Department of Physiology, The University of Tennessee Health Science Center, Memphis, Tennessee 38163
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ABSTRACT |
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C1297, 2002; 10.1152/ajpcell.00351.2001.We
have shown previously that depletion of polyamines delays
apoptosis induced by camptothecin in rat intestinal epithelial
cells (IEC-6). Mitochondria play an important role in the regulation of
apoptosis in mammalian cells because apoptotic signals
induce mitochondria to release cytochrome c. The latter
interacts with Apaf-1 to activate caspase-9, which in turn activates
downstream caspase-3. Bcl-2 family proteins are involved in the
regulation of cytochrome c release from mitochondria. In
this study, we examined the effects of polyamine depletion on the
activation of the caspase cascade, release of cytochrome c
from mitochondria, and expression and translocation of Bcl-2 family
proteins. We inhibited ornithine decarboxylase, the first rate-limiting
enzyme in polyamine synthesis, with -difluoromethylornithine (DFMO)
to deplete cells of polyamines. Depletion of polyamines prevented
camptothecin-induced release of cytochrome c from
mitochondria and decreased the activity of caspase-9 and caspase-3. The
mitochondrial membrane potential was not disrupted when cytochrome
c was released. Depletion of polyamines decreased
translocation of Bax to mitochondria during apoptosis. The
expression of antiapoptotic proteins Bcl-xL and Bcl-2
was increased in DFMO-treated cells. Caspase-8 activity and cleavage of
Bid were decreased in cells depleted of polyamines. These results
suggest that polyamine depletion prevents IEC-6 cells from
apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c.
polyamines; -difluoromethylornithine; mitochondria; Bcl-2
protein; caspase-9
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INTRODUCTION |
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APOPTOSIS is a highly regulated form of programmed cell death. During apoptosis, chromatin condensation and nuclear fragmentation occur, and cells may detach from neighboring cells and separate into intact membrane-bound fragments called apoptotic bodies (15). Inflammation does not occur because the apoptotic bodies are rapidly phagocytosed by other cells, and no intracellular constituents are released (46). Apoptosis results from a complex set of biochemical events catalyzed by a family of proteases called caspases. Caspases are synthesized as proenzymes and are activated by proapoptotic signals through various pathways (41). Caspases cleave lamina, a rigid structure that underlies the nuclear membrane, causing chromatin condensation. Caspases also alter cell structures indirectly by cleaving proteins involved in cytoskeletal regulation. The substrates of caspases also include proteins involved in DNA repair, mRNA splicing, DNA replication, and proteins that protect cells from apoptosis (59). Apoptosis is required for all multicellular organisms to achieve and maintain homeostasis of cell numbers within their tissues (15). Increased apoptosis produces degenerative diseases of the central nervous system or immunodeficiencies, whereas failure to undergo apoptosis results in developmental abnormalities and cancer (22, 63). In the small intestine, spontaneous apoptosis during development is necessary to achieve the optimal number of stem cells. Intestinal epithelial cells (IEC) undergo apoptosis at the luminal surface. Expression of the Bcl-2/Bax family of proteins, change in cell-to-cell contact, disruption of cell-to-extracellular matrix attachment, integrin expression, cytokines, environmental factors, and chemical agents may contribute to the induction of apoptosis at the luminal surface (1, 2, 22, 38, 49).
Polyamines are involved in the process of apoptosis. The
accumulation of spermidine in L1210 cells overexpressing ornithine decarboxylase (ODC) induces apoptosis (43).
Exposure of human leukemia cells to polyamines triggers caspase
activation (52). In a cell-free model using a postnuclear
extract from U937 cells, the addition of spermine triggers the onset of
caspase activity in the presence of ATP and mitochondria or cytochrome
c (53, 55). The effect of polyamine depletion
on apoptosis differs depending on the cell type. Depletion of
polyamines increases apoptosis of head and neck squamous
carcinoma cells (5), human T lymphoblastic leukemia cells
(9), mouse mammary epithelial cells (42), and
human gastric cancer cells (57). Apoptosis, induced by tumor necrosis factor- and cycloheximide, is delayed by
the depletion of polyamines in rat IEC (32, 47) and mouse thyroid cells (23). In some cell lines, depletion of
polyamines either increases or decreases the sensitivity to
apoptosis depending on the nature of apoptotic stimuli
(32, 54).
Camptothecin, an inhibitor of DNA topoisomerase I, has been
widely used to induce apoptosis under experimental conditions and is in phase III clinical trials for the treatment of colon cancer
(10, 11). Camptothecin causes breaks in DNA strands (34). Therefore, DNA damage is the initial signal of
apoptosis induced by camptothecin. We have previously shown
that depletion of polyamines delays apoptosis induced by
camptothecin in IEC-6 cells (47). However, the mechanism
whereby depletion of polyamines protects IEC-6 cells from
apoptosis remains unclear. Loeffler and Kroemer
(35) suggest a three-stage model of apoptosis: a premitochondrial phase during which upstream apoptotic signal transduction cascades are activated, a mitochondrial phase during which
mitochondrial membranes are permeablized and cytochrome c
and other proteins are released, and a postmitochondrial phase during
which proteins released from mitochondria activate caspases and
nucleases. Once cytochrome c is released, it activates a
caspase cascade, resulting in apoptosis (12).
Therefore, cytochrome c release is the crucial step in
determining whether a cell undergoes programmed death. Bcl-2 family
proteins are implicated in the opening of the mitochondrial
permeability transition pore (PTP) and the subsequent release of
cytochrome c into cytosol. They play a pivotal role in
regulating cell life and death (4, 19). Bax and Bid are
proapoptotic members of the Bcl-2 family. Translocation of Bax and
truncated Bid to mitochondria induces the release of cytochrome
c from mitochondria into cytosol (20, 28). Bcl-2 and
Bcl-xL antagonize the function of Bax and Bid and protect cells from apoptosis (61). In this study, we
examined the effect of polyamine depletion on cytochrome c
release from mitochondria and the levels of Bcl-2 family proteins. We
inhibited ODC, the first rate-limiting enzyme in polyamine synthesis,
with -difluoromethylornithine (DFMO) to deplete cells of polyamines.
We found that the camptothecin-induced increases in caspase activity,
release of cytochrome c from mitochondria, translocation of
Bax to mitochondria, and cleavage of Bid were inhibited in cells
depleted of polyamines.
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MATERIALS AND METHODS |
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Chemicals and supplies. Medium and other cell culture reagents were obtained from GIBCO BRL (Grand Island, NY). Dialyzed fetal bovine serum (FBS) was purchased from Sigma (St. Louis, MO). Cell Death Detection ELISA Plus kit was purchased from Roche Diagnostics (Indianapolis, IN). Caspase colorimetric substrates Ac-LEHD-pNA, Ac-IETD-pNA, and Ac-DEVD-pNA were obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Rabbit anti-cytochrome c, anti-Bcl-2, and mouse anti-Bcl-xL antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Bax antibody was obtained from Pharmingen (San Diego, CA). Rabbit anti-Bid antibody was a gift from Dr. Xiao-Ming Yin (University of Pittsburgh, Pittsburgh, PA). Horseradish peroxidase-conjugated secondary antibody was purchased from Sigma. The enhanced chemiluminescence Western blot detection system was purchased from Dupont NEN (Boston, MA). DFMO was a gift from Merrel Dow (Cincinnati, OH).
Cell culture. The IEC-6 cell line was derived from normal rat small intestinal crypt cells developed and characterized by Quaroni et al. (45) and was obtained from the American Type Culture Collection (Rockville, MD) at passage 13. The stock was maintained in T-150 flasks in a humidified 37°C incubator in an atmosphere of 90% air-10% CO2. The stock medium consisted of DMEM with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin. The stock was passaged weekly at 1:5 dilution, and the cells were fed three times a week. Passages 16-21 were used in the experiments. The cells were routinely checked for mycoplasma and were always found to be negative.
Quantitative DNA fragmentation ELISA. Cells were grown in six-well culture plates for both DNA fragmentation ELISA and protein determination. After treatment with camptothecin, cells were lysed and centrifuged to remove the nuclei. An aliquot of the nuclei-free supernatant was placed in streptavidin-coated wells and incubated with anti-histone-biotin antibody and anti-DNA peroxidase-conjugated antibody for 2 h at room temperature. After incubation, the sample was removed and the wells were washed three times with incubation buffer. After the final wash was removed, 100 µl of the substrate, 2,2'-azino-di-[3-ethylbenzthiazolin-sulfonate], was placed in the wells for 20 min at room temperature. The absorbance was read at 405 nm using a plate reader. Results were expressed as absorbance at 405 nm per minute per milligram of protein.
Caspase activity. After treatment with camptothecin (or vehicle), cells were then harvested for determination of caspase activity. Briefly, 10 ml of DPBS were added to the flask, and the monolayer was scraped and collected into a 25-ml tube. The flask was washed once with 10 ml of Dulbecco's PBS (DPBS), and the wash was added to the 25-ml tube. The cells were pelleted by centrifugation at 800 g for 5 min. The supernatant was discarded, and the pellet was resuspended in 1 ml of cold DPBS and transferred to a microfuge tube. The cells were pelleted by centrifugation at 10,000 g at 4°C for 10 min. The supernatant was discarded, and the cells were lysed in 100 µl of ice-cold cell lysis buffer [50 mM HEPES, pH 7.4, 0.1% CHAPS, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1% NP40]. The assay for caspase activity was carried out in a 96-well plate. Into each well were placed 20 µl of cell lysate, 70 µl of assay buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 100 mM NaCl, 10 mM DTT, and 1 mM EDTA), and 10 µl of caspase-3, -8, or -9 colorimetric substrate. The 96-well plate was incubated at 37°C for 2 h, during which time the caspase in the sample was allowed to cleave the chromophore p-NA from the substrate molecule. Absorbency readings at 405 nm were made after the 2-h incubation, with the caspase activity being directly proportional to the color reaction. Protein was determined for each sample using the bicinchoninic acid method (BCA; Pierce, Rockford, IL), and a standard curve for p-nitroanilide was constructed. Caspase activity was expressed as picomoles of pNA released per microgram of protein per minute.
Preparation of mitochondria and cytosol. Mitochondrial and cytosolic fractions were prepared by a previously described method (26). Briefly, cells were harvested, washed, resuspended in ice-cold buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride) containing 250 mM sucrose, homogenized with a Dounce homogenizer by using a type B (loose) pestle, and centrifuged at 1,000 g for 10 min to separate nuclei and unbroken cells. The supernatant was then centrifuged at 10,000 g for 15 min to pellet heavy membranes (mitochondrial fraction). The pellet was washed three times with buffer A to eliminate contamination by other subcellular fractions. The supernatant from the 10,000 g spin fraction was further centrifuged at 100,000 g for 1 h at 4°C to produce a supernatant corresponding to the cytosolic fraction (S100).
Preparation of total protein. Cells were washed twice with cold DPBS, and 500 µl of RIPA buffer were added to the flask. The cells were then harvested using a rubber scraper, transferred to microfuge tubes, incubated on a rotator at 4°C for 30 min, and centrifuged at 14,000 g at 4°C for 10 min, and then supernatants were collected.
Western blots. One hundred micrograms of protein were separated on 15% SDS-polyacrylamide gel and transferred to nitrocellose membranes. The equal loading and transfer of mitochondrial protein was confirmed by staining the nitrocellulose membrane with PonceauS. Actin and PonceauS staining were used as loading controls for caspase-3 and procaspase-3. Membranes were blotted with 3% bovine albumin in PBS containing 3% Triton X-100 and were then probed with primary antibody for 1 h at room temperature. They were then washed with wash buffer, incubated further with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and rewashed with wash buffer. The immunocomplexes on the membrane were reacted for 1 min with a chemiluminescence reagent. Finally, the filters were placed in a plastic sheet protector and exposed to autoradiograph film for 30 or 60 s. The density of bands was quantitated by NIH Image software.
Statistics. Values are means ± SE. Statistical analysis was performed using ANOVA and appropriate post hoc testing. Absence of error bars indicates that the SE was too small to be seen as separate from the mean. P < 0.05 was regarded as significant.
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RESULTS |
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Effect of polyamine depletion on DNA fragmentation during
apoptosis.
Cells were grown in regular medium, in the presence of DFMO (5 mM), or
with DFMO plus 10 µM putrescine for 4 days and then treated with 20 µM camptothecin for 6 h. DNA fragmentation was dramatically
increased after apoptosis was induced (Fig.
1). DFMO treatment inhibited DNA
fragmentation by >80%. Putrescine prevented the effect of polyamine
depletion caused by DFMO.
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Effect of polyamine depletion on cytochrome c release from
mitochondria, caspase-3 activity, and caspase-9 activity.
Cells were grown in regular medium, in the presence of DFMO (5 mM), or
with DFMO plus putrescine for 4 days and then treated with 20 µM
camptothecin for 6 h. Camptothecin increased caspase-3 and -9 activities approximately sevenfold (Fig.
2). These increases, indicative of
apoptosis, were almost totally prevented by DFMO. In the
presence of exogenous putrescine, DFMO had no effect on caspase
activity. Figure 3 shows identical
results with caspase-3 by indicating the level of the active protein
itself. Polyamine depletion also prevented the movement of cytochrome
c from mitochondria to cytosol. As shown in Fig.
4, cells treated with camptothecin and
DFMO had high levels of mitochondrial cytochrome c compared with control cells receiving only camptothecin and with
camptothecin-treated cells that were exposed to putrescine along with
DFMO.
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Effect of polyamine depletion on Bcl-2 family proteins.
Cells were grown in regular medium, in the presence of DFMO (5 mM), or
with DFMO plus putrescine for 4 days and then treated with 20 µM
camptothecin for 6 h. Bax proteins were primarily distributed in
cytosol in untreated cells (Fig. 5). In
cells treated with camptothecin, a large portion of Bax was associated
with mitochondria, and the amount of Bax protein in the cytosol was
dramatically decreased. DFMO-treated cells had a lower level of Bax
associated with mitochondria compared with control cells. The amount of
Bax associated with mitochondria in the DFMO plus putrescine group was
similar to that found in control cells. The total amount of Bcl-xL in DFMO-treated cells was increased to 180% of
control, whereas the total amount of Bcl-2 was increased to 135% of
control (Fig. 6). The total amounts of
Bcl-xL and Bcl-2 in the DFMO plus putrescine group were
close to the levels in control cells. Thus depletion of polyamines
increased the levels of Bcl-xL and Bcl-2 proteins.
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Effect of polyamine depletion on caspase-8 activity and cleavage of
Bid protein.
After apoptosis was induced with camptothecin, caspase-8
activity increased approximately fivefold. Polyamine depletion nearly prevented the increase in caspase-8 activity. The addition of putrescine to cells treated with DFMO totally prevented the effect of
DFMO. (Fig. 7). Before apoptosis
was induced with camptothecin, only the 22-kDa Bid protein was detected
and there were no differences in the amount of Bid among the three
different treatment groups (Fig.
8A). After cells were treated
with camptothecin for 6 h, 15- and 13-kDa bands were detected in
addition to the 22-kDa band, indicating that cleavage of 22-kDa Bid
occurred during apoptosis. However, there was little cleavage
of Bid in the polyamine-depleted cells. Compared with control cells,
DFMO-treated cells had more Bid remaining in the 22-kDa form and less
in 15- and 13-kDa bands (Fig. 8B). The pattern of bands in
the DFMO plus putrescine cells was similar to that in control cells.
These results indicate that depletion of polyamines inhibited the
cleavage of Bid following an apoptotic stimulus.
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DISCUSSION |
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More than 14 caspases have been characterized in mammals, and many
are implicated in the apoptotic process. These cysteine proteases
are synthesized in the cell as inactive precursors composed of four
distinct domains. Each caspase is activated by proteolytic removal of
linker regions and by assembly of the large and small subunits into an
active enzyme complex (60). Mitochondria play an essential
role in many forms of apoptosis by releasing apoptogenic factors, such as cytochrome c (19, 27) and
apoptosis-inducing factor (AIF) (56) from the
intermembrane space into the cytoplasm. Cytochrome c binds
to the cytoplasmic protein Apaf-1 via the COOH terminus in the presence
of ATP, resulting in an oligomer complex (51). This
complex recruits procaspase-9 and induces the self-activation of
caspase-9 (33, 51). Active caspase-9 cleaves and activates downstream caspases-3, -6, and -8, leading to apoptosis
(41). Stefanelli et al. (54) found that DFMO
treatment did not influence caspase activity triggered by staurosporine
but inhibited it when the apoptotic stimulus was cycloheximide or
etoposide. The susceptibility of polyamine-deficient IEC-6 cells to
staurosporine-induced apoptosis increased significantly as
measured by changes in morphological features and internucleosomal DNA
fragmentation (32). In contrast, polyamine depletion by
DFMO promoted resistance to apoptotic cell death induced by the
combination of tumor necrosis factor- and cycloheximide
(47). The fate of cells may depend on which apoptotic signal pathways are activated and the effect that polyamines have on
those specific signal pathways. In certain situations, the inhibitory
effect of polyamine depletion on a specific signal pathway may be
overcome by overall activation of multiple apoptotic pathways. This
may explain why the effect of polyamine depletion on apoptosis
is controversial. In the current study, we found that DFMO treatment
inhibited apoptosis in response to camptothecin as measured by
DNA fragmentation (Fig. 1). Exogenous putrescine prevented the
inhibition of apoptosis and all other effects of polyamine
depletion. Previous studies have shown that intracellular polyamine
concentrations in DFMO plus putrescine-treated cells are the same as in
control cells (68). Thus the changes observed in cells
treated with DFMO were caused by the lack of polyamines and not by DFMO
itself. We have previously shown that the amount of inactive
procaspase-3 was not changed by depletion of polyamines (47). Our results in the current study indicate that
depletion of polyamines decreased the release of cytochrome
c from mitochondria into cytosol (Fig. 3) and subsequently
prevented the downstream activation of caspases-9 and -3 (Figs. 2 and
3) in response to camptothecin.
Each of the Bcl-2 family proteins contains at least one of the four conserved regions called the Bcl-2 homology domains (BH1-BH4). These domains enable the different members of the family to form either homo- or heterodimers and regulate each other (25). Bcl-2 family proteins regulate cytochrome c release from mitochondria. Overexpression of the antiapoptotic proteins Bcl-2 or Bcl-xL in many cell types exposed to various cytotoxic stimuli prevents cytochrome c release, caspase activation, and apoptosis (27, 62, 66). Conversely, overexpression of the proapoptotic member Bax triggers cytochrome c release from mitochondria in the absence of a death signal (14, 16, 48). Bcl-2 or Bcl-xL inhibits the induction of cytochrome c release by Bax (16, 48). The direct addition of Bax to isolated mitochondria also induces cytochrome c release (14, 16). During the process of apoptosis, Bax undergoes a conformational change at its NH2 terminus, exposing its membrane-seeking domains (26, 39), and consequently translocates from the cytosol to the mitochondria (26, 39, 44, 64). Bax inserts itself into the outer membrane of the mitochondria and becomes an integral membrane protein (18), consequently inducing the release of cytochrome c. In our experiments, Bax translocated from cytosol to mitochondria after apoptosis was induced (Fig. 5). Compared with control cells, DFMO-treated cells had less Bax protein associated with mitochondria and more Bax in cytosol. These results suggest that polyamine depletion inhibits the translocation of Bax to mitochondria and consequently prevents cytochrome c release. The increase in the levels of Bcl-xL and Bcl-2 proteins in DFMO-treated cells (Fig. 6) may also contribute to the inhibition of cytochrome c release during apoptosis. One study shows that DFMO inhibits protein tyrosine phosphorylation during apoptosis (23). It is possible that depletion of polyamines inhibits the phosphorylation and function of proteins that are involved in the regulation of Bax translocation.
Mitochondrial cytochrome c is a water-soluble protein
residing in the mitochondrial intermembrane space (37).
The molecular mechanisms responsible for the release of
cytochrome c from mitochondria to cytosol during
apoptosis remain unknown. One model proposes that release
depends on the opening of the PTP in the mitochondrial membrane
(61). The PTP is an oligoprotein channel, consisting of a
voltage-dependent anion channel in the outer membrane, an adenine
nucleotide translocator on the inner membrane, and the matrix protein
cyclophilin D (29). This model predicts that opening of
the PTP leads to a decrease of the electrochemical gradient across the
inner membrane, resulting in mitochondrial depolarization. Many studies
have shown that early apoptosis is accompanied by a reduction
in the mitochondrial membrane potential (m)
(24, 40, 69, 70). However, we did not observe any significant change in the
m in IEC-6 cells treated
with camptothecin for 6 h (data not shown). This finding is in
agreement with some other reports that cytochrome c release
during apoptosis is independent of depolarization of the
mitochondrial membrane (6, 8, 14, 16, 17, 27, 30, 66).
Krohn et al. (30) reported that cells treated for 16 h with staurosporine maintain the
m while showing signs of nuclear apoptosis and 20-fold increases in
caspase-3 activity. Some authors suggest that release of
cytochrome c into the cytosol may be related to a transitory
opening of the PTP (6). Inhibitors of caspases effectively
block the reduction in the
m but fail to block the
release of cytochrome c (6, 71), suggesting
that the reduction in the
m may be a consequence of
caspase activity and occurs only when cells enter the late stage of the
death process.
It is well established that activation of the initiator caspase-8 is triggered by the ligand binding of death receptors, a process mediated by an adapter molecule Fas-associated death domain (FADD) (41). Bid, a proapoptotic Bcl-2 family protein, is cleaved by caspase-8. The truncated Bid translocates to the mitochondria and induces cytochrome c release (12, 28, 67). However, the cleavage of Bid is also observed when cells are exposed to multiple death-inducing stimuli, such as staurosporine, ultraviolet radiation, cycloheximide, or etoposide (50). We observed that caspase-8 activity was increased after cells were treated with camptothecin (Fig. 7). Activated caspase-3 is essential for activation of caspase-8 and processing of Bid in staurosporine-mediated apoptosis (58), suggesting that caspase-8 is activated by caspase-3. In addition to caspase-8, other caspases such as caspase-3 can also cleave Bid. Bid truncated by caspases other than caspase-8 is equally potent in inducing cytochrome c release, suggesting that Bid plays a role in amplifying various apoptotic signals in addition to relaying the apoptotic signal from cell surface death receptors (7). Belka et al. (3) compared the role of caspase-8 and Bid activation during radiation- and CD95-induced apoptosis. They concluded that activation of caspase-8 and Bid cleavage in radiation-induced apoptosis occurs during the later execution phase rather than in the early initiation phase. Bid is cleaved into a 15-kDa band (7, 67), whereas 13- and 11-kDa truncated Bid bands have also been observed in some studies (13, 21, 31, 36, 65). In our study, we observed 15- and 13-kDa bands after apoptosis was stimulated (Fig. 8). Depletion of polyamines inhibited the increase in caspase-8 activity in response to camptothecin (Fig. 7). Attenuated caspase-8 activity in DFMO-treated cells may be due to the decrease in the activity of caspase-3. Therefore, the decreased cleavage of Bid in DFMO-treated cells (Fig. 8) could be attributed to the inhibition of the activities of both caspase-8 and caspase-3. Thus the inhibition of the amplifying loop of Bid cleavage in polyamine depleted cells at least partially contributes to the decrease in the release of cytochrome c from mitochondria.
In summary, following the apoptotic stimulus, camptothecin changes caused by polyamine depletion in IEC-6 cells suggest that increases in the levels of Bcl-xL and Bcl-2 proteins decrease the translocation of Bax to the mitochondria, resulting in inhibition of the release of cytochrome c from mitochondria into cytosol. The decrease in cytochrome c release prevents the activation of the caspase cascade and subsequent apoptosis. Cytochrome c release is also likely prevented by a decrease in the cleavage and subsequent activation of Bid.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. R. Johnson, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (E-mail: ljohn{at}physio1.utmem.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.
10.1152/ajpcell.00351.2001
Received 26 July 2001; accepted in final form 10 January 2002.
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