Mcl-1Down-regulationPotentiatesABT-737Lethalityby ... · that more than one antiapoptotic protein (e.g., Mcl-1 and Bcl-xL) cooperate to sequester multidomain proapoptotic proteins,

Mcl-1 Down-regulation Potentiates ABT-737 Lethality by

Cooperatively Inducing Bak Activation and

Bax Translocation

Shuang Chen,1Yun Dai,

1Hisashi Harada,

1Paul Dent,

2and Steven Grant

1,2,3

Departments of 1Medicine, 2Biochemistry, and 3Pharmacology, Virginia Commonwealth University and Massey Cancer Center,Richmond, Virginia

Abstract

The Bcl-2 antagonist ABT-737 targets Bcl-2/Bcl-xL but notMcl-1, which may confer resistance to this novel agent. Here,we show that Mcl-1 down-regulation by the cyclin-dependentkinase (CDK) inhibitor roscovitine or Mcl-1-shRNA dramati-cally increases ABT-737 lethality in human leukemia cells.ABT-737 induces Bax conformational change but fails toactivate Bak or trigger Bax translocation. Coadministrationof roscovitine and ABT-737 untethers Bak from Mcl-1 andBcl-xL, respectively, triggering Bak activation and Bax trans-location. Studies employing Bax and/or Bak knockout mouseembryonic fibroblasts (MEFs) confirm that Bax is requiredfor ABT-737 F roscovitine lethality, whereas Bak is primarilyinvolved in potentiation of ABT-737–induced apoptosis byMcl-1 down-regulation. Ectopic Mcl-1 expression attenuatesBak activation and apoptosis by ABT-737 + roscovitine,whereas cells overexpressing Bcl-2 or Bcl-xL remain fullysensitive. Finally, Mcl-1 knockout MEFs are extremely sensitiveto Bak conformational change and apoptosis induced byABT-737, effects that are not potentiated by roscovitine.Collectively, these findings suggest down-regulation of Mcl-1by either CDK inhibitors or genetic approaches dramaticallypotentiate ABT-737 lethality through cooperative interactionsat two distinct levels: unleashing of Bak from both Bcl-xL andMcl-1 and simultaneous induction of Bak activation and Baxtranslocation. These findings provide a mechanistic basis forsimultaneously targeting Mcl-1 and Bcl-2/Bcl-xL in leukemia.[Cancer Res 2007;67(2):782–91]

Introduction

Cell death decisions are regulated by the complex interplaybetween two groups of Bcl-2 family members: proapoptoticproteins (e.g., multidomain: Bax and Bak; BH3-only: Bad, Bim,Bid, and Noxa) and antiapoptotic proteins (e.g., Bcl-2, Bcl-xL,Bcl-w, Mcl-1, and Bfl-1/A1; refs. 1, 2). In disorders such asleukemia, increased expression of antiapoptotic proteins, such asBcl-2, is required for disease maintenance (3), confers drugresistance (4), and is associated with poor clinical outcome (5).These observations have prompted the development of small-molecule Bcl-2 inhibitors (refs. 6, 7; e.g., HA14-1) that disable Bcl-2,resulting in induction of apoptosis in leukemia cell lines (8).

Alternative strategies include the use of antisense oligodeoxynu-cleotides (e.g., G3139; ref. 6) or stabilized forms of BH3 peptides (9),among others. Recently, a novel inhibitor (ABT-737) of Bcl-2,Bcl-xL, and Bcl-w, which is significantly more potent than previouscompounds of this type, has been developed. This compound actsby mimicking the capacity of the BH3-only protein Bad to dock tothe hydrophobic groove of antiapoptotic Bcl-2 family proteins,thereby diminishing their ability to antagonize apoptosis (10).

ABT-737 lowers the apoptotic threshold for chemotherapeuticagents or ionizing radiation and has shown impressive precli-nical activity against hematopoietic malignancies as well as solidtumors in vitro and in vivo (10). Recently, ABT-737 was shown toovercome drug resistance (e.g., toward imatinib) in Bcr/Abl+

leukemic cells (11). However, ABT-737 has a low affinity for otherantiapoptotic Bcl-2 family proteins (e.g., Mcl-1 and A1; ref. 10)and thus may exhibit limited cytotoxic effects in cells with highendogenous levels of Mcl-1 (12). Moreover, ABT-737 efficiently killsinterleukin-3 (IL-3)–dependent cells (e.g., FL5.12) only after IL-3withdrawal (13), suggesting that additional death signals may berequired for lethality. Notably, Mcl-1 is a highly expressed anti-apoptotic protein (14) and a critical survival factor for variousmalignant hematopoietic cells (14, 15). Recent evidence suggeststhat more than one antiapoptotic protein (e.g., Mcl-1 and Bcl-xL)cooperate to sequester multidomain proapoptotic proteins, suchas Bak, thereby preventing its activation (16). Thus, the impairedcapacity of ABT-737 to induce apoptosis in tumor cells expressinghigh Mcl-1 levels may stem from a requirement for inhibitionof multiple antiapoptotic proteins. A corollary is that down-regulation/inhibition of ABT-737–nontargeted proteins (e.g., Mcl-1or A1) may enhance the lethality of this compound (12).

One candidate strategy to down-regulate/inhibit Mcl-1 involvesthe use of cyclin-dependent kinase (CDK) inhibitors. In preclinicalstudies, CDK inhibitors, including flavopiridol and the roscovitinederivative CYC202 (seliciclib), are potent inducers of apoptosis inmalignant hematopoietic cells, including leukemia cells (17, 18).Notably, results from several laboratories have established thatCDK inhibitors (e.g., flavopiridol and CYC202) act, at least in part,by inhibiting CDK9, a kinase intimately involved in transcriptioninitiation and elongation through activation of the positivetranscription elongation factor-b, resulting in down-regulation ofseveral short-lived proteins, including Mcl-1 (17, 19).

Here, we report that Mcl-1 down-regulation by either CDKinhibitors or a small hairpin RNA (shRNA) approach leads to adramatic increase in ABT-737–mediated apoptosis in humanleukemia cells. Our results also indicate that this phenomenonstems from a mechanism involving two levels of cooperationbetween antiapoptotic and multidomain proapoptotic proteins ofthe Bcl-2 family: (a) simultaneous untethering of Bak from Bcl-xL(by ABT-737) and Mcl-1 (e.g., by roscovitine) and (b) the resulting

Note: S. Chen and Y. Dai contributed equally to this work.Requests for reprints: Steven Grant, Division of Hematology/Oncology, Virginia

Commonwealth University/Massey Cancer Center, MCV Station Box 230, Richmond,VA 23298. Phone: 804-828-5211; Fax: 804-828-2178; E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-06-3964

Cancer Res 2007; 67: (2). January 15, 2007 782 www.aacrjournals.org

Research Article

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activation of both Bak and Bax, culminating in Bax mitochondrialtranslocation and engagement of the apoptotic cascade. Thesefindings may provide a theoretical framework for combinatorialapproaches that target diverse antiapoptotic proteins thatcooperate in the efficient induction of apoptosis in malignant cells.

Materials and Methods

Cells and reagents. Human leukemia U937, HL-60, and Jurkat cells were

provided by the American Type Culture Collection (Rockville, MD) andmaintained in RPMI 1640 containing 10% FCS as previously reported (20).

U937/Bcl-2 and U937/Bcl-xL were obtained by stable transfection of cells

with full-length Bcl-2 and Bcl-xL cDNA, respectively (21). U937 cells stably

overexpressing Mcl-1 were kindly provided by Dr. Ruth Craig (DartmouthMedical School, Hanover, NH; ref. 22). All experiments used logarithmically

growing cells (3–5 � 105 cells/mL). Peripheral blood samples were obtained

with informed consent according to the Declaration of Helsinki from theperipheral blood of three patients with acute myeloblastic leukemia (AML;

FAB subtype M2) undergoing routine diagnostic aspirations with approval

from the Virginia Commonwealth University Institutional Review Board.

Leukemic blasts were isolated as previously described (20). Wild-type (wt),Bax�/�, Bak�/�, and Bax�/�/Bak�/� (double knockout) mouse embryonic

fibroblast (MEF) were kindly provided by the laboratory of Dr. Stanley

Korsmeyer (Dana-Farber Cancer Institute, Boston, MA; ref. 23). Mcl-1�/�

MEFs were kindly provided by Dr. Joseph Opferman (St. Jude Children’sResearch Hospital, Memphis, TN). All experiments were initiated in cells

cultured at f60% confluence.

ABT-737 was kindly provided by Dr. Gary Gordon (Abbott Laboratories,Abbott Park, IL; ref. 10). It was dissolved in DMSO, aliquoted, and stored at

�80jC. Roscovitine and R-roscovitine were purchased from Calbiochem

(San Diego, CA), dissolved in sterile DMSO, aliquoted, and stored at �20jC.

In all experiments, the final concentration of DMSO did not exceed 0.1%.Assessment of apoptosis. The extent of apoptosis was evaluated by flow

cytometric analysis using Annexin V-FITC staining as described previously

(20). To analyze the extent of cell death in MEFs, cells were trypsinized and

harvested together with those in the culture supernatant and then stainedwith 5 Ag/mL 7-amino-actinomycin D (7AAD; Sigma, St. Louis, MO) in PBS

for 20 min at 37jC. The percentage of dead cells (7AAD+) was assessed by

flow cytometry using a Becton Dickinson FACScan (Becton Dickinson, San

Jose, CA).Immunoblot. For subcellular fractionation, cells were lysed in digitonin

lysis buffer (20). After centrifugation, the supernatant (S-100, cytosolic

fraction) and pellets (organelle/membrane fractions) were collected andsubjected to immunoblot. Samples (30 Ag protein for each condition) from

whole-cell pellets or subcellular fractions were subjected to immunoblot

following procedures previously described (20). Where indicated, the blots

were reprobed with antibodies against h-actin (Sigma) or a-tubulin(Oncogene, La Jolla, CA) to ensure equal loading and transfer of proteins.

The following antibodies were used as primary antibodies: anti-Mcl-1, anti-

caspase-9, and anti-caspase-3 (BD PharMingen, San Diego, CA); anti-Mcl-1,

anti-Bcl-2, anti-Bcl-xS/L, anti–cytochrome c , anti-AIF, anti-Bak, and anti-Bax (Santa Cruz Biotechnology, Santa Cruz, CA); anti–cleaved caspase-3

(Asp175), anti–cleaved poly(ADP-ribose) polymerase (PARP; Asp214), and

anti-Bcl-xL (Cell Signaling, Beverly, MA); anti–RNA polymerase II (pol II)and anti–phospho-pol II (Upstate, Lake Placid, NY); anti-human Bcl-2 onco-

protein (DAKO, Carpinteria, CA); anti-caspase-8 (Alexis, San Diego, CA);

anti-PARP (Biomol, Plymouth Meeting, PA).

Immunoprecipitation. The interaction between Bak and Mcl-1 or Bcl-xL was evaluated by coimmunoprecipitation analysis by using ExactaCruz

kits (Santa Cruz Biotechnology) as per manufacturer’s instructions. For

these studies, CHAPS buffer was employed to avoid artifactual associations

reported with other buffers (24). Briefly, cells were lysed by syringing with a23-gauge needle in lysis buffer [20 mmol/L Tris (pH 7.4), 135 mmol/L NaCl,

1.5 mmol/L MgCl2, 1 mmol/L EDTA, 10% glycerol] containing 1% CHAPS

(Pierce, Rockford, IL); 800 Ag of protein per condition was used for

immunoprecipitation with anti-Bak antibody (Santa Cruz Biotechnology),

and the immunoprecipitated protein was subjected to immunoblot analysisusing antibodies to either Mcl-1 (BD PharMingen) or Bcl-xL (Cell Signaling).

Analysis of Bak and Bax conformational change. Cells were fixed and

permeabilized using FIX & PERM Cell Permeabilization Reagents (Caltag

Lab, Burlingame, CA) as per manufacturer’s instructions. Fixed cells wereincubated with either anti-Bak (Ab-1 for U937 cells and Ab-2 for MEFs,

Calbiochem) or anti-Bax (clone 3 for U937 cells, BD Transduction Lab,

Lexington, KY; YTH-6A7 for MEFs, Trevigen, Gaithersburg, MD) on ice for

30 min and then with FITC-conjugated goat-anti-mouse IgG (SouthernBiotech, Birmingham, AL) for 30 min in the dark. After washing, the

samples were analyzed by flow cytometry. The results for each condition

were calibrated by values for cells stained with mouse IgG (Southern

Biotech) as the primary antibody. Values for untreated controls werearbitrarily set to 100%. In parallel, cells for each condition were stained with

antibodies to total Bak (Santa Cruz Biotechnology) for comparison.

RNA interference. The pSUPER.retro.puro vector containing the humanH1 RNA promoter for expressing shRNA was obtained from Oligoengine

(Seattle, WA). DdRNAi oligonucleotides (5¶-GATCCCCGCGGGACTGGC-

TAGTTAAACTTCAAGAGAGTTTAACTAGCCAGTCCCGTTTTTA-3¶; ref. 25)

were cloned into pSUPER.retro.puro vector (pSUPER/shMcl-1). U937 cellswere transiently transfected with the pSUPER/shMcl-1 construct and its

empty vector by using the Amaxa Nucleofector device (program V-001) with

Kit V (Amaxa GmbH, Cologne, Germany) as per manufacturer’s instructions.

Reverse transcription-PCR. Total RNA was isolated using RNeasy Minikit with QIAshredder spin column (Qiagen, Valencia, CA) as per

manufacturer’s instructions; 1 Ag per condition of total RNA was subjected

to reverse transcription-PCR (RT-PCR) reaction using One-Step RT-PCR kit(Qiagen) and Thermal Cycler (MJ Research, Inc., Reno, NV). The primers

forward, 5¶-ATCTCTCGGTACCTTCGGGAGC-3¶ and reverse, 5¶-CCTGATGC-

CACCTTCTAGGTCC-3¶ (26) were used for Mcl-1. PCR products of Mcl-1

(442 bp) were analyzed in 2% agarose gel with ethidium bromide staining.Statistical analysis. The values represent the means F SD for at least

three independent experiments done in triplicate. The significance of

differences between experimental variables was determined using the

Student’s t test. Analysis of synergism was done according to median dose-effect analysis using Calcusyn software (Biosoft, Ferguson, MO; ref. 20).

Results

CDK inhibitors transcriptionally down-regulate the expres-sion of Mcl-1 and synergistically interact with ABT-737 toinduce apoptosis in U937 cells. Earlier reports indicated thatvarious CDK inhibitors, including roscovitine (27), transcriptionallydown-regulate expression of short-lived proteins, such as Mcl-1.As shown in Fig. 1A , treatment with either CDK inhibitor atconcentrations >10 Amol/L resulted in significant declines in Mcl-1protein levels, but no change in expression of Bcl-2 and Bcl-xL wasobserved. This phenomenon was associated with inhibition ofphosphorylation of pol II CTD and a pronounced reduction inMcl-1 mRNA levels (Fig. 1B).

Attempts were then made to determine what effect CDKinhibitors would have on the response of cells to ABT-737. Whereasexposure to 12 Amol/L roscovitine alone modestly increasedapoptosis, ABT-737 at a concentration range of 150 to 750 nmol/Lhad very little effect (Fig. 1C). However, combined treatmentresulted in a dramatic increase in the loss of mitochondrialmembrane potential (DWm; data not shown) and apoptosis.Identical results were obtained with R-roscovitine. Moreover, lowerconcentrations (V10 Amol/L) of either roscovitine or R-roscovitine,which failed to down-regulate Mcl-1 (Fig. 1A), did not enhanceABT-737 lethality (data not shown). Median dose-effect analysis,employing apoptosis determined by Annexin V-FITC as an endpoint, yielded combination index values <1.0 (Fig. 1C, inset),denoting synergistic interactions.

Mcl-1 Down-regulation Enhances ABT-737 Lethality

www.aacrjournals.org 783 Cancer Res 2007; 67: (2). January 15, 2007



Exposure of U937 cells to roscovitine F ABT-737 resulted inmarked down-regulation of Mcl-1 protein, whereas ABT-737 by itselffailed to modify Mcl-1 expression (Fig. 1D). In contrast, no change inBcl-2 or Bcl-xL expression was observed (data not shown). Lastly,effects of cotreatment with ABT-737 and roscovitine were examinedin relation to mitochondrial events. Cotreatment with roscovitineand ABT-737 triggered a pronounced increase in cytochrome c andAIF release into the cytosolic fraction (Fig. 1D). Combined treat-ment also induced a modest but discernible increase in caspase-9and caspase-8 cleavage, and a marked increase in cleavage ofcaspase-3, accompanied by cleavage of PARP (Fig. 1D). However,transfection with a dominant-negative caspase-8 construct (21)failed to protect U937 cells from mitochondrial damage andapoptosis induced by ABT-737/roscovitine (data not shown),arguing against involvement of the extrinsic apoptotic pathway.

Together, these findings indicate that CDK inhibitors dramaticallyincrease ABT-737–mediated apoptosis in association with Mcl-1down-regulation.

Roscovitine markedly down-regulates Mcl-1 and increasesABT-737 lethality in various human leukemia cells, includingprimary AML cells. Parallel studies were done in human HL-60promyelocytic and Jurkat lymphoblastic leukemia cells. First, theseleukemia cells exhibited differing susceptibilities to ABT-737–mediated lethality (IC50: 1.3 Amol/L for U937, 420 nmol/L forJurkat, 190 nmol/L for HL-60). Immunoblot showed that Bcl-2 andMcl-1 protein levels varied between the cell lines, whereas Bcl-xLexpression was equivalent. Interestingly, HL-60 cells, which werethe most sensitive of the three lines, exhibited very low Mcl-1expression but higher levels of Bcl-2 (Fig. 2A, inset). These resultssuggest that levels of Mcl-1 and/or the ratio between Mcl-1 and

Figure 1. CDK inhibitors interact with ABT-737 to induce mitochondria-related apoptotic signaling events in association with down-regulation of Mcl-1. A, U937 cellswere exposed to 7.5 to 15 Amol/L of either roscovitine (Rosc ) or R -roscovitine (R-rosc ), after which immunoblot analysis was done to monitor expression of Bcl-2,Bcl-xL, and Mcl-1. B, immunoblot analysis was conducted to assess phosphorylation status of pol II (P-pol II) CTD after 24 h of treatment with roscovitine (12 Amol/L).Alternatively, total RNA was extracted, and RT-PCR was done to monitor Mcl-1 mRNA levels. C, U937 cells were treated with 150 to 750 nmol/L ABT-737(ABT ) F 12 Amol/L roscovitine or 300 nmol/L ABT-737 F 12 Amol/L R-roscovitine for 24 h, after which flow cytometry was conducted to determine the percentage ofapoptotic cells (Annexin V+). Moreover, U937 cells were exposed (24 h) to varying concentrations of ABT-737 and roscovitine alone and in combination at fixed ratio1:100. At the end of this period, the percentage of Annexin V+ cells was determined for each condition, and median dose-effect analysis was then employed tocharacterize the nature of the interaction between these agents (inset ). Combination Index (C.I. ) values <1.0 denote a synergistic interaction. Representative for threeseparate experiments. D, U937 cells were exposed (24 h) to 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine, and immunoblot was then done to evaluateexpression of Mcl-1 as well as cleavage of caspases (casp) and PARP. Alternatively, S-100 cytosolic fractions were prepared and subjected to immunoblot analysis.Primary antibodies were employed as indicated. CF, cleavage fragment; Cyt c, cytochrome c. A, B , and D, representative results from one experiment, and twoadditional studies yielded equivalent results.

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Bcl-2 expression may represent as determinants of ABT-737sensitivity in leukemia cells.

Cotreatment with marginally toxic concentrations of ABT-737and roscovitine markedly induced mitochondrial damage (data notshown) and apoptosis in Jurkat and HL-60 cells (Fig. 2A). Roughlyequivalent results were obtained when R-roscovitine was coad-ministered (data not shown). Moreover, roscovitine, administratedalone F ABT-737 dramatically down-regulated Mcl-1 in Jurkat cells,and combined treatment marked increased PARP cleavage (Fig. 2B).

Effects of this regimen on primary leukemia blasts isolated fromthree patients with AML (Fig. 2C) were similar to those obtained inleukemia cell lines. Furthermore, roscovitine F ABT-737 almostcompletely abrogated Mcl-1 expression in AML blasts and inducedpronounced PARP (Fig. 2D), indicating that ABT-737/roscovitineinteractions occur in association with Mcl-1 down-regulation inboth continuously cultured human leukemia cell lines differing intheir sensitivity to ABT-737 as well as in primary AML blasts.

Roscovitine enhances ABT-737–mediated Bax conforma-tional change, whereas Bak activation is induced only bycombined ABT-737 and roscovitine administration. Bax andBak are essential for mitochondrial outer membrane permeabiliza-tion (28, 29), a critical cell death determinant (30). Whereas Bak isnormally localized at its site of action (i.e., organellar membranes),Bax is monomeric and found in the cytosol of healthy cells.Following death stimuli, Bax undergoes conformational changeand translocates to organellar membranes. Activation of both Bakand Bax is associated with a conformational change, which can bedetected by antibodies recognizing only the active proteinconformers (16). The effect of ABT-737 F roscovitine on Bak/Baxconformational change was then examined. When U937 cells wereexposed to ABT-737 (150–500 nmol/L) alone, a clear dose-dependent increase in Bax conformational change was observed(Fig. 3A). Interestingly, Bax conformational change was unaccom-panied by Bax translocation (see below; Fig. 3C) nor did it induce

Figure 2. Roscovitine down-regulates Mcl-1 and promotes ABT-737 lethality in multiple human leukemia cell lines and primary AML cells. A, untreated U937, HL-60,and Jurkat cells were lysed and subjected to immunoblot analysis to detect protein levels of Bcl-2, Bcl-xL, and Mcl-1 (inset ). Jurkat and HL-60 cells were exposed for24 h to ABT-737 (Jurkat, 100–200 nmol/L; HL-60, 30–50 nmol/L) F 12 Amol/L roscovitine, after which the percentage of Annexin V+ cells was determined by flowcytometry. B, Jurkat cells were treated as described in (A), after which immunoblot analysis was done to monitor Mcl-1 expression and PARP cleavage. C, blasts wereisolated from the peripheral blood of three AML patients (designed as #1–3 ; FAB subtype M2) and then incubated (24 h) with 150 nmol/L ABT-737 F roscovitine(#1 and #2, 10 Amol/L; #3, 12 Amol/L). At the end of this period, the percentage of Annexin V+ cells was determined by flow cytometry. Columns, means for anexperiment done in triplicate; bars, SD. Representative experiment (patient #3 ). D, the blasts (patient #3) were incubated (24 h) with 150 and 300 nmol/LABT-737 F 12 Amol/L roscovitine and then lysed for immunoblot using indicated primary antibodies. C-PARP, cleaved PARP. A (inset ), B , and D, representative forthree separate experiments.





apoptosis (Fig. 1C). On the other hand, roscovitine (12 Amol/L)alone minimally induced Bax conformational change. Notably, cellscoexposed to roscovitine and ABT-737 displayed a further increasein Bax conformational change compared with cells treated ABT-737alone (Fig. 3A), an effect accompanied by marked increases in Baxtranslocation (see below; Fig. 3C) and lethality. These findingssuggest that ABT-737–induced Bax conformational change by itselfmay not be sufficient to trigger Bax translocation and apoptosis.

Parallel studies were then done in U937 cells to assess Bakconformational change. In sharp contrast to results involvingBax, ABT-737 by itself had little or no effect on Bak activation(Fig. 3A), whereas roscovitine also failed to induce Bak activation.However, combined treatment with ABT-737 and roscovitineinduced pronounced Bak activation. No change in total Bak wasnoted for any condition (data not shown). Together, these resultsindicate that (a) roscovitine enhances ABT-737–mediated Baxconformational change and cooperates to trigger Bax transloca-

tion, and that (b) ABT-737 alone is ineffective in triggering Bakconformational change; instead, roscovitine coadministration isrequired for ABT-737–mediated Bak activation.

Coadministration of ABT-737 and roscovitine disrupts theassociation of Bak with both Bcl-xL and Mcl-1 and induces Baxtranslocation. One of the mechanisms by which Mcl-1 opposesapoptosis is by binding/sequestering Bak and preventing itsactivation (31). Furthermore, there is evidence that Bcl-xL, butnot Bcl-2, Bcl-w, or A1, acts analogously to block Bak activation(16), and that both Mcl-1 and Bcl-xL must be neutralized (e.g., byNoxa and Bad, respectively) to displace Bak in order to trigger celldeath (16, 32). Consequently, the effects of ABT-737 and roscovitineon interactions between Bak and Bcl-xL or Mcl-1 were assessed.No change in total Bak levels was observed with any treatment(see below; Fig. 3C). U937 cells exposed to ABT-737 F roscovitinewere lysed in CHAPS buffer, and associations between Bak andMcl-1 or Bcl-xL were assessed (Fig. 3B). Treatment with ABT-737

Figure 3. Bax is necessary for induction of cell death by both ABT-737 alone and in combination with roscovitine, whereas Bak activation is only required for synergisticinteractions between these agents. A, U937 cells were treated (24 h) with 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine, after which cells were stained withanti–conformationally changed Bax (clone 3)/FITC-conjugated goat-anti-mouse IgG and subjected to flow cytometry. In parallel, Bak activation was monitored byflow cytometry after staining with anti–conformationally changed Bak (Ab-1)/FITC-conjugated goat-anti-mouse IgG. Representative histograms (dotted, untreatedcontrols). Values, mean FSD for three separate experiments done in triplicate. B, U937 cells were exposed for 24 h to 150 to 300 nmol/L ABT-737 F 12 Amol/Lroscovitine, after which cells were lysed in CHAPS buffer and subjected to immunoprecipitation (IP ) using anti-Bak and then immunoblotted (IB ) with either anti-Mcl-1 oranti-Bcl-xL antibody. For comparison, the right lanes (designed as C ) were loaded with whole-cell lysates. C, U937 cells were treated as described in (A ), afterwhich S-100 cytosolic and pelleted organellar membrane fractions were prepared and subjected to immunoblot analysis using an anti-Bax antibody. Alternatively, totallevels of Bax and Bak were monitored in whole-cell lysates. D, untreated wt, Bak�/�, Bax�/�, and Bax�/�/Bak�/� [double knockout (DKO )] MEFs were lysed andsubjected to immunoblot to detect expression of both proapoptotic (Bak and Bax) and antiapoptotic (Mcl-1, Bcl-2, and Bcl-xL) proteins (inset ). Various MEFs wereexposed (24 h) to 300 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine, after which the cells, including those in the culture supernatant, were collected for each condition.Cells were then stained with 7AAD and subjected to flow cytometry to determine the percentage of 7AAD+ cells. *, P < 0.01, significantly greater than values fortreatment with each concentration of ABT-737 alone. B, C , and D (inset ), representative results from one experiment, and two additional studies yielded equivalentresults.

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alone modestly but discernibly increased the amount of Mcl-1associating with Bak. Notably, this effect was largely abrogated byroscovitine treatment, presumably due to Mcl-1 down-regulation(Fig. 1). Reciprocal effects were noted when the amount of Bcl-xLcoimmunoprecipitating with Bak was monitored (i.e., roscovitinesubstantially increased the amount of Bcl-xL associating with Bak,whereas ABT-737 essentially reversed this phenomenon). Together,these findings suggest that cotreatment with roscovitine and ABT-737 antagonizes interactions of Bak with both Mcl-1 and Bcl-xL.

Lastly, the effect of coexposure to roscovitine and ABT-737 onintracellular Bax localization was examined. Treatment with eitherABT-737 or roscovitine alone had little or no effect on theintracellular disposition of Bax (Fig. 3C). However, ABT-737/roscovitine coadministration induced a major translocation ofBax from the cytosolic compartment to the pelleted organellarmembrane fraction. This suggests that roscovitine and ABT-737cooperate to untether Bak from Mcl-1 and Bcl-xL, leading to Bakactivation and accompanying Bax translocation to organellarmembranes. These findings also suggest that concomitantactivation of Bax and Bak may be responsible for the dramaticinduction of apoptosis in cells coexposed to roscovitine andABT-737.

Bax knockout in MEFs substantially diminishes the lethalityof ABT-737 F roscovitine, whereas Bak knockout primarilyblocks synergistic interactions between these agents. Toevaluate further the functional roles of Bax and Bak in the lethalityof ABT-737 and its interactions with roscovitine, Bax�/�, Bak�/�,and Bax�/�/Bak�/� (double knockout) MEFs were employed(Fig. 3D, inset). Immunoblot analysis revealed that antiapoptoticBcl-2 family protein levels (e.g., Mcl-1, Bcl-2, and Bcl-xL) wereroughly equivalent in each of the cell types. Coadministration ofroscovitine clearly increased the lethality of ABT-737 in wt MEFs(P < 0.01, compared with cells exposed to ABT-737 alone; Fig. 3D).However, the lethality of ABT-737 F roscovitine was substantiallyblunted in Bax�/� MEFs, indicating that Bax is critical for thisphenomenon. In marked contrast, ABT-737 was still able to inducecell death in Bak�/� MEFs; in fact, these cells were slightly moresensitive than wt cells. Significantly, coadministration of roscovi-tine failed to increase the lethality of ABT-737 in Bak�/� MEFs.Finally, Bax/Bak double knockout MEFs displayed essentially noresponse to any of these treatments. Together, these findings sug-gest that Bax is required for induction of cell death by both ABT-737 F roscovitine, whereas Bak activation, although dispensablefor ABT-737 lethality, is nevertheless required for ABT-737/rosco-vitine synergism. They also support the notion that cooperationbetween Bak and Bax is critical for ABT-737/roscovitine lethality.

Roscovitine down-regulates Mcl-1 expression and attenu-ates ABT-737 resistance in leukemic cells ectopically express-ing Bcl-2 or Bcl-xL. Because ABT-737 targets Bcl-2 and Bcl-xL (10),it might be predicted that the relative abundance of antiapoptoticproteins, such as Bcl-2, would be related to ABT-737 sensitivity.Consequently, the effect of Bcl-2 or Bcl-xL expression on thesusceptibility of cells to ABT-737 F roscovitine was examined usingU937 cells ectopically expressing Bcl-2 or Bcl-xL. Ectopic expres-sion of Bcl-2 or Bcl-xL provided significant protection from thelethal effects of etoposide (VP-16), a potent inhibitor of DNAtopoisomerase (Fig. 4A and B). Notably, Bcl-2 or Bcl-xL over-expression attenuated ABT-737–mediated lethality but did notaffect roscovitine cytotoxicity. However, Bcl-2 or Bcl-xL over-expression failed to protect cells from mitochondrial damage(i.e., loss of DWm; data not shown) and apoptosis induced by

roscovitine and ABT-737 coadministration (P > 0.05, for each ABT-737 concentration, compared with empty vector controls U937/pCEP or U937/3.1). Cotreatment with roscovitine/ABT-737 inducedan equivalent decline in Mcl-1 expression and enhanced PARPcleavage in U937/Bcl-2, U937/Bcl-xL, and controls but did notmodify Bcl-2 or Bcl-xL expression (Fig. 4C and D). Collectively,these findings indicate that although ectopic expression of eitherBcl-2 or Bcl-xL reduces human leukemia cell sensitivity to ABT-737,they are unable to prevent the ABT-737/roscovitine regimen fromdiminishing Mcl-1 levels and inducing apoptosis.

Ectopic expression of Mcl-1 but not Bcl-2 blocks Bakactivation and apoptosis triggered by combined exposure ofleukemia cells to ABT-737 and roscovitine. Because Bcl-2/Bcl-xLand Mcl-1 may play disparate functional roles in blocking apoptosis(32), studies were done employing U937 cells ectopically expressingMcl-1 (22). As in cells overexpressing Bcl-2 or Bcl-xL, ectopicexpression of Mcl-1 substantially protected cells from VP-16– andABT-737–mediated lethality (Fig. 5A). However, in striking contrastto the former cells, enforced Mcl-1 expression significantlydiminished mitochondrial damage (loss of DWm; data not shown)and apoptosis (Fig. 5A) induced by the ABT-737/roscovitineregimen (P < 0.001, versus U937/pCEP).

Immunoblot revealed that combined treatment with ABT-737/roscovitine clearly diminished Mcl-1 expression in U937/pCEP cellsbut failed to do so in their U937/Mcl-1 counterparts (Fig. 5B).Moreover, PARP cleavage induced by ABT-737/roscovitine wasalmost completely abrogated in U937/Mcl-1 cells. Basal Bcl-2expression was somewhat lower in U937/Mcl-1 cells comparedwith controls but was not modified appreciably in either linewith any treatment. Together, these findings argue strongly thatdown-regulation of Mcl-1 plays a functional role in potentiationof ABT-737–mediated lethality by roscovitine.

Finally, the effects of ectopic expression of Bcl-2 or Mcl-1 werecompared with respect to conformational change of Bax and Bakinduced by the ABT-737/roscovitine regimen. Consistent with theirinability to block ABT-737/roscovitine–mediated lethality, Bcl-2overexpression failed to attenuate conformational change of Bax orBak in cells exposed to ABT-737 and roscovitine in combination(Fig. 5C), although it did reduce Bax conformational changeinduced by ABT-737 alone (data not shown). Similar results wereobtained in cells ectopically expressing Bcl-xL (data not shown).However, in sharp contrast, ectopic expression of Mcl-1, which alsoattenuated Bax conformational change mediated by ABT-737 alone(data not shown), essentially abrogated Bak activation triggered bythe ABT-737/roscovitine regimen and also partially reduced Baxconformational change after exposure to the combination (Fig. 5D).These findings provide further evidence that disruption of Mcl-1function plays a critical role in ABT-737/ roscovitine interactionsassociated with Bax/Bak activation and apoptosis.

RNA interference or gene knockout of Mcl-1 dramaticallysensitizes cells to ABT-737 but abrogates the capacity ofroscovitine to potentiate Bak activation and lethality. Toevaluate further the functional role of Mcl-1 in Bax/ Bak activationas well as apoptosis mediated by ABT-737 F roscovitine, a shRNAstrategy and Mcl-1�/� MEFs were employed. First, U937 cells weretransiently transfected with a construct encoding shRNA againstMcl-1 mRNA, and immunoblot analysis documented Mcl-1 down-regulation (Fig. 6A, inset). Mcl-1 down-regulation by this approachdramatically sensitized human leukemia cells to ABT-737 lethality(P < 0.02–0.001, for each ABT-737 concentration, compared withthose transfected with empty vector; Fig. 6A).





Further studies were done in wt and Mcl-1�/� MEFs. Immuno-blots revealed that levels of Bcl-2, Bcl-xL, Bak, and Bax in Mcl-1�/�

cells were roughly equivalent to those in wt MEFs, whereas Mcl-1was absent (Fig. 6B, inset). Mcl-1�/� cells exhibited a dramaticincrease in sensitivity to ABT-737 (50–100 nmol/L) compared withwt cells (P < 0.001; Fig. 6B). Significantly, roscovitine was unableto enhance cell killing mediated by ABT-737 in Mcl-1�/� cells(P > 0.05). Moreover, Bak conformational change was observed onlyafter coexposure of wt MEFs to ABT-737 and roscovitine, consistentwith preceding findings in human leukemia cells. In strikingcontrast, ABT-737 alone dramatically induced Bak activation inMcl-1�/� cells, a phenomenon that was not further enhanced byroscovitine (Fig. 6C). Analogously, Mcl-1�/� cells displayed greaterBax conformational change following ABT-737 exposure comparedwith their wt counterparts (Fig. 6C). However, as in the case of Bak,roscovitine failed to increase ABT-737–mediated Bax conforma-tional change in Mcl-1�/� cells. Together, these results provide clearevidence that Mcl-1 is a critical determinant of both ABT-737actions as well as the capacity of roscovitine to potentiate ABT-737lethality through cooperative activation of Bak and Bax.

Discussion

ABT-737, a novel small-molecule Bcl-2/Bcl-xL/Bcl-w inhibitorcurrently in development as an anticancer agent, has a relativelylow affinity for the more divergent antiapoptotic Bcl-2 familyproteins (e.g., Mcl-1 and A1; ref. 10). ABT-737 is less efficient inkilling tumor cells exhibiting relatively high levels of Mcl-1 (12).Because Mcl-1 has a short half-life (i.e., <2 h; ref. 33), it isparticularly susceptible to down-regulation by agents that disruptits de novo synthesis. Consequently, attention has recently focusedon the capacity of several clinical relevant CDK inhibitors (e.g.,flavopiridol and the roscovitine derivative CYC202) to transcrip-tionally down-regulate Mcl-1 through CDK9 inhibition (17, 19).Moreover, we previously reported that flavopiridol enhanced thelethality of HA14-1, although the mechanism underlying thisinteraction was not determined (34). Consistent with thesefindings, roscovitine and its R enantiomer R-roscovitine inhibitedphosphorylation of RNA polymerase II CTD, diminished Mcl-1mRNA levels, and markedly down-regulated protein levels.Significantly, such actions correlated closely with synergisticinteractions with ABT-737. Although the possibility that other

Figure 4. Overexpression of Bcl-2 or Bcl-xL fails to protect cells from roscovitine/ABT-737–mediated lethality. A and B, U937/Bcl-2 (A) and U937/Bcl-xL (B ), as wellas their empty-vector controls (U937/pCEP and U937/pcDNA3.1), were treated (24 h) with 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine or 25 Amol/L VP-16 (6 h)for comparison, after which the percentage of Annexin V+ cells was assessed by flow cytometry. **, P < 0.001, significantly less than values for empty vector controls.C, U937/Bcl-2 and U937/pCEP cells were treated as described in (A ), after which cells were lysed and subjected to immunoblot for expression of Bcl-2 and Mcl-1 aswell as PARP cleavage. D, U937/Bcl-xL and U937/pcDNA3.1 cells were exposed for 24 h to ABT-737 (top, 300 nmol/L; bottom, 150–500 nmol/L) F 12 Amol/Lroscovitine, after which immunoblot analysis was done to monitor protein levels of Bcl-xL and Mcl-1 as well as PARP cleavage. B and D, representative for threeseparate experiments.

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activities (e.g., CDK disruption) contribute to the lethality ofcombination regimens involving CDK inhibitors cannot be com-pletely excluded (35), it is noteworthy that Mcl-1 down-regulationplays an important role in apoptosis in malignant hematopoieticcells (17, 19, 36). On the other hand, in certain transformed cells,Mcl-1 down-regulation, although required, seems to be incapableby itself of initiating apoptosis (31, 37), indicating that othercooperating factors may be necessary for cell death. These findingssuggest that interventions targeting more than one antiapoptoticprotein may be required for optimal cell killing.

Whereas resistance to ABT-737 may reflect the compensatoryactions of Mcl-1 (12), the present investigation was prompted byrecent evidence that that Bak activation requires simultaneousdisruption of its associations with Mcl-1 (e.g., by Noxa) and Bcl-xL(e.g., by Bad; ref. 16). Moreover, increased production of Noxa canoppose Mcl-1 antiapoptotic functions, leading to simultaneousactivation of Bax and Bak (38). The present results suggest thatcoadministration of ABT-737 and roscovitine recapitulate theactions of more physiologic proapoptotic BH3-only proteins.Specifically, ABT-737, by binding to hydrophobic groove withinthe Bcl-xL BH3 domain (10), untethers Bak from Bcl-xL, analogousto the actions of BH3-only proteins such as Bad (39). On the otherhand, Mcl-1 down-regulation by roscovitine, mimicking the actionsof Noxa in displacing Bak from Mcl-1 (16), reciprocally released Bakfrom Mcl-1 sequestration. Thus, coadministration of ABT-737 androscovitine markedly diminished the association of Bak with bothBcl-xL and Mcl-1, inducing Bak activation. In support of this

hypothesis, Bak activation was observed only when roscovitineand ABT-737 were administrated concomitantly, but not afterABT-737 alone. The notion of cooperativity in the regulation ofABT-737 lethality is further supported by the results obtained inMcl-1 knockout MEFs, in which ABT-737 alone, in contrast to itsactions in wt cells, markedly induced Bak activation. Significantly,roscovitine was unable to enhance ABT-737 lethality further inthese cells presumably because Mcl-1 was absent and levels couldnot be reduced further. These findings provide strong support forthe concept that disruption of more than one antiapoptotic proteinof the Bcl-2 family (i.e., Mcl-1 and Bcl-2/Bc-xL) represents a highlypotent apoptotic stimulus (12).

The finding that ABT-737 induced Bax conformational change butdid not trigger apoptosis by itself suggests that the lethality of theABT-737/roscovitine regimen involves not simply activation ofeither Bax or Bak, but cooperativity between these proteins.Untethering of Bak from both Mcl-1 and Bcl-xL allows Bak confor-mational change, homo-oligomerization (16), as well as possibleassociations with Bax (40). Nevertheless, there is evidence thatBax and Bak may interact to promote apoptosis (41). Resultsobtained with Bax and Bak knockout MEFs are fully compatible witha model in which Bax and Bak cooperate to trigger cell death. Forexample, Bax knockout cells displayed marked resistance to ABT-737 given alone or in combination with roscovitine. This suggeststhat the presence of Bax is essential for both ABT-737 lethality andsynergistic interactions with roscovitine. In striking contrast, Bakknockout cells remained fully sensitive to ABT-737–mediated cell

Figure 5. Ectopic expression of Mcl-1, but not Bcl-2, diminishes Bak activation and potentiation of apoptosis in cells coexposed to ABT-737 and roscovitine. A, U937/Mcl-1 and its empty-vector control (U937/pCEP) cells were exposed to 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine (24 h) or 25 Amol/L VP-16 (6 h) forcomparison, after which the percentage of Annexin V+ cells was determined by flow cytometry. **, P < 0.001, significantly less than values for U937/pCEP cells.B, U937/Mcl-1 and U937/pCEP cells were treated (24 h) with 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine, and then immunoblot analysis was done to monitorexpression of Mcl-1 and Bcl-2 as well as PARP cleavage. Representative for three separate experiments. C and D, U937/Bcl-2 (C ) and U937/Mcl-1 (D ) cells,as well as their empty-vector controls (U937/pCEP), were treated for 24 h with 300 nmol/L ABT-737 + 12 Amol/L roscovitine, after which conformational change of Baxor Bak was monitored by flow cytometry using anti-Bax (clone 3) or anti-Bak (Ab-1) antibody, respectively.





killing, indicating that Bak is not absolutely required for the lethalityof an agent targeting Bcl-2/Bcl-xL. However, it is significant thatroscovitine failed to potentiate ABT-737 lethality in these cells,arguing strongly that Bak is required for full engagement of theapoptotic program following disruption of the Bcl-2/Bcl-xL axis.Moreover, Bax translocation and cell death occurred only in humanleukemia cells coexposed to ABT-737 and roscovitine (Fig. 3C).Although the mechanisms responsible for potentiation of ABT-737–mediated Bax translocation by roscovitine are presently unclear,there are several plausible explanations. These include thepossibilities that (a) Mcl-1 may be involved in Bax regulation, eitherthrough a process involving ‘‘activator’’ BH3-only proteins such asBim and/or tBid (13, 42–45), or directly by itself (46); or that (b)activated Bak may promote Bax translocation in an as yet to bedefined way. Collectively, the present results support the notionthat simultaneous interruption of Mcl-1 and Bcl-xL function freesand activates Bak, which, in the setting of Bax conformationalchange, results in Bax translocation, leading in turn to full engage-ment of the apoptotic machinery.

It is noteworthy that overexpression of Bcl-2 or Bcl-xL failedto protect leukemia cells from the ABT-737/roscovitine regimen,reflecting the important contribution of Mcl-1 down-regulation

to the lethality of this regimen. The finding that ectopic expressionof Mcl-1 diminished potentiation of ABT-737 lethality by rosco-vitine highlights the central role of Mcl-1 down-regulation insynergism between these agents. This interpretation is furthersupported by results showing that roscovitine was unable toenhance ABT-737–mediated apoptosis in Mcl-1 knockoutMEFs. Moreover, overexpression of Mcl-1, but not Bcl-2/Bcl-xL,essentially abrogated Bak activation following exposure to ABT-737/roscovitine, strongly arguing that Mcl-1 plays a major role inregulating Bak. This concept is consistent with previous findingsindicating that Mcl-1 binds with considerably greater affinityto Bak compared with Bcl-xL (IC50 < 10 versus < 100 nmol/L;ref. 16).

Whereas recent studies suggest that ABT-737 and the morespecific Bcl-xL inhibitor A-385358 increase the antitumor activityof conventional cytotoxic drugs (10, 47), this phenomenon mayreflect a generic lowering of the apoptotic threshold. On the otherhand, the present results suggest that a mechanism-basedapproach combining agents that target distinct antiapoptoticmolecules (e.g., CDK inhibitors that down-regulate Mcl-1 expres-sion and small-molecule Bcl-2/Bcl-xL inhibitors like ABT-737)deserve attention. These findings also highlight the importance of

Figure 6. Knockout or down-regulation of Mcl-1 sensitize cells to cell death induced by ABT-737. A, U937 cells were transiently transfected with either emptyvector (EV) or shMcl-1/pSUPER for 24 h, after which cells were lysed and subjected to immunoblot analysis (inset ). After transfection with empty vector or shMcl-1,cells were incubated for 6 h and then exposed to the indicated concentrations of ABT-737 for an additional 24 h, after which the percentage of viable cells (7AAD�)was determined by flow cytometry after stained with 7AAD. *, P < 0.02; **, P < 0.001, significantly less than values for cells transfected with empty vector.B, immunoblot analysis was done to monitor levels of antiapoptotic (Mcl-1, Bcl-2, and Bcl-xL) and multidomain proapoptotic proteins (Bax and Bak) in untreated wtand Mcl-1 knockout MEFs (inset ). MEFs were exposed to 50 to 100 nmol/L ABT-737 alone or in the presence of 12 Amol/L roscovitine for 24 h, after which cells,including those in the culture supernatant, were harvested together for flow cytometry using 7AAD staining. **, P < 0.001, significantly greater than values for wt cells.C, MEFs (wt and Mcl-1�/�) were incubated (24 h) with 100 nmol/L ABT-737 F 12 Amol/L roscovitine, after which cells were stained with anti–conformationally changedBak (Ab-2) or Bax (YTH-6A7)/FITC-conjugated goat-anti-mouse IgG and subjected to flow cytometry.

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cooperative interactions between such agents at two separate butinterrelated levels in cell death induction: (a) release of Bak fromboth Bcl-xL and Mcl-1 and (b) simultaneous activation of bothBax and Bak, which may be essential for Bax translocation andABT-737 lethality. Whether a strategy combining CDK inhibitors,or other transcriptional repressors capable of down-regulatingMcl-1, with Bcl-2/Bcl-xL antagonists will result in enhancedtherapeutic efficacy will depend upon multiple factors, includingthe capacity of such agents to diminish Mcl-1 expression in vivo ,and whether the therapeutic index is enhanced. In this context, itis noteworthy that ABT-737 displays in vivo antitumor selectivityin preclinical studies (10). In any case, the present findingssuggest that in addition to combining Bcl-2/Bcl-xL antagonists

with conventional cytotoxic drugs, combination strategies involv-ing targeted agents that down-regulate Mcl-1, a protein that cancompensate for the loss of Bcl-2/Bcl-xL function, represents apotentially useful alternative approach.

Acknowledgments

Received 10/25/2006; accepted 11/28/2006.Grant support: National Cancer Institute grants CA63753, CA 93738, and CA

100866; Leukemia and Lymphoma Society of America award 6045-03; V Foundation;and Department of Defense.

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

We thank Dr. Joseph T. Opferman for Mcl-1 knockout MEFs.



References1. Green DR. At the gates of death. Cancer Cell 2006;9:328–30.

2. Letai A. BCL-2: found bound and drugged! Trends MolMed 2005;11:442–4.

3. Letai A, Sorcinelli MD, Beard C, Korsmeyer SJ.Antiapoptotic BCL-2 is required for maintenance of amodel leukemia. Cancer Cell 2004;6:241–9.

4. Konopleva M, Zhao S, Hu W, et al. The anti-apoptoticgenes Bcl-X(L) and Bcl-2 are over-expressed and con-tribute to chemoresistance of non-proliferating leukae-mic CD34+ cells. Br J Haematol 2002;118:521–34.

5. Del Poeta G, Venditti A, Del Principe MI, et al. Amountof spontaneous apoptosis detected by Bax/Bcl-2 ratiopredicts outcome in acute myeloid leukemia (AML).Blood 2003;101:2125–31.

6. Reed JC, Pellecchia M. Apoptosis-based therapies forhematologic malignancies. Blood 2005;106:408–18.

7. Green DR, Kroemer G. Pharmacological manipulationof cell death: clinical applications in sight? J Clin Invest2005;115:2610–7.

8. Wang JL, Liu D, Zhang ZJ, et al. Structure-baseddiscovery of an organic compound that binds Bcl-2protein and induces apoptosis of tumor cells. Proc NatlAcad Sci U S A 2000;97:7124–9.

9. Walensky LD, Kung AL, Escher I, et al. Activation ofapoptosis in vivo by a hydrocarbon-stapled BH3 helix.Science 2004;305:1466–70.

10. Oltersdorf T, Elmore SW, Shoemaker AR, et al. Aninhibitor of Bcl-2 family proteins induces regression ofsolid tumours. Nature 2005;435:677–81.

11. Kuroda J, Puthalakath H, Cragg MS, et al. Bim andBad mediate imatinib-induced killing of Bcr/Abl+

leukemic cells, and resistance due to their loss isovercome by a BH3 mimetic. Proc Natl Acad Sci U S A2006;103:14907–12.

12. Cory S, Adams JM. Killing cancer cells by flipping theBcl-2/Bax switch. Cancer Cell 2005;8:5–6.

13. Certo M, Moore VG, Nishino M, et al. Mitochondriaprimed by death signals determine cellular addiction toantiapoptotic BCL-2 family members. Cancer Cell 2006;9:351–65.

14. Kaufmann SH, Karp JE, Svingen PA, et al. Elevatedexpression of the apoptotic regulator Mcl-1 at the timeof leukemic relapse. Blood 1998;91:991–1000.

15. Derenne S, Monia B, Dean NM, et al. Antisensestrategy shows that Mcl-1 rather than Bcl-2 or Bcl-x(L)is an essential survival protein of human myeloma cells.Blood 2002;100:194–9.

16. Willis SN, Chen L, Dewson G, et al. Proapoptotic Bakis sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, untildisplaced by BH3-only proteins. Genes Dev 2005;19:1294–305.

17. Chen R, Keating MJ, Gandhi V, Plunkett W.Transcription inhibition by flavopiridol: mechanism ofchronic lymphocytic leukemia cell death. Blood 2005;106:2513–9.

18. Alvi AJ, Austen B, Weston VJ, et al. A novel CDKinhibitor, CYC202 (R -roscovitine), overcomes the defect

in p53-dependent apoptosis in B-CLL by down-regula-tion of genes involved in transcription regulation andsurvival. Blood 2005;105:4484–91.

19. MacCallum DE, Melville J, Frame S, et al. Seliciclib(CYC202, R -roscovitine) induces cell death in multiplemyeloma cells by inhibition of RNA polymerase II-dependent transcription and down-regulation of Mcl-1.Cancer Res 2005;65:5399–407.

20. Dai Y, Rahmani M, Pei XY, et al. Farnesyltransferaseinhibitors interact synergistically with the Chk1 inhib-itor UCN-01 to induce apoptosis in human leukemiacells through interruption of both Akt and MEK/ERKpathways and activation of SEK1/JNK. Blood 2005;105:1706–16.

21. Dai Y, Dent P, Grant S. Tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL) promotes mitochon-drial dysfunction and apoptosis induced by 7-hydrox-ystaurosporine and mitogen-activated protein kinasekinase inhibitors in human leukemia cells that ectop-ically express Bcl-2 and Bcl-xL. Mol Pharmacol 2003;64:1402–9.

22. Zhou P, Qian L, Kozopas KM, Craig RW. Mcl-1, aBcl-2 family member, delays the death of hematopoieticcells under a variety of apoptosis-inducing conditions.Blood 1997;89:630–43.

23. Wei MC, Zong WX, Cheng EH, et al. ProapoptoticBAX and BAK: a requisite gateway to mitochondrialdysfunction and death. Science 2001;292:727–30.

24. Hsu YT, Youle RJ. Nonionic detergents inducedimerization among members of the Bcl-2 family. J BiolChem 1997;272:13829–34.

25. Taniai M, Grambihler A, Higuchi H, et al. Mcl-1mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarci-noma cells. Cancer Res 2004;64:3517–24.

26. Hartley PS, Bayne RA, Robinson LL, Fulton N,Anderson RA. Developmental changes in expression ofmyeloid cell leukemia-1 in human germ cells duringoogenesis and early folliculogenesis. J Clin EndocrinolMetab 2002;87:3417–27.

27. Hahntow IN, Schneller F, Oelsner M, et al. Cyclin-dependent kinase inhibitor roscovitine induces apopto-sis in chronic lymphocytic leukemia cells. Leukemia2004;18:747–55.

28. Reed JC. Proapoptotic multidomain Bcl-2/Bax-familyproteins: mechanisms, physiological roles, and thera-peutic opportunities. Cell Death Differ 2006;13:1378–86.

29. Lucken-Ardjomande S, Martinou JC. Newcomers inthe process of mitochondrial permeabilization. J Cell Sci2005;118:473–83.

30. Green DR. Apoptotic pathways: ten minutes to dead.Cell 2005;121:671–4.

31. Cuconati A, Mukherjee C, Perez D, White E. DNAdamage response andMCL-1 destruction initiate apoptosisin adenovirus-infected cells. Genes Dev 2003;17:2922–32.

32. Chen L, Willis SN, Wei A, et al. Differential targetingof prosurvival Bcl-2 proteins by their BH3-only ligandsallows complementary apoptotic function. Mol Cell2005;17:393–403.

33. Yang-Yen HF. Mcl-1: a highly regulated cell death andsurvival controller. J Biomed Sci 2006;13:201–4.

34. Pei XY, Dai Y, Grant S. The small-molecule Bcl-2inhibitor HA14–1 interacts synergistically with flavopir-idol to induce mitochondrial injury and apoptosis inhuman myeloma cells through a free radical-dependentand Jun NH2-terminal kinase-dependent mechanism.Mol Cancer Ther 2004;3:1513–24.

35. Cai D, Latham VM, Jr., Zhang X, Shapiro GI.Combined depletion of cell cycle and transcriptionalcyclin-dependent kinase activities induces apoptosis incancer cells. Cancer Res 2006;66:9270–80.

36. Wittmann S, Bali P, Donapaty S, et al. Flavopiridoldown-regulates antiapoptotic proteins and sensitizeshuman breast cancer cells to epothilone B-inducedapoptosis. Cancer Res 2003;63:93–9.

37. Nijhawan D, Fang M, Traer E, et al. Elimination ofMcl-1 is required for the initiation of apoptosis followingultraviolet irradiation. Genes Dev 2003;17:1475–86.

38. Qin JZ, Xin H, Sitailo LA, Denning MF, Nickoloff BJ.Enhanced killing of melanoma cells by simultaneouslytargeting Mcl-1 and NOXA. Cancer Res 2006;66:9636–45.

39. Kelekar A, Chang BS, Harlan JE, Fesik SW, ThompsonCB. Bad is a BH3 domain-containing protein that formsan inactivating dimer with Bcl-XL. Mol Cell Biol 1997;17:7040–6.

40. Mikhailov V, Mikhailova M, Degenhardt K,Venkatachalam MA, White E, Saikumar P. Associationof Bax and Bak homo-oligomers in mitochondria. Baxrequirement for Bak reorganization and cytochrome crelease. J Biol Chem 2003;278:5367–76.

41. Lindsten T, Ross AJ, King A, et al. The combinedfunctions of proapoptotic Bcl-2 family members bakand bax are essential for normal development ofmultiple tissues. Mol Cell 2000;6:1389–99.

42. Clohessy JG, Zhuang J, de Boer J, Gil-Gomez G,Brady HJ. Mcl-1 interacts with truncated Bid andinhibits its induction of cytochrome c release and itsrole in receptor-mediated apoptosis. J Biol Chem 2006;281:5750–9.

43. Han J, Goldstein LA, Gastman BR, Rabinowich H.Interrelated roles for Mcl-1 and BIM in regulation ofTRAIL-mediated mitochondrial apoptosis. J Biol Chem2006;281:10153–63.

44. Gomez-Bougie P, Bataille R, Amiot M. Endogenousassociation of Bim BH3-only protein with Mcl-1, Bcl-xLand Bcl-2 on mitochondria in human B cells. Eur JImmunol 2005;35:971–6.

45. Kuwana T, Bouchier-Hayes L, Chipuk JE, et al. BH3domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilizationboth directly and indirectly. Mol Cell 2005;17:525–35.

46. Sedlak TW, Oltvai ZN, Yang E, et al. Multiple Bcl-2family members demonstrate selective dimerizationswith Bax. Proc Natl Acad Sci U S A 1995;92:7834–8.

47. Shoemaker AR, Oleksijew A, Bauch J, et al. A small-molecule inhibitor of Bcl-XL potentiates the activityof cytotoxic drugs in vitro and in vivo . Cancer Res 2006;66:8731–9.



2007;67:782-791. Cancer Res Shuang Chen, Yun Dai, Hisashi Harada, et al. TranslocationCooperatively Inducing Bak Activation and Bax Mcl-1 Down-regulation Potentiates ABT-737 Lethality by

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