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P032 Oxidative stress in red blood cells, platelets and polymorphonuclear leukocytes from patients with myelodysplastic syndrome
S58
Posters
FAS/FASL extrinsic pathway has been reported in erythroid pathological clone and is responsible for apoptosis of these cells. This deregulation is overcome using a dominant negative mutant of FADD that blocks the FAS/FADD mediated activation of caspase 8 (Claessens et al. 2005). Another study demonstrated that FADD interact with NACA, leading to negative regulation of apoptosis (Stilo et al. 2003). We previously showed that NACA is a positive regulator of erythropoiesis. Based on these data we have investigated, whether the ectopic expression of NACA has an effect on ES-MDS-deriving erythroid precursors. In vitro expansion of CD34+ progenitors cells from ES-MDS including low and intermediate IPSS (n=5), were transduced with either a NACA or GFP encoding lentivirus vector. Cells were then cultured for 15 days in either expansion (FLT3L, TPO, SCF, IL-3) or erythroid cell differentiation (EPO, SCF, IL-3, IL-6) cultures. Morphologic analysis of MGG stained cells, revealed that the proportions of basophilic, polychromatophilic and acidophilic erythroblast cells were significantly higher (from 20% to 50% of erythroid cells) in NACA-transduced ES-MDS cells in comparison with GFP-transduced cells, from which few mature cells were counted (~3%). The number of BFU-E measured using clonogenic progenitor assay in semi solid medium (for 3 of these patients), was low (from 4 to 9 BFU-E) in untransduced cells or GFP-transduced cells. This number was significantly higher (from 2 to 5 fold) in cells transduced with NACA. In contrast, NACA transduction of late stage MDS deriving erythroid precursor cells (n = 3), had no effect in erythroid differentiation. No mature cells were counted and the number of proerythroblaste was identical in either NACA or GFP transduced cells. These results demonstrate that NACA rescues erythroid differentiation of ES-MDSderiving erythroid precursors. This finding may be important for the choice of strategies to correct cytopenia in ES-MDS bone marrow. The molecular mechanism is currently under study. In particular we explore the hypothesis according which uncomplexed FADD (overexpressed in these cells) would activate the apoptotic pathway and could be overcome by ectopic expression of NACA.
P032 Oxidative stress in red blood cells, platelets and polymorphonuclear leukocytes from patients with myelodysplastic syndrome H. Ghoti1 , J. Amer2 , A. Winder1 , E. Rachmilewitz1 ° , E. Fibach2 . 1 Department of Hematology, The E. Wolfson Medical Center, Holon, 2 Department of Hematology, Hebrew University – Hadassah Medical School, Jerusalem, Israel *E-mail: [email protected] Myelodysplastic syndrome (MDS) is characterized by refractory cytopenias due to ineffective hematopoiesis. Some patients with severe anemia require multiple blood
transfusions and develop iron overload. Consequently, reactive oxygen species (ROS) are generated concomitant with a decrease in cellular antioxidants such as reduced gluthatione GSH. The generated oxidative stress contributes to cell damage, apoptosis and ineffective hematopoiesis. Using flow cytometry, we measured the oxidative state of RBC, platelets and PMN in 14 low-risk MDS patients and 25 normal donors. The results indicate that the majority of the patients had higher ROS in RBC (2.79-fold) and platelets (2.91-fold) and lower GSH in their RBC (3.4fold) and platelets (2.1-fold) than normal (p < 0.005) As for PMN, there were no significant differences in ROS, although GSH was significantly (p < 0.1) lower in MDS compared with normal donors. The oxidative stress in MDS cells could be ameliorated by a short in vitro treatment with the iron-chelators deferrioxamine and deferiprone, or with the anti-oxidant N-acetylcysteine. These results suggest that the decrease in transfusion requirements with increase in platelet and PMN counts in MDS patients treated with deferrioxamine may be due to indirect antioxidant effect of the iron chelator. Consequently/therefore, (suggesting that) treatment with a combination of iron-chelators and antioxidants might be more effective.
P033 The Akt/mTOR signal transduction pathway is activated in high risk myelodysplastic syndromes and influences cell survival and proliferation M.Y. Follo1 , C. Finelli2 ° , S. Mongiorgi1 , C. Bosi2 , A. Cappellini1 , F. Chiarini1 , V. Papa1 , M. Libra1 , G. Martinelli2 , M. Baccarani2 , L. Cocco1 , A.M. Martelli1 . 1 Department of Anatomical Sciences, Cellular Signalling Laboratory, University of Bologna; 2 Institute of Hematology “Ser`agnoli”, University of Bologna; Italy *E-mail: cfi[email protected] Introduction: The phosphoinositide 3 kinase (PI3K)/Akt signaling pathway is involved in many different cellular processes, including proliferation, differentiation, and apoptosis. One of the downstream targets of Akt is represented by the mammalian Target of Rapamycin (mTOR), a highly conserved Ser/Thr protein kinase that is essential for the regulation of cell growth and proliferation. Recent studies have demonstrated that mTOR is also
Posters
FAS/FASL extrinsic pathway has been reported in erythroid pathological clone and is responsible for apoptosis of these cells. This deregulation is overcome using a dominant negative mutant of FADD that blocks the FAS/FADD mediated activation of caspase 8 (Claessens et al. 2005). Another study demonstrated that FADD interact with NACA, leading to negative regulation of apoptosis (Stilo et al. 2003). We previously showed that NACA is a positive regulator of erythropoiesis. Based on these data we have investigated, whether the ectopic expression of NACA has an effect on ES-MDS-deriving erythroid precursors. In vitro expansion of CD34+ progenitors cells from ES-MDS including low and intermediate IPSS (n=5), were transduced with either a NACA or GFP encoding lentivirus vector. Cells were then cultured for 15 days in either expansion (FLT3L, TPO, SCF, IL-3) or erythroid cell differentiation (EPO, SCF, IL-3, IL-6) cultures. Morphologic analysis of MGG stained cells, revealed that the proportions of basophilic, polychromatophilic and acidophilic erythroblast cells were significantly higher (from 20% to 50% of erythroid cells) in NACA-transduced ES-MDS cells in comparison with GFP-transduced cells, from which few mature cells were counted (~3%). The number of BFU-E measured using clonogenic progenitor assay in semi solid medium (for 3 of these patients), was low (from 4 to 9 BFU-E) in untransduced cells or GFP-transduced cells. This number was significantly higher (from 2 to 5 fold) in cells transduced with NACA. In contrast, NACA transduction of late stage MDS deriving erythroid precursor cells (n = 3), had no effect in erythroid differentiation. No mature cells were counted and the number of proerythroblaste was identical in either NACA or GFP transduced cells. These results demonstrate that NACA rescues erythroid differentiation of ES-MDSderiving erythroid precursors. This finding may be important for the choice of strategies to correct cytopenia in ES-MDS bone marrow. The molecular mechanism is currently under study. In particular we explore the hypothesis according which uncomplexed FADD (overexpressed in these cells) would activate the apoptotic pathway and could be overcome by ectopic expression of NACA.
P032 Oxidative stress in red blood cells, platelets and polymorphonuclear leukocytes from patients with myelodysplastic syndrome H. Ghoti1 , J. Amer2 , A. Winder1 , E. Rachmilewitz1 ° , E. Fibach2 . 1 Department of Hematology, The E. Wolfson Medical Center, Holon, 2 Department of Hematology, Hebrew University – Hadassah Medical School, Jerusalem, Israel *E-mail: [email protected] Myelodysplastic syndrome (MDS) is characterized by refractory cytopenias due to ineffective hematopoiesis. Some patients with severe anemia require multiple blood
transfusions and develop iron overload. Consequently, reactive oxygen species (ROS) are generated concomitant with a decrease in cellular antioxidants such as reduced gluthatione GSH. The generated oxidative stress contributes to cell damage, apoptosis and ineffective hematopoiesis. Using flow cytometry, we measured the oxidative state of RBC, platelets and PMN in 14 low-risk MDS patients and 25 normal donors. The results indicate that the majority of the patients had higher ROS in RBC (2.79-fold) and platelets (2.91-fold) and lower GSH in their RBC (3.4fold) and platelets (2.1-fold) than normal (p < 0.005) As for PMN, there were no significant differences in ROS, although GSH was significantly (p < 0.1) lower in MDS compared with normal donors. The oxidative stress in MDS cells could be ameliorated by a short in vitro treatment with the iron-chelators deferrioxamine and deferiprone, or with the anti-oxidant N-acetylcysteine. These results suggest that the decrease in transfusion requirements with increase in platelet and PMN counts in MDS patients treated with deferrioxamine may be due to indirect antioxidant effect of the iron chelator. Consequently/therefore, (suggesting that) treatment with a combination of iron-chelators and antioxidants might be more effective.
P033 The Akt/mTOR signal transduction pathway is activated in high risk myelodysplastic syndromes and influences cell survival and proliferation M.Y. Follo1 , C. Finelli2 ° , S. Mongiorgi1 , C. Bosi2 , A. Cappellini1 , F. Chiarini1 , V. Papa1 , M. Libra1 , G. Martinelli2 , M. Baccarani2 , L. Cocco1 , A.M. Martelli1 . 1 Department of Anatomical Sciences, Cellular Signalling Laboratory, University of Bologna; 2 Institute of Hematology “Ser`agnoli”, University of Bologna; Italy *E-mail: cfi[email protected] Introduction: The phosphoinositide 3 kinase (PI3K)/Akt signaling pathway is involved in many different cellular processes, including proliferation, differentiation, and apoptosis. One of the downstream targets of Akt is represented by the mammalian Target of Rapamycin (mTOR), a highly conserved Ser/Thr protein kinase that is essential for the regulation of cell growth and proliferation. Recent studies have demonstrated that mTOR is also