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Apoptosis signaling to mitochondria by death receptors and DNA damaging anti-cancerregimens

Werner, A.B.

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Citation for published version (APA):Werner, A. B. (2004). Apoptosis signaling to mitochondria by death receptors and DNA damaging anti-cancerregimens.

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Download date: 31 Oct 2020

ChapterChapter 7

GeneralGeneral Introduction

GeneralGeneral Introduction

GENERALL INTRODUCTIO N

Studiess in the nematode Caetwrhabditis elegans, the fly Drosophila melanogaster, and mammaliann cells have demonstrated that the molecular mechanisms of the execution machineryy leading to apoptosis are highly conserved among metazoans. These mechanismss have been extensively reviewed1"5; therefore I wil l only describe some essentiall information in the following paragraphs of this chapter that is related to the workk performed in this thesis.

Cancerr and apoptosis Tumorigenesiss in mammals is a multistep process of about five to seven rate-limiting, stochasticc events, leading to an unlimited growth advantage of the tumor cells6. Most cancerss have acquired the same set of functional capabilities during their development, thee so-called 'hallmarks of cancer'. In every case, alterations in defined pathways providee the tumorigenic phenotype, like self-sufficiency in growth signals, insensitivity too anti-growth signals, tissue invasion and metastasis, limitless replicative potential, sustainedd angiogenesis and evading apoptosis7. Normal tissues, however, rely on extracellularextracellular growth signals, growth inhibitory signals, survival signals generated by attachmentt to the extracellular matrix and soluble survival factors, as well as a control mechanismm in the form of programmed cell suicide and limited life span. A great diversityy of genetic alterations can lead to the same effect within one or more of the definedd pathways that favor tumorigenesis. For instance, effector molecules can be amplifiedd or mutated into constitutively active forms, or inhibitory molecules could be downregulatedd or mutated into inactive forms or even completely deleted from the genome.. The cell type and the associated selection pressure will influence which kind of geneticc alteration will take place.

Anticancerr drugs can potentially kill cells in two fundamentally different ways; byy interfering with cellular processes that are essential for maintenance of viability or byy stressing the cell such that an endogenous physiological cell death mechanism is triggered,, that indirectly prompts the cell to kill itself. Initially, the development of chemotherapeuticc agents was based on the observation that tumor cells proliferate faster thann normal cells. Thus, drugs that interfered with DNA replication or cellular metabolismm were chosen. However, these drugs also affected rapidly dividing normal cellss of the bone marrow and gut and thereby reducing the efficacy of the therapies. Besidess this, drug resistance lowers the efficiency of treatments. Drug resistance can takee place because proteins within the cell can interfere with either drug accumulation (drugg efflux pumps like P-glycoprotein) or stability (detoxifiers like glutathione) thus

9 9

ChapterChapter I

inhibitingg drug-target interactions, or by mutations in proteins that are involved in

variouss stress signaling pathways leading to cell death or cell cycle arrest .

Apoptosis,, or programmed cell death, is a multistep cell death program that is

presentt in a latent form in all cells of the body and is characterized by the active

participationn of the dying cell. In 1972, Kerr and coworkers were the first who described

thiss kind of cell death, which is morphologically and biochemically different from

necrosis9.. An apoptotic cell undergoes cell shrinkage, membrane blebbing and nuclear

condensationn and fragmentation, and subsequent disassembly into apoptotic bodies.

Phagocytess engulf these apoptotic bodies thereby preventing harmful leakage of cell

contentt of the dying cell into the surrounding tissue and avoiding an inflammatory

response,, which is a characteristic for necrosis. Apoptosis plays a crucial role in normal

development,, in maintenance of tissue homeostasis and in an effective immune system,

byy eliminating cells that are redundant, damaged or infected. It is therefore not

surprisingg that dysregulation of this process is implicated in numerous pathological

conditions,, ranging from degenerative disorders to autoimmunity and cancer.

Alterationss in apoptotic pathway components or their regulators have been

detectedd in a variety of cancers, suggesting that loss of the ability of cells to undergo

apoptosiss might contribute to carcinogenesis. For instance, the p53 tumor suppressor

genee is the most frequently mutated gene in human tumors, and loss of p53 function can

bothh disable apoptosis and accelerate tumor development in transgenic mice10"13. If there

iss DNA damage, this is sensed by kinases ATM and Chk2, which phosphorylate and

stabilizee p53. P53 can bring about a cell cycle arrest to repair the damage or activate the

apoptoticc pathway via transcription dependent and independent mechanisms " " .

Also,, functional mutations or altered expression of upstream regulators of p53 (like

ATM,, Chk2, Mdm2 and pl9ARF) or downstream effectors (PTEN, Bax, Bak and Apaf-

1)) occur often in human tumors1722. Furthermore, dysregulation of Bcl-2 family

members,, like Bcl-2, Bcl-xL and Bad, is often observed in tumors. In fact, Bcl-2 was

Firstt identified based on its translocation in follicular lymphoma, and is overexpressed in

aa variety of cancers2324. Also post-mitochondrial mutations have been reported, like

silencingg of Apaf-1 in metastatic melanoma and leukemia cell lines or overexpression of

inhibitorr of apoptosis proteins (IAPs) or heat shock proteins2225i6. There is also a

correlationn between tumorigenieity and mutations in death receptors, like CD95 and

TRAILL receptors, or with dysregulation of downstream signaling components, like

caspase-88 and FLIP2710.

Thus,, there is a relevant link between apoptosis and cancer. Since apoptosis

resistancee is one of the 'hallmarks of cancer' and conventional anti-cancer therapies kill

cellss by inducing apoptosis, it is important to develop novel anti-cancer therapies that

cann activate apoptotic pathways that are still operational in most of the cancers and that

preferentiallyy selectively induce apoptosis in tumor cells and leave healthy cells

10 10

GeneralGeneral Introduction

untouched.. It is therefore crucial to know what the fundamental mechanisms are of

proteinss that participate in apoptotic pathways.

Caspases:: the engine of cellular destruction AA crucial event in apoptosis involves the activation of members of the caspase family of cysteinee proteases, which coordinate destruction of the cell through limited proteolysis off an array of intracellular proteins31 3\ To prevent unscheduled cell suicide, each caspase.. essentially present in all cells, is synthesized as a dormant pro-enzyme that

requiress processing at caspase cleavage sites to generate the active enzyme"

Caspases,, like Caspase-8,-9 and -10 that initiate apoptosis become activated by self-associationn and binding to activating adaptor or scaffold proteins via their long prodomain.. This results in autocatalysis and the formation of the active tetrameric enzyme.. Effector caspases like Caspase-3,-6 and -7 are activated by processing by initiatorr caspases or other proteases like Granzyme B. In turn, these activated effector caspasess cleave an array of substrates resulting in the execution of the actual cell death. So,, a chain reaction of caspase activation is the cell's death sentence.

Att least 14 distinct mammalian caspases have been identified, with their orthologuess present in species ranging from C. elegans to D. melanogaster. The involvementt of the C. elegans CED-3 caspase homologue in apoptosis was first discoveredd by Yuan et al.36. Since then, compelling evidence has demonstrated that the mechanismm of apoptosis is evolutionarily conserved and executed by this family of cysteinee proteases. Caspases are named like that, since they all cleave after an aspartate residuee in their substrates37. The function of caspases is either related to cytokine maturationn or induction of apoptosis38. Caspases involved in apoptosis are generally dividedd into two categories based on their primary structure; the initiator caspases with theirr long amino-terminal prodomains include Caspase-2,-8,-9,-10 and the effector caspases,, with short prodomains, include Caspase-3,-6,-739. However, classification of thee caspases based on their cleavage specificity gives a different view. Then Caspase-2,-33 and -7 are grouped together with a preference for Asp-GIu-X-Asp and the Caspases-6,-8,-99 and -10 have a preference for (Leu/Val)-Glu-X-Asp40.

Caspasess and their activation: extrinsic and intrinsi c apoptosis pathways Theree are two major caspase-activating cascades that regulate apoptosis in mammals: onee is initiated from the cell surface death receptors (extrinsic pathway) and the other is triggeredd by changes in mitochondrial membrane integrity (intrinsic pathway), see also Figuree 1. The initiator caspases all contain a protein-protein interaction motif in their prodomain,, either a caspase-recruitment domain (CARD) or two death effector domains

U U

ChapterChapter I Growth h

Figuree 1 - Apoptosis pathways. Somee of the known components of the extrinsic (death receptor) and intrinsic (mitochondrial)) apoptosis pathways are shown. Proteins in circles promote apoptosis, whereass proteins in squares have an inhibitory function. See text for explanation. Adapted fromm references 4 and 8.

(DED).. These motifs interact with similar motifs present on oligomerized adaptorproteins,, thus bringing multiple initiator caspases into close proximity with one anotherr and facilitating their autoactivation .

Thee extrinsic apoptosis pathway is induced upon activation of trimerized death

receptors,, belonging to the tumor necrosis factor (TNF) receptor superfamily, by the

trimerizedd cognate ligands. Death receptors share the presence of a death domain (DD)

inn their cytoplasmic tail. The best characterized death ligand / receptor interactions

includee TNFa, CD95 ligand (CD95L, FasL) and TNF-related apoptosis-inducing ligand

(TRAIL )) with TNF receptor-1 (TNF-R1), CD95 (Fas, APO-1) and TRAIL-R1-R2

(DR4/DR5),, respectively. The initiator Caspases-8 and -10 are activated in response to

ligationn of a death receptor. Following stimulation by e.g. CD95L, the DD in the

cytosolicc tail of the trimerized death receptor CD95 associates with the DD of the

adaptorr molecule FADD (Fas-associated death domain). FADD then recruits

procaspase-88 via a homophilic DFD interaction. The protein complex thus formed at the

taill of the death receptor, known as the death-inducing signaling complex (DISC)42'"

resultss in autocleavage and activation of Caspase-83;44. As illustrated in Figure 1, active

Caspase-88 can initiate either directly the activity of a cascade of effector caspases or via

thee mitochondria, involving the pro-apoptotic Bcl-2 family member Bid. The

importancee of the mitochondrial contribution is dependent on the cell type .

12 12

Genera!Genera! Introduction

Initiatorr Caspase-9 is instead activated by assembly into a multiprotein caspase-activatingg complex, called the apoptosome, which contains besides Caspase-9, thee Apaf-1 scaffold protein and Cytochrome c (Cyt c)4<M8. Diverse cellular stresses like radiation,, chemotherapeutic drugs, glucocorticoids and cytokine deprivation, provoke thee release of Cyt c from the mitochondria into the cytosol, in the intrinsic pathway, see alsoo Figure i49;50. Cyt c associates in an ATP dependent manner to the carboxy-terminus off Apaf-l and induces a conformational change in it5152. In this state Apaf-1 assembles intoo a heptameric structure that recruits procaspase-9 via a homophilic CARD interactionn to the complex, where it undergoes auto-proteolytic activation53. Then, activee Caspase-9 activates the effector caspases, which cleave several substrates, leadingg to cellular and nuclear morphological changes and ultimately cell death.

Exactlyy by which mechanism initiator caspases achieve catalytic competence inn their recruitment / activation complexes is still unresolved. Recently, it was suggested thatt within this proximity-induced activation model, dimerization of monomeric zymogenss is the key event, rather than proteolysis. Various groups have shown that activationn of Caspase-8 and -9 occurs via dimerization and that the internal proteolysis probablyy serves to partially stabilize the activated dimers or to release the dimers from thee complexes54"57. Dimerization causes a conformational change of the zymogen's catalyticallyy active pocket, making it accessible to substrates56. It is suggested that this activationn by dimerization is the general mechanism to activate all initiator caspases. Recently,, Kumar and colleagues have suggested something similar for Caspase-2 activation58.. They show that Caspase-2 may become recruited in a CARD-dependent mannerr to a novel Apaf-1-independent activation complex, where it becomes activated withoutt the requirement of cleavage. Until now, the individual components of this complexx have yet to be identified. Whether Caspase-2 can be really grouped among the initiatorr caspases is still not solved. Tts activation mechanism, which is dependent on its prodomain,, and various reports that demonstrate a link between Caspase-2 and release off mitochondrial pro-apoptotic factors upon DNA damage-induced death, make it likely39'62.. On the other hand, collective data on Caspase-2 deficient mice, its substrate specificityy and reports (see also chapter 4 in this thesis) placing Caspase-2 downstream off the mitochondria in various apoptotic pathways, suggests that Caspase-2 could act as effectorr caspase4063"65.

Inn case of the effector Caspases-3,-6 and -7, which do not have a long prodomainn and thereby can not oligomerize to an adaptor/scaffold complex, the activatingg event is proteolysis by an initiator caspase (-8,-9, or -10) or by other proteasess like Granzyme B67. In turn, these activated effector caspases cleave various proteinn substrates leading to the systematic and orderly disassembly of the dying cell. UpUp to now, more than 280 different caspase substrates are known. However, some substratess are just cleaved as bystanders, whereas others have a discrete function in propagationn of the cell death process by turning off cell-protective mechanisms and

13 13

ChapterChapter 1

activatingg pathways that lead to cell destruction68. These substrates include regulators of

cell-cyclee progression, DNA repair proteins and nuclear and cytoskeletal structural

proteins,, explaining the characteristic features of apoptosis.

Itt should be mentioned that also apoptosis-like programmed cell death can

occur,, which is caspase-independent. Both death receptors and DNA damage can trigger

thiss caspase-independent cell death program that can involve organelles, including

mitochondria,, endoplasmic reticulum (ER) or lysosomes that activate other

proteases64'70. .

BcI-22 family: regulators of the mitochondrial membrane integrit y Thee mammalian Bcl-2 protein family consists of both pro- and anti-apoptotic proteins,

whichh show sequence homology and structural similarity in their Bcl-2 homology {BH)

regionss . The anti-apoptotic members include besides Bcl-2, also Bcl-xi" \ Bfl-

1/A17'74.. Mcl-17\ Bcl-w76 and Boo/Diva/Bcl-B77~7g. They ail share three to four Bcl-2

homologyy (BH) domains and contain a hydrophobic carboxy-terminal tail which

localizess these members mainly to intracellular membranes like the outer mitochondrial

membrane,, the ER and the nuclear envelope80. These members protect cells from a wide

rangee of cytotoxic insults, like cytokine deprivation, UV and y radiation and

chemotherapeuticc drugs. The pro-apoptotic Bcl-2 family members can be divided into

twoo subgroups. The Bax type subgroup of pro-apoptotic members has two to three BH

domainss and is structurally very similar to the anti-apoptotic relatives81. The group

consistss of Bax82, Bak83"* 5, Bok/Mtd8687, Bcl-xs72, Bcl-G88 and Bcl-Rambo89. The

secondd pro-apoptotic subgroup contains proteins that only share the BH3 region with

thee anti-apoptotic members and are therefore called 'the BH3-only's'90. BH3-only

memberss identified so far are Bid91, Bad'52, Bik/Nbk". Bim/Bod94, Bmf9",

Harakiri/DP59fi:97,, Noxa98, Puma/Bbc3*M0\ BNIP3/NIP3102 and BNIP3L/Nix'°\ NMR

structurall analysis of the Bcl-xL/Bak BH3 peptide complex has revealed that the BH3

domainn of Bak binds to a hydrophobic cleft formed by the BH1, BH2 and BH3 domains

off BCI-XL'04. Anti-apoptotic and pro-apoptotic Bcl-2 family members may thus engage,

byy a BH3 domain-dependent mechanism, in the formation of heterodimers in which

theyy mutually antagonize each other's function.

Thee response of the mitochondrion to upstream stimuli is a critical control

pointt in apoptosis, which is regulated by the pro- and anti-apoptotic Bcl-2 family

memberss via a yet incompletely resolved mechanism. Several apoptotic stimuli, as

describedd earlier, induce loss of mitochondrial membrane integrity, which allows the

releasee of numerous mediators from the mitochondrial intermembrane space, like Cyt

c47,, Smac/DIABLOt05;,°6, Omi/HtrA2107, apoptosis inducing factor (A1F)108 and

endonucleasee G , see also Figure 1. These mediators all promote the apoptotic

machineryy via their own mechanism. Cyt c is a crucial component for the formation of

14 14

GeneralGeneral Inti'oductkm

thee apoptosome, which initiates the caspase cascade. As a further safeguard mechanism, Smacc and Omi prevent the inhibitor of apoptosis proteins (IAPs) from inhibiting the activityy of processed caspases. AIF and endonuclease G have been proposed to contributee to 'caspase-independent' nuclear changes and cell death. However, whether thesee released mediators are essential for the induction of apoptosis is not yet established.. For instance, absence of Cyt c solely attenuates stress-induced apoptosis110. However,, microinjection of Cyt c into various cell types induces apoptosis in a caspase dependentt manner1". Loss of Smac does not influence the apoptotic process112, althoughh one could argue that Omi might still block IAP function. Nevertheless, inhibitionn of IAPs does not initiate apoptosis106.

Recently,, also the ER is thought to participate in stress-induced apoptosis that iss imposed by disruption of calcium homeostasis or by excess of unfolded proteins"1. Bcl-22 can function on the ER and can protect against ER-stress induced apoptosis and againstt the cross talk between ER and mitochondria114"116. Also Bax and Bak can localizee to the ER and either one of the two is required for ER-stress induced apoptosis"7"120,, and for the associated cleavage of ER-localized mouse Caspase-121J). Whetherr Caspase-12 is required for ER-stress induced cell death is uncertain, since Caspase-122 deficient mice were partially resistant to ER-stress induced apoptosis121. Thee human counterpart of Caspase-12 is a Caspase-12-like protein with a premature stopp codon and several point mutations preventing the translated protein from having anyy caspase-like enzymatic activity 4 .

Inn mammals, activation of BH3-only proteins integrates diverse apoptotic stimulii into one single pathway by triggering Bax/Bak-mediated mitochondrial dysfunction.. The BH3-only proteins are considered to function as the cell death triggers90,, but pro-apoptotic Bax and/or Bak are also essential for the cell killing, which cann be antagonized by anti-apoptotic Bcl-2 family members122. Inactivation of Bax affectedd apoptosis only slightly and disruption of Bak had no evident effect, but inactivationn of both genes dramatically impaired apoptosis in many tissues and preventedd BH3-only proteins to ^n118-119122123 g0t the presence of either Bax or Bak seemss to be essential for apoptosis in many cell types and places the BH3-only proteins upstreamm in the pathway. Bax and Bak appear to permeabilize the outer mitochondrial membranee and allow the release of apoptogenic proteins, as mentioned above. In normal cells,, Bax is predominantly a cytosolic monomer, but during apoptosis it undergoes a conformationall change that exposes both the amino- and carboxy-termini, leading to mitochondriall translocation and oligomerization124"128. Bak is constitutively localized to thee mitochondrial membrane, but also changes conformation and forms larger aggregatess upon apoptotic stimulation129"13'. In vitro, Bax and Bak homo-oligomers can permeabilizee mitochondria and artificial protein-free liposomes and form pores through whichh Cyt c can pass132"134. Although tBid can induce Bax and Bak oligomerization and insertionn into the outer mitochondrial membrane, the exact mechanisms that cause

75 5

ChapterChapter 1

conformationall changes and oligomerizations are still unknown13516. Incubation of tBid

withh mitochondria in vitro does not allow for detectable Bid present in cross-linked Bak

oligomers,, which suggest a 'hit-and-run" activation mechanism131, or perhaps Bid

insteadd activates Bax/Bak indirectly. Though high concentrations of Bcl-2 proteins

preventt Bax oligomerization and channel-forming activity124, cross-linking studies

reveall no Bcl-2 / Bax complexes'". Alternatively, Bax might interact with components

off the existing voltage-dependent anion channel to create a larger channel * , but

severall studies have found no evidence for such complexes l - 4 1- , , l 4° .

Modell A Model B Model C

BH33 BH3 BH3 BH3 V V

e.g.. Bad e.g. tBid

Figuree 2 - Models for functional relationship of the different subgroups of the Bcl-2 family. (Model(Model A) The BH3-only proteins (BH3) directly engage and activate the pro-apoptotic Bax/Bakk proteins, once they have overwhelmed the anti-apoptotic Bcl-2-type proteins (Bcl-2).. (Model B) The anti-apoptotic Bcl-2 type proteins prevent, either directly or indirectly, activationn of pro-apoptotic Bax/Bak proteins. The BH3-only proteins (BH3) target only Bcl-22 type proteins and inactivate these members. (Model C) Certain BH3-only proteins like Bad targett only Bcl-2 type members (Bcl-2) and are thought to act as sensitizers, while other B113-onlyy proteins like tBid can engage both Bcl-2 type and Bax/Bak proteins and act as the activators.. At least the class of activators has to be activated to activate Bax/Bak proteins. Adaptedd from reference 1.

Inn principle, there are three main variations on the theme of the functional

relationshipp between the different subgroups of the Bcl-2 family, as schematically

representedd in Figure 2, but collective data is still not conclusive. In model A, Bcl-2

typee proteins sequester BH3-only proteins from Bax/Bak types, thereby preventing the

BH3-onlyy induced activation of Bax/Bak proteins. This model is in line with data on the

capabilityy of tBid to trigger Bax to permeabilize protein-free liposomes1 . However, so

farr only Bid and the BH3 peptide of Bid and Bim have been shown to bind Bax or Bak

directly,, and this model does therefore not explain how the other BH3-only proteins

performm their activation on Bax/Bak91: 31:,41;142. in model B, BH3-onIy proteins only

16 16

GeneralGeneral Introduction

targett Bcl-2-type members and relieve the inhibition of Bcl-2 on Bax/Bak-type members.. In favor of this model all BH3-only proteins known so far are able to bind to thee Bcl-2-type members. Although overexpression of Bcl-2 and BCI-XL inhibit apoptosis inducedd by BH3-only proteins, which is in agreement with this model, mutants of Bcl-2 andd BCI-XL that could not bind to Bim, Bad or Bid are no longer able to protect cells fromm apoptosis induced by these BH3-only proteins123. This suggests that there is an existingg cooperation between BH3-only proteins and the pro-apoptotic Bax/Bak proteinss which could no longer be prevented and partially supports model C. This modell is a combination of the previous two. Bcl-2-type members have a neutralizing functionn by sequestering the BH3-only members and thereby prevent activation of Bax/Bak-typee members. In addition, this model suggests a specialization within the BH3-onlyy group; there are members, like Bad that predominantly have a sensitizing functionn and members like tBid that have an activating function. Both can by sequesteredd by Bcl-2-type members but, the tBid-like members (including Bim) have a lowerr binding affinity for Bcl-2 than the Bad-like members and are subsequently easily displacedd by them from Bcl-2 and are free to directly activate Bax/Bak members. This modell is based on data from in vitro competition experiments with BH3 peptides141. Thiss is furthermore supported by data with a mutant of Bcl-xL that can no longer bind Baxx or Bak, but still is able to prevent apoptosis. This suggests that BCI-XL sequesters thee BH3-only members rather than directly regulating the activity of Bax/Bak143. A criticismm on this model is that it would require the activation of the two proposed classes off BH3-only members, but single BH3-only proteins such as Bad are potent stimuli as well11* 123. .

Regulationn of activity of the mitochondrial 'cell death triggers' Inn order to prevent inappropriate cell death, BH3-onIy proteins are kept inactive by a numberr of different mechanisms until it is the right moment to come into action144. Transcriptionall control, induced by the tumor suppressor protein p53, has been describedd for Noxa and Puma in response to DNA damage9810011". Puma expression is alsoo regulated in a p53-independent manner, by glucocorticoids and serum deprivation".. Mouse knockout studies for Puma show that Puma is required for the p53-dependentt apoptotic responses to y radiation and DNA-damaging drugs in thymocytes,, as well as to c-Myc and El A oncogenes in primary embryonic fibroblasts (MEFs)145'146.. Puma deficiency also protects lymphocytes from several p53-independent apoptoticc stimuli. Characterization of Noxa knockout mice also indicate a role for Noxa inn p53-dependent apoptosis, though less pronounced than found for Puma146147. Furthermore,, the expression of Bid and Bim can be transcriptionally regulated by p53 andd the forkhead transcription factor FKHR-L1, respectively148'149.

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ChapterChapter 1

Inn addition, post-transcriptional controls like sequestration or protein

modificationn are ways to hold BH3-only proteins in check. For example. Bid requires

cleavagee to produce an active carboxy-terminal fragment (tBid) that translocates to the

mitochondriaa to induce, among other factors, the release of Cyt c into the

cytoplasm11^1^"1^.. Bid proteolysis is achieved by Caspase-8 upon ligation of death

receptorss or by the serine protease Granzyme B that is released from lytic granules into

thee target cell by cytotoxic T and NK cells1"'. It is suggested that amino-terminal

myristoylationn on tBid facilitates the translocation to the mitochondria -> , where it is

attractedd to cardiolipin-enriched areas in the mitochondrial outer membrane "\ Bid-

deficientt mice appear normal, but are resistant to CD95-induced hepatocellular

apoptosis1^6,, which reflects the requirement of the mitochondrial contribution to

apoptosiss induction in these cells. Furthermore, it is suggested that Bid serves as a

chemosensitivityy gene, since Bid-deficient MEFs were more resistant to adriamycin and

5-FUU than wild type MEFs140, though no differences were found in response to

yy radiation, staurosporin or dexamethasoneIM '. In healthy cells, both Bim and Bmf are

sequesteredd on cytoskeletal structures; the microtubular dynein motor or the actin-based

myosinn V motor complex, respectively9"'57. Stress signals, like cytokine deprivation

andd calcium flux for Bim, and anoikis and actin depolarizing drugs for Bmf, trigger

theirr release, which is probably induced by their phosphorylationlf i . Loss of Bim in

micee causes extensive lymphoid and myeloid cell accumulation, indicating a role in

deletingg autoreactive lymphocytes in v("vo159;,6° and its lymphocytes are resistant to

cytokinee withdrawal or taxol treatment, but remain sensitive to y radiation, etoposide or

dexamethasone161.. Another BH3-only protein, Bad, is a target for phosphorylation by

proteinn kinase-A and -B/Akt (PK.B). In healthy cells, survival factors induce

phosphorylationn of Bad and subsequently allow its sequestration by 14-3-3 proteins in

thee cytosol. Cytokine deprivation may induce apoptosis by permitting

dephosphorylationn of Bad and its subsequent release162"166. Bad deficient mice, however,

havee no detectable abnormality and their neurons are normally sensitive to NGF

deprivationn in culture167. This suggests that Bad just contributes in this pathway by

perhapss supporting Bim, which is essential in this pathway161'168.

Regulationn of apoptosis pathways Cellss constantly receive signals from outside and inside the cell that can cause damage.

Too ensure that they can make the right decision, so either to die or to stay alive and

repairr the damage, the apoptotic process is strongly regulated. Both caspase activation

andd caspase activity are tightly controlled. Death receptor-induced caspase activation

cann be inhibited by cellular FLICE-inhibitory (c-FIip) proteins 16g. There are two splice

variantss of c-Flip present within the cell. c-Flip-short (c-Flips) resembles the viral

variantt of Flip. It contains two DED domains with which it can interact with FADD and

18 18

GeneralGeneral Introduction

byy that interfere with the recruitment and activation of procaspase-8 or -10 to the DISC fromm ligand-activated death receptors. c-FIip-long (c-FlipL) in addition contains a caspase-Iikee domain and thus shares more structural similarity to Caspase-8/-10. However,, c-FlipL is catalytically inactive due to mutations in the catalytic domain and cann interfere thus with the processing of procaspase-8 and -10, but not with its recruitmentt to the DISC. c-FIipL is a different kind of caspase regulator than c-Flips. Loww expression levels are required for the activation of Caspase-8, because it potently promotess procaspase-8 activation through heterodimerization24* . Whereas at high expressionn levels, c-FlipL acts as an apoptosis inhibitor248. High levels of c-FlipL protein aree detected in several malignant melanoma tumors and gastric, colon and bladder adenocarcinoma17""173. .

Receptor-mediatedd cell death can also be inhibited by the expression of decoy receptors.. These receptors took like death receptors but either completely lack the cytoplasmicc death domain or contain truncated variants, and they therefore specifically sequesterr ligands away from their functional receptors. Decoy receptors can inhibit apoptosiss induced by TRAIL (decoy receptors DcRl and DcR2l74l7~) and CD95L (decoyy receptor DcR3176;l77). The TRAIL decoy receptors DcRl and DcR2 are often downregulatedd in tumor cells but not in normal cells or differentially subcellularly localized1781799 and may thus protect normal cells from TRAIL-induced apoptosis. In contrast,, the CD95 decoy receptor DcR3 is overexpressed in some lung and colon tumors,, suggesting a possible role in tumor cell resistance to CD95L177.

Ass mentioned earlier, Bcl-2 family members regulate the mitochondrial membranee integrity and thereby the activation of inducer Caspase-9. Caspases can also bee directly inhibited by members of the IAP family. XIAP, cIAPl and cIAP2 for instance,, directly inhibit Caspase-3, -7 and -9 by binding via one of their baculovirus IAPP repeats (BIR) and masking the active site in the caspases180"184. Furthermore, the RINGG domains of XIAP, cIAPl and cIAP2 have ubiquitin ligase (E3) activity and can actt as adaptors to bind to the ubiquitin-conjugating enzymes to direct the transfer of ubiquitinn to caspases and mediate their proteasomal degradation185"187. The activity of thee IAP family of proteins is in their turn inhibited by the IAP-interacting regulatory proteinss Smac in humans1"1 and Diablo in mice106 and HtrA2/Omi1071 8"190 upon release off the mitochondria. And in the case of XIAP, data indicate that apoptotic stimuli like dexamethasonee and etoposide lead to ubiquitylation and subsequent degradation of XIA PP itself185. Aberrant expression or activity of lAPs has been implicated in human diseases,, like t(l I;18)(q21;q21) translocation of cIAP2 in mucosa-associated lymphoid tissuee (MALT) lymphoma191, which results in a chimeric cIAP2-MALTl protein that lackss the RING domain19 . Overexpression of cIAPl by amplification of the q22 region onn chromosome 11 is found in oesophageal squamous-cell carcinoma ". High protein expressionn levels of XIAP in Hodgkin's lymphoma-derived B cells play a crucial role in resistancee to apoptosis .

19 19

ChapterChapter 1

Alsoo PKB/Akt is a strong anti-apoptotic regulator. It phosphorylates a variety

off proteins, including several associated with cell death pathways, leading to diminished

apoptoticc cell death. PKB-mediated phosphorylation of the pro-apoptotic Bcl-2 family

memberr BAD leads to its sequestration by 14-3-3 proteins and its dissociation from the

mitochondria163'166.. Likewise, phosphorylation of Caspase-9 inactivates this protease,

thuss blocking the propagation of death signals originating in the mitochondria ". In

addition,, PKB promotes cell survival by phosphorylating and inhibiting the Forkhead

transcriptionn factor FKHR-L1 and thereby preventing the expression of the pro-

apoptoticc Bcl-2 family member Bimi48:1%. Furthermore, it can upregulate Bcl-2

expressionn through the cAMP-response element-binding protein197. Another regulator of

celll survival, the NF-KB transcription factor, is also a target of PKB198. PKB regulates

NF-KBB activation via accelerated degradation of the NF-KB inhibitory protein IKBGI,

resultingg in elevated levels of for instance Bcl-X] or c-Flipi,1^202. Additionally, NF-KB

regulatess the expression of numerous other anti-apoptotic proteins, like Bfl-l/Al 20"'204,

Bcl-2205,, cIAP2 and X1AP206207 or the serpin protease inhibitor 2A to protect against

lysosome-mediatedd apoptosis208.

Thee role of mitochondrial permeabilization: an evolutionary perspective Concomitantt studies in mammals and in lower eukaryotes, primarily C. elegans and D.

melanogastet\melanogastet\ have shown that the fundamental processes and protein families involved

inn apoptosis are evolutionarily conserved. Proteins from different species also activate

orr inhibit apoptotic processes within each other. However, the system in C. elegans is

veryy simple and less diversified compared to the mammalian situation. D. melanogaster

providess a system of intermediate complexity between nematodes and mammals.

Inn C. elegans, three proteins are required for programmed cell death: the pro-

apoptoticc BH3-only Bcl-2 family member EGL-1, the caspase-9 homologue CED-3 and

thee Apaf-1 adaptor homologue CED-4, whereas the anti-apoptotic Bcl-2 homologue

CED-99 is essential for cell survival3. In cells destined to survive, CED-9 binds directly

too the adaptor CED-4, and co-localizes on mitochondria, and prevents it from activating

thee CED-3 caspase. However, when programmed cell death is initiated, the BH3-only

proteinn EGL-1, which is the only initiator present in C. elegans, is transcriptionally

upregulatedd and binds to and inactivates CED-9209"2". This allows CED-4, which works

autonomouslyy in causing cell death, to move to the nuclear membrane and to

multimerizee with CED-3 and promote the autocatalytic activation of CED-3. Although

Apaf-11 molecules have carboxy-terminal WD40 repeats, CED-4 lacks these repeats. Cyt

cc binds these WD40 repeats and changes the conformation of Apaf-1 in such a manner

thatt it can recruit procaspase-9. Since CED-4 does not contain WD40 repeats it is

perhapss not so surprising that C. elegans does not require Cyt c to activate CED-3.

Thus,, in the model system of C. elegans, the BH3-only protein neutralizes the Bcl-2-

20 20

GeneralGeneral Introduction

typee inhibition on Apaf-1/CED4 and Caspase-9/CED3 is activated without any mitochondriall involvement.

Noo Drosophila homologues of BH3-only proteins have been identified thus far. However,, various apoptotic stimuli induce the upregulation of one or more of the cell deathh inducer proteins like Reaper, Hid, Grim and Sickle212"216 by the Hox transcription factor2177 or by the Drosophila p53 orthologue218. Genetic analysis indicates that the fly apicall caspase Drone is essential for the pro-apoptotic activity of the Reaper, Hid, Grim (RHG)(RHG) proteins. Drone competes with RHG proteins for binding to Drosophila inhibitor off apoptosis proteins (Diapl, and 2). Under normal conditions, Drone binds to Diapl andd is ubiquitinated by Diapl and its associated ubiquitin-conjugating E2 enzyme (Morguee or Ubcdl) and targeted for proteosomal degradation219'222. However, following upregulationn of RHG proteins e.g. Reaper displaces Drone from Diapl. Binding of Reaperr to Diapl leads to auto-ubiquitination and proteasome degradation of Diapl instead,, thus facilitating Drone stabilization and activation"1 ' ' ~"\ There is no indicationn for a role of Cyt c release in Drosophila apoptosis, although the Drosophila

Apaf-11 homologue Dark contains WD40 repeats that can bind Cyt c226;227. Moreover,

thee IAP antagonists (e.g. Reaper) do not reside in mitochondria, like in mammalian cells arguingg against a mitochondrial contribution in Drosophila. The Drosophila

homologuess of Bax-type proteins, Debcl, and of Bcl-2-type proteins, Buffy, both localizee to mitochondria where they associate and counteract each other and function upstreamm of caspase activation228"23'. Whether they act downstream of RHG proteins or inn a parallel pathway and are essential for caspase activation is still being resolved.

Anti-cancerr therapy on modulators of apoptosis Ass impaired apoptosis is one of the hallmarks of cancer, the search for therapeutic strategiess to enhance apoptotic cell death is widely growing. These studies focus for instancee on limiting the function of Bcl-2 and IAPs, or engaging the death receptor pathway.. The oncogenic potential of anti-apoptotic Bcl-2 family members was first characterizedd for Bcl-2. The translocation of Bcl-2 in follicular lymphoma caused a reductionn in cell death rather than enhanced proliferation24. Mouse studies confirmed thee oncogenic potential of elevated levels of Bcl-2, but since the chance of getting tumorss in these mice is rare, it is suggested that additional mutations are required for transformation232"234.. When additional oncogenes are co-expressed, for instance Myc, whichh promotes proliferation, a dramatic synergism is observed in the formation of malignancy235"238.. Also indirect mutations in oncogenes induced by mitogens are found too increase the expression levels of anti-apoptotic Bcl-2 family members, like activation off the NF-KB pathway, which induces the expression of Bcl-xL and Bfl-l/A l genes203. Too fully transform, cells e.g. have to bypass growth arrest as well as senescence, which cann be achieved by elimination of p53 function. Most spontaneous B lymphoid tumors

21 21

ChapterChapter J

thatt arise in myc transgenic mice have in addition to elevated levels of Bcl-2 and/or Bcl-

xii mutations that eliminate p53 function" '" .

Thee pro-apoptotic Bcl-2 family members (BH3 only's and Bax-like) probably

alll behave like tumor suppressors and thus need to be inactivated to allow

transformationn to occur. It is likely that these pro-apoptotic members have a substantial

redundancy,, so their tumor suppressor function may only arise in specific cell types.

Downregulationn of Bax is regularly found in haematopoietic malignancies241, however

bax-rwxWbax-rwxW mice acquire few spontaneous tumors242. Absence of both Bax and Bak greatly

enhancee the transformation compared to loss of either one of the two"4. Bim is a potent

tumorr suppressor at least in B cells. Although loss of Bim in itself does not enhance

tumorr incidence in mice within the first 12 months of their life, already loss of a single

allelee of Bim dramatically accelerates lymphoma in mice expressing a myc transgene in

thee B lineage243. Moreover, 50% of Bid deficient mice develop chronic myelomonocytic

leukaemiaa by two years of age244. Since Puma and Noxa are both transcriptional targets

off the tumor suppressor protein p53 and knock out studies of these genes indicate a role

forr them in p53-dependent apoptosis induction, these proteins are attractive candidates

forr being tumor suppressors as well. Nothing is known yet about tumor incidence in

thesee recently made knock out mice.

Deathh receptor-induced apoptosis pathways can directly activate effector

caspases,, thus they can bypass certain inhibitions at the mitochondrial level that

frequentlyy occur in tumor cells and are not affected by loss of functional alleles of p53.

Tnn particular, the death ligand TRAIL harbors potential as anticancer agent. In vitro, it

killedd the majority of malignantly transformed cell lines tested but not normal cells.

Andd it shows no toxicity when systemically administered to animals in mouse and

monkeyy preclinical models'74'"45. Toxicity of TNFa and CD95L precludes their use for

systemicc therapy.

Scopee of this thesis Conventionall anti-cancer regimens like y radiation and chemotherapeutic drugs, kill

cellss by activating apoptotic signaling pathways, but apoptosis resistance is one of the

'hallmarkss of cancer'. This irony has led to the urge to design novel anti-cancer

therapiess that can activate apoptotic pathways in such a manner that the block(s), in

resistantt cancer cells, in these pathways can be circumvented. Insight in the molecular

naturee of apoptosis pathways can help to design novel types of therapies that activate

apoptoticc pathways that are still functional plus induce apoptosis as selectively as

possiblee in tumor cells. Most apoptotic stimuli convey their signal on mitochondria,

whichh is a critical control point in apoptosis signaling. The research presented in this

thesiss was therefore aimed at defining key mediators involved in communication to the

mitochondriaa in apoptosis signaling induced by death receptors as well as DNA

22 22

GeneralGeneral Introduction

damagingg anti-cancer regimens. Jurkat T leukemic cells were mainly used as model system,, because these cells are sensitive to multiple apoptotic stimuli, including death receptorr stimulation and treatment with DNA damaging anti-cancer regimens like etoposidee and y radiation. Previously, our lab has generated CD95-resistant Jurkat variantt clones, which fail to release Cyt c and as a consequence do not succeed to undergoo apoptosis. These variant clones proved cross-resistant to apoptosis induction by etoposidee and y radiation as a consequence of lack of Cyt c release, indicating the presencee of common aspects in apoptosis signaling by CD95, etoposide and y radiation. Therefore,, we also investigated at which level the mitochondrial activation was blocked andd what the common and distinct aspects are in these pathways. Interestingly, TRAIL-inducedd Cyt c release and apoptosis signaling are still operational in the variant clones. Inn chapter 2, the communication to mitochondria is investigated in death receptor signalingg and the important mediators for TRAIL-receptor and CD95 signaling are described.. The pathways diverge downstream of tBid. Mitochondrial apoptosis signalingg in the variant cells is blocked downstream of tBid but upstream of the mitochondria.. Therefore, we subsequently looked for Bid interacting proteins to search forr potential inhibitors {chapter 3). We identified Bfl-1 as a strong Bid-interacting and inhibitoryy protein, but it appeared not to be the factor responsible for the common inhibitionn in the variant clones. However, its mechanism of action provided insight into aa general mechanism of mitochondrial regulation by Bcl-2 family members. Next, we examinedd apoptosis signaling to the mitochondria in response to DNA damaging anti-cancerr regimens and show the importance of aspartate-cleaved Bid as key mediator of thee apoptotic response {chapter 4). As a result, this clarifies the cross-resistance in the variantt clones. The work described in chapter 5 started with the observation that the p53 DrosophilaDrosophila target Reaper could induce Cyt c release in Xenopus egg extracts via an unknownn mechanism. We investigated how Reaper induced apoptosis in mammalian cellss and established that this occurs independently of the mitochondria via a novel describedd mechanism for Reaper. Finally, the results of all chapters are summarized and potentiall future perspectives are discussed in chapter 6.

23 23

ChapterChapter 1

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