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REVIEW ARTICLE
From the INSERM E9910, Institut Claudius Régaud,
Toulouse, France; and the Service d'Hématologie, Centre
Hospitalier Universitaire Purpan, Toulouse, France.
The anthracycline daunorubicin is widely used in the
treatment of acute nonlymphocytic leukemia. The drug has, of course, been the object of intense basic research, as well as preclinical and
clinical study. As reviewed in this article, evidence stemming from
this research clearly demonstrates that cell response to daunorubicin
is highly regulated by multiple signaling events, including a
sphingomyelinase-initiated sphingomyelin-ceramide pathway,
mitogen-activated kinase and stress-activated protein/c-Jun N-terminal
kinase activation, transcription factors such as nuclear factor The anthracycline daunorubicin (DNR) is one
of the major antitumor agents widely used in the treatment of acute
myeloid leukemias (AMLs). Cytotoxicity mediated by DNR is generally
thought to be the result of drug-induced damage to DNA. This damage is
mediated by quinone-generated redox activity, intercalation-induced
distortion of the double helix, or stabilization of the cleavable
complex formed between DNA and topoisomerase II.1 However,
how and why such events should bring about cell death remains unclear, especially when one considers that DNA interaction may not be a
prerequisite for anthracycline cytotoxicity.2-5 Hence, the exploration and understanding of the process of apoptosis has forced a
reconsideration of the mechanisms whereby myeloid leukemia cells
respond to DNR. In this context, we have shown that, within a narrow
concentration range (0.2-1 µM), DNR can trigger apoptosis in the
monocytic U937 or the myelocytic HL-60 AML cells but not in the
immature (CD 34+) KG1a, KG1, or HEL cells,6
the latter being more resistant to DNR than the former.7
These results suggested that DNR triggers apoptotic signals in
drug-sensitive AML cells and that inhibition of these signals may
contribute to drug resistance and, for example, to the inherent
resistance of immature AML cells. For this reason, others and we have
investigated the mechanism by which DNR activates apoptosis in
DNR-sensitive AML cells.
DNR activates the sphingomyelin cycle in sensitive
leukemic cells
On the basis of those studies, our group investigated the role of the
sphingomyelin-CER pathway in DNR-induced apoptosis. DNR exposure at
concentrations that induced apoptosis (0.5-1 µM) stimulated early
(5-10 minutes) sphingomyelin cycle (hydrolysis and resynthesis) and
subsequent CER generation in both U937 and HL-60 cells. The
concentration of endogenous CER generated (~10 µM, estimated by
cell volume) is clearly sufficient to induce apoptosis.8
Because these cells exhibit a mutated or deleted form of p53, it
appears that CER operated through a p53-independant mechanism. Further
studies have shown that DNR induced sphingomyelin hydrolysis associated
with the stimulation of N-SMase, whereas acidic SMase did not appear to
be involved.10 Moreover, we have recently reported that
DNR induced sphingomyelin cycle, CER generation, and apoptosis in
Epstein-Barr virus-transformed lymphoblastoid cell lines established
from patients with Niemann-Pick disease, a genetic disorder
characterized by the lack of acidic SMase activity.11 These results suggest that acidic SMase is dispensable for DNR-induced sphingomyelin hydrolysis.
In another study, Bose et al12 have proposed that DNR may
induce CER accumulation because of enhanced de novo synthesis through
CER synthase stimulation, a mechanism that has also been suggested for
TNF In this study, we have found that DNR exposure resulted in at least 4 sphingomyelin cycles (hydrolysis and resynthesis) with concomitant (4 cycles) CER production within the first 4 hours, after which
poly(ADP-ribose) polymerase cleavage, DNA laddering, and
apoptosis-associated morphologic changes occurred. Moreover, the
successive waves of CER production were not influenced by fumonisin B1,
a potent and specific CER synthase inhibitor.15 These
results confirmed that de novo CER synthase plays little if any role in
DNR-induced CER signaling. The fact that DNR induced several peaks of
CER production suggested that sphingomyelin hydrolysis products (ie,
CER or phosphorylcholine) might reactivate N-SMase. In fact, we have
found that cell-permeant CER may indeed stimulate N-SMase,
sphingomyelin hydrolysis, and endogenous CER production, suggesting
that CER may enhance its own production through feedback control of
N-SMase.15 Therefore, one can speculate that DNR activates
a single sphingomyelin cycle that is sufficient for inducing CER
autoproduction and that these repeated CER-mediated apoptotic signals
are perhaps needed for maintaining an apoptotic (cell suicide)
signaling pressure overcoming latent cell survival mechanisms. Finally,
it is noteworthy that DNR-induced topoisomerase II cleavable complexes
have not been described in the literature under these conditions (1 µM DNR treatment for 4 hours); of course this does not demonstrate
unambiguously that DNR interaction with DNA, or more specifically with
topoisomerase II, is not requisite for apoptosis signaling because it
can be argued that present experimental procedures lack sensitivity in
detecting minute amounts of DNR-topoisomerase-DNA complexes.
CER targets
Regulation of CER production CER production is limited by both N-SMase activity and the magnitude of sphingomyelin pool disposable for hydrolysis. Previous studies have shown that N-SMase activity is strongly influenced by protein kinase C (PKC) activity. For example, we have reported that phorbol ester- or phosphatidylserine-induced PKC stimulation resulted in the inhibition of N-SMase stimulation, sphingomyelin hydrolysis, CER production, and apoptosis induced by DNR in U937 cells.32 Conversely, PKC inhibitors were found to stimulate N-SMase.33 The amount of hydrolyzable sphingomyelin is another candidate for regulating CER production. Indeed, it has been reported that, whereas sphingomyelin is preferentially distributed within the outer leaflet of the plasma membrane (sphingomyelin transverse asymmetry), the sphingomyelin pool disposable for hydrolysis consists primarily in the sphingomyelin component that is associated to the plasma membrane inner leaflet.34 Thus, it is conceivable that reduction of hydrolyzable sphingomyelin pool because of altered transverse asymmetry may result in reduced CER production. In 2 studies, we have reported that, in KG1a AML cells, which are naturally resistant to DNR,7 mitoxantrone,35 and TNF ,36 the inner leaflet-associated sphingomyelin
pool was greatly reduced, compared with the sensitive U937 cells,
whereas KG1a and U937 cells exhibited similar total sphingomyelin
amount. Interestingly, in the resistant cells, cytotoxic effectors such
as TNF,36 DNR, and mitoxantrone (A Bettaiëb et
al, unpublished results, July 1997) failed to induce
sphingomyelin hydrolysis, CER generation, and apoptosis. Thus, it is
possible that, in some AML cells, the activation of yet undefined
sphingomyelin translocases results not only in modification of
sphingomyelin transverse asymmetry but also in reduced sphingomyelin
hydrolyzable pool, decreased CER production, and inhibition of
apoptosis.37 Confirming this hypothesis, it has recently
been shown that the CER generated from the plasma membrane
sphingomyelin gains access to a SMase because of phospholipid
scrambling.38
Regulation of CER metabolism Intracellular CER concentration results from the equilibrium between CER production and CER metabolism. CER metabolism also plays an important role in regulating intracellular CER concentration and therefore DNR cytotoxicity. Theoretically, CER produced by DNR may enter into 3 distinct metabolic pathways that all result in decreasing intracellular CER levels.First, CER can be transferred to a phosphorylcholine group to generate sphingomyelin (and diacylglycerol [DAG]) on sphingomyelin synthase stimulation. It is likely that this metabolic pathway is activated for sphingomyelin resynthesis after sphingomyelin hydrolysis during the sphingomyelin cycle. This enzyme therefore has the important ability to directly regulate, in opposite directions, CER and DAG levels within the cells.39 However, despite the great biological potential of sphingomyelin synthase, very little is known about location, distribution, and regulation of this enzyme.40 Whether sphingomyelin synthase plays a role in DNR-induced cytotoxicity remains to be investigated. Second, on ceramidase stimulation, CER can be catabolized into sphingosine that, in turn, can be converted into sphingosine-1-phosphate through sphingosine-1-kinase. In a recent study, we have reported that DNR induced CER production and apoptosis in cells derived from Farber disease, which are genetically deficient for lysosomal ceramidase, the major component of cellular ceramidase activity.41 Although we cannot totally exclude the implication of extralysosomal ceramidases,42 this result suggests that sphingosine plays little if any role in DNR-induced apoptosis. However, from studies performed by Cuvillier et al,43,44 sphingosine-1-phosphate has emerged as one of the most potent regulators of apoptosis. Indeed, sphingosine-1-phosphate inhibits apoptosis induced by CER and other effectors.43,44 The mechanism by which sphingosine-1-phosphate interferes with CER-induced apoptotic signaling is not fully understood. However, it has been reported that sphingosine-1-phosphate inhibits CER-induced JNK stimulation and caspase activities.44,45 Therefore, sphingosine-1-phosphate and sphingosine-1-kinase might play an important role in regulating DNR-induced apoptosis. In fact, it has been demonstrated that sphingosine-1-phosphate inhibits doxorubicin-induced apoptosis.46 From these studies, one can speculate that sphingosine-1-kinase stimulation may contribute to DNR resistance. Because sphingosine-1-kinase is potently stimulated by PKC, it is possible that sphingosine-1-phosphate overproduction represents another mechanism by which PKC exerts its protective function in DNR-treated cells. Third, CER can be transformed to glucosylceramide by a glucosylceramide synthase. Previous reports have indicated that glucosylceramide has no cytotoxic property or may even stimulate cell proliferation47 and that glucosylceramide synthase inhibitors have been found to display some antitumor activity.48,49 These results suggest that glucosylceramide synthase plays an important role in cellular protection. Indeed, it has been described that cells transduced by glucosylceramide synthase gene were highly resistant to anthracyclines50 and that enzyme activity was significantly boosted in multidrug-resistant (MDR) cells.51 Conversely, transfection of glucosylceramide synthase antisense reverses adriamycin resistance.52 DNR triggers the sphingomyelin cycle in MDR cells when used at high doses.53 Therefore, one can speculate that in DNR-treated MDR cells CER originated from sphingomyelin hydrolysis is rapidly converted to glucosylceramide because of glucosylceramide synthase overactivity.54,55 If this speculation is the case, glucosylceramide synthase appears as an attractive target for MDR reversal. In fact, inhibition of CER glycosylation pathway increases MDR cell sensitivity to cytotoxics.56 The same group has reported that most MDR modulators, including cyclosporin A, tamoxifen, and verapamil, are potent glucosylceramide synthase inhibitors, whereas the cyclosporin A analogue PSC 833 (Valspodar), a clinically used potent MDR reversal agent, increases intracellular CER concentration by stimulating CER synthase.51,57,58 These results suggest that those agents may exert their chemosensitizing effect not only through their P-glycoprotein binding capacity, as previously postulated, but also by facilitating CER accumulation. However, the mechanism by which CER modulates anthracycline-induced cytotoxicity in MDR cells remains to be determined. For example, there is no evidence that CER interferes with P-glycoprotein function or intracellular drug distribution in MDR cells.59 Regulation of CER apoptotic signaling pathway The sphingomyelin-CER pathway appears to be efficiently regulated downstream of CER generation. Bcl-2 inhibits apoptosis induced by DNR without interfering with DNR-induced sphingomyelin cycle activation.60 This result is consistent with previous studies which have shown that Bcl-2 inhibits apoptosis induced by cell-permeant CER.61 PKC is also a potent regulator of CER-induced apoptosis. Indeed, previous studies not only showed that PKC activators, including phorbol esters and DAG, could inhibit the ability of cell-permeant CER to induce apoptosis but also that PKC inhibitors enhanced CER-induced apoptosis.9,32,62,63 Therefore, PKC appears as a critical regulator of the sphingomyelin-CER pathway because it operates both upstream and downstream of CER production. This finding may have important implications in our understanding of AML-cell drug resistance. Indeed, a large variety of hematopoietic growth factors (HGFs), including interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or basic fibroblast growth factor, were found to stimulate PKC activity through phosphatidylinositol or phosphatidylcholine hydrolysis and subsequent DAG production.64 Thus, it is conceivable that autocrine production of HGFs by leukemic cells may result in DNR-induced sphingomyelin-CER pathway inhibition and thereby contribute to reduce DNR cytotoxicity in AML. The fact that HGFs were found to inhibit DNR-induced apoptosis,65,66 as well as the chemosensitizing effect of PKC inhibitors on fresh AML cell progenitors cultured in the presence of HGFs,67 supports this hypothesis. Moreover, we have recently found that Kit signaling, activated either by its natural ligand (stem cell factor) or by modification of its intracellular domain, inhibits DNR-induced SMase stimulation, CER production, and apoptosis through a PKC-dependent mechanism.68
During the treatment of the cells with anthracyclines,
nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavin reductase reduces the drug to a semiquinone radical, which can donate
its free electron to molecular oxygen and generate the superoxide
radical (O Previous studies have shown that DNR-induced apoptosis was inhibited by
ROS scavengers such as pyrrolidine dithiocarbamate and N-acetylcystein,
a thiol antioxidant and a glutathione precursor, and was enhanced by
buthionine-sulfoximine, which depletes the glutathione
store.6 These results encouraged us to investigate the
role of ROS in the DNR-stimulated sphingomyelin-CER-JNK apoptotic pathway. Indeed, the level of endogenous H2O2
generated is comparable to that of a cell treated with 12 µM
H2O2. In fact, further studies showed that
pretreatment with N-acetylcystein abrogated DNR-induced N-SMase
stimulation, sphingomyelin hydrolysis, and CER generation induced by
DNR, evoking a role for ROS in N-SMase regulation.23 This
hypothesis is supported by 2 other studies which showed that H2O2 stimulates sphingomyelin hydrolysis and
CER generation and that N-acetylcystein and pyrrolidine dithiocarbamate
were potent inhibitors of TNF Other studies have demonstrated that ROS also plays a role in CER-induced apoptosis. Indeed, permeant CER induced early and transient (5-10 minutes) H2O2 production, followed by a second wave of H2O2 detected at 1 to 3 hours that originated from mitochondrial oxidative metabolism disturbances. Furthermore, N-acetylcystein inhibited H2O2 production, JNK activation, AP-1 activation, and apoptosis induced by CER.23,74,75 These results suggest that ROS plays an important role in the sphingomyelin-CER apoptotic pathway triggered by DNR at 2 different levels: upstream CER generation by stimulating N-SMase activity and downstream CER activity by mediating CER-induced JNK activation. The results also suggest a novel function of cytosolic-originated ROS in the cytotoxicity mechanism of anthracyclines. Thus, antioxidants, including superoxide dismutase, catalase, glutathione peroxidase, or thioredoxin, may influence DNR-induced cytotoxicity. However, the fact that antioxidants inhibit apoptosis induced by
chemotherapeutic drugs that have not been documented to stimulate the
sphingomyelin cycle (ie, actinomycin D, camptothecin, etoposide, and melphalan),76 raises the possibility that ROS
interferes with other apoptotic pathways in DNR-treated cells.
Furthermore, there is mounting evidence that ROS activates or mediates
many other signaling pathways, which can contribute more generally to
the cellular response to DNR. For example, ROS modulates both PKC77-79 and tyrosine kinase activities,80-85
contributes to cell cycle block,86 stimulates Raf-1/ERK
mitogen-activated protein (MAP) kinases,84,87 and triggers
activation of critical transcription factors, including nuclear
factor-
Anthracyclines, as other genotoxic agents including alkylating
agents and ionizing radiation, have been shown to increase cellular DAG
levels and PKC activity.90 However, the source of DAG as
well as its functional role in the cellular response to the drug were
not determined. In another study, we have reported that, in U937 cells,
DNR (and mitoxantrone) transiently stimulated concurrently with CER
generation both DAG and phosphorylcholine production by phospholipase C
hydrolysis of phosphatidylcholine. Moreover, pretreatment of cells with
the xanthogenate compound D609, a potent inhibitor of
phosphatidylcholine hydrolysis, led to a sustained increase in CER
levels. This result suggests that DNR may trigger in parallel both cell
death (CER) and survival (DAG and phosphorylcholine) mediators and
that, in sensitive leukemic cells, CER overrides the protective
function of DAG and phosphorylcholine.91 These results
support the notion that reciprocal regulation through DAG and CER may
be implicated in the regulation of apoptosis.92 The
mechanism by which phosphatidylcholine-derived DAG influenced intracellular CER concentration in DNR-treated cells is not yet characterized; however, because phosphatidylcholine-derived DAG did not
appear to influence N-SMase stimulation, it is conceivable that DAG
enhanced CER metabolism by facilitating sphingomyelin synthesis.
Furthermore, it has been documented that phosphatidylcholine-derived DAG binds and stimulates Raf-1 kinase activity93,94 and,
through Raf-1, may activate the MEK1/ERK classical MAP kinase module, a
negative regulator of apoptosis induced by various
stresses.84,87,95-97 Phosphatidylcholine-derived DAG has
been found to stimulate some PKC isoforms such as PKC
Among other enzymes that are involved in signal transduction
pathways, phosphoinositide 3-kinase (PI3K) plays an important role.
PI3Ks are a family of enzymes that catalyze the phosphorylation of
inositol lipids at the D3 position of the inositol ring, generating new
intracellular second messengers (see Franke et al105 for review). The lipid products of PI3K are
phosphatidylinositol-3-phosphate (PtdIns-3-P),
phosphatidylinositol-3,4-biphosphate (PtdIns-3,4-P2), and phosphatidylinositol-3,4,5-triphosphate
(PtdIns-3,4,5-P3). PtdIns-3,4-P2 and
PtdIns-3,4,5-P3 have been demonstrated to interact with a
large variety of downstream effectors, including serine-threonine kinase Akt,106 calcium-insensitive PKC
NF- Despite many efforts, the mechanism by which DNR activates NF- Previous studies have shown that NF- The mechanism by which NF-
Doxorubicin, as other antitumor compounds such as methotrexate, cisplatin, fludarabine, or bleomycin as well as ionizing radiation, was shown to enhance the expression of Fas and Fas-L on the surface of certain leukemic or epithelial malignant cells. For this reason, it has been proposed that doxorubicin-induced apoptosis occurs through autocrine or paracrine induction of the Fas-dependent pathway.148-151 In these studies, this hypothesis was supported by 2 lines of evidence: (1) cell lines resistant to Fas were found insensitive to anticancer drug-induced apoptosis, and (2) doxorubicin-induced apoptosis was prevented by CD95-neutralizing antibodies. However, this issue remains highly controversial mainly because antagonistic anti-Fas antibodies do not always inhibit drug-induced cell death.152-154 Moreover, many cells either naturally resistant to Fas (ie, HL-60 cells) or selected for Fas resistance152-154 remain sensitive to doxorubicin-induced apoptosis. Therefore, convincing evidence now exists showing that Fas/Fas-L pathway is not a principal and necessary mechanism of anthracycline-induced apoptosis.154 Other investigators have reported that in carcinoma cells doxorubicin
induced the clustering of Fas receptor and its interaction with
Fas-associated death domain-containing protein (FADD) in a
Fas-L-independent fashion.155 This has also been
described for UV and ionizing radiation.156,157 FADD is an
adapter molecule, which is recruited to Fas cell death domain, and then
binds to and activates procaspase-8; active caspase-8, in turn,
triggers activation of a proteolytic cascade that leads through
caspase-7, caspase-3, and caspase-6 to apoptosis. Moreover, these
investigators showed that FADD overexpression facilitated drug-induced
apoptosis, whereas down-regulation of FADD by transient transfection of
an antisense construct decreased tumor cell sensitivity to the
drug.155 For this reason, it has been proposed that
doxorubicin-induced cell death involves the Fas/FADD pathway without
interfering with Fas-L production and that FADD expression may
significantly influence the cellular response to this drug. Fas
clustering by cytotoxic agents was also described for VP-16,
cisplatinum, and vinblastin155 as well as for UV
radiation.157 However, the role of FADD in doxorubicin-induced caspase activation and apoptosis may be a function
of the cellular model. Indeed, in doxorubicin-treated Jurkat leukemic T
cells, Wesselborg et al154 have shown a correlation between doxorubicin-apoptosis and cleavage of procaspase-8 to its
active p18 subunit; however, the expression of a dominant-negative FADD
construct selectively abrogated Fas but not drug-induced effects. This
result suggests that caspase-8 can be activated by doxorubicin in the
absence of Fas receptor signaling.154 Fas- and
FADD-independent caspase-8 activation have been observed in lymphoid
cells treated with ionizing radiation.158 Altogether these
results suggest that, whatever the role of FADD, caspase-8 activation
represents a critical step in the cascade resulting in terminal
proteolytic events, including caspase-3 and caspase-6 activation
responsible for the cleavage of poly(ADP-ribose) polymerase and nuclear
lamins, respectively. However, other investigators have questioned the
role of caspase-8. For example, Villunger et al153 have
reported that expression of cowpox virus cytokine response modifier A,
a potent inhibitor of distinct members of the caspase-protease family,
including caspase-8,159 did not influence
doxorubicin-induced apoptosis in T-acute lymphatic leukemia CEM cells,
whereas it prevented Fas-mediated apoptosis. Moreover, expression of
FLIP (FLICE-inhibitory protein), which binds to caspase-8 and
interferes with its function, was found to have no influence on
apoptosis induced by doxorubicin or by other antileukemic compounds or
by Whatever the intimate mechanism by which Fas and caspases are involved in the cellular response to anthracyclines, all these results have important clinical implications. First, on the basis of the stimulatory effect of anthracyclines on Fas-L production in some tumor cells and with the demonstration that Fas-L produced by tumor cells can kill the specific effector CTL and other activated T cells that express Fas,164 it can be speculated that chemotherapy may decrease cellular cytotoxicity of natural effectors and, therefore, may facilitate immune escape.165 Conversely, it has been proposed that drug-induced Fas expression may result in sensitization toward Fas-dependent cytotoxicity of cellular immune effectors.166-169 Second, previous studies have shown that, whereas short-term culture with doxorubicin enhanced Fas expression level, prolonged exposure to the drug may result in Fas reduction or even lack of expression, establishing an intriguing link between MDR phenotype and Fas resistance.170 Similar results have been obtained with mitoxantrone.171 Third, if Fas signaling molecules (or Fas itself) play an important role in anthracycline-induced apoptosis, it is conceivable that diminution of their expression and/or altered capacity to form the multimolecular death-initiating signaling complex constituted by Fas death domain, FADD, and procaspase-8 may impair drug cytotoxicity. In this perspective, it is important to note that AML cells (as well as immature progenitor cells) are generally insensitive to Fas commitment although most of them do express Fas, suggesting a disruption in Fas-mediated death signaling.172-174 Therefore, it is tempting to speculate that negative control of Fas signaling contributes not only to immune escape but also to decreased drug cytotoxicity in leukemia cells. Finally, one should not disregard the emerging evidence that genotoxic stress, such as anthracycline treatment, has been described to increase death receptors such as DR5 for TRAIL/Apo-2 ligand in leukemia cells lines such as U937 and HL-60.175,176 These observations strongly suggest that one may optimize the antileukemic activity of anthracyclines by combining them with Apo-2 ligand treatment.
Previous studies have shown that doxorubicin, as many other DNA-damaging agents, activates p53-DNA binding.177 On the basis of the crucial role of p53 in the execution of some forms of apoptosis, it has been therefore speculated that p53 could play an importan |
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Abstract
Introduction
B activation...
B,
as well as the Fas/Fas-ligand system. These pathways are themselves
influenced by a number of lipid products (diacylglycerol, sphingosine-1
phosphate, and glucosyl ceramide), reactive oxygen species, oncogenes
(such as the tumor suppressor gene p53), protein kinases
(protein kinase C and phosphoinositide-3 kinase), and external stimuli
(hematopoietic growth factors and the extracellular matrix). In light
of the complexity and diversity of these observations, a comprehensive
review has been attempted toward the understanding of their individual
implication (and regulation) in daunorubicin-induced signaling.
(Blood. 2001;98:913-924)
-radiation in bovine endothelial cells.
OH). By employing electron spin resonance together with
a spin-trap such as DMPO (5,5-dimethyl-1-pyrroline N-oxide),
anthracycline-induced 
or 
"atypical" isoforms that have been involved in cell proliferation
and/or survival.
, 
, and 
isoforms,
B activation in DNR-induced
apoptosis
) which
phosphorylate residues 32 and 36 of I