Mitochondrial GPx1 decreases induced but not basal oxidative damage to mtDNA in T47D cells

The production of oxyradicals by mitochondria (mt) is a source of oxidative damage to mtDNA such as 8-oxo-dG lesions that may lead to mutations and mitochondrial dysfunction. The potential protection of mtDNA by glutathione peroxidase-1 (GPx1) was investigated in GPx1-proficient (GPx-2) and GPx1-deficient (Hygro-3) human breast T47D cell transfectants. GPx activity and GPx1-like antigen concentration in mitochondria were respectively at least 100-fold and 20- to 25-fold higher in GPx2 than Hygro-3 cells. In spite of this large difference in peroxide-scavenging capacity, the basal 8-oxo-dG frequency in mtDNA, assessed by carefully controlled postlabeling assay, was strikingly similar in both cell lines. In contrast, in response to menadione-mediated oxidative stress, induction of 8-oxo-dG and DNA strand breaks was much lower in the GPx1-proficient mitochondria (e.g., +14% 8-oxo-dG versus +54% in Hygro-3 after 1-h exposure to 25 microM menadione, P < 0.05). Our data indicate that the mitochondrial glutathione/GPx1 system protected mtDNA against damage induced by oxidative stress, but did not prevent basal oxidative damage to mtDNA, which, surprisingly, appeared independent of GPx1 status in the T47D model.     

Hyperoxia Induces S-Phase Cell-Cycle Arrest and p21-Independent Cdk2 Inhibition in Human Carcinoma T47D-H3 Cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O₂, 40–64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21^Cip1. The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21^Cip1 or p27^Kip1 in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21^Cip1/p27^Kip1-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Activation of the G2 cell cycle checkpoint enhances survival of epithelial cells exposed to hyperoxia

Reactive oxygen species produced during hyperoxia damage DNA, inhibit proliferation in G1- through p53-dependent activation of p21(Cip1/WAF1/Sdi1), and kill cells. Because checkpoint activation protects cells from genotoxic stress, we investigated cell proliferation and survival of the murine type II epithelial cell line MLE15 during hyperoxia. These cells were chosen for study because they express Simian large and small-T antigens, which transform cells in part by disrupting the p53-dependent G1 checkpoint. Cell counts, 5-bromo-2'-deoxyuridine labeling, and flow cytometry revealed that hyperoxia slowed cell cycle progression after one replication, resulting in a pronounced G2 arrest by 72 h. Addition of caffeine, which inactivates the G2 checkpoint, diminished the percentage of hyperoxic cells in G2 and increased the percentage in sub-G1 and G1. Abrogation of the G2 checkpoint was associated with enhanced oxygen-induced DNA strand breaks and cell death. Caffeine did not affect DNA integrity or viability of cells exposed to room air. Similarly, caffeine abrogated the G2 checkpoint in hyperoxic A549 epithelial cells and enhanced oxygen-induced toxicity. These data indicate that hyperoxia rapidly inhibits proliferation after one cell cycle and that the G2 checkpoint is critical for limiting DNA damage and cell death.     

Exposure to power frequency magnetic fields suppresses X-ray-induced apoptosis transiently in Ku80-deficient xrs5 cells

In an attempt to determine whether exposure to extremely low frequency (ELF) electromagnetic fields can affect cells, Ku80-deficient cells (xrs5) and Ku80-proficient cells (CHO-K1) were exposed to ELF electromagnetic fields. Cell survival, and the levels of the apoptosis-related genes p21, p53, phospho-p53 (Ser(15)), caspase-3 and the anti-apoptosis gene bcl-2 were determined in xrs5 and CHO-K1 cells following exposure to ELF electromagnetic fields and X-rays. It was found that exposure of xrs5 and CHO-K1 cells to 60 Hz ELF electromagnetic fields had no effect on cell survival, cell cycle distribution and protein expression. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields for 5 h after irradiation significantly inhibited G(1) cell cycle arrest induced by X-rays (1 Gy) and resulted in elevated bcl-2 expression. A significant decrease in the induction of p53, phospho-p53, caspase-3 and p21 proteins was observed in xrs5 cells when irradiation by X-rays (8 Gy) was followed by exposure to 5 mT ELF magnetic fields. Exposure of xrs5 cells to the ELF electromagnetic fields for 10 h following irradiation significantly decreased X-ray-induced apoptosis from about 1.7% to 0.7%. However, this effect was not found in CHO-K1 cells within 24 h of irradiation by X-rays alone and by X-rays combined with ELF electromagnetic fields. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields following irradiation can affect cell cycle distribution and transiently suppress apoptosis by decreasing the levels of caspase-3, p21, p53 and phospho-p53 and by increasing bcl-2 expression.     

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G(1) when exposed to 95% oxygen. Instead, cells arrested in S and G(2). Stable expression of p53 restored induction of p21 and G(1) arrest without affecting mRNA expression of the other Cip or INK4 G(1) kinase inhibitors. To confirm the role of p21 in G(1) arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G(1) arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G(1) during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G(1) arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.     

Hyperoxia induces macrophage cell cycle arrest by adhesion-dependent induction of p21Cip1 and activation of the retinoblastoma protein

Hyperoxia induces growth arrest, apoptosis, necrosis, and morphological changes (spreading and adhesion) in various types of cells. The mechanism of hyperoxia-induced cell growth arrest has not been well elucidated, especially in macrophages. One possible mechanism is a role of cell adhesion in hyperoxia-induced cell cycle arrest. To evaluate this finding, macrophages were cultured in normoxia (21% O2) or hyperoxia (95% O2) in adhesion or low adhesion conditions. Incubation of macrophages in hyperoxia induced cell cycle arrest. The hyperoxia-induced cell cycle arrest was prevented by low adhesion conditions. To evaluate pathways potentially involved in hyperoxia-induced growth arrest, we measured extracellular regulated kinase and retinoblastoma protein activation and p21Cip1 and p53 accumulation. Hyperoxia strongly induced activation of extracellular regulated kinase and retinoblastoma protein as well as up-regulation of p21Cip1. These effects of hyperoxia were attenuated under low adhesion conditions, suggesting a role for integrin-dependent signaling. The induction of p21Cip1 and activation of retinoblastoma protein occurred via a p53-independent mechanism. These results suggest that adhesion-dependent pathways are required for hyperoxia-induced cell cycle arrest in macrophages.     

The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury.

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O2; 5% CO2) for 0-48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH+XO activity decreased 98% after 48 h of 95% O2 exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH+XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8-14C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH+XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K3Fe(CN)6). After 48 h of culture in air with allopurinol, cell XDH+XO activity was enhanced when assayed after reversal of inhibition with K3Fe(CN)6, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K3Fe(CN)6 oxidation, XDH+XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH+XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O2- and H2O2 during normoxia.     

Poly(ADP-ribose) polymerase-1 (PARP-1) controls lung cell proliferation and repair after hyperoxia-induced lung damage

Oxygen-based therapies expose lung to elevated levels of ROS and induce lung cell damage and inflammation. Injured cells are replaced through increased proliferation and differentiation of epithelial cells and fibroblasts. Failure to modulate these processes leads to excessive cell proliferation, collagen deposition, fibrosis, and chronic lung disease. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated in response to DNA damage and participates in DNA repair, genomic integrity, and cell death. In this study, we evaluated the role of PARP-1 in lung repair during recovery after acute hyperoxia exposure. We exposed PARP-1 -/- and wild-type mice for 64 h to 100% hyperoxia and let them recover in air for 5-21 days. PARP-1-deficient mice exhibited significantly higher lung cell hyperplasia and proliferation than PARP-1 +/+ animals after 5 and 10 days of recovery. This was accompanied by an increased inflammatory response in PARP-1 -/- compared with wild-type animals, characterized by neutrophil infiltration and increased IL-6 levels in bronchoalveolar lavages. These lesions were reversible, since the extent of the hyperplastic regions was reduced after 21 days of recovery and did not result in fibrosis. In vitro, lung primary fibroblasts derived from PARP-1 -/- mice showed a higher proliferative response than PARP-1 +/+ cells during air recovery after hyperoxia-induced growth arrest. Altogether, these results reveal an essential role of PARP-1 in the control of cell repair and tissue remodeling after hyperoxia-induced lung injury.     

Effects of continuous and episodic hyperoxia on stress and hepatic glutathione levels in one-summer-old rainbow trout (Oncorhynchus mykiss)

One-summer-old rainbow trout ( Oncorhynchus mykiss ) were exposed to continuous hyperoxia (173 ± 24%) and three hyperoxic/normoxic treatments for 14 days. Hepatic glutathione status as the indicator of oxidative stress, as well as classical stress indicators such as hemoglobin, hematocrit and plasma cortisol levels, were measured during normoxic, constantly hyperoxic and the following episodically hyperoxic oxygen treatment regimes: 12 h hyperoxia:12 h normoxia (12 HYP:12 NOR), 24 HYP:24 NOR and 48 HYP:24 NOR. Constant hyperoxia tended to shrink erythrocytes, but the 12 HYP:12 NOR treatment increased the number of erythrocytes and thus enhanced the oxygen carrying capacity of blood. Similarly, a trend toward an elevation in plasma cortisol concentrations was detected in 12 HYP:12 NOR treatment group. The finding that elevated hepatic glutathione (GSH) levels (P < 0.01), indicative of enhanced potential of the liver tissue to resist oxidative stress, coincided with elevated cortisol levels might suggest that in the 12 HYP:12 NOR treatment physiological processes were recruited to increase oxygen carrying capacity in blood and to elevate protection against oxyradicals. However, none of the episodic hyperoxia treatments or continuous hyperoxia caused mortality or resulted in better growth. These data indicate that continuous hyperoxia (173 ± 24%) and hyperoxic-normoxic treatments may be applied in intensive culture of rainbow trout provided that fish have at least 24 h in normoxia prior to the next bout of hyperoxia. Shorter recovery periods, like in a 12 HYP:12 NOR treatment, may result in the increased need of oxygen in tissues followed by an activation of glutathione dependent defence system against an increased oxygen load.     

Glutathione peroxidase-1 protects from CD95-induced apoptosis

Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis.     

Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow.

Integrin-associated protein (CD47/IAP) is a pentaspan molecule that regulates integrin functions. We prepared a CD47-deficient Jurkat T cell line to assess its role in the arrest of T cells on inflammatory endothelium. Under flow conditions, constitutive arrest of CD47-deficient cells is strongly decreased as compared to the original cell line, whereas reexpression of CD47 reestablishes their ability to stop. Moreover, cells transfected with a chimera made with the extracellular portion of CD47 and the transmembrane domain of CD7 or several truncated forms of CD47 show that the first transmembrane domain and a short cytoplasmic loop are sufficient for this process. CD47 effect is indirect and depends mainly on the alpha4beta1/VCAM-1 pathway, as shown by blocking antibodies. We detected on endothelium the two CD47 counter receptors known to date: thrombospondin and SIRP1alpha. Blocking experiments show that both are involved. Overall, CD47 participates in the constitutive arrest of T lymphocytes on inflamed vascular endothelium by up-regulating alpha 4beta1 integrins.     

Regulatory volume decrease is actively modulated during the cell cycle

Nasopharyngeal carcinoma cells, CNE-2Z, when swollen by 47% hypotonic solution, exhibited a regulatory volume decrease (RVD). The RVD was inhibited by extracellular applications of the chloride channel blockers tamoxifen (30 microM; 61% inhibition), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM; 60% inhibition), and ATP (10 mM; 91% inhibition). The level and time constant of RVD varied greatly between cells. Most cells conducted an incomplete RVD, but a few had the ability to recover their volume completely. There was no obvious correlation between cell volume and RVD capacity. Flow cytometric analysis showed that highly synchronous cells were obtained by the mitotic shake-off technique and that the cells progressed through the cell cycle synchronously when incubated in culture medium. Combined application of DNA synthesis inhibitors, thymidine and hydroxyurea arrested cells at the G1/S boundary and 87% of the cells reached S phase 4 h after being released. RVD capacity changed significantly during the cell cycle progression in cells synchronized by shake-off technique. RVD capacity being at its highest in G1 phase and lowest in S phase. The RVD capacity in G1 (shake-off cells sampled after 4 h of incubation), S (obtained by chemical arrest), and M cells (selected under microscope) was 73, 33, and 58%, respectively, and the time constants were 435, 769, and 2,000 sec, respectively. We conclude that RVD capacity is actively modulated in the cell cycle and RVD may play an important role in cell cycle progress.     

PUMA inactivation protects against oxidative stress through p21/Bcl-XL inhibition of bax death

The tumor suppressor protein p53 activates growth arrest and proapoptotic genes in response to DNA damage. It is known that negative feedback by p21(Cip1/Waf1/Sdi1) represses p53-dependent transactivation of PUMA. The current study investigates PUMA feedback on p53 during oxidative stress from hyperoxia and the subsequent effects on cell survival mediated through p21 and Bcl-X(L). Deletion of PUMA in HCT116 colon carcinoma cells increased levels of p53 and p21, resulting in a larger G(1) population during hyperoxia. P21-dependent increase in Bcl-X(L) levels protected PUMA-deficient cells against hyperoxic cell death. Bax and Bak were both able to promote hyperoxic cell death. Bcl-X(L) protection against hyperoxic death was lost in cells lacking Bax, not PUMA, suggesting that Bcl-X(L) acts to inhibit Bax-dependent death. These results indicate that PUMA exerts a negative feedback on p53 and p21, leading to p21-dependent growth suppressive and survival changes. Enhanced survival was associated with increased Bcl-X(L) to block Bax activated cell death during oxidative stress.     

Continuous enteral nutrition attenuates pulmonary edema in rats exposed to 100% oxygen.

Adult rats exposed to hyperoxia develop anorexia, weight loss, and a lung injury characterized by pulmonary edema and decreased lung liquid clearance. We hypothesized that maintenance of nutrition during hyperoxia could attenuate hyperoxia-induced pulmonary edema. To test this hypothesis, we enterally fed adult male Sprague-Dawley rats via gastrostomy tubes and exposed them to oxygen (inspired O(2) fraction >0.95) for 64 h. In contrast to controls, enterally fed hyperoxic animals did not lose weight and had smaller pleural effusions and wet-to-dry weight ratios (a measure of lung edema) that were not different from room air controls. Enterally fed rats exposed to hyperoxia had increased levels of mRNA for the Na(+)-K(+)-ATPase alpha(1)- and beta(1)-subunits and glutathione peroxidase. These findings suggest that maintenance of nutrition during an oxidative lung injury reduces lung edema, perhaps by allowing for continued expression and function of protective proteins such as the Na(+)-K(+)-ATPase.     

Interactions between CD47 and thrombospondin reduce inflammation

CD47 on the surface of T cells was shown in vitro to mediate either T cell activation or, in the presence of high amounts of thrombospondin (TSP), T cell apoptosis. We report here that CD47-deficient mice, as well as TSP-1 or TSP-2-deficient mice, sustain oxazolone-induced inflammation for more than four days, whereas wild-type mice reduce the inflammation within 48 h. We observe that prolonged inflammation in CD47-, TSP-1-, or TSP-2-deficient mice is accompanied by a local deficiency of T cell apoptosis. Finally, we show that upon activation normal T cells increase the expression of the proapoptotic Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein) and undergo CD47-mediated apoptosis. This finding is consistent with our previous demonstration of a physical interaction between BNIP3 and CD47 that inhibits BNIP3 degradation by the proteasome, sensitizing T cells to CD47-induced apoptosis. Overall, these results reveal an important role in vivo for this new CD47/BNIP3 pathway in limiting inflammation by controlling the number of activated T cells.