Positive and negative regulation of apoptotic pathways by cytotoxic agents in hematological malignancies

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Most chemotherapeutic drugs can induce tumor cell death by apoptosis. Analysis of the molecular mechanisms that regulate apoptosis has indicated that anticancer agents simultaneously activate several pathways that either positively or negatively regulate the death process. The main pathway from specific damage induced by the drug to apoptosis involves activation of caspases in the cytosol by pro-apoptotic molecules such as cytochrome c released from the mitochondrial intermembrane space. At least in some cell types, anticancer drugs also upregulate the expression of death receptors and sensitize tumor cells to their cognate ligands. The Fas-mediated pathway could contribute to the early steps of drug-induced apoptosis while sensitization to the cytokine TRAIL could be used to amplify the response to cytotoxic drugs. The Bcl-2 family of proteins, that includes anti- and pro-apoptotic molecules, regulates cell sensitivity mainly at the mitochondrial level. Anticancer drugs modulate their expression (eg through p53-dependent gene transcription), their activity (eg by phosphorylating Bcl-2) and their subcellular localization (eg by inducing the translocation of specific BH3-only pro-apoptotic proteins). Very early after interacting with tumor cells, anticancer drugs also activate lipid-dependent signaling pathways that either increase or decrease cell ability to die by apoptosis. In addition, cytotoxic agents can activate protective pathways that involve activation of NFκB transcription factor, accumulation of heat shock proteins such as Hsp27 and activation of proteins involved in cell cycle regulation. This review discusses how modulation of the balance between noxious and protective signals that regulate drug-induced apoptosis could be used to improve the efficacy of current therapeutic regimens in hematological malignancies.


The concepts used for designing most chemotherapeutic regimens currently used for treating hematological malignancies include the use of drugs with non-overlapping toxicity and either cell cycle-dependent or -independent activities, their administration at maximally tolerated doses, and exclusion of agents that did not demonstrate significant activity against the targeted tumor in phase I studies. These regimens include the ara-C-topoisomerase II inhibitor combination for acute myelogenous leukemias (AMLs), the cyclophosphamide–doxorubicin–vincristine–prednisone regimen for lymphoid malignant diseases and the adriamycin–bleomycin–vinblastine–dacarbazine regimen for Hodgkin's disease. Although these drug combinations have demonstrated their efficacy in a significant number of patients, the question of how to cure those with resistant disease remains open.

Various mechanisms have been shown to induce tumor cell resistance to cytotoxic agents by preventing efficient drug interaction with its specific intracellular target. These mechanisms include decreased accumulation of the cytotoxic drug in tumor cells as a consequence of an increased energy-dependent efflux (the so-called ‘multidrug resistance’), altered cellular metabolism of the drug and mutation of its main intracellular target (Figure 1a).1 In some cases, these observations have led to specific strategies aiming to reverse the resistant phenotype. One of the most tested strategy is inhibition of P-glycoprotein-mediated efflux of chemotherapeutic agents by combination with a non-cytotoxic drug, also known as a ‘multidrug resistance reversing agent’. Though several compounds have demonstrated some efficacy in improving the response of P-glycoprotein-expressing diseases to conventional therapy, eg quinine improved the response of P-glycoprotein-positive myelodysplatic syndromes to the Ara-C–anthracycline regimen, the ideal reversing agent for clinical applications has yet to be identified.23

Figure 1

 Two complementary views of the mechanisms that regulate tumor cell sensitivity to drug-induced cell death. (a) The anticancer drug enters the cell, is metabolized to a more active derivative, interacts with its intracellular target, which triggers cell death. Resistance can be due to increased drug efflux (1), altered metabolism (2), alteration of the target (3) and increased repair of the damage (4). (b) In addition to interacting with its intracellular target, the anticancer drug itself activates protective and noxious pathways that modulate intracellular drug accumulation, drug metabolism, drug–target interaction and downstream cell death pathways.

In recent years, it appeared that apoptosis was the predominant form of cell death triggered by cytotoxic drugs in hematopoietic tumor cells, at least in cells cultured in vitro. The inability of tumor cells to activate the apoptotic machinery in response to drug-induced damage defined an additional mechanism by which tumor cells could resist cytotoxicity due to anticancer agents. Apoptosis is not the only form of cell death triggered by chemotherapeutic drugs. When studied by using a clonogenic assay or an in vivo assay, the sensitivity of tumor cells that die by mechanisms other than apoptosis can be similar to that of tumor cells that undergo rapid apoptosis.4 Nevertheless, studies based on apoptosis induction have shown that anticancer drugs simultaneously activate several different pathways that positively and negatively regulate drug interaction with its target and damage-induced activation of the cell death process (Figure 1b and Table 1), suggesting that modulation of the balance between noxious and protective signals could provide new strategies to improve the efficacy of current chemotherapeutic regimens. This review will discuss the role of apoptotic pathways in determining the sensitivity of hematopoietic tumor cells to cytotoxic agents.

Table 1  Anti-apoptotic and pro-apoptotic signals that could modulate cytotoxic agent-induced apoptosis of hematological malignant cells

Two main pathways for drug-induced apoptosis

The mitochondrial pathway

Exposure of many cultured hematological cell lines to a cytotoxic agent induces the release of molecules contained in the intermembrane space of the mitochondria to the cytosol under the control of Bcl-2 and Bcl-2-related proteins.5 Various molecular mechanisms have been proposed to account for this event, including pore formation in the external mitochondrial membrane by proteins such as Bax, rapid loss of the mitochondrial transmembrane potential Δψm following permeability transition and disruption of the outer membrane as a result of mitochondrial swelling.678910 Among the released molecules is cytochrome c that, on entry in the cytosol, induces oligomerization of APAF-1 (apoptotic protease activating factor-1) and exposes its CARD (caspase recruitment domain) in the presence of ATP.1112 In turn, oligomerized APAF-1 binds to cytosolic procaspase-9 in a so-called apoptosome complex and induces processing and activation of this caspase. Activated caspase-9 then activates the downstream caspase cascade.13 Other molecules released from the mitochondria include several procaspases and the flavoprotein AIF (apoptosis inducing factor) that translocates to the nucleus and triggers caspase-independent nuclear changes.11 The anti-apoptotic properties of Bcl-2 and related proteins have been related to their ability to prevent these mitochondrial events, whereas the targeting of BH3 domain-only proteins of the Bcl-2 family such as Bid, Bim or Bad from various parts of the cell to the mitochondria was shown to activate the death process by inducing the mitochondrial release of proapoptotic molecules (Figure 2).141516

Figure 2

 Two pathways for drug-induced apoptosis. The main pathway implicates the mitochondrial release of cytochrome c, eg as a consequence of the translocation of a Bcl-2-related BH3-only protein from the cytosol to the mitochondria. In the cytosol, and in the presence of ATP, cytochrome c induces oligomerization of APAF-1 that, in turn, activates caspase-9 (the complex formed by cytochrome c/ATP/APAF-1/procaspase-9 has been designated apoptosome). Caspase-9 then activates the effector caspases such as caspase-3 that induce the cell demise. An additional pathway involves the death receptor Fas at the plasma membrane level. Trimerization of Fas, as a consequence of FasL upregulation or increased Fas expression, recruits and activates caspase-8 in the death-inducing signaling complex (DISC). Based on Fas-mediated death pathways current knowledge, caspase-8 could either directly activate caspase-3 or cleave a BH3-only protein (?) to activate the mitochondrial pathway to cell death.

The Fas-dependent pathway

Another pathway that could account for cytotoxic drug-induced apoptosis involves the death receptor Fas (APO-1/CD95), a 45-kDa type I-membrane death receptor protein that belongs to the TNF (tumor necrosis factor)-receptor superfamily. Fas exists as a cell surface and a soluble protein, both of which are generated by alternative splicing. Its natural ligand, Fas ligand (FasL), is a 40-kDa type II-membrane protein that can be released as a 26 kDa soluble form. Fas is expressed on many tissues throughout the body while FasL expression is much more restricted. The Fas/FasL system plays an important role in the normal development of T lymphocytes in the thymus by eliminating self-reactive lymphocytes. Fas/FasL interaction is also one of the mechanisms by which cytotoxic immune cells can kill Fas expressing target cells. Lastly, Fas/FasL interaction is responsible for activation-induced cell death of T lymphocytes.17

Binding of trimeric FasL to Fas induces trimerization of the receptor and activates a signal transduction pathway that can lead to cell death. This pathway is initiated by the formation of a DISC (death-initiating signaling complex) that involves an adaptator protein named FADD (for fas-associated death domain)18 and procaspase-8.19 FADD binds to the cytoplasmic region of Fas through homophilic interactions of their respective DD (death domains) and to the N-terminal domain of procaspase-8 through a second interacting region called the DED (death effector domain).1819 Oligomerization of procaspase-8 in the DISC results in activation of the proteolytic enzyme.19 Depending on the level of DISC formed in each cell type, caspase-8 can either directly activate the caspase cascade or cleave the carboxy-terminal part of a BH3 domain-only pro-apoptotic member of the Bcl-2 family designated Bid.202122 In turn, translocation of the truncated Bid to the mitochondria activates the previously described mitochondrial pathway to cell death (Figure 2).

It was proposed that ionizing radiation23 and various anticancer drugs242526 caused upregulation of FasL and its interaction with Fas at the surface of tumor cells. For example, immunohistochemical analysis of tumor tissues in patients with malignant lymphomas demonstrated increased expression of FasL after radiotherapy at doses of 4 to 10 Gy.27 At least in some cell types, drug-induced upregulation of FasL was associated with the presence of a functional wild-type p53 gene.26 Interaction of FasL with Fas at the surface of tumor cells defines an autocrine/paracrine pathway similar to that observed in activation-induced cell death of T lymphocytes (Figure 2).

However, the role of the FasL/Fas system in drug-induced apoptosis has been challenged by several studies in which the cell death process was not influenced by antagonistic antibodies or molecules that prevent FasL interaction with Fas. In addition, apoptosis induced by cytotoxic agents is altered in neuroblastoma cells in which the gene for caspase-8 is silenced by DNA methylation or gene deletion,28 but is not altered in embryonic fibroblasts from FADD and caspase-8 knock-out mice.2930 Several explanations could account for these observations. First, DISC formation could be dispensable for drug-induced cell death, depending on the cell type. Secondly, we and others have shown that anticancer drugs, UV radiation and other compounds increased the expression of Fas at the surface of treated cells and triggered the formation of the DISC via receptor oligomerization in the absence of FasL interaction with Fas.31 Third, Fas alone or the FasL/Fas interaction may be involved in the initiation phase of apoptosis triggered by anticancer drugs while requirement for this system may be bypassed at later stages, when alterations of the mitochondria occur as a direct consequence of specific damage induced by the drug.25

Fas gene mutations that characterize some congenital autoimmune lymphoproliferative syndromes (ALPS) inhibit Fas-mediated apoptosis by a dominant-negative mechanism.32 Several Fas gene mutations have also been described in lymphoid malignancies, including myelomas,33 T cell lymphoblastic leukemias34 and HTLV-I-related adult T cell leukemias (ATL).35 In this latter disease, mutated Fas was also demonstrated to behave as a dominant-negative mutant,36 which could account for the chemoresistance of ATL. Caspase-10 mutations, that account for some congenital ALPS without Fas mutation,37 have not been so far identified in human lymphoid tumors.

The central role of caspases in drug-induced apoptosis

The caspase family

Caspases are a family of mammalian cysteine aspartic proteases that play a central role in the death process.38 The 14 caspases so far identified in mammalian cells, of which 11 human enzymes are known, are synthesized as inactive proenzymes that must be cleaved at key aspartate residues to be activated.39 X-ray analyses have shown that activated enzymes form a tetramer containing two large and two small subunits.4041 Based on their structural and functional homologies, mammalian caspases have been classified in two sub-families. Members of the caspase-1 sub-family (caspase-1, -4, -5, -11, -12 and -14) are mainly involved in cytokine maturation and inflammation, though they could contribute to some apoptotic pathways. Members of the caspase-3 sub-family (caspase-2, -3, -6, -7, -8, -9, -10) play a central role in apoptosis. Further subdivision can be made in this latter sub-family, depending on the size of their prodomain. Those with a short prodomain (caspase-3, -6 and -7) function as downstream effectors of a proteolytic cascade in which the remainder with a long prodomain, function as upstream signal transducers. Activation of caspases with a long prodomain usually involves the recruitment of multiple homologous enzymes to an adaptor molecule such as APAF-1, FADD or RAIDD/CRADD.18424344 Then, the proteolytic cascade propagates the death signal by cleaving key cellular proteins on the carboxyl side of aspartate residues. For example, in an in vitro system in which cytochrome c and dATP was added to cell-free extracts, caspase-9 was shown to activate caspase-7 and -3. In turn, caspase-3 activates caspase-2, -6, -8 and -10.45 Thus, some caspases with a short prodomain, such as caspase-3, could be involved in a feedback amplification loop by activating certain caspases with a long prodomain.

The central role of caspases in drug-induced apoptosis is suggested by the observation that several procaspases are cleaved in their active fragments during the cell death process. In addition, extracts from drug-treated cells cleave peptide substrates that mimic the sequence specifically recognized by several enzymes of this family. In U937 cells, caspase-3 and caspase-6 appear to play a central role in apoptosis triggered by topoisomerase inhibitors while the various isoforms of caspase-2 modulate their activity (Refs 46474849 and Droin et al, submitted). In addition, data from caspase-95051 and APAF-15253 knock-out mice have suggested that the generation of a caspase-9-containing apoptosome complex was crucial for drug-induced apoptosis. Caspase-3 is required for some typical hallmarks of apoptosis such as DNA fragmentation and membrane blebbing.5455 The other caspases involved in the cell death process could vary, depending on the cell type and the apoptotic stimulus. The reasons why the cells use different caspases for undergoing cell death, depending on the cell context, is not clear, but several observations provide potential explanations.

Caspase cellular compartmentalization

It was shown recently that a fraction of procaspase-3 was present in the mitochondria from several tissues, in a complex with heat shock proteins (Hsp) Hsp60 and Hsp10.5657 Upon activation of the death process, procaspase-3 is activated and dissociated from the Hsp complex. A substantial portion of cellular procaspase-2 and −9 is also found in the intermembrane mitochondrial space from various organs, as well as in isolated liver mitochondria.58 In some cells such as cardiomyocytes and neurons, death signals induce the translocation of mitochondrial caspase-9 to the nucleus.59 Following Fas activation of liver cells, active caspase-7 was observed to migrate from the cytosol to mitochondria and microsomes.60 Lastly, several caspases were shown to translocate to the nucleus in response to death stimuli.38 It was suggested that sequestration of procaspases in various cellular compartments might restrain their activation and separate them from their substrate in living cells. However, the role of caspase compartmentalization in regulating cell response to apoptotic stimuli remains poorly understood.

Transcriptional regulation of caspases

While the basal level of caspase expression was shown to depend on the STAT1 transcription factor,61 we have observed that cytotoxic drugs upregulated the transcription of several molecules involved in cell death pathways, including Fas, FADD and caspases, in a STAT-1-independent manner, before undergoing cell death.4862 The role of this upregulation remains unclear since inhibitors of macromolecule synthesis, such as actinomycin D and cycloheximide, often do not interfere and even sensitize to drug-induced apoptosis. Little is known regarding the regulation of CASP gene transcription. One could imagine that accumulation of these proteolytic enzymes in tumor cells might sensitize these cells to apoptotic stimuli. As discussed below, upregulation of death receptors and downstream caspases in cells exposed to anticancer drugs has already suggested new therapeutic strategies combining the drugs with death receptors agonists or lymphokine- activated immune cells.

Another level of transcriptional regulation of caspase expression is alternative splicing of CASP gene mRNA that was observed for several of these enzymes. For example, the genes encoding procaspase-263 and procaspase-964 can generate short isoforms that prevent rather than facilitate apoptotic cell death. Procaspase-9b was shown to prevent activation of the long isoform of procaspase-9 by interfering with its recruitment in the apoptosome.6465 We have observed that addition of the short isoform of caspase-2 to cell-free extracts interfered with the activation of various caspases by cytochrome c and dATP (Droin et al, submitted).

Post-translational modifications of caspases

S-nitrosylation of the active-site cysteine of procaspase-3 was shown to inhibit recombinant enzyme activity in cell-free systems.66 This reversible nitrosylation was demonstrated to occur in the absence of nitric oxide donor in human B and T cells while the signal transduction pathway activated by Fas ligation induced both the denitrosylation of the active site and procaspase-3 cleavage in its active fragments.67 Another post-translational modification of caspases is the phosphorylation of caspase-968 which is mediated by the protein kinase B (also known as Akt) in response to growth factors such as IL-3. This phosphorylation, that prevents procaspase-9 activation at the level of the apoptosome, occurs simultaneously with the phosphorylation of Bad, that mediates its interaction with a 14-3-3 protein in the cytosol and prevents Bad from neutralizing Bcl-2 on the mitochondrial membrane.69

Endogenous inhibitors of caspases

Several endogenous proteins directly or indirectly interfere with caspase activation (Figure 3). Proteins known as inhibitors of apoptosis (IAPs) were initially identified in baculoviruses.70 These proteins are structurally characterized by at least one BIR (for baculoviral inhibitor of apoptosis repeat) domain that has been identified so far in five human proteins (NAIP, cIPA1, cIAP2, XIAP and survivin). One of these proteins, survivin, shows strong upregulation in a number of human tumors, eg in high grade lymphomas. This small IAP with a single BIR domain, that is highly expressed in embryonic tissues, but cannot be detected in normal adult differentiated tissues, correlates with cell survival in several human tumors.71 The majority of IAPs inhibit some members of the caspase family, either directly or indirectly, though they also demonstrated caspase-independent inhibitory mechanisms such as those mediated by activation of the transcriptional factor NF-κB.70 These proteins were demonstrated to block apoptosis induced by a variety of anticancer drugs (etoposide, cisplatin, taxol, actinomycin D, adriamycin) and ionizing radiation.717273 Interestingly, survivin associates with mitotic spindles and demonstrates cell-cycle specific expression in G2/M which is mediated by G1 transcriptional repressor elements in the survivin promoter.737475 In malignant cells, however, the protein is expressed in a cell-cycle-independent manner. Survivin specifically protects against taxol while it demonstrates limited inhibitory effect against vincristine. Conversely, plasmids encoding antisense survivin cDNA were claimed to sensitize tumor cells to anticancer drug-induced apoptosis.76 Thus, survivin is an exciting tumor marker that could also modulate cell response to anticancer drugs. Another protein that negatively interferes with caspase activation is the cellular FLICE-inhibitory protein (c-FLIP also called Casper/I FLICE/FLAME-1/CASH/CLARP/MRIT/Usurpin), an inactive homolog of caspase-8 and caspase-10 that may act as a competitive inhibitor by preventing the binding of these caspases to the cytosolic domain of death receptors77 (Figure 3).

Figure 3

 Site of action of the negative modulators of drug-induced apoptosis. Various inhibitors, whose activity can be directly modulated by cytotoxic agents, negatively interfere with the pathways leading to apoptosis, sometimes at different levels of these pathways (see text).

Modulation of drug-induced cell death by Bcl-2 and related proteins

The bcl-2 gene family

The bcl-2 gene family constitutes one of the most critical groups of apoptosis regulatory genes. The bcl-2 (B cell leukemia/lymphoma 2) gene was identified at the chromosomal breakpoint of the t(14;18)(q32;q21) that characterizes follicular B cell lymphomas.7879 Overexpressed Bcl-2 protein antagonizes apoptosis induced by a number of different stimuli, including anticancer drugs, and can also affect the cell cycle of selected cell types.8081 The Bcl-2 protein is overexpressed in various other lymphomas, acute leukemias and a series of human solid tumors.81 This overexpression might contribute to cell accumulation and, in combination with abnormal expression of other oncogenes such as c-myc, to malignant transformation.82 Accordingly, transgenic mice that overexpress Bcl-2 in their B cells progress to high-grade lymphomas.8384

A number of bcl-2-related genes have been identified that either prevent cell death and function as potential oncogenes (Bcl-2, Bcl-XL, Mcl-1, A1, Bcl-w, Bfl-1) or encourage cell demise and behave as tumor suppressor genes (Bax, Bak, Bcl-XS, Bad, Bim, Bid, Bik, Bok, Hrk).8081 For example, missense mutations in Bax, that were recently identified in primary human tumors including about 20% of the studied hematological malignancies such as acute lymphoblastic leukemias (ALL), could contribute to disease progression and drug resistance.85 In addition, the genome of several pathogenic viruses such as Epstein–Barr virus (EBV) encodes Bcl-2 homologs.80 Structural analysis of these proteins led to distinguish three subfamilies. Anti-apoptotic proteins possess up to four conserved Bcl-2-homology (BH) domains designated BH1 to BH4. Proapoptotic proteins can be divided into those with at least three BH domains such as Bax and Bak and more distantly related proteins that share only the BH3 domain with Bcl-2. In addition, some of these proteins contain a carboxy-terminal transmembrane domain that targets them to cellular membranes.81

Prognostic value of Bcl-2 proteins

The prognostic value of Bcl-2 and Bcl-2-related protein expression is far from clear. A number of studies have correlated Bcl-2 expression to poor clinical outcome in hematological malignancies, including follicular lymphomas, chronic lymphocytic leukemias and AMLs.8687 In addition, an increase in Mcl-1 expression was associated with the relapse of AML and ALL.88 However, these correlations were not confirmed in all series.86 Moreover, high expression of the pro-apoptotic Bax was associated with unfavorable prognosis in childhood ALLs and other types of tumors, possibly due to missense mutations of the gene.85 The large number of Bcl-2-related proteins with possible redundancies between these proteins in controlling apoptotic pathways could explain the contradictory results obtained in the various series.8081 Another explanation could be that, although overexpression of Bcl-2 prevents apoptosis and its cytoprotective effect extends to some types of necrosis, cells remain able to die through another death mechanism.899091

In vitro studies have demonstrated possible interaction between apoptosis suppressors and apoptosis inducers of the Bcl-2 family of proteins.81 Based on their possible heterodimerization, it was proposed that the ratio of anti-apoptotic to proapoptotic members of the Bcl-2 family could determine the sensitivity of cells to apoptotic stimuli as a death/life rheostat, including the response of tumor cells to damage triggered by an anticancer drug.8192 More recent studies indicate that Bcl-2 and Bax can independently regulate apoptosis.5 Overexpression of Bcl-2 blocks apoptosis with comparable efficacy in the absence of Bax, while Bax can promote apoptosis in the absence of Bcl-2. Thus, while an in vivo competition exists between death antagonists and agonists of the Bcl-2 family, at least some of these proteins regulate apoptosis independently of their physical interaction to each other.580

Anti-apoptotic activity of Bcl-2

Bcl-2 and several of its homologs possess a C-terminal hydrophobic transmembrane domain that inserts these molecules into several intracellular membranes, mainly the mitochondrial external membrane, the endoplasmatic reticulum and the nuclear envelope. The intracellular distribution of members of the Bcl-2 family can depend on the cell type and its differentiation and activation stage. Functional studies suggest that Bcl-2-related molecules exert their apoptosis-regulatory effects by modulating mitochondrial alterations that precede caspase activation. One of the proposed mechanism of action is the modulation of the mitochondrial permeability transition pore, also called mitochondrial megachannel, a multiprotein complex formed at the contact site between the mitochondrial inner and outer membranes.80 Indirect mechanisms of action have also been proposed. Bcl-2 interacts with a series of proteins involved in cell cycle regulation and gene transcription. For example, Bcl-2 binds and sequesters calcineurin, a calcium-dependent phosphatase. As a consequence of calcineurin binding to Bcl-2, the nuclear factor of activated T-lymphocytes (NFAT) is not dephosphorylated in response to cytotoxic drugs, then does not move to the nucleus to activate the transcription of FasL and trigger apoptosis through a FasL/Fas interaction.93

The survival advantage provided by Bcl-2 contributes to resistance against a wide variety of antitumor agents, including cyclophosphamide, cisplatin etoposide, mitoxantrone, adriamycin, ara-C and methotrexate.94 Bcl-XL also suppresses the apoptotic effect of a number of cytotoxic agents.95 The multidrug resistance induced by upregulation of these proteins might be reversed by their specific downregulation, while upregulation of pro-apoptotic members of the Bcl-2 family might induce chemosensitization. Antisense strategies using oligonucleotides and hammerhead ribozymes to inhibit Bcl-2 expression have demonstrated some efficacy in vitro96 and in vivo97, while tumor selective expression of Bax, eg through adenoviral gene transfer, showed highly selective toxicity on tumor cells in both in vitro clonogenic assays and animal models.98

Bcl-2 phosphorylation

Bcl-2 activity can also be regulated by phosphorylation at serine residues 70 and 87 that is associated with loss of its anti-apoptotic function.99100101102103104105106107 Bcl-2 phosphorylation is specifically induced by anticancer drugs that interact with monomeric tubulin and affect microtubule polymerization such as vinca alkaloids, and those that interact with polymerized tubulin and prevent depolymerization such as taxanes.100101 This effect is not seen with DNA damaging agents. Bcl-2 phosphorylation has been shown to prevent the protein from forming heterodimers with Bax. Several signal transduction pathways could be involved in antimicrotubule agent-mediated Bcl-2 phosphorylation.102103104105106107 One of these pathways involves Raf-1,102 an ubiquitously expressed serine/threonine kinase which is activated in cells treated with paclitaxel and whose expression is decreased in cell lines resistant to this drug. Raf-1-mediated phosphorylation of Bcl-2 could involve induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1, either as a consequence of p53 induction or not.103 Another kinase whose role in Bcl-2 phosphorylation has been discussed is protein kinase A, another kinase that is activated in response to microtubule damage.104 The role of p34cdc2 kinase and the microtubule-associated protein (MAP) kinases that include extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38, in modulating Bcl-2 phosphorylation is still unclear and could depend on the cell type and the cytotoxic agent.105106107 For example, a transient and early activation of JNK/SAPK rather than Raf-1 phosphorylation might account for Bcl-2 phosphorylation in K562 myelogenous leukemia cells exposed to the 2-methoxyestradiol that inhibits microtubule dynamics.107 Whatever the pathway, phosphorylation/dephosphorylation of Bcl-2 appears to be a molecular marker of cell survival or death in response to microtubule damaging anticancer drugs (Figure 4).

Figure 4

 Dual effect of microtubule disrupting agents on Bcl-2. Bim is normally sequestered to the microtubule dynein motor complex. Anticancer drugs that affect microtubule polymerization such as vinca alkaloids, and those that prevent their depolymerization such as taxanes provoke the release of Bim, allowing Bim to associate with Bcl-2 and Bcl-2-like proteins. Antimicrotubule agents also trigger Bcl-2 phosphorylation through several pathways, one of these involving the serine/threonine kinase Raf-1. Bcl-2 phosphorylation and Bcl-2 interaction with Bim both decrease the anti-apoptotic activity of Bcl-2.

Translocation of BH3-only proteins

An additional mechanism that regulates Bcl-2-related protein activity is their translocation from the cytoplasm to the mitochondria, eg as a consequence of their proteolytic cleavage108109 or dephosphorylation.110111 In healthy cells, the BH3-only proteins, that do not contain a transmembrane domain, are maintained by various mechanisms in a latent form in the cytoplasm until unleashed by apoptotic signals. One of these BH3-only proteins is Bid that is cleaved by caspase-8 in some of the cells exposed to Fas agonists.108 The truncated carboxy-terminal part of Bid translocates to the mitochondria and, in cooperation with Bax,109 releases cytochrome c that activates the caspase cascade. Another BH3-only protein is Bad which is inactivated by survival-promoting factors such as interleukin-3 through several phosphorylation pathways.110 Dephosphorylation of Bad upon growth factor deprivation induces its translocation from a cytosolic 14-3-3 protein to the mitochondria.110111 A third BH3-only protein, Bim, could play an important role in microtubule agent-induced apoptosis.112 Bim is expressed in many hematopoietic cell types and Bim−/− mice have shown that the protein might play a role in regulating blood leukocyte numbers including B and T lymphocytes, monocytes and granulocytes and in platelet shedding from megakaryocytes. Bim is normally sequestered to the microtubule dynein motor complex by interaction with dynein light chain LC8. Apoptotic stimuli provoke release of Bim and LC8, allowing Bim to associated with Bcl-2-like proteins.113 When treated with taxol, purified bim−/− pre-T cells demonstrated extended survival. These cells were also more resistant to dexamethasone and ionizing radiation than wild-type cells but demonstrated similar sensitivity to etoposide.113 Thus Bim is essential for responses to certain apoptotic stimuli that can be antagonized by Bcl-2 but is largely dispensable for others (Figure 4). It thus appears that different BH3-only proteins are required to execute particular death responses in individual cell types. In addition, cleavage of Bcl-2 by caspases during the apoptotic process can transform the anti-apoptotic protein into a pro-apoptotic factor.114

Transcriptional regulation of Bcl-2-related proteins

A last mechanism that modulates Bcl-2-related protein expression and activity is their transcriptional regulation. The transcription factor p53 was demonstrated to upregulate bax gene while downregulating bcl-2 gene expression.115116 A transcriptional regulation of the BH3-only protein Hrk has also been demonstrated.117 Thus, pro-apoptotic members of the Bcl-2 family behave as sentinels for cellular damage. To avoid toxicity in healthy cells, these sentinels are either transcriptionally silent or compartmentalized in various parts of the cell. Each of them monitor specific damage, eg Bim is on the lookout for microtubule disruption, Bax and Bad for metabolic stress and Bid for limited caspase-8 activation. Whether specific alteration of the pathway could account for specific resistance to a given anticancer agent is still undetermined.

Modulation of drug-induced cell death by heat shock proteins (Hsps)

Hsps are a set of highly conserved proteins with various subfamilies, based on their molecular weight.118 Some of these proteins are constitutively expressed in mammalian cells such as Hsp90 and Hsp60, while others are induced or activated in response to stressful stimuli. The constitutively expressed Hsps behave as chaperones for other cellular proteins, eg to maintain these proteins in an active conformation, to prevent premature folding of nascent polypeptides or to facilitate their intracellular translocation. Like the Bcl-2 family of proteins, Hsps include anti-apoptotic and pro-apoptotic proteins whose expression level could determine the cell fate in response to death stimuli. For example, Hsp60 and Hsp10 form a complex with procaspase-3 in the mitochondrial fraction of Jurkat T cells.5657119 In an in vitro system, recombinant Hsp60 substantially accelerates the activation of procaspases by cytochrome c in an ATP-dependent manner and accelerates the caspase cascade.5657 Thus, Hsp60 and Hsp10 released from the mitochondria upon apoptotic stimuli actively contribute to the death process. Overexpression of Hsp90 has also been shown to increase the rate of apoptosis in U937 myelogenous leukemia cells exposed to TNFα or cycloheximide.120

In contrast, overexpression of Hsp70 protects the cells from apoptosis, both upstream and downstream of the effector caspase activation121 (Figure 3). It was proposed that Hsp70 could protect the cells from energy deprivation and/or ATP depletion associated with cell death. Hsp70 could also chaperone altered protein products generated upon caspase activation. Thus, this protein can rescue cells in a later phase of the apoptosis signaling cascade than any other known survival-enhancing protein or drug. The constitutively expressed Hsc70 was also shown to interact with BAG-1, an anti-apoptotic protein that binds to Bcl-2, although the functional impact of this interaction remains elusive.122 Recently, it was shown that Hsp70 could interfere with the signal transduction pathway leading to activation of JNK in response to stress.123124125

Hsp27 belongs to the small Hsp subfamily, a group of proteins that vary in size from 15 to 30 kDa and share sequence homologies and biochemical properties such as phosphorylation and oligomerization. Hsp27 is constitutively expressed in many cell types and tissues, at specific stages of development and differentiation. When overexpressed in tumour cells, this protein increases their tumorigenicity126 and protects these cells against apoptotic cell death triggered by various stimuli, including hyperthermia, oxidative stress, staurosporine, FasL and cytotoxic drugs.127128 These stimuli often induce Hsp27 (and Hsp70) overexpression, providing an example of a protective signal triggered by an apoptotic stimulus while simultaneously activating a death pathway. Several mechanisms could account for Hsp27 anti-apoptotic activity. The protein could increase the anti-oxidant defense of cells by increasing glutathione cell content129 and neutralizing the toxic effects of oxidized proteins by its chaperone-like activity.130 Hsp27 also binds to activated protein kinase B/Akt, a protein that generates a survival signal in response to growth factor stimulation although the consequences of this binding on the protective activity of Akt remains unclear.131 In addition, we have recently demonstrated that Hsp27, like Bcl-2, could prevent the activation of procaspase-9 and procaspase-3 in U937 human leukemic cells exposed to anticancer drugs.132 However, in contrast to Bcl-2, Hsp27 does not prevent the release of cytochrome c from the mitochondria into the cytosol (Figure 3). Actually, Hsp27 binds to cytochrome c released from the mitochondria and prevents cytochrome c-mediated interaction of Apaf-1 with procaspase-9.133

Based on the role of Hsp27 and Hsp70 in the negative regulation of drug-induced cell death, their abundant expression in many human tumors and their role in tumorigenesis, it is an obvious challenge to neutralize their anti-apoptotic activity, eg by designing specific pharmaceutical compounds binding to the active site of the protein or inhibiting their expression by specific antisense oligonucleotides. This latter strategy was shown to sensitize tumor cells to anticancer drugs in vitro and to result sometimes in spontaneous apoptosis of tumor cells.121 Another potential target for modulating Hsp70 expression in tumor cell is the Ku autoantigen whose negative influence on anticancer drug-induced apoptosis could be mediated by Hsps.134 Conversely, pharmacological stimuli or cytokines that induce Hsp expression could be proposed to protect normal tissues from stressful stimuli.

Synergy between death receptors and cytotoxic drugs

Upregulation of Fas by anticancer drugs

Tumor cells have developed multiple mechanisms to resist to Fas-mediated cell death including reduction or loss of Fas from the leukemic cell surface,135 mutation of the cytoplasmic domain of Fas,32333435 secretion of the soluble form of Fas, overexpression of anti-apoptotic proteins of the Bcl-2 family136 and overexpression of the previously described inhibitor known as c-FLIP.77137 This latter protein is structurally related to procaspase-8 but lacks a catalytic active site and the residues that form the substrate binding pocket and competitively interacts with FADD, caspase-8 and possibly caspase-10.77 Interestingly, some tumor cells that are resistant to Fas-mediated signaling can be sensitized by treatment with cycloheximide, actinomycin D, or interferon-γ.138 We and others have shown that cytotoxic drugs and ionizing radiation could upregulate the expression of the receptor Fas at the surface of tumor cells, thereby increasing tumor cell response to Fas-mediated apoptotic signals.138139140141 Thus, drug-resistant tumor cell lines which are crossresistant against Fas triggering become highly sensitive towards Fas-induced apoptosis upon exposure to anticancer drugs.141 Resistance to Fas-mediated cell death decreases tumor cell susceptibility to lymphokine-activated killer cells142143 that could be restored by drug-induced sensitization to Fas agonists.138 This observation has suggested that combining FasL with anticancer drug treatment might be an efficient strategy to trigger apoptosis of tumor cells, which is true in cultured cells. Unfortunately, intravenous infusion of agonistic anti-Fas antibody induces the rapid killing of animals due to lethal liver damage. This damage is caused by induction of Fas-dependent apoptosis in hepatocytes which express high levels of Fas.144

Synergy between TRAIL and anticancer drugs

Since TNFα also induces severe toxic side-effects when administered systematically, it was believed that cytokines of the TNF superfamily could not be used in systemic anticancer therapy. Hope came from identification of the FasL and TNF homolog designated TNF-related apoptosis inducing ligand (TRAIL),145 also known as Apo-2 ligand.146 Like other members of the TNF family of cytokines, TRAIL is a type II transmembrane protein that can be cleaved at the cell surface to form a soluble molecule.147 Interestingly, a wide range of normal tissues expresses TRAIL mRNA while being resistant to TRAIL-induced cell death, but transformed cells are sensitive to the cytokine. In addition, intravenous infusion of a soluble trimerized form of recombinant TRAIL did not induce any significant side-effects in animals.148149 However, a recent observation indicates that TRAIL induces apoptosis in normal human hepatocytes in culture.150 Thus, whether soluble TRAIL may have potential utility for systemic therapy of malignant tumors in humans remains to be established.

The differential sensitivity of normal and tumor cells to TRAIL suggested restricted receptor expression. Two type I transmembrane proteins, with a death domain similar to that of Fas and TNF-R1 in their cytoplasmic carboxy-terminal portion, were identified as receptors for TRAIL and designated TRAIL-R1 (or DR4) and TRAIL-R2 (or DR5). Two other receptors, known as TRAIL-R3 (or DcR1/TRID) and TRAIL-R4 (or DcR2), bind TRAIL with an affinity comparable to that of TRAIL-R1 and TRAIL-R2 but differ by their cytoplasmic domain and do not mediate apoptosis upon ligation.151152 TRAIL-R3 is devoid of any transmembrane or cytoplasmic domain and is glycosyl-phosphosphatidylinositol-linked to the cell surface whereas TRAIL-R4 contains only a partial death domain. Thus, TRAIL-R3 and TRAIL-R4 could act as decoy receptors that determine whether a cell is resistant or sensitive to TRAIL. An alternative hypothesis for the differential sensitivity of normal and tumor cells to TRAIL involves the differential expression of c-FLIP in these cells, a protein that also plays a role in the resistance of resting T cells to Fas-induced cell death and in the progression and immune escape of tumors in vivo.77

The safety of TRAIL administered in vivo to animals either locoregionally or intravenously148149 and its efficacy in suppressing tumor growth suggested that TRAIL-based tumor therapy may be an efficient anticancer strategy if its inocuousness is confirmed in humans.150 In addition, the ability of subtoxic concentrations of chemotherapeutic drugs to restore TRAIL-mediated pathway to death in cell lines that are resistant to TRAIL-induced cytotoxicity153 suggested that combination of an anticancer drug with TRAIL may enforce the TRAIL-based therapy.154155156 Interestingly, the synergy of TRAIL with anticancer drugs such as etoposide was observed in multiple myeloma cell lines that have been selected for their resistance to cytotoxic drugs and Fas-mediated apoptosis.154 The mechanisms proposed to account for the anticancer drug/TRAIL synergy include the p53-dependent or independent transcriptional induction of TRAIL-R1 and -R2, the decreased expression of intracellular inhibitors of TRAIL-induced apoptosis such as c-FLIP77 and the upregulation of pro-apoptotic molecules such as FADD and procaspases (Lacour S et al, submitted). The precise mechanism could depend on the tumor cell type, the cytotoxic drug and the concentration used to sensitize tumor cells to TRAIL-induced cell death. Whatever this molecular mechanism, preliminary studies testing the combination of TRAIL with 5-fluorouracil or camptothecin in vivo have confirmed the great potential interest of this strategy in treating human tumors.149

The role of the cell cycle regulating protein p53

The DNA damage checkpoints

DNA damage induced by cytotoxic drugs activates the DNA-dependent protein kinase (DNA-PK)157 and the ataxia-telangiectasia mutated (ATM) protein, a widely expressed member of the family of protein kinases with similarities to phosphatidylinositol 3-kinases.158 A key downstream target of DNA-PK and ATM is the p53 gene product, a sequence-specific transcriptional activator and critical human tumor suppressor which is phosphorylated and stabilized by these proteins. In turn, p53 induces cell cycle arrest or apoptosis. Another downstream target for ATM is the non-receptor tyrosine kinase c-Abl.159 In response to DNA damage, activated c-Abl was demonstrated to activate p73, the product of one of the two p53-related genes that have been recently discovered.160161162 While no relationship has been described so far between DNA damage and the other p53-related protein known as p63, p73 is either stabilized or phosphorylated in response to alkylating agents and radiations, respectively.160161162

The observation that p53 is a tightly regulated transcription factor that is activated in response to DNA damage provided a biological basis for understanding its involvement in maintaining the integrity of the genome. p53-driven cell cycle arrest prevents cells with altered DNA from proliferating and p53-controlled apoptosis (eg through down-regulation of bcl-2 and upregulation of bax) selectively eliminates severely damaged cells.163164 Recently, p53 was shown also to contribute to DNA damage repair by upregulating the newly described p53R2 gene that encodes a subunit of the ribonucleotide reductase.165

Cells respond to DNA damage by stopping the cell cycle either at the G1 DNA damage checkpoint, before DNA replication, or at the G2 DNA damage checkpoint, just before mitosis. p53 is an essential component of the G1 DNA damage checkpoint (Figure 5). In the absence of p53, cell cycle arrest in response to DNA damage occurs in G2 which has important therapeutic consequences. In cancer cells that lack p53, agents that disrupt the G2 DNA damage checkpoint, such as 7-hydroxystaurosporine (UCN-01), selectively potentiate the cytotoxic effects of DNA damaging agents.166 While p53-independent interaction of one of the seven human members of the 14–3–3 family of proteins with cdc25C is required for initiating the G2 DNA damage checkpoint, p53 contributes to sustain this checkpoint by activating the 14–3–3σ protein.167 The consequence of 14–3–3σ activation is the nuclear export of p34cdc2/cyclin B1 complexes to prevent the G2 checkpoint bypass. p34cdc2 is maintained in an inactive state by phosphorylation on tyrosine 15 by the nuclear kinase Weel and/or on threonine 14 and tyrosine 15 by the cytoplasmic kinase Myt1. Components of the pathway such as 14–3–3/cdc25C and 14–3–3σ/p53 were proposed as therapeutic targets for anticancer drug development since their inhibition might enhance the antitumor activity of DNA damaging agents167 (Figure 5).

Figure 5

 DNA damage checkpoints. DNA damage activate ataxia-telangiectasia mutated (ATM) protein that, in turn, activates either the non-receptor tyrosine kinase c-Abl and p73 or p53. The p73 and the p53 proteins can induce cell cycle arrest in the G1 phase. p53 can also trigger apoptosis. p53-independent interaction of one of the seven human members of the 14–3–3 family of proteins with cdc25C is required for initiating the G2 DNA damage checkpoint. p53 could sustain the DNA damage G2 checkpoint by interacting with the 14–3–3σ protein (see text).

p53 status and cell sensitivity to DNA damaging agents

p53 status influences the decision of whether a cell undergoes apoptosis after a genotoxic insult and the rate at which the cell dies. However, the question remains open whether and how p53 status affects the overall sensitivity of cells to anticancer drugs inducing genotoxic damage. Studies performed in dominant oncogene-transformed fibroblasts from embryos of p53 wild-type and p53 knock-out mice have suggested a role of p53 mutations in radiation and anticancer drug resistance.168169 This observation was enforced by the highly significant association of p53 mutation with drug resistant in a series of 60 cell lines used for screening anticancer agents.170 This hypothesis was challenged when in vitro cell killing was measured with long-term colony-forming assays rather than short term assays measuring growth inhibition, functional changes such as trypan blue or propidium iodide uptake or apoptosis. Actually, in many leukemia and lymphoma cell lines that are highly sensitive to chemotherapeutic agents and radiations and die rapidly through apoptosis in response to drug-induced DNA damage, mutation of p53 could behave as a true resistance factor to some specific genotoxic insults. In other cells, short-term assays can be misleading and apoptosis does not necessarily predict overall sensitivity to DNA damaging agents.90

One of the reasons for which no clear correlation can be established between p53 status and cell sensitivity to DNA damaging agents is the role of wild-type p53 protein in nucleotide excision repair, eg by activating the recently described p53-target gene p53R2 that plays a crucial role in supplying deoxyribonucleotides for DNA repair.165 Due to this function, p53 can protect cells from death induced by DNA damaging agents such as radiations, anthracyclines and alkylating agents. Conversely, inactivation of p53 protein could enhance cell sensitivity to chemotherapeutic drugs that activate a p53-dependent nucleotide excision repair mechanism. Similarly, altered checkpoint due to mutation of p21WAFI/CIP1, one of the main targets of p53, was shown to sensitize tumor cells to DNA damaging agent-induced apoptosis, as a consequence of uncoupling between mitosis and S phase. Accordingly, xenografts established from tumor cells with altered p21 checkpoint were more sensitive to radiations than those obtained with wild-type p21, otherwise isogenic cell lines.171

It was proposed that engineered minimally transformed cells such as normal fibroblasts expressing dominant oncogenes are hypersensitive to DNA damage-induced apoptosis as compared to normal cells, due to ARF-mediated upregulation of p53.172 In this situation, mutation of p53 only suppresses this hypersensitive state artificially created by the dominant oncogene.173 In the majority of human tumors, however, this hypersensitivity to DNA damage induced apoptosis has been lost, due to p53 mutation, bcl-2 overexpression or other changes.90116174 Reactivation of these pathways would improve the therapeutic potential of current cancer therapy by restoring the apoptotically sensitive phenotype associated with initial transformation.

What is good for eliminating tumor cells can be bad for normal cells. Chemotherapy and radiations often damage healthy tissues while killing tumor cells. Healthy tissues from p53-deficient mice were shown to suffer less damage from gamma irradiation than the healthy tissues from normal mice. A new pharmacological compound, called pifithrin-α, was recently shown to protect vulnerable tissues by temporarily and reversibly blocking p53. A single injection of pifithrin-α rescued normal mice from a near-lethal radiation exposure without changing the response of p53-deficient tumor xenografts, providing a potential way to give patients optimal doses of chemotherapy, at least when bearing a p53-deficient tumor.175

The role of Rel/NF-κB/IκB proteins

A typical example of the dual nature of signals triggered by cytokines and anticancer drugs concerns a protein called nuclear factor kappa B (NF-κB).176 The observation that protein synthesis inhibitors such as cycloheximide could increase cell killing by TNFα or FasL had suggested that genes might be turned on by these cytokines and protect against cell death. The massive apoptosis of liver cells that induces the death of embryonic NF-κB knock-out mice suggested that NF-κB was an anti-apoptotic protein.177 Various strategies then demonstrated that turning off NF-κB could sensitize lymphoid and other tumor cells to death induced by TNFα and anticancer drugs.178

The transforming properties of mutant IκB (also known as Bcl-3) has been demonstrated in human chronic lymphocytic leukemia,179 while amplification or rearrangements of NF-κB members have been described in various hematological malignancies.180 In most normal cells, NF-κB is bound up in the cytoplasm by an inhibitor molecule called IκB.181 Serine phosphorylation of this inhibitor triggers its proteolytic degradation for NF-κB to be activated. Then, the protein participates in the transcriptional initiation of diverse genes. On one hand, NF-κB upregulates several survival factors in lymphoid cells, including the expression of some IAPs182183184 that, in turn, activate NF-κB in a positive feedback loop. For example, it was proposed that, following infection with the Epstein–Barr virus (EBV), the host cell surface late antigen LMP1 acts as a constitutively active receptor molecule triggering NF-κB activation, which could explain the high level of c-IAP1 in Reed–Sternberg cells that characterize Hodgkin's disease.185186 On the other hand, NF-κB can directly transactivate the cytotoxic ligand FasL in response to anticancer drugs or radiations.187 Upregulated FasL could then contribute to cell death by a previously described autocrine or paracrine pathway.

The role of PML in drug-induced leukemic cell death

PML is another protein involved in cell death regulation188189 whose expression can be modified in human leukemic cells.190 Expression of the pml gene can increase in response to interferons (IFN) and cytotoxic drugs.188190 The cell growth and tumor suppressor PML protein is typically concentrated in discrete speckled nuclear structures called PML nuclear bodies (NBs) or PML oncogenic domains (PODs).191192 These nuclear-matrix associated structures of unknown function contain a number of different proteins, several of which were isolated as transforming fusion proteins. In acute promyelocytic leukemia (APL), recurrent translocations fuse PML or PLZF, another protein localized in PODs, to the gene RARA encoding the retinoic acid receptor alpha.193 In t(15;17) APL, PML/RARα fusion disrupts the NB localization of PML.194 PML-RARα increases the survival of hematopoietic cell lines. It has been proposed that APL pathogenesis relies in part on transcriptional silencing of retinoic acid target genes through the tethering by PML/RARα of stabilized corepressor-histone acetyltransferase complexes and in part on the loss of PML-triggered growth suppression.195196197 Two therapeutic agents, retinoic acid and arsenic trioxide, that induce clinical remissions in APL through differentiation and apoptosis, respectively, induce the restoration of the normal NB pattern of PML and associated proteins.198199 PML expression or cellular localization is altered in a number of other situations that involve abnormal cell survival, including viral infections and other human cancers. Altogether, these observations suggested that PML could be involved in apoptosis regulation.200

Accordingly, cells derived from pml−/− mice are resistant to a variety of caspase-dependent apoptosis inducers, including DNA damaging agents, death receptor ligands and ceramides, suggesting a broad involvement of PML in death pathways.189 In addition, overexpression of PML was shown to induce apoptosis.188 Surprisingly, PML-induced apoptosis was observed to be caspase-independent and to be enhanced by the broad caspase inhibitor z-VAD-fmk.188 Thus, the pml gene product can modulate both caspase-independent and caspase-dependent cell death pathways. To account for this dual effect, it was proposed that PML acted as a pivotal checkpoint in a central cell death pathway.188189

PML-RARα can heterodimerize with and sequester PML, thus interfering with its function in apoptosis. Arsenic, which accelerates PML targeting to NBs and degrades PML, accelerates PML-triggered death. Thus, PML traffic to NBs was suggested to be the critical determinant for death induction. Accordingly, this traffic is associated with the recruitment of other proteins such as Bax and p27Kip1 that can modulate cell death.188201 Another protein that co-localizes within nuclear PODs is human Daxx, a protein initially described to bind Fas receptor. Interestingly, overexpression of Daxx increases cell sensitivity to Fas-mediated caspase-dependent apoptosis, probably by interacting with other proteins present in PODs, and arsenic trioxide acts synergistically with Daxx to increase this apoptotic response to Fas agonists.202

Lipid-dependent signaling pathways

In addition to specific damage induced in target cells, eg DNA damage, anticancer agents simultaneously activate several lipid-dependent signaling pathways that can either stimulate or prevent the activation of the cell death machinery in response to this damage. One of the most studied of these signaling pathways that could modulate cell sensitivity to chemotherapeutic drugs and ionizing radiation is the sphingomyelin-ceramide pathway, a pathway which is activated within a few minutes after drug interaction with the target cell203204205 (Figure 6).

Figure 6

 Lipid-dependent signaling pathways. Anticancer drugs can activate simultaneously several antagonistic lipid-dependent pathways. For example, by activating a sphingomyelinase, anticancer agents induce ceramide generation. By activating the JNK1/SAPK pathway, ceramide facilitates cell death by apoptosis. Simultaneously, drug-induced activation of a phospholipase C generates diacylglycerol that activates protein kinase C. This kinase inhibits the ceramide pathway both upstream and downstream of ceramide generation. The reality is still more complex since ceramide itself can activate both pro- (eg JNK1/SAPK) and anti- (eg PKC ξ) apoptotic pathways. Cross-talks between these pathways contributes to determine the fate of the cell.

Sphingomyelin was initially considered as a structural element in eukaryotic cell membranes. Then, hydrolysis of sphingomyelin leading to ceramide generation was identified to occur in response to a large number of natural and pharmacological effectors and to play a role in cell sensitivity to apoptosis induction by these effectors. Several chemotherapeutic drugs were shown to activate a sphingomyelinase in leukemic cells, including daunorubicin, mitoxantrone, etoposide, vincristine, dexamethasone, ara-C and cis-platinum.206207208209 In addition, hydrolysis of sphingomyelin was identified in cells exposed to ionizing radiation,210 FasL211 and TNFα.212 The pool of sphingomyelin and the nature of the sphingomyelinase involved in ceramide generation remain controversial and could vary, depending on the cell type and the death stimulus. Ceramide generated in response to apoptotic stimuli activates several intracellular targets in which the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) cascade may play an essential role in ceramide-mediated apoptosis.213

Protein kinase C activators including phorbol esters and diacylglycerol can negatively interfere with the sphingomyelin-ceramide pathway both upstream of ceramide generation by preventing sphingomyelinase activity and downstream of ceramide by inhibiting its ability to induce apoptosis214215 (Figure 6). Interestingly, most anticancer drugs as well as cytokines such as TNFα are capable of activating the production of diacylgycerol in parallel with activating ceramide generation. Thus, the cytotoxic activity of an anticancer agent can be modulated by the balance between the noxious pathway mediated by ceramide and the protection pathway mediated by diacylglycerol. This observation opens perspectives of pharmacological manipulations aimed at increasing the lethal pathways while inhibiting the protective signals.216 For example, neutralization of oncogenes such as c-Abl that may contribute to accelerate diacylglycerol turnover could sensitize the cells to drug-induced cell death.

Such an approach could be extended to other signaling pathways that could be activated either directly by the cytotoxic agent or by other therapeutic molecules such as growth factors given simultaneously. For example, the phosphatidylinositol 3′-kinase, that is implicated in mitogen signals and survival, can be activated by some anticancer drugs.217 Downstream targets of this kinase include the mitogen-activated protein kinase cascade,218 that antagonizes ceramide-induced JNK/SAPK activation, and the atypical κ isoform of protein kinase C that is also involved in cell survival and mitogen response.219


A few years ago, anticancer drug activity was mainly envisioned as the consequences of drug–target interaction: the more damage induced by the drug, the more efficacy in killing tumor cells. What the study of apoptosis has taught us is that anticancer drugs, in addition to inducing specific intracellular damage, generate a series of protective and noxious signals that modulate the death response to specific damage. For example, anticancer drugs very rapidly activate several antagonistic lipid-dependent pathways that modulate cell death. These drugs can also trigger accumulation of anti-apoptotic Hsps220 and activate the protective transcription factor NF-κB, while activating the mitochondrial pathway and the death receptor-dependent pathway to cell death, respectively. In addition, the caspase-mediated cleavage of intracellular proteins generates peptides that either amplify the death process (eg cleavage of gelsolin contributes to morphological changes associated with apoptosis) or negatively interfere with cell death pathways (the p27Kip1 N-terminal fragment prevents further caspase activation).221222 Arsenic-mediated relocalization of PML and Daxx in the nucleus could also sensitize some leukemic cells to anticancer drug- and cytokine-induced apoptosis. Lastly, anticancer drugs increase the sensitivity of some tumor cells to death receptor agonists such as FasL and TRAIL. Thus, analysis of the molecular mechanisms of cell death has dramatically increased the field of anticancer drug pharmacology and suggested new strategies for tumor cell sensitization or normal cell protection. Some of them might be tested in clinics in the future.


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Our group is supported by grants from INSERM, the Ligue Nationale Contre le Cancer (comittees: Côte d'Or, Saône et Loire, Nièvre), the Association pour la Recherche sur le Cancer (No. 9567), the Association pour la Recherche sur la Transfusion (ART) and the Association Régionale pour l'Enseignement et la Recherche Scientifique et Technologique en Champagne-Ardenne (ARERS). ND is the recipient of a grant from the Société Française d'Hématologie.

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  • apoptosis
  • cytotoxic agents
  • caspases
  • Bcl-2
  • death receptors
  • hematological malignancies

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