U.S. patent application number 13/073946 was filed with the patent office on 2011-10-06 for compound and method for regulating plasminogen activation and cell migration.
This patent application is currently assigned to TRANSFERT PLUS. Invention is credited to Richard Beliveau, Yanick Bertrand, Michel Demeule, Julie Jodoin, Jonathan Michaud-Levesque, Yanneve Rolland.
Application Number | 20110243952 13/073946 |
Document ID | / |
Family ID | 33435212 |
Filed Date | 2011-10-06 |
United States Patent
Application |
20110243952 |
Kind Code |
A1 |
Beliveau; Richard ; et
al. |
October 6, 2011 |
COMPOUND AND METHOD FOR REGULATING PLASMINOGEN ACTIVATION AND CELL
MIGRATION
Abstract
The invention relates to novel regulators of plasminogen
activation and their use for regulating cell migration,
plasminolysis, angiogenesis, fibrinolysis, for treating cancer and
thrombo-embolic diseases such as heart stroke. Furthermore, the
present invention relates to novel pharmaceutical compositions form
regulating cell migration, plasminolysis, angiogenesis and for
treating cancer. In particular, the present invention relates to a
method of regulating the activation of plasminogen comprising
contacting a solution of pro-urokinase (uPA) or tissue plasminogen
activator (tPA) and plasminogen with melanotransferrin (p97) for a
time sufficient to effect regulation thereof.
Inventors: |
Beliveau; Richard;
(Montreal, CA) ; Demeule; Michel; (Longueuil,
CA) ; Bertrand; Yanick; (Longueuil, CA) ;
Michaud-Levesque; Jonathan; (Montreal, CA) ; Rolland;
Yanneve; (Montreal, CA) ; Jodoin; Julie;
(Montreal, CA) |
Assignee: |
TRANSFERT PLUS
Montreal
CA
|
Family ID: |
33435212 |
Appl. No.: |
13/073946 |
Filed: |
March 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10556145 |
Aug 21, 2006 |
7919103 |
|
|
PCT/CA04/00697 |
May 7, 2004 |
|
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13073946 |
|
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60469000 |
May 9, 2003 |
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Current U.S.
Class: |
424/152.1 ;
424/94.1; 514/13.3 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/2881 20130101; C07K 2317/76 20130101; A61P 9/00 20180101;
C07K 2317/92 20130101; A61K 38/40 20130101; C07K 16/30
20130101 |
Class at
Publication: |
424/152.1 ;
514/13.3; 424/94.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 38/17 20060101 A61K038/17; A61K 38/43 20060101
A61K038/43; A61P 9/00 20060101 A61P009/00 |
Claims
1. A pharmaceutical composition comprising a therapeutically
effective amount of melanotransferrin (p97) or an enzymatically
active fragment thereof, unconjugated to any moiety, in association
with a pharmaceutically acceptable carrier.
2. A method of regulating angiogenesis, comprising administering to
an individual a pharmaceutically effective amount of a
pharmaceutical composition according to claim 1.
3. The method according to claim 2, wherein said administering is
carried out orally, parenterally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, intraarterially, transdermally
or via a mucus membrane.
4. A method for inhibiting angiogenesis caused by cells expressing
melanotransferrin (p97) at their surface, said method comprising
the step of administering to a patient in need thereof exogenous
soluble p97 or an active fragment thereof, said soluble p97 or
active fragment thereof being unconjugated to any moiety and
competing with the p97 expressed on the cell surface, activating
plasminogen in solution instead of membrane-bound plasminogen, thus
preventing cell migration and preventing angiogenesis.
5. The method of claim 4, wherein said cell is an endothelial
cell.
6. The method of claim 4, wherein said cell is selected from the
group consisting of human vascular or microvascular endothelial
cells.
7. A method of inhibiting angiogenesis, comprising administering to
an individual a therapeutically effective amount of a
pharmaceutical composition comprising one of melanotransferrin
(P97) or an active fragment thereof, said melanotransferrin or
fragment thereof being unconjugated to any moiety, in association
with a pharmaceutically acceptable carrier.
8. The method according to claim 7, wherein said administering is
carried out orally, parenterally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, intraarterially, transdermally
or via a mucus membrane.
Description
[0001] This application is a Divisional of co-pending U.S.
application Ser. No. 10/556,145 filed on Aug. 21, 2006 and claims
priority under 35 U.S.C. .sctn.120. U.S. application Ser. No.
10/556,145 is the national phase under 35 U.S.C. .sctn.371 of
International Application No. PCT/CA2004/000697 filed May 7, 2004
in Canada.
BACKGROUND OF THE INVENTION
[0002] (a) Field of the Invention
[0003] The invention relates to novel regulators of plasminogen
activation and their use for regulating cell migration and treating
cancer. Furthermore, the present invention relates to novel
pharmaceutical compositions form regulating cell migration and
treating cancer.
[0004] (b) Description of the Prior Art
[0005] Melanotransferrin (p97) possesses a high level of homology
(37-39%) with human serum transferrin, human lactoferrin and
chicken transferrin. It is a glycosylated protein that reversibly
binds iron and was first found at high levels in malignant melanoma
cells. Two forms of p97 have been reported, one of which is bound
to cell membranes by a glycosylphosphatidylinositol anchor while
the other form is both soluble and actively secreted. The exact
physiological role of either membrane-bound p97 or secreted p97 is
largely unexplored.
[0006] In the early 1980s, p97 was found to be expressed in much
larger amounts in neoplastic cells and fetal tissues than in normal
tissues. More recently, it was reported that p97 mRNA is widespread
in normal human tissues. p97 is also expressed in reactive
microglia associated with amyloid plaques in Alzheimer's disease.
Normal serum contains very low levels of p97, which were reported
to increase by 5- to 6-fold in patients with Alzheimer's
disease.
[0007] It was previously demonstrated that recombinant human
melanotransferrin (p97) is transported at high rate into the brain
using both an in vitro model of the blood brain barrier (BBB) and
in situ mouse brain perfusion (Demeule M, et al., 2002 J Neurochem
83:924-933). It was also shown that p97 transcytosis might involve
the low-density lipoprotein related protein (LRP). This receptor is
also known to mediate the internalization of the
urokinase:plasminogen activator inhibitor:urokinase receptor
complex (uPA:PAI-1:uPAR). Briefly, single-chain proenzyme-uPA is
activated upon binding to its cell surface receptor uPAR, which is
a glycosylphosphatidylinositol (GPO-anchored membrane protein.
After its activation, uPA (which catalyzes the conversion of
plasminogen to plasmin) is quickly inhibited by the plasminogen
activator inhibitor type-1 (PAI-1). The inactive uPA:PAI-1 complex
binds to uPAR and then is rapidly internalized by LRP. The
uPA:PAI-1 complex is degraded in lysosomes whereas the uPAR is
recycled at the cell surface. Other LRP ligands include pro-uPA,
PAI-1, receptor-associated protein (RAP) and a diverse spectrum of
structurally unrelated proteins.
[0008] Heart disease has topped the list of killer diseases every
year but one since 1900. (The exception was 1918, when an influenza
epidemic killed more than 450,000 Americans.) Stroke is the third
leading cause of death in the United States, following cancer. Much
of the progress is due to the development of effective medicines to
control blood pressure and cholesterol, according to officials of
the National Heart, Lung and Blood Institute. But, experts warn,
the war against heart disease and stroke is not yet won. Every 33
seconds, an American dies of either heart disease or stroke. Nearly
62 million Americans have one or more types of cardiovascular
disease, and these diseases cost our society more than $350 billion
a year.
[0009] Two strategies are presently used to restore the flow after
thrombosis: 1) clot dissolution with administration of plasminogen
activators and 2) clot permeation by surgical intervention. The
tissue-type plasminogen activator (tPA) and its conventional
substrate plasminogen, are key players involve in fibrinolysis.
Currently, tPA is used as a stroke therapy, however, its associated
adverse effects might limit its efficiency.
[0010] It would be highly desirable to be provided with novel
regulators of plasminogen activation and their use for regulating
cell migration and treating cancer.
[0011] It would also be highly desirable to be provided with novel
pharmaceutical compositions form regulating cell migration and
treating cancer.
[0012] It would be highly desirable to be provided with a new
treatment for thrombo-embolic disorders such as venous or arterial
thrombosis, thrombophlebitis, pulmonary and cerebral embolism,
thrombotic microangiopathy and intravascular clotting. Some of
these disorders will lead for example in heart and cerebral
strokes.
[0013] It would be also desirable to be provided with a new method
for increasing fibrinolysis or for preventing angiogenesis.
SUMMARY OF THE INVENTION
[0014] One aim of the present invention is to provide novel
regulators of plasminogen activation and their use for regulating
cell migration and treating cancer.
[0015] Another aim of the present invention is to provide novel
pharmaceutical compositions form regulating cell migration and
treating cancer.
[0016] A further aim of the present invention is to provide a new
treatment for thromboembolic disorders such as, for example,
without limitation, venous or arterial thrombosis,
thrombophlebitis, pulmonary or cerebral embolism, thrombotic
microangiopathy or intravascular clotting, some of which will lead
for example in heart or cerebral strokes.
[0017] An additional aim of the present invention is to provide a
new method for increasing fibrinolysis or for preventing
angiogenesis.
[0018] In accordance with one embodiment of the present invention
there is provided a method for increasing plasminogen activation,
said method comprising contacting a solution containing
pro-uroquinase plasminogen activator (pro-uPA) with
melanotransferrin (p97) or an enzymatically active fragment thereof
for a time sufficient to increase plasminogen activation.
[0019] In a preferred embodiment, p97 increase plasminogen
activation and fibrinolysis through tissue plasminogen activator
(t-PA).
[0020] In accordance with another embodiment of the present
invention there is provided a method for inhibiting plasminogen
activation, said method comprising the step of contacting
pro-uroquinase plasminogen activator (pro-uPA) with membrane bound
melanotransferrin (p97) for a time sufficient to prevent
plasminogen activation.
[0021] In accordance with a further embodiment of the invention,
there is provided a method for preventing cell migration, said
method comprising the step of contacting a cell expressing
melanotransferrin (p97) on its surface with exogenous soluble 97 or
an antibody, or an antigen binding fragment thereof, directed to
said p97 expressed on the surface of said cell, said soluble p97
competing with the p97 expressed on the cell surface, activating
plasminogen in solution instead of membrane-bound plasminogen, thus
preventing cell migration, said antibody, or active fragment
thereof binding p97 on the surface of the cell thus preventing
activation of membrane-bound plasminogen, preventing cell
migration.
[0022] In a preferred embodiment of the invention, the antibody is
a monoclonal antibody, and more preferably one of L235, HybC, HybE,
HybF, 9B6 or 2C7.
[0023] The cell can be for example, without limitation, an
endothelial cell or a tumor cell, such as one selected from the
group consisting of human vascular or microvascular endothelial
cells such as HMEC-1 and human melanoma cells such as SK-MEL28
cells.
[0024] Still in accordance with the present invention, there is
provided a method for treating cancer caused by cells expressing
melanotransferrin (p97) at their surface, said method comprising
the step of administering to a patient in need thereof exogenous
soluble p97 or an antibody an antibody, or active fragment thereof,
directed to said p97 expressed on the surface of said cell, said
soluble p97 competing with the p97 expressed on the cell surface,
activating plasminogen in solution instead of membrane-bound
plasminogen, thus preventing cell migration, said antibody, or
active fragment thereof binding p97 on the surface of the cell thus
preventing activation of membrane-bound plasminogen, preventing
cell migration, preventing cancer cells from spreading.
[0025] Further in accordance with the present invention, there is
provided a method for regulating capillary tube formation, said
method comprising the step administering to a patient in need
thereof soluble 97, wherein said soluble p97 prevents or reduces
capillary tube formation.
[0026] Also in accordance with the present invention, there is
provided a pharmaceutical composition for use in regulating
activation of plasminogen, said composition comprising a
therapeutically effective amount of melanotransferrin (p97) or an
enzymatically active fragment thereof in association with a
pharmaceutically acceptable carrier.
[0027] Preferably, p97 is soluble p97 for increasing activation of
plasminogen.
[0028] In accordance with the present invention there is also
provided a method of regulating the activation of plasminogen,
comprising administering to an individual in need thereof a
therapeutically effective amount of the aforementioned
pharmaceutical composition.
[0029] In accordance with the present invention there is also
provided a pharmaceutical composition for use in regulating cell
migration of a cell showing p97 activity, comprising a
therapeutically effective amount of one of p97, an enzymatically
active fragment thereof, or an antibody recognizing specifically
p97, or an antigen binding fragment thereof, in association with a
pharmaceutically acceptable carrier.
[0030] Further in accordance with the present invention there is
also provided a method of regulating cell migration of a cell
showing p97 activity, comprising administering to an individual in
need thereof a therapeutically effective amount of the
aforementioned pharmaceutical composition.
[0031] In accordance with the present invention there is further
provided a pharmaceutical composition for treating cancer
comprising a therapeutically effective amount of one of
melanotransferrin (p97), an enzymatically active fragment thereof,
or an antibody recognizing specifically p97, or an antigen binding
fragment thereof, in association with a pharmaceutically acceptable
carrier.
[0032] Also in accordance with the present invention there is
further provided a method of treating cancer, comprising
administering to an individual a therapeutically effective amount
of the aforementioned pharmaceutical composition.
[0033] The cancer can be, for example, without limitation, selected
from the group consisting of melanoma, prostate cancer, leukemia,
hormone dependent cancer, breast cancer, colon cancer, lung cancer,
skin cancer, ovarian cancer, pancreatic cancer, bone cancer, liver
cancer, biliary cancer, urinary organ cancer (for example, bladder,
testis), lymphoma, retinoblastoma, sarcoma, epidermal cancer, liver
cancer, esophageal cancer, stomach cancer, cancer of the brain and
cancer of the kidney.
[0034] In accordance with the present invention there is also
provided a pharmaceutical composition for use in regulating
angiogenesis comprising a therapeutically effective amount of
melanotransferrin (p97) or an enzymatically active fragment thereof
in association with a pharmaceutically acceptable carrier.
[0035] Still in accordance with the present invention there is also
provided a method of regulating angiogenesis, comprising
administering to an individual a pharmaceutically effective amount
of the aforementioned pharmaceutical composition.
[0036] In accordance with the present invention, there is provided
the use of p97, or an enzymatically active fragment thereof, or of
any of the aforementioned composition for the various uses
described herein or for the manufacture of medication for the
various use described herein.
[0037] For the purpose of the present invention the following terms
are defined below.
[0038] The term "p97" is also referred to in the present invention
as Melanotransferrin, MTf, or P97. All of these terms are being
used interchangeably. The term soluble p97 thus make reference to
soluble p97 or soluble melanotransferrin.
[0039] The term "cancer" is intended to mean any cellular
malignancy whose unique trait is the loss of normal controls which
results in unregulated growth, lack of differentiation and ability
to invade local tissues and metastasize. Cancer can develop in any
tissue of any organ. More specifically, cancer is intended to
include, without limitation, melanoma, prostate cancer, leukemia,
hormone dependent cancers, breast cancer, colon cancer, lung
cancer, skin cancer, ovarian cancer, pancreatic cancer, bone
cancer, liver cancer, biliary cancer, urinary organ cancers (for
example, bladder, testis), lymphomas, retinoblastomas, sarcomas,
epidermal cancer, liver cancer, esophageal cancer, stomach cancer,
cancer of the brain and cancer of the kidney. Cancer is also
intended to include, without limitation, metastasis, whether
cerebral, pulmonary or bone metastasis, from various types of
cancers, such as melanomas, or from any types of cancer mentioned
above.
[0040] The terms "treatment", "treating" and the like are intended
to mean obtaining a desired pharmacologic and/or physiologic
effect, e.g., inhibition of cancer cell growth. The effect may be
prophylactic in terms of completely or partially preventing a
disease or symptom thereof and/or may be therapeutic in terms of a
partial or complete cure for a disease and/or adverse effect
attributable to the disease. "Treatment" as used herein covers any
treatment of a disease in a mammal, particularly a human, and
includes: (a) inhibiting the disease, (e.g., arresting its
development); or (B) relieving the disease (e.g., reducing symptoms
associated with the disease).
[0041] The term "administering" and "administration" is intended to
mean a mode of delivery including, without limitation, oral,
rectal, parenteral, subcutaneous, intravenous, intramuscular,
intraperitoneal, intraarterial, transdermally or via a mucus
membrane. The preferred one being orally. One skilled in the art
recognizes that suitable forms of oral formulation include, but are
not limited to, a tablet, a pill, a capsule, a lozenge, a powder, a
sustained release tablet, a liquid, a liquid suspension, a gel, a
syrup, a slurry, a suspension, and the like. For example, a daily
dosage can be divided into one, two or more doses in a suitable
form to be administered at one, two or more times throughout a time
period.
[0042] The term "therapeutically effective" is intended to mean an
amount of a compound sufficient to substantially improve some
symptom associated with a disease or a medical condition. For
example, in the treatment of cancer, a compound which decreases,
prevents, delays, suppresses, or arrests any symptom of the disease
would be therapeutically effective. A therapeutically effective
amount of a compound is not required to cure a disease but will
provide a treatment for a disease such that the onset of the
disease is delayed, hindered, or prevented, or the disease symptoms
are ameliorated, or the term of the disease is changed or, for
example, is less severe or recovery is accelerated in an
individual.
[0043] The compounds of the present invention may be used in
combination with either conventional methods of treatment and/or
therapy or may be used separately from conventional methods of
treatment and/or therapy.
[0044] When the compounds of this invention are administered in
combination therapies with other agents, they may be administered
sequentially or concurrently to an individual. Alternatively,
pharmaceutical compositions according to the present invention may
be comprised of a combination of a compound of the present
invention, as described herein, and another therapeutic or
prophylactic agent known in the art.
[0045] It will be understood that a specific "effective amount" for
any particular individual will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, and/or diet of the individual,
time of administration, route of administration, rate of excretion,
drug combination and the severity of the particular disease
undergoing prevention or therapy.
[0046] Pharmaceutically acceptable acid addition salts may be
prepared from inorganic and organic acids. Salts derived from
inorganic acids include hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like. Salts
derived from organic acids include citric acid, lactic acid,
tartaric acid, fatty acids, and the like.
[0047] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents (such as phosphate buffered saline
buffers, water, saline), dispersion media, coatings, antibacterial
and antifungal agents, isotonic and absorption delaying agents and
the like. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in therapeutic compositions is contemplated.
Supplementary active ingredients can also be incorporated into the
compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1A-1B illustrates that (1A) a significant (>50%)
reduction in the transport of [125I]-p97 (25 nM) from the apical
(blood side) to the basolateral side (brain side) of bovine brain
capillary endothelial cell (BBCEC) monolayers is observed in the
presence of 640 nM receptor-associated protein (RAP) and that (1B)
no interactions are observed between RAP and BSA proteins and p97,
suggesting that the inhibition of [.sup.125I]-p97 transcytosis is
not related to protein interactions between p97 and RAP;
[0049] FIG. 2 illustrates that various monoclonal antibodies to p97
are still able to recognize p97 following their immobilization to a
sensor chip surface, indicating that the p97 protein remains intact
following immobilization;
[0050] FIG. 3A-3B illustrates that (3A) sensor chip surface
immobilized p97 can interact with pro-uPA but no interaction can be
detected between p97 and PAI-1 or between p97 and tPA and that (3B)
plasminogen also interacts with immobilized p97 whereas plasmin and
angiostatin, two plasminogen fragments do not;
[0051] FIG. 4 illustrates the effects of p97 on pro-uPA, tPA and
plasminogen and shows that (A) VLK-pNA hydrolysis by pro-uPA
increases when p97 is added to the reaction; (B) p97 elicits no
observable effect on tPA; (C) the interaction of p97 with pro-uPA
does not result in the cleavage of either protein; and (D) p97
alters the cleavage of glu-plasminogen by pro-uPA;
[0052] FIG. 5 illustrates plasminogen activation by p97 and shows
that (A) VLK-pNA hydrolysis is 4-fold higher when p97 is added to
pro-uPA and plasminogen; (B) p97 stimulates plasminogen cleavage by
pro-uPA in a dose-dependent manner; (C) p97 positively affects the
activation of plasminogen by pro-uPA by increasing the catalytic
efficiency of pro-uPA; and (D) the effect of p97 upon pro-uPA's
activation of plasminogen is specific and involves the epitope
recognized by the mAb L235;
[0053] FIG. 6 illustrates the inhibition of cell migration by mAb
L235, an antibody to p97, and shows that (A) the presence of mAb
L235, inhibits the migration of HMEC-1 and SK-MEL28 cells but not
HUVEC cells; and (B) p97 is highly expressed in lysates from HMEC-1
and SK-MEL29 cells and at lower levels in their respective
conditioned culture media, but is almost undetectable in HUVEC
cells;
[0054] FIG. 7 illustrates that (A) exogenous p97 inhibits the
migration of MHEC-1 cells and (B) SK-MEL28 cells; and (C) the level
of inhibition is 50% and 70%, respectively at 100 nM p97 for those
same cells respectively;
[0055] FIG. 8 illustrates the inhibition of plasminolytic activity
at the cell surface by soluble p97 and mAb L235 and shows that (A)
100 nM p97 results in 95% inhibition of plasminogen activation in
HMEC-1 cells; and (B) mAb L235 results in more than 50% inhibition
of plasminolytic activity;
[0056] FIG. 9 illustrates the stimulatory effect of p97 on
plasminogenolytic activity of single chain urokinase plasminogen
activator (sc-uPA), uPA and tissue plasminogen activator (tPA) in
vitro;
[0057] FIG. 10 illustrates that low density lipoprotein related
protein (LRP) and the urokinase activator receptor (uPAR) are down
regulated in p97 treated HMEC-1 cells;
[0058] FIG. 11 illustrates that soluble p97 inhibits the
morphogenic differentiation of HMEC-1 (11A) and HUVEC (11B) into
capillary-like structures, when grown onto Matrigel-coated wells in
the presence or absence of soluble p97 (10 nM or 100 nM) as
described in the Materials and Methods sections hereinafter, the
length of the total capillary network being quantified after 18
hours using a map scale calculator by measuring and summing the
length of all tubular structures observed in a chosen field. The
results were expressed as the percentage of capillary-like tubes in
soluble p97-treated cells compared to untreated HMEC-1 and HUVEC
cells (11C);
[0059] FIG. 12 illustrates that soluble p97 inhibits HMEC-1 cell
migration (12A and 12B) without affecting cell adhesion (12C);
[0060] FIG. 13 illustrates that soluble p97 down-regulates u-PAR
(13A) and LRP protein (13B) expression;
[0061] FIG. 14 illustrates that soluble p97 unaffects the u-PAR/LRP
system mRNA expression;
[0062] FIG. 15 illustrates that soluble p97 modulates the cell
surface levels of u-PAR (15A) and LRP (15B) and binding of
125I-uPA.cndot.PAI-1 (15C) complex on the HMEC-1 cell surface;
[0063] FIG. 16 illustrates that soluble p97 up-regulates Cav-1 and
down-regulates pERK 1/2 (16D) protein expression and wherein the
level in control cells (16B) and ERK 1/2 (16C) was unchanged;
[0064] FIG. 17 illustrates that soluble p97 down-regulates eNOS
protein expression (17A) as well as VEGFR-2 and VEGF-A mRNA levels
(17B);
[0065] FIG. 18 is a schematic representation of soluble p97
treatment effects on the u-PAR/LRP system;
[0066] FIG. 19 illustrates that soluble p97 enhance cell detachment
(19A), plasminolytic activity (19B) and plasmin formation in
HEMEC-1 (19C);
[0067] FIG. 20 illustrates inhibition of cell detachment (20A) and
plasmin formation (20B) by inhibitors;
[0068] FIG. 21 illustrates that cell detachment stimulated by
soluble p97 induces degradation of fibronectin in HMEC-1;
[0069] FIG. 22 illustrates the interaction between p97 and
plasminogen using biospecific interaction analysis in
real-time;
[0070] FIG. 23 illustrates the effects of p97 interaction with
plasminogen (Plg) on tPA-dependant plasmin activity, and more
specifically demonstrates that the presence of p97 increases the
plasminogen activation (23A), that the induction caused by p97 of
the plasminogen activity is inhibited by the monoclonal antibody
directed against p97 (23B), the plasminolytic activity of tPA in
the presence of p97 (23C), and that soluble p97 decreases the
apparent K.sub.m of tPA for plasminogen (23D);
[0071] FIG. 24 illustrates fibrin clot permeation in the presence
of p97 (24A), the size increase of the perforation as a function of
soluble p97 concentration (24B), and the intrinsic fibrinolytic
activity of soluble p97 (24C);
[0072] FIG. 25 illustrates the effects of p97 on plasma clot
fibrinolysis by tPA;
[0073] FIG. 26 illustrates the effect of p97 on clot strength and
fibrinolysis, and more specifically of a thromboelastogram of a
fibrin clot model (26A) and of a plasma recalcified after addition
of 2 nM CaCl.sub.2 (26B);
[0074] FIG. 27 illustrates that L235 (27A) and soluble p97 (27B)
inhibited membrane bound p97-induced CHO cell invasion.
[0075] FIG. 28 illustrates transendothelial invasion across the
blood-brain barrier of CHO cells transfected with (mMTf-CHO cells)
or without (Mock-CHO cells) membrane bound p97; and
[0076] FIG. 29 illustrates that (29A) the interaction of pro-uPA
and plasminogen with soluble p97 increases the activation of
plasminogen; this induction can be inhibited by the mAb L235 which
recognizes a conformational epitope on p97; (29B) the addition of
mAb L235 reduces the plasminolytic activity on HMEC-1 cell surfaces
and results in an inhibition of cell migration and (29C) the
interaction of plasminogen and pro-uPA with membrane-bound p97 is
diminished when exogenous, competing human recombinant p97 is
added, which also results in a decrease in the activation of
plasminogen and leads to an inhibition of cell migration.
DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods
[0077] Soluble human recombinant p97 which is produced by
introducing a stop codon following the glycine residue at position
#711 (of SEQ ID NO:1) and monoclonal antibodies (mAbs) directed
against p97 were kindly provided by Biomarin Pharmaceutical Inc.
(Novato, Calif.). TPA, PAI-1 and plasmin are from Calbiochem (La
Jolla, Calif.). Pro-uPA and plasminogen are from American
Diagnostica (Greenwich, Conn.). Angiostatin is purchased from
Angiogenesis Laboratories (Tucson, Ariz.) whereas uPA is from Roche
Biochemicals (Laval, QC). CM5 sensor chips are from BIAcore
(Piscataway, N.J.). The plasmin substrate
(D-val-leu-lys-p-nitraniline or VLK-pNA) and other biochemical
reagents are from Sigma (Oakville, ON).
[0078] Antibodies directed against .alpha.-LRP (8G1 clone) and
u-PAR (#3937) were from Research Diagnostics Inc. (Flanders, N.J.)
and American Diagnostica (Greenwich, Conn.), respectively.
Antibodies directed against Cav-1 (#C3721) and phosphorylated Cav-1
(pCav-1) (#61438) were from BD Transduction Laboratories
(Lexington, Ky.). The antibody directed against eNOS (#N30020) was
from BD Biosciences (Mississauga, ON) and the antibody directed
against GAPDH (#RGM2) was from Advanced Immunochemical Inc. (Long
Beach, Calif.). Antibodies directed against extracellular
signal-regulated kinase 1/2 (ERK 1/2) (#9102) and pERK 1/2 (#9101S)
were from Cell Signaling Technology (Beverly, Mass.). Other
biochemical reagents were from Sigma (Oakville, ON).
Blood-Brain Barrier Model and Transcytosis Experiments
[0079] The in vitro model of the blood-brain barrier (BBB) is
established by using a co-culture of bovine brain capillary
endothelial cells (BBCEC) and newborn rat astrocytes as previously
mentioned (Demeule et al., Journal of Neurochemistry, 83: 924-933,
2002). p97 is radioiodinated with standard procedures using an
iodo-beads kit and D-Salt Dextran desalting columns from Pierce, as
previously described (Demeule M, et al., 2002 J Neurochem
83:924-933). Transcytosis experiments are performed as follows: one
insert covered with BBCECs is set into a six-well microplate with 2
ml of Ringer-Hepes and is pre-incubated for 2 h at 37.degree. C.
[.sup.125I]-p97 (0.5-1.5 .mu.Ci/assay), at a final concentration of
25 nM, is then added to the upper side of the insert. At various
times, the insert is sequentially transferred into a fresh well to
avoid possible reendocytosis of p97 by the abluminal side of the
BBCECs. At the end of the experiment, [.sup.125I]-p97 is assayed in
500 .mu.l of the lower chamber of each well following TCA
precipitation.
Cell Culture
[0080] Cells are cultured under 5% CO.sub.2/95% air atmosphere.
Human microvascular endothelial cells (HMEC-1) are from the Center
for Disease Control and Prevention (Atlanta, Ga.) and are cultured
in MCDB 131 media (Sigma) supplemented with 10 mM L-glutamine, 10
ng/ml epidermal growth factor (EGF), 1 .mu.g/ml hydrocortisone and
10% inactivated foetal bovine serum (FBS). Human umbilical vein
endothelial cells (HUVEC) and SK-MEL28 are obtained from ATCC
(Manassas, Va.). HUVECs are cultured in EGM-2 medium (bullet kit,
Clonetics #CC-3162) and supplemented with 20% FBS. Melanoma
SK-MEL28 cells are grown in MEM supplemented with 1 mM Na-pyruvate,
100 U/ml penicillin-streptomycin, 1.5 g/L Na-bicarbonate and 10%
FBS.
BIAcore Analysis
[0081] p97, PAI-1 and plasminogen are covalently coupled to a CM5
sensor chip via primary amine groups using the N-hydroxysuccinimide
(NHS)/N-ethyl-N'-(dimethylaminopropyl)carbodiimide (EDC) coupling
agents. Briefly, the carboxymethylated dextran is first activated
with 50 .mu.l of NHS/EDC (50 mM/200 mM) at a flow rate of 5
.mu.l/min. p97, PAI-1 or plasminogen (5 .mu.g) in 20 mM acetate
buffer, pH 4.0 are then injected and the unreacted NHS-esters are
deactivated with 35 .mu.l of 1 M ethanolamine hydrochloride, pH
8.5. Approximately 8000 to 10000 relative units of p97, PAI-1 or
plasminogen are immobilized on the sensor chip surface. Ringer
solution or a 50 mM Tris/HCl buffer (pH 7.5) containing 150 mM NaCl
and 50 mM CaCl.sub.2 is used as the eluent buffer. Proteins are
diluted in the corresponding eluent buffer and injected onto the
sensor chip surface. Protein interactions are analyzed using both
the Langmuir binding model, which is the simplest model for 1:1
interaction between analyte and immobilized ligand, and a two-state
conformational change model which describes a 1:1 binding of
analyte to immobilized ligand followed by a conformational
change.
Enzymatic Assay and Cell Treatment with Soluble p97
[0082] The enzymatic activity of pro-uPA is measured using a
colorimetric assay. The reaction is performed in a final volume of
200 .mu.l in an incubation medium consisting of 50 mM Tris/HCl
buffer (pH 7.5), 150 mM NaCl, and 50 mM CaCl.sub.2. This incubation
medium also contains 15 .mu.g/ml VLK-pNA with or without
plasminogen. Enzymatic activity is assessed in the absence or
presence of p97. The reaction is started by the addition of
pro-uPA. In this assay, the cleavage of VLK-pNA results in a
p-nitraniline molecule that absorbs at 405 nm. The reaction product
is monitored at 405 nm using a Microplate Thermomax Autoreader
(Molecular Devices, CA).
[0083] HMEC-1 are grown to 85% confluency in 6-well plates and are
incubated 18 hrs under 5% CO.sub.2/95% air atmosphere in cell
culture medium with or without p97 (100 nM). Endothelial cells are
washed twice with Ringer solution and mechanically scraped from the
wells. Cells are counted and frozen at -80.degree. C. until used. A
volume corresponding to 100,000 cells is incubated in the plasmin
assay as above and plasmin activity is monitored at 405 nm for 60
min. HMEC-1 are also individualized by PBS citrate solution (138 mM
NaCl, 2.7 mM KCl, 1.47 mM KH.sub.2PO.sub.4, 8.1 mM
Na.sub.2HPO.sub.4-7H.sub.2O, 15 mM Na citrate pH 6.8) for 15 min.
Cells are washed twice in Ringer-Hepes solution (150 nM NaCl, 5.2
mM KCl, 2.2 mM CaCl.sub.2, 0.2 mM MgCl.sub.2-6H.sub.2O, 6 mM
NaHCO.sub.3, 5 mM Hepes, 2.8 mM Glucose, pH 7.4) and counted. A
volume corresponding to 100,000 cells is incubated in the plasmin
assay with mAb L235 (325 nM) or IgG control. Plasmin activity is
monitored at 405 nm for 480 min.
Cell Migration Assay
[0084] HMEC-1, HUVEC and SK-MEL28 cell migration is performed using
Transwell filters (Costar; 8 .mu.m pore size) precoated with 0.15%
gelatin for 2 hrs at 37.degree. C. The transwells are assembled in
24-well plates (Falcon 3097) and the lower chambers filled with 500
.mu.l of cell culture medium. To study the effect of p97, mAb L235
or mouse IgG on cell migration, HMEC-1, HUVEC and SK-MEL28 cells
are harvested by trypsinization and centrifuged. Approximatively
10,000 cells are resuspended in 100 .mu.l fresh DMEM medium with or
without p97 (native or boiled for 30 minutes at 100.degree. C.),
mAb L235 or mouse IgG and added into the upper chamber of each
transwell (lower chamber of the transwell also contains p97, mAb
L235 or non-specific mouse IgG). The plates are then placed at
37.degree. C. in 5% CO.sub.2/95% air for 18 hrs. Cells that had
migrated to the lower surface of the filters are fixed with 3.7%
formaldehyde in PBS (Ca.sup.2+/Mg.sup.2+ free), stained with 0.1%
crystal violet/20% MeOH, and counted (4 random fields per filter).
Photomicrographs at 100.times. magnification are taken using a
Polaroid Microcam or Nikon Coolpix.TM. 500 digital camera attached
to a Nikon TMS-F microscope.
Cell Adhesion Assay
[0085] HMEC-1 cell adhesion was performed using 96-well plate
precoated with 0.15% gelatin for 2 hrs at 37.degree. C. To study
the effect of soluble p97 on cell adhesion, HMEC-1 cells were
harvested by trypsinization. 1.times.10.sup.4 cells were
resuspended in 100 .mu.L of fresh medium with or without soluble
p97 and added into each well. Cells were then incubated for 2 hrs
at 37.degree. C. After incubation, adherent cells were washed twice
in PBS (Ca.sup.+2/Mg.sup.+2 free) and stained with 0.1% crystal
violet/20% MeOH. Then, cells were lysed in 1% sodium dodecyl
sulfate (SDS) and cell lysates were measured at 595 nm using a
Microplate Thermomax Autoreader.TM. (Molecular Devices, Sunnyvale,
Calif.). After cell staining, adherent cells were visualized at a
100.times. magnification using a digital Nikon Coolpix.TM. 5000
camera attached to a Nikon TMS-F microscope.
Capillary Tube Formation on Matrigel
[0086] Matrigel (BD Bioscience, Mississauga, ON) was thawed on ice
and 50 .mu.L were added to a 96-well plate and incubated for 10 min
at 37 C. HMEC-1 or HUVEC cells were harvested by trypsinization.
2.5.times.10.sup.4 cells were resuspended in 100 .mu.L fresh medium
and added to Matrigel-coated wells for 30 min at 37 C. After cell
adhesion, the medium was removed and 100 .mu.L of fresh cell
culture medium with or without soluble p97 was added. Cells were
then incubated for 18 hrs at 37.degree. C. After incubation,
tubular structures were visualized at a 40.times. magnification
using a digital Nikon Coolpix.TM. 5000 camera attached to a Nikon
TMS-F microscope. The length of the total capillary network was
quantified using a map scale calculator by measuring and summing
the length of all tubular structures observed in a chosen
field.
Western Blot Analysis
[0087] HMEC-1 (3.times.10.sup.6 cells) were plated into a 75
cm.sup.2 culture flask and exposed to complete medium containing 0,
10 or 100 nM soluble p97. After 18 hours treatment, the cells were
washed twice with PBS (Ca.sup.+2/Mg.sup.+2 free) and solubilized in
lysis buffer (1% Triton-X-100.TM., 0.5% NP-40, 150 mM NaCl, 1 mM
ethylenediaminetetraacetic acid (EDTA), 10 mM Tris, 2%
N-octylglucoside, 1 mM orthovanadate, pH 7,5) for 30 minutes on
ice. Supernatant proteins were measured using a micro-BCA
(bicinchoninic acid) kit from Pierce (Rockford, Ill.). Conditioned
media and cell lysates of HMEC-1 were subjected to
SDS-polyacrylamide gel electrophoresis (SDS-PAGE), using 5%
acrylamide gels for the detection of LRP .alpha.-subunit, 10%
acrylamide gels for the detection of u-PAR and eNOS, 12% acrylamide
gels for the detection of GAPDH, Cav-1, pCav-1, ERK 1/2 and pERK
1/2. Separated proteins were transferred from polyacrylamide gels
to polyvinylidene difluoride membranes (PerkinElmer Life Sciences,
Boston, Mass.) using a Minitrans-Blot.TM. cell from Bio-Rad
(Mississauga, ON) for 90 minutes at 80 mA per gel. Following
transfer, Western blot analysis was performed. All immunodectection
steps were carried out in Tris-buffered saline/0.3% Tween, pH 8.0
(TBS-Tw (0.3%)). The primary antibody was diluted 1:250 for u-PAR,
.alpha.-LRP, GAPDH; 1:1000 for eNOS; 1:5000 for Cav-1, pCav-1, ERK
1/2 and pERK 1/2. The secondary antibody, used for u-PAR,
.alpha.-LRP, GAPDH, Cav-1, pCav-1 and eNOS immunodetection, was a
horseradish peroxidase-conjugated anti-mouse IgG from Jackson
Immunoresearch Laboratories (West Grove, Pa.) diluted 1:2500 in 5%
powdered skimmed milk in TBS-Tw (0.3%). Whereas, the secondary
antibody, used for ERK 1/2 and pERK 1/2 immunodetection, was a
horseradish peroxidase-conjugated anti-rabbit IgG from Jackson
Immunoresearch Laboratories diluted 1:2500 in 5% powdered skimmed
milk in TBS-Tw (0.3%). Incubation with enhanced luminol reagent
(PerkinElmer Life Sciences, Boston, Mass.) and exposure to x-ray
film was used for protein detection. Protein levels were quantified
by laser densitometry using Chemilmager.TM. 5500 from Alpha
Innotech Corporation (San Leandro, Calif.). In addition,
fibronectin and plasminogen were immunodetected by Western blot
analysis in the cell media following HMEC-1 detachment.
Total RNA Isolation and Reverse Transcription Polymerase Chain
Reaction (RT-PCR)
[0088] Total RNA was extracted from cultured HMEC-1 using
TRIzol.TM. reagent from Invitrogen (Burlington, ON). RT-PCR
reactions were performed using SuperScript.TM. One-Step RT-PCR with
Platinum.RTM. Taq Kit from Invitrogen (Burlington, ON). RT-PCR
reactions were performed using specific oligonucleotide primers,
derived from human cDNA sequences for the low-density lipoprotein
receptor (LDL-R) gene family (that includes LDL-R, LRP, LRP 1B, LRP
2, LRP 8), u-PAR, VEGFR-2, VEGF-A and GAPDH (see Table 1 for primer
sequences). Gene product amplification was performed for 40 cycles
of PCR (94.degree. C. for 15 sec, 60.degree. C. for 30 sec
(55.degree. C. for LRP 2), 72.degree. C. for 1 min.). RT-PCR
conditions have been optimized so that the gene products were at
the exponential phase of amplification. Amplification products were
fractionated on 2% (w/v) agarose gels and visualized by ethidium
bromide.
TABLE-US-00001 TABLE 1 Polymerase chain reaction (PCR) primer
sequences and estimated product sizes for u-PAR,LDL-R family gene,
GAPDH, VEGFR-2 and VEGF-A. Product size Gene Primer sequences (bp)
LRP S 5'-AGAAGTAGCAGGACCAGAGGG-3' (SEQ ID NO: 2) 301 AS
5'-TCAGTACCCAGGCAGTTATGC-3' (SEQ ID NO: 3) LRP 1B S
5'-TCTCTCCCTTCTCCAAAGACCC-3' (SEQ ID NO: 4) 403 AS
5'-TCAATGAGTCCAGCCAGTCAGC-3' (SEQ ID NO: 5) LRP 2 S
5'-CGGAGCAGTGTGGCTTATTTTC-3' (SEQ ID NO: 6) 280 AS
5'-CAGGTGTATTGGGTGTCAAGGC-3 (SEQ ID NO: 7) LDL-R S
5'-GGACCCAACAAGTTCAAGTGTCAC-3' (SEQ ID NO: 8) 377 AS
5'-AAGAAGAGGTAGGCGATGGAGC-3' (SEQ ID NO: 9) LRP 8 S
5'-CCTTGAAGATGATGGACTACCCTCG-3' (SEQ ID NO: 10) 415 AS
5'-AAAACCCAAAAAAGCCCCCCCAGC-3' (SEQ ID NO: 11) u-PAR S
5'-ACCGAGGTTGTGTGTGGGTTAGAC-3' (SEQ ID NO: 12) 306 AS
5'-CAGGAAGTGGAAGGTGTCGTTG-3' (SEQ ID NO: 13) GAPDH S
5'-CCATCACCATCTTCCAGGAG-3' (SEQ ID NO: 14) 540 AS
5'-CCTGCTTCACCACCTTCTTG-3' (SEQ ID NO: 15) VEGFR- S
5'-AAAGACATTGCGTGGTCAGGCAGC-3' (SEQ ID NO: 16) 521 2 AS
5'-GGCATCATAAGGCAGTCGTTCAC-3' (SEQ ID NO: 17) 466 VEGF-A S
5'-CCAGCACATAGGAGAGATGAGCTT-3' (SEQ ID NO: 18) 394 AS
5'-GGTGTGGTGGTGACATGGTTAATC-3' (SEQ ID NO: 19) 262 S = sense
strand; AS = antisense strand.
Binding of .sup.125I-uPA.cndot.PAI-1 Complexe to HMEC-1 Soluble
p97-Treated Cells
[0089] First, u-PA was radioiodinated using standard procedures
with Na-.sup.125I (Amersham Pharmacia Biotech, Baie D'Urfe, QC) and
an iodo-beads kit from Pierce (Rockford, Ill.).
.sup.125I-uPA.cndot.PAI-1 complexe was formed by incubating PAI-1
(277 nM) with two-chain .sup.125I-uPA (277 nM) at a molar ration of
1:1 for 1 hour at 37.degree. C. HMEC-1 (6.times.10.sup.5 cells)
were plated onto multiwell (6 wells/plate) disposable plastic
tissue culture plate using fresh media. When confluence was reach,
the medium was removed and completed cell culture medium with or
without soluble p97 (100 nM) was added for 18 hours. Binding
experiments were performed at 4.degree. C. to limit possible
concomitant internalization during the binding interval. Briefly,
after cell treatment, cell monolayers were washed and the binding
was initiated by adding 10 nM of .sup.125I-uPA.cndot.PAI-1 complexe
in 1 mL of Ringer/HEPES containing 0.05% ovalbumine. After 1 hour
incubation, cells were washed three times and lysed with 1 mL NaOH
(0.3 M). Cell associated radioactivity was quantitated in 800 .mu.L
after trichloroacetic acid (TCA) precipitation. The protein content
of control and soluble p97-treated HMEC-1 cells was measured by
using Coomassie.RTM. Plus Protein Assay Reagent kit (Pierce,
Rockford, Ill.).
Fluorescence-Activated Cell Sorting (FACS) Analysis of Cell Surface
u-PAR
[0090] HMEC-1 (3.times.10.sup.6 cells) were plated onto 75 cm.sup.2
dishes using fresh media with or without soluble p97 (100 nM).
After 18 hours incubation, HMEC-1 cells were detached by incubation
with PBS-citrate buffer (138 mM NaCl, 2.8 mM KCl, 1.47 mM
KH.sub.2PO.sub.4, 8.1 mM Na.sub.2HPO.sub.4, 15 mM sodium citrate,
pH 7.4). HMEC-1 (1.times.10.sup.6 cells) were counted and
resuspended in the binding buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM
CaCl.sub.2, pH 7.4). Cell suspension was then incubated at
4.degree. C. for 15 minutes with anti-u-PAR antibody #3937 (1
.mu.g/mL), anti-.alpha.-LRP antibody (8G1 clone) (1 .mu.g/mL) or
with a non-specific IgG1 (1 .mu.g/mL). The cells were then washed
with binding buffer and incubated in the dark at 4.degree. C. for
15 minutes with goat anti-mouse Ig-Alexa488 (1 .mu.g/mL) (Molecular
Probes, Eugene, Oreg.). After two washes with binding buffer, the
cells were analyzed by flow cytometry on a Becton Dickinson
FACscan.TM. with a 488 nM Argon laser using predetermined
instrument settings. Cell surface levels of u-PAR and .alpha.-LRP,
corrected for the background fluorescence intensity measured in the
presence of a non-specific IgG1, were expressed as mean
fluorescence intensities.
Cell Detachment Assay
[0091] HMEC-1 were plated into a 6-wells plate and placed at
37.degree. C. in 5% CO.sub.2/95% air until confluence. Cells were
then exposed to serum free medium containing 150 nM plasminogen and
4 nM tPA, with or without 100 nM of melanotransferrin in the
presence or absence of 150 nM alpha2-antiplasmin, 1 .mu.M EGCG or
10 .mu.M Ilomastat. After 24 hours treatment HMEC-1 detachment was
visualized at a 100.times. magnification using a digital Nikon
Coolpix.TM. 5000 camera (Nikon Canada, Mississauga, ON) attached to
a Nikon TMS-F microscope (Nikon Canada).
Human Plasma
[0092] Human blood samples were collected into a citrated
Vacutainer.RTM. (Becton Dickinson, Franklin Lakes, N.J.) and
centrifuged at 300.times.g for 5 minutes at 4.degree. C. Plasma
were aliquoted in eppendorfs and used fresh or frozen at
-80.degree. C. until used.
Thromboelastography Analysis
[0093] Thromboelastography analysis was performed with citrated
plasma or artificial clot model using a computerized dual-channel
thromboelastograph (TEG) analyzer (model 5000; Haemoscope Corp.,
Niles, Ill.). For the artificial clot model fibrinogen (8.2 .mu.M),
glu-plasminogen (3.3 .mu.M) and tPA (4.5 nM) diluted in buffer A
were transferred into the analyzer cups. Artificial clots were
polymerized with thrombin (0.4 U/ml). For the plasma clot model,
350 .mu.l of citrated plasma were transferred into the analyzer
cups with tPA (4.5 nM). CaCl.sub.2 (0.2 M) was added to initiate
the polymerisation of plasma clot. The thromboelastograph analysis
for both artificial and plasma clots were performed in the presence
or absence of 1 .mu.M p97.
Radial Clot Lysis Assay
[0094] Radial clot lysis assay was performed. Briefly, fibrin-clots
were obtained by incubating fibrinogen (8.2 .mu.M), glu-plasminogen
(2 .mu.M) and 0.4 .mu.ml of thrombin in buffer A at 37.degree. C.
for 60 min in a 6-wells plate. Clot lysis was initiated by dropping
2 .mu.l of tPA (2 nM) with or without p97. Clots were incubated for
30 min at 37.degree. C. and dyed with chinese ink. Photomicrographs
at 40.times. magnification were taken using a digital camera Nikon
Coolpix 5000 camera (Nikon Canada, Mississauga, ON) attached to a
Nikon TMS-F microscope (Nikon Canada).
Data Analysis
[0095] Statistical analyses are made with the Student's paired
t-test using GraphPad Prism (San Diego, USA). Significant
difference is accepted for p values less than 0.05.
[0096] The present invention will be more readily understood by
referring to the following examples which are given to illustrate
the invention rather than to limit its scope.
Example I
Transcytosis of p97 through BBCEC Monolayers
[0097] Transcytosis experiments are performed at 37.degree. C. for
2 hrs. [.sup.125I]-p97 (25 nM) is added to the upper side of the
cell-covered filter in the absence or presence of RAP (650 nM) or
BSA (5 .mu.M). At the end of the experiment, radiolabelled proteins
are measured in the lower chamber of each well by TCA
precipitation. Results represent means.+-.SE (n=6) (FIG. 1A). In
the second part of the experiment (FIG. 1B), p97 is immobilized on
a sensor chip surface (CM5) as described in the Materials and
Methods section above and p97, RAP and BSA (5 .mu.g/100 .mu.l) are
injected over the immobilized p97.
[0098] The first evaluation was the transcytosis of p97 across an
in vitro model of the BBB at 37.degree. C. (FIG. 1A). A significant
(>50%) reduction in the transport of [.sup.125I]-p97 (25 nM)
from the apical (blood side) to the basolateral side (brain side)
of BBCEC monolayers was observed in the presence of 640 nM RAP.
Transcytosis of [1251]-p97 was unaffected by a 200-fold molar
excess of BSA. The permeability coefficient for sucrose is similar
in the absence or presence of RAP indicating that the integrity of
the BBCEC monolayers was unaffected by this protein. The results
with RAP also indicate that LRP is involved in p97 transcytosis
since it has been reported to be an LRP ligand, whereas BSA was
shown to bind to megalin, another member of the LDL receptor
family, probably via cubilin (Kozyraki R et al., 2001 Proc Natl
Acad Sci USA 98:12491-12496). To determine whether protein
interaction could occur between p97 and RAP, leading to a reduction
in p97 transcytosis, protein interactions were investigated by
using biological interaction analysis in real-time (FIG. 1B). For
this analytical approach, p97 was first immobilized on the surface
of a sensor chip. Using standard NHS/EDC coupling procedures about
8 to 10 ng/mm.sup.2 of p97 were immobilized. RAP or BSA (0.05
.mu.g/.mu.l) were then injected over immobilized p97. No
interactions could be observed between these proteins and p97,
indicating that the inhibition of [.sup.125I]-p97 transcytosis is
not related to protein interactions between p97 and RAP.
Example II
Pro-uPA and p97 Interaction
[0099] Biospecific Interaction Analysis between p97 and anti-p97
mAbs
[0100] Biospecific interaction analysis in real-time between p97
and various anti-p97 mAbs is performed as follows. p97 is
immobilized on a sensor chip (CM5) using standard coupling
procedures incorporating NHS, EDC and ethanolamine. Different mAbs
directed against p97 (HybC, HybE, HybF, L235, 2C7, 9B6), diluted to
0.05 .mu.g/.mu.l in Ringer/Hepes, are injected into the BIAcore at
a flow rate of 5 .mu.l/min. The surface plasmon resonance response
obtained for these mAbs is plotted (in relative units (RU)) as a
function of time. After each injection immobilized p97 is
regenerated with 0.2M glycine at pH 2 for 2 min (n=4).
[0101] To evaluate the impact of immobilization procedures on the
structural integrity of p97 different mAbs directed against various
conformational epitopes of p97 were injected over p97 (FIG. 2). The
surface plasmon resonance (SPR) signal generated by the interaction
between p97 and various mAbs varied from 250 relative units (RU) to
2500 RU. These data show that the mAbs could still recognize p97,
indicating that the protein is intact following its immobilization
on the sensor chip surface. Table 2 shows the kinetic parameters
estimated by the BlAevaluation software for antibody interactions
with p97. From these values, the affinity constant
(K.sub.A=k.sub.a/K.sub.d) of these mAbs for immobilized p97 ranged
from 0.08 to 1.6 nM.sup.-1 and for the relative affinities are
HybE<L235<9B6<2C7, HybC<HybF.
TABLE-US-00002 TABLE 2 Kinetics of interaction between immobilized
p97 and mAbs. K.sub.a K.sub.d K.sub.A = K.sub.a/K.sub.d K.sub.D =
K.sub.d/K.sub.a Antibodies .DELTA.RU (M.sup.-1s.sup.-1) (s.sup.-1)
(M.sup.-1) (M) L235 1055 .+-. 82 4.4 .times. 10.sup.4 5.3 .times.
10.sup.-5 0.9 .times. 10.sup.9 0.1 .times. 10.sup.-10 HybC 1509
.+-. 184 7.2 .times. 10.sup.4 4.5 .times. 10.sup.-5 1.6 .times.
10.sup.9 6.4 .times. 10.sup.-10 HybE 232 .+-. 52 0.9 .times.
10.sup.4 9.8 .times. 10.sup.-5 0.08 .times. 10.sup.9 0.01 .times.
10.sup.-10 HybF 2199 .+-. 150 8.0 .times. 10.sup.4 3.0 .times.
10.sup.-5 2.7 .times. 10.sup.9 3.8 .times. 10.sup.-10 9B6 2440 .+-.
13 1.2 .times. 10.sup.4 9.1 .times. 10.sup.-5 1.3 .times. 10.sup.9
7.9 .times. 10.sup.-10 2C7 2290 .+-. 87 5.9 .times. 10.sup.4 3.8
.times. 10.sup.-5 1.6 .times. 10.sup.9 6.5 .times. 10.sup.-10 The
difference between the relative units measured after and before
injection of mAbs directed against p97 are presented (.DELTA.RU) as
well as the apparent association (K.sub.a) and dissociation
(K.sub.d) constants. The affinity (K.sub.A) and dissociation
(K.sub.D) constants were calculated from the K.sub.a and
K.sub.d.
Molecular Interactions of p97 and Various Components of the
PA:plasmin System
[0102] Determining the molecular interactions between p97 and
various components of the PA:plasmin system was as follows. Pro-uPA
and tPA (0.05 .mu.g/.mu.l), diluted in Ringer/Hepes, are injected
onto immobilized p97 on a sensor chip at a flow rate of 5
.mu.l/min. The SPR response for these proteins is plotted in RU as
a function of time. p97 (0.05 .mu.g/.mu.l) is also injected over
immobilized PAI-1 (p97/PAI-1). Plasminogen, plasmin or angiostatin
(0.05 .mu.g/.mu.l) are also injected onto immobilized p97. The SPR
response for these proteins is plotted in RU as a function of time.
The results indicate that pro-uPA and plasminogen interact with
p97. After each injection the sensor chip surface with immobilized
p97 is regenerated by injecting 10 mM glycine, pH 2.2 for 2
min.
[0103] When pro-uPA and tPA (0.05 .mu.g/.mu.l) were injected over
immobilized p97, protein interaction occurred between pro-uPA and
p97 but not between tPA and p97 (FIG. 3A). About 8-10 ng/mm.sup.2
of PAI-1 was also immobilized onto another well of a sensor chip
surface using NHS/EDC coupling conditions. No interaction between
p97 and immobilized PAI-1 could be detected (FIG. 3A). However a
strong interaction could be observed when tPA was injected over
PAI-1, indicating that PAI-1 can still interact with tPA following
immobilization. In addition, plasminogen, plasmin and angiostatin
(0.05 .mu.g/.mu.l) were injected over immobilized p97 (FIG. 3B).
According to the SPR, plasminogen also interacts with immobilized
p97 whereas plasmin and angiostatin, two plasminogen fragments, do
not. The kinetic data obtained from binding of pro-uPA or
plasminogen to immobilized p97 biosensor surface were evaluated
using both the 1:1 Langmuir binding model and the two state
conformational change model. Interestingly, the two state
conformational change model was a better fit than the 1:1 Langmuir
binding model when comparing a single concentration of either
pro-uPA and plasminogen over p97 biosensor surface. Kinetic data
obtained with the two state conformational model are presented in
Table 3. Kinetic data for the interaction between pro-uPA and p97
shows an association constant (k.sub.a1) of 6.6.times.10.sup.3
M.sup.-1s.sup.-1 and a dissociation rate constant (k.sub.d1) of
1.7.times.10.sup.-3 s.sup.-1. Furthermore, the forward rate
constant (k.sub.a2=3.2.times.10.sup.-3 s.sup.-1) and backward rate
constant (k.sub.d2=7.1.times.10.sup.-4 s.sup.-1) for the
conformational change provide an apparent equilibrium dissociation
constant ((K.sub.D=k.sub.d1/k.sub.a1)/(k.sub.d2/k.sub.a2)) of 65
nM. The kinetic analysis of plasminogen interaction with p97 shows
an association constant (k.sub.a1) of 2.1.times.10.sup.4
M.sup.-1s.sup.-1. The dissociation rate constant
(k.sub.d1=4.3.times.10.sup.-2 s.sup.-1), as well as the forward
rate constant (k.sub.a2) of 6.0.times.10.sup.-2 s.sup.-1 and
backward rate constant (k.sub.d2) of 1.1.times.10.sup.-3 s.sup.-1,
are different from those seen for the pro-uPA interaction with p97.
However, the apparent equilibrium dissociation constant (K.sub.D)
between p97 and plasminogen is 350 nM, which is different from that
observed for the interaction of pro-uPA with immobilized p97.
TABLE-US-00003 TABLE 3 Kinetics of interaction between immobilized
p97 and pro-uPA or plasminogen using the two state conformational
model Immobilized k.sub.a1 k.sub.a2 k.sub.d1 k.sub.d2 KD proteins
Ligands (.times.10.sup.4 M.sup.-1s.sup.-1) (.times.10.sup.-3
s.sup.-1) (.times.10.sup.-3 s.sup.-1) (.times.10.sup.-4 s.sup.-1)
(.times.10.sup.-9 M) p97 pro-uPA 66.2 3.2 6.0 7.1 65 plasminogen
2.1 6.0 3.1 11.2 350
[0104] Kinetic parameters of Table 3 were based on a two state
conformational change binding model using the biosensorgram shown
in FIG. 3. This model describes a 1:1 binding of analyte to
immobilized ligand followed by a conformational change in the
complex. It is assumed that the conformationally changed complex
can only dissociate through the reverse of the conformational
change: A+B=AB=ABx. The dissociation constants (K.sub.D) were
derived using both association (ka) and dissociation (k.sub.d)
rates (K.sub.D=(k.sub.d1/k.sub.a1).times.(k.sub.d2/k.sub.a2). The
parameters are: k.sub.at, association rate constant for A+B1=AB1
(M.sup.-1s.sup.-1); k.sub.d1, dissociation rate constant for
AB1=A+B1 (s.sup.-1); k.sub.a2, forward rate constant for AB=ABx
(s.sup.-1); k.sub.d2, backward rate constant for AB=ABx (s.sup.-1).
The mean Chi.sup.2 values for the sensorgram fits were less than
0.4.
Effect of p97 on pro-uPA, tPA and Plasminogen
[0105] To evaluate the effect of p97 interaction on pro-uPA, tPA
and plasminogen, the serine activity (VLK-pNA hydrolysis) of 90 nM
pro-uPA and 75 nM tPA were measured in the absence (o) or presence
( ) of 70 nM p97 without plasminogen using a colorimetric assay,
both with and without p97 (FIGS. 4A and 4B). The reaction was
performed in a final volume of 200 .mu.l as described in the
Materials and Methods section above. In both FIGS. 4A and 4B,
controls were also performed with p97 (.box-solid.) but without
pro-uPA or tPA (n=9, for pro-uPA; n=6, for tPA). In the absence of
p97, only a slight activity was measured for both pro-uPA and tPA.
However, the VLK-pNA hydrolysis by pro-uPA goes from less than 50
AU/min in the absence of p97 to more than 450 AU/min when p97 is
added into the incubation (FIG. 4A). Addition of p97 to tPA elicits
no observable effect and p97 alone had no proteolytic activity
(FIG. 4B). The results from both SPR and enzymatic activity
indicate that the change in pro-uPA conformation induced by p97
increased its ability to degrade the plasmin substrate.
[0106] To determine whether interaction with p97 leads to a
cleavage of pro-uPA, the proteins were co-incubated for 5 min. at
37.degree. C. in the presence or absence of plasminogen. They were
then separated by SDS-PAGE under reducing conditions using a 12.5%
acrylamide gel and stained with standard Coomassie Blue. The
results are shown in FIG. 4C. The lanes of the gel are as follows
(FIG. 4C): 2 .mu.g of p97 (lane 1), 1 .mu.g pro-uPA (lane 2) and 2
.mu.g plasminogen (lane 3) were incubated for 5 min. at 37.degree.
C. alone as controls. Pro-uPA (2 .mu.g) was incubated at 37.degree.
C. for 5 min. with 2 .mu.g of p97 (lane 4). Plasminogen and Pro-uPA
were added without incubation (lane 5) and with 5 min. incubation
at 37.degree. C. (lane 6). Pro-uPA with 2 .mu.g of both p97 and
plasminogen were added without incubation (lane 7) or with 5 min.
incubation at 37.degree. C. (lane 8). Tc-uPA (2 .mu.g) was also
loaded as a control (lane 9). Under these conditions p97 and uPA
migrated as 97 kDa and 33 kDa bands, respectively, whereas pro-uPA
migrated as a single band at 55 kDa. No major degradation of either
protein could be detected, indicating that the incubation of
pro-uPA with p97 under the conditions used to perform the VLK-pNA
hydrolysis did not cleave either protein. Even after 6 hours
incubation at 37.degree. C., both proteins were stable. In the
presence of plasminogen, pro-uPA was cleaved after an incubation of
5 min. at 37.degree. C. and two major fragments of 33 kDa and 29
kDa could be observed. When p97 was added to the incubation medium,
the generation of these fragments did not change.
[0107] The impact of p97 on plasminogen fragmentation by pro-uPA
was further estimated using 6 hours incubation at 37.degree. C. and
the results are shown in FIG. 4D. The lanes of the gel are as
follows: 3 .mu.g of p97 (lane 1), glu-plasminogen (lane 2) and
lys-plasminogen (lane 3) were incubated alone for 6 hours at
37.degree. C. as controls. In lane 4, 34 of both glu-plasminogen
and p97 were also incubated for 6 hours at 37.degree. C. Pro-uPA
(20 ng) was added to plasminogen for the same period of incubation
at 37.degree. C. (lane 5). p97 was added to pro-uPA and plasminogen
for 6 hours at 37.degree. C. (lane 6) or 4.degree. C. (lane 7). In
lane 8, 3 .mu.g of angiostatin (lane 8) was also added as a
control. Proteins were separated on a 7.5% acrylamide gel under
non-reducing conditions and stained with Coomassie blue. When p97
is added to glu-plasminogen no apparent fragment was generated. In
contrast, the addition of a low amount (10 ng) of pro-uPA, which
could not be detected using standard Coomassie blue staining,
induced degradation of Glu-plasminogen with the appearance of
fragments which migrated at the same molecular weight as
Lys-plasminogen. Moreover, when p97 is added to glu-plasminogen and
pro-uPA, the degradation profile of glu-plasminogen is changed. In
the presence of p97 with glu-plasminogen and pro-uPA, higher levels
of bands migrating at the same molecular weight as lys-plasminogen
were observed and two other fragments appeared at 50 and 30 kDa.
These fragments do not seem to be related to angiostatin since they
migrated at a different molecular weight than did the control
angiostatin at 42 kDa. These results indicate that p97 alters the
cleavage of glu-plasminogen by pro-uPA.
Example III
Plasminogen Activation by p97
[0108] The interaction of p97 with pro-uPA was further
characterized by measuring the activation of plasminogen by pro-uPA
in the presence of p97 (FIG. 5). The plasminolytic activity of 1 nM
uPA was measured without (o) or with ( ) 70 nM p97 in the presence
of 30 nM plasminogen. The reaction was performed in a final volume
of 200 .mu.l as described in the Materials and Methods section
above. As a control, the enzymatic activity in the presence of p97
alone was also measured (.box-solid.). When p97 is added to pro-uPA
and plasminogen, the VLK-pNA hydrolysis is 4-fold higher after 180
min (FIG. 5A). Control experiments performed with p97 indicated
that this protein alone does not generate plasmin when it is added
to plasminogen.
[0109] The plasmin activity in the presence of various
concentrations of p97 was also measured (FIG. 5B). Plasmin activity
induced by pro-uPA was measured in the presence of various p97
concentrations. Since the generation of plasmin proceeds at a
constant rate under the assay conditions used, plotting the
experimental data as a function of time (t).sup.2 allowed for the
determination of the initial rate of plasmin formation. From these
linear curves, the initial plasmin activity measured in the absence
of p97 was subtracted from the activities obtained in the presence
of various p97 concentrations. Thus, the data represent the initial
rates of plasmin activity (corresponding to the slopes) in the
presence of various p97 concentrations. p97 stimulates the
plasminogen cleavage by pro-uPA in a dose-dependent manner with
half-maximal stimulation occurring at 25.+-.6 nM.
[0110] The effect of p97 on plasmin activity in the presence of
various concentrations of plasminogen was also measured (FIG. 5C).
Plasmin activity induced by pro-uPA was measured without (o) or
with ( ) 250 nM p97 and various concentrations of plasminogen.
Initial rates of plasmin activity calculated at several plasminogen
concentrations were plotted as a function of plasminogen
concentrations. The resulting experimental data were fitted using
nonlinear regression analysis. p97 decreased the apparent Km of
pro-uPA for plasminogen from 188.+-.22 to 102.+-.17 nM and
increased the Vmax from 6.9.+-.0.4 to 8.9.+-.0.6 AU/min. These
results indicate that p97 positively affects the activation of
plasminogen by pro-uPA by increasing the catalytic efficiency by a
factor of 2.4-fold.
[0111] To determine whether the induction of plasmin formation by
p97 was specific, the formation of plasmin by pro-uPA in the
presence of either the mAb L235 (directed against p97) or a
non-specific IgG was measured (FIG. 5D). The plasminolytic activity
of pro-uPA was measured in the presence of 70 nM p97 and 65 nM of
either mAb L235 (o) or non-specific mouse IgG ( ). One
representative experiment is shown and data represent the
means.+-.SD of values obtained from triplicates (n=3). MAb L235 (50
nM) inhibited the pro-uPA activation induced by p97 by 50%. These
results indicate that the effect of p97 upon pro-uPA's activation
of plasminogen is specific and involves the epitope recognized by
the mAb L235.
Example IV
Inhibition of Cell Migration by mAb L235
[0112] Since p97 affects the activation of plasminogen in vitro and
since the uPA/uPAR system is important in cell migration, it was
further investigated whether endogenous p97 might be associated
with this process. Cell migration of HMEC-1, SK-MEL28 cells or
HUVEC was measured using modified Boyden chambers as described in
the Materials and Methods section above. Because p97 was first
identified in melanoma cells (Brown J P et al., 1981 Proc Natl Acad
Sci USA 78:539-543), the impact of the mAb L235 on the migration of
human melanoma (SK-MEL28) cells was also measured (FIG. 6A). Cells
that had migrated to the lower surface of the filters were fixed
and stained with crystal violet. Images obtained from a
representative experiment are shown. Cells that had migrated in the
presence of 50 nM mAb L235 or a non-specific mouse IgG were also
counted. The results were expressed as the percentage of the
control measured in the presence of a non-specific mouse IgG and
represent the means.+-.SD (n=5 for HMEC-1; n=4 for SK- MEL28; n=3
for HUVEC). Statistically significant differences are indicated by
***p<0.001 (Student's t-test). In the presence of mAb L235 (50
nM), the migration of HMEC-1 and SK-MEL28 cells was inhibited by
54% and 48%, respectively. However, cell migration of HUVEC was
unaffected by this concentration of mAb L235.
[0113] Endogenous p97 was immunodetected in lysates or
serum-deprived culture media (18 hours) from HMEC-1, SK-MEL28 and
HUVEC cells. FIG. 6B shows the detection of endogenous p97 by
Western blot analysis. Proteins were separated by SDS-PAGE and were
electrophoretically transferred to PVDF membranes. p97 was detected
by Western blotting using mAb L235 and a secondary anti-mouse IgG
linked to peroxidase. p97 migrated under unreduced conditions at 73
and 60 kDa, as previously observed. It was highly expressed in
lysates from HMEC-1 and SK- MEL28 cells and at lower levels in
their respective conditioned culture media. In HUVEC cells, p97 was
however almost undetectable. In fact, the exposure time was at
least 30 times greater to detect a much lower level of p97 in HUVEC
compared to HMEC-1 and SK-MEL28 cells. These results indicate that
mAb L235, by interacting with endogenous p97, inhibits the
migration of HMEC-1 and SK-MEL28 cells. This also indicates that
the endogenous p97 in these cells is involved in cell
migration.
Example V
Effect of Exogenous p97 on Cell Migration
[0114] It was also estimated whether exogenous p97 could affect the
migration of HMEC-1 and SK-MEL28 cells. HMEC-1 and SK-MEL28 cell
migration was performed using modified Boyden chambers as described
in the Materials and Methods section above. Cells that had migrated
in the presence or absence of p97 (100 nM) to the lower surface of
the filters were fixed and stained with crystal violet. The results
are shown in FIGS. 7A and 7B. Cells that had migrated were also
counted and expressed as a percentage of the control cells,
measured in the absence of p97 (n=4, for HMEC-1; n=3, for
SK-MEL28). Exogenous p97, at 10 nM and 100 nM, inhibited the
migration of HMEC-1 cells by 34% and 50% (FIG. 7C). The migration
of SK-MEL28 cells was inhibited by 44% and 70% in the presence of
10 and 100 nM p97. Migration of HUVEC cells was unaffected by these
concentrations of p97. Moreover, this inhibition of cell migration
is not related to a reduction of endothelial or melanoma cell
adhesion since the same concentrations of p97 did not affect
adhesion on gelatin of either HMEC-1 or SK-MEL28 cells.
Example VI
Inhibition of Plasminolytic Activity at the Cell Surface by Soluble
p97 and mAb L235
[0115] The effect of p97 on plasminolytic activity was determined
as follows. HMEC-1 cells were treated for 18 hours with 100 nM p97
(+p97) or Ringer solution (Control). Following this treatment the
plasminolytic activity was measured using standard conditions, as
described in the Materials and Methods section above. When cells
were treated with p97 (100 nM), plasminogen activation was
inhibited by 95% (FIG. 8A). This marked reduction in the
plasminolytic capacity of these cells by soluble p97 could explain
the inhibition of HMEC-1 migration. The effect of mAb L235 on
plasminolytic activity of HMEC-1 was also determined. HMEC-1 cells
(1.times.10.sup.5 cells) were pre-incubated 1 hr. at 37.degree. C.
with Ringer solution (Ctl) or with 250 nM of either mAb L235 or
non-specific mouse IgG. Following this pre-incubation, the
plasminolytic activity was measured for 6 hrs by adding pro-uPA (1
nM) and plasminogen (50 nM) using standard conditions, as described
in the Materials and Methods section. The plasminolytic activity of
HUVEC was also measured using 1.times.10.sup.5 cells under the same
conditions. Data represent the means.+-.SD of three independent
experiments performed in triplicate. Statistically significant
differences are indicated by *** where p<0.001 (Student's
t-test). When HMEC-1 cells were treated with the mAb L235, the
plasminolytic activity was inhibited by more than 50% compared to
non-specific mouse IgG (FIG. 8B). This inhibition by the mAb L235
indicates that endogenous, membrane-bound p97 participates in
plasminogen activation in HMEC-1.
Example VII
Anti-Angiogenic Properties of p97
[0116] Angiogenesis, a complex multistep process that leads to the
outgrowth of new capillaries from pre-existing vessels, is an
essential mechanism in wound healing, embryonic development, tissue
remodeling, and in tumor growth and metastasis. This process
involves EC proliferation, migration and morphogenic
differentiation into capillary-like structures. One of the key
elements in cell migration is the urokinase-type plasminogen
activator receptor (u-PAR). The plasminogen activator (PA) family
is composed of urokinase-type plasminogen activator (u-PA) and
tissue-type plasminogen activator (t-PA); their inhibitors are the
plasminogen activator inhibitor type 1 and 2 (PAI-1; PAI-2). u-PAR
mediates the internalization and degradation of u-PA/inhibitor
complexes via the low-density lipoprotein receptor-related protein
(LRP), whereas LRP mediates the internalization and degradation of
t-PA/inhibitor complexes. Thus, the u-PAR/LRP system controls cell
migration by regulating plasminogen activation by PAs at the cell
surface. PAs are therefore involved in angiogenesis by enhancing
cell migration, invasion and fibrinolysis. Moreover, plasminogen
needs to be first converted to the two-chain serine protease
plasmin. When Glu-plasminogen, the native circulating form of the
zymogen, is bound to the cell surface, plasmin generation by PAs is
markely stimulated compared with the reaction in solution. Optimal
stimulation of plasminogen activation at the EC surface requires
the conversion of Glu-plasminogen to Lys-plasminogen.
[0117] Since soluble p97 interacts with plasminogen and
single-chain u-PA (scu-PA), the potential role of soluble p97 on
angiogenesis was further investigated. Herein, it is shown that
soluble p97 inhibits EC migration and tubulogenesis by affecting
both u-PAR and LRP expression as well as the binding of the
u-PA.cndot.PAI-1 complexe at the cell surface of human microvessel
EC (HMEC-1). To further understand the impact of soluble p97 on
morphogenic differentiation of EC into capillary-like structures,
the expression of key players associated with angiogenesis was also
determined.
Cell Culture
[0118] Cells were cultured under 5% CO.sub.2/95% air atmosphere.
Human dermal microvessel endothelial cells (HMEC-1) were from the
Center for Disease Control and Prevention (Atlanta, Ga.) and were
cultured in MCDB 131 supplemented with 10 mM L-glutamine, 10 ng/ml
EGF, 1 .mu.g/ml hydrocortisone and 10% inactivated foetal bovine
serum (FBS). HUVECs was obtained from ATCC (Manassas, Va.). HUVECs
were cultured in EGM-2 medium (bullet kit, Clonetics #CC-3162) and
20% inactivated FBS.
Enzymatic Assay
[0119] The enzymatic activity of p97, sc-uPA, uPA and tPA was
measured using a colorimetric assay (FIG. 9). The reaction was
performed in a final volume of 200 .mu.L in an incubation medium
consisting of 50 nM Tris/HCl buffer (pH 7.5), 150 mM NaCl and 50 mM
CaCl.sub.2. This incubation medium also contained 15 .mu.g/mL
L-val-leu-lys-p-nitraniline (VLK-pNA) and 25 nM glu-plasminogen.
The enzymatic activity was assessed with or without 100 nM soluble
p97. The reaction was started by the addition of 1 nM sc-uPA, uPA
or tPA. In this assay, the cleavage of VLK-pNA results in a
p-nitraniline molecule that absorbs at 405 nm. The reaction product
was monitored at 405 nm using a Microplate Thermomax Autoreader.TM.
(Molecular Device, CA).
Western Blot Analysis
[0120] In FIG. 10, HMEC-1 (3.times.10.sup.6 cells) were plated into
a 75 cm.sup.2 culture flask with fresh medium with or without 10
and 100 nM of p97. After 18 hours treatment, HMEC-1 were washed
twice PBS Ca.sup.+2/Mg.sup.+2 free and solubilized in lysis buffer
(1% Triton-X-100.TM., 0.5% NP-40, 150 mM NaCl, 1 mM EDTA, 10 mM
Tris, 2% N-octylglucoside, 1 mM orthovanadate, pH 7,5) for 30
minutes on ice. Supernatant proteins were measured using a
micro-BCA (bicinchonic acid) kit (Pierce). Conditioned media and
cell lysates of HMEC-1 were subjected to SDS-PAGE using 5%
acrylamide gel for the detection of .alpha.-LRP, 10% acrylamide gel
for the detection of uPAR. Separated protein were transferred
electrophoretically from polyacrylamide gel to PVDF transfer
membrane (PerkinElmer Life Sciences) in a Minitrans-Blot.TM. cell
(Bio-Rad) for 90 minutes at 80 mA per gel. Following transfert,
western blot analysis were performed. All immunodectection steps
were carried out in Tris-buffered saline/0.1% Tween, pH 8.0 [TBS-Tw
(0.1%)]. The primary antibody was diluted 1:250 for LRP and uPAR.
The secondary antibody, horseradish peroxidase-conjugated
anti-mouse IgG (Jackson), was diluted 1:2500 in 1% powdered skimmed
milk in TBS-Tw. Incubation with enhanced luminol reagent
(PerkinElmer Life Sciences) and exposure to x-ray film were used to
determined protein levels
Capillary Tube Formation on Matrigel
[0121] In FIG. 11, Matrigel was thawed on ice and added (50 .mu.l)
to a 96-well plate for 10 min at 37.degree. C. HUVEC or HMEC-1 were
harvested by trypsinization and spun down. About 25 000 cells were
resuspended and added to Matrigel-coated wells for 30 min at
37.degree. C. After cell adhesion, the medium was removed and 100
.mu.l of fresh cell culture medium with or without p97 was added.
Wells were then incubated for 18 hours at 37.degree. C. After
incubation, tubular structures were visualized using a Nikon TMS-F
microscope (at a maginification of x40). The length of the
capillary network was quantified using a map scale calculator.
[0122] In conclusion, as shown in FIG. 9, p97 stimulates the
plasminolytic activity of single chain urokinase plasminogen
activator (sc-uPA or pro-uPA), uPA and tissue plasminogen activator
(tPA) in vitro. In addition, as shown in FIG. 10, low density
lipoprotein related protein (LRP) and the urokinase activator
receptor (uPAR) are down regulated in p97 treated MHEC-1 cells.
Furthermore, as shown in FIG. 11, HMEC-1 and HUVEC capillary tube
formation is inhibited by low concentration of soluble p97.
Soluble p97 Inhibits the Morphogenic Differentiation of EC into
Capillary-Like Structures
[0123] The process of angiogenesis is associated with the
morphogenic differentiation of EC into microvascular capillary-like
structures. To investigate this crucial step of angiogenesis, many
studies have used an in vitro assay for tube formation on Matrigel.
In the present invention, HMEC-1 and HUVEC cells growth on Matrigel
generated a stabilized network of capillary-like structures. This
is shown by the complexity of the tubular network per field in
control cells observed after 18 hours. The effects of exogenous
soluble p97 on HMEC-1 and HUVEC morphogenic differentiation was
therefore determined into capillary-like structures (FIG. 11). The
generation of capillary-like tubular structure was strongly reduced
when soluble p97 was added during the experiments. Indeed, soluble
p97 at 10 and 100 nM reduced, by 53% and 47%, the capillary-like
tube formation of HMEC-1 (FIG. 11A) and reduced, by 38% and 35%,
the capillary-like tube formation of HUVECs (FIG. 11B). These
results indicate that soluble p97 inhibits the initiation of
capillary-like tube formation. In FIGS. 11A to 110, data represent
the means.+-.SD of results obtained from three different
experiments performed in triplicates. Statistically significant
differences are indicated by **p<0.01, ***p<0.001 (Student's
t-test). Photos (original magnification, X40) obtained from a
representative experiment are shown.
Soluble p97 Modulates HMEC-1 Cell Migration
[0124] Since soluble p97 affected plasminogen activation, it first
investigated whether soluble p97 might modulate cell migration.
Using modified Boyden chamber, HMEC-1 cell migration was examined
in the presence of soluble p97 (FIG. 12A). soluble p97, at 10 and
100 nM, inhibited the migration of HMEC-1 by 34% and 50%,
respectively. The inhibition of HMEC-1 cell migration is completely
lost when soluble p97 was boiled for 30 minutes at 100.degree. C.
prior to the migration assay (FIG. 12B). This result indicates that
a native conformation of soluble p97 is required to inhibit HMEC-1
cell migration. The adhesion of HMEC-1 on gelatin was found
unaffected by soluble p97 (FIG. 12C), indicating that the
inhibition of cell migration is unrelated to a reduction of
adhesive properties. In FIG. 12A), HMEC-1 cell migration was
performed using modified Boyden chambers as described in the
Materials and Methods sections. Cells that had migrated in the
presence or absence of soluble p97 to the lower surface of the
filters were fixed, stained with crystal violet and counted.
Results are expressed as a percentage of migration in soluble
p97-treated cells compared to untreated cells. Data represent the
means.+-.SD of four independent experiments performed in
triplicates. (B) HMEC-1 cell migration was performed as indicated
previously with native or boiled soluble p97. Data represent the
means.+-.SD of two independent experiments performed in
triplicates. (C) HMEC-1 cell adhesion was performed on gelatin as
described in the Materials and Methods sections. Cells that had
adhered to the gelatin in the presence or absence of soluble p97
were stained with crystal violet. Results are expressed as a
percentage of adhesion in soluble p97-treated cells compared to
untreated cells. Data represent the means.+-.SD of three
independent experiments performed in triplicates. In all
experiments, statistically significant differences are indicated by
***p<0.001 (Student's t-test) (original magnification,
X100).
Soluble p97 Up-Regulates u-PAR and LRP Protein Expression
[0125] To identify a potential mechanism by which soluble p97
inhibited in vitro EC migration and tubulogenesis, the effect of
soluble p97 on the protein expression of both the u-PAR system and
LRP was measured by Western blot (FIG. 13). HMEC-1 cells were
incubated for 18 hours with or without soluble p97. GAPDH was
immunodetected to ensure that the protein content between samples
was equivalent. Soluble p97 treatment significantly down-regulated
u-PAR and LRP expression. In fact, exposure of HMEC-1 to soluble
p97 at 10 and 100 nM reduced u-PAR expression in cell lysates by
20% and 40%, respectively (FIG. 13A). The same concentrations
decreased LRP expression by 20% and 50%, respectively (FIG. 13B).
In FIGS. 13A and 13B, HMEC-1 were treated for 18 hours with or
without soluble p97. Following this treatment, proteins from cell
lysates were resolved by SDS-PAGE. Immunodetections of u-PAR (13A)
and LRP (13B) were performed as described in the Materials and
Methods section. Results were expressed as a percentage of protein
expression detected in soluble p97-treated cells compared to
untreated cells. Data represent the means.+-.SD of results obtained
from three different experiments. Statistically significant
differences are indicated by *p<0.05, ***p<0.001 (Student's
t-test).
Soluble p97 Unaffects the u-PAR/LRP System mRNA Expression
[0126] Since soluble p97 modulated u-PAR and LRP protein
expression, the mRNA expression of LDL-R family gene and u-PAR were
estimated by RT-PCR in HMEC-1 treated or not with soluble p97 (FIG.
14). In FIG. 14, HMEC-1 were treated for 18 hours with or without
soluble p97. Total RNA was isolated from HMEC-1 and gene products
were amplified by RT-PCR as described in the Materials and Methods
section. Table 4 shows the primer sequences used for specific cDNA
amplification. Expression of the different members of the
LDL-receptor family was first investigated in untreated HMEC-1
cells. Under the conditions used for RT-PCR analysis, LRP, LRP 1B,
LDL-R and LRP 8 were clearly amplified (35 cycles) whereas LRP 2
and LRP 5 products were almost undetectable. Following soluble p97
treatment, the mRNA levels of LRP, LRP 1B, LRP 2, LDL-R, LRP 8 or
u-PAR was unchanged in treated cells as compared to control cells
(FIG. 14). An internal control, GAPDH mRNA, was also unaffected by
soluble p97. Since u-PAR and LRP gene expression were unaffected by
soluble p97, these results indicate that soluble p97 effects on
u-PAR and LRP expression takes place at the protein level.
Soluble p97 Modulates the Cell Surface Levels of u-PAR and LRP
[0127] In view of the fact that u-PAR and LRP expression is
affected by exogenous soluble p97 and that the amount of u-PAR and
LRP at the membrane surface is a key element in plasmin formation,
the u-PAR and LRP levels at the cell surface was determined by FACS
analysis following soluble p97 treatment (FIGS. 15A and 15B).
HMEC-1 cells were incubated for 18 hours with or without 100 nM of
soluble p97. Flow cytometric analysis of cell surface u-PAR (15A)
and LRP (15B) levels was performed as described in the Materials
and Methods section. Control (grey line:1) or treated HMEC-1 (bold
line:2) were labeled with anti-u-PAR antibody (#3937) or with
anti-.alpha.-LRP antibody (clone 8G1) and detected with goat
anti-mouse IgG-Alexa488. These results are representative of three
different experiments. Results were corrected for the background
fluorescence intensity measured with a non-specific IgG1 and
expressed as mean fluorescence intensities. Data represent the
means.+-.SD of three different experiments. Statistically
significant differences are indicated by ** p<0.001,
***p<0.001 (Student's t-test). The mean fluorescence intensity
associated with the detection of cell surface u-PAR is
significantly higher by 25% following soluble p97 treatment. Cell
surface LRP expression was also assessed by FACS analysis as in
control (grey line:1) and treated cells (bold line:2) (FIG.
15B).
[0128] In FIG. 15C, following cell treatment with soluble p97,
binding of .sup.125I-uPA.cndot.PAI-1 complexe was performed as
described in the Materials and Methods section. Data represent the
means.+-.SD of three different experiments. Statistically
significant differences are indicated by ***p<0.001 (Student's
t-test).
[0129] The mean fluorescence intensity associated with the
detection of cell surface LRP is significantly lower by 30%
following soluble p97 treatment. These results suggest that soluble
p97 treatment significantly increased u-PAR levels and decreased
LRP levels at the cell surface of HMEC-1. To find out whether u-PAR
at the cell membrane of HMEC-1 soluble p97-treated cell is free or
occupied by u-PA and/or uPA.cndot.PAI-1 complexe, a binding assay
of .sup.125I-uPA.cndot.PAI-1 complexe on HMEC-1 following soluble
p97 treatment (FIG. 15C) was next performed. HMEC-1 were incubated
for 18 hours with or without soluble p97 and the binding of
.sup.125I-uPA.cndot.PAI-1 complexe was then measured at 4.degree.
C. in control and treated cells. The cell associated radioactivity
after the binding of .sup.125I-uPA.cndot.PAI-1 complexe was reduced
by about 23% following soluble p97 treatment. This result suggest
that the free u-PAR at the cell membrane was decreased after
soluble p97 treatment.
Soluble p97 Up-Regulates Cav-1 and Down-Regulates pERK 1/2 Protein
Expression.
[0130] To further understand the effects of soluble p97 on in vitro
EC migration and tubulogenesis, the expression and phosphorylation
levels of proteins associated with angiogenesis (FIGS. 16 and 17)
was next measured. In this invention, HMEC-1 were incubated for 18
hours with or without soluble p97 (10 or 100 nM). Following this
treatment, proteins from cell lysates were solubilized and resolved
by SDS-PAGE. Immunodetection of Cav-1 (16A) and pCav-1 (16B) as
well as ERK 1/2 (16C) and pERK 1/2 (16D) was performed as described
in the Materials and Methods section. Results were expressed as a
percentage of protein expression detected in soluble p97-treated
cells compared to untreated cells. Data represent the means.+-.SD
of results obtained from three different experiments. Statistically
significant differences are indicated by ***p<0.001 (Student's
t-test). Since Cav-1 play an important positive role in the
regulation of EC differentiation, a prerequisite step in the
process of angiogenesis, the effects of soluble p97 on the
structural protein Cav-1 and its tyrosine phosphorylated state
(pCav-1) was examined by Western blot analysis (FIGS. 16A and 16B).
The Cav-1 level was increased by 50% and 37% following soluble p97
treatment in HMEC-1 at 10 and 100 nM, respectively (FIG. 16A). The
pCav-1 levels remained however unchanged in soluble p97-treated
HMEC-1 as compared to control cells (FIG. 16B). Because Cav-1 has
been previously implicated as a tonic inhibitor of the ERK 1/2 MAP
kinase cascade involved in angiogenesis, the effects of soluble p97
on ERK 1/2 protein expression and phosphorylation levels was
evaluated by Western blot analysis (FIGS. 16C and 16D). The ERK 1/2
level was unchanged following soluble p97 treatment in HMEC-1 (FIG.
16C). In contrast, the pERK 1/2 level was significantly decreased
by 25% and 40% following soluble p97 treatment in HMEC-1 at 10 and
100 nM (FIG. 16D), respectively. Thus, these results show that
soluble p97 affects differently the expression of Cav-1 and ERK
1/2, two proteins involved in the setting of angiogenesis.
Soluble p97 Down-Regulated eNOS Protein Expression as well as
VEGFR-2 and VEGF-A mRNA Expression.
[0131] Cav-1 is also known to be an endogenous inhibitor of eNOS, a
protein related to many physiological and pathological functions,
including angiogenesis. Since soluble p97 modulates Cav-1
expression, the effect of soluble p97 on eNOS protein expression
was assessed by Western-blot analysis (FIG. 17A). Soluble p97, at
10 and 100 nM, reduced eNOS levels by about 30% and 50%,
respectively. In FIGS. 17A and 17B, HMEC-1 were treated for 18 hrs
with or without soluble p97. Following treatment, proteins from
cell lysates were solubilized and resolved by SDS-PAGE.
Immunodetection of eNOS (17A) was performed as described in the
Materials and Methods section. Results were expressed as a
percentage of protein expression detected in soluble p97-treated
cells compared to untreated cells. Data represent the means.+-.SD
of results obtained from three different experiments. Statistically
significant differences are indicated by **p<0.01, ***p<0.001
(Student's t-test).
[0132] Furthermore, eNOS has been suggest to play a predominant
role in VEGF-induced angiogenesis. Because immunodetected levels of
eNOS are reduced in soluble p97-treated HMEC-1 cells, the effect of
soluble p97 on the mRNA levels of VEGF-A and its receptor, the
VEGFR-2 (FIG. 17B) was estimated by RT-PCR. Following an incubation
of 18 hours with or without 100 nM soluble p97, soluble p97 reduced
VEGFR-2 and VEGF-A mRNA levels in HMEC-1 cells. In FIG. 17B,
following treatment, total RNA was isolated from HMEC-1 and gene
products were amplified by RT-PCR as described in the Materials and
Methods section. Results obtained from a representative experiments
are shown (N=3). These results indicate that soluble p97 affects
the expression of key players associated with angiogenesis,
including the protein expression levels of eNOS as well as the mRNA
levels of VEGFR-2 and VEGF-A.
[0133] The results presented herein suggest a mechanism by which
soluble p97 inhibits HMEC-1 cell migration as well as HMEC-1 and
HUVEC capillary-like tube formation. Soluble p97 could affect the
turn-over of LRP and u-PAR leading to a decreased capacity of
plasminogen activation at the cell surface (FIG. 18). In addition,
soluble p97 treatment affects EC phenotype by affecting Cav-1, pERK
1/2, eNOS, VEGF-A and VEGFR-2.
[0134] In FIG. 18, the schematic representation summarizes the
results obtained in the present study after soluble p97 treatment.
{circle around (1)} soluble p97 treatment decreases the total u-PAR
and LRP expression levels in cell lysates, as assessed by
Western-blotting. {circle around (2)} Since the total LRP levels
decreased, the cell surface level of LRP also decrease. It is well
established that LRP mediates the internalization of u-PAR. {circle
around (3)} Since cell surface LRP levels decreased, it was
postulated that the LRP-mediated endocytosis of u-PAR also
decreased. {circle around (4)} The diminished rates of u-PAR
endocytosis increased the total u-PAR level at the cell surface, as
assessed by FACS analysis. {circle around (5)} Since u-PAR is not
internalized by LRP, soluble p97 decreased the free u-PAR level at
the cell surface. {circle around (6)} The decreases free u-PAR
level at the cell surface lead to a decreased capacity of EC to
activate plasminogen. The net effect of soluble p97 treatment on
the u-PAR/LRP system lead to an inhibition of EC migration and
morphogenic differentiation of EC into capillary-like
structure.
Soluble p97 Causes Endothelial Cell Detachment and Extracellular
Matrix Degradation
[0135] So far, It has been shown herein that soluble p97 stimulates
plasminogen activation both in vitro and on endothelial cells.
Increased plasmin formation has been implicated in endothelial cell
detachment. Therefore, the effects of soluble p97 on endothelial
cell adhesion in the absence and presence of plasminogen (FIG. 19)
was studied. While soluble p97 or plasminogen alone did not induce
cell detachment, co-treatment of the endothelial cells with
plasminogen, tPA and soluble p97 resulted in an increase cell
detachment compared to control or tPA and plasminogen combination
(FIG. 19A). The plasminolytic activity measured in FIG. 19B, showed
that it is strongly increased when soluble p97 is added to tPA and
plasminogen. Immuodetections of plasminogen and plasmin (FIG. 19C)
indicate that the addition of soluble p97 increases the generation
of plasmin which lead to matrix degradation and cell detachment. In
FIG. 19, addition of soluble p97 stimulates HMEC-1 detachment (19A)
and plasminolytic activity in cell media (19B) in presence of
plasminogen and tPA. Photos (original magnification, .times.100)
obtained from a representative experiment are shown. In FIG. 19C,
following the treatment with or without soluble p97, proteins from
cell media were resolved by SDS-PAGE and immunodetection of
plasminogen and plasmin was performed as described in the Materials
and Methods section. Immunodetections obtained from a
representative experiment are shown. Results were expressed as a
percentage of protein expression detected. Data represent the
means.+-.SD of results obtained from three different experiments.
Statistically significant differences are indicated by *p<0.05,
***p<0.001 (Student's t-test).
[0136] Inhibitors of plasmin (alpha2-antiplasmin) and MMPs (EGCG
and Ilomastat) block the effects of soluble p97 on endothelial cell
detachment (FIG. 20A). In FIG. 20A, HMEC-1 detachement was
performed in presence of three different inhibitors, namely,
.alpha.2-antiplasmin, EGCG, and ilomastat. Photos (original
magnification, .times.100) obtained from a representative
experiment are shown. In FIG. 20B, following the treatments,
plasminolytic activity in cell media was measured as described in
the Materials and Methods section. Data represent the means.+-.SD
of results obtained from three different experiments. Statistically
significant differences are indicated by *p<0.05, ***p<0.001
(Student's t-test). The observed detachment of endothelial cells is
mediated by extracellular matrix degradation. As an important
component of the extracellular matrix involved in cell attachment,
fibronectin is degraded by MMPs.
[0137] Fibronectin degradation was studied in lysates of soluble
p97-treated endothelial cells by Western blotting. Whereas only
small amounts of fibronectin degradation products were generated in
the presence of plasminogen alone, co-treatment with tPA and
soluble p97 potently increased fibronectin degradation (FIG. 21).
In FIG. 21, HMEC-1 lysate from soluble p97 stimulated detachment
were resolved by SDS-PAGE and immunodetection of fibronectin was
performed as described in the Materials and Methods section.
Immunodetections obtained from a representative experiment are
shown. Results were expressed as a percentage of protein expression
detected.
[0138] Overall, these results (FIGS. 20 and 21) indicate that
soluble p97 stimulates plasmin- and MMP-dependent endothelial cell
detachment.
[0139] Consequently, these are the first data indicating that
exogenous human recombinant soluble p97 have anti-angiogenic
properties, by affecting the morphogenic differentiation of EC into
capillary-like structures, by interfering with key proteins
involved in angiogenesis and by inducing EC detachment.
Example VIII
Melanotransferrin Increases Human Blood Clot tPA-Fibrinolysis
[0140] Regulation of plasminogen is a key element in blood clot
fibrinolysis. In the present invention, potential interactions
between human recombinant p97 with components of the plasminogen
activator system in relation with fibrinolysis were investigated.
By using biospecific interaction analysis, it is demonstrated
herein that p97 interacts with immobilized plasminogen. Kinetics
analysis of the biosensorgrams using two state conformation change
model shows an apparent equilibrium dissociation constant K.sub.D
of 2.6.times.10.sup.-7 M for this interaction (FIG. 22). Moreover,
soluble p97 increased the tPA-dependent plasminogen activation.
This induction by p97 is inhibited by the monoclonal antibody L235
directed against p97 indicating that the increase in the
plasminolytic activity is specific to p97 (FIGS. 23A, 23B and 23C).
p97 also enhanced the tPA fibrinolysis of plasma and fibrin clots
(FIG. 26). The thromboelastography of fibrinolysis and clot
strength were evaluated with or without p97 (FIG. 26). Complete
lysis time (CLT) was reduced in the IVM (in vitro model) and plasma
by 50% and 20% respectively when p97 was added to tPA. There was
also a difference in the fibrinolysis by tPA at 30 min (LY30) in
both models when p97 was added. The LY30 was enhanced by 5- and
2-fold in both artifical and blood clots, respectively. These
results indicates that p97, by interacting with plasminogen,
enhanced plasminogen activity by tPA reduced time of thrombolysis.
In conclusion, these results demonstrate the potential of the
present invention in new treatments of arterial disease and
thrombosis and to reduce the damages to occluded hearth
tissues.
Interaction between p97 and Plasminogen Using Biospecific
Interaction Analysis in Real-Time
[0141] Plasminogen was immobilized on BIAcore with standard
coupling procedures. Various concentrations of p97 were injected
over immobilized plasminogen. The estimated constant of
dissociation (K.sub.D) estimated from these curves for the
interaction between p97 and immobilized plasminogen is 275 nM. The
results of this experiment are shown in FIG. 22.
Melanotransferrin (p97) Increases the Plasminogen Activation by
Tissue Plasminogen Activator (tPA)
[0142] Hydrolysis of the peptide VKL was measured in the presence
of p97 alone, tPA and tPA+p97. As shown in FIG. 23A, in the
presence of p97 the plasminogen activation by tPA was increased by
4-fold. As shown in FIG. 23C, the plasminogen activation by tPA was
increased in a dose-dependent manner by p97 with half-maximal
stimulation occurring at 12.+-.3 nM.
Inhibition of the p97 Effect by the Monoclonal Antibody L235
[0143] The plasmin activity was measured in the presence of tPA and
p97 with the monoclonal antibody directed against p97 (mAb L235) or
a non-specific mouse IgG (mouse IgG). As shown in FIG. 23B, the
induction caused p97 of the plasminogen activation by tPA is
inhibited by the monoclonal antibody directed against p97
indicating that this induction is specific to p97.
p97 Increases Clot Fibrinolysis Induced by tPA
[0144] The effect of p97 on fibrinolysis was measured using a
thromboelastograph. In the thromboelastography analysis (TEG), 320
.mu.l of citrated plasma or artificial clot model (8.2 .mu.M
fibrinogen, 2 .mu.M glu-plasmingen and 0.4 .mu.ml thrombin) was
transferred into analyser cups with tPA (4.5 nM) and in the
presence or absence of p97 (1 .mu.M). The cups were placed in
computerized dual-channel TEG analyzer (model 5000; Haemoscope
Corp., Niles, Ill.). In one of the cups (channel 1), tPA was added,
in another cup (channel 2) p97 and tPA were added. All cups
containing 20 .mu.l 0.2M CaCl.sub.2 were prewarmed to 37.degree. C.
and analyzed simultaneously. The TEG variables collected from each
sample included: CLT (clot lysis time), G (clot strength or Shear
elastic modulus in dyn/s.sup.2, defined as G=(5000 A)/(100-A)),
LY30 and LY 60 (percent of clot lysis at 30 and 60 min after
maximum clot strength is achieved). As shown in FIG. 26A, when p97
was added to the artificial clot, the clot lysis at 30 min was
increased by 5-fold. As shown in FIG. 26B, in the presence of p97,
the lysis at 30 min of human blood clot by tPA was increased by
2-fold.
[0145] Because soluble p97 interacts with glu-plasminogen, the
inventors have investigated whether human recombinant p97 might
affect fibrinolysis and clot permeation. To show that soluble p97
could modulate fibrinolysis, the impact of human recombinant
soluble soluble p97 on plasminogen activation by tPA (FIG. 23A) was
first determined. After 180 minutes, the addition of soluble p97
increased by 6-fold the plasminogen activation by tPA measured by
the hydrolysis of the VLK-peptide. Soluble p97 alone has no
proteolytic or plasmin-like activity. The induction of
tPA-dependent plasminogen activation by soluble p97 was also
measured in the presence of the mAb L235 directed against soluble
p97 or a non-specific IgG (FIG. 23B). The mAb L235, at 50 nM,
inhibited by 80% the effect of soluble p97 on plasminogen
activation by tPA. These results suggest that the effect of soluble
p97 on plasminogen activation is rather specific and involves the
conformational epitope recognizes by the mAb L235. In addition,
plasmin activities measured as a function of time allowed us to
extract initial rates. These rates were plotted as a function of
soluble p97 concentrations (FIG. 23C). Soluble p97 stimulated the
tPA-dependent conversion of plasminogen to plasmin in a
dose-dependent manner with half-maximal stimulation occurring at
53.+-.22 nM. The effect of soluble p97 on plasmin formation by tPA
in the presence of various concentrations of plasminogen (FIG. 23D)
was further evaluated. Initial rates of plasmin activity plotted as
a function of plasminogen concentrations indicate that soluble p97
decreases the apparent K.sub.m of tPA for plasminogen by 5-fold
from 280 to 52 nM. In FIG. 23A, the plasminolytic activity of tPA
(60 ng) was measured without (.smallcircle.) or with 1 .mu.g/ml p97
( ) in the presence of Plg (0.5 .mu.g). The reaction was performed
in a final volume of 200 .mu.l as described in the Materials and
Methods section. The plasminolytic activity in the presence of p97
alone was also measured (.box-solid.). In FIG. 23B, the
plasminolytic activity of tPA was measured in the presence of p97
(5 .mu.g/ml) and either the mAb L235 (.smallcircle.) or a
non-specific mouse IgG ( ). The reaction was performed in a final
volume of 200 .mu.l as described in the Materials and Methods
section. In FIG. 23C, plasmin activity induced by tPA was
determined by measuring VLK-hydrolysis in the presence of various
p97 concentrations. In FIG. 23D, initial rates of VLK-hydrolysis
during Plg activation by tPA were measured without (.smallcircle.)
or with 50 nM p97 ( ) in the presence of various concentrations of
Plg. Data are shown as means of 3 experiments.
[0146] To further characterize the soluble p97 effects on the
action of tPA in fibrinolysis, the effect of soluble p97 on a
radial tPA-fibrinolysis assay (FIG. 24) was evaluated.
[0147] The addition of soluble p97 to tPA enhances its action and
leads to an increase perforation of the fibrin-clot (FIG. 24A).
Surprisingly, in this experiment performed without tPA, soluble p97
in the presence of plasminogen creates a perforation of the
fibrin-clot. Moreover, the size of the perforation increases as a
function of soluble p97 concentration (FIG. 24B). In absence of
plasminogen and tPA, the fibrin-clot is unaffected by soluble p97
alone. To determine whether soluble p97 has an intrinsic
fibrinolytic activity, the release of fibrin fragments from clots
labeled with [.sup.125I]-fibrin (FIG. 24C) was measured. In spite
of its ability to perforate the clot, soluble p97 alone does not
generate [.sup.125I]-fibrin fragments. However, soluble p97 in the
presence of plasminogen increases the release of [.sup.125I]-fibrin
fragments by 2.5 fold following plasminogen activation by tPA.
[0148] The impact of soluble p97 on clot fibrinolysis by tPA was
also measured ex vivo (FIG. 25). The addition of soluble p97
increases by 2.5-fold the action of tPA. In FIG. 25, the
fibrinolytic activity of tPA (1 nM) on plasma clot fibrinolyis was
measured ex vivo in the presence of increasing concentrations of
p97.
[0149] In the blood coagulation system, the tissue-type plasminogen
activator (tPA) is associated with fibrinolysis. tPA, mainly
express by endothelial cells, cleaves the circulating plasminogen
to the active proteinase plasmin which is the major enzyme
responsible for the proteolytic degradation of the fibrin fiber.
Currently, tPA is a stroke therapy which efficacy may be limited by
neurotoxic side effects. Since soluble p97 potentialize plasminogen
activation by tPA, the impact of soluble p97 on clot formation and
lysis by thromboelastography analysis (TEG) has been evaluated
using first an artificial fibrin-clot model (FIG. 26A). This model
allowed to monitor the effect of soluble p97 on tPA-fibrinolysis in
the absence of plasmin inhibitor. The fibrin clot is formed by the
action of thrombin on fibrinogen and this clot also contains
glu-plasminogen (2 .mu.M). In FIG. 26, representative tracing
showing effects of p97 (1 uM) on the fibrinolysis of clot formation
under shear by TEG. In FIG. 26A illustrates a thrombelastogram of
the fibrin clot model and FIG. 26B illustrates a Thromboelastogram
of plasma recalcified after addition of 2 mM CaCl.sub.2. The
results shown here are representative of 3 experiments. The
monitoring of the TEG paramaters indicates that the addition of
soluble p97 increases the thrombolytic activity of tPA (Table 4).
In particular, when soluble p97 (1 .mu.M) is added to tPA, the
lysis of the clot after 30 min (LY30) after its complete formation
is 5 times higher whereas the complete lysis time (CLT) is 50%
shorter. The impact of soluble p97 on fibrin-clot dissolution using
human citrated plasma (FIG. 26B) was further evaluated. For these
analysis, CaCl.sub.2 is added to initiate the polymerisation of
plasma clot. The TEG parameters obtained for these experiments
(Table 4) indicate that the addition of soluble p97 to tPA causes a
30% decrease in the clot strength (G), increases twice the
fibrinolysis rate and reduces the CLT by 20%.
TABLE-US-00004 TABLE 4 Effects of p97 on thromboelastograph
parameters Conditions Parameters tPA tPA+ a. Artificial fibrin-clot
1. G d/sc 498 .+-. 7 446 .+-. 17 2. Lys (30) % 6.5 31.9 3. CLT min
54.7 30.3 b. Fibrin-clot with citrate- treated serum 1. G d/sc
13465 .+-. 1586 9560 .+-. 1626 2. Lys (30) % 4.3 .+-. 0.7 11.8 .+-.
4.0 3. CLT min 68.3 .+-. 1.6 49.1 .+-. 6.3
[0150] G (d/sc) is the maximum strength of the clot at maximum
amplitude of the TEG trace.
[0151] The present findings are significant for several reasons.
First, it was discovered that soluble p97, by interacting with
plasminogen, enhances its activation by tPA. Furthermore, it is
established that protein-protein interaction could positively
regulate the activity of an enzyme by inducing a conformational
change which lead to the exposure of active cryptic site. In
addition, the data presented here in the radial clot lysis assay
and the TEG analysis provide further evidence that soluble p97
positively regulates the tPA-dependent fibrinolysis by mainly
decreasing the clot strength and time of lysis. Overall, the data
indicate that soluble p97 increases the efficacy of the
anti-thrombolysis agent tPA.
[0152] Second, perforation of the clot by soluble p97 without any
release of fibrin fragments indicates that soluble p97 interaction
with plasminogen induces a change in the fibrin-clot structure.
Soluble p97 greatly facilitates the tPA action, leading to a
localized and accelerated fibrinolysis.
[0153] In conclusion, the data presented herein indicates that
human recombinant soluble p97 is as a switch activator of
plasminogen since its interaction with plasminogen leads to an
increase in the clot permeation and fibrinolysis by tPA.
Thrombolysis with blood clot dissolving agent like tPA can reduced
mortality in acute myocardial infraction.
Example IX
Inhibition of Angiogenesis by Melanotransferrin
[0154] During angiogenesis, cells must proliferate and migrate to
finally invade the surrounding extracellular matrix (ECM).
Moreover, metastasis is associated with tissue remodeling and
invasion. In fact, when processing from migration to invasion, an
additional complexity is added, as invasion comprises not only cell
locomotion, but also the active penetration of cells into ECM.
Cell Culture
[0155] Cells were cultured under 5% CO.sub.2/95% air atmosphere.
Ovary hamster cells expressing or not the membrane type
melanotransferrin (respectively mMTf-CHO and mock-CHO cells) were
cultured with Ham F12 suplemented with 1 mM HEPES and 10% of calf
serum (CS).
Cell Invasion Assay
[0156] Invasion was performed with CHO transfected with membrane
bound Mtf (p97) (mMtf-CHO) or with the vector only (MOCK-CHO) using
Transwell filters (Costar, Corning, N.Y.: 8 .mu.m pore size)
precoated with 50 .mu.g Matrigel (BD Bioscience). The transwell
filters were assembled in 24-well plates (Falcon 3097, Fisher
Scientific, Montreal, Quebec, Canada) and the lower chambers filled
with 600 .mu.L cell culture medium containing 10% calf serum with
or without 100 nM soluble p97 as well as 50 nM IgG1 or L235. To
study the effect of soluble p97 and L235 on cell invasion, CHO
cells were harvested by trypsinization and centrifuged.
1.times.10.sup.5 cells were resuspended in 200 .mu.L cell culture
medium without serum and containing or not 100 nM soluble p97 as
well as 50 nM IgG1 or L235 and added into the upper chamber of each
Transwell. The plates were than placed at 37.degree. C. in 5%
CO.sub.2/95% air for 48 hours. Cells that have invaded to the lower
surface of the filters were fixed with 3.7% formaldehyde in PBS,
stained with 0.1% crystal violet/20% MeOH, and count (4 random
fields per filter) with Norten Eclipse digital software.
Transendothelial Invasion Assay
[0157] Mock-CHO and mMTf-CHO cells were seeded onto the
<<blood brain barrier in vitro model>> at 100 000
cells/mL in presence of 5 mM Hoescht in supplemented Ham F12 medium
with or without 50 nM of L235 (antibody directed against
melanotranferrin). Cells were then incubated for 48 hours at
37.degree. C. 5% CO.sub.2. After the incubation, cells were fixed
in 3.7% formaldehyde in phosphate-buffered saline (PBS,
Ca.sup.+2/Mg.sup.+2 free) for 30 min and the plate were kept in the
dark. The formaldehyde was then removed and cells that had migrated
on the lower surface of the filter were then visualized with a
Nikon Eclipse TE2000-U.TM. microscope-stage automatic thermocontrol
system (Shizuoka-ken, Japan) at a 100.times. magnification using a
Q IMAGING RETIGA.TM. camera, and counted with the program Northern
Eclipse (Mississauga, Ontario).
[0158] As can be seen on FIG. 27, these results suggest that
endogenous membrane bound p97 stimulates CHO cell invasion. The
invasion is inhibited by L235, indicating that membrane p97
participates directly in cell invasion. Moreover, recombinant
soluble p97 could inhibit the invasion of these cells by competing
with endogenous membrane bound p97.
[0159] In FIG. 27, cell invasion assay were performed as described
in Material and Methods. Cell invasion assay was performed in
presence of 50 nM L235 (27A) and 100 nM soluble p97 (27B). Data
represents means.+-.SDs. ***P<0.001, *P<0.05 (Student t
test).
Transendothelial Invasion on the BBB In Vitro Model
[0160] Since soluble p97 affected plasminogen activation, the
inventors investigated whether soluble p97 might modulate brain
invasion. Using the blood-brain barrier (BBB) in vitro model, CHO
cell invasion was examined. Following a 48 hours incubation,
mMTf-CHO cells expressing the membrane associated melanotransferrin
show a higher invasive character through the BBB model,
comparatively to control cells (mock-CHO cells). Following the
addition of L235, an antibody raised against the melanotransferrin,
the invasive potential of membrane bound p97 transfected cells seem
to be stopped, demonstrating a important role for endogenous
membrane bound melanotransferrin in mechanisms leading to cell
invasion. The results are illustrated in FIG. 28. In FIG. 28, cells
that migrated were visualized by fluorescent microscopy and counted
(4 random fields per filter) with Norten Eclipse digital software
as described hereinabove.
Discussion
[0161] The data clearly show that both pro-uPA and plasminogen
interact with p97 and that these interactions are specific since no
interaction between p97 and other proteins including tPA, PAI-1,
plasmin, angiostatin, BSA, or ovalbumin could be measured. These
results are the first to describe potential interactions between
p97 and proteins of the uPA system.
[0162] In addition to its interaction with pro-uPA and plasminogen,
p97 stimulates plasminogen activation by decreasing the K.sub.m of
pro-uPA for plasminogen and by increasing the V.sub.max of the
reaction. The conversion of pro-uPA to two-chain uPA occurs by
proteolytic cleavage of a single peptide bond (Lys158-Ile159 in
human uPA). This conversion can be catalyzed by plasmin or several
other proteases such as plasma kallikrein, blood coagulation factor
XIIa, cathepsin B, cathepsin L and prostate-specific antigen. In
the present invention the SPR assay, the enzymatic assay and
electrophoresis experiments all indicate that p97 induces a
conformational change that increases pro-uPA activity without any
apparent cleavage of pro-uPA. The two-state conformational model
gave the best fits for the interactions of both pro-uPA and
plasminogen with immobilized p97 on the BIAcore. Such good fits of
experimental data to a multi-state model of interaction are an
indication that a conformational change is taking place.
Interestingly, the fragments of plasminogen generated by adding p97
were different from the plasminogen degradation by pro-uPA alone.
These biochemical analyses further suggest that p97 could also be
seen as a cofactor in uPA-dependent plasminogen activation.
[0163] The uPA/uPAR system has been involved in several
pathological and physiological processes which require cell
migration, such as tumor cell invasion and metastasis. Several
reports showed that the uPA/uPAR system plays a key role in signal
transduction as well as in regulation of melanoma cell migration
and angiogenesis. As shown in the present invention, when p97 is
added to both compartments of the Boyden chamber migration of
HMEC-1 is inhibited by more than 50%. Thus, given the important
role of plasmin, a protein like p97 which targets the formation of
plasmin and acts on the migration of endothelial cells as well as
of SK-MEL28 cells will thus affect angiogenesis and cancer
progression. It was also observed in the present invention that the
basal capacity for plasminogen activation by HMEC-1 decreased
following p97 treatment. A recent study demonstrated that the
expression of LDL receptor-related protein 1B (LRP1B), a new member
of the LDL receptor family, lead to an accumulation of uPAR on the
cell surface which event inhibits the migration of CHO cells. From
these results, it was proposed that LRP1B negatively regulates uPAR
regeneration and function whereas the net results of uPAR
regeneration seems to depend on the relative expression of the two
receptors.
[0164] Recently, it was shown that when glu-plasminogen is bound to
cell surfaces, plasmin generation by plasminogen activators is
markedly stimulated compared to the reaction in solution. This is a
key element for cell migration where the process of "grip and go"
would play an important role. The process of plasminogen activation
system is regulated by two different mechanisms: 1) cell
surface-binding sites which facilitate the productive catalytic
interactions with plasminogen and thereby increases plasmin
generation, and 2) protein inhibitors such as serpin inhibitors
which restrict the activities of the proteases. In light of this,
soluble p97 participates in the activation of plasminogen without
being in the pericellular environment (FIG. 29A). The present
invention also indicates that the migration and the plasminolytic
activity of cells expressing p97 are inhibited by mAb L235,
indicating that endogenous, membrane-bound p97 are involved in
these processes which are associated with cancer and angiogenesis
(FIG. 29B). Moreover, both the migration of HMEC-1 and the
plasminolytic activity are diminished when exogenous p97 is added,
indicating that soluble p97 affects the regulation of plasminogen
activation at the cell surface (FIG. 29C). Thus, by breaking the
equilibrium between soluble p97 and membrane bound p97, it is
possible to affect cell migration of HMEC-1 and SK- MEL28
cells.
[0165] In conclusion, these are the first results indicating that
p97 interacts with pro-uPA as well as with plasminogen and
regulates the activation of plasminogen by pro-uPA. As shown in the
present invention migration of HMEC-1 and SK-MEL28 cells is
inhibited by mAb L235 and soluble p97, indicating that active and
functional p97 participates in this process. Collectively, the
results thus indicate that the balance between membrane-bound and
soluble p97 could affect cell migration.
[0166] As mentioned above, these are the first data indicating that
exogenous human recombinant soluble p97 have anti-angiogenic
properties, by affecting the morphogenic differentiation of EC into
capillary-like structures, by interfering with key proteins
involved in angiogenesis and by inducing EC detachment.
[0167] Also as mentioned previously, the data presented herein
indicates that human recombinant soluble p97 can be seen as a
switch activator of plasminogen since its interaction with
plasminogen leads to an increase in the clot permeation and
fibrinolysis by tPA. Thrombolysis with blood clot dissolving agent
like tPA can reduced mortality in acute myocardial infraction.
However, damage can occur since the blow flow is restored by only
60% after 90 min. The results presented herein suggest that soluble
p97 could increase the efficiency of the thrombolytic agent (tPA)
when co-administrated. Furthermore, since the reoccluded clots are
usually more resistant to tPA, soluble p97 administration could
counter this adverse effect by increasing the therapeutic window of
tPA. According to the American Heart Association, two million
Americans suffer from atrial fibrillation, in which the two small
upper chambers of the heart quiver instead of beating effectively.
Blood in these quivering chambers can clot, travel and obstruct
blood circulation. This phenomenon can also happen in the vein,
where the clot would obstruct as well. Soluble p97 would enhance
tPA effectiveness and broaden its therapeutic window. P97 has also
the power to modify clot structure. Moreover, p97-containing gel
could also be used to control new blood vessel growth and to reduce
the need for coronary bypass surgery and provide effective
treatment for a debilitating cardiovascular disease.
[0168] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
Sequence CWU 1
1
191738PRTHomo Sapiens 1Met Arg Gly Pro Ser Gly Ala Leu Trp Leu Leu
Leu Ala Leu Arg Thr1 5 10 15Val Leu Gly Gly Met Glu Val Arg Trp Cys
Ala Thr Ser Asp Pro Glu 20 25 30Gln His Lys Cys Gly Asn Met Ser Glu
Ala Phe Arg Glu Ala Gly Ile 35 40 45Gln Pro Ser Leu Leu Cys Val Arg
Gly Thr Ser Ala Asp His Cys Val 50 55 60Gln Leu Ile Ala Ala Gln Glu
Ala Asp Ala Ile Thr Leu Asp Gly Gly65 70 75 80Ala Ile Tyr Glu Ala
Gly Lys Glu His Gly Leu Lys Pro Val Val Gly 85 90 95Glu Val Tyr Asp
Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val 100 105 110Val Arg
Arg Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys 115 120
125Ser Cys His Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val
130 135 140Gly Tyr Leu Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys
Asp Val145 150 155 160Leu Lys Ala Val Ser Asp Tyr Phe Gly Gly Ser
Cys Val Pro Gly Ala 165 170 175Gly Glu Thr Ser Tyr Ser Glu Ser Leu
Cys Arg Leu Cys Arg Gly Asp 180 185 190Ser Ser Gly Glu Gly Val Cys
Asp Lys Ser Pro Leu Glu Arg Tyr Tyr 195 200 205Asp Tyr Ser Gly Ala
Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val 210 215 220Ala Phe Val
Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr225 230 235
240Leu Pro Ser Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu
245 250 255Cys Arg Asp Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln
Cys His 260 265 270Leu Ala Arg Val Pro Ala His Ala Val Val Val Arg
Ala Asp Thr Asp 275 280 285Gly Gly Leu Ile Phe Arg Leu Leu Asn Glu
Gly Gln Arg Leu Phe Ser 290 295 300His Glu Gly Ser Ser Phe Gln Met
Phe Ser Ser Glu Ala Tyr Gly Gln305 310 315 320Lys Asp Leu Leu Phe
Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala 325 330 335Thr Gln Thr
Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met 340 345 350Lys
Gly Leu Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp 355 360
365Cys Val Leu Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val
370 375 380Ala Phe Arg Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val
Ser Ala385 390 395 400Lys Ser Pro Gln His Cys Met Glu Arg Ile Gln
Ala Glu Gln Val Asp 405 410 415Ala Val Thr Leu Ser Gly Glu Asp Ile
Tyr Thr Ala Gly Lys Lys Tyr 420 425 430Gly Leu Val Pro Ala Ala Gly
Glu His Tyr Ala Pro Glu Asp Ser Ser 435 440 445Asn Ser Tyr Tyr Val
Val Ala Val Val Arg Arg Asp Ser Ser His Ala 450 455 460Phe Thr Leu
Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe465 470 475
480Gly Ser Pro Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg
485 490 495Gly Phe Ile Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val
Ser Glu 500 505 510Phe Phe Asn Ala Ser Cys Val Pro Val Asn Asn Pro
Lys Asn Tyr Pro 515 520 525Ser Ser Leu Cys Ala Leu Cys Val Gly Asp
Glu Gln Gly Arg Asn Lys 530 535 540Cys Val Gly Asn Ser Gln Glu Arg
Tyr Tyr Gly Tyr Arg Gly Ala Phe545 550 555 560Arg Cys Leu Val Glu
Asn Ala Gly Asp Val Ala Phe Val Arg His Thr 565 570 575Thr Val Phe
Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala 580 585 590Glu
Leu Arg Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg 595 600
605Ala Glu Val Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro
610 615 620His Ala Val Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val
Tyr Gly625 630 635 640Leu Leu Asp Lys Ala Gln Asp Leu Phe Gly Asp
Asp His Asn Lys Asn 645 650 655Gly Phe Lys Met Phe Asp Ser Ser Asn
Tyr His Gly Gln Asp Leu Leu 660 665 670Phe Lys Asp Ala Thr Val Arg
Ala Val Pro Val Gly Glu Lys Thr Thr 675 680 685Tyr Arg Gly Trp Leu
Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met 690 695 700Ser Ser Gln
Gln Cys Ser Gly Ala Ala Ala Pro Ala Pro Gly Ala Pro705 710 715
720Leu Leu Pro Leu Leu Leu Pro Ala Leu Ala Ala Arg Leu Leu Pro Pro
725 730 735Ala Leu221DNAArtificial Sequenceprimer sequence
2agaagtagca ggaccagagg g 21321DNAArtificial Sequenceantisense
primer sequence 3tcagtaccca ggcagttatg c 21422DNAArtificial
Sequenceprimer sequence 4tctctccctt ctccaaagac cc
22522DNAArtificial Sequenceantisense primer sequence 5tcaatgagtc
cagccagtca gc 22622DNAArtificial Sequenceprimer sequence
6cggagcagtg tggcttattt tc 22722DNAArtificial Sequenceantisense
primer sequence 7caggtgtatt gggtgtcaag gc 22824DNAArtificial
Sequenceprimer sequence 8ggacccaaca agttcaagtg tcac
24922DNAArtificial Sequenceantisense primer sequence 9aagaagaggt
aggcgatgga gc 221025DNAArtificial Sequenceprimer sequence
10ccttgaagat gatggactac cctcg 251124DNAArtificial Sequenceantisense
primer sequence 11aaaacccaaa aaagcccccc cagc 241224DNAArtificial
Sequenceprimer sequence 12accgaggttg tgtgtgggtt agac
241322DNAArtificial Sequenceantisense primer sequence 13caggaagtgg
aaggtgtcgt tg 221420DNAArtificial Sequenceprimer sequence
14ccatcaccat cttccaggag 201520DNAArtificial Sequenceantisense
primer sequence 15cctgcttcac caccttcttg 201624DNAArtificial
Sequenceprimer sequence 16aaagacattg cgtggtcagg cagc
241723DNAArtificial Sequenceantisense primer sequence 17ggcatcataa
ggcagtcgtt cac 231824DNAArtificial Sequenceprimer sequence
18ccagcacata ggagagatga gctt 241924DNAArtificial Sequenceantisense
primer sequence 19ggtgtggtgg tgacatggtt aatc 24
* * * * *