Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line.
The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.
Armstrong
J S
JS
Department of Radiation Oncology, Stanford University, Stanford, California, CA 94305-5105, USA.
Steinauer
K K
KK
Hornung
B
B
Irish
J M
JM
Lecane
P
P
Birrell
G W
GW
Peehl
D M
DM
Knox
S J
SJ
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000172:deficiency / Q000378:metabolism*; D006801:Humans; D016393:Lymphoma, B-Cell / Q000378:metabolism / Q000473:pathology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology; D016328:NF-kappa B / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D051996:Transcription Factor RelA; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis
D000964:Antimetabolites, Antineoplastic; D003574:Cytochrome c Group; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D051996:Transcription Factor RelA; D016159:Tumor Suppressor Protein p53; D019328:Buthionine Sulfoximine; D005978:Glutathione; D004229:Dithiothreitol