The effect of alveolar oxygen tension on lung lipid peroxidation during lung ischemia was evaluated by using isolated rat lungs perfused with synthetic medium. After a 5-min equilibration period, global ischemia was produced by discontinuing perfusion while ventilation continued with gas mixtures containing 5% CO2 and a fixed oxygen concentration between 0 and 95%. Lipid peroxidation was assessed by measurement of tissue thiobarbituric acid-reactive products and conjugated dienes. Control studies (no ischemia) showed no change in parameters of lipid peroxidation during 1 h of perfusion and ventilation with 20% or 95% 02. With 60 min of ischemia, there was increased lipid peroxidation which varied with oxygen content of the ventilating gas and was markedly inhibited by ventilation with N2. Perfusion with 5-, 8-, 11-, 14-eicosatetraynoic acid indicated that generation of eicosanoids during ischemia accounted for -40-50% of lung lipid peroxide production. Changes of CO2 content of the ventilating gas (to alter tissue pH) or of perfusate glucose concentration had no effect on lipid peroxidation during ischemia, but perfusion at 8% of the normal flow rate prevented lipid peroxidation. Lung dry/wet weight measured after 3 min of reperfusion showed good correlation between lung fluid accumulation and lipid peroxidation. These results indicate that reperfusion is not necessary for lipid peroxidation with ischemic insult of the lung and provide evidence that elevated Po2 during ischemia accelerates the rate of tissue injury. (J. Clin. Invest. 1991. 88:674479.)

The oxidative pentose phosphate cycle (OPPC) is necessary to maintain cellular reducing capacity during periods of increased oxidative stress. Metabolic flux through the OPPC increases stoichiometrically in response to a broad range of chemical oxidants, including those that generate reactive oxygen species (ROS). Here we show that OPPC sensitivity is sufficient to detect low levels of ROS produced metabolically as a function of the percentage of O 2 . We observe a significant decrease in OPPC activity in cells incubated under severe and moderate hypoxia (ranging from <0.01 to 4% O 2 ), whereas hyperoxia (95% O 2 ) results in a significant increase in OPPC activity. These data indicate that metabolic ROS production is directly dependent on oxygen concentration. Moreover, we have found no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize hypoxia-inducible factor 1␣ (HIF-1␣) under moderate hypoxia. Myxothiazol, an inhibitor of mitochondrial electron transfer, did not prevent HIF-1␣ stabilization under moderate hypoxia. Moreover, the levels of HIF-1␣ that we observed after exposure to moderate hypoxia were comparable between 0 cells, which lack functional mitochondria, and the wild-type cells. Finally, we find no evidence for stabilization of HIF-1␣ in response to the non-toxic levels of H 2 O 2 generated by the enzyme glucose oxidase. Therefore, we conclude that the oxygen dependence of the prolyl hydroxylase reaction is sufficient to mediate HIF-1␣ stability under moderate as well as severe hypoxia.The primary role of the oxidative pentose phosphate cycle (OPPC) 2 in mammalian cells is to maintain the [NADPH/ NADPϩ] ratio, thereby helping to regulate the cellular redox equilibrium (1-3). Glucose-6-phosphate dehydrogenase (G6PD), the initial and rate-limiting enzyme of the OPPC, exists in a dimer-tetramer equilibrium with the tetramer being the catalytically active conformation. Each of four identical G6PD monomers contains a structural NADPϩ binding site (4, 5). When NADPϩ is bound at this site, formation of the active tetramer is favored. In non-stressed cells, the [NADPH]/ [NADPϩ] ratio is very high (approaching 1000) and flux through the OPPC is minimal. However, even a slight increase in [NADPϩ] can increase the number of active G6PD tetramers. Therefore, G6PD activity, by regulating flux through the OPPC, is uniquely sensitive to reactive oxygen species (ROS) as well as other chemical oxidants.The importance of the OPPC for the cellular response to ROS is evident from the elevated incidence of apoptosis that we observed in G6PD Ϫ Chinese hamster ovary cells following exposure to ionizing radiation (2). Likewise, Efferth et al. (6) observed an elevated incidence of oxidant-induced apoptosis in macrophages isolated from patients suffering from G6PD deficiency syndrome, while Fico et al. (42) observed H 2 O 2 -induced apoptosis in G6PD Ϫ mouse embryo fibroblasts. Notably, a 10-fold reduction in cloning efficiency was seen in G6PD Ϫ mouse embryo fibroblasts (MEFs) incubated in a...

Glucose-6-phosphate dehydrogenase (G6PD), the ratelimiting enzyme of the oxidative pentose phosphate cycle, regulates the NADPH/NADP ؉ ratio in eukaryotic cells. G6PD deficiency is one of the most common mutations in humans and is known to cause health problems for hundreds of millions worldwide. Although it is known that decreased G6PD functionality can result in increased susceptibility to oxidative stress, the molecular targets of this stress are not known. Using a Chinese hamster ovary G6PD-null mutant, we previously demonstrated that exposure to a thiol-specific oxidant, hydroxyethyldisulfide, caused enhanced radiation sensitivity and an inability to repair DNA double strand breaks. We now demonstrate a molecular mechanism for these observations: the direct inhibition of DNA end binding activity of the Ku heterodimer, a DNA repair protein, by oxidation of its cysteine residues. Inhibition of Ku DNA end binding was found to be reversible by treatment of the nuclear extract with dithiothreitol, suggesting that the homeostatic regulation of reduced cysteine residues in Ku is a critical function of G6PD and the oxidative pentose cycle. In summary, we have discovered a new layer of DNA damage repair, that of the functional maintenance of repair proteins themselves. In view of the rapidly escalating number of roles ascribed to Ku, these results may have widespread ramifications.

Ku protein is a heterodimer (Ku70 and Ku86) known to play an important role in V(D)J recombination, apoptosis, telomere fusion, and double-strand break repair. Its role in double-strand breaks is relevant to cancer therapy because lack of Ku86 causes one of the most radiationresponsive phenotypes (hamster cells, XRS5). Although it is known that the heterodimer is necessary for the various functions of this protein, the impact of targeting Ku in human cancer cells has not been shown due to lack of appropriate approaches. It is also not known whether complete knock-out of Ku protein is required to enhance the sensitivity of human cells to ; radiation as Ku protein is much more abundant in human cells than in hamster cells. In the current article, we have investigated the direct effect of Ku70 depletion in human cervical epithelioid (HeLa) and colon carcinoma (HCT116) cells. We specifically targeted Ku70 mRNA by use of small interfering RNA (siRNA). Of the five Ku70 siRNA synthesized, three inhibited the expression of Ku70 by up to 70% in HeLa cells. We have tested the effect of chemically synthesized siRNAs for target sequence 5 (CS #5) on the response of HeLa cells 72 hours after transfection to ; radiation and etoposide, as this showed the maximum inhibition of Ku70 expression. Ku70 siRNA induced a decrease in the surviving fraction of irradiated HeLa cells by severalfold. Similar sensitizing effects were observed for etoposide, a topoisomerase II inhibitor. Studies with HCT116 cells using the same Ku70 siRNA (CS #5) showed a direct correlation between expression of Ku70 and sensitization to radiation and etoposide treatments. [Mol Cancer Ther 2005;4(4):529 -36]

Polyamine homeostasis is critical for life and is accomplished via a balance of polyamine biosynthesis, degradation, and transport. Rapidly dividing cancer cells have been shown to have high polyamine transport activity compared to normal cells, likely due to their high requirement for polyamine metabolites. The polyamine transport system (PTS) is a therapeutically relevant target, as it can provide selective drug delivery to cancer cells. This report describes the synthesis and biological evaluation of multimeric polyamine derivatives as efficient PTS ligands. Arylmethyl-polyamine derivatives were synthesized to address two important concerns in PTS drug design: (a) PTS selectivity and (b) stability to amine oxidases. N(1),N(1')-[Naphthalene-1,4-diylbis(methylene)]bis{N(4)-[4-(methylamino)butyl])butane-1,4-diamine}, 3b, was found to have an optimal balance between these parameters and demonstrated excellent targeting of melanoma (e.g., MALME-3M) and breast cancer cells (e.g., T47D) over other cancer cell lines. These results provide a method to selectively target cancers via their intrinsic need for polyamine metabolites.