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BAD as a Prototype for a Novel Therapeutic Agent for the Treatment of Acute Leukemia
Aaron D. Schimmer
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of the Institute of Medical Sciences University of T oronto
@Copyright by Aaron D. Schimrner (2001)
National Library 1+1 of Canada Bibliothèque nationale du Canada
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BAD as a Prototype for a NoveI Therapeutic Agent for the Treatment of Acute Leukemia
Aaron D. Schimmer, PhD Thesis Institute of Medical Sciences. University of Toronto
200 1
Abstract
Inhibitors of Bcl-2 may be useful therapeutic agents for the treatment of a wide
variety of malignancies including leukemia. A potential prototype of such a compound is
the endogenous Bcl-2 and Bcl-xL binding protein BAD. Previous reports indicate that
BAD can overcome the anti-apoptotic effect of Bcl-xL but not Bcl-2. If BAD c m o t
induce apoptosis in cells over-expressing Bcl-2, it would limit the application of
molecules like BAD as novel anti-tumor agents. We reported that transient transfection
of BAD induced cell death in cells with and without over-expression of Bcl-2 or Bcl-XL
and thereby validated the punuit of molecules like BAD as novel therapeutic agents.
Small cytotoxic peptides that mimic BAD and overcome Bcl-2 mediated dnig
resistance may be useful in the treatment of malignant disease. One example is the BH3
domain of BAD that binds and inhibits Bcl-2 and Bcl-xL. A cell-permeable version of
the peptide waç created by hsing it to cpm, a decanoic fatty acid intemaiization
sequence. This fusion peptide induced rapid ce11 death and apoptosis. However, its
cytotoxicity was significantly impaired by the over-expression of Bcl-xL, which could
limit the usefulness of this compound in neating malignancies with increased levels of
Bcl-2 and Bcl-xL. Therefore, enhancing the ability of the BH3 domain of BAD to kill
cells over-expressing these swival factors might be therapeutically useful. One strategy
to enhance its toxicity is to capitalize on the inherent alpha helical shape of the peptide,
since some amphrpathic alpha hetices are potent naturally occhng antibiotics capable
disrupting negativeiy charged membranes.
We harnessed the aipha helical propenies of the BH3 domain of BAD by fusing it
to the Ante~apedia intemalkation sequence. We created a 37 amino acid fusion peptide
corresponding to the 21 amino acids of the human BH3 domain of BAD fused to the C-
terminus of the 16 amino acids of the ANT peptide (ANTBH3BA.û). The ANTBH3BAD
fusion peptide induced rapid ce11 death and apoptosis. Its alpha helical secondary
structure disrupted mitochondnal function and allowed the peptide to overcome Bcl-2
over-expression and caspase inhibiton.
Tabte of Contents . -
Abstract 11
Table of Contents i v Introduction
Surnmary - 7
Apoptotic pathways 5 Extnnsic pathway 8 lntrinsic pathway 1 O
Cythochrome c and the mithochondria 10 Bcl-2 family proteins 14
B &Y 18 Bd-2 18 BAD a BH3-only protein 19
Cross talk between the pathways 20 Regulation of apoptotic proteins through phosphorylation 20
Defects in apoptosis are important prognostically -- 77
Extrinsic pathway -- 7?
Intrinsic pathway 23 Bcl-2 24 Bax 25 Downstream Effectors 25
The apoptosis pathway as a target for novel therapeutic agents 27 Extrinsic pathway 3 1
TRAIL 3 1 Caspase-8 activators 3 1
Intrinsic pathway 32 Bcl-2 inhibitors 33 Second Messengers 35 Mi tochondria 37'
Conclusion 37 Re ferences 38
Bad ioduces apoptosis in cells over-expression Bcl-2 or Bcl-xl without loss of mitochondrial membrane potential 56
Abstract 57 Introduction 58 Materials and Methods 59
Cells 59 PIasmids 59 Transfection 60 Western Blot 61 Ce11 Death Assays 62
GFP expression 62 Floating cells 63 Clonogenic colony assay 63 Luci ferase Assay 64
Apoptosis assay Mitochondrial membrane potential
Results Selection of Bcl-2 and BcI-xL expressing clones BAD induces ce11 death in COS cells
GFP expression Floating celIs Clonogenic assay BAD induces ce11 death in hematopoetic ce11 lines B A D induces apoptosis but does not alter AYsi
Discussion References
The BH3 domain of BAD fused to the Antennapedia peptide induces apoptosis via its alpha helical structure and independent of Bcl-2
Abstract Introduction Expenmental Procedures
Peptides Real time surface plasmon resonance Ce11 lines Internalization of the peptides Cionogenic colony assay Annexin V assay Circular Dichroism Measurement of mitochondrial membrane potential,
reactive oxygen intemediates and cytosolic calcium Caspase-3 activation
Results Bcl-2 binds ANTBH3BAD and BHSBAD, but not ANT Intemalization of the peptides A i i H 3 B A D induces a rapid cell death ANTBH3BAD induces apoptosis ANTBH3BAD induces ce11 death independent of Bcl-2 Toxicity of the firsion peptide is due
to its alpha helical shape ANTHB3 BAD disrupts mitochondrial hnction ANTBH3BAD induces caspse-3 activation, but caspase inhibitors do not pmtect against ce11 death
P-glycoprotein protects against ANTBH3BAD toxicity Discussion Re ferences
Discussion Future Directions
List of Tables and Figures
Introduction Table 1. Mechanisms of mulitcimg resistance Figure 1. Pathways of apoptosis Figure 2. Activation of apoptosis through the TNF farnily
of death receptors Figure 3a. Electron transport chah Figure 3b. ATP and H+ flux across the mitochronàrial membranes Figure 4. Family of Bcl-2 proteins Table 2. The BH3 domains of the Bcl-2 family of proteins Table 3. Prototypic thenpeutic agents for treatment of acute leukemia Table 4a. Site of action of prototypic therapeutic agents for the
treatment of acute leukernia (intrinsic pathway) Table 4b. Site of action of prototypic therapeutic agents for the
treatment of acute leukemia (extnnsic pathway)
Bad induces apoptosis in cells over-expression Bcl-2 or Bcl-xl without loss of mi tohcondrial membrane po ten tiai
Figure la. Western blot dernonstrating expression of Bcl-2, Bcl-xL in COS cells
Figure1 b. Westem blot demonstrating expression of BAD in COS cells Figure 2. BAD induced ce11 death as measured by
decrease in GFP expression Figure 3. Dose response curve for BAD-induced apoptosis Figure 4. BAD induced ce11 death as measured by an increase
in the nurnber of fioating cells Figure 5. Westem blot demonstnting expression of BcI-2
and Bcl-xL in 3SD cells and KS62 cefls Figure 6. BAD induced cell death in K562 cells
in a dose dependent manner Figure 7. BAD induced apoptosis as demonstrated
by staining with H33342 Figure 8. BAD does not alter AYM Figure 9. BAD induced cytochrome c release
The BH3 domain of BAD fused to the Antennapedia peptide induces apoptosis via its alpha helicai structure and independent of Bcl-2
Table 1. Peptides Figure 1. Real time surface plasmon resonance Figure 2. Intemalization of the peptides Figure 3. Morphologie evidence of ceil death Figure 4. ANTBH3BAD induces ceIl death
Figure 5. Dose response m e IO8 Figure 6. Bcl-2 does not abrogate the toxicity of the fusion peptide 110 Figure 7. Circular Dichroism 112 Figure 8. ï h e non-helical mutant is non toxic 114 Figure 9. ANTBH3BAD induces loss of AYh1 115 Figure 10. zVAD Mls to prevent death 117 Figure 1 1. P-glycoprotein protects against ANTBH3BAD toxicity 119
Discussion Figure 1. Proposed mechanisrn of transduction of ANT internalizntion
sequence across ce11 membranes 132 Figure 2. Alpha helical stnicture 134 Table 1 .Cornparison of cytotoxic moiecules 136
vii
Apoptosis in acute leukemia - a translational view
Aaron D. Schimmer, David W. Hedley, Linda 2. Penn, and Mark D. Minden
Rincess Margaret Hospital, University Health Network, Toronto. ON, Canada
Running Title: Apoptosis in acute leukemia - a translational view
SUMMARY Acute myeloid leukemia and adult-onset acute lyrnphocytic leukernia are aggressive
diseases with generally poor prognoses. The primary cause of treatment failures in these
patients is the emergence of multi-drug resistance. Common mechanisms of resistant
disease are defects in apoptotic pathways. Apoptosis is a process by which cells undergo
organized self-destruction. Apoptosis is ultimately executed by caspases, a farnily of
cysteine proteases that cleave critical intracellular proteins and execute the apoptotic
program. At least two major pathways for caspase activation have been identified: a) the
extrinsic pathway which involves members of the TNF family of death receptors and b)
the intrinsic pathway involving cytochrome c release from rnitochondria. Defects in the
extrinsic or inuinsic pathway may confer prognostic information for adult patients with
acute leukemia suggesting ways that such information can be used in risk stratification.
In addition, emerging knowledge about apoptosis mechanisms has laid the foundation for
the eventual development of new therapies capable of overcoming blocks in apoptosis.
Here 1 review some of the recent advances in translating information about apoptosis
mechanisms into promising new stntegies for improved treatment of adult leukernia.
Acute leukemi-a is a neoplastic disease characterized by a rapid accmnufation of
primitive hematopoietic cells. Acute leukemia is sub-classified as myeloid or lymphoid
depending on the origin of the malignant cell. Acute lymphocyuc leukernia (AU) is
predominantly a disease of childhood with 75% of ail cases occumng in patients less than
15 years of agel. in contrast, acute myeloid leukemia (AML) is more common in adults
with an incidence that increases with as2. Both AML and adult AU. are aggressive
diseases with generally poor prognoses. For exarnple, patients with AML who are less
than 55 years of age and who have no adverse prognostic features have a cornplete
remission rate of 70-85% with standard induction chemotherapy, but their long-term
survival is only approximately 50%. The outcome in elderly patients is wone. Patients
older than 65 yean of age with AML treated with standard chemotherapy have a median
survivai of 6 months and only 7% rernain in remission at 3 years3.
The primary cause of marnent failures in adult patients with leukemia is the
emergence of resistant disease. Multiple mechanisms may account for why leukemic
cells become resistant to chemotherapy (Table 1). One common cause includes defects in
the cell's inherent programmed ce11 death (apoptosis) pathways. These defects
theoretically may impair the ability to achieve remission and cure with chemotherapy. In
an atternpt to overcome drug resistance and improve clinical outcornes, attention is
tuming to developing therapeutic agents that overcome defects in the apoptosis pathways.
Though mostly in preclinical stages of development, these efforts offer a glimpse into the
future armamentatium of agents that may eventually be available for treating leukemia.
In this review, I focus on translationai approaches to apoptosis research in adult
acute leukemia. More basic details of the apoptosis pathway have been reviewed
Table 1: Mechanisms of multidmg resistance
Decreased dmg delivery Decreased dmg uptake Increased drug export Decreased metabolism of dnig to active form Resistance of target to drug-action Increased repair of hg-induce DNA damage Increased d m g metabolisrn Increased ceIl survival Decreased apoptosis
etsewhere ' with s o m t articles m m t ~ t i n g on hdividud pehway components such as
the mitochon~iria~*~, the TNF superfamily of re~e~tors"~ , ~c1-2~"'~and c a ~ p a s e s ' ~ ~ ~ ~ . Here.
1 will discuss the apoptosis pathways concentrating on members with important
prognostic or therapeutic potential. 1 will then illustrate how defects in apoptosis confer
prognostic information in patients with acute leukemia. By identifying patients at highest
nsk of resistant disease or relapse, one rnay be able to offer them more aggressive or
altemate therapy at disease onset. Finally, 1 will discuss small molecules that overcome
blocks in apoptosis and that will likely have a major impact in the treatment of acute
leukemia.
Apoptotic pathways
Apoptosis is a process by which cells undergo organized self-destruction without
eliciting an inflarnmatory response. Morphologically, cells undergoing apoptosis display
membrane blebbing and nuclear and cytoplasrnic condensation. Molecularly, classical
apoptosis is caused by the activation of caspases. Caspases are a farnily of intracellular
cysteine proteases which lie in a latent (zyrnogen) state in cells but become activated in
response to a wide variety of ce11 death stimuli.
Growing evidence indicates that ce11 death with apoptotic features can occur
without caspase activation. The mechanisms of caspase-independent ce11 death still
requires clarification, however. Here, 1 will lirnit my discussion to caspase-rnediated ce11
death as understanding these pathways has lead to the development of agents that
enhance apoptosis and may be of therapeutic value.
Caspases are organized in a cascade with upstream (initiator) caspases responsible
for activating the downstream (effector) caspases. Upsnearn caspases contain long
prodomains that hteract with ttieir spMfic activators. The downstream caspases contairi
smaller prodomains and are activated by proteolytic cleavage by upstrearn caspases.
Once activated, the downstream caspases cleave specific protein substrates leading to the
execution of apoptosis,
At present, at least two major pathways of caspase activation have been revealed:
a) the extrinsic apoptosis pathway where the TNF- family of death receptors activate
upstream casPase-812'" and b) the inuinsic apoptosis pathway where cytochrome c is
released from the mi tochondna and activates upstrearn casPase-9 '5-18(Figure 1 ).
Both pathways culminate in the activation of the downstream effector caspase - caspase-
3 l9.?O . A third minor pathway of caspase activation involving Granzyme B has also been
revealed. Granzyme B is a senne protease synthesized in cytotoxic T lymphocytes. Via
the pore forming protein perfonn. cytotoxic T lymphocytes inject Granzyme B into target
cells. Granz yme B direct1 y cleaves and activates several caspases incl uding pro-caspase-
321-23 . As such, it bypasses both the intrinsic and extrinsic pathways of caspase
activation. While over fourteen caspases have been identified. 1 will restnct my
cornments to caspases 3,8 and 9 as exarnples of downstream effector, extrinsic pathway
initiator and intrinsic pathway initiator caspases respectiveiy.
Extrinsic Pathwav
TNF farnily of death receptors
1 Faddlmort- 1
Granzyme B - Caspase-3
Intrinsic Pathwav
mitochondria
cytochrome c
Apaf- 1 I
Figure 1: Pathways of apoptosis Two major pathways of apoptosis have been discemed -the intrinsic and extrinsic. Both pathways culminate in the activation of caspase-3 - the downstrearn effector caspase. A third minor pathway has also been identified whereby Granzyme B directly activates caspase-3.
Exrnnsic Path way:
The extrinsic pathway of apoptosis begins with the TNF family of cytokine
recepton that includes Fas (CD95). DR4 (Trail-R 1) and TNF-R 1 (CD UOa), and TNFRII
(Figure 2). These receptors differ in their ligand specificity, activating binding partners
and downstrearn effectors. Upon ligand binding. activated TNFRl and Fas receptors
recruit and bind the death effector protein ~addl~ort - lx -> . Bound Fadd/Mort- 1 recruits
procaspase-8 ? Pro-caspase-8 is converted to its active fonn and is released back into
the cytosol where it cleaves and activates the downstrearn effector caspase-3 19*20.
This receptor family also induces apoptosis through an alternative signal
transduction pathway. According to one proposed mode1, Fas receptors bind the adapter
protein Daxx that in tum activates the MAP3 kinase, ASK1. When activated, ASKl
launches a phosphorylation cascade that culminates in the activation of JNKZ4.
Activation of JNK leads to the phosphorylation of c-Jun that potentiates apoptosis
through an unknown mechanism. The TNFRI and TNFRII receptors also activate the
MAPK/JNK pathway by binding the adapter proteins TRADD and TRAF that activate
A S K - ~ ~ ~ ? Disputing this mode1 Michaelson et al. 28 reponed that Daxx knockout mice
Fas l
TNF-R 1
MEK cascade MEK cascade \
\ +
Figure 2:Activation of apoptosis through the TNF family of death recepton. Activated Fas and TNFR-1 launch the exuinsic pathway of apoptosis by recruiting the adapter protein Fadd/Mort- 1. In tum, caspase-8 is activated which cleaves procaspase-3. These receptors also induce apoptosis through an altemate pathway. Through the adapter pmteins Dam or TRADD and TRAF, a phosphoryiation cascade is initiated leading CO the activation of IMC Activated JNK phosphorylates the transcription factor c-Jun that Ieads to apoptosis through unknown mechanisms. The role of Daxx in this model, however is controversial (see tex t).
display widespread apoptosis and do not snMve beyond day IO of gestation. if Daxx
functions as a pro-apoptotic protein, then one would expect to see hyperproliferation in
these knock out mice rather than apoptosis. Furthexmore, Tom et al. 29 demonstrated that
human Daxx dues not bind Fas, but, rather. promotes Fas-mediated apoptosis through its
role as a transcriptional modulator in the nucleus. Therefore, activation of Fas can induce
apoptosis through multiple pathways, but some of the mechanisms still require
clarification.
Interestingly, activation of TNFR 1 can also inhibit apoptosis. Activated TNF-R 1
recmits adapter proteins such as, TRADD, and TRAF. These adapter proteins launch a
phosphorylation cascade that frees NFKB from its inhibitor MB. Once free, NFKB
rnigrates to the nucleus where it mediates the transcription of survival genes2'30.
lntrinsic Pathway
The intrinsic pathway for caspase activation is initiated by the release of
cytochrome c from the mitochondria. Cytochrome c is normal1 y sequestered between the
inner and outer membranes of the mitochonckia, In response to a variety of pro-apoptotic
stimuli, cytochrome c is released into the ~ ~ t o s o l ' ~ . Cytochrome c then binds and
activates Apaf- 1. Apaf- 1 activates procaspase-9, which in tum cleaves procaspase-316-18.
This pathway is potentiated by BaklBax and inhibited by Bcl-2 and its anti-apoptotic
family members.
Cytochrome c and the mitachondria
Cytochrome c release from the mitochondria can occur with and without loss of
the mitoc fionciriai membrane potential (AYM) and ph ysical mitochondrid swelling .
These differences relate to the site of mitochondrial damage. A description of the
mechanisms of mitochondiiai damage is important to understand the action of key
apoptotic regdators such as Bcl-2 and Bax. Furthemon, these mechanisms serve as the
basis for developing thetapeutic agents that enhance apoptosis by targeting the
mi tochondria.
Mitochondria contain two membranes-the inner and outer membranes -
sepsrated by the inter membrane space (Figure 3). The inner membrane sunounds the
mitochondrial matrix and is the site of electron transport and ATP synthesis. Under
normal conditions, respiratory chah complexes pump H+ ions across the relatively
impermeable inner mitochondnaf membrane. Through this process, a negative electncal
gradient, AYM, is established across this membrane. The AYM drives Fo-Fi ATP
synthase to produce ATP from ADP and Pi with the resultant influx of into the matrix.
The adenine nucleotide transporter (Ant) exchanges ATP for ADP across the inner
membrane. Dmage to the Ant creates a conforenational change leading to the formation
of a permeable pore in the inner membrane. When the inner membrane becomes
permeable, AYM is lost. the rnitochondnal matrix swells, and cytochrome c is
3132 released .
The outer mitochondnai membrane contains the voltage-dependent anion
channel (VDAC). VDAC is involved in rnitochondrial respiration and controls ATP
efflux From the mitochondrial outer membrane33". Disruptions of VDAC lead to
increased pcrmeability of the outer membrane and cytochrome c release. In this scenario,
NADH NAD+
Fig 3a
a) Electron transport chah The conversion of glucose to energy in the Krebs cycle requires the reduction of NAD+ to NADH. The reoxidization of NADH occurs through the electron transport chain in the inner mitochondrial membrane. The reaction can be summarized as: NADH + WC + 1/202 NAD' + H20
Mitochondria
ATP ADP
Figure 3b: ATP and flux across the mitochondnd membranes
The reoxidization of NADH to NAD+ in the eIectron transport chah produces H&J and releases energy. This energy is used to drive the production of ATP in the rnitochondrial mauix. The adenine nucleotide transporter (Ant) controls the flux of ADP, ATP across the inner mi tochondriaî membrane. The Voltage Dependent Anion Channel (VD AC) regulates the flux of ATP across the outer mitocho~al membrane. Darnage to VDAC or ANT can induce cytochrome c release and initiate the intrinsic pathway of apoptosis. Darnage to ANT is also accompanied by physicd rnitochondrial swelling and loss of ATM.
the inner membrane remains intact, so there is no ross of &YM or mitochondrid
swelling3'. Likewise, pore forming pro-apoptotic proteins such as Bax or Bak form
channels in the outer membrane independent of VDAC that increase the permeabilizaiton
of the outer membrane and induce cytochrome c release without damaging the inner
6.36.37 membrane .
Modulation of VDAC, which is reported to occur following growth factor
withdrawal, can also close this channel. When VDAC closes, it impairs the exchange of
mitochondrial ATP for cytosolic ADP. ATP then accumulates in the inter membrane
space dong with H+ ions which darnage the mitochondnal membranes. The increased
positive charge in the inter membrane space leads to a transient increase in ATM. AS
damage to the inner membrane ensues, however. there is swelling of the rnitochondrial
matrix. cytochrome c release and ultimately loss of A Y ~ ~ ~ .
BCL-2 f m i l y proteins:
The intrinsic pathway of apoptosis is regulated by the Bcl-2 family of anti- and
pro-apoptotic proteins (Figure 4). Membership in the Bcl-2 family is bved on shared
Bcl-2 homology (BH) domains BH1, BH2, BH3, and BH4. Currently, therapeutic agents
that either block or mimic these domains are being developed. The BHL and BH2
domains are present in al1 of the suMval factors as well as the death agonists Bax, Bak
and mtd These domains are Iikely involved in pore formation as their t h e dimensional
structure resembles the pore fonning region of the diphtheria toxin 39vj0. The ciifference
in the charge and amino acid sequences between the BHI and BH2 domains of the pro-
and anti-apoptotic proteins may lead to different pore properties and account for the
differences in function between these molecuIes.
Bcl-2, Bcl-xL, MCL-1 LOOP BH3 BHl BEI2 TM
BH4 BH3 BHI BH2 TM
Bax, Bak, mtd
Bcl-xs
Bk, Hrk, Bim, BIk
BAD. Bid
Figure 4: Family of Bcl-2 proteins
The Bcl-2 family of proteins are grouped based on their pro- or anti-apoptotic functions and their common Bcl-2 homology (BH) domains. Some members also share cornmon transmembrane domains (TM) and loop domains.
The BH.3 domain is a death-promoting region common to d l members of the Bcl-
2 family (Table 2). The BH3 domain contains a core sequence of 8 amino acids of which
the leucine at position 1 and the aspartic acid at position 6 are critical for
heterodimerization. Mutation of these residues prevents heterodimerization and
abrogates the toxicity of the death agonists4'43. In the anti-apoptotic membee, the BH3
domain foms a pocket with the BH1, and BH2 domains. This pocket serves two
purposes. Fint, it buries the BH3 domain so it cannot exert its death-promoting activity.
Second, the pocket is the docking site for the BH3 domains of the death agonists 39v40.
Heterodimerization with the death agonists inhibits the function of the survival factors, as
discussed below".
The BH4 domain is found primarily in the pro-survival factors and appears to be
involved in protein-protein interactions with regulatory proteins outside the Bcl-2 family.
This domain is involved in controlling progression of the ceIl cycle. It also appears to
stabilize the VDAC in the outer mitochondrial membraned5.
A regulatory loop domain between the EH3 and BH4 dornains is found in the
anti-apoptotic proteins BcI-2, Bcl-xL and MCl-1. Interestingly, cleavage of Bcl-2 at the
loop domain by caspase-3 releases the BH4 domain and tums Bcl-2 into a potent pro-
apoptotic protein '? Perhaps by cleaving the loop domain, the BHI, BH2, BH3 pocket is
disrupted leading to exposure of the death-promoting BH3 domain. The importance of
this cleaved isoform in regulating the apoptosis pathways is unknown.
Table 2: The BH3 domdms of the Bct-2 famiiy of prottms
Bd-2 = A L S P V P V V H L T L R Q A G D F S R R
B d - x L ~ E V I P M A A V K Q A L R E A G D E F E L
BAK 6 7 P S S T M G Q V G R Q L A I I G D D I N R
BAX 5 2 Q D A S T K K L S E C L K R I G D E L D S
BIK W C M E G S D A L A L R L A C I G D E M D V
BAD ~ ~ Y J N L W A A Q R Y G R E L R R M S D E F V D
EID , t J Q E D I 1 R N 1 A R H L A Q V G D S M D R
Core Homology Region
Bax
The Bax family of death agonists act at the mitochondria to induce apoptosis.
Three mechanisrns have been identified by which Bax induces apoptosis. Fint. at the
outer mitochondrial membrane, Bax accelerates the opening of VDAC with subsequent
release of cytochrome c3'. Second, at the inner mitochondrial membrane, Bax interacts
with Ant and enhances the pemeability of the inner mitochondriai membrane"3. Finally,
when expressed at high levels, Bax is capable of forming pores by direct oligornerization
mechanisms that disrupt the permeability of the mitochondnal membranes and induce
c ytoc home c release 3637.47
Bci-2
The Bcl-2 class of anti-apoptotic proteins are important inhibitors of the intrinsic
pathway of apoptosis and are cornmon targets of novel therapeutic agents. These proteins
inhibit apoptosis by interacting with BaxlBak, and the mitochondria. Bcl-2 c m inhibit
the action of Bax/Bak by forming inactivating heterodimersJ4. Early reports suggested
that BcI-2 functioned as an anti-oxidant by preventing reactive oxygen species
generation48'49. In retrospect, this function likely relates to Bcl-2's role at the
mitochondria. At the mitochondria, BcI-2 binds VDAC and stabilizes it, thereby
preventing permeabilization of the outer mitochondrial membranes0. These anti-
apoptotic molecules also fom pores that stabilize the outer mitochondrial membrane
preventing loss of AYM cytochrome c release and ultimately reactive oxygen species
production. In addition to its role as an anti-apoptotic protein, Bcl-2 has a separate
funetion in arresting ceth at aie GO p k of the cctt cyde. The BH4 domain of the
protein is responsible for this property, but the mechanism is poorly u n d e n t ~ o d ~ ' ~ ~ .
Bcl-2 also functions at the endoplasmic reticulum (ER), where it may have
distinct actions in preventing a p ~ ~ t o s i s ~ ~ . For example, in a rat-fibroblast ce11 line
expressing an activated c-myc allele, Bcl-2 targeted to the ER is as potent an inhibitor of
senun-withdrawal induced apoptosis as Bcl-2 confined to the rnitochon~iria~~. In
addition. BcI-2 reguiates calcium flux from the ER, although the effect of this function on
apoptosis is uncleds.
BAD a BH3-only protein:
The BW-only class of proteins are endogenous inhibitors of Bcl-2 and Bcl-xL
and are attractive prototypes for therapeutic agents. This class of compounds inhibits
Bcl-UBcl-xL through heterodimer formation. Among the BH3-only class of proteins,
BAD was the first identified and is the best characterized. BAD was first cloned from a
murine cDNA library through its ability to bind Bcl-2 in a yeast-two-hybrid assaYs6.
BAD heterodimerizes with Bcl-xL and Bcl-2 but not the pro-apoptotic members Bcl-xs,
Bax or ~ a k ? To date, the only known function of BAD is to bind Bc12 and Bcl-xL, and
block the anti-apoptotic action of these proteins"J7. BAD inhibits these anti-apoptotic
proteins by preventing them from binding ~ax/~ak'"" . BAD may also inhibit the ability
of Bcl-2 and Bcl-xL to bind Apaf-1. form pores in the mitochondrial membrane, or bind
and stabilize VDAC.
The BH3 domain of BAD is necessary for its ability to bind Bc12 and Bcl-xL and
induce apoptosis. Mutations within the BH3 domain of full Iength BAD inhibit the
binding of BAD to Bc12 and Bcl-xL and block BAD-induced apoptosis in ceI1s over-
txprtssing BCCXL~~. Frathemiore, peptides eomponding te the BH3 domein of BAD
competitively inhibit the binding of Bcl-xL to Bax. Mutations within these peptides
permit the binding of Bcl-xL to ~ a x ?
Cross ta lk between pathways
While the intrinsic and extrinsic pathways are presented as separate entities, cross
talk exists between them. For example, the two pathways cooperate to enhance apoptosis
through Bid. Bid, a member of the BH3-only family of pro-apoptotic proteins, is cleaved
and activated by caspase-8. Active Bid induces cytochrome c release and thereby
initiates the intrinsic pathway58sg. Conversely, BAR inhibits apoptosis in both pathways.
BAR binds and inhibits procaspase-8 and thereby blocks the extrinsic pathway. BAR
also inhibits the intrinsic pathway through an unknown rnechanism that involves
interactions with Bcl-2 and Bax. Physically, BAR bridges the two pathways by forming
a complex with procapsase-8 and Bcl-2, aithough the significance of this cornplex is
unknownm.
Regularion of apoptotic proteins through phosphorylation.
The apoptosis pathways are positive1 y and negativel y regulated through signal
transduction. Teleologically, this regulation allows tight control of apoptotic signals
avoiding unwanted ce11 death or proliferation. Therapeutically, these regdatory points
are valuable targets for the development of compounds that enhance apoptosis. Bcl-2 and
BAD are examples of proteins that are regulated by phosphorylation and whose
regulation is king exploited for dnig developrnent.
Three known sites of phosphorylation regdate Bcl-2; ser-17, ser-70 and thr-56.
Taxol induces phosphorylation of ser-17 and inhibits the anti-apoptotic function of Bcl-
261". Under Wfic conditions, phosphorylalion of ser-70 within the loop domain by
protein kinase C (PKC) activates the anti-apoptotic function of BCI-263. This finding is
controversial. however as other reports indicate that phosphorylation on ser-70 down-
regulates the action of Bel-2. Phosphorylation of Bcl-2 on thr-56 in the hop domain by
the ce11 cycle regulator CDC2 kinase is required for activation of Bcl-2's role as a ce11
cycle inhibitor. Mutation of thr-56 abrogates the role of Bcl-2 as a cell cycle inhibitor,
but does not alter the anti-apoptotic action of the protein; indicating that the function of
Bcl-2 as an inhibitor of apoptosis and a ce11 cycle inhibitor cm be ~i i s soc ia t ed~~~~.
Other sites of phosphorylation also appear important for the regulation Bcl-2 and
are currently king characterized. For example, ATRA inhibits Bcl-2 by inducing
phosphorylation on a senne distinct fiom the sites phosphorylated by taxol and PKC?
BAD is an example of a pro-apoptotic protein that is regulated by
phosphorylation. Phosphorylation of ser 112, ser 136 or ser-155 inhibits the function of
BAD. BAD is phosphorylated on ser-136 by Akt in the AktPUK pathway6s"6 and on
ser-112 by protein kinase C in the RasRaflMAPK p t h ~ a ~ ~ ' " ~ . Phosphorylation at these
sites promotes the binding of BAD to 14-3-3 proteins57. When bound to 14-3-3 proteins,
BAD is sequestered in the cytosol and unable to interact with Bcl-2 or Bcl-xL at the outer
mitochondrial membrane. The phosphorylation of ser-136 or ser-112 also induces a
conformational change in BAD that increases the accessibility of ser-155 within the BH3
domain to phosphorylation by protein kinase A~~'' . Phosphorylation of ser-155 directly
impairs the ability of BAD to bind Bcl-xL and BC~-Z"*".
Pathways that dephosphorylate BAD increase its activity and promote apoptosis.
CalcineUrin, a calcium dependent phosphatase, dephosphorylates BAD at both S112 and
5136 an& promotes BAD-hdnrrd apc~ptosis~~. The s t c a i c t mtssnrger cnamide piornotes
the accumulation of dephosphorylated forms of BAD by inhibiting
Defects in apoptosis are important prognostically
Defects in the apoptosis pathway render leukemic blasts resistant to multiagent
chemotherapy. These blocks translate clinically into reduced rates of remission, higher
rates of relapse, and reduced overall survival. Studies have identified specific blocks in
the extrinsic and intrinsic pathways that confer prognostic information in adult patients
with acute leukemia.
Ejltrinsic pathway .-
There is evidence to implicate defects in the extrinsic pathway of caspase
activation with chemoresistance in leukemia. Exposure of the T-ALL ce11 line CEM to
doxombicin induces apoptosis and up regulates Fas expression. A mutant CEM cell line
resistant to Fas ligand fails to up-regulate Fas or undergo apoptosis in response to
doxorubicin treat~nent~~. In contmt to this report, a Iurkat ce11 Iine resistant to Fas ligand
retained sensitivity to doxorubicin-induced apoptosis75. The discrepancy between these
two studies may reflect the numerous sites of blockage in the extrinsic pathway that can
induce resistance to Fas ligand. A defect at the level of the Fas receptor rnay render a ce11
resistant to Fas ligand, but if doxombicin triggers the pathway downstream of the
receptor, then the ce11 would rernain sensitive to doxorubicin. On the other hand, defects
in the extrinsic pathway downstream of where doxorubicin acts will render cells resistant
to both Fas ligand and doxombicin. Altematively, the discrepancy rnay result because
chemotherapeutic agents effect multiple pathways simultaneously. As cells acquire
defects in one pattrway they mmiask the dfem of the chemttierapeutic agent on the
other pathway.
Fas expression confea important prognostic information in patients with
leukemia. The Fas gene encodes two isoforms, full length Fas with a transmembrane
domain and a soluble fonn of Fas that lacks this transmembrane domain. Full length Fas
is the initiator of the extrinsic pathway of apoptosis. In contrast, soluble Fas is excreted
into the extracellular environment where it acts as a decoy and binds Fas ligand. As such,
soluble Fas is an inhibitor of apoptosis. In a study of 59 patients with adult T-cell
leukemia, levels of full length Fas expression did not influence patient survival.
However, increased serum levels of soluble Fas was associated with reduced survivai and
remained an important predictor in multivariate anal ysis even after accounting for clinicd
risk facto^-s7!
The prognostic importance of defects in the extrinsic pathway has not received
much attention to date. Perhaps, in the future, additional prognostic markers within this
pathway will be identified. For example, caspase-8 is inhibited by the dominant negative
inhibitor 1-FLICE. 1 - N C E over-expression has k e n documented in the
erythroleukemia ce11 line K562, but its incidence and prognostic importance in patients
with leukemia is unknownn.
lntnnsic Path way
Defects in the inninsic pathways of apoptosis also induce multidnig resistance
and confer important prognostic information. Most of the work on this pathway has
focussed on the Bcl-2 family of proteins.
BCL-2
Over-expression of Bcl-2 is an important cause of multi-drug resistance. As a
result, patients with AML and high b e l s of Bcl-2 have lower complete remission rates
and a lower overall survival compared to patients without high levels of Bcl-2. For
exarnple. in a study of 82 consecutive patients with AML treated with standard induction
chemotherapy, patients with high levels of BcI-2 expression by irnrnunofluroescent
staining had a complete response rate of 29% venus an 85% complete response rate in
patients without high levels of Bcl-2. Likewise. high level of Bcl-2 translated into
decreased overail survival. The predicted survival at 2 years in patients with increased
Bcl-2 was 15% venus 40% in patients without increased Bcl-2. Bcl-2 remained an
important predictor of sumival in multivariate analysis over other known prognostic
marken including age and WBC >30. The influence of cytogenetics was not reported,
however7*.
Surprisingly, some studies have indicated that increased levels of Bcl-2 are
associated wi th improved survival. In patients wi th breast cancer, increased Bcl-2 by
immunostaining comlated with improved disease-free sur~ival'~ and in childhood AU,
increased BcI-2 was associated with improved event-free survivalsO. These results may
reflect the importance of post-translational modification of Bcl-2. For example, PKC
activates B C I - ~ ~ ~ , so in the absence of PKC, the Bcl-2 that accumulates is non-hinctional,
confemng a better outcorne. In support of this hypothesis, lower levels of PKC
improved the prognosis in patients with AML8'.
Bax
In a study of 165 patients with newly diagnosed AMLal, levels of Bax expression
by irnmunoblotting did not comlate with response to induction chemotherapy or
survival. However, high ratios of Bcl2:Bax protein confemd a poor prognosis with
decreased rates of complete remission and overall survival. The prognostic
discrimination from the ratio of the proteins was greater than Bcl-2 levels aione. The
ratio also provided prognosîic information above cytogenetics. The study is flawed,
however, in that the patients selected were not consecutive and inclusion cnteria were not
predefined. In the future, by using more sensitive techniques to quantitate protein
expression, Bax may prove to be an independent prognostic marker.
Do wnstream Efectors
Levels of caspase inhibitors have been correlated with suMval in acute myeloid
leukemia (AML). Inhibitors of apoptosis proteins (IAP's) are a family of caspase
inhibiton that bind and inactivate caspases including caspases 3 and 9. As such, they
block apoptotic stimuli in both the intrinsic and extnnsic pathway. In a snidy of 78
patients with AML, 76 had detectable levels of XLAP. Patients with the lowest levels of
XIAP expression had the longest median swivala2. The importance of XIAP in the
context of other clinical prognostic factors is unknown, however.
In another study of patients with newly diagnosed AML, increased levels of
procaspase-3 in peripheral blood mononuclear cells correlated wiîh a poor prognosis.
However, patients with spontaneously increased levels of active caspase-3 at the time of
diagnosis showed improved swivals3. Likewise, in 45 consecutive adult patients with
ALL, incnased levels of active caspase-3 correlated with an improved rate of complete
remission? ni mttivariate andysts, tevets of caspase-3 w m no longer predictive of
survival, but the small sample size may have obscured the predictive effect. The poor
pmgnosis arnong patients with high levels of procaspase-3 may represent defects in
caspase activation and the subsequent accumulation of uncleaved caspase-3. The better
prognosis arnong patients with increased levels of cleaved caspase-3 may indicate
competent caspase activation pathways.
In contrast to these findings, Svingen et al.'' found no correlation between levels
of Apaf-1, prwaspase 3, 8, and 9 and response to induction chemotherapy in adult
patients with AML (n=42) or AIL (n=l8). A number of explanations may account for
the lack of correlation. First, the study had small sample size and thus, limited power to
detect a difference. However, the wide variation in the ievels of these apoptotoic
members suggests that simply increasing the number of patients would not reveal a
correlation. Altematively, this study used response to chemotherapy as their endpoint.
The overall response rate for patients with AML after the first induction was 53%, which
is lower than expected. Therefore, this group of patients may have had particularly
aggressive disease due to other molecular abnormalities that ovenhadow the importance
of procaspases or Apaf- 1.
The apoptosts padmay as a EInget lm uovet tnerapeuüc agents
Classical chemotherapeutic agents induce ce11 death through a variety of
mechanisms including directly or indirectiy affecting regulators of the apoptosis pathway.
Rather than review the mechanism of action of chemotherapeutic agents. however, 1 will
focus on small molecules that specificaily overcome blocks in the apoptosis pathway.
Anempts to develop these novel therapeutic compounds have targeted both the extrinsic
and intnnsic pathways of apoptosis (Table 3, figure 4).
Table 3: Prototypic therapeutic agents for treatment of leukemi a
Extrinsic Pathway
TNF receptor ligands TRALL
Caspase-8 activators CDDO
Intrinsic Pathway
Mitochondrial Disruption Alpha helix (KLALAKKLALAK)
Bcl-2 inhibitors Ant-BH3BAK cpm-BH3BAD HA- 14- 1 Antisense Bcl-2 oligonucleotides
Second Messengers PKC inhibitors
chelerythnne calphostin
PI3 kinase inhibitors W ortmanin Ly294002
AKT inhibitors
Intrinsic Pathw av
Cytochrome c
+ Apaf- 1
Figure 4a: Site of action of prototypic therapeutic agents for the treatment of acute leukemia (intrinsic pathway)
Figure 4b: Site of action of prototypic therapeutic agents for the treatment of acute leukemia (Extrinsic pathway)
Srnall molecules that target the extrinsic pathway cm induce caspase-3 activation
and overcome blocks confined to the intnnsic pathway, such as the over-expression of
Bcl-2 or Bcl-xL
TRAIL:
Treatment with TNF can induce apoptosis through the extrinsic pathway, but dso
produces severe toxicity to normal tissue8688. In contrast, TRAIL (AP02L), which is
sirnilar to Fas ligand and activates the DR3 and DR4 receptors, shows selectivity for
tumour cells despite the presence of the DR4 receptor on normal tissues89. In a study by
Ashkenazi et alw, soluble TRAIL induced ce11 death and apoptosis in a variety of
leukernic and solid tumor ce11 lines. In contrast, normal tissue including prostate,
fibroblasts, colon and smooth muscle cells were unaffected by exposure to TRAIL. In
nude rnice. intraperitoneal treatment with TRAIL induced apoptosis and shrinkage of
colon cancer xenografts. No observable toxicity from TRAIL was observed in these mice
or in studies of non-human primates. A m e n t report, however, has cautioned that
humans rnay experience significant hepatotoxicity after treatment with TRALL~~. TRAIL
induced apoptosis in normal human hepatocytes in culture, but was not toxic to rat,
mouse or monkey hepatocytes. Given these findings, TRAIL may have to be selectively
targeted to malignant cells to be a useful therapeutic agent.
Cmpase-8 activators
Compounds that activate the extrinsic pathway at the level of caspase-8 may
overcome blocks to apoptosis upstream of this level of regulation, such as at the level of
the TNF receptor family. Such compounds also can act independent of the level of
expression d the TNF receptor. One swh agent is 2-cyano-3,12-dioxodean-1,P-dien-2&
oic acid (CDDO). CDDO is a synthetic compound that is synthesized in an 1 1-step
reaction from oleanolic acid. This compound is a potent analogue of the naturally
occurring triterpenoids which are used as herbal remedies used to treat inflammation and
cancer. CDDO induces apoptosis in leukemic ceIl lines by activating procaspase-8. As
the compound targets the extrinsic pathway, over-expression of Bcl-xL has little effect on
the toxicity of this cornpound9'~92. Currently, it is unknown whether CDDO is a direct or
indirect activator of caspase-8. Potentially, direct caspase-8 activators would be very
useful clinically, because they could bypass both defects in both the inûinsic pathway and
blocks upstrewn of caspase-8 in the extrinsic pathway. The effects of CDDO on normal
tissue and bone marrow remain unknown, however, and this information would be
important before advancing this agent into clinical development.
lntrinsic Pathway
Most of the work in developing small molecules that target defects in apoptosis
have concentrated on the intrinsic pathway and Bcl-2 in particular.
Bcl-2 inhibitors
Bcl-2 is an attractive therapeutic target. It is over-expressed in a wide variety of
maiignancies including acute leukemia and its over-expression is often associated with
clinical treatment failures. Most intriguing, the temporary inhibition of Bcl-2 rnay not be
toxic to normal cells. In support of this hypothesis, Bcl-2 knockout mice survive until
birth with no histological abnormaiities in the brain, skin, intestines, or bone marrow
hematopoietic precursors. Beginning one week after birth, however, these mice begin to
develop rend dysfunction similar to polycystic kidney disease. By four weeks after birth,
the mice become lymphopnric prharify dat to the deah of mature T cellsg3.
Furthermore, inhibition of Bcl-2 does not automatically induce a compensatory
upregulation of other anti-apoptotic proteins. However, compounds that predominantly
inhibit Bcl-xL may be less useful therapeutically due to their potential toxicity to normal
tissue. In contrast to the Bcl-2 knockout mice, Bcl-xL knockout mice do not survive
beyond day 13 of gestation. These mice sustain massive neuronai ce11 death as well as
apoptosis of the hepatic haematopoietic and immature Furthermore. in
the most primitive hematopoietic precurson (CD34*,lin',CD38-, small size), Bcl-2 is
expressed in only 1-446 of cells. In contrast, Bcl-xL is expressed in greater than 95% of
these cells. As these primitive cells proliferate and differentiate, Bcl-2 expression is up
reg~la ted~~.
One strategy to create Bcl-2 inhibiton has focussed on developing small
molecules that mimic the action of the endogenous Bcl-2-binding death agonists. An
early prototype was the BH3 death domain of Bak. Hollinger et alg6.created a ce11
permeable version of BH3Bak by fusing it to the C terminus of the 21 amino acids
Antennapedia sequence that allows for entry into cells with high efficiency and low
toxicity. They demonstrated that this fusion peptide inhibited Bcl-2 and induced ce11
death and apoptosis in HeLa cells. Its effects on leukemic cells and its tumor specificity
were not reported. One might speculate, however that the BH3 domain on Bak might
show preferentiai killing of malignant cells and not induce apoptosis in normal tissues.
Bak induces apoptosis directly through its effects on the mitochondria, so the full-length
protein Uely would be toxic to normal cells. However, the role of its BH3 domain
seems confined to bindmg and intribitmg Bcf-2. and as sucit, rnight not induce apoptasis
in normal cells that do not have concurrent active death signals.
Compounds that rnimic the BH3-only class of death agonists may aiso be useful
novel therapeutic agents. BH3-only death agonists, such as BAD, inhibit the survival
proteins Bcl-2 and Bcl-xL, but do not appear to have independent death-promoting
activity. As such, they may show selectivity for malignant cells whose active death
signais are blocked by Bcl-Del-xL over-expression. Recently, the effects of a peptide
corresponding to the BH3 domain of BAD coupled to cpm, a fatty acid moiety
intemalization sequence have been repocted9'. The decanoic fatty acid cpm
(CH3(CH2)8CO) can shuttle peptides across ce11 membranes without untoward toxicity.
Like the peptide-based intemaiization sequences such as A N T ~ ' * ~ ~ , and TATI"
represents a novel vehicle for delivenng therapeutic peptides into celis.
The cpm-BH3BAD fusion peptide mimicked the action of its parent protein BAD.
It bound Bcl-2 in a ce11 free system and induced apoptosis in HL60 cells that over-
expressed Bcl-2. In contrast, cpm-BIUBAD had minimal effect on resting and
stimulatecl peripheral blood lymphocytes. When administered intraperitoneally into
SCID rnice, this peptide suppressed the growth of HL60 tumour xenografts without
inducing untoward toxicity. Based on the understanding of BAD. one rnight have
predicted that this compound would have been toxic to nomal tissue. BAD binds Bcl-xL
with higher aff5nity than BCI-~~ ' , and Bcl-xL is critical for the development of
hematopoietic precursoa and neural ce119*. So, one might have expected to see toxicity
to nonnal tissues as a result of its preferential inhibition of Bcl-xL.
fn a novet stratcgy to devetop a Bd-2 fibitor, Wang et d ' O 1 . screened a vimial
library of compounds. They modeled the binding of Bak's BH3 domain to the BHI,
BH2, BH3 pocket of Bcl-2. Then. using a cornputer simulation of binding, they screened
a virtuai library of 193,833 compounds for their potential ability to bind Bcl-2. From this
virtual database, 28 compounds were selected for physical testing. Of these 28, one
synthetic compound, HA-14-1 displayed an ability to bind Bcl-2 and induce apoptosis in
HL60 cells. The selectivity of this compound has not been reported, yet. Since the
binding pockets of Bcl-2 and Bcl-xL differ in sequence and structure. this synthetic
compound may show preferential toxicity to cells with high levels of Bcl-2 over those
with increased Bcl-xL.
A different strategy to inhibit Bcl-2 involves antisense oligonucleotides.
Liposomal antisense Bcl-2 oligonucleotides cm induce apoptosis in HL60 cells and
pnmary AMI, blasts. In addition, they c m sensitize the cells to matment with ~ r a - ~ ' " .
Antisense oligonucleotides have been used clinically in a phase I trial. In 21 heavily
pretreated patients with B-ce11 NHL, continuous subcutaneous infusion of an &mer Bcl-
2 antisense oligonucleotide yielded encouraging results. There was one complete
response, two partial responses and eight patients with stable disease. The only notable
toxicity was thrombocytopenia that was likely secondary to the phosphorothioate
backbone of the antisense rn01ecule~~~.
Second Messengers:
Targeting the proteins that regulate the activity of the Bcl-2 family of proteins
may be another useful strategy to developing novel therapeutic agents. Activation of BcI-
2 may require phosphorylation on ser-70 by PKC under certain àrcurnstances.
Thercfm, cumpwnQ that inhibit PKC may mhence apoptosis by blocking the
activation of Bcl-2. In HL60 cells, the PKC inhibitors chelerythnne and calphostin
induced apoptosis without changing levels of Bcl-2 ~ R N A ' ~ . However, the
phosphorylation status of Bcl-2 was not measured. Since PKC has many targets, it is
unclear if these inhibitoa functioned by blocking Bcl-2. Interestingl y, PKC activators.
such as bryostatin, also sensitized leukernic cells to ara-c induced apoptosis, which
highlights the importance of other pathways dependent of the activity of PKC"'.
Targeting the phosphorylation of BAD is another therapeutic strategy. The pro-
apoptotic action of BAD is inhibited by phosphorylation on ser-136 through the PDIAKT
pathway. Preven ting BAD-phosphoryiation may increase the acti vi ty of endogenous
BAD and promote apoptosis. Wortmanin and Ly294002 are P13K inhibitors and
sensi tize HL60 cells to undergo apoptosis after treatment with c hemotherapeutic agents.
While these agents block Akt activation, they do not alter phosphorylation of BAD or the
ability of BAD to heterodimerize with Bcl-2 or cl-dm. Therefore, these kinase
inhibitoa induce apoptosis but act through proteins other than BAD. Perhaps, these PU
kinase inhibitors act by enhancing the activity of caspase-9. Phosphorylation of caspase-
9 by Akt leads to inactivation1? Therefore, inhibition of AKT may prevent caspase-9
inactivation and enhance apoptosis.
In another attempt to prevent BAD-phosphorylation, AKT was targeted directty.
The C terminai fragment fiom the Protein Kinase C-Related Kinase-2 was identified as
an AICT inhibitor. Treatment with this protein inhibited AKT, blocked phosphorylation
of BAD, and sensitized 293 cells to apoptosislo8.
On-ect mirochmdnat t0Xms.-
Compounds that directly disrupt mitochondrial function and induce cytochrome c
release may serve as prototypes for therapeutic agents for the treatment of leukemia.
Alpha helical peptides are one such cornpound. Some amphipathic alpha helices are
potent naturally occurring antibiotics that disnipt the negatively charged bacterial
membranes. These antibiotics are not toxic to the mammalian ce11 outer membranes as
the membranes are only weakly negatively charged and stabilized by cholesterol. When
intemalized into mammalian cells, alpha helixes are exposed to the negatively charged
inner mitochondria that lacks cholesterol 109*110. Ellerby et al ' ". intemalized a 14 amino
acid alpha helical peptide (KLALAK)2 and demonstrated that it induced mitochondrial
swelling, caspase activation, and ce11 death. Using a tumour specific intemalization
sequence, they demonstrated that the alpha helix could cause regression of h a s t cancer
xenografis in nude mice without untoward toxicity to normal tissue. Potentially, alpha
helicai peptides could have value in the treatment of leukemia if targeted selectively.
CONCLUSION
Through basic investigation, the apoptosis pathway has ken pieced together.
Over tirne, M e r details will be elucidated, but the major players are likely in place.
Now, the goal is to translate this basic work into clinical practice. Prognostic marken are
king identified and prototypes for novel therapeutic agents are king developed. The
challenge ahead is to furthet the knowledge of prognostic markers and incorporate them
into current treatment protocols. On the therapeutic front, small molecules that overcome
defects in apoptosis hold promise for reversing multidrug resistance. Here the challenge
relates to enhancing tumour specificity, improving drug delivery, and moving the agents
intu c M c d me. The proof of principle has bem estabtished, however, and the future
looks brighter for the treatment of acute leukemia.
ACKNO WLEDGEMENT
We would like to thank JC Reed (Bumham Institute, La lolla, CA) for his heIpfuI advice
and discussion.
Authorization to include this material in the thesis has k e n granted by the CO-authors.
Contributions to manuscript: ADS conceived of the article, and wrote the paper. DWH and LZP reviewed the manuscript and provided critical feedback. MDM supervised the work and provided cri tical feedback
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BAD Induces Apoptosis in Cells Over-Expressing Bcl-2 or Bcl-xL without Loss of Mitochondrial
Membrane Potential
Aaron D. Schimmer, David W. Hedley, Nhu-An Pham, Sue Chow, Mark D.
Minden
Pnncess Margaret Hospital, University Hedth Network. Toronto. ON. Canada
Running TitIe: BAD induces apoptosis
ABSTRACT Inhibitors of BcI-2 may be useful therapeutic agents for the treatment of a wide variety of
malignancies including leukernia. A potential prototype of such a compound is the
endogenous Bcl-2 and Bcl-xL binding protein BAD. Previous reports indicate that BAD
can overcome the anti-apoptotic effect of Bcl-xL but not Bcl-2. If BAD cannot induce
apoptosis in cells over-expressing Bcl-2, it would limit the application of molecules like
BAD as novel anti-tumor agents. In this study, transient transfection of BAD induced cell
death in cells with and without over-expression of Bcl-2 or Bcl-xL. Forty-eight houn
after transfection, BAD increased cc11 death in COS, COS Bcl-2, and COS Bcl-XL cells.
In addition, BAD induced ce11 death in leukemic cell lines over-expressing Bcl-2 and Bcl-
xL. BAD-induced apoptosis was not accompanied by loss of mitochondrial membrane
potential. Therefore, in conclusion mnsient transfection of BAD directly induces
apoptosis in cells over-expressing Bcl-2 or Bcl-xL and validates the pursuit of molecules
like BAD as novei thenpeutic agents.
Key words: BAD, apoptosis, Bcl-2, Bcl-xL, rnitochondria, mitochondrial membrane
potential
INTRODUCTION The over-expression of Bcl-2, a common finding in rnalignant cells. is an
important mechanism by which they become resistant to a variety of foms of
chernotherapy"'. For example, leukernic blasts over-expressing Bcl-2 are resistant to
cytosine mbinoside. etoposide. dexamethasone. and ta~ol '*~. In addition, patients with
acute leukernia and high levels of Bcl-2 have lower complete remission rates and a lower
ovenll survival compared to patients without high levels of BCI-z718. Thus, compounds
that inhibit Bcl-2 may overcome multi-drug resistance and be useful in the treatment of a
wide variety of malignancies including acute leukemia. A prototype of such a molecule is
the endogenous Bcl-2 inhibitor. BAD (Bcl-2 Associated Death protein).
A family of prûteins including BAD^'", ~ i k " . ~ l k " , ~ i m l ~ , ~ i d ' ' and ~ r k ' ~ share
a cornmon BH3 domain and are endognous inhibiton of Bcl-2 . Of these family
mcmbers, BAD was the first identified and is the best characterized. BAD is a 29kDa
phosphoprotein that heterodimenzes with Bcl-xL and Bcl-2 but not the proapoptotic
members Bcl-xs, Bax or Bak 10.1 1.14.17.18 . To date the only known function of BAD is to
bind Bc12 and Bcl-xL, and block the anti-apoptotic action of these proteins. According to
the proposeci mechanism, blocking B c l - and Bcl-xL pemirs Bax or Bak
homodimerization and potentiates apoptosis.lO.l'.l'
Based on this rnechanism of action, BAD should induce apoptosis in cells over-
expressing Bcl-2 or Bcl-xL. Despite this widely held belief, the literature indicates that
BAD does not sensitize rnalignant ceils with high levels of Bcl-2 to apoptosis after
cytokine withdrawal. Yet, the same cells over-expressing Bcl-xL rire sensitized by BAD
over-expressionlO. if BAD is unobk to indoce apoptosis in cetk over-expressing Bct-2. it
would limit the application of molecules like BAD as novel therapeutic agents.
Here 1 report that transient transfection of BAD directly induces apoptosis in cells
with and without over-expression BcI-2 and Bcl-xL. These findings demonstrate that
BAD can overcome the anti-apoptotic action of Bcl-2 and validate the potential role of
molecules that mimic the action of BAD as broadly acting anti-tumor agents.
MATERIALS AND METHODS
Cells - Monkey kidney COS cells were naintained in Dulbeco's Modified Eagle's medium with
450mgL glucose (DMEM H21). Erythmleukemica K562 cells were grown in Minimal
Essential Medium alpha (alpha MEM). Murine myeloid precursor 32D cells over-
expressing Bcl-2 (32D Bcl-2) (a gift frorn S. Berger) were created by infection of 33D
cells with a retrovims containing munne Bcl-7. They were rnaintained in RPMI 1610
with 2 8 X63 conditioned medium containing IL-3. Al1 cell lines were supplemented
with 10% fetal calf serum (Wisent), penicillin and streptomycin. The cells were grown at
37'C in 3 5% C a incubator
PIasmids
Murine BAD with a hemaglutinin tag (gift from S.J. Korsmeyer) was subcloned into the
EcoRl sites of pcDNA3 (Invitrogen) and pTracer CMV (Invitrogen). The vector pTncer
CMV is a dual promoter plasrnid where the CMV promoter drives the gene of interest
and the SV40 promoter drives green fluorescent protein (GFP). Restriction enzyme
digestion and DNA sequencing conffrmed the correct sequences and orientations. f m m
Bcl-2 (gift from S.J. Korsmeyer) was obtained in prcCMV and human Bcl-xL with a
hemaglutinin tag (a gift from G. Nunez) was obtained in pcDNA3 (Invitrogen). In both
of these vecton, the CMV promoter drives expression of the gene of interest.
Transfec tion
COS cells were rnnsfecred with the desired plûsrnids using Lipofectarnine
(Gibco-BRL) according to the recommended protocol. COS cells (2x IO.') were seeded
onto six well plates i n ZmL of DMEM H2 1 with IO% fetd cdf serum. Twenty-four
houn later. they were transfected with Lipofectamine at a ratio of 2ug plasmid to lOul of
Lipofectarnine. The transfection medium was incubated with the cells for fÏve hours and
then replaced with fresh medium. The medium was changed again the next moming.
Using this transfection protocol, 1 could routine1 y achieve a transfection efficienc y of
85% as mesured by GFP expression after transfection of GFP in a pcDNA3 vector. GFP
expression after transfection of G W in pTracer was approxirnately 35%. The lower tevel
of GFP expression after transfection of pTncer is due to the weaker SV40 promoter
dnving the expression of GFP and the presence of two competing promoters in this
vector.
COS cells over-expressing BcI-2 (COS Bcl-2) and Bcl-xL (COS Bcl-xL) were
isolated by selecting cells with 0.3mg/ml of G418 after transfection. Stable clones were
selected 14 days after transfection with cloning rings. Over-expression of Bcl-2 and BcI-
xL in individual clones was confirmed by a Western blor assay. Single clones stably
expressirrg Bel-2 or Bci-fi were maint- in d m with 0.3rng/ml G4 18 unti1
needed for the experiments.
K562 cells were transfected with the desired plarnids using Lipofectamine. K562
cells (2x 106) were seeded onto six well plates in 0.8mL DMEM H I 1 without serum or
antibiotics. The cells were immediately tnnsfected with a ratio of Zug plasmid to 5uL of
Lipofectarnine. After a five hour incubation, 4mL of fresh alpha MEM +10% fetal calf
serum was added to the cells. With this protocol. I could rourinely achieve a transfection
efficiency of 5% as measured by GFP expression after transfection of GFP in a pcDNA3
vector.
32D cells were transfected by electropontion. 32D cells ( 5 . 5 ~ 106) were
electroporated (Gene Puiser II, Biorad. MA) with a capacitance of 975uF. 300 volts, and
20ug of plasmid in 1mL of PBS. After electroporation, the cells were left on ice for 30
minutes and then transferred to lOmL of complete medium. Using this transfection
protocol. 1 could routinely achieve a tnnsfection efficiency of 4% as measured by GFP
expression after transfection of GFP in a pcDNA3 vector.
Western Blot
Protein extracts were prepared from COS cells 24 hours after transfeciion. The cells were
washed with PBS and resuspended in lysis buffer (50mM Tris pH 8, 15OmM NaCL, O. 1%
SDS, 1% NP-40, 0.5% DOC, irnM pMSE lOug/mt aprotinin, and 5uM leupeptin).
Protein concentrations were determined by spectrophotometry. Equd protein
concentrations were electrophoresed on a standard SDS- 15% polyacrylamide gel and
transferred to a nitrocellulose membrane (Mandel Scientific, Ontario). Membranes were
washed twice in PBS with 1% non-fat milk and 0.1% Tween (PBS-T). They were
blockeb for one hour in PBS witk &O% non-far milk. Membranes were then probeci witk
polyclonal rabbit-BAD (1:300 dilution) ( a gift from S.J. Konmeyer), polyclonal mouse
anti-human Bcl-2 (1: 1000 dilution) (Dako), polyclonal nbbit anti-mouse Bcl-2 (1: 1000
dilution) (Pharmingen), monoclonal mouse anti-HA to detect HA-Bcl-xL
(Sug/rnL)(Boehnnger Mannheim), pol yclonal mouse an ti-Bcl-xL( 1 2 5 0 dilution)
(Pharmingen), polyclonal mouse anti pigeon cytochrome c ( 1 : 1000 dilution) (Sigma) or
polyclonal nbbit anti-actin (1:300 dilution) (Sigma) as a loading control. The
membranes were probed with the primary antibody for iwo houn at room temperature
and ihen washed twice in PBS-T. Secondary anti-mouse (L:SOOOdilution) or anti-nbbit
(1:5000 dilution) antibodies were applied for one hour. The membranes were washed
twice in PBS-T. The membranes were then developed using an enhanced
cherniluminescence (ECL) detection kit (Amersharn, Arlingon Heights, IL) according to
the manufacturen directions. The intensity of the autoradiognph bands were quanti tated
with a densitometer (Molecular D ynamics, Sunnyvale, CA)
Cell death assavs
GFP expression
GFP expression was assayed as a measure of cell ~iabilit~'~. COS ceUs were transfected
with pTracer-CMV BAD. the empty pTracer-CMV vector or BAD in the antisense
direction in pTracer-CMV. in pTracer, GFP and the gene of interest are in the same
plasmid. Thus, there is no need to adjust ratios of the marker plasmid to the plasrnid of
interest. Forty-eight hours after tnnsfection, GFP expression was measured by flow
cytornetry. Adherent cells were harvested, washed once in PBS, and then resuspended in
500ul of PBS. The cells were stained for five minutes at room tempenture with the
DNA-binding dye 7-runino-actinomy.Cin (7AAD) (fin& concentration 2ng/ut) (Sigma) as
a viability marker. After staining, GFP and 7AAD fluorescence were measured by flow
cytometry (FACScan; Becton, Dickinson, Immunocytometry system, CA, USA). The
Lysis II program was used to analyze these data. Viable cells were gated and the
percentage of gated cells expressing GFP was deterrnined. As transfected cells died, the
percentage of cells expressing GFP decreased. The number of cells expressing GFP after
transfecting pTracer-CMV BAD was expressed as a percentage of the number of cells
expressing GFP after transfection of the empty pTracer-CMV vector. GFP expression is
the mean of three transfections.
Floating cells
Ce11 death was measured by determining the number of celis that had detached from the
tissue culture plate and were floating in the rnedium19. As COS cells die. they detach
from the tissue culture plate and float in the medium above. Using a hematocytometer,
ce11 death was measured by counting the number of cells floating in the medium 48 houn
after transfection. The number of floating cells after transfection of pTrücer-CMV BAD
was expressed as a fold increase over the number of floating cells after transfection of the
ernpty uector,
Clonogenic colony assay
A clonogenic colony assay was performed after transfecting equal number of COS Bcl-2
cells with pTracer-CMV BAD or empty vector. Forty-eight hours after transfection
equal volumes of cells were plated into three sepante 100mm3 tissue culture plates. No
antibiotic selection was performed. One week after seeding, the medium was removed,
and the plates stained with methylene blue. The number of colonies formed was counted.
With rhis protocot, I coukt routinefy achieve a 50% ptating efficiency ;ifter mnsfectim of
cells with the empty vector.
Luciferase Assay
Luci ferase mtivity was measured as a marker of ce11 v i ab i~ i t~ '~ . K562 cells and 32D Bcl-
2 cells were CO-transfected with equal concentrations of BAD in pcDNA3 or empty
vector and the luci ferase reporter gene under control of the RSV promoter. Forty-eight
houn after tnnsfection, cells were harvested, and washed in PBS. Cytoplasmic protein
was extr~cted with lysis buffer (Promega, Madison, WI, USA). Luciferase assay
subsuate (Promega, WI, USA) was added to an aliquot of lysate and luciferase activity
was measured with a Lumat LB9507 luminometer (Berthold Systems Inc, Manyville, TN.
USA). As transfected cells died, the luci ferase activity decreased. The luci ferase activi ty
after transfection of B A D was expressed as a fold decrease compared to tnnsfection of
the empty vector. Luciferase activity represents the mean of three transfections.
Apo~tosis assay
We measured apoptosis by staining for fragmenad genomic DNA with the DNA-binding
dye Hoechst 33341 (H33342) stain. COS, COS Bcl-2 and COS Bcl-xL were transfected
with pTncer-CMV B A D or empty vector as described above. Forty-eight houn after
transfection, the adherent and fioating cells were harvested sepantely and stained with
H33342 for a final concentration of 20n.M for 30 minutes at 37 '~ . After staining, the
cells were mounted on slides and were examined under ultraviolet Iight for the presence
of apoptotic bodies. The percentage of apoptotic cells represents the mean of the at least
three transfections.
Mitoc hondnaf Membrane Potentiaf
Changes in Mitochondrial Membrane Potential (A y%*) were detected by staining the cells
with DiIC 1 (5 ) ( 1,1 '3.3,3',3'-hexamethylindodicarbocyanine)"." at a final concentration
of 4OnM and propidiurn iodide (final concentration Sug/rnL). DiIC l(5) concentntes in
the negatively charged mitochondrial rnatrix. As mitochondria lose their membrane
potential, the dye is no longer bound in the mitochondria and diffuses out of the cell.
Cells stained with DiICI(5) were incubated at 3 7 ' ~ for 30 minutes and then analyzed by
flow cytometry (Coulter Epics Elite, Coulter, Flonda) by exciting at 633nm and
rneasuring through a 675 _+ 20nm bandpass filter.
RESULTS
Selection of Bcl-2 and Bcl-xL ex~ressing clones
COS cells over-expressing Bcl-2 (COS Bcl-2) or Bcl-xL (COS Bcl-xL) were
selected two weeks after transfecting them with human BcI-2 or Bcl-xL expression
vectors. Bcl-2 and Bcl-xL over-expression were confirmed by Western blot (Figure la).
Bci-2 was detected with a polyclonal anti-human Bcl-2 antibody and Bcl-xL was detected
with a monoclonal anti-HA antibody against the HA tag on Bcl-xL. COS cells had
detectable basal levek of Bel-2 prior ta trmsfectim. The stable dones h3d 5 and 1 1 fotd
higher Bcl-2 and Bcl-xL expression respectively than the parent COS line as detemined
by densitometry of the Western blot.
COS COS + BCL- 2
BCL- 2 -
ACTlN - BCLXL
COS COS +BCLXL
ACTlN r
Fi pure 1 q Western blot demonstmting expression of Bcl-2, Bcl-xL in COS cells. COS cells were transfected with human Bcl-2 or Bcl-xL and stable clones were selected with G418. Protein lysates were separated on a 15% polyacrylmide gel and transferred to a membrane filter. Bcl-2 was detected with a polyclonai rabbit anti-human Bcl-2 antibody and Bcl-xL was detected with a monoclonal mouse anti-HA antibody directed against the HA tag on Bcl-xL. Membranes were pmbed with polycional rabbit anti-actin to demonstrate equal protein loading. Secondary anti-mouse or anti-rabbit antibodies were applied. The membranes were developed using an ECL detection kit.
BAD inchces cett death TI COS cetts
The effects of BAD on ce11 death and apoptosis were studied after transient tnnsfection.
BAD expression was detected 24 houn after tnnsfection by Western blot using a
polyclonal anti-BAD antibody (Figure I b). No BAD expression was detected in COS
cells pnor to transfection.
GFP expression
Cell death was measured by dernonstrating a decrease in the percentage of cells
expressing GFP by flow cytornetry. GFP expression peaked 24 hours after transfection of
pTracer-CMV and ce11 death due to BAD was maximal at 48 hours. In contrast. ceIl
death was minimal before 34 hours after transfection. For example, in a representative
experiment. 24 houn after tnnsfection of pTraccr-CMV BAD. the number of cells
expressing GFP was 83% of the empty vector. Therefore, I perfonned al1 measurements
of ce11 viability 48 houn after transfection. pTncer-CMV BAD or the empty vector were
tnnsfected into COS, COS Bcl-2 or COS Bcl-xL . Forty-eight houn after transfection,
the cells were analyzed by flow cytometry. The viable cells (7AAD negative) were gated,
and GFP expression among the gated cells was measured. The number of cells
expressing GFP after transfecting pTracer CMV- BAD was expressed as a pementage of
the number of cells expressing GFP after tnnsfection of the empty vector (Figure 2). The
percentage of GFP expression is the mean of the three transfections. Transfection of
pTncer-CMV BAD into COS, COS Bcl-2, or COS Bcl-xL reduced the number of cells
expressing GFP to 9.5+1.2%, 1021.8 and 8.621.5 % of the empty vector respectively.
These reductions were significant by the t-test (p = O.Oû3,0.0001. and 0.009
COS COS + BAD
Fieure 1 b Western blot demonstrating expression of BAD in COS cells. COS cells were transfected with murine BAD. Twenty-four hours after transfection, cellular protein was extracted. Protein lysates were separated on a 15% polyacryiamide gel and transfened to a membrane filter. BAD was detected with a polyclonal nbbit anti-BAD antibody. Membranes were probed with polyclonal nbbit anti-actin to demonstrate equal protein loading. Secondary anti-mouse antibodies were applied. The membranes were developed using an ECL detection kit.
Untreated
O empty vector
COS COS Bcl-2 COS Bcl-xL
cell line
Empty Vector BAD
Figure 2 BAD induced ce11 death as measured by a decrease in GFP expression. COS, COS Bcl-2 and COS Bcl-xL were transfected with pTracer-CMV BAD or empty vector. Forty-eight hours after transfection, adherent cells were harvested and GFP expression arnong viable celb was measured by flow cytometry. a) The number of cells expressing GFP d e r transfecting BAD was expressed as a percentage of the number of cells expressing GFP after transfecting the empty vector. The transfections were performed in ûiplicate. The results shown are the mean of three tram fec tions. Bars represent 1 standard error (SE) b) A representative flow cytometry experiment of COS cells transfected with pTracer- CMV BAD or empty vector.
respectively). Transfection of antisense BAD in pTncer CMV yielded GFP expression
equivalent to the empty vector (data not shown). The reduction in the percentage of cells
expressing GFP after BAD tnnsfection coincided with a reduction in the mean channel
fluorescence for GFP demonstnting that only cells weakly expressing GFP remained
after BAD transfection.
A dose response curve for BAD-induced ce11 death was constructed. COS cells were
tnnsfected with increasing amounts of pTracer-CMV BAD and GFP expression was
measured 48 hours after transfection. Empty vector was supplernented to maintain an
equivaleni DNA concentration of 2ug for the tnnsfections. Transfection of COS cells
with increasing amounts of pTncer-CMV BAD caused a corresponding decrease in GFP
expression (Figure 3).
I 1 I I
0.5 1 1.5 2
BAD cDNA (ug)
Figure 3
Dose response curve for BAD-induced apoptosis. COS cells were tmnsfected with increasing amounts of pTracer-CMV BAD. Empty vector was supplemented to maintain an equivalent DNA concentration of 2ug. Forty-eight houn after transfection, adherent cells were harvested the percentage of viable cells expressing GFP was determined by flow cytometry. The trûnsfections were performed in tnplicate and the results shown are the mean of three transfections. Bars represent 1 SD
Floating cells COS cells normally adhere to the tissue culture plate. but they round up. detach from the
plate, and float in the medium above when they lose viability. 1 measured ce11 death by
determining the number of ceils that were floating in the medium, An increase in tloating
cells has been reported as a marker of ceIl death after exposing ridherent cells to
chemotherapy2', heat shock2', c ytokines'4 and ionizing radiation? Desjardins et al".
determined that the detachment of HT29 cells from the tissue culture plate wlrs a late
event in the apoptosis cascade and correlated with fragmentation of the genornic DNA
into 180- 120bp oligomen.
Transfection of pTracer CMV -BAD into COS, COS Bcl-2, or COS Bcl-xL increased the
number of floating cells 8.7 2 1.7 fold, 5.2 t 1 and 5.2 21.3 fold respectively compared to
transfection of the empty vector (Figure 4). These increases were significant by the t-test
(p = 0.001.0.02, and 0.02 respectively). Transfection of antisense BAD did not increase
the number of floating cells compared to transfection of the empty vector. The floating
celIs were replated in fresh medium to confirm thrit they were nonviable. No cells
adhered ro the plate and no colonies grew after one week of observation. In contrast.
viable COS cells adhere to the tissue culture plate within 18 hours of plating and fom
colonies within a week.
L
L cn O L œ - a O $15 1 0 -
7 - .E 02 4 - 3 . 5 , O , € I 1 t - - QS O COS COS Bcl-xL COS Bcl-2
cell line
Figure 4
BAD induced ceIl death as rneasured by an increase in the number of floating cells. COS, COS Bcl-2, and COS Bcl-xL were tmnsfected with pTracer-CMV BAD or empty vector. Forty-eight hours after transfection, the number of floating cells were counted. The number of floating cells after transfection of BAD are expressed as ii fold increase over the number of floating ceils after transfection of the empty vector. The results shown are the mem of three transfections. Bars represent I SD.
Cionooerric assay
To funher study BAD-induced cell death in cells over-expressing Bcl-2, I performed a
clonogenic colony formation assay. Forty-eight hours after transfection of pTmcer-CMV
BAD or the empty vector into COS Bcl-2, the cells were harvested and equal volumes
were plated into 100mm3 plates. This volume was intended to represent 400 cells pnor to
transfection. One week later, the number of colonies was counted. Transfection with
BAD yielded 1 15 14 coionies whereris tnnsfection wi th the empty vector yielded 192 &
3 colonies. This difference was significant by ri t-test (p=0.0006). The reduction in the
number of colonies formed is less than the reduction in GFP expression. The reduction is
less, because tnnsfection efficiencies were approxirnately 35% as measured by GFP
expression after transfection of the empty pTracer CMV vector. Therefore, in the colony
formation assay, the majority of cells plated were not transfected. In contnst, only
successfull y transfected cells were assayed in the GFP expression experirnent.
BAD induces ce11 death in hematopoietic ceIl Iines
Next, 1 wished to determine whether BAD could induce death in hematopoietic ceIl lines.
K562 is a leukemic ce11 line with high levels of BCI-XL'~ and 32D BCI-2 is a murine
mye loid precunor ce l l line over-expressing ~ c l - ~ " ( ~ i ~ u r e 5). These cells were co-
transfected with equal concentrations of BAD in pcDNA3 or empty vector and the
luciferase reporter gene under control of the RSV promoter. Forty-eight houa after
tnnsfection, lucifemse activity was measured. Transfection of BAD into K562 and 32D
Bcl-2 cells reduced the luciferase activity 4.29.8 and 5S+L -9 fold respectively compared
to transfection of the empty vector. Transfection of BAD in the anti-sense direction
produced similar results as the empty vector.
Actin - Actin -
m Western blot demonstrating expression of %cl-2 and Bcl-xL in 32D Bcl-2 cells and K562 cells. Pro- tein lysates from 32D Bcl-2 cells and K562 cells were separated on a L5% polyacrylamide pl and transfened to a membrane filter. Bcl-2 was detected with a polyclonal nbbit anti-mouse BcI-2 anti- body and Bcl-xL was detected with a monoclonal rabbit anti-Bcl-x antibody. Membranes were probed with polyclonal rabbit anti-actin to demonstrate equd protein loading. Secondary anti-rabbit antibodies were applied. The membranes were developed using an ECL detection kit.
O ! I 1 1
O 0.25 0.5 1
BAD cDNA (ug)
Figure 6
BAD induced ce11 death in K562 cells in a dose dependent rnanner. K562 celis were transfected with increasing doses of BAD in pcDNA3 and the tuciferase reporter gene. Empty vector was supplemented to maintain an equal DNA concentration. Forty eight hours after trmsfection, the cells were harvested and luciferase activity was measured. Results are expressed as a percentage of the luciferase activity after trmsfection of empty vector. Each point represents the mean of three transfections. Bars represent 1 SD.
A dose response curve was constrrrcted for the ef fe ts of BAD an K562 cetls. As t he
dose of BAD cDNA was increased, there was a corresponding decrease in luciferase
activity (Fig 6).
BAD induces apo~tosis but does not alter 4 Y
WC wished to determine whether the increased toxicity observed after transient
transfection of BAD was due to an increase in apoptosis. Apoptosis was mecisured by
staining for fragmented genomic DNA with the H33342 stain (Figure 7). Forty-eight
hours after transfection, the adherent and floating cells were harvested sepuately and
examined for apoptotic bodies using H33342. Transfection of BAD into COS, COS Bcl-
2, and COS Bcl-xL increased the percentage of apoptotic floating cells compared to
transfection of the empty vector. In addition, transfection of BAD increased the number
of floating cells as stated above. BAD over-expression increased the number of apoptotic
cells still adherent to the tissue culture plate. The absolute percentage of apoptotic ceIls
still adherent to the tissue culture plate was relatively low, because the majority of the
cells on the plate were not successfully transfected and many of the apoptotic cells had
detached from the plate and were floating in the medium. Transfection of the empty
vector produced a basal level of apoptosis ihat was attributed to the ~ransfection
procedure and the Lipofectamine reagent.
Mitochondna play an important role in the apoptosis pathway. Feiitures of
mitochonàrial dysfunction in apoptosis are loss of AThi and release of cytochrome c into
the cytosol. Previous reports have indicated that apoptosis produced by Bax and Bak is
accompanied by loss of AY 51 and cytochrome c r e l e a ~ e ' ~ ~ ~ ~ . In addition,
unphosphorylated BAD translocates from the cytosol to the mitochondria where it
C
mmpty vector l BAD
I
COS COS Bcl-2 COS Bcl-xL
cell Iine
O empty vector
BAD
COS COS Bcl-2 COS Bcl-XL
cell line
Figure 7: BAD induced apoptosis as demonstr~ted by staining with H33342. COS, COS Bcl-2, and COS Bcl-xL were transfected with pTracer-CMV BAD or empty vector. Forty-eight hours after transfection, floating and adherent cells were harvested sepmtely. The cells were stained with H33342 and the cells observed under ultraviolet light for the presence of apoptotic bodies. The percentage of apoptotic cells (A) Roating cells and (B) adherent cells was determined. Results show represent the mean of at least t h e transfections. The di fference between the number of apoptotic adherent cells after transfection of BAD or the empty vector into COS, COS Bcl-2 or COS Bel-xL is significant by the t-test (p = 0.0003,0.003,0.022 respectively). The ciifference between the number of apoptotic floating cells after transfection of BAD or the empty vector into COS, COS Bcl-2 or COS Bcl-xL is significant by the t-test (p = 0.02,0.0001.0.032 respectively). Bars represent 1 SD
interacts with Bcl-2 and induces apoptosis30. The impact of BAD on A Y M and
cytochrome c release wüs not measured. Therefore, I wished to determine whether the
apoptosis induced by BAD is associated with these features.
Thirty hours after transfection of BAD cDNA . while the majority of the cells still
remained adherent to the tissue culture plate, adherent and floating COS cells were
harvested and stained with DiIC l(5). Loss of A Yzl was rneasured by fIow cytornetry.
No increase in the percentage of cells displaying loss of A Y,\! was observed after
transfection of BAD compared to transfection of the empty vector despite an increase in
the number of cells staining positive for propidium iodide (Figure 8). A subpopulation of
cells with loss of A was also not detected among the cells expressing GFP after
transfecting BAD in pTracer. Similar results were obtained when the cells were
harvested 48 hours after transfection. At this time point. more cells stained positive for
propidi um iodide, but no loss of AYM was observed. Li kewise, no increased loss of A <YAv
was observed in K562 cells transfected with BAD compared to empty vector.
Cytochrome c release was measured frorn COS cells after transfection with BAD
or empty vector. Thirty hours after transfection, the cells were harvested and the proteins
extracted. Cytochrome c wüs measured in the protein lysate by immunoblotting (Figure
9). Transfection of BAD increased cytochrome c release 2.2 fold compared to
transfection of the empty vector as measured by densitometry. The increase in
cytochrome c release after transfection of the empty vector compared to untreated cells
likely reflects the basal level of apoptosis produced by the transfection procedure.
untreated empty vector BAD
Figure 8 BAD does not alter A PM. Thirty hours after transfection of BAD in pcDNA3 or empty vector, the cells were harvested and stained with propidium iodine and DiICl(5). Loss of A YM was measured by flow cytometry. A representative experiment is shown.
cytochrome c
Actin
untreated empty vector
Fimue 9. BAD induces cy toc~ome c release. COS cells were transfected with BAD in pcDNA3 or empty vector. fhirty hours afler transfection, the floating and adherent ceils were harvested and the proteins extracted. Equal concentrations of SDS protein lysates were separated on a 15% polyacrylamide gel and transfened to a membrane filter. Cytochrorne c was detected with a polyclonal mouse anti-pigeon cytochrome c antibody. Membranes were probed with polyclonaI rabbit anti-actin to demonstrate equal protein loading. Secondary anti-mouse or anti- rabbit antibodies were applied. The membranes were developed using an ECL detection kit.
We adopted a transient transfection mode1 to assess the impact of BAD on cells over-
expressing Bcl-2 and Bcl-xL. 1 began my studies with COS cells because they can be
transfected with high efficiency and thus, they serve as a useful system in which to assess
ceIl viability after transient tnnsfection. Using this system. 1 demonstrated that BAD
induced apoptosis in ce1 1s over-ex pressing Bcl-2 and Bcl-xL. Having refuted the notion
that Bcl-2 over-expressing ceils are nsistant to BAD-induced apoptosis, I extended my
work to dernonstrate that BAD induced cell death in malignant hematopoietic ce11 lines
over-expressing these survival factors.
The ability of BAD to induce apoptosis in cells over-expressing Bcl-2 is
predicted, but kas not been shown previously. In fact, just the opposite is sugpsted in the
first description of BAD by Yang et al1'., who reported that BAD could modulate
apoptosis in cells over-expressing Bcl-xL but not Bcl-2. E U . 13 cells stably over-
expressing BAD and Bcl-xL underwent apoptosis efter IL-3 withdnwal. In contrast, the
same ce11 line with stable over-expression of BAD and Bcl-2 remained viable after
cytokine withdnwal. The protective effects of Bcl-2 were supported by Ottilie et al9. In
a vansient transfection assay, BAD induced cell death in 293 cells over-expressing Bcl-
xL, but could not induce ce11 death in the same cells over-expressing Bcl-2. In the
experiments with cells over-expressing Bcl-2, there was a high background of ce11 death
due to the transfection method and this high background may have obscured the effects of
BAD. As a consequence of these studies, other reports of BAD and apoptosis have only
evaluated the effects of BAD in cells over-expressing B C I - X L " + ~ ~ .
A few possibiIities rnay exphin why I was able to demonstrate BAD-indnced
apoptosis in cells over-expressing Bcl-2 when others have not. First. the high levels of
BAD expression in my transient transfection mode1 rnay have overcome the relative
lower affinity of BAD for cl-z9. Second the use of a transient transfection assay may
have allowed us to see IeveIs of apoptosis not apprecirtted in stable transfection
expenments used by others. Dunng the selection of stable ce11 lines. BAD may have
induced apoptosis in a portion of cells. such that, at the end of selection, only weak
expressen or resistant cells remained. Finally, it is possible that B A D may induce
apoptosis through a Bcl-2 independent pathway and this pathway becomes more evident
when BAD is expressed at high leve1s.
Mitochondrial dysfunction is a well described phenornenon in the apoptosis
pathway. An element of mitochondrial dysfunction is loss of the inner mitochondrial
membrane potential-termed loss of A Y,%[. Bax and Bak- induced apoptosis is associated
with loss of A Wang et al3' demonstnted that calcineurin dephosphorylates
BAD and permits its translocation to the mitochondnal outer membrane where it interacü
with Bcl-2. Translocation of BAD was associated with apoptosis. However, they did not
mesure A Yibf or cytochrome c release in this report. fn my study, BAD induced
cytochrome c release without loss of A Y1bf thiny houn or more after transfection.
Apoptosis and cyrochrome c release have been reporred in the absence OF loss of A
For example, the BH3-only family memben Bid and Bik induce cytochrome c release and
induce the formation of apoptotic bodies without dtentions in A y,vI3'. It would appear,
therefore, that the interaction of BH3-only family mernbers with BcI-2 at the
mitochondria induces apoptosis rhrough a pathway that does not invotve dismption of the
mitochondnal membrane electrical gradient.
Whether mitochondnal disruption leads to loss of AY likely depends on the site
of damage within the mitochondna. For example, damage to the adenine nucleotide
trmsporter that pumps protons and ATP across the relatively impermeable inner
mitochondnal membrane and maintains AYhf, leads to permeabilization of this
-7-34 membrane, mitochondnal swelling, loss of AYu, and ultimately cytochrome c release3-
In contrast, damage to the voltage-dependent anion channel at the outer mitochondnal
membrane c m lead to cytochrome c release wi thout disrupting the inner membrane
remiuns or inducing loss of A Y ~ ~ ' . Through a similar mechanism. the pore forming pro-
apoptotic proteins Bax or Bak can form channels that increase the permeabilization of the
outer membrane and induce cytochrome c release without damaging the inner
36-38 membrane .
There are a number of limitations to this study. First. 1 did not demonstrate that
Bcl-2 or Bcl-xL protected COS cells from other apoptotic stimuii. However, in a
previous report. COS cells over-expressing Bcl-2 or Bcl-xL were protected from
etoposide-induced apoptosis3g. Furthetmore. in my study, BcI-2 and Bcl-xL offered some
protection against BAD-induced ce11 death as measured by enumerating the increase in
floating cells after transfection of BAD. Second, cytochrome c release was measured in
cytosolic extracts of cells lysed with RIPA buffer. Theoretically. the detergents in the
RIPA buffer may have solubilized the mitochondria and released cytochrome c.
However, untransfected ceIIs and cells transfected with empty vector of BAD cDNA were
treated equally and no cytochrome c release was detected in the untreated controls. To
could have been lysed by mechanicd rneans and a tontrol antibody directed against COX
TV (an inner mitochondnal membrane protein) could have been used. Third. this study
Iacked a positive control such as cyanide rn-chlorophenylhydrazone (CCCP) to
demonstrate that COS celIs cm undergo loss of AYbi.
From my study. 1 conclude that BAD directly induces apoptosis in cells over-
expressing Bcl-2 and Bcl-xL. Furthermore, BAD-induced apoptosis is associated with
cytochrome c release but not loss of AYM suggesting that i t facilitates damage to the
mitochondria confined to the outer membrane. Since B A D is capable of inducing
apoptosis in cells over-expressing Bcl-2 it furthen the goal of using BAD as a prototype
to develop broadly acting therapeutic agents to induce apoptosis in malignant cells.
Attempts to develop therapeutic compounds that mimic BAD are currently underway.
Small synthetic molecules capable of transversing ce11 membranes that act like BAD by
binding and inhibiting Bcl-2 could be useful therapeutically40. Altematively, peptides
that correspond to the short death domains of pro-apoptotic proteins could be delivered
into cells using intemalization sequences such as ~ n t e n n a ~ e d i a ~ ' or fatty acid moieties
ACKNOMZEDGEMENTS
We thank Trude Nicklee for assistance photographing the cells stained with H33342.
Authorization to include this material in the thesis has been granted by the CO-authors and the publishen of Leukemia and Lymphoma. Contribution to manuscript: ADS conceived of the study, executed and analyzed the experiments and wrote the paper. DWH, NP and SC assisted in the design, execution and interpretation of the experiments pertaining to flow cytometry. They also reviewed the manuscript and provided cntical feedback. MDM supervised the work and provided cntical feedback that enhanced the design and execution of the snidy.
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THE BH3 DOMAIN OF BAD FUSED TO THE ANTENNAPEDIA PEPTIDE INDUCES APOPTOSIS VIA ITS ALPHA HELICAL
STRUCTURE AND INDEPENDENT OF BCL-2
Aaron D ~chimmer'. David W ~ e d l e ~ l , Sue chowl, Nhu-An Pham, ' Avijit chaknbarttyl, Denis ~ouchard'~'. Tak W. ~ a k l * ' , Michael R TI-US', Mark D ind den'
1. Princess Margaret Hospital, University Health Network, Toronto, ON, Canada 2. Arngen Institute, Toronto. ON, Canada
Running Title: The BE33 domain of BAD induces apoptosis via its alpha helical structure
ABSTIEACT Since the over-expression of Bcl-2 is a common cause of multi-drug resistance,
cytotoxic peptides that overcome the effects of Bcl-2 may be clinically useful. 1
haniessed the death-promoting alpha heiicd properties of the BH3 domain of BAD by
fusing it to Antennapedia (ANT) domain, which allows for cell entry (ANTBH3BAD).
Treatment of 32D cells with the ANTBH3BAD peptide results in a 99% inhibition of
colony formation. No significant toxicity is observed after treatment wi th ANT or
BH3BAD alone. A mutant fusion peptide unable to bind Bcl-2 induces cell denth as
effectively as the wild type ANTBH3BAD. Furthemore, 32D cells over-expressing Bcl-
2 show no resistance to the ANTBH3BAD peptide. Therefore. the toxicity of the peptide
was independent of the Bcl-2 pathway. I demonstrated that the toxicity of the peptide is
due to its alpha helicity that disrupts mitochondrial function. Since this peptide
overcomes major forms of dnig resistance. it may be therapeutically useful if
appropriatel y targeted to rnalignant cells.
Key words: apoptosis, alpha helix, rnitochondria, Bcl-2, caspases
INTRODUCTION
Since the over-expression of BcI-7 is a comrnon mechanisrn by which malignant
cells become resistant to multiple chemothenpeutic agents1", cytotoxic peptides that
overcorne BcI-3 mediated dmg resistwce may be useful in the treatment of malignanr
disease. One example is the alpha helical BH3 domain of BAD that binds and inhibits
Bcl-2 and Bcl-xL. Wang et aL8 created a cell-permeable version of the peptide by fusing
it to cpm, a decanoic fatty acid intemalization sequence. This fusion peptide induced
npid cell death and apoptosis. However, its cytotoxicity was significantly impaired by
the over-expression of Bcl-xL, which could limit the usefulness of this compound in
treating malignancies with increased levels of Bcl-2 and Bcl-xL. Therefore. enhancing
the ability of the BH3 domain of BAD to kill cells over-expressing these survivai factors
might be therapeutically useful. One strategy to enhance its toxicity is to capitalize on
the inherent alpha helical shape of the peptide, since some amphipathic alpha helices are
potent naturally occurring antibiotics. These compounds dismpt negatively charged
bacterial membranes, but are not toxic to the marnmalian cells, because their membranes
are only weakly negatively charged and stabilized by cholestero19-". When internalized
into mamrnalian cells. hoivever, the alpha helical peptide (KLALAK)2 induced ceil death
and apoptosis by disrupting the negatively charged mitochondria".
Here, 1 have harnessed the alpha helicd properties of the BH3 domain of BAD by
fusing it to the Antennapedia intemalization sequence. 1 have created a 37 amino acid
fusion peptide comesponding to the 21 amino acids of the human BH3 domain of BAD
fused to the C-terminus of the 16 arnino acids of the ANT peptide (ANTBH3BA-D).
ANT is an intemalization sequence that successfully translocates across a variety of ce11
membrimes with high efficimcy md h w toxi~it~'~~~~. f report bat the ANTBH3BAD
fusion peptide induces rapid ceIl death and apoptosis. Its alpha helical secondary
structure disrupts mitochondrial function and allows the peptide to overcome caspase
inhibitors and Bcl-2 over-expression.
EXPERIMENTAL PROCEDURES
Peptides
Peptides (Table 1) were synthesized comrnercially to my specifications (Amgen,
Colorado) and were purified to greater than 95% punty by reverse phase HPLC. Peptides
were resuspended in DMSO and stored at -20'~.
Real Tirne Surface Plasmon Resonance
Binding of Bcl-2 to the peptides was assessed using a BIAcore sensor system
(BIAcore, Uppsala, Sweden). ANTBH3BAD' ANT, BH3BAD. the LI LSA D 1 19R
mutant peptide, and BSA were diluted in t OrnM Na acetate pH 5 and their amine groups
were coupled to a CM5 sensor surface activated with 37.5mg/mL N-Ethyl-N'-
(3'dimethylaminopropyi) carbodiimide hydrochloride (EDC) and 7.5mg/rnL N-
hydroxysuccinimide (NHS). Peptides were added to saturate the chip. Remaining
reactive groups on the chip were then inactivated with LM ethanolamine pH 8.5. GST-
Bcl-2 (Santa Cruz) was diluted in running buffer (0.011M Hepes pH 7.4,O. 15 NaCI, 3mM
EDTA, and 0.005% polysorbate 20 (vlv)) and injected ont0 the sensor chip at varying
concentrations. All experirnents were performed at 2 5 ' ~ . Between injections of GST-
BCL-2, the chip was washed with running buffer and regenerated with LM NaCI and
50miui NaOH. Data were anaiyzed with tbe BIAevaluation software
Peptides
1 Intemîlization sequence
BH3 BAD
1 Non-helix mutant 1
BH3 BAD sequence - -
NLWAAQRYGRELRRMSDEFVD
NLWAAQRYGRELRRMSDEFVD
NLWAAQRYGREARRMSREFVD
NLWPPQRYGREPRRMSDEFVD
Cet/ tirres
HeLa cells were grown in Dulbeco's Modified Eagle Medium wi th high glucose
(DMEM H2 1). The T-ALL ceII line CEM and the vinblastine resistant CEM ce11 line
over-expressing p-glycooprotein (CEM VBL) were grown in RPMI 1640. A 32D ce11
line over-expressing Bcl-2 (a gift from S. Berger) was created by infecting murine
hematopoetic 32D cells with a retrovirus containing murine Bcl-2. Over-expression of
Bcl-2 was confirmed by Western blot. 32D cells were maintained in WMI 1640 and 2%
WeHi-3B conditioned medium as a source of IL-3. AI1 cell lines were supplemented
with 101 fetal calf serum, penicillin and streptomycin. Cells were grown at 3 7 ' ~ with
5% CO2 in a humid atmosphere.
Intemalkation of the Peptides
Biotinylated ANT or ANTBH3BAD peptides were added to 1x10~ 32D cells at a
final concentration of 50pM in semm free DMEM H11 (SF-DMEM). After 20 minutes,
t he cells were washed twice in PBS and fixed in 4% paraformaldehyde in PBS (pH 7.0)
for 10 minutes at roorn temperature. After fixation. celis were washed in PBS and
permeabilized with LI Triton X- 100 in PBS for 10 minutes at room temperature. Cells
were again washed in PBS and incubated with 3% bovine serum albumin in PBS for 1
hour. Streptavidin-FITC (5uL) (Pierce) in 3% bovine serum albumin in PBS was added
to the cells in a totd volume of 500uL and incubated at room tempenture for 30 minutes.
Cells were washed in PBS and andyzed by flow cytometry (FACScan: Becton Dickinson
Immunocytometry S ystems, CA) or mounted on glass slides and viewed with a
fluorescence (Leica DMLB) or a confocai fluorescence (Zeiss LSM 5 IO) microscope.
Ctmogenic Cutorry Assay
Suspension cells (5x IO*) in SF-DMEM H21 were treated with the peptides for 3
houn. Equal volumes of cells were plated in quadmplicate into 96 weli plates in
complete medium, with 0.85% methylcellulose. One week after seeding, the number of
colonies formed was counted. HeLa cells ( t x l d ) in DMEM H21 with 10% FCS were
seeded into 6 well plates. Twenty four houn Iater, they were treated with the peptides for
3 houn in SF-GhEM H21. Following treatment with the peptides, equal volumes of
cells were plated in triplicate into 100mm3 tissue culture plates. One week after seeding,
the medium was removed, the plates stained with methylene blue, and the number of
colonies fonned were counted. Yeast (S. cerevisiae) were grown overnight i n YPD broth
at 3 0 ' ~ . The next morning, 3 r d of culture was dded to 7mL YPD broth and grown at
3 0 ' ~ to i n OD 595 of 0.3-0.35. One ml of culture wûs diluted in PBS. Peptides were
added and incubated with the yeast for 2 hours at room temperature. Equal volumes of
cells were plated in triplicate onto YPD plates and incubated at 30 '~. Four days later, the
number of colonies formed were counted.
Annexin V Assay
Peptides were added to lx 106 32D cells in SF-DMEM at a final concentntion of
50pM for 30 minutes. ETïC conjugated Annexin V (5uL) (Pharmingen) and propidium
iodide (PI) (Sigma) (final concentntion SugImL) were added. The cells were incubated
for 20 minutes at room temperature in the dark and fluorescence was measured by flow
cytometzy (FACScan; Becton, Dickinson, hmunocytometry system, CA, USA). The
Lysis II program was used to analyze these data. Cells staining positive for Annexin V
and negative for PI were deemed apoptotic.
Circrltar Dicfrroim
ANT and ANTBH3BAD peptides were dissolved in O. 1M Na211P04 buffer, 0.1M
NarHP04 buffer and 1% SDS or O. IM Na2HP04 buffer and 40% (vlv) TFE at
approxirnately lrng/mL. Concentrations of the peptides were detemüned by tryptophan
absorbance16. Peptides were dispensed into a 1 cm path length cuvette. CD spectra were
recorded at room temperature on an AVIV CD spectrometer mode1 62A DS. Spectn
were collected with an averriging tirne of 10 seconds per point. Each point represents the
averaged e 1 lipticity value recorded over a 1 nm spectral interval.
Measlu-enlent of Mitochondrial Membrane Potential, Reactive 0-rygen In temediates and
Cytosolic Calcirim
Changes in Mitochondrial Membrane Potential (AThl) were detected by staining
the cells with DiIC l(5) (I,1'3,3,3',3'-hexiunethylindodicarbocyanine) at a final
concentration of 40nM and propidium iodide (final concentration 5ug/mL) as described
previously'7'19. Cells stained with DiICI(5) were incubated at 3 7 ' ~ for 20 minutes and
then iinalyzed by flow cytometry (Coulter Epics Elite, Coulter, Florida) by exciting at
633nm and measuring through a 675 t 20nm bandpass filter. While measunng changes
in AYbf over tirne. stained cells were maintained at 37'~.
Changes in reactive oxygen intermediate production, and cytosolic calcium were
detected by staining the cells with 5pM carboxy-DCFDA (carboxy-dichlorofIuroescin
diacetate), and 3pM indo- 1 AM, respectively. Carboxy-DCF excitation was at 488 nm
and fluorescence was measured using a 525 nm t lOnm bandpass filter. Ionized calcium
was obtained by electronic ratio rneasurement of indo-l AM ernission at 405nm and
5Xnm at an excitation of 360nm.
To rissess changes in h !&in the preseirce of a pmeabiked ceil membrane, 32D
cells were treated according to the rnethod of Pham et alf7. Bnefly, cells were suspended
in respiratory buffer (0.25M sucrose, 2rnM KH2P04, 5mM MgCl*, ImM EDTA, O. 1 %
fatty acid free BSA, 1 mM ADP, and 70mM MOPS (pH 7.4)). Digitonin (4 pg/mL final
concentration) was added to the cells for 5 minutes. Successful permeabilization was
documented by detecting entry of propidium iodine into the cells. Mitochondrial
function was driven by adding 5rnM succinate, a cornplex II subswte. BHSBAD
peptides were added at a final concentration of 50 ph4 and cells were incubated at 3 7 ' ~
for 15 minutes. A Kbtwas then measured by flow cytometnc analysis after staining with
40 nM DiIC l(5).
Caspase-3 Activation
Caspase-3 activation was rneasured with the Apo-Alert Caspase-3 nuorescent
assay kit (Clontech. CA) according to the manufacturers instructions. Cells ( l x lo6) were
treated with the peptides (50pM) for 2 houn and lysed. The caspase-3 substnte DEVD-
AMC was added to the lysate and incubated for one hour at 37'~. Release of free AMC
was measured in a fluorometer (Photon Techonology International) with a 400 nm
excitation filter and a 505 nm emission filter,
RESULTS
Bcl-2 binds ANTBH3BAD and BH3BAD, bris not ANT
To dernonstrate that .4NTBH3BAD binds Bcl-2, the interaction between Bcl-2
and the peptides was analyzed with teal time surface plasmon resonance detection.
ANTBWBAD, BH3BA.D and ANT peptides and BSA were coupled to a biosensor chip
through their amine groups. GST-Bcl-2 was injected at increasing concentrations onto
the sasor ch-@ and bmdirrg ro the peptides aba i r the BSA mntrot was m d . GST-
Bcl-2 bound avidly to ANTBH3BAD (kD = l x l ~ ' ~ ~ , by BiAevaluation software,
(BIAcore. Uppsala, Sweden)) and BH3BAD in a dose-dependent manner and did not
bind significantly to ANT (Figure 1). Bcl-2 remained tightly bound to the peptides with
minimal dissociation. GST done did not bind to the peptides.
Internali~atiori 0 f th e peptkles
ANT is an internalization sequence that facilitates the transport of peptides across
ce11 membranes with high efficiency. To study the intemalization and localization of the
peptides, 32D cells were treated with biotinytated venions of ANT and ANTBH3BAD.
In a representative experiment, 89% and 100% of 32D cells treated with N-terminally
biotinyated ANT or BH3BAD respectively intemalized the peptides as measured by flow
cytometry. The peptides were distributed diffusely throughout the nucleus and cytoplasm
as detennined by confocal fluorescent microscopy (Figure 2).
=INTBH3Brl D Indtces u Rapid Ce&/ Death
To assess the impact of the peptides on ce11 viability, HeLa cells were treated with
50pM of ANT, BH3BA.D or ANTBH3BAD for 3 hours. Cells treated with
ANTBH3BAD displayed rnorphologic evidence of ce11 death characterized by ce11
shrinkage, membrane blebbing, and detachment from the tissue culture plate. In contrast.
no significant morphological changes were seen atter treatment with 50pM ANT or
ANTBH3BAD. Cells treated with ANT or BH3BAD showed no evidence of toxicity
(Figure 3).
O 100 200 300 400 500 600 700
Time (seconds)
-- -
O 100 -200 300 400 500 600 700
Time (seconds)
Figure 1: Red time surface plasmon resonance BWBAD, ANT, ANTBH3BA.D and BSA were coupled to a CM5 BIAcore sensor chip. GST-Bcl-2 was injected ont0 the chip at increasing concentrations. The sensor surface was regenerated with 1M NaCl and 50mM NaOH between each injection. Binding to the peptides above the BSA control was measured as an increase in Response Units above the BSA control. A representative sensogram &ter the injection of GST-Bcl-2 (2pM) is shown (a). A dose response curve for the binding of GST-Bcl-3 to ANTBH3BAD is presented (b)
ANT
Figure 2: Intemaiization of the peptides Biotinylated versions of ANT and ANTBH3BAD were obtained. 33D cells (1x106 cells) were treated with the peptides a i a final concentration of 50pM for 20 minutes. The cells were washed, fixed in 4% parafonnaldehyde, permeabilized with 1 % Triton-X 100 and probed with Streptavidin- FITC to detect the biotinylated peptides. Cells were viewed under a confocal microscope with fluo- rescent Iight to determine the distribution of the peptide.
ANT BH3BAD
Figure 3: Morphologie evidence of ce11 death. HeLa cells (1x10~ cells) were seeded into 12 well plates in DMEM H21 + 10% fetal cdf serum. Twenty-four hours later, BEUBAD, ANT and ANTBH3BAD were added to the cells in SF-DMEM HX at a final concentration of SOW. Three hours after incubation, morphologie evidence of ce11 death was observed among the cells treated with ANTBH3BA.D. Cells treated with ANT or BH3BAD showed no evidence of toxicity.
To quantitate the effects of the peptides m cet1 snrvivat, H e h , 32D wd yeast were
treated with the peptides at a final concentration of 50ph4 and cell survival was measured
by a colony formation assay. Drnmatic decreases in cell survival were seen in al1 three
cell types after treatment with ANTBH3BAD. No significant toxicity was observed in
cells treated with ANT or BH3BAD alone (Figure 4). Therefore. ANTBH3BAD induces
ce11 death, but the toxicity is not related to the ANT domain. The BH3BAD peptide
alone is not toxic because it is not internalized.
Dose response curves for ANTBH3BAD-induced ce11 death were constructed.
32D cells were treated with increasing concentrations of ANTBH3BAD peptide for 3
hours and ceIl survival wris rneasured in a colony formation assay. As the peptide
concentration increased, there was a corresponding decrease in ce11 survival wiis observed
(Figure 5). The dose response curve was steep. as minimal toxicity was observed below
a peptide concentration of 1OpM and nearly complete growth inhibition was seen at a
concentration of 50pM.
ANTBH3BAD Induces Apoptosis
Next, 1 determined whether the cell death induced by ANTBH3BAD occumd via
apoptosis. 32D cells were treated with the peptides for 30 minutes and stained with FITC
labeled anti-Annexin V and PI. The percentage of apoptotic cells was determined by
flow cytomeuic analysis. After treatment with ANTBH3BAD 97+1%, of cells were
apoptotic based on Annexin V staining. In contrat, only 1522% of cells treated with
ANT and 1312% of cells treated with BH3BAD were apoptotic by Annexin V staining.
untreated BH3BAD ANT ANTBHSBAO
peptide (5OpM)
untreated BH38AD ANT ANTBH3BAD
peptide (50pM)
HeLa
S. cerevisia e
untreated BH3BAD ANT ANTBH3BAD
Figure 4: ANTBH3BAD induces celi death HeLa cells, 32D cells and S. cerevisiae were treated with BBBAD, ANT, or ANTBH3BA.D at a final concentration of 50p.M for three hours. After the incubation, equal volumes of cells were plated in a colony formation assay as descrïbed in "Experimental Procedures". The nurnber of colonies formed was counted. The results are a mean of at least three expenments. Bars equal SD
Figure 5: Dose response c w e A dose response curves for ANTBWBAD-induced ce11 death was consmicted. 32D cells were incubated with increasing concentrations of ANTBK3BAD for 3 hours. After incubation, equal volumes of cells were plated in a colony formation assay as described in "Experimental Frocedures". One week later, the number of colonies formed was counted.
ANTBH3BAD hdr~ces Ceif Deuth ttrdepmdent of Bct-2
We studied the effects of Bcl-2 over-expression on the toxicity of ANTBH3BAD.
32D cells with and without over-expression of rnurine Bcl-2 (5x10~ cells) were treated
with ANTBH3BAD and ce11 viability was measured by trypan blue exclusion. Over-
expression of Bcl-2 did not abrogate the toxicity of rny peptide (Figure 6). In contrast,
32D cells over-expressing Bcl-2 were protected from IL-3 withdrawal (data not shown),
indicating that Bcl-2 can protect this ce11 line from other apoptotic stimuli.
To confirrn the lack of impact of Bcl-2 on the toxicity of ANTBH3BAD. a mutant
fusion peptide was prepared in which residues LI 14 and Dl 19 in the BH3 domain of
BAD were substituted with A 1 14 and R 1 19. In a BIAcore binding assay. GST-Bcl-2 did
not bind to this mutant peptide. Although incapable of binding Bcl-2. the mutant peptide
was as toxic as the wild type indicating that the toxici ty of ANTBH3BAD is independent
of the Bcl-2 pathway.
Figure 6: BcI-2 does not abrogate the toxicity of the fusion peptide 32D cells and 32D cells over-expressing Bcl-2 were treated with ANTBMBAD at a final concentration of 10pM, 30pM and SOpM. Six hours after treatment, an equal volume of trypan blue (0.4% in 0.9% normal saline) was added for 10 minutes. At least 100 cells were scored. The percentage of cells staining positive for trypan blue was determined. The results represent the mean of three experiments. Bars equai SD
Tmicitp of the Fusion Pepnde is chte ta its Atphct Helicul Shape
Certain alpha helical peptides are potent antirnicrobial agents that induce ce11
death b y disrupting the negativel y charged bacterial ce11 membranegg10. When
intemalized into eukaryotic cells, afpha helical peptides induce apoptosis by disrupting
the negatively charged mi tochondna". ANTBH3BAD is predicted to have an alpha
helical conformation. To evaluate the contribution of the helical shape to its toxicity. 1
obtained, a non-helicai mutant of AiVTBH3BAD by substituting A106, A107, and Li 14
in BH3BAD with prolines. 1 tested the impact of this mutation on helical structure by
circula. dichroisrn (CD) (Figure 7). In 1OOmM phosphate buffer (pH 7). both the wild
type and non-helical mutant ANTBH3BAD peptides displayed a CD spectrum consistent
with a random coil. However, the wild type peptide adopted an alpha helical
conformation in 1% SDS and 40% (v/v) TFE, but the non-helical mutant mainly
remained as a random coil in these solutions. In addition, the ANT and BH3BA.D
peptides alone adopted alpha helical secondary structure in these solutions.
-30000 I l 1 1 1 1
200 210 220 230 240 250 260
Wavelength (nm)
Figure 7: Circular D i c b i s m secondary structure of ANTBH3BAD and the non-helical mutant were determined
by circular dichmism. The peptides were dissolved in 0.1M Na&P04 buffer (A), 1% SDS (B), or 40%(v/v) TFE (C). CD spectra were recorded at room temperature. Each point represents the averaged ellipticity value recorded over Inm spectrai intervais.
= ANTBH3BAD, - = non-helical mutant
A cotony formation a s s q was performedwittr the nm-hetic;it mutant to detemrine the
contribution of the alpha helicity to the toxicity of ANTBH3BAD (Figure 8). The non-
helical mutant was not toxic to 32D cells demonstrating that the toxicity of
ANTBH3BAD depends on i ts he 1 ica1 configuration.
ANTBHjBAD Disnlpts Mitochondriaf Funcrion
Alpha helical peptides induce swelling of isolated mitochondna". Therefore, to
confirm that the cytotoxcity of ANTBWBAD wûs related to its helical configurütion. 1
investigated the effects of the peptides on intracellular mitochondrial function.
HaIlmarks of mitochondrial dysfunction include Ioss of mitochondnal membrane
potential (A Yb/) and increased production of reactive oxygen intermediates (ROI).
Chariges in A !&and ROI production were measured over time by flow cytometry after
treating 32D cells with the peptides. ANTBH3BAD rapidly decreased A Tt1, whereas no
significant change was observed after treatment with ANT or BH3BAD (Figure 9). The
non-helical mutant also did not induce loss of A y\,. The population with decreased A q t l
also showed increased ROI production. The increased ROI and decreased A were not
accompanied by significant increases in cytosolic calcium. Therefore. the loss of A
mt secondary to large increases in cytosolic calcium from disruption of the endoplasmic
reticulum.
To ensure that the toxicity of ANTBMBAD is due to the alpha helical properties
of BH3BAD and not an artifact of the fusion peptide, 1 investigated the effects of
BH3BAD on A YArin pemeabilized cells. 32D cetls were suspended in respiratory buffer
to mimic the environment of the cytoplasm. The plasma membrane was permeabilized
with digitonin and mitochondrial activity was driven with succinate. BH3BAD (50 pM)
control ANTBH3BAD non-helica wild type mutant
Peptide (50pM)
Figure 8: The non-helical mutant is non toxic 32D cells (5 x 104 cells) were treated with ANTBH3BAD or the non-helical mutant at a final concentration of 50w. ARer a three hour incubation, equai volumes of cells were plated into complete medium with 0.85% methyl cellulose. One week after seeding, the nurnbcr of colonies formed was counted. Bars equal SD
O I! *--+--+--*
I i I 1
20 30 40 50
Time (min)
CCCP BH3BAD r
ANT
- ANTBH3BAD - -+-- ANT - BH3BAD
Figure 9: PLNTBH3BAD induces loss of A FM 32D cells (5xl0~cells) were ueated with BWBAD, ANT, ANTBH3BAD, at a final concentration of 50pM. Cells were also treated with the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) (final concentration 100phQ to induce complete Ioss of ATM and thereb y serve as a positive control. A YM was detected b y staining with DiIC1 (40nm). Changes in A YM were m e a s u ~ d at various times after the addition of the peptides by flow cytometry. A representative time course for the change in A YMis shown (a). A representative flow cytomeaic expriment after the addition of the peptides is presented (b).
was addecl to the pmeabikzed cetts for 15 mhtes mdcharrges in b Yhfw.,werr measured
by flow cytornetry. BH3BAD induced a 4.6 & 0.3 fold loss of A compared to
treatment with digitonin and succinate alone. Therefore, when intemalized into cells and
exposed to the mitochondria, BH3BA.D also displays an ability to induce loss of A %j.
ANTBH3BAD Induces Caspase-3 Activation, but Caspase Inh ibitors do no f Protect
Against Ce21 Death
Mitochondnal darnage can activate caspases. To determine i f the cytotoxici ty of
ANTBH3 B AD is caspase-dependent, 1 s tudied the e ffect of ANTB H3B AD on caspase
activation. Treatment with ANTBH3BAD induced a 5fl.9 fold increase in caspase-3
activation compared to untreated cells. In contrat, no increase in caspase-3 activity was
seen after treatment with ANT or BH3BAD. Next, 1 investigated the effects of the
genenl caspase inhibitor zVAD-fmk on ANTBHfBAD-induced caspase activation and
cytotoxicity. Pretreatment with LOO pM zVAD-fmk for one hour prevented
ANTBH3BAD-induced caspase-3 activation. Retreûtment with zVAD-frnk also
prevented the death of 32D cells after withdrawal of L-3 indicating that zVAD-fmk is
capable of inhibiting apoptotic stimuli in this ce11 line. Interestingly, pretreatment with
zVAD-fmk did not inhibit ANTBH3BA.D-induced ce11 death (Figure 10). Therefore,
ANTBH3BA.D induces caspase activation, but in the presence of caspase inhibitors. it
rernains cytotoxic due to caspase-independent mechanisrns.
Figure 10: zVAD-fmk fails to prevent cell death
32D cells (5xldcells) were pretreated with 1ûûp.M zVAD-mik for one hour followed by the addition of ANTBH3BA.D (30pM and 50pM). After three hours, ce11 viability was determined by nypan blue exclusion. The results represent the mean of three experiments. Bars equal SD
Over-expression of p-glycoprotein is another important mechanism of multidmg
resistance and is a poor prognostic marker in patients with ~eukernia'~~". Peptides and
proteins can act as substntes of p-g~ycoprotcin2'-". Therefore, I studied the effects of p-
glycoprotein over-expression on the toxicity of the peptides. CEM cells and CEM VBL
cells over-expressing p-glycoprotein were treated with varying concentrations of
ANTBH3BAD. Compared to the CEM parent ce11 line. CEM VBL cells displayed
resistance to ANTBH3BAD that was most apparent at a final concentration of lOpM
ANTBH3BAD. Pretreatment of CEM VBL with the p-glycoprotein inhibitor
cyclosporine (final concentration I O p M ) for one hour restored the toxicity of
ANTBH3BAD to levels seen in the wild type CEM Iine (Figure I 1). Therefore, p-
glycoprotein limits the toxicity of this peptide, but the effect can be reversed with p-
glycoprotein inhibitors.
DISCUSSION As a prototype for a novel therapeutic agent, 1 studied the effects of the BH3
domain of human BAD fused to the ANT intemalization sequence. This fusion peptide
induced a rapid ce11 death in al1 cells tested including yeast. The peptide was toxic
because its alpha helical shape dismpted mitochondriai Function.
In a ceIl free system. the ANTBH3BAD peptide bound BcI-3 with higher affinity
than the BH3BAD peptide alone. A similar finding was reported with BH3BAD fused to
the fatty acid moiety cpms. The increased binding likely reflects the additionai
interaction of the positively charged ANT sequence with Bcl-2.
1 0 0 - V1 Q1 - O Cern wt
8 1 0 - - I cem vbl O O
1 i- I 1 i
ANTBHBBAD peptide (PM)
untreated CYa ANTBHJBA-D cya + ANTBH3BAD
treatment (cya I O u M , ANTBH3BAD 1 O p M )
Figure 1 1 : P-glyocprotein protects against ANTBHîB AD toxicity
a) CEM and the p-glycoprotein over-expressing ce11 line CEM VBL were treated with AN?', BWBAD, or ANTBH3BAD for three hours at various concentrations. Cells were then seeded in complete medium with 0.85% methylcellulose. One week after seeding the nurnber of colonies formed was counted. b) CEM VBL cells were pretreated with cyclosporine (cya) at a final concentration of i0p.M for one hour. ANTBH3BAD was added at a final concentration of 10m. Three hours after incubation, the cells were seeded in complete medium with 0.85% methylcellulose. One week d e r seeding the number of colonies fomed was counted.
Despite being capable of binding Bcl-2, the cytotoxicity of ANTBH3BAD was
independent of the Bcl-2 pathway. Cells over-expressing Bcl-2 showed no resistance to
the peptide and a mutant fusion peptide unable to bind Bcl-2 was as toxic as the wild
type. 1 could not demonstrate Bcl-2-dependence because the alpha helicai propenies of
the peptide overcarne the protective role of Bcl-2.
The cytotoxicity of ANTBH3BAD was not an artifact of the fusion peptide. but
nther related to the helical properties of BH3BAD. As evidence to this point. BH3BAD
was capable of inducing loss of A !&in permeablized cells.
Previous studies of full iength BAD^*'^ or the BH3 domain of BAD^^'.' have not
reported toxicity attributable to the alpha helical configuration. These studies
demonsüated that over-expression of Bcl-2 md Bcl-xL inhibits apoptosis induced by the
full-length protein and peptide. Furthemore, mutants that do noi bind BcI-2 are not
ic6-8 J- ' A number of explanations rnay account for my ability to detect toxicity due
to the alpha helicity of BH3BAD. First. fusing the BH3 domain of BAD to the A1NT
intemalization sequence rnay have targeted the peptide more effectively to the
mitochondria where its alpha helical shape could dismpt the inner membrane. BH3BAD
peptides coupied to the cpm fatty acid rnoiety were confinecf to the cytoptasm. In
contrast, my peptide was distributed diffusely throughout the cell. Second. the
concentration of BAD used in the previous studies rnay have been too low to detect the
effects of the alpha helix. If BH3 BAD-mutants unable to bind Bcl-2 or Bcl-xL were
used at higher concentrations, the cytotoxicity from the alpha helical shape rnight have
becorne apparent. Finally, the full-length protein BAD rnay constrain the death
promoting attributes of the alpha helicai BH3 domain.
In a previous report by ~ t f e rbye t at" the 14 arrrirro acid alpha hetix (KIALAK)2
induced ce11 death and apoptosis. The peptide induced mitochondrial swelling and
caspase-3 activation in isolated mitochondria added to cytosolic extracts but the effect on
A cbIwas not reported. In addition, the effects of Bcl-2 and p-glycoprotein on the toxicity
of (KLALAK)? were not studied. Here, 1 have demonstrated that ANTBH3BAD disrupts
rnitochondria and induces loss of A q t f in intact and permeabilized cells. Unlike the
(KLALAK)? peptide, the toxicity of ANïBH3BAD was not inhibited by the general
caspase inhibitor zVAD-fmk. ZVAD-frnk c m inhi bi t mi tochondnal induced caspüse
activation. but i t does not prevent the peptide from darnaging the mitochondrin. As
mitochondriai damage continues, the ability of the mitochondria to synthesize ATP is
lost. and, as a consequence, cells undergo a metabolic and caspase independent death2'-19.
The finding of toxicity not affected by Bcl-2 suggests that alpha helical molecules
such as ANTBH3BAD can be potent mti-cancer agents capable of overcoming important
mechanisms of multidrug resistance. To see if other mechanisms of multidnig resistance
impact the toxicity of the peptide. I studied the effects of p-glycoprotein. Over-
expression of p-glycoprotein protected against the toxicity of ANTBH3BA.D and this
protection was revened by adding the p-glycoprotein inhibitor cyclosporine. P-
giycoprotein is best known for its role in extruding chemotherapeutic agents, but it also
7' 24.303 1 exports peptides and large proteins such as IL-2 and gamma interferon-'
Therefore, p-glycoprotein may protect the cells from the toxicity of ANTBH3BA.D by
exporting it out of the cells. Alternatively. it may be abrogating the peptide's toxicity
through its d e in inhibiting apoptosis. P-glycoprotein inhibits apoptosis in response to
Fas ligand, ultraviolet irradiation, and serum starvation, and the addition of venpamil can
restore the apoptotic signab ~fttieçestirnut?~~~. Cumntly, it is unknown how it directly
protects cells from these apoptotic stimuli.
There are a number of limitations to this study. Fint. 1 did not demonstrate that
the nonhelical mutant was intemalized into cells. Theoreticaily, the mutations in the BH3
domain of BAD could have produced a fusion peptide that could not be internalized.
However, ANT-mediated internalization does not appear dependent on the fusion partner.
Multiple peptides with a variety of secondary structures have been intemalized with the
ANT intemalization sequence and there are no reported cases where the fusion partner
inhibits this intemalization. Future experirnents would include obtaining a biotinylated
version of the non-helical mutant and studying its ability to transverse cell membranes.
Second. the circular dichroism data indicate that the peptide is a random coi1 in the
aqueous buffer. Only in the presence of SDS or TFE does the peptide acquire a helicd
shape. The degree of helicity in the presence of ce11 membranes has not been tested.
Therefore. it is unknown what conformation the peptide has when inside the cell. Third. 1
did not demonstrate that the ANTBH3BAD peptide directly interacts with the
mitochondriril membrane. Although the peptide is distributed di ffusel y throughout the
cytoplasm and nucleus, we did not isolate rnitochondria membranes to determine if they
contained the peptide. Therefore the toxicity of the peptide may be due to novel
mechanisms and the effects on the rnitochondria are a secondary phenomenon. In the
future, 1 propose to isolate mitochondria from peptide treated cells to determine whether
the peptide is partitioned in this cellular fraction.
In conclusion, I describe a cytotoxic effect of the BH3 domain of BAD is likely
due to its alpha helical properties and have harnessed this cytotoxic action by fusing
two major foms of dmg resistance - Bcl-2 and caspase inhibitors. As such this
compound may have clinical utility if it cm be selcctively targeled. This work also
furthers the understanding of how aipha helical peptides induce apoptosis in mrunmalian
cells and investigates factors that impact their toxicity.
ACKNO WLEGMENT
We would like to thank R Lutz and E. Hollinger (Immunogn, MA) for their helpful
advice and discussion.
Authorization to inciude this material in the thesis has been granted by the CO-authors and the publishers of Ce11 Death and Differentiation.
Contribution to manuscript: ADS conceived of the study, executed and anaiyzed the expenments and wrote the paper. DWH. NP and SC assisied in the design, execution and interpretation of the experiments pertaining to flow cytometry. AC assisted in the design. execution and interpretation of the expenments pertaining to circular dichroism. DB assisted in the design. execution and interpretation of the experiments pertaining to surface plasmon resonûnce. Al1 authon reviewed the manuscript and provided cntical feedback. MDM supervised the work and provided criticai feedback that enhanced the design and execution of the study.
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In an attempt to develop a novel therapeutic agent for the treatment of acute
leukemia. I tumed to the endogenous Bcl-2 and Bcl-xL-binding protein BAD. BAD
induces apoptosis by binding and inhibiting Bcl-2 and cl-XL'.'. In the proposed model,
BAD inhibits these survival factors and permits ce11 death signals to act u n ~ ~ ~ o s e d . ~ ~ .
Thus, BAD acts passively to induce cell death and does not have direct death-promoting
activity like Bax, Bak or caspase-3. Therefore, i t was hypothesized that BAD might
show selective toxicity for malignant cells because these cells have active death stimuli
that are blocked by the over-expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL.
In contrat, normal cells may not have active death signals and may not undergo
apoptosis after temporaril y inhibiting these survival factors.
According to the widely accepted model, BAD induces apoptosis in cells over-
expressing Bcl-2 or Bcl-xL. Despite this widely held betief, the literature indicates that
BAD does not sensitize malignant cells with high levels of Bcl-2 to apoptosis after
cytokine withdnwal. Yet, the s m e cells over-expressing Bcl-xL are sensitized by BAD
over-expression1.'. If BAD is unable to induce apoptosis in cells over-expressing Bcl-2.
it would limit the appiication of molecuIes like BAD as novel therapeutic agents as Bcl-2
often propagates neoplastic growth, and Bcl-xL is more important than Bcl-2 for normal
tissue d e v e ~ o ~ r n e n t ~ ~ .
In my study, using a transient transfection model, 1 demonstrated that BAD
induced apoptosis in cells over-expressing Bcl-2 and Bcl-xL. 1 also extended the results
to leukemic ceIl lines with high levels of these survivai factors. While these effects of
BAD rnay be predicted and confirm the mainstream d o p a , they had not been previously
repmed. This worir validates t h pursoit of d L molecules that mirnie BAD as
potential novel thenpeutic agents.
A peptide corresponding to the 71 amino acid BH3 domain of BAD is an exarnple
of a small molecule that mimics its parent protein and binds and inhibits Bcl-2 and Bcl-
xL. Wang et aL6 created a cell-permeable version of this peptide by fusing it to cpm, a
decanoic fatty acid internalization sequence. This fusion peptide induced rapid ce11 death
and apoptosis. However. its cytotoxicity was significantly impaired by the over-
expression of Bcl-xL, which could limit the usefulness of this compound in treating
malignancies with increased levels of Bcl-2 and Bcl-xL. Therefore, enhancing the ability
of the BH3 domain of BAD to kil1 cells over-expressing these survival factors rnight be
thenpeutically useful. One stntegy to enhance its toxicity is to capitalize on the inherent
alpha helical shape of the peptide, since some amphipathic alpha helices are potent
naturally occumng antibiotics.
We harnessed the alpha helical propenies of the BH3 domain of BAD by fusing i t
to the Antennapedia intemalization sequence. I created a 37 amino acid fusion peptide
corresponding to the 2 1 amino acids of the human BH3 dornain of BAD fused to the C-
terminus of the 16 arnino acids of the ANT peptide (PLNTBH3BAD). The ANTBH3BA.D
fusion peptide induced rapid ce11 death and apoptosis. Its alpha helical secondary
structure disrupted mi tochondrial function and ailowed the peptide to overcome caspase
inhibitors and Bcl-2 over-expression.
Previous studies of full length BAD'*' or the BH3 domain of BAD^.^ have not
reported toxicity attributabIe to the alpha helical configuration. A number of
explmations may account for my ability to detect toxicity due to the alpha helicity of
BH3BAD. Fmt, fmmg the BH3 domain of BAD to tht ANT intmdization sequence
rnay have targeted the peptide more effeLuvely to the mitochondna where its alpha
helical shape could dismpt the inner membrane. BH3BAD peptides coupled to the cpm
fatty acid moiety were confined to the cytoplasm6. In contrast, my peptide was
distributed diffusely throughout the cell. Second, the concentration of BAD used in the
previous studies rnay have been too low to detect the effects of the alpha helix. If BH3
BAD-mutants unable to bind Bcl-2 or Bcl-xL were used at higher concentrations, the
cytotoxicity from the alpha helical shape might have become apparent. Finally, the full-
length protein BAD rnay constrain the death promoting attributes of the alpha helical
BH3 domain.
In a previous study, the BH3 domain of B A fused to ANT behaved like full-
length Bak and induced apoptosis that was inhibited by Bcl-xL over-expression and
caspase inhibitoa9. Unlike ANTBH3BAD, no toxicity attnbutable to the alpha helical
configuration was observed. The difference in action between ANTBH3BAD and
ANTBH3Bak rnay be due to the greater predicted alpha helicity of the BH3 domain of
BAD than the BH3 domain of Bak.
We dernonstrated that transient transfection of BAD cDNA induced apoptosis and
cytochrome c release without affecting AYu. In contrast, the BH3BA.û peptide induced
loss of A\YM in permeabilized cells. A similar discrepancy was reponed with the BH3-
only farnily members Bik and id". The full-length proteins induced cytochrome c
release and apoptosis without altering AYM, but peptides corresponding to their BH3
domains, induced loss of ATM and cytochrome c release in isolated mitochondna. A
number of explanations rnay account for the difference between the full-length protein
and the peptide. Fint. the &Il-tength protein may constrain the effea of the BH3
domain. Loss of AYM is related to disruption of the inner mitochondnai membrane and
the full- length protein may prevent the BH3 domain from acting at this site1'.".
Alternatively. the concentration of the full-length protein may have been too low to
produce an effect on ATM.
In rny study. the ANT intemalization sequence was used to transport the
BH3BAD peptide across ceil membranes. The ANT intemalization sequence is derived
from the 42.8kDa Antennapeida transcription factor that controls the development of the
Drosophila embryo. In 199 1, Joliot et a1 made the serendipitous discovery that
Antennapedia can transverse ce11 membranes with high eficiency and low toxicity13.
Later, a 16 amino acid region of the third alpha helix was reported to be sufficient for ce11
transduction1". The mechanism by which the ANT transverses ce11 membranes is not
entirely clear. Intemalization occun both at 4*C and 37OC indicating that the mechanism
is not energy dependent. Revening the sequence of ANT and using a peptide with D-
enantiomers did not effect intemalization, demonstrating that the mechanism is not
dependent on a specific receptor. The alpha helical stnicture of the peptide also does not
account for its ability to translocate, because dismpting the alpha helix does not hamper
intemalization. Currently, i t is believed that the positive charge on the ANT peptide
facilitates its translocation by interacting with the negative charged phospholipids in the
ce11 membrane. The peptide forrns inverted micelles with the plasma membrane and
thereby passes into the cytosol (Figure l)15.
1 peptide 1
T t T T T l T
Figure 1 : Proposed mechanism of transduction of ANT internakation sequence across ceII rnem branes. The positive charge on the ANT peptide interacts with the negatively charged phospholipids in the cell membrane. The peptide forms an inverteci micelle and thereby passes through into the cytosol
We enhanced the toxicity of the BH3BAD by hmessing Its alpha helical properties.
Alpha helixes are corkscrew-shaped secondary structures that contain 3.6 amino acid
residues per tum and nse 1.5 A per residue. Alpha helixes have phi and psi angles of
approximately -60" and -50' respectively where the phi angle refers ro the mgle between
the WCa bond and the psi angle refen to the C C' bond (Figure 2). The helical
structure is stabilized by hydrogen bonds that form between c'=O of residue n and the
NH of residue n 4 . The amino acids Ala (A), Glu (E), Leu (L) and Met (M) favour at
helical structure, whereas the nnged form of proline disrupts the a helical shape. In the
helical configuration, the amino acid side chains face outwards. When one side of the
alpha helix contains hyârophobic side chains and the other side of the helix contains
hydrophilic (polar or charged) side chains. the alpha helix is deemed amphipathic.
Some amphipathic alpha helices are toxic because they can insert thernselves into
negatively charged membranes and disrupt their f u n ~ t i o n ' ~ ' ~ . Two models have been
proposed to explain this ability. In the "barrel-stave model", the alpha helix forms pores
by inserting perpendicuiariy into the membrane with the hydrophobie side chains facing
outwards. The pores facilitate leakage across the membrane and ultimatel y membrane
disruption. In the "carpet model", large arnounts of peptide bind transvenely to the
surface of the membrane and act like a detergent to form holes and soiubilize the
membrane2'.
Figure 2: Alpha Helical structure An alpha helix is a secondary peptide structure charactenzed by phi and psi angles of - 60° and -50' respectively.
The ANTBH3BAD peptide can be compared to other cytotoxic molecules
including dipthena toxin". Shiga t o ~ i n ~ ' ~ calicheami~in~~, gelonin toxin3 and the alpha
helix (KLALAK)2 able 1). These molecules differ in the mechanism by which they
induce ce11 death. Diphtheria toxin2'. Shiga toxin2' and gelonin?s induce ce11 death by
inhibiting protein synthesis at the level of the ribosome, but differ in their specific
molecular targets. Calichamicin cleaves DNA and thereby induces cell death'". In
contrast, (KLALAK)~'~ and ANTBH3BAD induce ce11 death by disrupting mitochondnal
function. ANTBH3BAD is unique in that it is unaffected by Bcl-2 over-expression and
caspase inhibitors. ZVAD-fmk inhibits the toxicity of (KLALAIQ and Bcl-2 over-
expression abrogates the effects of dipthena toxin. The influence of caspase inhibitors
and Bcl-2 on the other cytotoxic compounds has not been reported. Of this group of
compounds, only Shiga toxin displays selective toxicity for malignant cells. Shiga toxin
requires the CD77 receptor for intemalization. CD77 is found on the surface of most
myeloma and low-grade lymphoma cells, but is not present on normal hematopoietic
7 7 precursors--. In contrast. the other cytotoxic molecules in this group are equally toxic to
n m a i cetts anci need ta be setectiveiy targeted to be therapeuticaiiy useful.
Table 1: Cornparison of cytotoxic molecules
Inhibited by P-
gl ycoprotein
Nme
Diptheria Toxin
Gelonin Toxin
Shiga Toxin
Calicheamicin
(KLALAIQ2
AIWBH3BA.D
Inhibited by BcI-2
Size
58kDa
30kDa
58kDA
1 SkDa
2.2 D a 4.8 kDa
Inhibited by
caspase inhi bitors
Mechanism
Inhibit elongation factor 2 Inhibit nbosornal subunit 60s Inhibit elongation factor 1 Cleaves DNA Disrupts rnitochondria Disrupts mitochondna
Specific for
malignant cel1s
N
N
Y
N
N
N
To extend the work on ANTBH3BAD and continue the pursuit of this rnolecule
as a novei therapeutic agent, it would have to be selectively targeted to leukemic cells.
Selective targeting may spare normal celis from the toxicity of the peptide. In addition,
an anti-tumor effect rnay be achieved with Iower concentrations of the peptide because i t
would not have to equilibrate into al1 cells.
One approach to targeting the peptide to AML cells is to conjugate it to anti-
CD33. The CD33 antigen is found on the surface of AML blasts in over 80% of cases. It
is not expressed normally on non-hematopoietic cells, but is found on the surface of early
myeloid progeniton. monocytes. and dendritic cells. Other toxins, such as calichamicin"
and gelenonin=, have been conjugated to anti-CD33. These conjugated immunotoxins
show specific anti-tumor properties without untoward toxicity in mice or humans. For
example, CMA-676, a humanized anti-CD33 monoclonal antibody linked to caiichamicin
was evaluated in a phase II study of 23 patients with AML in first relapse. In this study.
10 patients responded to the immunotoxin and 3 achieved a complete response. Toxicity
was manageable and consisted of fever, chills, nausea, and tnnsient pancytopenia. There
was no evidence of mucositis or allopecia".
Sorne potentiai probiems may exist in using anti-CD33 to target ANTBH3BAD.
First. due to the ANT intemalization sequence, the irnrnunoconjugate may still transverse
al1 cells and not be selective. This problem might be overcome by deleting the two N
terminal amino acids from the ANT sequence that are required for intemalization. This
mutant would retain its alpha helical shape, but would not be intemdized. Internalization
of the imrnunotoxins occurs through endocytosis, so an ANTBH3BAD would have to be
abie to exit the nidocytotic vesicles to be effective. It is unclew, ~ O W ~ V ~ F , whether an
ANTBH3BA.D with a mutated intemalization sequence would be able to exit from the
endocytotic vesicles into the cytoplasm.
Additional future work will involve studying the apoptosis cascade in patients
with AML and in particular the pathways of caspase activation. At its most basic level.
treatment failures in AML represent a failure of the leukemic cells to undergo apoptosis
in response to chemothenpy. Failure to activate caspases may account for some of this
resistance to apoptosis. Defects in caspase activation and caspase inhibiton have been
described in AML ceIl lines and patient samples, but. the evidence, in many cases. is
circumstantial and a systematic evaluation of the caspase cascade has not been performed
for AML. Furthemore, the functional status of the caspase cascade in patients with
AML is unknown.
This future work will determine whether functional blocks in the caspase
activation pathways exist in patients with AML and delineate the defects responsible for
these blocks. In addition, it will attempt to modulate defective pathways to overcorne
sites of blockage and restore competency to the activation cascade. The hypothesis is that
defects in caspase activation will be common in patients with AML. Since inhibition of
caspase activation renden cells resistant to chemotherapy. one cm also hypothesize that
patients with chemoresistant disease will display defects in one or more of the pathways
whereas patients with chernosensitive disease and subsequent durable remissions are
more Ii kel y to have intact activation pathways. Through these expenments. new
prognostic markea for AML may be developed. By modulating the pathways and
restomig competency to the cascade stnitegia for novel h g development €0 crovercome
drug resistance in AML may become evident.
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