CD30 Activates Both the Canonical and Alternative NF-κB Pathways in Anaplastic Large Cell Lymphoma Cells
2007; Elsevier BV; Volume: 282; Issue: 14 Linguagem: Inglês
10.1074/jbc.m608817200
ISSN1083-351X
AutoresCasey W. Wright, Julie M. Rumble, Colin S. Duckett,
Tópico(s)T-cell and Retrovirus Studies
ResumoCD30 is a member of the tumor necrosis factor receptor superfamily whose expression is up-regulated on anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) cells. Many different outcomes of CD30 stimulation have been reported, including cell cycle arrest, apoptosis, and activation of the prosurvival transcription factor, NF-κB, although this last activity is much less well defined in ALCL cells. In order to better understand the signaling properties of CD30 in cancer, we established a system for the stimulation of CD30 with its physiological ligand. Using this system, CD30 was stimulated on ALCL and HL cells, and the subsequent CD30 signaling properties were characterized. We show that a fraction of ALCL cells rapidly underwent apoptosis following CD30 stimulation, whereas HL cells were unaffected. The surviving ALCL cells exhibited robust activation of both the canonical and alternative NF-κB pathways as measured by nuclear translocation of RelA, p50, RelB, and p52, and this culminated in the transactivation of classical NF-κB-responsive genes. With prolonged CD30 stimulation, ALCL cells underwent cell cycle arrest that correlated with expression of the cell cycle inhibitor p21waf1. Furthermore, p21waf1 expression and cell cycle arrest were found to depend predominantly on the canonical NF-κB pathway, since it was reversed by RNA interference-mediated suppression of RelA. In contrast, suppression of the p100/p52 NF-κB subunit had little effect on p21waf1. These data reveal that in ALCL cells, in contrast to other cell types, CD30 stimulation elicits p21waf1-mediated arrest through the canonical but not the alternative NF-κB pathway. CD30 is a member of the tumor necrosis factor receptor superfamily whose expression is up-regulated on anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) cells. Many different outcomes of CD30 stimulation have been reported, including cell cycle arrest, apoptosis, and activation of the prosurvival transcription factor, NF-κB, although this last activity is much less well defined in ALCL cells. In order to better understand the signaling properties of CD30 in cancer, we established a system for the stimulation of CD30 with its physiological ligand. Using this system, CD30 was stimulated on ALCL and HL cells, and the subsequent CD30 signaling properties were characterized. We show that a fraction of ALCL cells rapidly underwent apoptosis following CD30 stimulation, whereas HL cells were unaffected. The surviving ALCL cells exhibited robust activation of both the canonical and alternative NF-κB pathways as measured by nuclear translocation of RelA, p50, RelB, and p52, and this culminated in the transactivation of classical NF-κB-responsive genes. With prolonged CD30 stimulation, ALCL cells underwent cell cycle arrest that correlated with expression of the cell cycle inhibitor p21waf1. Furthermore, p21waf1 expression and cell cycle arrest were found to depend predominantly on the canonical NF-κB pathway, since it was reversed by RNA interference-mediated suppression of RelA. In contrast, suppression of the p100/p52 NF-κB subunit had little effect on p21waf1. These data reveal that in ALCL cells, in contrast to other cell types, CD30 stimulation elicits p21waf1-mediated arrest through the canonical but not the alternative NF-κB pathway. The tumor necrosis factor receptor superfamily member CD30 is highly expressed on the surface of many leukemia and lymphoma cells, including Hodgkin lymphoma (HL) 2The abbreviations used are: HL, Hodgkin lymphoma; ALCL, anaplastic large cell lymphoma; TRAF, TNF receptor-associated factor; NF-κB, nuclear factor κB; NPM, nucleophosmin; ALK, anaplastic lymphoma kinase; TNF, tumor necrosis factor; CHO, Chinese hamster ovary; EMSA, electrophoretic mobility shift assay; CDKI, cyclin-dependent kinase inhibitor; siRNA, short interfering RNA; PBS, phosphate-buffered saline; PI, propidium iodide; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol. (1Stein H. Mason D.Y. Gerdes J. O'Connor N. Wainscoat J. Pallesen G. Gatter K. Falini B. Delsol G. Lemke H. Schwarting R. Lennert K. Blood. 1985; 66: 848-858Crossref PubMed Google Scholar), anaplastic large cell lymphoma (ALCL), multiple myeloma, and adult T-cell leukemia (2Chiarle R. Podda A. Prolla G. Gong J. Thorbecke G.J. Inghirami G. Clin. Immunol. 1999; 90: 157-164Crossref PubMed Scopus (147) Google Scholar, 3Younes A. Carbone A. Int. J. Biol. Markers. 1999; 14: 135-143Crossref PubMed Scopus (46) Google Scholar, 4Younes A. Aggarwall B.B. Cancer. 2003; 98: 458-467Crossref PubMed Scopus (62) Google Scholar). The fact that CD30 is present normally on only a very small fraction of activated B- and T-cells makes it an attractive target for therapeutic intervention. The physiological role of CD30 is unclear, although it has been shown to contribute to negative selection of T-cells (5Amakawa R. Hakem A. Kundig T.M. Matsuyama T. Simard J.J. Timms E. Wakeham A. Mittruecker H.W. Griesser H. Takimoto H. Schmits R. Shahinian A. Ohashi P. Penninger J.M. Mak T.W. Cell. 1996; 84: 551-562Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 6Chiarle R. Podda A. Prolla G. Podack E.R. Thorbecke G.J. Inghirami G. J. Immunol. 1999; 163: 194-205PubMed Google Scholar, 7DeYoung A.L. Duramad O. Winoto A. J. Immunol. 2000; 165: 6170-6173Crossref PubMed Scopus (44) Google Scholar) and has been shown to serve as a co-stimulatory molecule on T-cells (8Del Prete G. De Carli M. D'Elios M.M. Daniel K.C. Almerigogna F. Alderson M. Smith C.A. Thomas E. Romagnani S. J. Exp. Med. 1995; 182: 1655-1661Crossref PubMed Scopus (167) Google Scholar, 9Croft M. Nat. Rev. Immunol. 2003; 3: 609-620Crossref PubMed Scopus (733) Google Scholar). The tumor necrosis factor receptor superfamily can be subdivided into two groups, defined by their signaling properties (10Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar). The group that includes CD30 (11Gedrich R.W. Gilfillan M.C. Duckett C.S. Van Dongen J.L. Thompson C.B. J. Biol. Chem. 1996; 271: 12852-12858Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 12Duckett C.S. Gedrich R.W. Gilfillan M.C. Thompson C.B. Mol. Cell Biol. 1997; 17: 1535-1542Crossref PubMed Google Scholar, 13Aizawa S. Nakano H. Ishida T. Horie R. Nagai M. Ito K. Yagita H. Okumura K. Inoue J. Watanabe T. J. Biol. Chem. 1997; 272: 2042-2045Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) signals through recruitment of one or more of the tumor necrosis factor (TNF) receptor-associated factors (TRAFs) directly to their cytoplasmic tails (14Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (932) Google Scholar, 15Cheng G. Cleary A.M. Ye Z.S. Hong D.I. Lederman S. Baltimore D. Science. 1995; 267: 1494-1498Crossref PubMed Scopus (442) Google Scholar). The other group signals through an ∼65-residue motif known as a death domain located in the cytoplasmic tail, which mediates proapoptotic signals through heterotypic interactions with components of the prodeath machinery. This second group also recruits adaptors via their cytoplasmic death domains that mediate interactions with the TRAFs (16Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar). In both groups of receptors, TRAF recruitment is thought to result in activation of the nuclear factor κB (NF-κB) family of transcription factors (17Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2580) Google Scholar). NF-κB transcription factors are important regulators of genes whose products are necessary for the innate and adaptive immune response and for the survival and proliferation of certain cell types (reviewed by Chen and Greene (18Chen L.F. Greene W.C. Nat. Rev. Mol. Cell Biol. 2004; 5: 392-401Crossref PubMed Scopus (1040) Google Scholar)). Five different proteins constitute the NF-κB family, which homodimerize and/or heterodimerize to form active transcription factors with different target gene specificity. The different NF-κB factors include RelA, RelB, c-Rel, and the precursor and processed products of the NFκB1 (p105/p50) and NFκB2 (p100/p52) genes. Aberrant regulation of the alternative NF-κB pathway subunit p100/p52 contributes to cancer, which has been linked to the cell cycle-regulatory function of the protein. Interestingly, a recent report demonstrated that p100/p52 is a regulator of the p21waf1 promoter in U-2 OS cells (19Schumm K. Rocha S. Caamano J. Perkins N.D. EMBO J. 2006; 25: 4820-4832Crossref PubMed Scopus (107) Google Scholar). A second characteristic of ALCL, besides high surface expression of CD30, is the expression of the oncoprotein NPM-ALK, which is the product of a chromosomal translocation t(2;5)(p23;q35) that fuses nucleophosmin (NPM) with anaplastic lymphoma kinase (ALK) (20Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1969) Google Scholar). NPM-ALK can associate with the cytoplasmic tail of CD30 (21Hubinger G. Scheffrahn I. Muller E. Bai R. Duyster J. Morris S.W. Schrezenmeier H. Bergmann L. Exp. Hematol. 1999; 27: 1796-1805Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), and a recent study reported that the presence of NPM-ALK in ALCL cells abrogates CD30-mediated NF-κB activation (22Horie R. Watanabe M. Ishida T. Koiwa T. Aizawa S. Itoh K. Higashihara M. Kadin M.E. Watanabe T. Cancer Cell. 2004; 5: 353-364Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Alternatively, other studies that have examined CD30 signaling observed effects ranging from apoptosis to cell cycle arrest and are suggestive of NF-κB activation in ALCL cells (23Smith C.A. Gruss H.J. Davis T. Anderson D. Farrah T. Baker E. Sutherland G.R. Brannan C.I. Copeland N.G. Jenkins N.A. Grabstein K.H. Gliniak B. McAlister I.B. Fanslow W. Alderson M. Falk B. Gimpel S. Gillis S. Din W.S. Goodwin R.G. Armitage R.J. Cell. 1993; 73: 1349-1360Abstract Full Text PDF PubMed Scopus (513) Google Scholar, 24Mir S.S. Richter B.W. Duckett C.S. Blood. 2000; 96: 4307-4312Crossref PubMed Google Scholar, 25Levi E. Pfeifer W.M. Kadin M.E. Blood. 2001; 98: 1630-1632Crossref PubMed Scopus (21) Google Scholar, 26Nishikori M. Ohno H. Haga H. Uchiyama T. Cancer Sci. 2005; 96: 487-497Crossref PubMed Scopus (30) Google Scholar). In order to investigate CD30 signaling in a more physiological manner, a CD30-responsive system was established that allowed a detailed analysis of the kinetics and specific cellular responses in ALCL and HL cells. We show here that CD30 stimulation alone causes apoptosis in a fraction of ALCL cells with observed reduction in cytoplasmic TRAF2 protein levels. In surviving cells, TRAF2 was found to translocate to a detergent-insoluble fraction of the cell, and this translocation correlated with both canonical and alternative NF-κB activation as measured by cytoplasmic processing of p105 and p100, NF-κB nuclear translocation, and transactivation of NF-κB-responsive genes. Up-regulation of NF-κB-responsive genes suggested a cell cycle-progressive consequence for CD30 signaling. In contrast to CD30 ligand (CD30L/CD153), exposure of ALCL cells to TNF activated only the canonical NF-κB pathway and was a poor activator of NF-κB-responsive genes. The sustained robust activation of NF-κB following prolonged physiological CD30 stimulation resulted in cell cycle arrest, which correlated with induction of p21waf1. Surprisingly, the canonical, but not the alternative, NF-κB pathway was primarily responsible for the CD30-induced expression of p21waf1, since short interfering RNA (siRNA) targeted against RelA, but not p100/p52, resulted in a reduction of CD30-induced p21waf1. Given these results, we conclude that, regardless of the presence of NPM-ALK, CD30 signaling in ALCL results in strong NF-κB activation, directly leading to cell cycle arrest. Plasmids and Cell Lines—The coding sequence of CD30L was subcloned into pcDNA5/FRT/TO (Invitrogen) and then co-transfected with a recombinase expressing plasmid into Flp-In Chinese hamster ovary (CHO) cells (Invitrogen) according to the manufacturer's instructions. This generated a cell line expressing CD30L and the hygromycin resistance marker to allow for selection of stable CD30L+ CHO cells with 500 μg/ml hygromycin B. Following selection, stable CD30L+ CHO cells were stained with a fluorescein isothiocyanate-conjugated anti-CD30L antibody and sorted by sterile flow cytometry. One cell was placed into each well of a 96-well plate and selected with hygromycin B. Forty-two clonal stable CHO lines expressing CD30L were obtained from this process. Of these, six lines were chosen, following screening as described above, based on high levels of CD30L surface expression. Of the remaining six lines, one had high and uniform CD30L expression (Fig. 1B). CHO cells (stable vector control) and CD30L+ CHO cells were maintained in F-12 nutrient medium (Invitrogen) supplemented with 10% fetal bovine serum (Mediatech) at 37 °C, 5% CO2. The Hodgkin cell lines, L428 and KM-H2, and the ALCL cell lines, Michel and Karpas 299, have been described previously (24Mir S.S. Richter B.W. Duckett C.S. Blood. 2000; 96: 4307-4312Crossref PubMed Google Scholar) and were propagated in RPMI medium (Mediatech) supplemented with 10% fetal bovine serum and 2 mm Glutamax at 37 °C, 5% CO2. Physiological CD30 Stimulation—Michel, Karpas 299, L428, and/or KM-H2 cells were resuspended in a 1:1 mixture of RPMI and F-12 nutrient media at a final concentration of 1 × 106 cells/ml. One ml of the lymphoma cells was incubated for the specified time with either CHO cells (negative control) or with CD30L+ CHO cells that had been seeded at 0.8 × 106 cells/well (0.5 × 106 cells/well for cell cycle analysis) in 6-well plates the previous day. Following CD30 stimulation, the lymphoma cell lines were removed from the CHO cells with gentle pipetting and collected by centrifugation at 200 × g for 5 min. The medium was aspirated, and the cells were washed once with 1 ml of phosphate-buffered saline (PBS). The cells were centrifuged at 200 × g for 5 min, the PBS was aspirated, and the cells were resuspended according to the experimental specifications listed below. Propidium Iodide Exclusion and Cell Death Analysis—CD30 on Michel, Karpas 299, L428, and KM-H2 cells was stimulated for 6 h as described above. The lymphoma cell lines were washed once with PBS and resuspended in 0.5 ml of PBS containing 2 μg/ml propidium iodide (PI). PI-positive cells were detected by flow cytometry. Cell death analysis following CD30 stimulation with agonistic antibodies M67 and M44 was performed as described (24Mir S.S. Richter B.W. Duckett C.S. Blood. 2000; 96: 4307-4312Crossref PubMed Google Scholar), except that 6-well plates were used, and each well was coated with 20 μg of either IgG1 control antibody or agonistic antibody. 1 × 106 Michel or Karpas 299 cells were incubated with IgG1, M67, or M44 for 24 h followed by washing once in PBS and resuspending in 0.5 ml of PBS containing 2 μg/ml PI. PI-positive cells were detected by flow cytometry. TRAF2 Translocation Analysis—CD30 on Karpas 299 and L428 cells was stimulated for 3 h as described above. The lymphoma cell lines were washed once in PBS and resuspended in Triton X-100 lysis buffer (25 mm HEPES, 100 mm NaCl, 10% glycerol, 1% Triton X-100, 1 mm PMSF, and 1 mm DTT) supplemented with additional protease inhibitors and incubated for 20 min on ice to ensure complete lysis. The soluble and insoluble Triton X-100 fractions were separated by centrifugation at 20,800 × g in a refrigerated microcentrifuge at 4 °C for 10 min. The soluble fractions were moved to a fresh microcentrifuge tube, and the insoluble pellets were washed once in 1 ml of Triton X-100 lysis buffer and centrifuged at 20,800 × g at 4 °C for 10 min. The Triton X-100 lysis buffer was aspirated, and the insoluble pellets were resuspended in Laemmli buffer (0.0625 mm Tris-HCl, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 1 mm PMSF, and 1 mm DTT). The soluble and insoluble fractions were analyzed by immunoblotting for the presence of TRAF2 protein. Antibodies and Immunoblotting—Lysates from control or CD30-stimulated lymphoma cells were prepared with radioimmune precipitation lysis buffer (PBS containing 1% Nonidet P-40, 0.5% (w/v) deoxycholic acid, 0.1% SDS, 1 mm PMSF, and 1 mm DTT) supplemented with additional protease inhibitors and incubated for 20 min on ice to ensure complete lysis unless stated otherwise. Protein samples were resolved on denaturing NuPAGE 4–12% polyacrylamide gradient gels (Invitrogen), transferred to nitrocellulose (Invitrogen), and blocked with 5% powdered milk (w/v) in TBS containing 0.05–0.2% Tween 20, depending on the primary antibody used. The membranes were incubated with the specified antibodies, washed, and then incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare). Peroxidase activity was detected by the enhanced chemiluminescence Western blot analysis system (GE Healthcare). Antibodies against TRAF2 (Transduction Laboratories), RelA and RelB (a kind gift from Nancy Rice), p50 and p52 (Upstate Biotechnology), p21waf1 (Transduction Laboratories), cyclin-dependent kinase 4 (CDK4; Transduction Laboratories), and β-actin (Sigma) were used. Electrophoretic Mobility Shift Assay—CD30 on Karpas 299 or L428 was stimulated for 3 h as described above except that 5 × 106 Karpas 299 cells were layered onto CHO and CD30L+ CHO cells previously seeded in 10-cm cell culture dishes at a density of 3.5 × 106 cells/plate. The lymphoma cells were washed once with PBS, and half were used for nuclear extract preparation, whereas the remaining cells were used for total RNA isolation. All steps were performed cold. Karpas 299 cells were washed once with buffer A (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.1 mm PMSF, and 0.5 mm DTT) and pelleted by centrifugation at 200 × g for 5 min. Buffer A was aspirated, and the pellet was resuspended by gentle pipetting in 30 μl of buffer A supplemented with 0.1% Nonidet P-40 and incubated on ice for 10 min. The nuclear pellet was isolated by centrifugation at 20,800 × g in a refrigerated microcentrifuge at 4 °C for 10 min, resuspended in 20 μl of cold buffer C (20 mm HEPES, pH 7.9, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.1 mm PMSF, 0.5 mm DTT, and 25% glycerol), and rotated at 4 °C for 15 min. The nuclear extract was clarified by centrifugation at 20,800 × g for 15 min, and 10 μl was transferred to a fresh tube and diluted with 60 μl of modified buffer D (20 mm HEPES, pH 7.9, 50 mm KCl, 0.2 mm EDTA, 0.1 PMSF, 0.5 mm DTT, and 20% glycerol), flash frozen, and stored at –80 °C. Two complementary oligonucleotides containing NF-κB consensus binding sites, κBEMSAoligo1 (5′-TGCAAGGGACTTTCCGCTGGGGACTTTCC-3′) and κBEMSAoligo2 (5′-TGCAGGAAAGTCCCCAGCGGAAAGTCCCT-3′), were annealed, and 50 ng was radiolabeled in a Klenow reaction in the presence of [α-32P]dCTP. The radiolabeled probe was then purified over a Sephadex column in TE containing 50 mm NaCl. To test for the presence of NF-κB in the nuclear extracts, prepared as described above, 2 μl of nuclear extracts were incubated for 20 min at room temperature with 1 μg of poly(dI-dC)·poly(dI-dC) in modified buffer D (minus glycerol) for a total volume of 20 μl. 0.2 μl of 32P-radiolabeled probe was added, and the entire reaction was separated on a nondenaturing 4% polyacrylamide gel. Autoradiography was performed overnight at –20 °C. For supershift analysis, 1–4 μg of antibody was incubated with the nuclear extract and poly(dI-dC)·poly(dI-dC) prior to the addition of 32P-radiolabeled probe. Real Time Reverse Transcription-PCR—CD30 on Karpas 299 or L428 cells was stimulated for 3 h (from 6 to 36 h for cell cycle analysis; see below) as described above. The lymphoma cells were washed with PBS followed by total RNA isolation using the RNeasy minikit (Qiagen) according to the manufacturer's instructions. 100 ng of total RNA was subjected to a reverse transcription reaction using random hexamer primers and MultiScribe™ Reverse Transcriptase (Applied Biosystems). 1 μl of the resulting cDNA was analyzed with the indicated target assay using the Applied Biosystems 7500 real time PCR system. Each target assay was normalized to glyceraldehyde-3-phosphate dehydrogenase levels and performed in triplicate. Cell Cycle Analysis—CD30 on Karpas 299 or L428 cells was stimulated for 6, 12, 24, and 36 h as described above. The lymphoma cells were washed with PBS and collected by centrifugation at 200 × g for 5 min, and each sample was normalized to 0.5 × 105 cells in 0.5 ml of PBS. The cells were fixed in 50% ethanol overnight at –20 °C. The following day, the cells were collected by centrifugation at 200 × g, the PBS/ethanol mixture was decanted, and the cells were resuspended in 0.5 ml of PBS containing 50 μg/ml PI and 100 μg/ml RNase A. The DNA content of the cells was analyzed by flow cytometry. RNA Interference—1 × 107 Karpas 299 cells were transfected with 16 μg of either a control (green fluorescent protein) siRNA (sense strand AAGACCCGCGCCGAGGTGAAG), siRNA targeted against p100 (sense strand AAGGCTGGTGCTGACATCCAT), or an siRNA targeted against RelA (sequence has been described (27Zhou A. Scoggin S. Gaynor R.B. Williams N.S. Oncogene. 2003; 22: 2054-2064Crossref PubMed Scopus (156) Google Scholar)). The indicated siRNA was transfected using a Bio-Rad Gene Pulser II electroporator set on infinite resistance, 300 V, and 950 microfarads. Twenty-four h post-transfection, dead cells resulting from the transfection procedure were removed by centrifuging the cells at 400 × g for 20 min on a Ficoll-Paque PLUS (GE Healthcare) density cushion. Forty-eight h post-transfection, CD30 was stimulated for 24 h on the transfected cells as described above. Following CD30 stimulation, total RNA was isolated from a fraction of the cells and subjected to real time reverse transcription-PCR. The remaining cells were divided and used for whole cell lysate preparation and Western blot analysis and cell cycle analysis. Physiological CD30 Stimulation Triggers Apoptosis in ALCL Cells—Much of our understanding of CD30 function is based on the use of agonistic CD30-specific antibodies or otherwise nonphysiologic means to activate CD30 signaling cascades. Although immobilized agonistic antibodies hypothetically mimic CD30L and are a convenient tool for stimulating CD30, there are conflicting data reported for different CD30 activating antibodies. These studies suggest that different epitopes on CD30 recognized by the various agonistic antibodies may contribute to different signaling outcomes. Since CD30L is normally expressed as a membrane-bound ligand on macrophages, T-cells, and B-cells, we decided to examine CD30 signaling using a membrane-bound form of CD30L to avoid the epitope-agonistic antibody approach. To this end, a system to stimulate CD30 via its physiological ligand was established, and the subsequent role of CD30 signaling in lymphoma cells was investigated. CHO cells were chosen to serve as a vehicle for stable expression of CD30L for two reasons. First, their strong adherent properties allow for convenient layering and removal of suspension CD30+ lymphoma cell lines for the purpose of CD30 stimulation without removing the CHO cells themselves (Fig. 1A). Second, because CHO cells are derived from a different species, there is less likely to be contaminating stimulants, such as secreted cytokines or membrane-bound receptors/ligands. Analysis of the stable CD30L+ CHO cells by flow cytometry and Western blot revealed that CD30L was expressed on the cell surface versus no detectable CD30L expression on stable vector control CHO cells as expected (Fig. 1B) (data not shown). We have previously shown that stimulation of CD30 on ALCL cells with agonistic antibodies M67 and M44 resulted in a percentage of cells succumbing to death (24Mir S.S. Richter B.W. Duckett C.S. Blood. 2000; 96: 4307-4312Crossref PubMed Google Scholar). Thus, to examine whether a physiological CD30 signal causes cell death, ALCL (Michel and Karpas 299) or HL (L428 and KM-H2) cell lines were layered onto control CHO cells or CD30L+ CHO cells for 6 h (Fig. 1A). Following incubation, the lymphoma cells were removed, and cell viability was evaluated by propidium iodide exclusion. As can be seen in Fig. 2A, stimulation of CD30 on both ALCL cell lines tested resulted in ∼20% cell death when normalized to control cells. These results are comparable with the death observed following CD30 stimulation with M44 or M67 (Fig. 2B). Conversely, HL cell viability was barely affected upon physiological CD30 stimulation (Fig. 2A), which has previously been attributed to the constitutive prosurvival signals of NF-κB in these cells (24Mir S.S. Richter B.W. Duckett C.S. Blood. 2000; 96: 4307-4312Crossref PubMed Google Scholar). CD30 Signaling Includes TRAF2 Translocation and NF-κB Activation—Ectopic expression of constitutively active CD30 in HEK293 cells or CD30 cross-linking of ALCL and HL cells with an agonistic antibody has been reported to result in the degradation of TRAF2 (TNF receptor-associated factor 2) (24Mir S.S. Richter B.W. Duckett C.S. Blood. 2000; 96: 4307-4312Crossref PubMed Google Scholar, 28Duckett C.S. Thompson C.B. Genes Dev. 1997; 11: 2810-2821Crossref PubMed Scopus (192) Google Scholar), an adaptor molecule necessary for the activation of NF-κB. In order to examine whether this phenomenon occurs following CD30 stimulation with its physiological ligand, ALCL (Karpas 299) or HL (L428) cell lines were layered onto control CHO cells or CD30L+ CHO cells for 3 h. Following CD30 stimulation, the lymphoma cells were removed, and Triton X-100-soluble fractions were prepared and analyzed by Western blot. Analysis of cell lysates using an antibody directed against TRAF2 revealed that TRAF2 disappeared from the Triton X-100-soluble fractions of Karpas 299 cells as early as 1 h and was completely lost at 3 h poststimulation of CD30 (Fig. 3A) (data not shown). Loss of TRAF2 was seen to a much lesser degree in the L428 soluble fraction following CD30 stimulation (Fig. 3A). In conjunction with TRAF2 loss from the soluble fraction, TRAF2 accumulated in the Triton X-100-insoluble fraction of the cell (Fig. 3A). Because the presence of NPM-ALK in ALCL cells has been shown to inhibit CD30-mediated NF-κB signaling by blocking the recruitment and aggregation of TRAF proteins (22Horie R. Watanabe M. Ishida T. Koiwa T. Aizawa S. Itoh K. Higashihara M. Kadin M.E. Watanabe T. Cancer Cell. 2004; 5: 353-364Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), we asked whether the observed TRAF2 translocation following a CD30 response correlated with NF-κB activation, as has been shown in other signaling situations (29Legler D.F. Micheau O. Doucey M.A. Tschopp J. Bron C. Immunity. 2003; 18: 655-664Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). In fact, the NF-κB precursors, p105 and p100, were processed in the cytoplasm of Karpas 299 cells stimulated for 3 h using our system (Fig. 3, B and C), suggesting that NF-κB was being activated following CD30 stimulation. Processing of p105 can occur co-translationally (30Lin L. DeMartino G.N. Greene W.C. Cell. 1998; 92: 819-828Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), which satisfies the basal necessity of NF-κB activity required by the cell. However, p105 processing has also been shown to occur post-translationally following a stimulant-dependent response (31Heissmeyer V. Krappmann D. Wulczyn F.G. Scheidereit C. EMBO J. 1999; 18: 4766-4778Crossref PubMed Scopus (173) Google Scholar, 32Lang V. Janzen J. Fischer G.Z. Soneji Y. Beinke S. Salmeron A. Allen H. Hay R.T. Ben-Neriah Y. Ley S.C. Mol. Cell Biol. 2003; 23: 402-413Crossref PubMed Scopus (107) Google Scholar), as in the case of CD30 stimulation shown here. To our knowledge, this is the first example of CD30-induced p105 processing, and it suggests that physiological CD30 stimulation increases the cellular pool of the p50 subunit. In L428 cells, there is a loss of p105 protein following CD30 stimulation (Fig. 3B), but because of the large amount of p50 present in these cells, it is difficult to determine whether p105 is being processed to p50 as in ALCL cells or if it is being degraded, a step that leads to activation of other signaling pathways in addition to releasing NF-κB subunits for nuclear translocation (33Beinke S. Deka J. Lang V. Belich M.P. Walker P.A. Howell S. Smerdon S.J. Gamblin S.J. Ley S.C. Mol. Cell Biol. 2003; 23: 4739-4752Crossref PubMed Scopus (101) Google Scholar, 34Beinke S. Robinson M.J. Hugunin M. Ley S.C. Mol. Cell Biol. 2004; 24: 9658-9667Crossref PubMed Scopus (171) Google Scholar). Similar exposure of ALCL and HL cells to membrane-bound CD30L resulted in activation of the alternative NF-κB pathway, as seen by the loss of p100 and the accumulation of the active p52 subunit (Fig. 3C). It has been reported recently that CD30 stimulation, either by overexpressing CD30 or using agonistic antibodies, results in p100 processing (26Nishikori M. Ohno H. Haga H. Uchiyama T. Cancer Sci. 2005; 96: 487-497Crossref PubMed Scopus (30) Google Scholar, 35Nonaka M. Horie R. Itoh K. Watanabe T. Yamamoto N. Yamaoka S. Oncogene. 2005; 24: 3976-3986Crossref PubMed Scopus (35) Google Scholar). The findings shown here clearly indicate that p100 processing occurs following stimulation of CD30 with its physiological ligand. Furthermore, in L428 cells that had been exposed to CD30L, p100 is processed to p52 at a higher rate than the constitutive processing already presen
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