Downregulation of a human colonic sialyltransferase by a secondary
bile acid and a phorbol ester
Ming
Li1,
Ravi
Vemulapalli1,
Asad
Ullah1,
Leighton
Izu2,
Michael E.
Duffey2, and
Peter
Lance1
1 Department of Medicine,
Division of Gastroenterology, Buffalo Veterans Affairs Medical Center;
and 2 Department of Physiology,
State University of New York at Buffalo, Buffalo, New York
14215
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ABSTRACT |
Fecal constituents such as bile acids and
increased sialylation of membrane glycoproteins by
-2,6-sialyltransferase (HST6N-1) may contribute to colorectal
tumorigenesis. We hypothesized that bile acids and phorbol ester
[12-O-tetradecanoylphorbol-13-acetate (TPA)]
would upregulate HST6N-1 in colonic cells. However, deoxycholate (DOC)
(300 µmol/l), a secondary bile acid, and TPA (20 ng/ml) decreased
expression of an ~100-kDa glycoprotein bearing
-2,6-linked sialic
acid in a colon cancer cell line (T84) in vitro. HST6N-1 mRNA levels
were reduced ~80% by treatment (
24 h) with DOC or TPA but not by
cholate, a primary bile acid. Treatment (24 h) with DOC or TPA
decreased activity of this enzyme to 30% and 13% of control,
respectively. These effects of DOC and TPA were transcriptional and
were mediated by Ca2+ and protein
kinase C, respectively. Thus DOC and TPA both downregulated, and did
not upregulate,
-2,6-sialyltransferase expression in vitro, but by
different transduction pathways. As colorectal tumors grow, their
progressive removal from the fecal milieu that normally downregulates
this enzyme may favor invasion and metastasis.
glycosyltransferase expression; gene expression regulation; colorectal neoplasia
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INTRODUCTION |
MOST MEMBRANE PROTEINS and many secreted proteins bear
oligosaccharides that are heterogeneous and exhibit tissue-specific patterns of expression. Sialic acids, in
-2,3- or
-2,6-linkage to
a penultimate galactose residue, occupy terminal positions in many
N-glycan oligosaccharides of colonic
and other membrane glycoproteins. Sialylated
N- and
O-glycans have been implicated in the
development and metastatic spread of colorectal carcinoma (3, 9, 22).
Highly specific glycosyltransferases catalyze the posttranslational
addition of the individual sugars that comprise
N-glycan and other oligosaccharides;
sialic acids are transferred to acceptor oligosaccharides by one of the
sialyltransferases, members of the glycosyltransferase family of
enzymes. It is widely accepted that tissue-specific or
disease-associated oligosaccharide expression is primarily a function
of variations in glycosyltransferase expression (20). We reported that
levels of
-galactoside
-2,6-sialyltransferase (HST6N-1) mRNA were more than threefold greater in human
adenocarcinomatous tissue than adjacent histologically normal colon
(16). Whether increased colonic epithelial expression of this enzyme is
related causally to neoplastic transformation and progression is
unknown, as are the oligosaccharide products and mechanisms involved.
Knowledge of the agents and mechanisms that regulate
glycosyltransferase expression is scarce.
n-Butyrate, a product of colonic bacterial fermentation detectable in portal blood, causes
differentiation-related changes in colonic and hepatic cells in vitro
(21, 27). We reported that culture of human colonic (16) and hepatic
(24) cells in the presence of
n-butyrate for less than 24 h caused >80% reduction in HST6N-1 mRNA expression by posttranscriptional mechanisms.
Normal adults discharge into the small intestine ~30 g/day of
conjugated (primary) bile salts that are mostly incorporated with
cholesterol and lecithin into mixed micelles and large vesicles (7).
Reabsorption of bile salt monomers in the terminal ileum is normally
efficient, but unabsorbed dihydroxy (secondary) bile acids, such as
taurodeoxycholate (TDC), stimulate colonic secretion of electrolytes
and water, causing diarrhea. Deconjugation by colonic bacteria to
deoxycholic acid (DOC) and other free secondary bile acids further
increases secretory potency. The intracellular mediator for the action
of bile salts on colonic epithelial cells was shown to be
Ca2+ (6). Application of TDC to
isolated T84 cells (a human colon cancer cell line) activated
K+ and
Cl
conductances that were
obligatory for secretion via an inositol 1,4,5-trisphosphate
(IP3)-mediated release of
Ca2+ from intracellular stores
(5).
In addition to causing diarrhea, secondary bile acids are thought to be
an etiological risk factor for colorectal cancer (25). Higher fecal
concentrations of DOC were reported in patients with colorectal cancer
and adenomatous polyps compared with control subjects (1, 26). Bile
acids are tumor promoters in experimental animal models of colon cancer
(19). Colonic epithelial cell hyperproliferation in response to
cytotoxicity has been proposed as the mechanism responsible for the
tumor-promoting effects of bile acids (15, 28).
We hypothesized that altered membrane sialylation through increased
HST6N-1 expression could be one of the cellular effects of colonic bile
acids with tumor-promoting consequences. Unexpectedly, however, DOC and
12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester
and another tumor promoter, caused selective downregulation rather than
upregulation of HST6N-1 expression. Both agents altered HST6N-1 gene
expression by direct transcriptional mechanisms. The transduction
pathways for the bile acid and phorbol ester signals, respectively,
were mediated by Ca2+- and protein
kinase C (PKC)-dependent mechanisms.
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MATERIALS AND METHODS |
Reagents
n-Butyrate, cholic acid, DOC, the
Ca2+ ionophore A-23187, and TPA
were obtained from Sigma Chemical (St. Louis, MO).
Sambucus nigra agglutinin
(S. nigra) was from E. Y. Laboratories (San Mateo, CA). Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) standards were from Bio-Rad Laboratories
(Hercules, CA). Enhanced chemiluminescence Western blotting detection
reagents were from Amersham (Arlington Heights, IL).
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) was from Calbiochem (San Diego, CA). Fura 2-AM was purchased from Molecular Probes (Eugene, OR). [
-32P]dATP (3,000 Ci/mmol) and
[
-32P]UTP (3,000 Ci/mmol) were from ICN Biomedicals (Costa Mesa, CA). Cytidine
monophosphate-N-acetyl-[4,5,6,7,8,9-14C]neuraminic
acid
(CMP-[14C]NeuAc)
(1.8 mCi/mmol) was from New England Nuclear Life Sciences (Boston, MA).
Bisindolylmaleimide (GF-109203X) was from LC Laboratories (Woburn, MA).
Tissue culture reagents were purchased from GIBCO Laboratories (Grand
Island, NY). All other reagents were of the highest quality
commercially available.
Cell Culture
T84 was purchased from American Type Culture Collection (Bethesda, MD),
and NCM460 was a generous gift from Dr. M. P. Moyer (University of
Texas, San Antonio, TX) (18). T84 cells were cultivated in Dulbecco's
modified Eagle's medium (DMEM)-Ham's F-12 medium supplemented with
5% fetal bovine serum (FBS) and 110 mg/l sodium pyruvate. NCM460 cells
were cultivated in high-glucose DMEM with 10% FBS. Penicillin (100 units) and streptomycin (100 µg/ml) were routinely added to cultures,
and both cell lines were cultivated at 37°C in a humidified 5%
CO2 incubator.
Lectin Blotting
Cell cultures were treated with DOC or TPA for different time periods
or incubated for the same time periods in medium not supplemented with
DOC or TPA (control cultures) and then washed extensively in ice-cold
phosphate-buffered saline (PBS) and incubated for 20 min at 4°C in
lysis buffer as described previously (8). Lysates were removed by
scraping and centrifuged at 10,000 g
for 30 min at 4°C to remove insoluble material. The protein content of the resulting supernatants was quantitated by the method of Bradford
(2). SDS-PAGE was performed with the use of supernatant sample volumes
that had been adjusted to give the same amount of protein in all lanes.
After electrophoresis, proteins were electroblotted to membranes that
had been incubated with S. nigra. Glycoproteins bearing
-2,6-linked sialic acids were visualized on
these membranes by incubation with chemiluminescent reagents, according
to the manufacturer's instructions, and autoradiography.
Cell Viability
Cell viability was determined by trypan blue exclusion. Cells were
seeded at the same time from a single parent culture. Cultures were
incubated without (control) or with DOC or TPA. Timing of the addition
of TPA or DOC was staggered. Treated (12, 24, or 48 h) and control
cultures were harvested at the same time, after careful washing to
remove cells that had detached during incubation. Harvested cells were
incubated with trypan blue and counted using a hemacytometer. From each
culture, four fields of duplicate preparations were counted for the
percentage of cells that excluded the dye.
Isolation of RNA and Northern Analysis
Isolation of total RNA, gel electrophoresis, Northern blotting,
hybridization with radiolabeled cDNA probes, and quantitation of mRNA
levels were performed as described previously (16, 24). Cells were
cultivated to confluence, treated with test compounds, or maintained in
medium unsupplemented with test compounds and harvested for isolation
of total RNA. The following cDNA probes were used: human HST6N-1 cDNA
was isolated previously by us (14), human
-1,4-galactosyltransferase
(GalT) cDNA was a gift from Dr. M. N. Fukuda [Masri et al.
(17)], human
N-acetylglucosaminyltransferase (GnT
I) cDNA was a gift from Dr. H. Schachter [Schachter et al. (23)], and rat
-galactoside
-2,3-sialyltransferase (ST3N) cDNA was a gift from Dr. J. C. Paulson [Wen et al. (29)].
The densities of DNA-RNA hybrids were determined by spectrophotometric scanning of autoradiographs, and results were normalized for intensity of staining with ethidium bromide.
Sialyltransferase Assay
Specific
-galactoside
-2,6-sialyltransferase enzyme activity was
assayed as described (8, 24), with modifications. T84 cells, either
untreated (control) or treated with DOC, cholate, or TPA, were washed
four times in ice-cold PBS, scraped from culture dishes in 0.4 ml of
sodium cacodylate (50 mmol/l) buffer at pH 6.5 (150 mmol/l NaCl, 1%
Triton X-100, and 20% glycerol), and homogenized. The homogenate was
centrifuged at 10,000 g for 30 min at
4°C. The reaction mixture for each assay contained 0.21 nmol
CMP-[14C]NeuAc, 19.4 nmol CMP-NeuAc, 1 mmol/l 2,3-dehydro-2-deoxy-NeuAc, 5 mmol/l
MnCl2, 50 mmol/l sodium
cacodylate, 150 mmol/l NaCl, and 195 µg of
asialo-
1-acid glycoprotein as
acceptor, in a final volume of 60 µl. Reactions, performed in
duplicate, were initiated by the addition of 20 µl of supernatant
from centrifuged cell homogenate and incubated at 37°C for 1 h. The
radioactive reaction product was isolated by chromatography on Sephadex
G-50 and quantitated by liquid scintillation spectrometry. The protein
content of cellular homogenates was quantitated by the method of
Bradford (2). Specific sialyltransferase enzyme activities of treated
and control cultures were compared using Student's
t-test.
Nuclear Transcriptional Assay
Nuclei were isolated, and the run-on protocol was carried out as
described previously (13, 24). Equal amounts of nascent radioisotopically labeled RNA transcripts (5 × 107 cpm/3 ml) were hybridized for
3 days to 2 µg of nitrocellulose-bound cDNA.
Intracellular
Ca2+
Measurements
Cells on glass coverslips were loaded at room temperature with fura
2-AM (4 µmol/l) for 20 min in
Ca2+-free solution, followed by
incubation for 1 h in the usual
Ca2+-containing medium for T84
cells. The coverslips were then placed in a Plexiglas chamber and
mounted on the stage of an inverted microscope (Nikon Diaphot) equipped
for epifluorescence using a ×40 oil-immersion lens, as described
previously (5). Fura 2 fluorescence images at 340-nm excitation
wavelengths were captured with a silicon intensified
target video camera and analyzed using imaging software
(Image 1/FL, Universal Imaging). Average whole cell ratio values were
determined. For chelation of intracellular free
Ca2+, cells were incubated for 20 min in Ca2+-free solution
supplemented with BAPTA-AM (20 µmol/l), followed by incubation in
regular medium without BAPTA-AM for 1 h.
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RESULTS |
Terminal
-2,6-Linked Sialic Acids and
Sialyltransferase Expression
Lectin affinity of T84 cell lysates.
Lysates of cells cultured in the absence or presence of DOC or TPA were
electrophoresed and electroblotted to membranes that were incubated
with S. nigra, a lectin with specific
affinity for
-2,6-linked sialic acid (Fig.
1). Three bands were detected, corresponding to sialylated glycoproteins with approximate sizes of
100, 80, and 75 kDa. With the length of exposure required for demonstration of the ~100-kDa band in Fig. 1, top, the
more intense ~80- and 75-kDa bands resemble a single broad band, but
they are readily distinguishable in Fig. 1, bottom.

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Fig. 1.
S. nigra lectin blots of T84 cell
glycoproteins. Lysates were prepared from cells incubated without (C,
control) or with (T, treated) deoxycholate (DOC; 300 µmol/l)
(top) or
12-O-tetradecanoylphorbol-13-acetate (TPA) (20 ng/ml)
(bottom) for time periods as shown.
Equal amounts of lysate protein from each preparation were loaded in
lanes of sodium dodecyl sulfate-polyacrylamide gels, resolved by
electrophoresis, and electroblotted on membranes as described in
MATERIALS AND METHODS. Molecular mass
markers (top) are -galactosidase
(116 kDa), phosphorylase B (97 kDa), bovine serum albumin (66 kDa),
ovalbumin (45 kDa), and carbonic anhydrase (31 kDa).
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Intensity of the ~80- and 75-kDa bands was unaffected by incubation
with DOC. From comparison of treated and control lanes, decreased
expression of the ~100-kDa glycoprotein was first evident 4 h after
exposure to 300 µmol/l DOC. Decreased expression of this
glycoprotein, relative to control, was most pronounced at 24 and 48 h
in the DOC experiment. Diminished expression of the ~100-kDa band,
compared with control, was first seen at 12 h in TPA-treated cells and
was still apparent at 24 h.
An overall decline in levels of sialylated glycoproteins was evident at
the extremes of both sets of experiments. This can be seen from reduced
intensity of the ~100-kDa bands in the control lanes of the DOC
(top) and TPA
(bottom) experiments, respectively, by 72 and 48 h compared with control ~100-kDa bands at earlier time
points. Exhaustion of the medium, which was not changed for control or
treated cultures, causing a generalized reduction of cellular synthetic
capacity in the longer experiments, is the likely explanation. Because
DOC or TPA treatment caused reductions of product level in <24 h, and
considerably earlier than the more generalized decline in levels of
sialylated glycoproteins, cell cultures were treated for ~24 h in
subsequent studies of the mechanisms of action of these compounds.
Sialyltransferase (HST6N-1) mRNA levels and activity in cells
treated with DOC and TPA.
Expression of the sialyltransferase responsible for transfer of sialic
acid to colonic glycoproteins in terminal
-2,6-linkage was studied
by Northern analysis (Fig. 2). HST6N-1 mRNA
level was reduced ~80% by exposure to 300 µmol/l DOC for 24 h, and
inhibition was first seen after 2 h. TPA caused threshold and maximal
(~85%) inhibition of HST6N-1 mRNA levels, respectively, at
concentrations of 5 and 20 ng/ml. The inhibitory effect of TPA was
first seen after exposure for 4 h.

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Fig. 2.
Effects of a secondary bile salt (DOC) and phorbol ester (TPA) on
glycosyltransferase mRNA levels in T84 cells.
A: dose response. Total cellular RNA
was isolated from T84 cells incubated for 24 h in presence of DOC or
TPA at concentrations indicated, or from control cells cultured for 24 h without DOC or TPA (0, far left lane). Electrophoresis, blotting, and
hybridization of RNA with HST6N-1, GalT, or GnT I cDNA probes were
performed as described in MATERIALS AND
METHODS. Densities of DNA-RNA hybrids were determined
by spectrophotometric scanning of autoradiographs, and results were
normalized for intensity of staining with ethidium bromide.
B: time course. Northern analysis of
HST6N-1 mRNA levels was performed on total RNA from cells incubated
with TPA (20 ng/ml) or DOC (300 µmol/l) or without either agent
(Ctrl), for times indicated.
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Downregulation of
-galactoside
-2,6-sialyltransferase activity on
exposure of T84 cells to DOC or TPA followed reductions in HST6N-1 mRNA
level (Table 1). Relative activity after
exposure for 24 h to DOC and TPA, respectively, was 30% and 13% of
control. Specific sialyltransferase activity in T84 cells incubated
with cholate was undiminished at 12 h and was reduced to 57% of
control after 24 h.
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Table 1.
-Galactoside
-2,6-sialytransferase enzyme activity in T84 cells
after treatment with cholate, DOC, or TPA
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Terminal N-glycan sialic acids are
linked to galactose residues, which are linked in turn to
N-acetylglucosamines.
N-Acetylglucosamine and galactose
sugars, respectively, are transferred sequentially to oligosaccharide
chains by the actions of
N-acetylglucosaminyltransferase (GnT
I) and galactosyltransferase (GalT) enzymes. To investigate the
specificity of HST6N-1 downregulation by DOC and TPA, HST6N-1, GalT,
and GnT I mRNA levels were assessed in the same Northern blot (Fig.
2A). DOC, at a threshold
concentration of 200 µmol/l, and TPA, at a threshold of 5 ng/ml,
caused modest increases in GalT mRNA level. Slight decreases in GnT I
mRNA level were discernible in T84 cells incubated with DOC or TPA.
Specificity of Bile Acid Effects
Comparison of primary and secondary bile acids.
DOC, a secondary bile acid, has greater secretory potency and, it is
thought, tumor-promoting activity than primary bile acids such as
cholate (19). Therefore, the effects of DOC and cholate on HST6N-1 mRNA
expression by T84 cells were compared. DOC caused reductions as before,
but HST6N-1 mRNA levels were unaltered by incubation of cells for up to
24 h in cholate concentrations of up to 300 µmol/l (Fig.
3).

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Fig. 3.
Effects of primary (cholate) and secondary (DOC) bile salts on HST6N-1
mRNA levels in T84 cells. Total cellular RNA was isolated from T84
cells incubated in presence of cholate (300 µmol/l) or DOC (300 µmol/l), or from control cells cultured without bile salts for time
periods indicated. Northern analysis of HST6N-1 mRNA levels and
normalization were performed as described in MATERIALS
AND METHODS and Fig. 2 legend.
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DOC treatment of nonneoplastic colonic cells.
The applicability to nonneoplastic colonic epithelium of bile acid
effects reported in colon cancer cell lines in vitro is uncertain. The
NCM460 cell line was established from normal human colonic epithelial
cells (18). As in cancer cell cultures, NCM460 cell HST6N-1 mRNA
expression was downregulated by incubation with DOC but not with
cholate (Fig. 4).

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Fig. 4.
Effects of cholate and DOC on HST6N-1 mRNA levels in nonneoplastic
(NCM460) colonic cells. Cells were incubated with DOC (300 µmol/l) or
cholate (300 µmol/l) or without either agent (Control), for times
indicated.
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Cell viability and morphology.
T84 cell viability, after treatment with DOC or TPA according to a
similar protocol to that used for the lectin affinity experiments depicted in Fig. 1, was determined by trypan blue exclusion (Table 2). Cell viability was
94% after all
treatment periods up to 48 h with either compound. Cell morphology was
not affected by cholate, but DOC caused pronounced changes (Fig.
5). Morphological changes caused by DOC
were reversible (data not shown); DOC-treated T84 cells continued to
grow after return to DOC-free medium and within 24 h were almost
indistinguishable in appearance from cultures that had never been
exposed to DOC. As further confirmation of reversibility, HST6N-1 mRNA
levels returned almost to pretreatment levels 24 h after DOC-treated
cells were shifted to DOC-free medium; by 48 h, pretreatment HST6N-1
mRNA levels had been exceeded (data not shown).

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Fig. 5.
Effects of cholate and DOC on T84 cell morphology. Cell cultures were
examined under an inverted microscope (×100). Appearance of cells
cultured in absence of bile acids did not alter over a 24-h period
(Control, 0 and 24 h). Culture in presence of cholate (300 µmol/l)
did not alter cell morphology (Cholate, 24 h). Incubation with DOC (300 µmol/l) for 24 h caused profound changes (DOC, 24 h); cells became
rounded, and cytoplasmic granules and vacuoles appeared.
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DOC and TPA Regulation of HST6N-1 Gene Expression
Nuclear transcriptional assays.
Possible mechanisms for DOC- and TPA-mediated reduction of HST6N-1 mRNA
expression include decreased transcription, reduced processing of
nuclear HST6N-1 mRNA precursors and increased degradation of mature
transcripts. The influence of these agents on the rate of HST6N-1
transcription was assessed by the nuclear run-on reaction (Fig.
6). The effects of DOC and TPA on
transcription of other glycosyltransferases (GalT, GnT I, and ST3N)
were examined to evaluate further the specificity of potential
regulatory effects. Linkages synthesized through the actions of GalT
and GnT I were described above.
-Galactoside
-2,3-sialyltransferase (ST3N) is an alternative sialyltransferase to
HST6N-1 that transfers sialic acid to galactose acceptors in an
-2,3- rather than an
-2,6-linkage.

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Fig. 6.
Nuclear transcriptional assays of T84 cells incubated with DOC or TPA.
T84 cells were incubated with DOC or TPA for 4 or 24 h. Cell nuclei
were separated, and the run-on protocol was performed as described in
MATERIALS AND METHODS. Nascent
radioisotopically labeled transcripts were hybridized with cDNAs for
HST6N-1, GalT, GnT I, ST3N, and actin.
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Densitometric analysis of the autoradiograph showed that HST6N-1
transcription was decreased to 30% of control after exposure of T84
cells to DOC or TPA for 24 h, but ST3N transcription was unaffected.
GalT transcription was increased to 230% and 150% of control,
respectively, in cells exposed to DOC for 4 and 24 h, but was unaltered
by TPA. GnT I transcript levels after 24-h exposure to DOC and TPA,
respectively, were 72% and 80% of control. Both DOC and TPA increased
transcription of the actin gene.
Signal-Transduction Pathways of DOC and TPA Effects
DOC-mediated increase of intracellular
Ca2+.
Activation of K+ and
Cl
conductances in T84 by
TDC (750 µmol/l), a secondary bile acid, was reported in T84 cells
(5). The mechanism of this action was via
IP3-mediated release of
intracellular Ca2+. Before
investigating whether HST6N-1 downregulation by DOC was Ca2+ mediated, we confirmed that
the secondary bile acid DOC could cause a rise in intracellular
Ca2+ concentration
([Ca2+]i).
As shown in Fig. 7, DOC (300 µmol/l)
caused a rapid increase in the ratio of
F340 to
F380, indicative of increased
[Ca2+]i.
This response occurred <10 min after exposure to DOC. A transient decrease and increase in the fluorescence ratio followed the initial rise. This behavior is similar to the rise in
[Ca2+]i
and Ca2+ oscillations seen on
exposure of T84 cells to TDC at the higher concentration of 750 µmol/l (5).

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Fig. 7.
Effect of DOC on intracellular
Ca2+ concentration
([Ca2+]i).
Increases in
[Ca2+]i
were assessed by measurement of fura 2 fluorescence ratio
(F340/F380)
in a single T84 cell as described in MATERIALS AND
METHODS. The example shown was one of 4 experiments,
all with similar results. The time of introduction of DOC (300 µmol/l) is indicated, and exposure to DOC continued until
"Wash" at the time indicated.
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Ca2+-mediated
regulation of HST6N-1 expression.
Exposure to DOC in the absence of extracellular
Ca2+ prevented downregulation of
HST6N-1 mRNA expression in T84 cells that had been preincubated with
BAPTA-AM to chelate intracellular
Ca2+ (Fig.
8). However, when intracellular
Ca2+ was not chelated, lack of
extracellular Ca2+ did not prevent
downregulation of HST6N-1 expression, indicating that this action of
DOC occurred as a consequence of the release of intracellular
Ca2+. Downregulation of HST6N-1 by
TPA was not Ca2+ dependent.

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Fig. 8.
Ca2+-dependent regulation of
HST6N-1 expression. T84 cells were incubated with DOC (300 µmol/l) or
TPA (20 ng/ml), or without either agent (Control) for 2 h. Experiments
were conducted in medium that contained
Ca2+ (bath
Ca2+, +) or lacked it (bath
Ca2+, ). In addition, either
there was no interference with
[Ca2+]i
[Cell Ca2+ free
(+)] or cells were preincubated with BAPTA-AM (20 µmol/l)
[Cell Ca2+ chelated
( )] as described in MATERIALS AND
METHODS. Isolation of RNA, Northern analysis, and
normalization of HST6N-1 mRNA levels were performed as described in
MATERIALS AND METHODS.
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To further substantiate the role of increased
[Ca2+]i
in regulating HST6N-1 expression, T84 cells were treated with a
Ca2+ ionophore, A-23187, to
increase
[Ca2+]i
(Fig. 9). HST6N-1 mRNA expression was
reduced by >80% with exposure to A-23187 (5 µmol/l) for 4 h.

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Fig. 9.
HST6N-1 downregulation after elevation of
[Ca2+]i.
T84 cells were incubated in presence of a
Ca2+ ionophore, A-23187 (5 µmol/l), and bath Ca2+ (1 mmol/l) for time periods indicated. A-23187, but not bath
Ca2+, was omitted from control
cultures. Isolation of RNA, Northern analysis, and normalization of
HST6N-1 mRNA levels were performed as described in
MATERIALS AND METHODS.
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PKC-mediated regulation of HST6N-1 expression.
Many of the tumor-promoting actions of phorbol esters are mediated by
PKC (4, 12), and bile salts have been shown to activate PKC under
certain conditions in vitro (11). Therefore, we investigated whether
GF-109203X, a selective inhibitor of PKC (10), blocked downregulation
of HST6N-1 by DOC or TPA in T84 cells (Fig. 10). GF-109203X (1 µmol/l) prevented inhibition of HST6N-1 mRNA expression by TPA but
not DOC. The effect of GF-109203X on the inhibitory actions of
n-butyrate was examined to define further the specificity of PKC-mediated regulation of HST6N-1 expression; n-butyrate-mediated
downregulation of HST6N-1 was unaffected by inhibition of PKC.
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DISCUSSION |
Wide variations between different tissue and cell types in levels of
expression of the genes that encode
N-glycan sialyltransferases have been
thoroughly documented. However, relatively little is known of the
responsiveness of these and other glycosyltransferase genes to
extracellular signals or the extent to which levels of expression vary
over time within the same tissue or cell type. In this study, a
secondary bile acid (DOC) and a phorbol ester (TPA) caused >80%
downregulation of HST6N-1 gene expression in the T84 human colon cancer
cell line (Fig. 2) and reduced levels of activity and product of this
enzyme. An ~100-kDa glycoprotein that was isolated from control cells
by means of its terminally expressed
-2,6-linked sialic acids could
no longer be detected after exposure of T84 cells to DOC for 48-72
h (Fig. 1); structural analyses of the oligosaccharides of this
glycoprotein are in progress. From nuclear transcriptional assays (Fig.
6), it was evident that the effects of DOC and TPA on HST6N-1
expression were, at least in part, due to reduced primary transcription
of this gene.
Several findings indicate that DOC- and TPA-mediated inhibition of
HST6N-1 expression was relatively selective. Concurrent with HST6N-1
downregulation, both DOC and TPA caused modest upregulation of GalT
mRNA expression (Fig. 2A). DOC
increased GalT gene transcription, and both DOC and TPA increased
transcription of the actin gene (Fig. 6).
The effects of DOC and TPA on T84 cells were not lethal and were
reversible. Trypan blue exclusion was unimpaired by both agents.
Morphological changes and downregulation of HST6N-1 mRNA expression
were fully reversible within 24 to 48 h of shifting cells to DOC-free
medium.
Different intracellular pathways mediated downregulation of HST6N-1
expression by DOC and TPA. Independent of the effect of DOC,
corroborating evidence that an increase in
[Ca2+]i
can lead to downregulation of HST6N-1 expression came from experiments
with A-23187; within 4 h of exposure to this
Ca2+ ionophore, which causes a
rise in
[Ca2+]i
by allowing bath Ca2+ to enter
cells, HST6N-1 mRNA levels fell by >80%. DOC, a deconjugated secondary bile acid, caused accumulation of cytosolic
Ca2+ (Fig. 7), as had been shown
previously for a conjugated secondary bile acid, TDC (5). We
demonstrated that release of intracellular Ca2+ was obligatory for the
downregulatory effect of DOC on HST6N-1 mRNA expression (Fig. 8).
However, downregulation of HST6N-1 expression could occur in the
absence of extracellular (bath)
Ca2+, provided sufficient
intracellular Ca2+ was available.
The effect of TPA on HST6N-1 expression was mediated, as expected, by
PKC (Fig. 10) but activation of PKC by
bile acids, as reported by Huang et al. (11) in an in vitro, cell-free
model, did not appear to contribute to the effect of DOC on HST6N-1. Near-total inhibition of HST6N-1 mRNA expression in T84 cells by TPA or
DOC confounded our initial hypothesis that such agents, thought to
enhance neoplastic transformation, would induce this enzyme. As in the
colon cancer cells, both agents also caused profound downregulation of
HST6N-1 in a human colonic epithelial cell line, NCM460, that was
derived from a normal colon (Fig. 4).

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|
Fig. 10.
Protein kinase C-mediated regulation of HST6N-1 expression. T84 cells
were incubated with DOC (300 µmol/l),
n-butyrate (5 µmol/l), or TPA (20 ng/ml) for 24 h. Each treatment was performed without and with
GF-109203X (GF) at concentrations of 1 and 5 µmol/l. Isolation of
RNA, Northern analysis, and normalization of HST6N-1 mRNA levels were
performed as described in MATERIALS AND
METHODS.
|
|
We reported previously that n-butyrate
downregulates HST6N-1 expression in T84 cells (16); thus DOC is the
second fecal constituent that we have shown can alter sialyltransferase
expression in short-term cultures of colonic cells in vitro. Our
results in vitro raise the possibility that fecal constituents and
other agents could regulate sialyltransferase expression in vivo but the physiological and pathophysiological significance of these potential effects is unknown.
Downregulation of HST6N-1 expression by DOC in malignant (T84) as well
as nonneoplastic (NCM460) cells suggests that malignant transformation
in colonic cells is not necessarily associated with aberrant or lost
regulation of N-glycan
sialyltransferase expression. We propose the following hypothesis.
-Galactoside
-2,6-sialyltransferase expression of normal and,
initially, neoplastic colonic epithelial cells is downregulated by
secondary bile acids, short-chain fatty acids, and, potentially, a
variety of other fecal constituents. With malignant transformation and
invasion, cells of a neoplastic clone are progressively removed from
the fecal milieu. This leads to HST6N-1 disinhibition and increased
-2,6-sialylation of specific membrane proteins that could confer a
selective advantage in subsequent steps of the metastatic cascade.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
DK-43649 (to P. Lance) and CA-09051 (to M. Li and P. Lance), by a Merit
Review Award from the Department of Veterans Affairs (to P. Lance), and
by Cystic Fibrosis Foundation Grant 5874 (to M. E. Duffey).
 |
FOOTNOTES |
Address for reprint requests: P. Lance, Division of Gastroenterology,
Buffalo General Hospital, 100 High St., Buffalo, NY 14203.
Received 11 August 1997; accepted in final form 24 December 1997.
 |
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