Sensitizing effects of cadmium on TNF-{alpha}- and TRAIL-mediated apoptosis of NIH3T3 cells with distinct expression patterns of p53

Byung Ju Kim1, Mi-Suk Kim1, Ki-Bae Kim1, Ki-Woo Kim1, Yeon-Mi Hong1, In-Ki Kim1, Han-Woong Lee2 and Yong-Keun Jung1,3

1 Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong Puk-gu, Kwangju 500-712, Korea and
2 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor necrosis factor (TNF)-{alpha} and TNF-related apoptosis inducing ligand (TRAIL) share a common signaling pathway. Here we show a novel potentiating effect of cadmium on TNF-{alpha}- or TRAIL-mediated cell death via distinct signaling. TNF-{alpha} or TRAIL sensitized otherwise resistant NIH3T3 embryo fibroblast cells to death, when exposed to cadmium. The potentiating effects elicited by TNF-{alpha} or TRAIL on cell death were NF-{kappa}B- and SAPK/JNK-independent and were not diminished by the expression of Bcl-2. TNF-{alpha} potentiated the cadmium-induced accumulation of p53 but did not affect expression levels of Bax, Mdm2 and p21WAF/CIP. A similar pattern of p53 accumulation was also observed in Balbc/3T3 fibroblasts but not in human tumor cell lines, MCF7 and HeLa cells. The synergistic cell death evoked by TNF-{alpha} and cadmium was attenuated by transient expression of a dominant negative p53Val135 mutant in NIH3T3 cells and was not observed in p53(–/–) mouse embryo fibroblasts, indicating that p53 accumulation appears to contribute to cell death. In contrast, TRAIL did not further increase the cadmium-induced accumulation of p53 despite its potentiation effects on the cadmium-induced cell death. Expression of p53Val135 mutant did not reduce TRAIL- and cadmium-mediated cell death. Taken together, these results suggest that TNF-{alpha} and TRAIL potentiate the cadmium-mediated cell death via distinct p53 expression patterns.

Abbreviations: ROS, reactive oxygen species; TRAIL, TNF-related apoptosis inducing ligand; TNF-{alpha}, tumor necrosis factor-{alpha}


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor necrosis factor (TNF)-{alpha} is a pleiotropic cytokine associated with various cellular responses, lethal effect such as septic shock, inflammation and apoptosis in susceptible tumor cells (1–3). TNF-{alpha} signaling is transduced through 55 (TNF-R1) and 70 kDa (TNF-R2) receptors and its associated factors including protein kinases and apoptotic molecules, e.g. caspase-8, TRADD, FADD/MORT, Daxx, RIP and TRAFs. During initiation of apoptosis, FADD couples with TNF-R–TRADD complexes leading to activation of caspase-8 (4), whereas for stress signaling and immune responses, Daxx and RIP activate JNK and NF-{kappa}B, respectively (5,6). Oxygen radical formation, lipid peroxidation and oxidative damage are also mediated by TNF-{alpha}, but the mechanism is not yet known (7–9).

Cellular stresses such as genotoxic injury and oxidative stress, viral proteins such as SV40 T antigen and adenovirus E1a, and conditional expression of cellular factors such as myc and ras all induce p53 expression (10,11). p53 accumulation is likely to be mediated via multiple pathways, most of which are post-transcriptional, involving both increased translation and prolonged half-life. Wide array of protein kinases and phosphatases have been implicated in the regulation of p53 (12–15). On the other hand, discrete receptor-mediated upstream signaling cascades linked to stabilization of p53 remain largely unknown.

p53, acting as a sequence-specific and DNA binding protein, is known to activate p21WAF/CIP, Bax, p85 and MDM2, which are linked to cell cycle arrest and apoptosis (16–18). Increased levels of Bax and p85, a regulator of PI3 kinase, contribute to p53-dependent apoptosis resulting from DNA damage and oxidative stress, respectively. p53 also increases levels of TNF receptor family in response to genotoxic stress; Fas/Apo1, KILLER/DR5, TRID and TRUNDD are induced in several cell types by genotoxic stress, which serves to link p53, with the caspase cascade in apoptosis (19–22). Consequently, loss of p53 function through mutation would be expected to permit inappropriate survival of damaged cells and thus accumulation of mutations (23–25). On the other hand, apoptosis evoked by ionizing radiation may be triggered via a p53-dependent, but transcription-independent, pathway (26) or as shown in p53 knockout (–/–) mice, by a p53-independent pathway (27).

Cadmium is a widespread environmental pollutant that is also classified as group 1 carcinogens. Cadmium is absorbed by inhalation and ingestion and has a long biological half-life (>25 years). In industrial or contaminated areas, cadmium has chronical effects on lung, prostate, kidney and stomach. While cadmium shows the carcinogenic effects, it also triggers an apoptosis-like form of cell death in many cell types including T lymphocytes (28), LLC-PK1 cells (29), canine proximal tubules (30) and rat testicular tissue (31). Recently, it was reported that cadmium induced cell death through caspase activation, which was suppressed by Bcl-2 (32).

We have characterized TNF signaling leading to p53 accumulation, given the importance of p53 level in the aforementioned responses to cellular stress. In this study, we showed that in the presence of cadmium, TNF-{alpha}- and TNF-related apoptosis inducing ligand (TRAIL) sensitized fibroblast cells and tumor cells to death. TNF-{alpha}, but not TRAIL, potentiated the cadmium-induced accumulation of p53 in a cell-specific manner. These potentiation effects of TNF-{alpha} on apoptotic process were not suppressed by the expression of Bcl-2 but significantly inhibited by the expression of a dominant negative mutant of p53.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Stable cell lines and cell cultures
NIH3T3, BalbC3T3, MCF7 and HeLa cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Stable NIH3T3 cell lines expressing bcl-2 (NIH3T3/Bcl-2) were developed as described previously (33). Briefly, NIH3T3 cells were transfected for 24 h with pBabe-bcl-2 plasmid, after which the transfectants were selected by incubating with 1 µg/ml puromycin for 3 weeks. Each resistant colony was cloned and expression of bcl-2 was assessed by western blot. Mouse embryonic fibroblasts (MEF) from intercross between p53 heterozygote null mice were prepared from embryos at day 13.5 of development, and cultured in DMEM with 10% FBS. MEFs were used at early passage (3–5) for transfection.

Reagents, plasmids and antibodies
TNF-{alpha} and etoposide were provided by Sigma (St Louis, MO). TRAIL was a kind gift from Genetech (San Francisco, CA). LTRp53cG-Val construct, expressing a temperature-sensitive p53 mutant, was a generous gift from P.Hinds (Harvard Medical School). Antibodies against p53 (Pab240), p53 (DO-1), Bax (N-20), Mdm2 (SMP-14) and p21WAF/CIP (C-19) were from Santa Cruz Biotechology (Santa Cruz, CA); Bcl-2 antibody was from Dako (Carpinteria, CA); {alpha}-tubulin antibody was from Sigma; and phospho-SAPK/JNK and SAPK/JNK antibodies were from New England Biolabs (Hertfordshire, UK).

DNA transfection and cell viability assay
Cells were subcultured 1 day before each experiment and then exposed to TNF-{alpha} or TRAIL in the presence of cadmium. Cell viability was assessed by trypan blue exclusion. In ectopic expression analysis, cells were transfected with pEGFP-N1 (Clontech, Palo Alto, CA) and either pcDNA3 or p53Val135 for 24 h, after which they were treated with TNF-{alpha} or TRAIL and/or cadmium. Cell viability was evaluated under fluorescence microscope based on the morphology of GFP-positive cells. Transfections were performed using LipofectAMINE PLUSTM reagent following a protocol from Gibco BRL (Grand Island, NY). In a typical cotransfection, 0.3 µg of pEGFP-N1 and 0.9 µg of appropriate plasmids (1:3 ratio) were transfected into cells in one well of a 6 well plate.

Western blot analysis
Cells were dissolved in protein lysis buffer containing 60 mM Tris–Cl at pH 6.8, 1% sodium dodecyl sulfate (SDS), 10% glycerol and 0.5% ß-mercaptoethanol. The cell lysates were then subjected to polyacrylamide gel electrophoresis and transferred to PVDF membranes (Bio-Rad, Hercules, CA) using a Semi-Dry Transfer system (Bio-Rad). The membranes were incubated with primary antibodies in TBST buffer (20 mM Tris–Cl at pH 7.5, 150 mM NaCl, 0.2% Tween-20) containing 2% non-fat dried milk. After incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), the proteins were visualized on X-ray film using Enhanced Chemiluminescence (ECLTM, Amersham, UK). Densitometric analysis was performed with Imaging Densitometer (Bio-Rad).

Hoechst 33258 staining
Morphological changes in the nuclear chromatin of cells were detected by staining with the DNA-binding fluorochrome Hoechst 33258 (bis-benzimide; Sigma). Briefly, cells grown to 80% confluence on cover glass were fixed for 10 min in 4% paraformaldehyde and permeabilized for 2 min in 100% ethanol. After washing twice with PBS, the cells were incubated in TBST buffer containing 0.1 µg/ml Hoechst 33258 and then mounted on slide glass for observation under a fluorescence microscope.

Total RNA isolation and reverse transcription PCR (RT–PCR)
Total RNA was isolated from NIH3T3 cells using Trizol reagent according to the manufacturer's instructions (Gibco BRL). RT was carried out as follows. Briefly, total RNA (10 µg) was reverse transcribed in a total volume of 50 µl using M-MLV Reverse Transcriptase (Gibco BRL) and oligo (dT) primer (Research Genetics, Huntsville, AL) at 37°C for 1 h. RT products (5 µl) were used for PCR amplification using the primers of mouse TNF-R1 (533 bp) (upstream primer: 5'-AAG TGC CAC AAA GGA ACC TAC TTG GT-3', downstream primer: 5'-GGG ATA TCG GCA CAT TAA ACT GAT GA-3') and mouse ß-actin (430 bp) (upstream primer: 5'-GAG GGA AAT CGT GCG TGA CAT-3', downstream primer: 5'-ACA TCT GCT GGA AGG TGG ACA-3').

NF-{kappa}B activity assays: luciferase and ß-galactosidase assays
Cells grown to 80% confluence were transiently transfected with 1 µg of either pGL2-Luc or NF-{kappa}B-Luc reporter plasmid (34) using the LipofectAMINE (Gibco BRL); 0.2 µg of a ß-galactosidase expression plasmid (pCMVßgal) was also introduced to normalize variations in transfection efficiency. One day later, cells were treated with TNF-{alpha} and/or cadmium, and cell extracts were prepared according to the manufacturer's instructions (Promega, Madison, WI). Luciferase activity was measured using a luminometer (Lumat LB9501, Berthold, Germany) and ß-galactosidase activity was measured using an ELISA reader (Molecular Device, Sunnyvale, CA) at 420 nm.

JNK/atress-activated protein kinase assays
JNK activity was determined using Phospho-SAPK/JNK (Thr183/Tyr185) Antibody Kit according to manufacturer's instructions (New England Biolabs). Cells were dissolved in protein lysis buffer and the cell lysates were subjected to polyacrylamide gel electrophoresis and transferred to PVDF membranes (Bio-Rad). The membranes were subjected to western blot analysis using phospho-SAPK/JNK antibody and the blots were stripped and reprobed with SAPK/JNK antibody for protein loading control.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cadmium-mediated sensitization of NIH3T3 cells to TNF-{alpha}-induced death: an uninhibitable pathway by Bcl-2
TNF-{alpha} exerts cytotoxic effects toward a subset of tumor-derived and virus-infected cells, but most cells are resistant to it. To examine whether cadmium sensitizes otherwise resistant cells to death, NIH3T3 cells were treated with cadmium, a genotoxic agent, in the presence of TNF-{alpha} and cell viability was then determined (Figure 1Go). Exposing NIH3T3 cells to 10–20 µM cadmium or 30 ng/ml TNF-{alpha} induced basal levels of cell death affecting 10–15 and 3% of cells, respectively. In contrast, when applied together, cadmium and TNF-{alpha} synergistically increased death rate to 50–60% (Figure 1AGo). Nuclear morphology of the dying cells was examined by staining with Hoechst 33258, a fluorescent dye that labels DNA (Figure 1BGo). Note that the nuclei of cells exposed to either cadmium or TNF-{alpha} appeared uniform and healthy, whereas the nuclei of cells incubated with cadmium and TNF-{alpha} together showed the condensed and fragmented chromatin characteristics of apoptosis.



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Fig. 1. Sensitization of NIH3T3 cells to TNF-{alpha}-induced cell death in the presence of cadmium. NIH3T3 cells were left untreated (control) or treated with cadmium (Cd, 10 and 20 µM), TNF-{alpha} (30 ng/ml) or both for 17 h. (A) Cell viability was determined by trypan blue exclusion; bars depict the means ± SE of percentages of dead cells measured by at least five independent experiments. (B) Cell nuclei were visualized under a fluorescence microscope after staining with Hoechst 33258. (C) NIH3T3 cells stably expressing bcl-2 were exposed to cadmium and TNF-{alpha} as described in (A) and cell viability was determined from three independent experiments. Expression of bcl-2 in NIH3T3/Bcl-2 cells was examined by western blot analysis.

 
Bcl-2 exerts antiapoptotic effects against various apoptotic stimuli, including DNA damage and oxidative stress. We reported previously that Bcl-2 suppressed cadmium-induced apoptosis in Rat-1 fibroblast cells (32). Thus, in order to determine whether Bcl-2 could block the potentiating effects of TNF-{alpha} on cadmium-induced cell death, NIH3T3 cells permanently expressing bcl-2 (NIH3T3/Bcl-2) were established. Determination of cell viability showed that the death rates of NIH3T3/Bcl-2 cells exposed to cadmium and TNF-{alpha} were not diminished (Figure 1CGo).

Potentiation of cadmium-induced accumulation of p53 by TNF-{alpha} in NIH3T3 cells
Expression of p53 is induced by cadmium (35,36). We therefore examined whether the sensitizing effect of TNF-{alpha} on the cadmium-induced cell death reflected changes in the expression level of p53. Western blot analysis showed that p53 accumulation was detectable in NIH3T3 cells exposed to cadmium alone (10–11% of death rate) (Figure 2Go). Interestingly, p53 accumulation in cells showing 47–58% of death rates was potentiated ~4-fold by treatment with TNF-{alpha} in the presence of cadmium, while TNF-{alpha} alone was less effective.



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Fig. 2. Potentiation of the cadmium-induced accumulation of p53 by TNF-{alpha}. NIH3T3 cells were incubated for 17 h with cadmium in the presence or absence of TNF-{alpha}. After determining death rates, cell extracts were analyzed by western blot using antibodies against p53, p21WAF/CIP, Bax, Mdm2 and tubulin. Fold induction of p53 was calculated by normalizing the expression level with tubulin (p53/tubulin).

 
Whether the TNF-{alpha}-induced accumulation of p53 led to transactivation of p53-regulated genes was investigated by assessing expression of p21WAF1/CIP, a cyclin-dependent kinase inhibitor, and Bax, an apoptosis-promoting protein. Both participate in and coordinate cell cycle inhibitory and apoptotic activities of p53. Cadmium treatment did dramatically induce p21WAF1/CIP, but its accumulation was not further increased by TNF-{alpha}; indeed, the cytokine seemed rather to decrease accumulation of p21WAF1/CIP. Levels of the Bax were not changed in NIH3T3 cells exposed to both cadmium and TNF-{alpha}. Also, levels of Mdm2, a p53 down-stream destabilizer responsible for degradation of p53, were not changed like Bax (Figure 2Go). These results indicate that Mdm2 may not be responsible for p53 accumulation triggered by cadmium and TNF-{alpha}, and that Bax is not transactivated to mediate downstream activity of p53 in these cells (Figure 2Go).

No effects of cadmium and TNF-{alpha} on the synergistic expression of TNF-R1 and activation of NF-{kappa}B and SAPK/JNK during cell death
As TNF-R1 (55 kDa) is known to transduce the death signaling evoked by TNF-{alpha}, we addressed whether the expression of TNF-R1 was induced by cadmium (Figure 3AGo). RT–PCR analysis showed that cadmium or TNF-{alpha} treatment did not increase the expression level of TNF-R1, indicating that the sensitization effects to TNF-{alpha} does not result from the increased expression of TNF-R1. TNF-{alpha} or cadmium also activates the transcription factor NF-{kappa}B and the stress-activated protein kinases (SAPK/JNK). Thus, the effects of TNF-{alpha} on the cadmium-mediated activation of NF-{kappa}B and SAPK/JNK were examined in NIH3T3 cells. As shown in Figure 3BGo, we could not observe any significant synergistic effects of TNF-{alpha} and cadmium on the activation of NF-{kappa}B compared with TNF-{alpha} or cadmium alone. Similarly, cadmium-induced activation of SAPK/JNK was not further increased in the presence of TNF-{alpha} (Figure 3CGo). These results suggest that NF-{kappa}B and SAPK/JNK activation may not play a potential role in the death process of NIH3T3 cells exposed to TNF-{alpha} and cadmium.



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Fig. 3. Effects of cadmium on the expression of the TNF-{alpha} receptor (TNF-R1) and the activities of NF-{kappa}B and SAPK/JNK. NIH3T3 cells were left untreated (control) or treated with TNF-{alpha} (30 ng/ml), cadmium (10 µM), or both for 14 h. (A) Total RNA was isolated, reverse transcribed and amplified by PCR. The PCR products were resolved on 0.8% agarose gel. (B) NF-{kappa}B activity was measured as described in the Materials and methods. (C) SAPK/JNK activation was analyzed by western blot using antibodies against a phospho-SAPK/JNK (Thr183/Tyr185) (p-JNK, upper panel) and SAPK/JNK (JNK, lower panel). Fold induction of SAPK/JNK activation was determined by normalizing phospho-SAP/JNK level with JNK protein (p-JNK/JNK).

 
Sensitization of NIH3T3 cells to TRAIL-mediated death by cadmium
TRAIL is cytotoxic to tumor cells but not to normal cells, which prompted us to further examine the sensitization effect of cadmium on TRAIL-mediated cell death. Like TNF-{alpha}, TRAIL could not induce apoptosis of NIH3T3 cells but synergistically increased death rates of NIH3T3 cells exposed to cadmium from 20 to 69% (Figure 4AGo). Expression of Bcl-2 in NIH3T3 cells did not reduce the potentiating effects of TRAIL on the cadmium-induced apoptosis (Figure 4BGo). Unlike TNF-{alpha}, western blot analysis showed that synergistic increase of p53 accumulation was not observed in NIH3T3 cells exposed to both TRAIL and cadmium (Figure 4CGo). These results imply that though both TRAIL and TNF-{alpha} exert similar potentiating effects on the cadmium-induced cell death, cadmium distinguishes TRAIL from TNF-{alpha} in a signal leading to p53 accumulation.



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Fig. 4. Potentiation of cadmium-induced death of NIH3T3 cells by TRAIL. NIH3T3 cells (A) and NIH3T3/Bcl-2 cells (B) were left untreated or treated with TRAIL in the presence or absence of cadmium for 24 h. Cell viability (means ± SE) was determined from at least three independent experiments and cell extracts were analyzed by western blot using antibodies against p53 and tubulin (C). Fold induction of p53 was normalized by the tubulin (p53/tubulin).

 
Cell type-specific effects of TNF-{alpha} on the cadmium-induced accumulation of p53
We then examined the potentiation effects of TNF-{alpha} on the accumulation of p53 in other cell types. Exposure of BalbC3T3 cells, a mouse embryo fibroblast, to TNF-{alpha} and cadmium evoked a synergistic induction of p53, which also reflected death rates (Figure 5Go). Further examination of human tumor cells including MCF7 cells, breast carcinoma, and HeLa cells cervical carcinoma, showed that TNF-{alpha} synergistically increased the death rates in those cells exposed to cadmium (Figure 5Go). However, unlike fibroblast cell type, TNF-{alpha} did not further increase p53 levels in these tumor cells, indicating that the potentiating effects of TNF-{alpha} on the accumulation of p53 appear to be cell type-specific.



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Fig. 5. Distinct effects of TNF-{alpha} on the cadmium-induced accumulation of p53 in different cell types. BalbC3T3, MCF7 and HeLa cells were untreated or incubated with TNF-{alpha} in the presence or absence of cadmium for 17, 24 and 40 h, respectively. p53 level in the cells showing the indicated viability was determined with western blot analysis.

 
Contribution of p53 to TNF-{alpha}- and cadmium-induced cell death
We then assessed contribution of p53 to cell death evoked by treatment with TNF-{alpha} and cadmium. To examine a role of p53 in cell death, NIH3T3 cells were transiently transfected with pEGFP-N1 along with p53Val135, a dominant negative mutant of p53. The transfectants were then exposed to cadmium and TNF-{alpha}, and viability of the cells was assessed based on the morphology of GFP-positive cells (Figure 6A and BGo). Dying cells became round and eventually detached from the culture plate. Expression of p53Val135 apparently reduced the incidence of TNF-{alpha}- and cadmium-induced cell death from 31 to 16% in NIH3T3 cells. In contrast, the expression of p53Val135 did not reduce death rate of NIH3T3 cells exposed to TRAIL and cadmium together (Figure 6CGo). These results indicate that p53 contributes to the death process elicited by TNF-{alpha} and cadmium.



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Fig. 6. Attenuation of TNF-{alpha}- and cadmium-mediated cell death by overexpressing the dominant negative mutant of p53 (p53Val135). NIH3T3 cells were transiently cotransfected with pEGFP-N1 and either pcDNA3 or p53Val135 and 1 day later cells were exposed to TNF-{alpha} or TRAIL in the presence or absence of cadmium. Viability of cells treated with TNF-{alpha} for 17 h (A) or TRAIL for 24 h (C) was determined based on the morphology of GFP-positive cells (means ± SE). (B) Cells were visualized under a fluorescence microscope.

 
To further investigate the contribution of p53 to both TNF-{alpha}- and cadmium-induced cell death, p53(–/–) MEF cells were exposed to TNF-{alpha} and cadmium (Figure 7Go). No synergistic cell death was observed in p53(–/–) MEF cells compared with wild-type MEF cells (Figure 7AGo). To exclude a possibility that p53Val135 itself affected cell death regardless of p53, p53(–/–) MEF cells were transiently transfected with p53Val135 and subsequently exposed with TNF-{alpha} and cadmium (Figure 7BGo). Determination of cell viability showed that the p53Val135 mutant itself did not reduce cell death triggered by TNF-{alpha} and cadmium in p53(–/–) MEF cells. Taken together, these results indicate that increased expression of p53 is required to mediate the sensitizing signaling of cell death evoked by TNF-{alpha} and cadmium.



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Fig. 7. Lack of TNF-{alpha}-mediated sensitizing effects on cadmium-induced cell death in p53 knockout mouse embryo fibroblasts [p53(–/–) MEF]. (A) Wild-type MEF and p53(–/–) MEF cells were left untreated or treated with cadmium, TNF-{alpha}, or both for 17 h and cell viability (% of cell death, means ± SE) was determined by trypan blue exclusion. (B) p53(–/–) MEF cells were cotransfected with pEGFP-N1 and either pcDNA3 or p53Val135 and cells were then exposed to TNF-{alpha}, cadmium, or both for 17 h. Cell viability was determined based on the morphology of GFP-positive cells.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Normal cells are usually resistant to cytotoxic cytokines: TRAIL is especially tumor-specific. However, we identified for the first time that TNF-{alpha} and TRAIL sensitized cytokine-resistant cells to death in combinations with cadmium. While TNF-{alpha} was recently shown to induce accumulation of p53 in MCF-7 and ME180S cells, two TNF-sensitive cell lines (37,38), the present study showed that TNF-{alpha} potentiated cadmium-induced accumulation of p53 with cell type-specific manner as a necessary step leading to cell death.

Under normal conditions of cell growth, p53 protein has a relatively short half-life and is rapidly targeted for ubiquitin-mediated degradation. In response to TNF-{alpha} and genotoxic stress, the half-life of p53 may be increased by modification through phosphorylation, defining the specificity of p53 activity in response to different types of cellular stress. However, a signaling cascade linking a downstream mediator of TNF receptor such as Daxx, RIP, TRADD or TRAFs to synergistic accumulation of p53 remains to be clarified.

The fate of cells experiencing genotoxic stress depends largely on the cell type, the environment and the extent of the damage. p53 may at least sensitize cells to cytotoxicity of TNF-{alpha} and TRAIL by increasing expression level of their receptors probably mediated by positive feedback mechanism of p53. However, lack of synergistic increase of TNF-R1 expression by TNF-{alpha} and lack of increased expression of p53 by TRAIL in the presence of cadmium let us to propose the presence of other common elements in the death signaling of the cells we have examined. The common elements constituting the death signal triggered by cadmium and TNF-{alpha} are not clearly understood. SAPK/JNK and NF-{kappa}B activities are not likely to be a common mediator of cell death, although those frequently mediate a signal of cell death and survival. Cadmium elicits a number of cellular stresses associated with its cellular toxicity, including generation of reactive oxygen species (ROS) (39,40). We also measured ROS using a fluorescent dye in NIH3T3 cells after treatment with TNF-{alpha} and cadmium but could not observe any synergistic increase of ROS generation during cell death triggered by TNF-{alpha} and cadmium (Y. Jung, unpublished data). Recently, Kim et al. (32) have shown that Bcl-2 suppressed the cadmium-induced death in Rat-1 cells. However, the potentiation effects elicited by TNF-{alpha} and TRAIL on cadmium-mediated cell death were Bcl-2-independent, while Bcl-2 gene family members were known to exert antiapoptotic effects against various apoptotic stimuli, including DNA damage and oxidative stress (41–43).

The cellular context also influences activation and accumulation of p53. Thymocytes and lymphoid or myeloid cell lines subjected to ionizing radiation undergo p53-dependent apoptosis (44,45), while fibroblasts receiving similar doses of radiation undergo cell cycle arrest but retain higher viability (46). The reason for this selectivity is not known, but expression of several genes associated with p53-dependent apoptosis, including Bax, is induced by DNA damage in only a subset of cell lines and may be associated with sensitivity to cell death (47,48). When viable cells were treated with cadmium, one of the first responses was induction of p21WAF/CIP, an inhibitor of CDKs. p21WAF/CIP expression, which is also regulated by growth factor, is protective against cell death, and proteolysis of p21WAF/CIP may be necessary to alter cell cycle progression and induce apoptosis (49,50). Thus, cell type selectivity in the potentiation effects of cytokines and cadmium on p53-mediated cell death deserves more extensive characterization.

In industrial areas, the damages triggered by exposure to cadmium may be highly susceptible to abnormal cell death elicited by cytokines including TNF-{alpha} or TRAIL. Inappropriate onset of or increased sensitivity to apoptotic stimuli may give rise to a number of clinical conditions including cancer, autoimmunity and neurodegenerative disorders (51–54). In summary, cytokines and cadmium together sensitize a subtype of cells to inappropriate cell death. Further characterization of the signal modulating cell death and p53 accumulation is important to achieve better understanding of the responses to cellular stress and their clinical implications.


    Notes
 
3 To whom corrrespondence should be addressed Email: ykjung{at}eunhasu.kjist.ac.kr Back


    Acknowledgments
 
We thank R.Oita for his critical reading of this manuscript. B.J.K. was supported by the Brain Korea 21. This work was supported by the grant of the National Research Laboratory (to Y.J.) and Life Phenomena and Function Research Group Program (2000) and Protein Network Research Center from the Korean Ministry of Science and Technology.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 18, 2001; revised November 14, 2001; accepted May 10, 2002.