(Received for publication, October 23, 1996, and in revised form, January 3, 1997)
From the Division of Human Genetics, Department of Forensic Medicine, Kurume University School of Medicine, Kurume, Fukuoka 830, Japan
The expression of the ABO antigens on erythrocyte
membranes is regulated by H gene (FUT1)-encoded
(1,2)fucosyltransferase activity. We have examined the expression of
the FUT1 in several tumor cell lines, including erythroid
lineage and normal bone marrow cells, by Northern blot and/or reverse
transcription-polymerase chain reaction (RT-PCR) analyses. RT-PCR
indicated that bone marrow cells, erythroleukemic cells (HEL), and
highly undifferentiated leukemic cells (K562) that have erythroid
characteristics expressed the FUT1 mRNA while four
leukemic cell lines did not. The FUT1 mRNA was also
demonstrated in gastric, colonic, and ovarian (MCAS) cancer cell lines
by RT-PCR. Northern blot analysis indicated that a 4.0-kilobase
FUT1 transcript was expressed in some of these tumor cell
lines. Rapid amplification of 5
cDNA end (RACE) analysis suggested
that the FUT1 transcript had several forms generated by two
distinct transcription start sites and alternative splicing. The
results of RT-PCR using specific primers for each starting exon
suggested that two transcription initiation sites (exon 1A and exon 2A)
of the FUT1 were identified in gastric cancer cells and in
ovarian cancer cells. Only exon 1A was identified as a transcription
start site in another gastric cancer cell line, two colonic cancer cell
lines, and in K562 cells, whereas only exon 2A was identified in HEL
cells and in bone marrow cells. These two transcription start sites
were located 1.8 kilobases apart. Therefore, two distinct promoters
appeared to be present in the FUT1. The distinct promoters
of the FUT1 and alternative splicing of the
FUT1 mRNA may be associated with time- and
tissue-specific expression of the FUT1.
The ABO(H) histoblood group antigens are oligosaccharides (1), and
their biosynthesis is regulated by several glycosyltransferases that
add monosaccharides to a precursor molecule in a sequential fashion (2,
3). The (1,2)fucosyltransferase1 that
forms the H antigen, an essential precursor of the A and B antigens,
plays a regulatory role in the tissue expression of ABO antigens. These
antigens are found not only on erythrocyte membranes but also in most
epithelial cells and in body fluids. Several lines of evidence have
indicated that at least two distinct
(1,2)fucosyltransferases are
present in human tissues (4-9). One is the H gene
(FUT1)-encoded
(1,2)fucosyltransferase (H enzyme) and the
other is the Secretor gene (FUT2)-encoded
(1,2)fucosyltransferase (Se enzyme). The H enzyme regulates the
expression of the H antigen mainly on erythrocyte membranes, while the
Se enzyme regulates the expression of the H antigen mainly in
epithelial cells and in body fluids such as saliva (2, 3, 10).
It is well known that the glycosylation patterns including ABH antigens
are changed during embryonic development, cell maturation, and
malignant transformation (1, 11). In early embryos, the ABH antigens
are expressed on cell surfaces of red blood cells and of endothelial
and epithelial cells of most organs. The surface expression of the ABH
antigens on epithelial cells reaches a maximum at about nine weeks and
thereafter decreases. Some cells such as the neurons, muscle cells, and
bone cells completely lose the capacity to synthesize ABH antigens. On
digestive mucosa, a decrease in the cell surface expression of ABH
antigens coincides with the onset of mucus secretion (12). One of the
well examined digestive mucosa for the expression of ABH and related
antigens is colorectal mucosa. While the ABH, Lewis y, and Lewis b are expressed in fetal distal colorectal mucosa, these antigens disappear in adult distal colorectal mucosa (13). However, these antigens are
re-expressed in colorectal carcinoma (13). The expression of H, Lewis
y, and Lewis b antigens in colorectal carcinoma are thought to be
regulated by both H and Se enzymes (13), and the level of the
FUT1 transcript was increasing during malignant
transformation in colorectal mucosa (14). Therefore, analyses of the
gene structure and promoter region that regulate the expression of the
(1,2)fucosyltransferase genes are important for understanding stage-
and tissue-specific expression of the H and H-related antigens.
Recently, the FUT1, FUT2, and a FUT2-pseudogene (Sec1) have been isolated (15, 16). These genes and a pseudogene share a high degree of DNA sequence homology and are located within a 100-kb region on chromosome 19q13.3 (17), suggesting that they were generated by gene duplication from the same ancestor gene and then subsequently diverged. Recently, Kelly et al. (18) reported the gene structure and transcription initiation site of the FUT1 in A431 cells. Here, we have examined the expression of the FUT1 and found several forms of FUT1 transcripts in several tumor cell lines including the erythroid lineage. We have identified two distinct transcription start sites of the FUT1 that were different from one previously described (18).
Human cancer cell lines MCAS (ovarian cancer), COLO201 and WiDr (colon cancer), KATOIII and MKN74 (gastric cancer), and HEL (erythroleukemia) cells were obtained from the Health Science Research Resources Bank, Osaka, Japan. Human hematopoietic cell lines K562 (chronic myelogenous leukemia, blast crisis), HL60 (promyelocytic leukemia), U937 (histiocytic lymphoma), BALL-1 (B-cell lymphoblastic leukemia), and MOLT-4 (T-cell lymphoblastic leukemia) cells were a kind gift from Dr. K. Sagawa (Kurume University). Total cytoplasmic RNA was isolated from these cells using the acid guanidinium thiocyanate/phenol/chloroform method (19). For Northern blot analysis, total RNA (20 µg) was denatured, divided into 10 µg/lane, and separated by 1.2% formaldehyde-agarose gel electrophoresis and then transferred onto a nylon membrane (Hybond N+, Amersham International, Tokyo) (20, 21). The membrane was divided to prepare duplicate membranes. One membrane was stained by 0.04% methylene blue in 0.5 M sodium acetate (pH 5.2) (22). The other membrane was hybridized with digoxigenin-labeled FUT1 antisense RNA probe for the total protein coding region. The digoxigenin-labeled RNA probe was prepared using the DIG RNA labeling kit (Boehringer Mannheim, Mannheim, Germany). Hybridization was carried out at 68 °C with 5 × SSC, 50% formamide, 2 × Denhardt's reagent, and 0.5% SDS overnight. The final wash was carried out at 68 °C with 0.2 × SSC and 0.1% SDS for 40 min. Detection of hybridized bands was performed using a DIG luminescent detection kit for nucleic acids (Boehringer Mannheim).
PCR AmplificationAll PCR amplifications (23) were performed in a 50-µl reaction mixture containing 10 pmol of each primer, 1.2 units EX Taq DNA polymerase (Takara, Kyoto, Japan), 200 µM dNTP, and 1 × EX Taq buffer.
Reverse Transcription PCR (RT-PCR)Synthesis of single
strand cDNA of several kinds of cultured cells was performed on
total RNA (2 µg) using SUPERSCRIPT preamplification system (Life
Technologies, Inc.) according to the manufacturer instructions. 1 µl
(total 20 µl) of resultant single strand cDNA was used as the
template for PCR. The RT-PCR primers for the amplification of
FUT1 (15) and glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) gene (24) are listed in Table I. The temperature
profile of all RT-PCRs was as follows: denature at 94 °C for 20 s, annealing at 65 °C for 1 min, extension at 72 °C for 1 min,
and 25 cycles for G3PDH and 30 cycles for FUT1. The starting
exon-specific RT-PCR was performed using each distinct starting
exon-specific primer (Table II) and the 3-FUT1 primer
(Table I) (denature at 98 °C for 10 s, annealing at 68 °C
for 1 min, extension at 72 °C for 2 min, and 30 cycles). For
identification of starting exons of the FUT1 mRNA in the
normal erythroid progenitor cells, first strand cDNA synthesis was
performed on 500 ng of human bone marrow poly(A)+ RNA
(Clontech, Palo Alto, CA), as mentioned above. Then the starting exon-specific PCR was performed. The products were analyzed by 1.0%
agarose gel electrophoresis and stained with ethidium bromide. No PCR
product was amplified in the control reactions without reverse
transcriptase.
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Poly(A)+ RNA was isolated from total cellular
RNA (100-500 µg) by Oligotex-dT30 (Takara). Double-stranded cDNA
synthesis and adapter ligation were performed using the Marathon
cDNA amplification kit (Clontech). 5 µl of the double strand
cDNA (250-fold diluted) were used as the template for the RACE
analysis. The first PCR was performed using 3-FUT1 primer (Table I)
and the AP 1 primer (provided by the supplier). Nested PCR was
performed using FUT1 nest primer (Table I) and the AP 2 primer
(provided by the supplier). The temperature profile of all PCR for RACE
analysis was as follows: denature at 98 °C for 10 s, annealing
and extension at 68 °C for 3 min, and 30 cycles. The 5
-RACE product
was cloned into pGEM using the pGEM-T vector system I (Promega,
Madison, WI) for sequencing.
The genomic DNA of the FUT1 (from exon 1 to
exon 3) was amplified by PCR using 100 ng of genomic DNA as the
template. The 5-Ex1 (upper primer, Table II) and FUT1 nest (lower
primer, Table I, and see Fig. 3C) primers were used for
amplification of the FUT1 (denature at 98 °C for 10 s, annealing and extension at 68 °C for 4 min, and 30 cycles). The
PCR product of the FUT1 genomic DNA between exons 1 and 3 was cloned into pGEM for DNA sequencing.
Construction of Luciferase Reporter Gene Plasmid
To create
constructs of the luciferase fusion genes, a 0.7-kb
PvuII-AatI fragment (see Fig. 5) of pGEM
containing the 2.8-kb PCR fragment between exons 1 and 3 of the
FUT1 was subcloned into pGL2-enhancer vector (Promega). The
plasmid DNA was purified with Qiagen tips (Qiagen Inc., Chatsworth, CA)
and transfected into MCAS cells using Trans-IT (Takara) as described
previously (20, 21). After 48 h, the cells were lysed, and the
luciferase activity was measured using the luciferase assay system
(Promega) and a luminometer (Lumat LB9501, Berthold, Wildbad, Germany).
The relative promoter activity was indicated by relative light units
obtained from transfection with pGL2-control vector as 100%.
Transfection efficiency was normalized as described previously
(21).
DNA Sequencing
Double-stranded plasmid DNA containing the FUT1 fragment was sequenced in both orientations using an AutoRead DNA sequencing kit and an ALF DNA sequencer (Pharmacia, Uppsala, Sweden) or using an ABI PRISM dye terminator cycle sequencing ready reaction kit and an ABI 373 sequencer (Applied Biosystems).
Northern
blot analysis indicated that about 4.0 kb of the transcripts of the
FUT1 were detected in total RNA from HEL and MCAS cells but
not from WiDr cells (Fig. 1A). The transcript
with the same size was also detected in total RNA from KATOIII cells but not from COLO201 and MKN74 cells (data not shown). However, the
FUT1 transcript was detected in all these cell lines (Fig. 1B) and in undifferentiated leukemic K562 cells by RT-PCR
analysis, whereas other leukemic cells (HL60, U937, BALL-1, and MOLT-4) tested did not express the FUT1 (data not shown). The
results indicated that epithelial cancer cells expressed
FUT1 mRNA, while many leukemic cells did not express the
mRNA. However, erythroid lineage HEL cells (25) and
undifferentiated leukemia K562 cells, which has erythroid
characteristics (26), expressed the FUT1 mRNA.
Identification of the 5
To isolate the 5-end of FUT1 cDNA,
RACE was performed using 1 µg of either poly(A)+ RNA from
HEL or MCAS cells or of human bone marrow Marathon-Ready cDNA
(Clontech). Several different sizes of 5
-RACE products of the
FUT1 were amplified from cDNA prepared from bone marrow,
HEL, and MCAS cells (Fig. 2). DNA sequence analysis of
15 clones of the 5
-RACE products of the FUT1 from each of
the cells indicated that the bone marrow and the HEL cells had two and
four different forms by alternative splicing with a single
transcription initiation site, respectively, while the MCAS cells had
seven forms with two distinct transcription initiation sites and
alternative splicing (Fig. 3, B and
C). Although we could not identify accurately the 5
-ends of
the FUT1 by RACE analysis, the longest 5
-ends of each starting exon are shown in Fig. 3C. To analyze the
transcription initiation sites in each cell line, RT-PCR was carried
out using a primer specific for each of the two distinct starting exons (5
-Ex1 primer for exon 1A or 5
-Ex2 primer for exon 2A, see Table II).
The results also indicated that both exon 1A and exon 2A were
transcription start sites in MCAS and KATOIII cells, while only exon 1A
was a start site in MKN74, WiDr, and COLO201 cells (Fig.
4). In hematopoietic cells, only exon 1A was used as a
transcription start site in K562 cells, while only exon 2A was used in
HEL cells (Fig. 4). As in HEL cells, exon 2A but not exon 1A was a
transcription start site of the FUT1 mRNA in normal bone
marrow cells (Fig. 4). Since the mature peripheral leukocytes (data not
shown) and four leukemic cell lines did not express FUT1
mRNA by RT-PCR analysis, the results suggested that normal
erythroid progenitor cells in bone marrow used only one promoter
present in the 5
-flanking region of FUT1 exon 2.
Identification of the FUT1 Gene Structure
To identify the FUT1 gene structure, the genomic DNA sequence between exons 1 and 3 of the FUT1 was amplified by PCR. DNA sequence analysis indicated that the first and second introns of the FUT1 were 1654 and 202 bp, respectively, and the all exon/intron junctions of these genes were compatible with GT/AG rule (Fig. 3C). The gene structure of the FUT1 is shown in Fig. 3A.
Analyses of DNA Sequence and Promoter Activity of the 5Since the 5-flanking region of
exon 2 (or first intron) of FUT1 appeared to act as a
promoter, we isolated this region and DNA sequence analysis was
performed. The 5
-flanking region of the exon 2 of the FUT1
contained a TATA-like sequence (27), three possible Sp 1 binding sites,
two possible Ap 2 binding sites, two GATA consensus sequences, and a
Myc consensus sequence (28) (Fig. 5).
The pGL2-enhancer vector containing 5-flanking regions of the exon 2 of the FUT1 (between nucleotides
662 and 45 bp of the exon
2 of the FUT1, Fig. 5) showed promoter activity about
20-fold that of pGL2-control vector with SV40 early promoter and
enhancer after transfection of each plasmid into MCAS cells (data not
shown). The results suggested that this region acted as a promoter and that the exon 2A was one of transcription initiation sites of the
FUT1 in MCAS cells.
In the present study, we examined the structures of cDNA and
genomic DNA of the FUT1. The FUT1 cDNA had
several forms created by two distinct transcription initiation sites
and alternative splicing of 5-untranslated exons. Kelly et
al. (18) have reported the transcription start site of
FUT1 gene of A431 cells using the primer extension method.
However, the transcription start site reported previously was present
within exon 3C in this study, and the site was unlikely to be a
transcription start site in MCAS and in HEL cells. The reason for this
discrepancy is unknown, but one possibility may be that it was due to a
difference in cell types.
A recent study has suggested that the re-expression of the H and H-related antigens such as Lewis b and Lewis y in colorectal tumors was regulated by H and Se enzymes (13). In addition, the expression of the FUT1 transcript was increasing during malignant transformation in colorectal mucosa (14). Our results indicated that the FUT1 transcript was expressed in not only colonic cancer cells but also gastric cancer cells. The expression of the H and H-related antigens in normal digestive mucosa, such as gastric and proximal colonic mucosa, are thought to be regulated by the Se enzyme but not the H enzyme (1). Our results suggest that the expression of the FUT1 mRNA and thereafter the expression of the H enzyme are increasing during malignant transformation in digestive mucosa.
The ABH antigens are expressed in embryonic but not in adult muscular,
bone, and neuronal tissues (12). Recently, as well as the
FUT1 gene, some other glycosyltransferase genes with
distinct promoters and alternative 5-ends have been reported, such as the
(2,6)sialyltransferase gene (29-31), murine
(1,4)galactosyltransferase gene (32), human
N-acetylglucosaminyltransferase V gene (33),
(1,3)fucosyltransferase (34) gene, and the human
(1,4)-N-acetylgalactosaminyltransferase gene (35). Kozak
(36) discussed that transcription of a single gene by multiple
promoters may provide additional flexibility in the regulation of gene
expression. Such promoters could have tissue- and developmental
stage-specific activity. In fact, as shown in this study, the promoter
usage and splicing patterns of the FUT1 mRNA in
erythroleukemic cells (HEL) and in normal erythroid progenitor cells
were different from those in undifferentiated leukemic cells (K562),
which has erythroid characteristics, suggesting that changes in
promoter usage and splicing patterns appear during differentiation of
the erythroid lineage. Thus, the alternative use of multiple promoters
and alternative splicing of the glycosyltransferase genes may be
associated with tissue- and stage-specific glycosylation patterns.
The tissue expression pattern of the ABO antigens is different among
vertebrate species (2). The expression of these antigens in the
digestive mucosa has been observed from amphibians to higher mammals,
while only human and some higher anthropoid primates but not old world
monkeys express these antigens on erythrocyte membranes. Recently,
rabbit homologues of human FUT1, FUT2, and a
pseudogene of FUT2 (Sec1) have been isolated (38,
39). In addition, we have had indications that the old-world African
green monkey had three functional (1,2)fucosyltransferase genes that were homologous to human FUT1, FUT2 and
Sec1.2 Thus, the expression of
ABO antigens in digestive mucosa was likely regulated by
(1,2)fucosyltransferase encoded by the FUT2 homologues in
many vertebrate species. On the other hand, although enzymatic
properties of the rabbit and monkey FUT1 homologue-derived enzymes are similar to the H enzyme (38), they cannot express ABO
antigens on their erythrocyte membranes. A possible reason for this
phenomenon may be a difference in the promoter characteristics. The
present study suggested that the FUT1 had two distinct
promoters and that normal erythroid lineage cells and erythroleukemia
HEL cells used only one promoter present in the 5
-flanking region of
exon 2. Thus, it is conceivable that an erythroid-specific promoter of
the FUT1 became functional in cells of erythroid lineage after separation from the ancestors of apes to that of old world monkeys. Rare individuals (Bombay and para-Bombay phenotypes) fail to
express the ABO antigens on their erythrocyte membranes because of a
lack in H enzyme activity (1). Although non-sense and mis-sense
mutations have been found in the coding region of the FUT1
of Caucasian and Japanese H-deficient individuals (18, 37), no DNA
sequence difference was observed in the coding region of the
FUT1 of Indian Bombay phenotype (40). The erythroid-specific promoter may be inactivated in Indian H-deficient individuals. Thus,
analyses of the promoter present in the 5
-flanking region of exon 2 of
Indian Bombay individuals or a corresponding region of the monkey may
provide further information for understanding tissue- and
species-specific expression of the H and ABO antigens.
In the present study, we identified several forms of the
FUT1 transcript generated by two transcription start sites
and alternative splicing in 5-untranslated exons. Our results
suggested that dual promoters regulated the stage- and tissue-specific
expression of the FUT1 transcript and thus regulated the
expression of ABO and related histoblood antigens in many human
tissues.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ and GeneBankTM/EBI Data Bank with accession numbers D87935-D87941[GenBank][GenBank][GenBank][GenBank][GenBank][GenBank][GenBank], D87943[GenBank], and D87944[GenBank].
We are greatly indebted to Dr. Kimitaka Sagawa, Department of Blood Transfusion of our university for providing several leukemic cell lines.