Introduction
The liver exclusively synthesizes bile acids from
cholesterol via two biosynthetic pathways. The "classical"
pathway begins with the 7α-hydroxylation of cholesterol and
operates entirely in the liver [ 1 ] . The "alternative"
pathway begins with the hydroxylation of the cholesterol
side chain. Although this reaction occurs in a wide variety
of cells, the completion of bile acid synthesis takes place
in liver cells. The classical pathway is responsible for
the bulk of the bile acids synthesized by the liver [ 2 3 ]
.
The major bile acid species produced by the liver are
cholic and chenodeoxycholic acids. These bile acids are
conjugated to either taurine or glycine before secretion
into bile [ 4 ] , which is subsequently released into the
small intestine. Bile acids are reclaimed from the lumen of
the intestine and returned to the liver via portal blood
for reuse [ 1 ] . The recovery of bile acids from the
intestine is an efficient process that involves both
passive transport along the entire axis of the intestine,
and active transport in the terminal ileum [ 5 ] . The
apical sodium/bile acid co-transporter (asbt) mediates the
active uptake of conjugated bile acids in the ileum [ 5 6 ]
. In hepatocytes, both sodium-dependent and
sodium-independent transport systems on the sinusoidal
membranes extract bile acids from portal blood. Of these,
the sodium/bile acid co-transporting polypeptide (ntcp) is
considered to be quantitatively the most important
transporter of conjugated bile acids in these cells [ 7 8 ]
. The transport of bile acids from the basolateral to the
canalicular membrane of liver cells is a poorly understood
process. Intracellular bile acid binding proteins have been
postulated to participate in the transcellular transport as
well as to buffer against the cytotoxic effects of bile
acids. ATP-dependent transporters on the canalicular
membranes of hepatocytes mediate the secretion of bile
acids into bile [ 9 ] .
Recently, it was discovered that bile acids are the
natural ligands for a nuclear receptor termed farnesoid x
receptor (FXR; NR1H4) [ 10 11 12 ] . Therefore, bile acids
may be important regulators of gene expression in the liver
and intestines. To date, the genes that have been shown to
be responsive to regulation by FXR encode proteins involved
in the biosynthesis and transport of bile acids [ 13 ] .
Bile acids have been shown to modulate a variety of other
cellular functions, such as secretion of lipoproteins from
hepatocytes [ 14 15 ] and translocation of bile acid
transporters to the hepatocyte canalicular membrane [ 16 ]
. In McNtcp cells, which are liver-derived cells engineered
to transport bile acids actively [ 17 ] ,
taurine-conjugated bile acids induce the formation of
intracellular vesicles that resembles structures observed
in cholestatic liver cells without apparent cellular
cytotoxicity [ 1 18 ] . In contrast, glycine-conjugated
bile acids promote apoptosis in these cells [ 19 ] . Thus,
different classes of conjugated bile acids stimulate
distinctive responses in McNtcp cells. Whereas
taurine-conjugated bile acids are well tolerated,
glycine-conjugated bile acids are extremely cytotoxic. Here
we examined if this differential response is limited to
cells that normally metabolize bile acids, and whether high
level expression of an intracellular bile acid binding
protein can attenuate the cytotoxic response of McNtcp.24
cells to glycine-conjugated bile acids.
Results
Differential cytotoxicity of glycine-conjugated
bile acids in McNtcp.24 cells
We previously showed that taurine-conjugated bile
acids, such as taurocholic (TCA) and
taurochenodeoxycholic (TCDCA) acids, were well tolerated
by McNtcp.24 cells [ 18 ] . In contrast,
glycine-conjugated bile acids, such as glycocholic (GCA)
and glycochenodeoxycholic (GCDCA) acids, were cytotoxic
to McNtcp.24 cells [ 1 ] concordant with what has been
documented in hepatocytes [ 19 ] . Consistent with the
induction of apoptotic cell death; GCDCA, but not TCA,
preferentially induced the activation of the initiator
caspase 8 in McNtcp.24 cells (Figure 1A). Indeed,
glycine-conjugated bile acids as a class of molecules
induced genomic DNA fragmentation in this cell line at
low concentrations (50 μM), while their taurine
counterparts did not (Figure 1B). The cytotoxicity of
glycine-conjugated bile acids cannot be attributed simply
to the preferential cellular accumulation of these bile
acids because McNtcp.24 cells can accumulate GCA and TCA
to similar levels (Torchia and Agellon, unpublished
observations).
There is considerable evidence to suggest that bile
acids activate a variety of cellular signaling pathways
in liver cells [ 20 ] . TCDCA is capable of activating
PI3K activity in McNtcp.24 cells and, direct elevation of
PI3K activity by transient expression of a plasmid
encoding PI3K can protect McNtcp.24 cells from
GCDCA-induced apoptosis [ 21 ] . TCA, like TCDCA, also
caused the elevation of PI3K activity in McNtcp.24 cells
(Figure 2A), suggesting that activation of PI3K may be a
general response to taurine-conjugated bile acids.
Treatment of McNtpc.24 cells with LY294002, a PI3K
inhibitor, prior to the addition of TCA or TCDCA resulted
in the loss of tolerance to these bile acids as
demonstrated by the activation of caspase 8 (Figure 2B).
On the other hand, preincubation of McNtcp.24 cells with
either TCA or TCDCA attenuated the GCDCA-mediated
activation of caspase 8 (Figure 2C), suggesting that the
activation of a PI3K-dependent survival pathway is
dominant over the death pathway. These results also
indicate that the differential sensitivity of the
liver-derived McNtcp.24 cells to conjugated bile acids is
due to the existence of a specific mechanism that is
capable of recognizing different classes of conjugated
bile acids. In these cells, taurine-conjugated bile acids
specifically increase PI3K activity which, in turn, leads
to the activation of a survival pathway [ 22 23 ] .
Expression of bile acid uptake activity in CHO
cells
To determine if non-liver derived cells also posses
differential sensitivity to bile acid-induced
cytotoxicity, we engineered CHO cells to take up bile
acids actively by expressing asbt. Colonies surviving the
selection process were assayed for asbt activity and
several clones showing a range of bile acid uptake
activities were selected for further analysis. Analysis
of total RNA from the selected clones (CHO.asbt) readily
showed the presence of the recombinant asbt mRNA (Figure
3A). Correspondingly, in CHO.asbt lysates, an
immunoreactive band of comparable size (~45 kDa) to rat
asbt protein can be easily detected using an anti-asbt
antibody (Figure 3B). The uptake of bile acids by the
CHO.asbt cells was saturable with an apparent K
m ranging between 22 to 25 μM (Figure
3C). The V
max values ranged from 0.323 to 2.769
nmol/mg protein/min and correlated with the abundance of
the recombinant asbt mRNA encoded by the transgene. The
uptake of 25 μM TCA by CHO.asbt.8 and CHO.asbt.35 cells
reached steady state within 10 min (Figure 3D), implying
the presence of a pathway for bile acid efflux in CHO
cells. Furthermore, it was possible to chase the
incorporated radiolabeled TCA from CHO.asbt.49 and
CHO.asbt.35 cells (Figure 3E). These results demonstrate
that CHO.asbt cells express a fully functional asbt
protein.
Cytotoxic potential of bile acids in CHO.asbt.35
cells
The increase of LDH activity in the culture medium of
CHO.asbt.35 and McNtcp.24 cells after treatment with
glycine- and taurine-conjugated bile acids were compared
to assess the cytotoxic potential of bile acids on these
cells. The CHO.asbt.35 cell line was chosen for this
study because this cell line has bile acid uptake
activity that was comparable to McNtcp.24 cells [ 17 ] .
LDH activity in the culture medium of McNtcp.24 cells
increased by 20 to 31% within 2 h of incubating the cells
in medium containing 100 μM of glycine conjugates of
cholic acid, chenodeoxycholic acid, and deoxycholic acid
(GCA, GCDCA, GDCA, respectively) (Figure 4A). GCDCA was
the most potent in inducing the release of LDH among the
bile acids tested whereas TCA had little effect as
compared to the no addition controls. In contrast,
incubation of CHO.asbt.35 cells in medium containing 100
μM of taurine- or glycine-conjugated bile acids resulted
in a time dependent increase of LDH activity the culture
medium (Figure 4B). GCA and GCDCA were equally potent in
increasing LDH activity, raising the level in the medium
of treated cells by 40% within 6 h. Taurine-conjugated
bile acids also raised the LDH activity in the medium of
treated cells to levels approaching GDCA-treated cells
(Figure 4B). Treatment of McArdle RH-7777 and CHO cells
with taurine- or glycine-conjugated bile acids had little
effect on the release of LDH (Figure 4Cand 4D),
indicating that bile acid uptake activity is necessary to
manifest their cytotoxic potential in both cell lines. In
addition, the cytotoxicity was specific to conjugated
bile acids since the addition of non-conjugated bile
acids to the culture medium of bile acid-transporting
cells had a negligible effect on the release of LDH into
the culture medium.
Mechanism of cell death
Members of the caspase family of cysteine proteases
have been implicated in the initiation and execution
phase of apoptosis [ 24 ] . In addition, several studies
have shown the activation of the caspase cascade by toxic
bile acids in hepatocytes [ 25 26 ] . We thus measured
caspase activity after bile acid treatment in CHO.asbt.35
cells. Activation of caspase 8 was detected in
CHO.asbt.35 cells after treatment with either taurine- or
glycine- conjugated bile acids (Figure 5A), but absent in
bile acid-treated CHO cells (Figures 5A). Analysis of
genomic DNA isolated from treated cells showed that TCA
and GCDCA induced DNA laddering in CHO.asbt.35 cells
(Figure 5B), indicating that both classes of conjugated
bile acids induce apoptosis in this non-hepatic derived
cell line. To further characterize the initiation of the
caspase cascade, we determined the time course of caspase
activation by bile acids in CHO.asbt.35 and McNtcp.24
cells. Activation of caspase 8 in both cell lines was
first detected by 30 min following GCDCA-treatment and
was maximal by 60 min (Figure 6). In contrast,
TCA-mediated activation of caspase 8 was first detected
at 60 min and reached the levels induced by GCDCA by 120
min.
Because the bile acid transporters used to establish
bile acid uptake activity in McNtcp.24 and CHO.asbt.35
cells were not the same, it was possible that the
sensitivity of CHO.asbt.35 cells to taurine-conjugated
bile acids was a consequence of asbt expression. To
address this possibility, asbt was expressed in McArdle
RH-7777 cells and then tested for response to glycine-
and taurine-conjugated bile acids. As shown in Figure 7A,
asbt conferred active bile acid uptake activity in
transfected cells. However, activation of caspase 8 was
evident only in the cells that were treated with GCDCA
(Figure 7B). These results demonstrate that the
sensitivity of this cell line to taurine- and
glycine-conjugated bile acids is not dependent on a
specific kind of bile acid transporter.
Activation of a PI3K-dependent pathway by
taurine-conjugated bile acids appears to decide the fate
of McNtcp.24 cells (Figure 2and ref. [ 21 ] ). Therefore,
we determined if PI3K activity was altered in bile
acid-treated CHO.asbt.35 cells. As shown in Figure 8,
PI3K activity in response to TCA or GCDCA was not evident
in CHO.asbt.35 cells, suggesting that the inability to
activate survival pathways by taurine-conjugated bile
acids commit these cells to apoptosis. These findings
also indicate that liver derived cells posses a mechanism
that responds to taurine-conjugated bile acids leading to
the activation of PI3K.
Cytotoxic potential of bile acids in
HBAB-expressing McNtcp.24 cells
It has been suggested that sequestration of bile acids
by intracellular bile acid binding proteins may also
protect cells from bile acid cytotoxicity. Thus, the
human bile acid binder (HBAB) was expressed in McNtcp.24
cells to determine if this class of proteins can protect
against GCDCA cytotoxicity. Figure 9Ashows the presence
of the recombinant mRNA encoding the recombinant HBAB in
clones that have incorporated the HBAB expression vector
into their genomes. In addition, the HBAB protein
abundance in the total lysates correlated with the
abundance of the HBAB mRNA (Figure 9A). Analysis of
radiolabeled TCA uptake (Figure 9B) showed that
expression of HBAB had minimal impact on bile acid uptake
despite the fact that the range of HBAB protein abundance
was up to 5-fold greater than that in the lowest HBAB
expressing cell. Moreover, the rates of decline in the
amount of radiolabeled TCA associated with the cells
after withdrawal of the radiolabeled bile acids from the
culture medium was comparable among the different clones
(Figure 9C), indicating that the rate of bile acid efflux
from these cells was also not affected by the presence of
HBAB.
Incubation of the BN clones with GCDCA increased the
LDH activity in the culture medium to a level similar to
that in the medium of GCDCA-treated McNtcp.24 cells
(Figure 10A). In addition, caspase 8 activity in bile
acid-treated BN.25, BN.46 and BN.49 was comparable to
that in McNtcp.24, which were elevated compared to that
in McArdle RH-7777 cells (Figure 10B). The time course of
caspase 8 activation in BN cells after treatment with 10
μM bile acids was similar to that observed for McNtcp.24
cells (Figure 10C). Therefore, the presence of an
intracellular bile acid binding protein did not protect
these cells from glycine-conjugated bile acid
cytotoxicity, even at low bile acid concentrations. These
results indicate that the activation of a signaling
cascade involving PI3K is the dominant mechanism
responsible for protecting McNtcp.24 cells from the
cytotoxicity of glycine-conjugated bile acids.
Discussion
In this study, we determined if the differential
sensitivity elaborated by the liver-derived McNtcp.24 cell
line to different classes of conjugated bile acids is
limited to cells that normally transport bile acids. We
previously demonstrated that taurine-conjugated bile acids
are not toxic to McNtcp.24 cells [ 18 ] . In contrast,
glycine-conjugated bile acids induce apoptosis in McNtcp.24
cells and primary mouse hepatocytes, through activation of
the Fas receptor in a FasL-independent manner [ 19 25 ] .
Moreover, GCDCA was demonstrated to activate both initiator
and effector caspases, and that the apoptotic response can
be blocked by Crm A, a caspase 8 inhibitor [ 25 ] . The
mechanism by which bile acids stimulate the activation of
Fas receptor remains unclear. In Fas deficient hepatocytes,
toxic bile acids were still able to induce apoptosis,
suggesting that bile acids are capable of stimulating
multiple pathways to initiate cell death [ 26 ] .
To gain further insight into the specificity of bile
acid cytotoxicity, we conferred active bile acid uptake
capacity in CHO cells, a cell type that does not normally
metabolize bile acids, by expressing a bile acid
transporter. CHO.asbt cells produce recombinant asbt with a
molecular mass and kinetic parameters that are similar to
previously reported values [ 6 ] . It is not known if CHO
cells have pathways capable of facilitating the efflux of
bile acids out of the cell. The apparent efflux observed in
CHO.asbt.35 could be attributed to the fact that asbt is
capable a bidirectional transport of bile acids [ 27 ] .
However, it is conceivable that an efflux mechanism that
recognizes bile acids exists in CHO cells and other
mammalian cells. Indeed, yeast and plants express proteins
capable of active bile acid transport [ 28 ] . As these
organisms do not normally synthesize bile acids, their
ability to transport bile acids may illustrate a
generalized mechanism for cellular transport of hydrophobic
organic anions.
CHO.asbt.35 cells were used as a model to assess the
cytotoxic potential of taurine- and glycine-conjugated bile
acids in non-hepatic derived and non-bile acid metabolizing
cells. Unlike McNtcp.24 cells, treatment of CHO.asbt clones
with taurine-conjugated bile acids resulted in cell death.
TCA was the most potent of taurine-conjugated bile acid
tested. It is unlikely that the observed cytotoxicity of
the conjugated bile acids could be attributed to the
generalized intracellular accumulation of bile acids
because unconjugated bile acids were transported by
CHO.asbt cells but did not cause significant release of LDH
into the culture medium (Torchia and Agellon, unpublished
observations). In contrast, glycine-conjugated bile acids
(GCDCA in particular) were highly effective at causing the
increase in LDH activity in the culture medium of both
McNtcp.24 and CHO.asbt.35 cells. Primary hepatocytes
exhibit differential sensitivity to taurine- and
glycine-conjugated bile acids in culture [ 29 ] . The bile
acid-transporting WIF-B cells, which are rat hepatoma-human
fibroblast hybrids [ 30 ] , remain viable even when grown
in TCA-containing culture medium for up to 14 days [ 31 ] .
In contrast, our study shows that taurine-conjugated bile
acids is toxic to a cell type that is not normally exposed
to bile acids. Liver cells, which normally metabolize bile
acids, apparently possess a mechanism that can distinguish
and differentially elaborate distinct biological activities
of taurine- and glycine-conjugated bile acids.
Both taurine- and glycine-conjugated bile acids induce
apoptotic cell death in CHO.asbt.35 cells. The involvement
of the caspase cascade in CHO.asbt.35 cells was illustrated
by the fact that conjugated bile acids activated caspase 8
in a time dependent manner. However, the different profile
of caspase 8 activation suggests that taurine- and
glycine-conjugated bile acids may trigger apoptosis by
different pathways. At present, it is not known how
conjugated bile acids activate caspases in CHO.asbt.35
cells. A protein that is immunoreactive to a Fas receptor
antiserum and has a molecular mass similar to the rat Fas
receptor is detectable in CHO cell extracts (Torchia and
Agellon, unpublished results). This leaves the possibility
that conjugated bile acids may activate the apoptotic
cascade through Fas receptor in this cell line, as
glycine-conjugated bile acids do in hepatocytes [ 25 ] .
However, the activation of caspases does not require a
specific kind of bile acid transporter since TCA is not
toxic to McArdle RH-7777 cells irrespective of whether
these cells internalize bile acids via ntcp or asbt.
The differential response of McNtcp.24 cells to
different classes of conjugated bile acids is apparently
due to the selective activation of either a PI3K-dependent
survival pathway [ 22 23 ] or caspase-dependent death
pathway [ 32 ] . Indeed, neither taurine- nor
glycine-conjugated bile acids stimulated PI3K activity in
CHO.asbt.35 cells and both classes of bile acids were
toxic. In addition, pre-treatment of McNtcp.24 cells with a
PI3K inhibitor rendered these cells susceptible to TCA- and
TCDCA- mediated cell death. The latter finding indicates
that taurine-conjugated bile acids are capable of inducing
apoptosis when PI3K is inhibited, probably by utilizing the
same mechanism activated by glycine-conjugated bile acids.
That TCA is able to activate PI3K in plasma membrane
fractions from McNtpc.24 agrees with a previous study
showing that TCA increases PI3K activity in canalicular and
sinusoidal membrane vesicles [ 16 ] . The activation of
PI3K in that report was also associated with recruitment of
sister of P-glycoprotein (the bile acid export pump) and
other multidrug resistance proteins to the canalicular
membrane [ 16 ] . Extended treatment of McNtcp.24 cells
does not appear to have detrimental and permanent
consequences on cellular function. The marked morphological
changes that occur during long term incubation with TCA [
18 ] dissipate after the withdrawal of the bile acid from
the culture medium, and the cells remain viable.
The significance of an intracellular and high affinity
bile acid binder in bile acid mediated cytotoxicity was
also evaluated. Several proteins capable of binding bile
acids in vitro have been identified (reviewed in ref. [ 1 ]
). It is thought that the major bile acid binding protein
in the cytosol of rat hepatocytes is 3α-hydroxysteroid
dehydrogenase, a member of the aldo keto reductase
supergene family [ 33 ] . However, the exact role of this
protein in intracellular transport of bile acids and
cytoprotection was undefined. HBAB, a member of the human
aldo keto reductase gene family, has been shown to bind
bile acids with high affinity (<1 μM) and is postulated
to play a similar role as 3α-hydroxysteroid dehydrogenase
in human hepatocytes [ 33 ] . Expression of HBAB in
McNtcp.24 cells was expected to increase the capacity of
these cells to sequester bile acids in the cytoplasm, and
thereby reduce their toxicity. High level expression of
HBAB had a negligible effect on the ability of the BN
clones to take up or secrete bile acids, suggesting that
HBAB has a minor role in the net transport of bile acids in
these cells. More importantly however, expression of HBAB
in McNtcp.24 cells had no effect on GCDCA-induced cell
death, even at low bile acid concentrations, indicating
that this class of protein does not provide cytoprotection
against toxic bile acids as previously suspected. This
finding is consistent with the idea that bile acids
directly modulate specific signaling cascades that control
cellular death and survival.
In summary, the failure of CHO.asbt cells to tolerate
taurine-conjugated bile acids has revealed the existence of
a mechanism that may be specific for cells that normally
metabolize bile acids. Since bile acids undergo
enterohepatic circulation, it would be reasonable to expect
the existence of the same mechanism in other cell types
that naturally transport bile acids, such as ileocytes and
cholangiocytes [ 34 35 ] . The last few years have brought
significant advances in our understanding of bile acid
transport in liver and intestinal cells. However, the
regulatory potential of bile acids is only beginning to be
realized. With the recent discovery that bile acids
represent the natural ligands for the nuclear receptor FXR
[ 10 11 12 ] , there is heightened interest in identifying
the genes that are regulated by bile acids. Insight into
how bile acids directly modulate a variety of other
cellular functions will be important in understanding the
significance of cellular responses to bile acids.
Materials and Methods
Materials
Tissue culture reagents were purchased from Canadian
Life Technologies (Burlington, ON, Canada). [
3H]-Taurocholic acid (2.6 Ci/mmol), [ 32P]-dCTP (3000
Ci/mmol), and [ 32P]-ATP (3000 Ci/mmol) were purchased
from Amersham Pharmacia Biotech (Baie d'Urfe, QC,
Canada). Bile acids were obtained from Sigma-Aldrich
Canada Ltd. (Oakville, ON, Canada) and
Calbiochem-NovaBiochem Corp. (San Diego, CA, USA). Other
reagents purchased from various commercial suppliers were
of analytical grade.
Cell lines
McNtcp.24 cells were cultured as described previously
[17]. Chinese hamster ovary K1 cells were cultured in
Ham's F12 medium supplemented with 10% fetal bovine
serum. To establish cell lines stably expressing asbt, a
cDNA encoding the hamster asbt [ 36 ] was cloned into
pBK-CMV expression vector (Stratagene, La Jolla, CA). The
prokaryotic promoter embedded in the 5'-untranslated
region of the encoded recombinant eukaryotic mRNA was
removed from the resulting plasmid. The human bile acid
binder (HBAB) cDNA [ 37 ] was cloned into a derivative of
pCep4 (Invitrogen Corp., Carlsbad, CA), that had been
previously modified by deleting the EBNA 1 gene [ 38 ] .
The lipofectamine reagent (Canadian Life Technologies)
was used to transfect the expression plasmid into CHO,
McArdle RH7777, and McNtcp.24 cells. The cell medium was
replaced 24 h post-transfection with medium containing
G418 (400 μg/ml) or hygromycin (500 μg/ml) to select for
stable transfectants. Surviving colonies were expanded
and assayed for the ability to take up [ 3H]-TCA from the
culture medium as described previously [ 17 ] or assayed
for the expression of HBAB by immunoblotting.
RNA and immunoblot analyses
Total RNA from CHO.asbt and BN cells was prepared and
analyzed by RNA blotting as previously described [ 17 ] .
Cell lysates were prepared by lysing cells in 50 mM
Tris-HCl (pH 7.4), 1% NP-40%, 0.25% sodium deoxycholate;
150 mM NaCl, 1 mM EGTA, 1 mM Na
3 VO
4 , 1 mM sodium fluoride, containing a
protease inhibitor cocktail (Sigma-Aldrich Canada Ltd.).
The lysates were fractionated by polyacrylamide gel
electrophoresis and then transferred to Immobilon-P
membrane (Millipore Ltd., Nepean, ON). Asbt was detected
using a rabbit antiserum directed against the last 14
amino acids of the carboxyl terminal of the hamster asbt
protein. HBAB protein was detected as previously
described [ 37 ] .
Bile acid uptake activity assay
Uptake studies in CHO.asbt, BN and McNtcp.24 cells
were done as previously described [ 17 ] . Protein
content was determined using the Bradford method [ 39 ]
with bovine serum albumin as the standard.
Lactate dehydrogenase (LDH) assay
Cells (10 4per well) were grown on 24 well multiwell
plates for 16 h. The cells were then washed with
phosphate buffered saline (PBS) and then incubated in
medium (Ham's F12 or Dulbecco's modified Eagle's medium
(DMEM)) containing 100 μM bile acids for 1 to 5 h as
detailed in the Figure legends. The media was collected
and adjusted to 0.1% Triton X-100. Cells remaining on the
dish were dissolved in medium (Ham's F12 or DMEM)
containing 0.1% Triton X-100. LDH activities in both the
cell lysates and media were measured as described
previously [ 40 ] .
Visualization of genomic DNA fragmentation
Cells (10 6per 60 mm dish) were grown for 16 h. After
washing with PBS, the cells were incubated with 50 μM
bile acids for 3 h at 37°C. Adherent and non-adherent
cells were dissolved in Tris-buffered saline (pH 7.6)
containing 10 mM EDTA and 0.5% sarkosyl, and 1 mg/ml
proteinase K. After digestion (~16 h) of cellular
proteins at 55°C, DNA was sequentially extracted with
phenol/chloroform and chloroform. The DNA was
precipitated, washed in 70% ethanol, and dissolved in
buffer containing 10 mM Tris-HCL (pH 7.5) and 1 mM EDTA.
The isolated DNA was treated with RNase A and then
analyzed by conventional agarose gel electrophoresis [ 41
] . DNA was visualized with ultraviolet light after
ethidium bromide staining.
Caspase activity assays
Cells (10 6per 60 mm dish) were grown for 16 h. After
washing with PBS, the cells were incubated with 50 to 100
μM bile acids for 1 to 2 h. After the incubation period,
the medium was discarded and adherent cells were
harvested in PBS and sedimented by centrifugation (500 ×
g). The cells were resuspended in buffer containing 50 mM
Tris-HCl (pH 7.5), 1% Nonidet P-40, and 150 mM NaCl.
Following 10 min incubation on ice, lysed cells were
centrifuged for 10 min, 10,000 × g at 4°C. The
supernatant was assayed for caspase activity in 20 mM
Piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.8), 100
mM NaCl, 1 mM EDTA, 1% CHAPS, and 10% sucrose with
Z-IETD-AFC for caspase 8 [ 42 43 ] . The release of the
fluorogenic-leaving group was detected by excitation at
400 nm and emission at 505 nm. Free AFC was used to
generate standard curve.
PI3K activity assay
Crude membrane fractions were isolated from CHO.asbt
and McNtcp.24 cells as described previously [ 44 ] . To
prepare lipid suspension, phosphatidylserine and
phosphatidylinositol (1:1) were sonicated in 25 mM
3-(N-morpholino)propanesulfonic acid (MOPS; pH 7.0), 1 mM
EGTA, 1 mM Na
3 VO
4 . Cell extracts were assayed in 25
mM MOPS (pH 7.5), 5 mM MgCl
2 , 1 mM EGTA, 1 mM Na
3 VO
4 , 130 μM ATP, 20 μCi [ 32P]-ATP
(3000 Ci/mmol) and 0.2 mg/ml sonicated lipids. The
reaction was allowed to proceed for 10 min at 37°C and
stopped by addition of 300 μl of 1N HCl:methanol (1:1).
Lipids were extracted twice with 350 μl chloroform and
the organic phases from both extractions were combined
then washed once with 700 μl 1 N HCl/methanol (1:1).
Lipids were dried, and then taken up in
chloroform:methanol (1:1). The samples were spotted along
with pure phosphoinositol phosphate standards (Avanti
Polar Lipids, Alabaster, AL, USA) onto 1.2% potassium
oxalate impregnated TLC plates [ 45 ] . The chromatogram
was developed in chloroform: methanol: water: aqueous
ammonia (90:70:17:3) [ 46 ] . The incorporation of [ 32P]
into phosphatidylinositol was quantitated by
phosphorimaging using a Fuji BAS1000 phosphorimager.
Visualization of standards was done by dippingTLC plates
in 10% CuSO
4 ,10% H
3 PO
4 and heating at 200°C for 10 min.
Statistical analysis
Each set of experiments was repeated at least twice.
The data is represented by means ± SD. Differences
between control and test cells were evaluated by a
Student's
t test. Differences in the slope of
regression lines were evaluated by analysis of variance.
P values of <0.05 were considered statistically
significant.
List of abbreviations used
asbt, apical sodium/bile acid cotransporter; GCA,
glycocholic acid; GCDCA, glycochenodeoxycholic acid; HBAB,
human bile acid binder; LDH, lactate dehydrogenase; PI3K,
phosphatidylinositol 3-kinase; TCA, taurocholic acid,
TCDCA, taurochenodeoxycholic acid
