Apoptosis induced by extracellular glutathione is mediated by H(2)O(2) production and DNA damage.
The biochemical basis of the anti-proliferative effect of exogenous glutathione was investigated in A2780 ovarian carcinoma cells. Previous observations have implicated gamma-glutamyl transpeptidase-mediated pro-oxidant reactions as a primary mechanism of the extracellular effects of glutathione. In 2 cell lines (A2780 and IGROV-1), glutathione led to H(2)O(2) production, but only A2780 cells, characterized by low expression of detoxification enzymes, were sensitive to the thiol compound. In A2780 cells, glutathione exposure resulted in DNA single-strand break formation, as measured by alkaline elution. Glutathione-induced DNA damage generated significant levels of apoptosis in A2780 cells, but not in IGROV-1 cells. The capability of glutathione to induce apoptosis was associated with cleavage of poly(ADP-ribose)polymerase and with generation of a low-molecular-weight form of the pro-apoptotic protein bax. In A2780 cells, glutathione exposure was followed by p21 and Bax induction and p53 up-regulation, as expected for genotoxic stress. Consistently, analysis of cell-cycle perturbations demonstrated the occurrence of G(2)M accumulation after exposure to glutathione, similar to what was observed for H(2)O(2). Taken together, these results indicate that the cytotoxic effect of extracellular glutathione, related to membrane metabolism, is mediated by production of H(2)O(2) leading to DNA damage and a cellular response involving p53. These findings might also provide insights into the cellular and molecular determinants of chemosensitivity to DNA damaging agents, as oxidative stress is implicated in p53-dependent apoptosis.
Perego
P
P
Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy. perego@istitutotumori.mi.it
Gatti
L
L
Carenini
N
N
Dal Bo
L
L
Zunino
F
F
D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000187:drug effects; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016158:Genes, p53; D005978:Glutathione / Q000494:pharmacology*; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008658:Inactivation, Metabolic; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology; D018384:Oxidative Stress; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D014407:Tumor Cells, Cultured / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004273:DNA, Neoplasm; D006131:Growth Inhibitors; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; D005978:Glutathione