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• W1971509320 abstract "Cell-surface protein CD10 is a prognostic marker for diffuse large B-cell lymphoma (DLBCL), where high expression of CD10 is found in the germinal center B-cell (GCB) subtype and CD10 expression is low or absent in the activated B-cell (ABC) subtype. As compared with the GCB subtype, patients with ABC DLBCL have a poorer prognosis after standard treatment, and ABC tumor cells have higher NF-κB activity. Herein, we show that increased expression of the NF-κB target micro-RNA miR-155 is correlated with reduced expression of transcription factor PU.1 and CD10 in several B-lymphoma cell lines. Moreover, electromobility shift assays and luciferase reporter assays indicate that PU.1 can directly activate expression from the CD10 promoter. Expression of a DLBCL-derived mutant of the adaptor CARD11 (a constitutive activator of NF-κB) in the GCB-like human BJAB cell line or v-Rel in the chicken DT40 B-lymphoma cell line causes reduced expression of PU.1. The CARD11 mutant also causes a decrease in CD10 levels in BJAB cells. Similarly, overexpression of miR-155, which is known to down-regulate PU.1, leads to reduced expression of CD10 in BJAB cells. Finally, we show that CD10 expression is reduced in BJAB cells after treatment with the NF-κB inducer lipopolysaccharide (LPS). Additionally, miR-155 is induced by LPS treatment or expression of the CARD11 mutant in BJAB cells. These results point to an NF-κB-dependent mechanism for down-regulation of CD10 in B-cell lymphoma: namely, that increased NF-κB activity leads to increased miR-155, which results in decreased PU.1, and consequently reduced CD10 mRNA and protein. Cell-surface protein CD10 is a prognostic marker for diffuse large B-cell lymphoma (DLBCL), where high expression of CD10 is found in the germinal center B-cell (GCB) subtype and CD10 expression is low or absent in the activated B-cell (ABC) subtype. As compared with the GCB subtype, patients with ABC DLBCL have a poorer prognosis after standard treatment, and ABC tumor cells have higher NF-κB activity. Herein, we show that increased expression of the NF-κB target micro-RNA miR-155 is correlated with reduced expression of transcription factor PU.1 and CD10 in several B-lymphoma cell lines. Moreover, electromobility shift assays and luciferase reporter assays indicate that PU.1 can directly activate expression from the CD10 promoter. Expression of a DLBCL-derived mutant of the adaptor CARD11 (a constitutive activator of NF-κB) in the GCB-like human BJAB cell line or v-Rel in the chicken DT40 B-lymphoma cell line causes reduced expression of PU.1. The CARD11 mutant also causes a decrease in CD10 levels in BJAB cells. Similarly, overexpression of miR-155, which is known to down-regulate PU.1, leads to reduced expression of CD10 in BJAB cells. Finally, we show that CD10 expression is reduced in BJAB cells after treatment with the NF-κB inducer lipopolysaccharide (LPS). Additionally, miR-155 is induced by LPS treatment or expression of the CARD11 mutant in BJAB cells. These results point to an NF-κB-dependent mechanism for down-regulation of CD10 in B-cell lymphoma: namely, that increased NF-κB activity leads to increased miR-155, which results in decreased PU.1, and consequently reduced CD10 mRNA and protein. The NF-κB signaling pathway is misregulated in a variety of human diseases including many chronic inflammatory diseases and cancers. As such, an understanding of the molecular details of NF-κB-dependent gene networks has implications for improved disease diagnoses and therapies. CD10, also known as the common acute lymphocytic leukemia antigen (CALLA) or neutral endopeptidase, is a cell-surface zinc metalloendopeptidase (1Shipp M.A. Vijayaraghavan J. Schmidt E.V. Masteller E.L. D'Adamio L. Hersh L.B. Reinherz E.L. Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 297-301Crossref PubMed Scopus (177) Google Scholar, 2Salles G. Chen C.Y. Reinherz E.L. Shipp M.A. Blood. 1992; 80: 2021-2029Crossref PubMed Google Scholar). The ability of CD10 to cleave signal peptides at the cell surface can affect cell proliferation, differentiation, and migration (2Salles G. Chen C.Y. Reinherz E.L. Shipp M.A. Blood. 1992; 80: 2021-2029Crossref PubMed Google Scholar, 3Sunday M.E. Hua J. Torday J.S. Reyes B. Shipp M.A. J. Clin. Invest. 1992; 90: 2517-2525Crossref PubMed Scopus (72) Google Scholar, 4Sumitomo M. Shen R. Nanus D.M. Biochim. Biophys. Acta. 2005; 1751: 52-59Crossref PubMed Scopus (90) Google Scholar). Expression of CD10 can be used as a diagnostic marker for a variety of cancers (5Ishimaru F. Potter N.S. Shipp M.A. Exp. Hematol. 1996; 24: 43-48PubMed Google Scholar, 6Langner C. Ratschek M. Rehak P. Schips L. Zigeuner R. Histopathology. 2004; 45: 460-467Crossref PubMed Scopus (49) Google Scholar, 7Deschamps L. Handra-Luca A. O'Toole D. Sauvanet A. Ruszniewski P. Belghiti J. Bedossa P. Couvelard A. Hum. Pathol. 2006; 37: 802-808Crossref PubMed Scopus (44) Google Scholar, 8Alizadeh A.A. Eisen M.B. Davis R.E. Ma C. Lossos I.S. Rosenwald A. Boldrick J.C. Sabet H. Tran T. Yu X. Powell J.I. Yang L. Marti G.E. Moore T. Hudson Jr., J. Lu L. Lewis D.B. Tibshirani R. Sherlock G. Chan W.C. Greiner T.C. Weisenburger D.D. Armitage J.O. Warnke R. Levy R. Wilson W. Grever M.R. Byrd J.C. Botstein D. Brown P.O. Staudt L.M. Nature. 2000; 403: 503-511Crossref PubMed Scopus (7946) Google Scholar, 9Wright G. Tan B. Rosenwald A. Hurt E.H. Wiestner A. Staudt L.M. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9991-9996Crossref PubMed Scopus (824) Google Scholar). Relevant to this study, CD10 is highly expressed in the germinal center B-cell (GCB) 6The abbreviations used are: GCBgerminal center B-cellDLBCLdiffuse large B-cell lymphomaABCactivated B-cellmiRmicro-RNABICB-cell integration clusterqPCRquantitative PCRLMP1latent membrane protein-1. molecular subtype of diffuse large B-cell lymphoma (DLBCL), whereas CD10 expression is low in the activated B-cell (ABC) subtype of DLBCL (8Alizadeh A.A. Eisen M.B. Davis R.E. Ma C. Lossos I.S. Rosenwald A. Boldrick J.C. Sabet H. Tran T. Yu X. Powell J.I. Yang L. Marti G.E. Moore T. Hudson Jr., J. Lu L. Lewis D.B. Tibshirani R. Sherlock G. Chan W.C. Greiner T.C. Weisenburger D.D. Armitage J.O. Warnke R. Levy R. Wilson W. Grever M.R. Byrd J.C. Botstein D. Brown P.O. Staudt L.M. Nature. 2000; 403: 503-511Crossref PubMed Scopus (7946) Google Scholar, 9Wright G. Tan B. Rosenwald A. Hurt E.H. Wiestner A. Staudt L.M. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9991-9996Crossref PubMed Scopus (824) Google Scholar). ABC DLBCLs also have a high NF-κB gene expression profile and a poorer clinical prognosis as compared with GCB DLBCLs (10Staudt L.M. Dave S. Adv. Immunol. 2005; 87: 163-208Crossref PubMed Scopus (205) Google Scholar). As such, reduced expression of CD10 and high NF-κB activity are both correlated with a less favorable DLBCL patient outcome (10Staudt L.M. Dave S. Adv. Immunol. 2005; 87: 163-208Crossref PubMed Scopus (205) Google Scholar, 11Hans C.P. Weisenburger D.D. Greiner T.C. Gascoyne R.D. Delabie J. Ott G. Müller-Hermelink H.K. Campo E. Braziel R.M. Jaffe E.S. Pan Z. Farinha P. Smith L.M. Falini B. Banham A.H. Rosenwald A. Staudt L.M. Connors J.M. Armitage J.O. Chan W.C. Blood. 2004; 103: 275-282Crossref PubMed Scopus (3140) Google Scholar, 12Iqbal J. Joshi S. Patel K.N. Javed S.I. Kucuk C. Aabida A. d'Amore F. Fu K. Indian J. Cancer. 2007; 44: 72-86Crossref PubMed Google Scholar). germinal center B-cell diffuse large B-cell lymphoma activated B-cell micro-RNA B-cell integration cluster quantitative PCR latent membrane protein-1. The correlation between high NF-κB activity and reduced CD10 expression has been observed in several other settings as well. We previously showed that overexpression of an activated mutant of the NF-κB family transcription factor REL in the GCB-like B-lymphoma cell line BJAB leads to reduced expression of CD10 (13Chin M. Herscovitch M. Zhang N. Waxman D.J. Gilmore T.D. Oncogene. 2009; 28: 2100-2111Crossref PubMed Scopus (25) Google Scholar). Infection of cells with Epstein-Barr virus (EBV) or human cytomegalovirus, both inducers of NF-κB, causes reduced expression of CD10 (14Carter K.L. Cahir-McFarland E. Kieff E. J. Virol. 2002; 76: 10427-10436Crossref PubMed Scopus (124) Google Scholar, 15Phillips A.J. Tomasec P. Wang E.C. Wilkinson G.W. Borysiewicz L.K. Virology. 1998; 250: 350-358Crossref PubMed Scopus (23) Google Scholar). Taken together, such results suggested to us that NF-κB or a target of NF-κB is involved in repressing CD10 gene/protein expression in certain B-cell lymphomas. Little is known about the control of CD10 transcription. Sequence analysis of the CD10 promoter/enhancer region revealed the presence of three consensus binding sites for transcription factor PU.1 (16Ishimaru F. Shipp M.A. Blood. 1995; 85: 3199-3207Crossref PubMed Google Scholar). PU.1 is a member of the Ets family of transcription factors and is required for proper B-cell development and differentiation (17Medina K.L. Pongubala J.M. Reddy K.L. Lancki D.W. Dekoter R. Kieslinger M. Grosschedl R. Singh H. Dev. Cell. 2004; 7: 607-617Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Additionally, increased PU.1 expression has been correlated with the GCB subtype of DLBCL (8Alizadeh A.A. Eisen M.B. Davis R.E. Ma C. Lossos I.S. Rosenwald A. Boldrick J.C. Sabet H. Tran T. Yu X. Powell J.I. Yang L. Marti G.E. Moore T. Hudson Jr., J. Lu L. Lewis D.B. Tibshirani R. Sherlock G. Chan W.C. Greiner T.C. Weisenburger D.D. Armitage J.O. Warnke R. Levy R. Wilson W. Grever M.R. Byrd J.C. Botstein D. Brown P.O. Staudt L.M. Nature. 2000; 403: 503-511Crossref PubMed Scopus (7946) Google Scholar). Therefore, we were interested in investigating whether PU.1 contributes to the regulation of CD10 expression in B-cell lymphoma. In this report, we provide evidence for a functional link between activation of NF-κB and reduced expression of CD10. We show that activation of NF-κB in the GCB-like DLBCL cell line BJAB leads to reduced CD10 expression. Our data are consistent with a pathway in which NF-κB-induced up-regulation of micro-RNA miR-155 leads to down-regulation of PU.1 protein levels, and consequently, reduced levels of CD10. Moreover, we show that there is a correlation between high miR-155 expression and low PU.1/CD10 levels in a variety of B-lymphoma cell lines. These results are significant in that they reveal a molecular mechanism for down-regulation of CD10, a key marker for predicting the clinical response of patients with DLBCL. Human A293, A293T, Bosc23, BJAB, SUDHL-4, BL41, Daudi, IB4, RC-K8, and chicken DT40 cells (gift of Céline Gélinas) (18Gupta N. Delrow J. Drawid A. Sengupta A.M. Fan G. Gélinas C. Cancer Res. 2008; 68: 808-814Crossref PubMed Scopus (15) Google Scholar), were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with either 10 or 20% heat-inactivated fetal bovine serum (FBS) (Biologos) as described (19Liang M.C. Bardhan S. Pace E.A. Rosman D. Beutler J.A. Porco Jr., J.A. Gilmore T.D. Biochem. Pharmacol. 2006; 71: 634-645Crossref PubMed Scopus (77) Google Scholar). The six human B-lymphoma cell lines used in this study are as follows: BJAB, EBV-negative, GCB-like DLBCL (20Menezes J. Leibold W. Klein G. Clements G. Biomedicine. 1975; 22: 276-284PubMed Google Scholar, 21Ngo V.N. Davis R.E. Lamy L. Yu X. Zhao H. Lenz G. Lam L.T. Dave S. Yang L. Powell J. Staudt L.M. Nature. 2006; 441: 106-110Crossref PubMed Scopus (511) Google Scholar); SUDHL-4, a B-lymphoma that has been characterized as both a follicular lymophoma (22Vaughn C.P. Crockett D.K. Lin Z. Lim M.S. Elenitoba-Johnson K.S. Proteomics. 2006; 6: 3223-3230Crossref PubMed Scopus (10) Google Scholar) and GCB-like DLBCL (23Davis R.E. Brown K.D. Siebenlist U. Staudt L.M. J. Exp. Med. 2001; 194: 1861-1874Crossref PubMed Scopus (880) Google Scholar); BL-41, EBV-negative Burkitt lymphoma (14Carter K.L. Cahir-McFarland E. Kieff E. J. Virol. 2002; 76: 10427-10436Crossref PubMed Scopus (124) Google Scholar); Daudi, EBV-positive, LMP1-negative Burkitt lymphoma (24Contreras-Salazar B. Ehlin-Henriksson B. Klein G. Masucci M.G. J. Virol. 1990; 64: 5441-5447Crossref PubMed Google Scholar); IB4, EBV-transformed lymphoblastoid cell line (14Carter K.L. Cahir-McFarland E. Kieff E. J. Virol. 2002; 76: 10427-10436Crossref PubMed Scopus (124) Google Scholar); and RC-K8, ABC-like DLBCL (25Kalaitzidis D. Davis R.E. Rosenwald A. Staudt L.M. Gilmore T.D. Oncogene. 2002; 21: 8759-8768Crossref PubMed Scopus (43) Google Scholar). For treatment of cells with lipopolysaccharide (LPS) (Sigma), BJAB cells were first cultured in DMEM containing 5% FBS for 24 h and then treated with 5 μg/ml of LPS for the indicated times. All primers used in the study were purchased from Invitrogen and are described under supplemental Table S1. pcDNA-PU.1 was a gift of Barbara Nikolajczyk (Boston University Medical School) and has been described previously (26Pongubala J.M. Van Beveren C. Nagulapalli S. Klemsz M.J. McKercher S.R. Maki R.A. Atchison M.L. Science. 1993; 259: 1622-1625Crossref PubMed Scopus (241) Google Scholar). To create pCD10-luc, a 717-bp region of the CD10 promoter, containing the three consensus PU.1 binding sites, was amplified by PCR from BJAB cell genomic DNA. The PCR product was digested with KpnI and XmaI, and this fragment was subcloned into the pGL3 promoter vector (Promega). For mutagenesis of the PU-1 site in the CD10 promoter, two overlapping primers were used that contained the desired mutations, as shown under supplemental Table S1. Transfections were performed using polyethylenimine (Polysciences, Inc.). On the day of transfection, cells were washed twice with TD buffer (25 mm Tris-HCl, pH 7.4, 137 mm NaCl, 5 mm KCl, 0.7 mm Na2HPO4) and were then incubated with a plasmid DNA:polyethylenimine ratio of 1:3 in 300 μl of DMEM for 15 min at room temperature. For transfection of Bosc23 cells with pSIREN and pVpack vectors, a plasmid DNA:polyethylenimine ratio of 2:3 in 300 μl of DMEM was used. The DNA mixture was added to 2 ml (35-mm plate) or 4.5 ml (60-mm plate) of DMEM containing 10% FBS and this mixture was then added to the cells. The next day, the medium was replaced with fresh DMEM containing 10% FBS. For reporter gene assays, cells were lysed 24 h later. The retroviral vector pMSCV has been described previously (27Gilmore T.D. Jean-Jacques J. Richards R. Cormier C. Kim J. Kalaitzidis D. Virology. 2003; 316: 9-16Crossref PubMed Scopus (9) Google Scholar). pMSCV-CARD11mut10 was created by subcloning a BamHI fragment containing the CARD11 mutant 10 cDNA (a gift of Georg Lenz, National Cancer Institute) into BglII-digested pMSCV. pMSCV-BIC was created by subcloning a BsrGI-EcoRI fragment from the BIC cDNA (a gift of Ricardo Aguiar, The University of Texas Health Science Center at San Antonio) into BsrGI-EcoRI-digested pMSCV. For overexpression of shRNAs, the retroviral vector pSIREN-RetroQ was used (a gift of Ulla Hansen, Boston University). The shRNA against HMGN1 (28Zhu N. Hansen U. Mol. Cell. Biol. 2007; 27: 8859-8873Crossref PubMed Scopus (19) Google Scholar) was excised from this vector and a BamHI-EcoRI fragment from the multiple cloning site of pSL1180 (Amersham Biosciences) was subcloned in to create pSIREN-MCS. All shRNAs (supplemental Table S1) were ligated into pSIREN-MCS using the BamHI-EcoRI sites. The shRNA against PU.1 (29Nagel S. Scherr M. Kel A. Hornischer K. Crawford G.E. Kaufmann M. Meyer C. Drexler H.G. MacLeod R.A. Cancer Res. 2007; 67: 1461-1471Crossref PubMed Scopus (63) Google Scholar) and the control shRNA (30Scherr M. Battmer K. Ganser A. Eder M. Cell Cycle. 2003; 2: 251-257Crossref PubMed Scopus (78) Google Scholar) have been previously described (also see supplemental Table S1). The retroviral, doxycycline-inducible system has been previously described (31Gossen M. Freundlieb S. Bender G. Müller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2026) Google Scholar). pRetroX-tight-CARD11mut10HA-Hyg was created by subcloning a BamHI/NotI fragment containing CARD11mut10 from the pMSCV-CARD11mut10 vector described above. Virus stocks were generated by transfecting A293T cells with pMSCV, pMSCV-CARD11mut10, pMSCV-BIC, pRetroX-tight-Hyg, or pRetroX-tight-CARD11mut10HA-Hyg plus helper plasmid pcL10a1, as described previously (13Chin M. Herscovitch M. Zhang N. Waxman D.J. Gilmore T.D. Oncogene. 2009; 28: 2100-2111Crossref PubMed Scopus (25) Google Scholar, 27Gilmore T.D. Jean-Jacques J. Richards R. Cormier C. Kim J. Kalaitzidis D. Virology. 2003; 316: 9-16Crossref PubMed Scopus (9) Google Scholar). Virus stocks for pSIREN-RetroQ vectors were generated in Bosc23 cells using helper vectors pVpack-GP and pVpack-VSV (28Zhu N. Hansen U. Mol. Cell. Biol. 2007; 27: 8859-8873Crossref PubMed Scopus (19) Google Scholar). Two days later, virus was harvested. Two ml of virus (in the presence of 8 μg/ml of Polybrene) was used to infect 106 BJAB cells using the spin infection method (13Chin M. Herscovitch M. Zhang N. Waxman D.J. Gilmore T.D. Oncogene. 2009; 28: 2100-2111Crossref PubMed Scopus (25) Google Scholar). Two days later, cells were selected with 2.5 μg/ml of puromycin (Sigma) or 400 μg/ml of hygromycin (Sigma), with fluid changing every 5 days in media containing puromycin or hygromycin for 2–4 weeks. Total RNA was isolated using TRIzol (Invitrogen). Semi-quantitative RT-PCR was performed as described previously (32Leeman J.R. Weniger M.A. Barth T.F. Gilmore T.D. Oncogene. 2008; 27: 6770-6781Crossref PubMed Scopus (20) Google Scholar) with the primer sets listed under supplemental Table S1. Semi-quantitative RT-PCR for miR-155 and 5S ribosomal RNAs was performed by first reverse transcribing the RNA using specific stem loop primers, which contain an annealing site for the universal reverse primer (supplemental Table S1) and the TaqMan miRNA Reverse Transcriptase kit (Applied Biosystems) as described previously (33Chen C. Ridzon D.A. Broomer A.J. Zhou Z. Lee D.H. Nguyen J.T. Barbisin M. Xu N.L. Mahuvakar V.R. Andersen M.R. Lao K.Q. Livak K.J. Guegler K.J. Nucleic Acids Res. 2005; 33: e179Crossref PubMed Scopus (3992) Google Scholar). miR-155 and 5S sequences were then amplified using a forward-specific primer and a universal reverse primer (supplemental Table S1). Real time PCR was performed using the 7900HT Fast real time PCR system (Applied Biosystems) with the Power SYBR Green PCR Master Mix (Applied Biosystems). Quantification of miR-155 RNA by real time PCR was performed three times with triplicate samples and values were normalized to the 5S values. The final values represent relative expression (miR-155/5S) as compared with control samples. Western blotting was performed as described (25Kalaitzidis D. Davis R.E. Rosenwald A. Staudt L.M. Gilmore T.D. Oncogene. 2002; 21: 8759-8768Crossref PubMed Scopus (43) Google Scholar). Whole cell extracts were prepared in AT buffer (20 mm HEPES, pH 7.9, 1 mm EDTA, 1 mm EGTA, 20 mm Na4P2O7, 1 mm DTT, 1% (v/v) Triton X-100, 20% (w/v) glycerol, 1 mm Na3VO4, 1 μg/ml of PMSF, 1 μg/ml of leupeptin, 1 μg/ml of pepstatin). Samples were boiled in SDS sample buffer (62.5 mm Tris-HCl, pH 6.8, 3.2% (w/v) SDS, 10% (w/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.1% (w/v) bromphenol blue). Samples containing equal amounts of protein were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Micron Separation Inc.). Antibodies against PU.1 (sc-352), CD10 (sc-58939), HA (sc-805), and β-tubulin (sc-9104) were purchased from Santa Cruz Biotechnology. The BCL2 antibody was obtained from BD Transduction Laboratories (number 610538). Anti-REL antiserum (number 265) was a kind gift of Nancy Rice, and antiserum against v-Rel was described previously (27Gilmore T.D. Jean-Jacques J. Richards R. Cormier C. Kim J. Kalaitzidis D. Virology. 2003; 316: 9-16Crossref PubMed Scopus (9) Google Scholar). Nitrocellulose filters were incubated with primary antiserum for 2–18 h at room temperature or 4 °C. The appropriate horseradish peroxidase-labeled secondary antiserum was added and immunoreactive proteins were detected with the SuperSignal Dura West Extended Duration Substrate chemiluminescence detection system (Pierce). EMSAs were performed using 10 μg of nuclear extract prepared from A293 cells, A293 cells transfected with pcDNA-PU.1, and BJAB cells stably transduced with MSCV, MSCV-CARD11mut10, or MSCV-RELΔTAD1 as described previously (13Chin M. Herscovitch M. Zhang N. Waxman D.J. Gilmore T.D. Oncogene. 2009; 28: 2100-2111Crossref PubMed Scopus (25) Google Scholar, 25Kalaitzidis D. Davis R.E. Rosenwald A. Staudt L.M. Gilmore T.D. Oncogene. 2002; 21: 8759-8768Crossref PubMed Scopus (43) Google Scholar). Nuclear extracts were incubated with 2 μg of poly(dI-dC), and 32P-labeled PU.1 or NF-κB site probes (supplemental Table S1) in binding buffer (25 mm Tris-HCl, pH 7.4, 100 mm KCl, 0.5 mm EDTA, 6.25 mm MgCl2, 0.5 mm DTT, 10% (w/v) glycerol) in a final reaction volume of 50 μl, as previously described (25Kalaitzidis D. Davis R.E. Rosenwald A. Staudt L.M. Gilmore T.D. Oncogene. 2002; 21: 8759-8768Crossref PubMed Scopus (43) Google Scholar). DNA-binding reactions were carried out for 30 min at room temperature. For supershifts, 3 μl of anti-PU.1 antibody (sc-352X, Santa Cruz Biotechnology) was incubated with the protein-DNA complexes for an additional 1 h on ice. Samples were resolved on 5% nondenaturing polyacrylamide gels. Gels were dried and protein-DNA complexes were detected by autoradiography. A293 cells in 35-mm plates were transfected with 1 μg of a pGL3-based CD10 promoter luciferase reporter plasmid (pCD10-luc or pCD10mutPU-1-luc), 0.5 μg of normalization plasmid pRSV-βgal, and 0.5 μg of pcDNA or pcDNA-PU.1 using polyethylenimine, as described above. Luciferase activity was measured using the Luciferase Assay System according to the manufacturer's instructions (Promega). Luciferase values were normalized to β-galactosidase values in all assays, as described previously (25Kalaitzidis D. Davis R.E. Rosenwald A. Staudt L.M. Gilmore T.D. Oncogene. 2002; 21: 8759-8768Crossref PubMed Scopus (43) Google Scholar). CD10 mRNA and protein levels are low in the less favorable ABC subtype of DLBCL (8Alizadeh A.A. Eisen M.B. Davis R.E. Ma C. Lossos I.S. Rosenwald A. Boldrick J.C. Sabet H. Tran T. Yu X. Powell J.I. Yang L. Marti G.E. Moore T. Hudson Jr., J. Lu L. Lewis D.B. Tibshirani R. Sherlock G. Chan W.C. Greiner T.C. Weisenburger D.D. Armitage J.O. Warnke R. Levy R. Wilson W. Grever M.R. Byrd J.C. Botstein D. Brown P.O. Staudt L.M. Nature. 2000; 403: 503-511Crossref PubMed Scopus (7946) Google Scholar, 9Wright G. Tan B. Rosenwald A. Hurt E.H. Wiestner A. Staudt L.M. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9991-9996Crossref PubMed Scopus (824) Google Scholar, 11Hans C.P. Weisenburger D.D. Greiner T.C. Gascoyne R.D. Delabie J. Ott G. Müller-Hermelink H.K. Campo E. Braziel R.M. Jaffe E.S. Pan Z. Farinha P. Smith L.M. Falini B. Banham A.H. Rosenwald A. Staudt L.M. Connors J.M. Armitage J.O. Chan W.C. Blood. 2004; 103: 275-282Crossref PubMed Scopus (3140) Google Scholar, 12Iqbal J. Joshi S. Patel K.N. Javed S.I. Kucuk C. Aabida A. d'Amore F. Fu K. Indian J. Cancer. 2007; 44: 72-86Crossref PubMed Google Scholar), which also has increased NF-κB activity and increased levels of the NF-κB target gene product miR-155 (23Davis R.E. Brown K.D. Siebenlist U. Staudt L.M. J. Exp. 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Pathol. 2005; 207: 243-249Crossref PubMed Scopus (578) Google Scholar), a micro-RNA that is processed from a non-coding RNA known as BIC (B-cell integration cluster gene). In contrast, GCB-like DLBCL primary tumors and cell lines express readily detectable levels of CD10 mRNA and protein, but have lower NF-κB activity and lower levels of miR-155 (13Chin M. Herscovitch M. Zhang N. Waxman D.J. Gilmore T.D. Oncogene. 2009; 28: 2100-2111Crossref PubMed Scopus (25) Google Scholar, 36Rai D. Karanti S. Jung I. Dahia P.L. Aguiar R.C. Cancer Genet. Cytogenet. 2008; 181: 8-15Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 37Rahadiani N. Takakuwa T. Tresnasari K. Morii E. Aozasa K. Biochem. Biophys. Res. Commun. 2008; 377: 579-583Crossref PubMed Scopus (50) Google Scholar). One direct target of miR-155 repression is the mRNA encoding transcription factor PU.1 (39Faraoni I. Antonetti F.R. Cardone J. Bonmassar E. Biochim. Biophys. Acta. 2009; 1792: 497-505Crossref PubMed Scopus (633) Google Scholar, 40Vigorito E. Perks K.L. Abreu-Goodger C. Bunting S. Xiang Z. Kohlhaas S. Das P.P. Miska E.A. Rodriguez A. Bradley A. Smith K.G. Rada C. Enright A.J. Toellner K.M. Maclennan I.C. Turner M. Immunity. 2007; 27: 847-859Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). Based on the above findings, we analyzed the levels of PU.1, CD10, and miR-155 among a panel of six B-lymphoma-like cell lines with varying levels of NF-κB-dependent gene expression. Two cell lines, IB4 (14Carter K.L. Cahir-McFarland E. Kieff E. J. Virol. 2002; 76: 10427-10436Crossref PubMed Scopus (124) Google Scholar) and RC-K8 (25Kalaitzidis D. Davis R.E. Rosenwald A. Staudt L.M. Gilmore T.D. Oncogene. 2002; 21: 8759-8768Crossref PubMed Scopus (43) Google Scholar), have gene expression profiles consistent with high levels of NF-κB activity; four cell lines, BJAB (13Chin M. Herscovitch M. Zhang N. Waxman D.J. Gilmore T.D. Oncogene. 2009; 28: 2100-2111Crossref PubMed Scopus (25) Google Scholar, 21Ngo V.N. Davis R.E. Lamy L. Yu X. Zhao H. Lenz G. Lam L.T. Dave S. Yang L. Powell J. Staudt L.M. Nature. 2006; 441: 106-110Crossref PubMed Scopus (511) Google Scholar), SUDHL-4 (23Davis R.E. Brown K.D. Siebenlist U. Staudt L.M. J. Exp. Med. 2001; 194: 1861-1874Crossref PubMed Scopus (880) Google Scholar), BL41 (41Cahir-McFarland E.D. Carter K. Rosenwald A. Giltnane J.M. Henrickson S.E. Staudt L.M. Kieff E. J. Virol. 2004; 78: 4108-4119Crossref PubMed Scopus (201) Google Scholar), and Daudi, are not known to have high NF-κB-driven gene expression. PU.1 and CD10 proteins were expressed at readily detectable levels in the four cell lines with low NF-κB activity, whereas PU.1 and CD10 protein levels were extremely low in the high NF-κB activity IB4 and RC-K8 cell lines (Fig. 1A). Moreover, as judged by qPCR, miR-155 was present at low levels in the four lymphoma cell lines with low NF-κB activity (Fig. 1B). In contrast, miR-155 was present at increased levels in the high NF-κB activity IB4 and RC-K8 cell lines, which had miR-155 levels ∼30–50 times higher than BJAB cells (Fig. 1B). These results indicate that PU.1 and CD10 levels are low in transformed B-lymphoid cell lines where miR-155 levels and NF-κB activity are high (e.g. IB4 and RC-K8). Based on this direct correlation between PU.1 and CD10 protein levels in six B-lymphoma-like cell lines and the presence of three consensus PU.1 binding sites (GAGGAA) in the CD10 upstream region (16Ishimaru F. Shipp M.A. Blood. 1995; 85: 3199-3207Crossref PubMed Google Scholar) (Fig. 2A), we next sought to determine whether the CD10 gene might be a direct transcriptional target for PU.1. We first assessed the ability of PU.1 to bind to the CD10 promoter by performing EMSAs using DNA probes containing each of the three putative PU.1 binding sites (PU-1, PU-2, and PU-3). Nuclear extracts were prepared from control A293 cells (Fig. 2B, lanes 2, 7, and 12) or A293 cells overexpressing PU.1 (Fig. 2B, lanes 3, 8, and 13), and these extracts were incubated with each of the three PU.1 site probes (PU-1, PU-2, or PU-3). We found that extracts from cells overexpressing PU.1 could specifically bind to the first PU.1 site (PU-1), whereas these extracts showed no binding to the PU-2 or PU-3 probes above the background binding seen with control A293 extracts (Fig. 2B). Additionally, binding to the PU-1 site was competed by an excess of cold probe (Fig. 2B, lane 4), and the PU-1 site protein-DNA complex was supershifted by a PU.1-specific antibody (Fig. 2B, lane 5). To assess the ability of PU.1 to activate transcription from the CD10 promoter, we placed a portion of the CD10 promoter region containing the three consensus PU.1 binding sites upstream of a luciferase cassette (pCD10-luc). A293 cells were co-transfected with pCD10-luc and an expression plasmid for PU.1 or a vector control, and we then measured the relative luciferase activity. Co-transfection of the PU.1 expression plasmid resulted in an ∼6-fold increase in expression from pCD10-luc as compared with the vector control (Fig. 2C). Mutation of the first PU.1 site (PU-1) in the CD10 promoter reduced the ability of PU.1 to transactivate the CD10 reporter plasmid by ∼35% (Fig. 2C). Western blotting confirmed that PU.1 was overexpressed in cells transfected with the pcDNA-PU.1 expression plasmid (Fig. 2C). Mutations in the scaffolding protein CARD11, which cause it to be a constitutive activator of NF-κB, have been identified in a subset of patients with DLBCL, primarily of the ABC subtype (42Lenz G. Davis R.E. Ngo V.N. Lam L. George T.C. Wright G.W. Dave S.S. Zhao H. Xu W. Rosenwald A." @default.
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• W1971509320 title "NF-κB Down-regulates Expression of the B-lymphoma Marker CD10 through a miR-155/PU.1 Pathway" @default.
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