|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
REVIEW ARTICLE
From the Princess Margaret Hospital, University Health
Network, Toronto, ON, Canada.
Acute myeloid leukemia and adult-onset acute
lymphocytic leukemia are aggressive diseases with generally poor
prognoses. The primary cause of treatment failures in patients with
these diseases is the emergence of multidrug resistance. Common
mechanisms of resistant disease are defects in apoptotic pathways.
Apoptosis is a process by which cells undergo organized
self-destruction. Apoptosis is ultimately executed by caspases, a
family of cysteine proteases that cleave critical intracellular
proteins and execute the apoptotic program. At least 2 major pathways
for caspase activation have been identified: (1) the receptor-mediated
pathway which involves members of the tumor necrosis factor (TNF)
family of death receptors and (2) the mitochondrial-mediated pathway
involving cytochrome c-release from the mitochondria. Defects
in the receptor- or mitochondrial-mediated pathway may confer
prognostic information for adult patients with acute leukemia,
suggesting ways that such information can be used in risk
stratification. In addition, emerging knowledge about apoptosis
mechanisms has laid the foundation for the eventual development of new
therapies capable of overcoming blocks in apoptosis. Here we review
some of the recent advances in translating information about apoptosis
mechanisms into promising new strategies for improved treatment of
adult leukemia. Acute leukemia is a neoplastic disease characterized by a rapid
accumulation of primitive hematopoetic cells. Acute leukemia is
subclassified as myeloid or lymphoid depending on the origin of the
malignant cell. Acute lymphocytic leukemia (ALL) is predominantly a
disease of childhood with 75% of all cases occurring in patients younger than 15 years of age.1 In contrast, acute myeloid
leukemia (AML) is more common in adults with an incidence that
increases with age.2 Both AML and adult ALL are aggressive
diseases with generally poor prognosis. For example, patients with AML
who are younger than 55 years of age and who have no adverse prognostic features have a complete remission rate of 70% to 85% with standard induction chemotherapy, but their long-term survival is only
approximately 50%. The outcome in elderly patients is worse. Patients
older than 65 years of age with AML treated with standard chemotherapy have a median survival of 6 months and only 7% remain in remission at
3 years.3 The primary cause of treatment failures in adult patients with leukemia
is the emergence of resistant disease. Multiple mechanisms may account
for why leukemic cells become resistant to chemotherapy (Figure
1). One common cause includes defects in
the cell's inherent programmed cell death (apoptosis) pathways. These
defects may impair the ability to achieve remission and cure with
chemotherapy. In an attempt to overcome drug resistance and improve
clinical outcomes, attention is turning to developing therapeutic
agents that overcome defects in the apoptosis pathways. Although mostly in preclinical stages of development, these efforts offer a glimpse into the future armamentarium of agents that may eventually be available for treating leukemia.
In this review, we focus on translational approaches to apoptosis
research in adult acute leukemia. More basic details of the apoptosis
pathway have been reviewed elsewhere4 with some articles
concentrating on individual pathway components such as the
mitochondria,5,6 the TNF superfamily of
receptors,7-9 Bcl-2,10,11 and
caspases.12,13 Here, we will discuss the apoptosis
pathways concentrating on members with important prognostic or
therapeutic potential. We will then illustrate how defects in apoptosis
confer prognostic information in patients with acute leukemia. By
identifying patients at highest risk of resistant disease or relapse,
one may be able to offer them more aggressive or alternate therapy at
disease onset. Finally, we will discuss small molecules that overcome
blocks in apoptosis and that will likely have a major impact in the
treatment of acute leukemia. Apoptosis is a process by which cells undergo organized
self-destruction without eliciting an inflammatory response.
Morphologically, cells undergoing apoptosis display membrane
blebbing and nuclear and cytoplasmic condensation. Molecularly,
classical apoptosis is caused by the activation of caspases. Caspases
are a family of intracellular cysteine proteases that lie in a latent
(zymogen) state in cells but become activated in response to a wide
variety of cell death stimuli.
Growing evidence indicates that cell death with apoptotic features can
occur without caspase activation14; the mechanisms of
caspase-independent cell death still require clarification. Here, we
will limit our discussion to caspase-mediated cell death, as
understanding these pathways has lead to the development of agents that
enhance apoptosis and may be of therapeutic value.
Caspases are organized in a cascade with upstream (initiator) caspases
responsible for activating the downstream (effector) caspases. Upstream
caspases contain long prodomains that interact with their specific
activators. The downstream caspases contain smaller prodomains and are
activated by proteolytic cleavage by upstream caspases. Once
activated, the downstream caspases cleave specific protein
substrates, leading to the execution of apoptosis.
At present, at least 2 major pathways of caspase activation have been
revealed: (1) the receptor-mediated apoptosis pathway where the TNF
family of death receptors activate upstream
caspase-812,13,15 and (2) the mitochondrial-mediated
apoptosis pathway where cytochrome c is released from the
mitochondria and activates upstream caspase-916-19 (Figure
2). Both pathways culminate in the
activation of a major downstream effector
caspase
Receptor-mediated pathway
This receptor family also induces apoptosis through an alternative
signal transduction pathway. According to one proposed model, Fas
receptors bind the adapter proteins that in turn activate the MAP3
kinase, ASK1. When activated, ASK1 launches a phosphorylation cascade
that culminates in the activation of c-Jun N terminal kinase
(JNK).25 Activated JNK phosphorylates substrates
such as c-Jun and p53 and induces apoptosis through multiple
mechanisms, including modifying and transcriptionally regulating
proteins in the Bcl-2 family.27-29 Therefore, activation
of Fas can induce apoptosis through multiple pathways, although some of
the mechanisms still require clarification.
The TNFRI and TNFRII receptors also activate the MAPK/JNK pathway
by binding the adapter proteins TRADD and TRAF that activate ASK-1.30,31 Interestingly, activation of TNFRI can also
inhibit apoptosis. Activated TNFRI recruits adapter proteins such as
TRADD and TRAF. These adapter proteins launch a phosphorylation cascade that frees NF Recently, the adapter protein Bcl-10 has been identified. This
molecule activates the receptor-mediated apoptosis pathway and the
NF Mitochondrial-mediated pathway
Triggers of mitochondrial-mediated apoptosis Cytochrome c-release from the mitochondria is often a starting point for discussions on caspase-mediated cell death. In reality, however, mitochondrial disruption is an end point for a multitude of diverse signals that culminate in cytochrome c-release and apoptosis. A detailed review of all agents and pathways that disrupt the mitochondria is beyond the scope of this article, but we will highlight a spectrum of triggers that activate the mitochondrial mediated pathway of apoptosis. For the purpose of this discussion, these triggers have been classified as activators, intermediates, and effectors depending on how directly they impact mitochondrial function (Figure 4).
Activators. Activators refer to exogenous cellular stresses such as ionizing radiation, chemotherapy, heat shock, and cytokine withdrawal, that ultimately lead to mitochondrial disruption.5,37-39 The pathways that connect the activators to the mitochondria are complex and often poorly understood. The complexity occurs because a given activator, such as chemotherapy, can up-regulate multiple intermediates that impact the mitochondria through multiple effectors. Some of the pathways have been discerned, however. Daunorubicin can induce apoptosis by activating caspase-840 and by increasing ceramide production.41 Ara-C acts on the mitochondria-mediated pathway of caspase activation as evidenced by its ability to induce cytochrome c-release and caspase-9 activation.42 Ultraviolet (UV) radiation induces mitochondrial-mediated apoptosis through the JNK signaling pathway. Knocking out the JNK1 and JNK2 isoforms in murine fibroblasts renders these cells resistant to UV radiation-induced cytochrome c-release and apoptosis. Intermediates. Cell signaling pathways and oncogenes can trigger apoptosis through the disruption of the mitochondria. Here, too, the pathways connecting the intermediate triggers with the mitochondria are complex because a given trigger can disrupt the mitochondria by up-regulating multiple effectors. The JNK cell signaling pathway is activated in response to a variety of insults, including radiation, heat shock, osmotic stress, and cytokine withdrawal.28 The JNK signaling pathway induces apoptosis in part by phosphorylating and stabilizing p53.27 Once activated, JNK also translocates to the mitochondria and phosphorylates Bcl-xL. This phosphorylation seems to inactivate Bcl-xL and potentiate apoptosis.29 Finally, JNK may mediate cytochrome c-release from the mitochondria by up-regulating the proapoptotic protein and mitochondrial-disrupter Bid.43 Ceramide is a component of the lipid signaling pathway that is synthesized de novo or through the catabolism of sphingomyelin in response to multiple cell stresses, including ionizing radiation, chemotherapeutic agents, and cytokines.44 Through conversion to GD3 ganglioside in the golgi apparatus, ceramide disrupts the mitochondrial membrane.45 In addition, ceramide induces apoptosis by activating the JNK pathway.46 In response to stimuli such as chemotherapy and radiation, p53 activates the mitochondrial-mediated pathway of apoptosis.47,48 p53 induces mitochondrial-mediated apoptosis through multiple mechanisms. It induces the transcription of the proapoptotic protein Bax.49 In addition, p53 enhances reactive oxygen species (ROS) production by transcriptionally activating genes such as PIG3.50 p53 may also disrupt the mitochondria independent of its function as a transcription factor. Studies indicate that within one hour of cell damage, p53 translocates to the mitochondria and triggers apoptosis through a mechanism that remains ill-defined, but is independent of the nucleus.51 The c-myc oncoprotein has diverse biologic properties including the induction of both proliferation and apoptosis.52 Myc deregulation can potentiate apoptosis by p53-dependent and independent mechanisms. Myc indirectly activates p53 by increasing expression of ARF.53 ARF activates p53 by biding and sequestering Mdm2, a protein that normally inhibits p53.54,55 Recently, a role for c-myc in potentiating the activation of Bax independent of an effect on Bax's translocation from the cytosol to the mitochondria has been identified.56 Rb, like p53, is a negative regulator of cell growth. Unlike p53, however, Rb overexpression inhibits apoptosis and can specifically block p53-mediated apoptosis.57Effectors. Direct effectors of mitochondrial disruption include both protein and nonprotein agents. Proapoptotic protein effectors of mitochondrial-mediated apoptosis include Bax, Bak, and Bid. Their effects on the mitochondria are discussed in greater detail below. Nonprotein effectors of mitochondrial dysfunction include calcium, ROS, GD3 ganglioside, and certain chemotherapeutic agents. High levels of intracellular calcium directly damage the mitochondria.45 Mitochondrial damage is also induced by increases in ROS. Blocks in the mitochondria's electron transport chain lead to increased ROS production.58 Increased ROS damages the mitochondrial membranes and induces cytochrome c-release.52 Certain chemotherapeutic agents act directly on the mitochondria. For example, arsenic induces loss of the mitochondrial membrane potential (  M) in intact cells
and a cell-free system.49 Arsenic also has indirect effects on the mitochondria by activating JNK and
p53.59,60
Cytochrome c and the mitochondria Cytochrome c-release from the mitochondria can occur with and without loss ofM and physical mitochondrial
swelling. These differences relate to the site of mitochondrial damage.
A description of the mechanisms of mitochondrial damage is important to
understand the action of key apoptotic regulators such as Bcl-2 and
Bax. Furthermore, these mechanisms serve as the basis for developing therapeutic agents that enhance apoptosis by targeting the mitochondria.
Mitochondria contain 2 membranes
The outer mitochondrial membrane contains the voltage-dependent anion
channel (VDAC). The VDAC is involved in mitochondrial respiration and
controls ATP efflux from the mitochondrial outer membrane.63,64 Disruptions of VDAC lead to increased
permeability of the outer membrane and cytochrome c-release. In this
scenario, the inner membrane remains intact, so there is no loss
of BCL-2 family proteins The mitochondrial-mediated pathway of apoptosis is regulated by the Bcl-2 family of antiapoptotic and proapoptotic proteins (Figure 6). Bcl-2 family proteins contain shared Bcl-2 homology (BH) domains BH1, BH2, BH3, and BH4. Currently, therapeutic agents that either block or mimic these domains are being developed. The BH1 and BH2 domains are present in all of the survival factors as well as the death agonists Bax, Bak, and Mtd. These domains are likely involved in channel formation as their 3-dimensional structure resembles the pore-forming region of the diphtheria toxin.68-70 The difference in the charge and amino acid sequences between the BH1 and BH2 domains of the proapoptotic and antiapoptotic proteins may lead to different pore properties and account for the differences in function between these molecules.
The BH3 domain is a death-promoting region common to all members of the
Bcl-2 family (Figure 7). The BH3 domain
contains a core sequence of 8 amino acids of which the leucine at
position 1 and the aspartic acid at position 6 are critical for
heterodimerization. Mutation of these residues prevents
heterodimerization and abrogates the toxicity of the death
agonists.71-73 In the antiapoptotic members, the BH3, BH2,
and BH1 domains form a pocket. This pocket serves 2 purposes. First, it
buries the BH3 domain so it cannot exert its death-promoting activity.
Second, the pocket is the docking site for the BH3 domains of the death
agonists.68,70 Heterodimerization with the death agonists
inhibits the function of the survival factors, as discussed
below.74
The BH4 domain is found primarily in the prosurvival factors and appears to be involved in protein-protein interactions with regulatory proteins outside the Bcl-2 family. This domain is involved in controlling progression of the cell cycle. It also appears to stabilize the VDAC in the outer mitochondrial membrane.75 A regulatory loop domain between the BH3 and BH4 domains is found in the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1. Interestingly, cleavage of Bcl-2 at the loop domain by caspase-3 releases the BH4 domain and turns Bcl-2 into a potent proapoptotic protein.76 Perhaps by cleaving the loop domain, the BH1, BH2, BH3 pocket is disrupted, leading to exposure of the death-promoting BH3 domain. The importance of this cleaved isoform in regulating the apoptosis pathways is unknown. Bax. The Bax family of death agonists act at the mitochondria to induce apoptosis. Three mechanisms have been identified by which Bax induces apoptosis. First, at the outer mitochondrial membrane, Bax accelerates the opening of VDAC with subsequent release of cytochrome c.65 Second, at the inner mitochondrial membrane, Bax interacts with Ant and enhances the permeability of the inner mitochondrial membrane.73 Finally, when expressed at high levels, Bax is capable of forming pores by direct oligomerization mechanisms that disrupt the permeability of the mitochondrial membranes and induce cytochrome c-release.66,67,77 Bcl-2.
The Bcl-2 class of antiapoptotic proteins are important inhibitors of
the mitochondrial-mediated pathway of apoptosis and are common targets
of novel therapeutic agents. These proteins inhibit apoptosis by
interacting with Bax/Bak, and the mitochondria. Bcl-2 can inhibit the
action of Bax/Bak by forming inactivating heterodimers.74
Early reports suggested that Bcl-2 functioned as an antioxidant by
preventing ROS generation.78,79 In retrospect, this
function likely relates to the role of Bcl-2 at the mitochondria. Bcl-2
binds VDAC and stabilizes it, thereby preventing permeabilization of
the outer mitochondrial membrane.80 These antiapoptotic
molecules also homodimerize and form small ion channels in synthetic
lipid vesicles.81 These channels may also exist in the
mitochondrial membrane where they stabilize the membrane and prevent
loss of BAD, a BH3-only protein. The BH3-only class of proteins are endogenous inhibitors of Bcl-2 and Bcl-xL and are attractive prototypes for therapeutic agents. This class of compounds inhibits Bcl-2/Bcl-xL through heterodimer formation. Among the BH3-only class of proteins, BAD was the first identified and is the best characterized. BAD was first cloned from a murine cDNA library through its ability to bind Bcl-2 in a yeast-2-hybrid assay.91 BAD heterodimerizes with Bcl-xL and Bcl-2 but not the proapoptotic members Bcl-xs, Bax, or Bak.91 To date, the only known function of BAD is to bind Bcl-2 and Bcl-xL, and block the antiapoptotic action of these proteins.71,92 BAD inhibits these antiapoptotic proteins by preventing them from binding Bax/Bak.71,92 BAD may also inhibit the ability of Bcl-2 and Bcl-xL to form channels in the mitochondrial membrane, or bind and stabilize VDAC. The BH3 domain of BAD is necessary for its ability to bind Bcl-2 and Bcl-xL and induce apoptosis. Mutations within the BH3 domain of full-length BAD inhibit the binding of BAD to Bcl-2 and Bcl-xL and block BAD-induced apoptosis in cells overexpressing Bcl-xL.72 Furthermore, peptides corresponding to the BH3 domain of BAD competitively inhibit the binding of Bcl-xL to Bax. Mutations within these peptides permit the binding of Bcl-xL to Bax.71Cross talk between pathways Although the mitochondrial-mediated and receptor-mediated pathways are presented as separate entities, cross talk exists between them. For example, the 2 pathways cooperate to enhance apoptosis through Bid. Bid, a member of the BH3-only family of proapoptotic proteins, is cleaved and activated by caspase-8. When cleaved, Bid interacts with ANT as well as Bax to permeabilize the mitochondrial membrane.93 In addition, cleaved Bid forms ion channels in liposomes, suggesting that it independently can disrupt the mitochondria and induce cytochrome c-release.94,95 Conversely, BAR inhibits apoptosis in both pathways. BAR was identified by screening for cDNAs capable of inhibiting Bax-induced apoptosis. BAR contains a protein-interacting domain that binds and inhibits procaspase-8 and thereby blocks the receptor-mediated pathway. BAR also inhibits the mitochondrial-mediated pathway through an unknown mechanism that involves interactions with Bcl-2 and Bax. Physically, BAR bridges the 2 pathways by forming a complex with procaspase-8 and Bcl-2, although the significance of this complex is unknown.96Regulation of apoptotic proteins through phosphorylation The apoptosis pathways are positively and negatively regulated through signal transduction. Teleologically, this regulation allows tight control of apoptotic signals avoiding unwanted cell death or proliferation. Therapeutically, these regulatory points are valuable targets for the development of compounds that enhance apoptosis. Bcl-2 and BAD are examples of proteins that are regulated by phosphorylation and whose regulation is being exploited for drug development.Three known sites of phosphorylation regulate Bcl-2: ser-17, ser-70, and thr-56. Taxol induces phosphorylation on multiple residues, including ser-17, and inhibits the antiapoptotic function of Bcl-2.97,98 Phosphorylation of Bcl-2 on thr-56 in the loop domain by the cell-cycle regulator CDC2 kinase is required for activation of the role of Bcl-2 as a cell-cycle inhibitor. Mutation of thr-56 abrogates the role of Bcl-2 as a cell-cycle inhibitor, but does not alter the antiapoptotic action of the protein, indicating that the function of Bcl-2 as an inhibitor of apoptosis and a cell-cycle inhibitor can be dissociated.83,84 Some aspects of Bcl-2 phosphorylation are controversial. One report indicates that phosphorylation of ser-70 by protein kinase C (PKC) activates the antiapoptotic function of Bcl-2,99 but another study reports that taxol treatment leads to phosphorylation on the same serine and inhibits its function.100 Other sites of phosphorylation also appear important for the regulation of Bcl-2 and are currently being characterized. For example, all-trans retinoic acid (ATRA) inhibits Bcl-2 by inducing phosphorylation on a serine distinct from the sites phosphorylated by PKC and the taxol-mediated pathway.101 BAD is an example of a proapoptotic protein that is regulated by phosphorylation. Phosphorylation of ser-112, ser-136, or ser-155 inhibits the function of BAD. BAD is phosphorylated on ser-136 by Akt in the Akt/PI3K pathway102,103 and on ser-112 by PKC in the Ras/Raf/MAPK pathway.104,105 Phosphorylation at these sites promotes the binding of BAD to 14-3-3 proteins.92 When bound to 14-3-3 proteins, BAD is sequestered in the cytosol and unable to interact with Bcl-2 or Bcl-xL at the outer mitochondrial membrane. The phosphorylation of ser-136 or ser-112 also induces a conformational change in BAD that increases the accessibility of ser-155 within the BH3 domain to phosphorylation by protein kinase A.106,107 Phosphorylation of ser-155 directly impairs the ability of BAD to bind Bcl-xL and Bcl-2.107,108 Pathways that dephosphorylate BAD increase its activity and promote apoptosis. Calcineurin, a calcium-dependent phosphatase, dephosphorylates BAD at both S112 and S136 and promotes BAD-induced apoptosis.109 The second messenger ceramide promotes the accumulation of dephosphorylated forms of BAD by inhibiting Akt.110
At a basic level, chemoresistance and treatment failures in acute leukemia represent a failure of the malignant clone to undergo apoptosis in response to chemotherapeutic agents. Resistance to apoptosis in acute leukemia is often due to aberrant gene expression. Chromosomal translocations are classic examples of aberrant gene expression in acute leukemia. The molecular basis of translocations in leukemia has been reviewed elsewhere111 and detailed discussions of the effect of chromosomal translocations on apoptosis and prognosis are beyond the scope of this article. Here, we highlight specific examples of how translocations impact apoptosis in acute leukemia. BCR-ABL The BCR-ABL fusion protein is created as a result of the t(9;22) translocation. This fusion protein is pathognomonic for chronic myeloid leukemia (CML) and is found in 25% of adult ALL where it confers a poor prognosis. This fusion protein dysregulates the ABL tyrosine kinase, leading to activation of several signaling pathways including Ras,112 AKT/PI3Kinase,113 NF B,114,115 and STAT.116 The BCR-ABL
fusion protein inhibits apoptosis, in part, by constitutively
activating STAT 5 that increases transcription of
Bcl-xL.117 The tyrosine kinase inhibitor STI 571 inhibits the ABL kinase and induces apoptosis in CML and leukemia
cells.118 Clinically, STI 571 has utility in the treatment
of CML119 and BCR-ABL-positive ALL.120
Mixed lineage leukemia-ELL Translocations involving the mixed lineage leukemia (MLL) gene on 11q23 are found in 4% of cases of AML and confer an intermediate prognosis.121 Over 25 fusion partners for MLL have been identified.111 Both the abnormal MLL gene and its fusion partners inhibit apoptosis, but the mechanism by which the abnormal MLL gene inhibits apoptosis is not well understood. The effects of the fusion partners are better characterized in some cases. For example, in the t(11;19) translocation, MLL is fused to the ELL/MEN gene. The dysregulated ELL gene inhibits apoptosis by binding p53 and thereby reducing its transcriptional activity.122PML-RAR  is present in most forms of AML M3.123
Overexpression of the fusion transcript in U937 leukemic cells inhibits
apoptosis in response to serum starvation and TNF. The mechanism by
which PML-RAR inhibits apoptosis is complex, but likely relates to inhibition of RAR-induced genes including STATs, Hox, and cell cycle
regulators. For a more detailed discussion of the PML-RAR fusion
transcript, the reader is referred to a recent review in this
journal.123
This review focuses on adult-onset acute leukemia, but we will briefly discuss childhood acute leukemia to illustrate how differences in disease biology translate into different clinical outcomes. Childhood and adult-onset ALL are clearly different diseases. Clinically, 80% of children survive long-term after treatment of ALL, but only about 30% of adults are alive 5 years after treatment.2 Biologically, childhood and adult ALL differ as well. For example, the spectrum of chromosomal translocations varies between these 2 groups. The t(9;22) translocation, for example, is present in 25% of adult ALL and is present in only 5% of childhood ALL.124 As discussed above, chromosomal translocations such as t(9;22) inhibit apoptosis and lead to treatment failures. Studies comparing expression levels of members of the receptor- and mitochondrial-mediated pathways of apoptosis between adult and childhood ALL have not been performed. However, it would not be surprising to find higher levels of antiapoptotic proteins in adults than in children given the higher prevalence of chemoresistant and relapsed disease among adults. In support of this hypothesis, longitudinal studies reported changes in levels of apoptotic proteins in children at the time of relapse compared with diagnosis. Bcl-2 expression increased at relapse compared with diagnosis, and levels of Bax, mutant p53, Fas, and active caspase-3 declined.125-127 All of these changes could contribute to more aggressive and less responsive disease. Studies specifically comparing apoptotic members in adult and childhood ALL are warranted because they will further our understanding of the biologic differences between these 2 diseases. With a better understanding of these differences, more risk-adapted therapy can be applied.
Defects in the apoptosis pathway render leukemic blasts resistant to multiagent chemotherapy. These blocks translate clinically into reduced rates of remission, higher rates of relapse, and reduced overall survival. Studies have identified specific blocks in the receptor-mediated and mitochondrial-mediated pathways that confer prognostic information in adult patients with acute leukemia. Receptor-mediated pathway There is evidence to implicate defects in the receptor-mediated pathway of caspase activation with chemoresistance in leukemia. Exposure of the T-ALL cell line CEM to doxorubicin induces apoptosis and up-regulates Fas expression. A mutant CEM cell line resistant to Fas ligand fails to up-regulate Fas or undergo apoptosis in response to doxorubicin treatment.128 In contrast to this report, a Jurkat cell line resistant to Fas ligand retained sensitivity to doxorubicin-induced apoptosis.129 The discrepancy between these 2 studies may reflect the numerous sites of blockage in the receptor-mediated pathway that can induce resistance to Fas ligand. A defect at the level of the Fas receptor may render a cell resistant to Fas ligand, but if doxorubicin triggers the pathway downstream of the receptor, then the cell would remain sensitive to doxorubicin. On the other hand, defects in the receptor-mediated pathway downstream of where doxorubicin acts will render cells resistant to both Fas ligand and doxorubicin. Alternatively, the discrepancy may result because chemotherapeutic agents effect multiple pathways simultaneously. As cells acquire defects in one pathway they unmask the effects of the chemotherapeutic agent on the other pathway.Fas expression confers important prognostic information in patients with leukemia. The Fas gene encodes 2 isoforms: full-length Fas with a transmembrane domain, and a soluble form of Fas that lacks this transmembrane domain. Full-length Fas is the initiator of the receptor-mediated pathway of apoptosis. In contrast, soluble Fas is excreted into the extracellular environment where it acts as a decoy and binds Fas ligand. As such, soluble Fas is an inhibitor of apoptosis. In a study of 59 patients with adult T-cell leukemia, levels of full-length Fas expression did not influence patient survival. However, increased serum levels of soluble Fas were associated with reduced survival and remained an important predictor in multivariate analysis even after accounting for clinical risk factors.130 The prognostic importance of defects in the receptor-mediated pathway has not received much attention to date. Perhaps in the future, additional prognostic markers within this pathway will be identified. For example, caspase-8 is inhibited by the dominant-negative inhibitor I-FLICE. I-FLICE overexpression has been documented in the BCR-ABL-positive cell line K562, but its incidence and prognostic importance in patients with leukemia is unknown.131 Mitochondrial-mediated pathway Defects in the mitochondrial-mediated pathways of apoptosis also induce multidrug resistance and confer important prognostic information. Most of the research on this pathway has focused on the Bcl-2 family of proteins.Bcl-2. Overexpression of Bcl-2 is an important cause of multidrug resistance. As a result, patients with AML and high levels of Bcl-2 have lower complete remission rates and a lower overall survival compared with patients with low levels of Bcl-2. For example, in a study of 82 consecutive patients with AML treated with standard induction chemotherapy, patients with high levels of Bcl-2 expression by immunofluroescent staining had a complete response rate of 29% versus an 85% complete response rate in patients with low levels. Likewise, high levels of Bcl-2 translated into decreased overall survival. The predicted survival at 2 years in patients with increased Bcl-2 was 15% versus 40% in patients with low levels of Bcl-2. Bcl-2 remained an important predictor of survival in multivariate analysis over other known prognostic markers, including age and white blood cell (WBC) count of more than 30. The influence of cytogenetics was not reported, however.132 Some studies have indicated that increased levels of Bcl-2 are not prognostic or even associated with improved survival. In patients with breast cancer, increased Bcl-2 by immunostaining correlated with improved disease-free survival,133 and in childhood ALL, increased Bcl-2 was associated with improved event-free survival.134 These results may reflect the importance of posttranslational modification of Bcl-2. For example, under certain circumstances PKC activates Bcl-2,99 so in the presence of PKC, the Bcl-2 that accumulates is functional, conferring a worse outcome. In support of this hypothesis, lower levels of PKC were associated with an improved prognosis in patients with AML.135Bax. In a study of 56 patients with newly diagnosed AML, increased levels of Bax expression by immunoblotting correlated with improved rates of overall survival.136 In contrast, a study of 165 patients with newly diagnosed AML135 reported that levels of Bax expression by immunoblotting did not correlate with response to induction chemotherapy or survival. However, high ratios of Bcl-2 to Bax protein conferred a poor prognosis with decreased rates of complete remission and overall survival. The prognostic discrimination from the ratio of the proteins was greater than Bcl-2 levels alone. The ratio also provided prognostic information above cytogenetics. Both studies are flawed, however, in that the patients selected were not consecutive and inclusion criteria were not predefined. Downstream effectors. Levels of caspase inhibitors have been correlated with survival in AML. IAPs are a family of caspase inhibitors that bind and inactivate caspases, including caspases 3 and 9. As such, they block apoptotic stimuli in both the mitochondrial-mediated and receptor-mediated pathways. In a study of 78 patients with AML, 76 had detectable levels of XIAP. Patients with the lowest levels of XIAP expression had the longest median survival.137 The importance of XIAP in the context of other clinical prognostic factors is unknown, however. Likewise, a trend to improved survival was seen in patients with low levels of the IAP inhibitor survivin.138 In another study of patients with newly diagnosed AML, increased levels of procaspase-3 in peripheral blood mononuclear cells correlated with a poor prognosis. However, patients with spontaneously increased levels of active caspase-3 at the time of diagnosis showed improved survival.139 Likewise, in 45 consecutive adult patients with ALL, increased levels of active caspase-3 correlated with an improved rate of complete remission.140 In multivariate analysis, levels of caspase-3 were no longer predictive of survival, but the small sample size may have obscured the predictive effect. The poor prognosis among patients with high levels of procaspase-3 may represent defects in caspase activation and the subsequent accumulation of uncleaved caspase-3. The better prognosis among patients with increased levels of cleaved caspase-3 may indicate competent caspase activation pathways. In contrast to these findings, Svingen et al141 found no correlation among levels of Apaf-1, procaspases 3, 8, and 9, and response to induction chemotherapy in adult patients with AML (n = 42) or ALL (n = 18). A number of explanations may account for the lack of correlation. First, the study had a small sample size and thus had limited power to detect a difference. However, the wide variation in the levels of these apoptotic members suggests that simply increasing the number of patients would not reveal a correlation. Alternatively, this study used response to chemotherapy as its end point. The overall response rate for patients with AML after the first induction was 53%, which is lower than expected. Therefore, this group of patients may have had a particularly aggressive disease due to other molecular abnormalities that overshadowed the importance of procaspases or Apaf-1.
Classical chemotherapeutic agents induce cell death through a
variety of mechanisms, including directly or indirectly affecting regulators of the apoptosis pathway. Rather than review the mechanism of action of chemotherapeutic agents, however, we will focus on small
molecules that specifically overcome blocks in the apoptosis pathway.
Attempts to develop these novel therapeutic compounds have targeted
both the receptor-mediated and mitochondrial-mediated pathways of
apoptosis (Figure 8).
Receptor-mediated pathway Small molecules that target the receptor-mediated pathway can induce caspase-3 activation and overcome blocks confined to the mitochondrial-mediated pathway, such as the overexpression of Bcl-2 or Bcl-xL.TRAIL. Treatment with TNF can induce apoptosis through the receptor-mediated pathway, but also produces severe toxicity to normal tissue.142-144 In contrast, TRAIL (APO2L), which is similar to Fas ligand and activates the DR3 and DR4 receptors, shows selectivity for tumor cells despite the presence of the DR4 receptor on normal tissues.145 In a study by Ashkenazi et al,146 soluble TRAIL induced cell death and apoptosis in a variety of leukemic and solid tumor cell lines. In contrast, normal tissue including prostate, fibroblasts, colon, and smooth muscle cells were unaffected by exposure to TRAIL. In nude mice, intraperitoneal treatment with TRAIL induced apoptosis and shrinkage of colon cancer xenografts. No observable toxicity from TRAIL was observed in these mice or in studies of nonhuman primates. Recently, Altucci et al147 demonstrated that ATRA induces apoptosis in acute promyelocytic leukemia (APL) cells through the production and paracrine action of TRAIL. Furthermore, the addition of exogenous TRAIL to APL cells and an ATRA-resistant cell line induced apoptosis. Therefore, clinically, TRAIL may have a role in the treatment of APL perhaps in synergy with ATRA and chemotherapy. TRAIL may also be quite useful in treating patients with ATRA-resistant disease. Caspase-8 activators. Compounds that activate the receptor-mediated pathway at the level of caspase-8 may overcome blocks to apoptosis upstream of this level of regulation, such as at the level of the TNF-receptor family. Such compounds also can act independent of the level of expression of the TNF receptor. One such agent is 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO). CDDO is a synthetic compound that is synthesized in an 11-step reaction from oleanolic acid. This compound is a potent analogue of the naturally occurring triterpenoids which are used as herbal remedies in the treatment of inflammation and cancer. CDDO induces apoptosis in leukemic cell lines by activating procaspase-8. As the compound targets the receptor-mediated pathway, overexpression of Bcl-xL has little effect on the toxicity of this compound.148 Currently, it is unknown how C |
Top
Introduction
Apoptotic pathways
caspase-3.
NAD+ + H20. (B) ATP and H+
flux across the mitochondrial membranes. The reoxidization of NADH to
NAD+ in the electron transport chain produces H20 and
releases energy. This energy is used to drive the production of ATP in
the mitochondrial matrix. The adenine nucleotide transporter (Ant)
controls the flux of ADP and ATP across the inner mitochondrial
membrane. The voltage dependent anion channel (VDAC) regulates the flux
of ATP across the outer mitochondrial membrane. Damage to VDAC or ANT
can induce cytochrome c-release and initiate the
mitochondrial-mediated pathway of apoptosis. Damage to ANT is also
accompanied by physical mitochondrial swelling and loss of
