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Background: COVID-19 virus has caused the world's deadliest pandemic. Early April 2020, the Delhi Government made it compulsory for people to wear face masks while going outdoors to curb disease spread. Prolonged use of surgical masks during the pandemic has been reported to cause many adverse effects. Intermittent hypoxia has been shown to activate erythropoietin (EPO leading to increased hemoglobin mass., Aim: To analyze whether face mask induced intermittent hypoxia has any effect on the hemoglobin levels of healthy blood donors., Materials and Methods: We retrospectively analyzed donor data from 1st July 2019-31st December 2020 for hemoglobin distribution across hemoglobin ranges and donor deferral on basis of hemoglobin. Study population was divided into two cohorts Group 1- (1st July 2019-31 st March 2020): before implementation of mandatory face masks Group 2- (1st April 2020-31 st December 2020): after implementation of mandatory face masks RESULTS: Mean Hb of blood donors in Group 2 (15.01 ± 1.1 g/dl) was higher than Group1 (14.49 ± 1.15 g/dl), (p < 0.0001). 47.1 % group2 donors had Hb of 16.1-18 g/dl compared to group1 (38.4 %). 52.9 % group 2 donors had Hb between 12.5-15 g/dl compared to 61.6 % Group 1 (p < 0.05). Deferral due to anemia was lesser in group 2 compared to group 1 (p < 0.00001). Group 2 had significantly higher deferral due to high Hb (>18 gm/dl) was than Group 1 (p = 0.0039)., Conclusion: This study including 19504 blood donors spanning over one and a half year shows that prolonged use of face mask by blood donors may lead to intermittent hypoxia and consequent increase in hemoglobin mass., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This review describes some of the physiological effects of recombinant human erythropoietin (EPO) in healthy humans. At the blood level EPO increases the arterial O(2) content not only by increasing red blood cell volume, but also by an equally important decrease in plasma volume. Well before that, EPO causes a prompt decrease in plasma levels of renin and aldosterone. Renal clearance studies suggest that EPO decreases renal proximal tubular reabsorption rate leading to activation of the tubuloglomerular feedback mechanism and a fall in glomerular filtration rate. Thus, treatment with EPO may result in suppression of endogenous EPO production through a decrease in intrarenal oxygen consumption. EPO elevates the arterial blood pressure even in healthy subjects. The receptor for EPO is present in many tissues. However, the functional effects of EPO in the skeletal muscle seem limited, and although it has been speculated that non-erythropoietic effects of EPO (angiogenesis, shift in muscle fibre types, cognitive effects) may be responsible for the increase in exercise performance, this has not been confirmed. EPO-induced haemodynamic effects call for careful monitoring during the administration period. The metabolic, hormonal and renal effects of EPO do not seem to range beyond physiologically acceptable limits and are reversible. Taken together, EPO seems safe to use for experimental purposes in healthy volunteers.

Fo Shou San (FSS) is an ancient herbal decoction comprised of Rhizoma Chuanxiong (RC; Chuanxiong) and Radix Angelicae Sinensis (RAS; Danggui) in a ratio of 2 : 3. It is mainly prescribed for patients having a blood deficiency. This combination is considered the most popular herb pair among Chinese medicines; however, the rationale of having these two chemically similar herbs within the decoction has historically not been made clear. Here, we attempted to reveal the chemical and biological properties of this decoction as a means to deduce its mechanism of action. The effects of FSS were determined in different cell culture models. With respect to stimulation of blood circulation, FSS inhibited ADP-mediated platelet aggregation in a dose-dependent manner. In order to reveal the hematopoietic effect of this decoction, FSS was applied onto cultured K562 human leukemia cells and Hep3B human hepatocellular carcinoma cells. Application of FSS in cultured K562 cells inhibited cell proliferation and subsequently induced the production of hemoglobin. Additionally, the mRNA expression of erythropoietin (EPO) was induced in a dose-dependent manner when FSS was applied to Hep3B cells. The current results reveal the effects of FSS in different cell models, paving a direction for mechanistic studies., (© Georg Thieme Verlag KG Stuttgart · New York.)

Objective: alpha-Hemoglobin stabilizing protein (AHSP) binds alpha-hemoglobin (Hb), avoiding its precipitation and its pro-oxidant activity. In the presence of betaHb, the alphaHb-AHSP complex is dismembered and betaHb displaces AHSP to generate the quaternary structure of Hb. The relationship between Hb formation and alterations in AHSP expression, which may affect human erythropoiesis, has not yet been described in human cells. Hence, in this study, we examined the effects of AHSP knockdown in hemin-induced K562 and erythropoietin-induced CD34(+) cells with particular reference to cellular aspects and gene expression., Materials and Methods: Short-hairpin RNA expression vectors aimed at the AHSP mRNA target sequence were cloned and transfected into K562 and CD34(+) cells. K562 and CD34(+) cells were stimulated to erythroid differentiation. Cells were examined in terms of gene expression using quantitative real-time polymerase chain reaction; reactive oxygen species (ROS) production, apoptosis, and Hb production through flow cytometry assays; and immunofluorescence assays for globin chains., Results: RNA interference-mediated knockdown of AHSP expression resulted in considerable alphaHb precipitation, as well as in a significant decrease in HbF formation. AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis., Conclusion: These findings strengthen the hypothesis that AHSP stabilizes the alphaHb chain, avoiding its precipitation and its ability to generate ROS, which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

In the present study, the zebrafish epo cDNA was cloned. The encoded protein displays 90%, 55% and 32% identity to the Epo from carp, fugu and human, respectively. Through RT-PCR, the expression of zepo mRNA was mainly in the heart and liver. In the COS-1 cell transfection experiments, the recombinant zEpo-HA protein was efficiently secreted into the culture medium as a glycoprotein and the carbohydrate moiety can be cleaved by the treatment of peptide-N-glycosidase F (PNGase F). Using the morpholino approach, we showed that zepo morphants displayed severe anemia leading to high mortality during development. Such an effect can be significantly rescued by zepo RNA. Furthermore, in the absence of functional zEpo, the expression of specific markers for adult globin genes, such as alphaA1- and betaA1-globin, but not the embryonic betae1-globin, was affected.

Erythropoietin, by binding to its receptor, stimulates definitive erythroblasts to accumulate hemoglobin (Hb) by up-regulating erythroid-specific genes and causes differentiation of erythroblasts into erythrocytes. In mouse decidua we have found the expression of transcripts for the erythropoietin receptor, the function of which has not yet been elucidated. Erythropoietin signaling was inhibited by the injection of a soluble form of the erythropoietin receptor capable of binding with erythropoietin into the mouse uterine cavity on day 4 of gestation, and pale and defective decidual bodies appeared three days later. These pale decidual bodies contained defective embryos without extension to the ectoplacental region, while normal reddish decidual bodies contained normal developing embryos and expressed embryonic and adult Hb with characteristic location of the respective hemoglobins in which an epsilon- or beta-globin signal was confirmed. Furthermore, blocking of erythropoietin signaling destroyed Hb-containing cells and resulted in apoptosis that caused embryonic death. Thus, erythropoietin-mediated Hb synthesis is essential for the survival of decidual cells. In addition, although no transcripts for GATA-1 and erythroid heme enzymes could be detected, genes for beta-globin, as well as non-specific delta-aminolevulinate synthase, were expressed and regulated in an erythropoietin-dependent manner. This is the first evidence that ectopic Hb synthesis exists and that erythropoietin coregulates erythroid (globin) and nonerythroid (delta-aminolevulinate synthase) genes.

The partial correction of ESRD anemia by recombinant human erythropoietin (EPO) has resulted both in generalized improvement in quality of life and physical activity and in reduced mortality and hospitalization rate. The question remains as to whether normalizing hemoglobin (Hgb) is desirable in patients with chronic kidney disease (CKD). This review provides an analysis and commentary on the available reports and, for the most part, randomized, controlled trials on the topic. In dialysis patients, normalization of Hgb is associated with improved quality of life and exercise capacity but not with reduced mortality and hospitalization rate. Moreover, no significant changes in the degree of left ventricular hypertrophy have been demonstrated. By contrast, an increased mortality rate has been reported for hemodialysis patients with overt cardiovascular disease (CVD) when randomly assigned to normal hematocrit by EPO. Data regarding patients who have CKD but are not yet on renal replacement therapy are scarce, and the effects of EPO on renal disease progression require further elucidation through controlled trials. The conclusion that can be drawn from the available studies is that Hgb >11 g/dl is the minimum required to achieve improved quality of life in patients with CKD, whereas values >12 g/dl are not recommended for patients with overt CVD. Finally, Hgb normalization might reasonably be restricted to a selected population of younger, employed, and active individuals, provided that they do not have CVD.

Anemia is highly prevalent in patients with cancer and its impact on quality of life and long-term outcome in these patients is well documented. Recombinant human erythropoietins, or epoetins, have been used to treat cancer-related or antitumor therapy-induced anemia for many years. Through a combination of clinical studies and extensive experience in the real-life clinical setting, epoetin beta has been shown to be efficacious and well tolerated, increasing hemoglobin levels, reducing the need for transfusion and improving quality of life. This favorable efficacy and safety profile has been demonstrated across a broad range of malignancy types, irrespective of the treatment used (platinum or non-platinum based). The effect of treatment with epoetin beta is rapid, with mean hemoglobin increases of 1 g/dl seen as early as 4 weeks following the start of therapy. Furthermore, there is no evidence that epoetin beta negatively affects overall survival or tumor progression in anemic patients with cancer. The approved 30,000 IU once-weekly dosing regimen (as opposed to the 10,000 IU three-times weekly regimen) provides greater convenience and may result in improved treatment compliance.

Background: In recent years there has been an increasing debate regarding the benefits of initiating erythropoietic treatment in patients with cancer when anemia is still relatively mild. To address this, a systematic review of studies was conducted that answered the following question: Is there a clinical benefit associated with early erythropoietic intervention (hemoglobin > or = 10 g/dL) for chemotherapy-induced anemia?, Methods: A systematic review of published literature and meeting abstracts was undertaken to identify relevant studies. Data were extracted from studies meeting prespecified eligibility criteria. For outcome measures not associated with significant heterogeneity, summary measures of relative risk associated with early erythropoietic intervention were estimated using the method of Mantel and Haenszel., Results: Eleven studies were eligible and were included in the review. Erythropoietic treatment effectively decreased transfusion incidence and the proportion of patients with hemoglobin < 10 g/dL compared with no treatment, with relative risk reductions of 0.50 (95% confidence interval [CI], 0.43, 0.59; 7 studies, P < 0.0001) and 0.40 (95% CI, 0.19, 0.83; 4 studies, P = 0.147), respectively. The findings from both prospective studies and planned subset analyses in which early and late intervention were compared also indicated a reduction in the relative risk of both transfusions and hemoglobin < 10 g/dL after early intervention (0.55 [95% CI, 0.42, 0.73; 5 studies, P < 0.0001] and 0.44 [95% CI, 0.33, 0.57; 2 studies, P < 0.0001], respectively)., Conclusion: Collectively, these findings suggest that optimal clinical benefit from erythropoietic treatment of chemotherapy-induced anemia may be achieved through early intervention., (Copyright 2005 American Cancer Society.)

Background: Darbepoetin alfa is an erythropoiesis-stimulating glycoprotein that functions by the same mechanism as recombinant human erythropoietin (rHuEPO), but has a three-fold longer serum half-life. Reduction in the frequency of darbepoetin alfa administration would be beneficial to patients with renal disease and their healthcare providers. This study evaluated the effect of extending the darbepoetin alfa dosing interval to once monthly in patients with chronic kidney disease (CKD) not receiving dialysis., Methods: This study was a multicenter, open-label study of 97 patients with CKD not on dialysis. Patients receiving stable subcutaneous doses of darbepoetin alfa once every two weeks were converted to darbepoetin alfa once monthly for 29 weeks. The proportion of patients who successfully maintained hemoglobin concentrations between 10.0 and 12.0 g/dl and the mean darbepoetin alfa dose were evaluated. Safety measurements (e.g. adverse events, laboratory parameters, blood pressure) and seroreactivity were assessed., Results: Hemoglobin concentration was maintained within the target range in 79% (95% confidence interval (CI) = 71% to 87%) of all patients receiving darbepoetin alfa and in 85% (95% Cl = 78% - 93%) of patients who completed the study period. The mean +/- standard deviation monthly darbepoetin alfa dose was similar between baseline (88.7 +/- 49.9 microg) and the evaluation period (86.6 +/- 78.8 microg). The safety profile for monthly darbepoetin alfa administration was comparable with that previously observed with more-frequent administration., Conclusion: Patients with CKD who are clinically stable on darbepoetin alfa administered once every two weeks can be safely and effectively converted to darbepoetin alfa administered once monthly.

It was hypothesized that pentoxifylline might improve the response to recombinant human erythropoietin (rh-Epo) in anemic renal failure patients. Sixteen patients with ESRD and rh-Epo-resistant anemia, defined by a hemoglobin of <10.7 g/dl for 6 mo before treatment and a rh-Epo dose of > or =12,000 IU/wk, were recruited. They were treated with oral pentoxifylline 400 mg o.d. for 4 mo. Ex vivo T cell generation of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) from the patients was assessed before treatment and 6 to 8 wk after therapy. A total of 12 of 16 patients completed the study. Before therapy, the 12 patients' mean hemoglobin concentration was 9.5 +/- 0.9 g/dl. After 4 mo of pentoxifylline treatment, the mean hemoglobin concentration increased to 11.7 +/- 1.0 g/dl (P = 0.0001). Baseline ex vivo T cell expression of TNF-alpha decreased from 58% +/- 11% to 31% +/- 23% (P = 0.0007) after therapy. Likewise, IFN-gamma expression decreased from 31% +/- 10% to 13% +/- 10% (P = 0.0002). Pentoxifylline therapy may significantly improve the hemoglobin response in patients with previously rh-Epo-resistant anemia in renal failure. This may occur due to inhibition of proinflammatory cytokine production, which could interfere with the effectiveness of rh-Epo.

Targeting BCR-ABL tyrosine kinase by treatment with the selective inhibitor imatinib (formerly STI571, Gleevec) has proved to be highly efficient for inhibiting leukemic growth in vitro. In addition, in clinical trials, imatinib has produced high response rates in patients with chronic myeloid leukemia (CML) in chronic phase and blastic crisis. However, episodes of severe cytopenia were also frequently observed, leading to discontinuation of therapy in some cases. Therefore, it is important to examine whether administration of cytokines overcomes the adverse effects of imatinib in in vitro systems. In this study, we examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) on TF-1/bcr-abl (which was generated by transduction of a bcr-abl fusion gene into the TF-1 cell line) as a model system for CML with blastic crisis. Imatinib induced apoptosis in TF-1/bcr-abl cells but not in the parental TF-1 cells. However, GM-CSF, a survival factor of the parental TF-1 cells, protected TF-1/bcr-abl cells from imatinib-induced apoptosis in a dose-dependent manner. Concomitantly, constitutive phosphorylation of Stat5 and FKHRL1 was significantly inhibited by imatinib, and the inhibition was canceled by the addition of GM-CSF, accompanied by upregulation of Bcl-xL and downregulation of p27/Kip1. In addition, although untreated TF-1/bcr-abl cells had lost responsiveness to both GM-CSF and EPO and showed autonomous growth, GM-CSF enhanced phosphorylation of Stat5 and FKHRL1 in these cells. Importantly, imatinib-treated TF-1/bcr-abl cells differentiated into hemoglobin-positive cells in the presence of EPO, as in the case for the parental TF-1 cells. Taken together, imatinib-treated CML cells may differentiate into mature cells in the presence of differentiation-inducing cytokines such as EPO.

Fixed dosing is potentially more convenient than weight-based dosing for both patients and physicians. Therefore, this open-label, randomized (1:1), multicenter study was conducted to compare the effectiveness, safety, and quality-of-life benefits of fixed vs. weight-based dosing of epoetin alpha in anemic cancer patients undergoing chemotherapy. Five hundred forty-six anemic patients undergoing platinum-based chemotherapy for solid malignancies were enrolled. Patients received epoetin alpha, either a fixed dose of 10,000 IU or a weight-based dose of 150 IU/kg, administered subcutaneously 3 times weekly for up to 12 weeks. Endpoints were transfusion requirements over days 29-84, change in hemoglobin (Hb) level from baseline, and change in quality-of-life (QOL) scores from baseline as measured using the Cancer Linear Analog Scale (CLAS). Five hundred and thirty-two patients received at least 1 dose of epoetin alpha, and 510 of these (255 in each treatment group) were considered evaluable for efficacy. At day 84, rates for freedom from transfusion were similar between the fixed-dose and the weight-based dose group (84% vs. 87%, respectively, p=0.32), as calculated by the lifetable method. These rates were also similar between patients in the 45-63 kg weight group receiving the fixed 10,000 IU dose or 7,000-9,000 IU on a per-weight basis (83% vs. 87%, respectively), and those in the 70-100 kg weight group receiving the fixed 10,000 IU dose or 11,000-15,000 IU on a per-weight basis (85% vs. 83%, respectively). Mean Hb increases from baseline to last observation were 2.10 g/dl [95% confidence intervals (CI95) 1.85-2.35] in the 10,000 IU group (from 9.64-11.74 g/dl) and 2.06 g/dl (CI95 1.82-2.30) in the 150 IU/kg group (from 9.70-11.76 g/dl). QOL results were similar for both groups and cumulative data have been reported. For 275 patients (in both groups combined) with CLAS QOL scores both at baseline and 29-98 days thereafter, the QOL index (average of scores for the 3 QOL parameters: energy level, ability to do daily activities and overall QOL) increased by 10.4 mm (CI95 7.5-13.2), from 46.2 mm at baseline to 56.6 mm at the final observation. QOL improvements were directly associated with Hb increases (p<0.001, multiple linear regression analysis) within all chemotherapy response classes. Epoetin alpha was well tolerated in both groups. Fixed (10,000 IU) and weight-based (150 IU/kg) dosing regimens of epoetin alpha demonstrated similar efficacy in maintaining freedom from transfusion, increasing Hb levels, and improving QOL in anemic cancer patients undergoing platinum-based chemotherapy. QOL improvements were directly associated with Hb increases. These findings support the use of a fixed-dose regimen of epoetin alpha, which may offer greater convenience for physicians and patients than weight-based dosing with this agent.

The erythroid Krüppel-like factor (EKLF) is essential for the transcription of betamaj globin in erythroid cells. We show here that RNA for this transcription factor did not alter during erythropoietin-induced differentiation of J2E cells; however, EKLF protein content decreased and was inversely related to globin production. This unexpected result was also observed during chemically induced maturation of two murine erythroleukemia cell lines. To explore the role of EKLF in erythroid terminal differentiation, an antisense EKLF construct was introduced into J2E cells. As a consequence EKLF RNA and protein levels fell by approximately 80%, and the cells were unable to manufacture hemoglobin in response to erythropoietin. The failure to produce hemoglobin was due to reduced transcription of not only globin genes but also key heme enzyme genes. However, numerous other genes, including several erythroid transcription factors, were unaffected by the decrease in EKLF. Although hemoglobin synthesis was severely impaired with depleted EKLF levels, morphological maturation in response to erythropoietin continued normally. Moreover, erythropoietin-induced proliferation and viability were unaffected by the decrease in EKLF levels. We conclude that EKLF affects a specific set of genes, which regulates hemoglobin production and has no obvious effect on morphological changes, cell division, or viability in response to erythropoietin.

To approach the goal of consistent long-term erythropoietin (Epo) expression in vivo, we developed an implantation procedure in which transduced autologous vascular smooth muscle was introduced into rats in a chamber created from a polytetrafluoroethylene (PTFE) ring placed under the serosa of the stomach. The implant became vascularized and permitted the long-term survival of smooth muscle cells expressing Epo. Hematocrits of treated animals increased rapidly and monitored over 12 months gave a mean value of 56.0 +/- 4. 0% (P < .001; n = 9), increased from a presurgery mean of 42.3 +/- 1. 6%. Hemoglobin levels rose from a presurgery mean of 15.2 +/- 0.4 g/dL and for 12 months were significantly elevated with a mean value of 19.5 +/- 1.3 g/dL (P < .001; n = 9). The hematocrit and hemoglobin levels of control animals receiving human adenosine deaminase (ADA)-expressing cells were not significantly different from baseline (P > .05; n = 5). In response to tissue oxygenation, kidney, and (to a lesser extent) liver are specific organs that synthesize Epo. Treated animals showed downregulation of endogenous Epo mRNA in kidney over a 12-month period. The PTFE implant provides sustained gene delivery, is safe, and is minimally invasive. It allows easy engraftment of transduced cells and may be applied generally to the systemic delivery of therapeutic proteins such as hormones and clotting factors., (Copyright 1998 by The American Society of Hematology.)

Changes in transferrin-receptor numbers and iron utilisation were monitored during erythropoietin-induced maturation of J2E erythroid cells. Uptake of transferrin and iron doubled 24 h after exposure to erythropoietin, due to a twofold rise in surface transferrin receptors. In addition, a tenfold increase in iron incorporation into haem was observed after erythropoietin stimulation, as iron taken up from transferrin was directed towards haem biosynthesis and away from storage in ferritin. The rise in iron chelation into haem correlated extremely well with haemoglobin synthesis. However, the increase in numbers of transferrin receptors was not essential for haemoglobin synthesis; rather, it was linked with a burst in proliferation stimulated by erythropoietin. We have shown previously that amiloride blocks erythropoietin-enhanced proliferation of J2E cells, but potentiates maturation [Callus, B. A., Tilbrook, P. A., Busfield, S. J. & Klinken, S. P. (1995) Exp. Cell Res. 219, 39-46]. Here we demonstrate that amiloride suppressed the hormone-induced increase in transferrin receptors, whereas the enhanced incorporation of iron into haem was not inhibited. Similarly, when sodium butyrate was used to induce differentiation of J2E cells, proliferation ceased and surface transferrin receptors remained unaltered, while haemoglobin production was accelerated. It was concluded from these experiments that the erythropoietin-stimulated rise in transferrin receptors during the final stages of J2E cell maturation is linked to cell division, and is not essential for haemoglobin synthesis.

In an increasing number of hematopoietic cytokine receptor systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late erythroid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO receptor in this differentiation-specific system. In studies of HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activation. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F) were expressed in SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglobinization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also may enforce mitogenesis.

Erythropoietin (EP) is required by late-stage erythroid progenitor cells to prevent apoptosis. Several lines of evidence suggest that it is this action of EP that regulates erythrocyte production in vivo. To study the control of apoptosis in mouse and human erythroblasts, the expression of members of the Bcl-2 family of proteins and the expression and activation of the apoptosis-linked cysteine protease Yama/CPP32/apopain were examined. These proteins have been implicated as regulators of apoptosis in several cell models. The Bcl-2 family members analyzed were Bcl-2, Bcl-X, Bax, Bad, Bak, A1, and Mcl-1. Bcl-X expression in proerythroblasts was highly EP-dependent. Bcl-X was strongly increased during the terminal differentiation stages of human and mouse erythroblasts, reaching maximum transcript and protein levels at the time of maximum hemoglobin synthesis. This increase in Bcl-X expression led to an apparent level of approximately 50 times the level in proerythroblasts. In contrast, neither mouse nor human erythroblasts expressed Bcl-2 transcript or protein. Bax and Bad proteins remained relatively constant throughout differentiation, but diminished near the time of enucleation. Bak protein was present in early erythroblasts, but diminished progressively during differentiation. EP deprivation in both mouse and human erythroblasts led to activation of the cysteine protease, apopain, as was indicated by cleavage of the proenzyme into its proteolytically active fragments. Apopain activation was detectable within 2 hours of EP deprivation in mouse erythroblasts. These findings suggest an important role for Bcl-X in late erythroid differentiation and for apopain in apoptosis of erythroblasts caused by deprivation of EP.

The erythroleukaemic cell line TF-1, infected with either the pBabe neo retrovirus or the retrovirus bearing the human erythropoietin (hEpo) gene, developed three growth factor-independent clones. Erythropoietin (Epo), interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) accelerated the proliferation of these clones. Autonomous growth of the clones was independent of Epo because it was not altered by Epo anti-sense oligonucleotides, nor was Epo detectable in culture supernatants. Cells from the mutant clones could not be induced by Epo to express glycophorin A and haemoglobin synthesis was markedly reduced. Haemin reversed the block in Epo-induced haemoglobin synthesis. Acquisition of growth factor-independence appears to be linked with the selective loss of differentiation capacity. These cells may provide a useful model for the study of the mechanisms involved in leukaemic transformation.

Erythropoietin (EPO) and its cell surface receptor (EPOR) play central roles in the proliferation and differentiation of mammalian erythroid progenitor cells. Recently both the tyrosine residues in the EPOR responsible for the activation of Stat5 and the role of Stat5 for EPO-dependent cell proliferation have been shown. Here, we describe the roles of Stat5 and of these tyrosine residues in the EPOR in the erythroid differentiation of murine hematopoietic cell line SKT6 which produces hemoglobin in response to EPO. Chimeric receptors carrying the extracellular domain of the EGF receptor and the intracellular domain of the EPOR were introduced into SKT6 cells. Like EPO, EGF equally activated Stat5 and induced hemoglobin. Activation of Stat5 and hemoglobin expression by EGF were markedly impaired in cells expressing the tyrosine mutated chimeric receptors. In addition, ectopic expression of the prolactin receptor, another cytokine receptor that activates Stat5, led to hemoglobin synthesis. Finally, hemoglobin synthesis was severely inhibited by overexpressing a dominant negative form of Stat5. These results collectively suggest that Stat5 plays a role in EPO-mediated hemoglobin synthesis in SKT6 cells.

Interaction of erythropoietin with its type 1 receptor is essential to the development of late erythroid progenitor cells. Through the ectopic expression of receptor mutants in lymphoid and myeloid cell lines, insight has been gained regarding effectors that regulate Epo-induced proliferation. In contrast, effectors that regulate Epo-induced differentiation events (e.g. globin gene expression) are largely undefined. For in vitro studies of this pathway, erythroleukemic SKT6 cell sublines have been isolated which stably and efficiently hemoglobinize in response to Epo. Epo rapidly activated Jak2, STAT5 and detectably STATs 1 and 3, while no effects on GATA-1, EKLF or STAT5 expression were observed. Finally, efficient hemoglobinization of SKT6 cells was shown to be mediated by chimeric receptors comprised of the EGF receptor extracellular domain and truncated cytoplasmic subdomains of either the Epo receptor or the prolactin Nb2 receptor. This work further establishes SKT6 cells as an important model for studies of Epo-stimulated differentiation, and shows that this signaling pathway is promoted by a limited set of membrane-proximal receptor domains and effectors.

Erythropoietin (EPO) plays a central role in the regulation of red blood cell (RBC) production. Since iron is an essential element for erythropoiesis and hemoglobin (Hb) synthesis, its importance is heightened in patients treated with epoetin alfa. Stimulation of erythropoiesis following the administration of epoetin alfa is associated with several changes in iron metabolism; indeed, plasma ferritin levels fall as a result of increased utilization of iron by the expanding erythroid marrow. The administration of epoetin alfa can therefore lead to a state of relative iron deficiency. Thus, iron supplementation is essential to maximize the effect of epoetin alfa-induced erythropoiesis.

To elucidate the role of phosphatidylinositol-3-kinase (Pl3K) in erythropoietin receptor (EPOR)-mediated signaling, we examined the effects of wortmannin (WT), a specific inhibitor of Pl3K on the proliferation of erythropoietin (EPO)-induced erythroid differentiation in K562 human erythroleukemia cells. Percentage of benzidine-positive cells synthesizing hemoglobin and level of glycophorin A expression in the cells were increased after EPO treatment. EPO-enhanced Pl3K activity was suppressed by WT treatment in a dose-dependent manner and constant inhibition of Pl3K by WT interfered with both hemoglobin synthesis and glycophorin A expression promoted by EPO. Wortmannin, however, did not inhibit hemin-induced erythroid differentiation. These findings in the present study suggest that Pl3K plays a crucial role in the transducing the erythroid differentiation signal through EPOR activated by EPO-binding ib K562 cells and that the signaling pathways involved in EPO-induced erythroid differentiation differ from those involved in hemin-induced differentiation.

A study was carried out to determine whether recombinant human erythropoietin can induce newborn-like hemoglobin synthesis in adult rats. A fixed dose of recombinant erythropoietin was administered each time intravenously in each rat for altogether 5 weeks. Blood samples drawn at 7-day intervals were analyzed by DEAE-cellulose chromatography. Hematological parameters like red blood cell counts, hematocrit values and reticulocyte counts were evaluated and compared. A significant changing pattern for certain hemoglobin components in red cells of erythropoietin-treated rats was measured compared to their baseline values. However, aspirin (a prostaglandin synthesis inhibitor) intake along with recombinant erythropoietin administration totally abolished the reversion of hemoglobin proportions toward newborn values, but not the increase in hemoglobin synthesis. These data reveal that concurrent prostaglandin synthesis is needed for reversing hemoglobin proportions in adult rats, but not for hemoglobin synthesis per se.

In order to elucidate the role of c-myb gene in erythroid differentiation of K562 cell induced by hemin (Hm) and erythropoietin (Epo), we constructed recombinant plasmid that could produce antisense myb RNA after induction with dexamethasone. During treatment with Hm, K562 cells constitutively expressed c-myb mRNA, and 50% of them began to synthesize hemoglobin (Hb). Expression of antisense myb RNA reduced the amount of c-myb mRNA, and the percentage of Hb-synthesizing cells was decreased to 20%. In the presence of Epo, c-myb mRNA declined and 20% of K562 cells synthesized Hb regardless of antisense myb RNA expression. It is suggested that constitutive expression of c-myb mRNA is necessary for Hm-induced differentiation, and that a decrease in the amount of c-myb mRNA induced by antisense myb RNA expression suppresses Hm-induced differentiation. The amount of c-myb mRNA in K562 cells was reduced during the differentiation induced by Epo. Expression of GATA-1 mRNA was almost constant during Hm-induced differentiation, but increased during Epo treatment. It is supposed that the mechanism of Hm-induced differentiation is distinguished from that of Epo-induced differentiation in K562 cells.

Erythropoietin (EPO) is the primary regulator of day-to-day red blood cell production. Secreted by peritubular capillary lining cells in the kidney, EPO circulates in the plasma to interact with target cells in the bone marrow to maintain or stimulate erythropoiesis. The primary target of EPO action is the intermediate-stage erythroid burst-forming unit and the erythroid colony-forming unit (CFU-E). The CFU-E is estimated to have 300 to 400 high-affinity EPO receptors per cell and, in healthy individuals, is the cell with the highest number of receptors in the body. There is some controversy as to whether EPO provides a mitogenic signal to the CFU-E or, rather, prevents programmed cell death (apoptosis). Iron is an essential element for hemoglobin synthesis and its importance has been emphasized in individuals receiving recombinant human erythropoietin (rHuEPO). The administration of rHuEPO to patients with chronic renal failure has resulted in a number of changes in iron metabolism, including the reversal of iron overload as iron is mobilized from storage sites for hemoglobin synthesis. In addition, higher doses of rHuEPO create a state of functional (or relative) iron deficiency that is characterized by a low percent transferrin saturation in the face of adequate iron stores. The value of aggressive iron supplementation in patients receiving rHuEPO has been demonstrated in clinical trials of rHuEPO administration in individuals storing blood for autologous use at the time of surgery.

UT-7 is a human leukemic cell line capable of growing in interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo) (Komatsu et al, Cancer Res 51:341, 1991). To study the effect of Epo on proliferation and differentiation of UT-7, we maintained the UT-7 cell culture for more than 6 months in the presence of Epo. As a result, a subline, UT-7/Epo, was established. The growth of UT-7/Epo could be supported by Epo but not by GM-CSF or IL-3. UT-7/Epo showed a greater level of heme content and ratio of benzidine-positive staining cells than did UT-7. Butyric acid promoted the synthesis of hemoglobin in UT-7/Epo, but not UT-7. Further, the mRNA concentrations of the c-myb oncogene and GM-CSF receptor beta-subunit were decreased substantially in UT-7/Epo cells. These findings showed that UT-7/Epo cells had progressed further in erythroid development than UT-7 cells, and suggested that long-term culture in Epo had promoted this differentiation. Whereas availability of the Epo receptor (Epo-R) for binding of Epo was reduced in UT-7/Epo cells compared with UT-7 cells, the Epo-R showed a similar affinity for Epo. This observation suggested that change(s) in postreceptor signaling step might be involved in the establishment and maintenance of the UT-7/Epo phenotype.

The murine allele temperature-sensitive (ts) p53Val-135 encodes a ts p53 protein that behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. This ts allele was introduced into the p53 nonproducer Friend erythroleukemia cell line DP16-1. The DP16-1 cell line was derived from the spleen cells of a mouse infected with the polycythemia strain of Friend virus, and like other erythroleukemia cell lines transformed by this virus, it grows independently of erythropoietin, likely because of expression of the viral gp55 protein which binds to and activates the erythropoietin receptor. When incubated at 32 degrees C, DP16-1 cells expressing ts p53Val-135 protein, arrested in the G0/G1 phase of the cell cycle, rapidly lost viability and expressed hemoglobin, a marker of erythroid differentiation. Erythropoietin had a striking effect on p53Val-135-expressing cells at 32 degrees C by prolonging their survival and diminishing the extent of hemoglobin production. This response to erythropoietin was not accompanied by down-regulation of viral gp55 protein.

The fetal mouse liver (FL) is an organ of intense, but transient, hematopoietic activity during mid-gestation, with erythropoiesis being predominant during days 11 through 16. It therefore seemed reasonable to expect that hematopoietic cytokines, such as erythropoietin (epo), interleukin-3 (IL-3), and stem cell factor (SCF), may play important roles in maintaining a homeostatic balance of erythropoiesis and apoptosis in liver during ontogeny. First, we determined the effects of these growth factors on hematopoiesis by measuring colony formation and hemoglobin synthesis of cultured FLs. Secondly, we determined the protection from apoptosis afforded by these cytokines, using electrophoretic analysis of DNA and by flow cytometry of FL cells deprived in culture of epo, IL-3, and SCF. Erythropoietin was necessary and alone sufficient for hemoglobin synthesis in colony-forming units-erythroid colonies, but IL-3 was a required cofactor to obtain maximal development of burst-forming units-erythroid colonies. SCF alone caused little colony formation in methylcellulose cultures of FLs, but when combined with epo and IL-3, it had dramatic effects both on the number of colonies and their size. Secondly, indices of apoptosis were determined by measuring DNA fragmentation caused by endogenous nuclease activity in apoptotic cells. Liver cells from cultures without cytokines showed the extensive degradation of DNA to low molecular weight nucleosomal oligomers, which is characteristic of apoptosis. Protection from apoptosis afforded by epo directly corresponded to the level of erythropoiesis in FLs of different gestational age. Erythropoietin was by far the most critical cytokine in sparing FL cells from apoptosis. Analyses of agarose gels showed that SCF and IL-3 alone had no apparent effect in reducing the amount of DNA in fragments, and when combined with epo they had no more protective effect than that provided by epo alone. However, using the more sensitive flow cytometric determination of cells with subdiploid amounts of DNA, SCF, and IL-3 alone had measurable protective effects that were less than those caused by epo. Thus, we show that normal, untransformed cells of the developing hematopoietic system not only require cytokines for proliferation and differentiation, but they have an initial and absolute requirement of them for protection from apoptosis.

Erythropoietin (epo) induced differentiation of the J2E erythroid cell line is characterized by haemoglobin synthesis, together with morphological changes and an immediate increase in proliferation. In this manuscript we have shown that the size of J2E cells decreased during differentiation and the nucleus to cytoplasm ratio was reduced appreciably. Furthermore, major ultrastructural alterations occurred-mitochondria, rough endoplasmic reticulum and Golgi apparatus decreased in size and number with maturation, while nuclei condensed considerably before extrusion. The use of mitotic indices, 3H-thymidine uptakes and flow cytometry confirmed that the immature J2E cells undergo enhanced replication shortly after epo stimulation. In addition, we demonstrated that cell division ceased as the cells entered the final stages of erythroid differentiation. Thus the J2E line provides a useful model, not only for haemoglobin synthesis, but for all aspects of erythroid terminal differentiation.

J2E erythroid cells proliferate and differentiate in response to erythropoietin (epo), the red blood cell specific hormone. Using methylcellulose colony assays and suspension cultures we have demonstrated that nearly all the cells stimulated by epo synthesized haemoglobin. To achieve maximum production of haemoglobin J2E cells had to be treated with epo for only 6 h; hormone added subsequently did not enhance haemoglobin synthesis. Although virtually all viable J2E cells produced haemoglobin, the cells matured morphologically at different rates. Thus, upon exposure to epo J2E cells become committed to erythroid terminal differentiation but proceed in an asynchronous manner.

The J2E erythroid cell line terminally differentiates in response to recombinant human erythropoietin (rHu-epo). Here we demonstrate that retrovirally produced monkey erythropoietin (Zen-epo) is also capable of inducing the final maturation steps of J2E cells in a dose-dependant manner. The hormone committed J2E cells to a pathway of accelerated proliferation, haemoglobin synthesis and morphological changes including enucleation. However, only 6 h exposure to Zen-epo was required to initiate maximum haemoglobin synthesis 72 h later. Significantly, the synthesis of haemoglobin was achieved by concomitantly activating globin production, haem synthesis and iron uptake. Methylcellulose assays revealed that the vast majority of clonogenic cells were able to respond to Zen-epo. These data show that retrovirally manufactured erythropoietin is an effective stimulus for the erythropoietic development of J2E cells in vitro.

We have studied the response of hemoglobin (Hb), hematocrit, reticulocytes, mean corpuscular volume, ferritin, serum iron, total iron-binding capacity (TIBC) and the transferrin saturation index in 9 patients on chronic hemodialysis (HD) with minimal aluminum overload who were treated with recombinant human erythropoietin (rHuEPO) when a single dose of deferoxamine (DFO; 40 mg/kg b.w.) was administered. Analytical determinations were performed basally and 48 h, 7 days and 14 days after a DFO test. Hb increased from a basal value of 10 +/- 0.28 to 10.7 +/- 0.33 (p < 0.05), 10.4 +/- 0.33 (p < 0.05) and 10.1 +/- 0.31 g/dl (NS), respectively, and similar increases were seen with the hematocrit. Serum iron increased from 52 +/- 7.62 to 89.2 +/- 14.48 (p < 0.01), 94 +/- 18.73 (p < 0.01) and 85 +/- 14.01 micrograms/dl (p < 0.01), respectively. TIBC and ferritin did not change but the transferrin saturation index increased significantly. DFO produces an immediate improvement of the anemia in HD patients treated with rHuEPO and who have minimal aluminium accumulation: it should be related to an increased iron availability to erythroid precursors either releasing stored iron or decreasing aluminum-bound transferrin.

This study investigates which factors influence the response of administered recombinant human erythropoietin (Re-HuEPO) with respect to the increase of haemoglobin in patients with end-stage renal disease. Pharmacokinetic parameters of administered Re-HuEPO in patients with end-stage renal disease and considerable differences in the amount of Re-HuEPO required ("Re-HuEPO-need") to obtain an increase of haemoglobin, revealed a pattern of dose-dependent first-order elimination without significant interindividual differences between the patients. As variable immunological inhibitors of erythropoietin are also absent, the administered Re-HuEPO seems to be equally available to the erythron in the various patients. In vitro incubation experiments with bone marrow cells show that the sera from patients with end-stage renal disease contain inhibitors of the erythropoietin-induced stimulation of bone marrow cells. As the patients' sera differ with regard to the degree of inhibition of erythropoietin bioactivity, this inhibition may also be responsible for the interindividual differences in amount of erythropoietin required. Besides a reduced endogenous production of erythropoietin, these inhibitors of the bioactivity of erythropoietin may also contribute to the pathogenesis of anaemia in patients with chronic renal failure.

The J2E cell line is a novel erythroid cell line that differentiates in response to erythropoietin (Epo), the physiologic stimulus for erythropoiesis. After exposure to Epo, the cells synthesize hemoglobin, and we show here that this process is tightly linked to increases in cellular proliferation and DNA synthesis. The hormone-induced terminal differentiation also results in morphologic alterations and the accumulation of transcripts for alpha, beta maj, and beta min globins. c-myc messenger RNA levels increase rapidly after exposure to Epo and precede the increase in cell division, while c-myb undergoes a transient decrease. Differentiation of J2E cells can also be achieved with sodium butyrate, but, in contrast with Epo, this is associated with a retardation of replication and a sudden decrease in c-myc levels. These results show that, in this system, chemically induced differentiation differs from terminal maturation promoted by Epo and that the processes of proliferation and differentiation in J2E cells can be uncoupled.

The development of end-stage renal disease (ESRD) in patients with sickle-cell anaemia results in increased transfusion dependence, increasing the risk of iron overload. Correction of anaemia with recombinant human erythropoietin (rHuEpo) in dialysis patients might also result in stimulation of haemoglobin F production, which protects against sickling, although very high doses were required to achieve this effect in non-uraemic animals. rHuEpo was administered to three transfusion-dependent patients with ESRD and homozygous sickle-cell disease (initial dose 100 U/kg twice weekly, increasing to 125 U/kg at 6 weeks, and to 150 U/kg at 9 weeks in two patients). This resulted in reticulocytosis and increased circulating erythroid blast-forming units. Total haemoglobin was predominantly HbA (i.e. transfused blood) at the start of the study, reflecting transfusion dependence, but after 3 months' treatment was between 60 and 94% HbS. No sickling crises occurred. Haemoglobin F remained at less than 3% of total haemoglobin. One patient was withdrawn at 10 weeks with CAPD peritonitis. The other two patients completed 12 weeks' treatment without transfusion but final Hb concentrations were 4.5 and 5.5 g/dl. Whether larger doses of rHuEpo will be more successful in managing such patients remains unclear. No effect on HbF production can be expected.

We have established a new nonlymphoid leukemic cell ine from a patient with myelodysplastic syndrome (MDS), which progressed to overt leukemia. The parental cell line and a subline derived from this line have absolute dependency on several cytokines for their long-term survival and growth. The parental line designated F-36P requires granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) for continuous growth, while a subline designated F-36E can be maintained in the presence of erythropoietin (Epo) alone. When these cytokines are depleted, both the parental and the subline cells die within several days, even in medium supplemented with fetal calf serum (FCS). F-36E, maintained in the presence of Epo, constitutively synthesizes hemoglobin at a significant level. F-36P, which is usually maintained in the presence of GM-CSF or IL-3, can be induced to synthesize hemoglobin when GM-CSF or IL-3 is substituted by Epo. The surface marker profile shows that the F-36P cells are positive for the leukocyte common antigen (CD45) and some common multilineage markers such as CD13, CD33, and CD34, and negative for T- and B-cell antigens and mature myelomonocytic antigens. However, some monoclonal antibodies recognizing erythroid and platelet glycoproteins react with these cells. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in a multipotent stem cell. It is also evident that the F-36 cells can be induced to differentiate into the erythroid lineage in the presence of Epo. This, to our knowledge, is the first description of a human leukemic cell line that can be stimulated to synthesize hemoglobin by Epo.

Human K562 leukemia cells have been induced to differentiate along the erythroid lineage by aclacinomycin (ACM), an anthracyclic antitumor drug. During differentiation over 3 days in culture, the expression and the nature of erythropoietin (EPO) receptors have been analyzed using 125I-labeled bioactive recombinant human EPO. Aclacinomycin at 20 nM, the concentration inducing optimum differentiation, progressively increased EPO-specific binding. On day 3, EPO binding was nine-fold higher than that of the controls (1031 +/- 101 cpm/5 x 10(6) cells versus 112 +/- 15 cpm); with various concentrations of ACM, the increase in EPO binding appeared to parallel the recruitment of hemoglobin-producing cells. However, at 95% of growth inhibition, EPO binding remained constant while the percentage of differentiated cells decreased. Specific binding was reversible, saturable, and proportional to cell number; bound EPO was displaced by unlabeled EPO. Scatchard analysis of the equilibrium binding data suggested the existence of a single class of EPO receptors with an apparent Kd of 867 +/- 458 pM, corresponding to 400 +/- 142 receptors per cell. Affinity cross-linking of 125I-EPO using disuccinimidyl suberate followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions demonstrated that EPO was preferentially cross-linked to a protein of approximately 116 kD. Taken together, these results demonstrate that, in addition to cytostatic properties, antitumor drugs such as ACM can modulate the expression of differentiation factor receptors on the surface of leukemic cells.

Erythropoietin (Epo) and dimethyl sulfoxide (DMSO) are believed to induce the differentiation of transformed erythroid cells by different signal transduction pathways. We have now obtained evidence for the interaction of these pathways. We used a Rauscher murine erythroleukemia cell line with a relatively low (8% to 10%) hemoglobinization response to Epo alone. Pretreatment of these cells for 1 day with DMSO followed by its removal and the addition of Epo resulted in a marked enhancement of the Epo specific hemoglobinization. We have designated this effect "DMSO priming." This priming effect of DMSO on the Epo response was both time-dependent and DMSO concentration-dependent. DMSO priming potentiated the Epo response in three ways. Firstly, DMSO priming increased the total number of Epo responsive cells from 8% to 10% to 40% to 60%. Secondly, DMSO priming reduced the time required to reach the optimal Epo-induced response from 4 days to 2 days. Thirdly, the Epo dose-response curve was left-shifted approximately 20-fold. DMSO priming was also associated with a marked increase in Epo receptor density characterized by an apparently new receptor population and by the appearance of positive cooperativity between receptors. Our results suggest that the DMSO priming effect is due to potentiation of the Epo signaling pathway, thus resulting in a much more rapid and dramatic Epo-induced hemoglobinization response.

The recently derived J2E cell lines differentiate in response to erythropoietin. However, during the course of growth in culture, a subclone has emerged which fails to synthesize hemoglobin or show accelerated proliferation following hormonal stimulation. The inability to respond to erythropoietin is not due to autocrine production of the hormone by these cells. Moreover, it appears unlikely that the block occurs at the level of the erythropoietin receptor as the number of receptors per cell was only marginally reduced and both ligand binding and internalization were normal. It is likely, therefore, that the intracellular signal transduction mechanism is faulty or a defect in hemoglobin production has developed.

Two main aspects of the in vitro mouse foetal liver cell assay for Erythroid Stimulating Factor (ESF) in human sera have been investigated. The haem extraction process has been shown to give specific and quantitative recovery of 59Fe labelled haem from haemoglobin thus confirming that the material assayed in human sera is stimulating the synthesis of this protein. The extraction procedure can be simplified considerably by prior mixing of the reagents without significantly influencing the results. Several serum constituents (citrate, testosterone, B12, folic acid and iron) have been investigated over a range of concentrations for possible effects on the cultures. Generally only small effects on haem synthesis were observed. It is concluded that variations in the levels of these factors in sera from treated patients will not produce any significant alterations in the estimated ESF concentrations.

The stimulatory effects of crude (148 U/mg protein) and purified (15,920 U/mg protein) human urinary erythropoietin (Ep) on heme and globin synthesis of rat bone marrow cells in short term (2-3 day) suspension culture were compared. Freshly isolated, unfractionated marrow cells responded equally well to both Ep preparations. Velocity sedimentation at unit gravity was used to separate the marrow cells into a fast-moving (population I) and a slow-moving (population II) fraction. Of the 2 preparations tested, only crude Ep was found to stimulate heme and globin synthesis of population I cells. Population II cells were unresponsive to both Ep preparations. Preincubation of the rat marrow cells in the absence of Ep for 48 h resulted in the disappearance of most of population II, the remaining cells only responded to the crude Ep preparation. Direct remixing experiments involving various proportions of population I and II cells also showed that addition of the purified Ep preparation could only bring about an increase in hemoglobin synthesis in those cultures which contained a sufficient number of population II cells. Cultures deficient in population II cells to which the crude Ep preparation was added did not show this requirement, suggesting that this preparation contained a component which could substitute for the presence of population II cells. This demonstrates that the action of Ep and of a second factor (or of a cell to cell signal) is required to increase hemoglobin synthesis in short term rat marrow cell cultures.

The erythropoietin (epo)-dependent burst forming activities (BFA) of foetal calf serum (FCS), mouse bone marrow cells (BFA-cell) and lectin-stimulated mouse spleen conditioned medium (BFA-mscm) were investigated. Burst enhancement by BFA-mscm was independent of FCS concentration above 5% FCS. The activity of BFA-cell was directly proportional to FCS concentration. There was a high correlation for the relationship between the logarithm of total cell concentration and the logarithm of burst number for each concentration of FCS. Burst formation in the presence of BFA-cell showed a high epo requirement whereas plateau numbers of bursts were evident at concentrations of 1 to 2 units of epo/ml in cultures containing BFA-mscm. The epo-independent developmental activities (EIDA) of BFA-cell and BFA-mscm were examined in methylcellulose cultures to which the addition of epo was delayed for up to four days. Under all conditions examined, the number of bursts formed in the presence of BFA-cell declined with increasing delay of epo addition. Studies on the kinetics of haemoglobin synthesis in such experiments demonstrated that this decline in colony number was associated with a delay in haemoglobin synthesis. Burst formation in cultures containing various levels of BFA-mscm was independent of the time of epo addition. These results indicate that the burst enhancing ability of BFA-mscm is directly related to its EIDA whereas bone marrow cells appear to lack this activity.