Introduction

Although allogeneic stem cell transplantation has dramatically changed the outcome for conditions such as acute and chronic leukemias, the lack of donors and treatment-related toxicity limit the use of such transplantation. In contrast, autologous bone marrow (BM) and peripheral blood stem cells (PBSCs) are readily available, and the regeneration of host hematopoiesis avoids the toxicity that develops from graft-versus-host disease. In addition, autologous transplantation can be performed on older patients.¹ Autologous BM and PBSC transplantations have proven to be effective treatment modalities for a number of hematopoietic malignancies, and promising results have been obtained for the treatment of selected solid tumors.^2,3,4 The major cause of treatment failure of autologous transplantation is still relapse of the underlying malignancy, which occurs in 40–75% of patients depending on the tumor type and remission status at the time of transplantation.⁵ Gene marking studies have shown that relapse can be caused by the reinfusion of residual malignant cells that can contribute to relapse after transplantation.^6,7 These findings have led to an increased interest in procedures, which can selectively remove cancer cells from autologous hematopoietic cells to be used for transplantation, without damaging the reconstituting hematopoietic cells. Methods applied to this problem include monoclonal antibodies targeted to cancer cell surface markers,⁸ cancer-specific monoclonal antibodies conjugated to toxins,⁹ in vitro incubation with cytotoxic drugs,¹⁰ lectin agglutination,¹¹ phototherapy,¹² and biological modifiers.¹³ Unfortunately, many of these systems for eradicating cancer cells also lead to the destruction of clonogenic progenitor cells, and thereby cause a prolongation of neutropenia after high-dose chemotherapy (HDC) and autologous hematopoietic stem cell transplantation (AHSCT).

One promising strategy for removing cancer cells from autologous BM or PBSCs is to selectively purge or eradicate cancer cells with TRAIL. TRAIL is a type II transmembrane protein that was initially identified because of the homology of its extracellular domain with CD95L, tumor necrosis factor (TNF), and lymphotoxin .^14,15,16 TRAIL is a potent inducer of apoptosis in a variety of transformed or cancer cells of human and mouse origin, but does not seem to be cytotoxic to normal cells.^16,17,18 A relatively high portion of the tumor cell lines tested are sensitive to the cytotoxic effects of TRAIL in vitro,^16,18 and evidence for the safety and potential efficacy of TRAIL therapy against breast and colon cancer was obtained from a severe combined immunodeficiency mouse model.^19,20 Recently, some reports have shown that the combination of chemotherapeutic agents and TRAIL could augment TRAIL-induced apoptosis in various cancer cells in vitro.^21,22,23 These studies suggest that TRAIL may have a potential use for cancer treatment. Unfortunately, Jo et al.²⁴ demonstrated that TRAIL induced apoptosis in normal human hepatocytes in culture, but not in hepatocytes isolated from other species. However, apoptosis and cell death in human hepatocytes were massive and rapid, and occurred in more than 60% of the cells exposed to TRAIL for 10 h. However, TRAIL is not cytotoxic to purified CD34+ hematopoietic stem cells in vitro. Furthermore, it has no adverse effect on the formation of CFU-GM and BFU-E colonies, at a concentration that kills >80% of yeloma cells.²⁵ Although TRAIL could be cautiously used systemically in human because of its hepatotoxicity, it may be possible to eradicate tumor cells in autologous BM or PBSCs during HDC without stem cell damage.

The aim of this study was to evaluate the potential use of the TRAIL to eradicate leukemia cells from autografts without damaging the self-renewing capacity of hematopoietic stem cells. For this purpose, we investigated sensitivity to TRAIL or TRAIL combined with a low dose of doxorubicin and their purging effect upon leukemic cell lines and normal human UCB mononuclear cells without stem cell damage. In addition, the expressions of the TRAIL receptors, DR4 and DR5, and the caspase family members were investigated after treatment with TRAIL, or TRAIL and a low-dose doxorubicin in leukemia cell lines and UCB mononuclear cells. Our results provide a rationale for the use of TRAIL as an ex vivo purging agent for autologous transplantation in hematologic malignancies.

Materials and methods

Cell lines and UCB mononuclear cells isolation

The human acute leukemia cell lines, Jurkat and Molt-4, were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in RPMI 1640 (GIBCO-BRL, Gaithersberg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO-BRL) and 1% penicillin–streptomycin. UCB was obtained at the end of full-term deliveries, after cramping and cutting the cord, and draining the blood into sterile collection tubes containing the anticoagulant citrate-phosphate dextrose. Mononuclear cells were isolated from the cord blood by Ficoll–Hypaque (1.077 g/cm³, Sigma, St Louis, MO, USA) density centrifugation (400 g for 25 min). The interfacial mononuclear cells were collected and washed twice with Ca²⁺-, Mg²⁺-free phosphate-buffered saline (PBS; Life Technologies, Grand Island, NY, USA).

Reagents

Recombinant human TRAIL was purchased from ALEXIS Co. (San Diego, CA, USA) and doxorubicin purchased from the Sigma Chemical Co. The TRAIL that we used was a trimer. Rabbit polyclonal antibody for human DR4 (TRAIL-R1) was obtained from Serotec Inc. (Raleigh, NC, USA) and DR5 (TRAIL-R2) from ALEXIS Co. Rabbit polyclonal antibody for human caspase 3, mouse monoclonal antibody for human caspase 9, and mouse polyclonal antibody for human caspase 8 were obtained from BD Pharmingen (Mountain View, CA, USA), and mouse polyclonal antibody for human caspase 7 from New England Biolabs (Beverly, MA, USA). The MTT assay kit for cell proliferation assay was purchased from Roche Diagnostics GmbH (Mannheim, Germany).

TRAIL- and chemotherapy-mediated toxicity

To assess TRAIL-mediated cytotoxicity upon the UCB mononuclear cells, Jurkat cell line, and Molt-4 cell line were plated at 5 10⁴ cells/well in 96-well microtiter plates. TRAIL was added at the indicated concentrations and the cells were cultured at 37°C for 3, 6, 9, 12, and 24 h. Cell viability was assessed by using the MTT dye reduction assay, as described previously.²⁵ To assess the effects of chemotherapy on the TRAIL-mediated apoptosis of the TRAIL-resistant Molt-4 cell line and UCB mononuclear cells, cells were plated as above. TRAIL and doxorubicin were added at the indicated concentrations and the cells were cultured at 37°C for 24 h. Cell viability was then assessed by using MTT assay as described.

Determination of TRAIL receptor status and caspase expression by RT-PCR and Western blotting

mRNA was isolated from 5 10⁶ cells by using a Micro-Fast Track Kit (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized with a cDNA Cycle Kit using AMV Reverse Transcriptase with oligo(dT) primers (Invitrogen). A volume of 2 l of the resultant cDNA was used in 50 l PCR mixtures, containing 0.5 M of each relevant primer, 200 M dNTPs, 2.5 mM MgCl₂, 1 reaction buffer and 2 U of Taq DNA polymerase (Roche, Indianapolis, IN, USA). The housekeeping gene -actin was used as an internal control. The sequences of specific primers used in this experiment were as follows: DR4 (TRAIL-R1) F, 5'-CTGAGCAACGCAGACTCGCTGTCCAC-3'; DR4 (TRAIL-R1) R, 5'-TCCAAGGACACGGCAGAGCCTGTGCCAT-3'; DR5 (TRAIL-R1) F, 5'-GCCTCATGGACAATGAGATAAAGGTGGCT-3'; DR5 (TRAIL-R2) R, 5'-CCAAATCTCA-AAGTACGCACAAACGG-3'; TRID (TRAIL-R3) F, 5'-GAAGAATTTGGTGCCAATGCCACTG-3'; TRID (TRAIL-R3) R, 5'-CTCTTGGACTTGGCTGGGAGATGTG-3'; TRUNDD (TRAIL-R4) F, 5'-CTTTTCCGGCGGCGTTCATGTCCTTC-3'; TRUNDD (TRAIL-R4) R, 5'-GTTTCTTCCAGGCTGCTTCCCTTTGTA-3', FLIP F, 5'-AATTCAAGGCTCAGAAGCGA-3'; FLIP R, 5'-GGCAGAAACTCTGCTGTTCC-3'; -actin F, 5'-GTGGGGCGCCCC-AGGCACCA-3'; -actin R, 5'-CTCCTTAATGTCACGCACGATTTC-3'.

The sizes of expected products were 506, 502, 612, 453, 226, and 539 bp, respectively. Samples were amplified using the GeneAmp PCR System 9600 (Perkin-Elmer, Foster City, CA, USA). The program consisted of one cycle of 95°C for 1 min, 55°C for 1 min, 72°C for 1 min; 30 cycles of: -95°C for 30 s, 55°C for 35 s, 72°C for 1 min; and finally 10 min at 72°C. PCR-amplified products were run on a 1.5% agarose gel containing 0.5 g/ml ethidium bromide and visualized under UV light. For the Western blot analysis, cells were lysed in ice-cold PBS containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2.0 g/ml aprotinin, and protease inhibitor cocktail (Roche Diagnostics GmbH) by passing them through a 21 gauge needle. The lysed cells were centrifuged at 12 000 rpm to remove cellular debris, and the protein concentrations of the extracts were determined by colorimetric bicinchoninic acid analysis (Pierce, Rockford, IL, USA). Proteins were separated by 10% SDS-PAGE, and the proteins were electrophoretically transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 8% nonfat dry milk in TTBS (50 mM Tris-Hcl (pH 7.5), 150 mM NaCl, 0.1% (v/v) Tween 20) for 1 h at room temperature and incubated in primary antibody diluted to 1:1000 in TTBS/8% non-fat dry milk for 4 h at room temperature. The membrane was then washed three times with TTBS for 15 min and subsequently incubated in HRP-conjugated goat anti-rabbit IgG (DAKO, Denmark) or HRP-conjugated goat anti-mouse IgG (Bio-Rad, Hercules, CA, USA) diluted to 1:3000 in TTBS/8% nonfat dry milk for 1 h at room temperature. The membrane was then washed three times, as described above and developed using the ECL detection system (Amersham Corp., Arlington Heights, IL, USA).

Mixture experiments of Jurkat, Molt-4, and UCB mononuclear cells to TRAIL or TRAIL with low-dose doxorubicin

Mixtures of UCB mononuclear cells (1 10⁵ cells) and Jurkat or Molt-4 cells (1 10⁶, 5 10⁵, 1 10⁵, 5 10⁴, 1 10⁴ cells) were exposed to 100 ng/ml of TRAIL or TRAIL and a low dose of doxorubicin (0.1 M) for 24 h. The selective tumor cell purging effect was evaluated by flow cytometry (FACScan, Becton-Dickinson, Mountain View, CA, USA). Control mixtures were also cultured for 24 h without TRAIL or TRAIL with doxorubicin. Jurkat and Molt-4 cells were transfected with green fluorescent protein (GFP) using retroviral vectors and selected GFP-positive cells were used for the marker. The selective tumor cell purging effect was evaluated by flow cytometry. GFP-positive cells after 24 h incubation were assessed on a flow cytometry at 488 nm excitation and 515 nm emission for fluorescein detection. 8000–10000 cells were counted for each analysis.

Limiting dilution assay of Jurkat cells and Molt-4 cells cultured with UCB mononuclear cells

Mixtures of UCB mononuclear cells (1 10⁵ cells) and Jurkat or Molt-4 cells (1 10⁶, 1 10⁵, 1 10⁴, 1 10³, 1 10² cells) were exposed to 100 ng/ml of TRAIL or TRAIL and a low dose of doxorubicin (0.1 M) for 24 h. Colony-forming cells were cultured in 1.3 ml of 0.9% methylcellulose in IMDM, 30% FBS, 1% bovine serum albumin, 10^-4 m 2-mercaptoethanol, 2 m L-glutamine, 10% agar leukocyte conditioned medium, 3 U/ml recombinant human erythropoietin (MethoCult^TM H4431, Stem Cell Technologies Inc, Vancouver, British Colombia, Canada). Jurkat and Molt-4 cells were transfected with GFP for distinguishing CFU-GM cells from UCB mononuclear cells. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂. The colonies were then counted on days 10–14 using an inverted fluorescent microscope.

Results

UCB mononuclear cells are resistant to TRAIL and TRAIL with doxorubicin

In the first set of experiments, we investigated the sensitivity of UCB mononuclear cells, Jurkat cell line, and Molt-4 cell line to TRAIL by using the MTT assay (Figure 1). As has been previously reported,²⁴ Jurkat cells were found to be more sensitive to TRAIL treatment in a time- and dose-dependent fashion, which resulted in more than a 90% reduction in the number of Jurkat cells after 24 h of incubation with 200 ng/ml of TRAIL. In contrast, Molt-4 cells showed about a 20% reduction in numbers after 24 h of incubation with 200 ng/ml of TRAIL. Moreover, UCB mononuclear cells showed no TRAIL-induced reduction (Figure 1a). We evaluated the cell killing effect of TRAIL with variable doses of doxorubicin on UCB mononuclear cells and Molt-4 cells. TRAIL and 0.1 M of doxorubicin enhanced cytotoxic effect upon the Molt-4 cells resulting in about a 60% reduction in viable cell numbers after 24 h of incubation. In the case of a high dose of doxorubicin (0.5 and 1 M) in combination with TRAIL, the doxorubicin-dependent cell killing predominated. UCB mononuclear cells were still resistant to TRAIL with doxorubicin, but cell viability was slightly lower for the higher dose of doxorubicin in combination with TRAIL (Figure 1b).

Figure 1.

TRAIL induced cytotoxicity and apoptosis. (a) TRAIL induced cell death in UCB mononuclear cells, Jurkat cell line, and Molt-4 cell line. Cells were exposed to the indicated concentrations of TRAIL for 3, 6, 9, 12, and 24 h. Viability was assessed by MTT assay, and results are given as percentages of the controls. Data points show the averages for triplicate results from a representative experiment; bars, s.d. (b) The effects of doxorubicin on TRAIL-mediated apoptosis of TRAIL-resistant Molt-4 cells and UCB mononuclear cells. TRAIL and doxorubicin, at the indicated concentrations, were added and the cells were cultured for 24 h. Viability was assessed by using the MTT assay as described in Materials and methods.

Full figure and legend (50K)

TRAIL receptors expression of UCB mononuclear cells and T-cell leukemia cell lines

To determine the expressions of TRAIL receptors in UCB mononuclear cells, Jurkat cells, and Molt-4 cells, a RT-PCR assay was performed (Figure 2). The death signaling receptors DR4 and DR5, inhibitory receptor DcR1, and caspase inhibitor FLIP were found to be expressed in both T-cell leukemia cell lines and UCB mononuclear cells, but the inhibitory receptor DcR2 was only expressed in UCB mononuclear cells.

Figure 2.

mRNA expression of TRAIL receptors (DR4, DR5, DcR1, and DcR2) and caspase inhibitor FLIP in T-cell leukemia cell lines and UCB mononuclear cells. Examples show Jurkat cells (a), Molt-4 cells (b), and UCB mononuclear cells (c). -actin was used as an internal control.

Full figure and legend (110K)

Change in TRAIL receptor (DR4, DR5) expression following TRAIL treatment

DR4 expression in Jurkat cells following treatment with 100 ng/ml of TRAIL was increased after incubation for 24 h, but DR5 expression was unchanged by Western blot analysis (Figure 3a). DR5 expression in Molt-4 cells was not changed after treatment with TRAIL, but was increased after treatment with a low dose of doxorubicin (0.1 M) or TRAIL with a low-dose doxorubicin. TRAIL with a low dose of doxorubicin showed stronger DR5 expression than doxorubicin alone. DR4 expressions were unchanged after treatment with TRAIL, low dose of doxorubicin, or TRAIL with low-dose doxorubicin in Molt-4 cells. The expression of DR4 and DR5 in UCB mononuclear cells was unchanged by TRAIL treatment, a low dose of doxorubicin, or TRAIL and a low-dose doxorubicin (Figure 3b).

Figure 3.

Changes in the protein expressions of DR4 and DR5. (a) UCB mononuclear cells and Jurkat cell lines were exposed to 100 ng/ml of TRAIL for 3, 6, 9, 12, and 24 h. Cell lysates were prepared from cell type and an equal amount of protein was loaded on 10% SDS-PAGE gel. Western blot analysis was performed with anti-DR4 and DR5 antibody. -actin was used as an internal control. (b) UCB mononuclear cells and Molt-4 cells exposed to 100 ng/ml of TRAIL, 0.1 M of doxorubicin, or TRAIL plus doxorubicin for 24 h. Western blot analysis was performed as described in Materials and methods.

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Activation of caspases in TRAIL-sensitive Jurkat cells after treatment with TRAIL

To confirm that TRAIL-induced apoptosis was the result of the activation of caspases, we measured caspase activation in Jurkat cells treated with 100 ng/ml of TRAIL, by Western blot. Initiator caspase 8 and 9 were activated during TRAIL-induced apoptosis in Jurkat cells (Figure 4a). The cleaved form of caspase 8 was detected as early as 3 h after treatment with TRAIL and gradually increased through 6, 9, and 12 h. Caspase 9 was also activated in Jurkat cells treated with TRAIL. The cleaved form of caspase 9 was detected at 9 and 12 h after exposure to TRAIL. Effector caspase 3 and 7 were also activated in Jurkat cells after treatment with TRAIL (Figure 4a). The precursor form of caspase 3 was cleaved to the 17- and 12-kDa fragments from 3 h after treatment with TRAIL, and caspase 7 was cleaved after 9 h of treatment with TRAIL. The activations of caspase 3 and 7 peaked at 9–12 h.

Figure 4.

Activation of caspases in TRAIL-induced apoptosis. (a) TRAIL-sensitive Jurkat cells were exposed to 100 ng/ml of TRAIL for 3, 6, 9, 12, and 24 h and Western blot analysis was performed for the expression of caspases 3, 7, 8, and 9. -actin was used as an internal control. (b) TRAIL-resistant Molt-4 cells and UCB mononuclear cells were exposed to 100 ng/ml of TRAIL, 0.1 M of doxorubicin, or TRAIL plus doxorubicin for 24 h and Western blot analysis was performed.

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Activation of caspases in TRAIL-resistant Molt-4 cells after treatment with TRAIL and a low dose of doxorubicin

TRAIL resistant Molt-4 cells and UCB mononuclear cells were treated with 100 ng/ml of TRAIL, a low dose of doxorubicin (0.1 M), or TRAIL and a low dose of doxorubicin for 24 h, and caspases (caspase 3, 7, 8, and 9) activation was measured by Western blot analysis. The expression of cleaved caspases was more potent after treatment with TRAIL and a low dose of doxorubicin than with TRAIL alone or by a low dose of doxorubicin alone (Figure 4b). UCB mononuclear cells treated with TRAIL and a low dose of doxorubicin for 24 h still did not express cleaved caspases. These results show that TRAIL-mediated apoptosis occurs via caspases activation, and that normal hematopoietic cells are resistant to TRAIL even in the presence of doxorubicin.

Mixtures of Jurkat cells and UCB mononuclear cells to treatment with TRAIL

Mixtures of GFP-transfected Jurkat cells in 1 10⁵ UCB mononuclear cells at different ratios (10:1, 5:1, 1:1, 1:5, and 1:10) were treated with 100 ng/ml TRAIL for 24 h. GFP-positive cells were markedly decreased (more than 90%) after treatment with TRAIL even in a flask inoculated with 10⁶ Jurkat cells in 10⁵ UCB mononuclear cells but UCB mononuclear cells were not decreased (Table 1, Figure 5). In case of TRAIL-resistant Molt-4 cells, GFP-positive cells also decreased more than 80% after treatment with TRAIL and a low-dose doxorubicin. UCB mononuclear cells still maintained even in TRAIL and a low-dose doxorubicin (Table 1). This showed that TRAIL or TRAIL with a low-dose doxorubicin can selectively remove leukemia cells when mixed with hematopoietic cells.

Figure 5.

Jurkat cells after being exposure mixed with UCB mononuclear cells to the TRAIL for 24 h. Mixtures of UCB mononuclear cells (1 10⁵ cells) and Jurkat cells (1 10⁶, 5 10⁵, 1 10⁵ cells) were exposed to 100 ng/ml of TRAIL for 24 h and followed by using flow cytometry. Jurkat cells were transfected with GFP using retroviral vectors. GFP-positive cells after 24 h incubation were assessed by flow cytometry by exciting at 488 nm and monitoring fluorescein emission at 515 nm. In total, 8000–10 000 cells were counted for each analysis.

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Table 1 - Percentage of Jurkat and Molt-4 cells after exposure of 1 10⁵ UCB mononuclear cells to the TRAIL or TRAIL with a low dose doxorubicin for 24 h.

Full table

Potential efficacy of TRAIL purging by limited dilution assay

We used a limited dilution assay to confirm the purging effect of TRAIL-sensitive Jurkat cell lines and TRAIL-resistant Molt-4 cell lines. We also tested the toxicity of the purging system to clonogenic hematopoietic cells in these experiments (Figure 6). The dilution assay showed that an eradication rate of more than 4 logs is obtained when Jurkat cell lines are treated with 100 ng/ml of TRAIL for 24 h (Table 2). In case of TRAIL-resistant Molt-4 cell lines, the eradication rate was about 3 logs when using 100 ng/ml of TRAIL with a low dose of doxorubicin (0.1 M) (Table 3). The number of CFU-GM colonies generated from UCB mononuclear cells was not changed even when the UCB mononuclear cells were exposed to TRAIL and a low dose of doxorubicin (Table 2 and 3). This showed that TRAIL can decrease the level of TRAIL-sensitive cells by more than 4 logs without stem cell damage when used for autologous BM or PBSCs purging.

Figure 6.

Colony forming assay with mixture of Jurkat cells and UCB mononuclear cells. Jurkat cells were transfected with GFP for distinguishing CFU-GM from UCB mononuclear cells. Cells were exposed to 100 ng/ml of TRAIL for 24 h. (a) Colony from UCB mononuclear cell and (b) colony from Jurkat cell.

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Table 2 - Jurkat and UCB clonogenic cells after exposure of a mixture of Jurkat cells in a 1 10⁵ UCB mononuclear cells to the TRAIL for 24 h.

Full table

Table 3 - Molt-4 and UCB clonogenic cells after exposure of a mixture of Molt-4 cells in a 1 10⁵ UCB mononuclear cells to the TRAIL with a low-dose doxorubicin for 24 h.

Full table

Discussion

The use of autograft for hematopoietic support has two possible disadvantages. First, any beneficial graft-versus-tumor effect is eliminated in this setting, where the curative effect is based solely on chemotherapy and/or radiotherapy. Second, BM and PBSCs are frequently contaminated with malignant cells.^1,28 Moreover, retroviral marking studies in autologous transplantation, used to treat both hematopoietic and solid tumor neoplasms, have shown that the presence of neoplastic cells in the infused hematopoietic cells can contribute to relapse after transplantation.^6,7,29 Fields et al³⁰ reported that patients infused with autologous hematopoietic cells, that are free of contaminating breast cancer cells (BCCs) by PCR assay, have a longer disease-free period than do those patients infused with autologous cells that are positive by PCR assay from BCCs. We therefore decided to test if TRAIL could be used to eradicate leukemia cells without altering the hematopoietic cells. We chose a TRAIL that induces apoptosis in a variety of transformed or cancer cells of human and mouse origin, but which does not seem to be cytotoxic to normal cells.

We tested whether hematopoietic stem cells and leukemia cell lines show different sensitivities to treatment with TRAIL. MTT assays on leukemia cell lines exposed to TRAIL showed that Jurkat cells are very sensitive to TRAIL but that Molt-4 cells are resistant. This is consistent with findings that tumors derived from the same tissue type display different sensitivities to TRAIL in vitro.^21,22,31 We also used doxorubicin in combination with a fixed concentration of TRAIL in the Molt-4 cells. Molt-4 cells were significantly sensitized to TRAIL-induced apoptosis by doxorubicin. Treatment of TRAIL-resistant cell lines with metabolic inhibitors of protein synthesis can convert them to TRAIL-sensitive cell lines, suggesting that the antitumor activity of TRAIL may be enhanced in vitro by using it in combination with chemotherapeutic agents that are known to disrupt a transformed cell's metabolism or mitotic activity.^18,21,22,23 The concentrations of doxorubicin used in this study (0.1–1 M) to sensitize cells to TRAIL-induced apoptosis were within a range clinically achievable in patients.³¹ However, many normal tissues typically affected by doxorubicin, for example, bone marrow, gastrointestinal mucosa, and cardiac tissue, may also be sensitized to TRAIL-induced apoptosis by doxorubicin. In our study, UCB mononuclear cells were found to be resistant to TRAIL alone and TRAIL with doxorubicin at 0.1 M, but cell viability was found to be decreased slightly in the presence of higher doses of doxorubicin (0.5 and 1 M) in combination with TRAIL. This suggests that high doses of doxorubicin may alter hematopoietic cells. Therefore, we used 0.1 M of doxorubicin in combination with TRAIL in our experiments upon TRAIL-resistant cell lines.

We measured the expressions of four TRAIL receptors (DR4, DR5, DcR1, and DCR2) and of the caspase inhibitor FLIP by RT-PCR in two leukemia cell lines and in UCB mononuclear cells to determine differences that might confer TRAIL sensitivity. The death signaling receptors DR4 and DR5, inhibitory receptor DcR1, and caspase inhibitor FLIP were expressed both in leukemia cell lines and UCB mononuclear cells, but inhibitory receptor DcR2 was only expressed in UCB mononuclear cells. This result suggests that DcR2 may regulate TRAIL resistance in hematopoietic stem cells. Differential expression of decoy receptors is considered to be one of the mechanisms that determines sensitivity to TRAIL-induced cell death,^32,33 although some reports fail to show a correlation between TRAIL resistance and the expressions of DcR1 and DcR2 mRNA.³⁴ We also measured changes in the expression levels of DR4 and DR5 during the treatment of Jurkat cells and Molt-4 cells with TRAIL and a low dose of doxorubicin. We found that TRAIL-sensitive Jurkat cell lines showed the increased expression of DR4 but that DR5 expression did not change after treatment with TRAIL. In the case of Molt-4 cells, TRAIL plus low dose of doxorubicin increased the expression of DR5 more than doxorubicin alone. These results show that DR4 expression is increased during TRAIL treatment, and that this induces apoptosis in TRAIL-sensitive Jurkat cells and that TRAIL-resistant Molt-4 cells become sensitive to TRAIL when combined with doxorubicin via DR5 activation. This finding is consistent with previous reports in other malignancies.^{23,35,36,37,38} Caspase activation is a common feature of cell death through apoptosis. To confirm that TRAIL-induced apoptosis is the result of caspases activation, we investigated caspases activation in Jurkat cells treated with 100 ng/ml of TRAIL by western blot. Initiator caspase 8, 9 and effector caspase 3 and 7 were found to be activated in Jurkat cells after treatment with TRAIL. In TRAIL-resistant Molt-4 cells, the expression of cleaved caspases was more potent after treatment with TRAIL and a low dose of doxorubicin than with TRAIL alone or with a low dose of doxorubicin alone. These results demonstrate that TRAIL-mediated apoptosis occurs via caspases activation, and that chemotherapeutic agents that themselves activate caspases also augment TRAIL-induced apoptosis. This finding has therapeutic implications because it suggests that the relative resistance to apoptosis may be overcome by combining agents that independently partially activate caspases.^21,23,39 The exact mechanism of this augmentation of caspase activation is not known. Although UCB mononuclear cells express the mRNA of TRAIL receptors, the death signaling receptors DR4, DR5, and the caspases were not activated by treatment with TRAIL or with TRAIL plus a low dose of doxorubicin in this study. These findings may explain the mechanism of TRAIL resistance of hematopoietic stem cells. Although many investigators have shown that normal cells^16,17,18,23 and hematopoietic stem cells²⁵ are resistant to TRAIL, our data suggest that hematopoietic stem cells are also resistant to TRAIL in the presence of a chemotherapeutic agent, which enhances the apoptosis of TRAIL-resistant cancer cells.^21,22,23

Finally, we tested the purging of contaminating tumor cells in hematopoietic stem cells after treatment to TRAIL in vitro. Direct analysis of mixtures of Jurkat cells and UCB mononuclear cells after exposure to TRAIL for 24 h using flow cytometry, showed that a dramatic reduction of Jurkat cells occurred even in experiments with 10 times more Jurkat cells than UCB mononuclear cells. We used Jurkat cells, which constantly expressed GFP, for flow cytometry because unselected UCB mononuclear cells also contain Jurkat cell markers and this system could give us more accurate results than is possible using other intrinsic markers. The reason we used unselected UCB mononuclear cells rather than CD34+ cells is that AHSCT is generally performed with harvested BM or mobilized PBSCs in the clinical setting rather than selected CD34+ cells. Molt-4 cells were also tested with this system. Molt-4 cells decreased more than 80% after exposure to TRAIL with a low-dose doxorubicin. The limited dilution assay, used to determine the effects of TRAIL on Jurkat cells, showed an eradication rate of more than 4 logs after incubating with TRAIL for 24 h. TRAIL-resistant Molt-4 cells showed a 3 log eradication rate using TRAIL plus a low dose of doxorubicin. These results suggest that TRAIL-sensitive tumor cells can be effectively eradicated by TRAIL in the autologous transplantation setting, but show that TRAIL, even combined with a low dose of doxorubicin, is still not enough for purging of the TRAIL-resistant tumor cells. Further investigation is necessary to increase the sensitivity to TRAIL of resistant leukemia cells in combination with another cytotoxic agents^40,41,42 or to investigate the possibilities of vector-mediated gene transfer strategies.⁴³ The most important reason for purging is to save the hematopoietic reconstituting cells. Unfortunately, many of the purging systems used for eradicating tumor cells also lead to the destruction of clonogenic progenitor cells, and thereby, cause a prolongation of neutropenia after intensive therapy and autologous transplantation.⁴⁴ We tested the toxicity of our purging system to clonogenic hematopoietic cells and no significant decrease in the numbers of CFU-GM in colonies was detected even when they were treated with TRAIL plus a low dose of doxorubicin.

Many investigators have reported studies with TRAIL, but the mechanism that decides TRAIL sensitivity remains unclear and clinical trials of TRAIL are uncertain because of hepatotoxicity in human.²⁴ Our results demonstrate that TRAIL is not toxic to hematopoietic stem cells in vitro and that TRAIL purging can eradicate TRAIL-sensitive tumor cells in vitro.

We conclude that TRAIL might be a useful drug for the purging of contaminated leukemia cells in HDC and AHSCT in patients with TRAIL-sensitive leukemia.

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