Apoptosis Cell Death Pathways

From: Contemporary Hematology Chronic Lymphocytic Leukemia: Molecular Genetics, Biology, Diagnosis, and Management Edited by: G. B. Faguet © Humana Press Inc., Totowa, NJ

for high-throughput screens to identify new agents. Two main pathways for apoptosis, termed the intrinsic (innate pathway) and extrinsic pathways, have been identified (Fig. 1). In the extrinsic pathway, signals from stimulated death receptors on the cell surface activate caspases, which function as the ultimate effectors and lead to the biochemical and morphological changes of apoptosis. In contrast, the intrinsic pathway is activated by endogenous molecules and is dependent on mitochondrial release of cytochrome c into the cytosol, which in turn activates terminal caspases and cellular death.

The BCL-2 family of proteins plays a central role in regulation of the intrinsic pathway, where their relative balance at the mitochondria determines cell survival or apoptosis. At least 20 members of the BCL-2 family have been identified in mammalian species to date, including proteins with anti-apoptotic effects (such as BCL-2, Bcl-XL, Bcl-W, Mcl-1, Boo/Diva, and Al/Bff-1) and others (such as Bax, Bcl-Xs, Bad, Hrk, Bim, Bik, Blk, APR/Noxa, and Bcl-Gs), which have pro-apoptotic properties. Functionally, the BCL-2 proteins determine the structural integrity of the mitochondrial membranes, endoplasmic reticulum, and nuclear envelope, thereby regulating the release of cytochrome c. The Bax protein promotes apoptosis by blocking the inhibitory activity of BCL-2 as well as by directly targeting mitochondrial membrane and inducing release of caspase activating proteins. Clinical responses to chemotherapy and radiation therapy are affected by defects in Bax, because DNA-damaging agents elicit a cell stress response that promotes apoptosis. Such responses may be mediated through induction of p53, which in turn binds to the Bax gene product and directs its transcription. Thus mutations that inactivate Bax can contribute to poor responses to anticancer agents. The increased survival of B-CLL lymphocytes has been partially ascribed to constitutive expression of BCL-2 (2). Moreover, high levels of BCL-2 protein or high ratios of BCL-2/Bax have been associated with more aggressive behavior of B-CLL, including progressive disease, chemotherapy resistance and decreased survival (3,4).

The Bim protein may play a role in resistance to the vinca alkaloids through its association with microtubules. The Bim isoforms are sequestered in microtubules, and their disruption allows Bim to translocate to the surface of mitochondria, bind BCL-2/Bcl-XL and other related anti-apoptotic proteins, and trigger cytochrome c release and apoptosis (Fig. 1). Another potentially relevant pathway in CLL involves the apoptotic protease activating factor I (APAF-1) protein, which, when activated by cytochrome c, activates caspase-9 and produces apoptosis (5) (Fig. 1). Recent data indicate that expression of Apaf-1 may be variable in CLL, suggesting that differences in Apaf-1 levels may affect the sensitivity of CLL to apoptosis (6). B-CLL has been reported to be profoundly resistant to Fas-induced apoptosis, suggesting that defects in the Fas pathway may also contribute to chemotherapy resistance (7).

The nuclear factor-KB (NF-kB) transcription factor plays an important role in apoptosis and has been linked to CLL. Normally, NF-kB is sequestered and bound to IkB (inhibitor of kappa B) and cannot enter the nucleus to increase expression of anti-apoptotic genes like BCL-2 and IAP's (inhibitor of apoptotic proteins; Fig. 1). In response to a death stimulus, tumor necrosis factor (TNF) family receptors can trigger NF-kB activation through interaction with TNF-asso-ciated factor (TRAF) family proteins, which phosphorylate IkB. Thus, a negative feedback mechanism has been demonstrated resulting from TNF-a signaling in which NF-kB activation suppresses the signals for cell death (8). Interestingly, increased TRAF levels are found in 50% of untreated and 80% of previously treated B-CLL (9). Elevated levels of NF-kB are found in CLL, possibly related to stimulation of CD40, a TNF family receptor, by the CD40 ligand, which is found on 15-30% of B-CLL cells (10,11).

Fig. 1. Potential apoptotic mechanisms of drug (shown in boxes) actions are summarized. The extrinsic and intrinsic apoptosis pathways leading to the activation of effector cysteine proteases (caspases), ultimately leading to apoptosis, are summarized. Also depicted is the antigen-dependent cellular cytotoxicity (ADCC) pathway for rituximab. Arrows represent positive regulation and bars (±) represent negative regulation. See text for details. Apaf-1, apoptotic protease-activating factor-1; APO2, apolopoprotein 2; Cyc, cytochrome C; FADD, Fas-associated death domain (Mort-1); CTL, cytotoxic T-lymphocytic; IAP, inhibitor of apoptotic protein; IKK, inhibitor of kappa B kinase; NF-kB, nuclear factor-kB; NK, natural killer (cell); PKC, protein kinase C; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand.

Fig. 1. Potential apoptotic mechanisms of drug (shown in boxes) actions are summarized. The extrinsic and intrinsic apoptosis pathways leading to the activation of effector cysteine proteases (caspases), ultimately leading to apoptosis, are summarized. Also depicted is the antigen-dependent cellular cytotoxicity (ADCC) pathway for rituximab. Arrows represent positive regulation and bars (±) represent negative regulation. See text for details. Apaf-1, apoptotic protease-activating factor-1; APO2, apolopoprotein 2; Cyc, cytochrome C; FADD, Fas-associated death domain (Mort-1); CTL, cytotoxic T-lymphocytic; IAP, inhibitor of apoptotic protein; IKK, inhibitor of kappa B kinase; NF-kB, nuclear factor-kB; NK, natural killer (cell); PKC, protein kinase C; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand.

The signal transducer and activator of transcription (STAT) proteins play a central role in mediating the response of hematopoietic cells to a diverse spectrum of cytokines. STAT proteins link cytokine receptor stimulation to gene transcription by acting as both cytosolic messengers and nuclear transcription factors. STAT proteins are activated through phosphorylation by JANUS kinase (JAK) kinases and other tyrosine kinases, and form dimers, which translocate to the nucleus and modulate gene expression. The serine residues of STAT proteins may also be phosphorylated and amplify the transcriptional activation mediated by tyrosine phosphorylation. In a recent study, inappropriate STAT-1 and STAT-3 serine phosphorylation was found in 23 patients with CLL, whereas none was found in normal control patients, suggesting that STAT proteins are involved in the pathobiology of CLL (12). Therapeutically, fludarabine administration in CLL patients depletes STAT-1 protein and mRNA, but not other STAT proteins, suggesting that the STAT signaling pathway is an attractive target for the development of specific inhibitors (13).

Apoptosis is an important if not principal mechanism of CLL cell death following effective therapeutic interventions, suggesting that these pathways are attractive targets for drug devel opment (Fig. 1). BCL-2, which appears to play a central role in the survival of CLL cells through inhibition of apoptosis, may be inhibited through a variety of strategies, some of which have entered clinical trials. A promising approach involves antisense oligonucleotides, which are complementary DNA strands that bind BCL-2 mRNA, prevent protein translation, and decrease mRNA degradation (14). Clinically, Genasense®, a BCL-2 antisense oligonucleotide (Fig. 1), has shown promising activity in CLL and is currently in a randomized phase III trial with fludarabine and cyclophosphamide. Using X-ray crystallographic and nuclear magnetic resonance analysis, the three-dimensional structure of BCL-2 and its homologs have been modeled to create small-molecule drugs that dock in the binding pocket of the BH3 domain (Fig. 1), which is responsible for dimerization, and inhibit its activity (15). It may also be possible to inhibit BCL-2 gene expression through drugs such as flavopiridol (Fig. 1), a protein kinase inhibitor that variably downregulates the levels of the anti-apoptotic proteins BCL-2 and Mcl-1, while having little effect on the expression of pro-apoptotic proteins such as Bax and Bak (16).

The TNF family ligand TNF-related apoptosis-inducing ligand (TRAIL), which binds and induces apoptosis through the death receptors DR4 and DR5, will soon enter clinical trial (17,18) (Fig. 1). Antibodies to CD40L, which block stimulation of CD40 and subsequent NF-kB activation, as well as small-molecule inhibitors of the NF-KB-activating IkB kinases (IKK)a and IKKP (Fig. 1), are under development. In vitro, the CD20 monoclonal antibody rituximab activates apoptosis through the intrinsic pathway and downregulates IAPs (Fig. 1) and the anti-apoptotic Mcl-1 protein. Clinically, rituximab displays synergy with cytotoxic agents, potentially through its unique action on the apoptotic pathways (19). The potential significance of the dATP/ATP binding site on Apaf-1 as a target for development of small-molecule inhibitors is suggested by the finding that purine analogs, such as fludarabine, can increase the catalytic efficiency of Apaf-1 by as much as 50-fold (5).

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