Cloning, Expression, and Characterization of a Novel
UDP-galactose:
-N-Acetylglucosamine
1,3-Galactosyltransferase (
3Gal-T5) Responsible for Synthesis of
Type 1 Chain in Colorectal and Pancreatic Epithelia and Tumor Cells
Derived Therefrom*
Soichiro
Isshiki
§¶,
Akira
Togayachi
¶
,
Takashi
Kudo
,
Shoko
Nishihara
,
Masahiko
Watanabe§,
Tetsuro
Kubota§,
Masaki
Kitajima§,
Norihiko
Shiraishi**,
Katsutoshi
Sasaki**,
Toshiwo
Andoh
, and
Hisashi
Narimatsu

From the
Division of Cell Biology, Institute of Life
Science, and
Department of Bioengineering, Faculty of
Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo
192-8577, the § Department of Surgery, Keio University
School of Medicine, Shinjuku-ku, Tokyo 160-8582, and ** Tokyo Research
Laboratories, Kyowa Hakko Kogyo Company Limited, 3-6-6 Asahi-machi,
Machida-shi, Tokyo 194-8533, Japan
 |
ABSTRACT |
The sialyl Lewis a antigen is a well known tumor
marker, CA19-9, which is frequently elevated in the serum in
gastrointestinal and pancreatic cancers.
UDP-galactose:N-acetylglucosamine
1,3-galactosyltransferase(s) (
3Gal-Ts) are required for the
synthesis of the sialyl Lewis a epitope. In the present study, a novel
3Gal-T, named
3Gal-T5, was isolated from a Colo205 cDNA
library using a degenerate primer strategy based on the amino acid
sequences of the four human
3Gal-T genes cloned to date.
Transfection experiments demonstrated that HCT-15 cells transfected
with the
3Gal-T5 gene expressed all the type 1 Lewis
antigens. In gastrointestinal and pancreatic cancer cell lines, the
amounts of
3Gal-T5 transcripts were quite well correlated with the
amounts of the sialyl Lewis a antigens. The
1,3Gal-T activity toward
agalacto-lacto-N-neotetraose was also well correlated with
the amounts of
3Gal-T5 transcripts in a series of cultured cancer
cells, and in Namalwa and HCT-15 cells transfected with the
3Gal-T5 gene. Thus, the
3Gal-T5 gene is
the most probable candidate responsible for the synthesis of the type 1 Lewis antigens in gastrointestinal and pancreatic epithelia and tumor
cells derived therefrom. In addition,
3Gal-T5 is a key enzyme that
determines the amounts of the type 1 Lewis antigens including the
sialyl Lewis a antigen.
 |
INTRODUCTION |
CA19-9 in serum is a well known tumor marker, which is frequently
used for the clinical diagnosis of cancer, in particular, colorectal,
pancreatic, and gastric cancers (1, 2). The 1116NS19-9
(19-9)1 monoclonal antibody
detects a CA19-9 antigen, of which the antigenic epitope has been
defined as the carbohydrate structure of sialyl Lewis a
(sLea) (2-4). Besides its usefulness as a tumor marker,
sLea antigen is known to be a ligand for selectins (5, 6).
Clinical statistical analysis demonstrated that cancer patients who
express abundant sLea antigens have a worse prognosis as to
liver metastasis than patients who do not express sLea
antigens (7, 8). Thus, it is of interest that sLea antigens
may confer some metastatic capacity on cancer cells.
At least three glycosyltransferases are required for the synthesis of
the sLea epitope. First,
N-acetylglucosamine-
1,3-galactosyltransferase (
3Gal-T)
transfers a galactose (Gal) to an N-acetylglucosamine (GlcNAc) with a
1,3-linkage, resulting in the synthesis of a type 1 chain, Gal
1,3GlcNAc, and then galactose-
2,3-sialyltransferase (ST3Gal) transfers a sialic acid (SA) to the Gal residue of the type 1 chain with an
2,3-linkage, resulting in sialyl-type 1 (sialyl Lewis
c; sLec) chain, SA
2,3Gal
1,3GlcNAc, synthesis.
Finally,
1,3/4-fucosyltransferase (Fuc-TIII, FUT3, Lewis enzyme)
transfers a fucose (Fuc) to the GlcNAc residue of the sialyl-type 1 chain with an
1,4-linkage to complete the synthesis of the
structure, SA
2,3Gal
1,3(Fuc
1,4)GlcNAc. In previous studies, we
demonstrated that Fuc-TIII (FUT3) is the only enzyme determining the
expression of sLea antigens in colorectal cancer (3, 4),
and that ST3GalIV, one of the ST3Gals, mainly participates in the
sLea synthesis in colorectal cancer (9).
Regarding
3Gal-Ts, we have reported for the first time the cloning
of a
3Gal-T gene from human WM266-4 melanoma cells using an expression cloning method (10). The recent rapid growth of data
bases of expressed sequence tags (ESTs) and the Human Genome Project
enabled us to find novel genes homologous to the original one. Thus,
three human
3Gal-T genes homologous to the original one
were cloned very recently (11, 12). The four
1,3GalTs, including the
original one, are named
3Gal-T1 to -T4 (12). Expression studies on
the four human
3Gal-Ts demonstrated that two of them,
3Gal-T1 and
T2, apparently transfer Gal to GlcNAc with a
1,3-linkage resulting
in type 1 chain synthesis, but
3Gal-T4 transfers Gal to an
N-acetylgalactosamine (GalNAc) residue, resulting in the
synthesis of the type 3 chain, Gal
1,3GalNAc (12). The human
3Gal-T4 did not transfer Gal to a GlcNAc residue for the type 1 chain synthesis (12). The human
3Gal-T4 is likely to be the human
homologue of the rat GM1/GD1 synthase (13),
since the amino acid sequence of human
3Gal-T4 shows very high
homology, 79.4%, to that of the rat GM1/GD1
synthase, and the human
3Gal-T4 apparently transfers Gal to the
GalNAc residue of asialo-GM2 and GM2, resulting in the
asialo-GM1 and GM1 synthesis, respectively (12). The activity of human
3Gal-T3 has not been detected toward any
of the acceptor substrates used in their study (12). Three mouse
3Gal-T genes have been cloned and named
m
3GalT-I, m
3GalT-II, and
m
3GalT-III, corresponding to human
3Gal-T1,
3Gal-T2, and
3Gal-T3, respectively (14).
m
3Gal-TII and m
3GalT-III were found to exhibit the
3Gal-T
activity toward both GlcNAc and GalNAc residues; however, they showed
quite low activities for the type 1 chain synthesis, i.e.
about 3% of the activity of m
3GalT-I (14).
It has not been elucidated which
3Gal-T determines the expression of
the sLea epitopes in gastrointestinal and pancreatic
cancers. The tissue distributions of the four
3Gal-Ts were
determined by Northern analysis (11, 12), it being found that neither
3Gal-T1 nor -T2 is expressed in the pancreas, which indicated that
there may be unknown
3Gal-T(s) synthesizing the type 1 chain in the
pancreas. They did not examine the expression of those
3Gal-Ts in
the gastrointestinal tissues, such as colon and stomach, which
frequently produce the sLea antigens when they become cancerous.
In this study, we first noticed that none of the four human
3Gal-Ts
cloned to date,
3Gal-T1 to -T4, is responsible for the sLea expression in gastrointestinal and pancreatic cancers,
and successfully cloned a novel
3Gal-T gene, named
3Gal-T5, from Colo205 cells.
3Gal-T5 is the most
probable candidate participating in the synthesis of the
sLea epitopes, i.e. CA19-9 antigens, in
gastrointestinal and pancreatic cancer cells.
 |
EXPERIMENTAL PROCEDURES |
Tumor Cell Lines and Monoclonal Antibodies--
Various tumor
cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum. The monoclonal antibodies
used in this study were as follows: 1116NS19-9 (19-9), which was used
for the detection of tumor marker CA19-9, anti-sLea (IgG)
(2); DU-PAN-2, anti-sLec (IgM) (4, 15); 7LE,
anti-Lea (IgG) (16); and TT42, anti-Leb (IgM),
of which the specificity against Leb was recently defined
by us, and will be published elsewhere. 19-9 and TT42 were kind gifts
from Fujirebio Inc. (Toyko, Japan), and Diagnostic Division, Otsuka
Pharmaceutical Co. Ltd. (Tokushima, Japan), respectively. DU-PAN-2 and
7LE were purchased from Kyowa Medex Co., Ltd. (Tokyo, Japan), and
Seikagaku-Kogyo Co., Ltd. (Tokyo, Japan), respectively.
Cloning of the Four Cloned
3Gal-T Genes from Various Human
cDNA Libraries and Construction of Expression Plasmids--
The
cDNA encoding
3Gal-T1 was cloned by the expression cloning
method used in our previous study (10). We found three sequences homologous to that of
3Gal-T1 in the EST data bases. The full-length cDNAs encoding the other three homologous sequences were cloned from various human cDNA libraries using probes encoding the
fragment sequences in the EST data base. They were identical to
3Gal-T2, -T3, and -T4, which were reported by Amado et
al. (12), and Kolbinger et al. (11).
Thus, the four human cDNAs encoding the respective full-length open
reading frames (ORFs) of
3Gal-T1, -T2, -T3, and -T4 were subcloned
into the pAMo vector for expression in cultured cells (17, 18).
Construction of a cDNA Library from Colo205 Cells--
Total
cellular RNA was isolated from Colo205 cells using the acid guanidium
thiocyanate-phenol-chloroform method (19).
Poly(A)+-rich-RNA was isolated with
OligotexTM-dT30 (Super) (Roche, Tokyo, Japan).
Complementary DNAs were synthesized with oligo(dT) primers from
poly(A)+-rich-RNA using a Superscript Choice System for
cDNA Synthesis (Life Technologies, Inc.). A cDNA library was
constructed by inserting size-fractionated cDNAs (more than 1.5 kilobase pairs) into an expression vector, pAMo, using SfiI
adaptors (17, 18). We obtained about 1 × 106
independent clones as a cDNA library and extracted plasmid DNAs from the library.
PCR for Cloning of a Fragment Encoding a Novel
3Gal-T--
On
alignment of the amino acid sequences of the four cloned
3Gal-Ts, we
found conserved amino acid sequences at three positions, and named them
Motifs 1, 2, and 3 (Table I). The
conserved amino acid sequences of Motifs 1, 2, and 3 were employed for
the design of degenerate primer sequences, i.e. primers at-1
(5' primer for Motif 1), 5'-GCI AT(A/C/T) (A/C)GI CA(A/G) ACI TGG
GG-3'; at-2 (3' primer for Motif 2), 5'-(A/G)TC (A/G)CT (A/G)TC IGT
(C/T)TT CAT IAC (A/G)TA-3'; at-3 (5' primer for Motif 2), 5'-TA(C/T)
GTI ATG AA(A/G) ACI GA(C/T) TCI GA(C/T)-3'; and at-4 (3' primer for Motif 3), 5'-(A/G)CA IA(A/G) ICC IAC (A/G)TA IAC (A/G)TC (C/T)TC-3', respectively.
The cDNAs of the Colo205 cDNA library described above were used
as templates for PCR amplification to obtain a DNA fragment. Two PCRs
were performed with two sets of degenerate primers, respectively, i.e. the first PCR was performed with primers at-1 and at-2,
and the second PCR with primers at-3 and at-4. The amplified PCR
products were inserted into a pBluescript SK (
) (pBS) vector
(Stratagene, La Jolla, CA), and the DNA fragments obtained were
sequenced by the dideoxynucleotide chain termination method using an
ALF DNA sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden). Two
fragment DNAs contained novel nucleotide sequences, which, however,
were homologous to the corresponding regions of the cloned
1,3GalT genes. On an additional PCR involving Colo205
cDNAs as templates using primers encompassing the two fragment
sequences, both fragments were found to be encoded by one species of cDNA.
Cloning of Full-length cDNAs Encoding a Novel
3Gal-T--
The two DNA fragments obtained through the two PCRs,
i.e. those with the Motif 1 and 2 primers, and the Motif 2 and 3 primers, respectively, were mixed and used as the probe for
hybridization to isolate full-length cDNA clones. We screened the
Colo205 cDNA library and isolated several distinct clones having
inserts of different sizes. All inserts encoded the same sequence of
one species of cDNA, this sequence being found to be homologous to those of the known four
3Gal-Ts. Thus, we named this novel gene the
human
3Gal-T5 gene. After the cDNA sequences had been
completed, we searched the data base of the Human Genome Project to
determine whether the same sequence or homologous ones were registered
or not. We found a genome sequence completely identical to the cDNA sequences in the data base, which was very recently registered (June
2nd, 1998). Its registration number is AF064860. By comparison between
the cDNA sequences and the genome one, we determined the genomic
organization of the
3Gal-T5 gene.
Identification of Alternatively Spliced Isoforms of
3Gal-T5
Transcripts and the Transcription Initiation Site--
By the 5'-rapid
amplification of cDNA ends (5'-RACE) method using two primers,
i.e. si-1 (5'-GAAAGGATTTAGACTGTACATGC-3'), this sequence
being positioned close to the ATG codon in the ORF, and si-2
(5'-GTGAATTCCTCTTTCTCTGCTG-3'), we obtained five different types of
amplified fragments, and subcloned them into a pBS (
) vector for
sequencing. Thus, five isoforms, isoforms 1, 2, 3, 4, and 5, of
3Gal-T5 transcript were identified. Further RT-PCR experiments were
performed to determine the abundance of each isoform of the
3Gal-T5
transcript expressed in Colo205 cells, for which the following primers
were employed, primers si-1, si-2, si-3
(5'-TGAAAGGAACAAAATCCAATGAT-3'), and si-4
(5'-AGAACCCTGACTAATACACCTGGA-3').
Quantitative Analysis of the five
1,3GalT Transcripts in Human
Tumor Cell Lines and Human Tissues by Competitive RT-PCR--
The
principle of the competitive RT-PCR method was described in detail in
our previous papers (9, 18). Competitor DNA plasmids each carrying a
small deletion within the respective full-length ORF cDNA were
constructed by appropriate restriction endonuclease digestion as shown
in Table II. For instance, a competitor DNA plasmid of the
3Gal-T1 gene was prepared by deleting the 212-bp
BanII-EcoRV fragment from the standard plasmid
DNA containing the full-length cDNA of
3Gal-T1.
Total cellular RNA was isolated from various tumor cell lines and human
tissues. Complementary DNAs were synthesized with an oligo(dT)
primer from 6 µg of DNase I-treated total RNA in a 20-µl (total
volume) reaction mixture using a SuperscriptTM
Preamplification System for First Strand cDNA Synthesis (Life Technologies, Inc.). After cDNA synthesis, the reaction mixture was
diluted 50-fold with H2O and then stored at
80 °C
until use.
The competitive RT-PCR was performed with AmpliTaq GoldTM
(Perkin Elmer) in a 50-µl (total volume) reaction mixture comprising 10 µl of standard plasmid DNA or sample cDNA, 10 µl of
competitor DNA at the optimal concentration, which differs with the
transcript, and 0.2 µM amounts of each primer of the
gene-specific primer sets listed in Table
II. The PCR buffer for the competitive
RT-PCR comprised 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl2, 0.2 µM of each
dNTP, and 0.001% (w/v) gelatin. PCR was performed with a pre-PCR heat
step at 95 °C for 11 min, followed by the optimal number of PCR
cycles, each of which comprised 1 min at 95 °C, 1 min at the
optimal annealing temperature (Table II), and 2 min at 72 °C. After
the competitive RT-PCR, a 10-µl aliquot was electrophoresed in a 1%
agarose gel and the bands were visualized by ethidium bromide staining.
The intensities of the amplified fragments were quantified by scanning
positive pictures using the public domain NIH Image
program.2 Measurement of the
-actin transcript in each sample was performed using the same
competitive RT-PCR method as for the
3Gal-T transcripts. Each value
for the
3Gal-T and
-actin transcripts was plotted on the
respective standard curve to obtain the actual amount of each
transcript. The actual amount of each
3Gal-T transcript was divided
by that of
-actin for normalization.
Transfection Experiments to Express the Five Human
3Gal-T
Genes in Namalwa (Burkitt Lymphoma) and HCT-15 Cells--
Each of the
five
3Gal-T genes subcloned into the pAMo vector was
stably transfected by the electroporation method into Namalwa or HCT-15
cells. These cells were selected in the presence of Geneticin (G418)
(Life Technologies, Inc.) at a concentration of 0.8 mg/ml in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum.
Stable transformant cells were obtained after 25 days of exposure to
Geneticin. Cell homogenates of stable transformants expressing each of
the
3Gal-T genes were subjected to assaying of
3Gal-T
activity. The levels of the transcripts expressed in the transformant
cells were measured by means of competitive RT-PCR to normalize the
3Gal-T activity. The stable transformants of HCT-15 cells were
subjected to limiting dilution to obtain single transformant clones.
Flow Cytometry Analysis--
The expression of type 1 Lewis
antigen epitopes, i.e. the Lea, Leb,
sLec, and sLea epitopes, on the surface of the
cultured tumor cells and the cells transfected with each of the
3Gal-T genes was examined by flow cytometry analysis
using an Epics Elite (Coulter, Tokyo, Japan). The transfected cells
(1 × 106) were incubated with a first antibody (10 µg/ml) for 1 h on ice, and then washed twice with PBS (pH 7.4)
containing 1% BSA and 0.1% sodium azide, followed by incubation with
fluorescein isothiocyanate-conjugated anti-mouse IgM or IgG (Bio-Rad).
Then, the cells were washed again with PBS-BSA and finally subjected to
flow cytometry analysis.
Western Blotting Analysis--
Cell pellets were solubilized in
20 mM HEPES buffer (pH 7.2) containing 2% Triton X-100 by
brief sonication. Proteins separated on 6% SDS-polyacrylamide gel
electrophoresis were transferred to an Immobilon PVDF membrane
(Millipore, Bedford, MA) in a Transblot SD cell (Bio-Rad). The membrane
was blocked with PBS containing 5% skim milk at 4 °C overnight and
then incubated with 10 µg/ml 19-9. The membrane was stained according
to the manual with the ECL Western blotting detection reagents
(Amersham Pharmacia Biotech).
Assaying of
3Gal-T
Activity--
Lacto-N-neotetraose (LNnT) was
pyridylaminated as in the previous study (17). The pyridylaminated-LNnT
(LNnT-PA) was digested with 20 milliunits/ml streptococcal
-galactosidase (Seikagaku-Kogyo) to remove the galactose residue at
the nonreducing end. Thus, agalacto-LNnT-PA was prepared, and used for
assaying
3Gal-T activity. Namalwa and HCT-15 cells tranfected stably
with each of the
3Gal-T genes, and various cultured cells
were solubilized in 20 mM HEPES buffer (pH 7.2) containing
2% Triton X-100. The
1,3Gal-T activity was assayed in 14 mM HEPES buffer (pH7.4), 75 µM UDP-Gal, 11 µM MnCl2, 0.01% Triton X-100, and 25 µM acceptor substrate. After incubation at 37 °C for
2 h, the enzyme reactions were terminated by boiling for 3 min.,
followed by dilution with water. After centrifugation of the reaction
mixtures at 15,000 rpm for 5 min., 10 µl of each supernatant was
subjected to high performance liquid chromatography analysis on a
TSK-gel ODS-80TS column (4.6 × 300 mm; Tosoh, Tokyo,
Japan). The reaction products were eluted with 20 mM
ammonium acetate buffer (pH 4.0) at the flow rate of 1.0 ml/min at
25 °C and monitored with a Jasco FP-920 fluorescence spectrophotometer (Jasco, Tokyo, Japan).
 |
RESULTS |
Discrepancy between the Expression Levels of three
3Gal-T
Transcripts, i.e. the
3Gal-T1, -T2, and -T3 Transcripts, and the
Amounts of Type 1 Lewis Antigens Expressed in Various Tumor
Cells--
Various tumor cell lines derived from different human
tissues were examined as to the transcript levels of the four
3Gal-T genes that were cloned previously, and their
expression levels were compared with the amounts of type 1 Lewis
antigens, i.e. the sLea (19-9), Lea
(7LE), and Leb (TT42) antigens, expressed in these cancer
cells (Fig. 1). Flow cytometry analysis
revealed that Colo205, Colo201, and SW1116 (colon cancer) cells, and
Capan-2 (pancreatic cancer) cells expressed large amounts of type 1 Lewis antigens, these results being consistent with those of a previous
study (20). All the above cell lines except for Capan-2 were strongly
stained with the three antibodies, i.e. 19-9
(anti-sLea), 7LE (anti-Lea), and TT42
(anti-Leb) antibodies. Capan-2 cells were also strongly
stained with 19-9, but not with 7LE or TT42. However, these four types
of cells did not express
3Gal-T1 or
3Gal-T2 transcripts at all.
The expression of
3Gal-T1 was abundantly detected in PC-1 (lung
cancer) cells, and faintly detected in Jurkat (T cell leukemia) and
PC-3 (prostatic cancer) cells.
3Gal-T2 was expressed in Namalwa and
SK-N-MC (neuroblastoma) cells at intermediate levels, and faintly
expressed in some cell lines from gastrointestinal cancers.
3Gal-T3
was abundantly expressed in some gastric cancer cells, i.e.
KATO III and MKN45 cells, and PC-3 cells, and intermediately expressed
in Capan-1 and Capan-2 (pancreatic cancer) cells, HCT-15 cells, and
SK-N-MC and SK-N-SH (neuroblastoma) cells; however, it was not detected
at all in Colo201 or Colo205 cells.

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Fig. 1.
Quantitative analysis of transcripts of
3Gal-T1, -T2, -T3, and -T4 in various human tumor cells by
competitive RT-PCR, and flow cytometry analysis for type 1 Lewis
antigens on these cells. A, for the quantitation of
each transcript, each single-stranded cDNA was amplified together
with 200 ag/µl of the respective competitor DNA. The human -actin
transcripts were quantified with 200 fg/µl of the competitor DNA. The
value for each 3Gal-T transcript was divided by that for the
respective -actin transcript. B, the positive peaks
observed on flow cytometry analysis are indicated as to +++,
depending on the intensity. NT, not tested.
|
|
We observed a significant discrepancy between the expression of the
three
3Gal-Ts, i.e.
3Gal-T1, -T2, and -T3, and the
expression of the type 1 Lewis antigens in these cell lines. In
contrast, the expression of
3Gal-T4 appeared to be correlated with
the type 1 Lewis antigen expression, i.e. the cells
expressing type 1 Lewis antigens, i.e. Colo201, Colo205,
SW1116, HT-29, and Capan-2 cells, also expressed substantial amounts of
the
3Gal-T4 transcript.
3Gal-T1, -T2, and -T3 could synthesize the type 1 chain (11, 12,
14); however, they were not correlated with the expression of type 1 Lewis antigens in the present study. The expression of
3Gal-T4
seemed to be correlated with the expression of type 1 Lewis antigens in
some tumor cells; however, it could not synthesize the type 1 chain
(12). From these results, we concluded that none of the four
3Gal-Ts
is responsible for type 1 chain synthesis, resulting in
sLea (CA19-9) antigen expression, in gastrointestinal and
pancreatic cancer cells.
Cloning and Sequence of a Novel cDNA Homologous to the Cloned
3Gal-Ts--
As described under "Experimental Procedures," we
obtained two DNA fragments encoding novel sequences, which, however,
are homologous to the corresponding regions of the four cloned
3Gal-T genes. The sequences of the two DNA fragments were
found to be encoded by a single cDNA species. We named this gene
3Gal-T5. By use of the DNA fragments as probes,
full-length cDNA clones were obtained from the Colo205 cDNA
library. Complementary DNA sequencing analysis revealed that the
3Gal-T5 cDNA contains an ORF encoding a protein of 310 amino
acids (Fig. 2). The position of the AUG
start codon was assigned according to the Kozak consensus sequence
(21). A hydropathy profile based on the Kyte and Doolittle method (22)
indicated that the ORF encodes a type II membrane protein, which is a
typical feature of glycosyltransferases (data not shown). The three
motifs of amino acid sequences, Motifs 1, 2, and 3, which we employed
for the design of degenerate primers in this study, were conserved in
the sequence of
3Gal-T5. Four cysteine residues were conserved in
the five
3Gal-Ts, which indicates that some of these cysteines are
essential for maintenance of the tertiary structures of
3Gal-Ts.
Fifty-four of the 310 amino acid residues of
3Gal-T5 were conserved
in comparison with the sequences of the other four
3Gal-Ts. Three
possible N-glycosylation sites were found in the primary
sequence of
3Gal-T5.

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Fig. 2.
Nucleotide sequence of the 3Gal-T5
cDNA and the predicted amino acid sequence of 3Gal-T5. A
putative transmembrane domain is boxed. Possible
N-glycosylation sites are indicated by double
underlines. Possible polyadenylation signals are
single underlined. Exon-intron junctions are
indicated. The four squares indicate cysteine residues
conserved in the five 3Gal-Ts. The shaded
amino acid letters indicate the three
motifs conserved in the five 3Gal-Ts.
|
|
Genomic Structure of the
3Gal-T5 Gene and Alternatively Spliced
Isoforms of Transcripts--
By comparison of the full-length cDNA
sequence with the genome sequence, which has been registered in the
Genome Project Database (registration no. AF064860), the chromosomal
localization and the genomic structure of the
3Gal-T5
gene were determined (Fig. 3A). According to the
description in GenBank, this gene is localized to human chromosome
21q22.3. The ORF of the
3Gal-T5 gene was found to be
encoded by a single exon, as in the cases of the four cloned
3Gal-T genes. The A nucleotide of the translation
initiation codon, ATG, was found to be the first nucleotide of exon 4 encoding the ORF.

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Fig. 3.
Genomic structure of the
3Gal-T5 gene. A, the four exons are
shown as boxes, and introns as lines. The
restriction sites in exon 2, XbaI, and in exon 3, BsmI, are indicated. ORF is indicated by a
hatched box. B, structures of five
isoforms of 3Gal-T5 transcripts. *, ratio means the abundance as a
percentage of abundance of each isoform in Colo205 cells. C,
RT-PCR analysis to determine abundance of the five isoforms of
3Gal-T5 transcripts. Molecular weight markers (100-bp ladders) are
on the leftmost lane.
|
|
The sequence of the 5'-flanking region was extended by the 5'-RACE
method using Colo205 transcripts. All subclones obtained with the
5'-RACE method had a common nucleotide sequence at the 5' end,
i.e. all of them started at nucleotide position 85153 in the
genome sequence AF064860 (Table III).
Thus, five isoforms of
3Gal-T5 transcripts, isoforms 1, 2, 3, 4, and
5, were identified with the 5'-RACE method (Fig. 3B). All
intron sequences at the exon-intron junctions complied with the
acceptor and donor site sequences of splicing rule, i.e. the
GT-AG rule (Table III). Each transcript of the five isoforms in Colo205
cells was quantified by determining the intensity of its band on a
RT-PCR gel (Fig. 3C). As shown in Fig. 3A, exon 2 or 3 contains an XbaI or BsmI restriction site,
respectively. The amplified bands on RT-PCR were digested with
XbaI or BsmI to confirm exon 2 or 3, respectively. As shown in Fig. 3C, two bands were obtained
on RT-PCR using the primer set, si-2 and si-1. The upper and lower
bands correspond to isoform 1, consisting of exons 1, 3, and 4, and
isoform 2, consisting of exons 1', 3, and 4, respectively, and both
materials were digested by BsmI, but not by XbaI.
The ratio of the band intensities of isoforms 1 and 2 was about 1 to 1. The other three isoforms, isoforms 3, 4, and 5, were not detected on
this RT-PCR, which indicated that isoforms 3, 4, and 5 are minor
transcripts in Colo205 cells. RT-PCR with primers si-4 and si-1 gave a
single band for isoform 3, which was digested by both BsmI
and XbaI. A band for isoform 4 was not detected on this
RT-PCR, because the amount of isoform 4 transcripts may be very small.
RT-PCR with primers si-2 and si-3 gave a single band for isoform 5. Isoform 1 and 2 were abundant among the five isoforms, and both
isoforms amounted to approximately 50% of the total transcripts of the
3Gal-T5 gene, respectively. The other three isoforms,
i.e. isoforms 3, 4, and 5, were only present in trace
amounts.
Correlation of the Expression Levels of the
3Gal-T5 Transcripts
with the Amounts of Type 1 Lewis Antigens in Various Tumor
Cells--
As can be seen in Fig. 4, the
expression levels of
3Gal-T5 transcripts and the amounts of CA19-9
antigens in various cancer cells were determined by the competitive
RT-PCR method and Western blot analysis, respectively. The results of
flow cytometry analysis in Fig. 1 are well consistent with those of
Western blot analysis in this section, i.e. the four types
of cells, i.e. Colo205, Colo201, SW1116, and Capan-2 cells,
that were stained strongly with 19-9 on flow cytometry also gave strong
positive bands, which were smear ones with high molecular weights
indicating they are mucins, with 19-9 on Western blotting (Fig. 4).
These four cell lines also expressed abundant
3Gal-T5 transcripts
(Fig. 4). The other cell lines, HT-29, WiDr, and Capan-1 cells,
intermediately expressed the
3Gal-T5 gene.

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Fig. 4.
Quantitative analysis of 3Gal-T5
transcripts by competitive RT-PCR, and Western blot analysis of CA19-9
antigens in various cancer cells. A, the expression
levels of 3Gal-T5 transcripts in various cancer cells are shown as a
bar chart. The results of actual gel
electrophoresis after competitive RT-PCR are shown under the
bars. B, Western blot analysis of various cancer
cells with 19-9 (anti-sLea).
|
|
The Ability of Type 1 Chain Synthesis of
3Gal-T5 in Transfected
Cells--
Namalwa cells do not possess the type 1 chain and lack
1,3/4-fucosyltransferase (Fuc-TIII), which is the only enzyme
capable of the synthesis of type 1 Lewis antigens, such as the
Lea, Leb, and sLea antigens.
Therefore, Namalwa cells transfected stably with the
3Gal-T5 gene were stained with DU-PAN-2
(anti-sLec), which recognizes the precursor structure,
SA
2,3Gal
1,3GlcNAc, of the sLea epitope (3, 4). As can
be seen in Fig. 5, Namalwa cells transfected stably with the
3Gal-T5 gene gave positive
peaks with DU-PAN-2. HCT-15 cells were chosen as the host cells for the
transfection experiment with the
3Gal-T5 gene for the
following reasons. First, they are cancer cells derived from colon
tissue. Second, they are known to express substantial amounts of
Fuc-TIII and ST3GalIV (data not shown), but not to express
3Gal-T5
at all (Fig. 4). A single transformant clone of HCT-15 cells, which had
been transfected stably with the
3Gal-T5 gene, was
obtained by the limiting dilution method and named HCT-3GT5H. Flow
cytometry analysis of HCT-3GT5H cells apparently showed positive peaks
with the antibodies against all type 1 Lewis antigens (Fig. 5). These results confirmed that
3Gal-T5 can synthesize the type 1 chain in
transformant cells, i.e. not only in Namalwa cells but also in colon cancer (HCT-15) cells.

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Fig. 5.
Flow cytometry analysis of Namalwa cells and
HCT-15 cells transfected stably with the 3Gal-T5 cDNA. The
shaded peak in each panel represents the results
of staining without the first antibody as a negative control. The
results for mock-transfected Namalwa or HCT-15 cells are presented in
the left panels. The results for Namalwa-3GT5 or
HCT-3GT5H cells, which were stably transfected with the
3Gal-T5 gene, are presented in the right
panels.
|
|
3Gal-T Activity toward Agalacto-LNnT in Various Tumor Cells and
HCT-3GT5 Cells--
Agalacto-LNnT-PA was used as an acceptor substrate
to measure
3Gal-T activity, resulting in the synthesis of
lacto-N-tetraose-PA (LNT-PA). The
3Gal-T activity,
i.e. the LNT-PA synthesizing activity, in Namalwa cells
transfected with the
3Gal-T5 gene, Namalwa-3GT5, was
strongest among all samples of transfected cells and cultured cancer
cells examined. Thus, the activity of Namalwa-3GT5 is expressed as
100% activity, and the
3Gal-T activities of the other samples relative to that of Namalwa-3GT5 cells are presented in Table IV. The amounts of transcripts for
individual
3Gal-T genes were measured by competitive
RT-PCR, and the relative amounts of individual transcripts normalized
as to the amounts of
-actin transcripts are shown in Table IV. The
level of
3Gal-T activity synthesizing LNT-PA was quite parallel to
the amounts of
3Gal-T5 transcripts, i.e. the cell
homogenate of Colo205 showed the strongest activity among the cultured
cancer cells, followed by those of SW1116 and Capan-2. This indicated
that the LNT-PA synthesizing activity is mainly directed by
3Gal-T5.
Mock-transfected HCT-15 (HCT-mock) cells did not exhibit any activity,
but HCT-3GT5H cells exhibited strong activity almost equal to that of
Colo205 cells. HCT-3GT5L cells, which expressed almost one-third of the
amount of
3Gal-T5 transcripts in the HCT-3GT5H cells, showed
one-third of the
3Gal-T activity of HCT-3GT5H cells. The cells
expressing substantial amounts of transcripts for the other four
3Gal-T genes, i.e. the
3Gal-T1,
-T2, -T3, and -T4 genes, did not
exhibit LNT-PA synthesizing activity at all. Namalwa cells stably
expressing
3Gal-T1, -T2, -T3 and -T4, which were named Namalwa-3GT1,
-3GT2, -3GT3, and -3GT4 cells, respectively, also did not exhibit
any LNT-PA synthesis activity.
The above results confirmed that the
3Gal-T activity synthesizing
LNT-PA is mainly directed by
3Gal-T5 in these cells, not by the
other four
3Gal-Ts.
Tissue Distribution and Quantitative Measurement of the
3Gal-T5
Transcripts--
As can be seen in Fig.
6,
3Gal-T5 transcripts were
substantially detected in the stomach, jejunum, colon, and pancreas,
which are known to express sLea antigens frequently when
they become cancerous. On the other hand, they were hardly detected in
the lungs, liver, spleen, adrenal glands, and peripheral blood
leukocytes, which rarely produce sLea antigens when they
become malignant. This strongly suggested that
3Gal-T5 is
responsible for the type 1 chain synthesis, resulting in the
sLea antigen synthesis in gastrointestinal and other
tissues.

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Fig. 6.
Quantitative analysis of 3Gal-T
transcripts in various human tissues by competitive RT-PCR. For
the quantitation of 3Gal-T5 transcripts, each single-stranded
cDNA was amplified together with 200 ag/µl of the respective
competitor DNA. The human -actin transcripts were quantified with
500 fg/µl of the competitor DNA. The amount of -actin differed
with the tissue.
|
|
Multiple Sequence Alignment (ClustalW) of the Five Members of the
Human
3Gal-T Family--
Multiple amino acid sequence alignment of
the five
3Gal-Ts was constructed by ClustalW method (Fig.
7). Three conserved amino acid motifs,
which were employed for design of the degenerate primers, and four
conserved cysteine residues were indicated in Fig. 7. One possible
N-glycosylation site was also conserved in all five
h
3Gal-Ts.

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Fig. 7.
Multiple sequence alignment (ClustalW) of the
five human 3Gal-Ts. Multiple amino acid sequences of the five
human 3Gal-Ts are shown. Introduced gaps are shown as
hyphens. Three conserved motifs used for design of
degenerate primers were shaded. Putative transmembrane
domains are boxed, the four conserved cysteine residues are
squared, and the conserved possible
N-glycosylation site is double underlined.
Asterisks indicate the amino acids conserved in the five
3Gal-Ts.
|
|
A phylogenetic tree of the five
3Gal-Ts was constructed by means of
the neighbor-joining method based on the amino acid sequences (data not
shown) (23). The position of
3Gal-T5 in the tree is closer to those
of
3Gal-T1 and -T2, which can synthesize the type 1 chain as
reported by us and others (10-12), than to those of
3Gal-T3 and
-T4. These three enzymes,
3Gal-T1, -T2, and -T5, form a subfamily on
the phylogenetic tree.
 |
DISCUSSION |
Expression of the sLea and sLec epitopes,
which are mainly carried on the carbohydrate chains of mucins, is
frequently elevated in gastrointestinal and pancreatic cancers. The
sLea antigens are known to be some of the factors
determining the prognoses of colorectal and gastric cancer patients (7,
8, 24). In this sense, it is very important to identify the
3GalT(s) responsible for type 1 chain synthesis, which results in the synthesis of the sLec and sLea epitopes in
gastrointestinal and pancreatic cancers. The novel
3Gal-T,
i.e.
3Gal-T5, isolated in the present study was
demonstrated to be responsible for the type 1 chain (including the
sLea and sLec antigen) synthesis in
gastrointestinal and pancreatic cancers by the following evidence. 1)
3Gal-T5 could synthesize type 1 chains, leading to expression of
Lea, Leb, sLec and sLea
in transfected cells, i.e. in Namalwa-3GT5 and HCT-3GT5H
cells. 2)
3Gal-T5 was expressed in cultured colon and pancreatic
cancer cells expressing substantial amounts of type 1 chains, whereas the other three
3Gal-Ts, i.e.
3Gal-T1, -T2, and -T3,
were not expressed in these cells. 3) The expression levels of
3Gal-T5 transcripts were well correlated with those of type 1 Lewis
antigens in cultured cancer cells. 4) In the transfection experiment
involving Namalwa cells, only
3Gal-T5 exhibited the LNT-PA
synthesizing activity, whereas the other four
3Gal-Ts did not. 5)
The expression level of
3Gal-T5 transcript in cultured cancer cells
was well correlated with the LNT-PA synthesizing activity.
Kolbinger et al. demonstrated that
3Gal-T2 was able to
synthesize type 1 chains in CHO cells through a transfection experiment (11). Amado et al. detected the
3Gal-T activities of
3Gal-T1 and -T2 (12). Hennet et al. demonstrated the
3Gal-T activities of three mouse
3Gal-Ts, i.e.
m
3GalT-I, -II and -III (14). In contrast, we could not detect LNT-PA
synthesis activity in the Namalwa cells transfected stably with each of
the
3Gal-T1, -T2, or -T3 genes.
This was probably due to the amounts or structures of the enzymes used
or the sensitivity of the assay system. In a previous study, we
detected the activity of
3Gal-T1 using purified
3Gal-T1 that was
expressed as a secreted form fused with the IgG binding domain of
Staphylococcus aureus protein A (10). The other three
groups, i.e. Hennet et al. (14), Kolbinger
et al. (11), and Amado et al. (12), assayed
3Gal-T activity by measuring radioisotope incorporation using
recombinant enzymes produced in soluble forms with a baculo-expression
system or the S. aureus protein A fusion system, whereas we
used cell homogenates as enzyme sources and a pyridylaminated acceptor
substrate in the present study.
We apparently detected LNT-PA synthesizing activity in
Namalwa-3GT5 cells and cultured cancer cells expressing
3Gal-T5,
whereas Namalwa cells transfected with the other four
3Gal-T genes and cultured cells endogenously expressing
3Gal-T1 (PC-1),
3Gal-T2 (Namalwa), or
3Gal-T3 (MKN45) did not
exhibit LNT-PA synthesizing activity at all. This indicated that
3Gal-T5 possesses the strongest activity as to type 1 chain
synthesis among the four
3Gal-Ts, i.e.
3Gal-T1, -T2,
-T3, and -T5. The
3Gal-T activity synthesizing LNT-PA in Colo205,
SW1116, and Capan-2 cells decreased in accordance with the amount of
the
3Gal-T5 transcript expressed in these cells (Table IV), and the
activity was not detected in cells that did not express
3Gal-T5.
These findings strongly indicated that the endogenous LNT-PA
synthesizing activity in cell lines such as Colo205, SW1116, etc., is
mainly directed by
3Gal-T5. Holmes reported that partially purified
3Gal-T(s) from Colo205 cells exhibit preferential activity toward
lactotriaosylceramide (Lc3), GlcNAc
1-3Gal
1-4Glc
1-1Cer (25). Valli et al.
found that the
3Gal-T activity in homogenates of human colorectal
cancer cell lines is correlated with the expression levels of type 1 Lewis antigens (26). Their results are consistent with the
3Gal-T5 activity demonstrated in this study in these cultured cell lines. The
3Gal-T activity detected in their studies may be attributed to that
of
3Gal-T5.
Western blot analysis revealed that the amounts of
3Gal-T5
transcripts were also well correlated with those of sLea
antigens on proteins, probably on mucins in colorectal and pancreatic cancer cells. This means that
3Gal-T5 utilizes carbohydrate chains on proteins as acceptor substrates. In the future, the substrate specificities of
3Gal-T5, as well as the other
3Gal-Ts, should be
examined by employing substrates as analogous as possible to physiological carbohydrate structures.
3Gal-T5 was demonstrated to be physiologically expressed in a set of
gastrointestinal and other tissues. The substantial amounts of
3Gal-T5 transcripts are expressed in stomach, jejunum, colon, and
pancreas, strongly suggesting that
3Gal-T5 is responsible for
expressing sLea antigens when those tissues become cancerous.
We previously determined the expression levels of 12 glycosyltransferase genes, i.e. those of five
1,3-fucosyltransferases (Fuc-TIII, -TIV, -TV, -TVI, and -TVII), four
ST3Gals (ST3Gal I, -II,- III, and -IV), one ST6Gal (ST6Gal I),
4Gal-T1, and core2-GlcNAc transferase, in colorectal cancer tissues
in order to correlate them with the amounts of the sLex and
sLea antigens (9). Although Fuc-TIII and ST3Gal IV are
essentially required for the sLea synthesis in colorectal
cancers, no single enzyme among the 12 was correlated with the amounts
of sLea antigens. Therefore, we conjectured in the previous
study that the combinatorial up-regulated expression of multiple
enzymes determines the amounts of the antigens. However, it is a
noteworthy finding in the present study that the expression levels of
3Gal-T5 were well correlated with the amounts of sLea
antigens and the other type 1 Lewis antigens in the cultured cancer
cells. This means that
3Gal-T5 is a key enzyme determining the
expression levels of type 1 Lewis antigens including sLea
antigens in these cells. The above, together with the results of the
present study, indicated that
3Gal-T5 and ST3Gal IV are responsible
for sLec synthesis, and Fuc-TIII is further required for
sLea synthesis in colorectal cancers in addition to
3Gal-T5 and ST3Gal IV.
On the other hand, the type 2 chain, Gal
1,4GlcNAc, was found to be
expressed in all cell lines examined in the present study, since
4Gal-T1 is a ubiquitous enzyme, and was substantially expressed in
all cell lines (data not shown).
It is of interest to determine whether or not the up-regulation of the
3Gal-T5 gene expression determines the levels of
sLea antigens in native cancer tissues. If this is the
case, transcriptional regulation of the
3Gal-T5 gene will
be an attractive subject in the future. In this study, we determined
the transcription initiation site of the
3Gal-T5 gene in
Colo205 cells. The nucleotide sequence in the upstream region was
examined for the binding sites of transcription factors using the
TFSEARCH (transcription factor search)
program,3 based on the data
bases deposited by Heinemeyer et al. (27). We searched the
1-kilobase pair upstream region from the transcription initiation site
of the
3Gal-T5 gene, but found no TATA box. Within 150 bp
upstream of the transcription initiation site, two CdxA sites, an AP-1
site, and a myeloid zinc finger 1 protein, MZF1, site were found. AP-1
and MZF1 have been reported to be potential targets of neoplastic
transformation (28, 29). CdxA, known as a chicken
homeobox-containing gene related to caudal in
Drosophila, was previously shown to be expressed in the
endoderm-derived gut epithelium during early embryogenesis (30). In the
future, we will examine whether or not these transcription factors
function in regulation of the
3Gal-T5 gene.
Finally, the results of the present study strongly indicate that
3Gal-T5 is the most probable candidate responsible for the sLea antigen synthesis in gastrointestinal and pancreatic
cancer cells. In the future, it will be interesting to determine
whether or not expression of the
3Gal-T5 gene changes
some characteristics of cancer cells, especially those related to malignancy.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Nobuyuki Imai, Dr. Kyoko
Takeuchi, Dr. Satoshi Nakagawa, Sachiko Kodama, Hiromi Inagaki, Reiko
Koda, and Hiromi Ohara of Kyowa Hakko Kogyo Co. for cloning and
sequencing of the four
3Gal-T genes,
3Gal-T1, -T2, -T3, and
-T4. We also thank Dr. Satoshi Ito, of Fujirebio Inc., and
Dr. Tetsuya Tachikawa, of Diagnostic Division, Otsuka Pharmaceutical
Co. Ltd. for the kind gifts of the 19-9 and TT42 antibodies, respectively.
 |
FOOTNOTES |
*
This work was supported in part by Grant-in-aid for
Scientific Research on Priority Areas 10178104, from the Ministry of
Education, Science, and Culture of Japan.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.
The nucleotide sequence reported in this paper has been submitted
to the DDBJ/GenBankTM/EBI Data Bank with accession number
AB020337.
¶
These authors contributed equally to this work and should be
considered as first authors.

To whom correspondence and reprint requests should be
addressed: Div. of Cell Biology, Inst. of Life Science, Soka
University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan. Tel.:
81-426-91-9466; Fax: 81-426-91-9315; E-mail:
hisashi{at}scc1.t.soka.ac.jp.
2
The NIH Image program was developed
at the National Institutes of Health and is available through the
Internet by anonymous FTP from zippy.nimh.nih.gov or on a floppy disk
from the National Technical Information Service, Springfield, VA (part
no. PB95-500195GEI).
3
The TFSEARCH program was developed by Y. Akiyama
and is available via the World Wide Web
(http://www.rwcp.or.jp/ lab/pdappl/papia.html).
 |
ABBREVIATIONS |
The abbreviations used are:
19-9, 1116NS19-9;
sLea, sialyl Lewis a;
sLex, sialyl Lewis x;
Lea, Lewis a;
Leb, Lewis b;
PCR, polymerase
chain reaction;
RT-PCR, reverse transcription-polymerase chain
reaction;
Fuc, fucose;
SA, sialic acid;
ORF, open reading frame;
5'-RACE, 5'-rapid amplification of cDNA ends;
EST, expressed
sequence tags;
PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;
bp, base pair(s);
3Gal-T, UDP-galactose:
-N-acetylglucosamine:
1,3-galactosyltransferase;
LNT, lacto-N-tetraose;
LNnT, lacto-N-neotetraose.
 |
REFERENCES |
-
Magnani, J. L.,
Steplewski, Z.,
Koprowski, H.,
and Ginsburg, V.
(1983)
Cancer Res.
43,
5489-5492[Abstract]
-
Magnani, J. L.,
Nilsson, B.,
Brockhaus, M.,
Zopf, D.,
Steplewski, Z.,
Koprowski, H.,
and Ginsburg, V.
(1982)
J. Biol. Chem.
257,
14365-14369[Abstract/Free Full Text]
-
Narimatsu, H.,
Iwasaki, H.,
Nishihara, S.,
Kimura, H.,
Kudo, T.,
Yamauchi, Y.,
and Hirohashi, S.
(1996)
Cancer Res.
56,
330-338[Abstract]
-
Narimatsu, H.,
Iwasaki, H.,
Nakayama, F.,
Ikehara, Y.,
Kudo, T.,
Nishihara, S.,
Sugano, K.,
Okura, H.,
Fujita, S.,
and Hirohashi, S.
(1998)
Cancer Res.
58,
512-518[Abstract]
-
Nguyen, M.,
Strubel, N. A.,
and Bischoff, J.
(1993)
Nature
365,
267-269[CrossRef][Medline]
[Order article via Infotrieve]
-
Takada, A.,
Ohmori, K.,
Yoneda, T.,
Tsuyuoka, K.,
Hasegawa, A.,
Kiso, M.,
and Kannagi, R.
(1993)
Cancer Res.
53,
354-361[Abstract]
-
Nakayama, T.,
Watanabe, M.,
Katsumata, T.,
Teramoto, T.,
and Kitajima, M.
(1995)
Cancer
75,
2051-2056[Medline]
[Order article via Infotrieve]
-
Nakayama, T.,
Watanabe, M.,
Teramoto, T.,
and Kitajima, M.
(1997)
Anticancer Res.
17,
1379-1382[Medline]
[Order article via Infotrieve]
-
Kudo, T.,
Ikehara, Y.,
Togayachi, A.,
Morozumi, K.,
Watanabe, M.,
Nakamura, M.,
Nishihara, S.,
and Narimatsu, H.
(1998)
Lab. Invest.
78,
797-811[Medline]
[Order article via Infotrieve]
-
Sasaki, K., Sasaki, E., Kawashima, K., Hanai, N., Nishi, T. & Hasegawa,
M. (July 5, 1994) Japanese Patent 0618759 A 940705
-
Kolbinger, F.,
Streiff, M. B.,
and Katopodis, A. G.
(1998)
J. Biol. Chem.
273,
433-440[Abstract/Free Full Text]
-
Amado, K.,
Almeida, R.,
Carneiro, F.,
Leverly, S. B.,
Holmes, E. H.,
Nomoto, M.,
Hollingsworth, M. A.,
Hassan, H.,
Schwientek, T.,
Nielsen, P. A.,
Bennett, E. P.,
and Clausen, H.
(1998)
J. Biol. Chem.
273,
12770-12778[Abstract/Free Full Text]
-
Miyazaki, H.,
Fukumoto, S.,
Okada, M.,
Hasegawa, T.,
Furukawa, K.,
and Furukawa, K.
(1997)
J. Biol. Chem.
272,
24794-24799[Abstract/Free Full Text]
-
Hennet, T.,
Dinter, A.,
Kuhnert, P.,
Mattu, T. S.,
Rudd, M. P.,
and Berger, E. G.
(1998)
J. Biol. Chem.
273,
58-65[Abstract/Free Full Text]
-
Hamanaka, Y.,
Hamanaka, S.,
Shinagawa, Y.,
Suzuki, T.,
Inagaki, F.,
Suzuki, M.,
and Suzuki, A.
(1994)
FEBS Lett.
353,
48-52[CrossRef][Medline]
[Order article via Infotrieve]
-
Torrado, J.,
Correa, P.,
Ruiz, B.,
Bernardi, P.,
Zavala, D.,
and Bara, J.
(1992)
Gastroenterology
102,
424-430[Medline]
[Order article via Infotrieve]
-
Kudo, T.,
Ikehara, Y.,
Togayachi, Y.,
Kaneko, M.,
Hiraga, T.,
Sasaki, K.,
and Narimatsu, H.
(1998)
J. Biol. Chem.
273,
26729-26738[Abstract/Free Full Text]
-
Sasaki, K.,
Kurata, K.,
Funayama, K.,
Nagata, M.,
Watanabe, E.,
Ohta, S.,
Hanai, N.,
and Nishi, T.
(1994)
J. Biol. Chem.
269,
14730-14737[Abstract/Free Full Text]
-
Chomczynski, P.,
and Sacchi, N.
(1987)
Anal. Biochem.
162,
156-159[CrossRef][Medline]
[Order article via Infotrieve]
-
Yago, K.,
Zenita, K.,
Ginya, H.,
Sawada, M.,
Ohmori, K.,
Okuma, M.,
Kannagi, R.,
and Lowe, J. B.
(1993)
Cancer Res.
53,
5559-5565[Abstract]
-
Kozak, M.
(1989)
J. Cell Biol.
108,
229-241[Abstract]
-
Kyte, J.,
and Doolittle, R. F.
(1982)
J. Mol. Biol.
157,
105-132[Medline]
[Order article via Infotrieve]
-
Saitou, N.,
and Nei, M.
(1987)
Mol. Biol. Evol.
4,
406-425[Abstract]
-
Nakamori, S.,
Furukawa, H.,
Hiratsuka, M.,
Iwanaga, T.,
Imaoka, S.,
Ishikawa, O.,
Kabuto, T.,
Sasaki, M.,
Kameyama, M.,
Ishiguro, S.,
and Irimura, T.
(1997)
J. Clin. Oncol.
15,
816-825[Abstract]
-
Holmes, E. H.
(1989)
Arch Biochem. Biophys.
270,
630-646[Medline]
[Order article via Infotrieve]
-
Valli, M.,
Gallanti, A.,
Bozzaro, S.,
and Trinchera, M.
(1998)
Eur. J. Biochem.
256,
494-501[Abstract]
-
Heinemeyer, T.,
Wingender, E.,
Reuter, I.,
Hermjakob, H.,
Kel, A.,
Kel, O.,
Ignatieva, E.,
Ananko, E.,
Podkolodnaya, O.,
Kolpakov, F.,
Podkolodny, N.,
and Kolchanov, N.
(1998)
Nucleic Acid Res.
26,
362-367[Abstract/Free Full Text]
-
Bohmann, D.,
Bos, T. J.,
Admon, A.,
Nishimura, T.,
Vogt, P. K.,
and Tjian, R.
(1987)
Science
238,
1386-1392[Medline]
[Order article via Infotrieve]
-
Hromas, R.,
Morris, J.,
Cornetta, K.,
Berebitsky, D.,
Davidson, A.,
Sha, M.,
Sledge, G.,
and Rauscher, F. R.
(1995)
Cancer Res.
55,
3610-3614[Abstract]
-
Frumkin, A.,
Haffner, R.,
Shapira, E.,
Tarcic, N.,
Gruenbaum, Y.,
and Fainsod, A.
(1993)
Development
118,
553-562[Abstract/Free Full Text]
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