Identification of Apoptosis-associated Proteins in a Human Burkitt Lymphoma Cell Line: CLEAVAGE OF HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A1 BY CASPASE 3

Ekkehard Brockstedt,Anke Rickers,Susanne Kostka,Andreas Laubersheimer,Bernd Dörken,Brigitte Wittmann-Liebold,Kurt Bommert,Albrecht Otto

doi:10.1074/jbc.273.43.28057

Abstract

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified proteins that are involved in anti-IgM antibody-mediated apoptosis using a subclone of the human Burkitt lymphoma cell line BL60. Apoptosis-associated proteins were detected by high resolution two-dimensional gel electrophoresis on a micropreparative scale. Comparison of the high resolution two-dimensional gel electrophoresis protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots including protein modifications. Analysis of the predominantly altered proteins was performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. Analysis was significantly improved by using new micropreparative high resolution two-dimensional gels employing high protein concentrations. The following 12 apoptosis-associated proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, FUSE-binding protein, dUTPase, lymphocyte-specific protein LSP1, UV excision repair protein RAD23 homologue B (HHR23B), 60 S acidic ribosomal protein P0 (L10E), heterochromatin protein 1 homologue alpha (HP1alpha), nucleolin, lamin, neutral calponin, and actin. Fragmentation of actin, hnRNP A1, hnRNP C1/C2, 60 S acidic ribosomal protein P0, lamin, and nucleolin could be inhibited by benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, a selective irreversible inhibitor of CPP32 (caspase 3).

Highlights

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection
Different proteins like HP1␣, FBP, dUTPase, HHR23B (UV excision repair protein RAD23 homologue B), LSP1, ribosomal protein P0, and neutral calponin seem to be involved in anti-IgM antibody-induced apoptosis since alterations of these proteins could be inhibited by Z-DEVD-FMK
Induction of Apoptosis by Anti-IgM Antibody and Separation of Apoptotic Cells by Magnetic Cell Sorting—To determine proteins involved in the regulation of anti-IgM antibody-induced apoptosis, we used a subclone of a Burkitt lymphoma cell line (BL60-2)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The human Burkitt lymphoma cell line BL60-2 was cultured in RPMI 1640 medium, 10% heat-inactivated fetal calf serum, penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM glutamine, 10 mM HEPES (pH 7.4), 20 nM bathocuproinedisulfonic acid, and 50 ␮M ␣-thioglycerol at 37 °C and 5% CO2 as described previously [16]. Inhibition of apoptosis was performed by incubating the cells with 200 ␮M Z-DEVDFMK (Calbiochem-Novabiochem, Bad Soden/Taunus, Germany) 30 min prior to addition of anti-IgM F(ab) essentially as described previously [16]. Electrophoresis of the analytical SDS-polyacrylamide gel (30 ϫ 23 ϫ 0.075 cm) was performed using a two-step increase of current, starting with 15 min at 65 mA, followed by a run of 7– 8 h at 85 mA, until the dye reached the end of the gel. Electrophoresis of the micropreparative SDS-polyacrylamide gel (30 ϫ 23 ϫ 0.15 cm) was performed using a two-step increase of current, starting with 15 min at 130 mA, followed by a run of 7– 8 h at 170 mA, until the dye reached the end of the gel. Amino acid sequences were compared with the SWISS-PROT Data Bank (release of June 1997) and with the deduced amino acid sequences of the GenBankTM/EMBL Data Bank (release of June 97)

RESULTS AND DISCUSSION

Neutral calponin

Additions and Corrections