1. Oxidative stress and hypoxia in normal and leukemic stem cells
Ugo Testa, Catherine Labbaye, Germana Castelli, Elvira Pelosi 
Experimental Hematology 
Volume 44, Issue 7, Pages (July 2016)
DOI: /j.exphem
Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

2. Figure 1
Schematic outline of the structure of the various HIF isoforms. HIF is a heterodimeric basic helix-loop-helix structure that is composed of HIF-1α, HIF-2α, or HIF-3α, the α subunit, and the aryl hydrocarbon nuclear translocator (ARNT), the beta subunit. Alpha and beta subunits contain a nuclear localization signal (NLS), followed by a basic helix-loop-helix domain near the C-terminus, which is involved in DNA binding, and by two distinct PAS domains, PASA and PASB, which are involved in ARNT dimerization. The structure of alpha subunits is the characterized by the presence of an oxygen-dependent degradation domain (ODDD) that regulates oxygen-dependent stability through the hydroxylation of two prolyl (P) residues (HIF-1α and HIF-2α) or one (HIF-3α) and by a N-terminal transactivation domain (NTAD). In HIF-1α and HIF-2α, there is also a C-terminal transactivation domain (CTAD) containing an asparaginyl residue (N) involved in transcriptional activation and, between the NTAD and CTAD, there is an intervening inhibitory domain (ID) that represses the transcriptional activity of the two transactivation domains (hypoxia inhibits the repressive activity of ID). In the C-terminal of HIF-3α, the CTAD is not present and is replaced by a leucine zipper (LZIP) domain that is involved in protein–protein interaction. At variance with HIFαs, HIF-1β and HIF-1β2 have a CTAD without an asparaginyl residue but lack the ODDD, NTAD, and LZIP domains.
Experimental Hematology  , DOI: ( /j.exphem )
Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

3. Figure 2
Mechanisms of ROS production. (A) ROS production and scavenging. ROS produced within the cells include O2*−, H2O2, and OH*). Superoxide is mainly generated from the oxidation of NADPH by NOXs or mitochondria through electron chain complexes I and III. The scavenging system involves first the reduction of O2*− to H2O2 either by catalase or GPX coupled with GSH oxidation. H2O2 can also spontaneously oxidize iron (Fe2+) to form the highly reactive OH*. (B) ROS production from membrane-bound NOX. NOXs consume NADPH to generate O2*− and, subsequently, H2O2. (C) ROS production from the electron transport chain. The electron transport chain complexes I–IV, localized at the level of the mitochondrial inner membrane, mediate a number of reactions in which electrons from NADH and succinate pass through the electron transport chain to oxygen. In these reactions, each electron donor passes electrons to a more electronegative acceptor, which in turn donates these electrons to another acceptor, up to the last step, where electrons are passed to oxygen. The passage of electrons between donor and acceptor releases energy that is used to generate a proton gradient across the membrane by pumping protons (H+) into the intermembrane space. Under normal conditions, O2 acts as the final acceptor at the level of complex IV; however, approximately 0.1–0.2% of the O2 consumed by mitochondria generates ROS through the premature flow to O2, a phenomenon mainly occurring at the level of complexes I and III.
Experimental Hematology  , DOI: ( /j.exphem )
Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

4. Figure 3
ROS production during stem cell differentiation. Quiescent stem cells have low levels of ROS due to the pronounced expression in these cells of the antioxidant molecular machinery induced by various factors such as Foxo 3, p53, ATM, and HIF-1. These factors also drive a peculiar energetic metabolism in these cells. Pro-oxidant activity promotes a rise of the ROS level and HSC differentiation and, during this process, there is a change in the energetic metabolism, promoting oxidative phosphorylation and mitochondrial biogenesis.
Experimental Hematology  , DOI: ( /j.exphem )
Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

5. Figure 4
Schematic diagram showing the main steps of cellular GSH metabolism. GSH is synthesized from glutamate and cysteine through two enzyme-controlled steps involving first the synthesis of γ-glutamil-cysteine and then of reduced GSH. GSH is present in the cell in part in its reduced form and in part in its oxidized form: the reduction reaction involving the transformation of GSSG in GSH is mediated by GSH reductase, whereas GSH oxidation involves GPX.
Experimental Hematology  , DOI: ( /j.exphem )
Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

6. Figure 5
Schematic representation of the metabolic routes related to energy production. The main metabolic pathways of glucose metabolism are shown, including glycolysis, PPP, FAO, OXPHOS, and glutaminolysis. Glycolysis starts with glucose uptake and ends with pyruvate synthesis, where this compound either undergoes the conversion to lactate or acetyl-CoA, entering into the mitochondria the Krebs cycle. Leukemia cells usually exhibit upregulated glycolysis rates and blocking of some enzymes of the glycolytic pathway, such as PKM2, LDHA, and PDK, is able to inhibit the leukemogenetic process. The PPP branches from glycolysis after the production of glucose 6-phosphate (G6P) and, during its oxidative phase, G6P is oxidized to produce NADPH and ribose-5-phosphate (R-5-P). The activity of the PPP is often increased in leukemic cells. FAO is briefly outlined, involving fatty acid uptake, mediated by proteins such as carnitine palmitoyltransferase (CPT1) and then FAO with consequent production of NADH and FADH2, oxidized in the oxidative phosphorylation electron transport chain, and generation of acetyl-CoA, entering the Krebs cycle to produce citrate. Leukemic cells show a shift to the oxidation of nonglucose carbon sources, mainly represented by fatty acids, through their oxidation. Glutamine conversion to glutamate is often increased in leukemic cells and represents an important mechanism to sustain cellular energy requirement and synthesis needs from TCA metabolites.
Experimental Hematology  , DOI: ( /j.exphem )
Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

7. Figure 6
Schematic representation of glutamine metabolism. Cellular glutamine uptake is mediated by the membrane glutamine transporter SLC1A5. Cellular glutamine can be then metabolized through a glutaminolysis process or be involved in a biochemical pathway of mTORC1 activation by leucine. The first pathway involves the initial hydrolysis of glutamine to glutamate, which mediated by the enzyme glutaminase (GLS). Glutamate is then transformed to α-KG by the enzyme glutamate dehydrogenase 1 (GDH1). An enhanced activity of this glutamine metabolic pathway determines an increase of fumarate levels in cancer cells, determining an inhibitory effect on GPX activity with consequent increased ROS production. In the second pathway, cellular glutamine is required to sustain the activity of the bidirectional membrane-transported SLC7A5/3A2, which is required to mediate the uptake of leucine, which is required to sustain mTORC1 activation.
Experimental Hematology  , DOI: ( /j.exphem )
Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

8. Figure 7
AMPK cellular targets. AMPK is a heterotrimer serine/threonine kinase, acting as a main regulator of several cellular processes and particularly of energy homeostasis in normal and tumor cells. AMPK is an energy sensor activated by various cellular stress conditions, such as glucose deprivation, hypoxia, and oxidative stress. Activated AMPK acts by regulating various cellular process, inducing an activation of autophagy (through mTORC1 inhibition and ULK1 [Unc.51-like kinase 1] activation), an inhibition of mTORC1 (mammalian target of rapamycin complex 1), an inhibition of ACC (acetyl-CoA carboxylase), with consequent inhibitory effects on fatty acid biosynthesis and as an inhibitor of thioredoxin-interacting protein (TXNPIP), with consequent increase of glucose uptake, stimulation of the glycolysis and of the PPP (with consequent increased NADPH production), redox protection, and cell survival. This last pathway is activated in LSCs and is essential for their survival in the hypoxic BM microenvironment.
Experimental Hematology  , DOI: ( /j.exphem )
Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

9. Figure 8
Mechanism of action of mutated isocitrate dehydrogenase. (A) Schematic outline of the chemical reactions catalyzed by the wild-type IDH1 and IDH2 enzymes and mutant IDH1 and IDH2 present in some AMLs. Wild-type IDH1 and IDH2 enzymes catalyze the NADP+-dependent oxidative decarboxlation of isocitrate into α-KG and NADPH. Mutant IDH1 and IDH2 have lost the normal catalytic activity and gained a new function, consisting of catalyzing the transformation of α-KG into D-2-HG. (B) The main pathogenetic events are induced by the accumulation of the metabolite D-2-HG. The accumulation of D-2-HG into the leukemic cells is responsible for the induction of epigenetic changes, the inhibition of cellular differentiation, and dysregulation of energetic metabolism, with consequent hypersensitivity of leukemic cells to BCL2 inhibitors.
Experimental Hematology  , DOI: ( /j.exphem )
Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions