Apoptosis in cancer—implications for therapy

Apoptosis in Cancer—Implications for Therapy Henning Schulze-Bergkamen and Peter H. Krammer Resistance towards apoptosis is a key factor for the survival of a malignant cell. Cancer results if there is too little apoptosis and cells grow faster and live longer than normal cells. In addition, defects in apoptosis signaling contribute to drug resistance of tumor cells. Thus, one of the main goals for oncologic treatment is to overcome resistance of tumor cells towards apoptosis. The exciting challenge in oncology is to translate the growing knowledge of apoptotic pathways into clinical applications. In this review we address the role of apoptosis signaling in tumorigenesis and drug resistance of tumor cells and discuss therapeutic approaches interfering with apoptosis pathways. Semin Oncol 31:90-119. © 2004 Elsevier Inc. All rights reserved.

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OR EVERY CELL there is a time to live and a time to die. Cell death occurs by two main types, necrosis and apoptosis, the latter also known as programmed cell death. Apoptosis is important for normal development and tissue homeostasis. A dysregulation of this process can result in inappropriate cell death contributing to the pathogenesis of various human diseases such as cancer. Apoptosis is a common property of all multicellular organisms. The phenomenon of apoptosis was first described as a unique process associated with typical morphological changes by Carl Vogt in 1842. This process was first called apoptosis in 1972.1 Apoptosis plays a fundamental role in physiologic processes, especially in mammalian development and immune reactions.2,3 During embryonic growth, apoptosis is the main mechanism for the removal of unnecessary tissue. In the adult organism, apoptosis regulates the balance between cell proliferation and cell death. When cells are no longer needed, aged, infected, or have become seriously damaged, they are eliminated by apopto-

From the Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany. Supported by the Deutsche Forschungsgemeinschaft (DFG), Deutsche Krebshilfe/Dr Mildred Scheel-Stiftung, Tumorzentrum Heidelberg/Mannheim, the Medical Faculty of the University of Heidelberg, and the National Cancer Center, Bethesda, MD. Address reprint requests to Peter H. Krammer, Tumor Immunology Program, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. © 2004 Elsevier Inc. All rights reserved. 0093-7754/04/3101-0011$30.00/0 doi:10.1053/j.seminonol.2003.11.006 90

sis.4 In the immune system, potentially autoreactive and useless immune cells are mainly cleared by apoptosis.5 The apoptotic event is characterized by several distinct morphological changes. Among these changes are shrinkage of the apoptotic cell, membrane blebbing (zeiosis), degradation of DNA in the nucleus by specific endonucleases,6 and finally the breakage of the cell into small vesicles. Phagocytic cells like macrophages and dendritic cells then engulf the cell fragments.7,8 In contrast to necrotic cell death, apoptosis is characterized by a minimum of inflammatory reactions. This is partly due to the secretion of anti-inflammatory cytokines by phagocytic cells and the rapid engulfment of the cell fragments.9 APOPTOSIS SIGNALING

On the molecular level the cell death program can be divided into three parts: initiation, execution, and termination of apoptosis. Apoptosis can be induced by various stimuli, including growth factor withdrawal, UV light or irradiation, cytotoxic drugs, and death receptor ligands. There are two major signaling routes in mammalian cells leading to apoptosis, the extrinsic and the intrinsic pathway (Fig 1). Extrinsic Death Pathway The extrinsic death pathway is initiated by binding of death receptor ligands to specific death receptors on the cell surface (Fig 2). The growing subfamily of death receptors belongs to the tumor necrosis factor (TNF) receptor superfamily.10 This superfamily is characterized by a sequence of two to five cysteine-rich extracellular repeats. The death receptors share a homologous, intracellular death domain (DD) of about 80 amino acids, which is essential for the transduction of the apoptotic signal.11,12 So far, six members of the death receptor subfamily are known: TNF-R1 (CD120a), CD95 (APO-1, Fas), DR3 (APO-3, LARD, TRAMP, WSL1), TRAIL-R1 (APO-2, DR4), TRAIL-R2 (DR5, KILLER, TRICK2), and DR6.13 Among these, CD95 has been characterized most extensively.14 CD95 is a widely expressed glycosylated cell surface molecule of approximately 45 to 52 kd (335 amino acids). It is a type I transmembrane protein that is expressed in most tissues of Seminars in Oncology, Vol 31, No 1 (February), 2004: pp 90-119

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Fig 1. The two main pathways for the initiation of apoptosis. Apoptosis can be triggered by two alternative pathways. The extrinsic pathway is initiated by binding ligands to specific death receptors on the cell surface. The intrinsic pathway is initiated at the mitochondria by various stimuli such as cytotoxic drugs. Initiator caspases such as caspase-8, -9, and -10 then activate executioner caspases such as caspase-3, -6, and -7. Executioner caspases cleave death substrates (see text) and, finally, apoptosis occurs. The mitochondrial pathway can also be activated from outside the cell without stimulation of death receptors in a fashion that is not completely understood. Apaf-1, apoptotic protease activating factor-1; cyt c, cytochrome c; DISC, death-inducing signaling complex.

Fig 2. Death receptors and their ligands. A schematic representation of various death receptors is depicted. The names of the receptors are given below the scheme, ligands at the top. The question mark indicates the unknown ligand for DR6 (death receptor 6). APO-2L, APO-2 ligand; CD95L, CD95 ligand; DcR1, 2, 3, decoy receptor 1, 2, 3; DR3, 4, 5, death receptor 3, 4, 5; LARD, lymphocyte-associated receptor of death; LIGHT, a cytokine that is homologous to lymphotoxins; LIT, lymphocyte inhibitor of TRAIL; LT, lymphotoxin; TNF, tumor necrosis factor; TL1A, endothelial cell-derived TNF-like factor; TNFR1/2, tumor necrosis factor receptor 1/2; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R1-4, TRAIL receptor 1-4; TRICK2, TRAIL receptor inducer of cell killing 2; TRID, TRAIL receptor without an intracellular domain; TRUNDD, TRAIL receptor with a truncated death domain.

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Fig 3. CD95 (APO-1/Fas) signaling in type I and type II cells. In type I cells, large amounts of active caspase-8 initiate the death signal and lead to a rapid processing and activation of caspase-3 and other caspases. In type II cells only reduced DISC formation is seen. Reduced amounts of caspase-8 are activated and the apoptotic signal is amplified via the mitochondria. Mitochondria can be activated by truncated Bid (cleaved by caspase-8). The apoptosome is the focal point of activation of caspase-9 (see text).

mammals.15,16 The ligand of CD95 is called CD95L (FasL, APO-1L, CD178), a 40-kd protein.17-19 The ligands of death receptors belong to the TNF superfamily of proteins. They are type II transmembrane proteins except TNF-␣.20 CD95L is expressed mainly on activated T, B, and natural killer (NK) cells, as well as in immune-privileged sites such as testis and eyes.17 In addition to a membrane-bound form, a soluble form of CD95L exists, which is generated by extracellular metalloproteinase activity.21-23 Several groups reported pro-apoptotic properties of the soluble CD95L,24 whereas others ascribe the capacity to induce apoptosis solely to the membrane-bound form.22,25 The death receptor ligand TNF-related apoptosisinducing ligand (TRAIL, APO-2L), has recently been the focus of interest due to its potential for clinical applications (see below). Five members of the TNF receptor superfamily are known to interact with TRAIL.26 TRAIL-R1 and -R2 contain a cytoplasmic death domain and are capable of transducing an apoptotic signal upon ligation with TRAIL.27-29 The other three receptors, TRAIL-R3 (LIT, TRID), -R4, and a soluble receptor called osteoprotegerin (OPG, TRAIL-R5), lack death

domains and may serve as decoy receptors to regulate TRAIL-mediated cell death. The crucial point of death receptor signaling is the formation of a multimeric complex of proteins triggered by receptor cross-linking with either agonistic antibodies30 or their natural ligands. The structure formed is called the death-inducing signaling complex (DISC).31 The DISCs of CD95, TRAIL-R1, and -R2 consist of trimerized CD95, TRAIL-R1, or -R2, respectively, the serine-phosphorylated adapter Fas-associated death domain protein (FADD/Mort1), caspase-8, and caspase10.32,33 Depending on the cell type, either of two different pathways is activated downstream of CD95 after DISC formation (Fig 3). In type I cells the death signal is propagated by a cascade, which is initiated by the activation of large amounts of caspase-8 at the DISC.34 In type II cells DISC formation is poor, and the propagation of the apoptotic signal depends on its amplification via mitochondria. Caspase-8 cleaves the Bcl-2 family member Bid after DISC formation and truncated Bid leads to mitochondrial membrane permeabilization (MMP).35 Although activation of mito-

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chondria occurs both in type I and type II cells, it is dispensable for the progression of type I cells into apoptosis, because caspase activity is sufficient for cleavage of death substrates. Signaling of apoptosis by the different members of the death receptor family seems to follow the same basic rules and is initiated by the same sequential steps: ligand binding, receptor trimerization36 or oligomerization of pre–ligand-binding assembly domains (PLADs),37 DISC formation including recruitment of the adapter molecule FADD into the DISC, association of procaspase-8, and the formation of active caspase-8 after autocatalytic cleavage.32,33 Intrinsic Death Pathway The intrinsic pathway is initiated at the mitochondrial level. Mitochondria are central players in apoptosis induction and act as integrating sensors of various death stimuli. BH3-only members of the Bcl-2 family (see below) initiate the mitochondrial signaling cascade by sensing damage to the cells.38 After activation, BH3-only proteins are released to neutralize Bcl-2 or its homologs. Subsequently, BH3-only proteins and Bax-like proteins (see below) promote mitochondrial membrane leakage and cause MMP. BH3-only proteins may act indirectly by interacting with Bax or Bak.39 Induction of apoptosis proceeds via the release of cytochrome c from the intermembranous space of mitochondria.40 Once cytochrome c is released into the cytosol, the apoptosome is assembled, a multi-protein complex in which Apaf-1 serves as an oligomerization platform for assembly and autoproteolytic activation of caspase-9.41 After assembly of the apoptosome, caspase-9 activates further downstream caspases. Upon mitochondrial activation, other proteins like Smac/ DIABLO (second mitochondria-derived activator of caspase/directed IAP binding protein with low pI) are released from mitochondria facilitating caspase activation by sequestering caspase inhibitors.42 In addition, the apoptosis-inducing factor (AIF) is released; AIF a protein thought to induce apoptotic morphological changes of the nucleus in a caspase-independent manner.43 Among the stimuli that trigger the intrinsic pathway are reactive oxygen species (ROS), survival factor insufficiency, and DNA damage, caused, for example, by chemotherapeutic drug treatment.44,45

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Execution and Termination of Apoptosis The activation of caspases (cysteine aspartases) is central to most apoptotic pathways including those initiated by cytotoxic drugs. Activation of caspases has been shown after a variety of apoptotic stimuli, including death receptor stimulation46 and mitochondrial activation. Caspases are cysteine aspartyl proteases and cause cell death by cleaving a variety of intracellular substrates. They are constitutively expressed in cells as inactive precursors (zymogens) and are activated by proteolytic cleavage at defined aspartate residues. The active enzyme is a heterotetrameric complex of two large and two small subunits. The so-called “initiator caspases,” such as caspase-8, -10, and -9, are recruited to large protein complexes such as the DISC or the apoptosome upon activation of the extrinsic or the intrinsic death pathways, respectively (see above). These caspases cleave and activate “executioner” caspases, mainly caspase-3, -6, and -7. By mutual cleavage of other executioner caspases amplification of pro-apoptotic proteolysis can occur.47 Moreover, the activity of executioner caspases leads to cleavage of cellular substrates, resulting in the characteristic biochemical and morphological changes of apoptosis. Cleavage of nuclear lamin is involved in chromatin condensation and nuclear shrinkage. Processing of ICAD (DFF45, inhibitor of caspase-3–activated DNase) causes the release of CAD (DFF40, caspase-activated deoxyribonuclease), which subsequently fragments DNA in the nucleus. In addition, the degradation of cytoskeletal proteins like actin and plectin by caspases leads to cell fragmentation. In the termination phase, apoptotic bodies are rapidly engulfed by phagocytes. Phagocytes bind to signals on the surface of apoptotic bodies; for example, to phospatidylserine or to surface oligosaccharides.48 Phosphatidylserine, which is normally exclusively localized to the inner leaflet of the plasma membrane, is exposed on the cell surface early in apoptosis. However, the signals mediating engulfment and phagocytosis of apoptotic cells are still largely unknown.49 The prototypic apoptosis pathways require caspase activity. Opposing this established hypothesis, caspase-independent apoptosis models were established recently.50-53 These models share some, but not all, characteristics of classical apoptosis

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Fig 4. Proteins of the Bcl-2 family. Proteins of the Bcl-2 family share homology in the Bcl-2 homology regions (BH1 to BH4). BH1 to BH4 are conserved sequence motifs. Anti-apoptotic Bcl-2 family members possess all four BH homology regions. They are primarily localized in mitochondria and stabilize mitochondrial membranes. Pro-apoptotic family members can either lack the BH4 domain or lack BH1, 2 and 4 (“BH3-only proteins”). Family members such as Bax and Bid can translocate from the cytosol to mitochondrial membranes leading to mitochondrial membrane permeabilization. Several functional domains of Bcl-2 are shown. 1-7 indicate helices identified in Bcl-xL, in which a core of two hydrophobic helices, 5 and 6, is surrounded by five amphipathic helices. A1/Bfl-1 shows a limited homology to the BH4 domain and is therefore denoted in brackets. TM, transmembrane domain.

pathways. Therefore, they have been called “apoptotic-like” cell death or “paraptosis.” REGULATION OF APOPTOSIS

The apoptotic cell death machinery, which is inherent in every mammalian cell, needs to be tightly controlled. This control takes place at different levels and is ensured by various anti- and pro-apoptotic proteins. The mechanisms mediating this control are relevant both in homeostasis and in tumorigenesis. Regulation of Death Receptors Cell surface expression of death receptors or their ligands can be altered. CD95 expression is transcriptionally induced by the tumor-suppressor protein p53.54 The same applies for TRAIL-R2.55 FLIPs (FADD-like interleukin-1␤-converting enzyme-like protease [FLICE/caspase-8]–inhibitory proteins) directly interfere at the level of death receptors.56 In human cells two splice variants—a long form (FLIPL) and a short form (FLIPS)— have been identified. Both forms share structural homology with procaspase-8 but lack catalytic activity. The structural homology allows FLIP mol-

ecules to bind to the DISC and thereby prevent the activation of the initiator caspase-8.24,57 Moreover, several kinases involved in survival signaling pathways, such as phosphatidylinositol-3 kinase (PI3K), can inhibit CD95 signaling at the DISC level.58 However, the mechanism mediating this inhibition remains elusive. Regulation of Mitochondria Many survival signaling pathways act by stabilizing mitochondrial integrity and the suppression of cytochrome c release. Mitochondrial integrity is a fundamental issue when dealing with apoptosis resistance of tumor cells (see below). A well-established family of proteins that has an important impact on mitochondrial integrity is the Bcl-2 family. Bcl-2 family members are capable of influencing the permeability of the mitochondrial membrane45,59,60 and set the threshold for activation of the apoptotic machinery. The members of this protein family can be roughly subdivided into anti- and pro-apoptotic proteins. Common to all Bcl-2 family members are the Bcl-2 homology regions (BH1-4) (Fig 4). These proteins are predominantly localized to the outer

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Fig 5. Regulation of mitochondrial activation. Various stimuli such as cytotoxic drugs can initiate mitochondrial activation. BH3-only proteins sense cell damage and, subsequently, promote mitochondrial membrane permeabilization, probably through interaction with Bax or Bak. Activation of mitochondria proceeds via the release of cytochrome c (cyt c) into the cytosol, where it binds to Apaf-1 (apoptotic protease activating factor 1) to form the apoptosome. At the apoptosome, caspase-9 is cleaved and activated. Apoptosis can be blocked on different levels by anti-apoptotic proteins, including the antiapoptotic Bcl-2 family members Bcl-2, Bcl-xL, and Mcl-1, and the inhibitors of apoptosis proteins (IAPs), which are inhibited by Smac/DIABLO.

mitochondrial membrane. However, Bcl-2 also localizes to other cellular membranes.61 Pro-apoptotic members of the Bcl-2 family can alter the membrane permeability of mitochondria (Fig 5). Bak and Bax induce MMP when added to purified mitochondria as recombinant proteins.45,62,63 They are thought either to form channels in the outer mitochondrial membrane or to alter the activity of existing channels, thereby leading to changes of the membrane potential. Bak, which is normally associated with the outer mitochondrial membrane (OMM), can be activated in two ways: pro-apoptotic proteins such as Bid or Bim can bind to and activate Bak, or other pro-apoptotic proteins, Bad and Bik, can bind to Bcl-2, which sequesters Bid, thereby allowing activation of Bak by Bid. In contrast to Bak, Bax is mostly found in the cytosol of unstimulated cells. Upon induction of apoptosis, it translocates to the OMM and causes MMP.64 Anti-apoptotic members of the Bcl-2 family, Bcl-2 representing the paradigm, reside in the OMM and counter the effect of pro-

apoptotic members. However, recent work indicates that Bcl-2 can act independently of the apoptosome to regulate caspase activation.65 The mitochondrial FK506-binding protein 38 (FKBP38), a member of the immunophilin family, has recently been described to anchor Bcl-2 and Bcl-xL to mitochondria and thereby to inhibit apoptosis.66 Regulation of Caspases Another family of regulatory proteins is the inhibitor of apoptosis (IAP) family.67 IAP family members are frequently found to be overexpressed in human cancer.68 In humans eight members of the IAP family have been identified so far, including those best-studied, XIAP, c-IAP1, c-IAP2, and survivin. IAPs are characterized by a 70 –amino acid zinc-finger fold domain called baculoviral IAP repeat (BIR). IAPs are endogenous inhibitors of caspases and therefore counteract apoptosis. They may also function as ubiquitin ligases, promoting the degradation of target caspases. Some of the

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proteins that are released from mitochondria by the MMP, such as Smac/DIABLO,42,69 can bind to IAPs and thereby relieve their caspase inhibition.70,71 APOPTOSIS IN TUMORIGENESIS

Progression of cancer is a multistep process. Mutations occurring in the genome of precancerous cells accumulate, finally leading to dysregulated cell cycle progression and proliferation. New insights into the biology of cancer have fostered a growing appreciation of the role of apoptosis in tumorigenesis. A number of genes that encode components of the apoptotic machinery are directly targeted by activating or inactivating genetic lesions in cancer cells. A classic example for an activating genetic lesion is the chromosomal translocation t(14;18), common among follicular B-cell lymphomas, which leads to high levels of the anti-apoptotic protein Bcl-2.72 Two main prerequisites are responsible for the occurrence of cancer: (1) a deregulation of cell proliferation and (2) a suppression of apoptosis. These two conditions enable a cell to become malignant and to expand.44,73,74 Deregulation of cell proliferation is based on autonomy from exogenous mitogens and a refractoriness to growth inhibitory signals. However, deregulated proliferation alone is not sufficient for tumor development but leads to cell death.73 This is at least in part due to the fact that overexpression of growth-promoting oncogenes such as c-MYC, E1A, or E2F1 (see below) sensitizes cells to apoptosis.75 In addition, proliferating cells depend on survival signals such as growth factors, cytokines, and hormones. A lack of these signals usually triggers apoptosis, a phenomenon termed “death by neglect.” Thus, tumor development requires mechanisms to suppress apoptosis.76-78 Altered Survival Signaling Several alterations in survival signaling pathways have been identified in tumors. The viability of a somatic cell requires survival signals triggered by soluble factors, as well as direct interactions with neighboring cells and the extracellular matrix. Cancer cells show a reduced dependence on exogenous growth stimulation, indicating autonomy from these proliferative/mitogenic signals. Conceivable ways to achieve this autonomy would be autocrine stimulation or overexpression and

self-signaling of growth factor receptors. Moreover, changes in the expression of several attachment receptors such as integrins can also contribute to increased proliferation.79,80 Binding of these surface receptors links the cell to the extracellular matrix, leading to the activation of signaling pathways that mediate apoptosis resistance, such as the PI3K/AKT pathway.81 During metastasis, tumor cells are also resistant to a special type of apoptosis, called anoikis,82 allowing them to travel to distant sites. Anoikis occurs when normally adherent cells lose contact to the extracellular matrix. After detachment, pro-apoptotic Bcl-2 family members such as Bim and Bmf are released from the cytoskeleton and induce mitochondrial activation.83,84 Another prerequisite for proliferation and survival of tumor cells is the stabilization of telomeres. Normal diploid cells only have limited replicative capacity. After 60 to 70 divisions, cells enter a senescent state and die by apoptosis. The finite number of cell divisions is determined by the length of the telomeres at the chromosome ends, shortened during each round of replication.85 When telomere length is critically reduced, cell cycle arrest or apoptosis is triggered by activation of p53.86,87 In tumor cells telomeres are stabilized by telomerases or a mechanism called alternate lengthening of telomeres (ALT). The pro-survival PI3K/AKT pathway contributes to apoptosis resistance and tumorigenesis in various cancer cells. PI3K is activated by survival signals such as growth factors. Active PI3K generates the phosphorylated lipid phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3], leading to recruitment of the kinases PDK1 and PDK2 (PtdInsP3-dependent kinase 1/2) and Akt (also known as protein kinase B or PKB) to the plasma membrane. In the complex formed, PDK1 and PDK2 activate Akt by phosphorylation. The isoforms of Akt are known as Akt1, 2, and 3.88 Activated Akt can induce strong survival signals; for example, by phosphorylation and inactivation of pro-apoptotic proteins such as Bad.89 In addition, Akt promotes cancer cell invasion in vitro through upregulation of the insulin-like growth factor (IGF)-1 receptor.90 Several mutations of the PI3K pathway leading to constitutive activation have been described91,92; for example, the loss of the signaling suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10).93-95 PTEN inhibits activation of downstream

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effectors such as Akt by dephosphorylating inositol phospholipid intermediates of the PI3K pathway.96 Loss of PTEN has been suggested as causing enhanced apoptosis resistance of tumor cells.97 PTEN mutations have been described in sporadic malignancies such as brain and colorectal cancers.98,99 Moreover, the Cowden syndrome, which is characterized by a high incidence of breast and thyroid cancers, is based on mutations of the PTEN gene.100 Ras Proteins Another group of proteins that are tightly linked to survival signaling and malignant transformation are Ras proteins. Ras proteins, including their well-described isoforms K-Ras, N-Ras, and H-Ras, are small guanosine triphosphate (GTP)binding proteins. Ras proteins are constitutively active in most human tumors either due to mutations in the RAS genes themselves or due to alterations in downstream or upstream signaling components.101 They contribute to important characteristics of malignancy such as deregulation of tumor cell growth and resistance to apoptosis.102,103 Ras proteins become activated following triggering of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR).104 EGFR has been found to be overexpressed in most carcinomas. A wide range of extracellular stimuli can lead to activation of Ras. Ras itself activates downstream kinases such as Raf or mitogen-activated protein kinase (MAPK) kinase MEK, which subsequently leads to phosphorylation and activation of the MAPK extracellular signal-regulated kinase 1 and 2 (ERK1 and 2). ERK phosphorylates and thereby activates downstream targets like the transcription factor c-Jun, a member of the activator protein-1 (AP-1) family.105 c-Jun is upregulated in many tumors, such as hepatocellular carcinomas (HCC). In nontransformed hepatocytes, c-Jun prevents apoptosis by antagonizing p53 activity. Thus, induction of c-Jun activity might contribute to the development of HCC.106 In addition, crosstalk between the Ras and the PI3K/ Akt pathways enables growth signals to evoke various survival signals concurrently within the cell.107 Along with the PI3K/Akt pathway, another effector pathway of Ras leads to inhibition of the forkhead transcription factors (FKHR), which are implicated in induction of apoptosis via activation of BIM and CD95L.108 In addition, Ras can

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interfere with p53 pathways by induction of the p53-inactivator Mdm-2.109 Alterations of the p53 Pathway The TP53 gene (the gene that encodes human p53) has become a focus of intense investigation since it became clear that TP53 mutations are the most frequent genetic alterations found in human cancer.110 Increasing numbers of sequences obtained from human malignancies add to a database of more than 10,000 somatic tumorigenic TP53 mutations.111,112 The role of TP53 in tumorigenesis is also supported by the observation that p53-/mice show a high incidence of tumor development.113 Moreover, germ-line mutation of one p53 allele in humans gives rise to the Li-Fraumeni cancer susceptibility syndrome.114 p53 exerts a tumor-suppressive role at multiple stages of carcinogenic progression. Wild-type (wt) p53 has inhibitory effects toward the growth of abnormal cells and has been termed “guardian of the genome” in its role to prevent cancer development. p53 transduces the diversity of signals arising from stress and damage into tumor-suppressive apoptotic or growth-arresting responses.115 This results in a strong selective advantage for cancer cells that have lost p53 function.116 p53 can be activated by kinases such as DNA-dependent protein kinase (DNA-PK) and the kinase ATM (ataxia-telangiectasia, mutated). ATM phosphorylates p53, which leads to the abrogation of p53 binding to the ubiquitin ligase Mdm-2 and activation of p53. Subsequently, p53 regulates the expression of several pro- and anti-apoptotic genes at transcriptional and post-translational levels. Inhibition of p53 pathways may occur either through inactivating mutations of TP53 itself, through perturbation of the p53-activating pathways in response to stress, or through defects in the downstream mediators of p53-induced apoptosis. Several genetic lesions have been identified that inhibit events downstream of p53, such as the inactivation of Mdm-2, an inhibitor of p53. In tumors with functional p53, these defects can block p53-mediated cell cycle arrest and apoptosis. Another possibility to inhibit p53 in tumor cells is the loss of ARF (alternative reading-frame product of the p16/ INK4 locus),117,118 which occurs in many earlystage cancers, where TP53 mutations are rarely found. Lymphomas from mice lacking ARF expression are highly invasive and display defects in

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apoptotic pathways.119 ARF interacts with Mdm-2 and prevents it from ubiquitinating p53 and targeting it for destruction in the proteasome.120 In both adenomas and carcinomas of the colon, inactivation of ARF occurs through methylation of the ARF promotor.121,122 Another lesion influencing signaling events of p53 is the lack of the apoptosis stimulating protein of p53 (ASPP) family. ASPP proteins—ASPP1 and ASPP2—act as potent activators of p53. ASPP expression is frequently downregulated in breast carcinomas with wt p53, thus explaining why wt p53 fails to suppress tumor growth in these cancers.123 Recently, an inhibitor of ASPP, inhibitory member of the ASPP family (iASPP), has been found to be overexpressed in several cancers containing wt p53 and normal levels of ASPP1 and ASPP2. Inhibition of iASPP by RNA interference induces p53-dependent apoptosis in human cells, implying its oncogenic properties.124 Several other inhibitory factors for p53 signaling have been described. One of these, the macrophage migration inhibitory factor (MIF), is overexpressed in various tumors125 and has been demonstrated to inhibit p53-dependent gene expression and apoptosis.126 Recently, two p53-related genes, p63127-130 and p73,131 have been identified. Dominant negative forms of p63 and p73, which are often encountered in large amounts in tumor cells, can compromise p53 function.132 Altered Death Receptor Signaling Apoptosis suppression in tumor cells has also been described at the level of death receptors. Downregulation of the death receptor CD95 has been observed in many tumors, such as HCC,133 melanomas,15 and neoplastic colon epithelium.134 This downregulation is, at least in part, due to mutations in the tumor-suppressor gene TP53, which regulates CD95 via an intronic enhancer element,54,135,136 Moreover, several mutations in the CD95 gene have been reported in myeloma and T-cell leukemia cells.137,138 In families with germ-line CD95 mutations, which cause the socalled autoimmunoproliferative syndrome, the incidence of lymphomas is increased.139 Another mechanism for abrogating death-receptor stimulation in tumor cells has been revealed in a panel of lung and colon carcinoma cell lines. A nonsignaling decoy receptor for CD95L, called DcR3, is upregulated, thus sequestering CD95L from CD95.140 A splice variant of CD95, soluble CD95

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(sCD95), has been shown to compete for binding of CD95L. sCD95 is expressed in various malignancies, and its serum levels correlate with a poor prognosis, such as in melanoma.141,142 Decreased expression or mutations have also been shown for TRAIL-R1 and -R2 in different tumors.143,144 Fresh isolates from human tumors, such as melanomas, are frequently resistant to TRAIL, probably due to a decreased expression of TRAIL receptors.145 In oropharyngeal and non-small cell lung cancers, a frequent deletion of the chromosomal region 8p21-22 affects the TRAIL-R2 gene.146 Some tumor cell lines (eg, melanoma and murine B-cell lymphoma lines) were shown to express high levels of c-FLIP.56,147 In Epstein-Barr virus (EBV)-positive Burkitt’s lymphoma cell lines, enhanced levels of c-FLIP were correlated with resistance to CD95-mediated apoptosis.148 Some tumorigenic viruses such as human herpesvirus 8 (HHV8) encode the viral analogue of FLIP, called viral FLIP (v-FLIP).149-151 In advanced Kaposi sarcomas, expression of v-FLIP is increased, thus inhibiting death-receptor–mediated apoptosis.152 Therefore, v-FLIP might play a role in the persistence and oncogenicity of some viruses.153 Stabilization of Mitochondria Decreased sensitivity of mitochondria towards intracellular apoptosis stimuli is an important factor for the survival of transformed cells.154 This decrease in mitochondrial sensitivity is observed frequently. In some cases the cellular events causing this phenotype have been identified successfully. Mitochondria, such as from isolated leukemia cells, have been shown to be resistant to Ca2⫹-induced changes in the transmembrane potential.155 Moreover, tumor cells have been reported to overexpress apoptosis-inhibitory components within mitochondria; for example, the adenine nucleotide translocator ANT-2.156 ANTs are the most abundant mitochondrial inner membrane proteins and catalyze the exchange of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) between cytosol and mitochondria. The anti-apoptotic proteins Bcl-2 and Bcl-xL protect against mitochondrial activation by inhibiting pro-apoptotic proteins, thereby preventing MMP.45,62,157 Overexpression of Bcl-2 has been described among various tumor types and has been correlated with a progressive state of tumor disease.158-164 In Bcl-2 transgenic mice, somatic

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translocations activating the oncoprotein c-Myc have frequently been observed.165 The c-Myc protein is both a stimulator of cell proliferation and an inducer of apoptosis. Tumor promotion by cMyc is most effective when accompanied by the overexpression of anti-apoptotic proteins such as Bcl-2 that nullifies its cell death–promoting activity while leaving its proliferative action intact.166,167 Bcl-2 also cooperates with another recently identified oncoprotein, BAG-1.168,169 Expression of BAG-1 is frequently altered in malignant cells170 and leads to protection from diverse apoptotic stimuli,171,172 Bcl-xL is upregulated in myeloma cells by constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling.173 IAP Family Another important role in regulation of sensitivity towards apoptosis is played by proteins of the IAP family. IAPs are overexpressed in various tumors and contribute to tumorigenesis. The IAP family protein survivin is found in virtually all human tumors that have been studied, but it cannot be detected in nontransformed adult tissues.68,174 Survivin expression probably represents an early event in tumorigenesis. Premalignant skin lesions show expression of survivin, indistinguishable from the later stages of skin cancer.175 In retrospective studies, patients with overexpression of survivin had a reduced apoptotic index in vivo.176-178 Some members of the IAP family have been shown to bind caspases directly and thereby suppress apoptosis. Another anti-apoptotic mechanism displayed by survivin is interference with p53-dependent apoptosis.179 In a xenograft tumor model expression of phosphorylation site-deficient mutants of survivin induced mitochondrial activation and cytochrome c release and suppressed tumor growth in vivo.180,181 Apart from its effect on apoptosis, survivin plays a major role in cell cycle control.71 cIAP2 is also implicated in tumorigenesis. In 50% of marginal cell lymphomas of the mucosa-associated lymphoid tissue (MALT lymphoma), a translocation t(11;18)(q21;q21) affecting cIAP2 has been found.182 Inactivation of Pro-apoptotic Proteins Tumor cells have not only been shown to overexpress anti-apoptotic proteins such as Bcl-2 or Bcl-xL, but they may also inactivate pro-apoptotic

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proteins by downregulation or mutation. In different tumors loss of expression as well as mutations in the BH domains of Bax have been identified.183,184 Several studies in transgenic mice confirmed the role of Bax as a tumor suppressor. Mouse fibroblasts lacking both Bax and Bak are resistant to a variety of apoptotic stimuli, such as cytotoxic drugs.185 In Bax-deficient mice tumor growth is accelerated, and Bax has been proven to be necessary for p53-mediated apoptosis induction.186 Injection of tumors expressing either wt or mutant Bax into nude mice showed a significant proliferative advantage of tumor cells with mutant Bax.187 Another way to block mitochondrial activation, is the loss of Apaf-1. Loss of Apaf-1 is a frequent event in melanoma and leukemias and contributes to their apoptosis resistance.188,189 The mechanisms leading to Apaf-1 deficiency are unclear, but may involve DNA methylation.190 Another pro-apoptotic molecule, XAF1 (XIAP-associated factor 1), is downregulated in various tumor cell lines.191 XAF1 acts via binding to XIAP1 and antagonizes its anti-apoptotic function at the level of caspases. Downregulation of caspases is also involved in tumorigenesis. Deletion or methylation of the gene encoding the initiator caspase-8 is frequently observed in neuroblastoma192 and lung cancer cells.193 Nuclear Factor-␬B–Dependent Mechanisms Nuclear factor B (NF-␬B) was originally discovered because of its role in regulating gene expression in immune and inflammatory responses.194 Subsequent studies have shown that NF-␬B plays a role in tumorigenesis, including such effects as transformation-associated apoptosis, cell cycle progression, regulation of cell adhesion, and angiogenesis.195,196 Activation of NF-␬B is illustrated in Fig 6. Constitutive activation of NF-␬B has been shown in various hematologic malignancies, as well as in epithelial tumors and contributes to tumor cell survival by exerting anti-apoptotic effects.195,197 Activation of NF-␬B has been shown to protect tumor cells from the apoptotic cascade induced by TNF-␣ and other stimuli.198 NF-␬B has been reported to inhibit apoptosis by its effect on the transcription of genes important in apoptosis signaling, such as c-FLIP, cIAP1, and cIAP2, and members of the Bcl-2 family such as A1/BFL1 and Bcl-xL.199 Cells with chromosomal rearrangements or other types of DNA damage are normally

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Fig 6. Activation of NF-␬B. Five mammalian reticuloendotheliosis family (Rel)/nuclear factor ␬B (NF-␬B) proteins are known: RelA (also known as p65), c-Rel, RelB, NF-B1 (also known as p105) and NF-␬B2 (also known as p100). NF␬B1 and 2 are processed resulting in mature p50 and p52 proteins, respectively. The family members need to dimerize in order to fulfill their function as transcription factors. The most commonly detected NF-␬B dimer is p50-RelA. Activity of these dimers is regulated by inhibitor of ␬B (I␬B) proteins (including I␬B␣, ␤, ␥, and ⑀), which cause their retention in the cytoplasm. All NF-␬B proteins contain a Rel homology domain, which is necessary for both dimerization and binding to DNA. Upon stimulation, the I␬B kinase (IKK) complex, consisting of two catalytic subunits, IKK, and one regulatory subunit, IKK s24, phosphorylates the IB proteins. Subsequently, IB is ubiquitinated and degraded by the proteasome, resulting in the nuclear translocation of NF-␬B proteins and transcriptional activation of target genes, eg, genes encoding anti-apoptotic proteins. LPS, lipopolysaccaride; IL-1, interleukin-1; cIAPs, cellular inhibitors of apoptosis proteins; cFLIP, cellular FADD-like interleukin-1– converting enzyme-like protease inhibitory proteins.

eliminated by apoptosis, often initiated by action of p53. NF-␬B has been shown to antagonize the function of p53, possibly by competing for transcriptional coactivators.200 Prevention of p53-mediated apoptosis by NF-␬B thus increases the pool of genetically altered cells. Constitutive activation of NF-␬B can be achieved by various means. In viral infections causing malignant transformation, viral oncoproteins are often found to be activators of NF-␬B. For example, the Tax oncoprotein of the human T-cell leukemia virus-1 (HTLV-1) activates the IKK complex, resulting in the activation of NF-␬B signaling pathways.201 Moreover, NF-␬B is activated by most of the known oncogene products consistent with its pivotal role in tumorigenesis. Chaperones Hsp70 is the major stress-inducible heat-shock protein and is abundantly and preferentially ex-

pressed in cancer cells. Hsp70 is capable of inactivating Apaf-1 and AIF and protects cancer cells from a variety of apoptotic stimuli despite a functional apoptotic signaling cascade.202,203 Inhibition of Hsp70 by antisense technique induces apoptotic cell death in a variety of tumor cell lines in vitro.204 Escape From the Immune Response The immune system can attack proliferating tumor cells by antibodies and cells. The main antitumor effectors of the immune system on the cellular level are cytotoxic T cells and NK cells. One mechanism by which these effector cells can kill tumor cells is induction of apoptosis via triggering of CD95 expressed on tumor cells.205 Another mechanism is the secretion by these effector cells of a membrane-permeabilizing protein called perforin and proteolytic enzymes known as gran-

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Fig 7. Activation of p53 upon cytotoxic drug treatment. p53 is a key factor in apoptosis induction in tumor cells. p53 is inhibited by Mdm-2, a ubiquitin ligase that targets p53 to the proteasome for degradation. Mdm-2 is inhibited by ARF (alternative reading-frame product of the p16/INK4 locus). ATM (ataxia telangiectasia, mutated) can activate p53 by phosphorylation. Following DNA damage caused by cytotoxic drugs or irradiation, p53 can be activated either directly, by activation of ATM or ARF, or by inactivation of Mdm-2. After activation, p53 transactivates pro-apoptotic genes. Ub, ubiquitin.

zymes.206 Granzymes have been demonstrated to activate caspases in target cells.153 Moreover, granzyme B can directly activate mitochondria by cleaving Bid.207 Tumor cells have developed strategies to resist killing by effector cells such as cytotoxic T cells. In tumor cells expression of the serine protease inhibitor PI-9/SPI-6, which inhibits granzyme B, has been observed.208,209 Resistance of tumor cells towards death-receptor– or granzyme-mediated cell death leads to the escape of tumor cells from the attacking immune system. In addition, tumor cells themselves might be able to directly induce apoptosis in immune cells. A number of tumors express functional CD95L constitutively or after chemotherapy. This might enable tumor cells to kill antitumor lymphocytes and to suppress antitumor immune responses, a mechanism called tumor counter attack.133,210,211 However, rejection of tumors expressing CD95L by chemoattracted neutrophils has also been observed.212,213 DRUG-INDUCED APOPTOSIS IN TUMOR CELLS

Most existing cytotoxic drugs interfere with the machinery of DNA synthesis and cell division. Multiple mechanisms of action are known for cytotoxic drugs, including DNA intercalation (eg, doxorubicin), inhibition of topoisomerase II (eg, etoposide), inhibition of topoisomerase I (eg, topotecan), DNA cross-linking (eg, cisplatin), and DNA alkylation (eg, cyclophosphamide). Although the primary intracellular sites of action of cytotoxic drugs are diverse, there are some effects common to all.214 Induction of apoptosis is one of

the main mechanisms by which cytotoxic drugs eliminate cancer cells.215 Thus, the success of a chemotherapeutic regimen depends on intact apoptotic signaling. The apoptotic pathways induced by cytotoxic drugs depend on the concentrations used for chemotherapy since different concentrations can trigger qualitatively distinct response mechanisms. Although most of the cytotoxic drugs mediate cell death by ultimately activating caspases,216 the exact molecular mechanisms linking the primary drug targets to the termination phase of apoptosis remain unclear. However, several apoptotic pathways have been described as being activated upon drug treatment so far. p53 and Drug Treatment Activation of the tumor-suppressor gene TP53 is frequently involved in drug-induced and irradiation-induced apoptosis (Fig 7).217,218 This involvement is why alterations in p53 pathways frequently influence sensitivity of tumor cells towards therapy-induced apoptosis. p53 is induced after DNA damage by phosphorylation through ATM. Alternatively, inactivation of Mdm-2 by phosphorylation or sequestration can activate p53. Post-translational modification, including phosphorylation, sumoylation, and acetylation of p53 after genotoxic stress, have also been shown to enhance its transcriptional activity. p53 can activate transcription of pro-apoptotic proteins involved in mitochondrial signaling events, including Bax, NOXA, PUMA and p53AIP1,219-221 and death receptors such as CD95, TRAIL-R1, and TRAIL-R2.54,222,223 The specific target genes probably differ widely among different tumor types.224

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In addition, p53 exerts pro-apoptotic effects independent of transcription, such as relocalization of death receptors to the cell surface.225 Mitochondrial Activation Upon Drug Treatment Several observations have shown a major role of mitochondria in the induction of apoptosis by cytotoxic drugs. Many cytotoxic drugs induce apoptosis via a direct effect on mitochondria without primarily involving death receptors.154,226 In vitro, various cytotoxic drugs cause MMP. MMP has also been demonstrated in cell-free systems, such as after treatment with paclitaxel and etoposide.227,228 In addition, cytotoxic drugs are capable of inducing pro-apoptotic proteins targeting mitochondria such as Bax and Bad. Triggering of Death Receptors Upon Drug Treatment One of the key apoptotic events leading to cancer cell death after drug treatment is the activation of death receptors such as CD95.229 In liver tumor cells, cytotoxic drugs such as the nucleotide analogue 5-fluorouracil (5-FU) have been described to induce CD95 transcriptionally in a p53dependent fashion.54,230,231 In addition, cytotoxic drugs engage the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) pathway in liver tumor cells to upregulate expression of CD95L (Fig 8).232 SAPK, a subclass of mitogenactivated protein kinases, regulates the activity of AP-1 transcription factors. AP-1 transcriptionally activates pro-apoptotic genes such as CD95L or TNF.232,233 By interfering with the interaction of CD95 and CD95L, cytotoxic drug-induced apoptosis could be blocked in leukemia cells in vitro.229,234,235 Furthermore, activation of CD95 mediates thymineless stress-induced apoptosis, triggered by 5-FU and leucovorin, such as in colon carcinoma cells in vitro236 or in thymocytes in vivo.237 However, in some systems, inhibition of CD95-mediated apoptosis using blocking antibodies against CD95238 or CD95L,239 or by the expression of DN-FADD,240 does not protect tumor cells from cytotoxic drug-induced apoptosis. A cooperative effect of cytotoxic drugs with other death receptors such as TRAIL-R1 and -R2, TNF-R1, DR3, and DR6 has also been described. An important mechanism of cytotoxic drug action is the upregulation of TRAIL-R2, thus sensitizing tumor cells for TRAIL-induced apoptosis.241,242

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Several other pathways have been described to be activated upon treatment with cytotoxic drugs. Clearly, single predominant effector pathways cannot be identified. The engaged pathways depend on different factors such as the type of drug and its dose, pretreatment, tumor type, tumor environment, and many other factors. MECHANISMS OF DRUG RESISTANCE

Therapy resistance of tumor cells is a common clinical problem in human cancer. Chemotherapy and irradiation kill tumor cells mainly by induction of apoptosis.229,243-245 Thus, resistance to apoptosis is a principal mechanism through which tumor cells are enabled to resist cancer therapy. However, drug resistance can also occur independent of defects in apoptosis pathways. In general, drug resistance can occur on at least five different levels (Fig 9). Absence of Drug-Target Interaction Chemotherapeutic drugs might be prevented from interacting with their targets mainly due to molecular transporters. Molecular transporters are capable of actively expelling cytotoxic drugs from tumor cells.246 The two main transporters found to confer drug resistance to tumor cells are the MDR1 gene products P-glycoprotein and MRP (multidrug resistance-associated protein).247 They both belong to the ATP-binding cassette superfamily of transporter proteins.248 P-glycoprotein acts as an ATP-dependent drug efflux pump, and its overexpression is associated with a poor prognosis for patients with cancer. Detectable levels of P-glycoproteins have been found in all types of hematologic malignancies.249 P-glycoproteins also protect cells from other apoptotic stimuli such as death receptor ligands.250 Absence of Drug Activation or Accumulation Drug resistance may be caused by the absence of drug activation or accumulation. Methotrexate (MTX), for example, has been used for the treatment of acute lymphocytic leukemia (ALL) and colorectal cancer. Once inside the cell, MTX is modified by the sequential addition of glutamate residues catalyzed by the enzyme folylpolyglutamate synthase (FPGS). This modification step leads to the accumulation of MTX in tumor cells. However, in some types of ALL, the accumulation

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Fig 8. Activation of CD95 upon cytotoxic drug treatment. Cytotoxic drug treatment of tumor cells leads to induction of CD95L expression following activation of the SAPK (stress-activated protein kinase)/JNK (Jun N-terminal kinase) pathway. In addition, CD95 (APO-1/Fas) is induced in a p53-dependent fashion. Concomitant upregulation of CD95 and CD95L enables the cell to induce apoptosis in neighbouring cells or condemns it to die by suicide. MEKK-1, mitogen-activated protein kinase kinase kinase 1.

of MTX is impaired, thus rendering these cells less sensitive to MTX treatment.251 Unavailability of Drug Targets Tumor cells can also lack appropriate targets for cytotoxic drugs. For example, decreased expression of uridine-cytidine monophosphate kinase (UMPK) in colon cancer cells has been associated with tumor cell resistance towards treatment with 5-FU.252 In addition, mutant forms of dihydrofolate reductase (DHFR) with reduced affinity for MTX have been identified in tumor cells.253,254

Overexpression of Drug Targets Overexpression of a drug target, such as thymidylate synthase (TS), can also contribute to resistance. TS is a target for cytotoxic drugs such as 5-FU, oral 5-FU prodrugs (eg, capecitabine), and other folate-based drugs (eg, raltitrexed). Overexpression or induction of TS is linked to resistance to TS-targeted cytotoxic drugs.255,256 DNA-Repair Mechanisms There are three major ways to repair damaged DNA after treatment with cytotoxic drugs: (1) nucleotide excision repair (NER), (2) base exci-

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Fig 9. Levels of drug resistance. Drug resistance can be mediated on at least five different levels. 5-FU, 5-fluorouracil.

sion, and (3) homologous double-stranded repair. Alterations in these pathways do not only play a role in tumorigenesis, but they also influence drug sensitivity of tumor cells. For example, the alkylating agent melphalan causes interstrand crosslinks and thereby activates the homologous double-stranded repair pathway. An enhanced removal of interstrand cross-links correlates with melphalan resistance of tumor cells.257,258 Defects in Apoptotic Pathways After drug-target interaction, several apoptosis pathways are activated. However, cytotoxic drugs, as well as irradiation therapy, often fail to activate the apoptotic program in tumor cells. Some tumors, such as small cell lung cancer, respond dramatically to chemotherapy. However, most patients with this cancer suffer from tumor relapses, and relapsing tumors often appear to be more resistant to chemotherapy than the primary tumor. The understanding of the mechanisms leading to apoptosis resistance of tumor cells is of general interest for cancer research. Anti-apoptotic mechanisms that contribute to tumorigenesis also raise the threshold for therapyinduced apoptosis in tumor cells. Therefore, tumorigenesis and drug resistance should be discussed in close conjunction. In the following paragraph, exemplary studies demonstrating the impact of apoptosis signaling pathways on therapy resistance are addressed.

The loss of p53 function is a central event in tumorigenesis. Moreover, the functionality of p53 plays a pivotal role for the fate of a tumor cell after treatment with cytotoxic drugs. Cancers that retain wt p53 expression are more likely to respond to chemotherapy than other tumor types. Without functional p53 or functional downstream targets, tumor cells are capable of evading apoptosis despite DNA damage. Lymphomas from mice deficient in p53 expression are highly invasive and markedly resistant to chemotherapy in vitro and in vivo.119 In cancers such as leukemia, testicular cancer, and Wilm’s tumors, loss of p53 expression results in chemoresistance and often occurs when the cancer is in relapse.259,260 Furthermore, specific mutations in TP53 are associated with resistance to doxorubcin treatment and early relapse in patient with breast carcinoma.261 The p53-inhibitor iASPP confers resistance to cytotoxic drugs like cisplatin.124 In addition, in the absence of functional p53, cytotoxic drug-induced upregulation of death receptors such as CD95 and TRAIL-R2 is impaired.54 However, defects in signaling pathways downstream or upstream of p53 may explain the observation that many types of normal and cancerous cells with wt p53 do not undergo apoptosis despite genotoxic stress.259,260 Furthermore, the PI3K/Akt-pathway plays a potential role in determining multidrug resistance in various cancers, such as hematologic malignancies. For example, in cell lines of acute leukemia, inhibition

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of this pathway significantly enhanced the sensitivity to cytotoxic drugs.262 Several mechanisms of drug resistance directly interfere with mitochondrial activation. Anti-apoptotic members of the Bcl-2 family have been shown to prevent apoptosis induced by anticancer drugs in vitro.45,263 For example, upregulation of Bcl-2 is involved in chemotherapy resistance of small cell lung cancer cell lines.164 Overexpression of Mcl-1 also renders tumor cell lines resistant to cytotoxic drugs.264 It is of interest that Mcl-1 expression is increased in leukemia cells at the time of relapse after initial chemotherapy. This might point to a selection of leukemia cells expressing high levels of this protein during chemotherapy.265 Even mitochondrial genes have been reported to be involved in drug resistance. In several tumor cell lines, mutations in mitochondrial genes reduce the efficiency of cytotoxic drugs. This point applies in particular to those agents that elicit the production of ROS by the respiratory chain.265 A reduction of Bax expression is associated with lower response rates to cytotoxic drug treatment; for example, in patients with breast carcinoma.266 In retrospective trials, patients whose tumors expressed higher levels of the IAP family member survivin, had reduced sensitivity to therapy and decreased survival.267,268 Downregulation or loss of caspases also contributes to drug resistance. This has been shown for doxorubicin treatment of neuroblastoma cells, in which caspase-8 is often inactivated.192 The loss of Apaf-1 contributes to chemoresistance of melanoma cells.188 The activation of the tyrosine kinase receptor EGFR has been associated with resistance of breast cancer cells to cytotoxic drug treatment.269 A recent report has provided evidence for crosstalk of Akt and SAPK signaling, leading to inhibition of apoptosis upon anticancer treatment. Akt inactivates ASK1, which normally transduces stress signals to the JNK pathway.270 Tumors with constitutive NF-␬B activity are highly resistant to cytotoxic drugs and irradiation. Moreover, inhibition of NF-␬B dramatically increases the sensitivity to such treatment.197 One reason for the sensitizing effect of NF-␬B inhibition is the downregulation of anti-apoptotic proteins. Another mechanism by which NF-␬B confers resistance to drug treatment is the induction of Mdr-1 expression in tumor cells.271 The extracellular matrix might also play a role in the drug

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resistance of in vivo tumor cells. Adhesion of small cell lung cancer cells to the extracellular matrix leads to activation of integrin signaling and confers resistance to chemotherapy.272 THERAPEUTIC APPROACHES

Most of the existant cytotoxic drugs exert antimitotic effects and do not target specific apoptotic signaling pathways. In addition, they induce significant toxicity, particularly in tissues with high rates of proliferation, such as the gut epithelium and the hematopoietic system. Insights into the molecular events leading to the apoptosis resistance of tumor cells now offer a variety of new strategies for the treatment of patients with cancer. Apoptosis-modulating agents to treat cancer have shown efficacy in preclinical animal models and are currently being tested in clinical trials. Targeting Death Receptors Over the past few decades, much effort has been devoted to the search for factors that can specifically induce apoptotic death of cancer cells. Death receptor ligands harbor some potential as cancer therapeutic agents, because they trigger apoptosis in many types of tumor cells by activating death receptors. Death receptors are interesting candidates for anticancer therapy, because their activation might circumvent certain forms of resistance to apoptosis that frequently occur in tumor cells, such as the inhibitory potential of anti-apoptotic Bcl-2 family members.34,273,274 Provided that the death receptors are expressed on the surface of tumor cells, death-receptor–induced apoptosis is not dependent on functional alleles of the transcription factor p53, representing the most common mutation in cancer.275 In tumors with wt p53, death receptors like TRAIL-R2 can be upregulated by chemotherapy or irradiation.54,55,276 Thus, combination therapies with death receptor ligands and chemotherapy or irradiation might be promising approaches.277,278 The death ligand TNF-␣ was originally found to induce hemorrhagic tumor necrosis and tumor regression in mouse tumors in vivo and to kill cancer cells in vitro.279 However, the therapeutic application of TNF-␣ is hampered by its high systemic toxicity such as ischemic and hemorrhagic lesions in various tissues.280 Recently, tumor-targeted TNF-␣ gene delivery in murine tumor models without systemic TNF-related toxicity was described.281 Agonistic antibodies against

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the death receptor CD95 (APO-1/Fas) have also been proposed as anticancer agents,30 but intraperitoneal administration of an agonistic CD95 antibody had lethal effects in mice due to fulminant liver failure.282 Thus, the high sensitivity of hepatocytes towards CD95-mediated apoptosis forms an obstacle for the development of CD95activating agents in cancer therapy. The death ligand TRAIL (APO-2L) has attracted great attention as a promising anticancer reagent and is soon to enter clinical trials. This is mainly due to the fact that TRAIL almost specifically targets transformed cells. Recombinant, soluble TRAIL derivatives induce apoptosis in a large panel of tumor cell lines but not, or only to a minor extent, in nontransformed cells.283 The resistance of nonmalignant cells to the cytotoxic potential of TRAIL may partially be mediated by a high level of surface expression of TRAIL decoy receptors that compete for ligand binding and reduce the activation of the pro-apoptotic TRAIL receptors.284 The potential usefulness of TRAIL would be its ability to even kill transformed cells with mutations of TP53. Preclinical studies with mice and monkeys using TRAIL as an anticancer agent have been promising,283,285 as administration of soluble, recombinant TRAIL induced significant tumor regression without severe side effects. However, not all cancer cells are sensitive to TRAIL treatment. DR5 mutations have been described in some cancers of the neck and lung and in Hodgkin’s lymphoma.275 Another frequent cause for TRAIL resistance of tumor cells is the expression of c-FLIP.56,286 By using RNA interference to deplete cells of c-FLIP, tumor cells could be sensitized to induction of apoptosis by TRAIL in vitro.287 Moreover, Bax mutations contribute to TRAIL-resistance in colon cancer cells.288 Many tumor cell lines that are only modestly sensitive towards TRAIL-induced apoptosis become highly sensitive when cotreated with cytotoxic drugs.289 In tumor xenograft experiments, TRAIL acts synergistically with cytotoxic drugs against various tumor cell types. Thus, combination therapies applying TRAIL and cytotoxic drugs in parallel offer promise for cancer treatment. Combination therapies have been found to be effective in tumors with wt p53.241 An additional way of breaking the resistance of tumor cells towards TRAIL-induced apoptosis is the use of proteasome inhibitors that can efficiently interfere with NF-␬B activation.290

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However, in a study with human keratinocytes, proteasome inhibition also led to sensitization of primary, nontransformed cells to TRAIL. Thus, a combination therapy of TRAIL and proteasome inhibitors might also lead to severe side effects.291 Targeting Mitochondria Decreased sensitivity of mitochondria towards apoptotic stimuli contributes to tumor cell survival. Thus, one main goal of cancer treatment is to resensitize mitochondria in tumor cells. Bcl-2 is frequently overexpressed in tumor cells of different tissue origin and confers resistance to anticancer treatment. Inhibition of Bcl-2 has been attempted through antisense technique and ribozyme constructs and has proven to sensitize cancer cells to chemotherapy-induced apoptosis.292 Another approach for interfering with mitochondrial apoptosis pathways in tumor cells is to disrupt the interaction between pro- and anti-apoptotic Bcl-2 family members. Several small molecules, so called BH3-mimetics, mimic the BH3 domain (eg, Bax), leading to enhanced activation of caspase-9. The molecule Ha-14-1 binds to the pocket of Bcl-2 and induces apoptosis.293 Other molecules disrupt the interaction between Bcl-xL and Bak by occupying the BH3-binding pocket of Bcl-xL. Peripheral benzodiazepine receptor (PBR) ligands represent another group of mitochondria-permeabilizing agents, which can induce apoptosis independent of Bcl-2 expression levels.154,294 PBRs reside in the outer membrane of mitochondria and are frequently overexpressed in tumor cells, contributing to their resistance towards apoptosis.295 Since mitochondrial hyperpolarization is a shared feature of many tumor cell lines, other molecules that have a high affinity to hyperpolarized mitochondria and exert mitochondriotoxic effects have been developed.296 As deficiency in Apaf-1 is a frequent cause of apoptosis resistance, upregulation of Apaf-1 protein levels by either gene transfer or demethylation treatment have been shown to increase the sensitivity of tumor cells to apoptosis.297 IAPs such as XIAP are involved in the resistance of tumor cells towards apoptosis. Because XIAP blocks apoptosis in the effector phase downstream of mitochondria, where many signaling pathways converge, it may be a promising target for new treatment strategies. Smac/DIABLO is released from mitochondria in response to apoptotic stimuli and promotes apoptosis by antagonizing

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IAPs.42,69,298 Thus, cell-permeable Smac agonists, which target the cytoplasmic compartment, may represent a new group of anticancer agents, especially in combination with other apoptosis-inducing agents. Peptides derived from the sequence of Smac have proven to sensitize various tumor cells in vitro. Moreover, in an orthotopic model of glioma in mice, a synthetic Smac peptide collaborated with TRAIL in effectively inducing tumor regression.299 Survivin may also represent an attractive target for cancer therapy.71 Ablation of survivin by antisense technique or ribozyme transfection effectively induced cell death in various human tumor cell lines in vitro and in in vivo murine tumor models.300-302 In addition, due to its differential expression in tumors versus normal tissues, it may be an attractive target for immunotherapy.303 Targeting the p53 Pathway Chemotherapeutic drugs and irradiation therapy usually require function of the TP53 tumorsuppressor gene for antitumor activity. Therefore, the restoration of the p53 pathway may be an approach for therapy of tumors with defects in this pathway. The first approach is the reintroduction of functional p53 into tumor cells, as it has been shown that reintroduction of p53 alone already induces apoptosis in different tumor cells.116 Approaches to reintroduce functional p53 include the use of gene-transfer vectors of retroviral and adenoviral origin.304 Adenoviral or retroviral gene transfer of wt p53 has entered clinical trials.305 Tumor disease stabilization was observed in a limited number of patients when p53 was applied alone or in combination with chemotherapy. The use of adenoviruses has also been considered to eliminate cancer cells in the bone marrow before autologous transplantation, because cancer cells in transplanted marrow are thought to contribute to tumor relapse after chemotherapy.306 Another approach for targeting p53-pathways is the rescue of mutant p53 function.112 Recent studies provide evidence that small molecules that influence p53 conformation can restore p53 activity in some cases and induce apoptosis in tumor cells.307 This approach is based on the fact that many p53 mutations cause alterations in the DNA-binding site of p53, which may be reconstituted by small molecules. Another way to treat tumors with p53 mutations is by depleting mutant p53, such as by

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geldanamycin.308 Geldanamycin exerts its effects by inhibiting the molecular chaperone hsp90, which contributes to the stability of mutant p53 and other signaling proteins.309 Some tumors that express wt p53 do not undergo apoptosis despite DNA damaging treatment. In these tumors, inhibition of negative regulators of p53 such as Mdm-2 and iASPP may be promising.124 Selective proteasome inhibitors such as PS-341,310 which are currently in clinical trial, cause stabilization and accumulation of p53. It is still unclear, however, if the apoptosis-inducing potency of proteasome inhibitors might not be due to the accumulation of other pro-apoptotic proteins such as Bax.311 Another pro-apoptotic gene, E1A, has entered clinical trial.312-314 E1A is an adenoviral gene with tumor suppressive effects in certain types of cancer. It sensitizes tumor cells to chemotherapy and irradiation.315-317 So far, E1A has been delivered into tumor cells mainly by adenoviral vectors or cationic liposomes. Indeed, several approaches have been performed in vitro to optimise gene delivery into tumor cells.318 Interference With Survival Signaling Interference with survival signaling may be another approach to overcome apoptosis resistance of tumor cells. An example is imatinib, which leads to inhibition of the kinase BCR-ABL and has improved the treatment of chronic myelogenous leukemia (CML).319,320 BCR-ABL is a constitutively active cytoplasmic tyrosine kinase present in virtually all patients with CML and in a minority of patients with ALL. The PI3K/Akt survival-signaling pathway represents another target for cancer treatment. In this context, inhibition of PTEN, which specifically antagonizes the action of PI3K and is upregulated in various human cancers, may provide an adjuvant treatment for several malignancies. In pancreatic cancer cells PTEN has recently been demonstrated to be upregulated by activation of a member of the peroxisome proliferator-activated receptor family, PPAR.321 Thus, PPAR ligands such as rosiglitazone, which is used for the treatment of diabetes type II, may represent a new therapeutic agent for pancreatic cancer. An alternative strategy would be to restore expression of PTEN in tumor cells by adenovirus-mediated gene transfer.214 Further potential targets in the PI3K/Akt pathway include the upstream tyrosine kinase receptors, activated

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Ras, PI3K, and the downstream targets Akt and PDK1. Inhibition of PI3K induces apoptosis in many tumor cell lines, regardless of their PTEN status. Interference with Akt expression is another possibility to eliminate survival signals. In support of this approach, dominant-negative Akt has been shown to reverse Ras-dependent survival signaling.81 Moreover, antisense Akt reduces growth of cell lines containing BCR-ABL.322 Several agents have been developed against different components of Ras-dependent pathways. Direct therapeutic intervention in Ras-activation is being attempted by the use of farnesyl transferase inhibitors, which prevent membrane localization and thereby activation of Ras.323,324 Other attempts include the use of antisense oligonucleotides against Ras and Raf.325 In addition, several inhibitors of kinases involved in Ras signaling pathways have entered clinical trial.326 The Ras pathway as well as other oncogenic pathways are often activated by aberrant activation of upstream receptor tyrosine kinases (RTKs). RTKs are a class of cell surface receptors with an intrinsic tyrosine kinase activity, including EGFR, and its close relative ERBB2, IGF-1R, platelet-derived growth factor receptor (PDGFR), and others. They have all been identified as potential therapeutic targets in cancer therapy.327 One example for targeting EGFR, is trastuzumab, a monoclonal antibody that binds HER2 (human EGFR-related gene 2)/neu on the surface of tumor cells and is used for the treatment of breast cancer.328 Trastuzumab the first genome research-based therapeutic that targets kinases and has been approved for routine clinical use. In the meantime, several small-molecule tyrosine kinase inhibitors targeting EGFR are being tested in clinical trial.329,330,331. Clinical trials employing inhibitors targeting other RTKs, such as PDGFR,331 are also ongoing. Activation of NF-␬B confers resistance of tumor cells to cancer treatment. Therefore, inhibition of NF-␬B may be an approach for cancer teatment. However, it is difficult to develop inhibitors that exert their effects predominantly in cancer cells, since NF-␬B is widespread and involved in a plethora of cellular processes. Present studies are focused on cancer treatment with systemic inhibitors of NF-␬B in combination with chemotherapy or the death-receptor ligand TRAIL. Since activation of NF-␬B interferes with TRAIL-induced apoptosis, NF-␬B–suppressing agents may sensitize

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tumor cells to TRAIL-induced apoptosis. A recent study has shown that the use of a semisynthetic analog of vitamin E leads to inhibition of NF-␬B and sensitizes tumor cells to TRAIL-induced killing.332 Moreover, a new class of retinoid-related agents was described to inhibit the activation of the NF-␬B regulator IKK by TNF-␣.333 Another way of inhibiting NF-␬B would be the application of proteasome inhibitors (see below). OTHER APPROACHES

Inhibitors of DNA Methylation Cytosine methylation is an epigenetic modification of DNA involved in the control of gene expression. Methylation of CpG islands of tumor suppressor genes and elevated levels of the DNA(cytosine-5)-methyltransferases play an important role in the inactivation of tumor-suppressor genes.334 Inhibition of DNA methylation sensitizes cancer cells to cytotoxic drug-induced apoptosis in vitro.188,226 Inhibition of DNA methyl transferases by antisense oligonucleotides has entered phase II clinical trial.335 Inhibition of Cyclin-Dependent Kinases Inhibitors of cyclin-dependent kinases (CDKs) have recently emerged as novel anticancer drugs, because they are able to disrupt several proliferation and survival pathways.336 For example, flavopiridol, a flavone inhibitor of different CDKs, has been shown to induce apoptosis in tumor cell lines and is currently in clinical trial.337 Histone Deacetylase Inhibition Histone deacetylase inhibitors such as suberoylanilide hydroxamic acid and Trichostatin-A represent a new class of anticancer agents exhibiting pro-apoptotic activities in vivo with minor side effects in preclinlical studies.338 GENERAL APPROACHES

Proteasome Inhibition The ubiquitin-proteasome system is an important regulator of apoptosis. Specific proteasome inhibitors are currently under intense investigation as anticancer agents.339 They have proven to induce apoptosis of tumor cells both in vivo and in vitro.340 The mechanism by which proteasome inhibitors exert their pro-apoptotic effects are still largely unknown. Proposed major mechanisms are

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inhibition of NF-␬B, increase of p53 activity,341 and accumulation of pro-apoptotic Bcl-2 family members.311 Importantly, proteasome inhibitors can reduce resistance of drug-resistant tumor cells to apoptosis.342 Several studies in mice showed potential application of proteasome inhibitors in cancer therapy.340 Proteasome inhibitors alone or in combination with other anticancer agents are being studied in both phase II and III trials in myeloma.343 Data concerning phase I and phase II trials of combinations with other anticancer agents will be forthcoming over the next several years. Application of Antibodies Antibody-based therapies are increasingly applied in oncology.327 Monoclonal antibodies (MAbs) have been applied either alone or in combination with chemotherapy or irradiation in several clinical trials. Antibodies may exert their antitumor effects in at least three different ways: (1) via induction of apoptosis (eg, agonistic, monoclonal antibodies specific for death receptors)30; (2) via competition with receptor ligands (eg, antibodies that specifically bind to growth receptors without activating them)344; and (3) by preventing the expression of oncogenic or anti-apoptotic genes. One of several obstacles of antibody treatment is the immunogenicity of xenogeneic antibodies and, if humanized immunoglobulins are used, the possible production of anti-idiotype antibodies.327 Structural modifications of antibodies may further improve their antitumor efficacy. Examples are the application of bispecific antibodies or small antibody fragments. For the treatment of lymphomas, MAbs directed against CD20 (such as rituximab, which is the first Mab approved by the Food and Drug Administration for the treatment of human malignancies345) have been coupled with radiosiotopes as a means of transporting the source of radiation specifically to the targeted cells.346 Antisense Therapy for Cancer Antisense oligonucleotides are a new class of highly specific agents targeting apoptosis pathways in cancer cells. They are short, synthetic stretches of DNA and hybridize with complementary mRNA strands. Thus, mRNA strands are prevented from being translated into a protein. Several transcripts that are overexpressed in tumor

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cells and contribute to apoptosis resistance are possible targets for antisense therapy, such as antiapoptotic members of the Bcl-2 family (including Bcl-2 itself) and members of the IAP family such as survivin. So far, phosphorothioate oligonucleotides, which are stable against cellular nucleasemediated degradation, are the gold standard for antisense therapy and have been shown to be relatively nontoxic.347 Antisense therapy for Bcl-2 has entered phase III trial for the treatment of melanoma and myeloma.347 In metastatic melanoma and prostate cancer, a Bcl-2 antisense oligonucleotide (Genasense, G3139, Genta, Berkley Heights, NJ), has been studied in phase I/II trials.347,348 RNA Interference Another way of blocking the expression of antiapoptotic genes in tumor cells is the use of the RNA interference (RNAi) technique. Here, double-stranded RNA (dsRNA) induces sequencespecific, post-transcriptional gene silencing.349 RNAi is now being widely used to downmodulate the expression of genes in cell culture.350 Recently, RNAi, based on retroviral vectors, was used to inhibit expression of an oncogenic K-RAS(V12) allele in human tumor cells. This might be the beginning of clinical trials of RNAi-based therapies.351 In Vivo Studies of Apoptosis Induction It would be of interest to study the induction of apoptosis in cancer cells in vivo upon treatment with anticancer drugs. Such studies would help to develop new effective drugs for cancer treatment and monitor the efficacy of the treatment. However, so far studies are limited mainly to histologic evaluation, which only allows rough simulation of the amount of late-stage apoptotic cells in tissues. In vivo monitoring of apoptosis is hampered by the fact that apoptotic cells are rapidly cleared by phagocytosis.352 However, there may be promising attempts to monitor apoptosis induction in vivo in a real-time fashion, and these attempts might help to improve treatment strategies targeting apoptosis pathways.352 Gene Expression Profiling Until several years ago, gene expression analysis was confined to a limited set of genes. This situation has undergone fundamental change with the

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development of microarray hybridization technologies for parallel analysis of the whole genome. In oncology, gene expression studies already have had a significant effect on tumor diagnosis, identification of drug targets, and elucidation of mechanisms important for tumorigenesis.353,354 So far, gene expression profiling by cDNA microarrays has yielded hundreds of candidate genes that are differentially expressed in cancers.355,356 In addition, cDNA microarrays have revealed differences in genomic and transcriptional profiles between different tumor types and have been used to identify new tumor subtypes.357,358 Furthermore, microarray analysis of individual tumor samples may influence treatment strategies in cancer patients. For example, gene expression profiling of breast cancer samples has been used to establish a set of genes which is predictive for poor prognosis. This may help to select patients who would benefit from a more intensive drug therapy.359 In conclusion, several agents that target defective apoptotic signaling in cancer cells are tested in clinical trials. They refer to at least four different mechanisms of apoptosis resistance: (1) Bcl-2 antisense oligonucleotides are currently being tested in phase III trials for chemorefractory malignancies; (2) gene therapies with p53 and E1A, which trigger expression of pro-apoptotic genes, are being used to sensitize tumor cells to irradiation therapy; (3) proteasome-inhibiting drugs that block NF-␬B activation; and (4) specific kinase inhibitors that interfere with survival signaling. Significant progress has been made in recent years toward understanding the molecular basis of apoptosis regulation and its dysregulation in cancer. However, before strategies can be developed that restore increased sensitivity of tumor cells towards apoptosis, a number of general problems have to be considered. Tumors in a progressive stage are highly heterogenous despite their clonal origin. It is likely that different cells within a tumor have acquired different mechanisms of apoptosis resistance. Moreover, even in a single tumor cell, resistance may occur in a redundant manner. One possibility to address this problem would be the combination of different treatment strategies. Another severe general problem is that therapeutic approaches targeting apoptosis pathways have not yet proven to be tumor specific. The ideal cancer therapy would exclusively target transformed cells without harming normal cells.

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Keeping in mind that most of the cells in the human body contain the entire program for the apoptosis machinery, this problem will remain a major challenge for years to come. CONCLUSIONS

Conventional treatment strategies of chemotherapy and irradiation have limited effectiveness and often fail to cure patients with advanced cancer. This failure is at least partly caused by the resistance of many cancers to apoptosis induction. The elucidation of molecular mechanisms conferring this resistance in recent years has provided insight into the possibilities to bypass these mechanisms, thus potentially allowing the elimination of resistant cancer cells. Research being currently conducted in this exciting field is likely to be beneficial for the future treatment of cancer. ACKNOWLEDGMENT We thank Cornelius Fritsch, Andreas Krueger, Ru¨ diger Arnold, and Christian R. Frey for critical reading of the manuscript.

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