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Int. J. Cancer (Pred. Oncol.): 74, 7–14 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
METANESTIN, A GLYCOPROTEIN WITH METASTASIS-ASSOCIATED EXPRESSION
IN TRANSITIONAL CELL CARCINOMA OF THE URINARY BLADDER
Masakazu TAKEMOTO,1,2,3* Tsutomu SHIRAHAMA,1 Teruo MIYAUCHI,2,4 Tetsusi MATSUSAKO,1 Norio KANEDA,3 Hisako MURAMATSU,2,3
Masayuki OZAWA,2 Yoshitada OHI1 and Takashi MURAMATSU2,3
1Department of Urology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan
2Department of Biochemistry, Faculty of Medicine, Kagoshima University, Kagoshima, Japan
3Department of Biochemistry, Nagoya University School of Medicine, Nagoya, Japan
4Japan Immunoresearch Laboratories, Takasaki, Gumma, Japan
A 60-kDa glycoprotein named metanestin was identified by
molecular cloning. The glycoprotein had a twice-repeated
motif of Pro-Gly-Pro-Gly and carried metastasis-associated
carbohydrate epitopes. The antibody to the protein portion
of metanestin strongly reacted with lymph-node metastasis
of transitional-cell carcinoma of the urinary bladder, but the
reactivity to the primary tumor was generally weaker and the
normal urothelium was scarcely reactive. Thus both metanestin and the carbohydrate on it showed metastasis-associated
expression. In addition, we identified a 100-kDa glycoprotein
with a 4-times-repeated motif of Pro-Ala-Pro-Ala. The antibody to the 100-kDa glycoprotein showed reactivity similar to
that to metanestin. Int. J. Cancer 74:7–14.
r 1997 Wiley-Liss, Inc.
The most important issue in management of patients with
urinary-bladder carcinoma is lymph-node metastasis. When invasive urinary-bladder carcinoma does not penetrate the muscle layer
(pT3a), the 5-year survival rate of patients is 50 to 60%. In cases of
more advanced carcinoma (pT3b, T4), this figure decreases markedly, to 15 to 20%, demonstrating the importance of lymph-node
metastasis (Wishnow et al., 1987; Pagano et al., 1991; Kakehi et
al., 1992.). Therefore, clarification of the mechanism of lymphnode metastasis is important for developing effective methods of
treatment of urinary-bladder carcinoma.
Various reports have described expression of carbohydrate
antigens, especially blood group-related antigens, in malignant
tumors. Some of the carbohydrate antigens are known to play
important roles in metastasis of carcinomas (Hoff et al., 1989;
Muramatsu, 1993.). In blood-borne metastases of colon carcinomas, sialyl Lex antigen and sialyl Lea antigen (blood-group-related
carbohydrate antigens) are considered to function as ligands of
E-selectin, and to enhance tumor metastasis (Hoff et al., 1989;
Takada et al., 1993). Physiologically, these adhesion molecules is
known to play an initial role in adhesion of leukocytes to
endothelial cells of blood vessels (Lasky, 1992).
We have studied the expression of blood-group-related carbohydrate markers, namely Lex, sialyl Lex and binding sites for Lotus
tetragonolobus agglutinin (LTA) in urinary-bladder carcinomas
and have reported that strong expression of these markers correlated with the occurrence of lymph-node metastasis and, consequently, poorer prognosis (Matsusako et al., 1991; Shirahama et
al., 1992a,b, 1993). Furthermore, we prepared monoclonal antibodies (MAbs) to LTA-binding glycoproteins isolated from urinarybladder carcinoma cells, and one of these antibodies, MM4, reacted
with an antigen (MM4 antigen) whose expression in primary
tumors correlated with the presence of lymph-node metastasis.
MM4 antigen was also strongly expressed in metastatic tumor nests
in lymph nodes (Matsusako et al., 1992). The aim of the present
investigation was to determine the antigenic epitope and to
characterize the molecule carrying the epitope.
MATERIAL AND METHODS
Cells
The BOY transitional-cell-carcinoma cell line, established in our
laboratory (Kayajima et al., 1989), was cultured in Dulbecco’s
modified Eagle’s minimum essential medium containing 10% FCS
(ICN, Costa Mesa, CA), 50 IU/ml ampicillin/50 IU/ml streptomycin (Flow, Irvine, UK), and amino acids (1/100 V/V) (Flow) and
fungison (GIBCO, Grand Island, NY) under 12% CO2 at 37°C.
After washing twice with Dulbecco’s PBS without Ca21, Mg21
[PBS(—)], cells were harvested mechanically and stored at 270°C
until use.
Construction of cDNA library
Total RNA was extracted from tumor tissues by the guanidiumisothiocyanate/cesium-chloride method (Maniatis et al., 1982).
Poly (A)1 RNA was isolated on an oligo (dT) cellulose column.
First-strand cDNA was synthesized using oligo (dT) and random
primers. Second-strand cDNA was synthesized using DNA polymerase, and the double-stranded cDNA was blunt-ended. After the
endogenous EcoRI sites were protected by EcoRI methylase,
EcoRI adapters were ligated to the synthesized cDNA. After
digestion with EcoRI, cDNA was separated from adapters by
chromatography on Sepharose 4B (Pharmacia, Milwaukee, WI).
The cDNA was ligated to lgt 11 arms and packaged.
Antibodies and screening of the cDNA library
The MAb MM4, established and characterized in our laboratory
(Matsusako et al., 1992), was used for screening. The library was
screened by the procedure of Young and Davis as modified by Schuh et
al. (1986). Phage clones with extended cDNA sequence were screened
by the plaque hybridization method (Maniatis et al., 1982).
Sub-cloning and DNA sequence analysis
Phage DNA was prepared by the plate lysate method (Maniatis et
al., 1982), and the cDNA inserts were cloned into Bluescript SK
(—). DNA sequencing was performed by the dideoxy-chaintermination method (Sanger et al., 1977). Homology was searched
using the data bases of EMBL-GDB (European Molecular Biology
Laboratory/Gene Data Bank).
Preparation of BNN 103 lysogen
The fusion protein produced by the recombinant lgt 11 clones, ST-1
and ST-2, was prepared as described (Schuh et al., 1986). BNN 103
lysogen was cultured in LB (Luria-Bertani) medium at 30°C until
optimal density was reached; phage production was induced at
42°C for 20 min and the cells were cultured at 37°C for 2 hr.
Preparation of antibody against a synthetic peptide
A synthetic peptide, GPAPASAGGDHSPAPAS (amino acids
51–67 in Fig. 1b), was prepared and conjugated to branched
*Correspondence to: Masakazu Takemoto, Department of Biochemistry,
Nagoya University School of Medicine, 65 Tsurumai-cho Showa-ku,
Nagoya 466, Japan. Fax: 81-52-744-2065.
Received 26 April 1996; Revised 23 July 1996
8
TAKEMOTO ET AL.
FIGURE 1 – Nucleotide and deduced protein sequence. (a) Partial sequence of cDNA of metanestin (ST-1-derived protein). The sequence of the
initially isolated clone (ST-1) is shown underlined and the repeating motif by dashed lines. (b) Partial sequence of cDNA of a 100-kDa protein
(ST-2-derived protein). The sequence of initially isolated clone (ST-2) is underlined. Double underline shows Poly A signal. The characteristic
repeating motif is shown by dashed lines.
A METASTASIS-ASSOCIATED GLYCOPROTEIN
polylysine oligomer (Peptide Institute, Suita, Osaka, Japan). New
Zealand White rabbits were immunized with 1 mg of the conjugated
peptide mixed with Freund’s complete adjuvant. Three booster immunizations were performed at 2-week intervals, and anti-serum was
collected 10 days after the last immunization. Five milligrams of the
synthetic peptide were coupled to 1 ml of CNBr-activated Sepharose
(Pharmacia) according to the manufacturer’s protocol. The antiserum (2
ml) was dialysed against 20 mM Tris-HCl buffer (pH 7.0) and applied to
an affinity column with the peptide-Sepharose mentioned above, which
was equilibrated with 20 mM Tris-HCl buffer (pH 7.0). After washing
with 10 to 20 volumes of Tris-HCl buffer, antibody was eluted with 0.1
M glycine-HCl buffer (pH 2.5) and immediately neutralized with 1.5 M
Tris-HCl buffer (pH 8.8). The antibody, anti-PAPA antibody, was mixed
with BSA (final concentration, 1 mg/ml) and stored at 280°C until use.
9
Glutathione-S-transferase(GST) fusion protein was prepared for
purification of antibody against MBP fusion protein. The cDNA
(nucleotides 611-1255 in Fig. 1a) was sub-cloned into a GST-fusionprotein vector pGEX-5X-3 (Pharmacia). E. coli JM 109 cells were
Preparation of fusion protein and antibody
The cDNA of ST-1 (nucleotides 462-813 in Fig. 1a) was
sub-cloned into a maltose-binding-protein(MBP) fusion protein
vector pMAL-c2 (Bio-Labs, Beverly, MA). E. coli TB-1 cells were
transformed with recombinant plasmid. The cells were cultured and
induced to produce the fusion protein, which was collected by
centrifugation. The pellets were suspended in column buffer (20
mM Tris-HCl, pH 7.4, 0.2 M NaCl, 1 mM EDTA, 10 mM
b-mercaptoethanol, 1 mM NaN3 ), and frozen and thawed. The
suspended cells from 1 liter of culture were disrupted by sonication
and centrifuged at 14,000 g for 20 min. The supernatant was
applied to an amylose-resin column (30 ml) (Bio-Labs), which was
then washed with 10 volumes of column buffer. The fusion protein
was eluted with 10 mM maltose in the column buffer. One hundred
micrograms of this protein was mixed with Freund’s complete
adjuvant and used for immunization.
FIGURE 3 – Northern-blot analysis of mRNA corresponding to ST-1
and ST-2. (a) ST-1 mRNA; 5 µg of Poly A1 RNA from BOY cells were
analyzed. (b) ST-2 mRNA; 20 µg of total RNA from BOY cells were
analyzed.
FIGURE 2 – Western-blot analysis to detect MM4 antigenic epitope
in fusion proteins. Bacterial pellet was lysed in SDS-PAGE sample
buffer and applied to each lane. SDS-PAGE was performed using 8%
polyacrylamide gels. Lane 1, extract of BNN 103 lysogen which had
b-galactosidase gene fused with the initially isolated insert of ST-2
(underlined in Fig. 2b) and expressed the 118-kDa fusion protein. Lane
2, extract of BNN 103 lysogen which had b-galactosidase gene fused
with the initially isolated insert of ST-1 (underlined in Fig. 2a) and
expressed the 128-kDa fusion protein. Lane 3, extract of BNN 93
expressing 116-kDa b-galactosidase. (a) Staining with Coomassie
brilliant blue; (b) staining with MM4 MAb.
FIGURE 4 – Western-blot analysis to detect antigenic proteins in
extracts from BOY cells; 400 µg of protein was applied to each well of
8% polyacrylamide gels. After SDS-PAGE, the gel was stained by
specific antibodies. (a) Anti-metanestin (ST-1 protein) revealed a
60-kDa band. (b) anti-PAPA antibody reacted with a 100-kDa band.
TAKEMOTO ET AL.
10
FIGURE 5 – Expression of metanestin in clinical samples as revealed by immunohistochemical analysis using anti-metanestin antibody. (a)
Normal epithelium (bar, 100 µm); (b) primary tumor (bar, 50 µm); (c) metastasized lymph node at low magnification (bar, 200 µm). Almost all
metastatic tumor cells were stained. (d) Metastasized lymph node at high magnification (bar, 50 µm). All samples were taken from one patient.
Concentration of the antibody was 7 µg/ml.
TABLE I – STAINING OF PRIMARY TUMOR AND METASTATIC LYMPH NODES
BY ANTI-METANESTIN AND ANTI-100-kDa-PROTEIN ANTIBODIES
Number of cases
Strong staining1
Anti-metanestin
Primary tumor
Lymph-node metastasis
Anti-100-kDa protein
Primary tumor
Lymph-node metastasis
No or weak staining2
9
6
15
1
12
8
6
2
1Strong
staining, more than 20% of tumor stained.–2Weak or no
staining, less than 20% of tumor stained.
transformed with the recombinant plasmid. The cells were cultured
and induced to produce fusion protein, which was then collected by
centrifugation. The pellets from 1 liter of culture were suspended in
PBS (—) and disrupted using sonicator. After centrifugation at
14,000 g for 20 min, the supernatants were applied to glutathioneSepharose column (10 ml) (Pharmacia) equilibrated with 10 bed
volumes of PBS (—). The column was drained and washed with 30
bed volumes of PBS (—) and eluted with elution buffer (10 mM
glutathione, 50 mM Tris-HCl, pH 8.0). The protein solution was
dialysed against coupling buffer (0.5 M NaCl, 0.1 M NaHCO3, pH
8.3) and used for coupling to CNBr-activated Sepharose. The
column was used to purify the antibody against the MBP fusion
protein, as described for purification of the antibody to synthetic
peptide.
Western-blot analysis
BOY cells were lysed in Laemmli’s sample buffer (Laemmli,
1970). Proteins were separated by SDS-PAGE on 8% gels and were
electrophoretically transferred to nitrocellulose membranes. The
membrane was reacted with mouse anti-MM4 MAb or affinitypurified rabbit antibodies, followed by HRP-labeled goat antimouse or rabbit IgG as described (Matsusako et al., 1992). Color
was developed using 0.05% 4-chloro-naphthol in 0.1 M Tris-HCl
buffer, pH 7.6, with 0.05% H2O2.
Northern-blot analysis
Total cellular RNA was extracted by the guanidium-isothiocyanate/cesium-chloride method (Maniatis et al., 1982). RNA was
denatured with glyoxal and dimethyl sulfoxide, separated on 1%
agarose gels and transferred to nylon membranes in 20 3 SSC.
DNA fragments were labeled with 32P and used as probes (metanestin: nucleotide 611-1255 in Fig. 1a; a 100-kDa protein, nucleotide
63-645 in Fig. 1b). Hybridization was performed under conditions
of low stringency (5 3 SSC, 50% formamide, 42°C) for 20 hr, and
washes were performed with 0.1 3 SSC, 0.1% SDS, at 55°C.
Immunohistochemical staining
Transitional-cell-carcinoma tissues of the bladder were resected
by radical cystectomy in Kagoshima University Hospital from
1994 to 1995; in all, 24 primary tumors of the bladder and 10
metastasized lymph nodes were analyzed, while some specimens
were available only for analysis of expression of one antigen. The
specimens were fixed with PBS(—)-buffered folmaldehyde and
then embedded in paraffin. They were cut into sections 4 µm thick,
A METASTASIS-ASSOCIATED GLYCOPROTEIN
11
FIGURE 6 – Expression of antigenic epitopes of a 100-kDa (ST-2) protein in clinical samples as revealed by immunohistochemical analysis
using anti-PAPA antibody. (a) Normal epithelium (bar, 50 µm); (b) primary tumor (bar, 50 µm); (c) metastasized lymph node (bar, 50 µm). Only
umbrella cells were faintly stained in normal epithelium, while primary tumors and the metastatic lesions were strongly stained. All samples were
taken from one patient. Concentration of the antibody was 10 µg/ml.
TAKEMOTO ET AL.
12
and endogenous peroxidase was blocked in methanol for 30 min.
Specimens were then rinsed with PBS(—), incubated with normal
goat serum (Nichirei, Tokyo, Japan) for 15 min at room temperature, then reacted with affinity-purified antibodies for 1 hr. After
washing with PBS (—), sections were reacted with biotinylated
goat anti-rabbit IgG (Nichirei) for 10 min at room temperature. The
specimens were incubated with avidin-biotin-peroxidase (Nichirei)
for 5 min, and color was developed with 0.05% 3,38-diaminobenzidine and 0.008% hydogen peroxide in 0.01 M Tris-HCl buffer, pH
7.4 for 5 min. The sections were washed with distilled water,
counterstained with hematoxylin, dehydrated, washed with xylene,
and mounted with permount. Negative control sections were
incubated with normal rabbit serum instead of antibody. The
positive control in each experiment was BOY transitional-cellcarcinoma cells, which were collected, fixed, embedded and
sectioned as described above.
RESULTS
Predicted partial sequences of 2 proteins with MM4 antigen
We screened an expression library constructed from BOY
bladder-carcinoma cells with MM4 antibody, and obtained 2
clones, ST-1 and ST-2. Repeated screening failed to detect additional clones, and we tentatively concluded that protein sequences
specified by the 2 clones included the MM4 antigenic epitope.
Indeed, b-galactosidase fusion proteins of the inserts of the 2
clones reacted with the antibody, but b-galactosidase itself did not
(Fig. 2). Thus, we decided to characterize the proteins encoded by
these clones. Using the inserts as probes, we screened the library
and obtained extended cDNAs. DNA sequences derived from the
two clones (Fig. 1) did not overlap and were thus considered to
encode different proteins.
The length of the extended insert of ST-1 was about 1.2 kb, and
that of ST-2 was 1.0 kb. Northern-blot analysis of Poly(A)1 RNA
from BOY cells revealed that mRNA corresponding to ST-1 was 5
kb, while that to ST-2 was 3.5 kb (Fig. 3). Thus, these were partial
clones.
Both ST-1 and ST-2 had extended open reading frames. By
analyzing the predicted protein sequences, we found that ST-1derived protein (ST-1 protein) contained 2 repeats of a Pro-Gly-ProGly motif (Fig. 1a) and ST-2-derived protein (ST-2 protein) had 4
repeats of a Pro-Ala-Pro-Ala motif. The initial clones, which
reacted with MM4 MAb, each had 2 of Pro-Gly-Pro-Gly or
Pro-Ala-Pro-Ala sequences (Fig. 1a,b, underlined sequences).
Glycine and alanine are homologous amino acids; addition of a
methyl group to glycine yields alanine. Since there are no other
identical or homologous sequences between the predicted protein
sequences, we conclude that the repeated Pro-Gly(or Ala)-ProGly(Ala) motif is the antigenic epitope of MM4 antigen.
The predicted partial sequences of ST-1 protein and ST-2 protein
were not related to any other protein sequences so far reported.
That of ST-1 is rich in proline, serine and threonine. Since mucin is
also known to be rich in these amino acids, ST-1 appears to be a
new mucin-type glycoprotein. The partial sequence of ST-2 has a 4
times-repeated sequence of SXXXNHGPAPA. Occurence of repeated structure is a characteristic of mucin, and ST-2 protein is
also considered to be a mucin-type glycoprotein.
ST-1 protein, re-named metanestin, was preferentially expressed
in metastatic nests in lymph nodes
Since the predicted open reading frame of ST-1 encodes a
42-kDa polypeptide chain, we prepared a rabbit antibody against
ST-1-maltose-binding protein fusion protein. The affinity-purified
antibody reacted strongly with a 60-kDa protein from BOY cell
extract (Fig. 4a). MM4 MAb also reacted principally with a 60-kDa
band among LTA-binding glycoproteins isolated from BOY cells
(Matsusako et al., 1992). Thus, we concluded that ST-1 encodes a
partial sequence of the 60-kDa protein. Although a band of 80 kDa
faintly reacted with the anti-ST-1-fusion-protein antibody, this was
considered to be due to cross reactivity.
The antibody to ST-1 fusion protein scarcely reacted with normal
urothelium (Fig. 5a), and reactivity to primary tumors of transitionalcell carcinoma of the urinary bladder was generally weak (Fig. 5b,
Table I). However, in 6 out of 7 cases of lymph-node metastasis, the
staining was generally strong (Fig. 5c,d, Table I). Among the 6
strongly positive cases in lymph-node metastasis, in 4 cases the
primary cancers did not react with the antibody, or reacted with it
weakly. These results indicated that ST-1 protein was expressed in
metastatic nests of lymph nodes, but was generally expressed only
weakly or not at all in primary tumors. From the data of Table I, we
conclude that the stronger expression of metanestin in lymph-node
metastasis than in primary tumors is statistically significant
( p 5 0.0329, Fisher’s exact probability test). Thus, ST-1 protein is
a novel protein which is preferentially expressed in metastatic nests
in lymph nodes as compared with the primary tumor or with normal
urothelium. We re-named ST-1 metanestin, because of its preferential expression in metastatic nests in lymph nodes.
TABLE III – EXAMINATION OF POSSIBLE CORRELATION BETWEEN ANTIGEN
EXPRESSION AND TUMOR STAGE
Antigen
Metanestin
TABLE II – EXAMINATION OF POSSIBLE CORRELATION BETWEEN ANTIGEN
EXPRESSION AND TUMOR GRADES
Antigen
Metanestin
100-kDa protein
Tumor grades
Primary tumor
G1
G2
G3
Lymph-node metastases
G1
G2
G3
Primary tumor
G1
G2
G3
Lymph-node metastases
G1
G2
G3
Number of cases
Total
Strongly positive
2
6
16
1
3
5
0
0
7
0
0
6
1
8
9
0
5
7
0
2
8
0
1
7
100-kDa protein
Tumor stages
Primary tumor
T1
T2
T3a
T3b
T4
Lymph-node metastases
T1
T2
T3a
T3b
T4
Primary tumor
T1
T2
T3a
T3b
T4
Lymph-node metastases
T1
T2
T3a
T3b
T4
Number of cases
Total
Strongly positive
10
2
5
6
1
2
2
1
4
0
1
0
1
4
1
1
0
1
3
1
6
1
4
6
1
2
1
3
5
1
1
0
3
5
1
1
0
1
5
1
A METASTASIS-ASSOCIATED GLYCOPROTEIN
A protein with PAPA motif also showed
tumor-associated expression
Since insert of ST-2 was small, we prepared an antibody against
a synthetic peptide with the repeating motif, PAPA. The antibody
reacted with a 100-kDa protein on Western blots of BOY cell
extract (Fig. 4b). The antibody faintly reacted with umbrella cells
(Fig. 6a), but not with basal cells of the urothelium. Both primary
tumors and metastatic lymph nodes reacted with the antibody (Fig.
6b,c). However, strong staining was also more frequently observed
in lymph-node metastasis than the primary tumor (Table I). Thus,
the 100-kDa protein with the PAPA motif also showed tumorassociated and metastasis-associated expression, although these
tendencies were not as clear as those of metanestin, and no
statistically significant conclusion was reached. We also examined
the possible correlation between expression of metanestin or the
100-kDa protein and tumor grades (Table II), but at the present
stage of analysis no clear-cut correlation was found. Similarly,
antigen expression and tumor stages were not clearly correlated
(Table III).
DISCUSSION
In transitional-cell carcinoma of the urinary bladder, we have
found metanestin, a 60-kDa protein with tumor-associated and
metastasis-associated expression, and elucidated its partial protein
structure. Previous studies from our laboratories showed that the
60-kDa glycoprotein is the predominant carrier of carbohydrate
epitopes, Lex and sialyl Lex, whose strong expression correlates
with metastatic potential of the cancer cells (Matsusako et al.,
1991, 1992). The 60-kDa glycoprotein corresponds to metanestin,
identified herein. A 100-kDa protein with a repeated PAPA motif
was also found to show tumor- and metastasis-associated expression, although these tendencies were less clear than those of
metanestin. Comparison of protein sequences of metanestin and the
100-kDa protein revealed that the metastasis-associated antigenic
epitope MM4 is a peptide, with twice-repeated PG (or A) PG (or A)
motif. In other words, the MM4 antigenic epitope was concluded to
be present both on metanestin and on the 100-kDa protein.
Our results indicate that both the carbohydrate antigens and core
proteins with the antigens show metastasis-associated expression.
It is possible that expression of core protein in the tumor is a factor
regulating the expression of an immunochemically detectable
13
carbohydrate antigen: the core protein may provide multiglycosylation sites, permitting production of multivalent carbohydrate
antigens, which will bind to an antibody, especially Ig M with high
affinity. Indeed, increased expression of Lex-related carbohydrate
antigen in tumor cells may not be simply explained by the level of
glycosyltransferase activities (Holmes and Macher, 1993). Both
metanestin and the 100-kDa glycoprotein with the PAPA sequence
were considered to be mucin-type glycoproteins, either from their
amino-acid composition or from the presence of the repeating
motif. Mucin-type glycoproteins have many glycosylation sites,
and are suitable for carriers of carbohydrate antigens with high
affinity (Shimizu and Shaw, 1993). Ligands of L-selectin are also
carried by mucin-type glycoproteins (Lasky et al., 1992).
Urinary-bladder carcinoma belongs to a group of cancers in
which lymph-node metastasis is prevalent. Blood-borne metastasis
occurs only in terminal stages or in a few cases of early cancer
(Kyun et al., 1987). We have found that lymph-node metastasis
occurs in 4.2% of cases in pL 0, 10% in pL 1 and 42.9% in pL 2
(data not shown). Thus, lymph-node metastasis is clearly correlated
with lymphatic-vessel invasion: cancer cells are considered to enter
into lymphatic vessels, and, through the lymphatic fluid, to
metastasize to lymph nodes. Although E-selectin in blood vessels
has been implicated in blood-borne metastasis, we do not know
whether a selectin is present in lymphatic vessels. Thus, the
metastasis-associated carbohydrate epitopes and their carrier, especially the 60-kDa glycoprotein metanestin, may function either as a
ligand of hitherto undetected selectin in lymphatic vessels or by an
entirely unrelated mechanism. In any event, demonstration of
proteins with metastasis-associated expression will contribute
significantly to understanding of the molecular mechanisms underlying lymph-node metastasis of urinary-bladder carcinoma. Metanestin may become clinically useful in detecting small lymph-node
metastases of urinary-bladder carcinomas and in designing immunotherapy of the metastatic tumors.
ACKNOWLEDGEMENTS
We thank Ms. K. Yoshioka, K. Saito, C. Mashima, A. Horisawa
and A. Miyata for secretarial assistance. This work was supported
by grants from the Ministry of Education, Science and Culture of
Japan.
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