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Tumor Biology |
B Activation Confers Sensitivity to Tumor Necrosis Factor-
by Impairment of Cell Cycle Progression in Human Glioma Cells1
Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601 [G.O., T.N., H.S.]; and Department of Neurosurgery, Nagoya University School of Medicine, Nagoya 466-8550 [G.O., K.S., M.M., J.Y.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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has been shown to exert cytotoxic or cytostatic effects on tumor cells, but susceptibility to TNF- varies among different types of cells. TNF- activates a transcription factor, nuclear factor-
B (NF-B), which induces a wide variety of genes and causes pleiotrophic responses. In this study, the relationship between susceptibility to TNF- and activation of NF-B was investigated in six human malignant glioma cell lines. Cell proliferation analysis revealed that only one cell line, SK-MG-1, was sensitive to TNF- and that the other five, including U-251MG, were resistant. Electrophoretic mobility-shift assay showed that TNF- strongly activated a subtype of NF-B, the p50-p65 heterodimer, in all of the resistant cell lines tested. However, this activation was weak in the sensitive cell line, SK-MG-1. Activation of NF-B by TNF- in the resistant cell lines resulted in a significant increase of a reporter gene expression driven by NF-B site, suggesting a possibility that activation of p50-p65 confers resistance to TNF-. To test this hypothesis, we established a stable cell line that expresses an inducible dominant negative NF-B (p65 DN) protein in one of the TNF--resistant cell lines, U-251MG. In the established clone, induction of p65 DN protein decreased TNF--dependent increase in the DNA binding of p50-p65 heterodimer and NF-B-dependent reporter gene activity. Although no growth inhibition of this clone was observed by TNF- treatment, induction of p65 DN together with TNF- resulted in a significant decrease in cell number. Cell cycle analysis revealed that this growth inhibition was due to the impairment of cell cycle progression. These results indicate that an active NF-B complex, such as the p50-p65 heterodimer, plays a crucial role in the progression of cell cycle in malignant glioma cells. Refractoriness to TNF- treatment could be prevented by inhibiting NF-B activation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3
or IFN-ß is one of the other therapeutic options (1
, 2)
. However, the median survival time of the patients with this disorder has been estimated as 
2 years, even when these therapies are combined (3
, 4)
.
TNF- was originally reported as a factor that induced hemorrhagic necrosis of a certain mouse sarcoma in vivo (5)
. Subsequently, it was shown to exert cytotoxic or cytostatic effects on a variety of tumor cells in vitro (6
, 7)
. TNF- induces apoptosis or necrosis in certain tumor cells in experimental conditions (8
, 9) , and it is also used to treat malignancies in clinical trials (10
, 11)
. However, the cytotoxic/cytostatic spectrum of TNF- is limited in some conditions.
TNF- has two types of receptors: p55 TNF-R1 and p75 TNF-R2. TNF-induced trimerization of these receptors induces the recruitment of signaling proteins such as TNF-R1-associated death domain protein, TNF-R1-associated protein, and TNF-R2-associated protein via their intracellular domains (12)
. These signal transducers activate several pathways, which result in a wide range of biological responses. These pathways include the activation of NF-B, Jun NH2-terminal kinase, and factors associated with apoptosis of the cells (12)
.
NF-B was first identified as a regulator of the expression of the light-chain gene in murine B lymphocytes (13)
. Subsequently, it has been demonstrated in a variety of cells to regulate the expression of many genes involved in immune and inflammatory responses (14
, 15)
. The NF-B complex is a dimer composed of the NF-B/Rel family proteins such as p50, p52, p65, c-Rel, and Rel B, and they share a highly conserved NH2-terminal 300-amino acid domain that is required for DNA binding, dimer formation, and nuclear translocation. The common combination in NF-B complex is a p50-p65 heterodimer. In cytosol, the p50-p65 heterodimer is complexed with IB protein, which inhibits the translocation of NF-B complex into nucleus. Stimuli such as TNF-, lipopolysaccharide, and interleukin 1 dissociate NF-B complex from IB protein through phosphorylation and degradation of IB and translocate it into the nucleus. This active NF-B complex binds to the NF-B-binding site of its responsive genes and induces their transcription. Recent studies suggest that this transcription factor also affects cell proliferation (16, 17, 18, 19, 20)
.
In this study, we focused on elucidating the mechanism of cellular resistance to TNF- in human glioma cells. Six malignant glioma cell lines were divided into two groups, the TNF--sensitive and -resistant cell lines, on the basis of TNF--dependent suppression of proliferation. In TNF--resistant cell lines, an active form of NF-B such as p50-p65 heterodimer was strongly induced by TNF- treatment, whereas a TNF--sensitive cell line expressed p50 homodimer constitutively with only a transient and weak induction of p50-p65 heterodimer. Reporter gene analysis confirmed that NF-B activated by TNF- in the resistant cell lines was functional. To study whether this active NF-B contributes to cellular resistance to TNF-, we established a cell line from a TNF--resistant cell line, in which NF-B dominant negative protein (p65 DN) was inducible by the Drosophila steroid hormone ecdysone. Induction of p65 DN decreased the binding of TNF--induced p50-p65 heterodimer and inhibited the cell proliferation. This inhibition was due to impairment of cell cycle progression. Inhibition of NF-B activation in TNF--resistant cells may confer susceptibility to this cytokine.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TNF- Receptor-binding Assay.
The TNF- receptor-binding assay using 125I-labeled TNF- was described previously (21)
. The dissociation constant (Kd) was calculated by Scatchard plot analysis.
Cellular Proliferation Assay.
The effect of recombinant human TNF- (kindly provided by Asahi Chemical Industry, Tokyo, Japan) on cellular proliferation was studied in each cell line. Approximately 1 x 103 cells were cultured per well in Falcon 96-well plates (Becton Dickinson Labware, Franklin Lakes, NJ). After overnight culture, TNF- (1000 units/ml) was added (day 0), and the cell viability was determined by WST assay kit (Dojindo, Kumamoto, Japan) on days 0, 3, and 6.
Preparation of Nuclear Extracts.
The cells were treated with 1000 units/ml TNF- for 1, 3, 6, and 24 h. After harvesting, the pelleted cells were resuspended in 1 ml of buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.1% NP40, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride] and incubated for 10 min at 4°C. After centrifugation, the nuclear pellet was resuspended in 50 µl of buffer B [20 mM HEPES (pH 7.9), 400 mM KCl, 1 mM EDTA, 20% glycerol, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride] and incubated for further 10 min on ice. The supernatant was then collected as the nuclear extract. Protein concentration was determined by Bradford protein assays (Bio-Rad, Hercules, CA).
EMSA.
EMSA was carried out as described previously (22)
. Oligonucleotide probes for NF-B binding were designed to include the NF-B binding site in the promoter region of the immunoglobulin light-chain gene (Ig-B; Ref. 23
). The sequences were as follows: sense, 5'-TCGAGCAGAGGGGAC-TTTCCGAGAG-3'; and antisense, 5'-TCGACTCTCGGAAAGTCCCCTCT-GC-3'. For supershift analysis, antibodies against NF-B subfamilies (anti-p50, p52, p65, c-Rel, or Rel B antibodies; Santa Cruz Biotechnology, Santa Cruz, CA) were used. The intensity of bands was determined using the densitometric analysis program (NIH Image Version 1.44).
Reporter Gene Transfection Assay.
Construction of a luciferase reporter gene (NF-B-responsive luciferase plasmid: pGL3-3Bpro) was described previously (24)
. The cells were cultured in six-well plates. At 70% confluence, the cells were transfected with 2 µg of the reporter plasmid and 0.1 µg of the pEBV-ß-galactosidase expression plasmid (Invitrogen, San Diego, CA) by using the SuperFect lipofection kit (QIAGEN, Hilden, Germany). The DNA-SuperFect reagent mixtures were added to the cells with 1 ml of medium and incubated for 2 h. After the medium was removed, the cells were treated with 1000 units/ml TNF- for 24 h, and the luciferase and ß-galactosidase activities in the cell extract were determined by luminometer (model LB9501; Berthold, Bad Wildbad, Germany). Luciferase reporter gene activity was corrected by ß-galactosidase activity.
Preparation of a Plasmid Expressing p65 DN under the Control of Ecdysone.
To study whether the transcriptionally active NF-B contributes resistance to TNF-, we constructed a dominant negative form of NF-B (p65 DN; p65 with COOH-terminal deletion). Full-length p65 cDNA (25)
in pGEM7 vector (Promega, Madison, WI) was digested with NdeI and HindIII, blunted by Klenow fragment of DNA polymerase I, and self-ligated to construct p65 DN cDNA (corresponding to amino acids 1–292). The p65 DN cDNA was ligated into CDM8-N-tag vector which was constructed by modifying pCDM8 vector (Invitrogen, San Diego, CA) with NH2-terminal T7 tag epitope. The p65 DN cDNA with T7 tag was subcloned into pIND vector (Invitrogen).
Establishment of p65 DN-inducible Stable Cell Line.
Recombinant pIND vector harboring p65 DN cDNA was transfected into U-251MG with pVgRXR by using the SuperFect lipofection kit. Transfected cells were cultured in a medium containing 100 µg/ml G418 (Life Technologies, Inc., Grand Island, NY) and 100 µg/ml Zeocin (Invitrogen) for the selection of transformants by pIND-p65 DN and pVgRXR plasmids, respectively. After 8 weeks, stable transformants were cloned with cloning rings (Iwaki, Chiba, Japan).
Trypan Blue Dye Staining.
To assess the percentage of dead cells, we performed trypan blue dye staining. The cloned transformants were cultured in Falcon 24-well plates (Becton Dickinson Labware) at 2 x 104 cells/well and treated with 1000 units/ml TNF- and 1 µM muristerone A, an ecdysone analogue (Invitrogen), for 48, 96, and 144 h. The floating and adhesive cells were then collected and stained with 5 mg/ml trypan blue, and both stained and nonstained cells were counted.
Detection of Apoptotic Cells.
For detection of apoptosis, two different methods were used. The cloned cells were treated with 1000 units/ml TNF- and 1 µM muristerone A for 6, 12, 24, 72, and 144 h, and the cells were used for apoptosis assays, as described below.
To detect DNA fragmentation, we used the method described by Sellins et al. (26) , with minor modifications. To detect apoptosis-induced translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layers (27) , we used the Annexin V FITC kit (Immunotech, Marseilles, France), following the supplier’s protocol. The samples were analyzed by flow cytometry (Coulter EPICS XL; Coulter Electronics, Hialeah, FL).
Cell Cycle Analysis.
The cloned cells at 50% confluence were treated with 1000 units/ml TNF- alone or together with 1 µM muristerone A for 24 h. Then the cells were synchronized by incubation with 3 µM aphidicolin (Sigma Chemical Co., St. Louis, MO) for 24 h and replaced with fresh medium containing 10% FBS. The cells were collected by trypsinization at 0 and 6 h after the medium change. The cells were fixed in 70% ethanol for 20 min at 4°C, pelleted (5 min of 3000 x g at 4°C), washed twice with PBS, and resuspended in 0.1 ml of PBS with 40 units/ml RNase A. After an incubation for 40 min at 37°C, they were stained with 50 µg/ml propidium iodide for 10 min at 4°C, and DNA contents were determined by Coulter EPICS XL. The percentages of cells in G0/G1, S, and G2-M of the cell cycle were analyzed and quantitated with the Multicycle software (Coulter Electronics).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Is Cytostatic for Only One Cell Line. treatment was analyzed in six human glioma cell lines using the WST assay kit (Fig. 1)
. TNF- exerted no remarkable cytotoxic or cytostatic effect on five glioma cell lines (U-251MG, U-251SP, U-251nu/nu, T98G, and AO2). In only one cell line, SK-MG-1, was cytostatic effect demonstrated with 1000 units/ml TNF-. The growth on day 6 in SK-MG-1 was suppressed to 54% of the growth of control cells.
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type I and II receptor-binding affinities were calculated by TNF- receptor-binding assays. The dissociation constants of type I and type II receptors were not different between the TNF--sensitive and -resistant cell lines (data not shown), indicating that the signaling pathways after receptor level are involved in the different cytotoxic effects.
TNF- Strongly Activates a p50-p65 Heterodimer in the Resistant Cell Lines.
To analyze whether TNF- activates NF-B in these six malignant glioma cell lines, we performed EMSA (Fig. 2A)
. It was demonstrated that two protein DNA complexes were present in T98G and AO2 without TNF- stimulation, whereas no binding complex was observed in U-251MG, U-251SP, or U-251nu/nu. In these five resistant cell lines, TNF- markedly induced the binding of these two complexes. In particular, the induction of the slower-migrating complex lasted for 6–24 h in all of the resistant cells. A supershift analysis using the nuclear extract from U-251MG treated with TNF- for 24 h revealed that both the faster-migrating (open arrowhead) and the slower-migrating (closed arrowhead) bands were supershifted by an anti-p50 antibody (Fig. 2B)
. The slower-migrating complex was also supershifted by an anti-p65 antibody, but other antibodies did not affect the mobility of these two complexes. The same results were obtained by using the nuclear extract from other cell lines (data not shown). These results suggest that the faster- and slower-migrating bands represent a p50 homodimer and a p50-p65 heterodimer, respectively.
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-sensitive SK-MG-1, only the faster-migrating complex, corresponding to p50 homodimer, was present without TNF- treatment (Fig. 2A)
. Addition of TNF- slightly increased the binding of this complex. In contrast to the resistant cell lines, the slower-migrating band (closed arrowhead, p50-p65 heterodimer) was transiently induced at 1–6 h after the addition of TNF-.
Because p50-p65 heterodimer has a strong transcriptional activity (28)
, these results suggest that an active form of NF-B, p50-p65 heterodimer, is predominantly induced in the TNF--resistant cell lines.
TNF- Induces NF-B-dependent Reporter Gene Activity in the Resistant Cell Lines.
To assess whether TNF--induced NF-B DNA binding activity in EMSA correlates with the transcriptional activation of NF-B-responsive genes, the NF-B-responsive luciferase reporter gene was transiently transfected into the TNF--resistant and -sensitive cell lines. In the five TNF--resistant cell lines, marked increases (3–17-fold) in the luciferase activity were observed with TNF- treatment (Fig. 3)
. In contrast, the TNF--sensitive SK-MG-1 cells showed extremely low luciferase activity without treatment, and TNF--dependent increase of reporter gene activity could not be observed. These results suggest that NF-B-dependent activation of the reporter gene in TNF--resistant cell lines is due to induction of the p50-p65 heterodimer.
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-resistant U-251MG cells were transfected with pIND-p65 DN and pVgRXR plasmids. After 8 weeks of selection with G418 and Zeocin, four clones were obtained. The cells from these four clones were cultured in the absence or presence of muristerone A for 24 h. The expression of p65 DN protein in the nuclear extract was analyzed by EMSA. In only one clone, named Clone 2, was NF-B-binding activity induced with muristerone A in a dose-dependent manner (Fig. 4A)
. The complex induced by muristerone A was supershifted by anti-T7 antibody (Fig. 4B
, Lane 2). An anti-p50 antibody caused a faint supershifted band (Lane 3). An antibody recognizing the NH2 terminus of p65 (p65-n) supershifted muristerone-induced band, whereas an antibody against COOH terminus (p65-c) did not (Lanes 4 and 5). These results suggest that the induced complex includes mostly p65 DN (COOH-terminal deletion of p65 with T7-epitope tag) and little p50.
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alone, revealed two protein DNA-binding complexes (Fig. 5A)
. They were characterized as p50 homodimer and p50-p65 heterodimer by supershift analysis (data not shown), which is compatible with the findings in the parent cells (U-251MG). In the U-251MG cells, muristerone A did not affect the TNF--induced NF-B activation (data not shown). As shown in Fig. 5A
, treatment of Clone 2 with muristerone A followed by TNF- increased the density of a faster-migrating complex (open arrowhead) and decreased the binding of slower-migrating complex (arrow). As will be described below, this slower-migrating complex was the p50-p65 heterodimer. Densitometric analysis revealed that muristerone A decreased the binding of p50-p65 heterodimer in a dose-dependent manner (Fig. 5B)
.
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B binding complex in Clone 2 after the treatment with muristerone A and TNF-, we performed supershift analysis (Fig. 5C)
. The faster-migrating band (open arrowhead) was supershifted by the antibody against T7 tag and by the antibody recognizing NH2 terminus of p65 (p65-n) but not by the antibody to COOH-terminus of p65 (p65-c). It is, thus, suggested that the faster-migrating band mainly contains p65 DN homodimer induced by muristerone A. The slower-migrating complex (arrow) was supershifted by anti-p50 antibody and anti-p65-c antibody, indicating that it contains p50-p65 heterodimer.
Transcriptional activity of NF-B was analyzed by reporter gene transfection assay in Clone 2 (Fig. 5D)
. Addition of TNF- alone markedly increased the luciferase activity. However, this TNF--induced increase was suppressed by preincubation with muristerone A in a dose-dependent manner. These results suggest that the muristerone A-induced p65 DN decreases the binding of p50-p65 heterodimer induced by TNF- and inhibits the NF-B-dependent induction of the gene.
Induction of p65 DN Protein Confers the Sensitivity to TNF- in the Resistant Cell Line, U-251MG.
Clone 2, in which p65 DN induced by muristerone A inhibited NF-B-dependent activation, was used for cell proliferation analysis. The proliferation rate of Clone 2 was similar to that of the parent cell line, U-251MG (data not shown). TNF- and muristerone A did not affect the proliferation of Clone 2 (Fig. 6)
. When p65 DN was induced by muristerone A, the cell proliferation was inhibited by addition of TNF-. This inhibitory effect was dependent on the dose of muristerone A.
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B Impairs Cell Cycle Progression.. One possibility is that induction of p65 DN together with TNF- may induce apoptotic cell death (16
, 18, 19, 20
, 29)
. Another is that p65 DN with TNF- may affect the cell cycle by inhibiting growth.
The first possibility was examined by trypan blue staining to assess viable and dead cells. The percentages of dead cells treated with muristerone A and TNF- for 48, 96, and 144 h were 4, 3, and 4%, respectively. Furthermore, there was no significant difference in the percentages of dead cells between muristerone A-/TNF--treated and nontreated cells. In accordance with these results, two different methods to detect apoptosis, analysis of DNA fragmentation and flow cytometrical analysis using Annexin V revealed no evidence of apoptotic cells at 6 h to 6 days under muristerone A/TNF- treatment (data not shown).
The second possibility was examined by analysis of cell cycle using a flow cytometer-assisted cell sorter (Fig. 7)
. The cells treated with muristerone A and TNF- were synchronized with aphidicolin, which prevents eukaryotic cells from entering S phase. After medium was replaced with fresh medium containing 10% FBS, Clone 2 proceeded rapidly into S phase and progressed to G2-M (Fig. 7
, Clone 2). TNF- or muristerone A alone did not affect the cell cycle progression (Fig. 7
, Clone 2+TNF- and Clone 2+muristerone A). Under induction of p65 DN by muristerone A, TNF- treatment arrested the cell cycle at G0/G1 (Fig. 7
, Clone 2+TNF-+muristerone A). Note that TNF- arrested the growth of TNF--sensitive SK-MG-1 (Fig. 7
, SK-MG-1+TNF-), whereas it did not affect the growth of TNF--resistant U-251MG (Fig. 7
, U-251MG+TNF-). These results suggest that cytostatic effect of TNF- through impairment of cell cycle progression is suppressed by activation of NF-B and that inhibition of NF-B activation exerts growth arrest of the cells with TNF-.
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DISCUSSION |
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B is a transcription factor that consists of a dimer complex of NF-B/Rel family proteins. It regulates a wide range of genes involved in inflammation, immune response, and stress (14
, 15) . NF-B/Rel family proteins have been reported to include five subunits (p50, p52, p65, c-Rel, and Rel B) in mammals. However, transcriptional activity of each subunit varies, depending on differences in the COOH-terminal structure regulating transactivation. p50, which lacks a transactivation domain, does not have a functional activity, whereas p65 has a strong transcriptional activity (28)
. Although the functional role of p50 homodimer remains to be clarified, the p50-p65 heterodimer, the common form of NF-B, is transcriptionally active. In the TNF--resistant cells, the persistent induction of p50-p65 heterodimer correlated well with transcriptional activation of NF-B-luciferase reporter gene. In contrast, TNF- caused only a transient induction of p50-p65 heterodimer and did not activate the luciferase reporter gene in SK-MG-1, a TNF--sensitive cell line. These results suggest that induction of active NF-B may interfere with the sensitivity of the glioma cells to TNF- and that inhibition of activated NF-B could convert the TNF--resistant cells to the sensitive. This possibility was tested by establishing a stable cell line expressing p65 DN under the control of muristerone A, in one of the TNF--resistant cell lines, U-251MG. In an established clone (Clone 2), muristerone A induced p65 DN homodimer in a dose-dependent manner. This induction diminished the DNA binding of p50-p65 heterodimer and decreased TNF--induced reporter gene activation. From EMSA analysis, it is suggested that the dominant negative effect of p65 DN protein is due to competition between muristerone A-induced p65 DN homodimer and TNF--induced endogenous NF-B complex on NF-B sites. Using Clone 2 in which p65 DN could be inducible by muristerone A, we demonstrated that inhibition of NF-B confers susceptibility to TNF- upon the resistant cells.
The role of NF-B in the cell proliferation or in exerting cytotoxicity remains controversial. NF-B was thought to mediate apoptotic pathway because it was activated by certain cytotoxic stimuli. For example, in neuronal cells, the activation of NF-B results in cell death (30
, 31)
. However, in TNF--sensitive breast carcinoma MCF-7 cell line, the inhibition of NF-B did not alter the sensitivity to TNF- (32)
. On the other hand, it has been shown that activation of NF-B inhibits cell apoptosis and that its inactivation results in cell death. Knockout mice missing Rel A (p65) died before birth, apparently due to a massive death of liver cells (17)
, suggesting that NF-B can protect embryonic liver cells from committing suicide. Another report showed that the inhibition of NF-B caused apoptotic death of the B cells, in which NF-B was constitutively active (20)
. The activation of NF-B by TNF-, ionizing radiation, or an anticancer drug, daunorubicin, was also found to protect the cells from death (19)
. The inhibition of NF-B nuclear translocation in human fibrosarcoma cell line using superrepressor IB enhanced apoptotic killing by these stimuli (19)
. From these reports, it was indicated that TNF- and other cytotoxic stimuli trigger an apoptotic pathway but that they also activate a molecule that can block this very pathway. NF-B is supposed to be a candidate transcription factor that induces the key molecule to prevent apoptosis (18
, 19
, 29) . To our surprise, the susceptibility to TNF- conferred by p65 DN protein did not involve apoptosis of the tumor cells. This led us to examine whether TNF- causes cell cycle arrest when NF-B activity is inhibited in Clone 2.
TNF- has been known to have a cytostatic effect as well as a cytotoxic effect. This cytostatic effect induced by TNF- was shown to be concomitant with G0/G1 arrest in several cells (33
, 34) . In accordance with these reports, our results demonstrated that TNF- treatment resulted in cell cycle arrest in TNF--sensitive SK-MG-1 cells but not in TNF--resistant U-251MG cells, in which NF-B was activated. Inhibition of active NF-B in Clone 2 by inducing p65 DN with muristerone A caused TNF--dependent cell cycle arrest. It was reported that NF-B activity was induced during the G0-to-G1 transition after serum stimulation, supporting a role for NF-B in the G0-to-G1 transition (35)
. In a Hodgkin’s lymphoma cell line in which NF-B is constitutively active, the inactivation of NF-B by mutant IB expression altered the cell cycle and inhibited the cell proliferation without induction of apoptosis (16)
. In these cells, additional stimulation with serum starvation produced apoptosis. Although a direct role of NF-B in promoting cell cycle progression has not been well established yet, our results also support the hypothesis that NF-B may play important role in cell cycle progression, resulting in the resistance to the cytostatic effect of TNF-. Inhibition of NF-B activity can, thus, confer susceptibility to TNF- in resistant cells.
In addition to TNF-, certain chemotherapeutic agents and irradiation also induce NF-B activation in malignant cells (19
, 36)
. This activation may contribute to the cell resistance to the agents by preventing apoptosis or cell cycle arrest. It is speculated that the inhibition of NF-B might confer the sensitivity of cells to these agents, either through apoptotic pathway or cell cycle arrest.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by Grants-in-Aids from the Ministry of Health and Welfare. 
2 To whom requests for reprints should be addressed, at Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Phone: 81 (52) 789-3867; Fax: 81 (52) 789-3887; E-mail: tnagaya{at}riem.nagoya-u.ac.jp
3 The abbreviation used are: TNF, tumor necrosis factor; TNF-R1 and -R2, TNF receptors 1 and 2, respectively; NF-B, nuclear factor B; EMSA, electrophoretic mobility-shift assay; FBS, fetal bovine serum.
Received 12/21/98. Accepted 7/ 1/99.
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 F. Chen, V. Castranova, and X. Shi New Insights into the Role of Nuclear Factor-{kappa}B in Cell Growth Regulation Am. J. Pathol., August 1, 2001; 159(2): 387 - 397. [Abstract] [Full Text]
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) or presence (
) of 1000 units/ml TNF-
