From the Cancer Biology Laboratories, Department of Molecular Medicine, Cornell University College of Veterinary Medicine, Ithaca, New York 14853
Received for publication, January 18, 2001, and in revised form, April 19, 2001
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ABSTRACT |
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Adhesion of blood-borne cancer cells to the
endothelium is a critical determinant of organ-specific metastasis.
Here we show that colonization of the lungs by human breast cancer
cells is correlated with cell surface expression of the
Colonization of secondary organs by blood-borne cancer cells marks
the final, usually fatal stage in a long, multistep cascade of tumor
progression that is propelled by an array of acquired, cumulative,
genetic abnormalities and promoting tissue microenvironmental cues
(1-5). Increasing evidence suggests that tumor cell targeting of
preferred, secondary organs for metastasis is mediated by distinct endothelial cell adhesion molecules (6-8). These molecules are expressed constitutively (organ-specifically) on the endothelial cell
luminal surface of select vascular compartments (e.g.
capillaries, arterioles, and/or venules) (7). By binding blood-borne
cancer cells at high affinity, these molecules mediate vascular arrest of tumor cells under hydrodynamic conditions (6-9) and, as shown recently, promote intravascular growth to form tumor colonies at these
secondary target sites (10, 11). Using a unique large vessel
endothelial cell system, in which an organ-specific vascular phenotype
can be induced by growing "neutral" bovine aortic endothelial cells
on matrix extracts of that organ (12), a lung-specific endothelial cell
adhesion molecule, termed Lu-ECAM-1
(lung-endothelial cell
adhesion molecule-1) was isolated, purified,
and cloned by our laboratory (13-15). Lu-ECAM-1 is the prototype of a
newly discovered mammalian family of proteins (termed CLCAs, for
Cl In this report, hCLCA21
cloned from a lung cDNA library (19) is identified as the human
counterpart of Lu-ECAM-1. We show that hCLCA2 is expressed by
endothelia from different lung vascular compartments and that lung
colonization of established human breast cancer cell lines is dependent
upon the tumor cells' ability to interact with hCLCA2. Breast cancer
cell adhesion to hCLCA2 is mediated by the Antibodies and Reagents--
Anti-Lu-ECAM-1 mAb 6D3 was
produced in BALB/c mice (22) and selected for
adhesion blocking of B16-F10 melanoma cells to Lu-ECAM-1-expressing
bovine aortic endothelial cells (13, 14). Rabbit polyclonal antibodies
(pAbs) 4 and 18 were generated against the hCLCA2 peptides
KANNNSKIKQESYEKANV (amino acids 94-111) and ESTGENVKPHHQLKNTVTVD
(amino acids 498-517), respectively. Antibodies against the
Cell Cultures--
Human breast cancer cell lines MDA-MB-231,
-435, -468, and -453 and MCF7 were from the ATCC (Manassas, VA).
MDA-MB-435L2 was from Dr. J. E. Price (The University of Texas
M.D. Anderson Cancer Center, Houston, TX) (20), and MDA-MB-435
transfected with wild type human RT-PCR Analyses--
Total RNA isolated from HMVEC-L, HAEC, and
HUVEC was reverse-transcribed, and an 800-base pair hCLCA2 product
amplified by PCR, using ttctctacaacatgacccaaaggagc and
catgggaaagctgtggtgaaag as 5' and 3' primers, respectively, and
Taq polymerase (Life Technologies) (19). The full-length
2.9-kilobase pair open reading frame of hCLCA2 was amplified from
HMVEC-L RNA, using primers corresponding to the 5'
(ttctctacaacatgacccaaaggagc) and 3'
(gacactttggatatttatttataataattttgttc) ends of the hCLCA2 open reading
frame. Both primer sets were tested on cloned plasmid templates to
ascertain that they would not recognize other CLCA homologs. RNA
extracted from HEK293 cells and processed in parallel served as
negative control.
Expression, Myc Tagging, Immunoprecipitation, and Purification of
hCLCA2--
Lung expression of hCLCA2 was analyzed by staining
sections of paraffin-embedded, formaldehyde (4%)-fixed tissue with
rabbit anti-hCLCA2 antiserum (pAb 4) at a 1:100 dilution (13, 22). Preimmune serum used at the same dilution served as control. Myc-tagged hCLCA2 constructs were generated as described and transfected into 80%
confluent HEK293 using the LipofectAMINETM Plus protocol (Life
Technologies) (19). Immunoprecipitated and immunopurified hCLCA2 were
from extracts of transfected HEK293 cells (48 h after transfection),
using anti-Myc mAb 9E10-conjugated goat anti-mouse IgG Dynabeads (or
Protein G beads). Surface expression of hCLCA2 was confirmed by cell
surface biotinylation (100 µg/ml Biotin N-hydroxysuccinimide) (25). Western blot analyses were done with anti-Myc mAb 9E10 (19).
Isolation, Purification, and Phosphorylation of the
Gel Overlay (Far Western)--
Transfection of K-Balb/3T3 Cells with FACS Analyses, Adhesion, and Lung Colony Assays--
FACS
analyses, adhesion assays, and lung colony assays were performed as
previously described in detail by our laboratory (12-14, 25).
hCLCA2 Expression by Endothelia of the Lung Vasculature--
Human
CLCA2 was cloned from a human lung cDNA library, and its amino acid
sequence, protein processing, transmembrane topography, and channel
properties are described elsewhere (19). Northern blot hybridization
and/or RT-PCR revealed epithelial expression of hCLCA2 in the mammary
gland and trachea (19), while RT-PCR and immunohistochemistry
demonstrated endothelial cell expression in the lungs (Fig.
1). In the latter, hCLCA2 protein was
expressed selectively in endothelia of small pulmonary arteries,
arterioles, and subpleural and interlobular venules (Fig. 1,
A-F), while endothelia in other tissues including brain,
liver, pancreas, kidney, alimentary tract, testis, ovary, adrenal
gland, thyroid, and skeletal muscle were negative (data not shown).
Strong hCLCA2 expression was also observed in cultured HMVEC-L lung
microvascular endothelial cells, while a weak hCLCA2 expression was
recorded for endothelial cells derived from human aorta (HAEC) and
umbilical vein (HUVEC) (Fig. 1G). Expression of hCLCA2
protein in HMVEC-L was confirmed by FACS, using the same polyclonal
antibody that had been employed in the immunohistochemical studies
(Fig. 1H).
hCLCA2 Mediates Adhesion of Human Breast Cancer Cells via the
Since isolation of the Specificity of the
In a second series of experiments, we show that selective cleavage of
the Lung Metastasis Is Inhibited by Effect of in Vivo Selection for Lung Metastatic Efficiency Versus
As shown in Fig. 3, Transfection of Kirsten-Ras-transformed Balb/3T3 Cells with
In this report, we describe a novel adhesion receptor/ligand pair
that mediates colonization of the lungs by human breast cancer cells
and possibly other cancer cell types. The pair consists of lung
endothelial cell hCLCA2 and breast cancer cell Molecular cloning of hCLCA2 from a lung cDNA library and
biochemical and functional characterization of hCLCA2 protein have shown that the molecule shares many of the characteristics of the other
CLCA family members (18). The adhesion function of CLCA channel
proteins is perplexing but not without precedent. Studies involving the
cystic fibrosis transmembrane conductance regulator have shown that
this chloride channel protein is also a cellular adhesion receptor for
Pseudomonas aeruginosa (16) and Salmonella typhi
(17). In cancer metastasis, a novel concept is the possible involvement
of a CLCA-mediated Cl The While an early stage of tumor progression (benign, well differentiated
tumor) may have accounted for lack of metastasis in These considerations have important experimental consequences for
metastasis research, since transfection of a gene suspected to play a
primary role in metastasis into a tumor cell line that is nonmetastatic
may not yield the expected result, since the introduced gene, even if
it appropriately associates with other membrane proteins to achieve
proper function, may not be sufficient in endowing tumors with mastery
over the complete metastatic cascade. Therefore, we have relied in our
transfection studies on a cell line that has a low lung metastatic
potential and, thus, expresses a gene array that is conducive to lung
metastasis including a low level of In conclusion, we have provided molecular evidence in support of the
observed link between 6
4 integrin and adhesion to human
CLCA2 (hCLCA2), a Ca2+-sensitive chloride channel protein
that is expressed on the endothelial cell luminal surface of pulmonary
arteries, arterioles, and venules. Tumor cell adhesion to endothelial
hCLCA2 is mediated by the
4 integrin, establishing for
the first time a cell-cell adhesion property for this integrin that
involves an entirely new adhesion partner. This adhesion is augmented
by an increased surface expression of the
6
4 integrin in breast cancer cells
selected in vivo for enhanced lung colonization but
abolished by the specific cleavage of the
4 integrin
with matrilysin.
4 integrin/hCLCA2 adhesion-blocking antibodies directed against either of the two interacting adhesion molecules inhibit lung colonization, while overexpression of the
4 integrin in a model murine tumor cell line of modest
lung colonization potential significantly increases the lung metastatic
performance. Our data clearly show that the
4/hCLCA2
adhesion is critical for lung metastasis, yet expression of the
4 integrin in many benign breast tumors shows that this
integrin is insufficient to bestow metastatic competence on cells that
lack invasiveness and other established properties of metastatic cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channel proteins,
Ca2+-activated) (13), which, similar to the
cystic fibrosis transmembrane conductance regulator (16, 17), serve the
dual function of mediating chloride conductance and cell-cell adhesion
(16-18). Lu-ECAM-1 protein, like all other members of the CLCA family, is synthesized as an ~125-kDa precursor protein that, upon membrane incorporation, is rapidly processed into N-terminal 90-kDa and C-terminal 35-kDa components (15, 18). The 90-kDa polypeptide is
responsible for the adhesion qualities of CLCAs, promoting the
Ca2+-dependent adhesion of a variety of lung
metastatic cancer cells but not cancer cells that metastasize to other
organ sites (12-15).
4 integrin,
which is prominently expressed in breast cancer cells that are able to
colonize the lungs upon tail vein injection of nude mice (20).
Cell-to-cell adhesion assays, adhesion-blocking assays with antibodies
generated against either of the two interacting molecules, and
overexpression of
4 in a murine model tumor cell line
are used to confirm involvement of the
4 integrin/hCLCA2
adhesion mechanism in lung metastasis. Together, our data confirm that
the
6
4 integrin is a lung
metastasis-associated gene (21) and establish for the first time a
cell-to-cell adhesion property for the
4 integrin that
involves an entirely new integrin adhesion partner.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 integrin ectodomain included mouse mAb 3E1 (Life Technologies, Inc.), rabbit pAb H101 (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), and rabbit pAb 81435 directed against TA3/HA mouse
mammary carcinoma cells (Dr. E. Roos, The Netherlands Cancer Institute,
Amsterdam) (23). Rat anti-human
6 integrin mAb GoH3 was
from PharMingen (San Diego, CA), mouse anti-human
1 mAb
2253 from Chemicon (Temecula, CA), and mouse anti-human Myc mAb
9E10 and anti-phosphotyrosine mAb from Calbiochem. Human placental and
murine EHS laminins as well as all other reagents were from Sigma.
4 integrin or tailless
4 integrin (
4
cyt) were from Dr.
A. M. Mercurio (Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA) (24). Human lung microvascular endothelial cells (HMVEC-L) and human aortic endothelial cells (HAEC) were from
Clonetics (San Diego, CA). Human umbilical vein endothelial cells
(HUVEC), human embryonic kidney 293 cells (HEK293), and Kirsten
Ras-transformed Balb/3T3 (K-Balb/3T3) cells were from the ATCC.
HMVEC-L were grown in EGM-2-MV BulletKit medium (Clonetics), and all
others were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies).
4 Integrin Ligand of hCLCA2--
The tumor cell ligand
of hCLCA2 was isolated from lysates of surface-biotinylated MDA-MB-231
breast cancer cells bound to a monolayer of transiently transfected
Myc-hCLCA2-HEK293 cells by co-immunoprecipitation with anti-Myc mAb
9E10. In brief, MDA-MB-231 cells (5 × 104 tumor
cells/cm2 of HEK293 monolayer surface) were allowed to
adhere to Myc-hCLCA2-HEK293 monolayers (or control vector-transfected
HEK293 monolayers) during a 20-min incubation period. After removing
unbound tumor cells by washing, bound tumor cells and HEK293 cells were
extracted in lysis buffer (26, 27), and extracts were subjected to
immunoprecipitation with anti-Myc mAb 9E10. Immunoprecipitates were
resolved by SDS-PAGE (6% polyacrylamide), blotted to nitrocellulose,
and probed with streptavidin-HRP or anti-
4 mAb 3E1
followed by HRP-conjugated secondary antibody. Alternatively, the
hCLCA2 tumor cell ligand was isolated by affinity chromatography from
surface-biotinylated MDA-MB-231 cells using Myc-hCLCA2-conjugated
anti-Myc mAb 9E10/goat anti-mouse IgG Dynabeads. Beads were boiled in
SDS-sample buffer, and proteins were resolved by SDS-PAGE and blotted
to nitrocellulose. Blots were probed with streptavidin-HRP,
anti-
4 mAb 3E1, or anti-Myc mAb 9E10. Purification of
4 was accomplished with anti-
4 mAb 3E1-conjugated Protein G beads from lysates of MDA-MB-231 cells (108 cells/preparation). To further corroborate the
specificity of the
4/hCLCA2 adhesion, MDA-MB-231 cancer
cells were labeled with H332PO4
(0.5 mCi/ml) in phosphate-free DMEM for 4 h at 37 °C and then seeded onto BSA-, fibronectin-, hCLCA2-, laminin-, and
poly-L-lysine-coated dishes and incubated for 20 min at
37 °C. Cell lysates were immunoprecipitated with
anti-
4 mAb 3E1, and precipitates were resolved by
SDS-PAGE, treated with 1 M KOH for 2 h at 55 °C,
and autoradiographed to visualize
4 integrin tyrosine
phosphorylation (28).
4 (immunopurified
from MDA-MB-231),
1 (
5
1),
and
3 (
v
3) (both from
Chemicon) integrins were resolved by SDS-PAGE, blotted to
nitrocellulose, denatured with 2.5 M guanidine, and
renatured in 5% milk in Tris-buffered saline containing 0.1% Tween 20 (29). Blots were incubated with Myc-tagged hCLCA2 (overnight; 4 °C), and bound hCLCA2 was detected with anti-Myc mAb 9E10, followed by
HRP-conjugated secondary antibody. Controls included blots probed with
antibody alone. Positions on the gel and loading quantities of the
integrins were determined in parallel Western blots.
4
cDNA--
Wild-type
4 cDNA cloned into the
expression vector pRC-CMV was from Dr. F. G. Giancotti. K-Balb/3T3
cells at 70% confluence were stably transfected with
4
cDNA by electroporation and selected for G418 resistance. Controls
were K-Balb/3T3 cells transfected with vector alone. Cells were used
for (a) FACS to quantify
4 surface expression
(25), (b) adhesion to immunopurified mCLCA1 (14), and
(c) lung colony assays (12-14).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Endothelial cell expression of hCLCA2.
A-F, sections (2 µm thick) from paraffin-embedded,
formaldehyde-fixed lung tissue blocks were stained with rabbit
anti-hCLCA2 pAb 4 at a dilution of 1:100 (A, C,
and E) or rabbit preimmune IgG (B, D,
and F). Bound antibody was detected by HRP-conjugated
goat anti-rabbit IgG antibodies and diaminobenzidine as substrate. A
positive staining reaction is observed in small arteries
(A), arterioles (C), and venules of interlobular
septa (E). Comparable vessels stained with preimmune serum
are negative (B, D, F).
Bar, 100 µm. G and H, expression of
hCLCA2 in cultured endothelial cells is shown by RT-PCR amplification
of an 800-base pair hCLCA2 product from total RNA of HMVEC-L
(lane 1), HAEC (lane 2),
and HUVEC (lane 3) but not HEK293
(lane 4) (see "Experimental Procedures") and
of the full-length 2.9-kilobase pair hCLCA2 open reading frame from
HMVEC-L RNA (G) and by FACS analysis of HMVEC-L stained with
rabbit anti-hCLCA2 pAb 4 (open histogram) or
rabbit preimmune IgG (closed histogram) and
fluorescein isothiocyanate-conjugated goat anti-rabbit IgG
(H).
4 Integrin--
The selective expression of hCLCA2 on
endothelia of lung blood vessels, which recently were implicated with
location of tumor cell arrest and early intravascular micrometastasis
formation by in situ epifluorescence microscopy (11),
suggested that hCLCA2 could serve as the human counterpart of Lu-ECAM-1
and might play a major role in lung metastasis of blood-borne human
cancer cells. To test this hypothesis, we selected three human breast
cancer cells with different biological behaviors for adhesion to
recombinant Myc-tagged hCLCA2 immunopurified from transfected HEK293
cells. The first cell line was MDA-MB-231, which efficiently colonizes the lungs of nude mice following tumor formation from cancer cells injected into mammary fat pads (orthotopic tumor xenografts) or intravenous injection; the second cell line was MDA-MB-435, which only
forms lung metastases from orthotopic tumor xenografts but not after
intravenous injection; and the third cell line was MCF7, which is
nonmetastatic by either of the two modalities (20). Consistent with the
proposed role of hCLCA2 in lung metastasis, only MDA-MB-231 cells
adhered in strong numbers to recombinant hCLCA2 (Fig.
2A). Adhesion correlated with
the amount of Myc-tagged hCLCA2 protein present in elution fractions
from anti-Myc mAb 9E10 immunoaffinity columns and was dependent upon
serum activation of tumor cells (Fig. 2B). To identify the
tumor cell molecule that served as the ligand for hCLCA2,
surface-biotinylated MDA-MB-231 cancer cells were allowed to bind to
confluent monolayers of Myc-tagged hCLCA2- or
vector-transfected HEK293 cells, yielding tumor cell adhesion values of
~75% for Myc-hCLCA2-HEK293 monolayers and 25% for
vector-transfected HEK293 monolayers. Myc-hCLCA2-HEK293 monolayers were
extracted together with bound tumor cells, and extracts were subjected
to immunoprecipitation with anti-Myc mAb 9E10. Precipitated proteins
resolved by SDS-PAGE and blotted to nitrocellulose were then probed
with streptavidin-HRP. A single band of molecular size 205 kDa was
identified that by Western analysis with anti-
4 mAb 3E1 was shown to
be
4 integrin (Fig. 2C). Subsequent
immunoprecipitation of Myc-hCLCA2-HEK293/MDA-MB-231 cell extracts with
anti-
4 pAb H101 and Western probing of the precipitate with anti-Myc
mAb 9E10 identified hCLCA2, further confirming the
4
integrin/hCLCA2 adhesion (Fig. 2D). Controls conducted with
MDA-MB-231 cancer cells bound unspecifically to vector-transfected
HEK293 monolayers did not yield any precipitate. Expansion of our
initial hCLCA2/tumor cell adhesion studies to MDA-MB-435L2, MDA-MB-468,
and MDA-MB-453 breast cancer cell lines supported the close correlation
between surface expression of the
4 integrin, hCLCA2
adhesion, and lung colonization (Fig. 3,
A-D). For example, the consistently high lung colonization
potential of the MDA-MB-231 cell line correlated with high levels of
4 integrin expression and hCLCA2 adhesion, while modest
lung colonization of the MDA-MB-435L2 cell line was associated with
modest
4 expression and hCLCA2 adhesion (Fig. 3,
A-D). All other breast cancer cell lines were unable to
form lung colonies and, with the exception of the MDA-MB-468 cell line, expressed low or nondetectable levels of the
4 integrin
and adhered poorly to hCLCA2 (Fig. 3, A-D). In the
metastatically incompetent MDA-MB-468 cell line, an intermediate level
of
4 expression correlated with a well differentiated,
near normal cellular phenotype in vitro and slow adenomatous
growth in vivo, implying that the MDA-MB-468 cell line
represents an early stage in tumor progression.
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Fig. 2.
The 4
integrin mediates adhesion of breast cancer cells to endothelial
hCLCA2. A, adhesion of MDA-MB-231, MDA-MB-435, and MCF7
human breast cancer cells to hCLCA2 was tested in microtitration plates
coated with ~3 µg/ml recombinant hCLCA2 (overnight; 4 °C).
B, adhesion of MDA-MB-231 cells to Myc-tagged hCLCA2 from
successive elution fractions of an anti-Myc mAb 9E10-immunoaffinity
column. Protein G beads conjugated with anti-Myc mAb 9E10 were
incubated with extracts from HEK293 cells transfected with Myc-tagged
hCLCA2 (overnight; 4 °C), and bound protein was eluted with 200 mM glycine (pH 2.8) in 150 mM NaCl and 0.5%
octyl-
-glucoside. Elutes were collected in 1-ml fractions in 0.1 volume of 1 M Tris (pH 11) to yield a final pH of 8.2. Each
fraction was evaluated for MDA-MB-231 adhesion and protein content by
Western blotting with anti-Myc mAb 9E10. Adhesion values correlate well
with the amount of hCLCA2 protein in the column fraction. C,
surface-biotinylated or untreated MDA-MB-231 cells bound to HEK293 cell
monolayers transfected with Myc-hCLCA2 (lanes 1)
or HEK293 cell monolayers transfected with vector alone
(lanes 2) (see "Experimental Procedures")
were extracted in lysis buffer containing 5 mM EGTA.
Extracts were subjected to immunoprecipitation with anti-Myc mAb 9E10,
and SDS-PAGE-resolved and blotted precipitates were probed with
streptavidin-HRP (left panel),
anti-
4 mAb 3E1 (middle panel), or
anti-Myc mAb 9E10 (right panel). D,
anti-
4 pAb H101 immunoprecipitate from extracts of
MDA-MB-231 bound to Myc-tagged hCLCA2-transfected (lanes
1) or vector-transfected (lane 2)
HEK293 monolayers were probed by Western blot with anti-Myc mAb 9E10
(left panel) or anti-
4 mAb 3E1
(right panel).
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Fig. 3.
The 4
integrin expression correlates with hCLCA2 adhesion and lung
colonization: FACS analyses of MDA-MB-231 (A),
MDA-MB-435 (435) and MDA-MB-435L2 (435L2) (B),
MDA-MB-468 (C), and MCF7 (D).
Tumor cells were stained with either mouse anti-
4 mAb
3E1 (open histogram) or mouse IgG
(gray filled histogram) and
fluorescein isothiocyanate-conjugated goat-anti mouse IgG secondary
antibody and then subjected to FACS analyses. The percentage of tumor
cell adhesion to hCLCA2-coated dishes (3 µg/ml) and the number of
lung colonies formed by each breast cancer cell line are displayed as
insets.
4 integrin-hCLCA2 complex
from MDA-MB-231/Myc-hCLCA2-HEK293 extracts and hCLCA2 affinity
purification of the
4 integrin from MDA-MB-231 extracts
(data not shown) were unable to rule out participation of an unknown,
intermediary molecule in the binding of hCLCA2 to
4
integrin, the
4/hCLCA2 partnership was further examined
by Far Western analysis. To accomplish this, adhesion receptor and
ligand were first immunopurified from hCLCA2-transfected HEK293 cells
and MDA-MB-231 cells, respectively, and their purity was assessed by
SDS-PAGE and silver staining. After subjecting the SDS-PAGE resolved,
blotted
4 integrin to cycles of denaturing and
renaturing, blots were probed with Myc-tagged hCLCA2, and hCLCA2-binding to
4 was visualized by anti-Myc
antibodies. hCLCA2 strongly and specifically bound to
4
integrin, but not to the control
integrin subunits
1
and
3 (Fig. 4A,
lanes 1, 3, and 5). Control
blots incubated with anti-Myc antibody alone were negative (Fig.
4A, lanes 2, 4, and
6). Western blotting (Fig. 4A, lanes
marked W) confirmed positions and equal loading amounts of
the three
integrins. To further scrutinize the specificity of the
4/hCLCA2 adhesion, Far Western analyses were also
conducted with blot-immobilized hCLCA2 (Fig. 4B,
lane W) that was probed with soluble
4 integrin immunopurified from MDA-MB-231 cell extracts. Binding of the
4 integrin to hCLCA2 was confirmed by
staining with anti-
4 pAb H101 (Fig. 4B,
lane 1), while blots that were stained with
antibody alone in the absence of
4 integrin were negative (Fig. 4B, lane 2).
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Fig. 4.
Documentation of the
4 integrin/hCLCA2 adhesion by far
Western analysis. Immunopurified integrins
4
(
6
4),
1
(
5
1), and
3
(
v
3) (A) and hCLCA2
(B) were resolved by SDS-PAGE and blotted to nitrocellulose
and then probed immediately with the respective anti-
integrin
antibodies (A) or anti-Myc mAb 9E10 (B) to
confirm equal protein loading (W). After successive
denaturing and renaturing cycles (29), parallel blots were incubated
with immunopurified Myc-hCLCA2 (lanes 1,
3, and 5) or 1% BSA (lanes
2, 4, and 6) (A) or with
immunopurified
4 integrin (lane 1)
or 1% BSA (lane 2) (B) and probed
with anti-Myc mAb 9E10 (A) or anti-
4 pAb H101
(B), respectively. In A only the
4
integrin is able to bind Myc-hCLCA2 (lane
1). Note that the
4 used in this study
appears as a triplet of 205, 180, and 150 kDa (W), since it
has been extracted from MDA-MB-231 cells in lysis buffer in the absence
of 5 mM EGTA (26, 27). In B, immobilized hCLCA2
strongly binds the
4 integrin (lane
1), but not anti-
4 antibody alone
(lane 2).
4 Integrin/hCLCA2
Adhesion--
The specificity of the
4 integrin/hCLCA2
adhesion was confirmed by adhesion blocking experiments involving
antibodies directed against either of the interacting adhesion
molecules. Dramatic adhesion inhibition was seen only for anti-hCLCA2
pAb 18 and anti-
4 mAb 3E1, while all other antibodies tested had no
effect on the
4/hCLCA2 adhesion (Fig.
5, A and B).
Neither of the two anti-hCLCA2 antibodies had any effect on the
adhesion of MDA-MB-231 cells to human placenta-derived laminin used as
a control substrate, while both anti-
4 antibodies 3E1
and 81435 inhibited laminin adhesion of MDA-MB-231 cells (Fig. 5,
A and B). To exclude a possible participation of
the
6
1 integrin, expressed in all human
breast cancer cell lines used in this study, in the hCLCA2 adhesion, functional anti-
1 antibodies (mAb 2253) were tested and
found to be ineffective in blocking the adhesion between MDA-MB-231 cells and hCLCA2 but effective in blocking the adhesion to placenta laminin (Fig. 5C).
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Fig. 5.
Specificity of the
4 integrin/hCLCA2 adhesion.
A-C, MDA-MB-231/hCLCA2 and MDA-MB-231/laminin adhesion
inhibition experiments were done in microtitration plates coated with 3 µg/ml hCLCA2 or 7.5 µg/ml placental laminin, using anti-hCLCA2
antibodies (pAbs 4 and 18) (A), anti-
4
antibodies (3E1; 81435, 450-9D) and anti-
6 antibodies
(GoH3) (B), and functional anti-
1 antibodies (2253)
(C). Anti-hCLCA2 pAb 18 and anti-
4 mAb 3E1 specifically
block the adhesion of MDA-MB-231 to hCLCA2 but not anti-
1 mAb 2253. D, matrilysin treatment of MDA-MB-231 cells abolishes
adhesion to hCLCA2 but not to placental laminin and causes degradation
of the 205-kDa
4 integrin (Western blot, lane
2) but not the
1 integrin (Western blot,
lane 4). Western lanes 1 and 3 are untreated controls. E, MDA-MB-231
cancer cells were labeled in phosphate-free DMEM containing
H332PO4 at a final concentration of
0.5 mCi/ml for 4 h at 37 °C and then seeded onto BSA (10 µg/ml)-, fibronectin (5 µg/ml)-, hCLCA2 (3 µg/ml)-, placental
laminin (7.5 µg/ml)-, and poly-L-lysine (1 mg/ml)-coated
dishes, incubated for 20 min at 37 °C, and lysed. Lysates were
immunoprecipitated with anti-
4 mAb 3E1, and precipitates
were resolved by SDS-PAGE and treated with 1 M KOH for
2 h at 55 °C. Tyrosine-phosphorylated proteins are visualized
by autoradiography. *, Student's t test; p < 0.01 (mean ± S.D. from four experiments).
4 integrin ectodomain with matrilysin (30) totally abolishes MDA-MB-231 adhesion to hCLCA2 yet had a negligible effect on
the adhesion to placental laminin (Fig. 5D). These adhesion data were supported by Western analyses showing specific cleavage of
the 205-kDa
4 protein but not the
1
integrin in matrilysin-treated tumor cells. Finally, we examined
whether the MDA-MB-231
4 integrin was activated
selectively when tumor cells were plated onto surfaces coated with
hCLCA2 (Fig. 5E). Data showed prominent tyrosine
phosphorylation of
4 integrin in tumor cells bound to
hCLCA2 and to placental laminin (31, 32). In contrast, fibronectin
generated only a weak tyrosine phosphorylation reaction, and BSA and
poly-L-lysine had no effect (Fig. 5E).
4/hCLCA2
Adhesion-blocking Antibodies--
To test whether the
adhesion-inhibitory effects of anti-hCLCA2 and anti-
4
integrin antibodies extended to an inhibition of lung metastasis, we
performed lung colony assays in nude mice with the lung metastatic
breast cancer cell line MDA-MB-231 in the presence of these antibodies.
Prior to conducting these assays, we established that human MDA-MB-231
cells were able to adhere to mCLCA1 (Table
I), the mouse counterpart of hCLCA2, and
that this adhesion was inhibited with anti-Lu-ECAM-1 mAb 6D3 (22) (cross-reacts with mCLCA1) and anti-
4 integrin mAb 3E1.
Anti-
4 mAb 3E1 was preincubated for 30 min and injected
together with tumor cells, while mAb 6D3 was injected with tumor cells
without preincubation. Control experiments were conducted in the
presence of nonimmune mouse IgG. Mice sacrificed 15 weeks later
revealed that both antibodies effectively blocked the colonization of
the lungs by MDA-MB-231 cells, causing an 84% inhibition of lung
metastasis with mAb 6D3 and a 100% inhibition with mAb 3E1 relative to
mIgG-treated controls (Table I).
Inhibition of lung colonization by the human breast cancer cell line
MDA-MB-231 with anti-mCLCA1 and anti-4 integrin antibodies
4 Integrin Transfection--
To test whether in
vivo selection for increased lung metastatic performance was
associated with increased
4 expression, we compared the
6,
1, and
4 expression
patterns of the selected cell line MDA-MB-435L2 (20) with those of the
parental MDA-MB-435 cell line and the
4-transfected
MDA-MB-435
4 cell line (24). The parental MDA-MB-435 cell line
exhibited strong expression of the
6 and
1 integrin subunits but only background levels of the
4 integrin. Accordingly, these tumor cells adhered
strongly to both placental and EHS laminins but poorly to hCLCA2
(5 ± 3%; Fig. 6A). The
selected MDA-MB-435L2 expressed comparable levels of the
6 and
1 integrin subunits and a modest
increase in surface expression of the
4 integrin. In
accordance with this expression pattern, MDA-MB-435L2 cells adhered
strongly to the two laminins and exhibited an increased adhesion
to hCLCA2 (25 ± 3%; Fig. 6A). These data were
contrasted with those from the parental MDA-MB-435 cell line that had
been transfected with human
4 integrin and then selected
for antibiotic resistance and by FACS for efficient stable expression
of
4 (24). Transfectant cells expressed significantly higher levels of
4 integrin than MDA-MB-435L2 cells and
adhered in higher numbers to hCLCA2 but in similar numbers to the two laminins, since the expression levels for both
6 and
1 remained unchanged. Transfection of MDA-MB-435 cells
with tailless
4 integrin (
4
cyt)
underscored requirement of the "complete"
4 integrin subunit in hCLCA2 binding, since adhesion to hCLCA2 did not improve relative to that of parental cells (7 ± 3%; Fig. 6A),
albeit the expression level of the truncated
4 was equal
to that of wild-type
4 expression in MDA-MB-231 cells.
Consistent with published data, adhesion of
4
cyt-transfected MDA-MB-435 cells to murine EHS laminin was also abolished (24), but not to human placental laminin
(33).
View larger version (32K):
[in a new window]
Fig. 6.
Effect of in vivo selection
versus 4-transfection on
hCLCA2 adhesion of human breast cancer cells. A,
MDA-MB-231, MDA-MB-435, MDA-MB-435L2, MDA-MB-435
4, and
MDA-MB-435
4
cyt were analyzed for
6,
1, and
4 expression by FACS, and the
expression patterns were contrasted with adhesion to hCLCA2 and EHS
(bars 1) and placental (bars
2) laminins (coated at 3 µg/ml, 20 µg/ml, and 7.5 µg/ml, respectively). In vivo selection of MDA-MB-435 for
enhanced lung colonization (MDA-MB-435L2) (20) and stable
4 transfection of MDA-MB-435
(MDA-MB-435
4) (24) increases
4 expression
and adhesion to hCLCA2, while adhesion to the two laminins remains
unchanged. Stable transfection of MDA-MB-435 with tailless
4 (MDA-MB-435
4
cyt) (24) increases
4 expression as detected with anti-
4 mAb
3E1 (directed against the extracellular domain of the
4
integrin), but
4
cyt-transfectants adhere in similarly
poor numbers to hCLCA2 as parental cells. Adhesion of
MDA-MB-435
4
cyt to EHS laminin (bar
1) is abolished, but not adhesion to placental laminin
(bar 2). B, extracts from surface-biotinylated
MDA-MB-231, MDA-MB-435
4,
MDA-MB-435
4
cyt, and MDA-MB-435 cells were subjected
to immunoprecipitation with anti-
4 pAb H101
(upper panel) or anti-
6 mAb GoH3
(lower panel). Immunoprecipitates were resolved
by SDS-PAGE (6%) and blotted to nitrocellulose and then probed with
Streptavidin-HRP. Lane 1, MDA-MB-231;
lane 2, MDA-MB-435
4;
lane 3, MDA-MB-435; lane 4,
MDA-MB-435
4
cyt.
4 cell surface expression and hCLCA2
adhesion correlated well with lung colonization of the established cell
lines MDA-MB-231, MDA-MB-435L2, and MDA-MB-435. Median and range of the
number of lung colonies were 30 (5-100), 5 (0-17), and 0 (0-3),
respectively. To our surprise, however, the
4-transfected cell line MDA-MB-435
4 was
unable to produce lung colonies following a 15-week incubation period
in nude mice, although the parental cell line MDA-MB-435 is known to
produce lung metastases after orthotopic tumor growth in mammary fat
pads of nude mice and in vivo selection of these cells
yielded a cell line with transiently enhanced lung colonization
potential (MDA-MB-435L2) (20), which was lost gradually with increasing
passage number. To explore whether differences in the quality of the
4 integrin expression on the surface of MDA-MB-231 and
MDA-MB-435
4 cancer cells may have accounted for the
discrepancy in the metastatic behavior, we examined the association
between the
4 integrin and its presumed
6
partner in the two cell lines. Surface-biotinylated cancer cells were
first subjected to immunoprecipitation with anti-
4 pAb
H101. As expected, the amounts of
4 detected in
streptavidin-HRP-probed blots were comparable with that identified by
FACS (Fig. 6, A and B). Next, the same tumor cell
extracts were subjected to immunoprecipitation with
anti-
6 mAb GoH3, and precipitates were analyzed for
4-co-immunoprecipitation. Surprisingly, only the
4 of MDA-MB-231 cells was effectively co-precipitated
with
6, while negligible amounts of
4
were co-precipitated from MDA-MB-435
4 cells and none from MDA-MB-435
(Fig. 6B, lanes 1-3). Since
4
cyt is also effectively co-immunoprecipitated with
6 from MDA-MB-435
4
cyt extracts (Fig.
6B, lane 4), our data imply that
wild-type
4 transfected into MDA-MB-435 cells may interact with an intrinsic protein that affects co-immunoprecipitation with
6 and metastasis but not in vitro
adhesion to hCLCA2.
4 Promotes Adhesion to hCLCA2 and Lung
Colonization--
To determine whether overexpression of the
4 integrin in a cell line that expresses low levels of
4 integrin and, accordingly, has modest, yet consistent,
lung metastatic capabilities, we chose a murine over a human tumor
model. The former had the significant advantage that the metastatic
performance could be tested in a syngeneic rather than a heterogeneic
animal. Moreover, the induction time of generating macroscopically
detectable lung colonies was only 3-4 weeks in syngeneic animals
(13-15) versus a minimum of 15 weeks in a human/mouse model
(20). The cell line we selected was the Kirsten-Ras-transformed
Balb/3T3 cell line, which expressed low levels of the
4
integrin and consistently produced a moderate number of lung colonies
upon tail vein injection. These tumor cells were transfected with
4 integrin cDNA or vector alone, and stable
transfectants were selected based on antibiotic resistance. Expression
of the
4 integrin was confirmed by FACS and surface biotinylation, both methods indicating a significantly increased surface expression of
4 integrin, which was
co-immunoprecipitable with
6, in
4-transfected relative to vector-transfected cells (Fig.
7A). Prior to conducting a
lung colony assay, adhesion assays were performed with mCLCA1, the
mouse counterpart of hCLCA2. Adhesion of
4-transfected
K-Balb cells to mCLCA1 was 66 ± 5% relative to 9 ± 4% for
mock-transfected K-Balb cells (Fig. 7B). These adhesion data
paralleled the metastatic performance of the two cell lines. The
4-transfected K-Balb cells injected at 2 × 105 cells/mouse generated a median number of >100 colonies
(69->100), while the mock-transfected cell line only generated 24 (15-29) lung colonies (Fig. 7B). This difference was also
reflected in the average lung weights of the two experimental groups,
measuring 0.71 ± 0.22 g in
4 transfectants
and 0.45 ± 0.02 g in mock transfectants.
View larger version (28K):
[in a new window]
Fig. 7.
Effect of stable
4 transfection of K-Balb/3T3 cells on
mCLCA1 adhesion and lung colony formation. A,
Kirsten-Ras-transformed Balb/3T3 (K-Balb/3T3) cells were transfected by
electroporation with
4 integrin cDNA, and stable
expressors were selected by G418 resistance. Wild-type K-Balb/3T3 cells
(gray filled histogram),
mock-transfected K-Balb/3T3 cells (thin open
histogram), and
4-transfected K-Balb/3T3
cells (thick open histogram) were
stained with rabbit anti-
4 pAb H101 and fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody and
then analyzed by FACS. Inset, anti-
4 pAb H101
immunoprecipitates from extracts of surface-biotinylated
4-transfected K-Balb/3T3 cells (a,
lane 1) and mock-transfected K-Balb/3T3 cells
(a, lane 2) and anti-
6
mAb GoH3 immunoprecipitates from surface-biotinylated,
4-transfected K-Balb/3T3 cells (b,
lane 1). B, lung colony formation by
4-transfected K-Balb/3T3 cells. Balb/c mice (6-weeks
old; male) were injected into the lateral tail vein with 2 × 105 tumor cells plus 0.3 ml of DMEM/mouse, and lung weights
(g) and number of lung colonies were determined 3 weeks after tumor
cell injection. Data were evaluated by Student's t test
(unpaired data). *, p < 0.01 (relative to
K-Balb-Mock).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
integrin. Human CLCA2 is expressed by endothelia lining arterial and
venous branches, all presumably derived from the bronchial artery,
while prominent expression of the
4 integrin has been
associated with the invasive and metastatic phenotypes of breast cancer
cells (24, 34-37). The location of MDA-MB-231 breast cancer metastases in mouse lungs is consistent with the vascular expression pattern of
the mouse counterpart of hCLCA2 (mCLCA1) (10) as well as the recently
established pattern of lung metastases by in situ epifluorescence microscopy (11). The binding interaction between the
two lung metastasis-promoting adhesion molecules is documented by
co-immunoprecipitation of the adhesion receptor/ligand pair from
extracts of hCLCA2-transfected HEK293 monolayers to which MDA-MB-231
cells were bound, by hCLCA2 affinity chromatography, and by adhesion
and metastasis inhibition experiments using functional antibodies.
Participation of an "intermediary binding molecule" in the
4 integrin/hCLCA2 adhesion was excluded by Far Western analyses, using blotted immunopurified
4 or Myc-hCLCA2
and the corresponding purified adhesion partner as probe. Strong and
specific binding between blotted
4 integrin and
Myc-hCLCA2 indicated that the
4 integrin was able to
recognize its endothelial cell receptor even after undergoing a
vigorous denaturing/renaturing treatment, suggesting that the usually
required interaction between
and
integrin subunits (38, 39)
and/or other interacting cell surface and intracellular molecules (40,
41) is not a mandate for the
4 integrin/hCLCA2 adhesion
function in vitro. This behavior is similar to that recently
reported for the
4 integrin/Shc adhesion, using blotted
4 integrin under denaturing/renaturing Far Western conditions (42).
conductance in cancer cell
extravasation by induction of apoptosis in the endothelium of the
target organ. Support for such involvement came from recent
observations that breast cancer cells seeded atop a monolayer of
hCLCA2-expressing HUVEC apparently induce apoptosis in apposed
endothelial cells (43). Endothelial apoptosis appears to involve
expression and activation of chloride channels (44, 45), leading to
intracellular acidification and, in turn, activation of endonucleases
and chromatin digestion (44). The advantage of a selective induction of
apoptosis in those endothelial cells to which tumor cells are bound is
obvious, since reduction of the endothelial cell by apoptotic
vesiculation may create an avenue for invasion of perivascular tissues
by tumor cells. The notion that these events are initiated by
4/hCLCA2 adhesion is supported by our preliminary
observation that endothelial cells incubated with immunopurified
4 integrin rapidly undergo apoptosis. The apoptotic
index of
4-treated endothelial cells was 26%, relative to 4% in untreated endothelial
cells.2
4 integrin has previously been linked to metastatic
disease (13, 46-49) and is confirmed here as a lung
metastasis-associated gene in breast cancer. Consistent with the
involvement of multiple genes in metastasis (1-8), the
4 integrin, like other metastasis-associated genes
including MMP-2, CD44,
v
3
integrin, and
6 integrin (50), is by itself incapable of
conferring mastery of the complex, multistep cascade
of metastasis. This is exemplified by the MDA-MB-468 breast cancer cell
line, which expresses the
6
4 integrin at
relatively high levels and, accordingly, is able to adhere to hCLCA2
in vitro but fails to produce metastases upon tail vein
inoculation (Fig. 3). When this cell line is compared with a
metastatically competent,
4-expressing cell line such as
MDA-MB-231, the former expresses a phenotype that is comparable with
the spontaneously immortalized, nontumorigenic
4
integrin-expressing breast epithelial cell line MFC-10A (51), while the
latter expresses an aggressive, invasive, and metastatic phenotype
(20). This difference is manifested by the formation of a
contact-inhibited, cobblestone-like monolayer in vitro and
adenomatous growth in vivo by MDA-MB-468 cells but anaplastic, crisscrossed, and multilayered growth in vitro
and the formation of invasive and metastatic tumors in vivo
by MDA-MB-231 cells (20). Genotype analyses indicate that the latter
cell line expresses an array of gene abnormalities that have been
associated with metastasis such as overexpression of
c-erbB-2 (52), MTA1 (53, 54),
MT1-MMP (55), vimentin (56),
6 integrin (57), and VEGF (58) and down-regulation or loss of E-cadherin
(59), nm23-H1 (60), and MUC1 (61), while most of
these genes are expressed at normal or near normal levels in MDA-MB-468
cells (50, 53, 55, 56, 62). These data imply that
4
integrin expression leads to lung metastasis only in those cancer cells possessing a genotype that is otherwise compatible with metastasis. A
similar scenario as described for the
4 integrin has
also been reported for other metastasis-associated proteins including
MMP-2, CD44,
v
3 integrin,
6 integrin, Rho GTPases, and fibronectin, which are
prominently expressed in many highly invasive and metastatic tumor
cells but individually are also detected in benign, nonmetastatic tumor
cells and even normal cells (25, 63-67).
4-expressing MDA-MB-468 cells, an altered modulation of
the
4 integrin by lateral associations with other
membrane (and/or cytoplasmic) proteins (reviewed in Refs. 40 and 41) in
4-transfected MDA-MB-435 cells versus
MDA-MB-231 cells may have been responsible for the observed discrepancy
in the metastatic behavior of the two cell lines. Although we have as
yet no evidence of such a differential lateral association of the
4 integrin in the two cell lines, we show here that
anti-
6 antibodies fail to co-immunoprecipitate the
4 integrin from
4-transfected MDA-MB-435
cell extracts but effectively do so from MDA-MB-231 cell extracts (as
well as from extracts of immortalized normal breast epithelial cells
MCF-10A and benign breast tumor cells MDA-MB-468; data not shown). This differential partitioning of the
4 integrin in
MDA-MB-231 and MDA-MB-435
4 cells, which incidentally
express virtually identical amounts of
6 and
1 integrins, and only a slightly reduced level of
4 in the MDA-MB-435
4 cell line is
difficult to explain. However, it is possible that under our extraction
conditions (1% Triton X-100), lateral association of the
4 integrin with an as yet undetermined membrane or
cytoplasmic protein in
4-transfected MDA-MB-435 cells
may have weakened the binding interaction between the
6
and
4 integrin subunits, resulting not only in failure of the two integrin subunits to co-immunoprecipitate but also in
inability to metastasize. Control co-immunoprecipitation of
6 and
4
cyt suggests that such
association is mediated by the cytoplasmic tail of the
4
integrin. Alternatively, our data may have been affected by the
unlikely event that the
4 integrin subunit may associate
with an as yet unidentified
chain to permit cell surface expression
in
4-transfected MDA-MB-435 tumor cells.
4 integrin
expression. When the
4 integrin is overexpressed in
these cells, the number of lung colonies generated from intravenously injected tumor cells increases proportional to the level of
6 co-immunoprecipitable
4 integrin.
Consistent with involvement of the
4 integrin gene in
metastasis, blockage of the
4/hCLCA2 adhesion abrogates
metastasis. Similar effects are achieved by blocking other
metastasis-associated genes each facilitating a different step in the
metastatic cascade, e.g. RhoC, metalloproteinases, heparatinase, angiogenic factors, dipeptidyl peptidase IV (68-75).
4 expression and malignant
progression (reviewed in Ref. 21), ascribing a key role to the adhesion mechanism between
4 integrin and vascular endothelial
cell hCLCA2 in lung metastasis. Although only a few examples of
integrin involvement in cell-to-cell adhesion are known
(e.g. leukocyte integrins
L
2 and
M
2 bind to endothelial cell ICAM-1,
integrins
4
1 and
7
1 to VCAM-1, and
E
7 to E-cadherin
(reviewed in Ref. 39)), we have identified and cloned for the first
time a
4 integrin-binding protein that is an integral
membrane protein and that is entirely new as an integrin-binding
partner (15, 18). Our discovery that hCLCA2 has an important function
in heterotypic cell-to-cell adhesion in addition to that in
Ca2+-sensitive chloride conductance extends the basic
knowledge about this protein and indicates that ion channels can have
multiple, seemingly unrelated functions (16-18, 44, 76).
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Renée A. Christopher for involvement in the preparation of the manuscript and Heather Archibald and the staff of the Image Lab for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by NCI, National Institutes of Health, Public Health Service Grants CA47668 and CA71626 (to B. U. P.).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: Cancer Biology
Laboratories, Dept. of Molecular Medicine, Cornell University College of Veterinary Medicine, Ithaca, New York 14853. Tel.: 607-253-3343; Fax: 607-253-3708; E-mail: bup1@cornell.edu.
Published, JBC Papers in Press, April 24, 2001, DOI 10.1074/jbc.M100478200
2 B. U. Pauli, H.-C. Cheng, and M. Abdel-Ghany, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: hCLCA2, human CLCA2; mCLCA1, mouse CLCA1; mAb, monoclonal antibody; pAb, polyclonal antibody; HMVEC-L, human lung microvascular endothelial cells; HAEC, human aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; HEK, human embryonic kidney; RT, reverse transcriptase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; HRP, horseradish peroxidase; FACS, fluorescence-activated cell sorting; DMEM, Dulbecco's modified Eagle's medium.
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