Eugenia Prus

Cellular copper content modulates differentiation and self‐renewal in cultures of cord blood‐derived CD34+ cells

Summary.  Several clinical observations have suggested that copper (Cu) plays a role in regulating haematopoietic progenitor cell (HPC) development. To further study this role we used an ex vivo system. Cord blood‐derived CD34+ cells were cultured in liquid medium supplemented with Kit‐ ligand, FLt3, interleukin 6 (IL‐6), thrombopoietin and IL‐3. Under these conditions, Cu content, measured by atomic absorption, was 7 ng/107 cells. Modulation of intracellular Cu was achieved by supplementing the cultures with the Cu chelator tetraethylenepentamine, which reduced cellular Cu (4 ng/107 cells), or ceruloplasmin or Cu sulphate that elevated cellular Cu (18 and 14 ng/107 cells respectively). The results indicated that low Cu content delayed differentiation, as measured by the surface antigens CD34, CD14 and CD15, colony‐forming unit (CFU) frequency and cell morphology, while high Cu accelerated differentiation compared with Cu unmanipulated cultures. As a result, expansion of total cells, CFU and CD34+ cells in low Cu was extended (12–16 weeks), and in high Cu was shortened (2–4 weeks), compared with control cultures (6–8 weeks). These effects required modulation of intracellular Cu only during the first 1–3 weeks of the culture; the long‐term effects persisted thereafter, suggesting that the decision process for either self‐renewal or differentiation is taken early during the culture. This novel method of controlling cell proliferation and differentiation by copper and copper chelators might be utilized for ex vivo manipulation of HPC for various clinical applications.

Accumulation of gamma-globin mRNA in human erythroid cells treated with angelicin

The aim of the present study was to determine whether angelicin is able to increase the expression of γ‐globin genes in human erythroid cells. Angelicin is structurally related to psoralens, a well‐known chemical class of photosensitizers used for their antiproliferative activity in treatment of different skin diseases (i.e., psoriasis and vitiligo). To verify the activity of angelicin, we employed two experimental cell systems, the human leukemic K562 cell line and the two‐phase liquid culture of human erythroid progenitors isolated from normal donors. The results of our investigation suggest that angelicin, compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and γ‐globin mRNA accumulation of human leukemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of angelicin, increases of γ‐globin mRNA accumulation and fetal hemoglobin (HbF) production, even higher than those obtained using hydroxyurea, were detected. These results could have practical relevance, as pharmacologically‐mediated regulation of the expression of human γ‐globin genes, leading to HbF induction, is considered a potential therapeutic approach in hematological disorders, including β‐thalassemia and sickle cell anemia.

Prodrugs of butyric acid. Novel derivatives possessing increased aqueous solubility and potential for treating cancer and blood diseases

The synthesis and biological activities of acidic, basic and neutral types of butyric acid (BA) prodrugs possessing increased aqueous solubility are described. The compounds are butyroyloxyalkyl derivatives of carboxylic acids, which possess functionalities suitable for aqueous solubilization. The anticancer activity of the prodrugs in vitro was evaluated by examining their effect on the growth of human colon, breast and pancreatic carcinoma cell lines, and their solubility in aqueous media was determined. The most promising compounds, with respect to activity and solubility, were found to be the butyroyloxymethyl esters of glutaric 2a and nicotinic acids 4a and phosphoric acid as its diethyl ester 10a, which displayed IC(50) values of 100 microM or lower. These prodrugs are expected to release formaldehyde upon metabolic hydrolysis. The corresponding butyroyloxyethyl esters (2b, 4b and 10b) that release acetaldehyde upon metabolism were significantly less potent. A similar correlation was observed for growth inhibition of the human prostate carcinoma cell lines PC-3 and LnCap and for induction of differentiation and apoptosis in the human myeloid leukemia cell line HL-60. The higher biological activity of the formaldehyde-releasing prodrugs 2a and 10a was further confirmed when induction of hemoglobin (Hb) synthesis in the human erythroleukemic cell line K562 was measured. Moreover, a therapeutic index (IC(50)/ED(50)) of ca. 5 was observed. The acute i.p. toxicity LD(50) in mice for 2a, 2b, 10a and 10b was similar and in the range of 400-600 mg kg(-1). The results obtained support the potential use of the butyric acid prodrugs for the treatment of neoplastic diseases and beta-globin disorders.

Macrophages function as a ferritin iron source for cultured human erythroid precursors

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin‐containing red blood cells. The transferrin–transferrin receptor pathway is the main route for erythroid iron uptake. Using a two‐phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin‐free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor‐mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co‐cultured with macrophages under transferrin‐free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin‐dependent, receptor‐mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin‐free conditions by delivering essential iron in the form of metabolizable ferritin. J. Cell. Biochem. 103: 1211–1218, 2008.

Flow cytometry measurement of the labile iron pool in human hematopoietic cells

Iron is important for many biological processes, and its deficiency or excess is involved in pathological conditions. Although most iron is firmly bound (e.g., in hemoglobin), some, the labile iron pool (LIP), is bound to low‐affinity ligands. The level of LIP is regulated to meet the cells requirements for iron but prevent excess. We describe herein a multiparameter flow cytometry procedure for measuring LIP in various human hematopoietic cells. Peripheral blood and bone marrow (BM) cells were loaded with calcein‐AM, washed, and then incubated with or without the high‐affinity iron‐chelator Deferiprone (L1). Specific cell subpopulations were identified based on side‐light scattering and expression of surface antigens. LIP was determined based on the ability of L1 to bind and remove iron from calcein and thereby increase the fluorescence emitted by the cells. Blood cells differ in their LIP content in the order monocytes > PMN > RBC > lymphocytes. Analysis of BM cells indicated a similar tendency among precursors of the different lineages. The results also showed that among myeloid precursors, LIP increases along cell maturation. Flow cytometry might be useful for evaluating LIP in various diseases and for studying the efficacy of iron‐chelators.

Effects of rapamycin on accumulation of α-, β- and γ-globin mRNAs in erythroid precursor cells from β-thalassaemia patients

Abstract:  We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 β‐thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two‐phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the β‐thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to γ‐globin mRNA accumulation, being only minor for β‐globin and none for α‐globin mRNAs. The ability of rapamycin to preferentially increase γ‐globin mRNA content and production of HbF in erythroid precursor cells from β‐thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti‐rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in β‐thalassaemia and sickle cell anaemia.

Rapamycin-mediated induction of γ-globin mRNA accumulation in human erythroid cells

The present study aimed to determine whether rapamycin could increase the expression of γ‐globin genes in human erythroid cells. Rapamycin is a macrocyclic lactone that possesses immunosuppressive, antifungal and anti‐tumour properties. This molecule is approved as an immunosuppressive agent for preventing rejection in patients receiving organ transplantation. To verify the activity of rapamycin, we employed two experimental cell systems, the human leukaemia K562 cell line and the two‐phase liquid culture of human erythroid progenitors isolated from normal donors and patients with β‐thalassaemia. The results suggested that rapamycin, when compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and γ‐globin mRNA accumulation in human leukaemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of rapamycin, γ‐globin mRNA accumulation and fetal haemoglobin (HbF) production increased to levels that were higher than those obtained using hydroxyurea. These effects were not associated with inhibition of cell growth. Furthermore, rapamycin was found to increase HbF content in erythroid precursor cells from four β‐thalassaemia patients. These results could have practical relevance, because pharmacologically mediated regulation of the expression of human γ‐globin genes, leading to increased HbF, is considered a potential therapeutic approach in haematological disorders, including β‐thalassaemia and sickle cell anaemia.

Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

The labile iron pool in human erythroid cells

Although most cellular iron is firmly bound (e.g. in haemoglobin), some, the labile iron pool (LIP), is bound to low‐affinity ligands. The LIP is regarded as the crossroads of cellular iron traffic. Using multi‐parameter flow‐cytometry of cells treated with the metal‐sensitive sensor calcein and the cell‐permeable chelator deferiprone, we studied LIP in various human erythroid cell populations in peripheral blood, bone marrow and culture. Erythroid maturation was found to be associated with a decrease in the LIP. In the peripheral blood, nucleated erythrocytes (normoblasts) had 5·8‐fold and 8·8‐fold greater LIP than reticulocytes and erythrocytes respectively. Early reticulocytes had 2·5‐fold more LIP than late reticulocytes. In the bone‐marrow and in culture, LIP decreased by c. 30‐fold as early erythroid precursors matured to late precursors. Adding holo‐transferrin to iron‐depleted cultures elevated LIP by 3·9‐fold. We also show that in β‐thalassaemia, a disease associated with iron‐overload, erythrocytes and reticulocytes in the blood and erythroid precursors in culture have a significantly greater LIP than their normal counterparts. In conclusion, the LIP in erythroid cells is altered under physiological (maturation) and pathological (thalassaemia) conditions. The methodology presented might be useful for evaluating the LIP in various diseases and for studying the efficacy of iron‐chelators.

Resveratrol: Antioxidant activity and induction of fetal hemoglobin in erythroid cells from normal donors and β-thalassemia patients

Thalassemia and sickle-cell anemia (SCA) present a major public health problem in countries where the number of carriers and affected individuals is high. As a result of the abnormalities in hemoglobin production, cells of thalassemia and SCA patients exhibit oxidative stress, which ultimately is responsible for the chronic anemia observed. Therefore, identification of compounds exhibiting both antioxidant and hemoglobin-inducing activities is highly needed. Our results demonstrate resveratrol to be such a compound. This was shown both in the human K562 cell line, as well as in erythroid precursors derived from normal donors and β-thalassemia patients. Resveratrol was shown to exhibit antioxidant activity and to stimulate the expression of the γ-globin genes and the accumulation of fetal hemoglobin (HbF). To the best of our knowledge, this is the first report pointing to such a double effect of resveratrol. Since this natural product is already marketed as an antioxidant, future investigations should concentrate on demonstrating its potential to augment HbF production in experimental animal models (e.g., thalassemia and SCA mice) as well as in patients. We believe that the potential of clinical use of resveratrol as an antioxidant and HbF stimulator may offer a simple and inexpensive treatment to patients.