|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Institute of Human Morphology, "G.
D'Annunzio" University of Chieti; the Department of Morphology and
Embryology, Human Anatomy Section, University of Ferrara; the
Department of Clinical and Experimental Medicine, Microbiology Section,
and "L.A. Seragnoli" Institute of Hematology, University of
Bologna; and the Institute of Morphological Sciences, University of
Urbino, Italy; and the Institute of Human Virology, University of
Maryland Biotechnology Institute, Baltimore.
Cytotoxic activity of tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL/Apo-2 ligand), used alone or in
different combinations with either a low (1.5 Gy) or a high (15 Gy)
single dose of ionizing radiation (IR), was investigated on
erythroleukemic cells (K562, HEL, Friend, primary leukemic
erythroblasts) and on primary CD34+-derived normal
erythroblasts. Human recombinant TRAIL alone variably affected the
survival/growth of erythroleukemic cells; K562 cells were the most
sensitive. Moreover, all erythroleukemic cells were radio-resistant, as
demonstrated by the fact that cytotoxicity was evident only after
treatment with high-dose (15 Gy) IR. Remarkably, when IR and TRAIL were
used in combination, an additive effect was noticed in all
erythroleukemic cells. Augmentation of TRAIL-induced cell death by IR
was observed with both low and high IR doses and required the
sequential treatment of IR 3 to 6 hours before the addition of TRAIL.
Conversely, both TRAIL and IR showed a moderate cytotoxicity on primary
CD34+-derived normal erythroblasts when used alone, but
their combination did not show any additive effect. Moreover, the
cytotoxicity of IR plus TRAIL observed in erythroleukemic cells was
accompanied by the selective up-regulation of the surface expression of
TRAIL-R1 (DR4), and it was completely blocked by the z-Val-Ala-Asp
(OMe)-CH2 (z-VAD-fmk) caspase inhibitor. On the other hand,
the surface expression of TRAIL-R1 in CD34+-derived normal
erythroblasts was unaffected by IR, which induced the up-regulation of
the decoy TRAIL-R3. These data demonstrate that treatment with IR
provides an approach to selectively sensitize erythroleukemic cells,
but not normal erythroblasts, to TRAIL-induced apoptosis through the
functional up-regulation of TRAIL-R1.
(Blood. 2001;97:2596-2603) Tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (L), is a
new member of the TNF family of cytokines.1,2 Members of
this family are structurally related proteins that play important roles
in regulating cell death, immune response, and
inflammation.3 Like other members of the TNF family, TRAIL
is a type 2 membrane protein, with an intracellular amino-terminal
portion, an internal transmembrane domain, and a carboxyl terminus
external to the cell. In addition, a soluble form of TRAIL has been
identified,4 as previously shown for TNF- Although a role for this cytokine in physiological conditions has not
been clearly envisioned yet, we have recently demonstrated that TRAIL
shows a lineage-specific inhibitory activity on the survival/growth of
CD34+-derived normal erythroblasts, showing an intermediate
expression of surface glycophorin A.15 Owing to its high
expression at the bone marrow level, we have proposed that TRAIL likely
plays an important role in the negative regulation of normal
erythropoiesis.15 Because it has been proposed that TRAIL
selectively kills cancer cells with respect to their normal
counterparts,7,9,16-19 we have here investigated how
TRAIL, used alone or in combination with ionizing radiation (IR),
modulates the survival/growth of erythroleukemic cells in comparison to
normal erythroblasts. For this purpose erythroleukemic (K562, HEL,
Friend) cell lines, primary blasts from 2 patients affected by acute
erythroleukemia, and primary CD34+-derived glycophorin
A+ erythroblasts obtained from normal donors were treated
with TRAIL alone or in various combinations with IR.
Reagents
The functional activity of each TRAIL preparation used in
this study was tested on the TRAIL-sensitive Jurkat cell line (J32 clone). In previous experiments, maximal effects on Jurkat cell apoptosis were observed in the presence of 0.1 to 1 µg/mL TRAIL. In
contrast, equimolar concentrations of His6-tag alone did not show any
significant toxicity.20 Therefore, the concentration of 1 µg/mL TRAIL was chosen to perform the experiments in both human
and murine erythroleukemic cells, as well as in primary erythroleukemic
blasts and primary normal erythroblasts. In this respect, it should be
noted that Walczak et al11 have previously demonstrated
that human recombinant TRAIL is equally effective in inducing apoptosis
on both human and murine target cells.
The broad inhibitor of caspase proteases,
Cbz-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk),21 and the
peptide control Cbz-Phe-Ala-fluoromethyl ketone (z-FA-fmk), both from
Enzyme Systems Products (Dublin, CA), were dissolved in dimethyl
sulfoxide and stocked in aliquots at Cell lines and patient samples
Bone marrow (BM) specimens were obtained from 2 male (16 and 56 years) patients affected by acute erythroleukemia (M6 of the French-American-British classification of acute myelogenous leukemias). We obtained their informed consent to the study, in accordance with the Helsinki Declaration of 1975. BM aspirates showing blast counts greater than 70% were collected at diagnosis before any therapy was initiated. Mononuclear cells were separated on density gradient centrifugation (Ficoll/Hypaque-1077; Sigma) and immediately frozen. Aliquots containing 40 to 45 × 106 BM mononuclear cells were thawed at the time of the study, cultured for 24 hours in RPMI + 10% FCS, subjected to density gradient centrifugation (Ficoll/Histopaque-1077) to eliminate dead cells, and treated as described below. Purification of CD34+ cells and cultures of primary erythroid cells Cord blood (CB) specimens, collected according to institutional guidelines, were obtained during 6 normal full-term deliveries. CB mononuclear cells were isolated by density gradient centrifugation (Ficoll/Histopaque-1077) and allowed to adhere to plastic for 1 hour at 37°C. CB CD34+ cells were then isolated from mononuclear nonadherent cells using the magnetic cell sorting program Mini-MACS and the CD34 isolation kit (Miltenyi Biotech, Auburn, CA) in accordance with the manufacturer's instructions. The purity of CD34-selected cells was determined for each isolation by Facscan (Lysis II program; Becton Dickinson, San Jose, CA), using a mAb that recognizes a separate epitope of the CD34 molecule (HPCA-2; Becton Dickinson) directly conjugated to fluorescein isothiocyanate. The purity of CD34+ cells ranged from 90% to 98%.CD34+ cells were cultured in Ex-vivo (Biowhittaker,
Walkersville, MD) serum-free medium, supplemented with nucleosides (10 µg/mL each), 0.5% BSA, 10 Cell treatments Erythroleukemic cells and normal CD34+-derived normal erythroblasts were treated with TRAIL (1 µg/mL) or rHis6-tag (0.15 µg/mL) and/or irradiated at room temperature by a Mevatron 74 Siemens (Rotterdam, Holland) linear accelerator (photonic energy, 10 MV) administering 1.5 and 15 Gy (dose rate, 3 Gy/min). All experiments were performed on exponentially growing cells, showing a viability of at least 95%. Cytotoxic effects were evaluated by counting viable cells by trypan blue dye exclusion and measuring cell cycle and apoptosis as described below.Evaluation of cell cycle and apoptosis Samples containing 2 to 5 × 105 cells were harvested by centrifugation at 200g for 10 minutes at 4°C, fixed with cold 70% ethanol for at least 1 hour at 4°C, and treated as previously detailed.26 Briefly, samples were pelleted, treated with 0.5 µg RNAse (type I-A) (Sigma) and resuspended in PBS containing 50 µg/mL propidium iodide (PI; Sigma). Analysis of PI fluorescence was performed by FACScan with the FL2 detector in a linear mode using the Lysis II software (Becton Dickinson). For each sample, 10 000 to 20 000 events were collected. For cell-cycle analysis, only the inferred gap 1 (G1), synthesis (S) and gap 2 plus mitosis (G2 + M) peaks were considered. The proportions of cells in the G1, S, and G2 + M phases of the cell cycle were calculated as described.27 For simplicity, the G1 and S/G2 + M values have been provided. For quantitative evaluation of apoptosis, the subdiploid (less than 2n) DNA content was calculated as described27 and expressed as percentage of apoptotic versus nonapoptotic cells, regardless of the specific cell-cycle phase.Ultrastructural analysis of erythroleukemic cells was performed by transmission electron microscopy. Briefly, cell pellets were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 30 minutes at 4°C, rinsed in the same buffer, and processed for conventional Spurr embedding. Western blotting of TRAIL receptors For the analysis of TRAIL-R1 and TRAIL-R2 expression, 40 × 106 cells were resuspended in freshly made ice-cold lysis buffer (50 mM Tris, pH 7.5, 1% Triton X-114, 150 mM NaCl, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mM PMSF) for 15 minutes. Lysates were centrifuged at 15 000 rpm for 10 minutes at 4°C. After discharging the pellets (mainly containing cell nuclei and debris), the proteins contained in the aqueous and membrane-associated phases were separated by adding 1% Triton X-114 at 37°C for 3 minutes. After centrifugation at 15 000 rpm, the upper aqueous phase was discharged, and the lower phase containing membrane-associated proteins was resuspended in lysis buffer (without Triton X-114). One hundred micrograms membrane-associated proteins for each sample was migrated in 12% SDS-PAGE and blotted onto nitrocellulose filters. Blotted filters were blocked for 30 minutes in a 3% suspension of dried skim milk in PBS and incubated overnight at 4°C with 1 µg anti-TRAIL-R1 or anti-TRAIL-R2 goat IgG. Filters were washed and further incubated for 1 hour at room temperature with 1:1000 dilution of peroxidase-conjugated antigoat IgG (Sigma) in 0.1% BSA. Specific reactions were revealed with the enhanced chemiluminescence Western blotting detection reagent (Amersham).Phenotypic analysis of surface TRAIL and TRAIL receptors At various culture time intervals after irradiation, HEL, K562, and primary cells were analyzed for the surface expression of TRAIL, TRAIL-receptor (R)1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 by indirect staining with primary goat antihuman TRAIL, TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 IgG (all from R&D System, Oxon, United Kingdom), followed by phycoerythrin (PE)-conjugated rabbit antigoat IgG secondary antibody (Sigma). Briefly, staining was performed on 5 × 105 cells in 200 µL PBS containing 1% FCS and 5 µL each primary antibody on ice for 30 minutes. Cells were washed twice, supplemented with 3 µL PE-conjugated rabbit antigoat IgG on ice for 30 minutes, washed twice with PBS, and analyzed by FACScan. Aspecific fluorescence was assessed by using normal goat IgG and then by a second layer, as above.Samples were assayed in duplicate, and gates containing viable cells were used to collect 10 000 events. Data are presented as either percentage of positive cells or mean fluorescence intensity (MFI) values. Statistical analysis Data were analyzed using the 2-tailed, 2-sample t test (statistical analysis software; Minitab, State College, PA). P < .05 was considered significant. Interactions between TRAIL and IR were classified by the fractional inhibition method as follows: when expressed as the fractional inhibition of cell viability, additive inhibition produced by both inhibitors occurred when i1,2 = i1 + i2; synergism occurred when i1,2 > i1 + i2; and antagonism occurred when i1,2 < i1 + i2 .28
Low susceptibility of erythroleukemic cells to the cytotoxic activity of TRAIL or IR, used alone Because it has been demonstrated that the homotrimerization of TRAIL is critical for its tumoricidal activity,29 each His6-tagged recombinant TRAIL preparation was analyzed by SDS-PAGE analysis performed in both reducing and nonreducing conditions and visualized by silver staining. As shown in Figure 1A, a single band of approximately 32 kd, corresponding to monomeric TRAIL, was recovered when SDS-PAGE was performed under reducing conditions. On the other hand, bands of approximately 60 kd and 90 kd, reflecting multimeric forms of TRAIL were observed in nonreducing conditions. Thus, human recombinant TRAIL protein used in this study is predominantly trimeric in solution.
The cytotoxic effect of TRAIL protein was evaluated by 2 independent methods: count of viable cells by trypan blue dye exclusion and analysis of cell cycle and apoptosis by flow cytometry after PI staining. At both 6 and 24 hours after the addition of TRAIL (1 µg/mL), erythroleukemic cell lines showed distinct patterns of response to TRAIL from the relatively high (P < .01) sensitivity of K562 (up to 45% decrease with respect to control cells treated with His6-tag peptide) to the moderate (P < .05) sensitivity of Friend (28% decrease) and the modest sensitivity of HEL (19% decrease) (Figure 1B). These data indicate that erythroleukemic cells show a variable response to TRAIL and that the peak of cytotoxicity was at 24 hours. In parallel experiments, K562, HEL, and Friend cells were treated with
IR. The choice of 1.5 and 15 Gy IR doses was made on the basis of the
standard radiotherapeutic schemes used for the management of various
human cancers.30,31 In fact, a daily fraction of 1.5 Gy or
more is commonly delivered in human tumor radiotherapy,30
whereas 15 Gy can be reached at the end of a therapeutic
protocol32 or occasionally used as a single fraction for
palliative treatment. As shown in Figure
2A, relatively modest cytotoxic effects
(less than 30% decrease in the number of viable cells in comparison to
untreated control cells) were observed in the presence of low IR doses
(1.5 Gy) in all cell lines. After 24 hours, in the presence of higher
IR doses (15 Gy), the cytotoxicity ranged from a 15% decrease (for
HEL) to a 40% decrease for both Friend (P < .01) and
K562 (P < .01) cells (Figure 2B).
Augmentation of the TRAIL-mediated cytotoxicity on erythroleukemic cells by pretreatment with IR In the following experiments, the effects of different combinations of TRAIL and IR were investigated. The results are summarized in Figure 3. When cells were pretreated with TRAIL for 6, 18, or 24 hours before IR (1.5-15 Gy), no additive or synergistic effects were noticed between the 2 treatments, irrespective of the IR doses used in the cell lines examined (Figure 3A-C). On the other hand, when the cells were pretreated with IR for 3 to 6 hours before TRAIL, a potentiation of TRAIL-mediated cytotoxicity was observed (Figure 3A-C). As previously shown for the 2 treatments used alone, the behavior of the 3 erythroleukemic cell lines was not uniform. In fact, in K562, the combination of either 1.5 Gy (20% decrease in the number of viable cells with respect to control cells) or 15 Gy (40% decrease) plus TRAIL (40%-45% decrease) resulted in an additive cytotoxic effect (68%-72% decrease) (Figure 3A). Similar additive effects (approximately 70% decrease) were noticed in Friend cells treated with 15 Gy (41% decrease) followed by TRAIL (28% decrease) (Figure 3B). Of note, HEL cells showed the maximal response to this combination of treatments. In fact, TRAIL (19% decrease alone) showed a synergistic cytotoxic effect when used with either 1.5 Gy (0% decrease alone versus 58% decrease in association with TRAIL) or 15 Gy (16% decrease alone versus 70% decrease in association with TRAIL) (Figure 3C).
Selective induction of apoptosis in erythroleukemic cells but not in normal glycophorin A+ erythroblasts by the combination of IR followed by TRAIL The cytotoxic mechanism of IR plus TRAIL on erythroleukemic cell lines was next investigated by analyzing the cell-cycle profile and the degree of apoptosis after PI staining and flow cytometry examination. As shown in Figure 4 and Table 1, IR by itself produced profound perturbations of the cell cycle, characterized by a dose-dependent accumulation of the cells in the S/G2-M phases of the cell cycle. On the other hand, TRAIL by itself induced modest changes in the cell cycle of the cells examined (Table 1). Rather it induced a moderate increase (P < .01) of apoptosis in K562 and HEL, but not in Friend, cells (Figure 4, Table 2). In cells treated with the combination of IR followed by TRAIL, the percentage of cells distributed in the different cell-cycle phases was unchanged in comparison to cells treated with IR alone (Table 1). On the other hand, the percentage of apoptosis was significantly (P < .05) increased in erythroleukemic cells treated with IR plus TRAIL with respect to those treated with TRAIL or IR alone and in control cells treated with His6-tag peptide (Table 2). Thus, although at the time points examined the cytotoxic effects of IR on erythroleukemic cells were mainly due to cell-cycle arrest, IR sensitized these cells to TRAIL-mediated apoptosis. Ultrastructural examination confirmed the presence of apoptosis in cell lines treated with IR plus TRAIL, displaying initial aspects of chromatin clumping and condensation followed by the formation of micronuclei (Figure 5).
We were also able to examine the cytotoxic activity of TRAIL alone or
in combination with IR on primary blasts obtained at diagnosis from 2 patients affected by acute erythroleukemia and on primary
CD34+-derived normal erythroblasts. As shown in Table 1,
both primary erythroleukemic blasts and glycophorin A+
normal erythroblasts (Figure 6) showed an
accumulation of the cells in the S/G2-M phases of the cell
cycle after treatment with IR. On the other hand, TRAIL
Selective up-regulation of TRAIL-R1 (DR4) in human erythroleukemic cells but not in normal erythroblasts in response to IR It has been previously demonstrated that genotoxic agents are able to up-regulate TRAIL-R2 (DR5) and TRAIL-R3 in different cell types through p53-dependent and p53-independent pathways.15,32,33 Therefore, we next investigated whether IR treatment was able to modulate the surface expression of the different TRAIL receptors and of surface TRAIL.12 Because of the low number of primary blasts in the specimens obtained from 2 patients affected by acute erythroleukemia, these experiments could only be performed using human erythroleukemic cell lines and primary CD34+-derived normal erythroblasts.Although untreated K562 cells showed clearly detectable levels of
TRAIL-R1 (DR4) and TRAIL-R2 (DR5) on Western blot (Figure 7A) and flow cytometry (Figure 7B)
analyses, HEL cells were positive for TRAIL-R1 but negative for
TRAIL-R2 expression (Figure 7A-C). No cross-reactivity was noticed on
Western blot analysis between anti-TRAIL-R1 and anti-TRAIL-R2
polyclonal antibodies, which recognized a single band of approximately
60 and 45 kd, respectively, in agreement with the findings of other
authors.32 Both cell lines showed either absent or dim
expression of decoy (TRAIL-R3 and TRAIL-R4) receptors (Figure 7B-C) and
of surface TRAIL (data not shown). Interestingly, exposure to either
1.5 or 15 Gy IR induced the selective up-regulation of TRAIL-R1 (DR4)
after 6 hours in HEL cells (Figure 7C) and after 18 hours in K562 cells
(Figure 7B). The MFI of 3 independent experiments was 18 ± 3.5 in
untreated HEL cells versus 27.5 ± 4 in HEL cells irradiated for 6 hours with 1.5 Gy (P < .05), and it was 12.3 ± 2.5 in
untreated K562 cells versus 22.5 ± 3 in K562 cells irradiated for 18 hours with 1.5 Gy (P < .05). No other effects on the
expression of TRAIL-R2-4 or surface TRAIL could be observed up to 18 to
24 hours after irradiation in these erythroleukemic cell lines.
The pattern of surface expression of TRAIL receptors in primary glycophorin A+ normal erythroblasts (Figure 7D) was similar to that observed in HEL cells, in which the only surface receptor clearly detectable was TRAIL-R1 (Figure 7B). However, at variance with erythroleukemic cells, the exposure of glycophorin A+ erythroblasts to IR did not modify the MFI of TRAIL-R1 after either 6 or 18 hours. On the other hand, exposure to IR induced a clear-cut up-regulation of the decoy TRAIL-R3 in primary normal erythroblasts (Figure 7D). The MFI of 3 independent experiments was 16 ± 2.8 in untreated cells versus 29.5 ± 5.5 in cells irradiated for 18 hours with 1.5 Gy (P < .05). Block of the cytotoxic effects mediated by combination of IR and TRAIL by the z-VAD-fmk caspase inhibitor We then sought to investigate whether the cytotoxicity observed in erythroleukemic cells treated with the combination of IR and TRAIL was susceptible to the pharmacologic activity of caspase inhibitors.34 Preincubation of cultures with z-VAD-fmk tripeptide at 20 µM before treatment with IR plus TRAIL completely blocked the cytotoxic activity of the combination of the 2 agents (Figure 8). The control z-FA-fmk peptide (20 µM) did not show any toxic or protective effect on erythroleukemic cells. Specificity of the z-VAD-fmk peptide inhibitor was confirmed by its ability to suppress apoptosis also in the presence of anti-CD95 agonistic antibody (data not shown), which is known to rapidly activate the caspase cascade.35
In this study, we have demonstrated for the first time that, though IR alone exerted a variable but overall relatively modest cytotoxicity on erythroleukemic cell lines and primary malignant erythroblasts, the sequential combination of irradiation and TRAIL resulted in a synergistic cytotoxicity on these cells. The low sensitivity of erythroleukemic cells in response to radiotherapy36-39 was likely due to the high intracellular iron content of these cells compared to radio-susceptible tumor cells.36 Therefore, in spite of their radio-resistance, erythroleukemic cells are sensitized by IR to TRAIL-mediated cytotoxicity. It is also noteworthy that the additive or synergistic cytotoxic effects were observed when cells were pretreated with IR, corresponding to either a daily fraction of 1.5 Gy or a whole cycle of 15 Gy. These findings are particularly interesting because fractionated radiation plays an integral role in the treatment of human cancer. In fact, it was determined empirically that radiation delivered in small fractionated doses would produce less damage to normal tissue, providing greater tumor control than radiation delivered in large single doses.40 More important, the combination of IR plus TRAIL did not show any additive cytotoxic activity on CD34+-derived primary normal erythroblasts. A major limitation of radiation therapy and of chemotherapy is that
they require function of the p53 tumor-suppressor gene for antitumor
activity,41 but more than half the human tumors acquire
inactivating p53 mutations, making them resistant to conventional anticancer therapy. On the other hand, TRAIL, like other death-inducing ligands such as TNF- Although we and others have recently shown that recombinant human TRAIL
can exert cytotoxicity on normal tissues and cells, including
hematopoietic progenitor cells committed to the erythroid lineage15 and hepatocytes,44 it still
represents the most promising candidate among the death-inducing
ligands for systemic anticancer therapy. In this respect, our data
showing the ability of IR to augment TRAIL-mediated cytotoxicity in
erythroleukemic cells We have also addressed the mechanisms underlining the synergistic effect of IR and TRAIL on erythroleukemic cells by demonstrating that IR selectively up-regulates TRAIL-R1 (DR4) in neoplastic cells but not in normal erythroblasts. In this respect, it has been shown that TRAIL-R1 represents the major determinant of TRAIL sensitivity in cancer cells.48 Remarkably, TRAIL-R1 was also expressed on the surfaces of primary normal erythroblasts, but its expression did not change on exposure to IR. On the other hand, IR was able to substantially up-regulate TRAIL-R3 in primary normal cells. These findings strongly suggest that the differential sensitivity of erythroleukemic cells and normal erythroblasts to the combined treatment with IR plus TRAIL results from a differential modulation of TRAIL receptors by IR. In this respect, previous studies have demonstrated an extreme complexity of the expression and function of TRAIL receptors in various cell types.13 At least 5 TRAIL receptors belonging to the apoptosis-inducing TNF-receptor (R) family have been described so far. TRAIL-R1 (DR4) and TRAIL-R2 (DR5) transduce apoptotic signals on the binding of TRAIL, whereas TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), and osteoprotegerin are homologous to DR4 and DR5 in their cysteine-rich extracellular domains, but they lack intracellular death domains and apoptosis-inducing capability. Although the expression of TRAIL-R3 and TRAIL-R4 do not appear to be key factors in determining the resistance or sensitivity of tumor target cells to the effects of TRAIL,45,49 they have been proposed to function as decoy receptors, protecting normal cells from apoptosis.13 Consistently, we found that TRAIL-R3 and TRAIL-R4 were either not expressed or showed dim expression in K562 and HEL cell lines and that they were not affected by IR treatment. TRAIL-R3, however, was significantly up-regulated in normal erythroblasts after IR treatment. It should be emphasized that the ability of IR to selectively up-regulate TRAIL-R1 in erythroleukemic cells, but not in normal hematopoietic cells, is a completely novel finding. In fact, previous studies have shown that chemotherapeutic genotoxic agents can preferentially up-regulate the expression of TRAIL-R2 in glioblastoma cells, breast cancer cells, and T-lymphoma cells.16,31,50,51 Taken together, these studies and our present findings suggest that the modulation of TRAIL-R1 and TRAIL-R2, which play a major role in determining the cytotoxic response of cancer cells to TRAIL, is lineage specific and that functional TRAIL-R1 can be selectively up-regulated in erythroleukemic cells by IR.
Submitted July 13, 2000; accepted January 3, 2001.
Supported by local funds from the Universities of Chieti, Ferrara, and Bologna, and by MURST Cofin (G.Z., S.M.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Giorgio Zauli, Institute of Morphology, "G. D'Annunzio" University of Chieti, Via dei Vestini 6, 66100 Chieti Scalo (CH), Italy; e-mail: g.zauli{at}morpho.unich.it.
1. Wiley SR, Schooley K, Smolak PJ, et al. Identification and characterization of a new memeber of the TNF family that induces apoptosis. Immunity. 1995;3:673-682[CrossRef][Medline] [Order article via Infotrieve].
2.
Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A.
Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family.
J Biol Chem.
1996;271:12687-12690 3. Baker SJ, Reddy EP. Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene. 1996;12:1-9[Medline] [Order article via Infotrieve]. 4. Mariani SM, Krammer PH. Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage. Eur J Immunol. 1998;28:973-982[CrossRef][Medline] [Order article via Infotrieve].
5.
Gruss HJ, Dower SK.
Tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas.
Blood.
1995;85:3378-3404 6. Misawa K, Nosaka T, Kojima T, Hirai M, Kitamura T. Molecular cloning and characterization of a mouse homolog of human TNFSF14, a member of the TNF superfamily. Cytogenet Cell Genet. 2000;89:89-91[CrossRef][Medline] [Order article via Infotrieve]. 7. Snell V, Clodi K, Zhao S, et al. Activity of TNF-related apoptosis-inducing ligand (TRAIL) in haematological malignancies. Br J Haematol. 1997;99:618-624[CrossRef][Medline] [Order article via Infotrieve]. 8. Jeremias I, Herr I, Boehler T, Debatin KM. TRAIL/Apo-2 ligand-induced apoptosis in human T cells. Eur J Immunol. 1998;28:143-152[CrossRef][Medline] [Order article via Infotrieve]. 9. Herr I, Wilhelm D, Bohler T, Angel P, Debatin KM. JNK/SAPK activity is not sufficient for anticancer therapy-induced apoptosis involving CD95-L, TRAIL and TNF-alpha. Int J Cancer. 1999;80:417-424[CrossRef][Medline] [Order article via Infotrieve].
10.
Martinez-Lorenzo MJ, Anel A, Gamen S, et al.
Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles.
J Immunol.
1999;163:1274-1281 11. Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med. 1999;5:157-163[CrossRef][Medline] [Order article via Infotrieve]. 12. Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest. 1999;104:155-162[Medline] [Order article via Infotrieve]. 13. Ashkenazi A, Dixit VM. Apoptosis control by death and decoy receptors. Curr Opin Cell Biol. 1999;11:255-260[CrossRef][Medline] [Order article via Infotrieve]. 14. Zhao S, Asgary Z, Wang Y, Goodwin R, Andreeff M, Younes A. Functional expression of TRAIL by lymphoid and myeloid tumour cells. Br J Haematol. 1999;106:827-832[CrossRef][Medline] [Order article via Infotrieve].
15.
Zamai L, Secchiero P, Pierpaoli S, et al.
TNFrelated apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis.
Blood.
2000;95:3716-3724
16.
Sheikh MS, Burns TF, Huang Y, et al.
p53-dependent and -independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and tumor necrosis factor alpha.
Cancer Res.
1998;58:1593-1598
17.
Sedger LM, Shows DM, Blanton RA, et al.
IFN-gamma mediates a novel antiviral activity through dynamic modulation of TRAIL and TRAIL receptor expression.
J Immunol.
1999;163:920-926
18.
Keane MM, Ettenberg SA, Nau EK, Russell EK, Lipkowitz S.
Chemotherapy augments TRAILinduced apoptosis in breast cell lines.
Cancer Res.
1999;59:734-741 19. MacFarlane M, Manzoor A, Srinivasula SM, Fernandes-Alnemri T, Cohen GM, Alnemri ES. Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL. J Biol Chem. 1997;41:25417-25420.
20.
Zamai L, Ahmad M, Bennett IM, Azzoni L, Alnemri ES, Perussia B.
Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells.
J Exp Med.
1998;188:2375-2380 21. Cohen GM. Caspases: the executioners of apoptosis. Biochem J. 1997;326:1-16. 22. Ben-David Y, Bernstein A. Friend virus-induced erythroleukemia and the multistage nature of cancer. Cell. 1991;66:831-834[CrossRef][Medline] [Order article via Infotrieve]. 23. Tetteroo PA, Massaro F, Mulder A, Schreuder van Gelder R, von dem Borne AE. Megacaryoblastic differentiation of proerythroblastic K562 cell-line cells. Leuk Res. 1984;8:197-206[CrossRef][Medline] [Order article via Infotrieve].
24.
Martin P, Papayannopoulou T.
HEL cells: a new human erythroleukemia cell line with spontaneous and induced globin expression.
Science.
1982;216:1233-1235
25.
Horita BM, Andreu EJ, Benito A, et al.
Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xl.
J Exp Med.
2000;191:977-984
26.
Zauli G, Vitale M, Falcieri E, et al.
In vitro senescence and apoptotic cell death of human megakaryocytes.
Blood.
1997;90:2234-2243
27.
Secchiero P, Bertolaso L, Casareto L, et al.
Human herpesvirus 7 infection induces profound cell cycle perturbations coupled to dysregulation of cdc2 and cyclin B and polyploidization of CD4+ T cells.
Blood.
1998;92:1685-1698 28. Webb JL. Effects of more than one inhibitor. Enzyme Metab Inhibitors. 1963;1:487-512. 29. Hymowitz SG, O'Connell MP, Ultsch MH, et al. A unique zinc-binding site revealed by a high- resolution x-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry. 2000;39:633-640[CrossRef][Medline] [Order article via Infotrieve].
30.
Dahlberg WK, Azzam EI, Yu Y, Little JB.
Response of human tumor cells of varying radiosensitivity and radiocurability to fractionated irradiation.
Cancer Res.
1999;59:5365-5369 31. Rana R, Vitale M, Mazzotti G, Manzoli L, Papa S. Radiosensitivity of human natural killer cells: binding and cytotoxic activities of natural killer cell subsets. Radiat Res. 1990;124:96-102[CrossRef][Medline] [Order article via Infotrieve].
32.
Chinnaiyan AM, Prasad U, Shankar S, et al.
Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy.
Proc Natl Acad Sci U S A.
2000;97:1754-1759 33. Sheikh MS, Huang Y, Fernandez-Salas EA, et al. The antiapoptotic decoy receptor TRID/TRAIL-R3 is a p53-regulated DNA-damage-inducible gene that is overexpressed in primary tumors of the gastrointestinal tract. Oncogene. 1999;18:4153-4159[CrossRef][Medline] [Order article via Infotrieve].
34.
Katsikis PD, Garcia-Ojeda ME, Torres-Roca JF, Tijoe IM, Smith CA, Herzenberg LA.
Interleukin-1 beta converting enzyme-like protease involvement in Fas-induced and activation-induced peripheral blood T cell apoptosis in HIV infection: TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in HIV infection.
J Exp Med.
1997;186:1365-1372
35.
Boesen-de Cock JG, Tepper AD, van Blitterswijk WJ, Borst J.
Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.
J Biol Chem.
1999;274:14255-14261
36.
Lipinsky P, Drapier JC, Oliveira L, Retmanska H, Sochanowicz B, Kruszewski M.
Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: a study of L5178Y mouse lymphoma cell lines differentially sensitive to H2O2.
Blood.
2000;95:2960-2966 37. Di Pietro R, Centurione L, Santavenere E, et al. Ionizing radiation-induced apoptosis and DNA repair in murine erythroleukemia cells. Scanning Microsc. 1996;10:253-259[Medline] [Order article via Infotrieve]. 38. Schwenke K, Peterson HP, Wangenheim KH, Feinendegen LE. Induction of differentiation in erythroleukemic K562 cells by gamma-irradiation. Leuk Res. 1995;19:955-961[CrossRef][Medline] [Order article via Infotrieve]. 39. Di Pietro R, Centurione MA, Falcieri E, Centurione L, Santavenere E, Rana R. Dimethylsulfoxide-induced cell death of murine erythroleukemia cells exposed to ionising radiation. Cell Signal. 1998;10:205-209[CrossRef][Medline] [Order article via Infotrieve]. 40. Fertil B, Malaise EP. Intrinsic radiosensitivity of human cell lines is correlated with radioresponsiveness of human tumors: analysis of 101 published survival curves. Int J Radiat Oncol Biol Phys. 1985;11:1699-1707[Medline] [Order article via Infotrieve]. 41. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997;88:323-331[CrossRef][Medline] [Order article via Infotrieve]. 42. Tartaglia L, Goeddel D. Two TNF receptors. Immunol Today. 1992;13:151-153[CrossRef][Medline] [Order article via Infotrieve]. 43. Nagata S, Golstein P. The Fas death factor. Science. 1995;95:1449-1456. 44. Jo M, Kim T-H, Seol D-W, et al. Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand. Nat Med. 2000;6:564-567[CrossRef][Medline] [Order article via Infotrieve].
45.
Griffith TS, Rauch CT, Smolak PJ, et al.
Functional analysis of TRAIL receptors using monoclonal antibodies.
J Immunol.
1999;162:2597-2605
46.
Gibson SB, Oyer R, Spalding AC, Anderson SM, Johnson GL.
Increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and TRAIL.
Mol Cell Biol.
2000;20:205-212
47.
Gliniak B, Le T.
Tumor necrosis factor-related apoptosis-inducing ligand's antitumor activity in vivo is enhanced by the chemotherapeutic agent CPT-11.
Cancer Res.
1999;59:6153-6158
48.
Kim K, Fisher MJ, Xu SQ, el-Deiry WS.
Molecular determinants of response to TRAIL in killing of normal and cancer cells.
Clin Cancer Res.
2000;6:335-346
49.
Zhang XD, Franco A, Myers K, Gray C, Nguyen T, Hersey P.
Relation of TNF-related apoptosis- inducing ligand (TRAIL) receptor and FLICE- inhibitory protein expression to TRAIL-induced apoptosis of melanoma.
Cancer Res.
1999;59:2747-2753 50. Wu GS, Burns TF, McDonald ER. Induction of the TRAIL receptor KILLER/DR5 in p53-dependent apoptosis but not growth arrest. Oncogene. 1999;18:6411-6418[CrossRef][Medline] [Order article via Infotrieve].
51.
Gong B, Almasan A.
Apo2 ligand/TNF-related apoptosis-inducing ligand and death receptor 5 mediate the apoptotic signaling induced by ionizing radiation in leukemic cells.
Cancer Res.
2000;60:5754-5760
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  W. F. Tam, T.-L. Gu, J. Chen, B. H. Lee, L. Bullinger, S. Frohling, A. Wang, S. Monti, T. R. Golub, and D. G. Gilliland Id1 is a common downstream target of oncogenic tyrosine kinases in leukemic cells Blood, September 1, 2008; 112(5): 1981 - 1992. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zamai, C. Ponti, P. Mirandola, G. Gobbi, S. Papa, L. Galeotti, L. Cocco, and M. Vitale NK Cells and Cancer J. Immunol., April 1, 2007; 178(7): 4011 - 4016. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Briggs, K. E. Shults, L. A. Flye, S. A. McClintock-Treep, M. H. Jagasia, S. A. Goodman, F. I. Boulos, J. W. Jacobberger, G. T. Stelzer, and D. R. Head Dysregulated Human Myeloid Nuclear Differentiation Antigen Expression in Myelodysplastic Syndromes: Evidence for a Role in Apoptosis. Cancer Res., May 1, 2006; 66(9): 4645 - 4651. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Zeng, A. Miyazato, G. Chen, S. Kajigaya, N. S. Young, and J. P. Maciejewski Interferon-{gamma}-induced gene expression in CD34 cells: identification of pathologic cytokine-specific signature profiles Blood, January 1, 2006; 107(1): 167 - 175. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. K. Rowinsky Targeted Induction of Apoptosis in Cancer Management: The Emerging Role of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Activating Agents J. Clin. Oncol., December 20, 2005; 23(36): 9394 - 9407. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhang, R. M. Cheung, R. Komaki, B. Fang, and J. Y. Chang Radiotherapy Sensitization by Tumor-Specific TRAIL Gene Targeting Improves Survival of Mice Bearing Human Non-Small Cell Lung Cancer Clin. Cancer Res., September 15, 2005; 11(18): 6657 - 6668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Tourneur, S. Delluc, V. Levy, F. Valensi, I. Radford-Weiss, O. Legrand, J. Vargaftig, C. Boix, E. A. Macintyre, B. Varet, et al. Absence or Low Expression of Fas-Associated Protein with Death Domain in Acute Myeloid Leukemia Cells Predicts Resistance to Chemotherapy and Poor Outcome Cancer Res., November 1, 2004; 64(21): 8101 - 8108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Zauli, E. Rimondi, V. Nicolin, E. Melloni, C. Celeghini, and P. Secchiero TNF-related apoptosis-inducing ligand (TRAIL) blocks osteoclastic differentiation induced by RANKL plus M-CSF Blood, October 1, 2004; 104(7): 2044 - 2050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. Spierings, E. G. de Vries, E. Vellenga, F. A. van den Heuvel, J. J. Koornstra, J. Wesseling, H. Hollema, and S. de Jong Tissue Distribution of the Death Ligand TRAIL and Its Receptors J. Histochem. Cytochem., June 1, 2004; 52(6): 821 - 831. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Chawla-Sarkar, J. A. Bauer, J. A. Lupica, B. H. Morrison, Z. Tang, R. K. Oates, A. Almasan, J. A. DiDonato, E. C. Borden, and D. J. Lindner Suppression of NF-{kappa}B Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL J. Biol. Chem., October 10, 2003; 278(41): 39461 - 39469. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Greil, G. Anether, K. Johrer, and I. Tinhofer Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools J. Leukoc. Biol., September 1, 2003; 74(3): 311 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. J. Spierings, E. G. E. de Vries, W. Timens, H. J. M. Groen, H. M. Boezen, and S. de Jong Expression of TRAIL and TRAIL Death Receptors in Stage III Non-Small Cell Lung Cancer Tumors Clin. Cancer Res., August 1, 2003; 9(9): 3397 - 3405. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Hietakangas, M. Poukkula, K. M. Heiskanen, J. T. Karvinen, L. Sistonen, and J. E. Eriksson Erythroid Differentiation Sensitizes K562 Leukemia Cells to TRAIL-Induced Apoptosis by Downregulation of c-FLIP Mol. Cell. Biol., February 15, 2003; 23(4): 1278 - 1291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Secchiero, A. Gonelli, P. Mirandola, E. Melloni, L. Zamai, C. Celeghini, D. Milani, and G. Zauli Tumor necrosis factor-related apoptosis-inducing ligand induces monocytic maturation of leukemic and normal myeloid precursors through a caspase-dependent pathway Blood, September 18, 2002; 100(7): 2421 - 2429. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
and CD95
(Apo-1/Fas) L.
and herpes
virus entry mediator ligand (HVEM-L).
20°C until used.
4 M BSA-adsorbed cholesterol,
10 µg/mL insulin, 200 µg/mL iron-saturated transferrin, and
5 × 10
used
either alone or in combination with IR
