Mutations in apoptosis genes: a pathogenetic factor for human disease

Mutation Research 488 (2001) 211–231

Review

Mutations in apoptosis genes: a pathogenetic factor for human disease Leonhard Müllauer∗ , Petra Gruber, David Sebinger, Judith Buch, Sabine Wohlfart, Andreas Chott Institute of Clinical Pathology, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria Received 18 September 2000; received in revised form 22 February 2001; accepted 22 February 2001

Abstract Cell death by apoptosis is exerted by the coordinated action of many different gene products. Mutations in some of them, acting at different levels in the apoptosis process, have been identified as cause or contributing factor for human diseases. Defects in the transmembrane tumor necrosis factor receptor 1 (TNF-R1) lead to the development of familial periodic fever syndromes. Mutations in the homologous receptor Fas (also named CD95; Apo-1) are observed in malignant lymphomas, solid tumors and the autoimmune lymphoproliferative syndrome type I (ALPS I). A mutation in the ligand for Fas (Fas ligand; CD95 ligand, Apo-1 ligand), which induces apoptosis upon binding to Fas, was described in a patient with systemic lupus erythematodes and lymphadenopathy. Perforin, an other cytotoxic protein employed by T- and NK-cells for target cell killing, is mutated in chromosome 10 linked cases of familial hemophagocytic lymphohistiocytosis. Caspase 10, a representative of the caspase family of proteases, which plays a central role in the execution of apoptosis, is defect in autoimmune lymphoproliferative syndrome type II (ALPS II). The intracellular pro-apoptotic molecule bcl-10 is frequently mutated in mucosa-associated lymphoid tissue (MALT) lymphomas and various non-hematologic malignancies. The p53, an executioner of DNA damage triggered apoptosis, and Bax, a pro-apoptotic molecule with the ability to perturb mitochondrial membrane integrity, are frequently mutated in malignant neoplasms. Anti-apoptotic proteins like bcl-2, cellular-inhibitor of apoptosis protein 2 (c-IAP2) and neuronal apoptosis inhibitory protein 1 (NAIP1) are often altered in follicular lymphomas, MALT lymphomas and spinal muscular atrophy (SMA), respectively. This article reviews the current knowledge on mutations of apoptosis genes involved in the pathogenesis of human diseases and summarises the gradual transformation of discoveries in apoptosis research into benefits for the clinical management of diseases. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis inducer; Apoptosis inhibitor; Autoimmunity; Inflammation; Lymphoma; Cancer

1. Introduction Apoptosis is a distinct form of cell death that proceeds along a genetically determined execution programme. It exhibits a characteristic morphology ∗ Corresponding author. Tel.: +43-1-40400-3651; fax: +43-1-405-3402. E-mail address: [email protected] (L. Müllauer).

[1] and features unique biochemical alterations [2]. An increasing number of genes involved in the execution of the apoptosis programme is identified and concepts of different, although interacting, apoptosis signalling pathways are delineated [3–5] (Fig. 1). One major apoptosis pathway involves cell surface ‘death receptors’ (DR) that transmit an apoptosis signal on binding of a specific ‘death ligand’ [3]. The largest known family of DRs is represented

1383-5742/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 ( 0 1 ) 0 0 0 5 7 - 6

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Fig. 1. Simplified scheme of major apoptosis pathways. DD: death domain, DED: death effector domain.

by tumor necrosis factor receptors (TNF-Rs) [6]. The best characterised members are TNF-R1 (also called p55, CD120a), TNF-R2 (p75, CD120b), Fas (CD95, Apo-1), death receptor 3 (DR3), death receptor 4 (DR4, TRAIL-R1) and death receptor 5 (DR5, TRAIL-R2) [3] (Fig. 1). The ligands that activate these receptors are structurally related molecules with homologies to tumor necrosis factor ␣ (TNF␣) [3,7–10]. Upon ligand binding an intracellular ‘death domain’ of the receptor interacts with a homologous domain in an adaptor protein, which recruits specific proteases, the so-called caspases [11–13] (Fig. 1). Caspases (for ‘cysteine aspartase’) are cysteine dependent proteases that exert a limited proteolyses by cleavage of their substrate after specific aspartate residues. They reside as inactive pro-forms within the cell and become activated by autocleavage when recruited to a DR signaling complex. Activated upstream caspases subsequently initiate a cascade of downstream effector caspases which cleave a plethora of cellular proteins and thereby ultimately cause cell death [11–13].

A second major apoptosis pathway involves mitochondria [14–16]. A key molecule in mitochondrial cell death is cytochrome c. When released from mitochondria in response to cell damage it binds to the cytoplasmic adaptor molecule Apaf-1 [14–16]. The Apaf-1 then recruits pro-caspase 9, which becomes activated by autoprocessing and triggers a cascade of downstream caspase reactions (Fig. 1). Members of the bcl-2 family are involved in the regulation of mitochondrial cell death [17–19]. Anti-apoptotic members like bcl-2 and bcl-xL inhibit cytochrome c release from mitochondria [20,21]. Pro-apoptotic members like Bax may act by forming pore complexes in the outer mitochondrial membrane [22]. At least a third major pathway of apoptosis induction seems to exist that does not primarily involve DRs or mitochondria. This pathway is represented by the nuclear protein p53 [23–26]. The p53 is activated in response to DNA damage. It blocks cells with damaged DNA in the G1 and G2 phase of the cell cycle [27]. If the DNA damage is severe, and dependent on

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Table 1 Apoptosis genes mutated in human diseases Gene

Affected disease

Tumor necrosis factor receptor 1 (TNF-R1) Fas (CD95; Apo-1) Fas ligand Perforin Caspase 10 bcl-10 p53 Bax bcl-2 c-IAP2 NAIP1

Familial periodic fever syndrome Autoimmune lymphoproliferative syndrome type I (ALPS I); malignant lymphoma; bladder cancer Systemic lupus erythematodes (only one case identified) Familial hemophagocytic lymphohistiocytosis (FHL) Autoimmune lymphoproliferative syndrome type II (ALPS II) Non-Hodgkin’s lymphoma Various malignant neoplasms Colon cancer; hematopoetic malignancies Non-Hodgkin’s lymphoma Low-grade MALT lymphoma Spinal muscular atrophy

cell type and oncogene composition of a cell, p53 initiates apoptosis by mechanisms that partially rely on the transcription of apoptosis executionary genes like Bax [18] and genes whose products generate reactive oxygen species [28]. A deregulation of apoptosis is implicated in the pathogenesis of various human diseases [29]. In malignant neoplasms, the balance of apoptosis and proliferation is shifted towards proliferation, either by increased mitosis and/or reduced apoptosis [30]. A failure to delete autoreactive lymphocyte clones contributes to the genesis of autoimmune diseases [31]. In neurodegenerative pathologies, such as Parkinson’s disease and spinal muscular atrophy (SMA), neuronal cells are abnormally prone to cell death [32]. For some human diseases defects in apoptosis genes have been identified as causative or contributing pathogenetic factor (Table 1). In various malignant neoplasms pro-apoptotic molecules like Fas [33–40], Bax [41,42], p53 [43] and the newly identified bcl-10 [40,44–48], as well as anti-apoptotic proteins like bcl-2 [49–53] and the caspase inhibitor cellular inhibitor of apoptosis 2 (c-IAP2) [54,55] are mutated. Benign lymphoproliferative diseases, often combined with autoimmune symptoms, are associated with alterations of Fas [56–60], FasL [61], or caspase 10 [62]. Hereditary fever syndromes [63] and chromosome 10 linked cases of familial hemophagocytic lymphohistiocytosis [64] are caused by defects in TNF-R1 and the cytotoxic molecule perforin, respectively [65,66]. Deletion of neural apoptosis inhibitory protein 1 (NAIP1), a caspase inhibitory protein, may

modify the severity of SMA, which is caused by loss of the survival motor neuron 1 (SMN1) gene [67,68]. The identification of mutations in the apoptosis genes described, greatly contributes to the understanding of the physiologic significance of the molecules involved, provides useful diagnostic disease markers and offers the chance to design therapies based on the molecular nature of the apoptosis defect. 2. Mutation of death receptors (DRs) 2.1. Tumor necrosis factor receptor (TNF-R) Tumor necrosis factor (TNF) is a cytokine with pleiotropic biological activities. It stimulates immune cells to secrete cytokines, causes endothelial cells to express adhesion molecules for leucocyte binding and exerts a pyrogenic effect [6,7]. The TNF induces also apoptosis in some cell types but usually only when new protein synthes´ıs is blocked [6,7]. It is produced predominantly by activated macrophages and in response to infection. The TNF binds two different transmembrane receptors, TNF-R1 (also named p55, CD120a) and TNF-R2 (p75, CD120b) [6,7]. The TNF receptors are present on nearly all cell types examined. The TNF-R1 can exert almost all biological activities attributed to TNF-R2, usually at much lower densities of receptors. The TNF-R2 seems to fulfil mainly an accessory role in enhancing TNF-R1 effects. In most cases, only TNF-R1 triggering is responsible for cytotoxic activity [6,7].

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The TNF trimerises TNF-R1 upon binding and thereby facilitates the association of the receptors intracellular death domains [6,7] (Fig. 1). Subsequently an adaptor molecule, termed TNFR-associated death domain (TRADD) binds and recruits several signaling molecules to the activated receptor: TNFR-associated factor-2 (TRAF2) and receptor interacting protein (RIP) stimulate pathways leading to activation of the transcriptional activators nuclear factor-␬B (NF-␬B) and activating protein 1 (AP-1) [6,7] (Fig. 1). This leads to the induction of pro-inflammatory and immunomodulatory genes and suppression of apoptosis, whereas binding of the adaptors Fas associated death domain (FADD; also named MORT-1) and RAIDD (RIP-associated ICH-1/CED-3-homologous protein with a death domain; CRADD) can mediate apoptosis. This is achieved by recruitment of caspases 8 (FLICE) and 10 (FLICE 2) by FADD and caspase 2 by RAIDD [6,7,69,70] (Fig. 1). The importance of TNF for balanced inflammatory reactions is illustrated by the recently identified TNF-R1 mutations in a dominantly inherited periodic fever syndrome [65]. Affected patients exhibit self-limited episodes of fever, serosal and synovial inflammation, myalgia, periorbital edema, scrotal pain and inguinal hernia. Six different missense mutations were identified in the TNF-R1 gene in seven affected families [65]. Four mutations produced missense changes in the first, two mutations in the second cysteine-rich extracellular domain of the TNF-R1 protein. Altogether, these six mutations were observed in 40 of 43 symptomatic family members; the three variant negative individuals all belonged to the same family and were assessed to represent genetic heterogeneity or misdiagnosis. The TNF-R1 mutations were nearly completely penetrant, with only two of 42 mutation-positive individuals being asymptomatic. The six mutations were not detected in any of 40 healthy relatives studied, or in over 100 ethnically matched control subjects [65]. The mutated TNF-R1 was expressed on leukocytes of patients with inherited periodic fever syndrome and was functional. But the clearance of the receptor from the membrane, which is regulated in part by metalloproteinase cleavage, was impaired [65]. This explains the much reduced blood levels of a soluble form of TNF-R1 in affected individuals. Shed TNF receptors retain ligand-binding activity and may have

an anti-inflammatory effect by competing for TNF binding with membrane receptors. This suggests that the symptoms of familial periodic fever syndrome are caused by disturbed downregulation of TNF responses to inflammatory stimuli. 2.2. Fas (CD95; Apo-1) Fas (CD95; Apo-1) is a transmembrane receptor protein [3,71]. Binding of its natural ligand, Fas ligand (FasL), triggers apoptosis [8,9,71]. The apoptosis signal is transmitted via an intracellular ‘death domain’ that interacts with a homologous motif in the adaptor protein FADD which recruits pro-caspase 8 [3,71] (Fig. 1). Aggregation of pro-caspase 8 within the Fas signaling complex triggers activation by autocleavage. Mature caspase 8 then initiates a sequence of further downstream caspase activations. An alternative Fas mediated apoptosis pathway has been identified that involves mitochondria. The pathway employed depends on the cell type (type I and II cells) and is partially dependend on the bcl-2 family member BID [19]. BID is cleaved by caspase-8 in vivo and its cleavage fragment p15 translocates to mitochondria and perturbs the integrity of the outer mitochondrial membrane with release of cytochrome c — an activity similar to the pro-apoptotic bcl-2 family member Bax (see Section 4.4). Since type II cells, but not type I cells, depend on the mitochondrial branch of the pathway, apoptosis in these cells can be blocked by overexpressed bcl-2 or bcl-xL [72]. Fas is widely expressed in different cell types and tissues. A physiologic role for Fas has been demonstrated in the immune system. The depletion of autoreactive T-cells [73] and the elimination of activated lymphocytes at the end of an immune response depends on Fas–FasL interactions [8,9]. The importance of Fas and FasL in the immune system is illustrated by the mouse strain MRL/lpr–lpr (lymphoproliferation), which harbors a spontaneous mutation in Fas and exhibits lymphoproliferation with massively enlarged lymph nodes, splenomegaly, and variably autoimmune disorders depended on the genetic background [74]. Similar symptoms are observed in patients with the auto-immune lymphroproliferative syndrome type I (ALPS I), also named Canale–Smith syndrome, in association with mutations in the Fas

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gene [56–60,75]. The ALPS I is a rare inherited disease with manifestation in childhood. Affected patients show lymphoproliferation and autoimmune disease with varying intensity of symptoms (splenoand hepatomegaly, lymph node enlargement — but rarely lymphomas, hyperimmunglobulinemia, lymphocytosis — although inconstant). Lymphocytes from ALPS individuals are resistant or less sensitive to cell death induction via Fas. Characteristic is an expansion of a rare T-cell receptor ␣/␤ expressing CD4-/CD8-T lymphocyte subset [56–60,75]. Lymphoproliferative manifestations resolve with age, whereas immunological disorders frequently persist. This observation suggests that Fas-mediated apoptosis plays a more important role in lymphocyte homeostasis in early childhood than later on in life. Despite the expression of Fas on a wide range of tissues, Fas-deficient patients do not suffer from non-immune manifestations. The mode of inheritance is variable. In some pedigrees an autosomal recessive inheritance is observed. In contrast, in other pedigrees, a clear autosomal dominant inheritance pattern is found. In others, only some carriers present with clinical symptoms. But homozygous Fas-null patients always suffer from severe clinical symptoms [75]. The Fas mutations in ALPS are dispersed among the nine exons of the gene but there is a clustering in the intracellular ‘death domain’. The observed mutations are mostly missense or truncating mutations. The severity of symptoms depends on the location of the mutation. Generally mutation of the intracellular region results in a more pronounced pathology than an alteration of the extracellular portion [57,59,60]. Fas mutations were also identified in malignant human diseases. Plasmacytomas harbor Fas mutations at a frequency of 10% (5/48) [33]. In a screen of different subtypes of B- and T-cell non-Hodgkin’s lymphomas Fas mutations were detected in 11% (16/150) of cases [36]. But mutation frequencies were much higher in lymphomas with extranodal manifestation and accompanying autoimmune diseases, such as hemolytic anemia and Sjögren’s syndrome. Somatic mutations of the Fas gene were also detected in Hodgkin and Reed–Sternberg cells in classical Hodgkin’s disease and can be acquired in human B-cells as a side-effect of the germinal center reaction [37,76]. A particularly high incidence of Fas mutations was detected in bladder cancer (12/43) [38]. Ten of the 12 identified

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mutations were located in the ‘death domain’ and eight of these 10 mutations showed an identical G to A transition at bp 993 (codon 251), indicating a potential mutation hotspot in bladder cancers. In human colon and gastric cancers with a microsatellite mutator phenotype Fas mutations were detected in mononucleotide tracts at a frequency of 10% [40]. However, in other malignancies, such as malignant melanoma (3/44) [39], hepatoblastoma (0/38 – only the death domain was analysed) [77], T- (2/101; 2/35) [34,35] and B-cell acute lymphoblastic leukemia (0/37) [78] Fas mutations seem to be rare. A mutated Fas molecule may help tumor cells to escape immune surveillance by turning resistant to FasL, which is utilised by attacking T-and NK-cells. Furthermore, Fas resistancy may allow tumor cells to overexpress and utilise FasL themselves and mount a counterattack on Fas-sensitive immune cells and/or aid in tissue invasion by killing stromal and parenchymal cells [79–81]. 3. Mutation of death ligands/cytotoxic effector molecules 3.1. Fas ligand Fas ligand (FasL) induces apoptosis on binding to its receptor Fas [8,9]. It is localised to the cell membrane. A soluble form with less cytotoxic activity than the membrane bound form is generated by metalloproteinase cleavage [8,9]. The FasL is mainly produced by T- and NK-cells but is also detected in non-lymphoid cells, such as Sertoli cells of the testis, different ocular cell types and various malignant neoplasms [8,9,79–81]. The so-far recognised physiological functions of FasL are largely confined to the immune system. It is employed by T- and NK-cells to kill target cells. Since lymphocytes upregulate FasL and Fas upon activation, Fas mediated apoptosis also contributes to the peripheral elimination of autoreactive T-lymphocytes [73] and the depletion of lymphocytes in the endphase of an immune response by auto- and paracrine FasL–Fas interactions [8,9]. Furthermore, FasL aids in the generation of the ‘immune privileged’ status of the testis, eye and placenta — sensitive tissues where FasL may promote apoptosis of any infiltrating inflammatory cells [82]. Malignant cells may

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utilise FasL to counteract an attack by Fas-sensitive immune cells and perhaps to facilitate tissue invasion [79–81]. The C3H/HeJ-gld/gld mice harbour a missense mutation in the Fas ligand gene which greatly decreases the ability of FasL to induce apoptosis [74]. The FasL mutation results in a lymphoproliferative syndrome that resembles the human autoimmune disease systemic lupus erythematodes (SLE). However, a screen of 75 patients with SLE by single-strand conformational polymorphism analysis for potential mutations of the extracellular domain of FasL revealed an alteration in one patient only [61]. Genomic DNA of this patient contained a heterozygous 84-bp deletion within exon 4 of the FasL gene. The PBMC from this patient revealed decreased FasL activity, decreased activation induced cell death, and increased T-cell proliferation after activation. However, concluding from this report, overall FasL germline mutations seem to be uncommon in SLE. The results, however, do not exclude the presence of sporadic FasL mutations in single autoreactive lymphocyte clones that may contribute to the generation of auto-immune symptoms and that would not be detected by the methods employed. 3.2. Perforin Perforin is a membrane pore forming protein employed by cytotoxic T- and NK-cells for target cell killing [83]. Perforin acts in context with a family of granule associated serine proteases termed granzymes [83,84]. Granzyme B is necessary for rapid induction of apoptosis, while other members of the granzyme family exert a delayed cytotoxic effect. Perforin ‘punctures’ the target cell membrane. The effects are an uptake of secreted, soluble granzymes by reparative endocytosis and osmotic swelling [84]. Internalised granzyme B preferentially activates caspase 3 to initiate the caspase cascade. Caspase 3, in turn, removes an inhibitory pro-domain of caspase 7 allowing activation by granzyme B. The outcome is rapid cross-activation of caspases, leading to cellular destruction [84] (Fig. 1). The spectrum of perforins physiological functions has been extended from a mere cytotoxic molecule that eliminates unwanted cells (e.g. virus-infected cells and tumor cells) to the down-regulation of

human immune responses by the discovery of defective perforin in familial hemophagocytic lymphohistiocytosis (FHL) [66]. The FHL is characterized by an accumulation of activated lymphocytes, monocytes and non-Langerhans cell histiocytes, with the latter exhibiting phagocytosis of blood cells [85]. The FHL is associated with overproduction of inflammatory cytokines, including ␥ interferon, interleukin-1 and interleukin-6 and TNF. Defective T- and NK-cells cytotoxicity is reported frequently [86]. It is a rare disease of young children presenting with fever, often associated with viral infection, pancytopenia, hepato-splenomegaly and frequent destructive organ infiltration, often with severe neurological symptoms. In familial cases an autosomal recessive pattern of inheritance is observed. Cytogenetic studies demonstrated the genetic heterogeneity of the disease with involvement of a gene locus either on chromosome 9q21.3-22 [87] or chromosome 10q21-22 [64]. In a study of eight unrelated 10q21-22 linked FHL patients nine independent mutations in exons 2 and 3 of the perforin gene were detected [66]. The homozygous premature stop codon of four patients would give rise to a truncated, nonfunctional perforin protein. The missense mutations found in the other four patients may affect the synthesis, stability, or function of perforin as T-cells from FHL patients showed no or strongly reduced perforin protein expression. The CD8+ T-cells from FHL patients with a premature stop codon displayed no cytotoxicity and cells from patients with missense mutations showed a greatly reduced capacity to lyse target cells [66]. The identification of defects in the perforin gene in 10q21-22 linked FHL gives a rational basis for therapy with allogeneic bone marrow transplantation, which achieved a 5-year survival of 66% versus 10% for treatment with chemotherapy alone [85]. 4. Mutation of intracellular apoptosis inducers 4.1. Caspase 10 (Mch 4/FLICE 2) Caspase 10 (also named Mch 4/FLICE 2) takes a proximal position in a cell DR triggered caspase cascade [88] (Fig. 1). It is recruited to the activated receptors Fas and TNF-R1 via the adaptor molecule

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FADD [88]. Aggregation of pro-caspase 10 within the DR signaling complex promotes activation by auto-cleavage with release of an amino-terminal pro-domain and the formation of heterodimers consisting of two subunits each. The caspase 10 gene on chromosome locus 2q23 is mutated in human autolymphoproliferative syndrome (ALPS) type II [62]. Patients with ALPS II exhibit prominent non-malignant lymphadenopathy, hepatosplenomegaly, hyperimmunegammaglobulinemia with multiple autoantibodies, autoimmune-hemolytic anemia and lymphocytosis with accumulation of normally rare CD4-/CD8-T-cells [62]. So far, caspase 10 mutations were identified in two distinct families. The modes of inheritance were dominant in one family and recessive in the other. In both families the mutation was a single base substitution leading to an amino acid change in the subunit which contains the active site of the enzyme [62]. The mutations resulted in decreased catalytic activity and inefficient autoprocessing. Apoptosis induction by multiple DRs (Fas, TNF-R1, DR3, DR4, and DR5) was diminished, thus, indicating that caspase 10 is not only involved in Fas and TNF-R1 signaling but is essential for apoptosis signaling via multiple DRs. Other apoptosis pathways, activated by UV irradiation or staurosporine, were unaffected by caspase 10 mutations [62]. In contrast to Fas mutations leading to ALPS I (see Section 2.2), the caspase 10 defects in ALPS II cause a disorder not only of lymphocyte but also of dendritic cell homeostasis. Lymph nodes of an affected ALPS II individual demonstrated accumulation of T lymphocytes and dendritic cells in paracortical areas. Normal dendritic cells are usually very sensitive to TRAIL, the ligand for DR4 and DR5, but less responsive to Fas and TNF␣ [62,89]. However, dendritic cells with caspase 10 mutations exhibited resistance to TRAIL. Therefore, the distinct accumulation of dendritic cells in ALPS II may be caused by unresponsiveness to TRAIL. Dendritic cells are potent stimulators of B- and T-cells [90]. Their turn-over is essential for the regulation of immune reactions [90]. The persistence of antigen presenting dendritic cells in ALPS type II patients may block the downregulation of immune responses, and thus, contribute to the genesis of hyperimmunoreactions.

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4.2. B-cell lymphoma/leukemia 10 (bcl-10) The B-cell lymphoma/leukemia 10 (bcl-10) is a cellular homologue of the equine herpesvirus-2 E10 protein [91,92]. The two proteins share amino acid homologies in an amino-terminal ‘caspase recruitment domain’ (CARD). The CARDs have been shown to function in apoptosis signaling and mediate protein–protein interactions [93]. Distal to the CARD bcl-10 shows no significant homology with E10 or any other known protein. The bcl-10 induces lymphocyte apoptosis in transgenic mice [94] and when transfected in MCF7 human breast carcinoma and 293 human embryonic kidney epithelial cells [45,46]. But in comparison with other apoptotic mediators, such as TNF-R1, bcl-10 is only weakly pro-apoptotic in transfection assays [45]. The mode of cell death induction by bcl-10 is not clear yet. It may involve caspase activation as the carboxy-terminal region of bcl-10 binds pro-caspase-9 and promotes autoproteolytic activation of the pro-caspase [91,92]. A recent report describes that bcl-10 also binds to TRAF-2, an anti-apoptotic protein, associated with TNF-Rs [94] (Fig. 1). Low levels of bcl-10 expression promoted the binding of TRAF2 to c-IAPs (cellular-inhibitor of apoptosis proteins), a further component of the TNF-R signaling complex with apoptosis suppressing activity. Conversely, bcl-10 overexpression inhibited TRAF-2 and c-IAP interaction. Excessive bcl-10 may, therefore, block TRAF-2 signaling and consecutively downstream activation of NF-␬B, the expression of which has been associated with suppression of apoptosis in most experimental systems [94–98]. The bcl-10 gene is involved in the chromosomal translocation t(1;14)(p22;q32) which is observed in some mucosa-associated lymphoid tissue (MALT) lymphomas [45,46], the most frequent type of extranodal lymphoma that often develops in a setting of chronic inflammation and/or autoimmune disease. The translocation brings the intact coding region of bcl-10 from chromosome 1p22 into the vicinity of immunoglubulin genes on chromosome 14q32. The bcl-10 cDNAs derived from MALT lymphomas with t(1,14)(p22;q32) exihibited three types of derivation from the wild type cDNA sequence that resulted from (a) the utilisation of three alternative splice sites at the exon 3 to 4 boundary; (b) nucleotide insertions and

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deletions, particularly within homopolymeric runs of eight A’s and seven T’s that would in most cases result in protein truncation distal to the CARD; and (c) point mutations [45,46,48]. Interestingly, several clones from an individual tumour shared identical mutations, in addition to containing unique abnormalities, suggesting that additional mutations accumulate in bcl-10 during tumorigenesis. Ongoing mutation is a feature of rearranged IgVH genes after a B-cell has encountered antigen, resulting in affinity maturation of antibody, but has also been seen in other genes in B-cells [99,100]. Therefore, bcl-10 may be exposed to Ig somatic hypermutation by the t(1;14). The functional consequences of the bcl-10 mutations truncating in the CARD were loss of apoptosis induction and NF-␬B activation, whereas truncation distal to the CARD caused also loss of the proapoptotic function but retained NF-␬B activation. Furthermore, truncated mutants gained a pro-proliferative function, which is absent in wild-type bcl-10 [45,46]. Loss of bcl-10 pro-apoptosis may confer a survival advantage to MALT B-cells, and constitutive NF-␬B activation may provide both anti-apoptotic and proliferative signals. The bcl-10 mutations were not only detected in MALT lymphomas but in various human neoplasias. Overall, 45% of 155 analysed cases of B- and T-cell lymphomas of various histological subtypes displayed cDNA alterations [45] and 9.5% (4/42) of follicular lymphomas [48] exhibited mutations in genomic DNA independently of the presence of a t(1;14)(p22;q32). Furthermore, 10/87 various solid tumor cell lines (including male germ cell tumors and mesotheliomas) showed truncating cDNA mutations [45]. However, the above results were questioned by several research groups that reported the lack of bcl-10 mutations in genomic DNA of B-cell lymphomas, male germ cell tumors, mesotheliomas, and various carcinomas, although these malignancies are frequently associated with 1p22 deletions and bcl-10 cDNA alterations were reported previously for some of them [101–106]. It was, thus, concluded that bcl-10 mutations are rare and that the putative tumor suppressor gene on 1p22 is not bcl-10, but rather an as yet unidentified gene, because of the lack of bcl-10 genomic DNA alterations. The discrepancies between different reports on the incidence of bcl-10 mutations in malignant neoplasias may partially depend on the use of cDNA

versus genomic DNA for mutation analysis. At least some of the bcl-10 abnormalities, originally described as mutations, seem to represent post-transcriptional modification of the bcl-10 RNA and are not encoded in genomic DNA [107]. The bcl-10 may, therefore, undergo what has been termed ‘molecular misreading’, a process where mutated transcripts are produced from a correct DNA sequence [108]. Recently, bcl-10 nucleotide insertions and deletions were reported to be also present in cDNA from peripheral blood leukocytes of normal individuals [109]. Apparently, bcl-10 cDNA abnormalities are not necessarily associated with or sufficient for malignant transformation. 4.3. p53 The p53 is a nuclear protein that functions as a transcription factor capable of regulating a range of downstream genes [23–26]. It plays a central role in the coupling of cell damage to cell-cycle arrest and to the induction of apoptosis. In response to cellular stress exerted by hypoxia, the presence of an activated oncogene or DNA damage, the cellular p53 level increases and p53 is activated by modification [110,111] and turns on the transcription of its target genes [23–26]. The p53 cell cycle arrest pathways involve p21 (WAF1, Cip-1) and GADD45. The p21 is a cyclindependent-kinase (cdk) inhibitor. It binds to a number of cyclin — cdk complexes and PCNA to block cell cycle progression in G1 and G2. GADD45 also binds to PCNA and arrests the cell cycle in G2; it is also engaged in DNA nucleotide excision repair [23–27]. The current model of p53 function holds that blockage of cell cycle progression allows repair of damaged DNA, and thus, suppresses propagation of DNA alterations. The p53 apoptosis pathways involve many different gene products. Depended on the cell type, augmentation of insulin like growth factor binding protein 3 (IGF-BP3) and Bax expression contribute to apoptosis induction by p53 [23–26]. IGF-BP3 blocks IGF survival and mitotic signaling. Bax binds and thereby antagonizes bcl-2 and probably facilitates cytochrome c release by forming pores into mitochondrial membranes [22]. Recently, a novel pro-apoptotic bcl-2 family member, named Noxa [112], that localizes to mitochondria, was proposed to represent a mediator of p53 induced apoptosis. An additional route by which p53 may signal to the mitochondria and

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induce apoptosis is through the increase of the levels of reactive oxygen radicals (ROS). The p53 induces genes that encode proteins that catalyse redox reactions and consequently generate ROS, which can trigger cytochrome c release from mitochondria [28]. Fas may also contribute to p53 mediated apoptosis. The p53 responsive elements have been identified in the Fas gene and Fas is induced by p53 in response to DNA damage. In addition, p53 may facilitate the transport of Fas from the Golgi to the membrane [113]. Intriguingly, NF-␬B was recently shown to be essential for p53 mediated apoptosis, although NF-␬B alone proved incapable of apoptosis induction [114]. A number of factors affect the decision of a cell to enter either a p53-mediated cell cycle arrest or an apoptotic pathway. The p53 mediated apoptosis seems to prevail under conditions in which the DNA damage is severe, survival factors are limiting, or an oncogene is activated [23–25]. The p53 mutations are the most common genetic alteration in a broad range of malignant neoplasms. They are found in 50–55% of all human cancers [43]. The mutations are clustered in regions of p53 that are essential for binding to DNA in a sequence-specific fashion. In about 70% of cases with the familial Li — Fraumeni cancer syndrome an inherited mutant p53 allele, followed by somatic loss of the remaining wild-type allele, leads to cancer. Affected relatives typically develop sarcomas, breast cancer, brain tumors, leukemia and adrenal cortical tumors, often at an early age [115,116]. The p53-dependent apoptosis can modulate the toxic effects of anticancer agents. In breast cancers, p53 mutations were associated with de novo resistance to doxorubicin treatment [117]. Other tumors that frequently contain p53 mutations (melanoma, lung cancers, colorectal tumors, bladder and prostate cancers) often respond poorly to radiation or chemotherapy. Childhood acute lymphoblastic leukemias, which most often have wild-type p53 respond well to chemotherapy [23]. When these tumors relapse, ineffectiveness of therapy correlates with the acquisition of p53 mutations [23]. In general, p53 mutations are more frequent in aggressive disease and are associated with poor survival, e.g. p53 alterations are rare in the chronic phase of CML but are frequent in blast crisis, and the transformation of indolent follicular lymphoma to diffuse

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aggressive disease correlates with p53 mutations in 25–30% of cases [49]. 4.4. Bax Bax is a bcl-2 homologous protein [17,18]. It resides in the cytoplasm or is loosly attached to cellular membranes. Upon receipt of an apoptotic stimulus it inserts into the mitochondrial outer membrane. Its mode of cell death induction is not clear yet, although the generation of pores in membranes may be important as Bax forms pores in lipid membranes in vitro and can cause the release of cytochrome c from mitochondria [17–19,22] (Fig. 1). It seems to act as a homodimer, the formation of heterodimers, e.g. with bcl-2 is not necessary for its apoptosis inducing function, although the ratio of Bax and other bcl-2 family members determines susceptibility to cell death stimuli. Bax deficient mice indicate that several normal developmental cell deaths (neurons, lymphocytes, ovarian granulosa cells, spermatogonia) depend on Bax and that it suppresses tumorigenesis [118,119]. The transcription of Bax is triggered by p53 [18] and in different experimental systems Bax mediates approximately 50% of p53 dependent cell deaths [119,120]. Bax mutations were detected in about 50% of human colon carcinomas with a microsatellite mutator phenotype (MMP) [41]. The MMP is caused by defects in DNA mismatch repair genes and is associated with genetic instability that leads to the accumulation of deletions and insertions in short nucleotide repeat sequences, referred to as microsatellites. Almost all cancers associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC) and some sporadic colon cancers exhibit a MMP [121]. The Bax mutations detected in colon cancers were frameshift mutations in a tract of eight deoxyguanosines (G)8 spanning codons 38–41 in the third coding exon. These mutations were absent in MMP negative tumors and were significantly less frequent in (G)8 repeats from other genes. Interestingly, colon carcinomas with a MMP typically do not contain p53 mutations. Therefore, it seems that Bax mutations may eliminate the selective pressure for p53 mutations during colon tumorigenesis [41]. Mutations in Bax were furthermore detected in 21% (6/28) of cell lines of different hematopoetic

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malignancies [42]. Approximately half of the mutations were again frameshifts confined to the (G)8 tract and were associated with loss of immunodetectable Bax protein. In addition to the frameshift mutations, single amino acid substitutions were found with loss of the pro-apoptotic function. However, Bax mutations were not detected in acute lymphoblastic leukemias in vivo, whereas leukemic cell lines frequently exhibited microsatellite instability and harbored frameshift mutations in Bax [122,123]. Thus, it is likely that mutations in DNA mismatch repair genes and Bax mutations are selected for during the establishment of leukemic cell lines. 5. Mutation of apoptotis inhibitory proteins 5.1. bcl-2 The bcl-2 is an anti-apoptotic protein that resides on the cytoplasmic face of the mitochondrial outer membrane, endoplasmic reticulum and nuclear envelope. It protects against various cytotoxic insults, e.g. gamma- and ultraviolet-irradiation, cytokine withdrawal, dexamethasone and chemotherapeutic drugs [17,18]. The bcl-2 is the prototype of a family of related proteins with either anti-apoptotic (bcl-2, bcl-xL ) or pro-apoptotic (bax, bcl-xS , bak, bad, bid) functions. Pro- and anti-apoptotic family members can form homo- and heterodimers and their relative concentration determines the susceptibility to a death stimulus [17,18]. The bcl-2 inhibits apoptosis by blocking the release of cytochrome c from mitochondria and thereby preventing Apaf-1 and consecutive caspase activation [19–21] (Fig. 1). The bcl-2 may also inhibit apoptosis by binding to pro-apoptotic molecules (Bax, bcl-xS ). The bcl-2 can also influence cell cycle progression. It promotes exit from the cell cycle under suboptimal growth conditions. This effect is genetically separable from its survival function and resides within the amino-terminal region [124]. The bcl-2−/− knockout mice are viable, but the majority die at a few weeks of age. They exhibit marked lymphocyte, neuronal and intestinal apoptosis, develop polycystic kidney disease and are hypopigmented because of decreased melanocytic survival [125]. Genetic alterations of the bcl-2 gene have been identified in hematologic malignancies. Most follicular

lymphomas are characterised by the t(14;18) translocation involving the bcl-2 locus on chromosome 18 and the heavy chain immunoglobulin (Ig) segments on chromosome 14. The translocation occurs nearly exclusively at two loci, a major breakpoint clustering region within the 3 non-coding region and a minor breakpoint clustering region within the 3 flanking region of the bcl-2 gene [50]. Less frequently, and in approximately 3–10% of chronic lymphocytic leukemias, the 5 flanking region of the bcl-2 gene is rearranged and preferentially linked to the Ig light chain genes on chromosome 2 or 22 [51,52]. In both instances, the bcl-2 rearrangement results in the deregulation and overexpression of bcl-2 protein, which prevents the cell from apoptosis. Many progressed follicular lymphomas display also missense mutations in the amino-terminal region of bcl-2. These coding changes may relieve cell cycle inhibition by bcl-2 [53]. The presence of a bcl-2 translocation, however, is not synonymous with neoplastic transformation. In fact, clonal bcl-2-Ig gene rearrangement products can be identified in normal lymphoid tissues and in normal peripheral blood samples [126]. Apparently, additional genetic alterations are necessary for malignant transformation. This has also been demonstrated in bcl-2/IgH transgenic mice which exhibit massive lymphoid hyperplasia but develop malignant lymphoma only after acquisition of secondary oncogenic events, such as rearrangement of the c-myc oncogene [17]. 5.2. Inhibitor of apoptosis proteins (IAPs) Inhibitor of apoptosis proteins (IAPs) are characterised by a domain of about 70 amino acids termed BIR (baculoviral IAP repeat) derived from the original discovery of this apoptosis suppressing motif in the genomes of baculoviruses [127]. To date, six IAP relatives have been identified in humans: c-IAP1 (also named API1, hIAP-2; MIHB), c-IAP2 (API2; hIAP-1; MIHC), NAIP, XIAP (hILP; MIHA), Survivin, and BRUCE [128–130]. Overexpression of c-IAP1, c-IAP2, NAIP, XIAP, or Survivin has been shown to suppress apoptosis induced by a variety of stimuli including TNF␣, Fas, staurosporin, etoposide/VP16, taxol and growth factor withdrawal. The c-IAP1, c-IAP2, XIAP and Survivin were shown to bind and potently inhibit the processing of caspases 3, 7 and 9, but not caspases 1, 6, 8, or 10 (Fig. 1). The c-IAP1 and

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c-IAP2 furthermore interact with TRAF1/TRAF2 heterodimers, a component of the TNF-R signaling complex engaged in activation of NF-␬B, which promotes cell survival in response to TNF [128–130] (Fig. 1). Recently, involvement of c-IAP2 and a novel gene on 18q21, termed MLT (for MALT-lymphoma associated translocation), in a key genetic lesion of MALT lymphomas, the chromosomal translocation (11;18)(q21;q21), was identified [54,55]. This translocation seems to be specific for low-grade MALT lymphomas. It has not been detected in high-grade MALT lymphomas or nodal marginal zone cell lymphomas, which cytologically and immunophenotypically resemble MALT lymphomas [55,131,132]. The native c-IAP2 protein contains three copies of the BIR at its amino-terminus and a CARD followed by a carboxy-terminal zinc binding RING finger domain [128–130]. The BIR domain-containing regions of c-IAP2 are sufficient for inhibition of caspases and suppression of apoptosis. The CARD may mediate protein–protein interactions, whereas the RING finger motif is involved in autoubiquitination and degradation of the protein [133]. In the t(11;18)(q21;q21) the c-IAP2 molecule is truncated after the BIR domains, which are fused in frame to the carboxy-terminal part of MLT. This truncation of c-IAP2 after the BIR domains could release their anti-apoptotic effect from negative regulation by the CARD and RING domains [54]. The function of the MLT gene is not yet known. It is characterised by several Ig-like C2-type domains and a domain similar to the murine Ig gamma chain VDJ4 sequence. The breakpoints on chromosome 18 are scattered throughout the MLT gene, but the VDJ4 related sequence is retained in frame in all fusion genes analysed, thus, pointing to a functional role in the fusion protein. The C2 domains are only present in a few of the studied cases, and thus, probably have no significance for lymphomagenesis [54,55]. Low-grade MALT lymphomas have been shown to display low levels of apoptosis and to escape from Fas-mediated apoptosis [134,135]. Although IAPs do not bind to caspase-8, the apical caspase in Fas signalling, they do bind to and inhibit its substrate caspase 3, thus, arresting the cascade of proteolysis and providing protection from Fas/caspase 8 induced apoptosis (Fig. 1). In the mitochondrial pathway for caspase activation c-IAP2 can bind directly to the apical caspase

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9, and prevent its activation by cytochrome c/APAF-1 complexes [128–130] (Fig. 1). Therefore, by blocking key caspases in different apoptosis pathways c-IAP2 may protect lymphoma cells from diverse apoptotic stimuli and thereby extend their life-span. 5.3. Neuronal apoptosis inhibitory protein 1 (NAIP1) Neuronal apoptosis inhibitory protein 1 (NAIP1) is a member of the IAP gene family. It contains three BIR domains but lacks CARD and RING Zn finger motifs [128–130]. It is ubiquitously expressed, with highest levels in the central nervous system and functions as an apoptosis suppressor that can block the activities of caspases 3 and 7. Overexpression of NAIP reduces death of hippocampal neurons following transient forebrain ischemia and deletion of the NAIP gene in mice greatly reduces the survival of pyramidal neurons in the hippocampus after kanaic acid-induced limbic seizures [136,137]. The NAIP1 and a variable number of truncated copies of the gene are located on human chromosome 5q in a segment to which the gene for SMA has been mapped [67]. Spinal muscular atrophy (SMA) is a common autosomal recessive disorder characterised by the loss of motor neurons in the anterior horn of the spinal cord and often in the brainstem, with the brain cortex being unaffected [67]. The SMA exists as a broad spectrum of disease from very severe infantile to mild chronic forms. Three major phenotypes are distinguished. The type I SMA (also called Werdnig– Hoffmann disease) is the most severe variant with onset at birth, severe muscle weakness and early death. The intermediate type II SMA manifests in early childhood. Affected children can sit but not stand or walk unaided. The type III (Kugelberg–Welander disease) patients show first symptoms after 18 months and are able to stand and walk, but the disease follows a slowly progressive course. The SMA region on chromosome 5q contains a large inverted duplication consisting of at least four genes, which are present in a telomeric and a centromeric copy: NAIP (NAIP1-telomeric; NAIP2-centromeric), survival motor neuron gene SMN (SMN1-telomeric; SMN2-centromeric); H4F5, a protein that may have a role in snRNPs biogenesis; and basal transcription factor subunit p44 [67].

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The SMN1 has been identified as the causative gene for development of SMA [138,139]. It is deleted in almost all SMA patients; the remaining individuals carry intragenic mutations. The severity of the SMA phenotype is proposed to be modified by other genes. One factor that may influence the phenotype is the presence of a variable number of copies of SMN2, which evolved by gene duplication as it differs from SMN1 in only five nucleotides. The SMN2 is poorly transcribed and exhibits defective mRNA splicing, which produces little complete SMN transcript. This explains why the SMA phenotype correlates with the copy number of SMN2. The more SMN2 copies a patient has, the more full-length SMN2 protein is present and the milder is the SMA phenotype [67,138,139]. The SMN protein is expressed in all tissues of mammalian organisms, but particularly high levels are found in motor neurons. It is located in the cytoplasm and the nucleus and functions in cytoplasmic assembly of snRNP particles [140]. It may also exert an anti-apoptotic effect as it interacts with bcl-2 and enhances the proteins apoptosis-suppressive activity [141]. Other candidates for a modifier of SMA located on chromosome 5q are H4F5 [142] and NAIP, whereas p44 deletions show no correlation with SMA [67]. The H4F5 exhibits homology to the rat matrin-cyclophilin, a protein that colocalises with snRNPs, which may suggest a function in the same biochemical pathway as SMN. The arguments for a role of NAIP in SMA phenotype modification are that the intact NAIP gene is deleted or interrupted on both chromosomes more frequently in SMA type I patients than in individuals with type II and III disease [68]. Furthermore, RT-PCR analysis indicated either absence of NAIP transcripts or revealed internally deleted and mutated forms of the NAIP message in type I individuals but not in unaffected individuals. It is suggestive to assume that motor neurons from SMA individuals with deletion of SMN1 and NAIP are prone to apoptosis and die earlier than they would with a SMN1 deletion only — resulting in a more severe phenotype. However, the role of NAIP in SMN pathogenesis remains controversial. First, no clear differences in the clinical course of SMA type I patients with or without NAIP deletion could be discerned. Second, some SMA type II and III patients also have NAIP gene defects [68]. A clarification may await the generation of proper

animal models with SMN and NAIP genes deleted conditionally, as constitutive SMN knock-out results in early embryonic lethality in mice [143,144].

6. Summary and future prospects Mutations in apoptosis genes contribute to the pathogenesis of human diseases. The spectrum of currently known diseases encompasses mainly non-malignant lymphoproliferative and inflammatory syndromes as well as malignant neoplasms (Table 1). All but one (NAIP1) of the outlined examples of mutated apoptosis genes result in reduced apoptosis and consecutive accumulation of immune or tumor cells. However, mutation is only one mechanism of apoptosis dysregulation. Alterations in the expression of apoptosis genes, e.g. FasL upregulation and/or Fas downregulation in tumor cells [79–81], by yet unknown mechanisms, or the silencing of caspase 8 expression in neuroblastomas by gene methylation [145], may also be involved in the pathogenesis of diseases. The identification of alterations in apoptosis genes contributes to the understanding of the function of the molecules involved, offers novel molecular tools for diagnosis and reveals potential targets for therapeutic intervention. The currently most promising results of apoptosis research that are on the verge of translation into clinical applications or have already entered the clinical setting are briefly outlined below. 6.1. Apoptosis gene mutations and their impact on the diagnosis of diseases The identification of mutations in TNF-R1, Fas, caspase 10 and perforin in patients with hereditary periodic fever syndrome, ALPS and FHL will greatly facilitate the molecular diagnosis of these diseases [56–60,62,65,66,75]. The translocation t(14;18) involving bcl-2 is already extensively employed for the diagnosis of follicular lymphomas. The translocation results in an overexpression of bcl-2 protein wich can be demonstrated immunohistochemically and utilised to differentiate reactive non-neoplastic lymphoid follicles which are bcl-2 negative from neoplastic follicles which usually stain strongly positive. The translocation is furthermore amenable to detection by PCR, which aids in the identification of “minimal residual

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disease” and early relapse after chemotherapy or bone marrow transplantation [146]. 6.2. Mutations in apoptosis genes and their impact on prognosis and response to therapy The effectiveness of chemotherapy depends, at least in some cell types, on the Fas/FasL system [147–151]. Cytotoxic agents induce the expression of Fas and FasL and the interaction of both molecules triggers apoptosis by auto- and/or paracrine action. In certain cell types cytotoxic agents induce also Fas and/or FasL, but the two molecules are dispensable for their cytotoxic effect [152–154]. However, in some of these cells FADD may be crucial for drug sensitivity [155]. Although the contribution of the Fas system to the cytotoxicity of chemotherapeutic agents remains a matter of controversy [151], knowledge of mutations in Fas and/or FADD may turn out useful to predict resistance to chemotherapy — at least in certain tumor types. Mutation of p53, as outlined in paragraph 4c, is linked with chemoresistence and transformation to more aggressive disease in a large number of tumor types. Knowledge of the p53 status in a given tumor can aid in prognosis assessment and guide the therapeutic decision process. For example, aberrations of p53 are associated with poor patient survival and represent an independent prognostic parameter in B-cell non-Hodgkin’s lymphoma [49,156]. In patients with metastatic breast cancer presence of a p53 mutation predicts a poor response to the anti-estrogen tamoxifen [157]. Likewise, the response of breast cancer to a DNA-damaging chemotherapy was found to depend on normal p53 [117,158]. The cytotoxicity of the microtubule stabilising agent paclitaxel, however, is related to defective p53 [158]. This may be explained by a lack of cell cycle arrest due to p53 deficiency which may support the efficacy of paclitaxel acting during mitosis. The bcl-2 contributes to resistance to chemotherapy [159]. In high-grade B-cell lymphomas bcl-2 protein expression appears to be a predictive of poor disease-free survival, and thus, represents a useful parameter for the identification of patient risk groups [160]. The chromosomal translocation t(11;18)(q21;q21) seems to be specific for low-grade MALT (mucosa

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associated lymphoid tissue) lymphomas, which represent a subtype of extranodal non-Hodgkin’s lymphoma [55,131,132,161]. The development of gastric MALT lymphomas is triggered by infection with Helicobacter pylori. This is impressively demonstrated by regression of up to 67% of early-stage and low-grade gastric MALT lymphomas after eradication of Helicobacter pylori by antibiotics [162]. The period to achieve regression of the lymphoma varies from a few months to 18 months [162]. Recent data indicate that lymphomas with a t(11;18)(q21;q21) are resistant to Helicobacter pylori eradication [163,164]. The detection of the transclocation may, therefore, turn out to be of great help in the management of patients with low-grade gastric MALT lymphoma and obviate the need for extended follow-up and repeated gastric endoscopy to evaluate response to antibiotic treatment. Patients with a t(11;18)(q21;q21) may, thus, profit from earlier initiation of a more aggressive therapy. 6.3. Apoptosis genes as therapeutic targets The TRAIL (TNF-related apoptosis inducing ligand) induces apoptosis on binding to death receptors 4 (DR4, TRAIL-R1) and 5 (DR5, TRAIL-R2) [3,10]. A surprising differential sensitivity exists between normal and malignant cells. Normal cells are usually resistant whereas most malignant cells are sensitive to killing by TRAIL [3,10]. The molecular basis for this difference is not clear yet. The utilisation of TRAIL as an anti-cancer weapon appears very promising and encouraging results have been obtained in animal models. The TRAIL was able to inhibit the growth of xenotransplanted tumors in immunodeficicient mice and achieved even regression of already established tumors [165,166]. No toxic side effects on normal tissue was observed in safety studies from mice to non-human primates [167]. The TRAIL, therefore, seems to differ from other members of the TNF-family like TNF␣ and FasL, which cause massive necrosis of various tissues including the liver. However, recent data indicate that TRAIL may be toxic to human hepatocytes too, although it has no detrimental effect on murine hepatocytes [168]. This might constitute a drawback for a systemic application of TRAIL. Caspases seem to be the key executioners of apoptosis, and thus, represent an attractive target for therapeutic intervention. Inhibition of caspases has already

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shown great promise in animal models of human diseases. Caspase family inhibitors attenuated neuronal death after focal ischemia in mice and limited myocardial infarct size as well as intestinal and hepatic tissue injury in murine ischemia–reperfusion injury models [169–172]. Transplantation of embryonic neural cells to patients with Parkinson’s or Huntington’s disease is hampered by the poor survival of grafted neurons. Treatment of embryonic nigral and striatal cells with caspase inhibitors reduced apoptosis in vitro and increased survival of neurons grafted to hemiparkinsonian rats and excitotoxically lesioned rat striatum (a model of Huntington’s disease) [173,174]. Caspase inhibiton seems also promising as an adjunct to antibiotic therapy. A broad-spectrum caspase inhibitor prevented hippocampal neuronal death in experimental pneumococcal meningitis and protected human neurons from the toxic effects of inflamed cerebrospinal fluid in vitro [175]. Furthermore, broad spectrum caspase inhibitors improved survival in a mouse model of sepsis and rescued mice from lethal LPS induced endotoxic shock [176,177]. Loss of functional p53 is the most common genetic alteration in human cancers. Restoration of normal p53 in cancer cells inhibits growth or induces apoptosis. Therefore, it seems attractive to design therapeutic strategies that restore p53 function. Gene replacement strategies that employ mostly viral vectors are currently most advanced [178]. But other approaches, e.g. “repair” of mutated p53 mRNA with ribozymes are also pursued [179]. The p53 gene replacement attempts have already entered phase I/II clinical trials, which demonstrated that p53 gene therapy is feasible and safe. In addition patients with advanced non-small cell lung cancer receiving monthly intratumoral injections of recombinant adenovirus containing human wild-type p53 had evidence of antitumoral effect with prolonged tumor stability or regression [180]. Furthermore, combined treatment of head and neck cancer with p53 gene replacement and chemotherapy achieved a high proportion of complete responses [181]. By 6 months, none of the responding tumors had progressed, whereas all non-injected tumors treated with chemotherapy alone had progressed. The bcl-2 renders cells resistant to a broad spectrum of apoptosis stimuli, including chemotherapeutic agents. The blockage of bcl-2 may, therefore, turn

malignant cells sensitive to treatment. Downregulation of bcl-2 expression has been successfully attempted with an 18-mer all-phosphorothionate antisense oligonucleotide, named G-3139. This molecule targets the first six codons of the human bcl-2 open reading frame. G-3139 treatment improves chemosensitivity to dacarbazin of human melanoma grown in severe combined immunodeficient (SCID) mice [182]. These results have already been translated into phase I clinical studies for the treatment of malignant melanoma [183]. The bcl-2 antisense therapy shows also potential for antitumor activity in other malignancies. In a phase I clinical trial of patients with relapsed follicular lymphomas subcutaneous infusions of G-3139 was applied as sole treatment. Disease stabilisation was seen in 43% (9 of 21) and improvements were observed in 14% (3 of 21, including one complete responder) [184]. Merkel cell carcinomas, an aggressive neuroendocrine skin tumor, expresses high levels of bcl-2. In a SCID xenotransplantation model of Merkel cell carcinoma a dramatic reduction of tumor growth or complete remission was achieved with G-3139, whereas the chemotherapeutic cisplatin had no significant anti-tumor effect [185]. Obviously, bcl-2 antisense treatment holds promise in the treatment of various malignant diseases. Modulation of other members of the bcl-2 gene family may soon follow. The anti-apoptotic protein Bcl-xL can be simultaneously downregulated with bcl-2 by a bi-specific antisense oligonucleotide that targets a region of homology between the two mRNAs [186]. Downregulation of two key anti-apoptotic proteins may thereby further enhance the chemosensitivity of malignant cells. The outlined examples of a gradual transformation of knowledge from basic apoptosis research into practical benefits for the clinical management of diseases may only be the tip of an iceberg. As the biochemical pathways of apoptosis are better understood new applications and treatment strategies will certainly evolve. Furthermore, the number of newly identified apoptosis genes is still increasing and several of them may turn out to be also altered in specific human diseases.

Acknowledgements We apologise to those many investigators whose original work was not properly cited because of

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