Leukemia (2001) 15, 921–928  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu In vitro susceptibility to TRAIL-induced apoptosis of acute leukemia cells in the context of TRAIL receptor gene expression and constitutive NF-kB activity C Wuchter 1 , D Krappmann 2 , Z Cai 3 , V Ruppert 1 , C Scheidereit 2 , B Do ¨ rken 1 , W-D Ludwig 1 and L Karawajew 1 1 Department of Hematology, Oncology, and Tumor Immunology, Robert-Ro ¨ ssle-Clinic, Charite ´, Humboldt-University of Berlin; 2 Max- Delbru ¨ ck-Center for Molecular Medicine, Berlin, Germany; and 3 Department of Hematology, Hospital of Zhejiang University, Hangzhou, PR China The TNF-related apoptosis-inducing ligand (TRAIL) is currently under evaluation as a possible (co-)therapeutic in cancer treat- ment. We therefore examined 129 cell samples from patients with de novo acute leukemia as to their constitutive suscepti- bility to TRAIL-induced apoptosis in vitro. Only 21 (16%) cell samples revealed at least 10% TRAIL-susceptible cells/sample as detected by flow cytometric annexinV staining after 24 h cul- ture compared with medium control. Precursor B cell ALL samples (11 (27%) of 41) were more TRAIL-susceptible com- pared with AML (5 (9%) of 54; P , 0.05) but not compared with precursor T cell ALL (5 (15%) of 34; P = 0.20). Furthermore, we examined constitutive mRNA expression levels of TRAIL recep- tors R1–R4 by semi-quantitative RT-PCR (n = 58). Expression levels were heterogeneous, however, there was no significant correlation between the expression of the signal-transducing receptors (R1, R2) as well as of the decoy receptors (R3, R4) and TRAIL sensitivity in this series. Constitutive NF-kB activity has been shown to influence TRAIL susceptibility of leukemic cells. In 39 leukemic cell samples examined, we found a gener- ally high NF-kB activity as detected by electrophoretic mobility shift assay which did not differ between TRAIL-susceptible and TRAIL-resistant cases. Finally, 49 acute leukemic cell samples were coincubated with doxorubicin in vitro. Doxorubicin sensit- ized four of 35 initially TRAIL-resistant samples and augmented TRAIL-induced apoptosis in two of 14 TRAIL-susceptible samples. In summary, constitutive TRAIL susceptibility differs between leukemia subtypes and does not correlate with mRNA expression levels of the TRAIL receptors R1–R4 as well as constitutive NF-kB activation status. The observed sensitiz- ation of leukemic cells to TRAIL by doxorubicin in vitro indi- cates that TRAIL should be further evaluated as to its possible role as an in vivo cotherapeutic in acute leukemia. Leukemia (2001) 15, 921–928. Keywords: apoptosis; acute leukemia; TRAIL; TRAIL receptors R1–R4; NF-kB Introduction Activation of apoptotic signalling pathways plays an important role in chemotherapeutic-induced cell death of tumor cells. 1–3 TRAIL, a member of the TNF family, was initially shown to induce surface receptor-mediated apoptosis in tumor cells but not in normal cells. 4,5 In several mouse models, in vivo administration of TRAIL induced apoptosis in a variety of human malignant cells. 6–9 Several chemotherapeutic agents induced and enhanced TRAIL susceptibility in tumor cells in vitro and in vivo. 9–11 Therefore, TRAIL may have potential as a cancer (co-)therapeutic in human malignancies. However, in light of recently published studies, the in vivo application of TRAIL might be associated with severe liver damage and cell death of normal brain cells in humans. 12,13 TRAIL also induced apoptosis in erythroblasts within normal hematopo- Correspondence: C Wuchter, Robert-Ro ¨ ssle-Clinic, Charite ´, Hum- boldt University of Berlin, Lindenberger Weg 80, 13125 Berlin, Germany; Fax: 0049-30-9417 1308 Received 11 September 2000; accepted 21 February 2001 iesis. 14 Thus, the use of TRAIL in human cancer therapy must be considered with caution and further investigations should be included in the preclinical evaluation of TRAIL. 12,13 TRAIL can bind to at least four different cell surface recep- tors: TRAIL-R1 (also called DR4) and TRAIL-R2 (also called KILLER/DR5) are receptors with two cysteine-rich extracellular ligand-binding domains and a cytoplasmic death domain that signals downstream caspase activation. TRAIL-R3 (also called DcR1 or TRID) and TRAIL-R4 (also called DcR2 or TRUNDD) are GPI-anchored surface receptors lacking a functional active intracellular death domain. These two receptors are thought to protect cells from TRAIL-mediated apoptosis as decoy receptors by competing with the functionally active TRAIL receptors R1 and R2 for binding to TRAIL. TRAIL receptor gene expression is at least partially controlled in a p53-depen- dent manner. 4,5,15 In human glioma cells, the cytotoxic drug etoposide increased TRAIL-R2 expression, leading to TRAIL- induced apoptosis in these tumor cells. 8 Expression levels of TRAIL-R1 and R2 were also upregulated by etoposide treat- ment in four different epithelial cell-derived tumor cell lines. Inactivation of the transcription factor NF-kB blocked the eto- poside-induced upregulation of R1 and R2 in these cells. 16 So far, no comprehensive study has investigated compara- tively TRAIL sensitivity in different acute leukemia subtypes. In precursor B cell ALL, a rather low TRAIL sensitivity was observed. 17 In this study, exogenous soluble CD40L failed to rescue TRAIL-sensitive leukemic cells from TRAIL-induced apoptosis. 17 TRAIL susceptibility was independent of MDR (‘multi-drug resistance’) protein overexpression and expression levels of the apoptosis-regulating molecules Bcl- 2 and Bax. 18 TRAIL effectively induced apoptosis in multiple myeloma cell lines independently of their Bcl-2 expression levels and was not cytotoxic to CD34 + /CD45 dim hematopo- ietic stem cells. 19,20 Primary tumor cells of hematopoietic ori- gin constitutively expressed TRAIL and were able to induce TRAIL-mediated apoptosis in Jurkat cells. 21 The human pro- myelocytic HL60 cell line showed a constitutive sensitivity to TRAIL-mediated apoptosis, which was downregulated during granulocytic differentiation induced by DMSO. 22 Recent stud- ies also indicated a major role of TRAIL in the regulation of immune processes. 23–26 Therefore, the functional TRAIL expression of leukemic cells may be involved in tumor cell evasion from immunosurveillance. 21 Inactivation of NF-kB led to an increase in TRAIL susceptibility of leukemia cell lines as well as primary acute leukemia cell samples. 27 Pretreatment of leukemic cell lines with chemotherapeutic drugs increased the sensitivity to TRAIL-induced apoptosis of the leukemic cells possibly due to an upregulation of TRAIL-R2. 28 To evaluate TRAIL susceptibility in a larger series of cell samples of patients with de novo acute leukemia, we exam- ined 54 AML, 41 precursor B cell ALL and 34 precursor T cell ALL samples for their in vitro susceptibility to TRAIL-induced apoptosis. To determine whether the expression of the TRAIL receptors R1–R4 and NF-kB activation status correlate with