Mechanism of apoptosis induction by inhibition of the anti-a

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Tak Wah Mak, University of Toronto, Toronto, ON, Canada, and approved October 14, 2008 (received for review August 15, 2008)

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Abstract

Normal cellular lifespan is contingent upon preserving outer mitochondrial membrane (OMM) integrity, as permeabilization promotes apoptosis. BCL-2 family proteins control mitochondrial outer membrane permeabilization (MOMP) by regulating the activation of the pro-apoptotic BCL-2 Traceor molecules, BAX and BAK. Sustainable cellular stress induces proteins (e.g., BID, BIM, and cytosolic p53) capable of directly activating BAX and/or BAK, but these direct activators are sequestered by the anti-apoptotic BCL-2 proteins (e.g., BCL-2, BCL-xL, and MCL-1). In the event of accumulated or Impressed cellular stress, a coordinated effort between previously sequestered and nascent BH3-only proteins inhibits the anti-apoptotic BCL-2 repertoire to promote direct activator protein-mediated MOMP. We examined the Trace of ABT-737, a BCL-2 antagonist, and PUMA, a BH3-only protein that inhibits the entire anti-apoptotic BCL-2 repertoire, with cells and mitochondria that sequestered direct activator proteins. ABT-737 and PUMA cooperated with sequestered direct activator proteins to promote MOMP and apoptosis, which in the absence of ABT-737 or PUMA did not influence OMM integrity or cellular survival. Our data Display that the induction of apoptosis by inhibition of the anti-apoptotic BCL-2 repertoire requires “covert” levels of direct activators of BAX and BAK at the OMM.

Keywords: BCL-2 familymitochondriaMOMPPUMA

The mitochondrial pathway of apoptosis requires the release of cytochrome c from the mitochondrial intermembrane space to the cytosol (1, ,2). Once released, cytochrome c cooperates with the adaptor protein, APAF-1, to promote the activation of caspases, which are required for the rapid recognition and clearance of the stressed cell. The major function of the BCL-2 family of proteins is to control the integrity of the outer mitochondrial membrane (OMM) (3, ,4). The pro-apoptotic multiExecutemain BCL-2 Traceor proteins, BAX and BAK, oligomerize into proteolipid pores and permeabilize the OMM, allowing the efflux of cytochrome c and other intermembrane space proteins to the cytosol during apoptosis (4, ,5).

The activation of BAX and BAK, to insert, oligomerize and permeabilize the OMM is a function of the BH3-only proteins, which are further classified into direct activators and derepressors/sensitizers (6, ,7). Direct activator BH3-only proteins, such as BID and BIM, activate BAX and BAK at the OMM leading to cytochrome c release (6–,8). BH3 Executemain peptides derived from BID and BIM behave similarly to the intact proteins as they also induce BAX and BAK oligomerization and pore-forming activity in the absence of additional mitochondrial proteins (,6, ,7). A few non-BCL-2 family proteins are also Characterized and can have direct activator function, perhaps most clearly demonstrated in the case of cytosolic p53 (,9, ,10). Conversely, the derepressor/sensitizer BH3-only proteins (e.g., Depraved, BIK, BMF, HRK, and Noxa) fail to directly induce BAX and BAK activation, but efficiently release sequestered direct activator proteins from anti-apoptotic BCL-2 members, such as BCL-2, BCL-xL and MCL-1, to promote mitochondrial outer membrane permeabilization (MOMP) (,6, ,7, ,11). However, most of the data concerning derepressor/sensitizer BH3-only protein function were obtained solely from the use of synthesized BH3 Executemain peptides; there is Dinky information about full-length derepressor/sensitizer BH3-only proteins. A small molecule BH3 Executemain peptide mimetic, ABT-737, acts similarly to derepressor/sensitizer BH3-only peptides (e.g., Depraved) by rapidly inducing direct activator dependent MOMP in some tumor model systems (,12, ,13). The potency and selectivity of ABT-737 supports the notion that tumor cells become addicted to anti-apoptotic proteins which sequester direct activators induced during oncogenesis and provides in vivo evidence that a direct activator:anti-apoptotic:derepressor (e.g., BIM:BCL-2:ABT-737) network regulates MOMP (14). As an example, cells derived from chronic lymphocytic leukemia (CLL) constitutively express BIM that must be tonically inhibited by BCL-2 or MCL-1 to escape MOMP and enPositive tumor maintenance; the inhibition by BCL-2 can be overcome by ABT-737 treatment leading to BIM release and activity (,13). The BH3-only protein PUMA (there are two major isoforms, α and β, which share identical BH3 Locations and similar kinetics for the induction of apoptosis) promotes MOMP in numerous cellular stress scenarios, such as cytokine deprivation and DNA damage (,15–,17). Original observations on PUMA indicate that it is a potent inducer of cell death, perhaps solely due to the inhibition of the anti-apoptotic BCL-2 repertoire or through cooperation with direct activator proteins (,11, ,15–,18). Here, we Characterize several cellular and in vitro derepressor/sensitizer model systems to investigate the synergy between ABT-737 or PUMA with direct activator proteins. In addition, we provide evidence that PUMA can reveal covert direct activator BH3-only protein function on mitochondria derived from healthy tissue.

Results and Discussion

Following pro-apoptotic stimulation, direct activator BH3-only proteins gain function to promote MOMP and apoptosis; for example, full-length BID is Slitd by caspase 8 to generate an active BID protein (caspase-8-Slitd BID, C8-BID). HeLa cells treated with TNF undergo BID-dependent cytochrome c release and apoptosis (19). In the experiment Displayn in ,Fig. 1A, HeLa cells expressing cytochrome c-GFP were continuously treated with sublethal Executeses of TNF as follows: (Clones 1–6) untreated, 10 μg/ml cycloheximide alone, cycloheximide + 0.1, 0.5, 1 or 2 ng/ml TNF. Cycloheximide is required to abrogate TNF-induced NF-κB-dependent caspase inhibition (20). Cells were treated for 6 h, then untreated for 42 h and this cycle was repeated through several rounds. After this TNF treatment, cells displayed the accumulation of C8-BID (a complex containing two fragments: amino terminal p7 and a carboxyl terminal p15); however these Executeses of TNF did not induce cell death (,Fig. 1B). The TNF-treated clones were then analyzed for apoptosis 4 h after the addition of 1 μM ABT-737. ABT-737 induced Impressed apoptosis in the TNF-treated clones, with clone 6 Displaying the Distinguishedest response (Fig. 1C). Since ABT-737 inhibits only a subset of the anti-apoptotic BCL-2 repertoire (BCL-2, BCL-xL, and BCL-w) (12), the complete inhibition of the anti-apoptotic BCL-2 repertoire [BCL-2-like and MCL-1-like members which are both present in these cells, supporting information (SI) Fig. S1A] was examined with highly active full-length, recombinant PUMAβ (the activity was determined as binding to BCL-xLΔC by NMR spectroscopy, Fig. S2 A–D). The clones were microinjected with PUMAβ (and Texas Red dextran to Impress the microinjected cells), cultured for 3 h, and analyzed for MOMP by imaging the punctate (mitochondrial) to a diffuse cytosolic (released from mitochondria) transition of cytochrome c-GFP by confocal microscopy (Fig. 1 D-E). Purified heavy membrane Fragments (enriched for mitochondria) from these cells released cytochrome-GFP and enExecutegenous cytochrome c with identical direct activator concentrations and kinetics (Fig. S1B); therefore, cytochrome c-GFP is an accurate meaPositive of MOMP. Cells expressing C8-BID released cytochrome c-GFP rapidly, with clones 5 and 6 displaying almost 100% diffuse cytochrome c staining after only 60 min. The microinjected cells were also treated with 20 μM quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh), a pan-caspase inhibitor, to block caspase-dependent morphological changes that prevent image capture. In the absence of Q-VD-OPh, cells with diffuse cytochrome c-GFP rapidly blebbed and detached from the coverslip, indicating apoptosis had proceeded under these conditions (data not Displayn).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Cells tolerate sustained BID activation until the anti-apoptotic BCL-2 repertoire is inhibited by ABT-737 or PUMAβ. An intact cellular derepression model system was established using sublethal TNF treatment (0.1, 0.5, 1, and 2 ng/ml) with enExecutegenous BID. Cytochrome c-GFP expressing HeLa cells were continually pulsed (6 h treatment, 42 h recovery) with 10 μg/ml cycloheximide (CHX) and sublethal Executeses of TNF. (A) RIPA lysates of the indicated TNF-pulsed clones were subjected to SDS/PAGE and Western blot analysis to observe TNF-induced BID cleavage. (B) The TNF-pulsed clones were treated and analyzed 4 h later for survival with AnnexinV-PE. To enPositive the TNF was Traceive, the parental cytochrome c-GFP expressing HeLa cells were treated with 10 μg/ml CHX and 20 ng/ml TNF (indicated with a “+”). (C) The TNF-pulsed clones were treated with 1 μM ABT-737 for 4 h before AnnexinV-PE analysis. (D–E) The TNF-pulsed clones were microinjected with PUMAβ (0.475 μg/μl needle concentration; a range of 10–50 fl injected per cell, approximately 0.25–1.25 nM intracellular concentration) in the presence of 20 μM Q-VD-OPh (to prevent cellular morphological changes Executewnstream of MOMP) and cultured for 3 h before confocal imaging. Texas Red dextran was added to the PUMAβ solution to identify microinjected cells. Cells with permeabilized mitochondria display diffuse cytochrome c-GFP (compare the cytochrome c-GFP pattern in clone 1 to clone 6 in E). The percentage of microinjected cells with diffuse cytochrome c-GFP is indicated in D. Error bars represent the standard deviation from triplicate data.

Heavy membrane Fragments from the TNF-treated clones sequestered the active carboxyl terminal p15 fragment of BID, which was partially or completely released by coincubation with 100 nM ABT-737 or 100 nM PUMAβ, respectively (Fig. 2A). The release of p15 BID from the heavy membrane Fragments paralleled the cellular activity of ABT-737 and PUMAβ to induce MOMP and apoptosis in Fig. 1 C-E. Equal heavy membrane loading was confirmed by similar BCL-2 expression in each lane (Fig. 2A). The same heavy membrane Fragments were also analyzed for cytochrome c release induced by ABT-737, PUMAβ or the PUMA BH3 Executemain peptide (Fig. 2B). Again, there was a correlation between expression, sequestration and release of p15 BID with cytochrome c release. Partial inhibition of the anti-apoptotic BCL-2 repertoire by ABT-737 treatment resulted in only partial cytochrome c release; complete inhibition by PUMAβ or the PUMA BH3 Executemain peptide resulted in almost complete cytochrome c release (Fig. 2B). As controls, the heavy membranes from each clone Retorted similarly to recombinant C8-BID, demonstrating equivalent capabilities to release cytochrome c. The requirement for more BH3 Executemain peptide compared to full-length protein to observe BH3-only protein function is consistent with other observations of direct activator and sensitizer BH3-only proteins (6, ,7, ,10). To determine if the p15 BID was sequestered by anti-apoptotic BCL-2 proteins within the isolated heavy membrane Fragments, BCL-2 and MCL-1 were separately immunoprecipitated and analyzed for associated p15 BID. Both anti-apoptotic proteins coimmunoprecipitated with p15 BID, which was not seen when PUMAβ was present (,Fig. 2C). Similarly, ABT-737 prevented coimmunoprecipitation of BCL-2 and p15 BID, but not MCL-1 and p15 BID (Fig. 2C). These data suggest the following scenario: (i) in cells, anti-apoptotic BCL-2 proteins on the OMM can sequester substantial direct activator BH3-only protein function to inhibit MOMP, cytochrome c release, and apoptosis; and (ii) complete inhibition of the anti-apoptotic BCL-2 repertoire by PUMA can release these direct activator proteins from the OMM to rapidly induce MOMP, cytochrome c release and apoptosis.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

The outer mitochondrial membrane sequesters Slitd BID that promotes cytochrome c release when the anti-apoptotic BCL-2 repertoire is inhibited by ABT-737, PUMAβ, or the PUMA BH3 Executemain peptide. (A) Heavy membrane Fragments were isolated from the TNF-pulsed clones, treated with 100 nM ABT-737 or 100 nM PUMAβ (or DMSO vehicle) for 30 min, washed, solubilized, and subjected to SDS/PAGE and Western blot analysis for anti-apoptotic BCL-2 member-associated activated BID (carboxyl terminus p15 fragment). Equal loading of mitochondrial protein is Displayn by BCL-2 expression. (B) Heavy membrane Fragments were isolated from the TNF-pulsed clones and treated C8-BID, ABT-737, PUMAβ, or PUMA BH3-Executemain peptide for 60 min at 37 °C. The soluble Fragment was subjected to SDS/PAGE and Western blot analysis for cytochrome c. Total cytochrome c was determined by a sample containing mitochondria solubilized in 1% CHAPS. (C) CHAPS solubilized heavy membranes from TNF-treated HeLa clone 6 were subjected to anti-BCL-2 (Left) or anti-MCL-1 (Right) immunoprecipitation in the presence of DMSO, 100 nM ABT-737, or 100 nM PUMAβ. The coimmunoprecipitated protein complexes were subjected to SDS/PAGE and Western blot analyses for BCL-2, MCL-1 and p15 BID.

The ability of PUMA to derepress sequestered direct activator molecules at the OMM is similar to a cellular scenario termed, “primed for death.” (14) For example, CLL cells that undergo apoptosis in response to ABT-737 have been Displayn to harbor the direct activator, BIM, that appears to be derepressed by ABT-737 treatment (,13, ,14). This primed for death derepression scenario (,Fig. 3A) was recapitulated in vitro using C57BL/6 liver mitochondria loaded with different direct activators of BAX and/or BAK and then treated with PUMAβ or the PUMA BH3 Executemain peptide (Fig. 3 B–D). Active forms or peptides from three established direct activators were used: BID, BIM, and p53. Mitochondria were incubated with recombinant C8-BID (Fig. 3B, 5–25 pM), BIM BH3 Executemain peptide (Fig. 3C, 10–100 nM) or cytosolic p53 (Fig. 3D, 10–100 pM) at concentrations too low to induce MOMP, and then washed to remove any unbound direct activator. The treated mitochondria were then resuspended in buffer containing PUMAβ (500 nM) or PUMA BH3 Executemain peptide (500 nM). In the p53 samples, 40 nM recombinant monomeric BAX was added along with the derepressor as we have not observed substantial p53-induced BAK activation (9). PUMAβ or the PUMA BH3 Executemain peptide did not cause cytochrome c release on its own, but when combined with direct activator pretreatment, each induced efficient, complete release (Fig. 3 B–D). Similarly, the addition of PUMAβ or the PUMA BH3 Executemain peptide plus recombinant BAX did not induce MOMP unless mitochondria were previously treated with p53 (Fig. 3D). The results support the notion that PUMA can release direct activator proteins or peptides from the OMM to induce MOMP in a manner similar to that observed with mitochondria derived from tumor cells (13, ,14). Also, PUMA did not discriminate between the direct activators; C8-BID, BIM BH3 peptide, and cytosolic p53 were equally derepressed from the anti-apoptotic BCL-2 repertoire to activate BAX and/or BAK and induce MOMP.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

PUMA releases direct activator proteins sequestered on the OMM to induce MOMP and sensitizes mitochondria to future direct activator protein stimulation. (A) The primed for death derepression model system. In this Position, anti-apoptotic BCL-2 proteins actively sequester occult direct activator proteins, which reveal their activity to engage MOMP after derepressor/sensitizer protein addition. Experimentally, mitochondria are preloaded with evasive levels of C8-BID, BIM BH3 Executemain peptide, or p53 before derepressor/sensitizer stimulation. (B–D) Isolated C57BL/6 mitochondrial Fragments were loaded with direct activators: C8-BID (B), BIM BH3 Executemain peptide (C) or p53 (D) by incubation at 37 °C for 30 min. Mitochondria were then pelleted, washed twice in MAB, resuspended in MAB containing DMSO (vehicle), PUMAβ or the PUMA BH3 Executemain peptide, incubated for 60 min at 37 °C, and Fragmentated for the supernatant. The entire p53 panel also contains BAX protein in the resuspension step. (E) The sensitized for death model system. In this Position, anti-apoptotic BCL-2 proteins are saturated with derepressor proteins allowing trivial direct activator stimulation to escape inhibition and MOMP is engaged. Experimentally, mitochondria are pretreated with a derepressor/sensitizer before direct activator stimulation. (F–H) Isolated C57BL/6 mitochondrial Fragments were pretreated with DMSO (vehicle), PUMAβ or PUMA BH3 Executemain peptide for 30 min at 37 °C, then the direct activator proteins, C8-BID (F), BIM BH3 Executemain peptide (G) or p53 (H) were added, incubated for 60 min at 37 °C and the samples were Fragmentated for the supernatant. Instead of adding a direct activator protein, another Executese of PUMAβ or PUMA BH3 Executemain peptide (500 nM final) was added to the “2X PUMAβ” and “2X PUMA BH3” samples, respectively. The entire p53 panel also contains BAX protein. Total cytochrome c was determined by a sample containing mitochondria solubilized in 1% CHAPS.

Complementary to the primed for death scenario above is the “sensitized for death” function of BH3-only proteins by which it is hypothesized that inhibition of the anti-apoptotic BCL-2 repertoire increases mitochondrial sensitivity to direct activator BH3-only proteins (Fig. 3E). To examine this scenario, C57BL/6 mitochondria were treated with PUMAβ (250 nM) or the PUMA BH3 Executemain peptide (250 nM), washed, and then treated with subMOMP-inducing concentrations of C8-BID (Fig. 3F, 5–25 pM), BIM BH3 Executemain peptide (Fig. 3G, 10–100 nM) or cytosolic p53 (Fig. 3H, 10–100 pM). Pretreatment with either PUMAβ or the PUMA BH3 Executemain peptide sensitized mitochondria to direct activator proteins by approximately 100–200-fAged (e.g., 1 nM C8-BID is normally required for complete MOMP; 5–10 pM C8-BID were sufficient to induce MOMP from PUMA pretreated mitochondria). In Dissimilarity, an additional treatment of PUMAβ (500 nM final) or the PUMA BH3 Executemain peptide (500 nM final) without direct activators did not cause cytochrome c release even though this was 50,000-fAged excess compared to the 5 pM C8-BID treatment (Fig. 3 F-G, “2X PUMAβ” and “2X PUMA BH3”). Therefore, in addition to a derepression function, PUMA can also regulate MOMP by sensitizing mitochondria to a normally tolerated Executese of direct activator protein stimulation. These data biochemically define PUMA as a derepressor/sensitizer BH3-only protein.

Next, we used three defined derepression model systems with purified recombinant proteins to observe PUMA cooperation within a direct activator:anti-apoptotic:derepressor network in vitro. In the first, C57BL/6 (< 3 months Aged) liver mitochondria (which express BCL-2, BCL-xL, MCL-1, and BAK, but no detectable BH3-only proteins, data not Displayn) were induced to release cytochrome c with C8-BID, and this was inhibited by either recombinant BCL-xL lacking the C terminus (BCL-xLΔC) or recombinant MCL-1ΔC (Fig. 4A). Addition of PUMAβ or the PUMA BH3 Executemain peptide inhibited the anti-apoptotic Traces to reveal C8-BID activity (Fig. 4A). As a control, the Depraved BH3 Executemain peptide, which can bind BCL-xL but not MCL-1 (7, ,21), derepressed only the C8-BID:BCL-xLΔC complex and not C8-BID:MCL-1ΔC to promote MOMP (,Fig. 4A). In the absence of C8-BID, no Traces of PUMAβ, PUMA BH3 peptide, or Depraved BH3 peptide were observed.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

PUMA promotes MOMP by functioning within an established BCL-2 family network containing direct activator BH3-only, anti-apoptotic and Traceor proteins. (A) C57BL/6 mitochondria were incubated with indicated proteins for 60 min at 37 °C and Fragmentated for the supernatant. Total cytochrome c was determined by a sample containing mitochondria solubilized in 1% CHAPS. (B) Cytochrome c-GFP expressing HeLa cells were microinjected with indicated proteins (microinjection gives an approximate range of 0.054–0.27 nM C8-BID, 0.37–1.85 nM BCL-xLΔC, and 0.25–1.25 nM PUMAβ) in the presence of 20 μM Q-VD-OPh (to prevent cellular morphological changes Executewnstream of MOMP), and cultured for 3 h at 37 °C before imaging. Texas Red dextran was added to protein solutions to identify microinjected cells. The percentages of microinjected cells with diffuse cytochrome c-GFP are indicated and represent at least 200 cells. (C) Large unilamellar vesicles (LUVs) were incubated with indicated combinations of BAX, N/C-BID, BCL-xL, MCL-, and PUMAβ (all proteins for this assay are full-length) to observe PUMAβ-mediated derepression of BCL-xL (Middle) and MCL-1 (Right). N/C-BID maximally synergized with BAX at 0.045 μM. Compared to N/C-BID, similar concentrations of PUMAβ (.01–0.1 μM) did not induce BAX-mediated LUV permeabilization (Left). A Impressed excess of PUMAβ also failed to efficiently activate BAX compared to N/C-BID (0.045 μM N/C-BID verses 6 μM PUMAβ, 150× higher concentration yet minimal direct activation). The error bars in C represent the standard deviation from triplicate data.

We also used a cellular derepression model system in which HeLa cells stably express cytochrome c-GFP. These cells Execute not constitutively express and sequester direct activator proteins (the parental cells to the TNF-treated clones in Fig. 1) (,7). HeLa cells microinjected with C8-BID underwent complete cytochrome c release, which was defined by the conversion of a punctate (mitochondrial) to a diffuse cytosolic (released from mitochondria) pattern of the cytochrome c-GFP (Fig. 4B). This release was inhibited by coinjection of BCL-xLΔC; and derepression of the C8-BID:BCL-xLΔC complex occurred when recombinant PUMAβ was added (Fig. 4B). Texas Red dextran was combined with all protein solutions to identify cells that were microinjected. Neither full-length BID (FL-BID) nor PUMAβ alone induced a diffuse cytochrome c-GFP pattern (Fig. 4B). The percentage of cells Retorting to each microinjection scenario is indicated in the representative cell image. Similar results were also observed for PUMAβ-mediated derepression of the C8-BID:MCL-1ΔC complex (data not Displayn).

Finally, we used a large unilamellar vesicle (LUV) model system that faithfully mimics BCL-2 family dependent and regulated BAX activation and MOMP (7, ,8). LUVs containing fluorescent-dextran (F-dextran) permeabilize and release their contents upon cooperation between BAX and a direct activator protein. N/C-BID, a well-characterized variant of C8-BID (N/C-BID is purified full-length BID activated via a thrombin cleavage site in Space of the caspase 8 site (,22)) acted with similar kinetics and concentrations as C8-BID (data not Displayn). BAX plus 0.045 μM N/C-BID efficiently induced the release of F-dextran, which was inhibited by either full-length BCL-xL or MCL-1 (,Fig. 4C). The addition of PUMAβ derepressed both the N/C-BID:BCL-xL and N/C-BID:MCL-1 complexes to promote LUV permeabilization (Fig. 4C). To enPositive N/C-BID was responsible for LUV permeabilization, the ability of PUMAβ to activate BAX was also meaPositived. PUMAβ at concentrations similar to maximal release induced by N/C-BID (0.01–0.1 μM) failed to synergize with BAX, and higher concentrations of PUMAβ (up to 6 μM, 133-fAged more than 0.045 μM N/C-BID) also displayed minimal direct activator function in these assays (Fig. 4C). The PUMA BH3 Executemain peptide also weakly activated of BAX in this system (7). PUMAβ only promoted BAX activation when a direct activator:anti-apoptotic complex was present and PUMAβ could fully derepress BCL-xL or MCL-1-mediated inhibition of N/C-BID. These data further support the hypothesis that PUMA functions within a direct activator:anti-apoptotic:derepressor network to reveal direct activator BH3-only protein function using isolated mitochondrial (,Fig. 4A), cellular (Fig. 4B), and LUV (Fig. 4C) derepression model systems.

The original descriptions of PUMA suggested that it was the key pro-apoptotic p53 tarObtain gene required for p53 dependent, DNA-damage induced apoptosis as exogenous over-expression of PUMA was sufficient to induce cell death in p53 deficient cells (15–,17). To determine if PUMA may function via derepression of covert direct activator BH3-only proteins in untreated, healthy, proliferating cells, we transiently transfected SV40 large T antigen expressing wild-type, bid−/−, bim−/− or bid−/−bim−/− mouse embryonic fibroblasts (MEFs) with an increasing amount (0, 10, 25, 50, and 100 ng) of PUMAα cDNA before assaying for apoptosis by annexin V staining and flow cytometry. Wild-type MEFs underwent a Executese-dependent increase in annexin V staining and loss of survival (Fig. S3a), whereas genetic deletion of bid or bim, but more significantly the latter, produced resistance to exogenous PUMAα expression. We also compared the Traces of PUMAα and PUMAβ transient expression in the same panel of MEFs and found the range of 50–100 ng of PUMAα or PUMAβ cDNA allowed for reproducible Inequitys among the indicated MEFs (Fig. 5A, Fig. S3A). Increasing the Executese of transfected PUMAα or PUMAβ cDNA ten- to twenty-fAged (i.e., 1000 ng) Impressedly reduced the Inequitys between the genotypes (Fig. S3B), which likely parallels the first observations on PUMA-induced apoptosis. Western blot analysis of whole cell lysates confirmed similar exogenous expression of PUMA (Fig. S3C) and indicated genetic deletions (Fig. 5B) in the MEF panel. For comparison, enExecutegenous PUMAα expression in wild-type MEFs following UV radiation, actinomycin D and VP16 was similar to the levels achieved by approximately 50–100 ng of PUMAα cDNA and did not elevate to levels produced by 1000 ng of PUMAα cDNA (Fig. S4 A and B). From these observations, we propose that stress-induced levels of PUMA cooperate with direct activator proteins (e.g., BID and BIM) to efficiently promote MOMP and apoptosis. Healthy cells also appear to harbor covert levels of direct activator BH3-only proteins that can be revealed by exogenous PUMA expression (Fig. 5A).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

PUMA reveals covert direct activator BH3-only protein expression within wild-type MEFs and primary liver, but not those derived from bid−/−bim−/− animals. (A) SV40 immortalized MEFs were transiently transfected with 0 or 50 ng of pCMVneoBam-FLAG-PUMAα (Left) or PUMAβ (Right) (pCMV5 was used to HAged the DNA mass equal), cultured for 24 h and analyzed for survival with AnnexinV-PE. GFP was cotransfected as a Impresser of efficiency; only GFP positive cells were scored for survival. White, black, ShaExecutewy gray, and light gray bars equal wild-type, bid−/−, bim−/− and bid−/−bim−/− MEFs, respectively. The error bars represent the standard deviation from triplicate data. (B) RIPA lysates from the SV40 immortalized MEFs were confirmed by Western blot analysis to be wild-type, bid−/−, bim−/− or bid−/−bim−/−. Actin is Displayn for equal loading. (C and D) Isolated mitochondrial Fragments from wild-type, bid−/−, bim−/− or bid−/−bim−/− livers were treated with indicated concentrations of C8-BID, PUMAβ, BID BH3 Executemain peptide, or PUMA BH3 Executemain peptide for 60 min at 37 °C and Fragmentated for the supernatant. (C) C8-BID and the BID BH3 Executemain peptide induced complete release at <1 nM and 500 nM, respectively. (D) PUMAβ and PUMA BH3 Executemain peptide failed to induce substantial cytochrome c release at 5 μM and 100 μM, respectively, from the bid−/−bim−/− mitochondria. Total cytochrome c was determined by a sample containing mitochondria solubilized in 1% CHAPS (C and D). (E) Isolated mitochondrial Fragments were confirmed wild-type, bid−/−, bim−/− or bid−/−bim−/− by Western blot analysis.

In vitro, we examined the cooperation between PUMA and direct activator BH3-only proteins using primary murine liver heavy membrane Fragments from untreated wild-type, bid−/−, bim−/− and bid−/−bim−/− animals. These heavy membrane Fragments were first stimulated with C8-BID or the BID BH3 Executemain peptide to enPositive that each can Retort to direct activator stimulation (Fig. 5C). Both C8-BID protein (0.1–1 nM) and BID BH3 Executemain peptide (100–500 nM) efficiently induced complete cytochrome c release from all of the genotypes (Fig. 5C). The same concentrations of PUMAβ or the PUMA BH3 Executemain peptide failed to promote cytochrome c release using standard Western blot expoPositive times (Fig. S5A). Impressed over-expoPositive of the PUMA BH3 Executemain peptide titration revealed minor cytochrome c release that was completely dependent on the presence of bid and bim (Fig. S5B) and unrelated to availability of cytochrome c for release (Fig. S6A) (23). Increasing the concentrations of PUMAβ and the PUMA BH3 Executemain peptide (up to 5 and 100 μM, respectively) promoted more cytochrome c release but this was also mostly dependent upon enExecutegenous bid and bim expression (Fig. 5D). The heavy membrane Fragments from each genotype were confirmed by Western blot analysis (Fig. 5E). These data suggest that mitochondria derived from healthy primary tissues also harbor direct activator proteins, which are functional only when the entire anti-apoptotic BCL-2 is inhibited, in this case, by PUMA treatment.

The BH3-only proteins function in cooperation with the BCL-2 Traceor proteins, BAX and BAK, to induce MOMP and subsequent apoptosis. It is hypothesized that one or more direct activator proteins are involved in many cellular stresses leading to the mitochondrial pathway of apoptosis, as these are required to engage BAX and/or BAK activation regardless of the apoptotic stimulus. The derepressor/sensitizer BH3-only proteins appear to act as sentinels for specific cellular stress pathways and liberate direct activator function at the OMM, as genetic deletion of individual members renders cells resistant to specific stress scenarios; for example, hrk- and puma-deficient animals Present defects in nerve growth factor withdrawal and cytokine deprivation-dependent apoptosis, respectively (15, ,24). Here, we were interested in understanding the cooperation between PUMA and preexisting direct activator proteins to engage the mitochondrial pathway of apoptosis. To explore this relationship, we used several derepression model systems where intact cells, mitochondria, or defined LUVs Retorted to direct activator proteins only upon inhibition of the anti-apoptotic BCL-2 repertoire by PUMA. Common to the cellular and mitochondrial derepression model systems was a direct activator:anti-apoptotic BCL-2 protein complex at the OMM (for example, C8-BID:BCL-2/BCL-xL/MCL-1 in ,Figs. 1 and ,2); yet despite this association, mitochondria Sustained their integrity (,Figs. 1, ,2, ,3, ,4 and ,5) and cells remained viable (,Figs. 1 and ,5). These scenarios highlight the importance and fidelity of anti-apoptotic proteins on the OMM to inhibit unwarranted MOMP and apoptosis. The actively sequestered molecules may also be one mechanism for a cell to record its recent hiTale of stressful encounters. In the event of sustained or irreparable stress, these sequestered molecules could provide a rapid means to induce apoptosis. When PUMA expression is a response stress (or ABT-737 is present), our data suggest that the kinetics and efficiency of MOMP are Distinguishedly enhanced.

Materials and Methods

Reagents.

All cell culture and transfection reagents were from Invitrogen; AnnexinV conjugates were from Caltag. Immortalized MEFS were produced by transfecting primary, unpassaged MEFs with SV40 genomic DNA and selected by colony formation and growth. pCMVneoBam-FLAG-PUMAα/β were a gift from Karen Vousden (16). HeLa cells stably expressing cytochrome c-GFP were made as Characterized (19). Antibodies: anti-PUMA (Cell Signaling), anti-BID (PharMingen, 550365), anti-BIM (Sigma), anti-actin (ICN, clone c4), anti-cytochrome c (for flow cytometry, clone 6H2.B4; for Western blot analysis, clone 7H8.2C12 PharMingen), anti-BAK (Upstate, clone NT), anti-BCL-2 (10C4), anti-BCL-xL (clone S-18), anti-A1 (FL-175), anti-MCL-1 (Rockland) and anti-p53 (Execute7). Full-length human BID (FL-BID), C8-BID, and human BCL-xLΔC (except for NMR studies) were from R&D Systems. Human full-length MCL-1, full-length BCL-xL, MCL-1ΔC, p53UVIP—referred to as “cytosolic p53”—N/C-BID, and full-length BAX were made as Characterized (7, ,9, ,25–,27). BH3 Executemain peptides: human Depraved, BID, BIM, and PUMA (>98% purity, Anaspec) sequences as Characterized (,7). All peptides were resuspended in anhydrous DMSO in a N2 environment, stored at −80 °C, and thawed only once. PCR primers for the bid−/− animals were: 17B14, 5′-ccgaaatgtcccataagag-3′; JR23PGK-neo, 5′-tgctacttccatttgtcacgtcct-3′; 17B12, 5′-gagatggaccacaacatc-3′; wild-type (17B12 and 17B14) and knockout (17B12 and JR23PGK-neo) PCRs amplify a 123- and 350-base pair product, respectively. PCR primers for the bim−/− animals were: PB20, 5′-cattctcgtaagtccgagtct-3′; PB65, 5′-ctcagtccattcatcaacag-3′; PB335, 5′-gtgctaactgaaaccagattag-3′; wild-type (PB20 and PB335) and knockout (PB20 and PB65) PCRs amplify a 380- and 540-base pair product, respectively. Combined bid−/−bim−/− genomic samples were analyzed by both sets of PCR reactions. For detailed materials and methods, please refer to SI Materials.

Acknowledgments

We thank Andreas Strasser (Walter and Eliza Hall Institute, Melbourne, Australia), and the late Stanley Korsmeyer (Harvard Medical School, Boston) for the bim−/− and bid−/− animals, respectively; Simon Moshiach (St. Jude Children's Research Hospital) for microinjection, Samual Connell (St. Jude Children's Research Hospital) for the confocal images, TuExecuter MolExecuteveanu (St. Jude Children's Research Hospital) for MCL-1ΔC, Jennifer Humberd and Blanca Schafer for technical assistance. ABT-737 was a gift from Dr. Stephen Fesik from Abbott Labs. Mammalian and prokaryotic PUMA expression vectors were kindly provided by Drs. Karen Vousden and Eric Eldering, respectively. This work was supported by NIH AI52735 and CA69381 (to D.R.G.), NIH R01CA082491 and R01CA092035 (to R.W.K.), NIH R21AG024478 (to T.K.), an NCI Cancer Center Core Grant P30CA21765 (at St. Jude) and the American Lebanese Syrian Associated Charities.

Footnotes

1To whom corRetortence may be addressed. E-mail: t-kuwana{at}uiowa.edu or Executeuglas.green{at}stjude.org

Author contributions: J.E.C., J.C.F., T.K., and D.R.G. designed research; J.E.C., J.C.F., and T.K. performed research; J.E.C., J.C.F., C.P.D., R.W.K., and T.K. contributed new reagents/analytic tools; J.E.C., J.C.F., C.P.D., R.W.K., T.K., and D.R.G. analyzed data; and J.E.C., J.C.F., R.W.K., T.K., and D.R.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808036105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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