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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2461-2468
Calreticulin and Calreticulin Fragments Are Endothelial Cell
Inhibitors That Suppress Tumor Growth
By
Sandra E. Pike,
Lei Yao,
Joyce Setsuda,
Karen D. Jones,
Barry Cherney,
Ettore Appella,
Kazuyasu Sakaguchi,
Hira Nakhasi,
Chintamani
D. Atreya,
Julie Teruya-Feldstein,
Peter Wirth,
Ghanshyam Gupta, and
Giovanna Tosato
From the Center for Biologics Evaluation and Research,
Rockville; the Laboratory of Cell Biology,
National Cancer Institute, DBS, Bethesda;
the Laboratory of Pathology, National Cancer
Institure, DCS, Bethesda; and the Laboratory of
Experimental Carcinogenesis, National Cancer Institute, DBS,
Bethesda, MD.
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ABSTRACT |
Several angiogenesis inhibitors are fragments of larger proteins
that are themselves not active as angiogenesis inhibitors. Vasostatin,
the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In
the present study, we examined whether the full-length calreticulin
molecule shares the antiangiogenic and antitumor activities of
vasostatin. Similar to vasostatin, calreticulin selectively inhibited
endothelial cell proliferation in vitro, but not cells of other
lineages, and suppressed angiogenesis in vivo. When inoculated into
athymic mice, calreticulin inhibited Burkitt tumor growth comparably
with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids
inhibited endothelial cell proliferation in vitro and Burkitt tumor
growth in vivo comparably with vasostatin. An internal calreticulin
fragment encompassing amino acids 120-180 also inhibited endothelial
cell proliferation in vitro and angiogenesis in vivo comparably with
calreticulin and vasostatin. These results suggest that the
antiangiogenic activities of vasostatin reside in a domain that is
accessible from the full-length calreticulin molecule and localize to
calreticulin N-terminal amino acids 120-180. Thus, calreticulin and
calreticulin fragments are inhibitors of angiogenesis that directly
target endothelial cells, inhibit angiogenesis, and suppress tumor
growth. This information may be critical in designing targeted
inhibitors of pathological angiogenesis that underlies cancer and other diseases.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
TUMOR GROWTH and metastasis formation are
dependent on the existence of adequate blood supply. As tumors grow
larger, adequate blood supply to the tumor tissue is often ensured by new vessel formation, a process named angiogenesis.1,2 In preclinical models, agents that target the tumor vasculature have been
shown to prevent or delay tumor growth and even to promote tumor
regression or dormancy.3-14 For example, antibodies to
 v 3, an integrin molecule that is
expressed at high levels on proliferating vessels, inhibited
angiogenesis and tumor formation on the chick chorioallantoid
membrane.8,15 Antibodies to vascular endothelial growth
factor (VEGF), a stimulator of angiogenesis that is produced by a
variety of tumor cells, reduced tumor growth in experimental models.6,14 Thrombospondin, an extracellular matrix protein secreted by endothelial cells and other cells, inhibited endothelial cell chemotaxis, neovascularization, and tumor growth.3,16 Angiostatin, a fragment of plasminogen, and endostatin, a fragment of
collagen XVIII, suppressed angiogenesis and the growth of a variety of
experimental tumors.9,13
Recent experiments have suggested distinct potential advantages of
tumor treatment with angiogenesis inhibitors. Resistance, commonly
observed with chemotherapeutic agents, was not noted even after
extended experimental treatment with the antiangiogenic protein
endostatin, suggesting that targeting the tumor vasculature can be
useful in the context of tumor cell resistance to chemotherapeutic agents.17,18 In addition, treatment of experimental tumors with the angiogenesis inhibitor angiostatin combined with radiotherapy displayed synergistic antitumor effects, suggesting that antiangiogenic agents may be useful in combination with agents that target more directly the tumor cells.19
Efforts in our laboratory aimed at identifying novel inhibitors of
endothelial cell proliferation in the culture supernatants of
Epstein-Barr virus (EBV)-immortalized cells resulted in the isolation
of vasostatin, the N-terminal domain of calreticulin, which includes
calreticulin amino acids 1-180.20 Vasostatin is a specific
inhibitor of basic fibroblast growth factor (bFGF)-induced endothelial
cell proliferation in vitro and a suppressor of bFGF-induced angiogenesis in vivo.20 When inoculated into athymic mice,
vasostatin prevented or significantly reduced experimental tumor
growth.20 Because other inhibitors of angiogenesis, such as
certain internal domains of prolactin and fibronectin, angiostatin, and
endostatin, are fragments of larger proteins that are themselves not
active as angiogenesis inhibitors,5,9,13,21 we sought to
examine whether the full-length calreticulin molecule displays the
antiangiogenic and antitumor properties of vasostatin. Additionally, we
wished to define further the vasostatin domain that is active in
endothelial cell and tumorigenesis assays. To this end, we have
compared vasostatin, calreticulin, a fragment of calreticulin lacking
the N-terminal amino acids 1-120, and an internal calreticulin peptide,
including amino acids 120 to 180 (Fig 1),
for their ability to inhibit endothelial cell proliferation and
angiogenesis.
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| Fig 1.
Schematic representation of full-length calreticulin and
calreticulin fragments expressed in E coli as fusion proteins
with MBP. MBP is depicted as a shaded box. The N-terminal (N),
proline-rich (P), and C-terminal (C) domains of calreticulin are noted.
Numbers above each box denote the amino acid numbers from mature
calreticulin.
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MATERIALS AND METHODS |
Cell proliferation assays.
Fetal bovine heart endothelial cells (American Type Culture Collection
[ATCC], Manassas, VA) were grown through passage 12 in Dulbecco's
Modified Eagle's Medium (DMEM) culture medium (BioWittaker, Walkersville, MD) containing 10% heat inactivated fetal bovine serum
(BioWittaker), 25 ng/mL bFGF (R&D Systems, Minneapolis, MN), and 5 µg/mL gentamicin (Sigma, St Louis, MO). For
proliferation assays, cells were trypsinized (Trypsin/EDTA; GIBCO BRL,
Gaithersburg, MD), washed, suspended in culture medium
(DMEM containing 10% heat inactivated fetal bovine serum and 5 µg/mL
gentamicin), plated (800 cells/well in 0.2 mL culture medium) in
triplicate onto 96-well plates, and incubated for 5 days. DNA synthesis
was measured by 3H thymidine deoxyribose uptake (0.5 mCi/well, 6.7 Ci/mmol; New England Nuclear, Boston, MA) during the last
20 to 23 hours of culture; cells were detached from wells by freezing
and thawing.22
Matrigel angiogenesis assay.
The Matrigel assay was performed as described.22,23
Matrigel, a crude extract of the Englebreth-Holm-Swarm tumor was
obtained from Collaborative Biomedical Products. (Becton Dickinson
Labware, Bedford, MA.) An aliquot (0.5 mL) of Matrigel alone or with
desired additives was injected subcutaneously (sc) into the
midabdominal region of female BALB/c nude mice, 6 to 8 weeks old. Five
mice were injected with each mixture. After 5 to 7 days, the animals were killed, Matrigel plugs were removed, fixed in 10% neutral buffered formalin solution (Sigma), and embedded in paraffin. Tissues
were sectioned (5 µ thickness) and slides stained with Masson's
trichrome (American Histolabs, Gaithersburg, MD).
Quantitative analysis of angiogenesis in Matrigel plugs used a
computerized semi-automated digital analyzer (40-10 System; Optomax,
Hollis, NH). The instrument was adjusted to evaluate a circular area
measuring 1.26 × 105 mm2 of Matrigel, and
within this area, to measure the area occupied by cells. For each plug,
12 to 15 distinct fields were evaluated. The fields were randomly
selected from each plug, and the operator was blind to the experimental
design. The average area occupied by cells/1.26 × 105
mm2 Matrigel field was calculated. Results are expressed as
the mean area occupied by cells/Matrigel field.
Production of recombinant calreticulin and calreticulin fragments.
The expression of human calreticulin, the calreticulin N-terminal
domain (amino acids 1-180), and the calreticulin N-terminal deletion
fragment lacking amino acids 1-120 ( 120-calreticulin) fused to
maltose-binding protein (MBP) in Escherichia coli was reported.24,25 For expression of the calreticulin fragment encompassing amino acids 120 to 180 calreticulin), the coding region
for this fragment was amplified by polymerase chain reaction and then
cloned (confirmed by sequencing) as an N-terminal translational fusion
protein with the MBP gene for expression in E coli. E coli were
grown in Luria-Bertani broth (Advanced Biotechnologies, Inc, Columbia,
MD) with 0.2% glucose and 100 µg/mL ampicillin (Sigma) to an OD600
of approximately 1.0, after which fusion protein expression was induced
with 0.3 mmol/L IPTG (GIBCO/BRL) for 2 to 2.5 hours. After lysis (1 µg/mL lysozyme in 10 mmol/L Tris; pH 7.5; 5% glycerol; 100 mmol/L
EDTA; 5 mmol/L B-ME), sonication, and centrifugation (30 minutes at
8,360g) of the bacterial suspension, the supernatant was loaded
onto a preequilibrated (20 mmol/L Tris; pH 7.5; 200 mmol/L NaCl; and l
mmol/L EDTA) 15 mL amylose resin column (New England Biolabs, Beverly,
MA). Bound material was eluted with 10 mmol/L maltose.
Protein-containing fractions were ultracentrifuged (2 hours at
104,000g); supernatant was retained. Separation of MBP from
calreticulin and vasostatin was accomplished by cleavage with factor
Xa, as described.24 Purification of cleaved calreticulin or
vasostatin from MBP was achieved by anion exchange chromatography using
a preequilibrated (20 mmol/L Tris; pH 8.0; 25 mmol/L NaCl) Resource Q
column (Amersham Pharmacia Biotech, Arlington Heights, IL). Bound material was eluted by a step-wise gradient
where MBP elutes at 100 to 150 mmol/L NaCl; Factor Xa elutes at
approximately 400 mmol/L NaCl; and calreticulin or vasostatin elute at
approximately 250 mmol/L NaCl. For construction of the glutathione S
transferase (GST)-calreticulin fusion construct, the coding region for
the mature calreticulin protein was cloned as a C-terminal
translational fusion with the GST gene for expression in E
coli. The coding region of human calreticulin was amplified and
cloned in frame with GST protein (confirmed by sequencing). The growth
of E coli and the induction and release of GST-calreticulin
from the bacteria was similar to that described for MBP-calreticulin,
except for concentration of ITPG (0.6 mmol/L). Purification of
GST-calreticulin was achieved by lysis of the bacteria, followed by
sonication and centrifugation. The supernatants (adjusted to pH 7.0)
were mixed with prewashed Glutathione Sepharose 4B (Bulk GST
purification module; Amersham Pharmacia Biotech) in phosphate-buffered
saline (PBS) with 1.0% Triton X-100. After 30 minutes incubation and washing the beads, bound protein was eluted with a 50 mmol/L Tris-HCl buffer containing 10 mmol/L Glutathione, pH 8.0. Eluted material was
ultracentrifuged (2 hours at 104,000g), and supernatant
retained. All protein lots for in vivo and in vitro experiments
(GST-calreticulin, control GST, MBP-calreticulin, MBP-vasostatin, MBP,
cleaved calreticulin, and vasostatin) were tested for endotoxin by the
Limulus Amebocyte Lysate (LAL) kinetic-QCLTM assay
(BioWittaker) and were found to contain less than 5 U/10
µg protein.
Protein sequencing.
Protein bands were excized from Coomassie-stained gel and destained.
The proteins were digested with trypsin (Promega, Madison, WI) in the
gel26 and the resulting fragments were separated by
microcapillary high performance liquid chromatography and analyzed in-line by ion-trap mass spectrometry (Finnigan model LCQ, San Jose,
CA).27
Mouse tumor model.
BALB/c nu/nu female mice, 6 weeks of age (National Cancer Institute,
Frederick, MD) maintained in pathogen-limited conditions, received 400 rad (1 rad = 0.01 Gy) total body irradiation and 24 hours later were
injected sc in the right abdominal quadrant with 107
exponentially growing human Burkitt lymphoma cells (CA46 cell line11) in 0.2 mL PBS. Immediately after the Burkitt
cells were inoculated sc, and continuing daily thereafter 6 d/wk, the
mice received sc injections proximal to the site of original cell
inoculation of test samples or appropriate controls. These included
purified GST-calreticulin, control GST, MBP-calreticulin,
MBP-vasostatin, MBP, or formulation buffer used to dilute test proteins
(sterile saline solution containing 50 mg/mL human albumin and 5 mg/mL mannitol; endotoxin <5 U/mL). Tumor size was estimated (in
mm2) twice weekly as the product of 2-dimensional caliper
measurements (longest perpendicular length and width). A subcutaneous
mass appearing at or proximal to the site of cell inoculation was
considered a tumor when it measured at least 0.16 cm2 in
surface area and increased in size by at least 30% over the following week.
Histology and immunohistochemistry.
Tumors and Matrigel plugs were fixed in 10% neutral buffered formalin
solution (Sigma), embedded in paraffin, sectioned at 4 µm, and
stained with hematoxylin and eosin, Masson's trichrome, or elastin van
Gieson reagent by standard methods. Immunohistochemical staining of
tumor vessels was performed as described28 on
paraffin-embedded samples that had been deparaffinized and rehydrated.
After treatment with trypsin for 10 minutes or heat for 25 minutes,
tissue sections were blocked with 3% goat serum in Tris-buffered
saline, after incubation (overnight at 4°C) with primary
antibody. Purified rabbit anti-von Willebrand factor (1:50 dilution)
and mouse anti-smooth muscle actin monoclonal antibody
(clone 1A4, 0.25 µg/mL) were purchased from DAKO (Carpinteria, CA).
After washing, biotynilated goat anti-rabbit or horse anti-mouse
secondary antibody (2 µmol/L; Vector Labs, Burlingame, CA) was
applied followed by VECTASTAIN ABC peroxidase complex (Elite ABC-kit;
Vector Labs). The sections were developed using peroxidase
3,3'-diaminobenzidine (DAB) substrate and counterstained with hematoxylin.
Statistical analysis.
Student's t-test was used to evaluate the significance of
group differences; Wilcoxon Rank Sums test was used to evaluate differences in tumor growth rates; Kruskal-Wallis Rank Sums test was
used to evaluate group differences in 3-way comparisons.
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RESULTS |
Effects of calreticulin on endothelial cell proliferation.
In initial experiments, we tested full-length calreticulin and a
truncated form of calreticulin lacking the N-terminal 1 to 120 amino
acids for their ability to inhibit endothelial cell proliferation in
vitro and compared with vasostatin. To this end, in addition to
purifying the recombinant N-terminal domain of calreticulin (amino
acids 1 to 18020), we purified human calreticulin from
E coli expressing the recombinant protein fused to either MBP
or GST. We also purified a truncated form of calreticulin, 120-calreticulin, from E coli expressing the protein fused
to MBP. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig 2),
GST-calreticulin (lane 1), GST (lane 2), MBP-calreticulin (lane 3), MBP
(lane 5), and MBP-120-calreticulin (lane 6) resolved as discrete
bands migrating at the expected relative positions. Calreticulin,
cleaved from MBP-calreticulin by treatment with factor Xa and
subsequently purified by anion exchange chromatography, resolved as a
doublet with a relative molecular weight of approximately 50 and 55 kD (lane 4). On Western blotting, both bands were
recognized by a rabbit antiserum against human calreticulin (not
shown). The lower band was subjected to trypsin digestion and the
tryptic fragments were sequenced by ion-trap mass spectrometry. By this
method, this band was identified as a cleavage fragment of calreticulin
encompassing at least amino acids 49 to 321.
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| Fig 2.
SDS-PAGE of recombinant purified proteins. 1:
GST-calreticulin; 2: GST; 3: MBP-calreticulin; 4: calreticulin cleaved
from MBP-calreticulin; 5: MBP; 6: MBP- 120-calreticulin.
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When tested in functional assays, recombinant purified MBP-calreticulin
inhibited the proliferation of fetal bovine heart endothelial cells
induced by bFGF (Fig 3). At a concentration of 1 µg/mL, MBP-calreticulin inhibited fetal bovine heart endothelial cell growth by 67%, whereas control MBP had minimal effects. Similar inhibition was noted with 1 µg/mL GST-calreticulin (Fig
4) and with vasostatin.20
Recombinant calreticulin that had been cleaved and purified from
MBP-calreticulin also inhibited the proliferation of fetal bovine heart
endothelial cells, whereas control MBP displayed minimal inhibition
(Fig 4). Furthermore, recombinant purified MBP-120-calreticulin
inhibited the proliferation of fetal bovine heart endothelial cells,
and the degree of inhibition was comparable with that of calreticulin
(Fig 5).
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| Fig 3.
Inhibition of endothelial cell proliferation by
MBP-calreticulin. Fetal bovine heart endothelial cells (800 cells/well)
were incubated for 5 days either in medium alone or medium supplemented
with bFGF (25 ng/mL), with or without recombinant purified
MBP-calreticulin or MBP (both at 1 µg/mL). The results of 16 experiments are expressed as mean cpm (±SD).
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| Fig 4.
Dose-dependency of inhibition of endothelial cell
proliferation by calreticulin. Fetal bovine heart endothelial cells
(800 cells/well) were cultured for 5 days in medium alone or medium
supplemented with bFGF (25 ng/mL). Recombinant purified
GST-calreticulin (1 µg/mL), control recombinant GST (0.03 to 1 µg/mL), calreticulin cleaved and purified from MBP-calreticulin (0.03 to 2 µg/mL), and MBP cleaved and purified from MBP-calreticulin (0.5 µg/mL) were added to endothelial cell cultures with bFGF (25 ng/mL).
Proliferation was measured by 3H thymidine incorporation
during the final 20 to 23 hours of culture; the results reflect mean
cpm/culture. (A) Reflects the mean of 9 experiments, each performed in
triplicate. (B) Reflects the mean of 2 experiments, each performed in
triplicate.
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| Fig 5.
Inhibition of endothelial cell proliferation by
120-calreticulin. Fetal bovine heart endothelial cells (800 cells/well) were cultured for 5 days in medium alone or medium
supplemented with bFGF (25 ng/mL). Recombinant purified
MBP-calreticulin, MBP-120-calreticulin, or MBP were added to
bFGF-supplemented cultures. Proliferation was measured by
3H thymidine incorporation during the final 20 to 23 hours
of culture. The results reflect the mean of triplicate cultures; SDs
within 12% of the mean.
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Because vasostatin (calreticulin N-domain, encompassing amino acids
1-180), full-length calreticulin, and 120-calreticulin (calreticulin
missing the N-terminal 1-120 amino acids) inhibited bFGF-induced
endothelial cell proliferation to a similar degree at similar
concentrations, we investigated whether the 61 amino acid calreticulin
fragment encompassing amino acids 120-180 (120-180 calreticulin) was
also active. To this end, we purified an MBP 120-180 calreticulin
fragment from E coli expressing the recombinant protein fused
to MBP (Fig 6A). When tested
for inhibition of endothelial cell proliferation, recombinant purified
MBP 120-180 calreticulin fragment inhibited the proliferation of bovine
heart endothelial cells induced by bFGF (Fig 6B). At a concentration of
0.5 µg/mL, the 61-amino acid calreticulin fragment inhibited fetal
bovine heart endothelial cell proliferation by 65%. A side-by-side
comparison of full-length MBP-calreticulin, MBP-vasostatin (1-180 calreticulin), MBP-120-calreticulin and MBP 120-180 calreticulin
fragment revealed that, on a molar basis, the 4 proteins display
similar endothelial cell growth inhibitory activity in vitro. Control
MBP was not inhibitory (Fig 7). These
results suggest that the antiangiogenic activity of calreticulin
resides in a domain between amino acids 120 and 180.
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| Fig 6.
(A) SDS-PAGE of recombinant purified proteins. 1: MBP
120-180 calreticulin fragment; 2: MBP. (B) Inhibition of endothelial
cell proliferation by MBP 120-180 calreticulin fragment. Fetal bovine
heart endothelial cells (800 cells/well) were incubated for 5 days
either in medium alone or medium supplemented with bFGF (15 ng/mL),
with or without recombinant purified MBP 120-180 calreticulin peptide
(1 µg/mL). The results of 8 experiments are expressed as mean
cpm (±SD).
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| Fig 7.
Comparative analysis of endothelial cell growth
inhibition by MBP calreticulin ( ),
MBP-120-calreticulin ( ), MBP-vasostatin ( ), MBP 120-180 calreticulin () fragment, and MBP ( ). Fetal bovine heart
endothelial cells (800 cells/well) were cultured for 5 days in medium
alone or medium supplemented with bFGF (15 ng/mL). Recombinant purified
fusion proteins were added to culture at 0.4 to 32 nmol/L
concentrations to bFGF-supplemented cultures. Proliferation was
measured by 3H thymidine incorporation during the final 20 to 23 hours of culture. The results reflect the mean of triplicate
cultures; SDs within 15% of the mean. The mean response of endothelial
cells was 2,217 cpm when cultured in medium alone, and 23,377 cpm when
cultured with bFGF alone.
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In contrast to its inhibitory effect on endothelial cell growth,
calreticulin at concentrations of 0.5 to 10 µg/mL had minimal effect
on the proliferation of a variety of other primary cells and cell lines
(not shown), including human peripheral blood mononuclear cells either
not stimulated or stimulated with phytohemagglutinin; B-cell-enriched
peripheral blood mononuclear cells stimulated with EBV;
T-cell-enriched populations stimulated with pokeweed mitogen; human
foreskin fibroblasts (H5 68); Burkitt lymphoma cells (CA46, BL41,
KK124, Ag876, SHO); lymphoblastoid cells (VDS-O line); T-cell line
(Molt-4); neuroblastoma cells (SK-N-MC); lung carcinoma cells (A-549);
breast adenocarcinoma cells (MDA-MB-468); acute promyelocytic leukemia
cells (HL-60); prostate adenocarcinoma cells (Tsu-Pr1, PC-3, Dul45);
Hodgkin's lymphoma cells (Hs445); colon adenocarcinoma cells (SW-480);
Wilms tumor cells (SK-NEP-1); and melanoma cells (A-375). The 61-amino
acid calreticulin fragment (120-180 calreticulin) at concentrations of
0.5 to 10 µg/mL had minimal effect on the proliferation of
lymphoblastoid and Burkitt lymphoma cells (not shown). These results
are similar to those derived with vasostatin.20 Thus,
calreticulin and vasostatin can selectively inhibit the proliferation
of endothelial cells in vitro.
Calreticulin and calreticulin fragments inhibit angiogenesis.
The murine Matrigel assay22,23 was used to evaluate the
effects of calreticulin and calreticulin fragments on angiogenesis in
vivo. When added to Matrigel at concentrations of 1.25 to 5 µg/mL,
GST-calreticulin displayed a concentration-dependent inhibition of
bFGF-induced neovascularization (Table 1,
experiment 1). When tested in the same experiment, MBP-calreticulin and
MBP-vasostatin used at the same concentration (5 µg/mL) inhibited
bFGF-induced neovascularization to a similar degree (Table 1,
experiment 2). In another experiment, calreticulin cleaved and purified
from MBP, MBP-120-calreticulin, and MBP 120-180 calreticulin
fragment similarly inhibited bFGF-induced neovascularization (Table 1, experiment 3). These results show that full-length calreticulin, the
N-terminal domain of calreticulin (amino acids 1-180, vasostatin), and
a calreticulin fragment encompassing amino acids 120-180 can all
function as angiogenesis inhibitors in vivo.
Calreticulin inhibits tumor growth in mice.
In previous experiments, vasostatin prevented or significantly reduced
Burkitt tumor growth in nude mice.20 Using the same experimental system, we now tested calreticulin for its ability to
prevent the growth of Burkitt lymphomas in athymic mice. Sublethally irradiated, 6-week-old female BALB/c nude mice were inoculated sc with
10 × 106 Burkitt cells (CA46 cell line). Beginning at the
time of cell inoculation, the mice received daily, 6 days/week for 14 days, sc injections (100 µL volume) of either control GST protein (20 µg/mouse) or GST-calreticulin (60 µg/mouse), adjacent to the site of cell injection (Fig 8A). As expected,
12/12 mice injected with control GST protein developed a tumor by day
17. By contrast, only 4/13 mice injected with GST-calreticulin
developed a tumor by day 17 (P = .005). The tumor-bearing
mice were killed, and the remaining 9 nontumor-bearing mice were
maintained untreated. Tumors eventually developed in 8 of 9 mice that
had received initial calreticulin treatment. The latest tumor developed
on day 36, 22 days after treatment had ended. One mouse remains tumor
free (>200 days).
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| Fig 8.
Calreticulin and calreticulin fragments inhibit tumor
growth. Burkitt lymphoma cells (CA46 cell line, 1 × 107
cells) were inoculated sc into BALB/c athymic mice, 6 weeks of age. (A)
Beginning on the day of cell inoculation and continuing thereafter
daily, 6 days/week, 12 mice were inoculated with control GST protein
( , 20 µg/d × 14 days), and 13 mice were inoculated with
GST-calreticulin (, 60 µg/d × 14 days). (B) Beginning on the
day of tumor appearance, 5 mice were inoculated with control MBP
protein (, 12.5 µg/d), 4 mice were inoculated with
MBP-calreticulin (, 12.5 µg/d), and 4 mice were inoculated with
MBP-vasostatin (, 12.5 µg/d). (C) Beginning on the day of cell
inoculation and continuing thereafter daily, 6 d/wk, 8 mice were
inoculated with control MBP (, 20 µg/d × 14 days), 9 mice were
inoculated with MBP-vasostatin (, 30 µg/d × 14 days), and 9 mice were inoculated with 120 calreticulin (, 30 µg/d × 14
days).
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We then examined the effects of calreticulin on established Burkitt
tumors and compared with vasostatin (Fig 8B). In a representative experiment, the rate of tumor growth appeared to be reduced in the
group of mice (4 mice) treated with MBP-calreticulin or MBP-vasostatin (both at the dose of 25 µg/mouse) compared with the control group (5 mice) treated with MBP (12.5 µg/mouse), but the difference did not
achieve overall significance (P = .089), in part because of
the small sample size. Growth rates were similar in the 2 groups of
mice treated with either MBP-calreticulin or MBP-vasostatin. All tumors
were removed on day 41 of treatment. The mean (±SD) weight of tumors
in the control group (5.55 ± 2.93 g) was greater than the weight of
tumors from mice treated with either MBP-calreticulin (2.74 ± 0.95 g) or MBP-vasostatin (3.01 ± 1.22 g).
In an additional experiment (Fig 8C), we tested
MBP-120-calreticulin (30 µg/mouse) for its ability to prevent
Burkitt tumor growth and compared its effects with those of
MBP-vasostatin (30 µg/mouse). After 14 days of treatment, all mice (8 of 8) inoculated with control MBP (20 µg/mouse) developed a tumor. By
contrast, only 3 of 9 mice inoculated with MBP-120-calreticulin,
and 3 of 9 mice inoculated with MBP-vasostatin developed a tumor
(P = .009). After treatment was suspended on day 14, the mice
were observed. One mouse from the group treated with
MBP-120-calreticulin and one mouse from the group treated with
MBP-vasostatin remain tumor free as of day 60. Thus, similar to
vasostatin, MBP-120-calreticulin can prevent Burkitt tumor growth.
The histology of Burkitt tumors treated with MBP-calreticulin (Fig
9), MBP-120-calreticulin (not shown),
and MBP-vasostatin20 was indistinguishable from controls
with respect to the morphology of the tumor cells and the number of
tumor cell mitoses. Immunohistochemical staining of vessels using
antibodies against smooth muscle actin (Fig 9) and factor VIII-related
antigen (not shown) revealed that Burkitt tumors treated with
MBP-calreticulin displayed fewer vessels than control tumors. To
quantitatively assess tumor vascularization, we counted all visible
microvessels at a magnification of 200 (ie, 0.74 mm2/field)
in tumor tissue sections. In 3 in vivo experiments, the number of
intratumoral and peritumoral microvessels was significantly (P < .05) reduced in animals treated with GST-calreticulin,
MBP-calreticulin, MBP-120-calreticulin, or MBP-vasostatin compared
with controls. Occasionally, calreticulin-treated tumors displayed
vascular alterations, including medial thickening, fibrinoid necrosis
and neutrophil infiltration of the vessel wall that were not noted in
control tissues (not shown). These vascular alterations were not
different from those noted in vasostatin20 or
MBP-120-calreticulin (not shown) treated mice. Similar to our
previous findings in vasostatin-treated animals,20 gross
and histological examination of liver, spleen, kidneys, heart, lung,
and lymph nodes from mice treated with either calreticulin or
MBP-120-calreticulin detected no abnormalities.
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| Fig 9.
Immunohistochemical detection of the tumor vasculature in
representative Burkitt tumors treated with MBP or MBP-calreticulin.
Paraffin-embedded tumor sections were stained with mouse anti-alpha
smooth muscle actin monoclonal antibody and counterstained with
hematoxylin. (A) Representative tumor tissue from a mouse treated with
MBP alone. (B) Representative tumor tissue from mouse treated with
MBP-calreticulin (original magnification ×20).
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| |
DISCUSSION |
Recently, we have reported that vasostatin, the N-domain of
calreticulin composed of amino acids 1 to 180, can inhibit endothelial cell proliferation, angiogenesis and tumor
growth.20 Here we show that full-length
calreticulin, a protein of 417 amino acids deduced from its cDNA, a
deletion fragment of calreticulin lacking the N-terminal 1-120 amino
acids (120 calreticulin), and an internal calreticulin fragment
composed of amino acids 120-180 can inhibit endothelial cell growth in
vitro. When compared with vasostatin for their ability to inhibit
endothelial cell growth in vitro, calreticulin, 120 calreticulin,
and the 120-180 calreticulin fragment inhibited endothelial cell growth
to a similar degree at similar concentrations. Like vasostatin,
calreticulin, 120 calreticulin, and the 120-180 calreticulin
fragment inhibited endothelial cell growth specifically and directly,
without a requirement for other cell types or extracellular matrix
proteins and with minimal effects on cell viability. When compared in
Matrigel-based angiogenesis assays, calreticulin, the 120-180 calreticulin fragment and vasostatin displayed similar inhibition. Like
vasostatin, calreticulin and 120 calreticulin could prevent or delay
significantly the development of Burkitt lymphoma. The similarities of
biological activities displayed by calreticulin, vasostatin, 120
calreticulin, and the 120-180 calreticulin fragment in endothelial cell
proliferation and neovascularization assays suggest that the
antiangiogenic activity of calreticulin resides in a domain within
amino acids 120-180.
Calreticulin, a highly conserved and ubiquitous protein, serves as one
of the major storage depots for calcium within the endoplasmic
reticulum and participates in calcium signaling.29,30 Structure-function analyses have distinguished the molecule in 3 domains. The highly acidic C-domain contains the KDEL signal sequence
for calreticulin retention in the endoplasmic reticulum and binds
calcium with high capacity.31 It can also associate with
the blood clotting factors IX, X, and prothrombin.32 The proline-rich P domain has extensive sequence homology to calnexin, a
protein of the endoplasmic reticulum proposed to serve as a chaperon,
and contains the high-affinity, calcium-binding site of
calreticulin.33 The N-domain, the most conserved domain
among calreticulins cloned so far, does not bind calcium but can bind the cytoplasmic domain of alpha subunits of integrins and the nuclear
receptors for glucocorticoid, androgen, and retinoic
acid.34-36 It also binds C1q, the recognition subunit of
the first component of the classical complement pathway.37
Additional activities of the N-domain of calreticulin that we have
recently recognized include inhibition of angiogenesis and tumor
growth.20 Here we show that full-length calreticulin,
120 calreticulin, and an internal calreticulin fragment composed of
amino acids 120 to 180 can also inhibit endothelial cell growth,
angiogenesis, or tumor growth.
A number of previously identified inhibitors of angiogenesis are
fragments of larger proteins, which are not themselves active as
angiogenesis inhibitors. An internal 16-kD fragment of prolactin was
reported to be an inhibitor of angiogenesis, but prolactin was found to
be inactive.5 Similarly, 2 heparin-binding fragments of
fibronectin inhibited endothelial cell growth, whereas intact fibronectin did not.21 Angiostatin, a fragment of
plasminogen, and endostatin, a fragment of collagen XVIII, are
previously recognized angiogenesis inhibitors. However, neither
plasminogen nor collagen XVIII were found to be active or to interfere
with the inhibitory action of their internal fragments.9,13
In contrast to these angiogenesis inhibitors, thrombospondin, a high
molecular weight extracellular matrix glycoprotein, displayed
antiangiogenic activity, as did some of its internal
fragments.38
Like vasostatin, calreticulin inhibited endothelial cell proliferation
directly and specifically, raising the possibility that it may bind to
a receptor on endothelial cells. Previously, calreticulin was reported
to bind specifically and reversibly to endothelial cells in vitro with
a kd of approximately 7.4 nmol/L.32 Endothelial cells were reported to release nitric oxide in response to
calreticulin.32 When injected in mice, calreticulin
localized selectively to the vascular endothelium, particularly in the
lungs.32 We do not know the mechanism by which calreticulin
or vasostatin inhibit angiogenesis and tumor growth. Although nitric
oxide was reported to suppress endothelial cell migration and
angiogenesis,39,40 we have found no evidence that nitric
oxide release mediates the effects of calreticulin or vasostatin based
on in vitro experiments with inhibitors of nitric oxide synthase (not
shown). In addition, we have found no evidence that C1q binding is
important to the antiangiogenic effects of calreticulin and vasostatin,
because C1q was not measurable in endothelial cell cultures (not
shown). We believe, however, that inhibition of endothelial cell growth is central to the inhibition of angiogenesis and tumor growth induced
by calreticulin and vasostatin.
We do not know whether calreticulin and its fragments are effective
inhibitors of all types of angiogenic endothelial cells or the full
spectrum of tumors that are inhibited by these agents. The observation
that full-length calreticulin, vasostatin, 120 calreticulin, and the
120-180 calreticulin fragment are all active inhibitors of endothelial
cell growth suggests that the active site resides within a 60-amino
acid internal fragment of calreticulin between amino acids 120-180. This observation raises the possibility that we may be able to identify
active fragments of calreticulin even smaller than vasostatin or the 61 amino acid internal calreticulin peptide that might be biologically
active as angiogenesis inhibitors. These molecules may be easier to
produce and deliver. There is considerable enthusiasm for the potential
application of inhibitors of angiogenesis to cancer
treatment.41 Calreticulin, 120 calreticulin, and
vasostatin hold promise as novel therapeutics for the treatment of
pathological angiogenesis in cancer and many other diseases.
| |
ACKNOWLEDGMENT |
We thank Drs Greg Pogue, Rob Duncan, Andrea Tenner, Malcolm Moos, Bob
Boykins, Venkatesha Basrur, Hynda Kleinman, Doug Roberts, Robert
Yarchoan, and Yoshi Aoki for their help in various aspects of this work.
| |
FOOTNOTES |
Submitted December 14, 1998; accepted May 28, 1999.
S.E.P. and L.Y. contributed equally to this work.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
This is a US government work. There are no restrictions on its use.
Address reprint requests to Giovanna Tosato, MD, Center
for Biologics Evaluation and Research, 1401 Rockville Pike, Rockville,
MD 20852; e-mail: Tosato{at}A1.CBER.FDA.GOV.
| |
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This is a US government work. There are no restrictions on its use.
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TOP
ABSTRACT
INTRODUCTION