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The Journal of Immunology, 1999, 162: 1510-1518.
Copyright © 1999 by The American Association of Immunologists

Lipopolysaccharide Inhibits TNF-Induced Apoptosis: Role of Nuclear Factor-{kappa}B Activation and Reactive Oxygen Intermediates

Sunil K. Manna and Bharat B. Aggarwal1

Cytokine Research Laboratory, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LPS, a component of the cell wall in Gram-negative bacteria, induces inflammation and septic shock syndrome by stimulating various inflammatory cytokines including TNF. How LPS affects the TNF-mediated cellular responses, however, is not understood. In this study, the effect of LPS on TNF-mediated apoptosis in human histiocytic lymphoma U-937 cells was investigated. We found that treatment of cells with LPS completely abolished TNF-mediated cytotoxicity and activation of caspase-3. LPS-chelating antibiotic, polymyxin B, suppressed the antiapoptotic activity, indicating the specificity of the effect. Within minutes, LPS through CD14 induced the activation of NF-{kappa}B, degradation of I{kappa}B{alpha} (inhibitory subunit of NF-{kappa}B) and I{kappa}Bß, and nuclear translocation of p65. An antioxidant, pyrrolidine dithiocarbamate, which blocked LPS-induced NF-{kappa}B activation, also abolished the antiapoptotic effects of LPS at the same time. Besides TNF, the apoptosis induced by taxol and okadaic acid was also sensitive to LPS-induced NF-{kappa}B activation, whereas that induced by H2O2, doxorubicin, daunomycin, vincristine, and vinblastine was NF-{kappa}B insensitive. Tumor cells that constitutively expressed NF-{kappa}B also showed resistance to the apoptotic effects of TNF, taxol, and okadaic acid, but sensitivity to all other agents, indicating the critical role of NF-{kappa}B in blocking apoptosis induced by certain agents. Overall, these results indicate that LPS induces resistance to the apoptotic effects of TNF and other agents, and that NF-{kappa}B activation, whether induced or constitutive, inhibits this apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
That Gram-negative bacteria and its component LPS have antitumor properties in vivo and that this property is mediated primarily through production of TNF are well established (1, 2). LPS or endotoxin is a glycolipid and is an integral component of the outer membrane of Gram-negative bacteria. LPS mediates a number of biologic manifestations of sepsis, including fever, hypotension, multiple organ failure, shock, and death (3). These effects of endotoxin are believed to result from an uncontrolled production of proinflammatory cytokines produced by cells of the reticuloendothelial system, particularly macrophages. LPS-dependent macrophage activation results in the release of TNF, IL-1, IL-6, IL-8, IL-10, and IL-12. LPS interacts with most cells through CD14, a 55-kDa glycophosphatidylinositol-anchored protein expressed on the surface of monocytes and neutrophils (4, 5). The binding of LPS to CD14 is enhanced by the LPS-binding protein present in the serum (5, 6). Mice who lack the CD14 gene show resistance to LPS-induced shock (7).

A unique property of LPS is that it can modulate a transient state of either hypersensitivity to itself or reduced responsiveness if single or repeated injections of small amounts of LPS are given. The latter phenomenon, called LPS tolerance, is controlled at the cellular level. The role of macrophages in LPS tolerance and reduced release of TNF from the macrophages of tolerant mice has been demonstrated (8).

Both LPS and TNF display several overlapping and nonoverlapping cellular responses. TNF induces apoptosis in a wide variety of tumor cells (for references, see 9 , whereas LPS is known to induce apoptosis only in certain types of endothelial cells (10). Like TNF, however, LPS also stimulates ceramide release (10, 11), activates ceramide-activated protein kinase (12) and caspase-1 (13), and induces the stress-activated protein kinase (SAPK/JNK) pathway (14, 15). Both LPS and TNF activate the NF-{kappa}B, but through pathways consisting of similar and dissimilar steps (16). For instance, the inhibitory subunit of NF-{kappa}B, I{kappa}Bß,2 is more profoundly affected by LPS than by TNF, whereas I{kappa}B{alpha} is affected equally by both agents (17).

Although most of the pathologic effects of LPS are mediated through the induction of TNF, how LPS modulates TNF signaling is not known. In the present study, we investigated the effect of LPS on TNF-induced apoptosis. Our results show that LPS completely blocked TNF-induced apoptosis. LPS activated NF-{kappa}B, and inhibition of NF-{kappa}B activation suppressed the antiapoptotic effects of LPS. LPS also inhibited apoptosis induced by taxol and okadaic acid, but not that induced by H2O2, doxorubicin, daunomycin, vincristine, and vinblastine. A similar pattern of sensitivity was noted in tumor cells that constitutively express activated NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

PMA, cycloheximide, glycine, LPS (Escherichia coli, 055:B5), polymyxin B sulfate, H2O2, okadaic acid, daunomycin, doxorubicin, vincristine, vinblastine, taxol, pyrrolidine dithiocarbamate (PDTC), and 3-(4,5-dihydro-6-(4-(3,4-dimethoxy benzoyl)-1-piperazinyl)-2(1H)-quinolinone (MTT) were obtained from Sigma (St. Louis, MO). Penicillin, streptomycin, RPMI 1640 medium, and FCS were obtained from Life Technologies (Grand Island, NY). Bacteria-derived human rTNF, purified to homogeneity with a sp. act. of 5 x 107 U/mg, was kindly provided by Genentech (South San Francisco, CA). Anti-poly(ADP)-ribose polymerase (PARP) Ab was obtained from PharMingen (San Diego, CA). Single-stranded NF-{kappa}B oligonucleotide was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), [{gamma}-32P]ATP from ICN Pharmaceutical (Costa Mesa, CA), and polynucleotide kinase from New England Biolabs (Beverly, MA). Anti-CD14 Abs were kindly provided by Dr. Nguyen T. Van (University of Texas, M. D. Anderson Cancer Center, Houston, TX).

Cell lines

The cell lines employed in this study included U937 (human histiocytic lymphoma), Jurkat (acute T cell leukemia), HuT-78 (cutaneous T cell lymphoma), HeLa (cervical epithelial), and H4 (glioma), all obtained from American Type Culture Collection (Manassas, VA). All were cultured in RPMI 1640 containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml), and were mycoplasma free, as tested by Gen-probe mycoplasma rapid detection kit (Fisher Scientific, Pittsburgh, PA).

Assay for NF-{kappa}B

Activated NF-{kappa}B was assayed following the method of Chaturvedi et al. (18). Nuclear extracts were prepared according to Schreiber et al. (19). Briefly, 2 x 106 cells were washed with cold PBS and suspended in 0.4 ml of hypotonic lysis buffer containing protease inhibitors. The cells were then lysed by the addition of 12.5 µl of 10% Nonidet P-40, the homogenate was centrifuged, and the pellet was resuspended in 25 µl ice-cold nuclear extraction buffer. After 30 min of intermittent mixing, the tube was centrifuged for 5 min in a microfuge at 4°C, and the supernatant (nuclear extract) was either used immediately or stored at -70°C for later use. The protein content was measured by the method of Bradford (20).

Electrophoretic mobility shift assays (EMSA) were performed by incubating 4 µg of nuclear extract with 16 fmol of 32P end-labeled 45-mer double-stranded NF-{kappa}B oligonucleotide from the HIV-LTR, 5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3', for 15 min at 37°C. The incubation mixture included 2–3 µg of poly(dI-dC) in a binding buffer (21). The DNA-protein complex formed was separated from free oligonucleotide on 6.6% native PAGE, and then the gel was dried. A double-stranded mutated oligonucleotide, 5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', was used to examine the specificity of binding of NF-{kappa}B to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide.

Visualization and quantitation of radioactive bands were conducted by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using Image-quant software.

Cytotoxicity assay

Cytotoxicity induced by different agents was detected by MTT dye uptake assay, as described recently from our laboratory (22). Briefly, cells (5000 cells/well) were incubated in the presence or absence of the indicated test sample in a final volume of 0.1 ml for 24 h at 37°C. Thereafter, 0.025 ml of MTT solution (5 mg/ml in PBS) was added to each well. After a 2-h incubation at 37°C, 0.1 ml of the extraction buffer (20% SDS, 50% dimethylformamide) was added. After an overnight incubation at 37°C, the OD at 590 nm were measured using a 96-well multiscanner autoreader (Dynatech MR 5000), with the extraction buffer as a blank.

Immunoblot analysis of PARP degradation

Apoptosis induced by different inducers was examined by proteolytic cleavage of PARP (23, 24). Briefly, cells (2 x 106/ml) were treated with various agents for various times at 37°C. After treatment, cell extracts were prepared by placing the cells on ice for 30 min in 0.1 ml buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, and 1 mM DTT. The lysate was centrifuged, and the supernatant was collected. Cell extract protein (50 µg) was resolved in 7.5% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted first with mouse anti-PARP Ab, and then detected by enhanced chemoluminescence (ECL; Amersham, Arlington Heights, IL). Apoptosis was represented by the cleavage of 116-kDa PARP into an 85-kDa peptide product.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Although most effects of LPS are mediated through TNF, there is yet no report on how LPS affects TNF signaling. The aim of the present study was to investigate the effect of LPS on TNF signaling. Since the binding of LPS to its cell surface receptor CD14 is accelerated if LPS-binding protein is present in the serum (6), we incubated freshly LPS with rabbit serum at 5 µg/ml for 1 h at 37°C and performed all of the experiments with this serum-activated LPS, referred to from hereon as SA-LPS. For most studies, we used a myeloid cell line, U-937, which expresses both forms of TNF receptors, and is known to respond to LPS and produce TNF and other cytokines.

SA-LPS blocks TNF-mediated apoptosis

To determine the effect of SA-LPS on TNF-induced apoptosis, U-937 cells were preincubated with SA-LPS (100 ng/ml) for 1 h and then incubated with different concentrations of TNF for 24 h at 37°C in a CO2 incubator. Thereafter, the cell viability was determined by MTT assay. The results in Fig. 1GoA show that there is a gradual decrease in cell viability with increasing concentration of TNF in untreated cells. However, when cells were pretreated with SA-LPS, the TNF-mediated cytotoxicity was completely blocked.



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  		FIGURE 1. Effect of SA-LPS on TNF-induced cytotoxicity and on caspase-3 activation. A, U-937 cells (1 x 104/100 µl) pretreated with 100 ng/ml SA-LPS (LPS was activated with serum for 1 h at 37°C) were incubated at 37°C in a CO2 incubator with different concentrations of TNF for 24 h. Cell viability was then determined by the MTT method, as described in Materials and Methods. The results are shown as the mean (± SEM) of OD from triplicate assays. B, Cells were incubated with SA-LPS (100 ng/ml) for 30 min, and then different concentrations of TNF were added for 24 h at 37°C. Then the cells were washed, the pellet extracted, and Western blotting performed using anti-PARP mAb. The upperband shown in the figure was at 116 kDa, which was degraded to 85 kDa.

 
The cytotoxic effects of TNF in most cells are mediated through the activation of caspase-3, which degrades various substrates including PARP (24). To determine whether SA-LPS inhibits the activation of caspase-3, U-937 cells were pretreated with SA-LPS (100 ng/ml) for 1 h, stimulated with different concentrations of TNF for 24 h, and then analyzed for PARP cleavage by Western blot analysis. The results in Fig. 1GoB demonstrate that TNF cleaved the PARP protein in a dose-dependent manner. The SA-LPS-pretreated cells, however, were completely protected from TNF-dependent PARP cleavage. These results indicate that SA-LPS also blocks TNF-induced activation of caspase-3.

SA-LPS activates NF-{kappa}B, degrades I{kappa}B, and translocates p65

How SA-LPS blocks TNF-induced apoptosis was further investigated. We hypothesized that the activation of NF-{kappa}B by LPS is responsible for suppression of TNF-induced apoptosis. To investigate this, we first determined the activation of NF-{kappa}B by LPS in U-937 cells. We compared the ability of serum-untreated and serum-activated LPS to activate NF-{kappa}B. For this, cells were incubated with different concentrations of LPS at 37°C for 1 h, and the nuclear extracts were prepared and then analyzed for NF-{kappa}B by EMSA. The results in Fig. 2GoA show that untreated LPS activated NF-{kappa}B by twofold at 1 µg/ml concentration. When LPS was preactivated with serum for 1 h at 37°C and then incubated with cells, a more potent activation of NF-{kappa}B at lower dose of LPS and after shorter treatment was observed. For instance, treatment of cells with 0.1 µg/ml SA-LPS for 30 min activated NF-{kappa}B by about eightfold (Fig. 2GoB). The NF-{kappa}B band observed consisted of p50 and p65, as it was supershifted by incubation of nuclear extracts only with p50 or p65 Abs, but not by irrelevant Abs (Fig. 2GoC). Furthermore, formation of this band could be blocked by the oligonucleotide containing the wild-type NF-{kappa}B binding site, but not by that containing the mutated binding site (Fig. 2GoC). It has been reported that agents such as TNF activate NF-{kappa}B transiently. Therefore, we also examined the duration of NF-{kappa}B activation by LPS. As shown in Fig. 2GoD, the activation of NF-{kappa}B by LPS occurred at 30 min, and it persisted at a steady level for up to 24 h. To determine whether LPS-induced NF-{kappa}B activation occurred through interaction with the cell surface receptor CD14, U937 cells were preincubated with anti-CD14 Ab for 1 h at 37°C, before activation of NF-{kappa}B by SA-LPS (100 and 1000 ng/ml). As shown in Fig. 2GoE, anti-CD14 completely abolished the NF-{kappa}B activation by either concentration of SA-LPS. Thus these results suggest that LPS-induced NF-{kappa}B activation is mediated through CD14 receptor.



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  		FIGURE 2. Effect of SA-LPS on activation of NF-{kappa}B. A, U-937 cells (2 x 106/ml) were preincubated with different concentrations of LPS at 37°C for 30 min and then tested for NF-{kappa}B activation, as described. B, Different concentrations of LPS were activated with serum (final serum concentration was 2%) at 37°C for 1 h. Then U-937 cells (2 x 106/ml) were preincubated with different concentrations of SA-LPS and serum (2%) control at 37°C for 30 min and then tested for NF-{kappa}B activation, as described. C, Supershift and specificity of NF-{kappa}B. Nuclear extracts were prepared from untreated or TNF (0.1 nM)-treated cells (2 x 106/ml), incubated for 15 min with different Abs and cold NF-{kappa}B oligo probe, and then assayed for NF-{kappa}B, as described. D, U-937 cells (2 x 106/ml) were preincubated with SA-LPS (1 µg/ml) for different time, as indicated in the figure, at 37°C and then tested for NF-{kappa}B activation, as described. E, Effect of anti-CD14 Ab on SA-LPS-induced NF-{kappa}B activation. U937 cells (2 x 106/ml) were preincubated with anti-CD14 Ab for 1 h at 37°C, then activated with SA-LPS (100 and 1000 ng/ml) for 30 min at 37°C, and tested for NF-{kappa}B activation, as described.

 
The transient activation of NF-{kappa}B has been shown to accompany degradation of I{kappa}B{alpha}, the inhibitory subunit of NF-{kappa}B, whereas persistent activation of NF-{kappa}B involves degradation of I{kappa}Bß (17). In this study, we examined the rate of degradation of both I{kappa}B{alpha} and I{kappa}Bß by Western blot analysis after treatment of cells with SA-LPS. The results shown in Fig. 3Go, A and B, indicate that both I{kappa}B{alpha} and I{kappa}Bß were degraded, I{kappa}B{alpha} a little faster than I{kappa}Bß (10 min versus 15 min). This degradation resulted in the translocation of p65 from the cytoplasm to the nucleus, as determined by Western blot analysis (Fig. 3GoC).



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  		FIGURE 3. Effect of SA-LPS on degradation of I{kappa}B{alpha} and I{kappa}Bß and on the nuclear translocation of p65. U-937 cells (2 x 106/ml) were incubated for different times with SA-LPS (100 ng/ml), and then assayed for I{kappa}Bs in the cytosolic fractions by Western blot analysis (A and B) and for p65 in cytoplasmic and nuclear extracts by Western blot analysis (C).

 
SA-LPS-induced NF-{kappa}B activation is needed to block TNF-induced apoptosis

From the above results, it is clear that SA-LPS can simultaneously block TNF-induced apoptosis and activate NF-{kappa}B. Whether these two events are related was further investigated. PDTC is a thiol compound and acts in part by chelating iron and in part by quenching reactive oxygen intermediates (25). It has been shown to block NF-{kappa}B activation induced by various agents (25). Thus, we investigated the effect of PDTC on LPS-induced NF-{kappa}B activation in U-937 cells. Cells were pretreated with various concentrations of PDTC for 1 h and then stimulated with 100 ng/ml SA-LPS for 30 min at 37°C, prepared the nuclear extracts, and assayed for NF-{kappa}B by EMSA. The results showed that PDTC at 75 µM can inhibit SA-LPS-induced NF-{kappa}B activation (Fig. 4GoA). Under these conditions, PDTC had no significant effect on TNF-induced NF-{kappa}B activation (Fig. 4GoB).



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  		FIGURE 4. A and B, Effect of PDTC on activation of NF-{kappa}B induced by SA-LPS and TNF. U-937 cells (2 x 106/ml) were incubated with different concentrations of PDTC, as indicated in the figure for 1 h, and then activated with SA-LPS (100 ng/ml) or with TNF (100 pM) for 30 min, and then assayed for NF-{kappa}B in nuclear fraction by EMSA, as indicated in Materials and Methods. C, Effect of PDTC on the antiapoptotic effects of SA-LPS. Cells (1 x 104/0.1 ml) were pretreated with 100 µM of PDTC for 1 h and then activated with 100 ng/ml SA-LPS for 30 min. The cells were then stimulated with different concentrations of TNF for 24 h at 37°C in a CO2 incubator. Cell viability was determined by the MTT method, as described in Materials and Methods. All determinations were made in duplicate. D, Effect of PDTC on the antiapoptotic effects of SA-LPS. U-937 cells, pretreated with 100 µM PDTC for 1 h, were treated with SA-LPS (100 ng/ml) for 30 min. Then cells were incubated with TNF (1 nM) for 24 h at 37°C in a CO2 incubator. Then the cells were washed, pellet extracted, and Western blot performed using anti-PARP mAb. The bands shown in the figure were at 116 kDa, which degraded into 85 kDa.

 
Whether inhibition of NF-{kappa}B activation by PDTC also inhibits the ability of SA-LPS to suppress TNF-induced apoptosis was examined. For this, U-937 cells were pretreated with 100 µM PDTC for 1 h and then activated with SA-LPS (100 ng/ml) for 30 min. Thereafter, cells were treated with different concentrations of TNF for 24 h and checked for cell viability by the MTT dye uptake method. The results shown in Fig. 4GoC indicate that TNF-induced cytotoxicity was blocked by SA-LPS and that PDTC blocked the anticytotoxic effects of SA-LPS. When SA-LPS was examined for suppression of TNF-induced caspase-3 activation, it was found that PDTC also inhibited the ability of SA-LPS to suppress caspase-3 (Fig. 4GoB). Thus, the activation of NF-{kappa}B by SA-LPS was perhaps responsible for blocking the apoptotic effects of TNF.

SA-LPS also blocks apoptosis induced by okadaic acid and taxol

Whether SA-LPS also blocks apoptosis induced by other agents was investigated. U-937 cells were pretreated with SA-LPS (100 ng/ml) for 30 min and then treated with daunomycin (10 µM), doxorubicin (10 µM), H2O2 (250 µM), okadaic acid (500 nM), TNF (1 nM), taxol (10 µM), vinblastine (10 µM), and vincristine (10 µM) for 24 h at 37°C, and then assayed for cell viability by the MTT method. As Fig. 5GoA shows, SA-LPS blocked cytotoxicity induced by TNF, taxol, and okadaic acid, but had no effect on the cytotoxic effects of daunomycin, doxorubicin, H2O2, vinblastine, or vincristine. These results indicate that NF-{kappa}B activation is quite selective, blocking apoptosis induced only by TNF, taxol, and okadaic acid.



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  		FIGURE 5. A, Effect of SA-LPS on cytotoxicity induced by diverse agents. U-937 cells (1 x 104/0.1 ml) were pretreated with SA-LPS (100 ng/ml) for 30 min and then activated with daunomycin (10 µM), doxorubicin (10 µM), H2O2 (250 µM), okadaic acid (500 nM), TNF (1 nM), taxol (10 µM), vinblastine (10 µM), and vincristine (10 µM) for 24 h at 37°C in a CO2 incubator. Thereafter, cell viability was determined by the MTT method, as described in Materials and Methods. All determinations were made in triplicate. B, Kinetics of activation of caspase-3 by diverse agents. U-937 cells (2 x 106/ml) were activated with daunomycin (10 µM), doxorubicin (10 µM), H2O2 (250 µM), okadaic acid (500 nM), TNF (1 nM), taxol (10 µM), vinblastine (10 µM), and vincristine (10 µM) for 2, 8, and 24 h at 37°C in a CO2 incubator. Thereafter, cells were washed, pellet extracted, and Western blot performed using anti-PARP mAb. The bands shown in the figure were at 116 kDa, which degraded into 85 kDa. C, Effect of SA-LPS on activation of caspase-3 induced by diverse agents. U-937 cells (2 x 106/ml) were pretreated with SA-LPS (100 ng/ml) for 30 min and then activated with daunomycin (10 µM), doxorubicin (10 µM), H2O2 (250 µM), okadaic acid (500 nM), TNF (1 nM), taxol (10 µM), vinblastine (10 µM), and vincristine (10 µM) for 24 h at 37°C in a CO2 incubator. Then the cells were washed and pellet extracted, and Western blot performed using anti-PARP mAb. The bands shown in the figure were at 116 kDa, which degraded into 85 kDa.

 
We also examined the effect of SA-LPS on the activation of caspase-3 by various agents. For this, U-937 cells were treated with different apoptotic inducers for 2, 8, and 24 h at 37°C, and cell extracts were prepared and assayed for PARP cleavage. Daunomycin, doxorubicin, taxol, and vincristine cleaved PARP (from 116 kDa to 85 kDa) at 8 h of incubation, whereas H2O2, okadaic acid, TNF, and vinblastine required 24 h of incubation (Fig. 5GoB). Pretreatment of cells with SA-LPS (100 ng/ml) for 30 min had no effect on PARP cleavage induced by daunomycin (10 µM), doxorubicin (10 µM), H2O2 (250 µM), vinblastine (10 µM), or vincristine (10 µM), but inhibited the cleavage induced by okadaic acid (500 nM), TNF (1 nM), and taxol (10 µM) (Fig. 5GoC). These results indicate that SA-LPS decreases the apoptosis induced by taxol and okadaic acid.

Tumor cell lines that constitutively express activated NF-{kappa}B are also resistant to the apoptosis induced by TNF, taxol, and okadaic acid

To further explore the relevance of NF-{kappa}B activation in prevention of apoptosis by different agents, we compared the ability of various agents to induce apoptosis in the HuT-78 cell line, which constitutively expresses activated NF-{kappa}B (23), with the Jurkat line, which does not. Both Jurkat and HuT-78 cells were incubated with daunomycin (10 µM), doxorubicin (10 µM), H2O2 (250 µM), okadaic acid (500 nM), TNF (1 nM), taxol (10 µM), vinblastine (10 µM), and vincristine (10 µM) for 24 h at 37°C, and then assayed for cell viability by the MTT method. The results in Fig. 6Go show that Jurkat cells were sensitive to the cytotoxic effects of all agents. In contrast, HuT-78 cells were resistant to the cytotoxic effects of TNF, okadaic acid, and taxol, but sensitive to all other agents. These results further confirm that NF-{kappa}B is involved in prevention of apoptosis induced only by TNF, taxol, and okadaic acid, and not other agents.



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  		FIGURE 6. Cytotoxic effects of diverse agents in Jurkat and HuT-78 cells. Jurkat and HuT-78 cells (1 x 104/0.1 ml) were activated with daunomycin (10 µM), doxorubicin (10 µM), H2O2 (250 µM), okadaic acid (500 nM), TNF (1 nM), taxol (10 µM), vinblastine (10 µM), and vincristine (10 µM) for 24 h at 37°C in a CO2 incubator. Cell viability was then determined by the MTT method, as described in Materials and Methods. All determinations were made in triplicate.

 
Polymyxin B blocks SA-LPS-induced NF-{kappa}B activation

LPS is a glycolipid, and its lipid moiety is known to be critical for its activity. Polymyxin B, a polycationic cyclic peptide, is known to bind to the lipid moiety of LPS and inactivate its activity (26). To ascertain this exquisite specificity for NF-{kappa}B activation, we treated U-937 cells with 10 µg/ml polymyxin B sulfate for 1 h at 37°C, then exposed the cells to SA-LPS (100 ng/ml) for 30 min or to TNF (100 pM), prepared the nuclear extracts, and analyzed the extracts for NF-{kappa}B activation by EMSA. The results in Fig. 7Go show that polymyxin B by itself had no effect on NF-{kappa}B activation, but it completely abrogated SA-LPS-induced NF-{kappa}B activation. This effect was specific, as TNF-induced NF-{kappa}B activation was unaffected.



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  		FIGURE 7. Effect of polymyxin B on activation of NF-{kappa}B induced by SA-LPS and TNF. U-937, Jurkat, HeLa, and H4 cells were pretreated with 10 µg/ml polymyxin B sulfate for 1 h at 37°C, treated with SA-LPS (100 ng/ml) for 30 min, and then stimulated with TNF (100 pM) for the next 30 min at 37°C. Then nuclear extracts were prepared and analyzed for NF-{kappa}B assay by EMSA, as indicated in Materials and Methods.

 
We found that SA-LPS could also activate NF-{kappa}B in Jurkat cells, but activation in HeLa and H4 cells was minimal (Fig. 7Go). This difference between various cell lines could be due to difference in the level of CD14 expression, one component of the LPS receptor. In contrast, we found that TNF activated NF-{kappa}B in all of the cell lines examined, and that this activation could not be suppressed by polymyxin B. Thus, these results indicate that the lipid moiety of LPS activated NF-{kappa}B.

Polymyxin B blocks the antiapoptotic effects of SA-LPS

We tested whether the lipid moiety of LPS is responsible for its antiapoptotic effects on TNF. U-937 cells were pretreated with 10 µg/ml polymyxin B sulfate for 1 h at 37°C, treated with SA-LPS (100 ng/ml) for 30 min, and then stimulated with TNF (1 nM) for the next 24 h; cytotoxicity was determined by the MTT method. The results shown in Fig. 8GoA indicate that the antiapoptotic effects of SA-LPS are completely reversed by polymyxin B. Thus, the lipid moiety of LPS is critical for its ability to block the TNF-induced apoptosis.



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  		FIGURE 8. A, Effect of polymyxin B on LPS-induced resistance of U-937 cells to TNF-induced cytotoxicity. U-937 cells were pretreated with 10 µg/ml polymyxin B sulfate for 1 h at 37°C, then treated with SA-LPS (100 ng/ml) for 30 min before exposure to TNF (1 nM) for 24 h at 37°C. Cell viability was determined by the MTT method, as described in Materials and Methods. All determinations were made in triplicate. B, Effect of dominant-negative I{kappa}B{alpha} on TNF-induced cytotoxicity in HuT-78 cells. Cells were transfected with dominant-negative I{kappa}B{alpha} by calcium phosphate method, cultured for 12 h, then incubated with 10,000 cells/well with TNF (10, 100, 1,000, and 10,000 U/ml) for 36 h and examined for cell viability by MTT method, as described in Materials and Methods. All determinations were made in triplicate.

 
DN-I{kappa}B{alpha} reverses TNF sensitivity of tumor cells that constitutively express activated NF-{kappa}B

To further explore the relevance of NF-{kappa}B activation in prevention of apoptosis, we used HuT-78 cells that are known to constitutively express activated NF-{kappa}B, and thus are resistant to the cytotoxic effects of TNF (23). These cells were transfected by calcium phosphate method with control and DN-I{kappa}B{alpha} cDNA contructs and then examined for TNF-induced cytotoxicity. As shown in Fig. 8GoB, the transfection of DN-I{kappa}B{alpha}, which suppresses NF-{kappa}B activation, sensitized the cells to TNF-induced cytotoxicity. These results futher suggest a critical role of NF-{kappa}B in TNF-mediated apoptosis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LPS, also called endotoxin, mediates septic shock by inducing production of various inflammatory cytokines such as TNF. Whether LPS modulates the cytotoxic effects induced by TNF or other agents is not known, and thus was the major focus of our study. We show that treatment of cells with LPS induced resistance to the apoptosis-inducing effects of TNF, taxol, and okadaic acid. Apoptosis induction by H2O2 and chemotherapeutic agents, including doxorubicin, daunomycin, vincristine, and vinblastine, was, however, unaffected. LPS induced the activation of NF-{kappa}B, nuclear translocation of p65, and degradation of I{kappa}B{alpha} and I{kappa}Bß. Several lines of evidence demonstrated that the cytoprotective effects of LPS are linked to the activation of NF-{kappa}B.

Our results show that LPS induces resistance to the apoptotic effects of TNF, and this may depend on the ability of LPS to activate NF-{kappa}B. The activation of NF-{kappa}B by LPS was found to be mediated through CD14. These results are consistent with a recent report (27). The role of NF-{kappa}B activation in TNF-induced apoptosis is highly controversial. Our results are in agreement with reports that indicate that NF-{kappa}B activation blocks TNF-induced apoptosis (28, 29, 30, 31, 32). There are other reports, however, that indicate that NF-{kappa}B activation is not involved in prevention of apoptosis by TNF (33, 34). Why the results differ is not clear. This may be due to the difference in kinetics of activation of NF-{kappa}B and of apoptosis. NF-{kappa}B activation in most instances is a much earlier event than apoptosis. TNF is known to induce transient activation of NF-{kappa}B, whereas LPS induces persistent activation (17). Similarly, activation of caspase-3 by TNF, one of the indicators of apoptosis, occurs within 2 h, whereas TNF-induced cytotoxicity requires 24 h. The difference between the reports may also be related to cell types. For instance, MCF-7 cells that are most sensitive to TNF-induced apoptosis lack caspase-3 (35), and NF-{kappa}B activation has no effect on apoptosis in these cells (34).

How LPS-activated NF-{kappa}B blocks TNF-induced apoptosis is not fully understood. The promoters of the cellular inhibitor of apoptosis and the zinc finger protein A20 genes contain NF-{kappa}B binding sites (31, 36), and the products of these two genes are known to block apoptosis (37, 38). It is possible that LPS induces the expression of these genes. It is also possible that the protective effect of LPS is NF-{kappa}B independent.

Recently, it was shown that JNK1 activation is necessary for the antiapoptotic activity of human inhibitor of apoptosis (IAP)-like protein (hILP) (39). LPS is a potent activator of JNK1 (14). In our studies we found that LPS activated JNK1 in U-937 cells (data not shown). Thus, the antiapoptotic effects of LPS could be due to JNK activation. This is consistent with a recent report that showed that inhibition of JNK activation increases TNF-induced apoptosis (40). Another report, however, showed that sustained JNK activation is needed for TNF-induced apoptosis (41). One more possible mediator of the antiapoptotic role of LPS is p38 mitogen-activated protein kinase. This kinase is activated by LPS (42), and has recently been demonstrated to block TNF-induced apoptosis (40) and NF-{kappa}B activation (43). Other reports indicate that p38 mitogen-activated protein kinase can also mediate apoptosis (44). Thus, although there are several potential mechanisms by which LPS could have antiapoptotic effects, our results favor NF-{kappa}B activation, as suppression of LPS-induced NF-{kappa}B activation blocked its effect on TNF-induced apoptosis. TNF induces apoptosis through interaction with its receptor and then sequential recruitment by the death domain of the cytoplasmic portion of the receptor of TNFR-associated death domain (TRADD), Fas-associated death domain (FADD), FADD-like IL-1 converting enzyme (FLICE) (also called caspase-8), and caspase-3 (for references, see 45 .

Our results show that LPS-induced NF-{kappa}B activation inhibits apoptosis induced by okadaic acid and taxol too. Ours is the first demonstration to indicate that NF-{kappa}B activation protects against apoptosis induced by these agents. The precise pathway leading to apoptosis induction by okadaic acid and taxol, however, is not known. In our studies, we found that both of these agents activated caspase-3. Several other chemotherapeutic agents (e.g., doxorubicin, daunomycin, vincristine, and vinblastine) that also induced apoptosis and activated caspase-3 were found, but were insensitive to the LPS-induced NF-{kappa}B activation. These results indicate that the NF-{kappa}B activation must block the apoptotic pathway at a step downstream from caspase-3.

It is unclear why LPS blocks apoptosis induced by okadaic acid and taxol, but not that induced by doxorubicin, daunomycin, vincristine, and vinblastine. It suggests that okadaic acid and taxol may induce apoptosis by a mechanism different from that of other agents. Both okadaic acid (an inhibitor of serine/threonine protein phosphatase) and taxol, a microtubule-binding diterpene, are inhibitors of microtubule formation and have been shown to induce the phosphorylation of bcl-2, resulting in its inactivation and thus leading to apoptosis (46, 47). In contrast to anticancer drugs that affect microtubule integrity, DNA-damaging anticancer drugs do not induce the phosphorylation of bcl-2, an antiapoptotic protein (48). Thus, it is quite possible that the apoptosis induced by agents through damage to the microtubules (e.g., TNF, okadaic acid, and taxol) is NF-{kappa}B sensitive, whereas apoptosis induced by DNA-damaging drugs (such as H2O2, doxorubicin, daunomycin, vincristine, and vinblastine) is NF-{kappa}B insensitive. The molecular basis for this distinction is unclear. Consistent with these results, a recent report from our laboratory shows that overexpression of Mn superoxide dismutase (SOD) induces resistance to the apoptotic effects of taxol and TNF, but not to that of doxorubicin, daunomycin, vincristine, and vinblastine, indicating distinct mechanisms (22). The expression of Mn-SOD gene is known to be regulated by NF-{kappa}B (49). Thus, it is possible that LPS-induced NF-{kappa}B activation leads to transcription of Mn-SOD, which in turn induces resistance to TNF, taxol, and okadaic acid.

Based on the effect of NF-{kappa}B on apoptosis, various agents that induce apoptosis can be grouped into two distinct categories: those agents that induce apoptosis that is NF-{kappa}B sensitive, including TNF, okadaic acid, and taxol; and a second group that includes agents that induce apoptosis that is NF-{kappa}B insensitive, including H2O2, doxorubicin, daunomycin, vincristine, and vinblastine. Our results show that leukemic tumor cell lines that constitutively express NF-{kappa}B are also resistant to agents TNF, taxol, and okadaic acid, but sensitive to H2O2, doxorubicin, daunomycin, vincristine, and vinblastine. Besides leukemic cells, a number of other tumors including melanoma, glioma, renal cell carcinoma, and breast carcinoma express constitutively activated NF-{kappa}B (50, 51, 52, 53). Based on our studies, we conclude that tumors from patients will be resistant to TNF, taxol, and perhaps certain other chemotherapeutic agents if they express constitutive NF-{kappa}B. In addition, like LPS, other agents that activate NF-{kappa}B may also induce resistance to apoptosis therapy.


   Acknowledgments
 
We thank Dr. Madan M. Chaturvedi and Mr. Walter Pagel for careful review of the manuscript. This research was conducted by The Clayton Foundation for Research.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Laboratory, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, P.O. Box 143, Houston, TX 77030. E-mail address: Back

2 Abbreviations used in this paper: I{kappa}B, inhibitory subunit of NF-{kappa}B; DN, dominant negative; EMSA, electrophoretic mobility shift assay; MTT, 3-(4,5-dihydro-6-(4-(3,4-dimethoxy benzoil)-1-piperazinyl)-2(1H)-quinolinone; PARP, poly(ADP) ribose polymerase; PDTC, pyrrolidine dithiocarbamate; SA-LPS, serum-activated LPS. Back

Received for publication June 25, 1998. Accepted for publication October 16, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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