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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Oncogene. Author manuscript; available in PMC 2009 October 23.
Published in final edited form as:
PMCID: PMC2701369

The von Hippel-Lindau protein sensitizes renal carcinoma cells to apoptotic stimuli through stabilization of BIMEL


von Hippel-Lindau (VHL) disease is caused by germ-line mutations in the VHL tumor suppressor gene and is the most common cause of inherited renal cell carcinoma (RCC). Mutations in the VHL gene also occur in a large majority of sporadic cases of clear-cell RCC, which have high intrinsic resistance to chemotherapy and radiotherapy. Here we show that VHL-deficient RCC cells express lower levels of the pro-apoptotic Bcl-2 family protein BIMEL and are more resistant to etoposide and UV radiation induced death compared to the same cells stably expressing the wild type VHL protein (pVHL). Re-introducing pVHL into VHL-null cells increased the half-life of BIMEL protein without affecting its mRNA expression, and over-expressing pVHL inhibited BIMEL polyubiquitination. Suppressing pVHL expression with RNA interference resulted in a decrease in BIMEL protein and a corresponding decrease in the sensitivity of RCC cells to apoptotic stimuli. Directly inhibiting BIMEL expression in pVHL-expressing RCC cells caused a similar decrease in cell death. These results demonstrate that pVHL acts to promote BIMEL protein stability in RCC cells, and that destabilization of BIMEL in the absence of pVHL contributes to the increased resistance of VHL-null RCC cells to certain apoptotic stimuli.

Keywords: VHL, BIMEL, renal cell carcinoma, apoptosis, protein stability


Renal cell carcinoma (RCC) is the most common form of kidney cancer arising from the renal tubule. The vast majority of cases are clear-cell RCC, and 75% of these are associated with mutations in the von Hippel-Lindau (VHL) gene (Cohen, 1999). Germ-line mutations in the VHL gene result in VHL disease, which is characterized by the development of clear-cell RCC, hemangioblastomas and pheochromocytomas (Kaelin, 2002; Nyhan et al., 2008). RCC associated with defects in VHL are highly resistant to conventional radiation and chemotherapies (Yagoda et al., 1995), which is recapitulated in vitro by the reduced susceptibility of VHL-deficient tumor cells to apoptotic stimuli (Qi and Ohh, 2003; Kim et al., 2004; Lee et al., 2005; Roe et al., 2006).

The VHL protein (pVHL) is best known as part of the E3 ubiquitin ligase that targets hypoxia-inducible factor (HIF) for degradation by the proteasome (Maxwell et al., 1999). HIF is a heterodimeric transcription factor comprised of an oxygen-sensitive α subunit and a constitutive β subunit that regulates the expression of genes that control angiogenesis, erythropoiesis, glycolysis and cell survival (Semenza, 2003). Under normoxic conditions, hydroxylation of proline in HIFα triggers its association with pVHL resulting in its polyubiquitination and degradation by the proteasome (Ivan et al., 2001; Jaakkola et al., 2001). Hypoxia suppresses the activity of the prolyl hydroxylases allowing HIFα to escape ubiquitination by pVHL and to become active as a transcription factor (Epstein et al., 2001; Bruick and McKnight, 2001). Besides HIFα proteins, a number of other proteins bind pVHL including p53, Card9, atypical protein kinase C (aPKC), multiple subunits of RNA polymerase II, myogenin, kinesin 2, and others (Frew and Krek, 2008). For some of these proteins, interaction with pVHL leads to their ubiquitination and degradation (Okuda et al., 2001; Fu et al., 2007). In other cases, pVHL serves as an adaptor linking proteins involved in transcription, cell adhesion, microtubule organization, and cell survival (Yang et al., 2007; Hergovich et al., 2003; Roe et al., 2006; Kurban et al., 2008).

Recently, Zantl et al. (2007) suggested that the poor apoptotic response of clear-cell RCC cells might be due to reduced expression of BIMEL, a pro-apoptotic BH3-only member of the Bcl-2 family (O’Connor et al., 1998). In their study (Zantl et al., 2007), BIMEL expression was readily detected in normal renal tubules but was markedly reduced in most clear-cell RCC samples analyzed. BIMEL is one of three major alternatively spliced forms of the BIM gene that collectively are important for cell death caused by many apoptotic stimuli (Bouillet et al., 1999). Regulation of BIMEL expression involves complex transcriptional and post-translational mechanisms, including phosphorylation-dependent regulation of its protein stability (Ley et al., 2005). Very little is known about the regulation of BIMEL in RCC cells and why its expression is frequently suppressed (Zantl et al., 2007). It is also not known whether the reduced levels of BIMEL in RCC are related to the status of pVHL in these cells.

Here we examined BIMEL expression in RCC cells with and without pVHL. Cells expressing pVHL had higher levels of BIMEL protein than the same cells lacking pVHL. Inhibiting pVHL expression with RNA interference led to a parallel decrease in BIMEL. Expression of pVHL reduced BIMEL polyubiquitination and extended its protein half-life, indicative of a role for pVHL in regulating BIMEL stability. pVHL-expressing RCC cells were also more sensitive to death induced by etoposide and UV radiation, which was reversed by inhibiting either pVHL or BIMEL with RNA interference. These results suggest that loss of pVHL may enhance the resistance of RCC to apoptosis-inducing therapies in part by destabilizing BIMEL.


pVHL expression in RCC cells correlates with increased sensitivity to apoptotic stimuli

The RCC line 786-0 contains mutations in both alleles of the VHL gene resulting in the absence of pVHL expression (Gnarra et al., 1994). To determine if the status of pVHL in RCC cells influences their sensitivity to cytotoxic stimuli, we examined the effects of the chemotherapeutic drug etoposide on 786-0 derived cell lines stably expressing wild type pVHL (WT7) or empty vector (PRC2) (Iliopoulos et al., 1995). Initial experiments showed that 50–100 μM etoposide caused a 30–40% decrease in the number of viable PRC2 cells at 24 h. When PRC2 and WT7 cells were treated with etoposide (50 μM), significantly more PRC2 cells remained alive after 24 h (Figure 1A). When PRC2 and WT7 cells were exposed to a second apoptotic stimulus, UV radiation (1000 J/m2), there was a 60% decrease in the number of viable PRC2 cells after 24 h compared to an 85% decrease in WT7 cells. To determine if the greater loss of WT7 cells reflected an increase in cell death, PRC2 and WT7 cells were exposed to etoposide or UV and then stained with the membrane impermeable DNA-binding dye propidium iodide. In both cases, we observed a 2-fold increase in WT7 cells stained with propidium iodide compared to PRC2 cells (Figure 1B, C). Thus, pVHL-expressing WT7 cells are more sensitive to etoposide and UV radiation induced death than VHL-null PRC2 cells.

Figure 1
Effect of pVHL expression on the sensitivity of RCC cells to etoposide and UV radiation induced cell death

To determine if the increased death of WT7 cells is directly related to pVHL expression, we stably introduced a short hairpin RNA targeting VHL (shVHL) or a non-targeting short hairpin RNA (shNT) into WT7 cells. pVHL levels in WT7-shVHL cells were approximately 50% the levels in WT7 and WT7-shNT cells (Figure 2A). Exposing the cells to etoposide and UV radiation revealed that WT7 and WT7-shNT cells were equally sensitive to both treatments (Figure 2B, C). In contrast, WT7-shVHL cells were more resistant to etoposide and UV radiation. In the case of etoposide, the number of viable WT7-shVHL cells at 24 h was nearly the same as PRC2 cells. Thus the increased sensitivity of WT7 cells to apoptotic stimuli depends on maintaining a threshold of pVHL expression.

Figure 2
The enhanced sensitivity of WT7 cells to death-inducing stimuli can be reversed by inhibiting pVHL expression

BIMEL protein but not mRNA levels are increased in RCC cells expressing pVHL

The intrinsic resistance of certain RCC cells to apoptosis was recently associated with reduced expression of BIMEL (Zantl et al., 2007). However, it is not known whether this is related to the status of the VHL gene. To determine if the different sensitivities of PRC2 and WT7 cells to etoposide and UV radiation might reflect a difference in BIMEL expression, we examined BIMEL protein levels in these cells and in VHL-deficient 786-0 cells. As expected, pVHL was detected in WT7 cells but not in PRC2 or 786-0 cells. BIMEL protein was also readily detectable in WT7 cells, but it was at much lower levels in PRC2 and 786-0 cells (Figure 3A). In contrast, BIMEL mRNA expression was not different suggesting that increased transcription or mRNA stability was not responsible for the higher levels of BIMEL protein (Figure 3B). To determine if the increased BIMEL protein in WT7 cells is a consequence of pVHL, we compared BIMEL levels in WT7-shNT and WT7-shVHL cells. BIMEL protein was at similar levels in WT7 and WT7-shNT cells but at much lower levels in WT7-shVHL cells and PRC2 cells (Figure 3C). Therefore, suppressing pVHL expression in WT7 cells leads to a marked decrease in BIMEL protein.

Figure 3
BIMEL protein levels in RCC cells correlates with expression of pVHL

To determine if inhibiting endogenous pVHL would cause a reduction in BIMEL protein, we stably introduced shNT and shVHL into ACHN cells, a renal adenocarcinoma cell line that retains the wild type VHL allele and produces wild type pVHL protein (Iliopoulos et al., 1995). Analysis of VHL mRNA levels by RT-PCR confirmed efficient knockdown in ACHN-shVHL cells but not in ACHN-shNT cells (Figure 3D). As in WT7 cells, the knockdown of VHL did not appear to affect BIMEL mRNA expression. However, ACHN-shVHL cells expressed much lower levels of BIMEL protein compared to either ACHN-shNT or parental ACHN cells (Figure 3E). Taken together, these data reveal that the absence or partial inhibition of pVHL expression results in a reduction in BIMEL protein but not mRNA expression.

BIMEL half-life is prolonged in RCC cells expressing pVHL

BIMEL protein levels are regulated in part by mechanisms that regulate its stability (Ley et al., 2005). To investigate whether pVHL affects BIMEL stability, we treated PRC2 and WT7 cells with a protein synthesis inhibitor for varying times and then analyzed BIMEL protein levels (Figure 4A). In PRC2 cells, BIMEL protein levels decreased 50% during the first 4-8 h after cycloheximide addition and another 50% by 16 h. In contrast, BIMEL protein levels were relatively constant in WT7 cells for the first 8 h after treatment and remained at nearly 70% of control levels at 16 h (Figure 4B). Results from 4 experiments revealed a half-life for BIMEL of ~5 h in PRC2 cells compared to >16 h in WT7 cells. pVHL levels in WT7 cells decreased only slightly over 16 h of cycloheximide treatment suggesting that it is relatively stable in these cells. BIMEL protein half-life was also markedly reduced in WT7-shVHL cells compared to WT7-shNT cells, confirming that its stability is closely linked to expression of pVHL (Figure 4C). These data strongly suggest that pVHL acts as a positive regulator of BIMEL protein stability.

Figure 4
BIMEL protein stability is enhanced in RCC cells expressing pVHL

pVHL and BIMEL co-immunoprecipitate

To examine if pVHL can interact with BIMEL, we attempted co-immunoprecipitation experiments in COS-7 cells over-expressing pVHL and BIMEL. In these experiments, pVHL was detected in anti-BIM immunoprecipitates and BIMEL was detected in anti-pVHL immunoprecipitates, but only when both proteins were co-expressed (Figure 5A). We did similar co-immunoprecipitation experiments using WT7 and PRC2 cells. As observed previously (see figure 3), BIMEL protein levels were higher in WT7 cells than in PRC2 cells (Figure 5B). Furthermore, the endogenous BIMEL in WT7 cells co-immunoprecipitated with pVHL. From these experiments, we estimate that about 2-5% of the total BIMEL co-immunoprecipitated with pVHL. As expected, no BIMEL protein was detected in anti-pVHL immunoprecipitates from PRC2 cells. The specificity of the interaction was also demonstrated by the presence of pVHL in anti-Bim immunoprecipitates from WT7 but not PRC2 cells. These results, together with those described above, raise the possibility that an interaction between BIMEL and pVHL may contribute to the regulation of BIMEL stability.

Figure 5
Co-immunoprecipitation of pVHL and BIMEL

pVHL decreases BIMEL polyubiquitination and degradation

In response to serum or growth factor stimulation, BIMEL gets targeted for polyubiquitination and degradation by the proteasome (Ley et al., 2005). We wondered whether pVHL might prolong BIMEL half-life through inhibiting its polyubiquitination. Before addressing this question, we first determined whether pVHL could influence BIMEL steady-state levels in transfected COS-7 cells as was observed in RCC cells. When BIMEL was expressed at relatively low levels, co-expressing pVHL resulted in a corresponding increase in BIMEL protein (Figure 6A), suggesting that the presence of pVHL could stabilize BIMEL in this system. However, in subsequent experiments that included a proteasome inhibitor, we found we could only detect higher molecular weight polyubiquitinated forms of BIMEL if greater amounts of BIMEL plasmid were transfected (data not shown). Although co-expressing pVHL under these conditions no longer increased the net amount of BIMEL (for example, see figure 5A), we reasoned that if pVHL were to influence polyubiquitination of a subset of the BIMEL protein, it would still do so under these conditions.

Figure 6
Effects of pVHL on BIMEL polyubiquitination and its stability after serum-stimulation

To determine if pVHL can affect BIMEL polyubiquitination, COS-7 cells were co-transfected with BIMEL and HA-tagged ubiquitin, with or without pVHL. Before lysis, some of the cells were treated with the proteasome inhibitor MG132. Lysates were immunoprecipitated with anti-BIM antibody and the precipitated proteins immunoblotted with anti-HA antibody to detect ubiquitinated forms of BIMEL. Addition of the proteasome inhibitor to cells transfected with BIMEL and HA-ubiquitin resulted in the appearance of a high molecular mass smear not seen in the absence of MG132 or in cells lacking ectopic BIMEL (Figure 6B), consistent with the accumulation of polyubiquitinated BIMEL. Introducing exogenous pVHL resulted in a concentration dependent decrease in polyubiquitinated BIMEL, suggesting that pVHL may stabilize BIMEL by inhibiting its ubiquitination and subsequent degradation. While these results indicate that the polyubiquitination of BIMEL can be suppressed in transfected COS-7 cells by pVHL, it remains to be determined whether pVHL causes a similar decrease in the polyubiquitination of BIMEL in RCC cells.

We next examined if pVHL could inhibit pathways that lead to degradation of BIMEL in response to serum stimulation. In serum starved cells, BIMEL protein levels increase in part due to an increase in its stability. When the same cells are stimulated with serum, BIMEL is rapidly targeted for degradation by the proteasome (Ley et al., 2003). When PRC2 and WT7 cells were starved of serum overnight, BIMEL levels increased in both cell types (data not shown). Compared to our earlier experiments involving cells maintained in the presence of serum (see figure 3A and C), the levels of BIMEL in serum-starved WT7 cells were only slightly higher than those in PRC2 cells (Figure 6C and D, compare lanes 1 and 5). Presumably, this is because pVHL has little effect on BIMEL in the absence of signals that target BIMEL for polyubiquitination. When serum-starved PRC2 cells were stimulated with fresh serum, BIMEL protein levels decreased markedly within 15 min (Figure 6C, lanes 1 and 3). In contrast, BIMEL levels in WT7 cells remained unchanged even after 30 min of serum stimulation (Figure 6C, lanes 5 and 8), suggesting that the presence of pVHL may inhibit BIMEL degradation under these conditions. Growth factor stimulation activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt protein kinase, both of which have been shown to phosphorylate BIMEL and stimulate its degradation by the proteasome (Ley et al., 2003; Qi et al., 2006). In PRC2 cells, however, the serum-induced decrease in BIMEL appears to be mediated by the MAP kinase kinase (MEK)/ERK pathway and not the phosphatidylinositol 3-kinase (PI3K)/Akt pathway since treatment with the MEK inhibitor U0126 and not the PI3K inhibitor LY294002 prevented the decrease in BIMEL (Figure 6D). Together these results suggest that the presence of pVHL can reduce BIMEL polyubiquitination and proteasomal degradation induced by ERK1/2 activation in response to growth factor stimulation.

The increased sensitivity of RCC cells expressing pVHL to apoptotic stimuli is reversed by inhibiting BIMEL expression

The results described above raise the possibility that the different sensitivities of PRC2 and WT7 cells to apoptotic stimuli are related to their differential expression of BIMEL. To test this hypothesis, WT7 cells were transfected with 4 different siRNA duplexes targeting BIMEL or a non-targeting siRNA control. siBIM duplexes 1 and 2 reduced the protein level of BIMEL in WT7 cells by ~50%, closer to although still greater than the levels detected in PRC2 cells (Figure 7A). Control cells and cells transfected with siBIM or non-targeting siRNA were then treated with etoposide or subjected to UV radiation. As observed previously, WT7 cells were more sensitive to etoposide and UV radiation induced death than PRC2 cells. Inhibiting BIMEL expression with either siBIM-1 or siBIM-2 significantly reduced the sensitivity of WT7 cells to etoposide (Figure 7B) or UV radiation (Figure 7C). We conclude that the increased sensitivity of pVHL-expressing RCC cells to apoptotic stimuli is in part due to pVHL-mediated stabilization of BIMEL protein in these cells.

Figure 7
Reduced sensitivity of pVHL-expressing RCC cells to apoptotic stimuli after siRNA-mediated knockdown of BIMEL


The results described above establish a link between pVHL and BIMEL and the vulnerability of RCC cells to certain apoptotic stimuli. Initially, we observed that BIMEL protein levels were greater in RCC cells expressing wild type pVHL than in the same cells lacking pVHL. Inhibiting pVHL expression with RNA interference produced a corresponding decrease in BIMEL protein, while exogenous pVHL inhibited the accumulation of polyubiquitinated BIMEL in cells treated with a proteasome inhibitor. Consistent with these results, the presence of pVHL correlated with an increase in BIMEL protein half-life, which was reversed when pVHL expression was inhibited. Finally, siRNA-mediated suppression of BIMEL in RCC cells expressing pVHL reduced the death of these cells after exposure to etoposide or UV radiation.

The finding that loss of pVHL results in destabilization of the pro-apoptotic protein BIMEL provides a mechanistic explanation for a recent report that BIMEL expression is frequently suppressed in clear-cell RCC clinical samples and cell lines (Zantl et al., 2007). In their study, BIMEL expression was reduced in 35 of 43 clear-cell RCC samples and in 6 of 9 RCC cell lines. The projected incidence of reduced BIMEL expression in RCC is similar to estimates that 60–75% of sporadic clear-cell RCC harbor mutations in VHL (Cohen, 1999; van Houwelingen et al., 2005). Zantl et al. (2007) also described an inverse correlation between BIMEL levels and resistance of RCC cells to apoptosis. Our results demonstrating a role for pVHL in regulating BIMEL stability establish a potential cause and effect relationship between mutations in VHL and a reduction in BIMEL in RCC. It will be important to investigate whether reduced BIMEL expression is a more general characteristic of clear-cell RCC associated with defective VHL.

Regulation of BIMEL is complex and involves transcriptional and post-translational mechanisms. Data presented here show that pVHL can influence BIMEL protein levels without affecting its mRNA expression. While the precise mechanism is unknown, the ability of pVHL and BIMEL to co-immunoprecipitate raises the possibility that polyubiquitination of BIMEL may be influenced by its interaction with pVHL. Similar mechanisms were proposed to explain the stabilization of p53 and Jade-1 by pVHL (Roe et al., 2006; Zhou et al., 2002). Alternatively, pVHL could promote BIMEL stabilization by interfering with the function of a ubiquitin ligase that normally degrades BIMEL. Recently, an E3 ligase consisting of CIS (cytokine-inducible Src homology 2 domain-containing protein), elongins B and C, and Cul2 was shown to mediate the degradation of BIMEL in ovarian and breast cancer cells (Zhang et al., 2008). Elongins B and C and Cul2 are also part of the pVHL E3 ligase (Iwai et al., 1999; Stebbins et al., 1999). Thus, the abundance of pVHL relative to CIS may be important for determining the ubiquitin ligase activity of the latter and thus the stability of substrates such as BIMEL. Interestingly, the substrate-binding β-domain of pVHL and not its elongin C-binding α-domain co-immunoprecipitates with BIMEL (data not shown), raising the possibility that complexes containing pVHL and BIMEL could also contain elongin C.

In response to a variety of apoptotic stimuli, BIMEL and related BH3-only proteins act by sequestering anti-apoptotic Bcl-2 proteins at the mitochondria, thus enabling the pro-apoptotic proteins Bax and Bak to form complexes that promote release of cytochrome c into the cytoplasm and activation of the apoptosome (Willis et al., 2007). In the absence of death inducing stimuli, survival-promoting growth factors and cytokines stimulate phosphorylation of serine-65 in BIMEL by ERK1/2 leading to its polyubiquitination and degradation (Ewings et al., 2007). Our results raise the possibility that pVHL may act by inhibiting ERK-dependent phosphorylation of BIMEL or the interaction between ERK-phosphorylated BIMEL and its ubiquitin ligase. While these hypotheses remain to be tested, co-immunoprecipitation experiments with truncated forms of BIMEL reveal that pVHL can interact with residues 41-97 of BIMEL, which include the critical ERK phosphorylation site at serine-65 (data not shown).

The ability of pVHL to promote BIMEL stabilization is not the only means by which pVHL sensitizes cells to apoptosis. pVHL was recently shown to promote the stabilization and transcription factor activity of p53 (Roe et al., 2006), which correlated with the increased susceptibility of pVHL-expressing tumor cells to certain apoptotic stimuli. In another study, over-expression of pVHL in 786-0 cells with a recombinant adenovirus caused cell death which was associated with an increase in Bax expression (Kim et al., 2004). In addition to enhancing death pathways, pVHL may also suppress cell survival pathways including those mediated by NF-κB (Qi and Ohh, 2003; Yang et al., 2007), atypical PKC (Okuda et al., 2001), JunB (Lee et al., 2005) and HIF (Semenza, 2003). Despite these and other reports that pVHL can sensitize RCC and other tumor cells to apoptosis, expression of pVHL in RCC cells can also decrease sensitivity to cell death under certain conditions (Devarajan et al., 2001; Schoenfeld et al., 2000; Gorospe et al., 1999). Given the multiple functions of pVHL in the regulation of protein stability, microtubule stability, extracellular matrix signaling, and transcriptional regulation (Frew and Krek, 2008), it is perhaps not surprising that pVHL influences cell survival in diverse ways and through multiple mechanisms.

In summary, we have identified a mechanism by which loss of pVHL contributes to the desensitization of RCC cells to apoptotic stimuli that involves destabilization of the pro-apoptotic protein BIMEL. The high incidence of mutations in VHL in inherited and sporadic clear-cell RCC, together with the recent finding that BIMEL expression is suppressed in a similarly high percentage of clear-cell RCC, suggest that pVHL-dependent regulation of BIMEL stability may be an important factor in the development of many kidney tumors.

Materials and methods

Materials and plasmids

Cell culture reagents were from Invitrogen (Carlsbad, CA). Etoposide, propidium iodide, and cycloheximide were from Sigma-Aldrich (St. Louis, MO). MG132 and LY294002 were from Biomol Research Laboratories (Plymouth Meeting, PA) and U0126 was from Promega (Madison, WI).

Plasmids expressing shRNA targeting human VHL and a non-targeting (scrambled sequence) control shRNA were obtained from Thomas Benzing (University of Cologne, Koeln, Germany) and are described in Table 1 and Figure S3a of Schermer et al. (2006). The plasmid expressing HA-ubiquitin was a gift from Patricia Hinkle (University of Rochester, Rochester, NY). The full-length BIMEL coding region was amplified from rat cDNA using Pfu DNA polymerase (Stratagene, La Jolla, CA). The complete VHL open reading frame was amplified from human smooth muscle cell cDNA. BIMEL and VHL cDNAs were subcloned into pcDNA3 (Invitrogen) and validated by automated DNA sequencing.

Cell culture

RCC 786-0, PRC2 and WT7 cells were obtained from William Kaelin (Dana Farber Cancer Institute, Boston, MA) and are described elsewhere (Lonergan et al., 1998). ACHN cells (CRL-1611) were from American Type Culture Collection (Rockville, MD). All cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The media for PRC2 and WT7 cells was supplemented with 10 μg/ml G418.

For irradiation, cells plated on 35 mm culture dishes in complete medium (>90% confluency) were exposed to UV (254 nm) at a dose of 1000 J/m2. After treatment, cells were returned to the incubator for 24 h before assessing viability. Transient transfections were done with LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. For experiments involving transfection of BIMEL plasmid, the caspase inhibitor boc-aspartyl(OMe)-fluoromethylketone (100 μM) (MP Biomedicals, Solon, OH) was added to the media in all wells to minimize cell death. For serum stimulation, fresh media containing 10% fetal bovine serum was added to cells previously cultured in the absence of serum for 12-14 h.

Viability assays

Equal numbers of cells were seeded into 35 mm dishes 24 h before initiating treatment. Twenty-four hours after addition of etoposide or irradiation with UV, cells were rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde. Cell nuclei were stained with Hoechst 33 342 (1 μg/ml in PBS, Invitrogen) and visualized under phase-contrast and fluorescence microscopy. For each treatment, three random fields of view encompassing a minimum of 200 cells were scored based on nuclear morphology as healthy (round nucleus containing diffusely stained chromatin) or dead/dying (pyknotic or misshapen nucleus with condensed chromatin). The number of healthy cells for a given treatment was normalized to the number of healthy cells in untreated controls. In some experiments, cells were stained with propidium iodide (1.5 μM in PBS) and then visualized under fluorescence microscopy. Cells that had lost plasma membrane integrity were able to take up propidium iodide and were presumed to be dead or dying. For a given treatment, cells in three fields of view were analyzed with the fraction of dead cells defined as the number of propidium iodide stained cells divided by the total number of cells. All viability experiments were repeated at least three times.

RNA interference

WT7 and ACHN cells were transfected with plasmids expressing shRNA targeting human VHL or a non-targeting scrambled control shRNA (plasmids described above). Stable transfectants were isolated by including 10 μg/ml blasticidin in the culture media. Drug-resistant colonies were pooled and expression of pVHL was assessed by immunoblotting.

siRNA oligonucleotides targeting human BIM and a non-targeting control siRNA were obtained from Thermo Fisher Scientific/Dharmacon (Waltham, MA). siRNAs (100nM) were transfected into WT7 cells with DharmaFECT according to the manufacturer’s instructions. Four different siRNAs targeting BIMEL were tested. siBIM 1 and 2 were most effective at decreasing BIMEL protein expression and thus were used in subsequent experiments.

Immunoblotting and immunoprecipitation

Cells were harvested in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA and a cocktail of protease inhibitors (Sigma-Aldrich). Cell lysates were clarified by centrifugation for 5 min and the protein concentration of the supernatants was determined using a modified Bradford assay (Bio-Rad, Hercules, CA). For immunoblotting, 25 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose, and incubated as described previously (Xie et al., 2005) with the following primary antibodies and dilutions: anti-BIM, 1:1000 (cat. #AAP-330, Assay Designs, Ann Arbor, MI); anti-VHL monoclonal, 1:500 (cat. #556347, BD Biosciences, San Jose, CA); anti-VHL polyclonal, 1:1000 (cat. #2738, Cell Signaling, Danvers, MA); anti-actin, 1:1000 (cat. #A2066, Sigma-Aldrich); anti-HA, 1:1000 (cat. #1583816, Roche Diagnostics, Indianapolis, IN); anti-FLAG M2, 1:1000 (cat. #F3165, Sigma-Aldrich); anti-α-tubulin, 1:1000 (cat. #T5168, Sigma-Aldrich). Goat anti-mouse and anti-rabbit secondary antibodies conjugated to horseradish peroxidase were used at a 1:10,000 dilution and immunoreactive bands were detected by chemiluminescence (SuperSignal, Thermo Fisher Scientific/Pierce, Rockford, IL). Densitometry was performed with NIH ImageJ software.

For immunoprecipitation experiments, 500 μg of clarified cell lysate were pre-absorbed with 50 μl of protein A Sepharose (GE Healthcare, Piscataway, NJ) for 1 h at 4°C. Lysates were then incubated overnight at 4°C with 1 μg of polyclonal anti-BIM antibody or monoclonal anti-pVHL antibody. Immune complexes were precipitated with 50 μl of Protein A Sepharose for 1 h and then washed three times with lysis buffer. Immunoprecipitated proteins were eluted in Laemmli sample buffer, separated by SDS-PAGE, and immunoblotted with the indicated antibodies as outlined above.

Reverse transcription—polymerase chain reaction (RT-PCR)

Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse transcription reactions and PCR were performed as described previously (Lipscomb et al., 1999). PCR amplifications were done over a range of cycle numbers to ensure that the analyses were carried out under non-saturating conditions. PCR products were separated by electrophoresis on 1.0% agarose gels and analyzed after staining with ethidium bromide.

Statistical analysis

Quantitative data are reported as the mean ± standard error of the mean (SEM) from at least three independent experiments. The means for various treatment groups were compared using analysis of variance (ANOVA) and Dunnett’s post-hoc test.


We thank Dr. William Kaelin for providing cell lines and Drs. Thomas Benzing and Patricia Hinkle for providing plasmids used in this study. This work was supported by grants NS034400 and NS058868 from the National Institutes of Health.


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