Glyceraldehyde-3-Phosphate Dehydrogenase: Nuclear Translocation Participates In Neuronal and Nonneuronal Cell Death (original) (raw)

1997, Proceedings of the …

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels increase in particulate fractions in association with cell death in HEK293 cells, S49 cells, primary thymocytes, PC12 cells, and primary cerebral cortical neuronal cultures. Subcellular fractionation and immunocytochemistry reveal that this increase primarily ref lects nuclear translocation. Nuclear GAPDH is tightly bound, resisting extraction by DNase or salt treatment. Treating primary thymocytes, PC12 cells, and primary cortical neurons with antisense but not sense oligonucleotides to GAPDH prevents cell death. Because cell-death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and nonneuronal systems, we propose that GAPDH is a general mediator of cell death and uses nuclear translocation as a signaling mechanism. MATERIALS AND METHODS Materials. Unless noted, chemicals were obtained from Sigma. Primary antibodies (Abs) were obtained from the following companies or as generous gifts: anti-MAP2 monoclonal Ab (Boehringer Mannheim) (dilution 1:300), anti-MAP2 polyclonal Ab (Dr. Y. Ihara, University of Tokyo, Japan) (dilution 1:300), anti-glial fibrillary acidic protein (GFAP) Ab (Dako) (dilution 1:1,000), anti-GAPDH monoclonal Ab (Chemicon) (dilution 1:200-1,200), anti-GAPDH polyclonal Ab (Charles River Breeding Laboratories) (dilution 1:200-1,200). Peroxidase-conjugated anti-mouse, rabbit, or goat IgG Abs were used as secondary Abs for immunoblot (Boehringer Mannheim; 1:2,000 dilution). FITC-conjugated anti mouse IgG Ab (dilution 1:200) (Jackson ImmunoResearch) for GAPDH and MAP2, FITC-conjugated anti-goat IgG Ab (dilution 1:200) (Jackson ImmunoResearch) for lactate dehydrogenase (LDH), and Texas red-conjugated antirabbit IgG Ab (dilution 1:200) (Jackson ImmunoResearch) for MAP2 and GFAP were used as secondary Abs for immunocytochemistry. For nuclear staining, Hoechst 33258 (1:500 dilution) or propidium iodide (1:1,000 dilution) (Molecular Probes) was used. Antisense phosphorothioate oligonucleotides to GAPDH were produced in a DNA analysis facility in the Johns Hopkins University. These rat sequences were 5Ј-GACCTTCAC-CATCTTGTCTA-3Ј for antisense 1, 5Ј-GTGGATGCAGG-GATGATGTT-3Ј for antisense 2, and 5Ј-TAGACAAGAT-GGTGAAGGTC-3Ј for sense oligo. Cell Culture. Primary thymocytes taken from the thymus of 1-to 2-month-old male Sprague-Dawley rats (Charles River Breeding Laboratories) were cultured in RPMI 1640 medium (GIBCO͞BRL) plus 10% fetal bovine serum (FBS). S49 cells were maintained in the same medium as for primary thymocytes. HEK293 cells were maintained in DMEM plus 10% FBS. After two washes with DMEM, the medium was replaced with serum-free DMEM to induce cell death. PC12 cells were grown and differentiated as described previously (23). Briefly, PC12 cells were maintained in DMEM plus 5% FBS and 10% horse serum (HS). Differentiation was initiated by addition of nerve growth factor (NGF) and changing FBS and HS concentrations to 2% and 1%, respectively. Five days after differentiation, the medium was completely replaced with medium lacking NGF and serum. Primary cortical cultures were prepared from fetal Sprague-Dawley rats (19-to 20-day gestation). The dissection and plating procedures were described previously (24). The dissociated cells were grown in B27-supplemented serum-free media (25). More than 95% of cells were neurons, as estimated by double staining using anti-MAP2 Ab and anti-GFAP Ab.