Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-{alpha} in IEC-6 cells

Sujoy Bhattacharya, Ramesh M. Ray, Mary Jane Viar, and Leonard R. Johnson

Department of Physiology, The University of Tennessee Health Science Center, College of Medicine, Memphis, Tennessee 38163

Submitted 6 May 2003 ; accepted in final form 9 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Intracellular polyamine homeostasis is important for the regulation of cell proliferation and apoptosis and is necessary for the balanced growth of cells and tissues. Polyamines have been shown to play a role in the regulation of apoptosis in many cell types, including IEC-6 cells, but the mechanism is not clear. In this study, we analyzed the mechanism by which polyamines regulate the process of apoptosis in response to tumor necrosis factor-{alpha} (TNF-{alpha}). TNF-{alpha} or cycloheximide (CHX) alone did not induce apoptosis in IEC-6 cells. Significant apoptosis was observed when CHX was given along with TNF-{alpha}, as indicated by a significant increase in the detachment of cells, caspase-3 activity, and DNA fragmentation. Polyamine depletion by treatment with {alpha}-difluoromethylornithine significantly reduced the level of apoptosis, as judged by DNA fragmentation and the caspase-3 activity of attached cells. Apoptosis in IEC-6 cells was accompanied by the activation of upstream caspases-6, -8, and -9 and NH2-terminal c-Jun kinase (JNK). Inhibition of JNK activation prevented caspase-9 activation. Polyamine depletion prevented the activation of JNK and of caspases-6, -8, -9, and -3. SP-600125, a specific inhibitor of JNK activation, prevented cytochrome c release from mitochondria, JNK activation, DNA fragmentation, and caspase-9 activation in response to TNF-{alpha}/CHX. In conclusion, we have shown that polyamine depletion delays and decreases TNF-{alpha}-induced apoptosis in IEC-6 cells and that apoptosis is accompanied by the release of cytochrome c, the activation of JNK, and of upstream caspases as well as caspase-3. Polyamine depletion prevented JNK activation, which may confer protection against apoptosis by modulation of upstream caspase-9 activation.

programmed cell death; intestine; caspases; putrescine; {alpha}-difluoromethylornithine; polyamines; cytochrome c


INTESTINAL CRYPT CELLS proliferate, migrate, and differentiate, traveling from the crypt to the villus tip in 48-72 h. The small intestine maintains a relatively constant mucosal mass and has a low incidence of epithelial cancer. Resistance to cancer is probably because of the relatively short half-life of the epithelial cells (3-5 days) as well as to the ability of the small intestine to eliminate senescent and genetically damaged cells by programmed cell death, which occurs on the villus and in the crypt. Although polyamines are intimately involved in growth-related processes, excessive accumulation may interfere directly with normal cell function (12, 30, 31, 52).

Our laboratory has examined the role of polyamines in the growth and repair of gastrointestinal mucosa in rats and in models using the cultured intestinal epithelial cell line IEC-6, a nontransformed line derived from rat crypt cells (33). Recently, we showed that depletion of polyamines led to cell cycle arrest and delayed apoptotic responses to the DNA topoisomerase inhibitor camptothecin and to tumor necrosis factor-{alpha} (TNF-{alpha}; see Refs. 34 and 35). Polyamine depletion decreased caspase-3 activity induced in response to both camptothecin and TNF-{alpha} (34). Polyamines have also been implicated in apoptosis. Overexpression of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis, causes apoptosis in IL-dependent 32D.3 murine myeloid cells upon IL-3 withdrawal (32). Overaccumulation of polyamines caused by deregulation of polyamine transport triggers apoptosis in ODC-overexpressing L1210 mouse leukemia cells (30, 45).

TNF-{alpha}, a pleiotropic cytokine, is a central mediator of diverse inflammatory processes. TNF-{alpha} induces cell growth arrest or apoptosis in some cell types, yet acts as a growth factor for others (9, 18). The signaling pathway by which TNF-{alpha} exerts its action leading to either apoptosis or cell proliferation remains unclear (21, 25, 46). TNF-{alpha} binds to two distinct membrane receptors, p55 and p75 TNF receptors (TNFR), both of which are expressed by intestinal epithelial cells (18, 20). TNFR has been implicated in the induction of apoptosis, activation of NF-{kappa}B, and the regulation of cell proliferation via mitogen-activated protein kinases or c-Jun NH2-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs; see Refs. 4, 17, 26, 36). The p55 TNFR1 is primarily involved in TNF-{alpha}-induced apoptosis (15, 43, 44, 49). Intracellular signal transduction from p55 TNFR1, which is the main TNF-{alpha} receptor in most cell types, occurs through a controlled series of protein-protein interactions. TNF-{alpha}-induced trimerization of the receptor recruits TNF-{alpha} receptor-associated death domain-containing protein (TRADD) to a region of TNFR1, to which cytotoxic function has been mapped, namely the death domain. TRADD act as an adapter to recruit the receptor to the downstream transducer, FAS-associated death domain-containing protein (FADD). FADD interacts with and activates the apoptotic proteases and triggers cell death (15, 16, 41).

Increasing evidence implicates mitochondria in apoptotic signaling. Bcl-2, an antiapoptotic protooncogene product, localizes in mitochondria (10, 19). Bcl-2, an acronym for the B-cell lymphoma/leukemia-2 gene, is a member of the Bcl-2 family, which contains both pro- and anti-apoptotic proteins. Overexpression or a high basal level of this protein in cancer cells inhibits apoptosis. The anti-apoptotic action of Bcl-2 has been proposed to be the result of 1) its antioxidant ability, 2) interaction with and neutralization of proapoptotic proteins such as Bax and, 3) inhibition of mitochondrial permeability. The molecular basis for increased mitochondrial permeability and apoptosis is now beginning to unfold. After TNF treatment, activated caspase-8 directly cleaves cytosolic Bid (a proapoptotic Bcl-2 family member). The COOH-terminal fragment of Bid translocates to mitochondria and induces the release of cytochrome c, which together with apoptosis-activating factor activates caspase-9 (7, 40).

We have previously shown that polyamine depletion prevents camptothecin- and receptor (TNF-{alpha})-mediated apoptosis in IEC-6 cells by downregulating caspase-3 activity (34). Caspase-3 is one of the so-called "executioner caspases" that enzymatically disassembles the cell. Addition of exogenous putrescine restores apoptosis, suggesting that polyamines are involved in the regulation of apoptosis in intestinal epithelial cells (34). Caspase proteins exist as inactive procaspases, and caspase-3 is activated by a series of reactions involving so called initiator caspases, such as caspases-1, -6, -8, and -9. The activities of this group of enzymes have not been examined in intestinal epithelial cells, and it is unknown whether polyamines influence this process. The induction of apoptosis by TNF-{alpha} is physiologically relevant, and the signal transduction pathway in response to TNF-{alpha} is well characterized (2, 3, 20).

For the first time, we have demonstrated the activation of upstream caspases in intestinal epithelial cells during apoptosis. Furthermore, we have shown that polyamine depletion inhibits TNF-{alpha}-induced JNK activation and subsequently prevents caspase-3 activation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. Medium and other cell culture reagents were obtained from GIBCO-BRL (Grand Island, NY). FBS and dialyzed FBS (dFBS, 10,000 molecular weight cut off) were purchased from Sigma (St. Louis, MO). TNF-{alpha} was obtained from PharMingen International (San Diego, CA). The enhanced chemiluminescence (ECL) Western Blot detection system was purchased from DuPont-New England Nuclear (Boston, MA). {alpha}-Difluoromethylornithine (DFMO) was a gift from the ILEX Oncology (San Antonio, TX). Anti-caspase-3 (CPP-32) and cytochrome c antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphospecific JNK and caspase-9 antibodies were purchased from Cell Signaling (Beverly, MA). SP-00125, a JNK inhibitor, was purchased from Tocris. The colorimetric caspase assay reagents were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). The Cell Death Detection ELISA Plus kit was purchased from Roche Diagnostics (Indianapolis, IN). ApoAlert mitochondrial membrane sensor kit was purchase from BD Biosciences (San Diego, CA). The IEC-6 cell line (ATCC CRL 1592) was obtained from American Type Culture Collection (Rockville, MD) at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (33). IEC-6 cells are nontumorigenic and retain the undifferentiated character of epithelial stem cells. Tests for mycoplasma were always negative. All chemicals were of the highest purity commercially available.

Cell culture. IEC-6 cell stock was maintained in T-150 flasks in a humidified, 37°C incubator in an atmosphere of 90% air-10% CO2. The medium consisted of DMEM with 5% heat-inactivated FBS and 10 µg insulin and 50 µg gentamicin sulfate/ml. The stock was passaged weekly and fed three times per week, and passages 15-22 were used. For the experiments, the cells were taken up with 0.05% trypsin plus 0.53 mM EDTA in Hanks' balanced salt solution without calcium and magnesium and counted by hemocytometer.

Apoptosis studies. Cells were plated (day 0) at a density of 6.25 x 104 cells/cm2 in DMEM/dFBS with or without the treatment compound(s) and with triplicate samples for each group. Cells were fed on day 2. On day 3, the normal culture medium was removed and replaced with serum-free medium. On day 4, TNF-{alpha} [20 ng/ml with or without 25 µg/ml cycloheximide (CHX)] was added to the serum-free medium for 3-6 h, with the appropriate vehicle added to controls. SP-600125 (25 µM) was added along with TNF/CHX treatment with appropriate vehicle for inhibition of JNK.

Cell number. Cells were grown in 75-cm2 flasks, and counts were determined separately for floating and attached cells. Floating cells were poured in a 25-ml tube, and the monolayer was washed one time with HBSS without calcium and magnesium. This wash was then combined in the tube with the floating cells. Attached cells were taken up with 0.05% trypsin plus 0.53 mM EDTA, followed by one wash with DMEM-5% FBS. Cells were counted on a Beckman model ZF Coulter counter. Floating cells were expressed as a percentage of the total cell count obtained by combining the number of floating and attached cells.

Caspase activity. After treatment on day 4, floating cells were poured off, combined with one wash with cold Dulbecco's PBS (DPBS), and counted. The attached cells were then harvested for determination of caspase activity. Briefly, 10 ml DPBS were added to the flask, and the monolayer was scraped and collected in a 25-ml tube. The flask was washed one time with 10 ml 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 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 ice-cold cell lysis buffer [50 mM HEPES, pH 7.4, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM DTT, 0.1 mM EDTA, and 0.1% Nonidet P-40]. The assay for caspase activity was carried out in a 96-well plate. In each well was placed 20 µl cell lysate, 70 µl assay buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 100 mM NaCl, 10 mM DTT, and 1 mM EDTA), and 10 µl caspase colorimetric substrate (2 mM Ac-YVAD-pNA, Ac-DEVD-pNA, Ac-VEID-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA prepared in assay buffer), a caspase-specific peptide that is conjugated to a chromogen, p-nitroanilide (p-NA). 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. Absorbance 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 (Pierce, Rockford, IL), and a standard curve for p-NA was constructed. Caspase activity was expressed as picomole p-NA released per milligram protein per minute.

Quantitative DNA fragmentation ELISA. Cells were grown in six-well culture plates for both DNA fragmentation ELISA and protein determination. After treatment, floating cells were discarded, and the attached cells were washed two times with DPBS. Briefly, 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) were 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 protein.

Mitochondrial permeability. Cells were grown for 4 days on Matrigel-coated glass coverslips and treated with TNF-{alpha} plus CHX for 1.5 h, as described earlier. Cells were washed with HBSS, covered with 1.0 ml diluted mitosensor reagent (final concentration: 5 µg/ml), and incubated at 37°C in a 5% CO2 incubator for 20 min. Cells were rinsed gently and examined with a fluorescent microscope using a band-pass filter (detects fluorescein and rhodamine).

Western blot analysis. Total cell protein (50 µg) from cell extracts prepared for the caspase-3 assay was separated on 15% SDS-PAGE and transferred to nitrocellulose membranes for Western blotting. Equal loading of protein was confirmed by staining the nitrocellulose membrane with Ponceau S. The membrane was then probed with an antibody directed against phospho-JNK, caspase-3 and -9, and cytochrome c proteins. The immunocomplexes were visualized by the ECL detection system.

Statistics. All data are expressed as means ± SE from representative experiments. All experiments were repeated three times, in triplicate. ANOVA and appropriate post hoc testing determined the significance of the differences between means. Values of P < 0.05 were regarded as significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Polyamine depletion prevents apoptosis induced by TNF-{alpha} and CHX. Confluent IEC-6 cells were treated with TNF-{alpha} (20 ng/ml) or CHX (25 µg/ml) alone or together for 6 h. Results in Fig. 1 show that cell detachment (A), caspase-3 activity (B), and DNA fragmentation (C) were significantly elevated in cells treated with both TNF-{alpha} and CHX compared with untreated controls. Neither TNF-{alpha} nor CHX alone induced detachment of cells or caspase-3 activity. CHX treatment alone increased the level of DNA fragmentation slightly. Induction of apoptosis or cell proliferation in response to TNF-{alpha} may also be concentration dependent. Therefore, we determined the dose response to TNF-{alpha} plus CHX in control cells to establish the conditions for the induction of apoptosis. Figure 2 shows that apoptosis was dose dependent and increased significantly up to a concentration of 20 ng/ml TNF-{alpha}. We also examined the time course of the induction of apoptosis by TNF-{alpha} plus CHX in control and polyamine-depleted cells using DNA fragmentation as an apoptotic index. Cells were grown in control medium for 4 days with or without DFMO. We have previously reported that polyamine depletion arrests cells in the G1 phase of the cell cycle. We grew cells to confluence, and they were serum starved for 24 h, at which time >80% of the cells are in the G1 phase in each group (35). Results in Fig. 3 show that DNA fragmentation began after 6 h of exposure to TNF-{alpha} plus CHX and increased further in a time-dependent manner in control cells. In polyamine-depleted cells, DNA fragmentation was delayed and did not begin to increase until after 9 h. By 12 h, the level of DNA fragmentation in control cells was fivefold greater than in cells depleted of polyamines.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1. Apoptosis in response to tumor necrosis factor-{alpha} (TNF-{alpha}) and cycloheximide (CHX). Cells were grown for 3 days in DMEM-5% dialyzed FBS (dFBS) and then serum deprived for 24 h before treatment with TNF-{alpha} (20 ng/ml) with and without CHX (25 µg/ml) for 6 h. A: number of floating cells. B: caspase 3 activity from attached cells. C: DNA fragmentation analysis from attached cells. A405, absorbance at 405 nm. *P < 0.05 compared with corresponding control value. Data are means values and SE of 9 observations.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Effect of TNF-{alpha} concentration on apoptosis. Cells were grown for 3 days in DMEM-5% dFBS and then serum deprived for 24 h before treatment with TNF-{alpha} (0-20 ng/ml) and CHX (25 µg/ml) for 3 h. DNA fragmentation was measured by cell death detection ELISA. *P < 0.05 compared with corresponding control value. Data are mean values ± SE of 9 observations.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. Time-dependent induction of apoptosis by TNF-{alpha} and CHX in the absence and presence of {alpha}-difluoromethylornithine (DFMO). Cells were grown for 3 days in DMEM-5% dFBS with or without 5 mM DFMO. Cells were serum deprived for 24 h before treatment with 10 ng/ml TNF-{alpha} + CHX (25 µg/ml) for 12 h. DNA fragmentation analysis was carried out at indicated time period by cell death detection ELISA. *P < 0.05 compared with corresponding control value. Data are mean values ± SE of 9 observations.

 

We next examined the effect of polyamine depletion on caspase activity and DNA fragmentation at 20 ng/ml TNF-{alpha} for 6 h to further understand the signaling events of apoptosis. Figure 4A indicates that polyamine depletion not only decreased apoptosis in response to TNF-{alpha} plus CHX but also decreased the basal level of DNA fragmentation. TNF-{alpha} increased apoptosis approximately fourfold over that seen in untreated controls, and DFMO totally prevented this response, decreasing apoptosis significantly below basal values. Although basal levels of caspase-3 activity in control and DFMO-treated cells were nearly identical, TNF-{alpha} plus CHX dramatically increased caspase-3 activity in control cells. Polyamine-depleted cells had only marginal increases in caspase-3 activity (Fig. 4B).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. Effect of polyamine depletion on apoptosis induced by TNF-{alpha} and CHX. Cells were grown for 3 days in DMEM-5% dFBS with or without 5 mM DFMO. Exogenous putrescine (Put; 10 µM) was added to one group of DFMO-treated cells. Cells were serum deprived for 24 h before treatment with 20 ng/ml TNF-{alpha} + 25 µg/ml CHX for 6 h. A: DNA fragmentation. B: caspase-3 activity from attached cells. *P < 0.05 compared with corresponding control value. Data are means ± SE of 9 observations.

 

Activation of the upstream caspase cascade in response to polyamine depletion. The above observations suggested that polyamine depletion might be affecting the upstream caspase cascade, which in turn leads to activation of caspase-3 by cleavage of procaspase-3. Cells grown in control, DFMO, or DFMO plus putrescine for 4 days were treated with TNF-{alpha} (20 ng/ml) plus CHX for 3 h, and caspase activity was determined using substrates specific for caspases-1, -3, -6, -8, and -9 (Fig. 5). There were no significant differences in the activities of any caspase from different groups in untreated cells. TNF-{alpha} plus CHX significantly increased the activities of caspases-3, -6, -8, and -9. Polyamine depletion with DFMO significantly reduced the increases of all caspases. The addition of exogenous putrescine to DFMO-treated cells prevented the reduction in caspase activities caused by polyamine depletion. The upstream caspases cleave procaspase-3 to its active form. We analyzed cell extracts from the above experiment by Western blotting to examine the processing of procaspase-3. Results in Fig. 6 show a decrease in the level of the procaspase form (32 kDa) with a concomitant increase in the 16- to 19-kDa activated form of caspase-3 in response to TNF-{alpha}. In polyamine-depleted cells, TNF-{alpha} did not increase the conversion of procaspase-3 to the active form. The exogenous addition of putrescine along with DFMO completely restored caspase-3 activation.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Effect of polyamine depletion and its prevention with added putrescine on the activity of caspases induced by TNF-{alpha} and CHX. Cells were grown for 3 days in DMEM-5% dFBS with or without 5 mM DFMO. Exogenous putrescine (10 µM) was added to one group of DFMO-treated cells. Cells were serum deprived for 24 h before treatment with 20 ng/ml TNF-{alpha} + 25 µg/ml CHX for 3 h. Caspase activity was determined by using specific substrates for caspases-1 (A), -3 (B), -6 (C), -8 (D), and -9 (E). *P < 0.05 compared with control and with TNF-{alpha}/CHX + DFMO. Data are means ± SE of 9 observations.

 


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6. Effects of polyamine depletion on the TNF-{alpha}-induced activation of caspase-3. Cells were grown for 3 days in DMEM-5% dFBS with or without 5 mM DFMO and 10 µM putrescine. Cells were serum deprived for 24 h before treatment with 20 ng/ml TNF-{alpha} + 25 µg/ml CHX for 3 h and lysed in cell lysis buffer. Protein (50 µg) was separated on SDS-PAGE for Western blot analysis using a caspase-3-specific antibody. The blot shown is representative of 3 observations.

 

Role of JNK activation in TNF-{alpha}- and CHX-induced apoptosis. The apoptotic response to cytokines and inhibitors of protein synthesis is primarily accompanied by the activation of JNKs, also referred to as SAPKs. Therefore, we first determined the state of JNK activation in response to TNF-{alpha} and CHX alone or in combination by Western blot analysis to detect the phospho-p54/p46 form of JNK. Activation of both JNK-p54/p46 was evident when cells were treated with CHX alone and CHX plus TNF-{alpha} compared with untreated cells (Fig. 7). Treatment of cells with TNF-{alpha} alone showed a marginal increase in phospho-JNK-p46 only. To determine the time course of JNK activation, IEC-6 cells were grown to confluence in the presence of DFMO and DFMO plus putrescine for 4 days and exposed to TNF-{alpha} plus CHX for 3, 6, and 9 h. Cell extracts were analyzed by Western blot to detect the phospho-JNK p46/p54 using a phosphospecific JNK antibody. Cells grown in control conditions showed time-dependent increases in the phosphorylation of p46 and p54 JNK, reaching a maximum at 9 h when >70% of the cells were detached from the plate. It was interesting to note that JNK1 (p46) phosphorylation was relatively higher than JNK2 (p54; Fig. 8). In contrast, activation of both JNK-p46 and -p54 in polyamine-depleted cells was prevented until 9 h compared with control (Fig. 8). Putrescine restored JNK activation in cells grown in the presence of DFMO (Fig. 8). Cell extracts from the same experiment were analyzed further to check the processing of caspase-3. As expected, activation of caspase-3 showed remarkable correlation with JNK activation in control cells and in cells grown in DFMO and putrescine. The time-dependent decrease in pro-caspase-3 (32 kDa) was followed by an increase in the active form (16-19 kDa; Fig. 9). Polyamine depletion completely prevented caspase-3 activation, which is evident by the presence of only procaspase-3 (Fig. 9). Restoration of JNK and caspase-3 activation by exogenous putrescine affirms the specificity of polyamines in the process of apoptosis.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 7. NH2-terminal c-Jun kinase (JNK) activation in response to TNF-{alpha} and CHX. Cells were grown for 3 days in DMEM-5% dFBS and then serum deprived for 24 h before treatment with TNF-{alpha} (20 ng/ml), CHX (25 µg/ml), or TNF-{alpha} + CHX for 3 h. Cell lysates equivalent to 50 µg protein from each sample were resolved on SDS-PAGE, transferred to membranes, and probed with phospho-JNK (Thr183/Tyr185) monoclonal antibody. The blot shown is representative of 3 observations.

 


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 8. Time-dependent activation of JNK after TNF-{alpha} and CHX in normal and polyamine-depleted cells. Cells were grown for 3 days in DMEM-5% dFBS with or without 5 mM DFMO and 10 µM putrescine. Cells were serum deprived for 24 h before treatment with 20 ng/ml TNF-{alpha} + 25 µg/ml CHX for 3 h and lysed in radioimmunoprecipitation buffer. Protein (50 µg) was separated on SDS-PAGE for Western blot analysis using a phospho-JNK (Thr183/Tyr185) monoclonal antibody. The blots shown are representative of 3 observations.

 


View larger version (71K):
[in this window]
[in a new window]
 
Fig. 9. Time-dependent activation of caspase-3 protein after TNF-{alpha} and CHX. Cells were grown for 3 days in DMEM-5% dFBS with or without 5 mM DFMO and 10 µM putrescine. Cells were serum deprived for 24 h before treatment with 20 ng/ml TNF-{alpha} + 25 µg/ml CHX for 3, 6, and 9 h and lysed in RIPA. Protein (50 µg) was separated on SDS-PAGE for Western blot analysis using a caspase-3-specific antibody. The blot shown is representative of 3 observations.

 

Inhibition of JNK prevents cytochrome c release and activation of caspase-9 and caspase-3. Although JNK activation, caspase activation, and induction of apoptosis were parallel in time, this does not mean that JNK is linked to caspase-3 activation. We used SP-600125, a specific inhibitor of JNK, to block the activation of the enzyme (5). We first established the effectiveness of the compound by evaluating the inhibition of JNK activation in response to TNF-{alpha} and CHX. The data in Fig. 10 demonstrate that the TNF-{alpha}- and CHX-induced activation of both p46 and p54 JNK was inhibited significantly by SP-600125. We used this compound to inhibit JNK activation and examined the activation of caspase-9 and caspase-3. TNF-{alpha} and CHX induced significantly high levels of caspase-9. The caspase-9 antibody used in this experiment recognizes the activated form of protein (40 kDa) and only weakly reacts with pro-caspase-9. SP-600125 almost totally prevented the activation of caspase-9 (Fig. 11A). TNF-{alpha} and CHX failed to activate caspase-9 in polyamine-depleted cells in the presence or absence of SP-600125, which is consistent with the decreased caspase-9 activity shown in Fig. 5.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 10. Inhibition of TNF-{alpha}- + CHX-induced JNK activation by SP-600125. Cells were grown for 3 days in DMEM-5% dFBS and then serum deprived for 24 h before treatment with TNF-{alpha} (20 ng/ml) with and without CHX (25 µg/ml). SP-00125 (25 µM) was added along with TNF-{alpha} + CHX for 3 h. Cell lysate equivalent to 50 µg protein from each sample was resolved on SDS-PAGE, transferred to membranes, and probed with phospho-JNK (Thr183/Tyr185) monoclonal antibody. The blot shown is representative of 3 observations.

 


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 11. Inhibition of JNK activation by SP-600125 prevents activation of caspase-9 (A) and caspase-3 (B). Cells were grown for 3 days in DMEM-5% dFBS with or without 5 mM DFMO and 10 µM putrescine. Cells were serum deprived for 24 h before treatment with 20 ng/ml TNF-{alpha} + 25 µg/ml CHX with and without 25 µM SP-00125 for 3 h and lysed in cell lysis buffer. Protein (50 µg) was separated on SDS-PAGE for Western blot analysis using caspase-9- and caspase-3-specific antibodies. The blots shown are representative of 3 observations.

 

Inhibition of caspase-9 by the JNK inhibitor and the fact that caspase-9 is an upstream activator of caspase-3 suggest that SP-600125 should also inhibit caspase-3 activity. Results in Fig. 11 show that the JNK inhibitor did not completely inhibit but significantly reduced caspase-3 activation (Fig. 11B). Polyamine depletion prevented activation of caspase-9 and caspase-3. Caspase-9 is activated in response to mitochondrial membrane permeability changes and the subsequent release of cytochrome c. We used the ApoAlert mitochondrial membrane sensor (mitosensor) assay to determine whether JNK activation is directly linked to mitochondrial membrane permeability changes and cytochrome c release. Mitosensor aggregates in the mitochondria of healthy cells and these aggregates fluoresce red. In apoptotic cells, mitochondrial membrane potentials are altered, and mitosensor cannot accumulate in mitochondria. It remains as a monomer in the cytoplasm and fluoresces green. The TNF-{alpha} and CHX treatment group had a significantly large number of green fluorescent (apoptotic) cells (Fig. 12A) indicative of an alteration in mitochondrial membrane permeability. In contrast, the DFMO treatment group had significantly fewer apoptotic green fluorescent cells. Addition of exogenous putrescine along with DFMO during growth prevented the changes in the mitochondrial membrane permeability. Inhibition of JNK activation (SP-600125 treatment) prevented the mitochondrial membrane permeability changes and significantly reduced the number of apoptotic green fluorescent cells (Fig. 12A). Changes in mitochondrial membrane permeability lead to cytochrome c release. Therefore, we analyzed the cytosol fraction of the cells treated with TNF-{alpha} plus CHX in the presence and absence of the JNK inhibitor SP-600125 by Western blot analysis using an antibody specific for cytochrome c. TNF-{alpha} and CHX increased cytochrome c levels in the cytoplasmic fraction compared with control cells. Addition of SP-600125 significantly decreased the release of cytochrome c in the cytoplasmic fraction of TNF-{alpha}plus CHX-treated cells (Fig. 12B), indicating the involvement of JNK in cytochrome c release from the mitochondria and in turn the activation of caspase-9 and caspase-3.



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 12. Inhibition of JNK activation by SP-600125 prevents mitochondrial permeability transition and cytochrome c release. A: cells were grown for 3 days in DMEM-5% dFBS with or without 5 mM DFMO and 10 µM putrescine. Cells were serum deprived for 24 h before treatment with 20 ng/ml TNF-{alpha} + 25 µg/ml CHX with and without 25 µM SP-00125 for 1.5 h. After being washed with HBSS, cells were processed as described in MATERIALS AND METHODS using the ApoAlert mitochondrial membrane sensor kit. A representative of 6 observations is shown. B: cells were grown for 3 days in DMEM-5% dFBS and serum deprived for 24 h. Cells were treated with 20 ng/ml TNF-{alpha} + 25 µg/ml CHX with and without 25 µM SP-600125. Serumstarved cells without any treatment were used as untreated control. Selective plasma membrane permeabilization with digitonin was used to examine release of cytochrome c from mitochondria to cytosol. Equal amounts of protein were resolved on 15% SDS-PAGE and analyzed by Western blot as previously described. A representative blot of 3 observations is shown.

 

Because the JNK inhibitor prevented the activation of caspase-9 and -3, we determined whether it also prevented DNA fragmentation, a terminal step in the process of apoptosis. Addition of SP-600125 alone or along with TNF and CHX completely blocked JNK activation, decreased the basal level JNK activation, and slightly increased DNA fragmentation (data not shown). Therefore, we chose to inhibit TNF- and CHX-induced JNK activation by the addition of SP-600125, 30 min and 1 h post-TNF and CHX treatment. Results in Fig. 13 show that DNA fragmentation was reduced significantly by SP-600125 when added 30 min after TNF and CHX treatment. Addition of SP-600125 after 1 h of exposure to TNF and CHX significantly reduced DNA fragmentation but not to the extent it did when added at the earlier time point.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 13. Inhibition of JNK activation prevents DNA fragmentation. Cells were grown as described in Fig. 10 and were treated with TNF-{alpha} + CHX for 3 h in the presence or absence of JNK inhibitor SP-600125. DNA fragmentation was measured by cell death detection ELISA. A: no treatment; B: TNF-{alpha} + CHX; C: vehicle; D: SP-600S125 (25 µM); E: SP-600S125 (25 µM) added 30 min after TNF-{alpha} + CHX addition; F: SP-600S125 (25 µM) added 1 h after TNF-{alpha} + CHX addition. *P < 0.05 compared with corresponding control value. Data are means ± SE of 6 observations.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Apoptosis, or programmed cell death, is a fundamental event in the regulation of cell number in all multicellular organisms. It is essential during development and tissue remodeling and for the prevention of malignancy (6, 14). The significance of apoptosis to the integrity of the gastrointestinal epithelium is well documented (31, 51). In the small intestine, spontaneous apoptosis during development is necessary to achieve the optimal number of stem cells. Increased expression of TNF-{alpha} mRNA and protein in inflammatory bowel disease and the use of anti-TNF-{alpha} antibodies in the treatment of Crohn's disease attest to the importance of this cytokine in pathogenesis (9, 42). Rummele et al. (37) demonstrated that TNF-{alpha} induced a maximum rate of cell death in intestinal crypt cells when both p55 and p75 TNFR were activated by high concentrations of TNF-{alpha}. In contrast, Dionne et al. (9) reported that TNF-{alpha} at 1-500 ng/ml increased IEC-6 cell proliferation. An earlier report from our laboratory showed that apoptosis induced both by DNA-damaging agents and by TNF-{alpha} plus CHX was delayed in polyamine-depleted cells, suggesting an important role for polyamines in this process (34). However, the signaling pathways influenced by polyamines in apoptosis have not been examined. DFMO and polyamine analogs are being evaluated as drugs for the treatment of breast, prostate, and colon cancer because of their inhibition of cell growth (8, 22). Growth inhibition of malignant cells using DFMO may prevent proliferation of cancerous cells, but polyamine depletion has both pro- and antiapoptotic effects, depending on cell type, species, and stimulus (28, 34). It is necessary to understand the mechanism by which polyamines modulate the apoptotic response in particular cell types to design treatment strategies. In our experiments, polyamines were depleted by inhibiting ODC with DFMO. Incubation of IEC-6 cells with DFMO causes putrescine to disappear within 6 h, spermidine within 24 h, and 60 percent of spermine within 4 days (23). Some spermine remains regardless of the length of exposure to DFMO, indicating that it is irreversibly bound. The effects of polyamine depletion are prevented by incubation with exogenous polyamines in the presence of DFMO during the 4-day growth period, indicating that all observed effects are because of the absence of polyamines and not because of DFMO itself (23). In these experiments, we chose to add putrescine, since it is the product of the ODC-catalyzed reaction. However, these effects are not primarily caused by putrescine itself, since it is converted rapidly to spermidine and then to spermine. We have shown earlier that maximal protection from TNF-{alpha}- and CHX-induced apoptosis was only observed after 4 days of DFMO treatment (34). Apoptosis was restored to that of control only when putrescine was added to the DFMO-treated cells during the 4 days of growth. Addition of putrescine, spermidine, or spermine along with TNF-{alpha} and CHX to the polyamine-depleted cells did not restore apoptosis (data not shown). This indicates that polyamine pools must be restored to prevent the effects of depletion.

TNF-{alpha} activates signaling pathways leading to new gene transcription, resulting in either cell proliferation or cell death. Cell death is a rare response in nontransformed cells, usually observed only when RNA or protein synthesis is blocked. Inhibition of transcription or translation activates SAPKs and induces apoptosis. Both transcriptional and translational inhibitors have been reported to sensitize cells to TNF-{alpha}-induced apoptosis (11, 48). Inhibitors also prevent TNF-{alpha}-responsive gene expression, which includes Bcl-2, the reactive oxygen species scavenger (manganese superoxide dismutase), and the cellular inhibitor of apoptosis protein, all of which prevent cell death (6, 50). The observation that many cells become sensitive to TNF-{alpha}-induced cell death when new protein synthesis is inhibited also implies that apoptosis is mediated by molecules that preexist in latent forms. It is now well established that a family of cysteine proteases, the caspases, mediates the induction of apoptosis by TNF-{alpha} (38, 39, 50). Results in Fig. 1 show that neither TNF-{alpha} nor CHX alone induced significant apoptosis. However, TNF-{alpha} along with CHX resulted in a significant increase in DNA fragmentation, caspase-3 activity, and detachment of cells from the substratum (Fig. 1). TNF-{alpha}, a pleiotropic cytokine, is released in response to stress and inflammation and signals proliferation or cell death, depending upon dose, exposure time, and cell type (1). We have examined the DNA fragmentation and caspase-3 activity of the attached cells as an index of the progression of apoptosis. An earlier study published from our laboratory showed that both TNF-{alpha} and camptothecin caused detachment of apoptotic cells from the substratum, as evidenced by caspase-3 activity and DNA fragmentation of floating cells (34). Other investigators have also reported the detachment-induced cell death is a recognized form of apoptosis in intestinal epithelial cells (13). At high concentrations, and in the presence of inhibitors of caspases, TNF-{alpha} has been shown to induce necrosis in many cell types, including IEC-6 cells (47, 37). Thus we examined DNA fragmentation of the attached cells to evaluate the TNF-{alpha} dose requirement for the induction of apoptosis. The degree of apoptosis as determined by nuclear fragmentation increased with increasing doses of TNF-{alpha} (Fig. 2). Thus, in subsequent experiments, we used 20 ng/ml TNF-{alpha} for 3 h to study the effect of polyamine depletion on the activation of the caspase cascade. Polyamine depletion significantly protected cells from TNF-{alpha}- plus CHX-induced apoptosis (Fig. 3). The time course of the development of apoptosis clearly showed that it was delayed in the absence of polyamines. These results indicate that polyamines are required for the normal onset of apoptosis and for the activation of caspase-3. Polyamine-depleted cells had large, significant decreases in DNA fragmentation and caspase-3 activity (Fig. 4, A and B) compared with control cells after 6 h of TNF-{alpha} and CHX treatment. These data suggest that polyamines regulate the activation of caspase-3 directly or modulate upstream signaling events.

The cascade of intracellular aspartate-specific cysteinyl proteases (caspases) is critical to apoptosis (Fig. 14). These intracellular caspases are present as inactive proenzymes that become activated after cleavage to subunits in response to apoptotic stimuli. Recently, models were proposed, distinguishing caspases as initiator caspases such as caspases-6, -7, and -8 and -9, which activate caspase-3, the major effector of apoptosis (1, 11, 24, 27, 49). TNF-{alpha} plus CHX significantly increased the activities of caspases-3, -6, -8, and -9 (Fig. 5). Polyamine depletion reduced activities of initiator caspases-8 and -9, resulting in decreases in the activity of the effector caspase (caspase-3). Caspases-8 participates in TNFR1 and Fas receptor-mediated signaling. These results suggest that polyamines regulate both upstream caspase cascades, leading to activation of caspase-3. Although significant caspase-3 induction and DNA fragmentation was observed after 6 h of treatment, we chose an earlier time point (3 h) to delineate the upstream signaling events.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 14. Schematic illustration depicting the role of polyamines in the regulation of TNF-{alpha}- and/or CHX-induced apoptosis in IEC-6 cells. TNF-{alpha}/CHX induces apoptosis in normal IEC-6 cells via death receptor-mediated activation of caspase-8 and JNK activation. JNK activation increased mitochondrial permeability probably by Bcl-2 phosphorylation and cytochrome c release. Increased cytochrome c in cytoplasm activates caspase-9 and in turn caspase-3 and induces apoptosis. SP-600125, a specific inhibitor of JNK, prevented cytochrome c release and caspase-9 activation. Polyamine depletion prevents apoptosis by inhibiting activation of caspase-8 directly or step(s) upstream of caspase-8 and JNK activation. TNFR, TNF-{alpha} receptor; FADD, FAS-associated death domain-containing protein.

 

Because CHX is required for TNF-{alpha}-induced apoptosis, and both TNF-{alpha} and CHX are known to induce the stress response, we measured the activity of SAPK/JNK. TNF-{alpha} plus CHX and CHX alone induced JNK activation compared with control or TNF-{alpha} alone (Fig. 7). JNK activation was correlated with caspase-3 activation (Figs. 8 and 9), suggesting that c-Jun kinase plays an important role in the induction of apoptosis in normal IEC-6 cells. Sustained activation of JNK has been reported to induce apoptosis in various cell lines (11, 24, 26). Although JNK activation has been implicated in apoptosis, the precise mechanism by which JNK activates apoptotic signaling is not clear. Inhibition of caspase-3 activation in the absence of polyamines provided a tool to study the mechanism by which JNK regulates apoptosis. Polyamine depletion prevented the activation of JNK and caspase-3 in response to TNF-{alpha} plus CHX (Figs. 8 and 9). This suggests that JNK regulates the upstream caspase cascade required for the activation of caspase-3. Inhibition of p46/p54 JNK by SP-600125 prevented caspase-9 and caspase-3 activation (Fig. 11) and the increase in mitochondrial permeability and cytochrome c release. Activation of caspase-8 is mediated by classical deathreceptor adaptor proteins such as TRADD or FADD, which finally results in the cleavage of Bid, a BH3 domain-containing proapoptotic member of the Bcl-2 family (11). Bid is cleaved by caspase-8, enabling it to translocate to the mitochondria and promote cytochrome c release, which further activates caspase-9 and finally caspase-3. Therefore, cleavage of Bid provides the link between the death receptor-mediated pathway and the mitochondrial pathway. Polyamine depletion also prevents Bax translocation and cytochrome c release (54). These results point out the role of polyamines in the regulation of both the extrinsic caspase-8 and intrinsic caspase-9 and cytochrome c release, pathways leading to apoptosis. However, the mechanisms through which polyamines regulate the upstream mediators are unknown and warrant further investigation. Our previous and present findings indicate that JNK regulates a step(s) upstream of caspase-9 and modulates the mitochondrial pathway by regulating cytochrome c release or the Bcl-2 family proteins (Fig. 13).

Earlier we also showed that polyamine depletion increased the expression of Bcl-XL and Bcl-2 (54). Partial inhibition of caspase-3 activation by the JNK inhibitor SP-600125 can be explained by the involvement of caspases other than caspase-9 (caspase-6 and caspase-8) that contribute to the JNK-independent activation of caspase-3 (Fig. 14). These data further suggest that multiple upstream effectors are involved in TNF-{alpha}-induced apoptosis in IEC-6 cells. Recent evidence suggests that JNK-mediated Bcl-2 phosphorylation modulates its function and in turn regulates the mitochondrial apoptotic pathway (53). Recently, Franzoso et al. (11) showed that NF-{kappa}B activation inhibits JNK activation and, thereby, prevented cell death by TNF-{alpha} (11). Polyamine depletion induced rapid activation of NF-{kappa}B in IEC-6 cells (29). Thus it is possible that NF-{kappa}B is responsible for inhibiting the JNK cascade in polyamine-depleted cells.

In summary, we have shown that apoptosis in intestinal epithelial cells involves multiple pathways leading to the activation of caspases-6, -8, and -9 as well as caspase-3. The entire cascade is prevented by polyamine depletion. Polyamine depletion prevented the activation of JNK in response to TNF-{alpha} plus CHX and provided conclusive evidence that JNK is the upstream mitochondrial apoptotic pathway regulator of caspase-9.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16505 and by the Thomas A. Erwin Endowment.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. Ray, Dept. of Physiology, The Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (E-mail: rray{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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Alnemri ES. Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J Cell Biochem 64: 33-42, 1997.[ISI][Medline]
  2. Ashkenzi A and Dixit VM. Death receptors: signaling and modulation. Science 281: 1305-1308, 1998.[Abstract/Free Full Text]
  3. Baker SJ and Reddy EP. Transducers of life and death: TNF receptor super family and associated proteins. Oncogene 12: 1-9, 1996.[ISI][Medline]
  4. Beg AA and Baltimore D. An essential role for NF-kappa B in preventing TNF-alpha-induced cell death. Science 274: 782-784, 1996.[Abstract/Free Full Text]
  5. Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, Leisten C, Motiwala A, Pierce S, Satoh Y, Bhagwat SS, Manning AM, and Anderson DW. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 98: 13681-13686, 2001.[Abstract/Free Full Text]
  6. Bertrand F, Desbois-Mouthon C, Cadoret A, Prunier C, Robin H, Capeau J, Atfi A, and Cherqui G. Insulin and apoptotic signaling involves insulin activation of the nuclear factor {kappa}B-dependent survival genes encoding tumor necrosis factor receptor-associated factor 2 and manganese-super oxide dismutase. J Biol Chem 274: 30596-30602, 1999.[Abstract/Free Full Text]
  7. Bradham CA, Qian T, Streetz K, Trautwein C, Brenner DA, and Lemasters JJ. The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release. Mol Cell Biol 18: 6353-6363, 1998.[Abstract/Free Full Text]
  8. Davidson NE, Hahm HA, McClosky DE, Woster PM, and Casero RA Jr. Clinical aspects of cell death in breast cancer: the polyamine pathways as new targets for treatment. Endocr J 6: 69-73, 1999.
  9. Dionne S, Ian D, Ruemmele FM, Levy E, St.-Louis J, Srivastava A, Levesque D, and Seidman EG. Tyrosine kinase and MAPK inhibition of TNF-{alpha}- and EGF-stimulated IEC-6 cell growth. Biochem Biophys Res Commun 242: 146-150, 1998.[ISI][Medline]
  10. Esposti MD, Hatzinisirious I, McLennan H, and Ralph S. Bcl-2 and mitochondrial oxygen redicals: new approaches with reactive oxygen species-sensitive probes. J Biol Chem 274: 29831-29837, 1999.[Abstract/Free Full Text]
  11. Franzoso G, Zazzeroni F, and Papa S. JNK: a killer on a transcriptional leash. Cell Death Differ 10: 13-15, 2003.[ISI][Medline]
  12. Grassilli EM, Desiderio A, Bellesia E, Salomoni P, Benatti F, and Franceschi C. Is polyamine decrease a common feature of apoptosis? Evidence from gamma rays- and heat-induced cell death. Biochem Biophys Res Commun 126: 708-714, 1995.
  13. Grossmann J, Mohr S, Lapetina EG, Fiocchi C, and Levin AD. Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 274: G1117-G1124, 1998.[Abstract/Free Full Text]
  14. Hale AJ, Smith CA, Sutherland LC, Stoneman VEA, Longthorne VL, Culhane AC, and Williams GT. Apoptosis: molecular regulation of cell death. Eur J Biochem 236: 1-26, 1996.[Abstract]
  15. Hsu H, Shu H-B, Pan M-G, and Goedeel DV. TRADD-TRF2 and TRADD-FADD interaction define two distinct TNF receptor 1signal transduction pathways. Cell 84: 299-308, 1996.[ISI][Medline]
  16. Hsu H, Xiong J, and Goeddel DV. The TNF receptor 1-associated protein TRADD signals cell death and NF-{kappa}B activation. Cell 81: 495-504, 1995.[ISI][Medline]
  17. Jobin C, Holt L, Bradham CA, Streetz K, Brenner DA, and Sartor RB. TNF receptor-associated factor-2 is involved in both IL-1{beta} and TNF-{alpha} signaling cascades leading to NF{kappa}B activation and IL-8 expression in human intestinal epithelial cells. J Immunol 162: 4447-4454, 1999.[Abstract/Free Full Text]
  18. Kaiser GC and Polk B. Tumor necrosis factor alpha regulates proliferation in a mouse intestinal cell line. Gastroenterology 112: 1231-1240, 1997.[ISI][Medline]
  19. Kuwana T, Smith JJ, Muzio M, Dixit V, Newmeyer DD, and Kornbluth S. Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c. J Biol Chem 273: 16589-16594, 1998.[Abstract/Free Full Text]
  20. Ledgerwood EC, Pober JS, and Bradley JR. Recent advances in the molecular basis of TNF signal transduction. Lab Invest 79: 1041-1050, 1999.[ISI][Medline]
  21. Liu Z-G, Hsu H, Goeddel DV, and Karin M. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-{kappa}B activation prevents cell death. Cell 87: 565-576, 1996.[ISI][Medline]
  22. Marton LJ and Pegg AE. Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol 35: 55-91, 1995.[ISI][Medline]
  23. McCormack SA, Viar MJ, and Johnson LR. Polyamines are necessary for cell migration by a small intestinal crypt cell line. Am J Physiol Gastrointest Liver Physiol 264: G367-G374, 1993.[Abstract/Free Full Text]
  24. Nagata Y and Todokor K. Requirement of activation of JNK and p38 for environmental stress-induced differentiation and apoptosis and of inhibition of ERK for apoptosis. Blood 94: 853-863, 1999.[Abstract/Free Full Text]
  25. Natoli G, Costanzo A, Guido F, Moretti F, Bernardo A, Burgio VL, Agresti C, and Levrero M. Nuclear factor {kappa}B-independent cytoprotective pathways originating at tumor necrosis factor receptor-associated factor2. J Biol Chem 273: 31262-31272, 1998.[Abstract/Free Full Text]
  26. Natoli G, Costanzo A, Ianni A, Templeton DJ, Woodgett JR, Balsano C, and Levrero M. Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF2 dependent pathway. Science 275: 200-203, 1997.[Abstract/Free Full Text]
  27. Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffen PR, Labelle M, Lazebnik YA, Munday NA, Raju SM, Smulson ME, Yamin TT, Yu VL, and Miller DK. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37-43, 1995.[ISI][Medline]
  28. Nitta T, Igarashi K, and Yamamoto N. Polyamine depletion induces apoptosis through mitochondria mediated pathway. Exp Cell Res 276: 120-128, 2002.[ISI][Medline]
  29. Pfeffer LM, Yang CH, Murti A, McCormack SA, Viar MJ, Ray RM, and Johnson LR. Polyamine depletion induces rapid NF-{kappa}B activation in IEC-6 cells. J Biol Chem 276: 45909-45913, 2001.[Abstract/Free Full Text]
  30. Potten CS. The significance of spontaneous and induced apoptosis in the gastrointestinal tract of mice. Cancer Metastasis Rev 11: 179-195, 1992.[ISI][Medline]
  31. Potten CS, Wilson JW, and Booth C. Regulation and significance of apoptosis in the stem cells of the gastrointestinal epithelium. Stem Cells 15: 82-93, 1997.[Abstract/Free Full Text]
  32. Pouline R, Pelletier G, and Pegg AE. Induction of apoptosis by excessive polyamine accumulation in ornithine decarboxylase-overproducing L1210 cells. Biochem J 311: 723-727, 1995.[ISI][Medline]
  33. Quaroni A, Wands J, Trelstad RL, and Isselbacher KJ. Epithelial cell culture from rat small intestine. J Cell Biol 80: 248-265, 1988.
  34. Ray RM, Viar MJ, Yuan Q, and Johnson LR. Polyamine depletion delays apoptosis of rat intestinal epithelial cells. Am J Physiol Cell Physiol 278: C480-C489, 2000.[Abstract/Free Full Text]
  35. Ray RM, Zimmerman BJ, McCormack SM, Patel TB, and Johnson LR. Polyamine depletion arrests cell cycle and induces inhibitors p21 waf1/cip1, p27kip1, and p53 in IEC-6 cells. Am J Physiol Cell Physiol 276: C684-C691, 1999.[Abstract/Free Full Text]
  36. Reinhard C, Shamoon B, Shyamala V, and Williams LT. Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by TRAF2. EMBO J 16: 1080-1092, 1997.[Abstract/Free Full Text]
  37. Ruemmele FM, Dionne S, Levy E, and Seidman E. TNF-{alpha}-induced IEC-6 cell apoptosis requires activation of ICE caspases whereas complete inhibition of the caspase cascade leads to necrotic cell death. Biochem Biophys Res Commun 260: 159-166, 1999.[ISI][Medline]
  38. Salveson GS and Dixit VM. Caspases: intracellular signaling by proteolysis. Cell 91: 443-446, 1997.[ISI][Medline]
  39. Salveson GS and Dixit VM. Caspase activation: the induced-proximity model. Proc Natl Acad Sci USA 96: 10964-10967, 1999.[Abstract/Free Full Text]
  40. Sandau KB and Brune B. Up-regulation of Bcl-2 by redox signals in glomerular mesanglial cells. Cell Death Differ 7: 118-125, 2000.[ISI][Medline]
  41. Shu HB, Halpin DR, and Goeddel DV. Casper is a FADD- and caspase-related inducer of apoptosis. Immunity 6: 751-763, 1997.[ISI][Medline]
  42. Targan SR, Hanauer SB, Van Deventer SJ, Mayer L, Present DH, Braakman T, DeWoody KI, Schaible TF, and Rutgeerts PJ. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor {alpha} for Crohn's disease: Crohn's disease cA2 study group. N Engl J Med 337: 1029-1035, 1997.[Abstract/Free Full Text]
  43. Tartaglia LA, Ayers TM, Wong GHW, and Goeddel DV. A novel domain within the 55 Kd TNF receptor signals cell death. Cell 74: 845-853, 1993.[ISI][Medline]
  44. Tartaglia LA, Rothe M, Hu YF, and Goeddel DV. Tumor necrosis factor's cytotoxic activity is signaled by the p55 TNF receptor. Cell 73: 213-216, 1993.[ISI][Medline]
  45. Tobias KE and Kahana C. Exposure to ornithine results in excessive accumulation of putrescine and apoptotic cell death in ornithine decarboxylase overproducing mouse myeloma cells. Cell Growth Differ 10: 1279-1285, 1995.
  46. Utaisincharoen P, Ubol S, Tangthawornchaikul N, Chaisuriya P, and Sirisinha S. Binding of tumor necrosis factor-alpha (TNF-{alpha}) to TNF-RI induces caspase(s)- dependent apoptosis in human cholaniocarcinoma cell lines. Clin Exp Immunol 116: 41-47, 1999.[ISI][Medline]
  47. Vercammem D, Beyaert R, Denecker G, Goossen V, Van Loo G, Declercq W, Grooten J, Fries W, and Vandenabeele P. Dual signaling of the fas receptor: initiation of both apoptotic and necrotic cell death pathways. J Exp Med 187: 1477-1485, 1998.[Abstract/Free Full Text]
  48. Wajant H, Hass E, Schwenzer R, Muhlenbeck F, Kreuz S, Schubert G, Grell M, Smith C, and Scheurich P. Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD). J Biol Chem 275: 24357-24366, 2000.[Abstract/Free Full Text]
  49. Wallach D, Boldin M, Varfolomeev E, Beyaert R, Vandenabeele P, and Fiers W. Cell death induction by receptors of the TNF family: towards a molecular understanding. FEBS Lett 410: 96-106, 1997.[ISI][Medline]
  50. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, and Baldwin AS Jr. NF-{kappa}B ant apoptosis: induction of TRAF1 and TRAF2 and c-IAP2 to suppress caspase-8 activation. Science 281: 1680-1683, 1998.[Abstract/Free Full Text]
  51. Wright NA and Alison M. Biology of Epithelial Cell Populations. Oxford, UK: Oxford Univ., vol. 2, 1985.
  52. Xie X, Tome ME, and Gerner EW. Loss of intracellular putrescine pool-size regulation induces apoptosis. Exp Cell Res 230: 386-392, 1997.[ISI][Medline]
  53. Yamamoto K, Ichijo H, and Korsmeyer SJ. BCL-2 is phosphorylated and Inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G2/M. Mol Cell Biol 19: 8469-8468, 1999.[Abstract/Free Full Text]
  54. Yuan Q, Ray RM, and Johnson LR. Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochrome c. Am J Physiol Cell Physiol 282: C1290-C1297, 2002.[Abstract/Free Full Text]