Departments of 1 Surgery and 2 Pathology, University of Maryland School of Medicine, and 3 Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201
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ABSTRACT |
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The maintenance of
intestinal mucosal integrity depends on a balance between cell renewal
and cell death, including apoptosis. The natural polyamines,
putrescine, spermidine, and spermine, are essential for mucosal growth,
and decreasing polyamine levels cause G1 phase growth
arrest in intestinal epithelial (IEC-6) cells. The present study was
done to determine changes in susceptibility of IEC-6 cells to
apoptosis after depletion of cellular polyamines and to further
elucidate the role of nuclear factor-B (NF-
B) in this process.
Although depletion of polyamines by
-difluoromethylornithine (DFMO)
did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced
apoptosis increased significantly as measured by changes in
morphological features and internucleosomal DNA fragmentation. In
contrast, polyamine depletion by DFMO promoted resistance to
apoptotic cell death induced by the combination of tumor necrosis
factor-
(TNF-
) and cycloheximide. Depletion of cellular
polyamines also increased the basal level of NF-
B proteins, induced
NF-
B nuclear translocation, and activated the sequence-specific DNA
binding activity. Inhibition of NF-
B binding activity by
sulfasalazine or MG-132 not only prevented the increased susceptibility
to STS-induced apoptosis but also blocked the resistance to
cell death induced by TNF-
in combination with cycloheximide in
polyamine-deficient cells. These results indicate that 1)
polyamine depletion sensitizes intestinal epithelial cells to
STS-induced apoptosis but promotes the resistance to
TNF-
-induced cell death, 2) polyamine depletion induces
NF-
B activation, and 3) disruption of NF-
B function is
associated with altered susceptibility to apoptosis induced by
STS or TNF-
. These findings suggest that increased NF-
B activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the
death stimulus.
programmed cell death; ornithine decarboxylase; growth arrest; IB; intestinal epithelium
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INTRODUCTION |
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THE EPITHELIUM OF THE INTESTINAL mucosa is continuously renewed from the proliferative zone within the crypts and has the most rapid turnover rate of any tissue in the body (21, 38). These undifferentiated epithelial cells divide and differentiate as they migrate to the luminal surface of the colon or up the villous surface in the small intestine. To maintain stable numbers of enterocytes, cell division is counterbalanced by apoptosis, a fundamental biological regulatory mechanism involving selective cell deletion to maintain tissue homeostasis (21, 22). Apoptosis is a genetically regulated form of programmed cell death defined by distinct morphological and biochemical features, including chromatin condensation, DNA fragmentation, and membrane blebbing (12, 14, 23, 53, 57). Apoptosis occurs in epithelial cells in the crypt area, where it maintains the critical balance in cell number between newly divided and surviving cells, and at the luminal surface of the colon and villous tips in the small intestine, where differentiated cells are lost (14, 23).
Normal physiological processes and pathological stimuli induce
apoptosis via different signal transduction cascades (12, 14, 23, 53, 57). Many of the genes that are activated in the
initiation of apoptosis are target genes of nuclear factor-B (NF-
B) (3, 4). The NF-
B family of transcription
factors consists of five different subunits in mammalian cells,
including p50, p52, p65/Rel A, c-Rel, and Rel-B, which are able to form homodimers or heterodimers (3). Under nonstress
conditions, NF-
B dimers are sequestered in most cells in the
cytoplasm by a member of the I
B family of proteins: I
B
,
I
B
, or I
B
(11, 40). Binding of I
B to
NF-
B blocks nuclear localization signals on NF-
B and prevents its
translocation to the nucleus (6, 26, 39, 54). In response
to a host of stimuli, I
B is phosphorylated and then degraded by the
ubiquitin-proteasome pathway (26, 39), which allows free
NF-
B to translocate to the nucleus to activate transcription of
specific genes involved in apoptosis.
Accumulating evidence has revealed that NF-B has a proapoptotic
or antiapoptotic function, depending on cell type and the death
stimulus (5, 29, 30, 46, 56). Overexpression of NF-
B in
chick bone marrow cells leads to apoptosis (1), and suppression of NF-
B activity by increasing I
B expression inhibits Sindbis virus-induced cell death in AT-3 prostate carcinoma cells (29, 30), indicating that NF-
B acts as a
proapoptotic transcription factor. Consistently, several
proapoptotic genes such as c-myc, p53, Fas ligand, and
the interleukin-1
-converting enzyme caspase-1 have NF-
B binding
sequences in their promoter regions (3, 24, 45, 69). In
contrast, NF-
B activity appears to be necessary for the activation
of genes that suppress some type of apoptosis. For example,
inhibition of NF-
B activity in immature B cells after addition of
anti-IgM or in liver during development markedly enhances apoptotic
cell death (7, 68). Antiapoptotic genes that are
regulated by NF-
B include manganese superoxide dismutase
(63) and the zinc finger protein A20 (40). However, the precise factors that determine the ability of NF-
B to
regulate these divergent biological actions are unknown.
Our previous studies (60-62) and others (20,
33, 38) have shown that the polyamines spermine and
spermidine and their diamine precursor putrescine are
absolutely required for the maintenance of normal intestinal mucosal
homeostasis and that intracellular polyamine levels are highly
regulated by the cell according to the state of growth. Decreasing
cellular polyamines by inhibition of ornithine decarboxylase (ODC), the
rate-limiting step in polyamine biosynthesis, with
-difluoromethylornithine (DFMO) suppresses mucosal growth in vivo
(33, 62) as well as in vitro (27, 63).
Because polyamines not only play a critical role in cell proliferation
(20, 38) but also are involved in apoptosis (16, 36), inhibition of the intestinal mucosal growth
after polyamine depletion could be due to a decrease in cell renewal and/or an increase in apoptosis. We (27) recently
demonstrated that depletion of cellular polyamines by DFMO results in a
significant increase in G1 phase growth arrest in
intestinal epithelial (IEC-6) cells but does not directly induce apoptosis.
The present studies asked whether inhibition of polyamine synthesis
alters susceptibility of intestinal epithelial cells to apoptotic
stimuli and further investigated the involvement of NF-B in this
process. First, we wanted to examine changes in susceptibility of IEC-6
cells to staurosporine (STS)- and tumor necrosis factor-
(TNF-
)-induced apoptosis after depletion of cellular
polyamines. Second, we examined whether polyamine depletion increased
NF-
B activity. Third, we wished to elucidate whether the observed
increase in NF-
B activity played a role in the alteration of
epithelial cell susceptibility to apoptotic stimuli.
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MATERIALS AND METHODS |
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Chemicals and supplies.
Disposable culture ware was purchased from Corning Glass Works
(Corning, NY). Tissue culture media and dialyzed fetal bovine serum
(FBS) were purchased from GIBCO BRL (Gaithersburg, MD), and
biochemicals were obtained from Sigma Chemical (St. Louis, MO). The
double-stranded oligonucleotides used in electromobility shift assay
and antibodies against NF-B were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). [
-32P]ATP (3,000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). DFMO was
a gift from Merrell Dow Research Institute of Marion Merrell Dow
(Cincinnati, OH).
Cell culture and experimental design. The IEC-6 cell line was purchased from the American Type Culture Collection at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (47). Originating from intestinal crypt cells as judged by morphological and immunological criteria, the IEC-6 cells are nontumorigenic and retain the undifferentiated character of epithelial crypt cells. Stock cells were maintained as previously described (42), and passages 15-20 were used in the experiments.
In the first series of studies, we examined whether depletion of cellular polyamines by treatment with DFMO could alter susceptibility to STS- and TNF-Assessment of morphology. After various experimental treatments, cells were photographed with a Nikon inverted microscope before fixation. Cells then were fixed with D-PBS containing Nonidet P-40, Hoechst-33342, and 4% formaldehyde as described previously (16). Hoechst-stained cells were visualized and photographed under ultraviolet excitation with a Nikon microscope, and the percentage of "apoptotic" cells was determined.
DNA fragmentation. DNA from treated cells was assayed using a modification of the method described by Armstrong et al. (2). Briefly, cells were lysed with 1.0 ml of digestion buffer and incubated at 50°C for 18 h. Samples were extracted twice with 1 vol of phenol-chloroform-isoamyl alcohol, precipitated with 7.5 M ammonium acetate and 100% ethanol, and resuspended in 10 mM Tris · HCl. Samples were then treated with RNase (40 µg/ml) in the presence of 0.1% SDS for 1 h at 37°C. Samples were reextracted, precipitated, and resuspended a second time as described above. Ten micrograms of DNA were loaded into each well and electrophoresed in 1.5% agarose gel. Gels were visualized by ultraviolet fluorescence and photographed with a Polaroid camera system.
Preparation of nuclear protein and electrophoretic mobility shift
assays.
Nuclear proteins were prepared by the procedure described previously
(70), and the protein contents in nuclear preparation were
determined by the method described by Bradford (10). The double-stranded oligonucleotides used in these experiments included 5'-AGTTGAGGGGACTTTCCCAGGC-3', which contains a consensus
NF-B binding site that is underscored. These oligonucleotides were radioactively end-labeled with [
-32P]ATP and T4
polynucleotide kinase (Promega, Madison, WI). For mobility shift
assays, 0.035 pmol of 32P-labeled oligonucleotides
(~30,000 cpm) and 10 µg of nuclear protein were incubated in a
total volume of 25 µl in the presence of 10 mM Tris · HCl (pH
7.5), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and 1 µg of poly(dI-dC). The binding reactions were allowed to proceed at
room temperature for 20 min. Thereafter, 2 µl of bromphenol blue
(0.1% in water) were added, and protein-DNA complexes were resolved by
electrophoresis on nondenaturing 5% polyacrylamide gels and visualized
by autoradiography. The specificity of binding interactions was
assessed by competition with an excess of unlabeled double-stranded
oligonucleotide of identical sequence.
Western blotting analysis.
Ten micrograms of cytoplasmic protein extracts were dissolved in SDS
sample buffer, boiled for 5 min, and then subjected to electrophoresis
on 10% acrylamide gels according to Laemmli (25). After
SDS-PAGE, the gels were transferred to nitrocellulose membranes for
1 h at 4°C. The blots were blocked with 5% nonfat dry milk in
PBS-0.1% Tween 20 (PBS-T) overnight at 4°C. Immunological evaluation was then performed for 1 h in PBS-T containing 0.2 µg/ml
affinity-purified polyclonal antibodies against NF-B subunits p50
and p65 (50) or I
B
protein. The blots were
subsequently washed with PBS-T and incubated for 1 h with goat
anti-rabbit IgG antibody conjugated to peroxidase at a dilution of
1:3,000 in PBS-T. After extensive washing with PBS-T, the blots were
developed for 30 or 60 s with enhanced chemiluminescence reagents (Amersham).
Immunohistochemical staining.
Immunohistochemical staining for NF-B protein was performed in
IEC-6 cells by the indirect immunoperoxidase method as described previously (27). The cells were incubated with rabbit
polyclonal antibody against the p65 subunit of NF-
B at a dilution of
1:100 in PBS containing 1% BSA for 1 h at room temperature and
then 1 h of incubation with biotinylated goat anti-rabbit IgG at a dilution of 1:500. Nonspecific slides were incubated without antibody against NF-
B. The bound antibody was visualized with avidin-biotin complexes. The slides were counterstained with hematoxylin, mounted, and viewed with an Olympus microscope.
Polyamine analysis.
The cellular polyamine content was analyzed by HPLC as described
previously (62). After the monolayers were washed three times with ice-cold D-PBS, 0.5 M perchloric acid was added, and the
monolayers were frozen at 80°C until ready for extraction, dansylation, and HPLC. The standard curve encompassed 0.31-10 µM. Values that fell 25% below the curve were considered not
detectable. Protein was determined by the Bradford method
(10). The results are expressed as nanomoles of polyamines
per milligram of protein.
Statistics. Values are means ± SE from six dishes. Autoradiographic results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined using Duncan's multiple range test (17).
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RESULTS |
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Effects of polyamine depletion on STS- and TNF--induced
apoptosis.
Administration of 5 mM DFMO, which totally inhibited ODC activity
(27, 63), almost completely depleted cellular polyamines in IEC-6 cells. The levels of putrescine and spermidine were
undetectable at 4, 6, and 8 days after DFMO treatment. Spermine was
less sensitive to the inhibition of ODC but was decreased by >60% in
cells exposed to DFMO for 4, 6, and 8 days (data not shown). Similar
results have been published previously (42).
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Effect of polyamines on NF-B protein levels and cellular
distribution.
To elucidate the mechanisms responsible for altered susceptibility of
polyamine-deficient IEC-6 cells to STS- and TNF-
-induced apoptosis, the role of NF-
B in this process was
investigated. Data presented in Fig. 3
clearly show that depletion of cellular polyamines
by treatment with DFMO not only significantly increased NF-
B protein
levels but also induced NF-
B nuclear translocation. The increase in
protein levels for NF-
B (p50 and p65 subunits) was noted 4 days
after DFMO exposure and remained elevated 6 and 8 days after exposure
(Fig. 3, A and B). The levels of NF-
B p50 protein in cells exposed to DFMO were ~1.8, ~2.1, and ~2.4 times the normal values (without DFMO) 4, 6, and 8 days after DFMO, respectively. Protein levels for the NF-
B p65 subunit were almost twice the normal values 4, 6, and 8 days after DFMO treatment. Spermidine (5 µM) given together with DFMO completely prevented the
increased levels of NF-
B proteins. Putrescine (10 µM) had an
effect equal to spermidine on NF-
B activation when it was added to
cultures that contained DFMO (data not shown).
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Changes in NF-B sequence-specific DNA binding activity in
polyamine-deficient cells.
Consistently, increased levels of NF-
B protein in cells exposed to
DFMO were paralleled by a marked increase in NF-
B binding activity
as measured by electrophoretic mobility shift assay (Fig. 4A). The NF-
B binding
activity was increased by nearly twofold 4 days after DFMO treatment
and by approximately threefold 6 and 8 days after DFMO (Fig.
4B). Spermidine (5 µM) given together with DFMO completely
prevented the increase in NF-
B binding activity. To evaluate the
specificity of the binding reaction in Fig. 4A, competitive
inhibition experiments were performed. As shown in Fig. 4C,
a and b, NF-
B binding activities in control
cells and cells exposed to DFMO were dose-dependently inhibited when
various concentrations of the unlabeled NF-
B oligonucleotide were
added to the binding reaction mixture. We also examined the effect of the unlabeled oligonucleotide containing a mutated NF-
B binding site
on NF-
B binding activity and found that the NF-
B-mutated oligonucleotide did not inhibit NF-
B binding activity in IEC-6 cells
(Fig. 4Cc). Figure 4Cd further shows that
preincubation of nuclear extracts with a specific antibody against the
NF-
B p65 subunit significantly inhibited the formation of NF-
B
binding complex. In contrast, preincubation with the control (anti-Myc) antibody had no inhibitory effect on NF-
B binding activity (data not
shown). These results indicate that altered susceptibility of IEC-6
cells to apoptotic stimuli after polyamine depletion is associated
with a significant increase in the NF-
B activity.
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Effect of polyamine depletion on IB.
To determine the involvement of I
B in the process of NF-
B nuclear
translocation after polyamine depletion, expression of I
B
protein
was examined in cells grown in the presence or absence of DFMO. As
shown in Fig. 5, depletion of cellular
polyamines by DFMO significantly inhibited the content of I
B
protein in IEC-6 cells. The level of I
B
protein was decreased by
~45 and ~65% 4 and 6 days after exposure to DFMO, respectively.
The I
B
protein content returned to normal levels when DFMO was
given together with spermidine. These results suggest that activation of NF-
B activity after depletion of cellular polyamines is mediated at least partially through the I
B pathway.
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Effects of NF-B inhibitors on STS- and TNF-
-induced
apoptosis in polyamine-depleted IEC-6 cells.
To investigate the role of induced NF-
B activation in the process of
altered susceptibility to apoptosis after polyamine depletion,
two potent and specific inhibitors of NF-
B, sulfasalazine (58) and MG-132 (18), were used in this
study. Inhibition of NF-
B activity by treatment with sulfasalazine
prevented the increased susceptibility of polyamine-deficient cells to
STS-induced apoptosis (Fig. 6).
Sulfasalazine at 1 mM slightly decreased the rate of apoptosis,
but these differences were not statistically significant. However,
sulfasalazine at 2 mM significantly blocked the increased sensitivity
of polyamine-deficient cells to STS-induced apoptosis. Typical
morphological features of programmed cell death (Fig. 6Ad)
and internucleosomal DNA fragmentation (Fig. 6B,
right) decreased markedly in DFMO-treated cells pretreated
with 2 mM sulfasalazine. The percentage of STS-induced apoptotic
cells was decreased from ~46% in DFMO-treated cells to ~18% when
DFMO-treated cells were pretreated with 2 mM sulfasalazine (Fig.
6C). In addition, inhibition of NF-
B activity by
sulfasalazine also prevented increased susceptibility of
polyamine-deficient cells to apoptosis induced by diclofenac or
indomethacin (data not shown). Sulfasalazine at 1 and 2 mM alone caused
no apoptosis in the absence of STS (Fig. 6C,
right).
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Effects of sulfasalazine and MG-132 on NF-B activity in
DFMO-treated cells.
To confirm the inhibitory effects of sulfasalazine and MG-132 on
NF-
B in polyamine-deficient cells, the activity of NF-
B was
measured in DFMO-treated cells exposed to sulfasalazine or MG-132.
Cells were grown in the presence of 5 mM DFMO for 6 days and then
treated with different concentrations of sulfasalazine or MG-132.
Exposure to sulfasalazine or MG-132 for 90 min decreased NF-
B
binding activity in polyamine-deficient cells (Fig.
9). When various doses of sulfasalazine
were tested, NF-
B binding activity was inhibited dose dependently,
with concentrations ranging from 1 to 4 mM. Significant inhibition of
NF-
B binding activity occurred at 2 mM, and the binding activities
were decreased by ~80% (Fig. 9, left). In cells treated
with MG-132, NF-
B activity was decreased by ~30 and ~80% at 1 and 10 µM, respectively (Fig. 9, right). There was no
apparent loss of cell viability in cells treated with DFMO alone or
DFMO + various doses of sulfasalazine or MG-132 before treatment
with STS or TNF-
(data not shown).
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DISCUSSION |
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Cellular polyamines have been essential for the maintenance of intestinal epithelial integrity, but few specific functions of polyamines in this process have been defined. The role of polyamines in apoptotic pathways has been rather controversial, depending on the cell type and death stimulus. Numerous studies have demonstrated that polyamines protect against apoptosis in certain cell types and that decreasing cellular polyamines induces programmed cell death (15, 16, 31, 36, 37, 43). The deregulation of cellular polyamine levels and abnormal polyamine metabolic enzyme activities are observed in cells undergoing apoptosis (15, 16, 31). A decrease in polyamine content by the superinduction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase results in a typical process of programmed cell death in non-small cell lung carcinoma NCI H57 cells (16, 37). Exposure to the polyamine oxidase inhibitor MDL-72527 reduces the levels of putrescine and spermidine and induces apoptosis in transformed hematopoietic cells (13). Similar findings are also observed in breast cancer cell lines (36).
On the other hand, contrary evidence has been reported, indicating that high levels of polyamines favor apoptosis and that inhibition of polyamine synthesis protects cells from apoptotic cell death. For example, excessive polyamine accumulation in ODC-overproducing L1210 cells induces characteristic features of apoptosis (44). Administration of the amino acid ornithine stimulates the accumulation of cellular putrescine and increases programmed cell death in ODC-overproducing mouse myeloma cells (55). Recently, Ray et al. (48) reported that depletion of cellular polyamines by treatment with DFMO decreased the apoptotic index induced by the DNA topoisomerase I inhibitor camptothecin in IEC-6 cells.
The findings reported here clearly indicate that polyamines are
involved in the regulation of susceptibility of intestinal epithelial
cells to apoptosis. The data also reveal that alteration in the
tolerance or the sensitization to apoptosis after polyamine depletion depends on the death stimulus. For example, the
susceptibility of intestinal epithelial cells to STS-induced
apoptosis increased dramatically after polyamine depletion. In
contrast, inhibition of polyamine synthesis by treatment with DFMO
prevents TNF--induced apoptosis in IEC-6 cells. Results
presented here further demonstrate that altered susceptibility of
intestinal epithelial cells to apoptotic stimulus after polyamine
depletion is associated with a significant increase in NF-
B
activity. Although the exact intracellular signaling pathway initiated
by depletion of cellular polyamines leading to NF-
B activation is
unclear, levels of I
B
protein decreased significantly in
polyamine-deficient cells. Because NF-
B normally exists in an
inactive form in the cytoplasm bound to I
B (11), the
downregulation of I
B
protein expression could be partially
responsible for the increased activation and nuclear translocation of
NF-
B in polyamine-depleted cells. It is not clear whether decreased
I
B
levels after polyamine depletion are due to an increase in
I
B
degradation by phosphorylation or a decrease in I
B
production.
Another study indicates that polyamines induce activation of NF-B.
Shah et al. (52) recently reported that natural spermine exerts a significant stimulatory effect on NF-
B binding activity in
breast cancer (MCF-7) cells. Spermidine and putrescine are also capable
of facilitating NF-
B binding, but to a lesser extent than spermine
and only at higher concentrations. The reasons for the different
responses of NF-
B to polyamines in IEC-6 and MCF-7 cells remain
unclear but may be related to the following facts. First, the results
are from two different experimental conditions. In our study, exogenous
spermidine was added to the culture medium with DFMO and kept in the
medium for 4, 6, and 8 days. In the studies with MCF-7 cells,
polyamines were added directly to the reaction mixtures of gel shift
assays. Second, there were significant differences in concentrations of
polyamines between these two studies: IEC-6 cells were exposed to 5 µM spermidine, and cellular extracts from MCF-7 cells were incubated
with polyamines ranging from 0.1 to 2 mM. Finally, the cell types are
different: the IEC-6 line is derived from the normal small intestinal
crypts, and the MCF-7 line is from breast tumor tissue.
Our results suggest that NF-B activation plays a critical role in
the process through which polyamine depletion alters the sensitivity of
IEC-6 cells to apoptotic stimulus. Inhibition of NF-
B activity
by treatment with sulfasalazine not only prevented the increased
susceptibility to STS-induced apoptosis but also blocked the
protection against apoptosis induced by TNF-
in combination with cycloheximide in polyamine-deficient cells. Although sulfasalazine may exert different effects on apoptosis through a process
independent of NF-
B, data obtained from the utilization of another
specific pharmacological inhibitor of NF-
B, MG-132, are similar to
those from the application of sulfasalazine. These findings from using two different NF-
B inhibitors provide the strong evidence supporting our hypothesis.
The observation that NF-B is involved in proapoptotic and
antiapoptotic processes is not surprising, because the function of
NF-
B in the regulation of apoptosis has been shown to depend on cell type and death stimulus (5, 29, 30, 46, 56). There
is evidence indicating that NF-
B protects dividing cells against
apoptotic cell death induced by TNF-
in certain cell types
(5, 56). The NF-
B p65 subunit knockout animals are more
sensitive to TNF-
-induced programmed cell death (68). Conversely, other studies have suggested that NF-
B promotes
apoptosis in different cell injury models (29, 30,
46). For example, quinolinic acid-induced apoptotic cell
death in striatal neurons is associated with a significant increase in
NF-
B activity, and prevention of NF-
B nuclear translocation
diminishes quinolinic acid-induced apoptosis (46).
Similar results from HL-60 cells also show that activation of NF-
B
activity by triphenyltin triggers apoptotic cell death
(31). Although the precise factors that determine the
ability of NF-
B to regulate these divergent biological outcomes are
unknown, the interaction of NF-
B with other effectors or regulators
of apoptosis may be involved in this process. It is possible
that polyamine depletion may induce expression of inactive forms of one
or more proapoptotic or antiapoptotic factors, which would be
activated by a specific death stimulus. Cross talk between NF-
B and
these activated factors could determine the function of NF-
B in the
regulation of apoptosis in polyamine-deficient cells.
How might NF-B mediate the increased susceptibility of intestinal
epithelial cells to apoptosis after polyamine depletion? NF-
B modulates transcription of many genes and has transcriptional cross talk with p53 (66, 69). The product of the p53 gene serves as a critical regulator of the cell cycle and of apoptotic mechanisms in normal and malignant cells (51). The
important contribution of p53 to the apoptotic process has been
well documented in various cell types and under different conditions
(45, 51, 69). We (27) recently reported that
polyamine depletion by DFMO significantly increases expression of the
p53 gene in IEC-6 cells. The remarkable parallelism that exists between
the stimulation of p53 gene expression and the increased susceptibility
to apoptosis elicited by NF-
B suggests the possibility that
activation of NF-
B after polyamine depletion sensitizes intestinal
epithelial cells to apoptosis in association with its ability
to regulate p53 gene expression. This contention is supported by recent
publications that indicate that activated NF-
B plays a role in
p53-mediated programmed cell death (49) and that NF-
B
mediates bcl-2 suppression in hypoxia-induced endothelial
apoptosis (35). In addition, other signaling
pathways may also be involved in STS-induced apoptosis. Polyamines have been shown to regulate intracellular Ca2+
concentration ([Ca2+]cyt) through
K+ channels, and depletion of cellular polyamines reduces
[Ca2+]cyt in IEC-6 cells (65).
Because [Ca2+]cyt has been implicated in
apoptosis through the regulation of bcl-2 expression and
protein kinase activity (8), the increased susceptibility
of polyamine-deficient cells to STS-induced cell death may result, at
least partially, from the decrease in
[Ca2+]cyt. It is possible that STS induces
apoptosis synergistically with the reduction of
[Ca2+]cyt.
The findings that activation of NF-B protects against
apoptosis induced by TNF-
in combination with cycloheximide
in polyamine-deficient cells are consistent with those from other
investigators, who demonstrated that NF-
B is a cell survival factor
(5, 56, 68). In general, NF-
B has been believed to
inhibit apoptosis in the caspase-dependent pathways, because
numerous reports documented the antiapoptotic action of NF-
B and
apoptosis caused by inactivation of NF-
B in a variety of
cell types (5, 56, 59, 68). Although the exact mechanism
through which activation of NF-
B prevents TNF-
-induced
apoptosis in DFMO-treated cells is unclear, it has been shown
that polyamine depletion decreases caspase-3 activity, which is
associated with delayed apoptosis after exposure to
camptothecin (48).
In summary, these results indicate that cellular polyamines play a role
in the regulation of apoptosis in intestinal epithelial cells
and that inhibition of polyamine synthesis alters susceptibility to
apoptotic stimuli. The present study also shows that depletion of
cellular polyamines significantly increases NF-B activity and
induces NF-
B nuclear translocation in IEC-6 cells. Inactivation of
NF-
B by treatment with sulfasalazine or MG-132 in
polyamine-deficient cells is associated with altered susceptibility to
apoptosis induced by STS or TNF-
+ cycloheximide. These
findings suggest that NF-
B can play a proapoptotic or
antiapoptotic role in intestinal epithelial cells, which is
dependent on the death stimulus.
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ACKNOWLEDGEMENTS |
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This work was supported by Merit Review Grants from the Department of Veterans Affairs (to J.-Y. Wang and B. L. Bass) and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57819 (to J.-Y. Wang).
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FOOTNOTES |
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Some of these data have been published in abstract form (28).
Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore VA Medical Center, 10 North Greene St., Baltimore, MD 21201 (E-mail: jwang{at}smail.umaryland.edu).
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.
Received 9 August 2000; accepted in final form 14 December 2000.
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