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 twodimensional 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 ϳ80 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 ␣ (HP1␣), 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).Apoptosis or programmed cell death plays a major role during development, homeostasis, and immune response in multicellular organisms. Inappropriate apoptosis may contribute to the pathology of many human diseases, including cancer, acquired immunodeficiency syndrome, and neurodegenerative disorders. Substantial progress has been made in understanding the control and mechanisms of apoptosis (1, 2). Nevertheless, major aspects of the apoptotic pathway remain undefined, and little is known about the molecular events controlling this process. It has proven difficult to identify the molecules involved in apoptosis by conventional biochemical and molecular approaches at the mRNA level. However, the process of apoptosis can be initiated by a variety of stimuli and results in defined morphological and biochemical changes (3) that may be easier studied at the protein level. Apoptosis is characterized by cellular and nuclear shrinkage, cytoplasmic blebbing, condensation of nuclear chromatin, and fragmentation of nuclear DNA (3, 4). Analysis of the apoptotic pathway in lymphocytes revealed several molecules as key controllers in the execution of apoptosis (5). Anti-IgM antibody-mediated apoptosis is thought to be controlled on at least two levels by the members of the bcl-2 gene family and the interleukin-1␤-converting enzyme/Ced3-like cysteine proteases now called caspases. A large number of caspases have been described (6 -8), but the individual roles of most intracellular proteases and their substra...

Apoptosis (programmed cell death) is instrumental in the process of controlling lymphocyte growth and selection. Negative selection, mediated by surface IgM (sIgM) signaling after encountering self antigen, eliminates autoreactive B cells. To identify proteins which are potentially involved in anti-IgM-mediated apoptosis, we used an anti-IgM-sensitive subclone of the human Burkitt lymphoma cell line BL60. After anti-IgM treatment and separation of apoptosis-committed cells, we performed high resolution two-dimensional gel electrophoresis (2-DE). Comparison of the 2-DE protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots. Subsequent analysis of these proteins was performed by mass spectrometry and Edman microsequencing. We report that one of these spots which disappears after sIgM cross-linking turned out to be D4-GDI. D4-GDI is an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPase. D4-GDI was rapidly truncated to a 23-kDa fragment in BL60 cells. By using a Rho-GDI-specific antiserum, which cross-reacts with D4-GDI, we observed the onset of cleavage after 8 h of stimulation with anti-IgM. Cleavage and apoptosis could be completely inhibited by z-DEVD-fmk, a selective irreversible inhibitor of CPP32 (caspase-3), whereas ac-YVAD-cmk, an inhibitor for interleukin-1beta-converting enzyme-like proteases, did not block cleavage of D4-GDI or apoptosis. Our results revealed the functional importance of caspases and a new target protein in the process of anti-IgM-mediated apoptosis.

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified RNA polymerase B transcription factor 3 (BTF3), which is associated with anti-IgM antibody-mediated apoptosis, using a subclone of the human Burkitt lymphoma cell line BL60. To identify the transcription factor BTF3, which is expressed only in minor amounts, we used preparative high-resolution two-dimensional gel electrophoresis (2DE) employing carrier ampholytes for isoelectric focusing. Comparison of the 2DE protein patterns from apoptotic and nonapoptotic cells showed BTF3 as a predominantly altered protein spot. The characterization of the differentially expressed transcription factor and 13 marker proteins described in this study were performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The proteome analysis was significantly improved by performing the newly developed preparative high-resolution two-dimensional gels employing high protein concentrations.

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