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 (gGCS), 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 24 h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3 h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24 h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21 waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a timedependent manner, beginning at 24 h 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 pancaspase 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 (DC m ) remained intact in most of the cells during the 72 h observation period, indicating that DC 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.

The ATM kinase has previously been shown to respond to the DNA damage induced by reoxygenation following hypoxia by initiating a Chk 2-dependent cell cycle arrest in the G 2 phase. Here we show that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage, detectable by either comet assay or 53BP1 focus formation. Hypoxia-induced activation of ATM correlates with oxygen concentrations low enough to cause a replication arrest and is entirely independent of hypoxia-inducible factor 1 status. In contrast to damage-activated ATM, hypoxia-activated ATM does not form nuclear foci but is instead diffuse throughout the nucleus. The hypoxia-induced activity of both ATM and the related kinase ATR is independent of NBS1 and MRE11, indicating that the MRN complex does not mediate the DNA damage response to hypoxia. However, the mediator MDC1 is required for efficient activation of Kap1 by hypoxiainduced ATM, indicating that similarly to the DNA damage response, there is a requirement for MDC1 to amplify the ATM response to hypoxia. However, under hypoxic conditions, MDC1 does not recruit BRCA1/ 53BP1 or RNF8 activity. Our findings clearly demonstrate that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks.Sensing and responding to DNA damage is crucial for maintaining cellular homeostasis and preventing the development of cancer. The cellular response to DNA damage can be divided into three parts: sensing the type of damage, activating DNA damage signaling pathways, and repairing the damage. The proteins involved in these three processes act as sensors, transducers, and effectors of the DNA damage response (41, 54). The ATM kinase is one of the key transducers of the DNA double-stranded break response. ATM has been identified as the product mutated or inactivated in ataxia telangiectasia (AT) patients and belongs to the phosphatidyl inositol-3-kinase-like kinase (PIKK) family, together with its family members ATR, DNA-PK, and mTOR. Elegant studies have demonstrated that ATM is present as an inactive dimer that undergoes rapid autophosphorylation on serine 1981 after DNA damage (4) and is recruited to sites of DNA strand breaks (2). Various proteins, such as the MRE11-Rad50-NBS1 (MRN) complex (49) and MDC1 (20, 43), have been described to be essential for the efficient response of ATM to DNA damage. Activated ATM phosphorylates downstream targets, such as p53, Chk2, and BRCA 1 and 2, that are involved in DNA repair, cell cycle control, and apoptosis.Low-oxygen tension or hypoxia is a common feature in all solid tumors (15). It is strongly associated with tumor development, malignant progression, metastatic outgrowth, and resistance to therapy and is considered an independent prognostic indicator for poor patient prognosis in various tumor types. Tumor hypoxia results from an imbalance between the cellular oxygen consumption rate of cells and the delivery of oxygen to cells (50). Interestingly, the level of tumor hy...

Exposure to hypoxia-induced replication arrest initiates a DNA damage response that includes both ATR-and ATM-mediated signaling. DNA fiber analysis was used to show that these conditions lead to a replication arrest during both the initiation and elongation phases, and that this correlated with decreased levels of nucleotides. The DNA damage response induced by hypoxia is distinct from the classical pathways induced by damaging agents, primarily due to the lack of detectable DNA damage, but also due to the coincident repression of DNA repair in hypoxic conditions. The principle aims of the hypoxia-induced DNA damage response seem to be the induction of p53-dependent apoptosis or the preservation of replication fork integrity. The latter is of particular importance should reoxygenation occur. Tumor reoxygenation occurs as a result of spontaneous changes in blood flow and also therapy. Cells experiencing hypoxia and/or reoxygenation are, therefore, sensitive to loss or inhibition of components of the DNA damage response, including Chk1, ATM, ATR, and poly(ADP-ribose) polymerase (PARP). In addition, restoration of hypoxia-induced p53-mediated signaling may well be effective in the targeting of hypoxic cells. The DNA damage response is also induced in endothelial cells at moderate levels of hypoxia, which do not induce replication arrest. In this situation, phosphorylation of H2AX has been shown to be required for proliferation and angiogenesis and is, therefore, an attractive potential therapeutic target. Clin Cancer Res; 16(23); 5624-9. Ó2010 AACR.

Several water-solubilized versions of the zinc ionophore 1-hydroxypyridine-2-thione (ZnHPT), synthesized as part of the present study, have been found both to increase the intracellular concentrations of free zinc and to produce an antiproliferative activity in exponential phase A549 human lung cancer cultures. Gene expression profiles of A549 cultures treated with one of these water-soluble zinc ionophores, PCI-5002, reveal the activation of stress response pathways under the control of metal-responsive transcription factor 1 (MTF-1), hypoxia-inducible transcription factor 1 (HIF-1), and heat shock transcription factors. Additional oxidative stress response and apoptotic pathways were activated in cultures grown in zinc-supplemented media. We also show that these water-soluble zinc ionophores can be given to mice at 100 Mmol/kg (300 Mmol/m 2 ) with no observable toxicity and inhibit the growth of A549 lung and PC3 prostate cancer cells grown in xenograft models. Gene expression profiles of tumor specimens harvested from mice 4 h after treatment confirmed the in vivo activation of MTF-1-responsive genes. Overall, we propose that water-solubilized zinc ionophores represent a potential new class of anticancer agents. [Cancer Res 2008;68(13):5318-25]