Emanuel Necas

A study of intracellular iron metabolism using pyridoxal isonicotinoyl hydrazone and other synthetic chelating agents

Abstract 1. 1. Rabbit reticulocytes with a high level of non-heme radioiron induced by preincubation with isonicotinic acid hydrazide and transferrin-bound 59Fe, were reincubated with various synthetic chelating agents and the amount of radioiron released from the cells was determined. Some substances, especially derivatives of pyridoxal or 2-hydroxybenzaldehyde and isonicotinic acid hydrazide or benzhydrazide, were found to mobilize significantly iron from 59Fe-labeled reticulocytes. The effectiveness of the compounds tested decreases in the following order: pyridoxal isonicotinoyl hydrazone, pyridoxal benzoyl hydrazone, 2-hydroxybenzal isonicotinoyl hydrazone, 2-hydroxybenzal benzoyl hydrazone, pyridoxal-valine Schiff base, pyridoxal. The efficiency of these compounds exceeded the ability of common chelators such as desferrioxamine, 2,2′-bipyridine, nitrolotriacetic acid, etc., to mobilize iron from reticulocytes. 2. 2. Iron mobilization from reticulocytes by pyridoxal isonicotinoyl hydrazone requires ATP to be produced by cells and is completely blocked by low temperature (4°C). Although the effect of desferrioxamine is also prevented by low temperature, modest iron mobilization due to this chelator seems to occur independently of ATP production in reticulocytes. 3. 3. Pyridoxal isonicotinoyl hydrazone mobilizes iron mainly from mitochondria and in part also from ferritin. Although 2,2′-bipyridine seems to enter reticulocyte mitochondria and bind iron there, this chelator is not able to release iron either from mitochondria or from the cells. 4. 4. Reticulocytes with a high level of non-heme radioiron are envisaged as a useful system for testing biological effectiveness of various iron chelators. 5. 5. Pyridoxal isonicotinoyl hydrazone was shown to be an effective in vivo chelator since its administration to mice decreased 59Fe radioactivity in liver, spleen and kidney.

TRAIL (Apo2L) suppresses growth of primary human leukemia and myelodysplasia progenitors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, APO2L) has been shown to induce apoptosis in a number of tumor cell lines as well as in some primary tumors whereas cells from most normal tissues are highly resistant to TRAIL-induced apoptosis. We have studied the susceptibility of primary malignant and normal bone marrow hematopoietic progenitors to TRAIL-induced apoptosis. Extracellular domain of human TRAIL with N-terminal His6 tag (His-TRAIL, amino acids 95–281) was produced in E. coli and its apoptosis-inducing ability was compared with the leucine-zipper containing TRAIL, LZ-TRAIL. Both variants of TRAIL had the same apoptosis-inducing ability. Clonogenic progenitor assays showed that His-TRAIL significantly reduced the number of myeloid colonies (CFU-GM) and clusters from patients with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndromes (MDS). His-TRAIL had no negative effect on the number of CFU-GM colonies and clusters derived from bone marrow cells of AML patients in complete remission, and lymphoma patients without bone marrow involvement, as well as those derived from normal cord blood cells. Moreover, we found that normal human stem cells treated with high doses of His-TRAIL maintain a repopulating potential when transplanted into NOD/SCID mice. To conclude, our data document that TRAIL does not affect normal human hematopoiesis but suppresses the growth of early primary leukemia and myelodysplasia progenitors.

MYB transcriptionally regulates the miR-155 host gene in chronic lymphocytic leukemia

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.

A Sudden Increase in Partial Pressure End-Tidal Carbon Dioxide (PETCO2) at the Moment of Return of Spontaneous Circulation

BACKGROUND Previous studies established that a level of partial pressure end-tidal carbon dioxide (P(ET)CO(2)) of 10 mm Hg divided patients undergoing advanced life support (ALS) into those likely to be resuscitated (values > 10 mm Hg) and those likely to die during ALS (values < 10 mm Hg). OBJECTIVE The study tested the significance of a sudden increase in the P(ET)CO(2) in signaling the return of spontaneous circulation (ROSC) during ALS. MATERIAL AND METHODS P(ET)CO(2) values were continuously recorded during ALS in out-of-hospital patients with cardiac arrest. Constant ventilation was maintained by an automatic device. There were 108 patients, representing two extreme outcomes of ALS, who were subdivided into two groups. The first group included 59 patients with a single ROSC followed by a stable spontaneous circulation. The second group included 49 patients with no signs of ROSC. RESULTS ROSC was associated with a sudden increase in P(ET)CO(2) that remained significantly higher than before ROSC. P(ET)CO(2) did not rise during the entire ALS in the second group of patients without ROSC and was lower than in the first group of patients. CONCLUSIONS In constantly ventilated patients, P(ET)CO(2) is significantly higher (about 10 mm Hg) after ROSC than before ROSC. A sudden increase in P(ET)CO(2) exceeding 10 mm Hg may indicate ROSC. Consequently, the rule of 10 mm Hg may be extended to include a sudden increase in continuously recorded P(ET)CO(2) by more than 10 mm Hg as an indicator of the possibility of ROSC.

Biliary iron excretion in rats following pyridoxal isonicotinoyl hydrazone.

Summary Biliary excretion of iron after administration of pyridoxal isonicotinoyl hydrazone (PIH), a recently identified effective iron‐chelating agent, was investigated in rats. PIH administered both intraperitoneally and orally was shown to increase significantly 59Fe excretion into bile of rats which had previously been injected with 59Fe‐transferrin to label hepatic parenchymal cells. 59Fe‐PIH appears in bile as early as 15 min after chelator administration and the peak of 59Fe‐radioactivity in bile is seen 1–5 h following intraperitoneal PIH injection. PIH, administered intraperitoneally, 125–250 mg/kg, increased 24 h biliary radioiron excretion about 35 times and in addition increased urinary and faecal iron excretion. When PIH was given immediately before 59Fe‐transferrin, 24 h cumulative biliary 59Fe excretion was even higher. PIH was also demonstrated to increase biliary excretion of radioiron released from 59Fe‐haemoglobin catabolysed in reticuloendothelial cells. The effect of PIH was confirmed by estimation of biliary iron concentration using the method of atomic absorption spectrophotometry. Repeated PIH administration to rats decreased 59Fe radioactivity in liver and kidney and increased urinary and faecal iron excretion.

Epigenetic silencing of the oncogenic miR-17-92 cluster during PU.1-directed macrophage differentiation.

The oncogenic cluster miR‐17‐92 encodes seven related microRNAs that regulate cell proliferation, apoptosis and development. Expression of miR‐17‐92 cluster is decreased upon cell differentiation. Here, we report a novel mechanism of the regulation of miR‐17‐92 cluster. Using transgenic PU.1−/− myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant Egr2 which, in turn, directly represses miR‐17‐92 expression by recruiting histone demethylase Jarid1b leading to histone H3 lysine K4 demethylation within the CpG island at the miR‐17‐92 promoter. Conversely, Egr2 itself is targeted by miR‐17‐92, indicating existence of mutual regulatory relationship between miR‐17‐92 and Egr2. Furthermore, restoring EGR2 levels in primary acute myeloid leukaemia blasts expressing elevated levels of miR‐17‐92 and low levels of PU.1 and EGR2 leads to downregulation of miR‐17‐92 and restored expression of its targets p21CIP1 and BIM. We propose that upon macrophage differentiation PU.1 represses the miR‐17‐92 cluster promoter by an Egr‐2/Jarid1b‐mediated H3K4 demethylation mechanism whose deregulation may contribute to leukaemic states.

Expression of genes regulating angiogenesis in human circulating hematopoietic cord blood CD34+/CD133+ cells

Objectives: Human CD34+ cells represent a heterogeneous population of immature cells which may differentiate to various cell types. The aim of the study was to determine angiogenesis regulating genes expression in CD34+ cells, their subpopulations, and during their differentiation induced by hematopoietic growth factors.

5-Azacitidine in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1 gene and cell differentiation capacity

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34+ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte–macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

Response of hematopoiesis to cyclophosphamide follows highly specific patterns in bone marrow and spleen.

Sublethal cyclophosphamide treatment induces unique regeneration patterns in bone marrow and the spleen of a mouse. Colony-forming units spleen (CFU-S)(day 8), CFU-granulocyte-macrophage (GM), nucleated cell counts, and their differentials in bone marrow, spleen, and peripheral blood were determined in mice treated with a single dose of cyclophosphamide. To study further the mechanisms underlying the unique patterns of hematopoietic regeneration after cyclophosphamide, mRNA levels for stem cell factor (SCF), Flt-3 ligand, and macrophage inflammatory factor (MIP)-1 alpha cytokines were determined in bone marrow and spleen. Granulocyte precursor cells were less depleted by cyclophosphamide compared to erythroid nucleated cells and lymphocytes both in bone marrow and spleen. Rapid expansion of granulopoietic cells increased the granulocytic/erythroid ratio significantly during regeneration. CFU-S in the bone marrow and the spleen showed different sensitivity in vivo but not in vitro to cyclophosphamide; CFU-GM were equisensitive in both sites. In bone marrow, an initial fast recovery of CFU-S and CFU-GM on days 2 to 3 was followed by a secondary deep decline in their numbers occurring between days 5 and 7. This decline was accompanied with a depression of CFU-S proliferation and with significantly increased CFU-S numbers in the peripheral blood. In the spleen, absolute CFU-S and CFU-GM numbers were increased several-fold at this time. Seven days after cyclophosphamide, the spleen contained 69% of the total body CFU-S compared to 4% in controls. Splenectomy did not abolish the secondary disease of CFU-S in the bone marrow, but it led to a marked elevation of circulating leukocytes and CFU-S. There was an eight-fold increase in the SCF mRNA level in the bone marrow 2 days after cyclophosphamide, corresponding with a high proliferation rate of CFU-S. No significant changes in mRNAs for Flt-3 ligand and MIP-1 alpha have been found. This in-depth analysis of murine hematopoietic responses to cyclophosphamide provides evidence for the complexity of the involved local and systemic regulations. This represents a significant challenge to experimental hematology, which could now be tackled with methods allowing the study of changes in the gene expression during cyclophosphamide-induced hematopoietic damage.

Chromatin remodeling gene SMARCA5 is dysregulated in primitive hematopoietic cells of acute leukemia.

We identified a subset of genes involved in chromatin remodeling whose mRNA expression changes in differentiating mouse erythroleukemia (MEL) cells. We furthermore tested their mRNA expression patterns in normal and malignant CD34+ bone marrow cells. SMARCA5, imitation switch gene homologue, was rapidly silenced during in vitro erythroid differentiation of MEL cells whereas it was up-regulated in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Moreover, SMARCA5 mRNA levels decreased in AML CD34+ progenitors after the patients achieved complete hematologic remission. We detected high levels of SMARCA5 mRNA in murine bone marrow and spleen and monitored its expression in these hematopoietic tissues during accelerated hematopoiesis following hemolytic anemia induced by phenylhydrazine. SMARCA5 expression levels decreased after the onset of accelerated erythropoiesis. Our data suggest that both in vitro and in vivo induction of differentiation is followed by down-regulation of SMARCA5 expression. In CD34+ AML progenitors over-expression of SMARCA5 may thus dysregulate the genetic program required for normal differentiation.