From EntreMed, Inc., Rockville, Maryland 20850
Received for publication, January 26, 2001, and in revised form, April 18, 2001
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ABSTRACT |
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The mechanism of action of Endostatin, an
endogenous inhibitor of angiogenesis and tumor growth, remains unknown.
We utilized phage-display technology to identify polypeptides
that mimic the binding domains of proteins with which Endostatin
interacts. A conformed peptide (E37) was identified that shares an
epitope with human tropomyosin implicating tropomyosin as an
Endostatin-binding protein. We show that recombinant human
Endostatin binds tropomyosin in vitro and to
tropomyosin-associated microfilaments in a variety of endothelial cell
types. The most compelling evidence that tropomyosin modulates the
activity of Endostatin was demonstrated when E37 blocked greater than
84% of the tumor-growth inhibitory activity of Endostatin in the
B16-BL6 metastatic melanoma model. We conclude that the E37
peptide mimics the Endostatin-binding epitope of tropomyosin and blocks
the antitumor activity of Endostatin by competing for Endostatin
binding. We postulate that the Endostatin interaction with tropomyosin
results in disruption of microfilament integrity leading to inhibition
of cell motility, induction of apoptosis, and ultimately inhibition of
tumor growth.
Angiogenesis, the formation of new blood vessels from existing
capillaries, is required for tumors to expand beyond 1-2
mm3 in size and is the principal determinant for tumor
growth (1, 2). Consequently, antiangiogenic molecules offer promise as novel therapeutic modalities for the treatment of cancer. Endostatin, a
20-kDa cleavage fragment of collagen XVIII first identified in the
conditioned medium of hemangioendothelioma cells (3), is a potent
inhibitor of tumor angiogenesis and growth in murine models, and
recombinant human (rh)1
Endostatin is currently being evaluated in the clinic (4, 6). Both
recombinant murine (rm) and rhEndostatin have been successfully
produced and shown to inhibit endothelial cell proliferation and
migration in vitro, as well as tumor growth in in
vivo tumor models (3, 5, 6). In fact, the first demonstration of tumor dormancy was achieved with systemic treatment of rmEndostatin (7).
It is increasingly recognized that alterations in the actin
cytoskeleton play a crucial role regulating the proliferation and
migration of endothelial cells (8, 9). Tropomyosins are a large family
of proteins; at least 20 different isoforms exist that are generated by
alternative splicing of a multigene family (Refs. 10, 11 and references
therein). They bind the The mechanism of action of the antiangiogenic activity of
Endostatin is unknown. To elucidate the mode of action of Endostatin, we screened a phage-display library to identify the binding domains of
proteins with which Endostatin potentially interacts. Here we present
evidence that human tropomyosin isoform 3 (hTM3) shares an epitope with
an Endostatin-binding peptide, demonstrate an interaction between
rhEndostatin and hTM3 in vitro, and show that rhEndostatin
protein binds tropomyosin-containing mircofilaments of endothelial
cells. We further show that the peptide mimotope of the hTM3
Endostatin-binding site blocks the antitumor activity of rhEndostatin
in vivo and suggest that the antitumor activity of
Endostatin results from an interaction with tropomyosin-containing microfilaments that leads to inhibition of microfilament function and
an induction of apoptosis.
Screening of the Phage-display and cDNA Libraries--
The
Ph.D.-C7C, a disulfide-constrained 7-mer phage-display library (New
England BioLabs, Beverly, MA), was screened as recommended by the
manufacturer. Following a third round of amplification, individual
phage were isolated, and the peptide sequences were deduced by DNA sequencing.
Peptide Synthesis and Generation of Anti-peptide Sera--
The
E37 (CTHWWHKRCGGGS) and control (CSNSDKPKCGGGS) peptides were
synthesized, cyclized at high dilution, and purified to at least 95%
purity by high-performance liquid chromatography (Infinity Biotech
Research and Resource, Aston, PA). The E37 peptide was coupled to
hemocyanin from keyhole limpets (Sigma) in the presence of
glutaraldehyde and used to immunize New Zealand White rabbits.
cDNA Library Screening--
The Lambda ZAP® II
(Stratagene, La Jolla, CA) bFGF-stimulated HUVEC cDNA library was
constructed by directional cloning of oligo (dT)-primed cDNA into
EcoRI and XhoI cloning sites. 1.4 × 106 recombinant phage were screened for immunoreactivity to
E37 peptide antisera.
Native and Recombinant Proteins--
Plasmid p
The cDNA encoding hTM3 cDNA was amplified by PCR using forward
primer 596 (5'-ATGCCATATGGACGCCATCAAGAAG-3') and reverse primer 597 (5'-ATGCAAGCTTTCACATGTTGTTTAACTCCAG-3') and cloned into pET-21a(+) (Novagen, Madison, WI). BL21 (DE3) cells were transformed with the p6T
plasmid and grown in Luria-Bertani broth containing 50 µg/ml
carbenicillin (Novagen, Madison, WI). The cells were induced with 5 mM isopropyl-1-thio- ELISA--
Plates were coated with 5 µg/ml of bovine serum
albumin or rhEndostatin and blocked. Dilutions of Surface Plasmon Resonance (BIAcore)--
Interactions between
purified soluble rhEndostatin and rhTM3 were evaluated by surface
plasmon resonance using the BIAcore 3000 (BiaCore, Piscataway, NJ). The
purified rhTM3 was immobilized on the flow cell of a CM-5 BIAcore
biosensor chip. The running buffer was 0.01 M HEPES, pH
7.4, 0.15 M NaCl, and 0.005% polysorbate 20 (v/v). The
competition experiments involving soluble rhTM3 were performed using
PBS, pH 7.4, as running buffer. All measurements were performed at
25 °C. To correct for differences in bulk refraction all protein
preparations were passed over an activated and blocked flow cell to
which no protein had been coupled. Effects due to bulk shift were
subtracted from experimental data.
Fluorescent Staining of Endothelial Cells--
To detect the
binding of Alexa-labeled rhEndostatin 1 × 104 HUVEC,
HMVEC, and HAECs were plated on 1.5% gelatin-coated Nalge Nunc Lab Tek
II chamber slides (Naperville, IL) and fixed in 10% neutral buffered
formalin followed by a methanol wash. The chamber slides were incubated
in PBS/1% calf serum containing 40 µg/ml (2 µM) Alexa
488-labeled rhEndostatin3 for
1 h at room temperature. The slides were washed in PBS, coverslips mounted using fluorescent mounting media (Kirkegaard and Perry Laboratories), and cells were photographed under Alexa 488 excitation wavelength. Co-localization studies were performed as above except cells were incubated in PBS/1% calf serum containing 1:200 mouse anti-tropomyosin TM311 (Sigma) and 40 µg/ml of Alexa 488-labeled rhEndostatin followed by incubation with goat anti-mouse IgG (H+L) conjugated to Alexa 594 (Molecular Probes, Eugene, OR). The same fields
were photographed under Alexa 488 and Alexa 594 wavelengths. Competition experiments were done by including 100 µM E37
or control peptide in the 40 µg/ml (2 µM)
Alexa-rhEndostatin solution used to stain the cell.
In Vivo Studies--
C57BL/6J mice (n = 5) were
injected intravenously (i.v.) with 5 × 104 B16-BL6
cells/mouse. Three days later treatment commenced. 100 µl of
citrate-phosphate buffer (66 mM sodium phosphate, 17 mM citric acid, 59 mM NaCl, pH 6.2) containing
1.5 nmol of rhEndostatin and 100 µl of PBS containing either 0, 15, 75, or 375 nmol of control or E37 peptide were mixed and injected
subcutaneously (s.c.) daily for 11 days. All mice were then sacrificed,
their lungs removed, and the number of surface metastases counted.
Results were analyzed for statistical significance using the 2-tailed Student's t test.
To determine the pharmacokinetics, C57BL/6J mice (3 per time point)
were dosed s.c. with 1.5 nmol of rhEndostatin alone or in combination
with 375 nmol of either E37 or control peptide. Sera was collected
0.17, 0.5, 1, and 2 h following s.c. administration. Serum drug
levels were determined using an Endostatin EIA kit (CytImmune Sciences,
College Park, MD) according to the manufacturer's instructions.
Pharmacokinetic analyses of the serum concentration versus
time data were performed by nonlinear least square minimization using
RSTRIP (Version 4.03, MicroMath Scientific Software, Salt Lake City, UT).
An Endostatin-binding Peptide Shares an Epitope with hTM3--
A
cyclic (disulfide bond-constrained) random 7-mer peptide phage-display
library was screened to identify peptides that interacted with
rhEndostatin (6). The cyclic, disulfide-bonded peptide, E37
(CTHWWHKRCGGGS) was found to bind rhEndostatin. A BLAST search of the
National Center for Biotechnology Information database failed to
identify proteins with any significant homology to the primary sequence
of the E37 peptide. To identify proteins that share common epitope(s)
with the E37 peptide, rabbit antisera was raised against KLH-coupled
E37 peptide and used to probe an FGF-2 stimulated human umbilical vein
endothelial cell (HUVEC) cDNA expression library. DNA sequencing of
9 clones recognized by E37 antisera identified seven as being unique
cDNAs of different lengths encoding non-muscle human tropomyosin
isoform 3 (hTM3), suggesting that E37 and hTM3 share a common epitope.
A comparison of the primary sequence of hTM3 with that of the E37
peptide failed to reveal significant homology, suggesting that the E37
peptide sequence represents a conformational mimotope of the
Endostatin-binding domain of tropomyosin. To confirm that the E37
peptide and human tropomyosin share an epitope, immunoblots of lysate
from rhTM3 expressing E. coli ( rhEndostatin and rhTM3 Interact in Vitro--
We characterized the
interaction between rhEndostatin and tropomyosin by ELISA and showed
that rhEndostatin binds rhTM3-expressing bacterial lysate (
Surface plasmon resonance, with the BIAcore 3000, was used to assess
the kinetics of rhEndostatin/rhTM3 binding. 3200 resonance units (RU)
of purified rhTM3 were immobilized on the surface of a sensor chip via
amine coupling and rhEndostatin injected over the chip as analyte. The
rhEndostatin showed a dose-dependent increase in RU,
demonstrating the real-time binding of rhEndostatin to immobilized
rhTM3 (Fig. 3A). The
dissociation constant (KD) for the
rhEndostatin/rhTM3 interaction was calculated to be ~100 µM using a steady-state model. When, as a negative
control, similar concentrations of rhAngiostatin K1-3 were passed over
the rhTM3 chip, no evidence of specific binding to rhTM3 was observed
(Fig. 3B), demonstrating the specificity of the
rhEndostatin/rhTM3 interaction. To further evaluate the specificity of
rhEndostatin/rhTM3 binding, competition experiments using soluble rhTM3
were performed. Soluble rhTM3 competed for the binding of rhEndostatin
to a flow cell containing 2000 RU of immobilized rhTM3 in a
dose-dependent manner (Fig. 3C), further
demonstrating the specificity of the rhEndostatin/rhTM3 interaction.
Endostatin Binding and Tropomyosin Co-localize to Actin
Microfilaments--
Incubation of formalin-fixed HUVECs, human
microvascular endothelial cells (HMVECs), and human aortic endothelial
cells (HAECs) with biologically active Alexa FluorTM 488-labeled
rhEndostatin showed that, given access to the cytosolic compartment,
rhEndostatin displays a pattern of binding identical to that observed
in cells immunostained for tropomyosin or filamentous actin (Fig.
4, A-C). Fluorescently
labeled rhEndostatin and anti-tropomyosin antibodies co-localized to
HUVEC microfilaments (Fig. 4, D-E). Similar results were
obtained using anti-actin antibodies (data not shown). Thus, in fixed
cells, Endostatin-binding, tropomyosin, and actin co-localize to
microfilament bundles of the cell cytoskeleton. We postulate that
Endostatin, which is specifically internalized by endothelial cells
(13), binds tropomyosin to inhibit microfilament function, and as a
consequence, tumor angiogenesis.
To determine whether the binding of Alexa-labeled rhEndostatin to the
HUVEC cytoskeleton was the result of an interaction with tropomyosin
competition experiments were performed. A 50-fold molar excess of the
E37 peptide, but not the control peptide inhibited the binding of
rhEndostatin to HUVEC microfilaments (Fig.
5, A-C), thus demonstrating
the role tropomyosin plays in Endostatin binding to the HUVEC
cytoskeleton. This observation further confirms the role of the E37
mimotope in the interaction between Endostatin and hTM3.
E37 Peptide Blocks the Antimetastatic Activity of rhEndostatin in
Vivo--
To further evaluate the biological significance of the
interaction between E37 and Endostatin, we assessed the effect of the E37 peptide on the antitumor activity of rhEndostatin. rhEndostatin has
been shown previously to inhibit the growth of pulmonary metastases in
the murine B16-BL6 experimental metastasis model in a
dose-dependent manner (6). Daily administration of 1.5 nmol
of rhEndostatin (1.5 mg/kg/day) inhibited the growth of experimental
B16-BL6 lung metastases by greater than 70% when compared with
buffer-treated control mice (Fig. 6). The
number of metastatic lesions on the lungs of the mice treated with up
to 375 nmol/day of E37 peptide in the absence of rhEndostatin was not
statistically different from that of the control group, demonstrating
that the E37 peptide alone has neither tumor promoting or inhibitory
activity in this assay. Co-administration of rhEndostatin with E37
peptide dramatically blocked the tumor growth inhibitory activity of
the rhEndostatin (Fig. 6). Co-injection of 1.5 nmol of rhEndostatin
with 15, 75, and 375 nmol of the E37 peptide resulted in a
dose-dependent decrease in the antitumor activity of the
rhEndostatin of 11, 30.5, and 84.5%, respectively. The number of
pulmonary metastases in mice co-treated with rhEndostatin and either 75 or 375 nmol of E37 peptide (50- and 250-fold molar excess,
respectively) were significantly higher than those on the lungs of mice
treated with either rhEndostatin alone or in combination with 75 or 375 nmol of control peptide, respectively (p < 0.02). The
incidence of metastatic lesions on the lungs of mice co-injected with
1.5 nmol of rhEndostatin and up to 375 nmol of a cyclic
disulfide-bonded control peptide, or in mice treated with 1.5 nmol of
rhEndostatin alone, was statically indistinguishable (p > 0.11). Thus, in the experimental B16-BL6 lung metastases model, the
antitumor activity of rhEndostatin is blocked in a
dose-dependent manner by co-injection of E37 peptide. We
conclude that because the E37 peptide mimics the binding pocket of TM3,
E37 peptide competes with tropomyosin for rhEndostatin binding,
resulting in disruption of the tropomyosin/rhEndostatin interaction and
the rescue of angiogenesis and tumor growth.
An alternative explanation for the inhibitory activity of E37 is that
co-administration of peptide with rhEndostatin results in the formation
of an insoluble precipitate at the site of injection, leading to
reduced bioavailability of rhEndostatin. To test this hypothesis, we
measured sera concentrations of rhEndostatin over time following
subcutaneous (s.c.) administration of 1.5 nmol of
rhEndostatin alone or in combination with either 375 nmol (250-fold molar excess) of the E37 or control peptide. rhEndostatin
co-administered with a 250-fold molar excess of E37 peptide had an area
under the curvelast (AUClast) equal to 85% of
that observed when rhEndostatin was administered alone or when
rhEndostatin was dosed with 375 nmol of control peptide (Table
I). Assuming 100% bioavailability of
rhEndostatin following its administration alone or with control peptide, then the bioavailable dose of rhEndostatin resulting from the
co-administration of rhEndostatin and E37 peptide would correspond to
1.3 nmol (1.25 mg/kg). Inference from the B16-BL6 experimental
metastasis model where a dose titration of rhEndostatin was used to
inhibit metastatic growth (6) suggested that a dose of 1.3 nmol would
inhibit lung metastases by 77%. In this study, 1.5 nmol of
rhEndostatin co-administered with a 250-fold molar excess of the E37
peptide resulted in an 11% inhibition of lung metastasis (Fig. 6).
Thus, despite having a lower AUClast, reduction of the
activity of rhEndostatin in the B16-BL6 metastasis model by the E37
peptide cannot be attributed to decreased levels of circulating
rhEndostatin. We thus conclude that the loss of tumor growth inhibition
was caused by inactivation of rhEndostatin that results from the
specific interaction of E37 with rhEndostatin.
We identified hTM3 as an Endostatin-binding protein. Using an
epitope-specific antibody (Fig. 1B) we further demonstrated that the Endostatin-binding epitope of hTM3 is not present in all
tropomyosins and consequently that Endostatin-binding is presumably not
a characteristic of all tropomyosin isoforms. This finding has broad
significance. Endostatin inhibits the growth of tumors by inhibiting
endothelial cell function (3, 5, 6). Systemic rhEndostatin therapy was
shown to induce a 50% reduction of intratumoral blood flow whereas
other non-tumor affected organs in the same animal were
unaffected.4 Furthermore,
other events that require angiogenesis such as wound healing remained
unaffected by rhEndostatin (15, 16). These observations suggest that
the antiangiogenic effects of Endostatin are tightly controlled. Our
finding that Endostatin may only affect specific isoforms of
tropomyosin provides an explanation for how this tight control could be
achieved and maintained. It is possible that specific isoforms of
tropomyosin are expressed and regulated in endothelial cells during
tumor angiogenesis. We speculate that these specific isoforms bind
Endostatin preferentially leading to potent inhibition of tumor growth.
We also postulate that expression of such isoforms of tropomyosin that
interact with Endostatin with different affinities and dissociation
constants provides the natural homeostatic balance of endothelial cell
growth under normal physiological conditions.
The binding of rhEndostatin to immobilized rhTM3 displays both rapid
rates of association and dissociation and a KD of
~100 µM (Fig. 3A). Similar binding kinetics
have been observed between T cell receptors and their cognate ligands
on the surfaces of antigen-presenting cells and between the
glycosylation-dependent cell-adhesion molecule-1 (GlyCAM-1)
and L-selectin (17-19). Kinetic analysis of these
interactions shows they have relatively low affinities that are also
the result of rapid dissociation rates. Such kinetics are suggestive of
highly dynamic interactions. It is possible that Endostatin may
have higher affinity for an as yet undefined isoform of tropomyosin or
that a third, as yet unidentified component is involved in stabilizing
the Endostatin/hTM3 interaction in vivo, and that in the
presence of this molecule(s) the KD of binding would
be significantly lower.
Whereas pharmacokinetic analysis indicates that parenteral
administration of rhEndostatin results in circulating levels in the
nM range (6), local concentrations in the tumor
microenvironment may be significantly higher. Planar imaging of mammary
tumor-bearing rats following i.v. injection of technetium
(99mTc) labeled rhEndostatin (100 µCi/rat) showed that
the tumor could be visualized from 0.5-4 h
post-injection.5 This
observation suggests that Endostatin targets the tumor microenvironment and indicates significantly higher Endostatin concentrations at the
tumor bed in comparison to circulating levels. These elevated local
concentrations may favor the interaction of Endostatin with hTM3 in the
tumor vasculature.
Whereas normally located intracellularly, tropomyosin has been
localized to the surface of colon epithelial cells in ulcerative colitis (20) but surface expression has not been reported for endothelial cells. This does not eliminate the possibility that tumor
endothelial cells more closely resemble damaged colonic epithelium in
this regard. Dixelius et al. (13) recently showed that
endothelial cells, but not fibroblasts internalized Endostatin by an
endocytic pathway. This observation together with well established differences that exist between the endothelial cells of normal and
tumor tissues, may explain the anti-endothelial cell activity of
Endostatin in vitro and the tumor specificity in
vivo. However, the precise mechanism(s) by which endocytosed
Endostatin gains access to the cytosolic compartment remains to be elucidated.
For the first time we present evidence that links a mechanism of action
to the in vivo antitumor activity of Endostatin.
Co-injection of the E37 peptide with rhEndostatin resulted in a
dose-dependent inhibition of the antitumor activity of
rhEndostatin in the B16-BL6 experimental metastasis assay (Fig. 6). We
postulate that the E37 peptide mimics the Endostatin protein-binding
epitope of hTM3 to compete for Endostatin binding and consequently
inhibits the antitumor activity of Endostatin.
We believe that the antiangiogenic effect of Endostatin is mediated via
an interaction with tropomyosin-containing microfilaments. Rearrangement of the actin cytoskeleton has been reported to be characteristic of, and sufficient to induce endothelial cell apoptosis (21). Taken together, the ability of rhEndostatin to induce endothelial
cell apoptosis (3, 22-24) and our observations that rhEndostatin binds
tropomyosin-containing mircofilaments, suggests that Endostatin
disrupts microfilament function to initiate apoptosis. Further
observations consistent with Endostatin exerting an effect upon the
actin cytoskeleton come from an endothelial cell monolayer-wound assay
(6, 25). In this assay, cell migration at the wound edge has been
correlated with increased incorporation of monomeric actin into
filaments and a reorganization of the actin cytoskeleton (26).
Endostatin binding to microfilaments may inhibit the cytoskeletal reorganization required for cell migration, leading to an inhibition of migration.
Whereas the mechanism by which the Endostatin and hTM3 interaction may
induce endothelial cell apoptosis is poorly defined, tropomyosin and
the cytoskeleton have critical roles in cell survival. Ingber and
co-workers (27-30) have demonstrated that actin-based microfilaments
play a crucial role in cell shape-regulated determination of cell fate
and that whereas growth factors and integrin signaling are required for
endothelial cell growth, they are not sufficient (31). Progression
through the cell cycle is inhibited following disruption of the
cytoskeleton or by release of cytoplasmic tension, implicating the
actin-based cytoskeleton and its ability to generate tension against
integrin-ECM contacts in the regulation of endothelial cell cycle
progression and cell fate (29, 32). Thus, we surmise that Endostatin,
which has been reported to regulate cell cycle progression in
endothelial cells (13, 23), may bind cytoskeletal tropomyosin leading
to a release of cytoskeletal tension and subsequent induction of cell apoptosis.
Our data indicate that Endostatin exerts its antitumor and
antiangiogenic effects as a result of its interaction with tropomyosin isoforms containing the E37 mimotope. We suggest that this interaction leads to disruption of the actin cytoskeleton, that blocks cell migration and contributes to the induction of apoptosis. Interestingly troponin I, better known for its role in muscle contraction where it
forms a regulatory complex with troponin T, troponin C, and tropomyosin, has been identified as cartilage-derived inhibitor of
tumor angiogenesis and metastasis (33). Troponin I is the actin-binding
component of the troponin complex, and thus may inhibit endothelial
cells via an interaction with microfilaments that disrupts cytoskeletal
function. Thus, troponin I and Endostatin may represent a family of
naturally occurring antiangiogenic proteins that block microfilament function.
We recognize that the antiangiogenic activity of Endostatin is complex
and potentially involves multiple mechanistic pathways, the convergence
of which results in the inhibition of endothelial cell migration,
proliferation, differentiation, and an induction of apoptosis. Our
study has identified tropomyosin as an Endostatin-binding protein, thus
providing a mechanism that may explain the antitumor activity of
Endostatin. Endothelial cells rapidly internalize Endostatin (13), and
we showed here that rhEndostatin and tropomyosin co-localize to the
microfilaments of formalin-fixed human endothelial cells (Fig. 4). The
biological relevance of the tropomyosin/rhEndostatin interaction to the
antiangiogenic activity of Endostatin was demonstrated by blocking
antitumor activity of rhEndostatin with a peptide mimotope of
tropomyosin. We postulate that the antiangiogenic activity of
Endostatin may result, at least in part, from internalization of
Endostatin by endothelial cells and its subsequent interaction with
tropomyosin; leading to a disruption of microfilament integrity, inhibition of cell motility and induction of apoptosis, and ultimately tumor growth inhibition.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical groove of actin filaments to
stabilize actin in the polymerized state directly influencing the
integrity of microfilaments and thus play a role regulating
reorganization of the actin cytoskeleton. Tropomyosins have been
identified in organisms as diverse as yeast and man and are core
components of the cell cytoskeleton. Many vertebrate non-muscle cells
express between five and eight isoforms of tropomyosin in a
tissue-specific manner, leading to speculation that tropomyosin
isoforms may have evolved to perform specific functions in the
microfilaments of non-muscle cells. Indeed, mutational analysis
indicates that tropomyosin isoforms have distinct functions and that
they play important roles in a variety of cellular functions, including
contraction, cytokinesis, intracellular transport, secretion, motility,
morphogenesis, and cell transformation (Ref. 11 and references therein).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
21-1 was
excised as a pBluescript phagemid (as recommended by Stratagene) from a
recombinant phage isolated from the HUVEC cDNA library based on its
recognition by E37 antisera. DNA sequencing of the cDNA insert of
p
21-1 showed it contained the complete hTM3 coding sequence fused
inframe downstream of the Lac Z gene. Escherichia coli
DH5
cells were transformed with p
21-1 and pBluescript plasmids
and crude bacterial lysates were prepared (12). Human cardiac
tropomyosin and muscle tropomyosins were purchased from Trichem
Resources (West Chester, PA) and Sigma, respectively.
-D-galactopyranoside,
harvested by centrifugation, resuspended in 1 M Tris-HCl,
pH 8, 1 M NaCl, and heated 45 min at 90 °C. The lysate
was cooled to room temperature, centrifuged, and the supernatant was
loaded onto a Sulfopropyl-Sepharose Fast Flow column (SP-FF; Amersham
Pharmacia Biotech), which had been equilibrated in a 50 mM
phosphate buffer, pH 7.5. The proteins were eluted with a linear NaCl
gradient, the recombinant (r) hTM3 fractions were acidified to a pH of
2 with trifluoroacetic acid, loaded onto a reverse phase C4 column
(Vydac Inc., Hesperia, CA) and eluted with an
acetonitrile/trifluoroacetic acid gradient. The fractions containing
hTM3 were concentrated and dialyzed against PBS. rhAngiostatin
K1-32 and rhEndostatin (6)
were produced in P. pastoris and purified to homogeneity.
21-1 or control
lysate were added and incubated for 1 h at 37 °C, followed by
incubation with 1:500 diluted anti-tropomyosin TM311 ascites fluid
(Sigma). Following incubation in 1:5000 diluted anti-mouse IgG (H+L)
conjugated to alkaline phosphatase (Promega, Madison, WI) wells were
developed using Blue Phos Phosphatase Substrate (Kirkegaard and Perry,
Gaithersburg, MD) and the A635 measured.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
21-1) were probed with either TM311 anti-tropomyosin monoclonal antibody (Sigma) or anti-E37 polyclonal sera (Fig. 1). The anti-E37
sera and TM311 anti-tropomyosin antibody specifically recognized common
bands of ~49 and 43 kDa in the rhTM3 expressing
21-1 lysate
whereas neither band was detected in immunoblots of control lysate
(Fig. 1). Furthermore, when a lysate of HUVECs was probed with anti-E37
sera or TM311 anti-tropomyosin antibody, a common band of ~38 kDa was
identified that co-migrated with E. coli expressed rhTM3
(data not shown). These findings demonstrate that the E37 peptide and
hTM3 share a common epitope. The anti-E37 sera however failed to
recognize tropomyosins purified from human cardiac tissue, rabbit
muscle, or chicken muscle (Fig. 1B). The amino acid
sequences of rabbit muscle tropomyosin and human cardiac tropomyosin
each share 86% identity with hTM3, diverging in two differentially
spliced exons, exon 6 (amino acids 189-212) and exon 9 (amino acids
258-284). The inability of the anti-E37 sera to recognize either
rabbit muscle or human cardiac tropomyosins suggests that the putative Endostatin-binding domain of hTM3 resides within either exon 6 or 9 of
hTM3. Because the E37 peptide was identified based on its ability to
bind rhEndostatin, the presence of the E37 mimotope may define the
specific isoforms of tropomyosin that bind Endostatin.
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Fig. 1.
The putative Endostatin-binding site of hTM3
is not present in all isoforms of tropomyosin. 50 µg each of
hTM3 expressing 21-1 and control E. coli lysate; and 250 ng each of purified cardiac (human) and muscle (rabbit and chicken)
tropomyosin proteins were resolved using SDS-PAGE under non-reducing
conditions and immunoblotted. Blots were reacted with TM311
anti-tropomyosin ascites fluid (A) and anti-E37 sera
(B), respectively. Although the TM311 anti-tropomyosin
antibody recognized all isoforms of tropomyosin tested, the anti-E37
sera recognized only the bacterially expressed hTM3 of the
21-1
lysate indicating that not all isoforms of tropomyosin contain the
putative Endostatin-binding site. Astersisks mark the
position of the ~49 and 43 kDa proteins detected in the
21-1
lysate by both TM311 and anti-E37 sera.
21-1)
(Fig. 2). This result provides evidence of a binding interaction between rhEndostatin and rhTM3.
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Fig. 2.
rhTM3 binds rhEndostatin in
vitro. The wells of a 96-well plate were coated with
either 5 µg/ml of rhEndostatin (closed symbols) or bovine
serum albumin (open symbols) and incubated with increasing
amounts of control E. coli lysate (circles) or
tropomyosin expressing 21-1 bacterial lysate (squares).
The amount of tropomyosin binding to each well was determined
spectrophotometrically following the addition of the TM311
anti-tropomyosin ascites fluid, alkaline phosphatase-conjugated
anti-mouse IgG and substrate. Error bars indicate mean ± S.D.
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Fig. 3.
Kinetics of the interaction of soluble
rhEndostatin with immobilized rhTM3. A, 0, 10, 20, 40, 80, and 160 µM rhEndostatin were injected at 5 µl/minute for 960 s through a biosensor flow cell that had been
activated and blocked as a control for bulk refractive index and
subsequently through a flow cell to which 3200 RU of rhTM3 had been
immobilized. The binding kinetics were recorded, and the differences
between the two curves plotted for each rhEndostatin concentration.
B, 0, 11.5, 23, and 46 µM rhAngiostatin were
injected at 5 µl/minute for 900 s over a flow cell containing
3200 RU of rhTM3 as above. Samples were run in duplicate, the data
corrected for bulk shift and the average of the 2 curves plotted.
C, injection of 5 µM rhEndostatin at 5 µl/minute for 240 s in the presence of 0, 6, 15, 30, and 45 µM soluble rhTM3 over a flow cell containing 2000 RU of
rhTM3. Samples were run in duplicate, the reference cell values
subtracted, and the average of each of the 2 curves plotted. The spikes
in RU seen at the beginning and end of each injection are due to the
lag in flow caused by the fact that the rhTM3 and reference cells are
non-adjacent.
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[in a new window]
Fig. 4.
RhEndostatin binding and tropomyosin
co-localize to the microfilaments of human endothelial
cells. HUVECs (A), HAECs (B), and
HMVECs (C) were incubated in the presence of 40 µg/ml
Alexa 488-labeled rhEndostatin and photographed at × 100 magnification under Alexa 488 (green) excitation wavelength.
D and E, HUVECs were incubated simultaneously in
anti-tropomyosin TM311 ascites fluid and 40 µg/ml Alexa 488-labeled
rhEndostatin followed by incubation in Alexa 594-conjugated goat
anti-mouse IgG (H+L). The same field was photographed under Alexa 488 (green) and Alexa 594 (red) excitation
wavelengths at × 100 magnification.
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[in a new window]
Fig. 5.
The inhibition of rhEndostatin binding to
HUVEC mircofilaments by E37 peptide implicates the role of
tropomyosin. HUVECs were incubated in the presence of 40 µg/ml
Alexa 488-labeled rhEndostatin alone (A), or in the presence
of a 50-fold molar excess of control peptide (B), or E37
peptide (C). Cells were photographed using the same exposure
times at × 100 magnification under Alexa 488 (green)
excitation wavelength.
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[in a new window]
Fig. 6.
The E37 peptide inhibits the antimetastatic
activity of rhEndostatin. C57BL/6J mice were injected via the tail
vein with 5 × 104 B16-BL6 melanoma cells on day 0. On
day 3, groups of five mice received daily subcutaneous doses of either
buffer control, 1.5 nmol (1.5 mg/kg/day) of rhEndostatin in combination
with 0, 15, 75, or 375 nmol of control or E37 peptide, or control or
E37 peptide alone for eleven days. All mice were sacrificed on day 14, and the number of pulmonary surface metastases in each animal counted.
Error bars indicate mean ± S.D.
Bioavailability of rhEndostatin administered in the presence of E37
peptide in mice
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Emily Kough, Michelle Johnson, Clara Dey, Dr. Barbara Nelson, and Art Hanson for excellent technical assistance. We are grateful to Drs. Donald Bottaro and William Fogler for helpful advice, discussions, and for critical review of the manuscript. We thank Dr. Jim Jung-Ching Lin at the University of Iowa, Iowa City, Iowa, the staff of EntreMed for advice and support, and Sauda Ayub for assistance in preparation of the figures.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: EntreMed, Inc., 9640 Medical Center Dr., Rockville, MD 20850. Tel.: 301-738-2485; Fax:
301-217-9594; E-mail: kims@entremed.com.
Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M100743200
1 The abbreviations used are: rh, recombinant human; rm, recombinant murine; hTM3, human tropomyosin isoform 3; PBS, phosphate-buffered saline; RU, resonance units; AUC, area under curve.
2 H. Liang and B. K. L. Sim, unpublished data.
3 D. L. Narum, S. R. Fuhrmann, B. Nelson, D. Bottaro, D. H. T. Chen, S. M. Plum, and B. K. L. Sim, manuscript in preparation.
4 D. R. Sørensen, T. A. Read, T. Porosol, B. R. Olsen, R. Timpl, T. Sasaki, P. O. Iversen, H. B. Benestad, B. K. L. Sim, and R. Bjerkvig, manuscript in preparation.
5 D. Yang, personal communication.
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