The MEK/ERK Pathway Acts Upstream of NFκB1 (p50) Homodimer Activity and Bcl-2 Expression in a Murine B-Cell Lymphoma Cell Line
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m212919200
ISSN1083-351X
AutoresJohn F. Kurland, David Voehringer, Raymond E. Meyn,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoIn a previously published report (Kurland, J. F., Kodym, R., Story, M. D., Spurgers, K. B., McDonnell, T. J., and Meyn, R. E. (2001) J. Biol. Chem. 276, 45380–45386), we described the NFκB status for two murine B-cell lymphoma cell lines, LY-as (apoptosis-sensitive) and LY-ar (apoptosis-refractory) and provided evidence that NFκB1 (p50) homodimers contribute to the expression of Bcl-2 in the LY-ar line. In the present study, we investigated the upstream signals leading to p50 homodimer activation and Bcl-2 expression. We found that in LY-ar cells, ERK1 and ERK2 were constitutively phosphorylated, whereas LY-as cells had no detectable ERK1 or ERK2 phosphorylation. Treatment of LY-ar cells with the MEK inhibitors PD 98059, U0126, and PD 184352 led to a loss of phosphorylated ERK1 and ERK2, a reversal of nuclear p50 homodimer DNA binding, and a decrease in Bcl-2 protein expression. Similarly, activation of the MEK/ERK pathway in LY-as cells by phorbol ester led to Bcl-2 expression that could be blocked by PD 98059. Furthermore, treatment of LY-ar cells with tumor necrosis factor-α, an IκB kinase activator, did not alter the suppressive effect of PD 98059 on p50 homodimer activity, suggesting an IκB kinase-independent pathway for p50 homodimer activation. Lastly, all three MEK inhibitors sensitized LY-ar cells to radiation-induced apoptosis. We conclude that the MEK/ERK pathway acts upstream of p50 homodimer activity and Bcl-2 expression in this B-cell lymphoma cell system and suggest that the use of MEK inhibitors could be useful clinically in combination with ionizing radiation to treat lymphoid malignancies. In a previously published report (Kurland, J. F., Kodym, R., Story, M. D., Spurgers, K. B., McDonnell, T. J., and Meyn, R. E. (2001) J. Biol. Chem. 276, 45380–45386), we described the NFκB status for two murine B-cell lymphoma cell lines, LY-as (apoptosis-sensitive) and LY-ar (apoptosis-refractory) and provided evidence that NFκB1 (p50) homodimers contribute to the expression of Bcl-2 in the LY-ar line. In the present study, we investigated the upstream signals leading to p50 homodimer activation and Bcl-2 expression. We found that in LY-ar cells, ERK1 and ERK2 were constitutively phosphorylated, whereas LY-as cells had no detectable ERK1 or ERK2 phosphorylation. Treatment of LY-ar cells with the MEK inhibitors PD 98059, U0126, and PD 184352 led to a loss of phosphorylated ERK1 and ERK2, a reversal of nuclear p50 homodimer DNA binding, and a decrease in Bcl-2 protein expression. Similarly, activation of the MEK/ERK pathway in LY-as cells by phorbol ester led to Bcl-2 expression that could be blocked by PD 98059. Furthermore, treatment of LY-ar cells with tumor necrosis factor-α, an IκB kinase activator, did not alter the suppressive effect of PD 98059 on p50 homodimer activity, suggesting an IκB kinase-independent pathway for p50 homodimer activation. Lastly, all three MEK inhibitors sensitized LY-ar cells to radiation-induced apoptosis. We conclude that the MEK/ERK pathway acts upstream of p50 homodimer activity and Bcl-2 expression in this B-cell lymphoma cell system and suggest that the use of MEK inhibitors could be useful clinically in combination with ionizing radiation to treat lymphoid malignancies. Nuclear factor κ B (NFκB) is traditionally described as a family of transcription factors that are activated during immune and inflammation responses, regulating the genes encoding cytokines such as IL-2, 1The abbreviations used are: IL, interleukin; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; GFP, green fluorescence protein; ar, apoptosis-refractory; as, apoptosis-sensitive; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; M3K, MAP kinase kinase kinase; IKK, IκB kinase; BIG, Bcl-2 IRES (internal ribosome entry site) GFP; EMSA, electrophoretic mobility shift assay. IL-6, IL-8, and granulocyte macrophage-colony stimulating factor (1Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar). NFκB has since been dubbed a central mediator of the human immune response and has been shown to control hundreds of genes in addition to those for cytokines, including cell adhesion molecule and immunoreceptor genes (2Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3447) Google Scholar). In the last few years, NFκB has additionally been recognized as a key regulator of anti-apoptotic genes, including the genes coding for the inhibitor of apoptosis proteins and members of the Bcl-2 family (3Baldwin A.S. J. Clin. Invest. 2001; 107: 241-246Crossref PubMed Scopus (1199) Google Scholar, 4Mayo M.W. Baldwin A.S. Biochim. Biophys. Acta. 2000; 1470: M55-M62PubMed Google Scholar). This control of anti-apoptotic genes has implicated aberrant NFκB activity as a cell survival signal for many cancer cell types. Cancers classified as having aberrant NFκB activity include Hodgkin's disease, chronic myelogenous leukemia, adult T-cell leukemia, acute lymphoblastic leukemia, melanoma, and solid tumors such as breast, colon, ovarian, pancreatic, thyroid, bladder, and prostate carcinomas (5Schwartz S.A. Hernandez A. Mark Evers B. Surg. Oncol. 1999; 8: 143-153Crossref PubMed Scopus (116) Google Scholar, 6Rayet B. Gelinas C. Oncogene. 1999; 18: 6938-6947Crossref PubMed Scopus (1008) Google Scholar). Often, chromosomal rearrangement or overexpression of Rel family members leads to aberrant NFκB activity, particularly in hematopoietic tumors (6Rayet B. Gelinas C. Oncogene. 1999; 18: 6938-6947Crossref PubMed Scopus (1008) Google Scholar). However, not all aberrant NFκB activity in cancer cells is a result of Rel family member overexpression, and it has been recognized that NFκB activity may also arise by the activation of signaling kinases that converge on the activation of I κ B kinase (IKK). IKK, in turn, phosphorylates IκB, initiating the degradation of IκB through the ubiquitin/proteasome pathway. This results in the release of bound NFκB subunits that then translocate to the nucleus and activate gene transcription (7Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4084) Google Scholar). NFκB-inducing kinase, a mitogenactivated protein kinase kinase kinase (M3K) that is constitutively activated in melanoma cells (8Dhawan P. Richmond A. J. Biol. Chem. 2002; 277: 7920-7928Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), exemplifies a signaling kinase that leads to aberrant NFκB activity in tumor cells upstream of IKK. There are a number of other kinases known to act upstream of IKK. These include members of the M3K family such as MEKK-1, MEKK-2, MEKK-3, and Tpl-2/COT (9Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. 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Lui W.Y. Hu C.P. Chang C. Chou C.K. Tsai S.F. Genomics. 1998; 50: 331-340Crossref PubMed Scopus (73) Google Scholar), and Tpl-2 with breast, colon, and gastric cancers (16Sourvinos G. Tsatsanis C. Spandidos D.A. Oncogene. 1999; 18: 4968-4973Crossref PubMed Scopus (76) Google Scholar, 17Ohara R. Hirota S. Onoue H. Nomura S. Kitamura Y. Toyoshima K. J. Cell Sci. 1995; 108: 97-103PubMed Google Scholar). Recently, Tpl-2 has also been found in malignancies associated with Epstein-Barr virus infection, where it acts as a mediator of latent membrane protein-1-induced NFκB activation (18Eliopoulos A.G. Davies C. Blake S.S. Murray P. Najafipour S. Tsichlis P.N. Young L.S. J. Virol. 2002; 76: 4567-4579Crossref PubMed Scopus (52) Google Scholar). Thus, cancers that have aberrant kinase activity upstream of IKK would be expected to have constitutively nuclear NFκB; this activity could, in turn, contribute to the expression of genes important to the survival of those cancers. As mentioned above, several M3K family members have been found to act upstream of IKK. These kinases additionally activate members of the mitogen-activated protein kinase (MAPK) cascade, leading to the activation of Jun N-terminal kinase (JNK), p38 MAPK, and/or extracellular signal-regulated protein kinase (ERK) (19Hagemann C. Blank J.L. Cell. Signal. 2001; 13: 863-875Crossref PubMed Scopus (245) Google Scholar). In melanoma cells, the NFκB activity downstream of NFκB-inducing kinase (NIK) appears to be dependent not only on the activation of IKK but also on NIK-regulated activation of the MEK/ERK cascade (8Dhawan P. Richmond A. J. Biol. Chem. 2002; 277: 7920-7928Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Other reports implicating downstream MAPK signaling kinases in the activation of NFκB include the demonstration that ERK5 and ERK2 cooperatively regulate NFκB activity in NIH 3T3 cells and the suggestion that persistent activation of NFκB by IL-1 is mediated by the MEK/ERK pathway (20Pearson G. English J.M. White M.A. Cobb M.H. J. Biol. Chem. 2001; 276: 7927-7931Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 21Jiang B. Brecher P. Cohen R.A. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1915-1920Crossref PubMed Scopus (73) Google Scholar). The activation of JNK may also contribute to NFκB activity through the induction of β-transducin repeat-containing protein, which mediates ubiquitination of phosphorylated IκB (22Spiegelman V.S. Stavropoulos P. Latres E. Pagano M. Ronai Z. Slaga T.J. Fuchs S.Y. J. Biol. Chem. 2001; 276: 27152-27158Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Thus, several different MAPK proteins may be capable of activating NFκB independently of IKK. It is possible, in terms of cell survival, that this parallel pathway plays an important and redundant role to IKK-induced NFκB activity. We previously described the NFκB status in two murine B-cell lymphoma cell lines, LY-as and LY-ar. The parent line, LY-as, was found to be lacking in NFκB activity, whereas the derived line, LY-ar, had constitutively nuclear NFκB1 (p50) homodimers that apparently contribute to the expression of the bcl-2 gene (23Kurland J.F. Kodym R. Story M.D. Spurgers K.B. McDonnell T.J. Meyn R.E. J. Biol. Chem. 2001; 276: 45380-45386Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In the present study, we used MEK inhibitors to establish a role for the MEK/ERK pathway upstream of p50 homodimer activity and Bcl-2 expression and demonstrate that MEK inhibitors sensitize normally radioresistant LY-ar cells to radiation-induced apoptosis. This study provides evidence that the activation of the MEK/ERK pathway could be an important step in the progression of lymphoma from an apoptosis-sensitive to an apoptosis-resistant phenotype. These data may therefore have clinical implications for the treatment of advanced cancers with therapeutic agents that induce apoptosis. Cell Culture and Treatments—LY-ar and LY-as murine B-cell lymphoma cell lines were maintained in RPMI 1640 culture medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) 200 mm l-glutamine, and 1% (v/v) 10,000 units/ml penicillin/streptomycin in an atmosphere of 5% CO2 at 37 °C. Cells were treated with MEK inhibitors at final concentrations of 50 μm PD 98059 (Calbiochem), 10 μm U0126 (Calbiochem), or 1 μm PD 184352 (a gift from Dr. Michael Andreeff, University of Texas M. D. Anderson Cancer Center, Houston, TX) in dimethyl sulfoxide. Treatment times for MEK inhibitors varied from 1 to 120 h. For experiments requiring 96 and 120 h treatment times, cells were spun down and resuspended in fresh medium at 72 h, at which time fresh MEK inhibitors were added. In some experiments, either TPA (Sigma) in 100% ethanol or recombinant murine TNFα (Sigma) was added to the culture medium at final concentrations of 50 ng/ml or 15 ng/ml, respectively. Fluorescence-activated Cell Sorter (FACS) Sorting and Analysis to Establish Stable LY-as Cell Lines Expressing a bcl-2 Construct—A retroviral infection strategy was adopted to deliver both human bcl-2 and the gene encoding green fluorescent protein (GFP) on a single construct containing an internal ribosomal entry site. Target LY-as cells were infected with retrovirus produced by PHOENIX-Ampho cells using standard polybrene-enhanced retroviral infection. Virus-treated LY-as cells were grown for 2–3 days and bulk-sorted to enrich for GFP expression. After several growth and sorting cycles, GFP-expressing cells were cloned through single-cell sorting by fluorescence-activated cell sorter. Stable clones that expressed both GFP and Bcl-2 (as detected by Western blot) were chosen for future experiments. Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared as described previously (24Dyer R.B. Herzog N.K. Biotechniques. 1995; 19: 192-195PubMed Google Scholar), and 15 μg of nuclear protein was incubated with 32P-labeled NFκB oligonucleotide as described by the manufacturer (Promega) except that .07 pmol of radiolabeled consensus oligonucleotide probe per reaction was used rather than .035 pmol. Bound probe was resolved from free probe using 4% native gels run at 180 V at 4 °C for ∼2.5 h. Gels were visualized by phosphorimaging. Western Blots—Whole-cell lysates were prepared in a lysis buffer containing 1% Triton X-100, 50 mm HEPES, pH 7.8, 150 mm NaCl, 1.5 mm MgCl2, 1.0 mm EDTA, 100 mm NaF, 1 mm Na3VO4, 2 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, 1% v/v protease inhibitor mixture (Sigma), 1% v/v phosphatase inhibitor mixture I (Sigma), 1% v/v phosphatase inhibitor mixture II (Sigma), and 10% glycerol. 25 μgof protein per lane was loaded on 5% stacking/10% resolving polyacrylamide gels and run for ∼1 h at 20 mA. Protein bands were then transferred onto polyvinylidene difluoride membrane at 100 V for1hat 4 °C. Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline with Tween 20 and probed with primary antibody overnight. Membranes were then washed in Tris-buffered saline with Tween 20 to remove excess primary antibody and probed with horseradish peroxidase-conjugated anti-Syrian hamster (Jackson Laboratories), anti-rabbit (Amersham Biosciences), or anti-mouse (Amersham Biosciences) secondary antibody for roughly 2–3 h. Blots were developed with ECL+ and visualized by fluorescence scanning and ImageQuant™ analysis software. Mouse Bcl-2 antibody (BD PharMingen) dilution was 1:2000 and secondary antibody dilution was 1:3000. Human Bcl-2 antibody (Dako) dilution was 1:1000, and secondary antibody dilution was 1:1000. Phospho-p44/p42 antibody (Cell Signaling Technology) dilution was 1:1000, and secondary antibody dilution was 1:2000. Total p44/p42 antibody (Cell Signaling) dilution was 1:2000, and secondary antibody dilution was 1:2000. Actin antibody (Chemicon) dilution was 1:4000, and secondary antibody dilution was 1:4000. DNA Fragmentation—Enzymatically induced DNA fragmentation as a result of apoptosis was quantified as described previously (25Story M.D. Voehringer D.W. Malone C.G. Hobbs M.L. Meyn R.E. Int. J. Radiat. Biol. 1994; 66: 659-668PubMed Google Scholar). Briefly, cells were prelabeled with 10 nCi/ml of [14C]thymidine and then incubated for 72 or 96 h in the presence of the MEK inhibitors described above. The cells were then exposed to 5 Gy of γ irradiation from a high dose-rate 137Cs unit (4–5 Gy/min) at room temperature. Cells were collected 4 h later, washed in phosphate-buffered saline, and lysed by incubation in 0.5 ml of lytic buffer (10 mm Tris, 1 mm EDTA, and 0.2% Triton X-100) for 20 min on ice. Insoluble chromatin was separated from soluble DNA fragments by centrifugation at 13,000 × g for 10 min. Soluble and insoluble fractions were transferred to scintillation vials containing 1 ml of Soluene 350 (Packard) and incubated overnight at 60 °C. Ten milliliters of Hionic Fluor (Packard) was added, and samples were counted in a liquid scintillation counter. DNA fragmentation was quantified as the percentage of the total radioactivity that appeared in the soluble fraction. LY-ar Cells Have Constitutively Phosphorylated ERK1 and ERK2 (p44 and p42), whereas LY-as Cells Lack Phosphorylated ERK1 and ERK2 Proteins—Two murine B-cell lymphoma cell lines, isolated from a mouse lymphoma (LY-TH) and grown in vitro, were used for these studies. LY-as and LY-ar cells have been described previously for their differences in radiationinduced apoptotic response (25Story M.D. Voehringer D.W. Malone C.G. Hobbs M.L. Meyn R.E. Int. J. Radiat. Biol. 1994; 66: 659-668PubMed Google Scholar). To determine whether the MEK1/MEK2 pathway was activated in these cell lines, Western blot analysis was performed using anti-phospho-p44 and -p42 antibodies. The two cell lines had equal expression of total p44 and p42 proteins, but only the LY-ar line possessed constitutively phosphorylated p44 and p42 (Fig. 1). These data indicate that LY-ar cells have a constitutively active MAPK pathway. Treatment of LY-ar Cells with MEK Inhibitors PD 98059, U0126, or PD 184352 Leads to Inhibition of ERK1 and ERK2 Phosphorylation—To reverse ERK1/ERK2 activation, LY-ar cells were treated with the MEK inhibitor PD 98059, which has been shown along with the inhibitors U0126 and PD 184352 to be highly specific for MEK kinases based on its inability to inhibit over 30 other kinases as part of a specificity screen (26Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3944) Google Scholar). Treatment with PD 98059 for 24 and 48 h led to decreases in phospho-p44 and -p42 as detected by Western blot (Fig. 2A). Similar results were obtained when LY-ar cells were treated with U0126 or PD 184352 for 48 h (Fig. 2B). To determine how rapidly p44 and p42 phosphorylation was diminished with MEK inhibitor treatment, we also treated LY-ar cells with PD 184352 for 1 h. This treatment also led to decreased p44 and p42 phosphorylation (Fig. 2C). It should be noted that the MEK inhibitors PD 98059 and U0126 additionally have been reported to inhibit ERK5 phosphorylation by inhibiting MEK5 activity (27Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar); however, Western blot demonstrated that whole-cell lysates from LY-as and LY-ar cells do not differ in their amounts of phosphorylated ERK5. Moreover, treatment of LY-ar cells with PD 184352, which has been shown to be more specific for inhibiting the phosphorylation of ERK1/ERK2 than ERK5 (28Squires M.S. Nixon P.M. Cook S.J. Biochem. J. 2002; 366: 673-680Crossref PubMed Scopus (90) Google Scholar), did not result in a loss of ERK5 phosphorylation (data not shown). Thus, the ERK1/ERK2 pathway that is activated in LY-ar cells is more likely to contribute to their radioresistant phenotype than the ERK5 pathway. Taken together, the data in Fig. 2 demonstrate that MEK inhibitors very rapidly reverse the phosphorylation of ERK1 and ERK2 in LY-ar cells and maintain MEK inhibition for long periods of time. Treatment of LY-ar Cells with MEK Inhibitors Leads to Reversal of Nuclear p50 Homodimer DNA Binding Activity— Based on reports suggesting that the MEK/ERK pathway acts upstream of NFκB activation (8Dhawan P. Richmond A. J. Biol. Chem. 2002; 277: 7920-7928Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 20Pearson G. English J.M. White M.A. Cobb M.H. J. Biol. Chem. 2001; 276: 7927-7931Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 21Jiang B. Brecher P. Cohen R.A. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1915-1920Crossref PubMed Scopus (73) Google Scholar), we performed an EMSA on nuclear lysates prepared from PD 98059-treated LY-ar cells to determine whether MEK inhibition would lead to a loss of nuclear p50 homodimer activity. DNA binding was lost by 24 h of treatment with PD 98059, and binding remained inhibited at 48 and 72 h (Fig. 3A). Similar results were obtained using nuclear lysates prepared from LY-ar cells treated for 24 h with U0126 or PD 184352 (Fig. 3B). Because ERK1/ERK2 phosphorylation was lost very rapidly when LY-ar cells were treated with PD 184352 (Fig. 2C), we also performed EMSA to determine whether the loss of p50/p50 binding correlated directly with the loss of ERK1/ERK2 activity. Indeed, DNA binding was diminished at both 1 and 3 h of treatment with PD 184352 with losses in p50/p50 DNA binding intensity of 20 and 40%, respectively (Fig. 3C). These data suggest that the MEK/ERK pathway acts upstream of p50 homodimer activation in LY-ar cells. Treatment of LY-ar Cells with MEK Inhibitors Leads to Decreases in Bcl-2 Protein Expression—Because our previous data suggested that p50 homodimer activity in LY-ar cells contributed to the expression of the bcl-2 gene (23Kurland J.F. Kodym R. Story M.D. Spurgers K.B. McDonnell T.J. Meyn R.E. J. Biol. Chem. 2001; 276: 45380-45386Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), we investigated whether the reversal of nuclear p50 homodimer DNA binding by MEK inhibitors correlated with a loss in Bcl-2 protein expression. Because of the long half-life of Bcl-2 protein, Western blot was performed on whole-cell lysates prepared from LY-ar cells treated with PD 98059 for up to 120 h. Treatment of LY-ar cells with PD 98059 led to decreases in Bcl-2 protein expression with roughly 2- and 4-fold decreases in expression at 48 and 120 h, respectively (Fig. 4A). Similar results were obtained when Western blot was performed on LY-ar cells treated for 72 h with U0126 or PD 184352, with roughly 2-fold decreases for each (Fig. 4B). Treatment of LY-as Cells with TPA Induces ERK1 and ERK2 Phosphorylation as well as Bcl-2 Protein Expression That Can Be Blocked by PD 98059 —We previously demonstrated that the treatment of LY-as cells with the phorbol ester TPA led to an increase in Bcl-2 protein expression (23Kurland J.F. Kodym R. Story M.D. Spurgers K.B. McDonnell T.J. Meyn R.E. J. Biol. Chem. 2001; 276: 45380-45386Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To determine whether TPA treatment would also lead to an activation of ERK1/ERK2, whole-cell lysates were prepared from TPA-treated LY-as cells and analyzed by Western blot for phosphop44 and -p42. Treatment of LY-as cells with TPA led to phosphorylation of ERK proteins, and this activation was blocked by simultaneous treatment with PD 98059 (Fig. 5A). TPA-induced Bcl-2 protein expression was also blocked by simultaneous treatment with PD 98059 as detected by Western blot (Fig. 5B). LY-as Cells Retrovirally Infected with Human bcl-2 Lack Phosphorylated ERK1/ERK2 and Nuclear NFκB but Are Resistant to Radiation-induced Apoptosis—To directly test the role of Bcl-2 expression in blocking radiation-induced apoptosis, LY-as cells were infected with retroviral vectors containing human bcl-2 cDNA. Virally infected cells were selected and cloned into stable cell populations by detection of GFP expression downstream of the bcl-2 open reading frame and an internal ribosome entry site (IRES). These cells are referred to as the BIG cell line (Bcl-2-IRES-GFP). As a control, LY-as cells were also infected with a retroviral vector expressing GFP alone. These cells are referred to simply as the GFP cell line. To determine whether Bcl-2 expression in LY-as cells caused activation of the MEK/ERK pathway, Western blot for phosphop44 and p42 was performed using whole-cell lysates from GFP and BIG cells. Neither GFP nor BIG cells possessed phosphorylated p44 and p42 proteins (Fig. 6A). In contrast to reports by ourselves and others (23Kurland J.F. Kodym R. Story M.D. Spurgers K.B. McDonnell T.J. Meyn R.E. J. Biol. Chem. 2001; 276: 45380-45386Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 29Catz S.D. Johnson J.L. Oncogene. 2001; 20: 7342-7351Crossref PubMed Scopus (436) Google Scholar, 30Heckman C.A. Mehew J.W. Boxer L.M. Oncogene. 2002; 21: 3898-3908Crossref PubMed Scopus (168) Google Scholar) suggesting NFκB family members directly regulate transcription of the bcl-2 gene, some researchers have suggested that Bcl-2 can act upstream of NFκB, providing evidence that bcl-2 transfection causes NFκB activation (31de Moissac D. Mustapha S. Greenberg A.H. Kirshenbaum L.A. J. Biol. Chem. 1998; 273: 23946-23951Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 32de Moissac D. Zheng H. Kirshenbaum L.A. J. Biol. Chem. 1999; 274: 29505-29509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 33Regula K.M. Ens K. Kirshenbaum L.A. J. Biol. Chem. 2002; 277: 38676-38682Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To determine whether Bcl-2 expression in LY-as cells led to NFκB activation, EMSA was performed using nuclear lysates from GFP and BIG cells. Both cell types were treated with TPA as a positive control for NFκB binding. Like their parent cell type LY-as, neither GFP nor BIG cells had constitutively nuclear NFκB DNA binding; however, NFκB activation by TPA remained intact in both cell lines (Fig. 6B). Although Bcl-2 expression did not lead to the activation of ERK1/ERK2 or NFκB in the LY-as cell background, we performed DNA fragmentation assays on irradiated BIG cells to determine the effect of Bcl-2 expression on apoptosis. As expected, the BIG cell line was resistant to radiation-induced apoptosis (Fig. 6C). These data are consistent with a model in which activation of the MEK/ERK and NFκB1 pathways is upstream of Bcl-2 expression in LY-ar cells and that Bcl-2 expression is sufficient for resistance to radiation-induced apoptosis in this B-cell lymphoma cell system. Treatment of LY-ar Cells with TNFα in the Presence of PD 98059 Suggests That the MEK/ERK Pathway, Not IKK, Is Sufficient for p50 Homodimer Activity—We previously demonstrated that TNFα treatment led to the nuclear translocation of the p50/p65 NFκB dimer in LY-ar cells (23Kurland J.F. Kodym R. Story M.D. Spurgers K.B. McDonnell T.J. Meyn R.E. J. Biol. Chem. 2001; 276: 45380-45386Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In this case, TNFα did not affect the level of constitutive p50 homodimers, suggesting that TNFα activated p50/p65 via a separate pathway from that responsible for p50 homodimer activation. TNFα is the prototypal agent for inflammatory cytokine-induced activation of NFκB through activation of IKK (7Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4084) Google Scholar). Because p50 homodimers were sensitive to MEK inhibitors (Fig. 3, A–C) and unaffected by a known IKK activator, TNFα, p50 homodimer activity in LY-ar cells appears IKK-independent. To further test this possibility, LY-ar cells were maintained in the presence of PD 98059 for 24 h, treated for an additional 1 h with TNFα while in the presence of PD 98059, and prepared for EMSA. PD 98059 inhibited p50 homodimer DNA binding in both PD 98059-treated LY-ar cells and TNFα/PD 98059-treated LY-ar cells (Fig. 7). Similar results were obtained in TNFα/PD 184352-treated LY-ar and TPA/PD 98059-treated LY-as cells (data not shown). These data indicate that MEK inhibition blocks p50 homodimer activity, even in the presence of a presumably activated IKK signal, suggesting that p50 homodimer activity in LY-ar cells is IKK-independent. It should be noted that TNFα-induced p50/p65 heterodimer activity was partially abrogated by PD 98059 (Fig. 7), suggesting that, in addition to its control of p50 homodimers, the MEK/ERK pathway contributes to the activation of p50/p65 downstream of IKK in LY-ar cells. MEK Inhibitors Sensitize LY-ar Cells to Radiation-induced Apoptosis—Because of the known changes to cell survival pathways caused by such treatments, we investigated whether MEK inhibitors could sensitize LY-ar cells to radiation-induced apoptosis. LY-ar cells were pretreated for 72 or 96 h with the MEK inhibitor PD 980589, U0126, or PD 184352, irradiated with 5 Gy ionizing radiation, and analyzed for apoptosis on the basis of DNA fragmentation 4 h later. It should be noted that within the 4 h time frame that apoptosis is induced, phosphorylated ERK1/ERK2 and Bcl-2 protein levels are not elevated by irradiation (data not shown). All three inhibitors sensitized normally apoptosis-resistant LY-ar cells to radiation-induced apoptosis by 72 h of treatment, reaching DNA fragmentation levels that were similar to those observed for the parent cell type, LY-as, by 96 h (Fig. 8). These results provide evidence that MEK inhibitors radiosensitize LY-ar cells by restoring apoptosis propensity. In this study, we examined the role of the MEK/ERK signal transduction pathway in the activation of NFκB1 (p50) homodimers and expression of Bcl-2 in two murine B-cell lymphoma cell lines. Using highly specific MEK inhibitors, we obtained evidence that activation of the MEK/ERK pathway is associated with p50 homodimer activity and Bcl-2 protein expression in LY-as and LY-ar cells. Further evidence suggested that the MEK/ERK pathway and not IKK mediates p50 homodimer activation. Lastly, we demonstrated that MEK inhibitors sensitize LY-ar cells to radiation-induced apoptosis. Our finding that the MEK/ERK pathway affects p50 homodimer DNA binding is, to our knowledge, the first indication of this activity. Although the MEK/ERK pathway has been implicated in the activation of other NFκB dimer forms (8Dhawan P. Richmond A. J. Biol. Chem. 2002; 277: 7920-7928Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 20Pearson G. English J.M. White M.A. Cobb M.H. J. Biol. Chem. 2001; 276: 7927-7931Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 21Jiang B. Brecher P. Cohen R.A. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1915-1920Crossref PubMed Scopus (73) Google Scholar), the constitutive activity of p50 homodimers is considered to be the result of a rearrangement of p50-p105 heterodimers into p50 homodimers by the IκB family member Bcl-3 (34Watanabe N. Iwamura T. Shinoda T. Fujita T. EMBO J. 1997; 16: 3609-3620Crossref PubMed Scopus (97) Google Scholar). IL-9- and granulocyte macrophage-colony stimulating factor-induced Bcl-3 protein expressions have been linked to enhanced p50 homodimer activity by a similar mechanism (35Richard M. Louahed J. Demoulin J.B. Renauld J.C. Blood. 1999; 93: 4318-4327Crossref PubMed Google Scholar, 36Zhang M.Y. Harhaj E.W. Bell L. Sun S.C. Miller B.A. Blood. 1998; 92: 1225-1234Crossref PubMed Google Scholar). The ability of Bcl-3 to interact with and control p50 DNA-binding activity appears to be phosphorylation-dependent (37Nolan G.P. Fujita T. Bhatia K. Huppi C. Liou H.C. Scott M.L. Baltimore D. Mol. Cell. Biol. 1993; 13: 3557-3566Crossref PubMed Google Scholar, 38Caamano J.H. Perez P. Lira S.A. Bravo R. Mol. Cell. Biol. 1996; 16: 1342-1348Crossref PubMed Scopus (88) Google Scholar). We have found no difference in the amount of Bcl-3 protein expression between LY-as and LY-ar cells by Western blot (data not shown), but it is possible that the MEK/ERK pathway controls Bcl-3 phosphorylation, thereby affecting p50 homodimer formation and/or DNA binding in LY-ar cells. An alternative mechanism by which p50 homodimers become activated is through IKK activation. The p50 precursor, p105, has been shown to be a target for IKK phosphorylation during cytokine-induced responses that lead to the activation of both p50/p65 and p50/50 dimer forms (39Heissmeyer V. Krappmann D. Wulczyn F.G. Scheidereit C. EMBO J. 1999; 18: 4766-4778Crossref PubMed Scopus (173) Google Scholar). By a similar mechanism, the constitutive p50 homodimer signal in LY-ar cells may not be a result of MEK/ERK-induced Bcl-3 activity but rather a result of increased p105 processing directly or indirectly controlled by activation of the MEK/ERK pathway. LY-as cells and LY-ar cells are characterized by profound differences in their apoptotic responses. Changes in NFκB signaling and Bcl-2 expression are apparently responsible for those differences. Indeed, Bcl-2 expression alone can render LY-as cells resistant to radiation-induced apoptosis (Fig. 6C). The data presented here establish a role for the MEK/ERK pathway upstream of both NFκB and Bcl-2 cell survival pathways, implicating the MEK/ERK pathway as a key mediator of apoptosis propensity for these lymphoma cells. These data therefore suggest that activation of the MEK/ERK pathway may be an excellent marker of disease progression, and our data demonstrating that MEK inhibitors sensitize LY-ar cells to radiation-induced apoptosis may be relevant to other advanced cancers displaying aberrant MEK/ERK activity. MEK inhibitors have been used by other groups to successfully sensitize cells to anti-cancer therapies. For example, PD 98059 and PD 184352 have been used to impair growth, abrogate clonogenicity, and sensitize acute myelogenous leukemia (AML) cells to chemotherapy-induced apoptosis (40Milella M. Kornblau S.M. Estrov Z. Carter B.Z. Lapillonne H. Harris D. Konopleva M. Zhao S. Estey E. Andreeff M. J. Clin. Invest. 2001; 108: 851-859Crossref PubMed Scopus (287) Google Scholar, 41Milella M. Estrov Z. Kornblau S.M. Carter B.Z. Konopleva M. Tari A. Schober W.D. Harris D. Leysath C.E. Lopez-Berestein G. Huang Z. Andreeff M. Blood. 2002; 99: 3461-3464Crossref PubMed Scopus (115) Google Scholar). Similar to our observations, decreases in the expression of members of the Bcl-2 family were observed. In addition to AML, the MEK/ERK pathway has also been implicated in Bcl-2 family member expression and survival in pancreatic and breast cancer cells (42Boucher M.J. Morisset J. Vachon P.H. Reed J.C. Laine J. Rivard N. J. Cell. Biochem. 2000; 79: 355-369Crossref PubMed Scopus (370) Google Scholar, 43Hu Y. Dragowska W.H. Wallis A. Duronio V. Mayer L. Breast Cancer Res. Treat. 2001; 70: 11-20Crossref PubMed Scopus (16) Google Scholar), and Sebolt-Leopold et al. (44Sebolt-Leopold J.S. Dudley D.T. Herrera R. Van Becelaere K. Wiland A. Gowan R.C. Tecle H. Barrett S.D. Bridges A. Przybranowski S. Leopold W.R. Saltiel A.R. Nat. Med. 1999; 5: 810-816Crossref PubMed Scopus (897) Google Scholar) have demonstrated that PD 184352 administered either intraperitoneally or orally inhibited growth of colon tumors in vivo by as much as 80% with no signs of toxicity. Therefore, small molecule approaches to inhibiting MEK activity may be useful clinically in the treatment of a variety of tumor types. In fact, PD 184352, also known as CI-1040 (45Herrera R. Sebolt-Leopold J.S. Trends Mol. Med. 2002; 8: S27-S31Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), is currently in clinical trials for patients with advanced cancer. Although previous studies have suggested that MEK inhibitors suppress the growth of various cancers and enhance the effectiveness of certain treatment modalities, some researchers have suggested that MEK inhibitors are not useful for sensitizing carcinoma cells to ionizing radiation (46Gupta A.K. Bakanauskas V.J. Cerniglia G.J. Cheng Y. Bernhard E.J. Muschel R.J. McKenna W.G. Cancer Res. 2001; 61: 4278-4282PubMed Google Scholar, 47Grana T.M. Rusyn E.V. Zhou H. Sartor C.I. Cox A.D. Cancer Res. 2002; 62: 4142-4150PubMed Google Scholar, 48Belka C. Knippers P. Rudner J. Faltin H. Bamberg M. Budach W. Anticancer Res. 2000; 20: 3243-3249PubMed Google Scholar). However, Vrana et al. (49Vrana J.A. Grant S. Dent P. Radiat. Res. 1999; 151: 559-569Crossref PubMed Scopus (43) Google Scholar) have used PD 98059 in combination with ionizing radiation to treat HL-60 cells, which resulted in a large increase in apoptosis and a large decrease in clonogenicity when compared with either treatment alone. That study, in combination with the work presented here and recent findings from Shonai et al. (50Shonai T. Adachi M. Sakata K. Takekawa M. Endo T. Imai K. Hareyama M. Cell Death Differ. 2002; 9: 963-971Crossref PubMed Scopus (55) Google Scholar) implicating the MEK/ERK pathway in radioresistance of lymphocytic leukemia cells, suggests that MEK inhibitors may be primarily useful for radiosensitizing hematopoietic cancers. Our study defines a novel upstream signal to NFκB1 activation, i.e. the MEK/ERK pathway; however, the upstream signals leading to MEK/ERK activation in LY-ar cells remain to be elucidated. We have performed a Western blot analysis to demonstrate that the M3K Tpl-2 is overexpressed in LY-ar cells as compared with LY-as cells (data not shown). Tpl-2 directly interacts with p105, and its overexpression leads to increased turnover of p105 into p50 subunits (51Belich M.P. Salmeron A. Johnston L.H. Ley S.C. Nature. 1999; 397: 363-368Crossref PubMed Scopus (186) Google Scholar). Here, p50 homodimer activity appeared to be strictly under control of the MEK/ERK pathway. In addition to its interaction with p105, Tpl-2 has been shown to activate MEK (52Salmeron A. Ahmad T.B. Carlile G.W. Pappin D. Narsimhan R.P. Ley S.C. EMBO J. 1996; 15: 817-826Crossref PubMed Scopus (269) Google Scholar). Therefore, Tpl-2 is a good candidate for the upstream signal leading to the sole activation of p50 homodimers in LY-ar cells. It would be interesting to identify a distal upstream signal in LY-ar cells. In T-cells, Tpl-2 has been implicated in NFκB signaling derived from cell surface interactions such as CD28 stimulation (13Lin X. Cunningham Jr., E.T. Mu Y. Geleziunas R. Greene W.C. Immunity. 1999; 10: 271-280Abstract Full Text Full Text PDF PubMed Google Scholar). It is possible that Tpl-2 has a similar function in LY-ar cells, and the identification of a receptor on LY-ar cells that may be engaged upstream of the observed changes in the MEK/ERK and NFκB signaling pathways is an aim we are actively pursuing. In summary, the MEK/ERK pathway is a subject receiving a lot of attention at the basic science level as well as in the clinic. The identification of signaling pathways associated with its activity will undoubtedly shed light on its role in cancer and will hopefully lead to novel strategies that reverse its effects. We thank Dr. Michael Andreeff for kindly providing PD 184352, Marvette Hobbs for technical assistance, and Katie Matias for assisting in the preparation of this manuscript.
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