa. Anal Biochem 166: 368-379 
Selvaraj P, Rosse W, Silber R, Springer T A (1988) The major Fc receptor in 
blood has a phosphatidylinositol anchor and is deficient in paroxysmal 
nocturnal haemoglobinuria. Nature 333: 565-567 
Silverstein R L, Leung L L K, Harpel P C, Nachman P (1984) Complex 
formation of platelet thrombospondin with plasminogen. Modulation of 
activation by tissue activator. J Clin Invest 74: 1625-1633 
Skriver L, Larsson L-I, Kielberg V, Nielsen L S, Andresen P B, Kristensen 
P, Dan.o slashed. K (1984) Immunocytochemical localization of 
urokinase-type plasminogen activator in Lewis lung carcinoma. J Cell Biol 
99: 753-758 
Skriver L, Nielsen L S, Stephens R, Dan.o slashed. K (1982) Plasminogen 
activator released as inactive proenzyme from murine cells transformed by 
sarcoma virus. Eur J Biochem 124: 409-414 
Stephens R W, Alitalo R, Tapiovaara H, Vaheri A (1988) Production of an 
active urokinase by leukemia cell lines: a novel distinction from cell 
lines of solid tumors. Leukemia Res 12: 419-422 
Stephens R W, Fordham C J, Doe W F (1987) Proenzyme to urokinase-type 
plasminogen activator in human colon cancer: in vitro inhibition by 
monocyte minactivin after proteolytic activation. Eur J Cancer Clin Oncol 
23: 213-222 
Stephens R W, Leung K-C, Pollanen J, Salonen E-M, Vaheri A (1987) 
Microplate immunocapture assay for plasminogen activators and their 
specific inhibitors. J Immunol Meth 105: 245-251 
Stoppelli M P, Corti A, Soffientini A, Cassani G, Blasi F, Assoian R K 
(1985) Differentiation-enhanced binding of the amino-terminal fragment of 
human urokinase plasminogen activator to a specific receptor on U937 
monocytes. Proc Natl Acad Sci USA 82: 4939-4943 
Stoppelli M P, Tacchetti C, Cubellis M V, Corti A, Hearing V J, Cassani G, 
Appella E, Blasi F (1986) Autocrine saturation of pro-urokinase receptors 
on human A431 cells. Cell 45: 675-684 
Stump D C, Lijnen H R, Collen D (1986a) Purification and characterization 
of single-chain urokinase-type plasminogen activator from human cell 
cultures. J Biol Chem 261: 1274-1278 
Stump D C, Thienpont M, Collen D (1986b) Urokinase-related proteins in 
human urine. J Biol Chem 261: 1267-1273 
Tarentino A L, Gomez C L, Plummer T H (1985) Deglycosylation of 
asparagine-linked glycans by Peptide-N-Glycosidase F. Biochemistry 24: 
4665-4671 
Thorsen S, Glas-Greenwalt P, Astrup T (1972) Differences in the binding to 
fibrin of urokinase and tissue plasminogen activator. Thrombos Diathes 
Haemorrh 28: 65-74 
Tryggvason K, Hoyhtya M, Salo T (1987) Proteolytic degradation of 
extracellular matrix in tumor invasion. Biochim Biophys Acta 907: 191-217 
Urano T, de Serrano V S, Gaffney P J, Castellino F J (1988) The activation 
of human (Glu.sup.1)plasminogen by human single-chain urokinase. Arch 
Biochem Biophys 264: 222-230 
Vassalli J-D, Hamilton J, Reich E (1977) Macrophage plasminogen activator: 
induction by concanavalin A and phorbol myristate acetate. Cell 11: 
695-705 
Vassalli J-D, Baccino D, Belin D (1985) A cellular binding site for the 
M.sub.r 55,000 form of the human plasminogen activator, urokinase. J Cell 
Biol 100: 86-92 
Vassalli J-D, Dayer J-M, Wohlwend A, Belin D (1984) Concomitant secretion 
of prourokinase and of a plasminogen activator-specific inhibitor by 
cultured human monocytes-macrophages. J Exp Med 159: 1652-1668 
Wun T-C, Ossowski L, Reich E (1982) A proenzyme form of human urokinase. J 
Biol Chem 157: 7262-7268 
Wun T-C, Reich E (1987) An inhibitor of plasminogen activation from human 
placenta. J Biol Chem 262: 3646-3653 
Yarden Y, Ullrich A (1988) Molecular analysis of signal transduction by 
growth factors. Biochemistry 27: 3113-3119 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 21 
(2) INFORMATION FOR SEQ ID NO:1: 
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(A) LENGTH: 16 amino acids 
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(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
LeuXaaXaaMetGlnXaaLysThrAsnGlyAspXaaArgValGluGlu 
151015 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 amino acids 
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(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
LeuXaaCysMetGlnCysLysThrAsnGlyAspCysArgValGluGlu 
151015 
HisAlaLeuGlyGlnXaaLeuXaaArgThrThrIleValXaa 
202530 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 92 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LeuArgCysMetGlnCysLysThrAsnGlyAspCysArgValGluGlu 
151015 
CysAlaLeuGlyGlnAspLeuCysArgThrThrIleValArgLeuTrp 
202530 
GluGluGlyGluGluLeuGluLeuValGluLysSerCysThrHisSer 
354045 
GluLysThrAsnArgThrLeuSerTyrArgThrGlyLeuLysIleThr 
505560 
SerLeuThrGluValValCysGlyLeuAspLeuCysAsnGlnGlyAsn 
65707580 
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8590 
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(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 99 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
LeuGluCysIleSerCysGlySerSerAspMetSerCysGluArgGly 
151015 
ArgHisGlnSerLeuGlnCysArgSerProGluGluGlnCysLeuAsp 
202530 
ValValThrHisTrpIleGlnGluGlyGluGluGlyArgProLysAsp 
354045 
AspArgHisLeuArgGlyCysGlyTyrLeuProGlyCysProGlySer 
505560 
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65707580 
ThrThrLysCysAsnGluGlyProIleLeuGluLeuGluAsnLeuPro 
859095 
GlnAsnGly 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 90 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ArgGlnCysTyrSerCysLysGlyAsnSerThrHisGlyCysSerSer 
151015 
GluGluThrPheLeuIleAspCysArgGlyProMetAsnGlnCysLeu 
202530 
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354045 
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505560 
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65707580 
AsnHisProAspLeuAspValGlnTyrArg 
8590 
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(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GCCAGACTGTGGGGAGGCACTCTCCTCTGGACCTAA36 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AlaArgLeuTrpGlyGlyThrLeuLeuTrpThr 
1510 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CCANNNNNTGG11 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
AGAGT5 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ACAGT5 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
AGACT5 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
ACTGT5 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CTAGTCTAGACTAG14 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
AGACTCTAGTCTAGACTAGACTGT24 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GACCTGGATATCCAGTA17 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
GluProGlyAlaAlaThrLeuLysSerValAlaLeuProPheAlaIle 
151015 
AlaAlaAlaAlaLeuValAlaAlaPhe 
2025 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CysLysAspSerSerIleValLeuThrLysLysPheAlaLeuThrVal 
151015 
ValSerAlaAlaPheValAlaLeuLeuPhe 
2025 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
ThrThrAspAlaAlaHisProGlyArgSerValValProAlaLeuLeu 
151015 
ProLeuLeuAlaGlyThrLeuLeuLeuLeuGluThrAlaThrAlaPro 
202530 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
ValSerAlaSerGlyThrSerProGlyLeuSerAlaGlyAlaThrVal 
151015 
GlyIleMetIleGlyValLeuValGlyValAlaLeuIle 
2025 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
ValLysCysGlyGlyIleSerLeuLeuValGlnAsnThrSerTrpLeu 
151015 
LeuLeuLeuLeuLeuSerLeuSerPheLeuGlnAlaThrAspLysIle 
202530 
SerLeu 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
TyrArgSerGlyAlaAlaProGlnProGlyProAlaHisLeuSerLeu 
151015 
ThrIleThrLeuLeuMetThrAlaArgLeuTrpGlyGlyThrLeuLeu 
202530 
TrpThr 
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