Hydroxyurea, a widely used cytotoxic/cytostatic agent that does not influence methylation of DNA bases, increases fetal hemoglobin production in anemic monkeys. To determine its effect in sickle cell anemia, we treated two patients with a total of four, 5-d courses (50 mg/kg per d, divided into three oral doses). With each course, fetal reticulocytes increased within 48-72 h, peaked in 7-11 d, and fell by 18-21 d. In patient I, fetal reticulocytes increased from 16.0 +/- 2.0% to peaks of 37.7 +/- 1.2, 40.0 +/- 2.0, and 32.0 +/- 1.4% in three successive courses. In patient II the increase was from 8.7 +/- 1.2 to 50.0 +/- 2.0%. Fetal hemoglobin increased from 7.9 to 12.3% in patient I and from 5.3 to 7.4% in patient II. Hemoglobin of patient I increased from 9.0 to 10.5 g/dl and in patient II from 6.7 to 9.9 g/dl. Additional single-day courses of hydroxyurea every 7-20 d maintained the fetal hemoglobin of patient I t 10.8-14.4%, and the total hemoglobin at 8.7-10.8 g/dl for an additional 60 d. The lowest absolute granulocyte count was 1,600/mm3; the lowest platelet count was 390,000/mm3. The amount of fetal hemoglobin per erythroid burst colony-forming unit (BFU-E)-derived colony cell was unchanged, but the number of cells per BFU-E-derived colony increased. Although examination of DNA synthesis in erythroid marrow cells in vitro revealed no decreased methylcytidine incorporation, Eco RI + Hpa II digestion of DNA revealed that hypomethylation of gamma-genes had taken place in vivo after treatment. This observation suggests that hydroxyurea is a potentially useful agent for the treatment of sickle cell anemia and that demethylation of the gamma-globin genes accompanies increased gamma-globin gene activity.

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Hydroxyurea enhances fetal hemoglobin production in sickle cell anemia.

Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo

Hematopoietic stem cells (HSC) can be harmed by disease, chemotherapy, radiation, and normal aging We show in this study that damage also occurs in mice repeatedly treated with very low doses of LPS Overall health of the animals was good, and there were relatively minor changes in marrow hematopoietic progenitors However, HSC were unable to maintain quiescence, and transplantation revealed them to be myeloid skewed Moreover, HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor This phenomenon was previously seen in normal aging Screening identified mAbs that resolve HSC subsets, and relative proportions of these HSC changed with age and/or chronic LPS treatment For example, minor CD150(Hi)CD48(-) populations lacking CD86 or CD18 expanded Simultaneous loss of CD150(Lo/-)CD48(-) HSC and gain of the normally rare subsets, in parallel with diminished transplantation potential, would be consistent with age- or TLR-related injury In contrast, HSC in old mice differed from those in LPS-treated animals with respect to VCAM-1 or CD41 expression and lacked proliferation abnormalities HSC can be exposed to endogenous and pathogen-derived TLR ligands during persistent low-grade infections This stimulation might contribute in part to HSC senescence and ultimately compromise immunity

Chronic Exposure to a TLR Ligand Injures Hematopoietic Stem Cells

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are members of a discrete family of cytokines that regulates the growth, differentiation, migration and effector function activities of many hematopoietic cells and immunocytes. These cytokines are involved in normal responses to infectious agents, bridging innate and adaptive immunity. However, in certain cases, the overexpression of these cytokines or their receptors can lead to excessive or aberrant initiation of signaling resulting in pathological conditions, with chronic inflammatory diseases and myeloid leukemias the most notable examples. Recent crystal structures of the GM-CSF receptor ternary complex and the IL-5 binary complex have revealed new paradigms of cytokine receptor activation. Together with a wealth of associated structure-function studies, they have significantly enhanced our understanding of how these receptors recognize cytokines and initiate signals across cell membranes. Importantly, these structures provide opportunities for structure-based approaches for the discovery of novel and disease-specific therapeutics. In addition, recent biochemical evidence has suggested that the GM-CSF/IL-3/IL-5 receptor family is capable of interacting productively with other membrane proteins at the cell surface. Such interactions may afford additional or unique biological activities and might be harnessed for selective modulation of the function of these receptors in disease.

The GM–CSF/IL‐3/IL‐5 cytokine receptor family: from ligand recognition to initiation of signaling

Friend erythroleukemia cells are induced to differentiate when treated with dimethyl sulfoxide (DMSO) and a number of other structurally unrelated agents, some of which are also known to alter the structure of DNA. In order to determine whether DMSO was responsible for DNA damage, the properties of [ 3 H]thymidine-labeled DNA from untreated cells and cells treated with DMSO were compared on alkaline sucrose gradients. Criteria for DNA degradation were: ( a ) an increase in the radioactivity of slowly sedimenting approximately 17S DNA, ( b ) a decrease in the amount of radioactivity in the heavy, major approximately 140S peak, and ( c ) a shift in the position of the heavy peak to a lower S value. Increasing damage was found with increasing doses of DMSO. The earliest change, which was detected at 3.5 hr, was an increase in radioactivity in the 17S peak. Therefore, breaks in DNA were detected prior to the stimulation of globin messenger RNA and hemoglobin synthesis. Other potent inducers, such as butyrate, caused similar DNA degradation. Agents that cause breaks in DNA (X-ray, bleomycin, and UV) also were found to stimulate partial erythrodifferentiation in these cells. “Folded genomes” were prepared from 24-hr control and DMSO-treated cultures labeled with either [ 3 H]- or [ 14 C]thymidine, respectively. Cosedimentation of these labeled folded genomes revealed a decrease of approximately 10% in the S value of the DMSO-treated samples as compared to the control. Reverse labeling, in which control samples contained 14 C and the DMSO samples contained 3 H, gave similar results. These changes are consistent with the accumulation of single-stranded nicks in the DNA of the folded genomes. Therefore, single-stranded scission in DNA may be an early step in the control of differentiation.

Breakage of DNA and alterations in folded genomes by inducers of differentiation in Friend erythroleukemic cells.

Since the last conference on erythropoiesis, much information has accumulated which has necessitated a revision of some of our notions as to the mechanism of red cell formation. It is still clear that the erythroid compartment is not a self-maintaining compartment, but is fed from a primitive stem cell pool. The work of Becker et a1.I and Whang et aL2 appeared to settle the long standing issue whether this is a unipotential or totipotential compartment. More recent evidence, however, raises the question as to whether there may not be two levels of stem cells, a more primitive totipotential stem cell compartment equivalent to the colony-forming unit of Till and McCulloch3 and a more differentiated precursor cell for each of the identifiable hematopoietic elements. This issue is by no means settled, but data will be presented favoring the latter explanation, i.e., two levels of precursor cells. The studies on the effect of erythropoietin in the hypertransfused rodent clearly demonstrated that erythropoietin is capable of differentiating the stem cell into an identifiable erythroid precursor. The mechanism by which this is accomplished is moot and the question of whether the action of erythropoietin is confined to the stem cell or whether it may also affect differentiated cells is at present undecided. In this paper it is our purpose to present data bearing on these points. The effect of erythropoietin has been studied in normal animals and in rats in which red cell production has been perturbed by hypertransfusion and/or vincristine. Critical to these considerations are studies on megakaryocytopoiesis using tritiated thymidine. These also will be reported briefly.

Regulation of erythropoiesis. XX. Kinetics of red cell production.

The ability of a single injection of killed, intact bacteria to effect an increase in the proliferative rate of hemopoietic stem cells was studied. The total numbers of colony forming units in bone marrow, spleen and peripheral blood as well as the proportion of CFU in cycle was assessed. Splenic CFU were observed to rise exponentially due initially to in situ proliferation and later to proliferation in bone marrow with migration via the blood to the spleen. The results are discussed in the light of current concepts of stem cell regulation.

Hemopoietic stem cell proliferation and migration following Bordetella pertussis vaccine.

Relationship of erythropoietin effectiveness to the generative cycle of erythroid precursor cell.

SummaryA single i.v. dose of hydroxyurea (0.9 g/kg) produced transient marrow erythroid depopulation and reticulocytopenia in the mouse. Within minutes after drug administration there was a virtually complete erradication of marrow DNA synthesis which persisted for approximately 2 hr. Erythroid cell cycle kinetics, estimated with colchicine, revealed an entry of erythroid cells into mitosis for 80 min followed by no further accumulation of metaphase forms until the seventh hour. Thereafter erythroid cells accumulated in mitosis at a normal rate. These findings are consistent with the hypothesis that hydroxyurea kills erythroid cells in DNA synthesis; the initial 80-min accumulation of erythroid mitoses represents the progression of G2 cells into mitosis. Unaffected cells in G1 at the time of drug administration require 7-8 hr to progress through DNA synthesis, G2, and enter mitosis. From these data it would appear that hydroxyurea may prove a useful agent in the study of erythropoiesis because of its sele...

The effect of hydroxyurea on differentiated marrow erythroid precursors.

Hydroxyurea, a cytotoxic agent which destroys cells in DNA synthesis, has been shown to evoke the differentiation of a small number of hemopoietic precursor cells in the erythroid series of erythropoietically suppressed hypertransfused mice. This effect does not appear to be mediated by erythropoietin (EP) since the simultaneous injection of anti-EP did not alter this response.

Bernard S. Morse

Papers

Regulation of erythropoiesis. XX. Kinetics of red cell production.

Hemopoietic stem cell proliferation and migration following Bordetella pertussis vaccine.

Relationship of erythropoietin effectiveness to the generative cycle of erythroid precursor cell.

The effect of hydroxyurea on differentiated marrow erythroid precursors.

Hydroxyurea-induced erythroid differentiation.