From the Department of Medicine and the Cancer
Center, Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, Massachusetts 02215 and ¶ ILEX Oncology, Inc.,
Products, Research and Development Division,
Boston, Massachusetts 02215
Received for publication, August 24, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Components of vascular basement membrane are
involved in regulating angiogenesis. Recently, tumstatin (the NC1
domain of Angiogenesis, the development of new blood vessels from
pre-existing ones, is required for tumor growth and metastasis (1, 2).
The tumor cell component and the endothelium of blood vessels function
in concert to coordinate the expansion of tumor tissue (1). These
events are mediated via an angiogenic switch generating an overwhelming
pro-angiogenic stimulus. Recent studies from several different
laboratories have shown that endogenous inhibitors of angiogenesis are
produced de novo to orchestrate a systematic regulation of
tumor uptake and growth (3-7).
Basement membranes are organized as thin layers of specialized
extracellular matrix that provide the supporting scaffold for epithelial and endothelial cells (8). Basement membranes not only
provide a mechanical support but also influence cellular behavior such
as differentiation, proliferation, and migration of various cells
including endothelial cells. Vascular basement membrane constitutes an
insoluble structural wall of newly formed capillaries and is speculated
to play an important role in regulating pro- and anti-angiogenic events
(6, 9, 10). Type IV collagen is one of the major macromolecular
constituents of basement membranes (11) and is expressed as six
distinct Recently, we identified that Production of Recombinant Deletion Mutants of Human Tumstatin and
Human Endostatin--
The sequence encoding deletion mutants
(Tum-1-5) of tumstatin was amplified using polymerase chain
reaction from the Synthetic Peptides--
Synthetic peptides CDCRGDCFC (RGD-4C)
and control peptides CNGRC were kindly provided by ILEX Oncology, Inc
(San Antonio, TX). These peptides were synthesized and characterized as
previously described (25).
Immunoblotting--
Recombinant Tum-5 was analyzed by SDS-PAGE
and immunoblotting as previously described (26). Goat anti-rabbit IgG
and anti-human IgG antibody conjugated with horseradish peroxidase were
purchased from Sigma. Monoclonal anti-polyhistidine tag antibody was
purchased from Invitrogen, and monoclonal anti-polyhistidine tag
antibody conjugated with peroxidase was purchased from Sigma.
Cell Lines and Culture--
Bovine pulmonary arterial
endothelial cells (C-PAE), human umbilical vein endothelial cells
(HUVEC), human prostate adenocarcinoma cell line (PC-3), and NIH3T3
fibroblasts were all obtained from American Type Culture Collection.
These cell lines were maintained in DMEM (C-PAE; Life Technologies,
Inc.) supplemented with 10% fetal calf serum (FCS), 100 units/ml of
penicillin, and 100 mg/ml of streptomycins; in EGM-2 (HUVEC; Clonetics,
San Diego, CA); or in F12K (PC-3; Cellgro). The melanoma cell line
WM-164 was obtained from Dr. Meenhard Herlyn at the Wistar Institute
(Philadelphia, PA) and maintained as previously described (20, 27).
DU-145 was purchased from American Type Culture Collection and
maintained in DMEM supplemented with 10% FCS.
Proliferation Assay--
A suspension of C-PAE cells (7,000 cells/well, passage 2-6) in DMEM containing 0.5% FCS was added onto
96-well plates precoated with fibronectin (Sigma, 10 µg/ml). After
24 h, medium was replaced with DMEM containing 20% FCS and
recombinant Tum-5. Then, after 36-48 h, methylene blue staining was
performed as previously described (5). Polymyxin B (Sigma, 5 µg/ml)
was used to inactivate any endotoxin that was still present (28),
although all samples tested were less than 50 enzyme units/mg in
endotoxin, a concentration that is not enough to affect
endothelial cell growth (data not shown). WM-164 cells were analyzed
using a similar protocol.
The BrdUrd incorporation assay was conducted using the BrdUrd
proliferation assay kit according to manufacturer's instructions (Calbiochem) with some modifications. Briefly, C-PAE cells were seeded
onto 96-well plates in DMEM containing 10% FCS. The next day the
medium was replaced with DMEM containing 2% FCS with or without Tum-5
or full-length tumstatin (293 cell expressed). The plates were then
incubated for 46 h at which time cells were pulsed for 2 h
with BrdUrd (10 nM). The cells/DNA were then fixed to the
wells, reacted with anti-BrdUrd primary and secondary antibodies and
then developed using a colorimetric reaction. The plates were read at
A450 nm on a Molecular Devices plate
reader. All groups represent triplicate samples.
MTT Assay--
Cell viability was assessed by MTT (Chemicon)
assay according to manufacturer instructions (Roche Molecular
Biochemicals) with some modifications. C-PAE cells (5,000 cells/well)
were plated in a 96-well plate in DMEM containing 5% FCS. The next
day, Tum-5 was added and incubated for 24 or 48 h, and cell
viability was evaluated as previously described (7).
Competition Proliferation Assay--
C-PAE cells were plated
onto 96-well plates and serum-depleted as described above. Tum-5 (final
concentration, 20 µg/ml) was incubated with varying concentrations of
human Cell Cycle Analysis--
C-PAE cells were growth arrested by
contact inhibition for 48 h. The 0 h value refers to the
percentage of cells in S phase at this time point. The cells (0.1 × 106/well) were harvested and plated into a 12-well plate
coated with fibronectin in 5% FCS and recombinant Tum-5 or
Tum-5/126C-A. After 21 h, the cells were harvested and then fixed
in 70% ice-cold ethanol. The fixed cells were rehydrated at room
temperature for 30 min in PBS containing 2% FCS and 0.1% Tween-20 and
then centrifuged and resuspended in 0.5 ml of the same buffer. RNase
digestion (5 µg/ml) was carried out at 37 °C for 1 h,
followed by staining with propidium iodide (5 µg/ml). The cells were
analyzed using a Coulter EPICS XL-MCL flow cytometer.
Annexin V-FITC Assay--
Annexin V, a
calcium-dependent phospholipid-binding protein with a high
affinity for phosphatidylserine, was used to detect apoptosis (29).
Briefly, cells (0.5 × 106/well) were seeded onto a
6-well plate in DMEM containing 10% FCS. On the next day, fresh medium
containing 10% FCS was added together with Tum-5 or 80 ng/ml of
TNF- Caspase-3 Assay--
Caspase-3 activity was determined as
previously described (7). Briefly, C-PAE cells (0.4 × 106/well) were plated in 60-mm Petri dishes precoated with
fibronectin (10 µg/ml). Cells were serum-starved (2% FCS) for
24 h, and then cells were stimulated with basic fibroblast growth
factor (3 ng/ml) in DMEM (2% FCS) also containing Tum-5 (10 µg/ml)
and incubated for 24 h. Controls received PBS buffer. TNF- Endothelial Tube Formation Assay--
The endothelial tube
formation assay was performed as previously described (7).
Matrigel-precoated 96-well plates were used, and HUVECs (5,000 cells/well) were seeded in the presence of Tum-5 or human endostatin.
Control cells were incubated with PBS. Cells were incubated for 24 h at 37 °C, and the number of tubes was counted using a microscope
(magnification of 3.3× ocular, 10× objective). Three wells were
viewed, and the number of tubes/well was counted and averaged by two
investigators blinded for the experimental protocols.
Cell Attachment Assay--
This assay was performed as
previously described (20, 30). 96-well plates were coated with 10 µg/ml of Tum-5 or vitronectin (Collaborative Biomedical Products) at
a concentration of 0.5-2.5 µg/ml overnight. Plates were blocked with
100 mg/ml of bovine serum albumin (Sigma) for 2 h. HUVECs or
C-PAEs (1.5 × 105 cells/ml, in M-199) were incubated
with either 10 µg/ml of antibody or synthetic peptide for 15 min.
Then, 100 µl of the cell suspension/well was added onto the plates
and incubated for 45 min at 37 °C. After washing, the number of
attached cells was determined with methylene blue staining. Control
mouse IgG1 and mouse monoclonal antibody to human Direct Binding Assay for Matrigel Plug Assay--
In vivo Matrigel plug assay
was performed as previously described (7). 5-6-week-old male C57/BL6
mice (Jackson Laboratories, Bar Harbor, ME) were obtained. All animal
studies were reviewed and approved by the animal care and use committee
of Beth Israel Deaconess Medical Center and are in accordance with the
guidelines of the Department of Health and Human Services. Matrigel
(Collaborative Biomolecules) was mixed with 20 units/ml of heparin
(Pierce), 50 ng/ml of vascular endothelial growth factor (R&D), and 5 µg/ml of Tum-5 or 10 µg/ml of Tum-1. The control group did not
receive recombinant proteins. The Matrigel mixture was injected
subcutaneously, and after 6 days mice were sacrificed, and the Matrigel
plugs were removed and fixed in 4% paraformaldehyde. The plugs were embedded in paraffin, sectioned, and hematoxylin and eosin
stained. Sections were examined by light microscopy, and the number of blood vessels from 4-7 high power fields (×400) were counted and averaged. All sections were coded and observed by an investigator who
was blinded for study protocols. Each group consists of four Matrigel plugs.
In Vivo Tumor Studies--
Male athymic nude NCRNU mice, 5-6
weeks old, weighing ~25 g were implanted with 2 × 106 PC-3 cells into the dorsal subcutis. The tumors were
measured using Vernier calipers, and the volume was calculated using
the standard formula (width2 × length × 0.52) (3,
4). The tumors were allowed to grow to ~50 mm3, and
animals were then pair-matched into groups of 6 mice. Initial doses
were given on the day of pair-matching (Day 1). Tum-5, Tum-5/126C-A, or
human endostatin was intraperitoneally injected twice daily at
doses ranging from 1-20 mg/kg for 20 days in sterile PBS. Only soluble
proteins were used. In addition, continuous delivery using Alzet
mini-pumps surgically implanted subcutaneously were used in one
treatment (Tum-5) group. Mice were weighed twice weekly, and tumor
measurements were taken twice weekly, starting on Day 1. Estimated mean
tumor volumes were plotted as a ratio
(V/Vo), where V = tumor volume on day of measurement and Vo = initial tumor volume. Upon termination (Day 21), the mice were weighed and sacrificed, and their tumors were excised and examined by light
microscopy and CD31 immunostaining. In this model, (the mean treated
tumor weight)/(mean control tumor weight) × 100% was subtracted
from 100% to give the tumor growth inhibition for each group. In all
experiments, the control group received vehicle injection.
CD31 Immunostaining--
Intratumoral microvessel density (MVD)
was analyzed on frozen sections of PC-3 tumor xenografts using a rat
anti-mouse CD-31 monoclonal antibody (Pharmingen, San Diego, CA) with a
standard streptavidin-biotin-peroxidase detection system (Vectastain
ABC Elite kit). Endogenous peroxidase activity was blocked using 1% H2O2/methanol for 30 min, and slides were then
subjected to antigen retrieval by incubating with proteinase K for 30 min at room temperature. Anti-mouse CD31 antibody was diluted 1:20 in
PBS-T (PBS containing 0.1% Tween-20) and incubated for 2 h after
sections were blocked with 5% normal goat serum/PBS-T. Normal rat IgG
was used as a negative control. Immunoperoxidase staining was carried
out utilizing the Vectastain ABC Elite reagent kit (Vector Labs,
Burlingame, CA). Sections were counterstained with Methyl green. MVD
was assessed at first by scanning the tumor at low power, then
identifying three areas at the tumor periphery that contain the maximum
number of discrete microvessels, and then counting individual
microvessels on a lower magnification (40×) field. The mean
microvessel density was compared among treatment groups and analyzed
using Student's t test.
Statistical Analysis--
All values are expressed as means ± S.E. Analysis of variance with a one-tailed Student's t
test was used to identify significant differences in multiple
comparisons. A level of p < 0.05 was considered statistically significant.
Expression and Purification of Tum-5--
Tum-5 was produced as a
fusion protein with a C-terminal 6-histidine tag in E. coli
using the expression plasmid pET 28a and in yeast using plasmid
pPICZ Anti-proliferative Effect of Tum-5--
The anti-proliferative
effect of Tum-5 and Tum-5/126C-A on C-PAE cells was examined in the
present study. Using both BrdUrd incorporation assay (Fig.
2A) and methylene blue
proliferation assay (Fig. 2B), Tum-5 (Fig. 2, A
and B) and Tum-5/126C-A (data not shown) revealed a
comparable dose-dependent inhibition. This inhibition is
comparable with full-length tumstatin (Fig. 2A). Tum-5 (Fig.
2B) and Tum-5/126C-A produced both in E. coli and yeast (data not shown) significantly inhibited 20% FCS-stimulated cell
proliferation in a dose-dependent manner with an
ED50 10-12 µg/ml. This anti-proliferative activity was
not observed on several nonendothelial cell lines including WM-164
cells (Fig. 2C).
Cell Cycle Analysis--
To further evaluate the inhibition of
endothelial cell proliferation by Tum-5, its effect on cell cycle
progression was analyzed. In contact inhibited cells (0 h), 5.8% of
cells were in S phase (Fig. 2D). When the cells were
stimulated with 5% FCS for 21 h, there was a 3.7-fold increase in
the percentage of cells in S phase (21.5%). Treatment with Tum-5
decreased the percentage of cells in S phase to 6.0% (basal level).
This effect of Tum-5 was dose-dependent (1 µg/ml, 19.3%;
10 µg/ml, 11.3%; 20 µg/ml, 6.0%). The percentage of cells in
G0/G1 was lowest in the 5% FCS control group,
and it was elevated upon Tum-5 treatment (data not shown). These
results strongly suggest that treatment of endothelial cells with Tum-5
causes G1 arrest of proliferating endothelial cells. Tum-5/126C-A also revealed an effect equivalent to Tum-5 in inducing G1 arrest of the cell cycle (Fig. 2D),
suggesting again that the 126C-A mutation has no effect on the activity
of Tum-5.
Effect of Tum-5 on Cell Viability--
Tum-5 and Tum-5/126C-A
significantly decreased cell viability in a dose-dependent
manner with an ED50 at 12 µg/ml, as assessed by the MTT
assay (Fig. 2, E and H). Tum-5/126C-A decreased
cell viability as potently as Tum-5, once again strongly suggesting that the terminal cysteine of Tum-5 is not essential for the
anti-angiogenic activity (Fig. 2E). Both of these
recombinant proteins did not exhibit any effect on nonendothelial cells
(PC-3, DU-145) (Fig. 2, F and G). These results
further suggest that Tum-5 is an endothelial cell-specific angiogenesis inhibitor.
Role of Secondary Structure (Disulfide Bonds) on the Activity of
Tum-5--
To establish the contribution of disulfide bonds and the
associated secondary structure on the anti-angiogenic activity of Tum-5, we reduced and alkylated Tum-5. Reduction of Tum-5 led to a
retarded migration of the protein band on SDS-PAGE gel, suggesting the
loss of disulfide bonds (data not shown). Cell viability assays with
reduced and nonreduced Tum-5 suggest that reduction of disulfide bonds
does not have any significant effect on the activity of Tum-5,
implicating that this activity is potentially contained within the
primary sequence of Tum-5 (Fig. 2H).
Endothelial Cell Apoptosis--
The induction of apoptosis in
endothelial cells by Tum-5 was examined using annexin V-FITC as
previously described (7). This assay detects the externalization of
membrane phosphatidyl serine, which occurs early during apoptosis,
because a FITC conjugate of annexin V binds naturally to
phosphatidylserine (29). Tum-5 at 5 µg/ml revealed a distinct shift
of annexin fluorescence peak after 18 h (Fig.
3A). The shift in fluorescence
intensity was similar for Tum-5, Tum-5/126C-A (data not shown), and the
positive control TNF- Tum-5 Increases the Activity of Pro-apoptotic Enzyme
Caspase-3--
Caspase-3 (CPP32), an intracellular protease activated
at an early stage of apoptosis, initiates cellular breakdown by
degrading structural and DNA repair proteins (38, 39). The protease activity of caspase-3 was measured spectrophotometrically by detection of the chromophore (p-nitroanilide) cleaved from the labeled
substrate (DEVD-pNA). Tum-5 (10 µg/ml)-treated cells exhibited a
4.5-fold increase in caspase-3 activity, comparable with maximal effect of TNF- Effect of Tum-5 on Endothelial Cell Tube Formation--
When
HUVECs are cultured on Matrigel matrix, they rapidly align and form
hollow tube-like structures (40). Human Tum-5 significantly inhibited
endothelial tube formation in a dose-dependent manner as
compared with control (Fig. 4,
A-C). The percentage of tube formation after treatment with
5 µg/ml of protein was: bovine serum albumin control, 22.7 ± 3.1%; human Tum-5, 2.1 ± 2.0%. Yeast produced human endostatin
was less potent in inhibiting tube formation when compared with yeast
produced human Tum-5/126C-A at molar equivalents (Fig.
4C).
Cell Attachment to Tum-5 Is Not Inhibited by RGD
Sequence--
Synthetic peptides RGD-4C and control peptides CNGRC
were previously reported to bind to vascular endothelial cells (25). RGD peptide at 5 µg/ml inhibits attachment of endothelial cells onto
vitronectin-coated plates, whereas CNGRC peptides does not exhibit this
property (Fig. 5A). Incubation
of endothelial cells with Tum-5 did not inhibit cell attachment onto
vitronectin-coated plates (Fig. 5A). When endothelial cells
were incubated with RGD peptide (10 µg/ml) or CNGRC peptide,
attachment of C-PAEs to Tum-5-coated plates was not decreased (Fig.
5B). This further suggests the notion that Tum-5 binds to
Tum-5 Binds to Endothelial Cells via
Direct Binding Assay for Tum-5 Binding to
Reversal of Anti-proliferative Effect of Tum-5 by Soluble
Effect of Human Tum-5 on Angiogenesis in Matrigel Plugs in C57BL/6
Mice--
To evaluate the in vivo effect of human Tum-5 on
the formation of new capillaries, we performed a Matrigel plug assay in
mice as previously described (7). A 91% reduction in the number of
blood vessels was observed with 5 µg/ml of human Tum-5 (Fig. 6D) on day 6 as compared with
the PBS control (Fig. 6B). Human Tum-1, lacking the
N-terminal 53 amino acids of tumstatin (7), inhibits neovascularization
by 95% (Fig. 6C). The number of vessels per high power
field was: Tum-1, 0.47 ± 0.16; Tum-5, 0.80 ± 0.16; and
control, 8.81 ± 0.35 (Fig. 6A).
Effect of Human Tum-5 on the Growth of Human Xenograft Tumors in
Nude Mice--
We examined the effect of soluble human Tum-5 and
Tum-5/126C-A on established primary human tumor models in nude mice. No evident toxicity was observed in any groups, as judged by weight change. Both human Tum-5 and human Tum-5/126C-A significantly inhibited
the growth of PC-3 human prostate carcinoma xenografts (Fig.
7A). Human Tum-5 at 1 mg/kg
had a tumor growth inhibition of 74.1% (p = 0.02), and
human Tum-5/126C-A had a tumor growth inhibition of 92.0%
(p = 0.001) as compared with the vehicle-injected control group. Continuous delivery of human Tum-5 (1 mg/kg, over 24 h) using Alzet mini-pumps implanted in the dorsal subcutis also
showed significant tumor growth inhibition (70.1%, p = 0.03). Human endostatin delivered at a dose of 20 mg/kg (twice
daily, bolus intraperitoneal) revealed insignificant tumor
growth inhibition as compared with the vehicle alone treated or Tum-5
group. These experiments suggest that at molar equivalents, human Tum-5
is at least 10-fold more potent than human endostatin in controlling the growth of human prostate xenograft tumors in mice. Further studies
are needed to firmly establish the head to head potency of human
endostatin in comparison with human Tum-5.
Tum-5 Decreased Neovascularization in Human PC-3 Xenograft Tumor in
Nude Mice--
We examined the effect of soluble human Tum-5 on
intratumoral MVD in PC-3 xenograft tumors by CD31 immunostaining. Tum-5
intraperitoneal injection (Fig. 7C) significantly decreased
MVD as compared with vehicle injected group (Fig. 7B). The
number of CD31-positive blood vessels per low power field (40×) was,
Tum-5, 6.33 ± 0.54, and control, 9.44 ± 1.05, p = 0.047 (Fig. 7D). These studies, of
course, only include tumors that were still present upon the full
course treatment of Tum-5. Groups treated with Tum-5/126C-A and the
Tum-5-pump group showed a similar decrease of MVD (data not shown).
Angiogenesis is essential for the progression of various
pathological disorders including diabetic retinopathy and rheumatoid arthritis, as well as tumor growth and metastasis (1). The switch to an
angiogenic phenotype requires both up-regulation of angiogenic
stimulators and down-regulation of angiogenesis inhibitors (1).
Vascular endothelial growth factor and basic fibroblast
growth factor are among the major inducers of angiogenesis. To date, a
number of angiogenesis inhibitors have been identified, and certain
factors such as angiostatin (3), endostatin (4), canstatin (5),
arresten (6), and tumstatin (7) are tumor-associated angiogenesis
inhibitors that are generated in vivo.
The predominant interest in our laboratory is focused on understanding
the anti-angiogenic cues originating from vascular basement membrane.
Our prevailing hypothesis centers on the notion that changes in the
extracellular matrix and vascular basement membrane during the
inductive and resolution phase of angiogenesis play an important role
in regulating the formation/creation of new blood vessels. These
changes are speculated to be modulated by growth factors produced by
proliferating tumor cells (10). In pursuit of understanding these
dynamic changes associated with angiogenesis, our laboratory recently
identified a novel anti-angiogenic protein domain derived from the Deletion mutagenesis identified a 54-132-amino acid region as pivotal
for the anti-angiogenesis activity of tumstatin. In the present study,
we recombinantly produced this molecule (Tum-5) in pET28a bacterial
expression system and pPICZ In this study, the anti-angiogenic activity of Tum-5 was compared with
recombinant full-length tumstatin expressed in 293-HEK cells in BrdUrd
incorporation assay. The activity of Tum-5 was almost equivalent to
full-length tumstatin. These results suggest that Tum-5 maintains equal
anti-angiogenic activity even upon 64% truncation. When Tum-5 was
produced in E. coli or yeast, it resulted in generation of
Tum-5 predominantly in aggregate form. Thus, to improve our production
yield, we replaced cysteine 126 with alanine to enable better
expression and solubility of Tum-5 domain and produce more soluble
protein for preclinical studies. This mutation resulted in enhanced
protein expression, but Tum-5/126C-A still possessed comparable
anti-angiogenic activity in vitro and in vivo, as
compared with Tum-5 and parent tumstatin. Collectively, these data
suggest that terminal cysteine of Tum-5 is not required in exerting its
anti-angiogenic activity. Our experiments to understand the mechanism
of action for Tum-5 are consistent with previous studies with
tumstatin, which document endothelial cell-specific apoptosis
associated with G1 arrest of endothelial cell cycle. Whether cyclins, cyclin-dependent kinases,
cyclin-dependent kinase inhibitors, and transcription
factors such as E2F are involved (41) needs further investigation.
Integrin binding assays using Tum-5 show that previously reported
In vivo experiments in C57BL/6 mice using Matrigel plugs
show that Tum-5 is effective in inhibiting serum-, vascular endothelial growth factor-, and basic fibroblast growth factor-induced
neovascularization. Studies using PC-3 xenograft tumors demonstrate
in vivo efficacy of Tum-5 in inhibiting, and in a few
instances, regressing pre-established tumors. When Tum-5 was
administered through a subcutaneous osmotic pump, tumor growth was
inhibited to a similar extent as two intraperitoneal injections
every day. These experiments suggest that human Tum-5 may have a
favorable half-life, potentially making continuous infusion not
necessary for maximal anti-tumor effect. Human endostatin was not
effective in inhibiting tumor growth at 20 mg/kg, in comparison with
human Tum-5, which inhibited tumor growth even at 1 mg/kg. This is
consistent with the result of the tube formation assay, which showed
less effect with human endostatin as compared with human Tum-5. These
results strongly suggest that at molar equivalent concentrations, human
endostatin is much less effective in inhibiting tumor growth when
compared with Tum-5.
Our studies indicate that tumstatin binds to
3 chain of type IV collagen) was identified as
possessing anti-angiogenic activity. In the present study, the
anti-angiogenic activity of tumstatin was localized to the putative
54-132-amino acid Tum-5 domain, and the activity mediated by
v
3 integrin interaction in an
RGD-independent manner. The recombinant Tum-5 produced in Escherichia coli and Pichia Pastoris
specifically inhibited proliferation and caused apoptosis of
endothelial cells with no significant effect on nonendothelial cells.
Tum-5 also inhibited tube formation of endothelial cells on Matrigel
and induced G1 endothelial cell cycle arrest. Moreover,
anti-angiogenic effect of Tum-5 was also examined in vivo
using both a Matrigel plug assay in C57BL/6 mice and human prostate
cancer (PC-3) xenografts in nude mice. The in vivo results
demonstrate that Tum-5 at 1 mg/kg significantly inhibited growth of
PC-3 tumors in association with a decrease in CD31 positive
vasculature. These in vivo studies also show that, at molar
equivalents, human Tum-5 is at least 10-fold more active than human
endostatin. In addition, these studies for the first time suggest that
through the action of endogenous inhibitors,
v
3 integrin may also function as a
negative regulator of angiogenesis. Taken together, these findings
demonstrate that Tum-5, a domain derived from tumstatin, is an
effective inhibitor of tumor-associated angiogenesis and a promising
candidate for the treatment of cancer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chains, namely,
1-
6 (12). These
-chains are
assembled into triple helices that further form a network to provide a
scaffold for other macromolecules to interact with the basement
membrane. These
-chains are composed of three domains, the
N-terminal 7 S domain, the middle triple helical domain, and the
C-terminal globular noncollagenous domain (NC1)1 (13). Type IV collagen
is thought to be important in endothelial cell proliferation and
behavior during the angiogenic process (5, 6, 9). The NC1 domain of
type IV collagen plays a crucial role in the assembly of type IV
collagen to form trimers and thus influences basement membrane
organization, which is important for new blood vessel formation (9, 11,
14). Synthetic peptides (amino acids 185-203) derived from NC1 domain
of
3 chain of type IV collagen (
3(IV)NC1) have been shown to
inhibit the proliferation of melanoma in vitro (15) and have
been found to bind to
v
3 integrin and
CD47/IAP (16).
3(IV)NC1, termed "tumstatin,"
possessed a novel anti-angiogenic activity (7, 17). Integrin
v
3 is potentially associated with
angiogenic vascular cells and plays a critical role in angiogenesis and
in promotion of endothelial cell survival (18, 19). In this regard, we
recently identified that tumstatin binds to
v
3 integrin in a RGD-independent manner,
and this binding is essential for its anti-angiogenic activity (20). In
the present study, the putative 54-132-amino acid anti-angiogenic
domain of tumstatin as determined by deletion mutagenesis (7) was
expressed as recombinant protein (Tum-5) using Escherichia
coli and yeast (Pichia pastoris). Tum-5 inhibits neovascularization and tumor growth in vivo using two
different model systems. Tum-5 also inhibits angiogenesis through
v
3 integrin interaction on endothelial
cells as previously shown for the full-length tumstatin. Results
obtained in the present study suggest that Tum-5 binds predominantly to
3 subunit of
v
3 integrin.
Additionally, Tum-5 interaction with
v
3
integrin is independent of vitronectin or fibronectin binding to
endothelial cells, suggesting that Tum-5 may function as an
angiogenesis inhibitor via negative regulation of
v
3 integrin on endothelial cells. In
addition, the present study also demonstrates that the anti-angiogenic
activity of Tum-5 is not dependent on disulfide bond linkage. These
results strongly implicate the use of this novel protein domain as a
possible drug candidate in the treatment of diseases dependent on angiogenesis.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3(IV)NCI/pDS vector as previously described
(7, 21). The resulting cDNA fragment was ligated into pET28a(+)
(Novagen, Madison, WI). Expression of recombinant protein in E. coli and purification using nickel-nitrilotriacetic acid-agarose
column (Qiagen) was performed as previously described (7). Amino acids
45-132 of tumstatin were expressed as Tum-5, which includes the
N-terminal 9 amino acids in addition to the previously identified
anti-angiogenic domain (54-132 amino acids) (7). The additional 9 amino acids were added to enhance the efficiency of protein expression.
Only soluble protein with a low endotoxin level (less than 50 enzyme units/mg) was used in these experiments. Recombinant
full-length tumstatin was expressed using 293 human embryonic kidney
cells as previously described (7). Recombinant Tum-5 was expressed in
yeast using P. pastoris with a method previously described
(22). pPICZ
A was used for the subcloning of Tum-5 so that Tum-5
would be fused to C-terminal 6-histidine tag. Site-directed mutagenesis
(C126 to A126) was induced to facilitate enhanced secretion of Tum-5
(Tum-5/126C-A). Human endostatin was expressed in yeast using P. pastoris with a previously described method (22). In some
experiments, Tum-5 was processed for reduction and alkylation as
described elsewhere (23, 24).
v
3 integrin protein (Chemicon) for
30 min at room temperature, and the mixture was added onto the cells
and incubated for 48 h. Proliferation assays were performed using
the methylene blue staining method as previously described (5). In
experiments with anti-
v
3 integrin
antibody, cells were plated and preincubated with the antibody for
1 h before Tum-5 (final concentration, 20 µg/ml) was added.
. After 18 h of treatment, floating and attached cells were
harvested and processed as described elsewhere (5).
(80 ng/ml) was used as a positive control. After 24 h, both the
supernatant and attached cells were combined, and an equal number of
cells (4 × 107 cells/ml) was processed following the
manufacturer's instruction (CLONTECH). A specific
inhibitor of caspase-3, DEVD-fmk was used for specificity. The
absorbance was measured in a microplate reader (Bio-Rad) at 405 nm.
Similarly nonendothelial cells (PC-3) were used and analyzed. This
assay was repeated three times.
1
integrin (clone P4C10) were purchased from Life Technologies, Inc.
Monoclonal antibody to human
v
3 integrin
(clone LM609) was purchased from Chemicon.
v
3
Integrin--
Direct ELISA was performed as described previously (31).
Tumstatin (293 cell expressed) or Tum-5 was coated onto a 96-well plate
in triplicate (100 ng/well), and an equal molar amount of binding
protein
v
3 integrin (Chemicon) was added.
Binding was established with monoclonal antibodies to
v
3 integrin (clone LM609, Chemicon). The
ELISA was developed with an alkaline phosphatase secondary antibody and
read in a plate reader at absorbance of 405 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
A. Tum-5 was expressed to encompass amino acids 45-132 to
include the putative amino acids 54-132 anti-angiogenic domain of
tumstatin. The E. coli expressed protein was isolated
predominantly as soluble protein after a refolding process, and
SDS-PAGE analysis revealed a monomeric band at 12 kDa (Fig.
1A). The eluted fraction
represented by lane 8 was used in the experiments described
in the present study. Tum-5 protein contains 5 cysteines, and to
decrease aggregation caused by the presence of 5th pair cysteine
residue, we generated Tum-5/126C-A, in which the cysteine residue at
position 126 of tumstatin was mutated to alanine. Tum-5/126C-A
expressed in E. coli was detected at the same molecular
weight size as Tum-5 (Coomassie Blue staining; Fig. 1B).
E. coli expressed Tum-5, and E. coli and yeast
(P. pastoris) expressed Tum-5/126C-A were immunodetectable
by anti-polyhistidine tag antibody (Fig. 1, C and
D). Tumstatin consists of 244 amino acids including 12 amino
acids from the triple helical portion located in the N-terminal
portion, and 232 amino acids derived from the NC1 domain. Goodpasture
(GP) syndrome is an autoimmune disease characterized by pulmonary
hemorrhage and/or rapidly progressing glomerulonephritis (32-34).
These symptoms are caused by the disruption of glomerular and alveolar
basement membrane through immune injury associated with autoantibodies
targeted against epitopes on
3 (IV) NC1 (32, 33).
Recently, the epitopes were identified in the N-terminal portion of the
NC1 domain (26, 35) and was further confined to be within the
N-terminal 40 amino acids (36, 37). Tum-5 consists of 45-132 amino
acids of tumstatin outside of the GP autoepitope. To further confirm
that Tum-5 is not potentially detectable by GP autoantibody, antisera
from patients with GP syndrome were used in Western blotting
experiments. GP antisera detected 293 cell expressed full-length
tumstatin with high sensitivity, although they failed to detect both
E. coli expressed Tum-5 and yeast expressed Tum-5/126C-A, as
shown for representative GP serum (Fig. 1E). These data
suggest that Tum-5 and Tum-5/126C-A produced in the present study do
not contain GP autoepitope, excluding the possibility that these
recombinant proteins might induce an autoimmune disorder.
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Fig. 1.
Recombinant production of Tum-5. The
recombinant Tum-5 protein was expressed and purified as described under
"Materials and Methods." Molecular mass markers
(MW) are shown at left of each panel.
A, SDS-PAGE (15%) Coomassie Blue staining. Lane
1, uninduced cells; lane 2, induced cells; lane
3, unbound fraction; lane 4, 10 mM
imidazole wash; lane 5, 25 mM imidazole wash;
lane 6, 50 mM imidazole eluted protein;
lane 7, 100 mM imidazole eluted protein;
lane 8, 250 mM imidazole eluted protein. The
arrowhead indicates the band of Tum-5 (12 kDa).
B, SDS-PAGE (15%) Coomassie blue staining of purified
Tum-5/126C-A expressed in E. coli. C, Western
blotting of E. coli expressed Tum-5 (lane 1) and
Tum-5/126C-A (lane 2) with horseradish peroxidase-conjugated
anti-polyhistidine tag antibody (Sigma, 1:2500 dilution). Each protein
was loaded 100 ng/lane. D, Western blotting of yeast
expressed Tum-5/126C-A (1.5 µg) with monoclonal anti-polyhistidine
tag antibody (Invitrogen, 1:2500 dilution). E, Western
blotting with using serum of a patient with Goodpasture's syndrome
(1:1000 dilution). SDS-PAGE was performed in nonreducing condition with
100 ng of protein/lane. Lane 1, 293 cell expressed
full-length tumstatin; lane 2, E. coli expressed
Tum-5; lane 3, yeast expressed Tum-5/126C-A.
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Fig. 2.
Tum-5 specifically inhibits proliferation of
endothelial cells, induces cell cycle (G1/S) arrest and
decreases cell viability. A, BrdUrd incorporation
assay. Tum-5 as well as full-length tumstatin expressed in 293 cells
decreased incorporation of BrdUrd in a dose-dependent
manner in C-PAEs. B, recombinant Tum-5 inhibited FCS (20%)
stimulated proliferation of C-PAE cells in a dose-dependent
manner. The difference between the mean value of
A655 (methylene blue staining) in Tum-5 treated
(5 and 10 µg/ml) and control (0 µg/ml) was significant
(p < 0.05). The anti-proliferative effect of Tum-5 (20 µg/ml) was not observed when control human melanoma cells (WM-164)
were used (C). D, Tum-5 and Tum-5/126C-A
(E. coli expressed) inhibit the progression of cell cycle
into S phase in C-PAEs. Growth arrested C-PAE cells were treated with
Tum-5 or mutant in the presence of 5% FCS for 21 h. The cells
were harvested and processed as described under "Materials and
Methods." The percentage of cells in S phase in growth arrested cells
was considered as the 0 h time point. This experiment was repeated
three times, and the representative data are shown. E-H,
MTT assay. Tum-5/126C-A decreased cell viability in C-PAEs
(E). This effect on cell viability was not observed in
control PC-3 and DU-145 cells (F and G). Tum-5
decreased cell viability in C-PAEs (H). This function of
Tum-5 was not altered by reduction and alkylation.
(data not shown).
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Fig. 3.
Tum-5 induces endothelial cell
apoptosis. Annexin V-FITC staining was performed on C-PAE cells
treated with Tum-5 for 18 h (A). Control cells received
PBS. FACS analysis was done to quantitate the percentage of cells
undergoing apoptosis (Annexin-V positive cells). Cells were stained
with propidium iodide (PI), and gating was performed to
analyze only annexin V positive and propidium iodide-negative cells.
FL-1 height represents the annexin fluorescence intensity as a log
scale. Tum-5 at 5 µg/ml induced a distinct shift of fluorescence
intensity peak as compared with the PBS control. Caspase-3 activity was
examined as described under "Materials and Methods." Increased
caspase-3 activity was observed by treating C-PAEs with 10 µg/ml of
Tum-5 (B). DEVD-fmk, a specific caspase-3 inhibitor, was
used to show the specificity. TNF- (80 ng/ml) was used as a positive
control. This increased activity of caspase-3 was not observed by
treating PC-3 cells with Tum-5 (C). These experiments were
repeated three times, and the representative data are shown.
(Fig. 3B). A specific inhibitor of caspase-3,
DEVD-fmk, decreased the protease activity to base line, indicating that the increase in the measured activity was specific for caspase-3 activity. In nonendothelial cells (PC-3), there was no difference in
caspase-3 activity between control and Tum-5-treated cells (Fig.
3C).
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Fig. 4.
Tum-5 inhibits endothelial tube
formation. HUVECs were allowed to form tubes on Matrigel-coated
plates incubated with or without Tum-5. A, control buffer
treated. B, 5 µg/ml of Tum-5 treated. C, number
of tube branches in low power field was counted (three independent
wells were counted and averaged). Tum-5 at 5 µg/ml significantly
decreased tube formation as compared with control. Yeast expressed
human endostatin was used at 5 and 20 µg/ml.
v
3 integrin in a RGD-independent manner
and thus independent of vitronectin binding. As expected, incubation of
cells with soluble Tum-5 decreased cell attachment onto Tum-5-coated
plates (Fig. 5B).
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Fig. 5.
Cell attachment assay, competitive
proliferation assay and ELISA. Cell attachment assay using
endothelial cells was performed as described under "Materials and
Methods." Antibodies or synthetic peptides used for incubation with
the cells are described at the bottom, and the protein used
for coating plates is described at the top (A-C).
A, attachment of C-PAEs on vitronectin coated plates was
inhibited by incubating with the RGD peptide. Incubation with Tum-5 did
not decrease cell binding. B, attachment of C-PAEs on
Tum-5-coated plates was not inhibited by incubating with the RGD
peptide. CNGRC peptide was used as a control. Incubation with Tum-5
decreased cell binding. C, attachment of HUVECs on Tum-5
coated plates was significantly inhibited by
anti- v
3,
1, and
3 integrin antibody. Control mouse IgG showed no
inhibition of cell attachment. D, vitronectin precoated
plates were incubated with 50 µl of soluble
v
3 integrin protein (Chemicon) for 30 min, and then C-PAEs were plated and incubated for 45 min. Attachment
of C-PAEs on vitronectin-coated plates was significantly inhibited by
incubating with
v
3 integrin protein at 1 and 2 µg/ml. E, direct binding assay for
v
3 integrin. Tum-5 or tumstatin was
coated on a 96-well plate, and binding to
v
3 integrin was assessed as described
under "Materials and Methods." F, Tum-5 (20 µg/ml) was
incubated with
v
3 integrin protein for 30 min and added onto C-PAE cells. After 48 h, cell number was
determined using methylene blue staining. Anti-proliferative effect of
Tum-5 was significantly decreased by
v
3
protein (final concentration, 0.5-1 µg/ml).
v
3 protein itself (1 µg/ml) did not
inhibit proliferation. G, C-PAE cells were preincubated with
anti-
v
3 integrin antibody for 1 h,
and then Tum-5 (20 µg/ml) was added. After 48 h, cell number was
determined using methylene blue staining. Anti-proliferative effect of
Tum-5 was significantly reversed by
anti-
v
3 antibody (1:30-1:100 dilution).
v
3 integrin antibody itself did not
inhibit cell proliferation.
v
3 and
1 Integrin--
We
examined the attachment of HUVECs and C-PAEs to Tum-5-coated plates in
the presence of integrin blocking antibodies. As shown in Fig.
5C,
v
3 antibody inhibited the
attachment of HUVECs by 51.0% and
1 antibody blocked by
48.4%, as compared with control IgG treatment. Cell attachment was
also decreased by
3 integrin antibody, and
interestingly,
v integrin antibody had no effect on cell
attachment. Incubating cells with both
v
3
and
1 integrin antibodies further decreased cell
attachment (but not complete inhibition) as compared with the
inhibitory effect by individual antibodies. This suggests that
additional binding sites for endothelial cells may still be present on
Tum-5. Comparable inhibition was also observed using C-PAE cells
instead of HUVECs (data not shown). Collectively, these results suggest
that Tum-5 binds to
v
3 and
1-containing integrin on endothelial cells. Interaction
of Tum-5 with
v
3 integrin is mediated
predominantly by
3 integrin subunit. In the previous
study, we showed that the
1-containing integrin binding
to tumstatin is
6
1 (20).
v
3 Integrin--
Binding of Tum-5 or
tumstatin to
v
3 integrin was assessed by
direct ELISA. ELISA plates were coated with Tum-5 or tumstatin and
incubated with equal molar concentration of
v
3 integrin. As shown in Fig.
5E, our results show that Tum-5 and tumstatin significantly
bind to
v
3 integrin as compared with
control group (bovine serum albumin-coated). The binding of tumstatin
is higher (1-fold) than Tum-5 presumably because of the additional
v
3 binding site present in the C-terminal region, as previously
reported (20).
v
3 Integrin Protein and
Anti-
v
3 Integrin Antibody--
To
further establish the role of
v
3 integrin
for Tum-5 activity, competition proliferation assays were performed as
previously described (20). To confirm the ability of
v
3 soluble protein in competing
v
3 receptors on the cell surface,
vitronectin precoated plates were incubated with
v
3-soluble protein, and the cell attachment assay was performed. Soluble
v
3 integrin protein at 1 and 2 µg/ml
significantly inhibited attachment of C-PAEs on vitronectin-coated
plates (Fig. 5D). Next, effective dosage of Tum-5 was
incubated with
v
3 integrin protein for 30 min and then added to C-PAEs stimulated with 20% FCS and growth
factors. After 48 h, cell proliferation was examined by methylene
blue staining. Anti-proliferative effect of Tum-5 was reversed
dose-dependently with increasing amount of
v
3-soluble protein (Fig. 5F).
The
v
3 protein at 1 µg/ml significantly
reversed Tum-5-induced anti-proliferative effect by 65.9%. Treatment
with
v
3 protein alone did not inhibit endothelial cell proliferation. Next,
anti-
v
3 integrin antibody (clone LM609)
was used in the competition assay to block
v
3 integrin on endothelial cells and
inhibit Tum-5 binding to this integrin. This antibody itself does not
inhibit proliferation of endothelial cells (20). The
v
3 integrin antibody at 1:30 dilution
significantly reversed Tum-5-induced anti-proliferative effect by
69.5% (Fig. 5G). In previous studies, we have shown that
control
5
1 integrin protein does not
inhibit the activity of tumstatin (20). These results suggest that the
anti-angiogenic activity of Tum-5 is mediated by binding to
v
3 integrin on the surface of endothelial cells.
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Fig. 6.
Matrigel plug assay. Sections of each
Matrigel plug stained by hematoxylin and eosin were examined by
light microscopy, and the number of blood vessels from 4-7 high power
fields was counted and averaged. Tum-5 (5 µg/ml, D)
significantly inhibited in vivo neovascularization, as
compared with controls (treated with PBS, B). Tum-1
treatment showed a similar effect (C). The difference
between the mean percentage value of Tum-1- or Tum-5-treated animals
and control animals was significant (A). Each column
represents the mean ± S.E. of 3-4 plugs/group. Representative
light microscopic appearance of the Matrigel plug (hematoxylin and
eosin staining, 200× magnification) in the control group are
shown in B. Marked neovascularization
(arrowheads) can be observed in the amorphous Matrigel plug.
There was less neovascularization observed in the Matrigel plug of
Tum-1 (C) and Tum-5 (D). Inset in
B, high magnification view (800× magnification) of blood
vessels. Inset in D, high magnification
view (800× magnification) of nonvascular infiltrating cells.
Arrows indicate the position magnified in insets.
Arrowheads indicate the blood vessels.
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Fig. 7.
Tum-5 suppresses tumor growth in PC-3
xenograft mice model. Twice daily intraperitoneal
injections of human Tum-5 (1 mg/kg) or human Tum-5/126C-A (1 mg/kg)
inhibited the growth of PC-3 xenografts as compared with the PBS
control. Treatment with human endostatin (20 mg/kg) did not show
significant decrease of tumor growth. Continuous administration of
Tum-5 (1 mg/kg/day) by using a subcutaneous osmotic pump was also
effective in inhibiting tumor growth. This experiment was started when
the tumor volumes were around 50 mm3. Each point represents
the mean ± S.E. of six mice. Frozen tumor sections were stained
by anti-CD31 antibody, and the number of vessels were counted and
averaged. B, control. C, Tum-5 (E. coli expressed) intraperitoneal injection (200× magnification).
Arrowheads show the CD31 positive blood vessels.
D, there were significantly less blood vessels observed in
the tumor section of Tum-5-treated group as compared with control
(p = 0.046).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3
chain of type IV collagen, associated with vascular basement membrane
(7). This protein domain, named tumstatin for its ability to cause
tumor "stasis," is an inhibitor of endothelial cell proliferation
and causes endothelial cell specific apoptosis (7). Subsequently, our
laboratory (20) and studies by Petitclerc et al. (17) show
that tumstatin (
3(IV)NC1) binds to endothelial cells via
v
3 integrin. We further show that the
binding to
v
3 is pivotal for the
anti-angiogenic activity associated with tumstatin and that the
activity is restricted to amino acids 54-132 within the 244-amino acid
tumstatin using deletion mutagenesis (20). Additionally, we show that
the
v
3 binding to tumstatin is mediated
via a mechanism independent of the RGD-containing amino acid sequence
(20). Petitclerc et al. (17) also show that tumstatin
(
3(IV)NC1) binds to endothelial cells via
v
3 integrin but speculate that it is
possibly via the RGD sequence present in the N terminus of the
(
3(IV)NC1) domain. This RGD sequence does not constitute the NC1
domain sequence but is derived from the triple helical region and
included in an original clone described by Neilson et al.
(21). Petitclerc et al. (17) used this clone to
recombinantly produce tumstatin (
3(IV)NC1) in 293 embryonic kidney
cells. When this sequence is removed using site-directed mutagenesis,
the
v
3 binding is preserved,
strongly suggesting a RGD-independent binding (20).
A yeast (P. pastoris) system
and evaluated for potential anti-angiogenic activity. Our experiments
suggest that the anti-angiogenic activity of tumstatin is localized to
this region using both in vitro and in vivo
assays. This activity is dependent on
v
3
integrin on endothelial cells, as is the activity of the parent
tumstatin molecule.
v
3 binding property of tumstatin, as well
as
1 binding, is still preserved. The result of
competition proliferation assay using soluble
v
3 integrin protein and
anti-
v
3 integrin antibody (LM609) with
Tum-5 suggest the involvement of
v
3
integrin binding in the anti-angiogenic activity of Tum-5. In this
regard, LM609 has been shown to inhibit angiogenesis (18). Cell binding
and proliferation assays in the present study suggest that Tum-5 binds to endothelial cells independent of an RGD sequence and vitronectin binding. Conceivably, tumstatin and Tum-5 can bind to
v
3 integrin on endothelial cells, whereas
the cells are still attached to vitronectin and fibronectin. Such
binding induces anti-proliferative and pro-apoptotic effect by
endogenous angiogenesis inhibitors such as tumstatin. This may
potentially explain the reason for the potent effect of tumstatin on
proliferating endothelial cells.
v
3 integrin, independent of vitronectin
binding, to
v
3 integrin. It is speculated that vitronectin binding to
v
3 integrin
may be responsible for the cell survival/proliferative signal to
endothelial cells (42). It is hence conceivable that tumstatin binding
to
v
3 integrin, in this context, may
counteract such survival/proliferative signals and drive the
endothelial cell toward apoptosis. Our studies constitute the first
report of a potential novel role for
v
3
integrin in negative regulation of angiogenesis. Such a novel
function for
v
3 integrin may implicate
its potential role in controlling and inhibiting tumor growth.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants DK-51711 and DK-55001 from the National Institutes of Health (to R. K.), by the 1998 Hershey Prostate Cancer Research Award (to R. K.), and by research funds from the Beth Israel Deaconess Medical Center.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.
§ Recipient of the 1999 Research Award for Young Scientists from the Inoue Foundation for Science of Japan.
Equity holders of Ilex Oncology, Inc., a company involved in
the clinical development of Tum-5.
** Supported by Deutsche Forschungsgemeinschaft Grant HO 2138/1-1.
Consultant for Ilex Oncology, Inc. To whom correspondence
should be addressed: Nephrology Div., Dept. of Medicine, RW 563a, Beth
Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-0445; Fax: 617-975-5663; E-mail:
rkalluri@caregroup.harvard.edu.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M007764200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NC1, noncollagenous 1; C-PAE, bovine pulmonary arterial endothelial; HUVEC, human umbilical vein endothelial cells; FCS, fetal calf serum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; BrdUrd, bromodeoxyuridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FITC, fluorescein isothiocyanate; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; MVD, microvessel density; GP, Goodpasture.
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