GGT activity was assayed with the method of Sze et al

GGT activity was assayed with the method of Sze et al. complexed by nitrilotriacetic acid (55Fe-NTA complex). The stimulation of GGT activity, by administration to cells of the substrates glutathione and glycyl-glycine, was generally reflected in a facilitation of transferrin-bound iron uptake. The extent of such facilitation was correlated with the intrinsic levels of the enzyme present in each cell line. Accordingly, inhibition of GGT activity by means of two independent inhibitors, acivicin and serine/boric acid complex, resulted in a decreased uptake of transferrin-bound iron. With Fe-NTA complex, the inhibitory effect C but not the stimulatory one C was also observed. Conclusion It is concluded that membrane GGT can represent a facilitating factor in iron uptake by GGT-expressing cancer cells, thus providing them with a selective growth advantage over clones that do not possess the enzyme. Background Iron is involved in several primary cellular functions C such as DNA synthesis, ATP generation, electron transfers, oxidation of substrates C and is therefore an essential factor for cell TSHR survival and replication [reviewed in [1]]. On the other hand, iron may also catalyze oxidation-reduction (redox) reactions, leading to the production of free radicals and potentially noxious oxidative stress. As a consequence, living organisms developed strictly regulated processes for iron transport, uptake and storage, and a balance between these mechanisms is essential for life [2]. In aqueous, non-acidic environments, iron exists in highly insoluble polymeric forms. Consequently, cells had to devise specific strategies to solubilize and absorb the metal. These include i) systems capable to effect the reduction of ferric ions to the more soluble ferrous form, and ii) the use of proteins capable to transport ferric iron as such, such as e.g. transferrins [3]. The main pathway for iron uptake by animal cells is through the plasmatic protein iron carrier, transferrin (Tf), and its specific receptor (TfR) located at the cell surface. Following the ligand binding, the Tf/TfR complex is internalized by a receptor-mediated endocytosis and iron is released by a process involving endosomal acidification through an influx of protons through an ATP-dependent proton pump [4,5]. Other studies have suggested that another critical step in cellular uptake of Tf-bound iron may be the reduction of ferric iron to ferrous [6], and several molecular species acting as reductants have been described, including the superoxide anion, ascorbic acid and thiol compounds [7]. However, the role of these processes in iron uptake from Tf remains controversial and still subject to debate. As recently pointed out by Kwok et al.[8], lines of evidence suggest that Fe3+ reduction takes place Prosapogenin CP6 Prosapogenin CP6 after uptake, as Fe is released from endosomes into the cytosol. Besides Tf-bound iron, animal cells can also obtain iron from small, non-protein, low molecular weight complexes. Such complexes can originate in conditions such as iron overload when the binding capacity of transferrin is saturated and free, “non-Tf-bound iron” (NTBI) is generated [9]. Interestingly, the involvement of free radical reactions in the uptake of NTBI has also been recently proposed [10]. Altogether, the precise molecular mechanisms by which iron is physiologically unloaded from its complexes and transported trough the cellular membranes in eukariotic cells are still in need of elucidation. With respect to the possibility that a reduction of Fe3+ to Fe2+ may represent a critical step in the process, thiol (-SH) compounds are known to reduce iron efficiently [11]. Such iron-reducing ability can be demonstrated for several molecules, including the well-known antioxidant tripeptide glutathione (gamma-glu-cys-gly; GSH) [12]. GSH C one of the main cellular antioxidants C as such cannot cross plasma membrane of most cell types, and thus the recovery of extracellular GSH is warranted by membrane gamma-glutamyl transpeptidase (GGT), an ecto-enzyme with the active site oriented toward the outer cell surface. GGT is capable to start the catabolism of extracellular GSH, and as in most cell types a continuous efflux of GSH to the extracellular space exists, in cells expressing GGT at their surface a continuous “GSH cycling” across the plasma membrane therefore occurs [13]. Recent studies of our and other laboratories have highlighted.Author M.C. was investigated by using 55Fe-loaded transferrin, as well as by monitoring fluorimetrically the intracellular iron levels in calcein-preloaded cells. Transferrin-independent iron uptake was investigated using 55Fe complexed by nitrilotriacetic acid (55Fe-NTA complex). The stimulation of Prosapogenin CP6 GGT activity, by administration to cells of the substrates glutathione and glycyl-glycine, was generally reflected in a facilitation of transferrin-bound iron uptake. The extent of such facilitation was correlated with the intrinsic levels of the enzyme present in each cell line. Accordingly, inhibition of GGT activity by means of two independent inhibitors, acivicin and serine/boric acid complex, resulted in a decreased uptake of transferrin-bound iron. With Fe-NTA complex, the inhibitory effect C but not the stimulatory one C was also observed. Conclusion It is concluded that membrane GGT can represent a facilitating factor in iron uptake by GGT-expressing cancer cells, thus providing them with a selective growth advantage over clones that do not possess the enzyme. Background Iron is involved in several primary cellular functions C such as DNA synthesis, ATP generation, electron transfers, oxidation of substrates C and is therefore an essential factor for cell survival and replication [reviewed in [1]]. On the other hand, iron may also catalyze oxidation-reduction (redox) reactions, leading to the production of free radicals and potentially noxious oxidative stress. As a consequence, living organisms developed strictly regulated processes for iron transport, uptake and storage, and a balance between these mechanisms is essential for life [2]. In aqueous, non-acidic environments, iron exists in highly insoluble polymeric forms. Consequently, cells had to devise specific strategies to solubilize and absorb the metal. These include i) systems capable to effect the reduction of ferric ions to the more soluble ferrous form, and ii) the use of proteins capable to transport ferric iron as such, such as e.g. transferrins [3]. The main pathway for iron uptake by animal cells is through the plasmatic protein iron carrier, transferrin (Tf), and its specific receptor (TfR) located at the cell surface. Following the ligand binding, the Tf/TfR complex is internalized by a receptor-mediated endocytosis and iron is released by a process involving endosomal acidification through an influx of protons through an ATP-dependent proton pump [4,5]. Other studies have suggested that another critical step in cellular uptake of Tf-bound iron may be the reduction of ferric iron to ferrous [6], and several molecular species acting as reductants have been described, including the superoxide anion, ascorbic acid and thiol compounds [7]. However, the role of these processes in iron uptake from Tf remains controversial and still subject to debate. As recently pointed out by Kwok et al.[8], lines of evidence suggest that Fe3+ reduction takes place after uptake, as Fe is released from endosomes into the cytosol. Besides Tf-bound iron, animal cells can also obtain iron from small, non-protein, low molecular weight complexes. Such complexes can originate in conditions such as iron overload when the binding capacity of transferrin is saturated and free, “non-Tf-bound iron” (NTBI) is generated [9]. Interestingly, the involvement of free radical reactions in the uptake of NTBI has also been recently proposed [10]. Altogether, the precise molecular mechanisms by which iron is physiologically unloaded from its complexes and transported trough the cellular membranes in eukariotic cells are still in need of elucidation. With respect to the possibility that a reduction of Fe3+ to Fe2+ may represent a critical step in the process, thiol (-SH) compounds are known to reduce iron efficiently [11]. Such iron-reducing ability can be demonstrated for several molecules, including the well-known antioxidant tripeptide glutathione (gamma-glu-cys-gly; GSH) [12]. GSH C one of the main cellular antioxidants C as such cannot combination plasma membrane of all cell types, and therefore the recovery of extracellular GSH Prosapogenin CP6 is normally warranted by membrane gamma-glutamyl transpeptidase (GGT), an ecto-enzyme using the energetic site oriented.