From the Department of Urology, § Cell
Biology and Anatomy, and ** Pathology, University of Miami School of
Medicine, Miami, Florida 33101 and the
Hamon Center for
Therapeutic Oncology Research, University of Texas Southwestern Medical
Center, Dallas, Texas 75390-8593
Received for publication, September 14, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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Hyaluronic acid (HA), a glycosaminoglycan,
regulates cell adhesion and migration. Hyaluronidase (HAase), an
endoglycosidase, degrades HA into small angiogenic fragments. Using an
enzyme-linked immunosorbent assay-like assay, we found increased
HA levels (3-8-fold) in prostate cancer (CaP) tissues when compared
with normal (NAP) and benign (BPH) tissues. The majority (~75-80%)
of HA in prostate tissues was found to exist in the free form. Primary
CaP fibroblast and epithelial cells secreted 3-8-fold more HA than
respective NAP and BPH cultures. Only CaP epithelial cells and
established CaP lines secreted HAase and the secretion increased with
tumor grade and metastasis. The pH activity profile and optimum (4.2; range 4.0-4.3) of CaP HAase was identical to the HYAL1-type HAase present in human serum and urine. Full-length HYAL1 transcript and
splice variants were detected in CaP cells by reverse
transcriptase-polymerase chain reaction, cloning, and sequencing.
Immunoblotting confirmed secretion of a ~60-kDa HYAL1-related protein
by CaP cells. Immunohistochemistry showed minimal HA and HYAL1 staining
in NAP and BPH tissues. However, a stromal and epithelial pattern of HA
and HYAL1 expression was observed in CaP tissues. While high HA
staining was observed in tumor-associated stroma, HYAL1 staining in
tumor cells increased with tumor grade and metastasis. The
gel-filtration column profiles of HA species in NAP, BPH, and CaP
tissues were different. While the higher molecular mass and
intermediate size HA was found in all tissues, the HA fragments were
found only in CaP tissues. In particular, the high-grade CaP tissues,
which showed both elevated HA and HYAL1 levels, contained angiogenic HA
fragments. The stromal-epithelial HA and HYAL1 expression may promote
angiogenesis in CaP and may serve as prognostic markers for
CaP.
The majority of newly diagnosed prostate cancer
(CaP)1 patients have
clinically organ-confined disease. The limited knowledge about which
CaP is aggressive and likely to progress, as well as when it will
recur, severely impedes individualized selection of therapy and
subsequent prediction of outcome (1). Routine biochemical
(i.e. prostate-specific antigen levels) and surgical and pathologic parameters (i.e. Gleason sum, margin, and
node status and seminal vesicle invasion) offer a glimpse of the
biological potential of the tumor (2-7). However, many of the CaP
patients (~50-60%) with clinically localized disease have
prostate-specific antigen levels between 4 and 10 ng/ml and a biopsy
Gleason score between 6 and 7, which limits the prognostic capability
of these markers (1, 2, 6). The prognosis of CaP patients can be
improved if molecules that associate with the biological potential of
CaP are identified (7). We have recently shown that both tumor-associated hyaluronic acid (HA) and tumor-derived hyaluronidase (HAase) possibly play a role in tumor progression.
HA is a nonsulfated glycosaminoglycan made up of repeating disaccharide
units, D-glucuronic acid and
N-acetyl-D-glucosamine (8). HA is a component of
extracellular matrix and is present in various tissues and tissue
fluids. It performs several functions in normal physiology.
Concentration of HA is elevated in several cancers including bladder,
colon, breast, and lung and Wilms' tumor (9-13). We have previously
shown that the urinary HA levels are 2.5-6.5-fold elevated in bladder
cancer patients and serve as a highly sensitive and specific marker for
detecting bladder cancer, regardless of the tumor grade (14, 15). In
tumor tissues, HA expands upon hydration and opens up spaces for tumor
cell migration. Tumor cells migrate on HA-rich matrix that is mediated
by cell surface HA receptors (e.g. CD44 and RHAMM; see Refs.
16-19). HA may also offer tumor cells some protection against immune
surveillance and chemotherapeutic agents (20). Small fragments of HA
(3-25 disaccharide units) are angiogenic (21). We have previously isolated such angiogenic HA fragments from the urine of high-grade bladder cancer patients and shown that these fragments induce endothelial cell proliferation (14). Furthermore, HA fragments of the
same length also induce endothelial cell migration and lumen formation
(22). Recent studies from our laboratory demonstrate that angiogenic HA
fragments interact with RHAMM on the surface of human endothelial cells
and induce the MAP kinase pathway (23, 24). Thus a regulated
degradation of HA in tumor tissues may be important for both tumor
metastasis and angiogenesis.
HAases are a family of enzymes which degrade HA (25). Initially termed
as a "spreading factor," the presence of HAase is crucial to the
spread of bacterial infections and toxins present in bee, snake, and
other venoms (26-28). In human, 6 HAase genes have been identified
(29-32). These genes cluster in two tightly linked triplets on human
chromosomes 3p21.3 (HYAL1, HYAL2, and HYAL3) and
7q31.3 (HYAL4, PH20, and HYALP1) (32). Among
these, HYAL1, HYAL2, and PH20 are relatively well
studied at the protein level. HYAL1 gene encodes a HAase
that is present in human serum, however, its cellular origin is unknown
(31). HYAL2 gene encodes a lysosomal HAase (30).
PH20 gene encodes the testicular-type HAase that shows a
broad (pH 3.2-9.0) pH activity profile (29).
In establishing the association of HAase to tumor biology, we initially
showed that HAase levels are elevated in CaP and these levels correlate
with CaP progression (i.e. metastatic > high-grade In this study, using biochemical and molecular biology techniques, we
have examined the expression of HA and HAase in prostate tissues and
cell culture. Furthermore, we have been able to identify and
characterize the type of HAase expressed in prostate cancer cells. In
addition, we have localized these molecules in prostate tissues by
immunohistochemistry. We also attempted to understand the function of
the tumor-associated HA-HYAL1 system.
Tissue Specimens--
Normal prostate (NAP) tissues from adults
(21-50 years) were obtained from organ donors. Neoplastic and BPH
tissues (~1 g) were obtained from patients undergoing open
prostatectomy. The tissue specimens were split and the mirror segment
was fixed in formalin, embedded in paraffin, and sectioned; then
hematoxylin and eosin staining evaluated the histologic grades of these
tumors. In this study, we have included data from only those specimens, which were histologically confirmed as normal, benign, and malignant.
Tissue Extracts--
Fresh or frozen (~0.5-1 g) specimens
were suspended in ice-cold homogenization buffer (5 mM
Hepes pH 7.2, 1 mM phenylmethylsulfonyl fluoride) and
homogenized for 30 s in a tissue homogenizer. The tissue extracts
were clarified by centrifugation at 40,000 × g for 30 min (14, 33, 41). The supernatants were designated as "Hepes
extracts." The tissue pellets were re-extracted in 50 mm sodium
acetate (pH 5.8), 6 M guanidine HCl, and 1 mM
phenylmethylsulfonyl fluoride. Following clarification by
centrifugation, the supernatants were designated as "guanidine
extracts." Both Hepes and guanidine extracts were assayed for HA and
protein concentration.
Primary Fibroblast and Epithelial Cultures--
Primary cultures
from prostate tissues were set up as described previously (33). For
culturing fibroblasts, collagenase-digested tissue fragments were
cultured in RPMI 1640 + 10% fetal bovine serum medium. The fibroblast
growth in cultures was confirmed by anti-vimentin staining. During
second passage, when the fibroblast cultures became ~60% confluent,
the cultures were washed extensively in PBS and incubated in serum-free
RPMI 1640 containing insulin, transferrin, and selenium (ITS solution,
Life Technologies, Inc., Gaithersburg, MD). The serum-free conditioned
medium (SF-CM) was collected after 2-3 days.
The prostatic epithelial explant cultures were set up in a prostate
epithelial cell growth medium, PrEGM (Prostate Epithelial Growth
Medium, BioWhitaker/Clonetics, San Diego, CA) as described before (33).
PrEGM is a serum-free growth medium. The epithelial cell growth in
cultures was confirmed by anti-cytokeratin staining (33). The SF-CM
from primary cultures was collected at second passage, 3 days after
subculturing, and concentrated 10-fold.
Tissue Culture--
Prostate cancer cell lines DU145 and LNCaP,
bladder cancer line HT1376, and human embryonic lung fibroblast (HL
fibroblast; passage 11) were obtained from the American Type Culture
Collection (Rockville, MD). The prostate cancer line PC3-ML was a gift
from Dr. M. E. Stearns, Medical College of Pennsylvania,
Philadelphia, PA, and the bladder cancer cell line 253J-Lung was kindly
provided by Dr. Colin Dinney, M.D. Anderson Cancer Center, University
of Texas, Houston, TX. All of these cell lines were cultured in RPMI 1640 + 10% fetal bovine serum and gentamycin. At ~60% confluence, the cultures were washed three times in PBS and incubated in serum-free RPMI + ITS. The SF-CM from these cultures was collected after 2-3 days.
Alternatively, CaP fibroblast and HL fibroblast were grown in the
culture medium (i.e. RPMI 1640 + 10% fetal bovine serum and
gentamycin) to 80-90% confluence and the conditioned medium was
collected. This medium was designated as S-CM, since it contained 10%
fetal bovine serum. The S-CM were collected from fibroblast cultures to
examine HAase activity, such S-CM have been used previously to
demonstrate HAase activity in fibroblast cultures at pH 3.7 (44).
Measurement of HA Levels--
HA levels in tissue extracts and
SF-CM were measured using an ELISA-like assay originally developed by
Fosang et al. (45), with modifications (14, 15). Briefly,
96-well microtiter plates were coated with 25 µg/ml human umbilical
cord HA (ICN Biomedicals, Costa Mesa, CA). The HA-coated wells were
incubated with various amounts of tissue extracts or SF-CM
(unconcentrated) from different cell types, in the presence of a
biotinylated HA-binding protein. The HA-binding protein was isolated
from bovine nasal cartilage according to the method described by
Tengblad (46), which utilizes HA affinity chromatography and
trypsinization to isolate the HA binding part of the proteoglycan
monomer. The purified HA-binding protein was biotinylated using
N-hydroxysuccinamido biotin (Sigma). The amount of
biotinylated HA-binding protein bound to the microtiter wells was
determined using an avidin-biotin detection system (Vector Laboratories, Inc., Burlingame, CA). The amount of HA present in each
sample (ng/ml) was determined using a standard graph. We routinely
normalize the amount of HA in biological fluids (e.g. urine)
or in culture CM to total protein. Normalization of HA levels in
biological fluids such as urine to total protein eliminates the
influence of the hydration status of an individual on HA levels (15).
For each sample, 3 different amounts, each in duplicate, were tested.
The results are expressed as mean ± S.E.
Measurement of HAase Levels--
HAase levels present in tissue
extracts and SF-CM/S-CM were measured using an ELISA-like assay similar
to that developed by Stern and Stern (47), with modifications (15, 41).
Briefly, 96-well microtiter wells were coated with 200 µg/ml human
umbilical cord HA. The HA-coated wells were incubated with various
amounts of culture CM at 37 °C for 16 h in HAase assay buffer
(0.1 M sodium formate, 0.15 M NaCl, pH 4.2, 0.2 mg/ml bovine serum albumin (BSA; ELISA-grade; Sigma). The HA remaining
on the wells after incubation was determined using the same
biotinylated HA-binding protein that is used in the HA-ELISA-like
assay, and an avidin-biotin detection system. In the avidin biotin
detection system, we do not include anti-keratan sulfate monoclonal
antibody to enhance the signal and routinely normalize the amount of
HAase activity (milliunits/ml) in any sample (CM, in this case) to
total protein (mg/ml). We also routinely normalize the amount of HAase
in biological fluids (e.g. urine) to eliminate the influence
of the hydration status of an individual on HAase levels. This is
especially important when determining urinary HAase levels of patients
with hematuria (i.e. blood in urine; Ref. 15).
The pH activity profile of HAase present in various CM was determined
as follows: 1) pooled serum from 3 normal adults (0.5 µl); 2) human
urine (2.0 µl) collected from 4 normal individuals (3 adults: 2 females and 1 male: age 25-40 years and 1 child: 7 years); 3) CM (4 µl, 10-fold concentrated) from Du145 (SF-CM), CaP fibroblasts
(established from a Gleason 7 CaP; SF-CM and S-CM), and HL fibroblast
cultures (SF-CM and S-CM). The indicated amounts of various samples
were added to HA-coated wells containing HAase assay buffer at
different pH values (2.5-7.0). Between pH 3.5 and 5.0, the HAase
activity was tested in buffers differing by 0.1 pH unit
(i.e. pH 3.5, 3.6, 3.7 ... 5.0). The control wells received the buffers of specified pH, identical to those added to the
sample wells. In addition, 10-fold concentrated RPMI + ITS (SF-medium
control) and RPMI + 10% fetal bovine serum + gentamycin (S-medium
control) were also tested at different pH values. These media served as
controls for SF-CM and S-CM collected from different cell types. The
results are expressed as (control Substrate (HA)-Gel Assay--
A method described by Gutenhoner
et al. (48) was used to detect the presence of HAase
activity in various samples (48). Aliquots (1.2 ml) of SF-CM and S-CM
were collected from DU145, CaP fibroblast, and HL fibroblast cultures.
These CM and S-medium control were concentrated ~10-fold (100 µl).
A 20-µl aliquot of each concentrated SF-CM, S-CM, S-medium control,
human serum (1.5 µl), 20 µl of 10-fold concentrated normal human
urine and ELISA-grade BSA (10 µg) were separated on an 8.5%
SDS-polyacrylamide gel containing 0.1 mg/ml HA. Four such gels were
prepared and simultaneously electrophoresed. Following electrophoresis,
the gels were soaked in 3% Triton X-100, to renature the HAase present
in various samples. Each gel was then incubated in HAase assay buffer
of pH 3.0, 3.7, 4.2, or 4.5 without BSA. Following incubation at
37 °C for 16 h, the gels were stained sequentially with 0.5%
Alcian blue and 0.15% Coomassie Blue solutions and then destained.
RT-PCR Cloning and Sequence Analyses--
Total RNA was
extracted from CaP cell lines, a Gleason 7 CaP primary epithelial
explant culture and bladder cancer lines, HT1376 and 253J-Lung using a
RNA extraction kit (Quiagen, Valencia, CA). Total RNA (1 µg) was
subjected to first strand cDNA synthesis using the
SuperscriptTM preamplification system and oligo(dT) primers
(Life Technologies, Inc., Gaithersburg, MD). The cDNA was amplified
using three different HYAL1-specific primer pairs. The
primers were designed based on the HYAL1 cDNA sequence deposited in
the GenBankTM data base (accession number HSU03056).
The sequences of the first primer pair were the following:
(a) HYAL1-L1 (the sequence between nucleotides 214 and 233),
5'-CTGGTGGAAGAGACAGGAAG-3'; (b) HYAL1-R1 (the reverse
complementary sequence between nucleotides 564 and 583),
5'-GGAGGCAGAGCTGAGAACAG-3'. The second primer pair was designed to
amplify the entire coding region of HYAL1. The sequence of the second
primer pair was the following: (a) HYAL1-L2 (the sequence between nucleotides 594 and 613), 5'-TTGTCCTCGACCAGTCCCGT-3'; (b) HYAL1-R2 (the reverse complementary sequence between
nucleotides 1,906 and 1,925), 5'-ATCACCACATGCTCTTCCGC-3'. The sequence
of the third primer pair was designed to amplify both the long and short forms of HYAL1 transcript. The primer sequences were the following: (a) HYAL1-L3, this sequence is between
nucleotides 27,274 and 27,294 in a human cosmid clone LUCA13 from
3p21.3 (GenBankTM accession number AC002455). The
cDNA clones, GenBankTM accession numbers AF173154
(spliced form) and HSU03056, which contain the entire HYAL1
coding sequence, lack the nucleotide base "C" present at the 5' end
in the HYAL1-L3 primer, and begin with the following T, as their first
nucleotide. Therefore, the sequence between nucleotides 2 and 21 of the
HYAL1-L3 primer matches with the sequence between nucleotides 1 and 20 of the cDNA sequences AF173154 and HSU03056. The sequence of the
HYAL1-L3 primer is, 5'-CTTCCTCCAGGAGTCTCTGGT-3'. (b)
HYAL1-R3, the reverse complementary sequence between nucleotides 247 and 267 in clone AF173154 and between nucleotides 732 and 752 in clone
HSU03056. This primer sequence is, 5'-TCTCCAGGCACCACTGGGTGT-3'. The PCR
conditions for HYAL1-L1/R1 primer pair were the following:
(a) initial melting at 94 °C for 5 min; (b) 35 cycles of 94 °C for 1 min, 62 °C for 30 s, and 72 °C for
1 min; (c) 72 °C for 10 min. For PCR analysis Taq polymerase (Promega Corp., Madison, WI) was used. The
PCR conditions for HYAL1-L2/R2 and HYAL1-L3/R3 primers were the
following: (a) 95 °C for 10 min (hot start);
(b) 10 cycles of 94 °C for 30 s (70-60 °C) for
30 s, i.e. annealing temperature dropping by 1 °C at
each cycle, 72 °C for 1 min; (c) 25 cycles of 94 °C
for 30 s, 60 °C for 30 s and 72 °C for 1 min;
(d) 72 °C for 7 min, final extension. The PCR mixture
contained 5% dimethyl sulfoxide and Ampli-Taq
GoldTM (PerkinElmer Life Sciences, Wellesley, MA).
Immunoblot Analysis--
Prostate epithelial cell culture SF-CM
were separated on an 8.5% SDS-polyacrylamide gel, under nonreducing
conditions, and then blotted onto a polyvinylidene difluoride membrane.
The blotted membrane was stained with 0.15% Coomassie Blue in 30%
methanol for 1 min and then destained to visualize and compare total
protein profile in each lane as described previously (23). This method rules out the possibility that any differences observed in the intensity of the HYAL1 band among various samples is simply due to
differences in sample loading and protein transfer (23). Following
visualization of the total protein profile, the blot was completely
destained, rehydrated, and blocked with 3% BSA in 20 mM
Tris-HCl, 0.15 M NaCl, and 0.05% Tween 20. The blot was probed with 5 µg/ml anti-HYAL1 antibody at 4 °C for 16 h. The anti-HYAL1 antibody was purified as the IgG fraction using protein G-Sepharose, according to the manufacturer's protocol (Amersham Pharmacia Biotech). The blot was then washed and incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:7500 dilution; Sigma), at room temperature for 2 h. The blot was then washed and
developed using an alkaline phosphatase color detection system, involving nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate substrates (Bio-Rad). To determine the specificity of the
immunoblot analysis, in some experiments, the HYAL1-(321-338)-MAP peptide (50 µg/ml) was included during the anti-HYAL1 antibody incubation. This MAP peptide was used as an antigen to generate the
anti-HYAL1 antibody (43). We have previously characterized this
antibody for its specificity and ability to detect partially purified
HYAL1 protein, and HYAL1 protein present in complex biological fluids
(e.g. urine) and bladder cancer cell CM (43).
Immunohistochemistry--
Immunohistochemical localization of HA
and HAase was carried out in 57 prostate tissues (NAP,
n = 5; BPH; n = 7, Gleason 5, n = 7; Gleason 6, n = 11; Gleason 7, n = 16; and Gleason HA Staining--
One set of the slides was incubated with 2 µg/ml biotinylated HA-binding protein at room temperature for 35 min.
The biotinylated HA-binding protein used in this experiment was the
same as that used in the ELISA-like assay for HA level measurement. The
specificity of HA staining was established by incubating tissue
specimens with 100 milliunits/ml of Streptomyces HAase (ICN
Biomedicals, Costa Mesa, CA) at 37 °C for 3 h, prior to
incubation with biotinylated HA-binding protein. Following incubation
with HA-binding protein, the slides were washed in PBS and processed as
described below.
HYAL1 Staining--
The second set of slides was incubated with
3.7 µg/ml anti-HYAL1 antibody (IgG fraction) at 4 °C for 16 h
to localize HYAL1 in prostate tissues. The concentration of the
anti-HYAL1 antibody used in these experiments was that concentration at
which the preimmune IgG displayed no staining of tissues
(i.e. no nonspecific staining). To further determine the
specificity of tissue staining observed by anti-HYAL1 antibody, in some
cases, 50 µg/ml HYAL1 MAP peptide-(321-338) was included
during incubation of tissue specimens with anti-HYAL1 antibody.
Following incubation with anti-HYAL1 antibody, the slides were washed
in PBS and incubated with a linking solution containing biotinylated
goat anti-rabbit IgG (Dako LSAB kit, Dako Laboratories, Carpinteria,
CA) at room temperature for 30 min.
Chromogen Treatment--
Both sets of slides were then
sequentially incubated with streptavidin peroxidase at room temperature
for 30 min and 3,3'-diaminobenzidine (DAB) chromogen substrate solution
(Dako Laboratories). The slides were then counterstained with
hematoxylin and mounted. Three independent readers evaluated the
slides. Two independent readers (D. R. and V. B. L.)
graded the slides in a blinded fashion. These readings were confirmed
by study pathologist (M. N.). The slides were graded with respect
to the staining intensity as, 0 (no staining), +1 (weak), +2
(moderate), and +3 (high).
Gel-filtration Chromatography--
The size profiles of HA
species present in the Hepes and guanidine extracts of NAP, BPH, and
low-grade and high-grade CaP tissues (n = 2/category)
were determined. Six milligrams of total protein in each extract were
applied to a Sephadex G-50 column (1.5 × 120 cm) equilibrated
with PBS. The column was eluted at 7 ml/h, and 3.6-ml fractions were
collected. The column fractions were assayed using the ELISA-like assay
for HA, as described above. The protein profile of the column was
determined by measuring absorbance at 280 nm. The column was calibrated
using human umbilical cord HA, and HA fragments (F1 (10-15
disaccharide units), F2 (2-3 disaccharide units), and F3 (~2
disaccharide units)) (14).
Measurement of HA Levels in Prostate Tissues
The concentration of HA in prostate tissues has not been
determined previously. We used the HA ELISA-like assay to measure HA
levels (µg/mg) in the extracts of NAP, BPH, and CaP tissues. In tissues, HA may exist in the free form or as bound to the link molecules (50, 51). Therefore, we extracted HA present in prostate
tissues sequentially, in a Hepes buffer (Hepes extract: free HA) and in
a 6 M guanidine/HCl buffer (guanidine extract: HA bound to
link molecules). Fig. 1A shows
HA levels present in Hepes extract. As shown in Fig. 1A, HA
levels in low-grade (Gleason sum 5 and 6; 13.8 ± 2.5) and
high-grade (Gleason sum
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
low-grade > benign prostatic hyperplasia (BPH)/normal)
(33). In cell culture studies, we observed that, primary explant
cultures of CaP cells secrete elevated levels of HAase. The elevated
levels of HAase have now been demonstrated in metastatic breast tumors and in several carcinoma lines (34-40). However, the identity of the
type of HAase expressed in most cancer tissues and cells is still
unknown. In bladder cancer we observed that, elevated urinary HAase
levels indicate the presence of G2 and G3 bladder cancer (41, 42).
Recently, we purified the first tumor-derived HAase from the urine of
bladder cancer patients and showed its similarity to HYAL1 (43). We
also observed the expression of HYAL1 at the transcript and
protein levels, in invasive bladder cancer cell lines, which secrete
high levels of a HAase in their conditioned media. This HAase activity
has a pH optimum in the range 4.1-4.3 (43).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sample)
A405; the control represents buffer only.
8, n = 11)
and one each of locally extended and metastatic CaP lesions, using a
method described previously.2
The Gleason scoring is a standard method of grading CaP tissues. Paraffin-embedded blocks were cut into 3-µm thick sections and placed
on positively charged slides. The specimens were deparafinized, rehydrated, and subjected to antigen retrieval, as described
before.2 For each specimens two slides were prepared, one
for HA and the other for HYAL1 staining, as described below.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7; 15.7 ± 2.9) CaP tissues are
7-8- and 3-4-fold elevated as compared with the HA levels in NAP
(2.1 ± 0.6) and BPH (4.2 ± 1.3) tissue extracts,
respectively. The differences in the HA levels present in CaP (low- and
high-grade) tissue extracts and NAP/BPH tissue extracts are
statistically significant (p < 0.001). The HA levels are also similarly elevated in the extracts of seminal vesicles invaded
with CaP (15.4 ± 2.5) as compared with those in normal seminal
vesicle extracts (2.6 ± 0.7) (p < 0.001). The
differences observed in HA levels among Hepes extracts prepared from
low-grade and high-grade CaP and seminal vesicles invaded with CaP
tissues are not statistically significant (p > 0.05).
These results demonstrate that the levels of free HA are elevated in
CaP tissues and the increase is not associated with Gleason sum and
metastasis.
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Fig. 1.
Determination of HA levels in prostate tissue
extracts. The NAP, BPH, and low-grade and high-grade CaP tissues
(n = 5/category) were sequentially extracted in a Hepes
buffer (Hepes extract) and in a guanidine HCl buffer (guanidine
extract). The HA concentration in each extract was determined by the
ELISA-like assay for HA as described under "Experimental
Procedures." The results are expressed as mean ± S.E.
A, Hepes extract; B, guanidine extract.
The guanidine HCl treatment further extracted some HA from each category of tissues. However, HA bound to the link molecules accounted for ~20-25% of the total HA present in prostate tissues. As shown in Fig. 1B, HA levels present in guanidine extracts of low-grade (3.5 ± 0.9) and high-grade (4.2 ± 0.7) CaP tissues are also 7-8- and 3-4-fold elevated as compared with the HA levels present in NAP (0.53 ± 0.11) and BPH (1.2 ± 0.3) tissues. The differences in the amount of HA bound to link molecules in CaP tissues (low-/high-grade) and in NAP/BPH tissues is statistically significant (p < 0.001). However, the differences in HA levels present in guanidine extracts of low-grade and high-grade CaP tissues are not statistically significant (p > 0.05). These results demonstrate that the amount of HA bound to link molecules is increased in CaP tissues, however, the increase occurs independent of Gleason sum.
Measurement of HA Levels in Prostate Fibroblast Cultures
The cellular origins of tumor-associated HA are varied. Both tumor
cells and fibroblasts isolated from tumor tissues have been shown to
produce HA in vitro (9, 52). To determine which cell types
might be producing HA in CaP tissues, we used the HA ELISA-like assay
to measure HA levels in the CM of stromal fibroblast and epithelial
explant cultures as well as in the CM of prostate cell lines. As shown
in Fig. 2A, HA levels secreted
by stromal fibroblast cultures from low-grade (17.9 ± 2.5) and
high-grade (18.4 ± 3.2) CaP were 3-4-fold more than that
secreted by fibroblast cultures set up from NAP (3.2 ± 1.1) and
BPH (6.4 ± 1.7) specimens, respectively. The differences in HA
levels secreted by tumor-derived stromal fibroblast and NAP/BPH stromal
fibroblast cultures were statistically significant (p < 0.001). Furthermore, the increased production of HA by tumor-derived
fibroblast cultures was unrelated to the grade of CaP (Fig.
2A). The measurement of HA in prostate epithelial explant
cultures showed that the HA levels produced by these cultures are
comparable to those produced by stromal fibroblast cultures. The BPH
epithelial explant cultures secrete 2.9-fold more HA (5.3 ± 1.9)
than that produced by NAP explant cultures (1.8 ± 0.3). The CaP
explant cultures (low-grade, 10.4 ± 2.2; high-grade, 11.7 ± 2.9), secrete ~2-fold and ~6-fold more HA when compared that
produced by BPH and NAP epithelial explant cultures, respectively (Fig.
2B). Among the established CaP cell lines, PC3-ML cells
secrete significantly higher levels of HA (14.7 ± 1.4) than those
secreted by DU145 (3.8 ± 0.5) and LNCaP (4.2 ± 0.2) cells.
The reason for the observed differences among various CaP cell lines,
with respect to HA production, is unknown, however, both DU145 and
LNCaP cells secrete significantly more HAase in their CM than PC3-ML
cells (see below). These results demonstrate that both stromal
fibroblasts and tumor epithelial cells isolated from CaP tissues
secrete elevated HA levels and this increased secretion is independent
of the tumor grade.
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Identification of HAase Secreted by CaP Cells
RT-PCR Analysis--
We have previously shown that CaP cells
secrete a HAase of apparent Mr ~ 55,000 in
their CM (33). However, the type of HAase secreted by CaP cells has not
been identified. Since we have previously detected HYAL1-type HAase
expression in invasive bladder cancer cells, we studied the expression
of HYAL1 in CaP cells by RT-PCR analysis using a HYAL1-specific primer
pair that was used to amplify HYAL1 PCR product from bladder cancer
cells (43). As shown in Fig.
3A, an expected 370-bp PCR
amplification product is visible in RNA preparations from a Gleason 7 CaP epithelial explant culture, and three established CaP cell lines,
LNCaP, PC3-ML, and DU145. The 370-bp amplified PCR product from Gleason
7 CaP explant culture and DU145 cells was cloned and sequenced. The
sequence of this product matched 100% with the known HYAL1 sequence
(GenBankTM accession number HSU03056). As expected, no PCR
product is amplified in the negative control lane (Fig.
3A).
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To further confirm the expression of HYAL1 transcript in CaP cells, we performed RT-PCR analysis on total RNA from CaP cells, using a HYAL1-specific primer pair that should amplify the entire 1.3-kb coding region in HYAL1 cDNA. As shown in Fig. 3B, the expected 1.3-kb PCR product is amplified from DU145, LNCaP, PC3-ML, and Gleason 7 CaP explant culture RNA samples. The bladder cancer lines HT1376 and 253J-Lung that express HYAL1 were used as positive controls. The same 1.3-kb PCR product is amplified from the RNA of both of these bladder cancer cells. The specificity of the 1.3-kb PCR product is further confirmed from the negative PCR control (Fig. 3B). The cDNA cloning and sequencing of this 1.3-kb PCR product from both the CaP cells and bladder cancer cells confirmed that the sequence of this product matches 100% with the known HYAL1 sequence (data not shown).
Based on the HYAL1 cDNA sequences deposited in the GenBankTM, the 5'-untranslated region between nucleotides 104 and 588 in the HYAL1 transcript (GenBankTM accession number HSU03056) appears to be alternatively spliced, giving rise to two HYAL1 transcripts. To examine which one of the two transcripts are expressed in CaP cells, we performed RT-PCR analysis using a HYAL1-specific primer pair that lies outside the boundary of the alternatively spliced region. Using this primer pair we expected a 267-bp PCR amplification product from the spliced HYAL1 transcript and a 752-bp PCR amplification product from the unspliced HYAL1 transcript, respectively. As shown in Fig. 3C, both ~260- and ~750-bp PCR products are amplified from Gleason 7 CaP explant culture, LNCaP, PC3-ML, and DU145 RNAs, suggesting that CaP cells express both the spliced and unspliced HYAL1 transcripts. Furthermore, the bladder cancer lines, HT1376 and 253J-Lung that were used as a positive controls, show the expression of the same two PCR products that are amplified from the spliced (~260-bp product) and the unspliced HYAL1 transcripts (~750-bp product) (Fig. 3C, lane 5). The sequence of the shorter product revealed that it contains 267 bp and it lacks the region between nucleotides 104 and 588, that is present in the longer 752-bp PCR product (data not shown). As expected, the negative control shows no amplification (Fig. 3C). These results demonstrate that CaP cells express both the spliced and unspliced HYAL1 transcripts.
Detection of HYAL1-related Protein in CaP Cells--
To examine
whether an HYAL1-related protein is expressed in CaP cells, we
performed immunoblot analysis on the CM of primary prostate epithelial
explant cultures, using a rabbit anti-HYAL1 antibody, as described
previously (43). In this experiment, 2 primary cultures from each
category (i.e. NAP, BPH, etc.) were analyzed. Since identical
results were obtained in both experiments, results of one experiment
are shown in Fig. 4. As shown in Fig. 4,
CM from NAP, BPH, and Gleason 5 CaP explant cultures do not show any
cross-reactivity with the anti-HYAL1 antibody. However, the CM of
Gleason 7 CaP explant culture, and that of LNCaP, PC3-ML, and DU145
show the presence of a ~60-kDa protein that cross-reacts with the
anti-HYAL1 antibody. The densitometric scanning showed that the
expression of this HYAL1-related protein is 7.5 to >10-fold higher
than that in NAP, BPH, and Gleason 5 CaP explant cultures. The
detection of the 60-kDa HYAL-related protein can be blocked when
HYAL1-(321-338)-MAP peptide is included during anti-HYAL1 antibody
incubation (data not shown).
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To confirm that the detection of the 60-kDa HYAL1-related protein in CaP CM correlates with the presence of HAase activity, the same CM were analyzed by the HAase ELISA-like assay. As shown in Table I, NAP (1.7 ± 0.5), BPH (2.2 ± 0.8), and Gleason sum 5 explant (3.9 ± 0.4) cultures secrete very little HAase activity (milliunits/mg) in their CM. These results are consistent with our previous observation (33). Furthermore, as expected, the Gleason 7 prostate explant culture (36.9 ± 9.5), LNCaP (84.2 ± 13.8), PC3-ML (16.1 ± 3.4), and DU145 (206.5 ± 11.4) cells secrete high levels of HAase activity in their CM (Table I). One of the reasons why PC3-ML cells appear to secrete lower amounts of HAase activity than LNCaP and DU145 cells, despite expressing comparable amount of HYAL1 protein, may be that the high levels of HA secreted by PC3-ML cells in their CM interferes with the HAase ELISA-like assay (15).
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Using the HAase ELISA-like assay we also investigated the secretion of HAase activity in the culture CM of fibroblast cultures set up from Gleason 5 and Gleason 7 specimens. In these cultures no HAase activity was detected (see below).
Determination of pH Activity Profile of HAase
We have previously shown that the HAase activity expressed in bladder cancer cells, which is HYAL1-related, has a pH optimum range between 4.1 and 4.3. However, the HAase activity present in human serum and normal human urine, that is also attributed to HYAL1, has been shown to have a pH optimum at 3.7-3.8 (53-56). Furthermore, HYAL1 purified from human plasma and urine, as well as, recombinant HYAL1 have been shown to have a pH optimum at 3.7 (53, 54). In addition, fibroblast cultures (human dermal, and fetal, as well as, fibrosarcoma) were shown to secrete a HAase that is active at pH 3.7 but not at pH 4.5 (44). Among these observations, some were based on the "in-gel HAase activity" detected by substrate (HA)-gel assay performed at pH 3.7 (44, 56). To understand the differences in pH activity profiles of HYAL1-related HAase reported in different laboratories, we compared the pH activity profiles of HAase expressed in various sources using both the HAase ELSA-like assay and substrate (HA)-gel assay.
HAase ELISA-like Assay--
We measured the pH activity profile of
HAase expressed in DU145 CM, human serum, and normal human urine. We
also measured whether HAase activity can be detected in the CaP
fibroblast and HL fibroblast culture CM. The CaP and HL fibroblast CM
were either serum-free (SF-CM) or contained 10% fetal bovine serum
(regular growth medium; S-CM). The S-CM was included in the analysis,
since Stair-Nawy et al. (44) have shown that HAase activity
is detected in fibroblast S-CM at pH 3.7 but not at pH 4.5. As shown in
Fig. 5, the HAase activity secreted in
DU145 SF-CM has a pH optimum at 4.2 (range 4.0-4.3). In comparison to
the optimum activity at pH 4.2, the enzyme is 55% active at pH 3.7 and
50% active at pH 4.5 (Fig. 5). The HAase expressed in human serum,
shows optimum activity at pH 4.0 and 4.1. The enzyme is 85% active
both, at pH 3.7 and pH 4.5 (Fig. 5). The HAase activity present in
normal human urine has a pH optimum at pH 4.1 and 4.2. The enzyme is 72% active at pH 3.7 and 69% at pH 4.5 (Fig. 5). The results
demonstrate that the HAase activity expressed in CaP cells has a pH
optimum similar to that in human serum and urine, and it is at pH 4.1 to 4.2. The HAase in all of these three sources is similarly active pH
3.7 and 4.5.
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The SF-CM or S-CM, both either unconcentrated or concentrated, from CaP fibroblast and HL fibroblast cultures do not show any HAase activity at any pH between 2.5 and 7.0. The SF- and S-medium controls have no HAase activity at any pH tested (Fig. 5). These results demonstrate that the CaP fibroblast and HL fibroblast cultures do not secrete any HAase activity, as determined by the HAase ELISA-like assay.
Substrate (HA)-gel Assay--
We next analyzed HAase activity in
various samples using a substrate (HA)-gel assay (48). The in-gel HAase
activity was assayed in HAase assay buffer adjusted to pH 3.0, 3.7, 4.2, and 4.5. As shown in Fig.
6A, at pH 3.0 "clear
bands" (that would appear to arise due to the presence of a HAase
activity that is digesting the HA in the gel) are present in
electrophoresed human serum, urine, DU145 SF-CM, CaP fibroblast S-CM,
HL fibroblast S-CM, S-medium control, and even pure ELISA-grade BSA.
The clear bands in lanes containing CaP fibroblast S-CM, HL fibroblast
S-CM, S-medium control, and BSA samples appear to have faster
electrophoretic mobility. However, in the unconcentrated samples, the
mobility of the clear bands is closer to ~60 kDa, the approximate
molecular weight of BSA (data not shown). The presence of such
tremendous HAase activity (as judged by the intensity of clear bands)
at pH 3.0 in various samples is not consistent with the HAase
ELISA-like assay results presented in Fig. 5. Furthermore, BSA is not
known to have any HAase activity and the HAase assay buffer used in the
ELISA-like assay contains BSA as a carrier protein. In addition, no
HAase activity is detected in S-medium control at any pH tested between
2.5 and 7.0 (Fig. 5). At pH 3.0, no HAase activity is detected in SF-CM
of CaP fibroblast and HL fibroblast cultures, even after 10-fold
concentration (Fig. 6A).
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At pH 3.7, the intensity of the clear band is higher in human serum (3.5-fold) and DU145 SF-CM samples. The urine sample not only shows an increase in the intensity of the clear band (3.8-fold), but a new band appears. The presence of two HAase species in urine has been reported previously (41, 54). The molecular weight of the HAase species in DU145 SF-CM is between the two urinary HAase species (Fig. 6B). Contrary to an increase in the intensity of clear bands observed in human serum, urine, and DU145 SF-CM, which is consistent with the data on the HAase ELISA-like assay, at pH 3.7 the intensity of the clear band is decreased by ~50% in CaP fibroblast S-CM, HL fibroblast S-CM, S-medium control, and BSA sample lanes, when compared with that at pH 3.0 (Fig. 6B). At pH 4.2, consistent with the HAase ELISA-like assay results, the intensity of "clear HAase activity bands" in human serum (1.7-fold), urine (1.8-fold), and DU145 SF-CM (1.4-fold) samples, is increased further when compared with that at pH 3.7 (Fig. 6C). However, the clear band detected at pH 3.0 and 3.7, in CaP fibroblast S-CM, HL fibroblast S-CM, S-medium, and BSA samples, almost all disappears at pH 4.2 (Fig. 6C). It should be noted that no HAase activity is detected at pH 4.2 in CaP fibroblast and HL fibroblast SF-CM (Fig. 6C). At pH 4.5, the HAase activity in human serum, urine, and DU145 is decreased by ~50% from that at pH 4.2, nonetheless, it is still significant (Fig. 6D). No clear bands are detected in CaP fibroblast S-CM, HL fibroblast S-CM, S-medium, and BSA samples at pH 4.5 (Fig. 6D).
These results demonstrate the following. 1) The HAase activity present in human serum, urine, and DU145 SF-CM has a similar pH optimum (4.1-4.2), when assayed using both the HAase ELISA-like assay and substrate (HA)-gel assay. 2) CaP fibroblast and HL fibroblast cultures do not secrete any HAase activity in SF-CM, as judged by the ELISA-like assay and the substrate (HA)-gel assay. The clear band that is present in S-CM from these cells, at pH 3.0 and 3.7, may be an artifact since the S-medium control and BSA alone also show the presence of the same clear band with comparable intensity. This clear band disappears, whereas, the intensity of the true HAase activity bands in serum, urine, and DU145 SF-CM samples increases at pH 4.2. 3) CaP fibroblast and HL fibroblast do not secrete any HAase activity that is active between pH 2.5 and 7.0.
Immunohistochemical Localization of HA and HAase in Prostate Tissues
To investigate the distribution of elevated HA and HAase (i.e. HYAL1) in CaP tissues, we utilized a biotinylated HA-binding protein and the anti-HYAL1 antibody to localize HA and HAase in prostate tissues. The biotinylated HA-binding protein has been utilized previously to localize HA in tumor tissues (10, 12).
HA Localization--
Deparaffinized archival NAP
(n = 5), BPH (n = 7), and CaP
(n = 45) tissues were sequentially incubated with the
biotinylated HA-binding protein, streptavidin peroxidase, and DAB
substrate to localize HA. As shown in Fig.
7A, little HA staining is
observed in the NAP specimen. Out of the five NAP tissues tested, 4 showed no HA staining (0) and 1 specimen showed +1 staining intensity (Table II). The BPH specimen, shown in
Fig. 7B, stains with +1 intensity and the staining is focal.
Furthermore, all of the staining is present in the stromal component
and none is observed in epithelial cells. Out of the 7 BPH specimens
that were stained, +1 and +2 staining intensity was seen in 5 and 2 specimens, respectively. As shown in Fig. 7, panels C-F, HA
staining in CaP specimens is significantly higher (intensity +2 to +3)
and diffuse, regardless of whether the specimen is from low-grade
(i.e. Gleason sums 5 and 6) or high-grade (i.e.
Gleason sums 7, 8 and 9) CaP. All of the HA staining in CaP specimens
is in the stroma and none in the tumor epithelial cells. Furthermore,
the stroma surrounding normal prostate glands present in the CaP
specimens shows only 0 to +1 staining (data not shown). Among the seven
Gleason sum 5 specimens that were stained, +2 and +3 staining intensity
was seen in 4 and 2 specimens, respectively, and no staining
(i.e. 0 intensity) was seen in 1 specimen (Table II).
Similarly, among Gleason 6 specimens (n = 11) that were
stained, 1, 2, 6, and 2 specimens showed 0, +1, +2, and +3 intensity,
respectively. All of the Gleason sum 7 (n = 16) and 8
(n = 11) specimens showed +2 and +3 HA staining
intensity (Table II). These results demonstrate that in prostate
tissues HA is localized in the stromal compartment and is significantly
elevated in the tumor-associated stroma.
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HAase Localization--
The same paraffin-embedded archival
prostate specimens that were used to localize HA were also used to
localize HYAL1-type HAase. As shown in Fig.
8, no staining for HYAL1 is observed in NAP, BPH, and Gleason sum 5 tissues (Fig. 8, panels A-C).
Out of the five NAP specimens that were stained, none showed HYAL1 staining (Table III). Among the 7 BPH
specimens that were stained, 4 showed no staining, whereas, 3 specimens
showed +1 staining intensity. Out of the 7 Gleason 5 specimens that
were stained, 6 showed no staining and 1 specimen showed +2 staining
intensity, respectively (Table III). As shown in Fig. 8, panel
D, the Gleason sum 6 specimen shows +1 intensity for HYAL1
staining and the staining is exclusively localized in tumor epithelial
cells. Among 11 Gleason sum 6 tissues that were stained, 4 specimens
showed +1 and 7 showed +2 intensity for HYAL1 staining (Table III). In
all of these Gleason 6 specimens, the normal prostate glands showed no
staining for HYAL1 (data not shown). The staining intensity for HYAL1
further increased (+2 to +3) in CaP specimens with Gleason sum 7 and
8. As shown in Fig. 8E, the tumor epithelial cells in
Gleason 7 specimen show +3 staining intensity for HYAL1. Among the 16 Gleason 7 CaP specimens examined, 2, 1, 10, and 3 specimens showed 0, +1, +2, and +3 staining intensity, respectively. Fig. 8F
shows HYAL1 staining in a Gleason 9 CaP specimen. All of the tumor
cells in this specimen are stained with +3 intensity and this is
further confirmed in the magnified view of the specimen (Fig. 8,
panel F, inset). Fig. 8F also shows perineural
invasion, and once again, the tumor cells surrounding the neuron are
stained with +3 intensity. As shown in Table III, all of the Gleason
8 specimens are stained with either +2 or +3 intensity. These results
demonstrate that HYAL1-type HAase is expressed exclusively in tumor
epithelial cells and the expression increases with higher grades of
CaP.
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Localization of HA and HYAL1 in Locally Extended and Metastatic CaP
Lesions--
To determine whether the intensity and the patterns of HA
and HYAL1 staining are the same in a primary CaP lesion and in the clinically progressed lesions, we localized both molecules in a Gleason
9 specimen, in seminal vesicles invaded with CaP (locally extended
lesion), and in a lymph node that is positive for CaP (i.e.
metastatic lesion). These three specimens were obtained from the same
patient. As shown in Fig. 9, +3 intensity
is seen for HA (panel A) and HYAL1 (panel B) in
the primary CaP lesion. HA is localized in stromal components, whereas,
HYAL1 is localized in tumor epithelial cells. In seminal vesicles
invaded with CaP, the stroma surrounding the seminal vesicle that is
invaded by CaP shows +3 staining intensity for HA (Fig. 9, panel
C). Interestingly, tumor cells in this locally extended CaP
lesion, as well as, the seminal vesicle itself stain with +3 intensity
for HYAL1 (Fig. 9, panel D). However, any clinical
significance of this observation cannot be evaluated at the present
time since the staining intensity of HYAL1 in normal seminal vesicles
is unknown. We are currently working on obtaining some normal seminal
vesicle tissues to clarify this issue. In the lymph node, the same
pattern of HA and HYAL1 staining is observed. The stroma in the lymph
node lesion stains with +3 intensity for HA and the tumor cells stain
with +3 intensity for HYAL1.
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These results demonstrate that both the divergent HA and HYAL1 staining pattern and the intensity of staining seen in primary CaP lesion are duplicated in the locally extended and metastatic CaP lesions.
Determination of Tissue HA Profiles
The stromal-epithelial pattern of HA and HYAL1 expression and
increased concentration of HYAL1 in higher Gleason sum CaP tissues, raises the question whether the tumor cell-derived HYAL1 might degrade
stroma-associated HA. If this were the case, angiogenic HA fragments
might be present in CaP tissues. To address this issue, we examined the
profiles of HA species present in the Hepes (free HA) and guanidine (HA
bound to link molecules) extracts of NAP, BPH, and low-grade and
high-grade CaP tissues, using gel-filtration chromatography. Two
tissues were tested per category in separate experiments. As shown in
Fig. 10A, which represents
the profiles of HA species present in the Hepes extracts, the NAP and
BPH tissues contain one major HA species that corresponds to the high
molecular mass HA. A minor intermediate size HA species (peak II) is
observed in the BPH sample. On the contrary, HA species of different
sizes are present in CaP tissues, which represent peaks I to IV.
Although, the high molecular mass HA appears to be a major species, a
significant amount of HA species present in low-grade CaP tissue sample
elute as peaks II and III. The high-grade CaP sample predominantly
contains high molecular mass (peak I) and intermediate size (peak II)
HA species. However, ~25% of the HA species elute at peak III, which represents the angiogenic HA fragments. The column profile of total
protein present in the Hepes extracts of various samples was similar
(Fig. 10A and data not shown).
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The profiles of HA species present in guanidine extracts of various
prostate tissue samples is very similar, with the majority of HA
eluting as high molecular mass HA (Fig. 10B). A small amount of intermediate size HA (peak II) is also present in high-grade CaP
sample (Fig. 10B). These results demonstrate that in CaP
tissues, the size distribution of HA existing in the free and bound
forms is different. Predominantly, the free HA is degraded into smaller species of different sizes, presumably due to the action of HAase. In
high-grade CaP tissues, tumor-associated free HA may be degraded by
HYAL1 into angiogenic HA fragments.
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DISCUSSION |
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In this study we have been able to demonstrate the expression of both HA and HAase in CaP. We also identified which tumor components (i.e. stroma and tumor cells) contribute to the elevated levels of these molecules in CaP. Tumor-associated HA is known to enhance tumor metastasis, and increased HA levels are observed in several human cancers (9-14). Our results presented here show that HA levels are significantly elevated (4-7-fold) in CaP tissues and the increase does not correlate with CaP aggressiveness or progression (Fig. 1). This finding is similar to what we had previously observed in bladder cancer. We observed that the tissue and urinary HA levels are elevated (2.5-6.5- fold) in bladder cancer patients, regardless of the tumor grade (14, 15). The data presented here show that both stromal fibroblasts and tumor cells produce elevated levels of HA under in vitro culture conditions (Fig. 2). It has been previously shown that fibroblasts and most kinds of tumor cells produce at least some HA in vitro (9).
Contrary to the cell culture studies, the histochemical analysis shows that in prostate tissues HA is localized almost exclusively in the stroma and its concentration is increased considerably in the tumor-associated stroma (Fig. 7). It is noteworthy that the majority of this HA in prostate tissues exists in the free form (Fig. 1). It is possible that in vivo, CaP cells induce surrounding stromal cells, through cellular signaling ("inductive interaction"), to synthesize and secrete increased amounts of HA. This hypothesis is supported by our observation that the tumor-associated stroma, and not the stroma surrounding normal prostate glands, shows increased HA staining (Fig. 7).3 In addition, our studies show that in a locally extended CaP lesion (i.e. seminal vesicle invaded with CaP) or metastatic lesion (i.e. lymph node positive for CaP), elevated HA staining is observed in the stroma. Normally these tissues have little HA in their stroma.3 The concept of increased HA production in tumor tissues due to tumor-stromal interaction has been suggested previously (52).
In colon and gastric cancers, in addition to the stromal components, tumor cells also stain for HA (12, 57, 58). The HA staining in tumor cells correlates with tumor invasiveness and appears to be an independent predictor for survival (12, 57, 58). In our study, by histochemical analysis, we observed distinct HA staining in tumor cells in a few CaP specimens. The functional significance of this finding is not yet known.4
Unlike the HA expression, secretion of the HYAL1-type HAase correlates with CaP progression (Figs. 4 and 8 and Table I). The molecular biological, biochemical, and immunohistochemical studies presented here reveal the expression of HYAL1-type HAase in CaP cells. Some of the earlier findings have demonstrated that HAase secretion correlates with the invasive/metastatic potential of tumor epithelial cells (40-42). The fact that HYAL1-type HAase is also expressed in invasive bladder tumor cells and in the urine of high-grade bladder cancer patients (43) suggests that HYAL1-type HAase may be expressed by invasive tumors of various tissue origins.
The pH activity profile and substrate (HA)-gel assay studies demonstrate that the HAase activity secreted by CaP cells is similar to the HAase activity present in human serum and urine (Figs. 5 and 6). The pH optimum of the HAase activity from these sources appears to be at ~4.2 (range 4.0-4.3). The enzyme is also 50-85% active at pH 4.5. We had previously reported that the pH optimum of the HAase activity secreted by CaP cells is 4.6 (33). However, in that study we did not examine the pH profile in the 0.1 pH unit intervals and had not compared the HAase activity between different sources in a side-by-side fashion. Nonetheless, it is important to note that the HAase activity secreted in human urine and serum, that is attributed to HYAL1, is active at pH 4.5 and to the same degree as seen at pH 3.7. These results are different from those reported previously (53-56). The HYAL1-related HAase activity in human serum and urine has been to shown to have a pH optimum at 3.7 (53-56). However, in these studies the pH activity profile using the ELISA-like assay was measured at 0.5 pH intervals and some of these results are also based on substrate (HA)-gel assay performed at pH 3.7 (53, 54, 56).
The results presented in Fig. 6 demonstrate that the substrate (HA)-gel assay may give rise to an artifact that can be mistaken for "true HAase" activity, if the assay is performed at pH < 4.0 and the sample contains other proteins such as BSA (or human serum albumin).3 Such artifacts may have been seen previously, when the HAase activity was assayed in serum-containing media (59, 60). Given the observation that substrate (HA)-gel assay gives rise to artifacts, and yet, it is a good assay to determine the molecular weight of the active HAase species, it may be important to: 1) assay the HAase activity by both the ELISA-like assay and substrate (HA)-gel assay; 2) perform the substrate (HA)-gel assay at pH > 4.0; 3) assay the HAase activity in serum-free culture CM. However, it is noteworthy that some commercially available ITS solutions (e.g. Sigma) contain BSA as a carrier protein, and therefore, it is important to include an appropriate medium control while assaying the samples.
The HAase activity expressed in CaP cells is distinctly different from that of the known membrane-bound and soluble forms of PH20 (61, 66). The CaP HAase is inactive at pH > 5.0. This is noteworthy, since expression of PH20 transcripts has been detected in CaP tissues by RT-PCR analysis (63). However, except for LNCaP cells, we did not detect PH20 mRNA expression in other CaP cells.4 Our results on the pH activity profile are consistent with the results of Podyma et al. (37) who tested optimum pH for HAase activity secreted by human lung carcinoma H460 cells. Using buffers differing in 0.5 pH intervals, they demonstrated that the HAase expressed by H460 cells has the same pH optimum as human serum, both at pH 4.0.
Our results demonstrate that CaP fibroblasts do not secrete any HAase
that has activity between pH 2.5 and 7.0. Similar results are observed
for HL fibroblasts. The substrate (HA)-gel assay results demonstrate
that this technique might generate artifacts that may be mistaken for
the true HAase activity at pH 3.7 and lower. However, at pH 4.2 this
artifact disappears, while the true HAase remains active. The artifact
may become problematic, if one is analyzing HAase activity in S-CM or
biological fluids that contain proteins such as the serum albumin (Fig.
6). It is possible that certain serum proteins may precipitate at
acidic pH (4.0).
At present, we do not know whether HYAL1-type HAase is the only HAase expressed in CaP cells. Together with prostate cancer, the expression of PH20 mRNA has been demonstrated in melanoma, glioblastoma, glioma, and colonic carcinoma cell lines, as well as in invasive breast carcinomas (34, 40, 63). However, the pH optimum (3.8 to 4.0) of the HAase activity present in the extracts of breast primary tumors, and regional metastases is similar to the pH optimum of HAase present in human serum, and CaP SF-CM, observed in this study and it is different from that of the known pH20 isoforms (35, 61, 62). Therefore, it may be necessary to demonstrate the expression of PH20 protein, PH20-related HAase activity along with PH20 mRNA expression in various tumors. In CaP cells, the detection of HYAL1-related protein by anti-HYAL1 peptide IgG is HYAL1 specific. This is because, the HYAL1 peptide sequence against which this antibody was generated, shares only 4, 5, and 3 amino acids with PH20, HYAL2, and HYAL3 sequences, respectively. Furthermore, the shared amino acids between the HYAL1 sequence and other HAases do not occur in a continuous order.
Due to the widespread acceptance of prostate-specific antigen as a biochemical screening marker, it is perceived that there is limited need for another tumor marker for CaP. However, the majority of men with clinically localized CaP have very similar prostate-specific antigen values (i.e. 4-10 ng/ml) and biopsy Gleason score (i.e. between 5 and 7) (1, 2, 6). Thus based upon these two parameters alone, it is difficult to identify which patients have aggressive disease. Certain molecules that regulate CaP growth and metastasis have shown promise as prognostic markers (64-70). Our data presented here show that both HA and HAase (i.e. HYAL1) associate with the biology of CaP and show a distinct stromal epithelial pattern of expression in CaP tissues. It is possible that either one or both of these molecules function as prognostic indicators for CaP.
The physiological consequence of elevated HA and HAase levels in tumors
may be stimulation of angiogenesis, due to the generation of small HA
fragments. Detection of angiogenic HA fragments in CaP tissues supports
this notion. We have previously shown that angiogenic HA fragments bind
to RHAMM-type HA receptor and subsequently induce the MAP kinase
pathway which stimulates endothelial cell proliferation (23). Given the
facts that HYAL1 is ~80% active at pH 4.5, and the interstitial
environment in malignant tumors is acidic (49), the tumor-associated
HA-HYAL1 system may aid in angiogenesis. The localization of HA and
HAase in CaP tissues, documentation of the divergent pattern of their
production, and identification of at least one of the prostate tumor
cell-derived HAase should help the determination of the clinical
relevance of these two markers in CaP.
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ACKNOWLEDGEMENTS |
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We thank Dr. W. Wu for helpful suggestions during IHC experiments. We thank the University of Miami's Transplant Organ Retrieval team (L. Olson, J. Ferreira, R. Cartaya, W. Bonilla, M. Ariza, R. Santana, and J. Contillo) for providing normal prostate tissues. We appreciate Soum Lokeshwar's assistance during this work. We gratefully acknowledge the invaluable assistance of Douglas Roach and Pamela Roza with illustrations.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R29 CA 72821 (to V. B. L.), R01 CA 71618 (to J. D. M.), and RO1 CA 61038 (to B. L. L.), and the Sylvester Comprehensive Cancer Center, and L. Austin Weeks Endowment funds, University of Miami.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: Dept. of Urology (M-800), University of Miami School of Medicine, P. O. Box 016960, Miami, FL 33101. Tel.: 305-243-6321; Fax: 305-243-6893; E-mail: vlokeshw@med.miami.edu.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M008432200
2 Hautmann, S. H., Lokeshwar, V. B., Schroeder, G. L., Civantos, F., Duncan, R. C., Gnann, R., Friedrich, M. G., and Soloway, M. S. (2001) J. Urol., in press.
3 V. B. Lokeshwar and D. Rubinowicz, unpublished results.
4 V. B. Lokeshwar and D. Rubinowicz, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CaP, prostate cancer; HA, hyaluronic acid; HAase, hyaluronidase; DAB, 3,3'-diaminobenzidine; CM, conditioned medium; SF-CM, serum-free conditioned medium; S-CM, serum containing conditioned medium; BSA, bovine serum albumin; HL fibroblast, human fetal lung fibroblast; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; bp, base pair(s); kb, kilobase pair(s); NAP, normal prostate; BPH, benign prostatic hyperplasia.
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