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INTRODUCTION |
Prostate cancer is the second leading cause of cancer
death in men (1). In metastatic prostate cancer, peripheral tumor cells
undergo phenotypic changes that facilitate invasion of surrounding organ tissues, entry into the lymphatic system and/or the bloodstream, and colonization of other tissues in the body. Their ability to establish growth in a remote site is dependent upon a specific recognition process involving an initial rapid adhesion of the circulating tumor cell to endothelial cells lining the bone marrow microvasculature (2), transmigration through the endothelial cell
barrier, and subsequent lodgment in the stroma (3, 4). Remote metastases in prostate cancer often occur in bone marrow, suggesting tissue or endothelial cell-specific factors may contribute to prostate cancer metastasis to bone (2, 5).
Bone marrow endothelial cells
(BMEC)1 maintain a
specialized endothelium that must allow cell trafficking in and out of
the bone marrow (3, 4, 6). In addition to regulating the egress of
mature myeloid and lymphoid cells, BMEC selectively allow
transmigration of progenitor cells from a circulating population, indicating that specific receptors regulate the movement of cells through the endothelium (7). Studies have shown that the transmigrating cells move directly through an endothelial cell in a process that involves specific adhesive interactions (Ref. 3 and references therein). BMEC constitutively express adhesion receptors such as VCAM-1
(8, 9), E-selectin (9), and P-selectin (10), which are not expressed in
large vein endothelia unless activated by cytokines (6). Specific
adhesive interactions between hemopoietic cells and BMEC thought to be
important for homing include endothelial lectins and progenitor
glycoproteins (11-14), VCAM/VLA-4 (11-13), and CD44/hyaluronan (15).
Initial adhesion may be mediated primarily through selectins and
glycoproteins. BMEC-associated chemokines such as SDF-1
(stromal-derived factor) can then stimulate integrin ligation, leading
to progenitor arrest (16, 17).
Because the bone marrow microvasculature presents the first site of
interaction for circulating tumor cells metastasizing to the bone
marrow, it is likely that mechanisms of metastasis may parallel those
employed by homing progenitors. In fact, tumor cells have been shown to
bind and transmigrate through BMEC (17-19). The adhesion molecules
implicated in these processes, CD44/hyaluronan, VLA-4/VCAM, and
LFA-1/ICAM, are also involved in homing and extravasation of
circulating lymphocytes and progenitor cells.
Hyaluronan (HA) is a ubiquitous high molecular weight glycosaminoglycan
polymer required for growth, development, cell motility, and cushioning
of joints (20, 21). Elevated levels of HA are associated with various
pathologies, such as arthritis, inflammation, and several cancers
(22-24), including prostate (25, 26). Melanoma cells selected for high
expression of HA were more metastatic when injected into nude mice than
cells that expressed low amounts of HA (27). Furthermore,
overexpression of HA biosynthetic enzymes in tumor cell lines has been
shown to increase tumorigenicity and metastatic potential (28, 29).
To investigate the molecular mechanism of initial adhesion to bone
marrow endothelium, we modeled adhesion in vitro using the
bone marrow endothelial cell lines BMEC-1 and trHBMEC and four prostate
adenocarcinoma cell lines, PC3, PC3M-LN4, DU145, and LNCaP. Highly
metastatic PC3 and PC3M-LN4 cells adhered rapidly to BMEC-1 but not to
large vein endothelial cells (HUVEC). DU145 and LNCaP cells, in
contrast, were poorly adherent to endothelial cells. Maximal BMEC
adhesion was inhibited by addition of excess exogenous hyaluronan, and
by hyaluronidase digestion of pericellular HA, found assembled
specifically on PC3 and PC3M-LN4 cells. Presence of pericellular HA was
correlated with elevated levels of HA synthesis and expression of HA
synthase. Our data relate HA synthase overexpression to metastatic
potential of prostate tumor cells and represent the first report of
such a correlation. Collectively, our results implicate tumor
cell-associated HA and up-regulation of HA synthase in prostate cancer
progression and may directly impact metastatic potential or
preferential tissue colonization of individual tumor cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Reagents--
PC3, DU-145, and LNCaP human
prostate adenocarcinoma cell lines were purchased from ATCC (Manassas,
VA). PC3 and DU145 cells were maintained in MEM supplemented with 10%
FBS, 1 mM sodium pyruvate and non-essential amino acids.
LNCaP cells were cultured in RPMI containing 10% FBS. The PC3
derivative cell line, PC3M-LN4, was kindly provided by Dr. Isaiah J. Fidler (M. D. Anderson Hospital Cancer Center, Houston, TX), and
was maintained in the media described above for PC3 cells. Prostate
carcinoma cells were plated 2 days prior to experiments and used at
~70% confluence. The BMEC-1 human bone marrow endothelial cell line
was a gift from Dr. S. Rafii (Cornell University Medical Center, New
York, NY) and were maintained in M199 containing 20% FBS (30). trHBMEC
human bone marrow endothelial cells were a gift from Dr. Karin
Schweitzer (Free University Hospital Amsterdam, The Netherlands) and
were maintained in RPMI containing 10% FBS (11). HUVEC were purchased
from Clonetics and cultured in endothelial cell growth medium, EGM-2,
as recommended by the vendor. Human bone marrow stromal cells (31) were
cultured and generously provided by Nisha Shah and Dr. Tucker LeBien
(University of Minnesota, Minneapolis, MN). Aggrecan was prepared as
previously described (32) from Swarm rat chondrosarcoma.
Cell Adhesion Assay--
Subconfluent prostate carcinoma cells
were PBS-EDTA-released, washed with adhesion medium (RPMI with 0.1%
BSA and 20 mM Hepes, pH 7.4, maintained throughout the
assay at 37 °C), and resuspended at 1 × 106
cells/ml in adhesion medium. Cells were incubated with 25 µg/ml calcein-AM (Molecular Probes, Eugene, OR), a compound that is converted
to a fluorophore only upon uptake and metabolism by living cells, for
20 min, washed with adhesion medium, and resuspended at 1 × 105 cells/ml. BMEC-1 or HUVEC were seeded 100% confluent
(about 40,000 cells/well) in 48-well plates for 2 days and washed twice
with adhesion medium prior to the assay. trHBMEC were similarly
prepared, but seeded only overnight prior to the assay. Prostate
carcinoma cells (300 µl/well) were added to the confluent endothelial
cell monolayers and incubated for 12 min at 37 °C unless otherwise indicated. Non-adherent cells were removed with two gentle washes of
adhesion medium. Viability of non-adherent cells was verified by trypan
blue exclusion. Adherent cells were solubilized with PBS containing 0.2 N NaOH/1% SDS and quantified in a Cytofluor II
fluorescence plate reader at 485/530 nm (Biosearch Inc., Bedford, MA).
Inhibition of Cell Adhesion--
Calcein-AM-labeled prostate
carcinoma cell suspensions, at 5 × 105 cells/ml, were
preincubated with 16 units/ml Streptomyces hyaluronidase (HAase, Calbiochem, San Diego, CA) for 25 min and diluted to 1 × 105 cells/ml with adhesion medium prior to the assay.
BMEC-1 monolayers were pretreated with 16 units/ml HAase in adhesion
medium for 25 min where indicated. In the case of determining the cell
type retaining surface HA, the HAase was removed from each cell type before the assay. In the other experiments, the HAase was present throughout the assay.
To determine the effect of exogenous HA on intercellular adhesion,
trHBMEC were seeded at 100% confluence overnight in 48-well tissue
culture plates. Prior to the assay, the cell monolayers were washed
twice in adhesion medium and preincubated for 30 min in 100 µl of
adhesion medium containing the indicated concentrations of high
molecular weight human umbilical cord HA (Sigma H1751). Prostate
carcinoma cells were labeled and pretreated in the absence or presence
of HAase as described above. Labeled cells were then washed,
resuspended in adhesion medium containing the appropriate concentration
of HA, and immediately added to the endothelial cell monolayers (30,000 cells/well).
Particle Exclusion Assay--
Pericellular HA matrices were
visualized as described previously (33). Briefly, prostate carcinoma
cells cultured in 48-well plates overnight prior to the assay were
treated for 25 min in the absence or presence of 16 units/ml
Streptomyces hyaluronidase in phenol red-free MEM with 0.1%
BSA at 37 °C. This medium was removed and cells were incubated 90 min with 2 mg/ml aggrecan in MEM/0.1% BSA at 37 °C. The aggrecan
solution was removed and 1 × 108 glutaraldehyde-fixed
sheep red blood cells (Accurate Chemical and Scientific Corp.) in
PBS/1% BSA were added, allowed to settle for 15 min and then viewed
with phase-contrast microscopy. The HA matrix was evidenced by halos
surrounding the cells from which the fixed erythrocytes were excluded.
Representative cells were photographed at 400× magnification. To
quantify matrix retention, outlines of matrices and cellular boundaries
from 20 individual cells of each type were traced and relative areas
calculated using IMAGE software (National Institutes of Health).
Relative matrix areas from similar tracings of each cell type following
HAase digestion were subtracted, and HA matrix thickness was reported as the ratio of matrix area to cell area for each cell type. A ratio of
1 indicates complete absence of pericellular clearing.
HA Synthesis Quantitation--
The concentration of HA in cell
culture supernatants was determined in a competitive binding assay
(34). 96-well Immulon microtiter plates were coated with human
umbilical cord HA at 25 µg/ml in 200 mM carbonate buffer
(pH 9.6) for 4 h at 37 °C. Excess HA was removed with four
washes of PBS/0.05% Tween 20. Prostate carcinoma cells (5000/well)
were plated overnight in 12-well plates. 24-h conditioned culture media
were harvested, and cell counts were determined by trypsin release and
manual counting in a hemacytometer. Serial dilutions of cell culture supernatant (100 µl of total volume in PBS/Tween 20) were combined with 100 µl of a 1 µg/ml solution of biotinylated hyaluronic
acid-binding protein (Seikagaku) and incubated in the HA-precoated
wells at room temperature overnight. The plate was washed 4× with
PBS/Tween 20, developed using an avidin-biotin HRP system (Vector
Laboratories ABC-HRP kit PK-4000) with OPD (Sigma P8287) as substrate,
and read at 490 nm. HA concentration was interpolated from a standard curve generated by plotting HA standards against absorbance values. The
mean HA concentration for each sample of culture supernatant was
calculated, and the results were normalized to cell number. Data are
presented as mass of HA (in µg) per 106 cells.
Determination of HA Synthase Expression--
HA synthase isoform
and relative level of expression in prostate carcinoma cell lines was
semi-quantitatively assayed by RT-PCR. Poly(A)+ RNA was
isolated from subconfluent PC3M-LN4, PC3, DU145, and LNCaP cell lines
with the Oligotex mRNA isolation kit (Qiagen) and quantitated by
Ribogreen fluorescence (Molecular Probes). Normal prostate
poly(A)+ RNA was purchased from
CLONTECH. 25 ng of each mRNA template was
reverse-transcribed with an oligo(dT) primer using the Superscript II
first strand cDNA synthesis kit (Life Technologies, Inc.). PCR
oligonucleotides specific for Has1, Has2, and Has3 messages were
designed from the sequence data base; exact sequences are given in
Table I along with relative positions in
the reported sequences and expected product sizes.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified
with each reaction to standardize conditions using oligos available
from Life Technologies. Cycling conditions for Has2 and Has3 were
optimized independently as follows: 1-min initial denaturation at
95 °C; 33 cycles (Has2) or 27 cycles (Has3) of 30-s denaturation,
30-s annealing at 60 °C, and 30-s polymerization at 72 °C; 5-min
final extension at 72 °C. 15 µl of each reaction was
electrophoresed on a 3% agarose gel, stained with ethidium bromide,
and digitally photographed. To determine relative expression levels,
digital images were integrated using Molecular Analyst software, and
band intensities were normalized to the corresponding GAPDH band.
Levels are reported as the fold expression relative to that determined
for normal prostate.
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RESULTS |
PC3M-LN4 Cells Adhere Rapidly and Specifically to BMEC-1 Bone
Marrow Endothelial Cells--
Prostate adenocarcinoma cells have been
reported to adhere preferentially to bone marrow-derived microvascular
endothelial cells relative to endothelial cells from a large vein
source (HUVEC). This preference implies a specific intercellular
recognition process dictated in part by heterogeneous expression of
endothelial cell surface adhesion receptors. To investigate the
molecular interactions underlying this process, we initially chose
PC3M-LN4, a human prostate adenocarcinoma cell line derived from PC3
cells. This subline was clonally selected for enhanced metastatic
propensity in mice and, in particular, was shown to be capable of
metastasis to bone upon intracardial injection (35). We determined a
time course for adhesion of PC3M-LN4 cells to BMEC-1 bone marrow
endothelial cells and compared it with adhesion to HUVEC. Within 10 min, 70% of the cells were adherent on BMEC-1 relative to 16% on
HUVEC (Fig. 1). After 30 min, nearly
100% of the cells adhered to BMEC-1 compared with only 25% on HUVEC.
PC3M-LN4 cells, therefore, demonstrate preferential rapid adhesion to
BMEC-1 bone marrow microvascular endothelial cells.

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Fig. 1.
Time course for adhesion of PC3M-LN4 prostate
carcinoma cells to BMEC-1 and HUVEC. Calcein-AM-labeled PC3M-LN4
cells were added in a single cell suspension to confluent BMEC-1
(closed symbols) or HUVEC (open symbols)
monolayers in a 48-well plate. At the indicated times, wells were
washed to remove nonadherent cells, and adherent cells were lysed and
quantified in a fluorescence plate reader. Results are presented as the
mean percentage of input cells from triplicate wells ± standard
error of the mean (S.E.).
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Hyaluronan Presented by the Prostate Carcinoma Cells Mediates
Adhesion to BMEC-1--
Several surface-borne adhesion molecules have
been implicated in homing of circulating cells to the bone marrow,
including VLA-4 (
4
1 integrin),
LFA-1 (
L
2 integrin), CD44 proteoglycan, and the high molecular weight glycosaminoglycan hyaluronan (HA). Pretreatment of PC3M-LN4 cells with blocking antibodies directed against
4,
1,
L, or
2 integrin subunits, or against CD44, had no effect on
adhesion of these cells to BMEC-1 (data not shown). To assess the
relevance of HA to prostate carcinoma cell adhesion, we pretreated
PC3M-LN4 or BMEC-1 cells individually or simultaneously with
hyaluronidase (HAase) enzyme and assayed initial rapid adhesion at a
12-min time point. As above, ~70% of PC3M-LN4 cells were adherent at
this time point in the absence of enzymatic digestion (Fig.
2). Pretreatment of BMEC-1 cells with
HAase had no effect on this adhesion. However, treatment of PC3M-LN4 or
both cell types reduced adhesion to about 35%, indicating that cell
surface HA promotes this rapid intercellular interaction. Furthermore, the HA required for maximum adhesion is carried by the prostate carcinoma cells.

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Fig. 2.
HA on the PC3M-LN4 cells is required for
adhesion to BMEC-1. BMEC-1 monolayers and/or calcein-AM-labeled
PC3M-LN4 cell suspensions were treated where indicated with 16 units/ml
hyaluronidase for 25 min. The hyaluronidase was removed, and the
PC3M-LN4 cells were added to the BMEC-1 for 12 min at 37 °C.
Nonadherent cells were removed by washing, and adherent cells were
quantified in a fluorescence plate reader. Each bar
represents the mean of triplicate wells assayed ± S.E., reported
as percentage of input cells.
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HA-mediated Adhesion of Prostate Carcinoma Cells Is Specific for
Bone Marrow-derived Endothelial Cells--
Because endothelial cell
types of different origin exhibit differences in HA binding (36), we
assayed the specificity of hyaluronidase-sensitive prostate tumor cell
adhesion to endothelial cells. PC3M-LN4 cells were preincubated in the
absence or presence of hyaluronidase and allowed to adhere to
BMEC-1, HUVEC, or bone marrow-derived stromal cells (BMSC). Consistent
with the above results, PC3M-LN4 cells adhered rapidly to BMEC-1 and
adhesion was inhibited by 50% in the presence of HAase (Fig.
3). Cells adhered weakly to HUVEC, and
adhesion was not inhibited by enzyme treatment. Although adhesion of
PC3M-LN4 cells to BMSC was efficient and rapid, it was not inhibited by
HAase digestion, suggesting cell surface HA was not mediating this
adhesion. Collectively, these results imply that heterogeneous
expression of HA receptors among endothelial cell types may dictate
specific recognition of prostate carcinoma cells, although different
molecules may dominate their interactions with other bone
marrow-derived cells.

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Fig. 3.
Enzymatic removal of HA inhibits PC3M-LN4
adhesion to BMEC-1 but not HUVEC or BMSC. Calcein-AM-labeled
PC3M-LN4 suspensions were pretreated for 25 min at 37 °C in the
absence (solid bars) or presence (open bars) of
16 units/ml Streptomyces hyaluronidase. The cells were
diluted 5-fold with adhesion medium and added to BMEC-1, HUVEC, or BMSC
monolayers in a 48-well plate for 12 min at 37 °C. Nonadherent cells
were removed by washing, and adherent cells were lysed and quantified
in a fluorescence plate reader. Each bar represents the
mean ± S.E. of quadruplicate wells assayed, reported as
percentage of input cells. Each assay was repeated three times.
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Rapid, Specific Prostate Carcinoma Cell Adhesion to Bone Marrow
Endothelial Cells Is Differentially Sensitive to Hyaluronidase and
Exogenous Hyaluronan--
To examine whether HA would generally
promote adhesion of prostate carcinoma cell lines to bone marrow
endothelial cells, we measured HAase-sensitive BMEC-1 adhesion of the
commercially available cell lines PC3, DU145, and LNCaP. As
demonstrated above with PC3M-LN4 cells, about 60-70% of PC3 cells
adhered to BMEC-1 monolayers and adhesion was inhibited 40-50% by
HAase treatment (Fig. 4A). In
contrast, DU145 and LNCaP cells adhered very poorly (about 25%), and
the low level of adhesion was not altered by HAase treatment. None of
the cell lines adhered well to HUVEC, and adhesion to HUVEC was not
sensitive to HAase (Fig. 4A, inset). These
results were replicated in similar experiments using another human bone
marrow endothelial cell line, trHBMEC. As presented in Fig.
4B, PC3 and PC3M-LN4 cells exhibited comparable levels of
HAase-sensitive adhesion to trHBMEC, whereas very few DU145 or LNCaP
cells were adherent, and their adhesion was unaffected by HAase
treatment. HA, therefore, is required for maximal rapid interaction
between prostate carcinoma cells and bone marrow endothelial cells.

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Fig. 4.
Adhesion of prostate carcinoma cell lines to
bone marrow endothelial cells is differentially HA dependent. PC3,
DU145, LNCaP (A and B), and PC3M-LN4
(B) cell suspensions were calcein-AM-labeled, pretreated in
the absence (solid bars) or presence (open bars)
of 16 units/ml Streptomyces hyaluronidase. Cells were then
diluted in adhesion medium and added to confluent washed monolayers of
BMEC-1 (A), HUVEC (panel A, inset
graph), or trHBMEC (B) in a 48-well plate for 12 min at
37 °C. Nonadherent cells were removed by washing, and adherent cells
were lysed and quantified in a fluorescence plate reader. Each
bar represents the mean ± S.E. of quadruplicate wells
assayed, reported as a percentage of input cells determined separately
for each cell type. Each assay was repeated three times.
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To further establish the requirement for direct HA recognition by the
BMEC as a component of preferential adhesion to prostate carcinoma
cells, we pretreated trHBMEC monolayers with increasing concentrations
of exogenous high molecular weight HA. PC3M-LN4 cells were incubated
with or without HAase, which was then removed. Cells were resuspended
in the respective concentrations of HA and added to the BMEC. As
before, about 80% of the cells were adherent after 12 min, and
adhesion was reduced to 35% by HAase digestion (Fig.
5). Preincubation of the BMEC with 10 µg/ml of HA modestly increased adhesion of untreated cells, but
rather strikingly increased adhesion of HAase-treated cells to about 60%. However, a dose-dependent inhibition was observed at
higher concentrations, with adhesion almost completely inhibited at 500 µg/ml HA. This effect was more greatly manifested in HAase-treated cells, suggesting the residual adhesion observed for these
cells may be due to incompletely digested HA or HA resynthesis during the time of the assay. When DU145 cells were similarly incubated with
pretreated trHBMEC (Fig. 5, inset graph), no effect of
exogenous HA was observed. This confirms that the inhibitory effect of
HA is not occurring indirectly by destabilizing the BMEC monolayer, and
that other receptor interactions are promoting the less rapid adhesion
of DU145 cells to BMEC.

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Fig. 5.
Effect of exogenous hyaluronan addition on
PC3M-LN4 adhesion to trHBMEC. Calcein-AM-labeled PC3M-LN4
suspensions or DU145 suspensions (inset graph) were
pretreated for 25 min at 37 °C in the absence (solid
bars) or presence (open bars) of 16 units/ml
Streptomyces hyaluronidase. Cells were then washed and
resuspended in the indicated concentrations of hyaluronan and added to
confluent monolayers of trHBMEC cells preincubated for 30 min in the
same hyaluronan concentrations. Nonadherent cells were removed after
incubation for 12 min at 37 °C and remaining adherent cells were
lysed and quantified by fluorescence plate reader. Each bar
represents the mean ± S.E. of quadruplicate wells assayed,
reported as a percentage of input cells determined separately for each
cell type.
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Prostate Carcinoma Cell Surface HA Retention Correlates to BMEC
Adhesion--
If HA was mediating the differential BMEC adhesion
observed among the various prostate carcinoma cell lines, then these
differences should be manifested in varied levels of HA retained on the
surface of prostate carcinoma cells. Therefore, we used a particle
exclusion assay to visualize the cell surface HA. In these experiments, surface-associated HA was first amplified by addition of aggrecan, a
large multivalent proteoglycan that specifically associates with HA at
the cell surface, surrounding the cell with a highly hydrated gel-like
envelope. HA is thereby detected as a pericellular clear zone upon
addition of fixed red blood cells, which cannot settle directly at the
cell surface. Cells that lack HA do not exhibit these cleared halos,
and the blood cell particles contact the cell perimeter. PC3M-LN4 cells
were surrounded by a large matrix (Fig.
6A) that disappeared
quantitatively after HAase treatment (Fig. 6A,
inset). Similarly, PC3 cell surfaces bore a matrix (Fig. 6B) that was removed by HAase treatment (Fig. 6B,
inset). In contrast, neither DU145 cells nor LNCaP cells
retained HA on the cell surface (Fig. 6, C and
D). Average ratios of matrix area with respect to cell area,
obtained by integrating individual tracings of each cell type (Table
II), were 1:1 for DU145 and LNCaP
and 2:1 for PC3 and PC3M-LN4. Presence of cell surface HA, therefore,
correlated with rapid, specific adhesion to BMEC. Conversely, its
absence corresponded to weak adhesion.

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Fig. 6.
Visualization of prostate carcinoma cell
surface HA. Subconfluent PC3M-LN4 (A), PC3
(B), DU145 (C), or LNCaP (D) cells
were incubated with 2 mg/ml aggrecan for 90 min, followed by addition
of 1 × 108 fixed red blood cells and incubation at
37 °C for 15 min. HA-aggrecan coats evidenced by halos surrounding
the cells were photographed at 400× magnification. Hyaluronidase
treatment eliminated the pericellular matrices of PC3M-LN4 (panel
A inset) and PC3 (panel B inset) cells, verifying their
HA composition.
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Table II
Elevated HA synthase expression correlates to prostate carcinoma cell
surface HA, adhesion to BMEC-1, and previously reported metastatic
potential
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Prostate Carcinoma Cell HA Synthesis Correlates to Presence of a
Pericellular HA Matrix and Adhesion to BMEC--
Elevated levels of HA
have been reported to correlate with progression of several tumor
types, including prostate. Furthermore, high levels of HA synthesis are
necessary and sufficient for production of a pericellular HA coat. To
address the possibility that the prostate carcinoma cells used in our
study could be synthesizing large amounts of HA but differentially
retaining it at the cell surface, we quantitated the HA synthesized by
each cell line. HA synthesis occurs at the plasma membrane, concurrent
with extrusion of HA from the cell, such that the majority of cellular
HA is shed into the culture medium. Overnight culture media from each cell type were accordingly analyzed for HA content by a competitive binding assay and HA level was normalized to cell count (Fig. 7). PC3 and PC3M-LN4 cell culture
supernatants were found to contain high levels of HA (~5 and 6 µg/106 cells, respectively, Table II) whereas DU145
culture medium had very little (1 µg/106 cells), and
levels in LNCaP culture were virtually undetectable. These results are
consistent with high levels of HA synthesis contributing to cell
surface HA retention, promoting adhesion to bone marrow endothelial
cells.

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Fig. 7.
HA synthesis and secretion by prostate
carcinoma cells in culture. Equal numbers of prostate carcinoma
cells were seeded overnight then given fresh culture medium. After an
additional 24 h, supernatants were harvested and cells were
trypsin-released and counted. HA-containing culture media were serially
diluted, incubated with biotinylated HA binding protein, and applied to
HA-coated plates. Bound HA binding protein was detected, following
extensive washes, with streptavidin-HRP conjugate and OPD substrate,
and quantified spectrophotometrically. Total HA in the culture media
was determined by interpolation from a concurrent HA standard curve and
plotted relative to cell number in the original culture.
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Elevated Levels of HA Synthase Expression by Prostate Carcinoma
Cells Correlate to HA Production and Adhesion to BMEC--
HA
synthesis is catalyzed by one or more isoforms of three homologous HA
synthase enzymes: Has1, Has2, and Has3. We used RT-PCR to determine
initially which isoform(s) were expressed by each prostate carcinoma
cell line, in an attempt to correlate elevated HA synthesis and BMEC
adhesion with presence of a specific message. We were unable to detect
Has1 message in any cell line using any set of oligonucleotide primers,
although we could amplify a product from a Has1 cDNA control
plasmid (data not shown). However, expression of Has2 and Has3 was
detectable in most of the cell lines. Because the presence or absence
of message for a particular isoform did not appear to correlate with
either HA matrix formation or BMEC adhesion, we developed a
semi-quantitative approach to look at relative message levels. Equal
amounts of input mRNA from normal prostate
(CLONTECH) and each of the prostate carcinoma cell
lines were reverse-transcribed and used as templates for PCR
amplification of Has2 (Fig.
8A, lanes 8-14) or
Has3 (Fig. 8B, lanes 8-14), concurrent with a
GAPDH housekeeping control message (Fig. 8, lanes 2-6). Product yields were normalized to GAPDH and presented as fold expression relative to normal prostate in Table II.

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Fig. 8.
HA synthase expression in prostate
adenocarcinoma cell lines. HA synthase isoform and level of
expression was determined for each of the four prostate carcinoma cell
lines PC3M-LN4, PC3, DU145, and LNCaP, and for normal prostate, by
RT-PCR as described under "Experimental Procedures." A,
RT-PCR amplification of Has2 and GAPDH control messages; B,
Has3 and GAPDH. Anticipated product sizes (in base pairs) are indicated
adjacent to the figure: Has2 is ~210 bp, Has3 is ~410 bp, and GAPDH
is ~980 bp. Lanes are numbered as follows: lanes
1 and 7, 100-base pair DNA ladder; lanes 2 and 8, normal prostate; lanes 3 and 9,
PC3M-LN4; lanes 4 and 10, PC3; lanes 5 and 11, DU145; lanes 6 and 12, LNCaP;
lane 13, Has2 control plasmid; lane 14, Has3
control plasmid.
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Results of this assay showed that PC3 (lanes 4 and
10) and PC3M-LN4 cells (lanes 3 and 9)
expressed higher levels of both Has2 and Has3 than DU145 (lanes
5 and 11), or LNCaP (lanes 6 and 12), consistent with higher levels of HA synthesis and rapid
adhesion to BMEC by those cell lines. Has2 was virtually absent in
DU145 cells and undetectable in normal prostate (lanes 2 and
8) or LNCaP cells. Has3 expression was dramatically
increased in the most highly metastatic line, PC3M-LN4, followed by PC3
and DU145. Has3 expression was very low in normal prostate or LNCaP
cells. Although DU145 cells appeared to express significant levels of
Has3, this cell line was found to be a heterogeneous population in
which about 2-10% carry surface associated HA (in contrast to PC3 and PC3M-LN4, in which >99% of the cells carry abundant surface HA). This
was visualized by phase contrast microscopy at the level of individual
cells using biotinylated HA binding protein, followed by
streptavidin-HRP detection with diaminobenzidine precipitation (data
not shown). The apparent Has3 cDNA level amplified from this cell
line may be anomalously high due to those specific cells and not
representative of the overall phenotype of the cell line, which
probably expresses lower message levels. Collectively, these results
demonstrate that HA synthesis correlates very well to HA synthase
expression level, which in turn may dictate adhesion to bone marrow
endothelial cells. Furthermore, it is clear that HA synthesis and HA
synthase expression are dramatically up-regulated in aggressive
prostate adenocarcinoma cells, with overall HA production and HAS
expression levels correlating directly to metastatic potential.
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DISCUSSION |
Bone metastasis is an eventuality of advanced stage prostate
cancer that results in severely reduced quality of life and ultimate morbidity. Metastasis is preceded by initial adhesion of circulating tumor cells to endothelial cells lining the vasculature of the secondary site. Because prostate carcinoma metastasizes to bone, we
modeled this initial event using prostate carcinoma cell lines and the
transformed bone marrow sinusoidal endothelial cell lines, trHBMEC and
BMEC-1. In this study we demonstrate that highly metastatic prostate
carcinoma cells adhere to bone marrow endothelial cells through
pericellular hyaluronan (HA). Unlike reports of other model system
interactions requiring this molecule, we find that the prostate
carcinoma cells present the HA recognized by the BMEC. The HA-mediated
adhesion shows endothelial source specificity, because PC3M-LN4 cells
do not adhere rapidly to HUVEC, another endothelial cell type.
Furthermore, HA-mediated adhesion exhibits specificity among
bone-derived cell types: Although adhesion of PC3M-LN4 cells to bone
marrow stromal cells is rapid, HA does not appear to be involved,
suggesting other receptor interactions contribute to this process.
Up-regulation of HA synthase in prostate tumor cells may promote bone
marrow metastasis by specifically arresting those cells on the bone endothelium.
The bone marrow microvasculature is a specialized network of venules
and fenestrated sinusoids permeable to low molecular weight
fluorophores but impenetrable by larger macromolecular conjugates and
cellular bodies (10). However, the bone marrow as the site of
hemopoiesis must be capable of progenitor cell flux across its
protective endothelial layer, a function not required or desirable in
other types of endothelium. Endothelial cell types exhibit
heterogeneity in cytokine response (11, 37), receptor expression (9,
11, 36), and signaling pathways (38). In the absence of specific
stimuli, endothelial heterogeneity alone is able to influence homing of
circulating progenitor cells to the bone marrow through differential
expression of selectins (10, 39, 40), glycoproteins (14), and VCAM-1
and CD44 (15). Receptor expression and adhesion of circulating
leukocytes to sites of inflammation is further regulated through
endothelial activation by inflammatory cytokines (41, 42). These cell surface differences may translate into preferential adhesion of circulating tumor cells to endothelial cells in specific tissues.
Transformed BMEC lines recently developed have facilitated exploration
of the mechanisms by which endothelial adhesion receptors may dictate
tumor preference for specialized endothelia. Results presented in this
study demonstrate that prostate cancer cells adhere rapidly to bone
marrow but not large vein endothelial cells. This is in agreement with
observations by other investigators that prostate adenocarcinoma cell
lines adhere preferentially to cell cultures enriched for BMEC over
components of the bone marrow or hepatic endothelial cells (2). In
another study, adhesion of prostate carcinoma cells to a cell line
immortalized from isolated BMEC was shown to be inhibited by
preincubation of BMEC with monoclonal anti-LFA-1 antibodies or
polyclonal anti-galectin-3 (43), but it was unclear which cell type
expressed LFA-1 because neither has been previously reported to do so.
We have used two transformed bone marrow endothelial cell
lines, BMEC-1 (30) and trHBMEC (11), which express the same cell
adhesion molecules and synthesize the same cytokines as the primary
endothelial cells with minor differences in level of expression, to
determine that HA on the prostate tumor cells mediates adhesion to
BMEC. However, this does not exclude the possibility of other
interactions. We expect that, like progenitor arrest, adhesion and
migration of prostate tumor cells on BMEC involves multiple adhesive
interactions that may or may not be interdependent.
HA is a ubiquitous polysaccharide component of extracellular and
cell-associated matrices (44, 45), essential for growth and motility.
HA is required for normal ductal branching in the developing prostate
gland (46), underscoring its vital role in cell migration (47). In some
cell types, this requirement entails assembly of an HA pericellular
matrix for proliferation and migration (33, 48). Cell-associated HA may
facilitate growth and motility by stimulating detachment of rounded,
dividing cells and the trailing edges of migrating cells, respectively. HA exhibits further functional complexity as an adhesion molecule involved in recruitment of circulating lymphocytes to inflamed tissues
through the action of cell surface HA receptors (38). The structure of
HA, consisting of many thousand repetitions of a disaccharide motif, is
well-suited to serve as a multivalent ligand for coordinate binding by
many simultaneous adhesion receptors or for providing a scaffold for
cellular movement.
Cellular behaviors contributing to cancer progression include
unrestricted growth, motility, and ability to circulate and colonize
new tissues. Because HA is a normal component of such processes, it is
not surprising that elevated levels correlate with cancer progression
(21, 49, 50). High levels of serum HA, for example, correlated with
disseminated carcinoma in general (51, 52) and, specifically, with
tumor progression to metastatic disease in malignant lymphoma (53) and
breast carcinoma (54). In human breast carcinoma, HA is more
concentrated in areas where the tumor is invading into the surrounding
tissue (22), and elevated stromal and cell-associated HA correlates
with malignancy (55). Elevated stromal and epithelial HA are also
indicative of poor survival rate in patients with ovarian and
colorectal cancers (23, 24). In animal models, tumor cells with high levels of HA expression were more metastatic than cells expressing lower levels of HA (27, 29, 56, 57). Interestingly, both high and low
HA-expressing cells have the same growth rate in vitro and
at the primary injection site (27). This correlation of HA with
metastasis but not growth rate suggests that HA may be more critical to
endothelial adhesion and/or the infiltration of the cells into tissues.
PC3M-LN4 cell adhesion to BMEC in vitro was both enhanced
and inhibited by the addition of high molecular weight HA. At low concentrations comparable to those secreted into the culture medium of
the cells during growth, adhesion of hyaluronidase-treated tumor cells
to BMEC was significantly enhanced by HA preincubation. HA prebinding
by the BMEC may restore adhesion by replacing the cross-bridging ligand
normally presented by the prostate tumor cells via its own cell surface
HA receptors. When BMEC were precoated with higher levels of HA,
PC3M-LN4 cell adhesion was almost entirely precluded, regardless of
HAase treatment. This suggests that BMEC and prostate HA receptors have
been saturated and are no longer able to cross-link. HA has been
previously reported to enhance/inhibit intercellular adhesion in this
fashion in development of chick limb buds (58). Rapid intercellular
adhesion was synergistically inhibited by HAase treatment and high
exogenous HA, suggesting the incomplete HAase effect is probably due to
HA resynthesis during the assay. By contrast, DU145 cell adhesion was
not affected by addition of HA at any concentration, and therefore,
these cells most likely lack active cell surface HA binding proteins.
PC3 (59) and PC3M-LN4 (35) prostate carcinoma cells are highly
metastatic in mouse models and are shown in this report to produce a
dense pericellular HA matrix that mediates adhesion to bone marrow
endothelial cells. DU145 (60) and LNCaP (61) cell lines, by contrast,
are poorly metastatic in mice, produce very little HA, and do not
adhere well to BMEC. It is worth noting the origins and characteristics
of the four cell types: PC3 is from a human bone metastasis; its
derivative, PC3M-LN4, metastasizes to mouse bone; DU145 and LNCaP are
from a human brain and a human lymph node metastasis, respectively, and
their interaction with bone has never been documented. The correlation
between high metastatic potential as reported in the literature,
up-regulated HA synthesis and expression of HA biosynthetic enzymes is
summarized in Table II. This correlation is consistent with a putative
role for HA as a component of prostate cancer metastasis. In fact, HA
overproduction is thought to be directly involved in prostate cancer
progression. Histological sections of normal adult prostate tissue
demonstrate the presence of HA in the prostate stroma (25, 46). In
cancerous human prostates, HA expression levels are increased on the
carcinoma cells and correspond to dedifferentiation of the cancer (25, 26).
Synthesis and secretion of HA is catalyzed in vertebrates by a family
of three HA synthases: Has1 (62), Has2 (63), and Has3 (64), each of
which is capable of conferring HA synthesis and pericellular HA
retention to transfected cells (for a review of HA synthases, see Ref.
65). HAS expression is ubiquitous, but isoforms exhibit temporal and
tissue-specific distribution. Targeted disruption of the
has2 gene is an embryonic lethal mutation in mice, which
fail to produce HA essential for pericardial endothelial cell migration
and endothelial/mesenchymal transformation during cardiac development
(66). Has2 is also specifically up-regulated in response to
wounding in a mesothelial cell model (67). Because HAS expression is
critical during periods of normal tissue remodeling, understanding its
dysregulation in tumors may be important in controlling tumor growth
and metastasis. HAS expression is regulated by glucocorticoids (68),
growth factors such as platelet-derived growth factor (69),
transforming growth factor
1 (70), and pro-inflammatory cytokines
(71). Expression of HAS appears to correlate directly to HA synthesis
(69), suggesting regulation occurs at the level of transcription. To
date, there is no evidence for post-transcriptional mechanisms.
Elevated HA in tumor cells is, therefore, probably a reflection of HAS
gene expression. In support of this, we have determined that Has2 and
Has3 are strongly up-regulated in highly metastatic prostate tumor
cells. HAS up-regulation in prostate cancer progression may be dictated
in part by factors such as those described above, produced and secreted
by prostate stromal or epithelial cells.
HA synthase enzymes have been implicated in tumorigenesis and
metastasis in mouse models. Overexpression of Has2 in fibrosarcoma cells yields significantly larger subcutaneous tumors (28). Mammary
carcinoma cells transfected with has1 were more metastatic than control cells (29). In a melanoma model, tumor cells selected for
high cell surface expression of HA were highly tumorigenic and
metastatic, whereas tumor cells bearing little or no surface HA,
although equally tumorigenic, did not metastasize (27). In the latter
model, however, has isoform expression was not
characterized. Our data present the first characterization of
has expression in prostate carcinoma cells and reveal a
possible correlation of Has3 overexpression with tumor cell metastatic
potential. Collectively, these results suggest involvement of HA in
tumor growth and metastasis, and imply that specific HA synthase
isoforms and/or expression levels of those isoforms may mediate these processes.
Both Has2 and Has3 are capable of synthesizing HA with an average
molecular mass of 1-2 million Da (72), the average size of the
exogenous HA used to enhance/inhibit adhesion (Fig. 5). This would
suggest that the products of both enzymes are capable of supporting
intercellular adhesion. DU145 cells, however, appear to express
elevated Has3 but synthesize little HA and retain no matrix. One
possible explanation may be that Has3 message is transcribed but not
translated in these cells or that the protein made is inactive.
Alternatively, there may be a requisite maximum threshold of HAS
expression for maintenance of a pericellular matrix. Levels of Has3
expression sufficient to promote matrix retention may occur in only a
subset of DU145 cells, with the remaining cells expressing it at lower
levels. This may also be the case for Has2 expression. If production of
an HA matrix enhances arrest in the bone marrow sinusoids, the cell
population would have significantly diminished propensity to do so,
relative to PC3 or PC3M-LN4 cells, which could then translate to
reduced bone metastatic proclivity. Both enzymes are up-regulated in
highly metastatic cells, which may implicate HAS up-regulation in
prostate cancer progression. Has3 overexpression most consistently
corresponds to aggressive potential, but intrinsic heterogeneity within
the cell lines renders assignment of such a correlation premature.
Another factor to consider is that DU145 cells may lack surface
receptors to anchor the matrix, because exogenous HA could restore
adhesion of HAase-inhibited PC3M-LN4 cells, which are normally able to
maintain an HA coat, but not enhance adhesion of DU145 cells. It is
important to recognize that, although HA produced by prostate tumor
cells may facilitate metastasis to bone marrow by initially attaching
to the endothelium in the bone marrow, changes in matrix-associated HA
binding proteins could also modify prostate tumor cell behavior.
Because HA is secreted as a free glycosaminoglycan and is not attached
to a core protein, its retention at the cell surface is achieved
through accessory proteins such as versican, which contribute to the
matrix, specifically binding and cross-linking the
multivalent HA into a dense network (33, 48). Elevated
levels of versican are associated with prostate carcinoma progression,
but it is not known if its HA binding properties are responsible
(73).
However, in addition to the HA binding proteins in the extracellular
matrix, HA is specifically recognized by a widely expressed transmembrane receptor, CD44. Bone marrow and umbilical vein
endothelial cells BMEC-1, trHBMEC, and HUVEC express cell surface
CD44 but exhibit different affinity for PC3M-LN4 cells. Preincubation
of BMEC-1 cells with various reported anti-CD44 blocking monoclonal antibodies failed to impact prostate tumor cell interaction (data not
shown). Nonetheless, regulation of CD44 activation state with respect
to HA binding occurs on many levels and probably contributes to
endothelial adhesive preference. CD44 and HA interactions may be
important for metastasis. CD44-mediated migration and invasion of
glioma cell lines is stimulated by HA (74) and inhibiting CD44/HA
interactions in vivo inhibits metastasis (75, 76). HA
clusters CD44 resulting in stimulation of signal transduction pathways
and engagement of adhesion molecules such as integrins (77), which
could either strengthen adhesion or lead to transmigration of the
prostate cells through the endothelium.
Alternatively, HA binding proteins on the surface of BMEC-1 other than
CD44 may contribute to rapid adhesion. One candidate protein is the
receptor for HA-mediated motility (RHAMM), which has not been reported
on BMEC surfaces but has been shown to mediate differential HA binding
by endothelial cells of different vascular origin (36). It is therefore
possible that differences in vascular endothelial RHAMM expression
could be promoting the recognition of prostate carcinoma cells bearing
surface-associated HA. Other recently described cell surface HA
receptors include the lymph vessel endothelial-specific LYVE-1 (78),
and the HA receptor for endocytosis thought to be specific for
clearance of circulating HA through the liver and spleen (79).
In this report, we have surveyed established prostate carcinoma cell
lines for common molecular interactions governing preferential adhesion
to bone marrow endothelial cells and discovered a correlation between
metastatic potential and elevated HA synthase. We have delineated a
mechanism in which prostate cancer cells adhere to bone marrow
endothelial cells via tumor cell-associated HA. It will be important to
extend these findings to establish whether this adhesive interaction
contributes to prostate cancer metastasis. With this goal, we are
currently manipulating HA levels on prostate carcinoma cells in HA
synthase transfectants, which should enable us to determine if HA
changes the metastatic potential of prostate carcinoma cells in an
animal model and whether a specific HA synthase isoform is responsible.
Furthermore, this approach will allow us to study the impact of HA
overproduction on activation of adhesion receptors and signal
transduction pathways in HA-mediated adhesion of prostate carcinoma
cells to bone marrow endothelium.