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Molecular Cancer Research 2:574-584 (2004)
© 2004 American Association for Cancer Research

Signaling and Regulation

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Mediated Activation of Mitochondria-Associated Nuclear Factor-{kappa}B in Prostatic Carcinoma Cell Lines1

Natalya V. Guseva, Agshin F. Taghiyev, Mary T. Sturm, Oskar W. Rokhlin and Michael B. Cohen

Department of Pathology, University of Iowa, Iowa City, Iowa

Requests for reprints: Michael B. Cohen, Department of Pathology, University of Iowa, 200 Hawkins Drive, C670, Iowa City, IA 52242-1087. Phone: 319-384-9609; Fax: 319-384-9613. E-mail: michael-cohen{at}uiowa.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
It has been suggested that some nuclear transcription factors may participate in the regulation of mitochondrial functions through transcriptional control of mitochondrial DNA. Very little is known about the response of transcription factors within mitochondria to the activation of death receptors. Recent publications indicate that nuclear factor-{kappa}B (NF-{kappa}B) is localized in mitochondria of mammalian cells. Because of the critical role of mitochondria in the execution of many apoptotic pathways, we suggest that NF-{kappa}B-dependent mechanisms operating at the level of mitochondria contribute to its role in regulating death receptor signaling. We have found NF-{kappa}B p65 and p50 subunits with DNA binding activity in the mitochondria of prostatic carcinoma cell lines. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) affects DNA binding activity of mitochondria-associated NF-{kappa}B but does not change the amount of p65 in mitochondria, which suggests activation of mitochondrial NF-{kappa}B without additional translocation of NF-{kappa}B subunits to mitochondria. We have also shown that TRAIL decreases mitochondrial genome encoded mRNA levels and inhibition of NF-{kappa}B prevents this decrease. TRAIL effects on mitochondrial NF-{kappa}B-DNA binding and mitochondrial genome encoded mRNA levels also depend on Bcl-2 overexpression. In addition, transcription factor activator protein-1 with DNA binding activity is also found in mitochondria of prostatic carcinoma cells and TRAIL treatment affects this binding. In summary, NF-{kappa}B is found in mitochondria of prostatic carcinoma cells, where it is thought to regulate mitochondria genome encoded mRNA levels in response to TRAIL treatment.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Recent studies suggest that some nuclear transcription factors may participate in the regulation of mitochondrial functions through transcriptional control of mitochondrial DNA (1). Very little is known, however, about the response of transcription factors within mitochondria to the activation of specific death receptors located on the cell surface.

The transcription factor nuclear factor-{kappa}B (NF-{kappa}B) is associated with oncogenesis and apoptosis (2) and has been extensively studied in the last several years. In unstimulated cells, NF-{kappa}B is sequestered in the cytoplasm by inhibitory molecules of the I{kappa}B family. Most agents that activate NF-{kappa}B employ a common pathway based on the phosphorylation of the two NH2-terminal serines in I{kappa}B, resulting in subsequent ubiquitination and degradation of these proteins by the 26S proteasome (3). The free NF-{kappa}B dimers translocate to the nucleus and regulate gene transcription by binding to target sequences in the regulatory regions of NF-{kappa}B responsive genes. However, more complex aspects of NF-{kappa}B regulation have also been reported. NF-{kappa}B shuttles into and out of the nucleus in unstimulated cells (4). Additionally, I{kappa}B can be degraded by a nonproteasomal mechanism following stimulation of cells with cytokines (5). Recently, a new function was reported for I{kappa}B{alpha} that can increase NF-{kappa}B-independent transcription through binding to histone deacetylases (6). Of particular interest are recent reports from two groups that NF-{kappa}B subunits were found in mitochondria (7, 8).

The activator protein-1 (AP-1) transcription factor is a dimeric complex that contains members of the Jun, Fos, ATF, and Maf protein families. AP-1 is activated by several different agents. Activation occurs both transcriptionally and post-translationally, and signaling occurs predominantly through the mitogen-activated protein kinase cascade (9). AP-1 regulates the expression of multiple genes essential for cell proliferation, differentiation, and apoptosis. Constitutive activation of endogenous AP-1 occurs in human tumors, suggesting that AP-1 plays an important role in human oncogenesis and might be a good target for anticancer therapy (10, 11). AP-1 has also been found in mitochondria (12, 13).

Mitochondria play a central role in cellular survival and apoptotic death (14-16). Distinct stress signals, such as irradiation, chemical substances, and changes in cell homeostasis, engage the mitochondria, resulting in the release of apoptogenic factors such as cytochrome c, Smac/DIABLO, and HtrA2/Omi into the cytosol. The ability of the mitochondrial pathway to induce apoptosis depends on involvement of the Bcl-2 family of proteins (17) that can either suppress or promote changes in mitochondrial membrane permeability required for release of cytochrome c and other apoptogenic proteins. Although most mitochondrial proteins are encoded by nuclear DNA, some are encoded by mitochondrial DNA and synthesized by a separate mitochondrial translation system. The human mitochondrial genome is circular with 16.8 kb of DNA. The mitochondrial genes encode 2 rRNAs, 22 tRNAs, and at least 13 proteins, which contribute to complex I, III, IV, and V of the electron transport system. A regulatory region associated with the origin of replication also serves as a promoter region for two large mitochondrial RNA transcripts that are processed into individual RNAs for the structural RNAs and mRNAs (18).

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family. TRAIL induces apoptosis in many transformed cells and could play an important role in cancer therapy. TRAIL can bind to four different receptors. Two of them, TRAIL-R1 and TRAIL-R2, contain cytoplasmic death domains, and ligation of these receptors triggers a series of protein-protein interactions that lead to the assembly of a death-inducing signaling complex at the cytoplasmic death domain. These receptors can transmit two distinct signaling cascades, leading either to activation of caspase-8 (or caspase-10) at the death-inducing signaling complex and apoptosis or to activation of NF-{kappa}B through adaptor proteins of death-inducing signaling complex (19-21). Expression of TRAIL receptors was shown on prostatic carcinoma cell lines PC3, DU145, and LNCaP. However, only PC3 is sensitive to TRAIL, and mitochondria play a critical role in TRAIL-induced apoptosis (22).

Here, we report that NF-{kappa}B complexes with DNA binding activity are found in the mitochondria of prostatic carcinoma cell lines. TRAIL affects DNA binding activity of mitochondria-associated NF-{kappa}B but does not increase the amount of p65 in mitochondria, suggesting activation of mitochondrial NF-{kappa}B without additional translocation of NF-{kappa}B subunits to mitochondria. We have also shown that TRAIL decreases mitochondrial genome encoded mRNA levels, and inhibition of NF-{kappa}B prevents this decrease. TRAIL effects on mitochondrial NF-{kappa}B-DNA binding and mitochondrial genome encoded mRNA levels are cell specific and also depend on Bcl-2. In addition, the transcription factor AP-1 with DNA binding activity is also found in mitochondria of prostatic carcinoma cells and TRAIL treatment affects this binding.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Effect of TRAIL on NF-{kappa}B Activity in Prostatic Carcinoma Cell Lines
PC3 and DU145 (but not LNCaP) display constitutive NF-{kappa}B activity (23). However, constitutive activation of NF-{kappa}B is not correlated with sensitivity of cells to TRAIL. PC3 (but not DU145 or LNCaP) are highly sensitive to TRAIL-induced apoptosis (22, 24).

To investigate the influence of TRAIL on NF-{kappa}B activation, cells were transfected with a luciferase reporter plasmid containing NF-{kappa}B binding sites and later treated with TRAIL. As can be seen from Fig. 1A, TRAIL inhibited NF-{kappa}B activation in PC3 but did not affect activity in DU145. Treatment of LNCaP with TRAIL induced NF-{kappa}B activation but to a very low extent when compared with PC3 or DU145. Electrophoretic mobility shift assay (EMSA) showed similar results for nuclear extracts of PC3, DU145, and LNCaP (Fig. 1B). Thus, TRAIL inhibits constitutive NF-{kappa}B activation in PC3, but it is not clear if this inhibition causes cell death or is a result of cell death. It also remains unclear whether TRAIL-mediated NF-{kappa}B activation in LNCaP plays a role in resistance to TRAIL treatment.



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  		FIGURE 1. TRAIL influence on NF-{kappa}B activation in prostatic carcinoma cell lines. A. Cells were transfected with pGL2-Luc-NF-{kappa}B reporter together with ß-galactosidase vector. After 36 hours of transfection, cells were treated with TRAIL (200 ng/mL) for 8 hours, and cell lysates were prepared in assay buffer and examined for luciferase and ß-galactosidase activity. Columns, mean luciferase activities normalized for ß-galactosidase expressions relative to untreated cells, which is taken as 100 for PC3 and DU145 or 1 for LNCaP; three replicates in one of three separate experiments with similar results. B. Cells were treated with TRAIL (200 ng/mL) for 8 hours and nuclear extracts were obtained as described in Materials and Methods. Proteins (5 µg) from mitochondrial fraction were incubated in binding buffer with 32P-end-labeled NF-{kappa}B probe and resulting protein-DNA binding was analyzed by EMSA. To verify specificity of bands, some extracts were preincubated for 15 minutes with p65 antibodies for supershift analysis. Arrows, specific NF-{kappa}B-DNA complexes.

 
Localization of NF-{kappa}B With DNA Binding Activity in Mitochondria
Because mitochondria play a critical role in TRAIL-mediated apoptosis in PC3 (22) and strong evidence of mitochondrial NF-{kappa}B localization in some cell types was reported recently (6, 7), we examined prostatic carcinoma cell lines for the presence of the major NF-{kappa}B subunits in mitochondria. As shown in Fig. 2A, Western blot analyses with specific antibodies to p65 and p50 showed the presence of these subunits in the mitochondrial fraction of all three cell lines. No differences in the amount of proteins were detected in the cell lines in either mitochondrial or cytosolic fractions. Cytochrome c was detected only in mitochondria and caspase-8 was detected only in the cytosol fraction. Cross-contamination with cytosol was also assessed using a specific enzyme marker (lactate dehydrogenase). Its activity in a mitochondrial fraction was 2.5% (data not shown). These results suggest mitochondrial localization of NF-{kappa}B. To test this possibility, we used two approaches. We assessed the sensitivity of p65 in the mitochondrial fraction to proteinase K compared with whole cell lysate. As can be seen from Fig. 2B, p65 in mitochondria remained unaffected, whereas p65 in whole cell lysate was degraded by proteinase K in a concentration-dependent manner. Additionally, mitochondria were treated with increasing concentrations of digitonin in Mito buffer, and after centrifugation, pellets and supernatants were subjected to a Western blot analysis. As shown in Fig. 2C, 0.4% digitonin induced a disappearance of p65 from mitochondria, concomitant to their appearance in the supernatant. Therefore, NF-{kappa}B seems to be inside the mitochondria.



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  		FIGURE 2. A. Localization of NF-{kappa}B in mitochondria of prostatic carcinoma cells. Mitochondria and cytosol from PC3, DU145, and LNCaP cells were obtained by differential centrifugation as described in Materials and Methods. Mitochondrial (7 µg) or cytosolic proteins (15 µg) were separated on 10% SDS-PAGE, blotted to nitrocellulose membrane, blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20, and incubated with mouse anti-p65, anti-cytochrome c, anti-caspase-8, or rabbit anti-p50 antibodies. The blots were counterstained with anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase. The immunoreactive bands were visualized by incubation of the membrane with an enhanced chemiluminescence reagent. B. Mitochondrial p65 NF-{kappa}B are resistant to proteolysis by proteinase K. Mitochondria or whole cell lysates of PC3 cells were incubated with the indicated doses of proteinase K for 10 minutes at 4°C. The p65 was visualized by Western blotting with specific antibodies. C. Release of p65 NF-{kappa}B from mitochondria after digitonin treatment. Mitochondria were isolated from LNCaP cells and incubated with 0.2% or 0.4% digitonin in Mito buffer. After centrifugation, supernatant (Sn) contained released proteins.

 
To determine whether the NF-{kappa}B components in mitochondria were able to bind DNA, mitochondrial extracts from PC3, DU145, and LNCaP were analyzed by EMSA. DNA-bound complexes in the extracts from mitochondria of PC3 and DU145 were detected (Fig. 3A). Preincubation of cell lysates with excess mutant probe (mutant competitor) did not affect protein-DNA binding, whereas excess unlabeled specific probe (wild competitor) completely removed DNA-bound bands. This confirms specificity of mitochondrial NF-{kappa}B-DNA binding. Mitochondrial extracts from LNCaP did not reveal NF-{kappa}B activity (data not shown). Because mitochondrial activation of NF-{kappa}B in PC3 and DU145 could be caused by nuclear contamination, we examined the purity of mitochondrial fractions by assessment of the nuclear protein poly(ADP-ribose) polymerase. As shown in Fig. 3B, poly(ADP-ribose) polymerase was detected only in total and nuclear lysates but not in the mitochondrial fraction. To exclude possible contamination of the mitochondrial fraction by cytosolic proteins or other cellular compartments such as the Golgi apparatus or endoplasmic reticulum, mitochondria were further enriched and purified on a Percoll/metrizamide gradient. Mitochondria from PC3 were prepared by subcellular fractionation and gradient purification. The band intensity of NF-{kappa}B-DNA binding was 15% higher after gradient purification (data not shown), confirming mitochondrial localization of NF-{kappa}B. In subsequent experiments, we used subcellular fractionation because a larger number of cells are required for gradient purification of mitochondria.



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  		FIGURE 3. A. Detection of DNA binding activity of NF-{kappa}B complexes from mitochondrial fraction. The DNA binding capacities of mitochondrial NF-{kappa}B were examined by EMSA. Mitochondria were isolated by differential centrifugation and proteins (5 µg) were incubated with 32P-end-labeled double-stranded oligonucleotide probe containing a NF-{kappa}B binding site. To ensure binding specificity, mitochondrial extracts were preincubated with an excess of cold (wt. comp.) or mutant (mut. comp.) NF-{kappa}B probe. NS, nonspecific bands. B. Assessment of the mitochondrial fraction purity. Total cell lysate, nuclei, and mitochondria after cell differential centrifugation were prepared from PC3 cells. Cell lysates or nuclear extracts (20 µg) and mitochondrial proteins (10 µg) were investigated in Western blot analysis with anti–poly(ADP-ribose) (PARP) polymerase antibodies.

 
Taken together, our data indicate that p65 and p50 localized in mitochondria of prostatic carcinoma cells and display DNA binding activity.

TRAIL Affects Mitochondrial NF-{kappa}B-DNA Binding and Mitochondrial Genome Encoded mRNA Levels
To determine whether TRAIL affects mitochondrial NF-{kappa}B-DNA binding, cells were treated with TRAIL for 2 hours and mitochondrial extracts were prepared and analyzed by EMSA. Results of these studies are shown in Fig. 4A. TRAIL treatment did not change the electromobility of mitochondrial NF-{kappa}B-DNA bands but increased the intensity of binding in PC3, decreased it in DU145, and induced it in LNCaP. To identify which components of NF-{kappa}B contributed to this binding, supershift analyses were done. The incubation of mitochondrial extracts from TRAIL-treated cells with anti-p65 and anti-p50 antibodies resulted in supershifted bands. These observations lead to the conclusion that mitochondrial NF-{kappa}B-DNA complexes after TRAIL treatment in all three cell lines consisting of p65 and p50 subunits. To compare TRAIL effects on mitochondrial and nuclear NF-{kappa}B-DNA binding, we also prepared EMSA with nuclear extracts from these cells. In contrast to mitochondria, TRAIL decreased NF-{kappa}B-DNA binding in nuclear extracts from PC3 after 2 hours and increased it in DU145. However, TRAIL also induced NF-{kappa}B-DNA binding in a nuclear fraction of LNCaP. The main complexes of NF-{kappa}B in the nuclear fraction are p65/p50. From these results, we suggest that TRAIL affects mitochondrial and nuclear NF-{kappa}B-DNA binding differently.



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  		FIGURE 4. Influence of TRAIL on mitochondrial and nuclear NF-{kappa}B-DNA binding. Cells were treated with TRAIL (200 ng/mL) for 2 hours and mitochondrial or nuclear extracts were obtained. Mitochondrial or nuclear proteins (5 µg) were incubated with 32P-end-labeled NF-{kappa}B probe for 30 minutes at room temperature and separated on a native 5% PAGE. Extracts were preincubated for 15 minutes with p65 and p50 antibodies for supershift analysis. X-ray films were exposed to the dried gels at –70°C for 14 hours for nuclear extracts and 24 hours for mitochondrial extracts. Arrows, specific DNA-protein complexes.

 
Because TRAIL treatment changed intensity of mitochondrial NF-{kappa}B-DNA binding, it was also of interest to examine if TRAIL affected mitochondrial genome encoded mRNA levels. To answer this question, we treated cells with TRAIL for 2, 4, and 6 hours and determined the amount of mitochondrial genome encoded cytochrome c oxidase (Cox) III mRNA by semiquantitative reverse transcription-PCR (RT-PCR). As shown in Fig. 5, we found a decrease of Cox III PCR fragments in PC3 and LNCaP cells after TRAIL treatment. However, TRAIL did not induce statistically significant changes of Cox III mRNA level in DU145 at any investigated time point.



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  		FIGURE 5. TRAIL decreases the level of Cox III mRNA level in PC3 and LNCaP but not DU145 cells. Semiquantitative RT-PCR was done for the fragments of the mitochondria-encoded Cox III gene using total RNAs prepared from prostatic carcinoma cells, either untreated or treated with TRAIL (200 ng/mL) for the indicated times. Intensity of ethidium bromide–stained Cox III RT-PCR fragments was normalized to intensity of GAPDH RT-PCR fragments. Densitometric analysis represents the percentage of RT-PCR fragment intensity of treated cells relative to untreated cells, which were taken as 100%. Columns, mean of three separate experiments; bars, SD.

 
TRAIL-Mediated Decrease of Mitochondrial Genome Encoded mRNA Levels Depends on NF-{kappa}B Activation
To further investigate the effect of NF-{kappa}B inhibition on TRAIL-mediated decrease of mitochondrial genome encoded mRNA levels, LNCaP was stably transfected with the pFI{kappa}B{alpha}AA expression vector or an empty vector containing a neomycin resistance gene (LNCaP-Neo). A mutant form of I{kappa}B{alpha} with mutations at Ser32 and Ser36 has been shown previously to effectively prevent I{kappa}B{alpha} phosphorylation and degradation (25). Western blot analysis confirmed the expression of mutant Flag-tagged I{kappa}B{alpha}AA (FI{kappa}B{alpha}AA) in the LNCaP-FI{kappa}B{alpha}AA (Fig. 6A). We probed the nuclear and mitochondrial extracts of LNCaP-FI{kappa}B{alpha}AA and LNCaP-Neo for NF-{kappa}B activity by EMSA to determine whether FI{kappa}B{alpha}AA could block mitochondrial NF-{kappa}B activation. As shown in Fig. 6B, the expression of FI{kappa}B{alpha}AA prevented TRAIL-induced activation of NF-{kappa}B in both nuclear and mitochondrial fractions.



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  		FIGURE 6. Inhibition of NF-{kappa}B activation by mutant FI{kappa}B{alpha}AA prevents TRAIL-induced decrease of mitochondrial gene expression. A. Expression of mutant FI{kappa}B{alpha}AA was investigated by Western blot analysis with anti-rabbit I{kappa}B{alpha} antibodies. FI{kappa}B{alpha}AA has a higher molecular weight and separated from endogenous I{kappa}B{alpha} in the gel. B. Expression of mutant FI{kappa}B{alpha}AA prevents NF-{kappa}B activation in both nuclei and mitochondria. LNCaP-FI{kappa}B{alpha}AA and control LNCaP-Neo cells were treated with TRAIL (200 ng/mL) for 2 hours. Nuclear proteins (5 µg) were incubated with 32P-end-labeled NF-{kappa}B probe for 30 minutes at room temperature and separated on a native 5% PAGE. Arrows, specific DNA-protein complexes. C. Total RNAs were prepared from LNCaP cells transfected with either control vector or FI{kappa}B{alpha}AA-containing vector after treatment with TRAIL (200 ng/mL) for different times. All RT-PCRs were done in the linear range for the fragment of the indicated gene compared with GAPDH as a reference control. Each experiment was done three times with the same results. D. Densities of Cox II, Cox III, and Cyt b PCR products (C) were quantified, normalized to GADPH, and expressed as a percentage of the density of untreated cells. E. FI{kappa}B{alpha}AA overexpression sensitized LNCaP cells to TRAIL treatment. Cells were treated with different concentrations of TRAIL for 48 hours and calcein assay was done as described in Materials and Methods.

 
To investigate the mitochondrial genome encoded mRNA level, LNCaP-Neo and LNCaP-FI{kappa}B{alpha}AA cells were treated for 2, 4, and 6 hours with TRAIL, and total RNA was isolated, reverse transcribed, and amplified with primers for three different mitochondrial DNA encoded genes: Cox III, Cox II, and cytochrome b (Cyt b). As can be seen from Fig. 6C and D, the level of PCR products for all three genes was about half in LNCaP-Neo after 2 hours of treatment with TRAIL compared with untreated cells and four to six times lower after 4 and 6 hours of treatment. The level of Cox II, Cox III, and Cyt b PCR products remained at the higher level in LNCaP-FI{kappa}B{alpha}AA at any investigated time point. As shown in Fig. 6F, inhibition of NF-{kappa}B sensitized LNCaP cells to TRAIL. After 48 hours of treatment of LNCaP-FI{kappa}B{alpha}AA cells with high concentration of TRAIL, ~45% of the cells were dead. Thus, inhibition of NF-{kappa}B prevents TRAIL-mediated decrease of mitochondrial genome encoded mRNA levels in LNCaP and indicates a role for NF-{kappa}B in this process.

Regulation of NF-{kappa}B Activation by Bcl-2 Overexpression
Because Bcl-2 is a very important protein in apoptosis-mediated mitochondrial events and Bcl-2 overexpression prevents TRAIL-induced cell death in PC3 (22), it was interesting to investigate the effect of Bcl-2 on NF-{kappa}B activation. To compare the effect of Bcl-2 overexpression on NF-{kappa}B activation in different cell lines, we also obtained LNCaP cells with stable overexpression of Bcl-2 (Fig. 7A). As shown in Fig. 7B, we found that constitutive activation of NF-{kappa}B was lower in PC3-Bcl-2 cells compared with control. However, TRAIL treatment did not result in a statistically significant inhibition of NF-{kappa}B activity in PC3-Bcl-2 cells. Interestingly, Bcl-2 overexpression in LNCaP did not affect basal level of luciferase activity but inhibited TRAIL-induced NF-{kappa}B activation. Thus, Bcl-2 overexpression affects TRAIL-mediated changes in NF-{kappa}B activation in prostatic carcinoma cell lines.



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  		FIGURE 7. Effects of Bcl-2 on NF-{kappa}B activation in prostatic carcinoma cell lines. A. Stable overexpression of Bcl-2 in LNCaP cells. Triton lysates were prepared from LNCaP-Hygro and LNCaP-Bcl-2 and cell proteins (20 µg) were analyzed by Western blot analysis. B. Cells were transfected with pGL2-Luc-NF-{kappa}B reporter together with ß-galactosidase vector. After 36 hours of transfection, cells were treated with TRAIL (200 ng/mL) for 8 hours and cells lysates were prepared in assay buffer and examined for luciferase and ß-galactosidase activity. Columns, mean luciferase activities normalized for ß-galactosidase expressions relative to untreated cells, which is taken as 100 for PC3 and 1 for LNCaP; three replicates in one of three separate experiments, which gave similar results.

 
Bcl-2 Overexpression Prevents TRAIL-Mediated Increase of Mitochondrial NF-{kappa}B Activation and Reduction of Mitochondrial Genome Encoded mRNA
Because Bcl-2 prevents TRAIL-mediated inhibition of NF-{kappa}B in PC3, it was interesting to investigate whether Bcl-2 affects mitochondrial NF-{kappa}B-DNA binding in these cells. As shown in Fig. 8A, TRAIL treatment increased NF-{kappa}B-DNA binding in mitochondria of PC3-Hygro that started after 2 hours of treatment and was strongest at 8 hours. TRAIL treatment increased the intensity of the DNA-bound band in PC3-Bcl-2 after 1 hour of treatment, but this intensity remained at the same level after 2, 4, and 8 hours of treatment; it was lower compared with PC3-Hygro at all time points. Thus, Bcl-2 overexpression prevents TRAIL-mediated increase of mitochondrial NF-{kappa}B-DNA binding.



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  		FIGURE 8. Bcl-2 overexpression prevents TRAIL-mediated activation of mitochondrial NF-{kappa}B and decrease of mitochondrial gene expression. A. Influence of Bcl-2 on NF-{kappa}B-DNA binding in mitochondria of PC3 cells. PC3-Hygro and PC3-Bcl-2 cells were treated with TRAIL for the indicated times. Mitochondrial proteins (5 µg) were incubated with 32P-end-labeled NF-{kappa}B probe in binding buffer for 30 minutes and EMSAs were done. B. Cells were treated with TRAIL (200 ng/mL) for 4 and 8 hours and p65 expression was investigated by Western blot analysis. Number below each band, relative intensity of band compared with untreated cytosol or mitochondria PC3-Hygro, which is taken as 100 for each group. C. Bcl-2 prevents decrease of mitochondrial gene transcription. PC3-Hygro and PC3-Bcl-2 cells were treated with TRAIL (200 ng/mL) for different times and total RNAs were prepared. All RT-PCRs were done for the fragment of the indicated gene compared with GAPDH as a reference control. Each experiment was done three times with the same results. D. Densities of Cox II, Cox III, and Cyt b PCR-products (C) were quantified and expressed as a percentage of the density of untreated cells.

 
The mechanism of induction of mitochondrial NF-{kappa}B-DNA binding activity by TRAIL and the role of Bcl-2 in this process is not clear. Apparently, TRAIL treatment induces translocation of cytosolic NF-{kappa}B into mitochondria or activates a mitochondrial pool of NF-{kappa}B without translocation. To investigate this question, Western blot analysis for p65 was carried out. As shown in Fig. 8B, TRAIL decreased the level of p65 in mitochondria of either PC3-Hygro or PC3-Bcl-2 cells after 8 hours by 32% and 25%, respectively. This result leads to the conclusion that TRAIL does not induce translocation of p65 in the mitochondria but stimulates activation of mitochondrial pool of NF-{kappa}B in PC3 cells, which is prevented by Bcl-2 overexpression.

To investigate how Bcl-2 overexpression affects mitochondrial genome encoded mRNA levels in PC3, we prepared cDNA from PC3-Hygro and PC3-Bcl-2 after TRAIL treatment and amplified fragments of Cox II, Cox III, and Cyt b. A notable decrease of mitochondria-encoded gene PCR products was observed in PC3-Hygro after 4 and 6 hours of TRAIL treatment (Fig. 8C and D) and this reduction was partially prevented in PC3-Bcl-2. Therefore, Bcl-2 overexpression prevents TRAIL-induced decrease of mitochondrial genome encoded mRNA levels in PC3.

TRAIL Treatment Increased AP-1-DNA Binding Activity in Mitochondria
Mitochondrial localization of AP-1, another important apoptosis-regulating nuclear transcription factor, was reported recently (12, 13). We found AP-1-DNA binding in extracts from mitochondria of untreated PC3, DU145, and LNCaP. To investigate the influence of TRAIL on mitochondrial AP-1-DNA binding, we did EMSA analysis. The dynamic of AP-1 activation in mitochondria of PC3 (Fig. 9) was almost the same as the dynamic of mitochondrial NF-{kappa}B activation (Fig. 8A). Maximum AP-1-DNA binding was observed after 4 hours of treatment and remained at the same high level at 8 hours. However, this increase of AP-1 activation was not found in PC3 with Bcl-2 overexpression. In contrast to mitochondrial NF-{kappa}B, the basal level of mitochondrial AP-1-DNA binding in PC3-Bcl-2 and PC3-Hygro was the same and had a very low intensity. TRAIL treatment increased AP-1 activation in mitochondria of DU145 and LNCaP after 4 and 8 hours to a much lower extent compared with PC3.



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  		FIGURE 9. Detection of AP-1 complexes with DNA binding activity in mitochondria of prostatic carcinoma cells. The DNA binding capacity of mitochondrial AP-1 was examined by EMSA. Mitochondrial proteins (5 µg) were incubated with 32P-end-labeled double-stranded oligonucleotide probe containing an AP-1 binding site. To ensure binding specificity, mitochondrial extracts were preincubated with an excess of cold AP-1 probe (wt. comp.), mutant probe (mut. comp.), or c-jun antibodies (c-jun Ab). NS, nonspecific bands.

 
Thus, AP-1 in the mitochondrial fraction of prostatic cell lines has DNA binding activity, TRAIL increases intensity of AP-1-DNA binding, and Bcl-2 overexpression prevents the effect of TRAIL.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Our results show that the p65 and p50 NF-{kappa}B subunits with DNA binding activity are localized in the mitochondria of prostatic carcinoma cells. Previously, we have described that mitochondria play a key role in TRAIL-induced apoptosis in PC3. The next logical follow-up is whether the mitochondrial pool of NF-{kappa}B plays a role in TRAIL-mediated apoptosis. Because NF-{kappa}B is a transcription factor and mitochondrial NF-{kappa}B can also bind to DNA, one of the possible roles for mitochondrial localization of NF-{kappa}B may be in modulating mitochondrial gene expression.

The first key consideration is whether TRAIL affects mitochondrial NF-{kappa}B-DNA binding in prostatic carcinoma cells. TRAIL increased mitochondrial NF-{kappa}B activity in PC3, decreased it in DU145, and induced it in LNCaP cells (Fig. 4A). The TRAIL-induced increase of mitochondrial NF-{kappa}B-DNA binding cannot be explained by translocation of cytosolic NF-{kappa}B to mitochondria because we did not find an increase of p65 in the mitochondrial fraction after TRAIL treatment (Fig. 8B). It is interesting to note that the effects of TRAIL on mitochondrial and nuclear NF-{kappa}B binding are inversely correlated for PC3 and DU145 and in parallel for LNCaP. Two hours of treatment with TRAIL increased NF-{kappa}B-DNA binding activity in mitochondria but decreased it in nuclei of PC3 and decreased it in mitochondria but increased it in nuclei of DU145. In LNCaP, TRAIL induced NF-{kappa}B-DNA binding in both mitochondria and nuclei (Fig. 4B). Crosstalk of nuclear and mitochondrial NF-{kappa}B activation is not understood and is possibly a key point in explaining of sensitivity (or resistance) to TRAIL.

The second consideration is whether TRAIL affects mitochondrial genome encoded mRNA levels. Eukaryotic mitochondrial biogenesis requires the coordinated expression of numerous genes encoded in two different genetic compartments, and most proteins involved in mitochondrial functions are synthesized in the cytoplasm through transcriptional regulation of the nuclear genome. Only some of the essential proteins involved in mitochondrial electron transport and oxidative phosphorylation are synthesized within mitochondria from the mitochondrial genome (26). An essential process in this coordinated interaction between the nucleus and the mitochondria is the transcription of mitochondrial DNA that requires nuclear proteins to interact with specific elements on the noncoding region of mitochondrial DNA. We found that Cox III, Cox II, and Cyt b mRNA decreased following TRAIL stimulation in PC3 and LNCaP. Therefore, the level of mitochondrial genome encoded mRNA is affected by TRAIL in prostatic carcinoma cell lines. We have also shown that inhibition of NF-{kappa}B in LNCaP prevented the decrease of mitochondrial genome encoded mRNA, suggesting involvement of NF-{kappa}B in regulating this process. The mechanism of this regulation by NF-{kappa}B is not clear. Direct binding of NF-{kappa}B to mitochondrial DNA has not been reported previously. Because only the steady-state levels of mRNA are determined by semiquantitative RT-PCR, we cannot be sure that TRAIL treatment affects mitochondrial genome encoded mRNA levels specifically through changes in mitochondrial transcription rates. Further, involvement of NF-{kappa}B in regulating mitochondrial genome encoded mRNA may be indirect. Because mutant FI{kappa}B{alpha}AA prevented NF-{kappa}B activation in both mitochondrial and nuclear fractions, we cannot exclude the possibility that NF-{kappa}B-dependent nuclear encoded proteins can regulate specific mitochondrial mRNA accumulation.

We also found a notable correlation between the increase of mitochondrial DNA binding and the reduction of mitochondrial genome encoded mRNA. Our data agree with Cogswell et al. (8) who showed NF-{kappa}B-dependent decrease of mitochondrial Cox III expression in HT1080 cells after tumor necrosis factor-{alpha} treatment.

Thus, TRAIL increases mitochondrial NF-{kappa}B activity in PC3 and decreases mitochondrial genome encoded mRNA levels that we suggest may be important for sensitivity of these cells to TRAIL. However, we do not have enough evidence to make this a definitive conclusion. Similar changes of mitochondrial genome encoded mRNA levels were found in TRAIL-resistant LNCaP cells after TRAIL treatment. Further, the overexpression of FI{kappa}B{alpha}AA prevented TRAIL-induced decrease of mitochondrial genome encoded mRNA in LNCaP and sensitized cells to TRAIL-induced cell death. However, Bcl-2 overexpression prevented the decrease of mitochondrial mRNA in PC3 as well as TRAIL-induced apoptosis. These data suggest that the role of TRAIL-induced changes in mitochondria genome encoded mRNA level might be cell specific.

Alternatively, mitochondrial NF-{kappa}B could also exert a transcriptionally independent regulatory function on apoptosis. The role of mitochondria in apoptosis is very important. A large variety of different signals, including death ligands, engage the mitochondria to release apoptogenic factors such as cytochrome c, Smac/DIABLO, HtrA2/Omi, apoptosis-inducing factor, and procaspases into the cytosol (16, 27, 28). Bottero et al. (7) described that I{kappa}B{alpha}-NF-{kappa}B complexes are present in the mitochondrial intermembrane space via interaction of the NH2-terminal domain of I{kappa}B{alpha} with the mitochondrial protein ANT (adenine nucleotide translocator) and may participate in regulating the release of apoptogenic factors from mitochondria.

The release of cytochrome c is controlled by proteins of the Bcl-2 family that are anchored in the outer mitochondrial membrane (17). Bcl-2 and Bcl-xL are antiapoptotic members of this Bcl-2 family and prevent cytochrome c release in contrast to the proapoptotic members Bax and Bak (17). We have shown previously that TRAIL induced loss of {Delta}{psi}m and cytochrome c release in PC3 and that Bcl-2 overexpression had a protective role (22). Here, we report that Bcl-2 prevents TRAIL-mediated increase of mitochondrial NF-{kappa}B activation and decrease of mitochondrial gene expression. Because TRAIL did not increase the amount of p65 in mitochondria, we believe that Bcl-2 somehow prevents the activation of mitochondrial NF-{kappa}B.

It is interesting that localization of other nuclear transcription factors such as p53, glucocorticoid receptors, and AP-1 in mitochondria has also been reported (12, 13, 29, 30). For example, kainite-induced AP-1 transcription factor binds to the noncoding region of the mitochondrial genome through interaction with sequences similar to the nuclear element recognized by the AP-1 complex in the mouse hippocampus (13). The functional significance of mitochondrial AP-1 complexes, however, remains unknown. Here, we report that AP-1 with DNA binding activity is associated with mitochondria of prostatic carcinoma cells. TRAIL treatment significantly increased AP-1-DNA binding in PC3, and Bcl-2 prevented this activation. The TRAIL-induced increase of mitochondrial AP-1 activation was lower in DU145 and LNCaP. Apparently, the regulation of mitochondrial genome encoded mRNA depends on communication of several nuclear transcription factors localized in the mitochondria and specific mitochondrial transcription factors.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture and Permanent Transfections
The human prostatic carcinoma cell lines PC3, DU145, and LNCaP were maintained in RPMI 1640 supplemented with 10% fetal bovine serum. Effectene transfection reagent (Qiagen, Inc., Valencia, CA) was used for transfection according to the manufacturer's instruction. Preparation of PC3-Bcl-2 and control PC3-Hygro cells was described previously (22). LNCaP cells were transfected with the plasmid pSFFV/Hygro with or without cDNA encoding Bcl-2 followed by hygromycin selection (22) and with the plasmid pOPRSVmcs1 with or without cDNA encoding FI{kappa}B{alpha}AA followed by neomycin selection. FI{kappa}B{alpha}AA with serine to alanine substitutions at positions 32 and 36 was a kind gift from Dr. Gail A. Bishop (University of Iowa, Iowa City, IA) and was described previously (31).

Transient Transfection and Luciferase Reporter Assay
Cells (1 x 106) were transfected with NF-{kappa}B reporter plasmid (1 µg, a kind gift from Dr. Bishop) together with pCMVß (0.5 µg, BD Biosciences Clontech, Palo Alto, CA). Effectene transfection reagent was used for transfection. Thirty-six hours after transfection, the cells were treated with TRAIL (200 ng/mL, PeptoTech, Rocky Hill, NJ) for 8 hours. Cells were harvested by trypsinization, washed in PBS, and lysed in reporter lysis buffer (200 µL, Promega, Madison, WI). Luciferase chemiluminescence activity was measured using the luciferase assay kit (Promega). Sample aliquots (20 µL) were assayed for light emission with a plate reader luminometer (MLX Dynex Technology, Inc., Franklin, MA). Transfection efficiencies were normalized by an amount of ß-glycosidase, which were measured using ß-glycosidase Reporter Gene Activity Detection Kit (Sigma Chemical Co., St. Louis, MO). The values of the luciferase assay were normalized with respect to the values of the ß-galactosidase assay for the relative comparison of each transfection.

Preparation of Nuclei and Mitochondria
Subcellular fractionation was done as described previously (22). Briefly, cells were lysed in ice-cold Mito buffer [20 mmol/L HEPES (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L DTT, 250 mmol/L sucrose, 0.1 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL pepstatin, leupeptin, aprotinin] by homogenization in a small glass homogenizer with a Teflon pestle. The homogenates were spun twice at 800 x g for 10 minutes to pellet nuclei. After a washing step, the nuclei were suspended in a hypotonic "low salt buffer" [20 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 20 mmol/L KCl, 0.2 mmol/L EDTA, 25% glycerol (v/v), 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride] and nuclear proteins were extracted by the slow addition of a "high salt buffer" [20 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 800 mmol/L KCl, 0.2 mmol/L EDTA, 25% glycerol (v/v), 1% NP40, 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride] and used for EMSA. Postnuclear supernatant was spun at 14,000 x g for 30 minutes at 4°C twice to pellet the mitochondria. The resultant supernatants and pellets were designated as cytosolic and mitochondrial fractions, respectively.

Highly purified mitochondria were prepared according to Storrie and Madden (32). Cells were harvested, washed in PBS, and homogenized in cell homogenization medium [150 mmol/L MgCl2, 10 mmol/L KCl, 10 mmol/L Tris-HCl (pH 6.7)]. Ice-cold cell homogenization medium (1/3 volume) containing 1 mol/L sucrose (final concentration of sucrose, 0.25 mol/L) was added to homogenate. Nuclei were pelleted by centrifugation at 1,000 x g for 5 minutes. Postnuclear supernatant was overlayed on hybrid Percoll/metrizamide gradient (layers from the bottom: 35% metrizamide, 17% metrizamide, 6% Percoll) and centrifuged for 15 minutes at 50,500 x g. Mitochondria were taken from the 17% metrizamide/35% metrizamide interface and washed twice with cell homogenization medium containing sucrose (0.25 mol/L). Mitochondrial proteins were extracted in nuclear extract buffers as described above for nuclei. Cross-contamination with cytosol was assessed using lactate dehydrogenase as a specific enzyme marker. The level of cross-contamination was calculated as the percentage of the total cellular enzyme activity in the different fractions.

Preparation of Total Cell Lysate and Western Blot Analysis
Cells were harvested, washed twice in PBS, and lysed in Triton X-100 buffer and Western blot detection of proteins was done as described previously (33). Membranes were blocked with 5% nonfat dry milk in PBS and incubated with the corresponding mouse monoclonal antibodies: anti-p65 of NF-{kappa}B, c-jun/AP-1 (Oncogene, Uniondale, NY), anti–poly(ADP-ribose) polymerase, anti-cytochrome c (PharMingen, San Diego, CA), caspase-8 (Upstate, Charlottesville, VA), or rabbit polyclonal p50 of NF-{kappa}B (Santa Cruz Biotechnology, Santa Cruz, CA).

Electrophoretic Mobility Shift Assay
Assays were done using double-stranded oligonucleotides 5'-AGTTGAGGGGACTTTCCCAGGC-3' or 5'-GCTTGATGAGTCAGCCGGAA-3' (Promega) as probes for detection of DNA binding activity of NF-{kappa}B or AP-1, respectively. Nuclear or mitochondrial protein (5 µg) was incubated with 32P-end-labeled double-stranded DNA probe for 30 minutes at room temperature in a binding buffer [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L KCl, 0.5 mmol/L EDTA, 0.1% Triton X-100, 12.5% glycerol (v/v), 0.1 mg/mL deoxyinosinic-deoxycytidylic acid, 0.2 mmol/L DTT]. For the supershift reaction, proteins were preincubated with anti-p65 (0.05mg/mL, BD Biosciences Transduction Labs, Lexington, KY), anti-p50, anti-p52, anti-c-Rel, and anti-RelB (0.1 mg/mL, Santa Cruz Biotechnology) antibodies. For competition reaction, 100-fold excess of unlabeled mutant or wild-type NF-{kappa}B probes was added to the samples before the addition of the labeled probe. Mutant double-stranded oligonucleotides were 5'-AGTTGAGGCGACTTTCCCAGGC-3' for NF-{kappa}B and 5'-GCTTGATCAGTCAGCCGGAA-3' for AP-1. Gel analysis was carried out in native 5% polyacrylamide gels. X-ray films were exposed to the dried gels at –70°C for 14 hours for nuclear extracts and 24 hours for mitochondrial extracts.

Semiquantitative RT-PCR
Cells were treated with TRAIL (200 ng/mL) for 2, 4, or 6 hours. RNA was purified with RNeasy Mini Kit (Qiagen). To avoid contamination by mitochondrial DNA, samples were treated with the RNase-free DNase (Qiagen) during purification. Equal amounts (1 µg) of total RNA from control and treated cells were reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) and cDNA (5 µL) was taken for amplification with primers for Cox II, Cox III, Cyt b, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cox II: forward primer 5'-AGCTTATCACCTTTCATCATCAC-3' and reverse primer 5'-GTACTACTCGATCAACGTC-3' (352 bp). Cox III: forward primer 5'-GCGCGATGTAACACGAGAAAG-3' and reverse primer 5'-GCGCGATGTAACACGAGAAAG-3' (242 bp). Cyt b: forward primer 5'-CTCGGCATGATGAAACTTCGG-3' and reverse primer 5'-AGCAGGAGGATAATGCCGATG-3' (281 bp). GAPDH: forward primer 5'-ACCACAGTCCATGCCATCAC-3' and reverse primer 5'-TCCACCACCCTGTTGCTGTC-3' (451 bp). All RT-PCRs were done in the linear range for each transcript compared with GAPDH as a reference control. The PCR products were analyzed on 6% PAGE. Ethidium bromide–stained gels were scanned (Photodyne, Northridge, CA) and analyzed with Scion imaging software (Scion Corp., Frederick, MD). Negative RT-PCR controls were done in the absence of RNA and/or reverse transcriptase. Control for contamination by DNA was prepared using RNA sample as a template in PCR reaction instead of cDNA.

Estimation of Cell Viability
To measure cell viability, we used calcein AM (Molecular Probes, Eugene, OR) as described previously (22).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. Gail A. Bishop for providing the FI{kappa}B{alpha}AA construct.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 NIH grant CA93870 (M.B. Cohen). Back

Received March 11, 2004; revised August 5, 2004; accepted August 6, 2004.


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

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