Molekularni patofyziologie vybranych dedicnych poruch erytropoezy. Soubor publikovanych praci s komentarem.

The publicated articles are in English.Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

DMT1-Mutant Erythrocytes have Shortened Life Span, Accelerated Glycolysis and Increased Oxidative Stress

Background/Aims: Deficiency of the divalent metal transporter 1 (DMT1) leads to hypochromic microcytic anemia. We have previously shown that DMT1 deficiency impairs erythroid differentiation and induces apoptosis of erythroid cells. Here we analyzed metabolic processes and survival of mature erythrocytes in order to address potential involvement of erythrocyte defect in the pathophysiology of the disease. Methods: FACS analysis was used to determine the half-life of erythrocytes (CFSE fluorescence), phosphatidylserine exposure (Annexin V binding), cytosolic Ca2+ (Fluo3/AM fluorescence) and reactive oxygen species (ROS; DCF fluorescence). Enzyme activities were determined by standard biochemical methods. The concentration of ATP and ADP was measured on HPLC-MS/MS. Results: We observed an accelerated clearance of CFSE-labeled DMT1-mutant erythrocytes from circulating blood when compared to wild-type erythrocytes. In vitro, DMT1-mutant erythrocytes showed significantly increased Annexin V binding after exposure to hyperosmotic shock and glucose depletion. Despite exaggerated anti-oxidative defense, higher ROS levels were present in DMT1-mutant erythrocytes. Accelerated anaerobic glycolysis and reduced ATP/ADP ratio detected in DMT1-mutant erythrocytes indicate enhanced demand for ATP. Conclusions: We propose that DMT1 deficiency negatively affects metabolism and life span of mature erythrocytes; two other aspects of defective erythropoiesis which contribute to the pathophysiology of the disease

Role of DNA Damage Response in Suppressing Malignant Progression of Chronic Myeloid Leukemia and Polycythemia Vera: Impact of Different Oncogenes

Inflammatory and oncogenic signaling, both known to challenge genome stability, are key drivers of BCR-ABL-positive chronic myeloid leukemia (CML) and JAK2 V617F-positive chronic myeloproliferative neoplasms (MPNs). Despite similarities in chronic inflammation and oncogene signaling, major differences in disease course exist. Although BCR-ABL has robust transformation potential, JAK2 V617F-positive polycythemia vera (PV) is characterized by a long and stable latent phase. These differences reflect increased genomic instability of BCR-ABL-positive CML, compared to genome-stable PV with rare cytogenetic abnormalities. Recent studies have implicated BCR-ABL in the development of a "mutator" phenotype fueled by high oxidative damage, deficiencies of DNA repair, and defective ATR-Chk1-dependent genome surveillance, providing a fertile ground for variants compromising the ATM-Chk2-p53 axis protecting chronic phase CML from blast crisis. Conversely, PV cells possess multiple JAK2 V617F-dependent protective mechanisms, which ameliorate replication stress, inflammation-mediated oxidative stress and stress-activated protein kinase signaling, all through up-regulation of RECQL5 helicase, reactive oxygen species buffering system, and DUSP1 actions. These attenuators of genome instability then protect myeloproliferative progenitors from DNA damage and create a barrier preventing cellular stress-associated myelofibrosis. Therefore, a better understanding of BCR-ABL and JAK2 V617F roles in the DNA damage response and disease pathophysiology can help to identify potential dependencies exploitable for therapeutic interventions

MLL-ENL Inhibits Polycomb Repressive Complex 1 to Achieve Efficient Transformation of Hematopoietic Cells

Stimulation of transcriptional elongation is a key activity of leukemogenic MLL fusion proteins. Here, we provide evidence that MLL-ENL also inhibits Polycomb-mediated silencing as a prerequisite for efficient transformation. Biochemical studies identified ENL as a scaffold that contacted the elongation machinery as well as the Polycomb repressive complex 1 (PRC1) component CBX8. These interactions were mutually exclusive in vitro, corresponding to an antagonistic behavior of MLL-ENL and CBX8 in vivo. CBX8 inhibited elongation in a specific reporter assay, and this effect was neutralized by direct association with ENL. Correspondingly, CBX8-binding-defective MLL-ENL could not fully activate gene loci necessary for transformation. Finally, we demonstrate dimerization of MLL-ENL as a neomorphic activity that may augment Polycomb inhibition and transformation