Erythropoietic Factor Research Articles

Clinical evaluation and comparison of erythropoietic growth factors for chemotherapy-induced anemia (CIA)

Background CIA is a significant problem in> 50% of cancer pts, impacting quality of life. Our objective was to evaluate & compare the use of epoetin (E) & darbepoetin (D) for CIA at our institution. Methods In this observational study, demographics, doses, hemoglobin (Hgb), transfusions, & chemotherapy use were followed× 12 wks in 108 adults (54 D, 54 E). By an intent to treat analysis, successful clinical response was defined as a Hgb (g/dL) increase≥ 2 or Hgb ≥12 at wk 12. Practice patterns were compared to clinical guidelines. Results The usual starting dose was 200 mcg QOW for D & 40K units Q7D for E. Demographics were similar between groups: mean (SD) age 52(1.4) yr, 57.8% female, 65.6% had solid tumors. Mean (SD) baseline Hgb was 10.2 (1.4) for D, & 10.0 (1.3) for E (p=0.3). At 12 wks, mean (SD) Hgb increase was: D, 1.1 (1.9); E, 0.8 (1.7), p=0.7. In all pts, the mean (SD) 12 wk Hgb was 11.0 (1.5); D, 11.4 (1.6); E, 10.7 (1.4) (p=0.04). The overall clinical response rate at the end of therapy was 38.9% (42.6% D, 35.2% E, p= 0.4). No dose changes occurred with D; 8 E pts were dose escalated. Therapy was rarely discontinued in non-responders. Conclusions The effectiveness of erythropoietic growth factors in clinical practice was lower than reported in clinical trials, due in part to underutilization of clinical guidelines & dose escalations. Both D & E provided equivalent responses in our population. Due to high cost of therapy, future plans are to implement a prospective monitoring program for use of these agents in our institution. Clinical Pharmacology & Therapeutics (2005) 77, P17–P17; doi: 10.1016/j.clpt.2004.11.068

Lyn Kinase Supports Primary Erythroid Progenitor Cell Development.

Several lines of investigation have implicated Lyn as an important positive effector of red cell development: Deregulated Src kinases promote erythroleukemia, and Lyn is the predominant Src kinase of erythroid cells; as critical erythropoietic factors, Kit and the Epo receptor each stimulate Lyn kinase; and in an insightful set of investigations in J2E cells, Lyn has been shown to be required for Epo-dependent late erythroid development. Based on these considerations, adult bone marrow-derived primary erythroid progenitor cells from Lyn −/− mice presently were assessed for their ex vivo growth, survival and differentiation potentials. Lyn −/− erythroid progenitors expanded efficiently in serum-free media, and showed essentially wild-type Epo dose-dependent proliferative responsiveness. When transferred to BSA/insulin/transferrin differentiation medium, however, Lyn −/− erythroid progenitor cells clearly faltered in their development to Ter119-high, CD71-low erythroblasts. For these Lyn −/− cells, annexin-V binding studies revealed that this defect was associated, in part, with a stage-specific loss in survival potential. Interestingly, however, this defect was not Epo-dose dependent. In addition, MACS-isolated Kit-positive early erythroid progenitor cells prepared from Lyn −/− mice (unlike preparations from wild-type mice) failed to support synergistic effects of SCF-plus-Epo in 3HdT incorporation assays. In response to phenylhydrazine, Lyn −/− mice exhibited expanded erythroid progenitor cell pools (including BFUe and CFUe), and this hyper-expansion may occur in response to the compromised survival of late Lyn −/− erythroblasts. Analyses of pp60-Src expression revealed elevated levels of activated PY416-Src specifically in Lyn −/− EPC, a finding that is consistent with the activation of apparent compensatory mechanisms. In contrast, no significant changes in the levels of GATA1 or other assessed erythroid defining factors were detected. In response to phenylhydrazine, Lyn −/− mice showed ≥2-fold enhanced splenomegaly, as well as enhanced frequencies of BFUe, CFUe and Ter119(+) cells. Overall, these studies in primary erythroid progenitor cells from Lyn −/− mice reveal a previously undiscovered positive role for Lyn as a late-stage specific positive effector of erythroid cell survival, and regulator of Epo receptor and Kit co-signaling.

Darbepoetin alfa: a new approach to the treatment of chemotherapy-induced anaemia

Chemotherapy-induced anaemia has important consequences on the quality of life and social function of cancer patients. The finding of erythropoietin (EPO) deficiency in these patients led to the therapeutic development of erythropoietic proteins. Darbepoetin alfa (Aranesp®, Amgen Inc, Thousand Oaks, California), a new erythropoietic growth factor, has eight more sialic acids than epoetin alfa. The increased sialic acid content confers a three-fold longer half-life and allows the drug to be administered less frequently than epoetin alfa. Darbepoetin alfa affects the same early haematopoietic cells as epoetin alfa and the endogenous hormone EPO. Preclinical pharmacokinetic studies suggest that the intrinsic pharmacological properties of darbepoetin alfa are comparable to those of epoetin alfa, but that the increased sialic acid content allows for less-frequent administration with superior performance. Darbepoetin alfa has been shown to have safe clinical efficacy in a variety of tumour settings and with several types of chemotherapy.

Posttransplant erythrocytosis

Posttransplant erythrocytosis (PTE) is defined as a persistently elevated hematocrit to a level greater than 51% after renal transplantation. It occurs in 10% to 15% of graft recipients and usually develops 8 to 24 months after engraftment. Spontaneous remission of established PTE is observed in one fourth of the patients within 2 years from onset, whereas in the remaining three fourths it persists for several years, only to remit after loss of renal function from rejection. Predisposing factors include male gender, retention of native kidneys, smoking, transplant renal artery stenosis, adequate erythropoiesis prior to transplantation, and rejection-free course with well-functioning renal graft. Just as in other forms of erythrocytosis, a substantial number (approximately 60%) of patients with PTE experience malaise, headache, plethora, lethargy, and dizziness. Thromboembolic events occur in 10% to 30% of the cases; 1% to 2% eventually die of associated complications. Posttransplant erythrocytosis results from the combined trophic effect of multiple and interrelated erythropoietic factors. Among them, endogenous erythropoietin appears to play the central role. Persistent erythropoietin secretion from the diseased and chronically ischemic native kidneys does not conform to the normal feedback regulation, thereby establishing a form of "tertiary hypererythropoietinemia." However, erythropoietin levels in most PTE patients still remain within the "normal range," indicating that erythrocytosis finally ensues by the contributory action of additional growth factors on erythroid progenitors, such as angiotensin II, androgens, and insulin-like growth factor 1 (IGF-1). Inactivation of the renin-angiotensin system (RAS) by an angiotensin-converting enzyme (ACE) inhibitor, or an angiotensin II type 1 AT1 receptor blocker represents the most effective, safe, and well-tolerated therapeutic modality.

Biological Significance of the Different Erythropoietic Factors Secreted by Normal Human Early Erythroid Cells

Evidence is accumulating that autocrine/paracrine regulatory mechanisms play an important role in regulating normal hematopoiesis. To support this, various growth factors, cytokines and chemokines are expressed and secreted by normal early and differentiated hematopoietic cells. In this review, we summarize recent advances in the identification and understanding of the role of autocrine/paracrine axes in normal human erythropoiesis. We will also address a biological significance of the secretion of (i) metalloproteinases which in addition to growth factors and cytokines are secreted by normal erythroid cells and (ii) membrane-derived microvesicles (MV), that are shed from the surface of maturating erythroblasts/reticulocytes, and as we postulate may also play a role in intercellular communication. We hypothesize that all these factors together play an important role in a crosstalk between erythroid cells and their environment. A better understanding of intercellular crosstalk operating in normal erythropoiesis and of the mechanisms regulating synthesis of these endogenously produced factors may allow us to develop more efficient therapeutic strategies to treat various erythropoietic disorders.

Biological significance of MAPK, AKT and JAK-STAT protein activation by various erythropoietic factors in normal human early erythroid cells.

The aim of this study was to identify signal transduction pathways activated by erythropoietin (EpO) and erythropoietin co-stimulatory factors (kit ligand), insulin-like growth factor, thrombopoietin, interleukin 3 and granulocyte-macrophage colony-stimulating factor) in normal human bone marrow CD34(+) cells and d 11 erythroid burst forming unit derived glycophorin+ cells. The activation of these signal transduction pathways was further correlated with various biological effects such as (i) cell proliferation, (ii) inhibition of apoptosis, (iii) activation of adhesion and (iv) secretion of the matrix metalloproteinases (MMPs) MMP-9 and MMP-2, and vascular endothelial growth factor (VEGF). We found that in human CD34(+) cells and erythroblasts erythropoietic factors may activate similar but different signalling pathways, and that activation of each of the JAK-STAT, MAPK p42/44 or PI-3K-AKT axes alone is not sufficient either to stimulate cell proliferation or inhibit apoptosis, suggesting that these processes are regulated by orchestrated activation of multiple signalling cascades. Accordingly, we found that although cell proliferation was more related to simultaneous activation of JAK-STAT and MAPK p42/44, the effect on cell survival correlated with activation of PI-3K-AKT, MAPK p42/44 and JAK-STAT proteins. We also demonstrated that differentiating normal human erythroid cells lose their adhesive properties and secrete angiopoietic factors such as MMP-9, MMP-2 and VEGF, and we postulate that this secretion by early erythroid cells may play a role in their maturation and egress from the haematopoietic niches of the bone marrow.

Relationship between routine hematological parameters, serum IL-3, IL-6 and erythropoietin and mild anemia and degree of function in the elderly.

To investigate the influence of functioning on unexplained senile anemia, we measured commonly used hematological parameters (serum iron, transferrin, iron saturation and ferritin) in addition to specific erythropoietic factors, such as interleukin-3 (IL-3), interleukin-6 (IL-6), and erythropoietin (EPO) in 48 elderly subjects aged 65-90 years. The subjects were divided into 3 groups: 1) 17 patients with unexplained mild anemia; 2) 17 non-anemic patients with newly acquired stroke and who previously were functionally active; 3) 14 functionally active patients with no major disease who served as controls. Anemia was defined as hemoglobin (Hb) values under 12.0 g/dL. The degree of functional ability was defined and scored by the "functional independence measure" (FIM) test. Data are presented as mean values +/- SD. The results revealed a correlation between the functional state and levels of Hb, iron and transferrin with unchanged iron saturation. Patients in the mild anemia group were found to be functionally declined (FIM = 57 +/- 19.4) with the relatively lowest mean iron (75.1 +/- 17 micrograms/dL) and transferrin levels (243 +/- 42.6 micrograms/dL). The stroke group (FIM = 62 +/- 17.7) had intermediate levels of iron (85.4 +/- 20.3 micrograms/dL) and transferrin (245 +/- 45.2), and with the continuation of the declined functional state the Hb level decreased significantly (13.7 +/- 0.9 to 12.0 +/- 1.0 g/dL, p < 0.001). The highest mean values of iron (102 +/- 27.9 micrograms/dL) and transferrin (322 +/- 42.7 micrograms/dL) were found in the control group (FIM = 122.7 +/- 5.8). The ferritin levels showed an opposite trend. IL-3 values were undetectable in the anemic and control groups, and were elevated in some patients in the stroke group. The lowest IL-6 level was observed in the anemic group, and the highest in the control group. Serial IL-6 assays in the stroke group showed an upward trend. Erythropoietin levels in all groups showed no difference.

Erythropoiesis after withdrawal of enalapril in post-transplant erythrocytosis

Enalapril effectively decreases hematocrits in patients with postrenal transplant erythrocytosis (PTE). We studied the effect of enalapril withdrawal on erythropoiesis in 18 patients with PTE who had been treated for 13 +/- 8 months. Hematocrit, reticulocyte count, plasma erythropoietin, and plasma insulin-like growth factor I were measured biweekly for six weeks. Red cell mass, plasma volume, transferrin saturation, and plasma angiotensin II were measured at withdrawal and six weeks later. Hematocrit increased by at least 0.04 in 13 patients ('responders') but changed by -0.08 to 0.01 in five patients ('nonresponders'). In the responder subgroup, hematocrit increased from 0.43 +/- 0.05 to 0.51 +/- 0.05 (P < 0.001), red cell mass increased from 25.4 +/- 5.9 to 28.9 +/- 5.9 ml/kg body weight (P < 0.001), and transferrin saturation decreased from 41 +/- 16 to 27 +/- 9 percent (P < 0.01). Reticulocyte count increased two weeks after withdrawal of enalapril. Plasma volume did not change significantly. No measurement changed in the nonresponder subgroup. Plasma levels of erythropoietin, total erythroid stimulating activity, insulin-like growth factor I, and angiotensin II did not change significantly in either subgroup. Enalaprilat did not inhibit erythropoiesis in cell culture. Thus, erythropoiesis increased in 13 of 18 patients after stopping enalapril and was independent of changes in circulating concentrations of several erythropoietic factors, including erythropoietin. The pathogenesis of PTE and mechanism underlying the beneficial effect of angiotensin converting enzyme inhibition remain undetermined.

THE N-TERMINAL SEQUENCE OF THE MAJOR ERYTHROPOIETIC FACTOR OF AN ANEPHRIC PATIENT IS IDENTICAL TO INSULIN-LIKE GROWTH FACTOR I.

The erythropoietic factors present in an anephric patient with nearly normal hematocrit were isolated from plasma by reversed-phase and gel permeation HPLC. The most active fraction was purified and the analysis of its N-terminal sequence was identical to the published sequence of the human insulin-like growth factor I (IGF I). Recombinant human IGF I had identical elution positions as the isolated erythropoietic factor on reversed-phase HPLC and the same molecular weight on gel permeation HPLC. Furthermore, hrIGF I stimulated erythroid colony formation in human bone marrow cultures as was previously shown for the isolated human erythropoietic factor. These results suggest that IGF I may replace erythropoietin as a stimulator of erythropoiesis in some patients with anemia and renal failure.

Effects of burst-promoting activity and erythropoietin from human urine on erythroid burst forming units in human peripheral blood.

Erythropoietin (Ep) was isolated from the urine of patients with aplastic anemia [Yanagawa et al, J. Biol. Chem., 259, 2707 (1984)] and burst-promoting activity (BPA) was extensively purified from the residue obtained after removal of Ep. These erythropoietic factors were studied for their effcects on erythroid burst-colony formation of human peripheral blood mononuclear cells in methylcellulose cultures. Reddish bursts were formed with the addition of Ep alone. Addition of BPA not only elevated the number of bursts but also greatly reduced the amount of Ep required for burst formation. The presence of BPA alone in cultures did not permit bursts to form but did permit the growth of small colonies that did not contain hemoglobin (Hb). Addition of Ep to these small colonies led to the formation of erythroid bursts. Administration of Ep to the cultures could be delayed for 6 days without decreasing the number of bursts if the cultures were initiated in the presence of BPA; in the absence of BPA, the erythroid precursors (BUF-E) were rapidly lost if Ep was not provided at the start of the cultures. BPA produced larger bursts than those formed in the presence of Ep alone. Microassays of Hb in the bursts indicated that BPA increased the amonut of Hb per burst. This increase could not be entirely explained by the augumentation in cell number per burst but was partly ascribable to the increased amount of Hb per cell.

L-triiodothyronine augments erythropoietic growth factor release from peripheral blood and bone marrow leukocytes

To investigate cellular mechanisms involved in thyroid hormone stimulation of erythropoiesis, we studied the response of erythroid burst-forming unit (BFU-E) proliferation to L-triiodothyronine (L-T3) in a serum-free culture system. When added directly to culture, L-T3 stimulates erythroid burst formation by normal human bone marrow cells. In contrast, granulocyte-macrophage colony formation is unaffected. Enhancement of erythroid burst formation by L-T3 required the presence of nylon wool adherent and/or B-4 antigen-positive light-density marrow populations. Addition of other erythropoietic factors including platelet-derived growth factor and insulinlike growth factor II did not abrogate this apparent cellular requirement. Pulse exposure of marrow and peripheral blood mononuclear cells (greater than 95% lymphocytes) to L-T3 accelerates the release of a soluble factor that augments BFU-E proliferation into serum-free liquid culture medium. Time-course studies show that this factor appears in conditioned medium (CM) coincidentally with erythroid burst-promoting activity (BPA). Furthermore, incubation of CM with an antibody known to react with and adsorb BPA from solution removes the inducible mitogen. Biochemical analysis of CM prepared from unexposed and L-T3 pulse-exposed cells indicates that the rate of protein appearance is accelerated by L-T3 in a fashion that immediately precedes growth factor release and that several polypeptides are quantitatively increased. We conclude that unlike erythropoietin, which is mitogenic for progenitor cells directly, L-T3 enhances BFU-E proliferation indirectly by augmenting the release of soluble BPA-like molecules from accessory cells in culture.

L-Triiodothyronine Augments Erythropoietic Growth Factor Release From Peripheral Blood and Bone Marrow Leukocytes

To investigate cellular mechanisms involved in thyroid hormone stimulation of erythropoiesis, we studied the response of erythroid burst-forming unit (BFU-E) proliferation to L-triiodothyronine (L-T3) in a serum-free culture system. When added directly to culture, L-T3 stimulates erythroid burst formation by normal human bone marrow cells. In contrast, granulocyte-macrophage colony formation is unaffected. Enhancement of erythroid burst formation by L-T3 required the presence of nylon wool adherent and/or B-4 antigen-positive light-density marrow populations. Addition of other erythropoietic factors including platelet-derived growth factor and insulinlike growth factor II did not abrogate this apparent cellular requirement. Pulse exposure of marrow and peripheral blood mononuclear cells (greater than 95% lymphocytes) to L-T3 accelerates the release of a soluble factor that augments BFU-E proliferation into serum-free liquid culture medium. Time-course studies show that this factor appears in conditioned medium (CM) coincidentally with erythroid burst-promoting activity (BPA). Furthermore, incubation of CM with an antibody known to react with and adsorb BPA from solution removes the inducible mitogen. Biochemical analysis of CM prepared from unexposed and L-T3 pulse-exposed cells indicates that the rate of protein appearance is accelerated by L-T3 in a fashion that immediately precedes growth factor release and that several polypeptides are quantitatively increased. We conclude that unlike erythropoietin, which is mitogenic for progenitor cells directly, L-T3 enhances BFU-E proliferation indirectly by augmenting the release of soluble BPA-like molecules from accessory cells in culture.

Erythropoietin, an autocrine regulator? Serum-free production of erythropoietin by cloned erythroid cell lines

Production of lymphoid and myeloid growth regulatory factors by hematopoietic cells is well documented. On the other hand, the major site of production of erythropoietin (Epo), which regulates physiologic red blood cell development, is thought to be the kidney. Here we report the isolation of multiple erythroleukemia cell lines that produce erythropoietic factors and present extensive biological, immunologic, and biochemical evidence to document that the active agent is Epo. The erythropoietic activity was neutralized by Epo antiserum and exhibited physical properties indistinguishable from those of human and sheep Epo. Positive lines produced between 0.1 and 1.5 U/mL of Epo, which stimulated erythropoiesis in vivo and in vitro in nine biological assays. Twenty sublines derived from single cells were inducible for hemoglobin and spectrin synthesis. All the sublines produced Epo. Production of the hormone continued when the cells were seeded in the absence of serum. Our finding that multiple independent isolates produce Epo raises the possibility that Epo production by erythroid precursors may play a role in normal erythropoiesis or, alternatively, that Epo gene activation may be a relatively common occurrence that contributes to, or is associated with, certain forms of virus-induced leukemias.

Erythropoietin, an Autocrine Regulator? Serum-Free Production of Erythropoietin by Cloned Erythroid Cell Lines

Production of lymphoid and myeloid growth regulatory factors by hematopoietic cells is well documented. On the other hand, the major site of production of erythropoietin (Epo), which regulates physiologic red blood cell development, is thought to be the kidney. Here we report the isolation of multiple erythroleukemia cell lines that produce erythropoietic factors and present extensive biological, immunologic, and biochemical evidence to document that the active agent is Epo. The erythropoietic activity was neutralized by Epo antiserum and exhibited physical properties indistinguishable from those of human and sheep Epo. Positive lines produced between 0.1 and 1.5 U/mL of Epo, which stimulated erythropoiesis in vivo and in vitro in nine biological assays. Twenty sublines derived from single cells were inducible for hemoglobin and spectrin synthesis. All the sublines produced Epo. Production of the hormone continued when the cells were seeded in the absence of serum. Our finding that multiple independent isolates produce Epo raises the possibility that Epo production by erythroid precursors may play a role in normal erythropoiesis or, alternatively, that Epo gene activation may be a relatively common occurrence that contributes to, or is associated with, certain forms of virus-induced leukemias.

Erythropoietin research, 80 years after the initial studies by Carnot and Deflandre

The discovery and present state of knowledge of the specific hormonal mechanism controlling erythropoiesis are briefly reviewed. 80 year ago, Carnot and Deflandre postulated the existence of a humoral erythropoietic factor (‘hémopoïétine‘) produced in response to anemia. The general approval of this concept required almost 50 years, when the factor was recognized to be a glycoprotein hormone and termed ‘erythropoietin’. Very recently, human erythropoietin has been purified and its amino acid sequence been identified. The hormone is mainly of renal origins. Its production is stimulated by tissue hypoxia of different causes. The mechanism of O 2 sensing and the exact site of erythropoietin synthesis in the kidney still remain to be clarified.

Immunological comparison of renin substrate with erythropoietin.

Immunological similarities between human renin substrate and erythropoietin were studied. Purified renin substrate was coupled to erythrocytes, and haemagglutination was demonstrated both with antisera against renin substrate and erythropoietin. Extensive purification of renin substrate was performed on fast protein liquid chromatography (FPLC), and fractions were collected and assayed for renin substrate and erythropoietin. Following FPLC, the major protein peak corresponded to renin substrate, whereas erythropoietin immunoactivity dissociated from this peak. Iodinated renin substrate and erythropoietin-containing fractions showed specific binding to both renin substrate and erythropoietin antisera. The results suggest that human erythropoietin or erythropoietin-like material may co-purify with renin substrate, and that preparative procedures like FPLC should be included to obtain pure angiotensinogen preparations. While renin substrate and erythropoietin, judging from the present results and cloned DNA sequences, appear completely unrelated, the possible relationship between renin substrate and other erythropoietic factors remains unsettled.

Evidence for a hepatic-renal antagonism in the production of hepatic erythropoietin in a phenylhydrazine-induced compensated hemolytic state in the rat.

A compensated hemolytic state was induced in rats by injection of phenylhydrazine (PHZ) over a 6-week period. The liver and kidney were perfused to determine the levels and time of appearance of a hepatic erythropoietic factor (HEF) which induces the production of hepatic erythropoietin (Ep) and its antagonist, a renal inhibitory factor (RIF). Erythropoietin assays on the perfusates have been previously reported. The amount of HEF in perfusates recovered from the livers of the PHZ-treated rats was significantly higher during the 4th to 5th weeks of treatment, coinciding with the time of increased liver production of Ep. During the 6th week of PHZ treatment, the titer of RIF in perfusates recovered from the kidney was markedly increased and the HEF titer was decreased to near the control level, suggesting inhibitory action of RIF on HEF synthesis and/or effect. These findings indicate that an HEF/RIF control mechanism regulates hepatic Ep production in this compensated hemolytic condition.

Characterization of erythropoietin produced by IW32 murine erythroleukemia cells

IW32 is a recently described murine erythroleukemia cell line that produces an erythropoietic factor similar to erythropoietin by in vivo and in vitro bioassays and without species specificity. Biochemical characteristics of IW32 erythropoietic factor and sheep or mouse plasma erythropoietins were compared. Murine colonies derived from erythroid colony-forming units (CFU-E) in plasma clot culture were used as the bioassay system. Both IW32 erythropoietic factor and sheep plasma erythropoietin were stable in the pH range of 3 to 10, after exposure to denaturing agents (8 mol/L urea, 4 mol/L guanidine hydrochloride, 1% sodium dodecyl sulfate), to a reducing agent (0.1 mol/L 2- mercaptoethanol) and to an oxidizing agent (5 mmol/L sodium metaperiodate). Only the combination of 0.1 mol/L 2-mercaptoethanol and 1% sodium dodecyl sulfate resulted in a significant loss of activity. IW32 erythropoietic factor and murine plasma erythropoietin were similarly precipitated by ethanol and ammonium sulfate. IW32 erythropoietic factor eluted as a single major peak after gel exclusion chromatography, with an estimated molecular weight of 45,000 daltons. Results were identical using supernatants from cultures in the presence of and absence of fetal calf serum. The supernatant of IW32 cells cultured without serum induced erythroid colonies after seven days on normal human bone marrow nonadherent mononuclear cells cultured in serum-free conditions. All these results made it very likely that IW32 cells produce an authentic erythropoietin. This cell line would be very useful for the study of murine erythropoietin.

Characterization of Erythropoietin Produced by IW32 Murine Erythroleukemia Cells

IW32 is a recently described murine erythroleukemia cell line that produces an erythropoietic factor similar to erythropoietin by in vivo and in vitro bioassays and without species specificity. Biochemical characteristics of IW32 erythropoietic factor and sheep or mouse plasma erythropoietins were compared. Murine colonies derived from erythroid colony-forming units (CFU-E) in plasma clot culture were used as the bioassay system. Both IW32 erythropoietic factor and sheep plasma erythropoietin were stable in the pH range of 3 to 10, after exposure to denaturing agents (8 mol/L urea, 4 mol/L guanidine hydrochloride, 1% sodium dodecyl sulfate), to a reducing agent (0.1 mol/L 2-mercaptoethanol) and to an oxidizing agent (5 mmol/L sodium metaperiodate). Only the combination of 0.1 mol/L 2-mercaptoethanol and 1% sodium dodecyl sulfate resulted in a significant loss of activity. IW32 erythropoietic factor and murine plasma erythropoietin were similarly precipitated by ethanol and ammonium sulfate. IW32 erythropoietic factor eluted as a single major peak after gel exclusion chromatography, with an estimated molecular weight of 45,000 daltons. Results were identical using supernatants from cultures in the presence of and absence of fetal calf serum. The supernatant of IW32 cells cultured without serum induced erythroid colonies after seven days on normal human bone marrow nonadherent mononuclear cells cultured in serum-free conditions. All these results made it very likely that IW32 cells produce an authentic erythropoietin. This cell line would be very useful for the study of murine erythropoietin.

Erythroid colony formation in primary proliferative polycythaemia, idiopathic erythrocytosis and secondary polycythaemia: sensitivity to erythropoietic stimulating factors.

This is a study of in vitro production of erythropoietic colonies from peripheral blood and bone marrow of normal subjects and patients with different polycythaemic conditions. Proliferative stimuli included: 1. Fetal calf serum (FCS) only. 2. FCS plus a source of erythropoietic-stimulating activity (ESA). 3. FCS + ESA + erythropoietin (Ep). It was found that normal subjects and patients with secondary polycythaemia (SP) exhibited full colony growth only in the presence of both ESA and Ep, while patients with primary proliferative polycythaemia (PPP) showed colony production with FCS alone, further enhanced in the presence of ESA and Ep. A group of patients with idiopathic erythrocytosis (IE), namely with an increase of red cell mass not accompanied by other signs of myeloproliferative disorder, and without underlying cause, showed a heterogeneous response to ESA which in some patients was significantly greater than in normal subjects or in SP patients. It appears therefore that sensitivity of erythropoietic colony formation to Ep and ESA may be helpful in differentiating among various forms of polycythaemia; this study also establishes the heterogenicity of the IE group.