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Keywords:

  • acute myeloid leukaemia;
  • apoptosis;
  • death receptor;
  • TNF-related apoptosis-inducing drug;
  • cytotoxic drug

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Actions of TRAIL on AML cells
  6. Actions of TRAIL and cytotoxic drugs on AML cells
  7. Molecular analysis of apoptosis induction by TRAIL and cytotoxic agents
  8. Processing of pro-caspase 8 and BID
  9. Discussion
  10. Acknowledgments
  11. References

Summary. We have studied the actions of tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) on cells isolated from patients with acute myeloid leukaemia (AML). Apoptosis induction was initially assessed by quantitative morphological analysis. Only 2/19 isolates showed a > 10% increase in apoptotic cells following TRAIL treatment. However, incubation with TRAIL combined with fludarabine, cytosine arabinoside or daunorubicin resulted in additive or super-additive apoptosis induction in approximately half of the isolates. Molecular evidence of super-additive apoptosis induction by TRAIL and cytotoxic agents was obtained by quantification of caspase 3 activation, detected by Western blot analysis of poly (ADP ribose) polymerase cleavage. The ability of TRAIL and daunorubicin to induce super-additive apoptosis correlated with the ability of these agents to activate caspase 8 and to augment cellular levels of the truncated pro-apoptotic form of the BCL-2 family member BID. Our data suggest that co-administration of TRAIL with conventional cytotoxic drugs may be of therapeutic value in some patients with AML.

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of a family of death ligands, which also includes TNF and FAS. Binding of these ligands to cognate death receptors (DRs) initiates pro-apoptotic signalling pathways (Ashkenazi & Dixit, 1998; Nicholson, 2000). Binding of TRAIL to DR4 or DR5 triggers recruitment of the adapter protein FADD (Fas-associated death domain) and pro-caspase 8 to the ligand-bound receptor (Chaudhary et al, 1997; Schneider et al, 1997; Bodmer et al, 2000; Kischkel et al, 2000; Sprick et al, 2000). Consequent cleavage and activation of caspase 8 then initiates the apoptotic signal. Caspase-8-mediated activation of effector caspases, such as caspase 3, initiates proteolysis of a restricted set of ‘death substrates’, including poly (ADP ribose) polymerase (PARP), resulting in the morphological and functional changes that are characteristic of apoptotic cell death (Wickremasinghe & Hoffbrand, 1999; Hengartner, 2000).

In contrast, the majority of cytotoxic drugs induce apoptosis by promoting cytochrome c release from mitochondria to the cytosol. Subsequent cytochrome c binding to the cytoplasmic apoptosis protease activating factor-1 (APAF-1) protein enables the latter protein to enhance the latent proteolytic activity of pro-caspase 9, resulting in the activation of caspase 3 (Wickremasinghe & Hoffbrand, 1999; Hengartner, 2000). In some cell types, the death-ligand-initiated and mitochondria-dependent mechanisms represent distinct apoptosis-inducing pathways. However, in cells in which initial death-ligand-induced cleavage of pro-caspase 8 is inefficient, cell killing requires the initial signal to be amplified via mitochondrial cytochrome c release. This crosstalk is achieved via cleavage of the pro-apoptotic BCL-2 family member BID by caspase 8. The truncated BID (tBID) translocates from the cytosol to the mitochondrion, resulting in cytochrome c release and activation of the APAF-1/caspase 9 mechanism (Krammer, 2000). Recent evidence has shown that TRAIL induces BID cleavage, collapse of the mitochondrial membrane potential and cytochrome c release in malignant cell lines (Lacour et al, 2001; Rohn et al, 2001; Rokhlin et al, 2001; Suliman et al, 2001; Thomas et al, 2000).

In addition to the levels of DR4 and 5 expression, several other determinants modulate the sensitivity of cells to TRAIL-induced killing. The decoy receptors DcR1 and DcR2, which bind TRAIL but are unable to transduce a death signal, can inhibit cell killing by sequestration of the death ligand (Ashkenazi & Dixit, 1998; Nicholson, 2000). The intracellular cFLIP [cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein], a catalytically inactive pseudo-caspase which blocks caspase 8 recruitment and activation by ligand-bound death receptors, also inhibits death signalling by TRAIL (Schneider et al, 1997; Ashkenazi & Dixit, 1998; Nicholson, 2000).

Studies on cell lines suggest that TRAIL may kill tumour cells more effectively than normal cells (Ashkenazi et al, 1999; Walczak et al, 1999). Furthermore, cytotoxic drugs and TRAIL induced the super-additive killing of solid tumour (Ashkenazi et al, 1999; Nagane et al, 2000), leukaemia (Wen et al, 2000) and multiple myeloma (Mitsiades et al, 2001) cell lines. This super-additive effect may be attributable to drug-induced augmentation of death receptor expression. Only a limited number of studies of TRAIL sensitivity have been carried out using cells freshly isolated from patients with haematological malignancies. A modest level of killing of B acute lymphoblastic leukaemia cells suggested that TRAIL may have a limited role as a single agent for treatment of this malignancy (Clodi et al, 2000). In a study of 21 acute myeloid leukaemia (AML) cell isolates (Wuchter et al, 2001), supra-additive killing by TRAIL and doxorubicin was observed in only one cell isolate.

Here we have studied the sensitivity of AML cells to TRAIL alone and in combination with fludarabine, 1-β-d-arabinofuranosylcytosine (Ara C) or daunorubicin. The data showed that only a minority of AML cell isolates were TRAIL sensitive. However, simultaneous exposure to TRAIL and cytotoxic drugs resulted in supra-additive killing of cells isolated from approximately half of the patients studied.

Reagents. Tissue culture materials were from Life Technologies, Paisley, Scotland. Propidium iodide (PI) and cytotoxic drugs were from Sigma, Poole, UK. TRAIL was from Peprotech, London, UK.

Patients and cells. Blood or bone marrow samples from patients with AML were obtained with informed consent. The clinical features of the patients are summarized in Table I. Mononuclear cells were obtained by centrifugation on Ficoll–Hypaque gradients (Nycomed, Oslo, Norway). All samples studied contained in excess of 80% blast cells. Cells were cultured immediately following isolation in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin and 100 µg/ml streptomycin.

Table I.   Clinical data on AML patients.
PatientSampleSubtypeSex/age (years)WBC (× 109/l)
  1. WBC, white blood cell count; PB, peripheral blood; BM, bone marrow; NA, data not available.

198PBNANANA
200PBM4F/37 35
204PBM4F/65NA
205PBM4F/61NA
208BMM1M/68 17·3
211BMM1F66NA
215PBM5F/40 90
216PBM1M/71 90
223PBM1F/71 50
228BMM4F/74 59
229PBM1F/76 60
232PBM1M/62 34
235PBMDSM/65NA
238PBNAF/62188
250PBNAM/76104
269PBM0F/72 47
271BMNAF/70 79
273BMM2F/70 63
289PBM4M/63NA

Assessment of apoptosis.  Apoptotic cells were estimated by counting the proportion of cells with condensed or fragmented nuclei in May–Grunwald-Giemsa-stained cytospin preparations (McGahon et al, 1994; Cotter & Martin, 1996). At least 1000 cells in four randomly selected fields were counted on each slide. The standard errors of these determinations were less than 10% of the mean values, and have been omitted from the figures.

Specific apoptosis induced by a given agent or agents was calculated using the following formula as previously described (Posovszky et al, 1999):

  • image

Interactions between TRAIL and cytotoxic agents were arbitrarily classified by the following criteria: super-additive interaction, apoptosis induction by TRAIL plus a cytotoxic agent exceeded the theoretical additive apoptosis induction by TRAIL and the cytotoxic agent applied singly by > 1·5-fold; inhibitory interaction, apoptosis induction by TRAIL plus cytotoxic agent was less than the theoretical additive apoptosis induction by TRAIL and the cytotoxic agent applied singly by > 20%; resistant, combinations of TRAIL plus cytotoxic agent induced < 5% specific apoptosis. Other interactions were considered to be essentially additive. The same criteria were used to classify responses measured by quantification of PARP cleavage.

Western blot.  Cell lysis and Western blot analysis were carried out as described (Jones et al, 2001). The following primary antibodies were used: pro-caspase 8 (monoclonal B9-2; Pharmingen, Oxford, UK), caspase 8 subunits (polyclonal, Santa Cruz, CA, USA), 85 kDa PARP fragment (p85 PARP, polyclonal, Promega, Southampton, UK), 116 kDa intact PARP (p116 PARP, monoclonal; Pharmingen, Cowley, UK), BID (polyclonal; Pharmingen), β-actin (monoclonal AC15; Sigma), p53 (monoclonal; Santa Cruz). Bands were visualized using appropriate horseradish peroxidase-linked secondary antibodies (Dako, Ely, UK) and the enhanced chemiluminescence system (ECL; Amersham Pharmacia, Little Chalfont, UK), and analysed using the GS700 densitometer and quantity one software (Bio-Rad, Hemel Hempstead, UK).

Reverse transcription polymerase chain reaction (RT-PCR). Total cell RNA was extracted by the guanidinium isothiocyanate–acid phenol method (Chomczynski & Sacchi, 1987). cDNA synthesis and PCR were carried out as described (Jones et al, 2001). The following primers were used: β-actin forward, 5′-TGC TAT CCA GGC TGT GCT AT-3′; β-actin reverse, 5′-GAT GGA GTT GAA GGT AGT TT-3′; BID forward, 5′-CGC TTG GGA AGA ATA GAG GCA G-3′; BID reverse, ACA CTT CTG GAA CTG TCC GTT CAG-3′; p21WAF1 forward, 5′-AGA TTT CTA CCA CTC CAA ACG CC-3′; p21WAF1 reverse, 5′-CCC TTC AAA CTG CCA TCT GTT TAC-3′.

PCR products were visualized on ethidium-bromide-stained 3% agarose gels. Bands were quantified by densitometry using the Gel Doc video camera and quantity one software (Bio-Rad). BID and p21WAF1 band intensities were normalized to the density of actin PCR bands generated from the same sample.

Actions of TRAIL on AML cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Actions of TRAIL on AML cells
  6. Actions of TRAIL and cytotoxic drugs on AML cells
  7. Molecular analysis of apoptosis induction by TRAIL and cytotoxic agents
  8. Processing of pro-caspase 8 and BID
  9. Discussion
  10. Acknowledgments
  11. References

The actions of 500 ng/ml TRAIL on freshly isolated AML cells ex vivo were studied by quantitative morphological analysis of apoptosis (Fig 1). Modest TRAIL-induced increases in the percentage of apoptotic cells were seen in 14 of the 19 isolates studied. Only three of the isolates (AML 205, 232 and 289) showed a greater than 10% increase in apoptotic cells in the TRAIL-treated samples relative to spontaneous apoptosis. In contrast, treatment of Jurkat cells with 500 ng/ml TRAIL induced 70% apoptotic cells following 24-h incubation (not shown), confirming the ability of the TRAIL preparation to induce cell killing.

image

Figure 1.  Actions of TRAIL on AML cells ex vivo. Apoptosis was quantified by morphological criteria. The standard errors of these determinations were less than 10% of the mean values shown. Incubations were in the absence (open bars) or presence (shaded bars) of 500 ng/ml TRAIL. AML isolates are identified by number (see Table I).

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Actions of TRAIL and cytotoxic drugs on AML cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Actions of TRAIL on AML cells
  6. Actions of TRAIL and cytotoxic drugs on AML cells
  7. Molecular analysis of apoptosis induction by TRAIL and cytotoxic agents
  8. Processing of pro-caspase 8 and BID
  9. Discussion
  10. Acknowledgments
  11. References

We then quantified apoptosis induced by co-incubation of AML cells with TRAIL and cytotoxic agents, and compared these values to the expected additive actions of TRAIL and each cytotoxic agent added alone. Data obtained using multiple AML isolates exposed to TRAIL in combination with fludarabine, Ara C or daunorubicin are summarized in Fig 2. To correct for spontaneous apoptosis, the percentage of apoptotic cells determined following each treatment was expressed as specific agent-induced apoptosis, as described in Materials and methods. The specific apoptosis induced by co-incubation with TRAIL and each cytotoxic agent is shown by an open bar. The composite bar shows the theoretical additive apoptosis expected by summing the separate actions of TRAIL alone (hatched bar) and of the cytotoxic agent alone (solid bar).

image

Figure 2.  Interactions between TRAIL and cytotoxic agents in the induction of apoptosis of AML cells ex vivo. The open bars show apoptosis induction following incubation with combinations of TRAIL (500 ng/ml) and the relevant cytotoxic agent [Flu, fludarabine (A); Ara C, 1-β-d-arabinofuranosylcytosine (B); DNR, daunorubicin (C)]. The composite bars denote the theoretical additive apoptosis induced by TRAIL alone (hatched area) and by the cytotoxic agent alone (solid area). The pattern of response to TRAIL plus cytotoxic agent is indicated thus: S, super-additive; A, additive; I, inhibitory; R, resistant (see Materials and methods).

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The types of interaction between TRAIL and cytotoxic agents are shown (as defined in Materials and methods), and were dependent on the particular AML isolate and the cytotoxic agent. Essentially additive interactions were seen in 3/15 isolates treated with fludarabine, 2/15 with Ara C and 2/19 with daunorubicin. Super-additive apoptosis induction was seen in 6/15 samples incubated with fludarabine, 7/15 with Ara C and 10/19 with daunorubicin. For example, cells from AML 208, which were resistant to either fludarabine or daunorubicin and only minimally sensitive to TRAIL alone, showed super-additive apoptosis induction when exposed to combinations of TRAIL and either cytotoxic agent (Fig 2). AML 216 also showed substantial super-additive apoptosis when fludarabine, Ara C or daunorubicin were combined with TRAIL. AML 229 was completely resistant to TRAIL alone, but showed super-additive apoptosis when TRAIL was combined with each of the three cytotoxic agents, the most pronounced effects being seen with daunorubicin.

In contrast, TRAIL inhibited the apoptotic response of other isolates to cytotoxic agents (Fig 2). For example, the observed response of AML 204 to TRAIL in combination with Ara C or daunorubicin was substantially reduced compared with the expected additive level of apoptosis. Some isolates were essentially resistant to the actions of TRAIL and cytotoxic agents. AML 250 showed minimal levels of apoptosis following incubation with TRAIL and each cytotoxic agent. In contrast, cells from AML 200 were resistant to TRAIL alone or to combinations of TRAIL and fludarabine, but showed low but clearly detectable apoptosis in response to TRAIL plus Ara C or daunorubicin.

The time dependence and dose–responses of cells from AML 235 are shown in Fig 3. This isolate was entirely resistant to apoptosis induction by TRAIL alone. At 24 h, cells incubated with TRAIL plus Ara C showed clear super-additive apoptosis at all levels of the drug tested (Fig 3A). Strikingly, 5 µmol/l Ara C alone induced no detectable apoptosis while 20% of apoptotic cells were seen in cultures incubated with Ara C plus 500 ng/ml TRAIL. Higher levels of apoptosis were induced by Ara C alone following 48-h incubation, and super-additive killing with TRAIL was less pronounced than at 24 h but was still evident (Fig 3B). At both 24 and 48-h incubation, a super-additive response with TRAIL was evident at 0·1 µmol/l daunorubicin (Fig 3C and D). A high level of apoptotic killing was induced by 1 µmol/l daunorubicin alone, with only marginal evidence of super-additivity with TRAIL.

image

Figure 3.  Dose and incubation time dependence of interactions between TRAIL and cytotoxic agents. Cells from patient 235 were incubated with the indicated level of cytotoxic agent in the absence (open circles) or presence (closed circles) of 500 ng/ml TRAIL. Spontaneous apoptosis in this experiment was 6% at 24 h and 13% at 48 h. Specific apoptosis was evaluated as described in Materials and methods. This isolate was completely resistant to killing by TRAIL alone.

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Molecular analysis of apoptosis induction by TRAIL and cytotoxic agents

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Actions of TRAIL on AML cells
  6. Actions of TRAIL and cytotoxic drugs on AML cells
  7. Molecular analysis of apoptosis induction by TRAIL and cytotoxic agents
  8. Processing of pro-caspase 8 and BID
  9. Discussion
  10. Acknowledgments
  11. References

The caspase-3-mediated cleavage of p116 PARP to p85 PARP provides a sensitive and unambiguous molecular criterion to assess apoptosis induction (Nicholson et al, 1995). In order to corroborate conclusions based on morphological analysis of apoptosis (Fig 2), we used an antibody specific for p85 PARP but unreactive with intact p116 PARP to assess the interactions between TRAIL and cytotoxic agents. Cells from AML 229 showed no evidence of TRAIL-induced generation of p85 PARP following 24-h incubation (Fig 4). While incubation with fludarabine alone resulted in no PARP cleavage, generation of the p85 fragment was clearly evident in cells co-incubated with TRAIL plus fludarabine. Super-additive PARP cleavage was also evident in cells treated with Ara C plus TRAIL, and was particularly prominent when daunorubicin was combined with the death ligand. Quantitative analysis of the Western blot data by scanning laser densitometry and normalization of the p85 band intensities with respect to the intensities of the p116 PARP band in the same lane confirmed that co-incubation with TRAIL enhanced the PARP cleavage response of AML 229 cells to all three cytotoxic drugs (Fig 4). PARP cleavage analysis (not shown) also confirmed the ability of cytotoxic drugs to synergize with TRAIL in inducing apoptosis of the AML 223 isolate (Fig 2).

image

Figure 4.  Interactions between TRAIL and cytotoxic agents in the induction of PARP cleavage. Cells isolated from AML 229 were incubated for 24 h with the indicated additions. Flu was at 17·5 µmol/l, Ara C at 40 µmol/l and DNR at 0·1 µmol/l. Relative levels of p116 PARP, p85 PARP and actin were determined by Western blotting. The Western blots were analysed by laser densitometry, and the ratio p85 PARP/p116 PARP was evaluated for each lane. Open bars show PARP cleavage induced by co-incubation with 500 ng/ml TRAIL plus a cytotoxic agent. The solid bars show the PARP cleavage induced by the cytotoxic agent alone. PARP cleavage induced by TRAIL alone was negligible.

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The synergy between TRAIL and daunorubicin revealed by morphological analysis of AML 271 (Fig 2C) was also confirmed by analysis of PARP cleavage (Fig 5A). In contrast, AML 273, which was resistant to TRAIL, daunorubicin or a combination of these agents (Fig 2C), showed no induction of PARP cleavage under any of the incubation conditions tested (Fig 5B). Therefore, analysis of PARP cleavage in a total of four isolates was concordant with morphological analysis of apoptosis induction in the same samples, and confirms the ability of TRAIL and cytotoxic drugs to synergize in killing a subset of AML.

image

Figure 5.  Processing of pro-caspase 8 and BID in AML cells incubated with TRAIL and DNR. Malignant cells isolated from two patients were incubated with 500 ng/ml TRAIL, 0·1 µmol/l DNR or TRAIL + DNR. Proteins were extracted at 6 h or 24 h and analysed by Western blotting. Normalized densities of bands are shown numerically. SU/Pro, caspase 8 subunits/pro-caspase 8.

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Processing of pro-caspase 8 and BID

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Actions of TRAIL on AML cells
  6. Actions of TRAIL and cytotoxic drugs on AML cells
  7. Molecular analysis of apoptosis induction by TRAIL and cytotoxic agents
  8. Processing of pro-caspase 8 and BID
  9. Discussion
  10. Acknowledgments
  11. References

The processing of pro-caspase 8 and of BID was studied in AML cells treated with daunorubicin and TRAIL. Cells from AML 271 showed specific apoptosis of 41% following 24-h incubation with TRAIL plus daunorubicin, compared with 1% with TRAIL alone and 16% with daunorubicin (Fig 2C). At 6-h or 24-h incubation, TRAIL alone induced no detectable processing of pro-caspase 8 to its active subunits, consistent with the resistance of this isolate to TRAIL-induced PARP cleavage and apoptosis (Fig 5A). Cells treated with daunorubicin alone for 24 h showed evidence of pro-caspase 8 processing as well as a more pronounced tBID immunoreactive band. The highest levels of both the caspase 8 subunits and tBID were seen in cells treated simultaneously with TRAIL and daunorubicin, with evidence of super-additive generation of both, relative to levels in cells treated with each agent alone (Fig 5A). A similar response pattern to TRAIL and daunorubicin of super-additive apoptosis induction (Fig 2C), pro-caspase 8 cleavage and tBID generation was also seen in cells from AML 269 (data not shown).

In contrast, cells from AML 273 were highly resistant to TRAIL and daunorubicin, either as single agents or in combination (Fig 2C). Furthermore, no processing of either pro-caspase 8 or BID was detected under any of the incubation conditions (Fig 5B). Our data are, therefore, consistent with the interpretation that induction of super-additive apoptosis by combined exposure to TRAIL and daunorubicin correlated with the ability of these agents to induce super-additive proteolytic processing of pro-caspase 8 and BID.

The Western blots of AML 271 (Fig 5A) suggest that the total amount of immunoreactive BID was apparently augmented in cells treated with daunorubicin alone and further increased upon treatment with daunorubicin plus TRAIL. Daunorubicin augmented expression of the transcription factor p53 (e.g. Figure 5A), whose pro-apoptotic target genes include BID (Sax et al, 2002). We, therefore, investigated the possibility that super-additive apoptosis induction by these two agents may be accounted for by the ability of daunorubicin to induce BID gene transcription. We used RT-PCR analysis to quantify BID transcripts following daunorubicin treatment of AML 289, an isolate that showed super-additive induction of apoptosis by TRAIL and daunorubicin (Fig 2C). We simultaneously quantified expression of the p53 target p21WAF1 as a positive control. Normalization to the intensity of actin bands showed that 4, 8 or 24-h treatment with daunorubicin failed to augment the constitutive level of BID expression (Fig 6). In contrast, transcripts of p21WAF1, undetectable in untreated cells, were dramatically elevated by daunorubicin treatment. These conclusions were confirmed in two additional PCR experiments and also by real-time PCR analysis using a LightCycler system (not shown). Therefore, the apparent increase in immunoreactive BID following daunorubicin treatment may be accounted for by greater reactivity of tBID compared with full-length BID towards the antibody used for Western blotting, rather than to augmented BID expression. This interpretation is compatible with the markedly different conformation adopted by BID following its cleavage by caspase 8 (Luo et al, 1998).

image

Figure 6.  Expression of BID and p21WAF1 transcripts in AML cells. Semi-quantitative RT-PCR analysis of actin (Act), BID and p21WAF1 transcripts in AML cells from patient 289, following treatment with DNR for 4, 8 and 24 h. The ratios of intensities of the BID and p21 bands relative to that of actin bands amplified from the same sample are shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Actions of TRAIL on AML cells
  6. Actions of TRAIL and cytotoxic drugs on AML cells
  7. Molecular analysis of apoptosis induction by TRAIL and cytotoxic agents
  8. Processing of pro-caspase 8 and BID
  9. Discussion
  10. Acknowledgments
  11. References

Here we have shown that malignant cells isolated from a majority (16/19) of patients with AML were resistant to apoptosis induction by TRAIL, in agreement with a previous report (Wuchter et al, 2001). However, the simultaneous exposure of AML to TRAIL and cytotoxic agents resulted in additive or super-additive apoptosis induction in a significant proportion of isolates. Super-additive apoptotic cell killing was observed when TRAIL was co-incubated with fludarabine, Ara C or daunorubicin. Western blot quantification of PARP cleavage provided unambiguous molecular evidence for super-additive interactions between TRAIL and cytotoxic agents. Our observations contrast with those of Wuchter et al (2001), who observed synergistic killing of only one of 21 AML isolates treated with doxorubicin and TRAIL. This discrepancy may result from the different cytotoxic agents used in our study or to the different methods used to assess cell killing. We stress that the additive or super-additive effects in our study were observed at 500 ng/ml TRAIL, half the concentration used in the earlier study.

In some AML isolates, we observed that apoptosis induction by TRAIL and cytotoxic agents was less than that expected by summation of apoptosis induction by each agent alone. The highest proportion of subadditive responses (5/15) was seen when TRAIL was combined with Ara C. TRAIL can induce the antiapoptotic transcription factor nuclear factor NFκB (Chaudhary et al, 1997; Schneider et al, 1997; Jeremias et al, 1998). It is, therefore, possible that NFκB induction by TRAIL may underlie the subadditive responses seen in a subset of isolates.

Western blot analysis of the AML samples studied here failed to reveal a relationship between the expression of DR4, DR5, DcR1, DcR2 or cFLIP and the susceptibility to apoptosis induction by either TRAIL alone or in combination with cytotoxic agents (data not shown). Treatment of the leukaemic cell lines HL60, Jurkat or U937 with cytotoxic drugs resulted in augmented expression of DR5 and enhanced susceptibility to TRAIL, suggesting that the synergistic actions of TRAIL and cytotoxic drugs were attributable to their ability to upregulate expression of the death receptor (Wen et al, 2000). However, we did not detect cytotoxic drug-induced changes in the expression of death receptors, decoy receptors or of cFLIP, which may have accounted for the induction of super-additive cell killing (not shown).

Apoptosis induction by TRAIL is dependent on activation of caspase 8 by trimerized death receptors (Krammer, 2000). However, TRAIL did not efficiently promote cleavage of either pro-caspase 8 or its pro-apoptotic target BID in AML isolates, compatible with the poor response of these cells to the death ligand. However, daunorubicin alone resulted in the modest cleavage of both pro-caspase 8 and BID in susceptible isolates, compatible with earlier reports that some cytotoxic drugs can activate caspase 8 in a death-receptor-independent manner (Wesselborg et al, 1999; Engels et al, 2000; Jones et al, 2001). Caspase 8 activation and BID cleavage were further augmented by co-treatment with TRAIL and daunorubicin, and this augmentation was compatible with the susceptibility of individual isolates to super-additive apoptosis induction by these agents. Our observations are consistent with the hypothesis that the ability of TRAIL alone to activate caspase 8 and BID cleavage is blocked in AML cells, and that treatment with daunorubicin facilitates reversal of this blockade, resulting in the observed super-additive actions. The mechanism underlying this facilitation is unclear at present, but our data suggest that it is not accounted for by daunorubicin-induced changes in expression of death or decoy receptors or of cFLIP (data not shown). We anticipate that ongoing studies using a global proteomics approach may contribute to elucidating the underlying mechanisms.

The toxicity of TRAIL to human liver cells has been demonstrated in a single in-vitro study (Jo et al, 2000), suggesting that its use in a clinical setting should be approached with caution (Nagata, 2000). However, it is currently unclear whether this hepatotoxicity is a feature of all preparations of TRAIL. Therefore, the ability of this agent to induce regression of both solid (Ashkenazi et al, 1999; Walczak et al, 1999) and haematological (Mitsiades et al, 2001) tumours, carried as xenografts in immunodeficient mice, with acceptable toxicity to normal organs suggest that this death-inducing ligand may play a significant role in the design of future therapeutic protocols. Our observations suggest that combinations of TRAIL and conventional cytotoxic drugs may be of value in the treatment of a proportion of AML patients. Although these patients cannot be readily identified either by the levels of expression of TRAIL receptors, decoy receptors or other proteins which modulate TRAIL signalling, it is plausible that suitable patients may be identified by empirical ex-vivo testing protocols.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Actions of TRAIL on AML cells
  6. Actions of TRAIL and cytotoxic drugs on AML cells
  7. Molecular analysis of apoptosis induction by TRAIL and cytotoxic agents
  8. Processing of pro-caspase 8 and BID
  9. Discussion
  10. Acknowledgments
  11. References
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