Bruton's Tyrosine Kinase as an Inhibitor of the Fas/CD95 Death-inducing Signaling Complex
1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês
10.1074/jbc.274.3.1646
ISSN1083-351X
AutoresAlexei Vassilev, Zahide Özer, Christopher S. Navara, Sandeep Mahajan, Fatih M. Uckun,
Tópico(s)Cell death mechanisms and regulation
ResumoBruton's tyrosine kinase (BTK) is a member of the Src-related Tec family of protein tyrosine kinases. Mutations in the btk gene have been linked to severe developmental blocks in human B-cell ontogeny leading to X-linked agammaglobulinemia. Here, we provide unique biochemical and genetic evidence that BTK is an inhibitor of the Fas/APO-1 death-inducing signaling complex in B-lineage lymphoid cells. The Src homology 2, pleckstrin homology (PH), and kinase domains of BTK are all individually important and apparently indispensable, but not sufficient, for its function as a negative regulator of Fas-mediated apoptosis. BTK associates with Fas via its kinase and PH domains and prevents the FAS-FADD interaction, which is essential for the recruitment and activation of FLICE by Fas during the apoptotic signal. Fas-resistant DT-40 lymphoma B-cells rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout underwent apoptosis after Fas ligation, but wild-type DT-40 cells or BTK-deficient DT-40 cells reconstituted with wild-type human btk gene did not. Introduction of an Src homology 2 domain, a PH domain, or a kinase domain mutant humanbtk gene into BTK-deficient cells did not restore the resistance to Fas-mediated apoptosis. Introduction of wild-type BTK protein by electroporation rendered BTK-deficient DT-40 cells resistant to the apoptotic effects of Fas ligation. BTK-deficient RAMOS-1 human Burkitt's leukemia cells underwent apoptosis after Fas ligation, whereas BTK-positive NALM-6-UM1 human B-cell precursor leukemia cells expressing similar levels of Fas did not. Treatment of the anti-Fas-resistant NALM-6-UM1 cells with the leflunomide metabolite analog α-cyano-β-methyl-β-hydroxy-N-(2, 5-dibromophenyl)propenamide, a potent inhibitor of BTK, abrogated the BTK-Fas association without affecting the expression levels of BTK or Fas and rendered them sensitive to Fas-mediated apoptosis. The ability of BTK to inhibit the pro-apoptotic effects of Fas ligation prompts the hypothesis that apoptosis of developing B-cell precursors during normal B-cell ontogeny may be reciprocally regulated by Fas and BTK. Bruton's tyrosine kinase (BTK) is a member of the Src-related Tec family of protein tyrosine kinases. Mutations in the btk gene have been linked to severe developmental blocks in human B-cell ontogeny leading to X-linked agammaglobulinemia. Here, we provide unique biochemical and genetic evidence that BTK is an inhibitor of the Fas/APO-1 death-inducing signaling complex in B-lineage lymphoid cells. The Src homology 2, pleckstrin homology (PH), and kinase domains of BTK are all individually important and apparently indispensable, but not sufficient, for its function as a negative regulator of Fas-mediated apoptosis. BTK associates with Fas via its kinase and PH domains and prevents the FAS-FADD interaction, which is essential for the recruitment and activation of FLICE by Fas during the apoptotic signal. Fas-resistant DT-40 lymphoma B-cells rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout underwent apoptosis after Fas ligation, but wild-type DT-40 cells or BTK-deficient DT-40 cells reconstituted with wild-type human btk gene did not. Introduction of an Src homology 2 domain, a PH domain, or a kinase domain mutant humanbtk gene into BTK-deficient cells did not restore the resistance to Fas-mediated apoptosis. Introduction of wild-type BTK protein by electroporation rendered BTK-deficient DT-40 cells resistant to the apoptotic effects of Fas ligation. BTK-deficient RAMOS-1 human Burkitt's leukemia cells underwent apoptosis after Fas ligation, whereas BTK-positive NALM-6-UM1 human B-cell precursor leukemia cells expressing similar levels of Fas did not. Treatment of the anti-Fas-resistant NALM-6-UM1 cells with the leflunomide metabolite analog α-cyano-β-methyl-β-hydroxy-N-(2, 5-dibromophenyl)propenamide, a potent inhibitor of BTK, abrogated the BTK-Fas association without affecting the expression levels of BTK or Fas and rendered them sensitive to Fas-mediated apoptosis. The ability of BTK to inhibit the pro-apoptotic effects of Fas ligation prompts the hypothesis that apoptosis of developing B-cell precursors during normal B-cell ontogeny may be reciprocally regulated by Fas and BTK. Apoptosis is a common mode of eukaryotic cell death which is triggered by an inducible cascade of biochemical events leading to activation of endonucleases that cleave the nuclear DNA into oligonucleosome-length fragments (1Whyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-305Crossref PubMed Scopus (6851) Google Scholar, 2Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2445) Google Scholar, 3Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). Several of the biochemical events that contribute to apoptotic cell death as well as both positive and negative regulators of apoptosis have recently been identified (1Whyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-305Crossref PubMed Scopus (6851) Google Scholar, 2Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2445) Google Scholar, 3Fraser A. Evan G. 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Therefore, an improved understanding of the molecular basis of apoptosis and the pro-apoptotic versus anti-apoptotic regulatory signals may provide further insights into the pathogenesis of human lymphoid malignancies and have important implications for treatment of leukemias and lymphomas. The Fas/APO-1 (CD95) cell surface receptor, a member of the tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; BTK, Bruton's tyrosine kinase; PTK, protein tyrosine kinase(s); DICS, death-inducing signaling complex; PH domain, pleckstrin homology domain; SH2 and SH3 domains, Src homology 2 and Src homology 3 domains, respectively; GST, glutathioneS-transferase; MBP, maltose-binding protein; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PI, propidium iodide. receptor family, is one of the major regulators of apoptosis in a variety of cell types (8Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4578) Google Scholar, 9Schulze-Osthoff K. Krammer P.H. Droge W. EMBO J. 1994; 13: 4587-4596Crossref PubMed Scopus (321) Google Scholar, 10Rieux-Laucat F. LeDeist F. Hivroz C. Roberts I.A. Debatin K.M. Fischer A. deVillartay J.P. Science. 1995; 268: 1347-1349Crossref PubMed Scopus (1198) Google Scholar, 11Fischer G.H. Cell. 1995; 81: 935-946Abstract Full Text PDF PubMed Scopus (1318) Google Scholar). Functional abnormalities of Fas have been associated with pathologic conditions of the immune system homeostasis, including lymphoproliferative disorders, immunodeficiencies, and autoimmunity (10Rieux-Laucat F. LeDeist F. Hivroz C. Roberts I.A. Debatin K.M. Fischer A. deVillartay J.P. Science. 1995; 268: 1347-1349Crossref PubMed Scopus (1198) Google Scholar, 11Fischer G.H. Cell. 1995; 81: 935-946Abstract Full Text PDF PubMed Scopus (1318) Google Scholar). Identifying the molecules that participate in the apoptotic death signal pathways linked to the Fas receptor and finding ways to modulate the activity of such molecules could provide the basis for innovative treatment programs. Ligation of the cell surface Fas molecule rapidly and dramatically induces apoptosis in many but not all Fas-positive cell types (8Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4578) Google Scholar). DT-40 is a chicken lymphoma B-cell line that we have used previously to elucidate the molecular mechanism of radiation-induced apoptosis (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar). Despite their abundant surface expression of Fas, DT-40 cells, similar to human B-cell precursor leukemia cells, are very resistant to the cytotoxic effects of Fas ligation, indicating the existence of potent negative regulators of Fas-mediated apoptosis. Bruton's tyrosine kinase (BTK) is a member of the Src-related Tec family of protein tyrosine kinases (PTK) (13Rawlings D.J. Witte O.N. Immunol. Rev. 1994; 138: 105-119Crossref PubMed Scopus (102) Google Scholar, 14Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (184) Google Scholar). Mutations in thebtk gene have been linked to severe developmental blocks in human B-cell ontogeny leading to human X-linked agammaglobulinemia (15Tsukada S. Saffran D.C. Rawlings D.J. Parolini O. Allen R.C. Klisak I. Sparkes R.S. Kubagawa H. Mohandas T. Quan S. Cell. 1993; 72: 279-290Abstract Full Text PDF PubMed Scopus (1193) Google Scholar,16Vetrie D. Nature. 1993; 361: 226-233Crossref PubMed Scopus (1290) Google Scholar) and less severe deficiencies in murine B-cells leading to murine X-linked immune deficiency (17Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Science. 1993; 261: 358-361Crossref PubMed Scopus (794) Google Scholar). Recent studies implicated BTK as a pro-apoptotic enzyme in B-lineage lymphoid cells exposed to ionizing radiation (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar) as well as mast cells deprived of growth factors (18Kawakami Y. Miura T. Bissonnette R. Hata D. Khan W.N. Kitamura T. Maeda-Yamamoto M. Hartman S.E. Yao L. Alt F.W. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3938-3942Crossref PubMed Scopus (129) Google Scholar). In murine B-cells, BTK has also been shown to act as an anti-apoptotic protein upstream of bcl-xL in the B-cell antigen receptor (but not the CD40 receptor) activation pathway (19Anderson J.S. Teutsch M. Dong Z. Wortis H.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10966-10971Crossref PubMed Scopus (116) Google Scholar). Because of the recently discovered but not well understood ability of BTK to act both as a positive and negative regulator of apoptosis after ionizing radiation, growth factor deprivation, or B-cell antigen receptor signaling and its abundant expression in DT-40 cells (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar), we investigated whether BTK plays a role in the pronounced resistance of DT-40 cells as well as human leukemic B-cell precursors against Fas-mediated apoptosis. Our study provides biochemical and genetic evidence that BTK is an inhibitor the Fas/APO-1 death-inducing signaling complex (DISC) in B-lineage lymphoid cells. BTK associates with Fas via its kinase and pleckstrin homology (PH) domains and prevents the FAS-FADD interaction, which is essential for the recruitment and activation of FLICE by Fas during the apoptotic signal. Notably, treatment of human leukemic B-cell precursors with a potent inhibitor of BTK abrogated the BTK-Fas association and sensitized the cells to Fas-mediated apoptosis. The establishment of BTK-deficient DT-40 lymphoma B-cell clones has been described previously (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar). To disrupt the btk gene, targeting constructs containing the neomycin resistance gene cassette (i.e. pcBTK-neo) or histidinol resistance gene cassette (i.e. pcBTK-hisD) were sequentially transfected into DT-40 cells. The targeting vectors, pcBTK-neo and pcBTK-hisD, were constructed by replacing the 0.7-kilobaseBglII-BamHI genomic fragment containing exons that correspond to human BTK amino acid residues 91–124 with theneo or hisD cassette. pcBTK-neo was linearized and introduced into wild-type DT-40 cells by electroporation. Screening was done by Southern blot analysis using a 3′-flanking probe (0.5-kilobase BglII-Bgl-II fragment). The neo-targeted clone was again transfected with pcBTK-hisD and selected with both G418 (2 mg/ml) and histidinol (1 mg/ml). Southern blot analysis of a BTK-deficient DT-40 clone confirmed the homologous recombination at both btk loci, and hybridization with a neo and hisD probe indicated that the targeted clone had incorporated a single copy of each construct. Lack of BTK expression in BTK-deficient DT-40 cells was confirmed by both immune complex kinase assays and Western blot analysis (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar). Mutations in the human btk cDNA were introduced by polymerase chain reaction using Pfu polymerase (Strategene) and confirmed by sequencing. Wild-type and mutantbtk cDNAs were subcloned into pApuro expression vector and electroporated into BTK-deficient cells. The PTK activity of BTK immune complexes, as measured by in vitroautophosphorylation, was abrogated by the catalytic domain mutation, reduced by the PH domain mutation, but not affected by the mutation in the Src homology 2 (SH2) domain. Equal amounts of BTK protein were detected by Western blot analysis in all of the BTK-deficient DT-40 clones transfected with wild-type or mutated human btkgenes, but no BTK protein was detectable in the untransfected BTK-deficient DT-40 cells (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar). The establishment and characterization of LYN-deficient DT-40 clones were reported previously (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar). In addition to these chicken lymphoma B-cells, we also used the following human B-lineage lymphoid cell lines: NALM-6-UM1, a BTK-positive human B-cell precursor (pre-B acute lymphoblastic leukemia) cell line; RAMOS-1, a BTK-deficient human Burkitt's/B-cell leukemia line; and KL2, a BTK-positive human Epstein-Barr virus-transformed normal B-lymphoblastoid cell line. Antibodies directed against BTK, SYK, and LYN have been described previously (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar, 20Dibirdik I. Kristupaitis D. Kurosaki T. Tuel-Ahlgren L. Chu A. Pond D. Tuong D. Luben R. Uckun F.M. J. Biol. Chem. 1998; 273: 4035-4039Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 21Uckun F.M. Tuel-Ahlgren L. Waddick K.G. Jun X. Jin J. Myers D.E. Rowley R.B. Burkhardt A.L. Bolen J.B. J. Biol. Chem. 1996; 271: 6389-6397Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Polyclonal antibodies to BTK were generated by immunization of rabbits with glutathioneS-transferase (GST) fusion proteins (Amersham Pharmacia Biotech) containing the first 150 amino acids of BTK. In addition, we used the following anti-BTK antibodies in Western blots of purified fusion proteins: polyclonal goat anti-BTK carboxyl terminus (Santa Cruz Biotechnology), polyclonal goat anti-BTK amino terminus (Santa Cruz Biotechnology), and polyclonal rabbit serum raised against the BTK SH2-SH3 domains (amino acids 219–377). Polyclonal anti-MBP (maltose-binding protein) antibodies were generated by immunizing rabbits. The rabbit polyclonal anti-Fas (sc-715 mixed 1:1 with sc-714), which cross-reacts with both human and chicken Fas proteins, goat polyclonal anti-FADD (sc-1171), goat polyclonal anti-TRADD (sc-1163), and goat polyclonal anti-FLICE (sc-6135) were purchased from Santa Cruz Biotechnology and used according to the manufacturer's recommendations. The monoclonal anti-Fas antibody (F22120) was obtained from the Transduction Laboratories, Inc. (Lexington, KY). Immunoprecipitations, immune complex protein kinase assays, and immunoblotting using the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech) were conducted as described previously (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar, 20Dibirdik I. Kristupaitis D. Kurosaki T. Tuel-Ahlgren L. Chu A. Pond D. Tuong D. Luben R. Uckun F.M. J. Biol. Chem. 1998; 273: 4035-4039Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 21Uckun F.M. Tuel-Ahlgren L. Waddick K.G. Jun X. Jin J. Myers D.E. Rowley R.B. Burkhardt A.L. Bolen J.B. J. Biol. Chem. 1996; 271: 6389-6397Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 22Mahajan S. Fargnoli J. Burkhardt A.L. Kut S.A. Saouaf S.J. Bolen J.B. Mol. Cell. Biol. 1995; 15: 5304-5311Crossref PubMed Scopus (137) Google Scholar, 23Uckun F.M. Burkhardt A.L. Jarvis L. Jun X. Stealey B. Dibirdik I. Myers D.E. Tuel-Ahlgren L. Bolen J.B. J. Biol. Chem. 1993; 268: 21172-21184Abstract Full Text PDF PubMed Google Scholar, 24Uckun F.M. Evans W.E. Forsyth C.J. Waddick K.G. Tuel-Ahlgren L. Chelstrom L.M. Burkhardt A. Bolen J. Myers D.E. Science. 1995; 267: 886-891Crossref PubMed Scopus (269) Google Scholar, 25Uckun F.M. Yang Z. Sather H. Steinherz P. Nachman J. Bostrom B. Crotty L. Sarquis M. Ek O. Zeren T. Tubergen D. Reaman G. Gaynon P. Blood. 1997; 89: 3769-3777Crossref PubMed Google Scholar). The BTK inhibitor α-cyano-β-methyl-β-hydroxy-N-(2, 5-dibromophenyl)propenamide) (LFM-A13; IC50 for BTK, 2.5 μm; IC50 values for epidermal growth factor receptor, insulin receptor, JAK-1, JAK-2, JAK-3, SYK, HCK, all >300 μm) was a kind gift from Dr. Yaguo Zheng from the Department of Chemistry at the Hughes Institute. The analytical physicochemical data for LMA-13 were: yield: 78% from tetrahydrofuran; mp: 148–150 °C; IR (KBr): 3,353, 2,211, 1,648, and 1,590 cm−1; UV-visible: 220, 245, and 296 nm; 1H NMR (dimethyl sulfoxide-d 6): δ 11.41 (s, 1H, NH), 8.57 (m, 1H, ArH), 7.55 (d, J = 8.7 Hz, 1H, ArH), 7.14 (q, J 1 = 6.0 Hz, J 2 = 2.4 Hz, 1H, ArH), 7.10 (s br, 1H, OH), 2.17 (s, 3H, CH3); electron ionization mass spectrometry m/z[M]+: 358. cDNAs encoding full-length BTK and its kinase or PH domains with polymerase chain reaction-generated 5′- and 3′-BamHI sites were cloned into the Escherichia coli expression vector pMAL-C2 with the isopropyl 1-thio-β-d-galactopyranoside-inducible Ptac promoter to create an in-frame fusion between these coding sequences and the 3′-end of the E. coli malE gene, which codes for MBP. cDNAs encoding the SH2, SH3, or SH2+SH3 domains with polymerase chain reaction-generated 5′- and 3′-BamHI sites were cloned into the E. coli expression vector pGEX-2t with the isopropyl 1-thio-β-d-galactopyranoside-inducible Ptac promoter to create an in-frame fusion between these coding sequences and the 3′-end of the E. coli GST gene. The generated recombinant plasmids were transformed into the E. colistrain DH5α. Single transformants were expanded in 5 ml of Luria-Burtain (LB) medium (1% tryptone, 1% NaCl, 0.5% yeast extract) containing ampicillin (100 μg/ml) by overnight culture at 37 °C. Expression of the fusion proteins was induced with 10 mmisopropyl 1-thio-β-d-galactopyranoside. The cells were harvested by centrifugation at 4,500 × g in a Sorvall RC5B centrifuge for 10 min at 4 °C, lysed in sucrose-lysozyme buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 10% sucrose, 1 mm EDTA, 20 mm lysozyme), and disrupted further by sonication. After removal of the cell pellets by centrifugation at 35,000 × g for 1 h at 4 °C, GST-BTK fusion proteins were purified by gluthathione-Sepharose chromatography (21Uckun F.M. Tuel-Ahlgren L. Waddick K.G. Jun X. Jin J. Myers D.E. Rowley R.B. Burkhardt A.L. Bolen J.B. J. Biol. Chem. 1996; 271: 6389-6397Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), whereas MBP-BTK fusion proteins were purified from the culture supernatants by amylose affinity chromatography (26Hsu D. Zhao X. Zhao S. Kazantsev A. Wang R.P. Todo T. Wei Y.F. Sancar A. Biochemistry. 1996; 35: 13871-13877Crossref PubMed Scopus (270) Google Scholar). Wild-type and BTK-deficient DT-40 cells treated with anti-Fas antibody (1 μg/ml, 24 h at 37 °C) were attached to poly-l-lysine-coated coverslips and fixed in ice-cold (−20 °C) methanol for 15 min. After fixation, the coverslips were washed for 15 min in phosphate-buffered saline (PBS) + 0.1% Triton X-100. Cells were stained with a rabbit polyclonal anti-tubulin antibody according to the manufacturer's recommendations (Sigma) to visualize their cytoplasms. DNA was labeled for 10 min with Toto-3, a DNA specific dye (Molecular Probes, Eugene OR) to visualize the apoptotic changes in the nuclei. MBP-BTK-electroporated BTK-deficient DT-40 cells and nonelectroporated BTK-deficient DT-40 cells were labeled with an antibody raised against MBP. The secondary antibody was a goat anti-rabbit fluorescein-conjugated antibody. In some experiments, cells were examined for Fas expression by confocal microscopy. In brief, cells were attached to poly-l-lysine-coated coverslips and fixed for 40 min in 2% paraformaldehyde in PBS. Cells were rinsed in PBS + 115 mmglycine to quench the formaldehyde and then blocked in PBS containing 2% bovine serum albumin (PBS+BSA). A monoclonal antibody raised against the extracellular domain of Fas (Transduction Labs, Lexington, KY) was added in PBS+BSA, and the coverslips were incubated for 40 min at 37 °C before rinsing again in PBS. A fluorescein-labeled secondary antibody (Zymed Laboratories Inc., San Francisco) diluted in PBS+BSA was then added to the coverslips, and they were again incubated for 40 min at 37 °C. After another wash, cellular DNA was labeled by incubation in 1 μm Toto-3 for 20 min at room temperature. Coverslips were inverted and mounted onto slides in Vectashield (Vector Labs, Burlingame, CA) to prevent photobleaching and were sealed with nail varnish. Slides were examined using a Bio-Rad MRC-1024 laser scanning confocal microscope mounted on an Nikon Eclipse E-800 upright microscope equipped for epifluorescence with high numerical aperture objectives (27Uckun F.M. Jun X. Narla R.K. Zeren T. Venkatachalam T. Waddick K. Rostostev A. Myers D.E. Clin. Cancer Res. 1998; 4: 901-912PubMed Google Scholar). Optical sections were obtained and turned into stereomicrographs using Lasersharp software (Bio-Rad). Representative digital images were saved to Jaz disk and processed using Adobe Photoshop software (Adobe Systems, Mountain View CA). Images were printed with a Fuji Pictography thermal transfer printer (Fuji Photo, Elmsford, NY). Digital data were archived and stored on CD-ROM. To induce apoptosis, cells were treated with an agonistic anti-Fas/APO-1 antibody (Bender MedSystems) at a 0.1, 0.5, or 1.0 μg/ml final concentration. MC540 binding (as an early marker of apoptosis) and propidium iodide (PI) permeability (as a marker of advanced stage apoptosis) were measured simultaneously in DT-40 cells 24 h after exposure to anti-Fas antibody, as described previously (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar). Whole cells were analyzed with a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA). All analyses were done using 488 nm excitation from an argon laser. MC540 and PI emissions were split with a 600 nm short pass dichroic mirror. A 575 nm band pass filter was placed in front of one photomultiplier tube to measure MC540 emission, and a 635 nm band pass filter was used for PI emission. To detect apoptotic fragmentation of DNA, DT-40, NALM-6-UM1, and RAMOS-1 cells were harvested 24 h after exposure to anti-Fas. DNA was prepared from Triton X-100 lysates for analysis of fragmentation (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar, 24Uckun F.M. Evans W.E. Forsyth C.J. Waddick K.G. Tuel-Ahlgren L. Chelstrom L.M. Burkhardt A. Bolen J. Myers D.E. Science. 1995; 267: 886-891Crossref PubMed Scopus (269) Google Scholar, 27Uckun F.M. Jun X. Narla R.K. Zeren T. Venkatachalam T. Waddick K. Rostostev A. Myers D.E. Clin. Cancer Res. 1998; 4: 901-912PubMed Google Scholar). In brief, cells were lysed in hypotonic 10 mmol/liter Tris-HCl, pH 7.4, 1 mmol/liter EDTA, 0.2% Triton X-100 detergent and subsequently centrifuged at 11,000 × g. To detect apoptosis-associated DNA fragmentation, supernatants were electrophoresed on a 1.2% agarose gel, and the DNA fragments were visualized by ultraviolet light after staining with ethidium bromide. In some experiments, MBP-BTK fusion proteins (100 μg/2.5 × 108 cells) were electroporated (420-V electrical field, 125 microfarads) into BTK-deficient DT-40 cells using a Bio-Rad gene pulser and the procedures of Bergland and Starkey (28Bergland D. Starkey J. Cytometry. 1991; 12: 64-67Crossref PubMed Scopus (29) Google Scholar) with slight modifications 4 h before Fas ligation and apoptosis assays. GST-BTK fusion proteins were noncovalently bound to glutathione-agarose beads (Sigma), and MBP-BTK fusion proteins were noncovalently bound to amylose beads under conditions of saturating protein, as described previously (21Uckun F.M. Tuel-Ahlgren L. Waddick K.G. Jun X. Jin J. Myers D.E. Rowley R.B. Burkhardt A.L. Bolen J.B. J. Biol. Chem. 1996; 271: 6389-6397Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26Hsu D. Zhao X. Zhao S. Kazantsev A. Wang R.P. Todo T. Wei Y.F. Sancar A. Biochemistry. 1996; 35: 13871-13877Crossref PubMed Scopus (270) Google Scholar). In brief, 50 μg of each protein was incubated with 50 μl of the beads for 2 h at 4 °C. The beads were washed three times with 1% Nonidet P-40 buffer. Nonidet P-40 lysates of BTK-deficient DT-40 cells, NALM-6-UM1 human leukemic B-cell precursors, and KL2 human Epstein-Barr virus-transformed lymphoblastoid cells were prepared as described (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar,21Uckun F.M. Tuel-Ahlgren L. Waddick K.G. Jun X. Jin J. Myers D.E. Rowley R.B. Burkhardt A.L. Bolen J.B. J. Biol. Chem. 1996; 271: 6389-6397Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and 500 μg of the lysate was incubated with 50 μl of fusion protein-coupled beads for 2 h on ice. The fusion protein adsorbates were washed with ice-cold 1% Nonidet P-40 buffer and resuspended in reducing SDS sample buffer. Samples were boiled for 5 min and then fractionated on SDS-polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon-P (Millipore) membranes, and the membranes were immunoblotted with anti-Fas (F22120, Transduction Laboratories), according to procedures described previously (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar, 20Dibirdik I. Kristupaitis D. Kurosaki T. Tuel-Ahlgren L. Chu A. Pond D. Tuong D. Luben R. Uckun F.M. J. Biol. Chem. 1998; 273: 4035-4039Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar,21Uckun F.M. Tuel-Ahlgren L. Waddick K.G. Jun X. Jin J. Myers D.E. Rowley R.B. Burkhardt A.L. Bolen J.B. J. Biol. Chem. 1996; 271: 6389-6397Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 25Uckun F.M. Yang Z. Sather H. Steinherz P. Nachman J. Bostrom B. Crotty L. Sarquis M. Ek O. Zeren T. Tubergen D. Reaman G. Gaynon P. Blood. 1997; 89: 3769-3777Crossref PubMed Google Scholar). In a series of experiments designed to examine the potential negative regulatory role of BTK in Fas-mediated apoptosis, we first compared the effects of Fas ligation on wild-type DT-40 cells with the effects of Fas ligation on a BTK-deficient subclone of DT-40 cells which was established by homologous recombination knockout (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar). To this end, we first used a quantitative flow cytometric apoptosis detection assay (12Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (168) Google Scholar). MC540 binding and PI permeability were measu
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