Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein pVHL is the cause of the familial VHL disease and the majority of sporadic renal clear cell carcinomas (RCCs). RCCs pose a significant problem for conventional cancer treatment protocols because of their highly recalcitrant characteristics to radio- and/or chemotherapies. In fact, the leading cause of morbidity and mortality of VHL patients is RCC. Recently, global gene profiling of RCC cells has revealed that sensitivity to tumor necrosis factor (TNF)-α-mediated cytotoxicity is pVHL dependent. Here, we report that although RCC cells devoid of functional pVHL (RC3) were resistant to the cytotoxic effects of TNF-α, reconstitution of these RCC cells with wild-type pVHL (WT8) restored their sensitivity to TNF-α cytotoxicity. The major TNF-α-inducible transcription factor nuclear factor (NF)-κB in the nuclear fraction capable of binding NF-κB-binding motifs was significantly increased in RC3 cells. Concordantly, the expression of NF-κB-target antiapoptotic genes c-FLIP, Survivin, c-IAP-1, and cIAP-2, which block the activities of caspases 8 and 3, were dramatically elevated in RC3 cells. Indeed, RC3 cells showed low caspases 8 and 3 activities. These results demonstrate that pVHL facilitates TNF-α-induced cytotoxicity in RCC cells, at least in part, through the down-regulation of NF-κB activity and subsequent attenuation of antiapoptotic proteins c-FLIP, Survivin, c-IAP-1, and c-IAP-2.

Functional inactivation of the VHL3 tumor suppressor protein is the cause of the rare familial VHL disease, which is characterized by the development of tumors in multiple organs, including the brain, spine, retina, adrenal gland, pancreas, epididymus, inner ear, and kidney. Biallelic inactivation of the VHL gene product (pVHL) is also responsible for the vast majority of sporadic kidney cancer called RCC. RCC is the predominant form of kidney cancer and, to date, remains to be the major cause of morbidity and mortality for VHL patients (1).

Cloning of the VHL gene did not indicate any discernable function because of the absence of homology to proteins or amino acid sequence motifs with known functions. Thus, in an effort to elucidate the putative tumor suppressor function(s) of pVHL, several groups have sought to identify cellular proteins that associated with pVHL (1). There are now several pVHL-associated proteins that suggest interesting but diverse functional roles of pVHL. pVHL directly binds elongin C and, via elongin C, associates with elongin B, Cul2, Nedd8, and Rbx1 (2). This multiprotein complex forms an E3 ubiquitin ligase complex and specifically targets prolyl-hydroxylated hypoxia-inducible factor α during normoxic conditions (2, 3). Under reduced oxygen tension, hypoxia-inducible factor α becomes stabilized and transactivates the expression of numerous hypoxia-inducible genes such as vascular endothelial growth factor, platelet-derived growth factor Β, glucose transporter 1, and erythropoietin, to trigger our physiological responses to hypoxia (2). In addition, pVHL binding to atypical PKC (4, 5), deubiquitinating enzyme (VDU)-1 (6), and homeodomain-containing protein Jade-1 (7) was shown to regulate their stability. pVHL also binds fibronectin and cytosolic protein-folding chaperonin CCT (8). Basement membranes in VHL−/− embryo or mouse embryo fibroblast and RCC cells devoid of functional pVHL are incapable of generating proper extracellular fibronectin matrix (9). It is thought that pVHL plays a role in the retrograde transport of malfolded fibronectin from the endoplasmic reticulum. In support, 10–15% of the cytosolic pVHL is associated with the endoplasmic reticulum (10). Furthermore, pVHL participates in the formation of β1-integrin fibrillar adhesions (11) and oxygen-dependent regulation of cyclin D1 (12). At a posttranscriptional level, pVHL affects the mRNA stability of hypoxia-inducible genes such as vascular endothelial growth factor and glucose transporter 1 (13). Moreover, pVHL regulates the abundance and polysomal association of RNA-binding heteronuclear ribonucleoprotein A2 (14). At a transcriptional level, pVHL binds Sp1 to mediate the transcription of tyrosine hydroxylase gene (15). Thus, pVHL seems to regulate, directly or indirectly, the expression of numerous genes with varied functional consequences at multiple levels ranging from transcription to posttranslation.

Recently, serial analysis of gene expression of RCC cells has revealed that sensitivity to TNF-α-mediated cytotoxicity is pVHL dependent (16). Here, we report that once recalcitrant RCC cells can be sensitized to TNF-α-induced cytotoxicity by reconstituting wild-type pVHL. This sensitization was due, in part, to (a) pVHL-dependent down-regulation of NF-κB activity, (b) attenuation of NF-κB-regulated antiapoptotic genes c-FLIP, Survivin, c-IAP-1, and c-IAP-2, and (c) restoration of the activities of caspases 8 and 3, the proapoptotic functions of which were inhibited by c-FLIP, Survivin, c-IAP-1, and c-IAP-2 in the absence of pVHL. Our findings support the role of pVHL as a positive mediator of TNF-α-induced apoptosis partially by suppressing the NF-κB-dependent antiapoptotic pathway.

Cell Lines

Human 786-O RCC subclones expressing wild-type pVHL (WT8), empty plasmid (RC3), and disease-causing pVHL (C162F) were described previously (3). The cells were maintained in DMEM supplemented with 10% FCS and 1.0 mg/ml G418 at 5% CO2, 37°C humidified incubator. ACHN cells obtained from American Type Culture collection (Manassas, VA) were cultured in DMEM with 10% FCS.

TNF-α Cytotoxicity Assays

Viability Analysis.

RC3, C162F, WT8, and ACHN cells were seeded in 6-well plates at 1 × 105 cells/well for at least 6 h. Cells were treated with TNF-α (Chemicon) at 12.5, 25, 50, 100, and 200 ng/ml for 72 h. Cell viability was monitored by counting the number of viable cells.

Apoptosis Analysis.

RC3 and WT8 cells were seeded in 24-well plate at 5 × 105 cells/well. Cells were treated with 50 ng/ml TNF-α for 24, 48, and 72 h and then harvested and resuspended in 100 μl of incubation buffer [10 mm HEPES (pH 7.4), 140 mm NaCl, and 5 mm CaCl2] containing 20 μl of Annexin V-FITC (Caltag Laboratories) and 1 μl of propidium iodide (1 mg/ml; Sigma) for 15 min. Cells were analyzed on Epics Elite flowcytometer (Beckman-Coulter).

EMSA.

RC3 and WT8 cells growing in 100-mm plates at 80% confluency were treated with 50 ng/ml TNF-α for 0, 0.3, 1.5, and 3 h. Nuclear extract isolation and EMSA procedures were performed as described previously (17). Briefly, the cells were washed with PBS twice and resuspended in 400 μl of buffer A [10 mm HEPES (pH 7.9), 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm DTT, and 0.5 mm phenylmethylsulfonyl fluoride] and incubated on ice for 15 min. Twenty-five μl of 10% NP40 were added, incubated on ice for 5 min, and vortexed. Lysates were centrifuged at 12,000 × g for 1 min, and the pellets were resuspended in 50 μl of buffer C [20 mm HEPES (pH 7.9), 0.4 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 0.5 mm phenylmethylsulfonyl fluoride, and protease/phosphatase inhibitor mixture (Roche Molecular Biochemicals)] and incubated on ice for 30 min. Nuclear extract was then isolated by centrifugation at 12,000 × g for 5 min. Fifteen μg of nuclear extract were incubated with 3 μg of poly(deoxyinosinic-deoxycytidylic acid) (Sigma) and double-stranded oligonucleotides for the consensus NF-κB-binding sites (5′-AGTTGAGGGGACTTTCCCAGGC-3′) labeled with γ-[32P]dATP by T4 polynucleotide kinase in a binding buffer [5 mm HEPES (pH 7.8), 50 mm KCl, 0.5 mm DTT, and 10% glycerol]. Protein-oligo mixtures were separated on a nondenaturing 5% polyacrylamide gel in 0.5× Tris-borate EDTA. For specific competition, proteins were preincubated with double-stranded unlabeled NF-κB-binding oligonucleotide or mutant oligonucleotide (5′-AGTTGAGGCGACTTTCCCAGGC-3′) at 100-fold molar excess.

Antibodies, Immunoblotting, and Immunoprecipitation.

Cells were lysed in EBC buffer [50 mm Tris-HCl (pH 8.0), 120 mm NaCl, and 0.5% NP40] supplemented with protease and phosphatase inhibitors (Roche Molecular Biochemicals). One hundred μg of protein were separated by SDS-PAGE and immunoblotted with anti-c-FLIP (Stressgen Biotechnologies Corp.) and anti-α-tubulin (Sigma) antibodies. Antibody for λPKC was kindly provided by Dr. Syu-ichi Hirai. IκB-α antibody was purchased from Upstate Biotechnology. Monoclonal and polyclonal anti-VHL antibodies IG32 and R98 were described previously (9). Immunoprecipitation was performed as described previously (3).

RPA.

To detect the differential expression of Survivin, c-IAP-1, and c-IAP-2 at the transcriptional level, the corresponding cRNA probes were synthesized. A 306-bp cDNA fragment of Survivin encompassing 3′ 31 bp of exon 2B, 118 bp of exon 3, 87 bp of exon 4, and 70 bp of 3′ untranslated region (nucleotides 4498–4528, 5158–5275, and 11955–12114 of GenBank accession no. U75285) were reverse transcription-PCR amplified and inserted into SmaI site of pBluescript II KS (+/−) plasmid in the sense direction. The resulted 372-bp Survivin cRNA probe (including 66-bp plasmid sequence) protected mRNAs at 175, 275, and 306 bp representing Survivin-ΔEx3, Survivin, and Survivin-2B. To generate the cRNA probes for c-IAP-1 and c-IAP-2, 250- and 269-bp cDNA fragments corresponding to the nucleotides 361–610 of c-IAP-1 (GenBank accession no. U45879) and 511–780 of c-IAP-2 (GenBank accession no. U45878) were PCR amplified from pCI-c-IAP-1 and pGC-c-IAP-2 plasmids, respectively, and inserted into SmaI site of pBluscript II KS (+/−) in sense direction. A fragment of the β-actin (nucleotide 704–833 of GenBank accession no. X00351) was amplified by PCR and cloned in the same way as described above. All plasmids were linearized with EcoRI and labeled with [α-32P]UTP using T7 RNA polymerase. RPA was performed on 15 μg of total RNA using the RPA III kit (Ambion, Inc.) according to the manufacturer’s instructions. Protected fragments were separated by electrophoresis in 6% acrylamide 7 m urea gels.

Caspases 8 and 3 Assays.

Cells were seeded in 100-mm plates at 80% confluency. Cells were treated with 50 ng/ml TNF-α for 0, 1, 2, 4, 8, and 24 h. Caspases 8 and 3 fluorometric assays were performed in 96-well plates according to the manufacturer’s (B&D Systems) instructions. The reactions were read on SpectraMAXGERMINI microplate spectrophotometer (Molecular Devices).

pVHL Sensitizes RCC Cells to TNF-α-Induced Cytotoxicity.

TNF-α is a proinflammatory cytokine that mediate complex biological responses ranging from inflammation, septic shock, antiviral responses, survival, and apoptotic cell death (18). We asked whether pVHL played a role in the sensitivity of RCC cells to TNF-α-mediated cytotoxicity. 786-O RCC subclones ectopically expressing empty plasmid (RC3) or wild-type pVHL (WT8) or disease-causing pVHL (C162F) and ACHN, which expresses endogenous pVHL, were analyzed for their sensitivity to increasing concentrations of TNF-α (Fig. 1,A). The expression of ectopic pVHL (WT8), tumor-derived mutant pVHL (C162F), and endogenous pVHL in ACHN cells are shown in Fig. 1,B. Cells devoid of functional pVHL or expressing disease-causing mutant pVHL (C162F) were significantly more resistant than cells expressing wild-type pVHL (WT8 and ACHN) at every dosage of TNF-α tested (Fig. 1 A). Microscopic examination of the adherent WT8 and ACHN cells showed marked increase in number of detached and condensed or “rounded-up” cells after 24 h of TNF-α treatment (data not shown). This was in stark contrast to RC3 and C162F cells, which showed very few detached cells.

We next asked whether this observed TNF-α-induced death in wild-type pVHL-expressing cells were attributable to apoptosis (Fig. 1 C). Annexin-V assay was performed on RC3 and WT8 cells after TNF-α treatment. Forty-eight and 72 h after treatment, ∼32 and 61% of WT8 cells, respectively, stained positive for Annexin-V, whereas only 2 and 12% of RC3 cells, respectively, were Annexin-V positive. Thus, RCC cells devoid of functional pVHL were highly resistant to TNF-α-induced cytotoxicity, and reconstitution of RCC cells with wild-type pVHL restored their sensitivity to TNF-α.

pVHL Suppresses NF-κB Accumulation in the Nucleus and the Expression of NF-κB Target Antiapoptotic Genes c-FLIP, Survivin, c-IAP-1, and c-IAP-2.

TNF-α can elicit both survival and apoptotic cell death signals (18, 19). As with other TNF-α family members such as Fas ligand and TNF-related apoptosis-inducing ligand, TNF-α recruits the FADD to the cytoplasmic tail of TNFR 1. This recruitment initiates the activation of caspase-signaling cascade, which ultimately leads to apoptotic cell death. However, TNF-α also activates NF-κB pathway, which functions to suppress apoptosis through the activation of various IAPs. This process involves the TNF-α-induced activation of aPKC, which then activates IKKβ. IKKβ phosphorylates IκB, leading to ubiquitination of ΙκB and subsequent degradation by the 26S proteasome. This releases NF-κB from the cytosol to the nucleus, where it activates the transcription of various IAPs to suppress apoptosis (Refs. 18, 19; Fig. 4).

The inhibition of aPKC isoform ζ in Xenopus oocytes has been shown to block the activation of NF-κB activity. In support, targeted disruption of aPKCζ gene resulted in the impairment of the NF-κB pathway (20). This was likely because of the inability to activate IKKβ in the absence of functional aPKCζ, which led to the extended sequestration of NF-κB in the cytosol by IκB. Recently, pVHL was shown to directly bind aPKC isoforms ζ and ι/λ and to target aPKCι/λ for ubiquitination (4, 5). We asked whether pVHL influenced the level and/or activity of NF-κB in the nucleus (Fig. 2). Nuclear fractions were prepared from RCC cells devoid of pVHL (RC3) and RCC cells reconstituted with wild-type pVHL (WT8). EMSA analysis of the nuclear fractions showed significantly higher basal level of NF-κB capable of binding NF-κB-binding DNA motifs in RC3 cells than WT8 cells (Fig. 2,A). Cells expressing tumor-derived pVHL (C162F) had similarly high levels of NF-κB in the nucleus. Upon TNF-α treatment, there was a rapid induction of NF-κB in the nucleus of WT8 cells, but the extent of this accumulation was dramatically lower than that observed in RC3 nucleus (Fig. 2 A). These results suggest that in the absence of pVHL, which can sequester or ubiquitinate aPKC, there are more aPKC ready to be activated upon TNF-α stimulation. This would account for the higher basal level of NF-κB and higher accumulation of NF-κB in the nucleus of cells devoid of pVHL. In accordance, a constitutive activation of NF-κB in various RCC cell lines was shown to prevent apoptotic induction (17). Furthermore, the basal level of NF-κB was found to be lower in cells that express wild-type pVHL such as Caki-1 and ACHN and significantly higher in cells expressing mutant pVHL such as 769P (17).

We next asked whether the increased accumulation of NF-κB in the nucleus of cells devoid of functional pVHL led to the corresponding activation c-FLIP, Survivin, c-IAP-1, and c-IAP-2, which are antiapoptotic genes regulated by NF-κB (18, 19, 20, 21, 22). c-FLIP and Survivin have been shown to block the activities of caspases 8 and 3, respectively (18, 22). c-FLIP is a caspase 8 homologue that contains two death effector domain domains, which is required for the association with FADD but lacks any proteolytic function (18, 22). Survivin, c-IAP-1, and c-IAP-2 are IAP family member that directly binds to and inhibits caspases 3 and 7, which function as terminal effector molecules in the apoptosis-protease cascade (21, 22).

Cells expressing pVHL (WT8) showed marked attenuation of c-FLIP protein expression, whereas cells devoid of functional pVHL (RC3 and C162F) showed strong expression of c-FLIP (Fig. 2,B). The expressions of two known c-FLIP isoforms [c-FLIPLong(L) at Mr 55,000 and c-FLIPShort(S) at Mr 28,000] were detected in RC3 and C162F cells (Fig. 2 B). Anti-Survivin immunoblot was likewise performed. However, the quality of anti-Survivin antibody was substandard and the resulting immunoblots were uninterpretable.

To detect the transcriptional regulation of NF-κB-target genes, we performed RPA to measure the transcript levels of Survivin, c-IAP-1, and c-IAP-2 (Fig. 2, C and D). Survivin cRNA probes were designed to distinguish between the three known splicing variants of Survivin at 175 bp (Survivin-ΔEx3), 275 bp (Survivin), and 306 bp (Survivin-2B). RC3 and C162F cells showed strong basal expression of Survivin, whereas WT8 cells failed to show any detectable levels of Survivin (Fig. 2,C). In these cells, Survivin-ΔEx3 transcripts were not detected and Survivin-2B transcripts were barely detectable in RC3 and C162F cells. The basal levels of c-IAP-1 and c-IAP-2 in WT8 cells were comparatively lower than in RC3 and C162F cells. Upon TNF-α stimulation, Survivin, c-IAP-1, and c-IAP-2 mRNA expressions increased marginally (when normalized to β-actin) in WT8 cells after several hours of exposure (Fig. 2,D). There was a slight increment of Survivin, and no detectable increase of c-IAP-1 and c-IAP-2 mRNA levels in RC3 cells upon TNF-α treatment (Fig. 2 D). These results suggest that pVHL suppresses the expression of NF-κB-driven antiapoptotic genes c-FLIP, Survivin, c-IAP-1, and c-IAP2.

pVHL Is Required for the Induction of TNF-α-Induced Caspases 8 and 3 Activities.

We asked whether the increased expression of antiapoptotic molecules c-FLIP and Survivin in RCC cells devoid of pVHL had corresponding suppression of proapoptotic Caspases 8 (also known as an initiator caspase) and 3 (effector caspase) activities (Fig. 3). After 24 h of TNF-α treatment, WT8 cells showed a significant increase in caspase 8 activity (4-fold; Fig. 3,A). In contrast, caspase 8 activity increased marginally in RC3 cells (2-fold; Fig. 3,A). Moreover, the relative activity of the effector caspase 3 activity was dramatically increased in WT8 cells (11-fold; Fig. 3,B), although there was a significantly lesser enhancement of caspase 3 activity in RC3 cells upon TNF-α treatment (3-fold; Fig. 3,B). These results demonstrate a marked suppression of caspase 8 and caspase 3 activities in RCC cells devoid of functional pVHL upon TNF-α treatment. In accordance, caspases 8 and 3 activities were restored upon the reconstitution of RCC cells with wild-type pVHL. Thus, increased expression of c-FLIP, Survivin, c-IAP-1, and c-IAP-2 in RCC cells devoid of wild-type pVHL may indeed account for the attenuation of caspases 8 and 3 activities, respectively, and explains, at least in part, why RCC cells are resistant to TNF-α-induced cytotoxicity. Furthermore, the increase of Survivin, c-IAP-1, and c-IAP-2 seen in WT8 cells (see above; Fig. 2 C) upon TNF-α treatment was insufficient in preventing the activation of the effector Caspase 3.

TNF-α can trigger both apoptotic pathway (via the caspase-protease pathway) and survival/antiapoptotic pathway (via the NF-κB pathway; Fig. 4). The balance of these two pathways is critical for the ultimate fate of a cell: death or survival. TNFR engagement of TNF-α triggers the activation of aPKC, which then phosphorylates IKKβ. The now active IKKβ phosphorylates IκB, which triggers its ubiquitination and subsequent degradation. This releases NF-κB from its inhibitor IκB and allows the translocation of NF-κB into the nucleus where it activates the transcription of numerous antiapoptotic genes, including c-FLIP and IAP family. Recently, pVHL has been shown to directly target aPKC for ubiquitin-mediated destruction (4, 5). Thus, it is plausible that aPKC, released from the destructive targeting in cells devoid of functional pVHL, would constitutively phosphorylate downstream IKKβ, leading to the destruction of IκB, and subsequent nuclear accumulation of NF-κB. Although our findings support increased NF-κB DNA binding activity and concordantly high basal expression level of NF-κB-driven genes c-IAP-1, cIAP-2, c-FLIP, and Survivin in RCC cells devoid of wild-type pVHL, our present analyses of aPKC and IKKβ were inconclusive (data not shown). Thus, the precise pVHL-dependent mechanism upstream of the nuclear accumulation of NF-κB is currently unknown.

In summary, we have found pVHL-negative RCC cells of having significantly high level of NF-κB DNA binding activity, resulting in transactivation of numerous antiapoptotic genes that inhibit the caspase-protease pathway. This likely explains why RCC cells lacking functional pVHL are highly resistant to TNF-α-mediated cytotoxicity and suggests a new tumor suppressor function of pVHL as a positive regulator of apoptosis by suppressing NF-κB-mediated antiapoptotic pathway. Involvement of pVHL in this process may represent a common function of pVHL lost in all or a subset of VHL diseases (i.e., type 1 or type 2A/B/C). Exploring this avenue of pVHL function may help to elucidate the molecular events that underlie the genotype-phenotype correlation in VHL disease.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work has been supported by the National Cancer Institute of Canada with funds from Terry Fox Run Grant 13030. H. Q. is a recipient of a Canadian Institute of Health Research postdoctoral fellowship. M. O. is a Canada Research Chair in molecular oncology.

3

The abbreviations used are: VHL, von Hippel-Lindau; pVHL, VHL tumor suppressor protein; RCC, renal clear cell carcinoma; PKC, protein kinase C; TNF, tumor necrosis factor; EMSA, electrophoretic mobility shift assay; IκB, inhibitor of nuclear factor-κB; RPA, RNase protection assay; FADD, Fas-associated death domain protein; TNFR, TNF receptor; IAP, inhibitor of apoptosis; aPKC, atypical protein kinase C; IKKβ, IκB kinase β; c-FLIP, cellular FLICE (FADD-like interleukin-1β converting enzyme)-inhibitory protein.

Fig. 1.

pVHL sensitizes RCC cells to TNF-α-induced cytotoxicity. A, 786-O RCC subclones expressing wild-type pVHL (WT8) or empty plasmid (RC3) or tumor-derived pVHL (C162F) and ACHN cells that expresses endogenous pVHL were seeded in 6-well plates at 1 × 105 cells/well. Cells were treated with the indicated increasing concentrations of TNF-α for 72 h. Cell viability was measured by counting the number of viable cells. Data represent average values obtained from three experiments, each performed in triplicates. B, the expression of pVHL in the cells. One hundred μg of whole cell extracts from WT8, RC3, and C162F were immunoblotted with R98 anti-pVHL antibody (Lane 1–3). Two mg of whole cell extracts from ACHN cells were immunoprecipitated IG32 monoclonal anti-pVHL antibody and immunoblotting with the polyclonal R98 antibody (Lane 4). C, WT8 and RC3 cells were treated with 50 ng/ml TNF-α for the indicated times. Annexin-V-FITC and propidium iodide staining were performed and measured on a flowcytometer to determine the percentage of cells undergoing apoptosis (shown on the top-right quadrant).

Fig. 1.

pVHL sensitizes RCC cells to TNF-α-induced cytotoxicity. A, 786-O RCC subclones expressing wild-type pVHL (WT8) or empty plasmid (RC3) or tumor-derived pVHL (C162F) and ACHN cells that expresses endogenous pVHL were seeded in 6-well plates at 1 × 105 cells/well. Cells were treated with the indicated increasing concentrations of TNF-α for 72 h. Cell viability was measured by counting the number of viable cells. Data represent average values obtained from three experiments, each performed in triplicates. B, the expression of pVHL in the cells. One hundred μg of whole cell extracts from WT8, RC3, and C162F were immunoblotted with R98 anti-pVHL antibody (Lane 1–3). Two mg of whole cell extracts from ACHN cells were immunoprecipitated IG32 monoclonal anti-pVHL antibody and immunoblotting with the polyclonal R98 antibody (Lane 4). C, WT8 and RC3 cells were treated with 50 ng/ml TNF-α for the indicated times. Annexin-V-FITC and propidium iodide staining were performed and measured on a flowcytometer to determine the percentage of cells undergoing apoptosis (shown on the top-right quadrant).

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Fig. 2.

TNFα-induced activation of NF-κB and the expression of NF-κB-regulated antiapoptotic molecules c-FLIP, Survivin, c-IAP-1, and c-IAP-2 are dependent on pVHL. A, nuclear proteins were extracted from WT8, RC3, and C162F cells that were treated with (Lanes 7–9 and 11–13) or without TNF-α (Lanes 1–6 and 10; 50 ng/ml) for the indicated times. The specificity was determined by competing with normal or mutant NF-κB oligonucleotides (Lane 1 and 2, respectively). B, differential expression of c-FLIP in WT8, RC3, and C162F cells. Cells were lysed, and 100 μg of cellular extracts were separated on SDS-PAGE and immunoblotted with anti-cFLIP antibody. The arrow indicates the Mr 55,000 c-FLIPL. Asterisk indicates a nonspecific binding protein. α-Tubulin immunoblot was performed as an internal loading control (bottom panel). C, pVHL down-regulates the expression of Survivin, c-IAP-1, and c-IAP-2 at the transcription level. Total RNA was isolated from the indicated cells, and RPA was performed using corresponding cRNA probes. β-Actin was used as internal control. D, TNF-α induction of Survivin, c-IAP-1, and c-IAP-2. WT8 and RC3 cells were treated with TNF-α (20 ng/ml) for the indicated times and RPA was then performed.

Fig. 2.

TNFα-induced activation of NF-κB and the expression of NF-κB-regulated antiapoptotic molecules c-FLIP, Survivin, c-IAP-1, and c-IAP-2 are dependent on pVHL. A, nuclear proteins were extracted from WT8, RC3, and C162F cells that were treated with (Lanes 7–9 and 11–13) or without TNF-α (Lanes 1–6 and 10; 50 ng/ml) for the indicated times. The specificity was determined by competing with normal or mutant NF-κB oligonucleotides (Lane 1 and 2, respectively). B, differential expression of c-FLIP in WT8, RC3, and C162F cells. Cells were lysed, and 100 μg of cellular extracts were separated on SDS-PAGE and immunoblotted with anti-cFLIP antibody. The arrow indicates the Mr 55,000 c-FLIPL. Asterisk indicates a nonspecific binding protein. α-Tubulin immunoblot was performed as an internal loading control (bottom panel). C, pVHL down-regulates the expression of Survivin, c-IAP-1, and c-IAP-2 at the transcription level. Total RNA was isolated from the indicated cells, and RPA was performed using corresponding cRNA probes. β-Actin was used as internal control. D, TNF-α induction of Survivin, c-IAP-1, and c-IAP-2. WT8 and RC3 cells were treated with TNF-α (20 ng/ml) for the indicated times and RPA was then performed.

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Fig. 3.

Activation of caspases 8 and 3 in RCC cells is dependent on pVHL. WT8 and RC3 cells were seeded in 100-mm plates at 80% confluency and treated with TNF-α (50 ng/ml) for the indicated times. Cells were lysed and analyzed for caspases 8 (A) and 3 (B) activities. The relative activity at 0 h was arbitrarily assigned a value of 1. Data represent average values obtained from three experiments.

Fig. 3.

Activation of caspases 8 and 3 in RCC cells is dependent on pVHL. WT8 and RC3 cells were seeded in 100-mm plates at 80% confluency and treated with TNF-α (50 ng/ml) for the indicated times. Cells were lysed and analyzed for caspases 8 (A) and 3 (B) activities. The relative activity at 0 h was arbitrarily assigned a value of 1. Data represent average values obtained from three experiments.

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Fig. 4.

Model of pVHL involvement in TNF-α-mediated signaling. TNF-α elicits apoptotic and antiapoptotic signals. In the apoptotic pathway, TNFR engagement of TNF-α triggers the activation of caspase-protease cascade leading to programmed cell death. In the antiapoptotic pathway, TNFR engagement of TNF-α triggers the activation of aPKC, which is in a complex with TRAF2, RIP, and P62/ZIP. Activated aPKC phosphorylates IKKβ, which phosphorylates IκB, triggering its ubiquitination and subsequent degradation. This releases NF-κB from its inhibitor IκB, allowing the translocation of NF-κB to the nucleus where it activates the transcription of numerous antiapoptotic genes, including c-FLIP, Survivin, c-IAP-1, and c-IAP-2, which are inhibitors of caspases 8 and 3. Thus, completing the antiapoptotic pathway. One possibility is that pVHL directly targets aPKC for ubiquitin-mediated destruction or pVHL affects the nuclear localization and activation of NF-κB independent of pVHL-medicated degradation of aPKC.

Fig. 4.

Model of pVHL involvement in TNF-α-mediated signaling. TNF-α elicits apoptotic and antiapoptotic signals. In the apoptotic pathway, TNFR engagement of TNF-α triggers the activation of caspase-protease cascade leading to programmed cell death. In the antiapoptotic pathway, TNFR engagement of TNF-α triggers the activation of aPKC, which is in a complex with TRAF2, RIP, and P62/ZIP. Activated aPKC phosphorylates IKKβ, which phosphorylates IκB, triggering its ubiquitination and subsequent degradation. This releases NF-κB from its inhibitor IκB, allowing the translocation of NF-κB to the nucleus where it activates the transcription of numerous antiapoptotic genes, including c-FLIP, Survivin, c-IAP-1, and c-IAP-2, which are inhibitors of caspases 8 and 3. Thus, completing the antiapoptotic pathway. One possibility is that pVHL directly targets aPKC for ubiquitin-mediated destruction or pVHL affects the nuclear localization and activation of NF-κB independent of pVHL-medicated degradation of aPKC.

Close modal

We thank the members of the Ohh lab for helpful comments and discussions. We also thank Dr. Wen-Chen Yeh for providing additional anti-c-FLIP antibody and for his technical advice on EMSA protocol. We thank Dr. Martin Holcik for providing c-IAP-1 and c-IAP-2 cDNAs.

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