leupeptins and Teratocarcinoma

Copper, a transition metal with essential biological functions, exerts neurotoxic effects when present in excess. The aim of the present study was to better elucidate cellular and molecular mechanisms of CuSO4 toxicity in differentiated P19 neurons. Exposure to 0.5 mM CuSO4 for 24 h provoked moderate decrease in viability, accompanied with barely increased generation of reactive oxygen species (ROS) and caspase-3/7 activity. Glutathione (GSH) and ATP contents were depleted, lactate dehydrogenase inactivated, and glyceraldehyde-3-phosphate dehydrogenase overexpressed. In severely damaged neurons exposed to only two times higher concentration, classical caspase-dependent apoptosis was triggered as evidenced by marked caspase-3/7 activation and chromatin condensation. Multifold increase in ROS, together with very pronounced ATP and GSH loss, strongly suggests impairment of redox homeostasis. At higher copper concentration protease calpains were also activated, and neuronal injury was prevented in the presence of calpain inhibitor leupeptin through the mechanism that affects caspase activation. MK-801 and nifedipine, inhibitors of calcium entry, and H-89 and UO126, inhibitors of PKA and ERK signaling respectively, exacerbated neuronal death only in severely damaged neurons, while ROS-scavenger quercetin and calcium chelator BAPTA attenuated toxicity only at lower concentration. In a dose-dependent manner copper also provoked transcriptional changes of genes involved in intracellular signaling and induction of apoptosis (p53, c-fos, Bcl-2 and Bax). The obtained results emphasize differences in triggered neuronal-death processes in a very narrow range of concentrations and give further insight into the molecular mechanisms of copper toxicity with the potential to improve current therapeutic approaches in curing copper-related neurodegenerative diseases.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Apoptosis Regulatory Proteins; Calpain; Caspases; Cell Line, Tumor; Chelating Agents; Chromatin; Copper Sulfate; Dizocilpine Maleate; Gene Expression Regulation; Glutathione; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Leupeptins; Mice; Neoplasm Proteins; Neurons; Nifedipine; Oxidation-Reduction; Oxidative Stress; Protease Inhibitors; Protein Kinase Inhibitors; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Teratocarcinoma

To bind DNA and to be retained in the nucleus, PBX proteins must form heterodimeric complexes with members of the MEINOX family. Therefore the balance between PBX and MEINOX must be an important regulatory feature. We show that overexpression of PREP-1 influences the level of PBX-2 protein maintaining the PREP-1-PBX balance. This effect has important functional consequences. F9 teratocarcinoma cells stably transfected with PREP-1 had an increased DNA binding activity to a PREP-PBX-responsive element. Because PREP-1 binds DNA efficiently only when dimerized to PBX, the increased DNA binding activity suggests that the level of PBX might also have increased. Indeed PREP-1-overexpressing cells had a higher level of PBX-2 and PBX-1b proteins. PBX-2 increase did not depend on increased mRNA level or a higher rate of translation but rather because of a protein stabilization process. Indeed, PBX-2 level drastically decreased after 3 h of cycloheximide treatment in control but not in PREP-1-overexpressing cells and the proteasome inhibitor MG132 prevented PBX-2 decay in control cells. Hence, dimerization with PREP-1 appears to decrease proteasomal degradation of PBX-2. Retinoic acid induces differentiation of F9 teratocarcinoma cells with a cascade synthesis of HOX proteins. In PREP-1-overexpressing cells, HOXb1 induction was more sustained (3 days versus 1 day) and the induced level of MEIS-1b, another TALE (three amino acid loop extension) protein involved in embryonal development, was higher. Thus an increase in PREP-1 leads to changes in the fate-determining HOXb1 and has therefore important functional consequences.

Topics: Amino Acid Sequence; Blotting, Northern; Cell Differentiation; Cell Nucleus; Cycloheximide; Cysteine Endopeptidases; Cytoplasm; Dimerization; DNA; Homeodomain Proteins; Immunoblotting; Kinetics; Leupeptins; Models, Biological; Molecular Sequence Data; Multienzyme Complexes; Myeloid Ecotropic Viral Integration Site 1 Protein; Neoplasm Proteins; Proteasome Endopeptidase Complex; Protein Binding; Protein Biosynthesis; Protein Synthesis Inhibitors; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Teratocarcinoma; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured