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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-06-1770.
NEOPLASIA
From the Department of Pediatrics, School of Medicine,
University of Yamanashi, Japan; the Department of
Immunology, Juntendo University School of Medicine, Tokyo,
Japan; and the Laboratory of Immunology, Saitama
Shakaihoken Hospital, Saitama, Japan.
Tumor necrosis factor (TNF)-related apoptosis-inducing
ligand (TRAIL) and Fas ligand (FasL) have been implicated in antitumor immunity and therapy. In the present study, we investigated the sensitivity of Philadelphia chromosome (Ph1)-positive leukemia cell
lines to TRAIL- or FasL-induced cell death to explore the possible
contribution of these molecules to immunotherapy against Ph1-positive
leukemias. TRAIL, but not FasL, effectively induced apoptotic cell
death in most of 5 chronic myelogenous leukemia-derived and 7 acute
leukemia-derived Ph1-positive cell lines. The sensitivity to TRAIL was
correlated with cell-surface expression of death-inducing receptors DR4
and/or DR5. The TRAIL-induced cell death was caspase-dependent and
enhanced by nuclear factor Tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL) is a proapoptotic member of the TNF
superfamily that also includes TNF- Chronic myelogeneous leukemia (CML) is a clonal myeloproliferative
expansion of transformed hematopoietic progenitor cells characterized
by Philadelphia chromosome (Ph1) created from t(9;22) (q34;
q11).19-21 Approximately 20% of adult22 and
5% of childhood23 acute lymphoblastic leukemia (ALL) and
2% of acute myeloblastic leukemia (AML)20 are also Ph1
positive. The Ph1 results in the juxtaposition of
bcr and abl genes, and this generates a chimeric protein termed BCR-ABL with a marked tyrosine kinase
activity.19,20 Virtually all cases of CML express a
210-kDa form of BCR-ABL (p210), while half of adult and most
of childhood Ph1-positive ALLs express a shorter form of BCR-ABL termed
p190. IFN- Although the clinical observations strongly suggest a pivotal role of
cytotoxic T lymphocytes (CTLs) in suppressing Ph1-positive leukemias,
the underlying effector mechanisms have not been well characterized. A
role of FasL has been suggested, but its contribution to the
elimination of Ph1-positive leukemia cells is still controversial. Selleri et al reported that Fas was expressed on CD34+
cells from CML patients and up-regulated by IFN- Leukemia cells
Reagents
3H-thymidine uptake assay Leukemia cell lines (2-5 × 104 cells/well) were cultured in triplicate in 200 µL RPMI1640 medium supplemented with 10% FCS in a flat-bottomed 96-well plate (Costar, Cambridge, MA). The plates were incubated for the indicated periods, pulsed for the last 6 hours with 3H-thymidine (1 µCi/well [0.037 MBq/well]), and harvested onto glass-fiber filters. Radioactivity incorporated into DNA was measured by liquid scintillation counting.The effects of rhsFasL and rhsTRAIL were determined by the last 6-hour
pulse of the 42-hour culture in the absence or presence of 3-fold
diluted concentrations (3.7, 11, 33, 100, and 300 ng/mL) of rhsFasL or
rhsTRAIL. In some experiments, a neutralizing anti-TRAIL monoclonal
antibody (mAb) (RIK-2; 10 µg/mL)13 or z-VAD-fmk (20 µM) was used to block the activity of rhsTRAIL and caspases,
respectively. In other experiments, cell lines were preincubated for 3 hours with LLnL (2.5 µM), SN50, or SN50M (100 µg/mL) and then
cultured in the absence or presence of rhsTRAIL. The effect of imatinib mesylate was also determined after 42-hour incubation in the absence or
presence of imatinib mesylate (1.0 µM). The percent inhibition by
TRAIL or imatinib mesylate was calculated as follows: {1 Viability and apoptosis assays The cytotoxic effects of FasL and TRAIL were examined by the dye exclusion assay. Cell lines (1 × 105 cells/well) were cultured in the presence of rhsFasL or rhsTRAIL at 100 ng/mL, harvested at 12, 24, and 36 hours, and the viability was determined by staining with trypan blue. The early apoptotic event in leukemia cell lines was examined by binding of Annexinne-V to surface-exposed phosphatidylserine. Cell lines (4 × 105 cells/mL) were cultured in the absence or presence of rhsTRAIL (100 ng/mL), harvested at 12 hours, stained with fluorescein isothiocyanate (FITC)-conjugated Annexin-V (MBL, Nagoya, Japan), and analyzed by flow cytometry (EPICS PROFILE; Coulter, Miami, FL). In experiments with primary leukemias, cells (2 × 104 cells/well) were incubated in the absence or presence of rhsFasL or rhsTRAIL at 100 ng/mL with or without a neutralizing anti-TRAIL mAb RIK-2 (10 µg/mL) in triplicate in a 96-well plate for 24 hours, and the viability was determined by staining with trypan blue. In some patient samples, apoptosis was also examined by binding of FITC-conjugated Annexin-V after 12-hour culture.Cell-surface expression of TRAIL receptors and Fas mAbs specific for DR4 (DJR1, mouse immunoglobulin G 1 [IgG 1]), DR5 (DJR2, mouse IgG 1), DcR1 (DJR3, mouse IgG 1), and DcR2 (DJR4, mouse IgG 1) were raised against soluble human IgG1 Fc fusion proteins containing the extracellular domain of each TRAIL receptor and identified by their specific reactivity with the respective fusion protein in enzyme-linked immunosorbent assay (ELISA). Leukemia cell lines, primary leukemias, and baby hamster kidney (BHK) cell lines stably expressing DR4, DR5, DcR1, or DcR2 cDNA (1 × 106 cells) were incubated with 1 µg of biotinylated control mouse IgG 1 or mAb for 30 minutes on ice. After washing, the cells were incubated with phycoerythrin-conjugated streptavidin (Biomeda, Foster City, CA) for 30 minutes on ice, and then analyzed by flow cytometry. The relative fluorescence intensity (RFI) was determined by calculating the ratio of mean fluorescence intensity for specific staining to that for control staining. For the Fas expression, each cell line was incubated with mouse antihuman Fas (4A5; MBL) or irrelevant mouse IgG for 30 minutes on ice, and subsequently with FITC-conjugated anti-mouse IgG, and analyzed by flow cytometry.Western blot analysis The nonidet P-40 lysates of cell lines were separated on a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions and then transferred to polyvinyl difluoride membranes as previously described.42 After blocking with 5% nonfat dry milk in 0.05% Tween-20 Tris (tris(hydroxymethyl)aminomethane)-buffered saline (TBS), membranes were incubated with mouse antihuman FADD (1:250 dilution; BD Transduction Laboratories, Lexington, KY), antihuman caspase-8 (1:1000 dilution; MBL), antihuman kinase domain of c-ABL (1:400 dilution; Pharmingen, San Diego, CA), or rat antihuman FLIP (FLICE [Fas-associating protein with death domain-like interleukin-1-converting enzyme]/caspase-8 inhibitory protein; 1:1000 dilution; Kamiya Biochemical, Seattle, WA) antibodies in 5% milk TBS at 4°C overnight. Membranes were incubated with horseradish peroxidase-conjugated goat antimouse or rat IgG (1:1000 dilution; MBL) at room temperature for 1 hour and were then developed using the enhanced chemiluminescence kit (Amersham Pharmacia Biotec, Buckinghamshire, United Kingdom).
Cytotoxic effect of FasL against Ph1-positive leukemia cell lines We first examined the susceptibility of 12 Ph1-positive leukemia cell lines to rhsFasL by the 3H-thymidine uptake for the last 6 hours of the 42-hour culture with various concentrations of rhsFasL. As shown Figure 1A, a marked growth inhibition was observed in a dose-dependent manner against well-characterized FasL-sensitive T-leukemia cell lines (Jurkat and MOLT4F).43 This growth inhibition was due to loss of cell viability, rather than cytostasis, as estimated by the trypan blue exclusion assay (Figure 1B). Although 1 of 5 CML-BC-derived (Nalm1) and 1 of 7 AL-derived (KOPM30) cell lines were moderately susceptible, the other 10 Ph1-positive cell lines were highly resistant to FasL as estimated by either the 3H-thymidine uptake (Figure 1A and Table 1) or the trypan blue exclusion assay (Figure 1B).
These results suggested that Ph1-positive leukemia cell lines are generally resistant to the FasL-induced cell death. Next, we analyzed the cell-surface expression of Fas by flow cytometry. As indicated in Figure 1C and summarized in Table 1, Fas was detectable on myeloid cell lines except for K562 but rarely detectable on lymphoid cell lines except for Nalm1. Accordingly, the relatively low expression of Fas could explain the resistance to FasL in lymphoid cell lines, while some factor other than the Fas expression might contribute to the resistance to FasL in myeloid cell lines. Cytotoxic effect of TRAIL against Ph1-positive leukemia cell lines We next investigated the cytotoxic effect of rhsTRAIL against Ph1-positive leukemia cell lines. Among the CML-BC-derived cell lines (Figure 2A, left panel), 2 cell lines (Nalm1 and KOPM28) showed a marked growth inhibition in a dose-dependent manner, whereas the other 3 cell lines (KOPM53, KOPN55bi, and K562) were rather resistant. Among the Ph1-positive AL-derived cell lines (Figure 2A, right panel), 3 cell lines (YAMN91, KOPN66bi, and KOPM30) were highly sensitive, 2 cell lines (YAMN73 and KOPN72bi) were moderately sensitive, but 2 cell lines (KOPN57bi and KOPN30bi) were resistant. To demonstrate that these antileukemic effects were really mediated by TRAIL, we performed the blocking experiment using a neutralizing anti-TRAIL mAb (RIK-2).13 As shown in Figure 2B, the growth inhibition in Nalm1 was totally abolished by RIK-2, substantiating the specific activity of rhsTRAIL. As summarized in Table 2, the sensitivity of Ph1-positive leukemia cell lines to TRAIL was not correlated with the type of disease (CML-BC or AL), the type of BCR-ABL fusion protein (p210, p203, or p190), or the type of lineage (myeloid or lymphoid).
To more directly evaluate the cytotoxic activity of TRAIL, we performed the dye exclusion assay (Figure 2C). Consistent with the growth inhibition as estimated by the 3H-thymidine uptake assay, 7 of 12 Ph1-positive leukemia cell lines were moderately or highly sensitive to the TRAIL-induced cell death. We also examined the binding of Annexin-V by flow cytometry after
12-hour treatment with rhsTRAIL (100 ng/mL). As shown in Figure 3, the Annexin-V-positive
population was increased to 95% in the highly sensitive cell line
(KOPM28), 72% in the moderately sensitive cell line (YAMN73), but only
13% in the resistant cell line (KOPM53), indicating that the
TRAIL-induced cell death in Ph1-positive leukemia cell lines was caused
by apoptosis.
To confirm that the apoptotic cell death induced by TRAIL was
dependent on the activation of caspases, we performed
the 3H-thymidine uptake assay in the presence
of z-VAD-fmk, a broad caspase inhibitor. As shown in Figure
4, the TRAIL-induced growth inhibition
was abrogated by z-VAD-fmk partially in Nalm1 and almost completely in
KOPM28, indicating that the activation of caspases was required for the
TRAIL-induced cell death in Ph1-positive leukemia cells.
Expression of TRAIL receptors on Ph1-positive leukemia cell lines To investigate whether the sensitivity of Ph1-positive leukemia cells to TRAIL depends on the expression of TRAIL receptors, we analyzed the cell-surface expression of DR4, DR5, DcR1, and DcR2 by flow cytometry. The specificity of each mAb is shown against BHK cell lines transfected with respective TRAIL receptor cDNAs (Figure 5A).
Representative cytofluorographic data on highly sensitive (KOPM28,
KOPM30), moderately sensitive (KOPN72bi), and resistant (KOPM53) cell
lines are shown in Figure 5B, and the RFI in each cell line is
summarized in Table 2. Among 4 TRAIL receptors, DR4 and DR5 were
detectable (RFI Correlation between the cell-surface DR4/DR5 expression and the percent inhibition by TRAIL (Figure 5C) revealed that all TRAIL-sensitive cell lines expressed DR4 and/or DR5 at significant levels. In contrast, the TRAIL-resistant cell lines except for K562 showed undetectable or low levels of DR4 and DR5. These results suggested that the sensitivity of Ph1-positive leukemia cells to TRAIL is mostly correlated with the cell-surface expression levels of DR4 and DR5. Cytotoxic effects of TRAIL and FasL against primary Ph1-positive leukemia cells To verify the antileukemic effects of TRAIL and FasL against primary leukemia cells, leukemic blasts from 10 Ph1-positive ALL cases and 2 CML-BC cases were tested. Each sample was cultured for 24 hours in the absence or presence of 100 ng/mL of rhsFasL or rhsTRAIL in combination with a neutralizing anti-TRAIL mAb, RIK-2, and the viability was determined by the trypan blue exclusion assay. As summarized in Table 3, viability of the cells was significantly reduced by the addition of TRAIL in 6 of 10 Ph1-positive ALL cases, while only 1 case was moderately sensitive to FasL. The specific activity of TRAIL was demonstrated by the significant recovery of viability with the RIK-2 treatment. Induction of apoptosis by TRAIL was also confirmed in leukemic blasts from patients 8 and 9 by Annexin-V binding on flow cytometry (Figure 6A). Importantly, primary leukemia cells from patient 3 were sensitive to TRAIL, while the cell line (KOPN57bi) established from this patient was resistant to TRAIL (Figure 2A and Table 2). Leukemic blasts from patients 4 and 8, the origin of TRAIL-sensitive YAMN91 and YAMN73, respectively, were sensitive to TRAIL, while those from patient 2, the origin of TRAIL-resistant KOPN30bi, were resistant. Regarding TRAIL sensitivity in leukemic blasts from CML-BC, only one case (patient 11) was evaluated and showed resistance.
Next, the cell-surface expression of DR4 and DR5 was analyzed by flow cytometry as indicated in Figure 6B and Table 3. In Ph1-positive ALL cases, the expression of DR4/DR5 was detectable on TRAIL-sensitive leukemia cells from patients 4, 8, and 9, but almost undetectable on TRAIL-resistant leukemia cells from patients 1, 2, and 5. In the CML-BC case (patient 12), both DR4 and DR5 were clearly detectable, although its TRAIL-sensitivity could not be evaluated because of excessive spontaneous cell death in control culture. Expression of molecules consisting of death-inducing signaling complex (DISC) Ligation of death receptors by FasL44 and TRAIL45,46 triggers a series of protein-protein interactions that leads to assembly of a DISC. Thus, expression levels of the molecules consisting of DISC are critical determinants for sensitivity. It is known that Fas and DR4/DR5 recruit FADD47,48 and caspase-849,50 into DISC, and that FLIP51 acts as a negative regulator of caspase-8.We therefore performed Western blot analysis of these molecules
in Ph1-positive leukemia cell lines (Figure
7). FADD, caspase-8, and FLIP were almost
ubiquitously expressed in Ph1-positive leukemia cell lines irrespective
of their differential sensitivities to FasL and TRAIL. These
observations indicated that an absence of FADD and/or caspase-8 or an
excessive expression of FLIP could not be a mechanism for resistance to
TRAIL and FasL.
Modulation of TRAIL sensitivity by NF-  B is constitutively
activated by BCR-ABL in Ph1-positive leukemias52 and that
NF-B could modulate TRAIL-induced cell death.6,9,53 We
attempted to examine whether the NF-B inhibitors could modulate the
TRAIL sensitivity of Ph1-positive leukemia cells. For this purpose, 10 leukemia cell lines (6 sensitive and 4 resistant) were preincubated for
3 hours with 2.5 µM proteasome inhibitor LLnL,54 which
is known to inhibit the activation of NF-B by blocking the
degradation of the IB inhibitory protein, followed by 42-hour
exposure to lower concentrations of rhsTRAIL (10 ng/mL for sensitive
and 50 ng/mL for resistant cell lines), and then the
3H-thymidine uptake was assessed for the last 6 hours
(Figure 8A). As expected from a
considerable role of NF-B in the BCR-ABL-mediated transformation,
the treatment with LLnL alone moderately repressed the
3H-thymidine uptake in most of Ph1-positive leukemia cell
lines. In the TRAIL-sensitive cell line KOPM28, for instance, the
treatment with either TRAIL or LLnL reduced the
3H-thymidine uptake to approximately 70% or 50%,
respectively, but the treatment with both TRAIL and LLnL almost
completely abrogated the 3H-thymidine uptake. Similar
results were obtained in all TRAIL-sensitive cell lines examined. In
contrast, LLnL did not enhance the proapoptotic activity of TRAIL in
all TRAIL-resistant cell lines, including K562, which considerably
expressed DR5. These results suggested that the inhibition of NF-B
activation by LLnL either augmented the TRAIL sensitivity or
synergistically acted with TRAIL in the process of apoptosis induction,
but could not convert the TRAIL-resistant cells to be TRAIL
sensitive.
A similar experiment was also performed in KOPM28 using another
NF- Comparison of antileukemic effects of TRAIL and imatinib mesylate Imatinib mesylate is a specific inhibitor of BCR-ABL tyrosine kinase activity41 and shows a potent cytotoxic effect on Ph1-positive leukemias.55,56 In a clinical trial, imatinib mesylate was reported to be very effective in CML patients who were resistant to the IFN- therapy.55
Thus, we compared the cytotoxic effects of TRAIL and imatinib mesylate
against 12 Ph1-positive cell lines (Figure
9). All these cell lines were established
from the patients who had not been treated with imatinib mesylate and
were not selected by imatinib mesylate treatment in vitro. All myeloid
cell lines (KOPM28, KOPM30, KOPM53, and K562; closed symbols) showed
high sensitivity to imatinib mesylate (percent inhibition, > 60%).
In contrast, only 1 of 8 lymphoid cell lines (KOPN57bi) was highly
sensitive, and 4 lymphoid cell lines (Nalm1, KOPN66bi, YAMN73, and
YAMN91) were naturally resistant to imatinib mesylate (percent
inhibition, < 40%). Two myeloid cell lines (KOPM28 and KOPM30)
showed high sensitivity to both TRAIL and imatinib mesylate. The 4 lymphoid cell lines resistant to imatinib mesylate were sensitive to
TRAIL, while 5 cell lines (myeloid 2; lymphoid 3) were resistant to
TRAIL but sensitive to imatinib mesylate. Most importantly, none of 12 cell lines were resistant to both TRAIL and imatinib mesylate. These results suggested that TRAIL and imatinib mesylate could be used complementarily to eliminate Ph1-positive leukemia
cells.
In the present study, to explore the possible contribution of FasL and TRAIL to the immune-mediated antileukemic effects against Ph1-positive leukemia cells, we first investigated whether FasL or TRAIL could induce cell death in 12 Ph1-positive leukemia cell lines. Consistent with a previous report,32 most of the Ph1-positive cell lines were resistant to FasL. In contrast, 2 of 5 CML-BC-derived and 5 of 7 Ph1-AL-derived cell lines were highly or moderately sensitive to TRAIL-induced apoptotic cell death. Similar results were obtained with primary Ph1-positive ALL cells. Consistent with our data, Plasilova et al recently reported that the growth of CML progenitors in chronic phase as well as in accelerated or blastic phases was significantly suppressed by recombinant TRAIL.57 We next analyzed the cell-surface expression of TRAIL receptors to understand the differences in TRAIL sensitivity among the Ph1-positive leukemia cell lines. All TRAIL-sensitive cell lines and primary cells expressed the death-inducing receptors DR4 and/or DR5 on their surface, whereas the TRAIL-resistant cell lines, except for K562, and primary cells expressed neither DR4 nor DR5. In addition, none of these cell lines expressed DcR1 and DcR2, which are thought to act as decoy receptors. These results suggested that the sensitivity of Ph1-positive leukemia cells to TRAIL is primarily determined by the cell-surface expression of DR4 and/or DR5. In addition to the receptor expression, regulators in the death-signaling pathway would be critical for the determination of sensitivity to FasL and TRAIL. We demonstrated that caspase-8, FADD, and FLIP were ubiquitously expressed in Ph1-positive leukemia cell lines irrespective of their differential sensitivities to FasL and TRAIL, suggesting that these molecules are not critically involved in the determination of sensitivity to FasL and TRAIL at least in their expression levels. We observed a marked discrepancy between sensitivity to TRAIL and FasL in several Ph1-positive cell lines despite expression of both death receptors. In particular, KOPM28 expressing both DR5 and Fas at highest levels showed a high sensitivity to TRAIL but not to FasL. Recently, a similar discrepancy has also been documented in some solid tumors.58,59 In addition, distinct intracellular signaling pathways in TRAIL- and FasL-mediated apoptosis have been reported.60 Thus, KOPM28 would serve as a useful subject to explore distinct intracellular signaling via TRAIL and FasL in further studies. It has been demonstrated that the binding of TRAIL to DR4 and DR5 as
well as DcR2 induced the NF- We previously demonstrated that IFN- Allo-SCT is a potentially curative therapy against CML and Ph1-positive ALL, and its effectiveness is thought to be achieved mainly by the immune-mediated GVL effect. We herein showed that TRAIL could efficiently induce apoptosis in Ph1-positive AL-derived cell lines as well as CML-BC-derived cell lines. In this regard, endogenously expressed TRAIL on CTLs or NK cells may be involved in the GVL effect. To address this hypothesis, the correlation between the in vitro TRAIL sensitivity of leukemia cells and the leukemia-free survival after allo-SCT in CML and Ph1-positive ALL patients has to be determined in future study. A significant number of CML patients cannot tolerate the IFN- Finally, we investigated the correlation between TRAIL sensitivity and
imatinib mesylate sensitivity. imatinib mesylate exerted a potent
antileukemic effect against Ph1-positive cells not only in patients
with CML in chronic phase who failed to the IFN-
Submitted June 14, 2002; accepted December 17, 2002.
Prepublished online as Blood First Edition Paper, December 27, 2002; DOI 10.1182/blood-2002-06-1770.
Supported in part by research grants from the Ministry of Education, Science, and Culture, Japan.
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: Takeshi Inukai, Department of Pediatrics, School of Medicine, University of Yamanashi, Tamaho, Nakakoma, Yamanashi 409-3898, Japan; e-mail: tinukai{at}res.yamanashi-med.ac.jp.
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© 2003 by The American Society of Hematology.
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  J. Jin, H. Liu, C. Yang, G. Li, X. Liu, Q. Qian, and W. Qian Effective gene-viral therapy of leukemia by a new fiber chimeric oncolytic adenovirus expressing TRAIL: in vitro and in vivo evaluation Mol. Cancer Ther., May 1, 2009; 8(5): 1387 - 1397. [Abstract] [Full Text] [PDF]
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K. Hara, M. Okamoto, T. Aki, H. Yagita, H. Tanaka, Y. Mizukami, H. Nakamura, A. Tomoda, N. Hamasaki, and D. Kang Synergistic enhancement of TRAIL- and tumor necrosis factor {alpha}-induced cell death by a phenoxazine derivative Mol. Cancer Ther., July 1, 2005; 4(7): 1121 - 1127. [Abstract] [Full Text] [PDF]
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C. Tecchio, V. Huber, P. Scapini, F. Calzetti, D. Margotto, G. Todeschini, L. Pilla, G. Martinelli, G. Pizzolo, L. Rivoltini, et al. IFN{alpha}-stimulated neutrophils and monocytes release a soluble form of TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) displaying apoptotic activity on leukemic cells Blood, May 15, 2004; 103(10): 3837 - 3844. [Abstract] [Full Text] [PDF]
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H.-J. Kolb, C. Schmid, A. J. Barrett, and D. J. Schendel Graft-versus-leukemia reactions in allogeneic chimeras Blood, February 1, 2004; 103(3): 767 - 776. [Abstract] [Full Text] [PDF]
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P. Secchiero, E. Melloni, M. Heikinheimo, S. Mannisto, R. Di Pietro, A. Iacone, and G. Zauli TRAIL regulates normal erythroid maturation through an ERK-dependent pathway Blood, January 15, 2004; 103(2): 517 - 522. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
[(cpm
of treated well)/(cpm of untreated well)]} × 100. The percent 3H-thymidine uptake was calculated as follows: [(cpm of
treated well)/(cpm of untreated well)] × 100.
1.5) on 5 and 8, respectively, of 12 Ph1-positive
leukemic cell lines. DcR1 and DcR2 were not detectable on any tested
cell lines.
induces apoptosis of leukemia cells by down-regulating the c-myc gene expression via blockade of the Tcf-4 activity.
Cell Death Differ.
2002;9:513-526