Identification of Apoptosis-associated Proteins in a Human Burkitt Lymphoma Cell Line

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 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...

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study

“…On the ground of the SRB assay results, IC 50 values of 0.5 and 0.3 lM were determined for auranofin on the sensitive and resistant cell lines, respectively, after 72-h drug exposure. In turn, the measured IC 50 values for Auoxo6 were found to be perfectly consistent with those previously reported by Casini et al [5] (1.8 and 4.9 lM for A2780/S and A2780/R, respectively). We also observed that very limited cell death was evident, for both compounds, at 24 h, this rendering a classic proteomic approach well feasible.…”

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“…Briefly, the cells were seeded in tissue-culture plates at 5 9 10 4 cells/mL (total volume 30 mL) and incubated overnight, then exposed to concentrations of the study compounds equal to 72-h-exposure IC 50 values for 24 h. At the end of the incubation the cells were washed with phosphate-buffered saline, then were scraped in RIPA buffer [50 mM Tris-HCl pH 7.0, 1% NP-40, 150 mM NaCl, 2 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, 100 mM NaF] containing a cocktail of protease inhibitors (Sigma). The cells were sonicated (10 s) and protein extracts were clarified by centrifugation at 8,000g for 10 min.…”

Structural Change of Ribosomes during Apoptosis: Degradation and Externalization of Ribosomal Proteins in Doxorubicin-Treated Jurkat Cells

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Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

“…Apoptosis studies on Burkitt lymphoma BL60 cells revealed various apoptosis-associated proteins not known in this process so far [117,118]. They gave clues about the apoptosis pathway, the caspases cascade and specific cleavage sites [119,120].…”