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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3587-3600
REVIEW ARTICLE
By
From the Department of Hematology, Royal Free and University College
School of Medicine, London, UK.
GENETIC CHANGES involving oncogenes and
tumor suppressor genes contribute to the deregulated expansion of
malignant cells. While some of these changes result in increased
proliferation, others contribute to an increase in cell numbers by
inhibiting apoptosis (programmed cell death).1 Because
cytotoxic drugs or irradiation result in cell killing by apoptosis, the
genetic changes underlying malignancy often reduce the ability of these agents to destroy malignant cells.1,2 The elucidation of the pathways involved in the regulation of apoptosis in normal and
malignant hematopoietic cells is therefore likely to contribute to the
development of improved therepeutic stategies in the treatment of
leukemia and lymphoma. This review first summarizes recent advances in
the understanding of the control of apoptosis. Examples of how this
control is altered in leukemic cells is then described.
Apoptosis is a tightly regulated form of physiological cell death which
is dependent on the expression of cell-intrinsic suicide machinery.3 Prominent morphological changes include cell
shrinkage, condensation of the nuclear chromatin, fragmentation of the
nucleus, and cleavage of chromosomal DNA at internucleosomal sites,
resulting in the generation of a characteristic ladder pattern of DNA
fragments on electrophoresis. Blebbing of the cell surface results in
the release of membrane-bound apoptotic bodies.3
Phosphatidylserine, which is normally located on the inner face of the
plasma membrane, becomes exposed on the outer surface and provides a
recognition signal for engulfment by phagocytes.4,5 Thus,
apoptosis results in the rapid and efficient removal of superfluous or
damaged cells.
Genetic studies in the nematode C elegans have resulted in the
identification of a set of genes involved in the regulation of
apoptosis.6 The ced-3 gene encodes a cysteine
protease, which is homologous to members of the caspase protease family that execute the apoptotic program in mammalian cells (see section 3.1). The ced-4 gene product is required for the activation of CED-3. This activation step is blocked by the CED-9 protein,
which is homologous to mammalian BCL-2. BCL-2 can substitute for CED-9 in blocking apoptosis in C elegans7,8 whereas
overexpression of CED-4 induces apoptosis in mammalian
cells,9 suggesting a high degree of conservation of the
mechanisms of apoptosis regulation. Therefore, the C elegans
model has been of value in the identification of the proteins that
control apoptosis in human cells (see sections 3 and 4).
The induction of apoptosis may conveniently be divided into three
stages: (1) the interaction of the inducing signal with the cell, (2)
biochemical transduction of the death signal, and (3) the execution of
apoptosis. Because different extracellular signals and signal
transduction pathways converge on a final common pathway during the
execution phase, this terminal stage of apoptosis will be summarized first.
3.1. The caspase family of proteases mediates the terminal stages of
apoptotic cell death.
The terminal stages of apoptosis involve the activation of a related
family of proteases, the caspases.10,11 These enzymes possess an essential cysteine residue within their active sites and
cleave substrates adjacent to aspartate residues. The cDNAs encoding 10 caspases have been cloned.10
3.2. Specific protease inhibitors block cell death by targetting
terminal caspases.
In vitro studies suggest that the inhibitor of apoptosis (IAP) family
of proteins may modulate cell death via abrogation of caspase activity.
3.3. Ligation of FAS or the tumor necrosis factor
(TNF) receptor results in the direct activation of
caspases.
FAS and the TNF receptor are structurally related transmembrane
receptor proteins. Their extracellular domains bind FAS ligand and TNF,
respectively, resulting in the formation of receptor trimers. The
cytoplasmic domain of FAS contains a "death domain" (Fig 2), whose elimination results in the
abrogation of cell killing.19 The death domain of FAS
recruits the death domain of the FADD (Fas-associated death domain)
protein following receptor trimerization.20 FADD also
contains a death-effector domain, which mediates interaction with
similar amino acid sequences in the pro-domain of pro-caspase 8.21,22 Trimerization of the FAS/FADD/pro-caspase 8 complex after ligand binding results in the cleavage of the pro-caspase and
generation of active caspase 8. Caspase 8 then cleaves
pro-caspase 3, probably via activation of an unidentified
intermediate caspase (Fig 2).
3.4. Apoptosis induction by the perforin/granzyme system.
Killing of target cells by cytotoxic T lymphocytes plays a major role
in defense against malignant and virus-infected cells, and contributes
to transplant rejection and autoimmune disease. Killing is preceded by
the release of the contents of cytotoxic T-cell granules, which contain
perforin and the serine proteases granzymes A and B. Perforin forms a
pore in the plasma membrane of the target cell, thereby allowing entry
of granzyme B into the cytosol.23 Granzyme B cleaves and
activates caspase 3 in cell-free systems.24,25 However, the
primary target of granzyme B in intact cells is likely to be caspase
10, whose activation results in the subsequent activation of caspase
3.26 In cell-free systems, the addition of granzyme B
initiates cleavage of several apoptosis-specific substrates and also
induces chromatin condensation. Abrogation of these events by selective
inhibitors suggests that activation of caspase 3 (and possibly of
caspase 7) may be important mediators of apoptosis induction by
cytotoxic T-cell-derived granzyme B.26 However, genetic
studies have shown that the granules of cytotoxic T cells contain
additional cytotoxic components in addition to perforin and granzymes A
and B.27
4.1. The BCL-2 protein family plays a central role in the regulation of
apoptosis.
The 26-kD BCL-2 protein protects cells from the induction of
apoptosis by diverse stimuli, including the withdrawal of survival factors, heat shock, and treatment with DNA damaging
agents.28-30 BCL-2 is the prototype of a family of related
proteins. Other anti-apoptotic family members include
BCL-XL, BCL-w, MCL-1, and A1. In contrast, the BAX, BAK,
and BAD proteins are examples of pro-apoptotic BCL-2 family members
whose overexpression promotes cell killing.30 The conserved
BH1 (BCL-2 homology 1) and BH2 domains of
the anti-apoptotic proteins form a hydrophobic cleft which binds the
BH3 domains of pro-apoptotic family members, at least in
vitro.31,32
4.2. Cytochrome c triggers caspase 3 cleavage via activation of
caspase 9.
Three proteins have been purified from the cytosol of HeLa cells which,
when recombined in the presence of adenosine triphosphate (ATP) (or
deoxy ATP), were necessary and sufficient for the cleavage and activation of pro-caspase 3. These proteins, originally designated as Apaf 1, 2, and 3 (Apaf = apoptotic
protease activating factor) have been
characterized.40,41 Apaf 1 contains a central domain with
homology to C elegans CED-4.40 The amino-terminal
domain of Apaf 1 is homologous to the CARDs (caspase
recruitment domains) of some caspases
(Fig 3). The carboxy terminus consists of
several WD repeats, which mediate interactions between certain
regulatory proteins. Apaf 2 was found to be identical to
cytochrome c,40 while Apaf 3 is identical to caspase
9.41
4.3. The BCL-2 protein family apparently regulates the release of
cytochrome c from mitochondria.
Cytochrome c is released from mitochondria during apoptosis induced by
diverse stimuli.11 Overexpression of BCL-2 or
BCL-XL inhibit cytochrome c release induced by etoposide,
actinomycin D, oxidative stress, Fas ligation, or interleukin-3 (IL-3)
withdrawal.43-45 The BAX protein, on the other hand,
triggers redistribution of cytochrome c in the absence of apoptotic
stimuli.46 Thus, BCL-2 and BCL-XL may prevent
apoposis by inhibiting cytochrome c release while BAX favors cell death
by promoting its relocation to the cytosol. However, it is unclear
whether these actions of the BCL-2 family result from the direct
actions of these proteins on mitochondria, which then initiate caspase
activation or whether cytochrome c release is secondary to BCL-2
family-regulated caspase activation and plays a subsequent role in
amplification of the apoptotic signal.47
4.4. Caspase action on mitochondria amplifies the initial apoptotic
signal via a positive feedback loop.
Caspases can themselves trigger cytochrome c release, because selective
inhibitors of these proteases can abrogate release in response to some
stimuli.45 Furthermore, death signals including ligation of
Fas, which directly activate caspases (section 4.2), may nevertheless
be amplified via caspase-mediated cytochrome c release.45
Recombinant caspases disrupt 4.5. Survival factors regulate apoptosis via phosphorylation of the
BAD protein.
The BAD protein is a pro-apoptotic member of the BCL-2
family.30,59 FL5.12 lymphoid cells depend on IL-3 for their
survival in vitro. In cells cultured in the presence of IL-3, the
BAD protein is phosphorylated on serine residues.
Phosphorylated BAD is sequestered via binding to the 14-3-3 protein and
is, therefore, unable to promote apoptosis.59 The BAD
protein rapidly becomes dephosphorylated in the absence of IL-3,
dissociates from 14-3-3, and triggers apoptosis
(Fig 5).
The p53 protein plays an important role in the coupling of DNA damage
to cell-cycle arrest and to the induction of apoptosis. In cells with
undamaged DNA, p53 protein levels are maintained at a low level as a
result of rapid turnover. An increase in stability after the induction
of DNA damage results in an increased level of p53.63 The
protein product of the ataxia telangiectasia (atm) gene
participates in a pathway that links the detection of DNA damage to the
upregulation of p53.64 However, the carboxy terminus of the
p53 protein itself can bind to damaged DNA,65 suggesting that both p53 and the putative damage detector may colocalize at the
site of DNA damage. The radiation resistance of thymocytes derived from
p53 "knockout" mice when compared with wild-type thymocytes66,67 emphasizes the importance of p53-dependent mechanisms in the induction of apoptosis after DNA damage induction. Upregulation of p53 also results in cell-cycle arrest. The pathways involved in this facet of p53 action have recently been
reviewed.63
5.1. Transcriptional activation by p53.
A tetramer of p53 molecules functions as a transcription factor that
binds to consensus sequences in the 5' untranslated regions of
specific target genes.63 The upstream region of the BAX
gene contains p53 consensus binding sites.68 Enforced p53
expression augments BAX expression, which is followed by apoptosis
induction.69 Genetic studies on apoptosis induction in
adriamycin-treated mouse fibroblasts suggest that BAX is an important
(but not the only) effector of p53-mediated apoptosis.70
However, thymocytes isolated from p53 "knockout" mice expressing
elevated levels of BAX are as resistant to etoposide-induced apoptosis
as are thymocytes from p53 "knockout" mice expressing normal
levels of bax.71 Therefore, mechanisms other than BAX
induction may mediate p53-dependent apoptosis, at least in some cell types.
5.2. Transcriptional repression by p53.
In addition to its transactivating properties, p53 represses
transcription from several promoters that lack p53 binding sites. BCL-2
can relieve this transcriptional repression and also protect cells from
apoptosis, suggesting that inhibition of transcription of specific but
as yet unidentified genes may contribute to the ability of p53 to
induce apoptosis.77 However, p53 is also able to induce
apoptosis via pathways that are not dependent on the regulation of gene
expression.78 Therefore, induction of apoptosis by p53 can
occur by diverse pathways depending on the cellular context. It is also
clear that some apoptotic pathways do not involve p53. For example,
thymocytes from p53 knockout mice are resistant to etoposide and
radiation but not to glucocorticoids.67 Furthermore, HL60
cells, which have lost both p53 alleles, are extremely sensitive to
apoptosis induction by drugs that induce DNA
strand-breaks.79 The p53 dependence of apoptotic pathways is also tissue dependent, because radiation-induced apoptosis is
compromised in the thymus of p53 knockout mice, but not in the
lung.80
5.3. p53 loss results in resistance to cytotoxic regimes.
When transplanted into imunodeficient mice, fibrosarcomas expressing
functional p53 show a high proportion of apoptotic cells and regress
after treatment with adriamycin or 5.4. The p53-related p73 gene product.
The p73 gene encodes a protein that is closely related to
p53.83 Overexpresion of p73 results in the induction of
some genes that are also targets of p53, and also induces apoptosis.
However, p73 expression is apparently not augmented after the induction of DNA damage.83 Therefore, there is at present no evidence implicating p73 in DNA damage-induced cell killing.
During hematopoiesis, the survival of progenitor cells is regulated
both positively and negatively by a complex, interacting network of
cytokines and adhesion molecules.84 Noncycling primitive CD34+ human hematopoietic progenitors require the
continuous presence of IL-3 or granulocyte-macrophage
colony-stimulating factor (GM-CSF) for survival in vitro. In contrast,
other cytokines including IL-6 and IL-11 trigger proliferation of these
progenitors.85 Stem cell factor, Flt ligand, and IL-3
suppress apoptosis in single-cell assays designed to test the direct
actions of cytokines on primitive progenitors. Thrombopoietin is more
effective in preventing apoptosis than any of these
cytokines.86 Cytokines show target cell selectivity in
preventing apoptosis. For example, stem cell factor selectively promotes survival of primitive hematopoietic cells, whereas IL-3 blocks
cell death in more committed progenitors.87 Flt ligand is
selective for progenitors committed to the myeloid
lineage.88
Treatment of leukemia cell lines with cytotoxic drugs results in the
release of cytochrome c43-45 and the activation of
caspases.100,101 Caspases are also activated after
cytotoxic treatment of freshly isolated B chronic lymphocytic leukaemia
(B-CLL) cells.102 However, the mechanisms that couple DNA
damage to more downstream regulatory events are largely unclear.
Evidence of a largely circumstantial nature suggests that some of the
mechanisms of apoptosis control described in sections 3, 4, and 5 are
deregulated in leukemia cells, thus contributing to their abnormal
expansion and, in some cases, to drug and radiation resistance.
Deregulation of apoptosis results from translocations involving genes
that encode cell death-regulating proteins. However,
microenvironmental factors also impinge on both the basal survival of
leukemia cells and their killing by cytotoxic regimes. Some of this
evidence is summarized next.
8.1. Translocation of the BCL-2 gene in non-Hodgkin's lymphoma (NHL).
The t(14;18) chromosomal translocation associated with NHL results in
the juxtaposition of the BCL-2 gene to the Ig heavy chain (IgH)
locus.103 Translocation results in enhanced levels of BCL-2
mRNA, which may be partially attributable to the presence of a powerful
transcriptional enhancer in the IgH locus.98 The efficiency
of splicing of BCL-2 exons is also increased as a result of their
fusion to Ig gene introns in t(14;18) cells. The resulting increase in
cellular levels of spliced BCL-2 open reading frames also contributes
to the upregulation of BCL-2 protein levels in NHL
cells.104 Transgenic mice carrying a BCL-2 gene expressed via the IgH gene enhancer overexpress BCL-2 specifically in B-lymphoid cells. These mice accumulate abnormal numbers of small,
nonproliferating B cells that show extended survival in
vitro.105,106 BCL-2 overexpression alone is insufficient
for lymphomagenesis. However, doubly transgenic mice in which
overexpression of both BCL-2 and c-MYC is targetted to B-lymphoid cells
rapidly develop tumors originating from primitive lymphoid-committed
lymphoid cells, suggesting that a second genetic event is necessary for
the malignant transformation of lymphocytes overexpressing
BCL-2.106
8.2. BCL-2 expression in CLL.
CLL cells show an extended life span in vivo. They proliferate very
slowly, suggesting that a failure to die by apoptosis contributes to
the accumulation of malignant cells in this disease.114 Translocations of the BCL-2 gene to Ig loci are detected in less than
2% of CLL cases.115 Nevertheless, CLL cells from some
patients express high levels of BCL-2 protein compared with
BAX.116,117 In vitro, malignant cells isolated from 30% of
CLL cases survive for several weeks in the absence of added
cytokines.118 However, malignant cells from the remaining
70% of patients undergo rapid apoptosis in culture, but are protected
by the addition of cytokines including IL-4 and IFN- 8.3. BCL-2 expression by acute myeloid leukemia (AML) and acute
lymphoblastic leukemia (ALL) cells.
Genetic changes that directly result in augmented expression of BCL-2
have not been described in AML. However, those AML patients in which
greater than 20% of blasts express detectable BCL-2 levels show
shorter survival and lower rates of achievement of complete remission
compared with patients whose malignant cells express low BCL-2
levels.126 Immunohistochemical staining of BCL-2 is more
intense in malignant cells from AML patients who fail to achieve
remission than in those who respond to chemotherapy.127 Malignant cells from patients showing high BCL-2 expression are drug
resistant in vitro.128 Therefore, high BCL-2 expression, resulting from unknown mechanisms, may confer drug resistance on AML
cells. However, other mechanisms may also confer protection even in the
absence of high BCL-2 expression, because some AML isolates with low
BCL-2 expression are also drug-resistant in vitro.128
Elevated expression of MCL-1 at relapse suggests that cytotoxic regimes
may result in the selection of AML clones expressing high levels of
this anti-apoptotic BCL-2 family protein.129
Deletion and/or mutation of p53 alleles results in the generation of
tumors with impaired expression of functional p53
protein.63 The distribution of p53 mutations in human
leukemia, which has been extensively reviewed 132, will be
briefly summarized here. In general, p53 alterations are more frequent
in aggressive disease and are associated with drug resistance and poor survival.
9.1. p53 mutations in myelodysplastic syndromes, CML, and CLL.
p53 changes are seen in 4% of myelodysplastic syndromes and are more
frequent in advanced stages.132 p53 mutations are rare in
the chronic phase of CML but are more frequent in blast
crisis.133 p53 mutations are detected in 10% to 15% of
CLL and are associated with poor response to therapy and shorter
survival.134-136 Mutations are more frequent (about 40%)
in Richter's immunoblastic transformation.137 However,
sensitivity of B-CLL cells to camptothecin analogs or fludarabine in
vitro did not correlate with the presence of p53 mutations.116
9.2. p53 mutations in lymphoma and ALL.
A high proportion (30%) of Burkitt's lymphoma and 55% of its
leukemic counterpart, L3 ALL, harbor p53 mutations in addition to
translocation and overexpression of the MYC oncogene.137
The transformation of follicular lympho |
TOP
INTRODUCTION
1. MORPHOLOGICAL AND...
m) across the
inner mitochondrial membrane and thereby regulate mitochodrial volume.
The opening of "megachannels" in the inner membrane allows the
passage of molecules of less than 1.5 kD, resulting in the dissipation
of 
radiation. In contrast, tumors
lacking p53 show few apoptoses and are resistant to adriamyin or
radiation.
(TGF-
.