Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.

No abstract

Peroxiredoxin 6 (Prdx6) is the prototype and the only mammalian 1-Cys member of the Prdx family. Major differences from 2-Cys Prdxs include the use of glutathione (GSH) instead of thioredoxin as the physiological reductant, heterodimerization with pGSH S-transferase as part of the catalytic cycle, and the ability either to reduce the oxidized sn-2 fatty acyl group of phospholipids (peroxidase activity) or to hydrolyze the sn-2 ester (alkyl) bond of phospholipids (phospholipase A 2 [PLA 2 ] activity). The bifunctional protein has separate active sites for peroxidase (C47, R132, H39) and PLA 2 (S32, D140, H26) activities. These activities are dependent on binding of the protein to phospholipids at acidic pH and to oxidized phospholipids at cytosolic pH. Prdx6 can be phosphorylated by MAP kinases at T177, which markedly increases its PLA 2 activity and broadens its pH-activity spectrum. Prdx6 is primarily cytosolic but also is targeted to acidic organelles (lysosomes, lamellar bodies) by a specific targeting sequence (amino acids 31-40). Oxidant stress and keratinocyte growth factor are potent regulators of Prdx6 gene expression. Prdx6 has important roles in both antioxidant defense based on its ability to reduce peroxidized membrane phospholipids and in phospholipid homeostasis based on its ability to generate lysophospholipid substrate for the remodeling pathway of phospholipid synthesis. Antioxid. Redox Signal. 15, 831-844.

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca 2؉ -independent phospholipase A 2 (aiPLA 2 ). The cDNA encoding aiPLA 2 was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH 2 terminus, did not exhibit aiPLA 2 activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H 2 O 2 at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA 2 activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA 2 activity. Mutation of Ser 32 to Ala abolishes aiPLA 2 activity, yet the NSGPx activity remains unaffected; a Cys 47 to Ser mutant is devoid of peroxidase activity but aiPLA 2 activity remains intact. These results suggest that Ser 32 in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA 2 , while Cys 47 in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, can protect cells against membrane oxidation through glutathione (GSH)-dependent reduction of phospholipid hydroperoxides to corresponding alcohols. However, purified native or recombinant enzyme in vitro generally lacks GSH peroxidase (GPx) activity because of oxidation of its single conserved cysteine. Reduction of the resultant oxidized cysteine is difficult because of its protected location within the homodimer formed by the oxidized protein monomers. Partial purification of 1-cysPrx from bovine lung revealed the presence of GST in an active preparation, while purification to homogeneity yielded enzyme that inactivated with time. We show that heterodimerization of 1-cysPrx with GSH-saturated GST results in glutathionylation of the oxidized cysteine in 1-cysPrx followed by subsequent spontaneous reduction of the mixed disulfide and restoration of enzymatic activity. Maximum activation of 1-cysPrx occurred with a 1:1 molar ratio of GSH-saturated GST and a 2:1 molar ratio of GSH to 1-cysPrx. Liposome-mediated delivery of oxidized recombinant enzyme into NCI-H441 cells that lack 1-cysPrx but express GST resulted in 1-cysPrx activation, whereas activation in MCF7 cells required co-delivery of GST. Our data indicate a physiological mechanism for glutathionylation of the oxidized catalytic cysteine of 1-cysPrx by its heterodimerization with GST followed by its GSH-mediated reduction and enzyme activation. P eroxiredoxins are a superfamily of nonheme and nonselenium peroxidases that are widely distributed throughout all phyla (1-4). Of the six mammalian peroxiredoxins, five (Prx I-V) contain two conserved cysteines that participate in intramolecular disulfide͞sulfhydryl redox cycling with thioredoxin resulting in reduction of H 2 O 2 and organic hydroperoxides into corresponding alcohols (2). By contrast, 1-cys peroxiredoxin (1-cysPrx or Prx VI) † has a single conserved cysteine (5) and does not use thioredoxin as reductant (5, 6). This peroxiredoxin is expressed in all tissues but at particularly high levels in brain, eye, testes, and lung (2,7,8). Expression of 1-cysPrx protein or mRNA is decreased in a mouse that is susceptible to experimental atherosclerosis (9) and is elevated in brains of patients with Parkinsonian dementia (10), sporadic Creutzfeldt-Jacob disease (11), and Pick disease (12), in lungs from newborns (13), in malignant mesothelioma (14), in the healing edge of skin wounds (15), and in experimental cellular premature senescence (16). 1-cysPrx can reduce phospholipid and other hydroperoxides (6) and protects against cellular membrane damage (17, 18). This enzyme has phospholipase A 2 activity (19), participates in the activation of neutrophil NADPH oxidase through its interaction with p67 phox (20), and prevent methemoglobin formation in erythrocyte hemolysates (21).1-cysPrx catalysis results in the peroxide-mediated oxidation of its Cys-47 to sulfenic acid as deduced from 1-cysPrx crystallization (22). Reduction of the sul...

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl-or sn-2-arachidonoylphosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM ؊1 min ؊1 ) were k 1 ‫؍‬ 1-3 ؋ 10 5 and k 2 ‫؍‬ 4 -11 ؋ 10 4 . In the presence of 0.36 mM GSH, apparent K m was 120 -130 M and apparent V max was 1.5-1.6 mol/min/mg of protein. Assays with H 2 O 2 and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7-8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K i 4 M. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.

No abstract

We have previously demonstrated the generation of reactive oxygen species (ROS) in cultured bovine pulmonary artery endothelial cells (BPAECs) and in isolated perfused rat lungs exposed to high K+ and during global lung ischemia. The present study evaluates the NADPH oxidase pathway as a source of ROS in these models. ROS production, detected by oxidation of the fluorophore, dichlorodihydrofluorescein, increased 2.5-fold in BPAECs and 6-fold in rat or mouse lungs exposed to high (24 mmol/L) K+. ROS generation was markedly inhibited by diphenyliodonium, a flavoprotein inhibitor, and by the synthetic peptide PR-39, an inhibitor of NADPH oxidase assembly, whereas allopurinol had no effect. With ischemia (1 hour), ROS generation by rat and mouse lungs increased 7-fold; PR-39 showed concentration-dependent inhibition of ROS production, with 50% inhibition at 3 micromol/L PR-39. ROS production in lungs exposed to high K+ or ischemia was essentially abolished in mice with a "knockout" of gp91(phox), a membrane-localized cytochrome component of NADPH oxidase; increased ROS production by these lungs after anoxia/reoxygenation was similar to control. PR-39 also inhibited ischemia and the high K+-mediated increase in lung thiobarbituric acid reactive substance. Western blotting of BPAECs and immunocytochemistry of BPAECs and rat and mouse lungs showed the presence of p47phox, a cytoplasmic component of NADPH oxidase and the putative target for PR-39 inhibition. In situ fluorescence imaging in the intact lung demonstrated that the increased dichlorofluorescein fluorescence in these models of ROS generation was localized primarily to the pulmonary endothelium. These studies demonstrate that ROS production in lungs exposed to ischemia or high K+ results from assembly and activation of a membrane-associated NAPDH oxidase of the pulmonary endothelium.