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Address correspondence to Stephan Mathas, Max-Delbrück-Center for Molecular Medicine, FG Dörken, D-13125 Berlin, Germany. Phone: 49-30-94062720; Fax: 49-30-94063124; email: mathas{at}rrk-berlin.de
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B. Despite expression of other NF-B–dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.
Key Words: lymphoma • CD95 antigen • TRAIL protein • NF-B • siRNA
The online version of this article contains supplemental material.
Abbreviations used in this paper: ALPS, autoimmune lymphoproliferative syndrome; AP-1, activator protein 1; C-8, caspase-8; C-10, caspase-10; c-FLIP, cellular FLICE-inhibitory protein; c-FLIPL, c-FLIP long; c-FLIPS, c-FLIP short; cHL, classical Hodgkin's lymphoma; CHX, cycloheximide; DISC, death-inducing signaling complex; FADD, Fas-associated death domain–containing protein; FLIP, FLICE-inhibitory protein; GC, germinal center; GFP, green fluorescent protein; HRS, Hodgkin/Reed-Sternberg; NF, nuclear factor; rhTRAIL, recombinant human TNF-related apoptosis-inducing ligand; siFLIP, c-FLIPS/L–specific small interfering RNA oligoribonucleotide; siRNA, small interfering RNA oligoribonucleotide; TRAIL, TNF-related apoptosis-inducing ligand.
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The death receptor system is an essential component in the regulation of homeostasis of the lymphoid system (7–10). It is assumed that the negative selection process of B as well as T cells in the germinal center (GC) and thymus, respectively, depends on the CD95 system. Several lines of evidence indicate the importance of this system for the balance between B cell proliferation and apoptosis. (a) Mice lacking functional CD95 expression suffer from lymphadenopathy, autoimmunity, and higher incidence of B cell lymphomas (11, 12). (b) Purified GC B cells rapidly undergo apoptotic cell death due to CD95 activation (13) and the CD95 system has been reported to drive negative selection of B cells in vivo (7). (c) Patients with autoimmune lymphoproliferative syndrome (ALPS) carry germline mutations of proteins involved in lymphocyte apoptosis (14, 15). These mutations have the impairment of CD95 function in common, which predisposes these patients to autoimmune disorders and lymphoma development. (d) Clonal deleterious somatic mutations of the CD95 gene were identified in lymphomas, in particular those deriving from GC B cells, suggesting a role in lymphomagenesis (for review see reference 16).
Classical Hodgkin's lymphoma (cHL), a common human lymphoma, has been proposed to be derived from GC B cells in the majority of cases (17). Currently, the molecular pathogenesis of cHL remains unclear. Our group identified the transcription factors nuclear factor (NF)-B (for review see reference 18) and activator protein 1 (AP-1; for review see reference 19) as important regulators of Hodgkin/Reed-Sternberg (HRS) cell biology (20–22). Most of the known proteins aberrantly expressed in HRS cells were shown to depend on NF-B, some of them being coregulated by NF-B and AP-1 (21–23). NF-B and AP-1 support proliferation of HRS cells, and NF-B is a key survival factor for these cells. Recently described genomic alterations of the NF-B system in the malignant HRS cells support the idea that at least NF-B plays a central role in the pathogenesis of cHL (17, 24).
Two characteristics of cHL are unique among human lymphomas. First, among tumor-forming cells the malignant HRS cells are rare. Usually they represent <1% of cells in affected lymph nodes. They are surrounded by benign cells, which consist, among others, of an abundant number of T cells. Second, HRS cells have lost their B cell phenotype, including Ig expression (25). In accordance with the current concept of B cell ontogenesis, in contrast to HRS cells, cells with nonfunctional Ig expression usually die from apoptosis. These characteristics of cHL raise two questions. How do HRS cells evade the control of the immune system, especially the cytotoxic T cell response? And, how do they survive despite the absence of Ig expression? A common answer for both questions might be the evasion from death receptor–induced apoptosis. In particular, the CD95 system is involved in both processes, the CTL-mediated target cell lysis including the control of tumor growth (26–28) as well as the negative selection in the GC (7, 9, 13). Indeed, despite expression of WT CD95 in most cases of cHL, HRS cells do not undergo apoptotic cell death after CD95 stimulation (29, 30). Although c-FLIP expression (29, 31) and alteration of C-8 expression (32) were described in HRS cells, the functional significance of these data is unclear.
The experiments described in this work were performed to functionally analyze the CD95 system in HRS cells. We report that in contrast to many other tumor entities, the CD95 system on HRS cells is rather up-regulated than down-regulated. This is evident from CD95 overexpression in HRS cell lines and from the immunohistochemical analysis of CD95, FADD, and c-FLIP on 59 cases of cHL. Upon CD95 stimulation, a strong DISC formation is observed in HRS cell lines. c-FLIP proteins, the expression of which strictly depends on NF-B, are rapidly incorporated into the DISC. Most importantly, although several other NF-B–dependent antiapoptotic proteins are up-regulated in HRS cells, the specific down-regulation of c-FLIP proteins by small interfering RNA oligoribonucleotides (siRNAs) is sufficient to render HRS cell lines sensitive to CD95 and TRAIL stimulation.
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Western Blotting.
The preparation of whole cell extracts and SDS-PAGE has been described (21). For Western blot analysis the following primary antibodies were used: monoclonal anti–c-FLIPS/L (Dave-2; Qbiogene and G-11; Santa Cruz Biotechnology, Inc.), polyclonal anti-IB
(C-21), polyclonal anti-CD95 (C-20), polyclonal anti-p65 (A; all from Santa Cruz Biotechnology, Inc.), monoclonal anti-FADD (Clone-1 and Clone A66-2; BD Biosciences), monoclonal anti–tubulin (MCA78A; Serotec), monoclonal anti–C-8 (C-15; provided by P. Krammer), and polyclonal anti–Bcl-xL (Transduction Laboratories). Filters were incubated with horseradish peroxidase–conjugated secondary antibodies. Bands were visualized using the enhanced chemiluminescence system (Amersham Biosciences).
Analysis of the CD95 DISC by Immunoprecipitation.
DISC immunoprecipitation was essentially performed as described by Scaffidi et al. (34). In brief, cells were left untreated or treated with 2 µg/ml anti–APO-1 antibody (IgG3; provided by P. Krammer). 10 min after the addition of the antibody, unbound anti–APO-1 antibody was removed by washing the cells once with ice-cold PBS. Cells were incubated in lysis buffer (30 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% [vol/vol] glycerol, and a protease inhibitor cocktail; Complete, Mini; Roche). As control, untreated cells were lysed and the lysate was supplemented with the anti–APO-1 antibody. 25 µl protein A–Sepharose beads were added to 700 µg of each lysate. Immunoprecipitation was performed for 1.5 h while rotating at 4°C. Protein A–Sepharose beads were washed five times with ice-cold lysis buffer and boiled for 5 min in SDS loading buffer. The supernatant was separated by SDS-PAGE and analyzed by Western blot analysis using monoclonal anti–C-8 (C-15, IgG2b; provided by P. Krammer), monoclonal anti-FADD (IgG1; BD Biosciences), and monoclonal anti–c-FLIPS/L (IgG1; Santa Cruz Biotechnology, Inc.) as primary antibodies followed by incubation with isotype-specific, horseradish peroxidase–conjugated secondary antibodies (all from Southern Biotechnology Associates, Inc.).
Analysis of Apoptosis.
Cells were stained with 5 µg/ml acridine orange (Sigma-Aldrich) and analyzed by fluorescence microscopy. The number of cells with fragmented or condensed nuclei was counted and the percentages were determined. All assays were performed in triplicate. Alternatively, cells were stained with FITC-conjugated annexin V and propidium iodide (Bender MedSystemsTM; Biozol) according to the manufacturer's recommendations. Samples were analyzed using a FACSCaliburTM flow cytometer and CELLQuestTM software (Becton Dickinson).
Immunohistology.
All cases were drawn from the files of the Consultation and Reference Center for Haematopathology at the Institute of Pathology, Benjamin Franklin Clinic, Free University of Berlin. Final diagnosis had been established after extensive immunohistological analysis according to the criteria of the World Health Organization classification (35). Representative areas of the lymph node specimens containing diagnostic HRS cells were sampled out in the form of a cylinder and arrayed on one recipient paraffin block (generated by S. Joos, DKFZ, Heidelberg, Germany). The 59 available specimens originated from 28 cases of nodular sclerosing and 31 cases of mixed cellularity cHL subtypes. The specificity of the CD95 antibody C-20 (Santa Cruz Biotechnology, Inc.) as well as the CD95L antibody G247-4 (BD Biosciences) has been described (36, 37). Specificity of the FADD antibody A66-2 (BD Biosciences), the c-FLIP antibody G-11 (Santa Cruz Biotechnology, Inc.), and the C-8 antibody C-15 (provided by P. Krammer) was controlled as described below (see Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20031080/DC1). All antibodies required a heat-induced antigen-demasking procedure before their application. Specifically bound antibodies were demonstrated using a streptavidin alkaline phosphatase method and FastRed as chromogen. For confocal microscopy, representative cHL cases were stained with the anti-CD30 antibody Ber-H2 (DakoCytomation), c-FLIP G-11 antibody (Santa Cruz Biotechnology, Inc.), or the respective IgG1 isotype control (BD Biosciences). Specifically bound antibodies were detected using anti–mouse Cy3-conjugated secondary antibodies. Nuclei were stained with DAPI. Immunofluorescence microscopy was performed on an LSM510 inverted laser scanning microscope (Carl Zeiss MicroImaging, Inc.). Cy3 and DAPI signals were recorded with 
exc = 543 nm (HeNe), LP em = 560 nm and exc = 364 nm (Argon laser), and BP em = 390–465 nm wavelengths, respectively.
Online Supplemental Material.
In Fig. S1, total RNA was prepared using RNeasy kit (QIAGEN). 10 µg total RNA was subjected to gel electrophoresis and transferred to a nylon membrane (Appligene). The membrane was hybridized (ExpressHyb solution; CLONTECH Laboratories, Inc.) with [-32P]dCTP–labeled random prime–labeled DNA probes (c-FLIP, GAPDH). Fig. S2 shows specificity controls of antibodies used for immunohistochemistry. Specificity of the FADD antibodies Clone-1 and Clone A66-2 (both BD Biosciences) as well as NCL-FADD (Novocastra) was analyzed by the staining of paraffin-embedded Jurkat cells and a mutant thereof lacking FADD expression. Only Clone A66-2 recognized FADD under these conditions. The other two antibodies failed to detect any specific signal. The c-FLIP antibodies Dave-2 (Qbiogene), G-11 and C-19 (both from Santa Cruz Biotechnology, Inc.), and c-FLIPL (Sigma-Aldrich) were tested on paraffin-embedded HEK293 cells transfected with c-FLIPS or c-FLIPL expression plasmids. Only antibody G-11 specifically stained the c-FLIP–transfected cells. The specificity of the C-8 antibody C-15 was controlled by the staining of paraffin-embedded Jurkat cells and a mutant thereof lacking C-8. Figs. S1 and S2 are available at http://www.jem.org/cgi/content/full/jem.20031080/DC1.
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c-FLIPS and c-FLIPL Are Up-regulated in HRS Cells; c-FLIP Expression is NF-B Dependent.
Because of the high amount of c-FLIP proteins detectable in DISC immunoprecipitates of HRS cell lines, we analyzed mRNA and protein levels of c-FLIP proteins in HRS and nonHodgkin cell lines. mRNA overexpression, most evident for two splice variants presumably corresponding to c-FLIPS (4), was found in HRS compared with nonHodgkin cell lines by Northern blot analysis (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20031080/DC1). As suggested by these data, we observed c-FLIP protein overexpression in HRS cell lines (Fig. 2 A). Remarkably, the robust c-FLIPS expression was restricted to HRS cell lines.
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B up-regulates expression of c-FLIP, a key mediator of the inducible resistance to death receptor–induced apoptosis (42). Therefore, we investigated the contribution of constitutive NF-B activity in HRS cells (22) to c-FLIP expression. L428 cells, which lack endogenous expression of the NF-B inhibitory protein IB (23), and HDLM-2 HRS cells were infected with an adenovirus encoding for the NF-B superrepressor IB
N, a stabilized form of IB. As previously described (22, 23), 24 h after infection the NF-B activity decreased dramatically (not depicted). Concomitantly, we observed a strong reduction of c-FLIPS/L mRNA and protein expression levels (Fig. 2 B and not depicted). 24 h after infection, c-FLIP expression had disappeared nearly completely, which identified, compared with other known NF-B–dependent genes in HRS cells, c-FLIP as one of the most strongly NF-B–regulated genes.
Treatment of HRS Cells with CHX Rapidly Down-regulates c-FLIP and Allows Activation of C-8 and Induction of Apoptosis by CD95 Stimulation.
The rapid loss of c-FLIP expression after NF-B inhibition prompted us to investigate the stability of c-FLIP proteins in HRS cell lines. Treatment of L428, HDLM-2, and L1236 HRS cells with CHX revealed that c-FLIPS and c-FLIPL are indeed short-lived proteins. Thus, as soon as 30–60 min after CHX treatment a dramatic decrease of protein expression was detectable (Fig. 3 A and not depicted). In contrast, even after 420 min of CHS treatment, no decrease of C-8, FADD, Bcl-xL, or NF-B-p65 protein expression level was observed (Fig. 3 A and not depicted).
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Specific Down-regulation of c-FLIP by siRNA Sensitizes HRS Cell Lines to CD95- and TRAIL-induced Apoptosis.
To directly analyze the function of c-FLIP proteins in death receptor–induced apoptosis of HRS cells, we specifically down-regulated c-FLIP by siRNAs, which were recently shown to strongly down-regulate c-FLIP expression (33). Experiments were performed with the cell lines L428 and L540Cy, which were the only HRS cell lines sufficiently transfectable for functional assays. 24 h after transfection, cells were analyzed for c-FLIPS/L expression and incubated with the anti-CD95 antibody CH-11 or recombinant human TRAIL (rhTRAIL), respectively (Fig. 4). Transfection of both cell lines with the siFLIP rapidly down-regulated endogenous c-FLIP expression (Fig. 4, A and B, right).
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15–20% of siK3-transfected cells underwent apoptotic cell death. However, in siFLIP-transfected cells the percentage of apoptotic cells was at least doubled. CH-11 or rhTRAIL induced apoptosis in 40–50 and 30–40% of cells, respectively. To prove that the observed increase in apoptosis is indeed caused by c-FLIP down-regulation, we transfected L540Cy cells with a mutated c-FLIPS-cDNA, which cannot be recognized by siFLIP. Despite siRNA mediated down-regulation of endogenous c-FLIP, this construct should maintain a c-FLIP expression level sufficient to block apoptosis sensitization. Indeed, transfection of the cells with the mutated cDNA resulted in a c-FLIP expression level comparable to endogenous c-FLIP expression despite cotransfection of siFLIP (Fig. 4 C, right). In contrast to endogenous c-FLIP, the mutated c-FLIP mRNA is not degraded by siFLIP, which is reflected by the decrease of the endogenous WT but not the ectopically expressed c-FLIPS protein transcribed from the mutated cDNA. Importantly, c-FLIP reconstitution reverted to a large extent the siFLIP-mediated sensitization for apoptosis induction by the CD95 system (Fig. 4 C, left). In summary, down-regulation of endogenous c-FLIP expression sensitized L428 and L540Cy cells for CD95 or TRAIL-R–induced apoptosis. These data indicate that c-FLIP proteins play a key role in the protection of HRS cells from death receptor–induced apoptosis.
Immunohistochemical Analysis of CD95, FADD, C-8, c-FLIP, and CD95L in cHL.
To draw valid conclusions from our in vitro experiments, we analyzed expression of CD95, FADD, C-8, c-FLIP, and CD95L on 59 samples of patients with cHL arrayed on a single paraffin block by immunohistochemistry (Fig. 5). Controls for antibody specificity were performed as we have described (refer to Material and Methods; Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20031080/DC1). We included the analysis of c-FLIP in this work because the c-FLIPL antibody used in previous studies (29, 31) gave no specific signal in our experiments, neither in a Western blot nor by immunohistochemical analysis (not depicted). In HRS cells of all except one case we detected a strong expression of CD95 (Fig. 5, A and B). Because staining was performed with a COOH-terminal antibody, these data confirm that CD95 mutations leading to a loss of the death domain are rare events in cHL (29). FADD expression was found in all cases (Fig. 5, C and D) and some cases presented FADD overexpression in HRS compared with surrounding cells. This is surprising because ectopic FADD overexpression was reported to induce apoptosis in other cell types (43). C-8 was detected in HRS cells of all cases, even though in many cases (32 out of 59) only very weak staining was observed (Fig. 5, E and F). c-FLIP expression was analyzed by an antibody recognizing c-FLIPS and c-FLIPL (Fig. 5, G and H). As suggested by the cell line data, we observed in all except four cases a c-FLIP overexpression in HRS compared with surrounding cells, which is in accordance with previously published data (29, 31). Finally, we analyzed CD95L expression in cHL. Only one case presented a strong staining of CD95L in HRS cells (Fig. 5 K), whereas HRS cells of the other cases did not stain for CD95L (Fig. 5 I). However, in all except three cases we detected CD95L in plasma cells and endothelia surrounding the HRS cells (Fig. 5 I). Thus, CD95L is produced by cells in the microenvironment of HRS cells. Taken together, all components of the CD95 system are expressed in primary HRS cells and regarding CD95, FADD, and c-FLIP, this system is rather up-regulated than down-regulated.
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Several of our experiments indicated that the resistance of HRS cells to death receptor–induced apoptosis is not due to structural, but to functional inhibition of death receptor pathways by c-FLIP proteins. First, a DISC is formed in HRS cells (Fig. 1 B). FADD, C-8, C-10, and most importantly, c-FLIPS and c-FLIPL coimmunoprecipitated with CD95 after CD95 activation. An exception is the cell line L1236, in which we did not observe a clear DISC formation. L1236 cells might be type II cells, or a recently described CD95 mutation in this cell line (29) might inhibit DISC formation, as shown for CD95 mutations identified in patients with ALPS (14). Second, treatment of HRS cell lines with CHX, which leads to the loss of c-FLIP expression, allowed activation of C-8 and induction of apoptosis after death receptor stimulation (Fig. 3, B and C). Third, selective down-regulation of c-FLIP proteins by siRNAs sensitized HRS cell lines to death receptor–induced apoptosis, including CD95- and TRAIL-induced apoptosis (Fig. 4).
The proposed mechanisms of c-FLIPS– or c-FLIPL–mediated inhibition of C-8 activation differ (6). Although c-FLIPS is supposed to completely inhibit activation of procaspase-8 at the DISC, c-FLIPL allows generation of the C-8 p43/p41 intermediate cleavage products, but no processing to active C-8 p18/p10. In line with these results we detected procaspase-8 and C-8 p43/p41 in the DISC (Fig. 1 B), but no active C-8 was detectable in whole cell lysates after CD95 ligation (Fig. 3 B). Activation of C-8 after CHX pretreatment and CD95 stimulation (Fig. 3 B) is most likely explained by the loss of c-FLIP expression (Fig. 3 A). c-FLIPs are short-lived proteins in HRS cells (Fig. 2 A). These results are in contrast to previously published data (31). In our hands, lower concentrations of CHX (10 and 1 µg/ml), which were used by Thomas et al. (31), also induced a rapid loss of c-FLIP in HRS cell lines (unpublished data). The discrepancy between these results might be due to methodical differences. Due to the short half-life of c-FLIP, a strong transcriptional activation is required for maintenance of high expression level. Indeed, c-FLIPs strictly depend on constitutive NF-B activity in HRS cells (Fig. 2). This finding is supported by data that show NF-B–dependent c-FLIP regulation in other cell types (42). Recently, FLIP proteins were reported to activate NF-B itself (46). In our experiments we did not observe an alteration of the NF-B activity after specific c-FLIP down-regulation (unpublished data), suggesting that c-FLIP does not contribute to the NF-B activation in HRS cells.
Thus, activated NF-B in HRS cells not only triggers proliferation and is a key survival factor for these cells (20, 22, 23), but also prevents tumor cell death mediated by the microenvironment. This is suggested by our data regarding c-FLIP expression in HRS cells and the interaction of CD95 in HRS and CD95L on surrounding cells. This interpretation is further supported by recently described animal models, which show that c-FLIP overexpression protects tumor cells from the in vivo immune response (27, 28, 47). Furthermore, NF-B was reported to render cells resistant to CD95- and TRAIL-induced apoptosis (48, 49). Consequently, the NF-B–dependent c-FLIP expression in HRS cells offers an explanation for HRS cell immune escape. Similarly, c-FLIP might protect HRS cells against apoptotic stimuli regarding the GC B cell origin because there is evidence that c-FLIP prevents GC B cell apoptosis (9, 13).
Remarkably, down-regulation of c-FLIP alone is sufficient for sensitization of HRS cells to death receptor–induced apoptosis, although the cells express other NF-B–dependent, antiapoptotic proteins (22). That CD95- and TRAIL-mediated induction of apoptosis after specific c-FLIP down-regulation is not observed in the whole cell population (Fig. 4) is most likely explained by the fact that c-FLIP down-regulation is not complete and not similarly effective in all cells of the population due to different transfection efficiencies.
In contrast to data shown for other tumor entities, in which CD95 often became down-regulated (50–52), CD95 is strongly expressed in HRS cells (Figs. 1 and 5; references 40 and 41). Furthermore, staining of CD95L suggests that CD95 signaling is triggered on primary HRS cells (Fig. 5). However, in other studies different frequencies of CD95L expression in HRS and surrounding cells were reported (41, 53). Therefore, we performed confocal microscopy of c-FLIP in primary HRS cells with the aim to directly analyze a DISC formation (Fig. 6). Although analysis was only possible in a limited number of cases and c-FLIP might be associated with other death receptors than CD95, the membranous staining pattern of c-FLIP in HRS cells argues for an activation of the CD95 system. In addition, activation of the CD95 system in primary HRS cells might occur in a ligand-independent fashion, as described for GC B cells (13), although this is not observed in HRS cell lines (see above). If c-FLIP proteins block CD95-mediated signaling toward apoptosis, what are the consequences of the strong CD95 expression, together with FADD and c-FLIP, which might facilitate signaling by these receptors? Recent studies revealed several functions of CD95 signaling in the context of blocked apoptosis induction (for review see reference 54). These data are in accordance with the dual function of other TNFR family members concerning apoptosis or survival triggering (1). Thus, in CD95-resistant cells, CD95 induction can trigger proliferation and differentiation (55, 56). Most excitingly regarding the biology of HRS cells, CD95 signaling activates transcription factor NF-B (57), can induce transcriptional activation of different cytokines (58, 59) including IL-1 and IL-6, and down-regulates Ig expression in B cells, which is independent of apoptosis-inducing signaling events (60). However, the lack of a suitable in vivo model for cHL makes it difficult to further analyze a possible contribution of CD95 signaling to the pathogenesis of cHL. There might be two indications that CD95 signaling could contribute to the pathogenesis of cHL. First, despite long-term culture of HRS cell lines, they maintain an elevated CD95 expression level (Fig. 1). The strong CD95 expression also holds true for primary cases of cHL (Fig. 5). Second, patients with ALPS carry dominant negative CD95 mutations (14), which block apoptosis induction but might allow other CD95-associated signaling events. These patients are predisposed to lymphoma development and, intriguingly, with the highest incidence among these lymphomas they develop cHL (15).
Finally, the combined strong expression of different members of the TNFR family, including CD30, CD40, RANK (61–63), and CD95, is unique among human lymphomas. This suggests a common mechanism for their up-regulation and points to an important role in the pathogenesis of cHL. Given the strong activation of NF-B and AP-1 in HRS cells (21, 22), it will be important to clarify the contribution of the TNFR family to activation of these transcription factors that are central for the biology of HRS cells.
Our data show that resistance of HRS tumor cells to death receptor stimulation is due to functional inhibition mediated by a strong, NF-B–dependent up-regulation of c-FLIP proteins. c-FLIP mediates protection of HRS cells from CD95 and TRAIL death receptor stimuli, but also probably other TNFR family members. The manipulation of death receptor systems by pharmacological agents that down-regulate NF-B activity and subsequently c-FLIP expression, or the specific down-regulation of c-FLIP proteins, might render HRS cells susceptible to death receptor–induced apoptosis and thus offer new therapeutic strategies for cHL.
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Acknowledgments |
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BN and Michael Hinz (Berlin) for preparing mRNA and protein extracts after adenovirus infection of HRS cell lines. We thank Toralf Kaiser (Berlin) for cell sorting, Claus Scheidereit (Berlin), Andreas Krueger, and Rüdiger Arnold (Heidelberg) for helpful discussion, and Harald Wajant (Würzburg) for critical reading of the manuscript. This work was supported in part by a grant from the Berliner Krebsgesellschaft to S. Mathas and a grant from the National Genome Research Network (NGFN) to B. Dörken.
Submitted: 1 July 2003
Accepted: 2 March 2004
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