Sequence and Helicity Requirements for the Proapoptotic Activity of Bax BH3 Peptides

Molecular Cancer Therapeutics 1343

Sequence and helicity requirements for the proapoptotic activity of Bax BH3 peptides

Sanjeev Shangary,1 Christopher L. Oliver,2 PC-3 cells. Our results define 15 amino acids as the minimal Tommy S. Tillman,3 Michael Cascio,3 and length required for Bax BH3 peptide biological activity and Daniel E. Johnson1,4 show that amino acids COOH terminal to the BH3 core sequence are less critical than those located NH terminal 1 2 3 2 Departments of Medicine, Otolaryngology, Molecular Genetics to the core. In addition, circular dichroism spectroscopy and Biochemistry, and 4Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania revealed that high a-helical content generally correlated with, but was not sufficient for, peptide activity. Taken together, these studies provide a basis for future optimiza- Abstract tion of Bax BH3 peptide as a therapeutic anticancer agent. [Mol Cancer Ther 2004;3(11):1343–53] Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL is commonly observed in human malignancies and contributes to chemotherapy and radiation resistance. Bcl- Introduction 2 and Bcl-X inhibit apoptosis by binding to proapoptotic L Apoptosis is a genetically defined form of cell suicide that proteins such as Bax, thereby preventing chemotherapy- is critically important for tissue homeostasis. The Bcl-2 induced or radiation-induced release of cytochrome c from protein family, which consists of both proapoptotic and mitochondria and subsequent activation of the caspase antiapoptotic members, acts to regulate apoptosis via protease cascade. Efforts to inhibit Bcl-2 or Bcl-XL governance of the ‘‘intrinsic’’ pathway of cell death. The function in tumor cells have focused on developing agents intrinsic or mitochondrial-mediated pathway is character- to inhibit the interactions of these proteins with proapo- ized by mitochondrial release of apoptogenic factors ptotic proteins. Peptides derived from the BH3 domains of including cytochrome c, Smac/DIABLO, and apoptosis- proapoptotic proteins have been shown to disrupt the inducing factor following treatment of cells with death interactions of Bcl-2 and Bcl-X with key binding partners L stimuli such as chemotherapy drugs or ionizing radiation in cell-free reactions and to promote cellular apoptosis. (1–5). The release of cytochrome c and Smac/DIABLO However, less is known about the targets of BH3 peptides leads to subsequent activation of the caspase protease in intact cells as well as the sequence, length, and cascade, which promotes the internal destruction of the cell conformational requirements for peptide biological activ- (1, 2, 4–7). Antiapoptotic Bcl-2 family members, including ity. In this report, we show that cell-permeable Bax BH3 Bcl-2 and Bcl-XL, inhibit apoptosis by preventing the peptides physically disrupt Bax/Bcl-2 heterodimerization in release of apoptogenic proteins into the cytosol (1–3), intact cells and that this disruption correlates with peptide- whereas proapoptotic proteins, including Bax and Bak, induced cell death. A point-mutant, control peptide that promote such release (8–10). In view of their ability to failed to disrupt intracellular Bax/Bcl-2 interactions also prevent apoptosis, it is not surprising that Bcl-2 and Bcl-X failed to promote apoptosis. To determine important L are commonly overexpressed in human malignancies (11– sequence, length, and structural requirements for peptide 14). Moreover, in several forms of cancer, Bcl-2 or Bcl-XL activity, we generated and systematically analyzed the overexpression has been shown to correlate with chemo- biological activities of 17 Bax BH3 peptide variants. therapy and radiation resistance as well as with poor Peptides were quantitatively examined for their ability clinical prognosis (11–14). These observations highlight the to inhibit Bax/Bcl-2 and Bax/Bcl-XL heterodimerization need to develop effective agents that specifically target Bcl- in vitro and to promote cytochrome c release from 2 or Bcl-X in the tumor cells. mitochondria isolated from Jurkat, HL-60, U937, and L Antisense molecules directed against mRNA for Bcl-2 or Bcl-XL have been used to down-regulate the expression of these proteins in cell culture and in vivo (15–17). In Received 7/13/04; revised 8/18/04; accepted 8/27/04. particular, G3139, an 18-nucleotide antisense molecule, has Grant support: Leukemia and Lymphoma Society of America Translational been shown to down-regulate Bcl-2 expression in tumor Research Award 6456 and NIH grant RO1 CA86980 (D.E. Johnson). cell lines (17) and has exhibited anticancer activity in The costs of publication of this article were defrayed in part by the clinical trials (13, 16, 18, 19). More recently, several agents payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to have been developed that target Bcl-2 or Bcl-XL function. indicate this fact. The application of a variety of techniques, including Requests for reprints: Daniel E. Johnson, Division of Hematology/ fluorescence polarization binding assays, nuclear magnetic Oncology, University of Pittsburgh, and University of Pittsburgh Cancer resonance shift assays, computer modeling, and cell-based Institute, Room 2.18c, Hillman Cancer Center, 5117 Center Avenue, Pittsburgh, PA 15213. Phone: 412-623-3245; Fax: 412-623-7768. cytotoxicity assays, has led to the identification of several E-mail: [email protected] small organic molecules that bind and/or inhibit Bcl-2 and C Copyright 2004 American Association for Cancer Research. Bcl-XL, including HA-14-1 (20), antimycin A3 (21), BH3Is

Mol Cancer Ther 2004;3(11). November 2004

(22), gossypol (23), and compound 6 (ref. 24; small organic Materials and Methods molecule inhibitors reviewed in refs. 13, 25, 26). In addition, Cell Lines and Reagents increased understanding of the three-dimensional struc- Vector-transfected Jurkat T leukemic cells and Jurkat cells tures of Bcl-2 family members, and the interactions that overexpressing Bcl-2 were generated as described previous- occur between these proteins, has led to the design of ly (47). Jurkat cells overexpressing Bcl-XL were a kind gift of peptide-based agents to inhibit Bcl-2 and Bcl-XL function Dr. Craig Thompson (University of Pennsylvania, Phila- (27–29). delphia, PA). Transfected Jurkat cell lines were maintained Structural and functional studies have revealed that in RPMI 1640 (Life Technologies, Gaithersburg, MD) protein-protein interactions among Bcl-2 family members containing 10% fetal bovine serum, 2 mmol/L L-glutamine, play a key role in the ability of these proteins to regulate 1% penicillin/streptomycin, 0.2% fungizone, and 0.5 mg/ apoptosis. Following treatment of cells with agents that j mL G418 in a humidified atmosphere of 5% CO2 at 37 C. activate the intrinsic pathway, Bax and/or Bak homooligo- HL-60 and U937 cells were cultured in RPMI 1640 and PC-3 merize in the mitochondrial outer membrane and facilitate prostate cancer cells were maintained in F-12K medium the release of apoptogenic factors (10, 30–32). Antiapop- (Mediatech, Inc., Herndon, VA) supplemented with 10% totic Bcl-2 and Bcl-XL can inhibit this process by hetero- fetal bovine serum, 2 mmol/L L-glutamine, 1% penicillin/ dimerizing with the Bax or Bak proteins (33–37). streptomycin, and 0.2% fungizone. Mutational analyses have determined that the BH3 domain Polyclonal anti-Bax antibody (N20) was obtained from of proapoptotic proteins is necessary and sufficient for Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal heterodimerization with antiapoptotic proteins (38–40). anti-Bcl-2 antibody (clone 124) was from DAKO (Carpin- Thus, it has been reasoned that peptides based on the BH3 teria, CA), polyclonal anti-caspase-3 was from Cell Signal- domains of proapoptotic proteins may have the capacity to ing Technology (Beverly, MA), anti-tubulin (clone DM1A) disrupt physical interactions between proapoptotic and was from Sigma Chemical Co. (St. Louis, MO), and anti– antiapoptotic Bcl-2 family members and thereby promote cytochrome c oxidase IV (clone 2OE8) was from Molecular apoptosis in Bcl-2-overexpressing or Bcl-XL-overexpressing Probes (Eugene, OR). Monoclonal antibodies recognizing cells. poly(ADP-ribose) polymerase (clone 4C10-5) and cyto- Previous studies have shown that peptides derived chrome c (clone 7H8.2C12) were purchased from PharMin- from the BH3 domains of Bak (19-mer), Bid (20-mer), Bad gen (San Diego, CA). (21-mer), and Bax (20-mer) are capable of inducing Peptide Synthesis apoptosis in a variety of cell line models (41–44). Peptides were synthesized on a Pioneer peptide synthe- Peptide-induced apoptosis was associated with activation sizer (Applied Biosystems, Foster City, CA) using a of the intrinsic cell death pathway and release of fluoren-9-ylmethoxycarbonyl synthesis protocol (Peptide cytochrome c from the mitochondria (41, 43, 45). The Synthesis Facility, Molecular Medicine Institute, University ability of Bax, Bid, and Bim BH3 peptides to provoke of Pittsburgh). Synthesis was done by stepwise addition of cytochrome c release from purified mitochondria suggests activated amino acids to the solid support (polyethylene that the peptides directly target Bcl-2 family members glycol-polystyrene resin) starting from the COOH termi- present in the outer mitochondrial membrane (41, 43, 45), nus. Activation of amino acids was done by DIPEA/ although direct targeting of Bcl-2 family members in intact HOBT/TBTU chemistry. Peptides were cleaved from the cells has not been firmly established. Furthermore, the resin using reagent R (90% trifluoroacetic acid, 5% ability of BH3 peptides to promote apoptosis in Bcl-2- thioanisole, 3% ethanedithiol, 2% anisole) and subjected overexpressing or Bcl-XL-overexpressing cells underscores the potential utility of these peptides as novel anticancer to multiple ether extractions. The crude peptides were agents (41–44, 46). However, future clinical application of purified to >95% purity by gel filtration (G-25 column) and the BH3 peptides, or derivatives thereof, is hindered by a reverse-phase high-performance liquid chromatography lack of understanding regarding the minimal sequence and (486 and 600E; Waters Corporation, Milford, MA) on an structural characteristics needed for biological activity. acetonitrile/trifluoroacetic acid gradient. Correct mass was Because shorter peptides are likely to exhibit improved confirmed by electrospray mass spectroscopy (Quattro II, delivery and stability, efforts are needed to needed to Fisons Inc., Valencia, CA). Peptides were reconstituted as optimize the size and sequence composition of proapop- 10 mmol/L stock solutions by dissolving in DMSO (Ant- totic BH3 peptides. In this report, we used in vitro Bax BH3 peptides) or 10 mmol/L Tris (pH 7.4; untagged heterodimerization assays and cytochrome c release assays peptides) and then stored as aliquots at 80jC. to determine the minimal length and essential sequence Intracellular Heterodimerization composition of Bax BH3 peptide. Additionally, we did To examine the effects of the Ant-Bax BH3, Ant-Bax BH3 circular dichroism (CD) spectroscopy to evaluate the CS, and Ant-Bax BH3 CS/LE peptides on intracellular Bax/ relationship between a-helical content and the proapoptotic Bcl-2 heterodimerization, coimmunoprecipitation assays activity of Bax BH3 peptides. Our results define an were done. HL-60 cells (2 106) were first incubated in optimized Bax BH3 peptide of 15 amino acids and indicate the absence or presence of peptides for 12 hours at 37jC. that a-helical content is important but not sufficient for Whole cell lysates were then prepared in coimmunupreci- potent proapoptotic activity. pitation lysis buffer [10 mmol/L HEPES, 140 mmol/L KCl,

Mol Cancer Ther 2004;3(11). November 2004

5 mmol/L MgCl2, 1 mmol/L EDTA (pH 7.4)] containing determined using the Bio-Rad protein assay kit (Hercules, 0.2% NP40. Anti-Bax was added to a final concentration of CA) and homogenization buffer was added to achieve a 2 Ag/mL and incubation was allowed to proceed for 2 final concentration of 2 mg/mL. Cytochrome c release hours at 4jC followed by addition of 20 AL of protein assays were done by incubating 100 Ag of mitochondria in A-agarose beads for 2 hours. The immunoprecipitates were the absence or presence of peptides for 1 hour at 37jC. boiled, electrophoresed on 12.5% SDS-PAGE gels, trans- Following incubation, reaction mixtures were centrifuged at ferred to nitrocellulose, and subjected to immunoblotting 10,000 g for 30 minutes at 4jC to pellet mitochondria, with anti-Bcl-2. whereas supernatants were carefully collected and sub- Protein Purification and In vitro Tr a n s l a t i o n s jected to a second centrifugation. Resulting supernatants Glutathione S-transferase (GST)-Bax protein was pro- and the mitochondrial pellets were analyzed by immuno- duced in Escherichia coli harboring pGEX-2T-Bax construct blotting for release and depletion of cytochrome c, (41). Bacteria were grown to an A600 nm of 0.6 to 0.8, respectively. induced with 0.5 mmol/L isopropyl-L-thio-B-D-galactopyr- CD Spectroscopy anoside for 3 hours at 37jC, and harvested by centrifuga- Far UV CD spectra were recorded on a model 202 CD tion. The pellet was resuspended in HKMEN buffer [10 spectrometer (Aviv Instruments, Lakewood, NJ) with a 0.1 mmol/L HEPES (pH 7.2), 140 mmol/L KCl, 5 mmol/L mm path length cuvette and thermostatically controlled MgCl2, 2 mmol/L EDTA, 0.5% NP40] containing 0.03% temperature of 25jC. The spectra were collected with a SDS, 100 Ag/mL lysozyme, 1 Ag/mL aprotinin, 1 Ag/mL 1-nm step size over the 280 to 185 nm wavelength range leupeptin, 1 mmol/L DTT, and 1 mmol/L phenylmethyl- using a 1-second time constant. For each peptide, at least sulfonyl fluoride. Following sonication, lysate was mixed five spectra were averaged and the background signal of the with glutathione-agarose beads to pull-down GST-Bax. buffer alone was subtracted. Peptide concentrations were Purified protein was eluted with 20 mmol/L reduced determined by quantitative ninhydrin assay using leucine glutathione prepared in 50 mmol/L Tris (pH 8.0) and then as a standard (49). The spectra were deconvoluted using a dialyzed against PBS. variable selection method, the CDSSTR algorithm (50–52), 35 35 In vitro translated S-Bcl-2 and S-Bcl-XL were synthe- and a reference protein data set consisting of 42 proteins sized from full-length cDNA (41, 48) using a TNT-coupled (5 denatured) in aqueous solution (protein reference data transcription/translation system (Promega, Madison, WI) set 6; refs. 52, 53) using the DICHROWEB server (54).5 according to the manufacturer’s instructions. Disruption of In vitro Heterodimerization Interactions Results The effect of Bax BH3 peptides on Bax/Bcl-2 or Bax/Bcl- Disruption of Intracellular Bax/Bcl-2 Heterodimeriza- XL heterodimerization interactions was examined in in vitro tion and Activation of Apoptosis Signaling by Bax BH3 assays as described previously (41). Briefly, for each assay, Peptides 5 Ag of GST-Bax were incubated with 10 ALofin vitro Previously, we and others have shown that peptides translated 35S-Bcl-2 or 35S-Bcl-X in a total reaction volume L derived from BH3 domains of proapoptotic proteins can of 50 AL. Incubations were done for 2 hours at 4jC in the physically disrupt heterodimerization interactions among absence or presence of peptides. Glutathione-agarose beads Bcl-2 family members in cell-free systems (41, 55, 56). were then added for 1 hour followed by washing of the Specifically, we have shown that a 20–amino acid Bax BH3 beads with HKMEN buffer. The washed beads were boiled peptide disrupts Bax/Bcl-2 heterodimerization interactions in SDS-PAGE sample buffer and the resulting supernatant with an IC of 15 Amol/L and Bax/Bcl-X interactions with proteins were electrophoresed on 12.5% SDS-PAGE gels 50 L an IC of 9.5 Amol/L (41). To verify that Bax BH3 peptides and then transferred to nitrocellulose membranes. Autora- 50 also disrupt heterodimerization interactions in whole cells, diography and densitometry were used to quantify the Bax BH3 peptides were introduced into whole cells. In amount of radioactive protein bound to GST-Bax. prior studies, we have achieved successful intracellular Isolation of Mitochondria and Cytochrome c Release delivery of peptides using a lipid-mediated delivery agent Assays (BioPorter peptide delivery system, Gene Therapy Systems, To isolate mitochondria, cells were washed with PBS and San Diego, CA; ref. 41). However, long-term exposure of then resuspended in homogenization buffer [20 mmol/L cells to the BioPorter agent resulted in high background HEPES (pH 7.4), 10 mmol/L KCl, 1.5 mmol/L MgCl2, levels of cell death. Therefore, for the current experiments, 1 mmol/L EDTA, 250 mmol/L sucrose] containing 1 mmol/L we employed Bax BH3 peptides fused to the 16–amino A DTT, 1.5 mmol/L phenylmethylsulfonyl fluoride, 3 g/mL acid peptide transduction domain from Drosophila Anten- A leupeptin, and 20 g/mL aprotinin. The resuspended cells napedia protein (termed Ant peptide; Fig. 1A). The Ant were homogenized in a type B Dounce homogenizer peptide was fused to the NH2 terminus of 20-mer Bax BH3 (Wheaton, Millville, NJ). Nuclei and other debris were peptide to generate Ant-Bax BH3. In addition, to minimize j removed by centrifugation at 600 g for 5 minutes at 4 C. the potential for intracellular oxidation of the peptide, we Supernatants were subjected to a second centrifugation synthesized a variant peptide called Ant-Bax BH3 CS in again for 30 minutes at 10,000 g to pellet mitochondria, which were then resuspended in homogenization buffer containing protease inhibitors. Protein concentrations were 5 http://www.cryst.bbk.ac.uk/cdweb/html/home.html

Mol Cancer Ther 2004;3(11). November 2004

In Fig. 1B, peptides were added to intact HL-60 cells for 12 hours followed by preparation of whole cell lysates and immunoprecipitation of cellular Bax. The immunoprecipitates were then analyzed by immunoblotting for the presence of coimmunoprecipitating Bcl-2. HL-60 cells were chosen for these experiments because they are known to express high levels of Bax and Bcl-2. As depicted, treatment of cells with 5 or 25 Amol/L Ant-Bax BH3 peptide efficiently disrupted intracellular interactions between Bax and Bcl-2. The Ant-Bax BH3 CS peptide was somewhat less effective than wild-type Ant-Bax BH3 peptide when used at 5 Amol/L but comparable with wild-type activity when used at 25 Amol/L. As expected, the Ant-Bax BH3 CS/LE mutant control peptide failed to disrupt Bax/Bcl-2 heterodimers even when used at 25 Amol/L. Taken together, these results verified that the ability of synthetic Bax BH3 peptides to disrupt heterodimerization interactions in cell-free reactions correlates with their ability to disrupt physiologically relevant interactions in whole cells. The inability of Ant-Bax BH3 peptide to achieve complete disruption of intracellular interactions (Fig. 1B), compared with complete disruption of heterodimerization interactions observed previously in in vitro assays (41), may point to functional inactivation of the peptide in the complex intracellular milieu or to limited cellular permeability. At the same time, the ability of Ant-Bax BH3 peptide to cause at least partial disruption of intracellular interactions showed that fusion of the Ant peptide transduction domain to the NH2 terminus of the Bax BH3 peptide did not profoundly interfere with the ability of the peptide to bind to the BH3 binding domain on the surface of the Bcl-2 protein. Figure 1. Cell-permeable Bax BH3 peptides disrupt intracellular Bax/Bcl- To determine whether Bax BH3 peptides activate 2 heterodimerization and activate apoptosis signaling in HL-60 cells. A, apoptosis signaling in whole cells, the cell-permeable amino acid sequences of three cell-permeable BH3 peptides. Mutated amino acids are underlined. B, HL-60 cells (1 106 cells/lane) were peptides were incubated with HL-60 cells for 12 hours plated at a density of 1 106 cells/mL and then treated in the absence followed by immunoblot analysis of caspase activation (Fig. (Control) or presence of 5 or 25 Amol/L of the indicated Ant-tagged 1C). Both Ant-Bax BH3 and Ant-Bax BH3 CS peptides peptides. After 12 hours at 37jC, whole cell extracts were prepared and cellular Bax immunoprecipitated with anti-Bax polyclonal antibody. The induced processing of the 32-kDa pro-caspase-3 zymogen immunoprecipitated proteins were resolved on a 12.5% SDS-PAGE gel, to an active enzyme form. The activation of caspase-3 by transferred to nitrocellulose, and probed with anti-Bcl-2 monoclonal both peptides was accompanied by cleavage of the caspase- antibody. To verify equivalent immunoprecipitation of Bax in the different samples, the blot was also probed with anti-Bax polyclonal antibody. C, 3 substrate proteins poly(ADP-ribose) polymerase and Bcl- HL-60 cells were plated at 1 106 cells/mL and then treated in the 2 to 85 and 23 kDa fragments, respectively. By contrast, absence (Control) or presence of 25 Amol/L of the indicated Ant-tagged Ant-Bax BH3 CS/LE mutant control peptide had no effect peptides. Following a 12-hour incubation, aliquots of the cells were used on caspase activation or the cleavage of caspase substrate to determine percentage cell death as assessed by trypan blue exclusion assay. In addition, whole cell lysates were prepared and subjected to proteins. Moreover, although Ant-Bax BH3 and Ant-Bax immunoblotting with anti-caspase-3, anti – poly(ADP-ribose) polymerase BH3 CS led to 62% and 79% cell death, respectively, the (PARP), and anti-Bcl-2. The locations of intact and cleaved forms of the control peptide did not affect cell viability. proteins are indicated. The blot was stripped and reprobed with anti- tubulin to verify equal loading. Disruption of Bax/Bcl-2 Heterodimerization by Bax BH3 Peptide Variants We next sought to determine the minimal length and which Cys62 (numbering based on human Bax sequence) in important sequence requirements for the biological activity the BH3 domain was changed to Ser62. A control peptide, of Bax BH3 peptide. Having verified that the activities of Ant-Bax BH3 CS/LE, was also synthesized in which Leu63 Bax BH3 peptides in cell-free assays accurately reflect was mutated to Glu63. Mutation of Leu63 to Glu63 in the activities observed in whole cells (ref. 41; Fig. 1), we did our full-length protein has been shown to markedly reduce Bax studies in vitro using peptides lacking the Ant peptide function (57) and we have shown that peptides with this transduction domain. A series of peptide variants (Table 1) mutation are ineffective at disrupting heterodimerization were synthesized and compared with parental Bax BH3 20- interactions in vitro (41). mer (Bax 1) for biological activity. Because homology

Mol Cancer Ther 2004;3(11). November 2004

Table 1. Properties of the Bax BH3 peptides

Peptide Sequence* Length Disruption of heterodimerization Cytochrome a-Helical content c b x (amino acids) IC50 (Amol/L) c release (%)

Bax/Bcl-2 Bax/Bcl-XL Jurkat HL-60 U937

Bax 1 parental STKKLSECLKRIGDELDSNM 20 4.7 2.0 + + + 62 Bax 2 AAKKLSECLKRIGDELDSNM 20 3.3 2.3 + + + 77 Bax 3 STKKLSECLKRIGDELDS-- 18 62.6 18.3 + + + 35 Bax 4 STKKLSECLKRIGDELDS-M 19 18.4 7.6 + + + 53 Bax 5 STKKLSECLKRIGDELD--M 18 17.1 2.1 + + + 45 Bax 6 STKKLSECLKRIGDEL---M 17 15.4 8.0 + + + 65 Bax 7 STKKLSECLKRIGDE----M 16 30.7 7.4 + + + 38 Bax 8 STKKLSECLKRIGD-----M 15 > > 46 Bax 9 --KKLSECLKRIGDELDSNM 18 450.1 148.0 + ND Bax 10 ---KLSECLKRIGDELDS-- 15 > > 36 Bax 11 ----LSECLKRIGDELDSN- 15 > > 25 Bax 12 STKKL-ECLKRIGDELDSNM 19 > > 37 Bax 13 STKKL--CLKRIGDELDSNM 18 > > ND Bax 14 STKKL---LKRIGDELDSNM 17 > > ND Bax 15 STKKLSECL---GDELDSNM 17 > > 16 Bax 16 STKKLSECLKRIGDEL---- 16 > 20.0 + + + 63 Bax 17 -TKKLSECLKRIGDEL---- 15 > 79.0 + + 82 Bax 18 -TKKLSECLKRIGDE----- 14 > > 39

*The highly conserved BH3 core sequence is italicized. c Average of three experiments. >, the IC50 value was >500 Amol/L. b Mitochondria isolated from vector/Jurkat, Bcl-2/Jurkat, and Bcl-XL/Jurkat cells showed similar responses to peptides. +, ability to promote cytochrome c release; , failure to promote release. xa-Helical content refers to the sum of ‘‘helix 1’’ and ‘‘helix 2’’ from the CDSSTR algorithm. ND, helicity was not determined.

between BH3 domains from different proapoptotic proteins while still retaining an ability to disrupt Bax/Bcl-2. is highest in the central 7–amino acid BH3 core (italicized However, in these deletions, we found that it was in Table 1), we focused on deleting less conserved amino important to retain the last COOH-terminal methionine acids outside of this core sequence. Briefly, peptide variants residue (compare Bax 16 versus Bax 6 or 7). Third, all were generated containing deletions at the NH2 terminus, internal deletions abolished peptide activity. The internal deletions in the COOH terminus, or a combination of deletion mutants were designed to remove amino acids deletions at both ends. In addition, several peptides were that are less conserved among BH3 domains (57, 58). Up to generated containing deletions of internal amino acids. Bax three contiguous internal amino acids were deleted in an 2 was designed to substitute two NH2-terminal residues effort to preserve the amphipathic nature of the BH3 with alanine residues (ST/AA). domain a-helix. However, none of the internal deletion The peptide variants were first examined for their abili- mutants retained an ability to disrupt Bax/Bcl-2. In ty to disrupt Bax/Bcl-2 heterodimerization interactions summary, these experiments defined a 16–amino acid in vitro when used at 200 Amol/L (Fig. 2A). Peptides that peptide (Bax 7) as the shortest peptide capable of efficiently displayed an ability to disrupt Bax/Bcl-2 were subsequent- disrupting Bax/Bcl-2 interactions. ly evaluated in greater detail to determine the IC50 value of Disruption of Bax/Bcl-XL Heterodimerization by Bax the peptide for disrupting Bax/Bcl-2 interactions (Fig. 2B; BH3 Peptide Variants Table 1). The findings from these experiments can be We next examined whether the variant Bax BH3 peptides summarized as follows: First, deletion of two or more could disrupt Bax/Bcl-XL heterodimerization interactions amino acids from the NH2 terminus abolished peptide (Fig. 3; Table 1). In general, the abilities of peptides to activity. For example, Bax 9, with two amino acids deleted disrupt Bax/Bcl-XL correlated with their abilities to disrupt f F at the NH2 terminus, exhibited an IC50 value of 450.1 Bax/Bcl-2. However, all of the active peptides showed F 30.3 Amol/L compared with an IC50 value of 4.7 1.5 significantly lower IC50 values for disrupting Bax/Bcl-XL Amol/L for parental Bax 1 peptide. Interestingly, substitu- than for disrupting Bax/Bcl-2. Peptides that failed to dis- tion of the two NH2-terminal amino acids with alanine rupt Bax/Bcl-2 were found ineffective at disrupting Bax/ residues did not affect activity, as Bax 2 exhibited an IC50 Bcl-XL, with two notable exceptions. Bax 16, a 16–amino value of 3.3 F 0.8 Amol/L. Second, deletion of up to four acid peptide, and Bax 17, a 15–amino acid peptide, while amino acids in the COOH terminus could be accomplished failing to disrupt Bax/Bcl-2 (Fig. 2), disrupted Bax/Bcl-XL

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assessed by immunoblotting of supernatant proteins (Fig. 4; Table 1). To determine the abilities of the peptides to overcome the inhibitory effects of Bcl-2 or Bcl-XL overexpression, experiments were done using mitochondria isolated from vector-transfected Jurkat cells, Bcl-2-overexpressing Jurkat cells, and Bcl-XL-overexpressing Jurkat cells. As shown, each of the peptides that disrupted Bax/ Bcl-2 or Bax/Bcl-XL interactions in vitro showed an ability to promote cytochrome c release. Similar profiles were seen when mitochondria from HL-60, U937, or PC-3 cells were used (Fig. 5; Table 1; data not shown). Expectedly, release of cytochrome c was accompanied by a corresponding loss of cytochrome c from the mitochondrial pellet. The specificity of the cytochrome c release was confirmed by immunoblotting for cytochrome c oxidase IV, a mitochon- Figure 2. Disruption of Bax/Bcl-2 heterodimerization by Bax BH3 peptide drial protein that is not released into the cytosol during variants. A, in vitro translated 35S-Bcl-2 (10 AL) was incubated with GST- apoptosis signal transduction. Bax (5 Ag) for 2 hours at 4jC in the absence (Control) or presence of 200 The results shown in Fig. 4 also showed that peptides Amol/L of the indicated peptides. Glutathione-agarose was then used to pull down the GST-Bax along with bound 35S-Bcl-2. Samples were capable of promoting cytochrome c release from mitochon- resolved on a 12.5% SDS-PAGE gel, transferred to nitrocellulose, and dria of vector-transfected Jurkat cells also promoted release subjected to autoradiography. Lane 1, 20% of the input 35S-Bcl-2; lane 2, from mitochondria of Bcl-2-overexpressing or Bcl-XL-over- control lane reflects incubation in the absence of peptide. To determine the homogeneity of the purified fusion proteins used in the experiment, 5 Agof expressing cells (Fig. 4; Table 1). Thus, Bcl-2 and Bcl-XL, purified GST or purified GST-Bax were resolved on a 10% SDS-PAGE gel which normally inhibit cytochrome c release, are function- followed by staining with Coomassie blue. B, peptides exhibiting an ability ally inactivated by peptides that disrupt the interactions of to disrupt Bax/Bcl-2 interactions were examined in quantitative fashion to these proteins with proapoptotic partners. Curiously, determine IC50 values. For each peptide tested, a dose-response curve was established for disruption of Bax/Bcl-2 interactions using the in vitro assay certain peptides (e.g., Bax 9 and Bax 17) with high IC50 described in A. For each peptide concentration, densitometry of the values for disruption of heterodimerization interactions autoradiograph was used to determine the percentage of Bax/Bcl-2 (Table 1) were still capable of promoting cytochrome c heterodimerization relative to that observed in the absence of peptide. Percentage disruption was calculated according to the equation: percent- release, indicating that partial inhibition of Bcl-2 or Bcl-XL age disruption = (35S-Bcl-2 densitometric signal in the presence of pep- may be sufficient to trigger the release of cytochrome c. tide) / (35S-Bcl-2 densitometric signal in the absence of peptide) 100. Interestingly, both Bax 16 (16-mer) and Bax 17 (15-mer), IC50 values were calculated from the point of 50% disruption of Bax/Bcl-2 heterodimerization and are shown in Table 1. which were capable of disrupting Bax/Bcl-XL but not Bax/

F F with IC50 values of 20 4.1 and 79 18.3 Amol/L, respectively (Fig. 3; Table 1). Thus, two different 16–amino acid peptides (Bax 7 and Bax 16) and one 15–amino acid peptide (Bax 17) were capable of disrupting Bax/Bcl-XL interactions. Induction of Cytochrome c Release by Bax BH3 Pep- tide Variants During apoptosis mediated by the intrinsic mitochondrial pathway, the release of cytochrome c into the cytosol constitutes a key step toward activation of the caspase protease cascade and ultimate cellular demise (1, 2, 6, 7). Bcl-2 and Bcl-XL prevent apoptotic cell death by inhibiting cytochrome c release (1, 2, 59), whereas proapoptotic proteins such as Bax promote cytochrome c release (8, 30, 60). It is thought that Bcl-2 and Bcl-XL may prevent cytochrome c release by binding and inhibiting Bax or other proapoptotic proteins. Previously, we have shown that the parental 20–amino acid Bax BH3 peptide (Bax 1), in addition to disrupting Bax/Bcl-2 and Bax/Bcl-XL, can Figure 3. Disruption of Bax/Bcl-XL heterodimerization by Bax BH3 promote cytochrome c release from isolated mitochondria peptide variants. A, in vitro assays of Bax/Bcl-XL heterodimerization were 35 (41). Therefore, we examined the activity of the variant done by incubating GST-Bax with S-Bcl-XL in the absence (Control)or peptides in cytochrome c release assays. presence of the indicated Bax BH3 peptides (200 Amol/L). Reaction mixtures were analyzed as described in Fig. 2A. B, dose-response curves Peptides were added for 1 hour to mitochondria isolated of peptide-mediated disruption of Bax/Bcl-XL interactions were done as from Jurkat T leukemic cells and cytochrome c release was described in Fig. 2B and IC50 values for the peptides are shown in Table 1.

Mol Cancer Ther 2004;3(11). November 2004

Figure 4. Induction of cytochrome c release from isolated Jurkat mitochondria by Bax BH3 peptide variants. Mitochondria were isolated from vector- transfected Jurkat cells (A; Vector/Jurkat), Bcl-2-overexpressing Jurkat cells (B; Bcl-2/Jurkat), and Bcl-XL-overexpressing Jurkat cells (B; Bcl-X L /Jurkat) as described in Materials and Methods. Isolated mitochondria (100 Ag/sample) were then incubated for 1 hour at 37jC in the absence (Control) or presence of the indicated peptides (200 Amol/L). Following incubation, the mitochondria were pelleted to obtain supernatant (released proteins) and pellet (mitochondria) fractions. The supernatant and pellet fractions were electrophoresed on 12.5% SDS-PAGE gels, transferred to nitrocellulose, and probed with anti – cytochrome c (Cyt c). The pellet fraction was also probed with anti – cytochrome c oxidase IV (Cyt c Ox IV) to verify the specificity of the cytochrome c release.

Bcl-2, promoted cytochrome c release from Jurkat mito- context of the full-length protein, can be extremely flexible chondria (Fig. 4). In a dose-response experiment, Bax 17 and often show little net structure in aqueous solution. To (15-mer) exhibited nearly equal potency to that of Bax 1 (20- determine whether helical content of the Bax BH3 peptides mer) parental (Fig. 6). Additional experiments showed that correlated with biological activity, we used CD spectros- Bax 17 (15-mer) also promotes cytochrome c release from copy to analyze peptide a helicity. In 10 mmol/L HL-60 mitochondria but not from mitochondria of U937 or potassium phosphate buffer (pH 7.4), the 20–amino acid PC-3 cells (Fig. 5; Table 1; data not shown). By contrast, Bax Bax BH3 parental peptide showed a CD spectrum 16 (16-mer) promoted cytochrome c release from mito- characteristic with that of a random coil, although we were chondria of all four cell types (Jurkat, HL-60, U937, and PC- unable to deconvolute it (data not shown). However, in a 3). Thus, although Bax 17 is the shortest peptide capable of 40% solution of trifluoroethanol (TFE), this peptide showed inducing cytochrome c release, the biological activity of this a strong total helicity of 62%. TFE/H2O mixtures promote 15–amino acid peptide seems to depend on the specific intrapeptide hydrogen bonds and are commonly used to cellular environment. determine the helix-forming propensities of small flexible The biological activity of Bax 9 also seems to be cell type peptides (61, 62). In previous studies, BH3-derived pep- specific. Surprisingly, Bax 9 promoted release of cyto- tides were examined by CD spectroscopy in 40% (v/v) TFE chrome c from Jurkat mitochondria despite only weak to test whether the helical content of the peptide correlated ability to disrupt Bax/Bcl-2 and Bax/Bcl-XL interactions with toxicity (44). Analogously, 40% TFE was used in our (Figs. 2 and 3). In experiments with HL-60, U937, or PC-3 studies to compare the relative helical content of all the mitochondria, the Bax 9 peptide did not cause cytochrome peptides. The CD spectra of the tested peptides are shown c release (Fig. 5; Table 1; data not shown). in Fig. 7. The percentage of a-helical content was quantified a-Helical Content of the Bax BH3 Peptide Variants as described in Materials and Methods and is shown in Nuclear magnetic resonance imaging and X-ray crystal- Table 1. All values obtained had a normalized root mean lography of full-length Bcl-XL, Bcl-2, and Bax proteins have square deviation (63) of <0.02, indicating that the reference revealed that BH3 domains exist in an a-helical conforma- database used in the CD studies was appropriate in fitting tion (27–29). However, short peptides, when taken out of the experimentally determined CD spectra.

Figure 5. Induction of cytochrome c release from isolated HL-60 and U937 mitochondria. Isolated mitochondria (100 Ag) were incubated with the indicated peptides for 1 hour at 37jC, and specific release of cytochrome c was assessed as described in Fig. 4.

Mol Cancer Ther 2004;3(11). November 2004

apoptosis pathway (1, 2, 6, 10). Antisense-mediated down- regulation of Bcl-2 or Bcl-XL enhances the sensitivity of cancer cells to apoptotic stimuli, and Bcl-2 antisense (G3139) is currently being investigated in clinical trials (13, 16, 18, 19). The peptides we have characterized and optimized target the function, rather than the expression, of Bcl-2 and Bcl-XL. Using cytochrome c release assays, our results clearly show the ability of Bax BH3 peptides to overcome the antiapoptotic action of Bcl-2 and Bcl-XL. Structural and molecular modeling studies have determined that the BH1, BH2, and BH3 domains of Bcl-2 and Bcl-XL form a hydrophobic groove on the surface of these proteins (27, 28). This groove is critical for the antiapoptotic activity of Bcl-2 and Bcl-XL and serves as a receptor-like Figure 6. Parental Bax 1 (20-mer) and Bax 17 (15-mer) peptides exhibit binding pocket for BH3 domains of proapoptotic proteins similar potencies at overcoming Bcl-2 overexpression. Isolated mitochon- (64–66). Thus, this BH3 binding pocket is an attractive dria (100 Ag) from Bcl-2-overexpressing Jurkat cells were incubated for 1 hour at 37jC in the absence or presence of increasing doses of Bax 1 or Bax 17 peptides. Cytochrome c release was assessed as described in Fig. 4.

The parental Bax BH3 peptide Bax 1 exhibited 62% a-helical content, whereas the a-helical content of the biologically active peptide variants Bax 2 to 7 ranged from 35% to 77%. The a-helical content of the inactive peptides tested (Bax 8, 10, 11, 12, 16, and 18) ranged from 16% to 46%. In general, the a-helical content of biologically active peptides (58 F 16% average helicity for peptides testing positive for cytochrome c release in Jurkat cells) was higher than that observed in biologically inactive peptides (33 F 11%). This suggests that a-helical content is important for the proapoptotic biological activity of the peptides. However, the results also indicate that a-helical content alone is not sufficient for biological activity. Bax 8, which was biologically inactive, exhibited higher helical content (46%) than the two active peptides [Bax 3 (35%) and Bax 7 (38%)] and similar content to a third active peptide [Bax 5 (45%)]. Moreover, two peptides that exhibited variable biological activities, Bax 16 and 17, were found to have high helical content. Bax 16, which was capable of disrupting Bax/Bcl-XL and promoting cytochrome c release in all cell lines tested but incapable of disrupting Bax/Bcl-2, exhibited 63% a helicity. Bax 17, which failed to disrupt Bax/Bcl-2 and only promoted cytochrome c release in certain cell types, exhibited the highest a-helical content at 82%. Thus, although helical content seems to roughly correlate with biological activity, it is not sufficient; the specific sequence composition of the peptide is also critical.

Discussion Overexpression of antiapoptotic members of the Bcl-2 family protein family, including Bcl-2 and Bcl-XL, is common in human malignancies and contributes to chemotherapy and radiation resistance. In several cancers, overexpression of Bcl-2 or Bcl-XL has been shown to correlate with poor clinical Figure 7. CD spectra of the Bax BH3 peptide variants. The CD spectrum prognosis (13). At the molecular level, overexpressed Bcl-2 of each peptide represents the average of at least five spectra of the or Bcl-X binds to proapoptotic proteins such as Bax, peptide in 40% (v/v) TFE. For clarity, the spectra are divided arbitrarily into L two panels. Optical signals are presented as Dq, which has units of mdeg thereby preventing cytochrome c release into the cytosol mol/L1 cm1. The a-helical content was determined as described in and activation of the intrinsic, mitochondrial-mediated Materials and Methods and is shown in Table 1.

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74 target for drug discovery efforts aimed at inhibiting Bcl-2 or Met . Deletion of amino acids NH2 terminal to the BH3 Bcl-XL function. We and others have shown that peptides core was not tolerated, although substitution of these derived from the BH3 domains of Bax, Bid, Bad, and Bak residues with alanine residues may be sufficient to inhibit heterodimerization among Bcl-2 family members in maintain activity. in vitro assays and promote cellular apoptosis (41, 42, 46, The ability of the peptides to promote cytochrome c 55, 56, 67). In addition, several small organic molecules release from isolated mitochondria further supports the have recently been identified that bind to Bcl-2 or Bcl-XL contention that Bcl-2 or Bcl-XL in the mitochondrial outer and promote apoptosis (20–24). membrane are the direct targets of these agents. Moreover, Although several studies have examined the ability of peptides that failed to disrupt Bax/Bcl-XL were incapable BH3 peptides to disrupt protein-protein interactions in of promoting cytochrome c release. Interestingly, Bax 16 cell-free systems, little is known about the ability of these (16-mer) and Bax 17 (15-mer), which disrupted Bax/Bcl-XL peptides to directly target and interfere with antiapoptotic but not Bax/Bcl-2, retained the capacity to promote proteins in intact cells. Therefore, we first asked whether cytochrome c release from Jurkat and HL-60 mitochondria. Bax BH3 domain peptides could disrupt intracellular This suggests that Bcl-XL represents the critical antiapop- heterodimerization between Bax and Bcl-2 and whether totic protein in these cell lines. However, we cannot rule this correlates with the proapoptotic activities of the out the possibility that the Bax BH3 peptides may be peptides. We found that cell-permeable Bax BH3 peptides binding and directly activating proapoptotic Bax or Bak. that promoted caspase activation and loss of cell viability Letai et al. (43) have shown that Bid BH3 peptide binds also disrupted Bax/Bcl-2 heterodimerization in intact cells. directly to Bak, inducing oligomerization and proapoptotic By contrast, a mutant peptide that failed to induce cell death activity. The inclusion of antibodies that block the BH3 also was deficient at disrupting Bax/Bcl-2 interactions. The binding domains of Bcl-2 and Bcl-XL in experiments ability of Bax BH3 peptide to disrupt Bax/Bcl-2 hetero- involving BH3 peptide treatment of isolated mitochondria dimerization in intact cells argues strongly that the peptide may help to resolve whether the peptides are directly directly targets Bcl-2 family members in the cell. In an targeting antiapoptotic (Bcl-2 or Bcl-XL) or proapoptotic attempt to improve the intracellular stability and activity of (Bax or Bak) Bcl-2 family members on the surface of the Bax BH3 peptide, we also changed Cys62 to Ser62. However, mitochondria. the serine-containing peptide was at best equivalent in The role of a-helical content in the proapoptotic activity activity to the wild-type cysteine-containing version, of BH3 peptides has been controversial. Structural analyses indicating no significant benefit to modifying this residue. have revealed that BH3 domains exist in an a-helical The usefulness of peptides as therapeutic agents is conformation when binding to the BH3 binding pocket of limited to some degree by the large size of peptide antiapoptotic proteins (29, 64, 65). Moreover, the BH3 compounds. The large molecular weight and the chemical domain a helix is amphipathic, with hydrophobic residues composition of peptides can contribute to poor solubility, interfacing with the binding pocket and polar residues cellular permeability, and stability. Thus, when considering exposed to the aqueous environment (57, 64, 65). Strikingly, a peptide-based compound as a therapeutic agent, it is first a previous report indicated that cell death resulting from important to define the minimal size and essential sequence Bad BH3 peptide can be attributed solely to the helical composition that is needed for biological activity. By content of the peptide in 40% TFE independent of any Bcl-2 reducing the size of the peptide compound, it also becomes binding capacity (44). Our results indicate a strong more feasible to generate peptidomimetic derivatives with correlation between the ability of a peptide to disrupt improved drug-like properties. Therefore, we undertook to either Bax/Bcl-XL or both Bax/Bcl-2 and Bax/Bcl-XL and optimize the size, sequence, and structural composition of the ability to promote cytochrome c release. In addition, biologically active Bax BH3 peptide. although helical content generally correlated with biolog- To generate an optimized Bax BH3 peptide, we did a ical activity, helical content alone was not sufficient to systematic analysis of peptide variants. We identified 15 induce cytochrome c release. Bax 17 (15-mer), which amino acids as the shortest peptide length capable of exhibited the highest a-helical content (82%), was incapable promoting cytochrome c release. These results are consis- of promoting cytochrome c release from mitochondria of tent with those of Diaz et al. (55), who observed disruption two of the four cell lines tested. Thus, the precise sequence of Bax/Bcl-XL heterodimerization in an in vitro plate composition, length, and helical content all seem to play binding assay using a 15–amino acid Bax BH3 peptide. important roles in determining the biological activity of the The 15–amino acid peptide (Bax 17) we identified peptides. disrupted Bax/Bcl-XL but not Bax/Bcl-2 and promoted In summary, our studies have defined an optimal cytochrome c release from mitochondria of two of the four length as well as critical sequence and structural require- cell lines tested. A 16–amino acid peptide (Bax 7), however, ments of the Bax BH3 peptide, laying the groundwork for disrupted both Bax/Bcl-XL and Bax/Bcl-2 and promoted future optimization of the peptide, or derivatives thereof, release of cytochrome c from mitochondria of all four as an anticancer agent. Future structural modifications, cell lines. Additional analyses indicated that deletion of including lactam bridging (68), incorporation of non- amino acids COOH terminal to the BH3 core sequence natural amino acids, and generation of peptidomimetic was well tolerated but revealed a critical importance of derivatives, should help to improve stability and potency.

Mol Cancer Ther 2004;3(11). November 2004

Additionally, it will be informative to determine whether 19. Jansen B, Wacheck V, Heere-Ress E, et al. Chemosensitization of malignant melanoma by BCL2 antisense therapy. Lancet 2000;356:1728 – 33. BH3 peptides synergize with any of the growing number of 20. Wang JL, Liu D, Zhang ZJ, et al. Structure-based discovery of an small molecule Bcl-2/Bcl-XL inhibitors to promote cell organic compound that binds Bcl-2 protein and induces apoptosis of tumor killing of Bcl-2-overexpressing or Bcl-XL-overexpressing cells. Proc Natl Acad Sci U S A 2000;97:7124 – 9. tumors. Moreover, it will be important to test whether 21. Tzung SP, Kim KM, Basanez G, et al. Antimycin A mimics a cell-death- sublethal doses of optimized BH3 peptides can be used to inducing Bcl-2 homology domain 3. Nat Cell Biol 2001;3:183 – 91. sensitize drug-resistant tumor cells to chemotherapeutic 22. Degterev A, Lugovskoy A, Cardone M, et al. Identification of small- molecule inhibitors of interaction between the BH3 domain and Bcl-xL. Nat agents. Cell Biol 2001;3:173 – 82. 23. Kitada S, Leone M, Sareth S, Zhai D, Reed JC, Pellecchia M. Discovery, characterization, and structure-activity relationships studies of Acknowledgments proapoptotic polyphenols targeting B-cell lymphocyte/leukemia-2 proteins. We thank the members of Dr. Thomas Smithgall’s laboratory (University of J Med Chem 2003;46:4259 – 64. Pittsburgh School of Medicine, Pittsburgh, PA) for providing assistance 24. Enyedy IJ, Ling Y, Nacro K, et al. Discovery of small-molecule inhibitors with the densitometric scanning. of Bcl-2 through structure-based computer screening. J Med Chem 2001; 44:4313 – 24. 25. Baell JB, Huang DC. Prospects for targeting the Bcl-2 family of proteins References to develop novel cytotoxic drugs. Biochem Pharmacol 2002;64:851 – 63. 1. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of 26. Wang S, Yang D, Lippman ME. Targeting Bcl-2 and Bcl-XL with cytochrome c from mitochondria: a primary site for Bcl-2 regulation of nonpeptidic small-molecule antagonists. Semin Oncol 2003;30:133 – 42. apoptosis. Science 1997;275:1132 – 6. 27. Muchmore SW, Sattler M, Liang H, et al. X-ray and NMR structure of 2. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of human Bcl-xL, an inhibitor of programmed cell death. Nature 1996;381: cytochrome c from mitochondria blocked. Science 1997;275:1129 – 32. 335 – 41. 3. Susin SA, Lorenzo HK, Zamzami N, et al. Molecular characterization of 28. Petros AM, Medek A, Nettesheim DG, et al. Solution structure of the mitochondrial apoptosis-inducing factor. Nature 1999;397:441 – 6. antiapoptotic protein bcl-2. Proc Natl Acad Sci U S A 2001;98:3012 – 7. 4. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that 29. Suzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer promotes cytochrome c-dependent caspase activation by eliminating IAP formation and intracellular localization. Cell 2000;103:645 – 54. inhibition. Cell 2000;102:33 – 42. 30. Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH. Pro- 5. Verhagen AM, Ekert PG, Pakusch M, et al. Identification of DIABLO, a apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores mammalian protein that promotes apoptosis by binding to and antagoniz- that result in the release of cytochrome c. Cell Death Differ 2000;7:1166 – 73. ing IAP proteins. Cell 2000;102:43 – 53. 31. Mikhailov V, Mikhailova M, Degenhardt K, Venkatachalam MA, White 6. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent E, Saikumar P. Association of Bax and Bak homo-oligomers in mitochondria. formation of Apaf-1/caspase-9 complex initiates an apoptotic protease Bax requirement for Bak reorganization and cytochrome c release. J Biol cascade. Cell 1997;91:479 – 89. Chem 2003;278:5367 – 76. 7. Zou H, Li Y, Liu X, Wang X. An APAF-1.cytochrome c multimeric 32. Wei MC, Zong WX, Cheng EH, et al. Proapoptotic BAX and BAK: a complex is a functional apoptosome that activates procaspase-9. J Biol requisite gateway to mitochondrial dysfunction and death. Science 2001; Chem 1999;274:11549 – 56. 292:727 – 30. 8. Kuwana T, Mackey MR, Perkins G, et al. Bid, Bax, and lipids cooperate to 33. Ruffolo SC, Shore GC. BCL-2 selectively interacts with the BID-induced form supramolecular openings in the outer mitochondrial membrane. Cell open conformer of BAK, inhibiting BAK auto-oligomerization. J Biol Chem 2002;111:331 – 42. 2003;278:25039 – 45. 9. Wei MC, Lindsten T, Mootha VK, et al. tBID, a membrane-targeted death 34. Griffiths GJ, Dubrez L, Morgan CP, et al. Cell damage-induced ligand, oligomerizes BAK to release cytochrome c. Genes Dev 2000;14: conformational changes of the pro-apoptotic protein Bak in vivo precede 2060 – 71. the onset of apoptosis. J Cell Biol 1999;144:903 – 14. 10. Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization of 35. Antonsson B, Montessuit S, Sanchez B, Martinou JC. Bax is present as BAX results in its translocation, mitochondrial dysfunction and apoptosis. a high molecular weight oligomer/complex in the mitochondrial membrane EMBO J 1998;17:3878 – 85. of apoptotic cells. J Biol Chem 2001;276:11615 – 23. 11. Gazitt Y, Fey V, Thomas C, Alvarez R. Bcl-2 overexpression is 36. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo associated with resistance to dexamethasone, but not melphalan, in with a conserved homolog, Bax, that accelerates programmed cell death. multiple myeloma cells. Int J Oncol 1998;13:397 – 405. Cell 1993;74:609 – 19. 12. Friess H, Lu Z, Andren-Sandberg A, et al. Moderate activation of the 37. Mikhailov V, Mikhailova M, Pulkrabek DJ, Dong Z, Venkatachalam MA, apoptosis inhibitor bcl-xL worsens the prognosis in pancreatic cancer. Ann Saikumar P. Bcl-2 prevents Bax oligomerization in the mitochondrial outer Surg 1998;228:780 – 7. membrane. J Biol Chem 2001;276:18361 – 74. 13. Shangary S, Johnson DE. Recent advances in the development of 38. Chittenden T, Flemington C, Houghton AB, et al. A conserved domain anticancer agents targeting cell death inhibitors in the Bcl-2 protein family. in Bak, distinct from BH1 and BH2, mediates cell death and protein binding Leukemia 2003;17:1470 – 81. functions. EMBO J 1995;14:5589 – 96. 14. Groeger AM, Esposito V, De Luca A, et al. Prognostic value of 39. Zha H, Fisk HA, Yaffe MP, Mahajan N, Herman B, Reed JC. Structure- immunohistochemical expression of p53, bax, Bcl-2 and Bcl-xL in resected function comparisons of the proapoptotic protein Bax in yeast and non-small-cell lung cancers. Histopathology 2004;44:54 – 63. mammalian cells. Mol Cell Biol 1996;16:6494 – 508. 15. Mercatante DR, Bortner CD, Cidlowski JA, Kole R. Modification of 40. Simonen M, Keller H, Heim J. The BH3 domain of Bax is sufficient for alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. interaction of Bax with itself and with other family members and it is analysis of apoptosis and cell death. J Biol Chem 2001;276:16411 – 7. required for induction of apoptosis. Eur J Biochem 1997;249:85 – 91. 16. Webb A, Cunningham D, Cotter F, et al. BCL-2 antisense therapy in 41. Shangary S, Johnson DE. Peptides derived from BH3 domains of Bcl-2 patients with non-Hodgkin lymphoma. Lancet 1997;349:1137 – 41. family members: a comparative analysis of inhibition of Bcl-2, Bcl-x(L) and 17. Kitada S, Miyashita T, Tanaka S, Reed JC. Investigations of antisense Bax oligomerization, induction of cytochrome c release, and activation of oligonucleotides targeted against bcl-2 RNAs. Antisense Res Dev cell death. Biochemistry 2002;41:9485 – 95.

1993;3:157 – 69. 42. Holinger EP, Chittenden T, Lutz RJ. Bak BH3 peptides antagonize Bcl-xL 18. Marcucci G, Byrd JC, Dai G, et al. Phase 1 and pharmacodynamic stud- function and induce apoptosis through cytochrome c-independent activa- ies of G3139, a Bcl-2 antisense oligonucleotide, in combination with che- tion of caspases. J Biol Chem 1999;274:13298 – 304. motherapy in refractory or relapsed acute leukemia. Blood 2003;101:425 – 32. 43. Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ.

Mol Cancer Ther 2004;3(11). November 2004

Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, 56. Ottilie S, Diaz JL, Horne W, et al. Dimerization properties of human serving as prototype cancer therapeutics. Cancer Cell 2002;2:183 – 92. BAD. Identification of a BH-3 domain and analysis of its binding to mutant 44. Schimmer AD, Hedley DW, Chow S, Pham NA, Chakrabartty A, BCL-2 and BCL-XL proteins. J Biol Chem 1997;272:30866 – 72. Bouchard D, et al. The BH3 domain of BAD fused to the Antennapedia 57. Wang K, Gross A, Waksman G, Korsmeyer SJ. Mutagenesis of the BH3 peptide induces apoptosis via its a helical structure and independent of domain of BAX identifies residues critical for dimerization and killing. Mol Bcl-2. Cell Death Differ 2001;8:725 – 33. Cell Biol 1998;18:6083 – 9. 45. Polster BM, Kinnally KW, Fiskum G. BH3 death domain peptide 58. Kelekar A, Chang BS, Harlan JE, Fesik SW, Thompson CB. Bad is a BH3 induces cell type-selective mitochondrial outer membrane permeability. domain-containing protein that forms an inactivating dimer with Bcl-XL.Mol J Biol Chem 2001;276:37887 – 94. Cell Biol 1997;17:7040 – 6.

46. Wang JL, Zhang ZJ, Choksi S, et al. Cell permeable Bcl-2 binding 59. Kharbanda S, Pandey P, Schofield L, et al. Role for Bcl-xL as an inhibitor peptides: a chemical approach to apoptosis induction in tumor cells. Cancer of cytosolic cytochrome c accumulation in DNA damage-induced apopto- Res 2000;60:1498 – 502. sis. Proc Natl Acad Sci U S A 1997;94:6939 – 42. 47. Dou QP, An B, Antoku K, Johnson DE. Fas stimulation induces RB 60. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. dephosphorylation and proteolysis that is blocked by inhibitors of the ICE Science 1998;281:1322 – 6. protease family. J Cell Biochem 1997;64:586 – 94. 61. Luo P, Baldwin RL. Mechanism of helix induction by trifluoroethanol: a 48. Maser RS, Antoku K, Scully WJ Jr, Cho RL, Johnson DE. Analysis of framework for extrapolating the helix-forming properties of peptides from the role of conserved cysteine residues in the bcl-2 oncoprotein. Biochem trifluoroethanol/water mixtures back to water. Biochemistry 1997; Biophys Res Commun 2000;277:171 – 8. 36:8413 – 21. 49. Tencza SB, Douglass JP, Creighton DJ Jr, Montelaro RC, Mietzner TA. 62. Roccatano D, Colombo G, Fioroni M, Mark AE. Mechanism by which Novel antimicrobial peptides derived from human immunodeficiency virus 2,2,2-trifluoroethanol/water mixtures stabilize secondary-structure forma- type 1 and other lentivirus transmembrane proteins. Antimicrob Agents tion in peptides: a molecular dynamics study. Proc Natl Acad Sci U S A Chemother 1997;41:2394 – 8. 2002;99:12179 – 84. 50. Compton LA, Johnson WC Jr. Analysis of protein circular dichroism 63. Mao D, Wachter E, Wallace BA. Folding of the mitochondrial proton spectra for secondary structure using a simple matrix multiplication. Anal adenosine triphosphatase proteolipid channel in phospholipid vesicles. Biochem 1986;155:155 – 67. Biochemistry 1982;21:4960 – 8.

51. Manavalan P, Johnson WC Jr. Variable selection method improves the 64. Petros AM, Nettesheim DG, Wang Y, et al. Rationale for Bcl-xL/Bad prediction of protein secondary structure from circular dichroism spectra. peptide complex formation from structure, mutagenesis, and biophysical Anal Biochem 1987;167:76 – 85. studies. Protein Sci 2000;9:2528 – 34.

52. Sreerama N, Woody RW. Estimation of protein secondary structure from 65. Sattler M, Liang H, Nettesheim D, et al. Structure of Bcl-xL-Bak peptide circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR complex: recognition between regulators of apoptosis. Science methods with an expanded reference set. Anal Biochem 2000;287:252– 60. 1997;275:983 – 6. 53. Sreerama N, Venyaminov SY, Woody RW. Estimation of protein sec- 66. Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are ondary structure from circular dichroism spectra: inclusion of denatured pro- required for inhibition of apoptosis and heterodimerization with Bax. Nature teins with native proteins in the analysis. Anal Biochem 2000;287:243 –51. 1994;369:321 – 3. 54. Lobley A, Whitmore L, Wallace BA. DICHROWEB: an interactive 67. Moreau C, Cartron PF, Hunt A, et al. Minimal BH3 peptides promote cell website for the analysis of protein secondary structure from circular death by antagonizing anti-apoptotic proteins. J Biol Chem 2003;278: dichroism spectra. Bioinformatics 2002;18:211 – 2. 19426 – 35. 55. Diaz JL, Oltersdorf T, Horne W, et al. A common binding site mediates 68. Yang B, Liu D, Huang Z. Synthesis and helical structure of lactam heterodimerization and homodimerization of Bcl-2 family members. J Biol bridged BH3 peptides derived from pro-apoptotic Bcl-2 family proteins. Chem 1997;272:11350 – 5. Bioorg Med Chem Lett 2004;14:1403 – 6.

Mol Cancer Ther 2004;3(11). November 2004

Sanjeev Shangary, Christopher L. Oliver, Tommy S. Tillman, et al.

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