TRAIL, caspases and maturation of normal and leukemic myeloid precursors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a membrane-bound cytokine molecule that belongs to the family of tumor necrosis factor (TNF). Members of this family share diverse biological effects, including induction of apoptosis and/or promotion of cell survival. Identification of TRAIL has generated considerable enthusiasm for its ability to induce apoptotic cell death in a variety of tumor cells, by engaging the death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5, while sparing most normal cells. Beside its anticancer activity, several studies have suggested a role for endogenously expressed TRAIL in hemopoiesis. In this review, we summarize the knowledge about the different lineage-specific roles of TRAIL and its receptors in hemopoiesis regulation. Moreover, the complex interplay among the signaling pathways triggered by TRAIL/TRAIL-receptors in myeloid cells is discussed in some detail.     

Dual role of ERK2/NF-κB signaling in TRAIL sensitivity

Targeting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling is a promising approach in cancer treatment. Although ERK and/or NF-κB signaling is involved in the expression of TRAIL receptors (TRAIL-R), the exact underlying mechanisms remain unknown. In this study, we evaluated the role of ERK2 and NF-κB in the cytotoxicity of TRAIL during cisplatin treatment. Cisplatin treatment of neuroepithelioma cells (SK-N-MC) significantly induced ERK2 activation and increased TRAIL cytotoxicity via the upregulation of death receptor 5 (DR5) expression. In partial ERK2 knockdown cell lines that maintained only basal levels of ERK2 activity, cisplatin treatment did not increase ERK2 activity or DR5 expression. These findings indicate that induced (rather than basal) ERK2 activity enhances TRAIL susceptibility via DR5 expression. In complete ERK2 knockdown cell lines with no basal ERK2 activity, DR4, DR5, and DcRs expression levels were increased, and additional treatment with cisplatin did not further increase TRAIL-R expression. Chemical inhibition of ERK2 also enhanced TRAIL cytotoxicity by upregulating DR4 and DR5 expression. These findings indicate that basal ERK2 activity suppresses TRAIL-R expression. Both basal and inducible ERK2 activities regulate TRAIL-R expression via the NF-κB signaling pathway. Overall, our findings suggest that the ERK2/NF-κB signaling pathway has a dual role in TRAIL susceptibility by differentially regulating TRAIL-R expression in the same cellular system.     

TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma

PURPOSE: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells, and a number of clinical trials have recently been initiated to test the safety and antitumoral potential of TRAIL in cancer patients. Four different receptors have been identified to interact with TRAIL: two are death-inducing receptors (TRAIL-R1 [DR4] and TRAIL-R2 [DR5]), whereas the other two (TRAIL-R3 [DcR1] and TRAIL-R4 [DcR2]) do not induce death upon ligation and are believed to counteract TRAIL-induced cytotoxicity. Because high levels of DcR2 expression have recently been correlated with carcinogenesis in the prostate and lung, this study investigated the importance of TRAIL and TRAIL receptor expression in breast cancer patients with invasive ductal carcinoma, taking various prognostic markers into consideration. METHODS AND MATERIALS: Immunohistochemical analyses were performed on 90 breast cancer patients with invasive ductal carcinoma using TRAIL and TRAIL receptor-specific antibodies. Age, menopausal status, tumor size, lymph node status, tumor grade, lymphovascular invasion, perineural invasion, extracapsular tumor extension, presence of an extensive intraductal component, multicentricity, estrogen and progesterone receptor status, and CerbB2 expression levels were analyzed with respect to TRAIL/TRAIL receptor expression patterns. RESULTS: The highest TRAIL receptor expressed in patients with invasive ductal carcinoma was DR4. Although progesterone receptor-positive patients exhibited lower DR5 expression, CerbB2-positive tissues displayed higher levels of both DR5 and TRAIL expressions. CONCLUSIONS: DR4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma.     

H-Ras regulation of TRAIL death receptor mediated apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists.     

DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer

Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL^wt), TRAIL-R1 (sTRAIL^DR4) and TRAIL-R2 (sTRAIL^DR5) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL^DR4) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL^wt. Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future.     

Sensitization of multidrug resistant human ostesarcoma cells to Apo2 Ligand/TRAIL-induced apoptosis by inhibition of the Akt/PKB kinase

Chemotherapeutic agents have been used for the treatment of patients with osteosarcoma (OS). However, inherent or acquired resistance to these agents is a serious problem in the management of OS patients. The emergence of the multidrug resistance (MDR) phenotype in cancer cells is often associated with the overexpression of P-glycoprotein, encoded by the multidrug resistance gene MDR-1. The administration of some of the most common chemotherapeutic agents to these cells becomes ineffective because of their P-gp-driven efflux from the cell. Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that is considered to induce death of cancer cells but not normal cells. Its powerful apoptotic activity is mediated through its cell surface death domain-containing receptors, TRAIL-R1/DR4 and TRAIL-R2/DR5, which in turn spread the signal in the cytosol through the activation of the caspase cascade. The Akt/PKB kinase is an important cell survival protein which is regulated by D3-phosphoinositides. High Akt expression and activity levels are well documented in many types of tumors, which very often show an altered PI3-K/Akt/PTEN pathway. In this study the U2OS human osteosarcoma cell line and its multidrug resistant (MDR) subline that overexpresses MDR-1 gene, MDR-U2OS, have been analyzed for their responsiveness to TRAIL. In conflict with the presence of active DR4 and DR5 receptors in both clones, U2OS cells exhibited only a low responsiveness to TRAIL, while the MDR-U2OS subline did exhibit a marked TRAIL sensitivity. An analysis of the post-receptor events showed that TRAIL responsiveness correlates with a reduced expression of endogenous Akt. In fact, expression in MDR-U2OS cells of a constitutively active Akt strongly decreased their sensitivity to TRAIL. The identification of Akt as a key modulator of TRAIL responsiveness could help to design TRAIL-based combinations for treatment of osteosarcoma. Moreover, the discovery that multidrug resistant osteosarcomas are highly sensitive to TRAIL-induced apoptosis indicates TRAIL as a new candidate for the treatment of multidrug resistant bone malignancies.     

<Emphasis Type="Italic">In vitro</Emphasis> effects of topotecan and ionizing radiation on TRAIL/Apo2L-mediated apoptosis in malignant glioma

The survival of patients with malignant gliomas is still unsatisfactory despite multimodality treatment, therefore new therapeutic strategies are required. Tumor necrosis factor apoptosis related ligand (TRAIL/Apo2L), a member of the tumor necrosis factor superfamily, may induce apoptotic cell death in several tumors, but not in normal cells, upon binding with specific receptors. In the present study, the expression and function of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) has been investigated in five human glioma cell lines (U87, U138, U373, A172, SW1783) in ex vivo tumors and in primary cultures obtained from the tumors. Our data show that gliomas preferentially express TRAIL R2 and that treatment with topotecan, a topoisomerase I inhibitor, significantly up-regulates its expression as detected by flow cytometry and western blotting. Moreover, in most cases, treatment with topotecan resulted in an increased sensitivity to TRAIL-dependent apoptosis, although cyclohexymide had to be added to induce apoptosis. On glioma cell lines, the effects of irradiation on TRAIL receptors were also analysed. In our experimental conditions, irradiation with 2Gy had a modest additive effect on TRAIL-dependent apoptosis and was not able to modulate TRAIL receptor expression.     

TRAIL as a target in anti-cancer therapy

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can initiate apoptosis through the activation of their death receptors. The ability of TRAIL to selectively induce apoptosis of transformed or tumor cells but not normal cells promotes the development of TRAIL-based cancer therapy. Accumulating preclinical studies demonstrate that the TRAIL ligand can effectively induce cancer cell apoptosis. Completed and ongoing Phases I and II clinical trials using TRAIL are showing clinically promising outcomes without significant toxicity. Importantly, TRAIL, DR4 and DR5 can all be induced by chemotherapeutics and/or radiation, which can sensitize cancer cells to TRAIL. Thus, understanding the regulation of the TRAIL apoptosis pathway can help develop more selective TRAIL-based agents for the treatment of human cancer.     

Modulation of death receptors by cancer therapeutic agents

Death receptors are important modulators of the extrinsic apoptotic pathway. Activating certain death receptors such as death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (i.e., DR4 and DR5) selectively kills cancer cells via induction of apoptosis while sparing normal cells. Thus, soluble recombinant TRAIL and agonistic antibodies to DR4 or DR5 have progressed to phase I and phase II clinical trials. Many cancer therapeutic drugs including chemotherapeutic agents have been shown to induce the expression or redistribution at the cell surface of death receptors including TRAIL death receptors. In addition, chemotherapeutic agents have also been shown to enhance induction of apoptosis by TRAIL or agonistic antibodies or overcome cell resistance to TRAIL or agonistic antibodies. Targeted induction of apoptosis by activation of the death receptor-mediated extrinsic apoptotic pathway should be an ideal therapeutic strategy to eliminate cancer cells. Therefore, death receptors, particularly TRAIL death receptors, have emerged as an important cancer therapeutic target. This article will focus on reviewing and discussing the modulation of death receptors by cancer therapeutic agents and its implications in cancer therapy.     

Androgens regulate TRAIL-induced cell death in prostate cancer cells via multiple mechanisms

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent for prostate cancer because it selectively induces apoptosis in cancer cells but not in normal cells. Previous reports have suggested that androgens regulate TRAIL-induced apoptosis in prostate cancer cells. However, there are discrepancies between these reports of how androgens affect TRAIL-induced cell death. To clarify the role of androgens on TRAIL-induced apoptosis in prostate cancer cells, we investigated the effects of androgen on TRAIL-induced cell death in a dose–response manner. Our results showed that although androgens sensitize LNCaP cells to TRAIL-induced apoptosis, this effect is dose-dependent and biphasic. We found that low levels of androgen are superior to high levels of androgen in term of sensitizing LNCaP cells to TRAIL. We also found that upregulation of DR5 (TRAIL-R2) expression by androgens is critical for sensitizing LNCaP cells to TRAIL. However, low levels of androgen are sufficient to induce DR5 expression and sensitize LNCaP cells to TRAIL-induced cell death. High levels of androgen alter the TRADD/RIP1 ratio, which may contribute to NF-κB activation and sequentially inhibit TRAIL-induced apoptosis.     

A mechanism of resistance to TRAIL/Apo2L-induced apoptosis of newly established glioma cell line and sensitisation to TRAIL by genotoxic agents

Most tumour cells are sensitive to TRAIL-induced apoptosis, but not normal cells; thus, cancer therapy using TRAIL is expected clinically. Several tumour cells are resistant to TRAIL-induced apoptosis, and various mechanisms of such resistance were reported in individual cases. In this study, we established a TRAIL-resistant glioma cell line, which completely lacked TRAIL receptors. In addition, this tumour cell line had wild-type p53 tumour-suppressive gene, suggesting new mechanisms for tumour cells to expand and escape from immune surveillance. The present study further explored the mechanisms that determine the sensitivity to TRAIL. We show that genotoxic agents such as cisplatin, doxorubicin and camptothecin, in addition to UV radiation, can induce TRAIL-R2 on the cell surface of TRAIL receptor-negative tumour cells. Newly synthesised TRAIL-R2 is functional, so apoptosis is effectively induced by TRAIL, but it is significantly inhibited by constitutive expression of dominant-negative p53. In addition, apoptosis induced by pretreatment of genotoxic agents and additional stimulation of TRAIL is efficiently inhibited by either antagonistic anti-TRAIL-R2 antibody or pan-caspase inhibitor z-VAD-FMK. Taken together, these findings suggest that resistance to TRAIL by lack of TRAIL receptors on glioma is restored by genotoxic agents, which support the new strategies for tumour killing by TRAIL-bearing cytotoxic cells in combination with genotoxic treatment.     

TRAIL receptor-selective mutants signal to apoptosis via TRAIL-R1 in primary lymphoid malignancies

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its agonistic antibodies, which are currently in early clinical trials for treating various malignancies, induce apoptosis through triggering of either TRAIL-R1 or TRAIL-R2. Based on studies using agonistic monoclonal antibodies, we recently proposed that primary chronic lymphocytic leukemic cells seem to signal apoptosis primarily through TRAIL-R1. We have now synthesized mutant forms of TRAIL specific for TRAIL-R1 or TRAIL-R2. The selectivity of these mutants to induce apoptosis in cell lines is due to selective binding to their cognate receptors resulting in apoptosis via formation of a death-inducing signaling complex. Using these mutants, we now unequivocally show that primary cells from patients with chronic lymphocytic leukemia and mantle cell lymphoma signal to apoptosis almost exclusively through TRAIL-R1. Thus, no significant therapeutic benefit can be anticipated from treating such patients with agents currently in clinical trials that signal predominantly through TRAIL-R2, such as HGS-ETR2 or Apo2L/TRAIL. Our study highlights the necessity to determine whether primary cells from a particular tumor signal via TRAIL-R1 or TRAIL-R2. Such information will provide a rational approach to optimize TRAIL therapy.     

On the TRAIL of a new therapy for leukemia.

The cytokine TRAIL (tumor necrosis factor alpha-related apoptosis-inducing ligand) as well as agonistic antibodies that bind to the TRAIL receptors, death receptor 4 (DR4) and DR5, are undergoing preclinical and early clinical evaluation as potential therapeutic agents for a variety of hematological and nonhematological malignancies. Here, we briefly review the normal biological function of TRAIL, the mechanism of cytotoxicity of TRAIL receptor ligands, and their effects on normal myeloid progenitors, myelodysplastic marrow and leukemic cells, including acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL), in vitro. Recent observations suggesting that DR4 is the predominant receptor for the cytotoxic effects of TRAIL in CLL and that histone deacetylase inhibitors synergize with TRAIL in CLL in vitro are described and discussed. Collectively, the reviewed studies not only illustrate the potential therapeutic usefulness of TRAIL and the agonistic antibodies, but also highlight the need for additional preclinical evaluation of these agents.     

Histone deacetylase inhibitors enhance lexatumumab-induced apoptosis via a p21Cip1-dependent decrease in survivin levels

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in malignant cells by binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Several agents that therapeutically exploit this phenomenon are being developed. We investigated the anticancer activity of two novel, highly specific agonistic monoclonal antibodies to TRAIL-R1 (mapatumumab, HGS-ETR1) and TRAIL-R2 (lexatumumab, HGS-ETR2) in colon cancer cell lines. Our analyses revealed that colon cancer cells display significantly higher surface expressions of TRAIL-R2 than TRAIL-R1, and are more sensitive to lexatumumab-induced apoptosis. The proapoptotic effects of lexatumumab in TRAIL-resistant HCT8 and HT29 cells were dramatically augmented by the histone deacetylase inhibitors trichostatin A or suberoylanilide hydroxamic acid. The presence of p21, but not p53, was critical for the synergy between lexatumumab and histone deacetylase inhibitors. The absence of p21 did not interfere with the formation of the death-inducing signaling complex by lexatumumab, suggesting the involvement of other apoptotic and/or cell cycle regulators. Indeed, treatment with suberoylanilide hydroxamic acid greatly reduced the expression of the inhibitor of apoptosis protein survivin and cdc2 activity in HCT116 p21(+/+) cells but not in the HCT116 p21(-/-) cells. Inhibition of cdc2 activity with flavopiridol decreased survivin expression and sensitized the p21-deficient cells to lexatumumab-induced apoptosis. Similarly, small interfering RNA-mediated knockdown of survivin also enhanced lexatumumab-mediated cell death. Therefore, survivin expression plays a key role in lexatumumab resistance, and reducing survivin expression by inhibiting cdc2 activity is a promising strategy to enhance the anticancer activity of lexatumumab.     

Mutation analysis and mRNA expression of trail-receptors in human breast cancer

The chromosome region 8p12-p22 shows frequent allelic loss in a variety of human malignancies, including breast cancer (BC). The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptors TRAIL-R1, -R2, -R3 and -R4 are located on 8p21-p22 and might be candidate tumor suppressor genes in this region. To evaluate the involvement of TRAIL receptors in breast carcinogenesis, we have analyzed the entire coding region of TRAIL-R2 and the death domain (DD) regions of TRAIL-R1 and -R4 for the detection of somatic mutations in a series of breast tumors, lymph node metastases and BC cell lines. Overall, we detected 1, 11 and 3 alterations in the TRAIL-R1, -R2 and -R4 genes, respectively. Although functional studies have not yet been performed, we assume that most of these alterations do not alter the function of TRAIL-receptors. Additionally, we analyzed individuals from BC families for the detection of TRAIL-R2 germline mutations. One alteration has been found in the Kozak consensus motif at position -4 with respect to the translation initiation AUG [1-4 (C-->A)]. We further studied the mRNA expression of TRAIL and the 4 TRAIL receptors. In BC cell lines, a strongly decreased mRNA expression of TRAIL, TRAIL-R1, -R3 and -R4 was found, whereas the expression of TRAIL-R2 was only slightly reduced. In breast tumors, a 1.2-3.6-fold reduction of mRNA signals of the 5 genes was observed. No correlation was found between the expression level of TRAIL and the receptor mRNAs and clinicopathologic variables and between the expression of TRAIL-R2 and TP53 mutation status and loss of heterozygosity (LOH) at 8p21-p22. Taken together, we cannot exclude the involvement of TRAIL-receptors in BC. Our mutation studies indicate that DD receptor mutations occur at low frequency and are not the primary cause for the altered mRNA expression of TRAIL and TRAIL-receptors in BC.     

High TRAIL-R3 expression on leukemic blasts is associated with poor outcome and induces apoptosis-resistance which can be overcome by targeting TRAIL-R2

Activation of the TNF-related apoptosis-inducing ligand (TRAIL) pathway can induce apoptosis in a broad range of human cancer cells. Four membrane-bound receptors have been identified. TRAIL-R1 and TRAIL-R2 contain a functional death domain; TRAIL-R3 and TRAIL-R4 lack a functional death domain and function as decoy receptors. Flow-cytometric analysis revealed that acute myeloid leukemic (AML) blasts expressed significantly more pro-apoptotic receptors compared to normal blasts. However, about 20% of AML patients highly expressed decoy receptor TRAIL-R3, which was strongly correlated to a shortened overall survival. TRAIL-R3 expression was also high on CD34+/CD38− cells, the compartment that harbors the leukemia initiating stem cell. Expression levels of pro-apoptotic TRAIL receptors were not correlated to the susceptibility for soluble TRAIL, which was generally low (mean level of cell death induction 14%). Cell death could be enhanced by down-modulation of TRAIL-R3, confirming its decoy function on AML blasts. Bypassing of TRAIL-R3 by treatment with antibodies directly targeting TRAIL-R2 resulted in higher rates of induced cell death (max. 80%).In conclusion, AML blasts do express pro-apoptotic TRAIL receptors. However, co-expression of decoy receptor TRAIL-R3 results in significant shortened overall survival. AML blasts could be targeted by anti-TRAIL-R2 antibodies, yielding a new therapeutic option for AML patients.     

Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert potent cytotoxic activity against many tumor cell lines but not against normal cells. It has been hypothesized that this difference in TRAIL sensitivity between normal and transformed cells might be due to the expression of the non-death-inducing TRAIL receptors (TRAIL-R) TRAIL-R3 and TRAIL-R4, presumably by competition for limited amounts of TRAIL. To assess the regulation of resistance versus sensitivity to TRAIL in primary as well as transformed keratinocytes, we examined TRAIL sensitivity, TRAIL receptor expression, and intracellular signaling events induced by TRAIL. Although TRAIL induced apoptosis in primary as well as transformed keratinocytes, a marked difference in sensitivity could be observed with primary keratinocytes (PK) being 5-fold less sensitive to TRAIL than transformed keratinocytes (TK). Yet both cell types exhibited similar TRAIL receptor surface expression, suggesting that expression of TRAIL-R3 and TRAIL-R4 may not be the main regulator of sensitivity to TRAIL. Biochemical analysis of the signaling events induced by TRAIL revealed that PK could be sensitized for TRAIL and, similarly, for TRAIL-R1- and TRAIL-R2-specific apoptosis by pretreatment of the cells with cycloheximide (CHX). This sensitization concomitantly resulted in processing of caspase-8, which did not occur in TRAIL-resistant PK. These data indicate that an early block of TRAIL-induced apoptosis was present in PK compared with TK or PK treated with CHX. Interestingly, cellular FLICE inhibitory protein (cFLIP) levels, high in PK and low in TK and several other squamous cell carcinoma cell lines, decreased rapidly after treatment of PK with CHX, correlating with the increase in TRAIL sensitivity and caspase-8 processing. Furthermore, ectopic expression of cFLIP long (cFLIP(L)) in TK by transfection with a cFLIP(L) expression vector resulted in resistance to TRAIL-mediated apoptosis of these cells. Thus, our results demonstrate that TRAIL sensitivity in PK is primarily regulated at the intracellular level rather than at the receptor level.     

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.

Both DR4 and DR5 have recently been identified as membrane death receptors that are activated by their ligand TRAIL to engage the intracellular apoptotic machinery. TRID (also named as TRAIL-R3) is an antagonist decoy receptor and lacks the cytoplasmic death domain. TRID protects from TRAIL-induced apoptosis by competing with DR4 and DR5 for binding to TRAIL. TRID has been shown to be overexpressed in normal human tissues but not in malignantly transformed cell lines. DR5 is a p53-regulated gene and we have recently reported that DR5 expression is induced in response to genotoxic stress in both a p53-dependent and independent manner (Sheikh et al., 1998). In the current study, we demonstrate that TRID gene expression is also induced by the genotoxic agents ionizing radiation and methyl methanesulfonate (MMS) in predominantly p53 wild-type cells, whereas UV-irradiation does not induce TRID gene expression. Consistent with these results, exogenous wild-type p53 also upregulates the expression of endogenous TRID in p53-null cells. Thus, TRID appears to be a p53 target gene that is regulated by genotoxic stress in a p53-dependent manner. Using primary gastrointestinal tract (GIT) tumors and their matching normal tissue, we also demonstrate for the first time that TRID expression is enhanced in primary tumors of the GIT. It is, therefore, possible that TRID overexpressing GIT tumors may gain a selective growth advantage by escaping from TRAIL-induced apoptosis.     

Expression of the TRAIL receptors in blood mononuclear cells in leukemia

TRAIL receptors are differentially expressed on restricted subpopulations of normal blood cells. In the present study, we investigated the utility of individual TRAIL receptors in evaluating the presence of circulating tumor cells in blood. Patients with chronic myeloid leukemia (CML) carrying the t(9;22) translocation were compared with patients in whom no translocation was detected, with patients with multiple myeloma and with a group of healthy individuals. TRAIL receptor expression was analyzed by RT-PCR in blood mononuclear cells. Blood mononuclear cells of healthy subjects expressed the TRAIL-R1 and TRAIL-R2 death receptors and the TRAIL-R4 decoy receptor while the other decoy receptor TRAIL-R3 was not detectable. This normal expression pattern was also observed in all cases with multiple myeloma and in almost all patients without translocation (42/43; 97.7%). However, in 24/56 (42.9%) of the translocation-positive patients, the expression pattern was completely different. In this group the TRAIL-R4 receptor alone or in combination with TRAIL-R1 disappeared from blood mononuclear cells, while the TRAIL-R2 was expressed at normal level, indicating that the loss of expression is specific for the TRAIL-R4 and TRAIL-R1. This expression pattern was also confirmed by real-time PCR. The differences between the translocation-positive and -negative groups for the TRAIL-R4 and TRAIL-R1 expression were highly significant (p=0.0001 and p=0.0004, respectively). However, the differential expression pattern did not correlate with the number of leukemic cells. Our results suggest a correlation between the presence of leukemic cells in circulation and the differential expression pattern of TRAIL receptors in blood mononuclear cells.