Rapid improvement in symptoms and physical function following ibrutinib initiation in chronic lymphocytic leukemia and the associated changes in plasma cytokines.

A prospective pilot study was carried out on 34 CLL patients treated with ibrutinib, evaluating the effects on symptoms and physical function with changes in plasma exosomes (EXs), ß2-microglobulin (ß2M) and 26 plasma cytokines. The revised Edmonton Symptom Assessment Scale (ESAS-R) demonstrated moderate fatigue, shortness of breath and a sense of unwellness before treatment, which significantly improved within 2 weeks of starting ibrutinib. These changes were associated with a rapid improvement in sit-to-stand and 4 m walking speeds. The plasma levels of CCL11, IL-7, -8 and -10 dropped initially while the levels of TNF-α/-ß, CCL3, CCL4, CCL17, and IL-16 continued to decline for 12 months. Despite the initial lymphocytosis, plasma ß2M levels fell but no consistent change in plasma EXs occurred. Thus, ibrutinib can produce a rapid and sustained improvement in symptoms and physical function in CLL, associated with a decline in multiple plasma cytokines.

Physician perceptions about living organ donation in patients with Amyotrophic Lateral Sclerosis.

OBJECTIVES: Patients with Amyotrophic Lateral Sclerosis (ALS) have expressed desire to become living organ donors but are unable to do so with current organ donation policies. Our objective is to assess ALS patient's interest in organ donation, and perceived concerns of this practice by ALS neurologists. PATIENTS AND METHODS: An electronic survey was administered to ALS neurologists across the United States regarding living organ donation in ALS patients prior to respiratory failure. RESULTS: 52 complete responses were received from 121 invites. 67% (35/52) of neurologists expressed no concerns about living organ donation in ALS patients, and 33% had concerns. The concerns related to respiratory failure, anesthesia exposure and renal dysfunction. With their concerns addressed, 71% of neurologists reported that they would endorse living organ donation. 49% of neurologists reported being asked by a patient for information regarding living organ donation. ALS neurologists felt that 22.8% of ALS patients (median 19%) would be interested in learning more about organ donation, while only 6% of neurologists broach this subject with their patients. CONCLUSION: Our results indicate that 1 in every 4 ALS patients may be interested in exploring options for living organ donation, and this topic is not routinely addressed by ALS clinics. These results indicate an unexplored area of patient interest. To honor a patient's wishes to donate, the transplant community will have to accommodate living organ donation from terminally ill patients, and address neurologist concerns. Such a practice could benefit two groups of patients.

Ferroptosis is induced following siramesine and lapatinib treatment of breast cancer cells.

Ferroptosis is an iron-dependent, oxidative cell death, and is distinct from apoptosis, necrosis and autophagy. In this study, we demonstrated that lysosome disrupting agent, siramesine and a tyrosine kinase inhibitor, lapatinib synergistically induced cell death and reactive oxygen species (ROS) in MDA MB 231, MCF-7, ZR-75 and SKBr3 breast cancer cells over a 24 h time course. Furthermore, the iron chelator deferoxamine (DFO) significantly reduced cytosolic ROS and cell death following treatment with siramesine and lapatinib. Furthermore, we determined that FeCl3 levels were elevated in cells treated with siramesine and lapatinib indicating an iron-dependent cell death, ferroptosis. To confirm this, we treated cells with a potent inhibitor of ferroptosis, ferrastatin-1 that effectively inhibited cell death following siramesine and lapatinib treatment. The increase levels of iron could be due to changes in iron transport. We found that the expression of transferrin, which is responsible for the transport of iron into cells, is increased following treatment with lapatinib alone or in combination with siramesine. Knocking down of transferrin resulted in decreased cell death and ROS after treatment. In addition, ferroportin-1 (FPN) is an iron transport protein, responsible for removal of iron from cells. We found its expression is decreased after treatment with siramesine alone or in combination with lapatinib. Overexpression FPN resulted in decreased ROS and cell death whereas knockdown of FPN increased cell death after siramesine and lapatinib treatment. This indicates a novel induction of ferroptosis through altered iron regulation by treating breast cancer cells with a lysosome disruptor and a tyrosine kinase inhibitor.

Lysosomotropic agents selectively target chronic lymphocytic leukemia cells due to altered sphingolipid metabolism.

Lysosome membrane permeabilization (LMP) mediates cell death in a variety of cancer cells. However, little is known about lysosomes and LMP in chronic lymphocytic leukemia (CLL). Owing to drug resistance and toxicity in CLL patients, better treatment strategies are required. Our results show that CLL cells were sensitive to the lysosomotropic agent siramesine. Furthermore, this drug was more effective in CLL cells, regardless of prognostic factors, compared with normal B cells. Siramesine caused LMP, lipid peroxidation and transcription factor EB nuclear translocation followed by mitochondrial membrane potential loss and reactive oxygen species release. Siramesine-induced cell death was blocked by lipid antioxidants, but not by soluble antioxidants or protease inhibitors. To determine whether CLL cells had altered lysosomes, we investigated sphingolipid metabolism as the lysosome is a hub for lipid metabolism. We found that CLL cells had more lysosomes, increased sphingosine-1-phosphate phosphatase 1 (SPP1) expression, and increased levels of sphingosine compared with normal B cells. Raising sphingosine levels increased LMP and cell death in CLL cells, but not in normal B cells. Together, these results show that excess sphingosine in CLL cells could contribute to their sensitivity toward LMP. Thus, targeting the lysosome could be a novel therapeutic strategy in CLL.

Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling.

Chronic lymphocytic leukemia (CLL) can be divided into groups based on biomarkers of poor prognosis. The expression of the tyrosine kinase ZAP-70 (member of the Syk tyrosine kinase family) in CLL cells is associated with shorter overall survival in CLL patients. Currently, there is a lack of targeted therapies for patients with ZAP-70 expression in CLL cells. The tyrosine kinase inhibitor gefitinib has been shown to be effective at induce apoptosis in acute myeloid leukemia through inhibition of Syk. In this study, we sought to test the efficacy of gefitinib in primary human ZAP-70+ CLL cells. We demonstrate that gefitinib preferentially induces cell death in ZAP-70-expressing CLL cells with a median IC50 of 4.5 µM. In addition, gefitinib decreases the viability of ZAP-70+ Jurkat T leukemia cells but fails to affect T cells from CLL patients. Western blot analysis shows gefitinib reduces both basal and B-cell receptor (BCR)-stimulated phosphorylation of Syk/ZAP-70, ERK, and Akt in ZAP-70+ CLL cells. Moreover, gefitinib inhibits the pro-survival response from BCR stimulation and decreases pro-survival proteins such as Mcl-1. Finally, ZAP-70 expression sensitizes Raji cells to gefitinib treatment. These results demonstrate that gefitinib specifically targets ZAP-70+ CLL cells and inhibits the BCR cell survival pathway leading to apoptosis. This represents the likelihood of tyrosine kinase inhibitors being effective targeted treatments for ZAP-70+ CLL cells.

Synergistic apoptotic response between valproic acid and fludarabine in chronic lymphocytic leukaemia (CLL) cells involves the lysosomal protease cathepsin B.

Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins.

Increased risk of second malignancies in chronic lymphocytic leukaemia patients as compared with follicular lymphoma patients: a Canadian population-based study.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) patients have an increased risk of other malignancies. This may be due to surveillance bias, treatment or immunosuppression. METHODS: Cohort study of 612 consecutively diagnosed CLL patients in a Canadian province, with comparisons to follicular lymphoma (FL) patients. RESULTS: Treated CLL patients had a 1.7-fold increased risk of second cancers compared with untreated CLL patients. As compared with untreated FL patients, untreated CLL patients had a two-fold increased incidence of second malignancies. CONCLUSION: Chronic lymphocytic leukaemia patients have an inherent predisposition to second cancers and the incidence is further increased by treatment.

BNIP3 acts as transcriptional repressor of death receptor-5 expression and prevents TRAIL-induced cell death in gliomas.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor, and current treatment modalities such as surgical resection, adjuvant radiotherapy and temozolomide (TMZ) chemotherapy are ineffective. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a novel cancer therapeutic agent for GBM because of its capability of inducing apoptosis in glioma cells. Unfortunately, the majority of glioma cells are resistant to TRAIL-induced apoptosis. The Bcl-2 nineteen kilodalton interacting protein (BNIP3) is a pro-cell death BH3-only member of the Bcl-2 family that is one of the highest expressed genes in hypoxic regions of GBM tumors. We previously found that BNIP3 is localized to the nucleus in GBM tumors and suppresses cell death in glioma cells. Herein, we have discovered when BNIP3 nuclear expression is knockdown in glioma cell lines and in normal mouse astrocytes, TRAIL and its death receptor, death receptor-5 (DR5) expression is increased. In addition, when nuclear BNIP3 expression is increased, the amount of TRAIL-induced apoptosis is reduced. Using a streptavidin pull-down assay, we found that BNIP3 binds to the DR5 promoter and nuclear BNIP3 binds to the DR5 promoter. Furthermore, nuclear BNIP3 expression in GBM tumors correlates with decreased DR5 expression. Taken together, we have discovered a novel transcriptional repression function for BNIP3 conferring a TRAIL resistance in glioma cells.

Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer.

Myeloid cell leukaemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. In breast tumours, increased Mcl-1 expression correlates with high tumour grade and poor patient survival. We have previously demonstrated that Her-2 levels correspond to increased Mcl-1 expression in breast tumours. Epidermal growth factor (EGF) receptor signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival. Herein, we determined the critical downstream signals responsible for the EGF mediated increase of Mcl-1 and their role in cell survival. We found that both Mcl-1 mRNA and protein levels are rapidly induced upon stimulation with EGF. Promoter analysis revealed that an Elk-1 transcription factor-binding site is critical for EGF activation of the Mcl-1 promoter. Furthermore, we found that knockdown of Elk-1 or inhibition of the Erk signalling pathway was sufficient to block EGF upregulation of Mcl-1 and EGF mediated cell survival. Using chromatin immunoprecipitation and biotin labelled probes of the Mcl-1 promoter, we found that Elk-1 and serum response factor are bound to the promoter after EGF stimulation. To determine whether Mcl-1 confers a survival advantage, we found that knockdown of Mcl-1 expression increased apoptosis whereas overexpression of Mcl-1 inhibited drug induced cell death. In human breast tumours, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels. These results indicate that the EGF induced activation of Elk-1 is an important mediator of Mcl-1 expression and cell survival and therefore a potential therapeutic target in breast cancer.

High incidence of chronic lymphocytic leukemia (CLL) diagnosed by immunophenotyping: a population-based Canadian cohort.

Incidence and outcomes of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) are not well established at the population level, especially since the widespread use of immunophenotyping. We studied the epidemiology of CLL in Manitoba (Canada) by combining data from a centralized flow cytometry facility and the provincial cancer registry for the period 1998-2003. Of 616 cases identified, 27% of patients identified by flow cytometry were not on the cancer registry. The age-adjusted incidence of 7.99/100,000 is substantially higher than the reported incidence in registry reports. We also noted differences in relative survival based on age and gender.

Superoxide is the major reactive oxygen species regulating autophagy.

Autophagy is involved in human diseases and is regulated by reactive oxygen species (ROS) including superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)). However, the relative functions of O(2)(*-) and H(2)O(2) in regulating autophagy are unknown. In this study, autophagy was induced by starvation, mitochondrial electron transport inhibitors, and exogenous H(2)O(2). We found that O(2)(*-) was selectively induced by starvation of glucose, L-glutamine, pyruvate, and serum (GP) whereas starvation of amino acids and serum (AA) induced O(2)(*-) and H(2)O(2). Both types of starvation induced autophagy and autophagy was inhibited by overexpression of SOD2 (manganese superoxide dismutase, Mn-SOD), which reduced O(2)(*-) levels but increased H(2)O(2) levels. Starvation-induced autophagy was also inhibited by the addition of catalase, which reduced both O(2)(*-) and H(2)O(2) levels. Starvation of GP or AA also induced cell death that was increased following treatment with autophagy inhibitors 3-methyladenine, and wortamannin. Mitochondrial electron transport chain (mETC) inhibitors in combination with the SOD inhibitor 2-methoxyestradiol (2-ME) increased O(2)(*-) levels, lowered H(2)O(2) levels, and increased autophagy. In contrast to starvation, cell death induced by mETC inhibitors was increased by 2-ME. Finally, adding exogenous H(2)O(2) induced autophagy and increased intracellular O(2)(*-) but failed to increase intracellular H(2)O(2). Taken together, these findings indicate that O(2)(*-) is the major ROS-regulating autophagy.

The role of Bcl-2 family member BNIP3 in cell death and disease: NIPping at the heels of cell death.

Bcl-2 nineteen-kilodalton interacting protein (BNIP3) is a BH-3-only Bcl-2 family member whose expression levels increase during stress such as hypoxia through hypoxia-inducing factor-1-dependent or -independent mechanisms. When BNIP3 expression is induced, it localizes to the mitochondria and triggers a loss of membrane potential, and an increase in the reactive oxygen species production, which often leads to cell death. Cells under normal growth conditions suppress BNIP3 expression through transcriptional repression. There is considerable debate in the literature regarding what type of cell death is induced by BNIP3. It has been observed that BNIP3 could induce necrosis, autophagy and/or apoptosis. In contrast, other studies indicate that BNIP3 could promote cell survival. Besides its cell death regulation, BNIP3 plays a key role in the pathogenicity of many diseases. In cardiac infarction, loss of BNIP3 expression has been shown to reduce the number of damaged cardiomyocytes after ischemia and reperfusion. BNIP3 expression also plays an important role in the deregulation of cell death in many cancers. In this review, we will discuss the different and often contradictory mechanisms of BNIP3 regulation of cell death and the role that BNIP3 may play in diseases.

Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells.

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H(2)O(2)) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H(2)O(2) or 2-ME-induced cell death. H(2)O(2) and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H(2)O(2) or 2-ME through overexpression of manganese-superoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H(2)O(2)- or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H(2)O(2) or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in non-transformed cells.

BNIP3 subfamily BH3-only proteins: mitochondrial stress sensors in normal and pathological functions.

The BNIP3 subfamily of BH3-only proteins consists of BNIP3 and BNIP3-like (BNIP3L) proteins. These proteins form stable homodimerization complexes that localize to the outer membrane of the mitochondria after cellular stress. This promotes either apoptotic or non-apoptotic cell death such as autophagic cell death. Although the mammalian cells contain both members of this subfamily, the genome of Caenorhabditis elegans codes for a single BNIP3 ortholog, ceBNIP3, which shares homology in the transmembrane (TM) domain and in a conserved region close to the BH3 domain of mammalian BNIP3 protein. The cell death activities of BNIP3 and BNIP3L are determined by either the BH3 domain or the C-terminal TM domain. The TM domain of BNIP3 is unique, as it is capable of autonomous stable dimerization and contributes to mitochondrial localization of BNIP3. In knockout mouse models, BNIP3L was shown to be essential for normal erythrocyte differentiation and hematopoietic homeostasis, whereas BNIP3 plays a role in cellular responses to ischemia/reperfusion injury in the heart. Both BNIP3 and BNIP3L play a role in cellular responses to stress. Under hypoxia, both BNIP3 and BNIP3L expression levels are elevated and contribute to hypoxia-induced cell death. In addition, these proteins play critical roles in disease states. In heart disease, both BNIP3 and BNIP3L play a critical role in cardiomyocyte cell death following ischemic and non-ischemic injuries. In cancer, expression of BNIP3 and BNIP3L is downregulated by promoter hypermethylation or by homozygous deletion of the gene locus in certain cancers, whereas their expression was increased in other cancers. In addition, BNIP3 expression has been correlated with poor prognosis in some cancers. The results reviewed here suggest that BNIP3 and BNIP3L may be novel therapeutic targets for intervention because of their pathological roles in regulating cell death in disease states.

Akt is transferred to the nucleus of cells treated with apoptin, and it participates in apoptin-induced cell death.

OBJECTIVES: The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is well known for the regulation of cell survival, proliferation, and some metabolic routes. MATERIALS AND METHODS: In this study, we document a novel role for the PI3-K/Akt pathway during cell death induced by apoptin, a tumour-selective inducer of apoptosis. RESULTS: We show for the first time that apoptin interacts with the p85 regulatory subunit, leading to constitutive activation of PI3-K. The inhibition of PI3-K activation either by chemical inhibitors or by genetic approaches severely impairs cell death induced by apoptin. Downstream of PI3-K, Akt is activated and translocated to the nucleus together with apoptin. Direct interaction between apoptin and Akt is documented. Co-expression of nuclear Akt significantly potentiates cell death induced by apoptin. Thus, apoptin-facilitated nuclear Akt, in contrast to when in its cytoplasmic pool, appears to be a positive regulator, rather than repressor of apoptosis. CONCLUSIONS: Our observations indicate that PI3-K/Akt pathways have a dual role in both survival and cell death processes depending on the stimulus. Nuclear Akt acts as apoptosis stimulator rather than as a repressor, as it likely gains access to a new set of substrates in the nucleus. The implicated link between survival and cell death pathways during apoptosis opens new pharmacological opportunities to modulate apoptosis in cancer, for example through the manipulation of Akt's cellular localization.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) up-regulates death receptor 5 (DR5) mediated by NFkappaB activation in epithelial derived cell lines.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/APO-2L) activates nuclear factor kappaB (NFkappaB). This activation is regulated by the recruitment of an adaptor protein Fas associating death domain (FADD) to TRAIL death receptors, death receptor 4 (DR4, TRAIL-R1) and death receptor 5 (DR5 TRAIL-R2). This leads to recruitment of caspase 8 and receptor interacting protein (RIP) to the receptor complex. Upon recruitment of caspase 8 and RIP, NFkappaB inducing kinase (NIK) becomes activated causing NFkappaB activation. The role of TRAIL induced NFkappaB activation in epithelial cells is unknown. Herein we demonstrate that TRAIL increases expression of DR5 in human embryonic kidney (HEK) 293, MCF-7 and MDA MB 231 epithelial cell lines while DR4 expression remains unchanged. Blockage of NFkappaB activation either by expression of dominant negative IkappaB or treatment with proteasome inhibitor lactacystin eliminates TRAIL induced DR5 expression. Expression of FADD dominant negative in HEK 293 cells that prevents the recruitment of caspase 8 and RIP to TRAIL death receptors also eliminates this increase. By over expression of the p65 subunit of NFkappaB that increases NFkappaB transcriptional activity, DR5 expression was increased compared to vector alone expressing cells. By blocking TRAIL induced NFkappaB activation, the sensitivity of cells to undergo TRAIL induced apoptosis was significantly decreased. Conversely, the amount of TRAIL induced apoptosis was increased in HEK 293 cells over expressing p65 subunit of NFkappaB. Finally blockage of NFkappaB activation eliminates the synergistic apoptotic response of TRAIL and etoposide. Thus, TRAIL mediated NFkappaB activation increases DR5 expression thereby amplifying the apoptotic response of TRAIL in epithelial derived cells.

MEKK2 gene disruption causes loss of cytokine production in response to IgE and c-Kit ligand stimulation of ES cell-derived mast cells.

Ligation of the high-affinity IgE receptor (FcepsilonRI) or of c-Kit stimulates cytokine production in mast cells. We show that MEK kinase 2 (MEKK2), a MAPK kinase kinase (MAP3K) that regulates the JNK and ERK5 pathways, is required for cytokine production in embryonic stem (ES) cell-derived mast cells (ESMC). Targeted disruption of the MEKK2 or MEKK1 gene was used to abolish expression of the respective kinases in ESMC. Transcription of specific cytokines in response to IgE or c-Kit ligand was markedly reduced in MEKK2(-/-) ESMC relative to wild-type ESMC. Cytokine production in MEKK1(-/-) ESMC was similar to that of wild-type ESMC, demonstrating the specificity of MEKK2 in signaling cytokine gene regulation. MEKK2(-/-) ESMC also lost receptor-mediated stimulation of JNK. In contrast, JNK activation in response to UV irradiation was normal, showing that MEKK2 is required for receptor signaling but not for cellular stress responses. MEKK2 is the first MAP3K shown to be required for mast cell tyrosine kinase receptor signaling controlling cytokine gene expression.

Increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and TRAIL.

Chemotherapeutic genotoxins induce apoptosis in epithelial-cell-derived cancer cells. The death receptor ligand TRAIL also induces apoptosis in epithelial-cell-derived cancer cells but generally fails to induce apoptosis in nontransformed cells. We show here that the treatment of four different epithelial cell lines with the topoisomerase II inhibitor etoposide in combination with TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) induces a synergistic apoptotic response. The mechanism of the synergistic effect results from the etoposide-mediated increase in the expression of the death receptors 4 (DR4) and 5 (DR5). Inhibition of NF-kappaB activation by expression of kinase-inactive MEK kinase 1(MEKK1) or dominant-negative IkappaB (DeltaIkappaB) blocked the increase in DR4 and DR5 expression following etoposide treatment. Addition of a soluble decoy DR4 fusion protein (DR4:Fc) to cell cultures reduced the amount of etoposide-induced apoptosis in a dose-dependent manner. The addition of a soluble TNF decoy receptor (TNFR:Fc) was without effect, demonstrating the specificity of DR4 binding ligands in the etoposide-induced apoptosis response. Thus, genotoxin treatment in combination with TRAIL is an effective inducer of epithelial-cell-derived tumor cell apoptosis relative to either treatment alone.