Transcription factor NF- B is constitutively activated in acute ... - Nature

3 downloads 100 Views 111KB Size Report
and strongly suggests a critical role of this factor for leukemia cell survival. Leukemia (2000) 14, 399–402. Keywords: acute lymphoblastic leukemia; transcription ...
Leukemia (2000) 14, 399–402  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu

Transcription factor NF-␬B is constitutively activated in acute lymphoblastic leukemia cells U Kordes1, D Krappmann1, V Heissmeyer1, WD Ludwig2 and C Scheidereit1 1

Max-Delbru¨ck-Centrum for Molecular Medicine; and 2Humboldt University of Berlin, Universita¨tsklinikum Charite´, Campus Berlin-Buch, Berlin, Germany

The pleiotropic transcription factor NF-␬B controls cellular apoptotic and growth processes and increasing evidence suggests a role in tumorigenesis. We describe here that constitutively activated NF-␬B complexes are found in the vast majority (39 out of 42 samples) of childhood acute lymphoblastic leukemia (ALL) without any subtype restriction. Electrophoretic shift analysis further demonstrates that these complexes are composed of p50-p50 and p65-p50 dimers. Proteasome inhibition in primary ALL cultures results in a hyperphosphorylated form of I␬B␣, indicating that activation of upstream kinases, which trigger I␬B␣ degradation, has led to nuclear translocation of NF-␬B. Careful inhibition of cellular proteolytic activities is of importance when analyzing extracts from primary ALL cells. Degradation of p65 and other proteins in ALL samples could be specifically suppressed by ␣-1 antitrypsin. Constitutive NF␬B activation is thus a common characteristic of childhood ALL and strongly suggests a critical role of this factor for leukemia cell survival. Leukemia (2000) 14, 399–402. Keywords: acute lymphoblastic leukemia; transcription factors; kinases

Introduction The NF-␬B family of transcription factors has important regulatory functions for both normal and malignant hematopoiesis in many species and plays important roles in the immune system.1,2 Murine knockout models of NF-␬B and I␬B genes have shown specific roles for lymphocyte development and function.3 Accumulating evidence indicates a role of NF-␬B and I␬B proteins in growth control and oncogenesis.4 Homo- and heterodimers of the NF-␬B subunits p50, p52, p65, c-Rel, and RelB are under control of the inhibitory I␬B proteins I␬B␣, I␬B␤, I␬B⑀, p105 and p100.5 Prototypic NF-␬B p50-p65 is sequestered in the cytoplasm by I␬B␣, ␤ or ⑀ and is released after stimulation through various signal transduction pathways which induce degradation of the I␬Bs. Tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1) and other agents induce phosphorylation of I␬Bs by I␬B kinases at conserved serines residues which triggers subsequent ubiquitination and destruction of the inhibitors by the proteasome.5 In most cell types, signalinduced translocation of NF-␬B into the nucleus and activation of transcription is only transient. However, constitutively active nuclear NF-␬B containing the c-Rel and RelB subunits, is found in mature B cells and correlates with enhanced turnover of I␬B␣.2 Constitutively activated NF-␬B p50-p65 has been observed in neurons and in primary cells or cell lines derived from Hodgkin’s disease (HD), adult T cell leukemia (ATL), breast carcinoma, melanoma and pancreatic adenocarcinoma.6–11 In the context of neoplastic diseases, it is relevant that NF-␬B p50-p65 suppresses apoptosis and mounts a pro-

Correspondence: C Scheidereit, Max-Delbru¨ch-Centrum for Molecular Medicine, Robert-Ro¨ssle Str. 10, D-13125 Berlin, Germany; Fax: 49 30 9406 3866 Received 19 July 1999; accepted 17 November 1999

tective response against anthracyclines and topo-II-isomerase inhibitors.12–14 NF-␬B further regulates cell adhesion molecules, tissue plasminogen activator, and cyclins,1,15,16 and controls the cell cycle by affecting the Rb pathway.16 We have previously shown that inhibition of NF-␬B indeed strongly increases apoptosis rate and slows down cell proliferation in Hodgkin/Reed–Sternberg cells.7 Constitutive NF-␬B activation in HD is caused by persistently activated I␬B kinases and, in some cases, by mutated I␬B␣.17–19 Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood and comprises a heterogeneous group of malignant clonal disorders originating from B- or less commonly from T-progenitor cells. ALL subgroups with distinct biological characteristics are frequently characterized by deregulated transcription factors or kinases, most prominently the fusion proteins TEL-AML1 encoded by t(12;21)(p13;q22) in presumedly good risk ALL, or BCR-ABL encoded by t(9;22)(q34;q11) and MLL-AF4 by t(4;11)(q21;q23) in high risk ALL.20 When screening various pediatric tumors for aberrant NF-␬B activities, we found that ALL cells at diagnosis typically contain constitutively activated p50-p65 heterodimers and p50 homodimers. Furthermore, kinases acting on I␬B␣ were activated in the vast majority of ALL samples investigated. Materials and methods

Cell culture Primary ALL samples from peripheral blood or bone marrow aspirates (Robert-Ro¨ssle-Klinik, MDC, Berlin) and mononuclear cells from normal peripheral blood were purified by Ficoll gradient centrifugation. A few samples were thawed and washed after cryopreservation. Cell viability was assessed by trypan blue exclusion and was mostly greater than 90%. An initial round of cryopreserved samples was obtained from the laboratory at the University-Childrens-Hospital, Charite´, Berlin (KH Seeger). ALL cells were kept in short-term culture in RPMI 1640 (Gibco, Karlsruhe, Germany), 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin. L1236 and HDLM-2 HD cells were grown in the same medium. SEM and uocb-1 ALL cells were kindly provided by R Marschalek, University Erlangen, and by T Look, SJCRH, Memphis, TN, USA, respectively. 50 nM phorbol-12-myristate-13-acetate (PMA; Sigma, St Louis, MO, USA) or 50 ␮g/ml N-acetyl-leucyl-leucyl-norleucinal (ALLN; Calbiochem, San Diego, CA, USA) was added where indicated.

Electrophoretic mobility shift assay (EMSA) and Western blotting Whole cell extracts were prepared as described21 with 3 ␮g/␮l ␣-1 antitrypsin in the extraction buffer. After washing cells

Constitutive NF-␬B in primary ALL cells U Kordes et al

400

with PBS, lysis buffer (20 mM HEPES pH 7.9, 350 mM NaCl, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM MgCl2, 10% glycerol, 1% Nonidet P-40, 1 ␮g/ml pefabloc, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin A, 10 mM NaF, 8 mM glycerophosphate, and 1 mM DTT) was added. After 10 min at 4°C the lysate was centrifuged for 5 min at 15 800 g in a microfuge. EMSA was performed as described.21 Western blots were analyzed by chemiluminescense following the manufacturer’s recommendations (New England Biolabs, Beverly, MA, USA). Antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) (I␬B␣, p65, c-Rel and RelB), Rockland (p50) and Upstate Biotechnology (Lake Placid, NY USA) (p52). 1 ␮g of purified recombinant wild-type I␬B␣ was added where indicated. Results and discussion The analysis of NF-␬B activity in ALL cells from patients at diagnosis was initially hampered by proteolysis in about half of the samples, even though a standard broad-spectrum inhibitor cocktail was used during extract preparation. This led to abrogation of both Oct-1 and NF-␬B DNA binding activities and to truncation of p65 and I␬B␣ proteins (Figure 1). By mixing extracts of the HD cell line HDLM-2 with a proteolytically active ALL extract lacking Oct-1 and NF-␬B activities and testing various protease inhibitors, we determined that ␣-1-antitrypsin was best in preserving the shift activity (Figure 1). We compared NF-␬B DNA binding activity in ALL samples to that found in HD cells (L1236) or in control mononuclear

Figure 1 NF-␬B and Oct-1 DNA binding activities (EMSA, upper panels) as well as I␬B␣ and p65 proteins (Western blots, lower panels) are affected by proteases present in whole cell extracts of a primary ALL sample prepared with a standard proteinase inhibitor cocktail. Specific NF-␬B- or Oct-1-DNA complexes and full length p65 or I␬B␣ proteins (filled triangles) in whole cell extracts from the HD cell line HDLM-2 (lane 1) are abrogated by incubation with a representative extract of primary ALL cells (lane 2); ⌬p65 and ⌬I␬B␣, truncated proteins. Proteinase inhibitors (lanes 3–14) were antipain dihydrochloride (4.8 mM), bestatin (2.7 mM), chymostatin (5.5 mM), E-64 (0.9 mM), leupeptin (0.33 mM), pepstatin (0.2 mM), phosphoramidon (3.3 mg/ml), pefabloc (26 mM), EDTA (18 mM), aprotinin (1.6 mg/ml) (all purchased from Boehringer), ␣-1 antitrypsin (Sigma) (3 mg/ml), DEVD-chlormethylketone (Bachem) (50 ␮M). Leukemia

cells from healthy probands, using ␣-1-antitrypsin in the extraction buffer (Figure 2). Results were standardized against Oct-1 shift activity as an internal control for each sample (Figure 2b). 39 of 42 samples invariably showed constitutively activated NF-␬B in comparison to unstimulated cell lines (not shown) or normal mononuclear cells (Figure 2a). Constitutive NF-␬B in ALL appears to be a general characteristic since there was no correlation with immunophenotype, disease at primary diagnosis or relapse, or good or poor prednisone responders. NF-␬B activation in ALL was similar to that in HD cell lines. Unstimulated peripheral blood mononuclear cells isolated from healthy probands displayed some faint NF-␬B activity, possibly accounting for the mature B cell population, but this activity could be strongly stimulated by phorbol-12myristate-13-acetate (PMA). In contrast, constitutive NF-␬B activities in HD cells and half of the ALL cells tested were refractory to strong superstimulation by PMA, indicating close to maximal NF-␬B activation (Figure 1a). Constitutive NF-␬B activity in ALL consisted of p50-p65 heterodimers and of p50 homodimers, as determined by supershift analysis with antibodies directed against p65, p50, p52, c-Rel or RelB (Figure 2c). Addition of wild-type recombinant I␬B␣ to the DNA binding reaction inhibited the p50-p65 but, as expected, not p50-p50 complexes, indicating that the observed constitutive activity was not due to mutations or modifications that render NF-␬B resistant to I␬B␣ (Figure 2c). Furthermore, p65 displayed a normal migration in Western blots (not shown). If constitutive NF-␬B is caused by activated signaling cascades acting upstream of I␬B␣ in ALL cells, phosphorylated I␬B␣ should be stabilized upon inhibition of the proteasome. ALL cells were kept in medium with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN). Cellular extracts were prepared and probed with an anti-I␬B␣ antibody in Western blots. Unlike in Hodgkin cells,17–19 I␬B␣ consistently had the wildtype length (Figure 2d and data not shown). In 18 out of 19 samples analyzed, a slower migrating phosphoform of I␬B␣ was stabilized in the presence of ALLN (Figure 2d), indicating that upstream kinases were indeed activated. Similarly, phosphorylated I␬B␣ was stabilized by ALLN in HD cells (Figure 2d and Ref. 17). We were not able to induce NF-␬B in control cell lines by supernatants of ALL cell cultures (data not shown) and consider it unlikely that NF-␬B activation is caused by an autocrine mechanism. Our data thus indicate that constitutive activation of NF-␬B in ALL is caused by ongoing signaling in these cells which leads to persistent activation of I␬B kinases. It will have to be addressed in future studies whether mutations in signaling molecules upstream of I␬B␣ have been accumulated in these cells. Mori et al11 reported that NF-␬B is constitutively activated in an apparently HTLV-1-Tax-independent manner in seven primary adult T cell leukemia (ATL) samples and in one ATLderived cell line. In contrast, we failed to detect constitutive NF-␬B activation in several transformed ALL cell lines analyzed (Reh, Blin, SEM, Uocb-1) (data not shown). It is possible that constitutive NF-␬B is activated in ALL and ATL by the same mechanisms. Hematopoietic tumors in adults contain rearrangements or amplifications of members of the NF␬B/I␬B gene families in up to 25%,4 however this is not the case for childhood ALL.22 The bcr-abl product of the highrisk translocation (9;22)(q34;q11) in ALL has been shown to activate NF-␬B through an as yet unknown target in the NF␬B/I␬B pathway.23,24 It is present in 5% of childhood ALL, 20% of relapsed childhood ALL and 30% of adult ALL, and

Constitutive NF-␬B in primary ALL cells U Kordes et al

401

Figure 2 (a) NF-␬B and DNA binding activities in whole cell extracts from ALL cells, HD cells (L1236) and normal mononuclear cells (control). EMSA was performed with the H2K-NF-␬B DNA binding site probe, free DNA is not shown. Six representative ALL samples (C-ALL, T-ALL, preB-ALL, proB-ALL) are shown. Cells were treated with PMA, as indicated. Constitutive NF-␬B was independent of immunophenotypic subtype and was found in 21 of 22 C-ALL, 2 of 2 pre-B-ALL, 3 of 3 pro-B-ALL, 2 of 2 B-ALL and 11 of 13 T-ALL. (b) Oct-1 DNA binding activities in unstimulated samples used in (a), determined by EMSA with an H2B octamer DNA binding site probe. Free DNA is not shown. (c) Supershift analysis of the NF-␬B activities in the representative ALL samples used in (a) with p65, p50, p52, c-Rel or RelB antibodies. Migration of p50-p65- and p50-p50-DNA complexes is indicated. Recombinant I␬B␣ was added as indicated. (d) Western blot analysis of I␬B␣ in extracts of C-ALL cells and HD cells (HDLM-2) after treatment with PMA (1 h) or ALLN (1.5 h). The migration of phosphorylated I␬B␣ (p-I␬B␣) in the ALLN treated samples and of hypophosphorylated I␬B␣ is indicated.

thus can account for NF-␬B activation in some, but not all ALLs. The clonal evolution of malignant lymphoblasts in ALL has to escape safeguards that control normal proliferation and differentiation in lymphopoiesis and ensure delivery of functional mature lymphocytes. While some transcription factors are deregulated by certain hallmark translocations in subtypes of childhood ALL and in fact allow stratification into different therapeutic regimens, no such factor has been shown to be uniformly involved. We propose that general constitutive acti-

vation of NF-␬B in ALL serves as an important switch that ensures survival of ALL cells either by blocking apoptosis or by enhancing proliferation. Acknowledgements We thank U Anderer, Charite´ Berlin, for pediatric tumor cell lines, R Marschalek, University Erlangen, and T Look, SJCRH, Memphis, TN, for ALL cell lines, KH Seeger, Virchow KliniLeukemia

Constitutive NF-␬B in primary ALL cells U Kordes et al

402

kum Berlin for ALL samples, respectively, and H Riehm and M Schrappe (chairmen of the ALL-BFM study) for continuous support. R Dettmer kindly provided highly purified, bacterially expressed I␬B␣. References 1 Baeuerle PA, Henkel T. Function and activation of NF-␬B in the immune system. Annu Rev Immunol 1994; 12: 141–179. 2 Wulczyn FG, Krappmann D, Scheidereit C. The NF-␬B and I␬B gene families: mediators of immune response and inflammation. J Mol Med 1996; 74: 749–769. 3 Attar RM, Caamano J, Carrasco D, Iotsova V, Ishikawa H, Ryseck RP, Weih F, Bravo R. Genetic approaches to study Rel/NF-␬B/I␬B function in mice. Semin Cancer Biol 1997; 8: 93–101. 4 Luque I, Gelinas C. Rel/NF-␬B and I␬B factors in oncogenesis. Semin Cancer Biol 1997; 8: 103–111. 5 May MJ, Ghosh S. Rel/NF-␬B and I␬B proteins: an overview. Semin Cancer Biol 1997; 8: 63–73. 6 Kaltschmidt C, Kaltschmidt B, Neumann H, Wekerle H, Baeuerle PA. Constitutive NF-␬B activity in neurons. Mol Cell Biol 1994; 14: 3981–3992. 7 Bargou RC, Emmerich F, Krappmann D, Bommert K, Mapara MY, Arnold W, Royer HD, Grinstein E, Greiner A, Scheidereit C, Dorken B. Constitutive NF-␬B/RelA activation is required for proliferation and survival of Hodgkin’s disease tumor cells. J Clin Invest 1997; 100: 2961–2969. 8 Sovak MA, Bellas RE, Kim DW, Zanieski GJ, Rogers AE, Traish AM, Sonenshein GE. Aberrant NF-␬B/Rel expression and the pathogenesis of breast cancer. J Clin Invest 1997; 100: 2952– 2960. 9 Shattuck-Brandt RL, Richmond A. Enhanced degradation of I␬B␣ contributes to endogenous activation of NF-␬B in Hs294T melanoma cells. Cancer Res 1997; 57: 3032–3039. 10 Wang W, Abbruzzese JL, Evans DB, Larry L, Cleary KR, Chiao PJ. The nuclear factor ␬B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. Clin Cancer Res 1999; 5: 119–127. 11 Mori N, Fujii M, Ikeda S, Yamada Y, Tomonaga M, Ballard DW, Yamamoto N. Constitutive activation of NF-␬B in primary adult T-cell leukemia cells. Blood 1999; 93: 2360–2368.

Leukemia

12 Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS Jr. NF-␬B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998; 281: 1680–1683. 13 Beg AA, Baltimore D. An essential role for NF-␬B in preventing TNF-␣-induced cell death. Science 1996; 274: 782–784. 14 Wang CY, Mayo MW, Baldwin AS Jr. TNF-and cancer therapyinduced apoptosis: potentiation by inhibition of NF-␬B. Science 1996; 274: 784–787. 15 Perkins ND, Felzien LK, Betts JC, Leung K, Beach DH, Nabel GJ. Regulation of NF-␬B by cyclin-dependent kinases associated with the p300 coactivator. Science 1997; 275: 523–527. 16 Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, Strauss M. NF-␬B function in growth control: regulation of cyclin D1 expression and G0/G1 to S phase transition. Mol Cell Biol 1999; 19: 2690–2698. 17 Krappmann D, Emmerich F, Kordes U, Scharschmidt E, Do¨rken B, Scheidereit C. Molecular mechanisms of constitutive NF-␬B/Rel activation in Hodgkin/Reed–Sternberg cells. Oncogene 1999; 18: 943–953. 18 Wood KM, Roff M, Hay RT. Defective I␬B␣ in Hodgkin cell lines with constitutively active NF-␬B. Oncogene 1998; 16: 2131– 2139. 19 Emmerich F, Meiser M, Hummel M, Jundt F, Mathas S, Dehmel G, Foss H-D, Krappmann D, Scheidereit C, Stein H, Do¨rken B. Overexpression of I␬B␣ without inhibition of NF-␬B activity and mutations in the I␬B␣ gene in Reed–Sternberg cells. Blood 1999; 94: 3129–3134. 20 Look AT. Oncogenic transcription factors in the human acute leukemias. Science 1997; 278: 1059–1064. 21 Wulczyn FG, Krappmann D, Scheidereit C. Signal-dependent degradation of I␬B␣ is mediated by an inducible destruction box that can be transferred to NF-␬B, bcl-3 or p53. Nucleic Acids Res 1998; 26: 1724–1730. 22 Liptay S, Seriu T, Bartram CR, Schmid RM. Germline configuration of nfkb2, c-rel and bcl3 in childhood acute lymphoblastic leukemia (ALL). Leukemia 1997; 11: 1364–1366. 23 Hamdane M, David-Cordonnier MH, D’Halluin JC. Activation of p65 NF-␬B protein by p210BCR-ABL in a myeloid cell line. Oncogene 1997; 15: 2267–2275. 24 Reuther JY, Reuther GW, Cortez D, Pendergast AM, Baldwin AS Jr. A requirement for NF-␬B activation in Bcr-Abl-mediated transformation. Genes Dev 1998; 12: 968–981.