Abstract

We hypothesized that inhibition of the FAS-mediated apoptosis pathway by FLICE-like inhibitory protein (c-FLIP) may contribute to oncogenesis in ALK+ anaplastic large-cell lymphoma (ALCL). Treatment with increasing concentrations of CH-11 (CD95/FAS agonistic antibody) had no effect on cell viability of 2 ALK+ ALCL cell lines, Karpas 299 and SU-DHL1, each expressing high levels of c-FLIP. However, inhibition of endogenous c-FLIP expression by specific c-FLIP siRNA in Karpas 299 and SU-DHL1 cells treated with CH-11 resulted in FAS-mediated cell death associated with increased annexin V binding, apoptotic morphology, and cleavage of caspase-8. In 26 ALK+ ALCL tumors, assessed for expression of DISC-associated proteins, CD95/FAS and c-FLIP were commonly expressed, in 23 (92%) of 25 and 21 (91%) of 23 tumors, respectively. By contrast, CD95L/FASL was expressed in only 3 (12%) of 26 ALCL tumors, although it was strongly expressed by surrounding small reactive lymphocytes. Our findings suggest that overexpression of c-FLIP protects ALK+ ALCL cells from death-receptor-induced apoptosis and may contribute to ALCL pathogenesis. (Blood. 2006;107:2544-2547)

Introduction

Anaplastic large-cell lymphoma (ALCL)1  frequently carries the t(2;5)(p23;q35)2  or other 2p23 locus rearrangements3  that result in overexpression of anaplastic lymphoma kinase (ALK). The t(2;5), the most common abnormality, creates a novel fusion gene, npm-alk. Among other mechanisms, NPM-ALK is believed to mediate its oncogenic potential through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and STAT3 signaling pathways resulting in downstream antiapoptotic signals.4-8 

The extrinsic apoptotic pathway is initiated by death surface receptors, a subgroup of the tumor necrosis factor receptor (TNFR) superfamily, that have a characteristic intracytoplasmic domain, designated the death domain (DD). Upon ligation, CD95 (FAS), the best characterized death surface receptor, oligomerizes at the cell membrane and recruits the adapter protein FADD (FAS-associated death domain protein) through DD-DD association. FADD, in turn, recruits procaspase-8 by its death effector domain (DED), initiating the formation of the death-inducing signaling complex (DISC). In the DISC, procaspase-8 molecules are located in close proximity and are autocatalytically processed to active caspase-8. In “type I” cells, death receptors induce strong caspase-8 activation, directly activating effector caspases, including caspase-3, to ensure apoptosis. In “type II” cells, low amounts of caspase-8 are activated, depending on a mitochondrial amplification mechanism through the cleavage of BID.9,10 

Death-receptor-induced apoptosis is inhibited by c-FLIP, a DED-containing protein with a nonfunctional caspaselike catalytic domain with antiapoptotic function in the DISC.11,12  The death receptor system has been involved in normal and abnormal immune processes,13,14  as well as in Hodgkin15,16  and non-Hodgkin lymphomagenesis,17  and it is currently attracting interest as a possible therapeutic target.18,19 

Here we show that overexpression of c-FLIP in ALCL may confer resistance to apoptosis induced by cell surface signals.

Study design

Cell lines

Two ALK+ (Karpas 299, SU-DHL1)20  cell lines known to carry the t(2;5) were used. As controls, a CD30+ T-cell lymphoma cell line, Mac2a (a gift from Dr K. Elenitoba-Johnson, Salt Lake City, UT), a Hodgkin lymphoma cell line, L-1236 (purchased from DSMZ, Braunschweig, Germany), and a mantle cell lymphoma cell line, Mino (a gift from Dr R. Ford, Houston, TX) were also used. The cell lines were maintained according to methods reported.20  Karpas 299 and SU-DHL1 cells were treated with the PI3K inhibitor LY294002, and the JAK3 inhibitors, WHI-P131 and WHI-P154, at the indicated concentrations. Whole-cell lysates were prepared 24 or 48 hours following treatment. Karpas 299 and SU-DHL1 cells also were infected with the HA-tagged, constitutively active, myrAkt adenovirus21  at a multiplicity of infection (MOI) of 20, previously shown to significantly increase Ser473p-AKT levels in vitro (not shown).

Induction and detection of apoptosis

Each cell line (1 × 106 cells/mL) was incubated with increasing concentrations of the anti-CD95/FAS agonistic CH11 antibody (Beckman Coulter, Fullerton, CA) for 24 hours in 6-well plates, and a dose-response curve of cell viability was generated. A 200-ng/mL concentration of CH-11 produced approximately 60% and 50% cell death 24 hours after treatment in control Mac2a and Mino cells, respectively (“Results”). Cell viability was evaluated using trypan blue staining in triplicate. Apoptosis was assessed with annexin-V staining (BD Biosciences Pharmingen, San Diego, CA) detected by flow cytometry (FACS Calibur; Becton Dickinson, San Jose, CA) and DAPI staining detected by immunofluorescence.8 

Western blot analysis and immunofluorescence

Lysates prepared from cells in log-phase growth or after apoptosis induction were collected, washed twice in cold PBS, and lysed at 4°C in lysis buffer as previously described.20  Western blot analysis was performed as reported elsewhere.20  The primary antibodies used were as follows: CD95/FAS (sc-715, polyclonal), CD95L/FASL (N-20, polyclonal), and c-FLIP (H-202, polyclonal) (Santa Cruz Biotechnology, Santa Cruz, CA); procaspase-8 (Ab-3, monoclonal) and cleaved caspase-8 (Asp384, 11G10, monoclonal) (Oncogene, La Jolla, CA); and β-actin (Sigma, St Louis, MO).

Cleaved caspase-8 (dilution, 1:50; Oncogene) was also detected using an immunofluorescence method. Briefly, cytospin cell preparations were incubated with the primary antibody overnight. Detection was achieved by incubation with a fluorescent antimouse antibody (Alexa fluor 488; Molecular Probes, Eugene, OR) at a 1:200 dilution for 1 hour. 4′,6′-diamidino-2-phenylidole (DAPI) (Molecular Probes) was used as counterstain.

Inhibition of c-FLIP expression by siRNA

The sequences of c-FLIP small interference (si) RNA targeting the human c-FLIP gene product were purchased from Ambion (Austin, TX): GGGACCUUCUGGAUAUUUUtt (sense) and AAAAUAUCCAGAAGGUCCCtg (antisense). Transient transfection of Karpas 299 and SU-DHL1 cells was performed using the Nucleofector solution “T” (Amaxa Biosystems, Gaithersburg, MD). Approximately 2 × 106 cells were transfected with 0, 5, and 10 μg c-FLIP siRNA using 100 μL T solution (Amaxa Biosystems) according to the manufacturer's protocol. Cells were harvested at 48 hours following transfection and whole-cell lysates were prepared for Western blot analysis. In addition, Karpas 299 and SU-DHL1 cells (each 1 × 106 cells/mL) were preincubated with 10 μg c-FLIP siRNA for 24 hours in 6-well plates and subsequently with 200 ng/mL CH11 (Beckman Coulter). Control transfections with the siRNA of 2 housekeeping genes, GAPDH and 4EBP1, were also performed.

Tissue microarray and immunohistochemical methods

Twenty-six ALK+ ALCL tumors obtained prior to therapy at The University of Texas M. D. Anderson Cancer Center between 1984 and 2003 were analyzed using immunohistochemistry after antigen retrieval20  and tissue microarrays as previously described.22  The panel of antibodies used included CD95/FAS (C-20, polyclonal; dilution, 1:200), CD95L/FASL (N-20, polyclonal; dilution, 1:200), and c-FLIP (H-202, polyclonal; dilution, 1:50) (Santa Cruz Biotechnology). Lymph nodes with reactive follicular hyperplasia served as controls. A 10% cutoff was used to define positivity of all proteins studied.

Figure 1.

ALK+ALCL cells are resistant to CD95/FAS-induced cell death as a result of c-FLIP overexpression. (A) Using trypan blue exclusion assay, cell viability did not change after treatment of 2 ALK+ ALCL cell lines, Karpas 299 and SU-DHL1, with increasing concentrations of the CH-11 antibody, an inducer of the extrinsic apoptotic pathway. These experiments were performed in triplicate, and the mean percentage of viable cells was calculated. Flow cytometry also showed no change in annexin V binding after treatment of Karpas 299 and SU-DHL1 cells with increasing concentrations of CH-11 (data not shown). By contrast, cell viability of the control CD30+ T-cell lymphoma cell line, Mac2a, decreased significantly after treatment of cells with increasing concentrations of the CH-11 antibody. Approximately 60% death of Mac2a cells was seen at a concentration of 200 ng/mL CH-11. Similar results were obtained for a control B-cell line, Mino (not shown). (B) Karpas 299 cells were transiently transfected with 5 μg or 10 μg c-FLIP siRNA, and whole-cell lysates were prepared at 48 hours after transfection. Immunoblots showed that endogenous c-FLIP expression was completely inhibited when 10 μg siRNA was used. (C) Transient transfection of Karpas 299 cells with 10 μg siRNA resulted in increased annexin V binding (from 21% to 39%) as shown by flow cytometry. Control transient transfections of Karpas 299 and SU-DHL1 cells with 10 μg or 20 μg siRNA (Ambion) of 2 housekeeping genes, GAPDH and 4EBP1, were also performed using the Nucleofector system (Amaxa Biosystems). No changes in cell viability and only minimal changes in the fraction of annexin V-positive cells were observed (not shown). These experiments were performed in triplicate. (D) Karpas 299 cells were initially transfected with 10 μg c-FLIP siRNA and, at 24 hours after transfection, the cells were treated with 200 ng/mL CH-11. As shown here, inhibition of c-FLIP expression significantly sensitized Karpas 299 cells to FAS-mediated apoptosis induced by CH-11. Cell viability was assessed by trypan blue exclusion studies. Bar graphs show results from experiments performed in triplicate using SU-DHL1 cells. Similar results were obtained using Karpas 299 cells. (E) Annexin V/propidium iodide (PI) staining assessed by flow cytometry showed a substantial increase of annexin V-positive (from 10% to 45%) and PI-positive SU-DHL1 cells. (F) Evidence of apoptotic morphology (nuclear condensation and fragmentation) in SU-DHL1 cells stained with 4′,6′-diamidino-2-phenylidole (DAPI) (top panel). Membranous and cytoplasmic expression of cleaved caspase-8 detected using an immunofluorescence method in SU-DHL1 cells that were treated with CH-11 and c-FLIP siRNA (bottom panel). Similar findings were seen using Karpas 299 cells. Immunofluorescence: × 400 original magnification. Images were obtained with a BX51 Olympus microscope and a DP12 Olympus camera (Olympus, Melville, NY). An Olympus universal semi-apochromat UPlan FI objective lens (40 ×/0.75 NA) was used. (G) Immunoblots showed that CH-11-induced apoptotic cell death is associated with cleavage of caspase-8 in Karpas 299 (and SU-DHL1) cells treated with c-FLIP siRNA. Error bars in panels A and D indicate standard error.

Figure 1.

ALK+ALCL cells are resistant to CD95/FAS-induced cell death as a result of c-FLIP overexpression. (A) Using trypan blue exclusion assay, cell viability did not change after treatment of 2 ALK+ ALCL cell lines, Karpas 299 and SU-DHL1, with increasing concentrations of the CH-11 antibody, an inducer of the extrinsic apoptotic pathway. These experiments were performed in triplicate, and the mean percentage of viable cells was calculated. Flow cytometry also showed no change in annexin V binding after treatment of Karpas 299 and SU-DHL1 cells with increasing concentrations of CH-11 (data not shown). By contrast, cell viability of the control CD30+ T-cell lymphoma cell line, Mac2a, decreased significantly after treatment of cells with increasing concentrations of the CH-11 antibody. Approximately 60% death of Mac2a cells was seen at a concentration of 200 ng/mL CH-11. Similar results were obtained for a control B-cell line, Mino (not shown). (B) Karpas 299 cells were transiently transfected with 5 μg or 10 μg c-FLIP siRNA, and whole-cell lysates were prepared at 48 hours after transfection. Immunoblots showed that endogenous c-FLIP expression was completely inhibited when 10 μg siRNA was used. (C) Transient transfection of Karpas 299 cells with 10 μg siRNA resulted in increased annexin V binding (from 21% to 39%) as shown by flow cytometry. Control transient transfections of Karpas 299 and SU-DHL1 cells with 10 μg or 20 μg siRNA (Ambion) of 2 housekeeping genes, GAPDH and 4EBP1, were also performed using the Nucleofector system (Amaxa Biosystems). No changes in cell viability and only minimal changes in the fraction of annexin V-positive cells were observed (not shown). These experiments were performed in triplicate. (D) Karpas 299 cells were initially transfected with 10 μg c-FLIP siRNA and, at 24 hours after transfection, the cells were treated with 200 ng/mL CH-11. As shown here, inhibition of c-FLIP expression significantly sensitized Karpas 299 cells to FAS-mediated apoptosis induced by CH-11. Cell viability was assessed by trypan blue exclusion studies. Bar graphs show results from experiments performed in triplicate using SU-DHL1 cells. Similar results were obtained using Karpas 299 cells. (E) Annexin V/propidium iodide (PI) staining assessed by flow cytometry showed a substantial increase of annexin V-positive (from 10% to 45%) and PI-positive SU-DHL1 cells. (F) Evidence of apoptotic morphology (nuclear condensation and fragmentation) in SU-DHL1 cells stained with 4′,6′-diamidino-2-phenylidole (DAPI) (top panel). Membranous and cytoplasmic expression of cleaved caspase-8 detected using an immunofluorescence method in SU-DHL1 cells that were treated with CH-11 and c-FLIP siRNA (bottom panel). Similar findings were seen using Karpas 299 cells. Immunofluorescence: × 400 original magnification. Images were obtained with a BX51 Olympus microscope and a DP12 Olympus camera (Olympus, Melville, NY). An Olympus universal semi-apochromat UPlan FI objective lens (40 ×/0.75 NA) was used. (G) Immunoblots showed that CH-11-induced apoptotic cell death is associated with cleavage of caspase-8 in Karpas 299 (and SU-DHL1) cells treated with c-FLIP siRNA. Error bars in panels A and D indicate standard error.

Results and discussion

ALK+ ALCL cells are resistant to FAS-induced apoptosis

Induction of the extrinsic apoptotic pathway using an agonistic anti-CD95/FAS antibody demonstrated no cell death in both ALK+ ALCL cell lines (Figure 1A). In contrast, substantial apoptotic cell death was observed after treatment of Mac2a and Mino cells with increasing concentrations of CH-11. Apoptotic death of Mac2a cells after CH-11 treatment was associated with cleavage of caspase-8 and down-regulation of c-FLIP (Figure S1, available on the Blood website; see the Supplemental Figures link at the top of the online article).

These results are consistent with previously published data by Dirks et al23  who described resistance to CD95/FAS-induced apoptosis in ALK+ ALCL cells. They also assessed for FAS gene mutations as a possible underlying mechanism, but they did not identify mutations in 3 ALK+ ALCL cell lines, including Karpas 299. Another possibility would be inhibition of DISC function through overexpression of c-FLIP. In both ALK+ ALCL cell lines (Karpas 299, SU-DHL1), the long (c-FLIPL) and short (c-FLIPS) isoforms of c-FLIP were expressed (Figure S2). Of note, the short c-FLIP isoform, which has been shown to totally prevent caspase-8 cleavage,24  was expressed at a relatively higher level in ALK+ ALCL cells than in control cell lines.

Specific inhibition of c-FLIP expression by siRNA induces CD95/FAS-mediated apoptosis in ALK+ ALCL

We show here that a major inhibitor of DISC function, c-FLIP, appears to be responsible for resistance of ALK+ ALCL cells to death-receptor-induced apoptosis. Endogenous c-FLIP expression was completely inhibited when 10 μg siRNA was used (Figure 1B). This was associated with a slight increase in the number of apoptotic cells (Figure 1C-D) that may be due to the endogenous FASL expression (Figure S2). However, preincubation of ALK+

ALCL cells with c-FLIP siRNA and treatment with CH-11 significantly induced apoptosis, associated with apoptotic morphology and cleavage of caspase-8 (Figure 1E-F). Therefore, inhibition of c-FLIP expression using c-FLIP siRNA sensitized ALK+ ALCL cells to FAS-induced apoptosis. In line with our data, 2 recent reports demonstrated sensitization to CD95/FAS-mediated cell death in Hodgkin lymphoma cell lines using a specific c-FLIP siRNA.15,16  However, additional factors contributing to protection of ALK+ ALCL cells from extrinsic apoptotic signals cannot be excluded.

Expression of extrinsic apoptotic pathway proteins in ALK+ ALCL tumors

Using a 10% cutoff, CD95/FAS and c-FLIP were overexpressed in 23 (92%) of 25 and 21 (91%) of 23 tumors, respectively, (Figure 2A,C). By contrast, CD95L/FASL was expressed by the neoplastic cells in only 3 (12%) of 26 ALCL tumors (Figure 2B). However, FASL was always detected at high levels in the reactive small lymphocytes and endothelial cells, suggesting that the microenvironment of these tumors is capable of producing FASL. These results concur with previous findings reported by Sigel and Hsi.26  In a series of 10 ALCL cases (including 2 ALK+), CD95/FAS was expressed in more than 90% of tumor cells in all cases, whereas CD95/FASL was expressed only in a subset of tumor cells.

Figure 2.

c-FL/P overexpression in ALCL. (A-C) Immunohistochemical detection of extrinsic apoptotic pathway proteins in reactive lymph node and ALK+ anaplastic large-cell lymphoma (ALCL) tumors. (A) CD95/FAS was weakly expressed in a subset of centroblasts within the germinal centers (GCs) of reactive lymph nodes (left). Strong cytoplasmic and membranous CD95/FAS expression was observed in most ALK+ ALCL tumors (right). (B) CD95L/FASL was strongly positive in the mantle and marginal zones of reactive lymph nodes (left). CD95L/FASL was not expressed in most ALK+ ALCL tumors (right). Infiltrating small reactive lymphocytes strongly expressed CD95L/FASL and served as internal positive controls in all tumor specimens. (C) c-FLIP expression was restricted to a subset of GC cells (left). Strong cytoplasmic expression for c-FLIP was observed in most ALK+ ALCL tumors (right). (D-E) c-FLIP up-regulation may result from activation of PI3K/Akt and JAK3/ALK oncogenic pathways. Immunohistochemistry: × 200 original magnification (20 ×/0.75 NA objective; left panels) or × 400 (40 ×/0.75 NA objective; right panels). Images were obtained with a BX51 Olympus microscope, a DP12 Olympus camera, and a universal semi-apochromat UPlan FI lens. (D, left panel) Karpas 299 and SU-DHL1 cells were treated with a PI3K inhibitor, LY294002, at concentrations of 0, 5, or 20 μg/μL. Whole-cell lysates were then prepared at 48 hours. Immunoblots showed that serine 473 phosphorylation of Akt is almost completely inhibited at a concentration of 20 μg/μL that is associated with a substantial decrease of c-FLIP levels. (D, right panel) Karpas 299 and SU-DHL1 cells were infected with the constitutively active, myrAkt adenovirus21  at an MOI of 20, which significantly increases Ser473pAKT levels in these cells. Expression of myrAkt was confirmed by Western blot analysis using a monoclonal antibody specific for the hemagglutinin (HA) tag. An adeno-β-Gal adenovirus construct expressing β-Gal served as a control in this experiment. Whole-cell lysates were prepared from control and infected cells 48 hours after infection. Immunoblots showed a dramatic increase of Akt phosphorylation, which was associated with an increase of c-FLIPS splice variant. (E) ALK+ ALCL cells were treated with 2 known JAK3 inhibitors, WHI-P131 and WHI-P154, at concentrations previously shown to inhibit JAK3 and ALK enzymatic activity. 25 Whole-cell lysates were prepared 24 hours following treatment. Immunoblots demonstrate that c-FLIP levels are decreased after treatment with WHI-P131 or WHI-P154 in a dose-dependent manner.

Figure 2.

c-FL/P overexpression in ALCL. (A-C) Immunohistochemical detection of extrinsic apoptotic pathway proteins in reactive lymph node and ALK+ anaplastic large-cell lymphoma (ALCL) tumors. (A) CD95/FAS was weakly expressed in a subset of centroblasts within the germinal centers (GCs) of reactive lymph nodes (left). Strong cytoplasmic and membranous CD95/FAS expression was observed in most ALK+ ALCL tumors (right). (B) CD95L/FASL was strongly positive in the mantle and marginal zones of reactive lymph nodes (left). CD95L/FASL was not expressed in most ALK+ ALCL tumors (right). Infiltrating small reactive lymphocytes strongly expressed CD95L/FASL and served as internal positive controls in all tumor specimens. (C) c-FLIP expression was restricted to a subset of GC cells (left). Strong cytoplasmic expression for c-FLIP was observed in most ALK+ ALCL tumors (right). (D-E) c-FLIP up-regulation may result from activation of PI3K/Akt and JAK3/ALK oncogenic pathways. Immunohistochemistry: × 200 original magnification (20 ×/0.75 NA objective; left panels) or × 400 (40 ×/0.75 NA objective; right panels). Images were obtained with a BX51 Olympus microscope, a DP12 Olympus camera, and a universal semi-apochromat UPlan FI lens. (D, left panel) Karpas 299 and SU-DHL1 cells were treated with a PI3K inhibitor, LY294002, at concentrations of 0, 5, or 20 μg/μL. Whole-cell lysates were then prepared at 48 hours. Immunoblots showed that serine 473 phosphorylation of Akt is almost completely inhibited at a concentration of 20 μg/μL that is associated with a substantial decrease of c-FLIP levels. (D, right panel) Karpas 299 and SU-DHL1 cells were infected with the constitutively active, myrAkt adenovirus21  at an MOI of 20, which significantly increases Ser473pAKT levels in these cells. Expression of myrAkt was confirmed by Western blot analysis using a monoclonal antibody specific for the hemagglutinin (HA) tag. An adeno-β-Gal adenovirus construct expressing β-Gal served as a control in this experiment. Whole-cell lysates were prepared from control and infected cells 48 hours after infection. Immunoblots showed a dramatic increase of Akt phosphorylation, which was associated with an increase of c-FLIPS splice variant. (E) ALK+ ALCL cells were treated with 2 known JAK3 inhibitors, WHI-P131 and WHI-P154, at concentrations previously shown to inhibit JAK3 and ALK enzymatic activity. 25 Whole-cell lysates were prepared 24 hours following treatment. Immunoblots demonstrate that c-FLIP levels are decreased after treatment with WHI-P131 or WHI-P154 in a dose-dependent manner.

The mechanisms underlying c-FLIP up-regulation in ALK+ ALCL cells most likely are complex. Although, it has been suggested that c-FLIP up-regulation depends on NF-κB activation in Hodgkin lymphoma,15,16  recent evidence shows that NF-κB is not active in ALK+ ALCL.27  Therefore, NF-κB-independent mechanisms may be involved. One possibility is that c-FLIP is up-regulated by the PI3K/Akt pathway,28  as Akt can be phosphorylated/activated by NPM-ALK.4,5,29  Here, we show that inhibition of PI3K/Akt down-regulates c-FLIP, and, inversely, forced expression of constitutively active Akt up-regulates c-FLIPS splice variant (Figure 2D). Another possibility is that ALK or other kinases interacting with ALK, such as JAK3, up-regulate c-FLIP. Indeed, we have shown that inhibition of JAK3 kinase activity, which has been shown to inhibit ALK activity,25  also down-regulates c-FLIP (Figure 2E).

Taken together, our results suggest that ALK+ ALCL cells are resistant to CD95/FAS-induced cell death due to c-FLIP overexpression. Up-regulation of c-FLIP may be a downstream target of oncogenic pathways involved in ALCL pathogenesis, thus providing a novel therapeutic target in ALCL patients.

Prepublished online as Blood First Edition Paper, November 22, 2005; DOI 10.1182/blood-2005-06-2601.

M.P.O. is a recipient of a scholarship provided by The Pontificia Universidad Católica de Chile.

The online version of the article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

We thank Dr Francois-Xavier Claret for providing us with the myrAkt adenoviral vector.

1
Delsol G, Ralfkiaer E, Stein H, Wright D, Jaffe ES. Anaplastic large cell lymphoma. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds.
World Health Organization Classification of Tumours: Pathology and Genetics of Tumors of Haematopoietic and Lymphoid Tissues
. Lyon, France: IARC Press;
2001
:
230
-235.
2
Morris SW, Kirstein MN, Valentine MB, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.
Science.
1994
;
263
:
1281
-1284.
3
Drexler HG, Gignac SM, von Wasielewski R, Werner M, Dirks WG. Pathobiology of NPM-ALK and variant fusion genes in anaplastic large cell lymphoma and other lymphomas.
Leukemia.
2000
;
14
:
1533
-1559.
4
Bai RY, Ouyang T, Miething C, Morris SW, Peschel C, Duyster J. Nucleophosmin-anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway.
Blood.
2000
;
96
:
4319
-4327.
5
Slupianek A, Nieborowska-Skorska M, Hoser G, et al. Role of phosphatidylinositol 3-kinase-Akt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis.
Cancer Res.
2001
;
61
:
2194
-2199.
6
Zhang Q, Raghunath PN, Xue L, et al. Multilevel dysregulation of STAT3 activation in anaplastic lymphoma kinase-positive T/null-cell lymphoma.
J Immunol.
2002
;
168
:
466
-474.
7
Zamo A, Chiarle R, Piva R, et al. Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death.
Oncogene.
2002
;
21
:
1038
-1047.
8
Amin HM, McDonnell TJ, Ma Y, et al. Selective inhibition of STAT3 induces apoptosis and G(1) cell cycle arrest in ALK-positive anaplastic large cell lymphoma.
Oncogene.
2004
;
23
:
5426
-5434.
9
Scaffidi C, Schmitz I, Zha J, Korsmeyer SJ, Krammer PH, Peter ME. Differential modulation of apoptosis sensitivity in CD95 type I and type II cells.
J Biol Chem.
1999
;
274
:
22532
-22538.
10
Peter ME, Krammer PH. The CD95(APO-1/Fas) DISC and beyond.
Cell Death Differ.
2003
;
10
:
26
-35.
11
Tibbetts MD, Zheng L, Lenardo MJ. The death effector domain protein family: regulators of cellular homeostasis.
Nat Immunol.
2003
;
4
:
404
-409.
12
Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP.
Nature.
1997
;
388
:
190
-195.
13
Takahashi Y, Ohta H, Takemori T. Fas is required for clonal selection in germinal centers and the subsequent establishment of the memory B cell repertoire.
Immunity.
2001
;
14
:
181
-192.
14
Thome M, Tschopp J. Regulation of lymphocyte proliferation and death by FLIP.
Nat Rev Immunol.
2001
;
1
:
50
-58.
15
Mathas S, Lietz A, Anagnostopoulos I, et al. c-FLIP mediates resistance of Hodgkin/Reed-Sternberg cells to death receptor-induced apoptosis.
J Exp Med.
2004
;
199
:
1041
-1052.
16
Dutton A, O'Neil JD, Milner AE, et al. Expression of the cellular FLICE-inhibitory protein (c-FLIP) protects Hodgkin's lymphoma cells from autonomous Fas-mediated death.
Proc Natl Acad Sci U S A.
2004
;
101
:
6611
-6616.
17
Lajmanovich A, Irisarri M, Molens JP, et al. Impairment of death-inducing signalling complex formation in CD95-resistant human primary lymphoma B cells.
Br J Haematol.
2004
;
124
:
746
-753.
18
Suh WS, Kim YS, Schimmer AD, et al. Synthetic triterpenoids activate a pathway for apoptosis in AML cells involving downregulation of FLIP and sensitization to TRAIL.
Leukemia.
2003
;
17
:
2122
-2129.
19
Sayers TJ, Brooks AD, Koh CY, et al. The proteasome inhibitor PS-341 sensitizes neoplastic cells to TRAIL-mediated apoptosis by reducing levels of c-FLIP.
Blood.
2003
;
102
:
303
-310.
20
Rassidakis GZ, Claret FX, Lai R, et al. Expression of p27(Kip1) and c-Jun activation binding protein 1 are inversely correlated in systemic anaplastic large cell lymphoma.
Clin Cancer Res.
2003
;
9
:
1121
-1128.
21
Fujio Y, Guo K, Mano T, Mitsuuchi Y, Testa JR, Walsh K. Cell cycle withdrawal promotes myogenic induction of Akt, a positive modulator of myocyte survival.
Mol Cell Biol.
1999
;
19
:
5073
-5082.
22
Rassidakis GZ, Jones D, Thomaides A, et al. Apoptotic rate in peripheral T-cell lymphomas: a study using a tissue microarray with validation on full tissue sections.
Am J Clin Pathol.
2002
;
118
:
328
-334.
23
Dirks W, Schone S, Uphoff C, Quentmeier H, Pradella S, Drexler HG. Expression and function of CD95 (FAS/APO-1) in leukaemia-lymphoma tumour lines.
Br J Haematol.
1997
;
96
:
584
-593.
24
Krueger A, Schmitz I, Baumann S, Krammer PH, Kirchhoff S. Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex.
J Biol Chem.
2001
;
276
:
20633
-20640.
25
Amin HM, Medeiros LJ, Ma Y, et al. Inhibition of JAK3 induces apoptosis and decreases anaplastic lymphoma kinase activity in anaplastic large cell lymphoma.
Oncogene.
2003
;
22
:
5399
-5407.
26
Sigel JE, Hsi ED. Immunohistochemical analysis of CD30-positive lymphoproliferative disorders for expression of CD95 and CD95L.
Mod Pathol.
2000
;
13
:
446
-451.
27
Horie R, Watanabe M, Ishida T, et al. The NPM-ALK oncoprotein abrogates CD30 signaling and constitutive NF-kappaB activation in anaplastic large cell lymphoma.
Cancer Cell.
2004
;
5
:
353
-364.
28
Panka DJ, Mano T, Suhara T, Walsh K, Mier JW. Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells.
J Biol Chem.
2001
;
276
:
6893
-6896.
29
Rassidakis GZ, Feretzaki M, Atwell C, et al. Inhibition of Akt increases p27Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma.
Blood.
2005
;
105
:
827
-829.

Supplemental data