Erythropoiesis in vitro. Role of calcium.

The in vitro plasma clot technique was employed to examine the role of calcium during the interaction of erythropoietin and mouse erythroid progenitor cells. Erythropoietin-induced erythroid colony formation was increased 24% by the carboxylic ionophore A23187 (10 nM), whereas a 35% increase was produced by the carboxylic ionophore Ro 2-2985/1 (1 nM). EGTA (3 mM) inhibited erythropoietin-induced erythroid colony formation. Inhibition of erythroid colony formation by EGTA could be reversed by Ca2+, but not by Mn2+, Mg2+, Zn2+, or Fe2+. At least 30 min exposure of marrow cells to erythropoietin in vitro was required for production of erythroid colonies. EGTA substantially inhibited erythropoietin-induced erythroid colony formation even when the marrow cells were exposed to the hormone for up to 2 h before addition of the chelator. Marrow cells incubated first in calcium-free medium with erythropoietin and then cultured in the presence of calcium but not erythropoietin, failed to form erythroid colonies although colony formation occurred when erythropoietin was provided. Taken together, the data indicate that calcium is required for both extracellular and intracellular events during the interaction of erythropoietin with its target cells.

Tumor-promoting phorbol esters support the in vitro proliferation of murine pluripotent hematopoietic stem cells.

The effect of tumor-promoting phorbol esters on the in vitro proliferation of mouse pluripotent hematopoietic stem cells (CFU-S) was examined using a short-term in vitro culture system and an 11-d spleen colony assay. Phorbol myristate acetate (PMA, 10(-7) M), but not the inert compound phorbol, supported the in vitro survival of day 11 CFU-S for 72 h in a manner similar to IL 3. PMA also enhanced the effect of IL 3 on the in vitro survival of day 11 CFU-S and as little as 1 h of exposure to PMA was sufficient for this purpose. The effect of PMA on CFU-S survival in vitro was not mediated by prostaglandins, did not require an established adherent cell population, and was observed at a concentration of 10(-9) M. PMA alone did not enhance the in vitro survival of day 11 CFU-S at very low concentrations of FCS but was still able to potentiate the effect of IL 3 on these cells. PMA also enhanced the in vitro survival of day 11 CFU-S from mice treated with 5-fluorouracil or from marrow cells exposed to merocyanine 540 and light. The interaction of PMA with day 11 CFU-S was not inhibited by a neutralizing antiserum to IL 3 but was inhibited by the protein kinase inhibitor H-7. Together, the data indicate that tumor-promoting phorbol esters interact with pluripotent hematopoietic stem cells. Like IL 3, their effect appears to be permissive and involves stem cells with marrow repopulating ability.

Chromate transport in human leukocytes.

Chromium is a trace metal of importance in human physiology and, in addition, as 51-chromate, has been extensively used as a label in the study of blood cell pool sizes and intravascular kinetics. The transport characteristics of 51-chromate were investigated in normal human leukocytes. Chromate uptake is unidirectional over a 1 hr incubation with extracellular chromate concentrations up to 200 mumoles/liter. Under these conditions, intracellular 51-chromium is in a form in which it is nonexchangeable. Influx is temperature sensitive with a Q(10) of approximately 2 and may be energy dependent since a variety of metabolic poisons strongly inhibit uptake. The unidirectional influx of chromate follows Michaelis-Menten kinetics; the maximum velocity is 52 mmumoles/g dry weight of cells per min and the chromate concentration at which influx velocity is half maximal is 87 mumoles/liter. This transport mechanism is highly specific for chromate; other divalent tetrahedral anions only slightly inhibit influx at concentrations up to 10 times that of chromate. Metavanadate, however, competitively inhibits chromate influx at equimolar concentrations. Exposure of cells to unlabeled chromate leads to inhibition of subsequent influx of 51-chromate. It is suggested that this is due to a primary inhibitory effect of chromate on cellular energy metabolism.

Sequential activation of splenic nuclear RNA polymerases by erythropoietin.

The spleen of the ex-hypoxic polycythemic mouse was employed to study the effect of erythropoietin on nuclear RNA polymerase activity. On the basis of ionic strength requirements and sensitivity to the fungal toxin alpha-amanitin, two major forms (I and II) of nuclear RNA polymerase were identified. Within 0.5 h after administration of erythropoietin, at a time when no morphologically identifiable erythroblasts were present in the spleen, there was an increase in the activity of polymerase II. By 2 h, polymerase II activity had declined to control levels. At 3 h, polymerase I activity began to increase, rising to a peak, 88% above control levels, by 12 h. During this period, early erythroblasts began to appear in the spleen. At 12 h, a second increase of similar magnitude occurred in polymerase II activity. Polymerase I activity fell to control levels by 18 h while polymerase II declined more slowly. These data indicate that stimulation of transcription is an early effect of erythropoietin. Multiple forms of RNA polymerase are involved and activation of these is sequential. Nuclear RNA polymerase activity is maximal during the period of early erythroblast proliferation and declines as these cells mature.

Interleukin 3 promotes the in vitro proliferation of murine pluripotent hematopoietic stem cells.

Medium conditioned by activated T lymphocytes stimulates the in vitro proliferation of pluripotent hematopoietic stem cells (spleen colony-forming units [CFU-S]) but the factors involved have not been identified. Because the lymphokine interleukin 3 (IL-3) enhances in vitro colony formation by committed hematopoietic progenitor cells, we examined the effect of IL-3 on the in vitro proliferation of CFU-S using an 11-d spleen colony assay. When mouse marrow cells were placed in liquid culture, CFU-S content declined progressively and by 96 h only 13% of the CFU-S remained. By contrast, after 96 h in the presence of 20 U/ml of IL-3, the number of CFU-S were the same as that in the initial inoculum. Although the number of CFU-S eventually declined, they could still be recovered after 264 h of culture. In the absence of IL-3, the number of CFU-S synthesizing DNA was negligible; in its presence, greater than 20% of the CFU-S were in cycle. IL-3 stimulated CFU-S proliferation at a concentration of 0.2 U/ml. The dose-response curve was similar to that observed for other biologic effects of the lymphokine, and as little as 1 h of exposure to IL-3 enhanced the survival of CFU-S in vitro. Treatment of marrow cells with anti-Thy 1.2 antibody and complement before exposure to IL-3 did not inhibit spleen colony formation, but treatment of the cells with anti-Thy 1.2 antibody and complement after exposure to IL-3 reduced CFU-S recovery after 96 h of culture by 45%. The cell composition of day 11 spleen colonies formed by IL-3-treated marrow cells was similar to that of colonies formed by untreated marrow cells. Finally, day 11 CFU-S persisting in the marrow of mice treated with 5-fluorouracil required IL-3 for proliferation in vitro. Taken together, these data indicate that IL-3 promotes the proliferation of CFU-S in vitro, increases the number of CFU-S synthesizing DNA, but does not alter their commitment program, and the target cell population includes CFU-S with self-renewal and marrow-repopulating ability.

The third International Congress on Myeloproliferative and Myelodysplastic Syndromes.

This meeting was convened by Richard T. Silver and co-chaired by Jerry L. Spivak. It was held from 27 to 29 October 2005 in Washington, DC. Thirty-one invited speakers from seven different countries participated in the conference, which was attended by more than 300 individuals from 23 countries. As in previous years, a clinical symposium for patients, held the day before the symposium, was sponsored by the Cancer Research and Treatment Fund, Inc., New York, NY 10021. This meeting report provides a summary of the five sessions prepared and highlighted by one of the session chairs. In addition to the formal presentations on the biology, clinical aspects and management of these diverse marrow stem cell disorders, there was considerable interest generated because of the availability of several new agents that have been recently approved. A special luncheon satellite symposium was devoted to the dramatic changes in the therapeutic options for the myelodysplastic syndromes, sponsored by MGI Pharma, Inc. The keynote address was presented by Dr. George Q. Daley from Harvard Medical School and the Children's Hospital Medical Center. He reviewed the molecular steps in the formation of the Philadelphia chromosome and some of the newly described mutations leading to resistance to chemotherapy (see Section 4).

Effects of the differentiating agent hexamethylene bisacetamide on normal and myelodysplastic hematopoietic progenitors.

Hexamethylene bisacetamide (HMBA; NSC 95580) is a potent polar-planar differentiating agent of leukemia and solid tumor cell lines in vitro at clinically achievable concentrations. HMBA is currently being studied in patients with myelodysplastic syndrome. Previous phase I trials have demonstrated that HMBA produces hematologic toxicity in morphologically normal bone marrows of patients with solid tumors. Because of concern that HMBA may produce more severe myelotoxicity in patients with myelodysplastic syndrome since these patients have limited hematopoietic reserves, we studied the effects of HMBA on myelodysplastic and normal hematopoietic progenitors in vitro. HMBA concentrations that are optimal for differentiation in vitro (2 to 5 mmol/L) and HMBA concentrations that are being achieved in clinical trials (1 to 2 mmol/L) inhibited the growth of granulocyte-macrophage colony-forming units and erythroid burst-forming units from all 15 patients with myelodysplastic syndrome and all 4 normal subjects, HMBA did not induce proliferation of myelodysplastic or normal progenitors at any concentration; rather, it produced nearly identical inhibition of normal and myelodysplastic hematopoietic progenitors. HMBA also produced quantitatively similar inhibition of clonogenic leukemic growth of two myeloid leukemia cell lines. For a differentiating agent to be effective, it will likely have to either produce both differentiation and proliferation of abnormal hematopoietic progenitors or show selective inhibitory effects on abnormal as compared with normal progenitors. Although the mechanisms responsible for the antiproliferative effects of HMBA cannot be determined from this study, similar inhibitory effects of HMBA on normal and abnormal hematopoietic progenitors suggest that HMBA may be of limited utility in producing and sustaining elevations of peripheral blood cell counts in patients with myelodysplastic syndrome.

Magnitude of calcium influx required to induce dehydration of normal human red cells.

Activation by [Ca2+]i of Ca2+-sensitive K+ channels has long been known to cause dehydration of red cells suspended in low-K, plasma-like media. However, the fundamental question of the extent to which Ca influx must be increased to trigger dense cell formation in conditions likely to arise in the circulation has not been established. We report here that in ionophore permeabilized red cells, increasing Ca influx above 0.7 mmol/litre cells per h induces the formation of subpopulations of dehydrated cells within 1-2 hours. The presence or absence of glycolytic substrates had little effect suggesting that ATP depletion was not large enough to significantly inhibit the pump within that period. Below maximal dehydrating Ca influxes of about 1.2 mmol/litre cells per h, the trend was for the fraction of dense cells formed to remain steady in time. As Ca influx was increased, both the rate of dense cell formation and the fraction of dense cells formed increased. These results are analyzed in relation to mechanisms and to possible states of increased Ca2+ permeability in physiological and physiopathological conditions.

Hexamethylene bisacetamide in myelodysplastic syndrome: effect of five-day exposure to maximal therapeutic concentrations.

The efficacy of hexamethylene bisacetamide (HMBA), a potent polar-planar solvent which is capable of differentiating leukemias and solid tumors in vitro at clinically achievable concentrations, was studied in 16 patients with severe myelodysplastic syndromes (MDS). An adaptive control dosing algorithm was used to maintain HMBA steady-state concentrations (Css) within a narrow therapeutic window (1-2 mM) for five days every four weeks. Despite achieving the target HMBA Css during at least two courses in each of 15 patients, HMBA did not produce clinically relevant improvements in blood cell counts nor in other functional indices. Instead, HMBA induced cytopenias in the majority of these patients, most of whom had preexisting cytopenias and limited hematopoietic reserves. These disappointing results correlated with concurrent in vitro bone marrow studies from these patients in which both the HMBA concentrations that were optimal for differentiation in vitro (2-5 mM) and the HMBA Css that were achieved in this study (1-2 mM) substantially inhibited the growth of granulocyte-macrophage colony-forming units and erythroid burst-forming units. Although the mechanism responsible for the anti-proliferative effects of HMBA on hematopoietic progenitors (cytotoxicity versus terminal differentiation) could not be determined, the induction of cytopenias and lack of significant clinical improvements suggest that HMBA is cytotoxic and will not be useful alone as a differentiating agent on this schedule of administration in the treatment of MDS.

Masked megaloblastic anemia.

In six patients, eight episodes of anemia associated with folic acid or vitamin B12 deficiency were unaccompanied by macrocytosis. Six of the eight episodes of anemia were complicated by illnesses of an inflammatory or infectious nature, two patients had iron deficiency, two appeared to have a thalassemia trait, and one had severe renal failure. In five of the eight episodes, erythropoiesis was not megaloblastic and there was insufficient anisocytosis or poikilocytosis to suggest an underlying vitamin deficiency state. Hypersegmented neutrophils were observed in all episodes, but a neutrophil lobe average of greater than 3.5 lobes per cell was observed only once, and in one patient, less than 5% of the circulating neutrophils were hypersegmented. Giant metamyelocytes, however, were present in the marrow in all of the episodes and provided an important clue to the presence of the vitamin deficiency state.

Protease activity in human urine erythropoietin preparations.

The presence of protease activity in 22 of 23 crude urine erythropoietin (Ep) preparations was identified by two different protease assays. Protease activity was enhanced at pH 4.5. Seven known protease inhibitors, two chelating agents, a sulfhydryl reagent and a chaotrophic agent failed to inhibit the protease activity of these Ep preparations. However, heating at 80 degrees C for 5 min uniformly eliminated protease activity without affecting Ep activity. In view of the sensitivity of cell surface proteins to proteolytic attack, the presence of protease activity in crude preparations of Ep should be considered when such preparations are employed in cell culture experiments.

Erythropoietin stimulates serine kinase activity in erythropoietin-dependent cells.

Protein phosphorylation is an early event that follows the interaction of erythropoietin (Epo) with its receptor, even though this receptor lacks a kinase domain. To further define the role of protein kinases in Epo-mediated signal transduction, the effect of Epo on serine-threonine kinase activity was examined in the Epo-dependent cell line, HCD-57, using a kinase renaturation assay. In HCD-57 cells synchronized in G0 phase by centrifugal elutriation, multiple serine-threonine kinases were constitutively active, and exposure to Epo was associated with an increase in the activity of kinases with apparent molecular masses of 170, 120, and 90-95 kD. Phosphoamino acid analysis established the covalent incorporation of 32P into serine and threonine for constitutively active kinases and into serine alone for the 90-95 kD kinase. Reelectrophoresis experiments established that 32P incorporation represented kinase autophosphorylation as opposed to protein substrate phosphorylation. Epo-associated serine kinase autophosphorylation was both hormone concentration and time dependent as well as restricted to the G0, G1, and S phases of the cell cycle. Cell fractionation studies localized the activity of the 90-95 kD serine kinase to the plasma membrane.

Protein kinases and phosphatases are involved in erythropoietin-mediated signal transduction.

To study the role of protein phosphorylation in erythropoietin (EPO)-mediated signal transduction, we examined the effects of tyrosine phosphatase and tyrosine and serine-threonine kinase inhibitors as well as activators of serine kinases on DNA synthesis and cell proliferation in the murine EPO-dependent cell line HCD-57. HCD-57 cells were obtained synchronized in G0 by centrifugal elutriation, and DNA synthesis was measured by incorporation of labeled thymidine into DNA. Half-maximal DNA synthesis was stimulated by 0.001 U/ml of EPO. Sodium orthovanadate (Na3VO4), a tyrosine phosphatase inhibitor, at 5 microM potentiated a subsaturating concentration of EPO. Na3VO4 alone stimulated HCD-57 DNA synthesis at concentrations of 0.1-20 microM. Zinc chloride, another tyrosine phosphatase inhibitor, also stimulated HCD-57 DNA synthesis at concentrations of 50-100 microM. Genistein, a tyrosine kinase inhibitor, blocked the effect of EPO at a concentration of 5 micrograms/ml. Bryostatin, a protein kinase C (PKC) activator, stimulated DNA synthesis in HCD-57 cells at concentrations of 10(-9)-10(-10) M, whereas the phorbol ester, phorbol 12,13-dibutyrate (PDBu), was stimulatory only at a concentration of 10(-11) M. Staurosporine, a PKC inhibitor, blocked the effect of EPO at a concentration of 10(-7) M, and H-7, a nonspecific protein kinase inhibitor, was not inhibitory. These agents also had similar effects on the in vitro proliferation of HCD-57 cells. Taken together, the data indicate that the EPO-mediated transition from G0 to S phase in HCD-57 cells involves the activation of both tyrosine and serine-threonine kinases and is modulated by tyrosine phosphatase activity.

Cell cycle-specific behavior of erythropoietin.

The murine erythropoietin-dependent erythroleukemia cell line, HCD-57, was employed to study the cell cycle-specific behavior of erythropoietin. Cell cycle duration for HCD-57 cells was approximately 12 hours and was uninfluenced by erythropoietin. Populations of HCD-57 cells synchronized in G1 by centrifugal elutriation were able to pass through one complete cell cycle in the absence of erythropoietin but, thereafter, arrested in G1 as identified by propidium iodide staining and flow cytometry. Analysis of cell cycle behavior using the metachromic dye acridine orange, however, revealed that HCD-57 cells pass through a G0 cell cycle phase and, like serum-deprived 3T3 cells, actually arrest in G0 when deprived of erythropoietin. Expression of the cell cycle regulatory protein p34cdc2 was invariant throughout the cell cycle in HCD-57 cells. p34cdc2 was constitutively phosphorylated in G0 cells, and this effect was not modified by erythropoietin. Erythropoietin receptor distribution was log normal in HCD-57 cells in each phase of the cell cycle. The affinity of these surface receptors for erythropoietin was essentially invariant throughout the cell cycle, but receptor expression was upregulated in G2M cells as compared with cells in G1 or S phase. Taken together, these data indicate that erythropoietin has an important role in the G0-G1 to S phase transition but, based on receptor expression, is involved in other phases of the cell cycle as well.

Erythropoietin.

Erythropoietin is a glycoprotein hormone produced in the kidneys and to a lesser extent in the liver which regulates the proliferation and differentiation of erythroid progenitor cells. The gene for erythropoietin is well conserved and the hormone is always present in the plasma even in the renoprival state, emphasizing its important physiologic role. Failure to produce adequate quantities of erythropoietin leads to severe anemia, a situation most often encountered in patients with end stage renal disease. With the application of recombinant DNA technology, the gene for erythropoietin has been molecularly cloned, sequenced and expressed in a biologically active form in mammalian cells. In recent clinical trials, the recombinant hormone has been demonstrated to correct anemia in patients with severe end stage renal disease and alleviate their transfusion requirements.

The clinical physiology of erythropoietin.

From the foregoing data, it is clear that erythropoietin production is tightly regulated not only under normal circumstances but also during hypoxia, unless the hypoxia is extreme. Various disease states can have a negative impact on erythropoietin production, but while altering it quantitatively, they do not appear to abrogate basic fundamental control mechanisms. Rather, the threshold stimulus for either the recruitment of cells to produce erythropoietin or the effective production of erythropoietin is altered, usually in a predictable fashion. However, a reduction in erythropoietin production inevitably leads to a reduction in erythropoiesis. Nevertheless, assuming that the bone marrow remains responsive, administration of exogenous erythropoietin will increase the red blood cell mass. The immunoassay for serum erythropoietin therefore provides a means for identifying those situations in which erythropoietin therapy should be effective, but the assay must be used critically, bearing in mind the physiology of the hormone and the threshold concept of erythropoietin production (Table 3).

Hematophagic histiocytosis. A report of 23 new patients and a review of the literature.

The clinical and laboratory features of 23 new patients as well as 50 previously reported patients with the syndrome of hematophagic histiocytosis are reviewed. The syndrome occurs more often in men than women but has no age predilection. Common presenting features include fever, hepatic and splenic enlargement, lymphadenopathy, and profound depression of blood counts. The clinical course is generally fulminant and may be complicated by coagulation abnormalities, hepatic dysfunction and renal failure. In the majority of patients, however, the syndrome is self-limited with resolution of the clinical and laboratory abnormalities within several weeks. Hematophagic histiocytosis generally occurs in patients who develop infections in the setting of preexisting immunologic abnormalities or neoplasms. Viral infections are most commonly involved, but virtually any other infectious agent can precipitate this syndrome. The characteristic morphologic feature of the hematophagic histiocytosis is the proliferation of mature histiocytes actively ingesting other blood cells. These hematophagic histiocytes most commonly involve the bone marrow but may also be present in the lymph nodes, spleen and liver. Other bone marrow abnormalities include hypocellularity with preservation of megakaryocytes, and myelofibrosis. The principal features of this syndrome that distinguish it from malignant histiocytosis are the cytologic maturity and degree of hematophagic activity of the histiocytes as well as its more favorable prognosis. In some patients, however, a clear distinction of malignant from reactive histiocytosis will not be possible until a clonal marker for malignant histiocytes is identified. Based in our experience, the syndrome of hematophagic histiocytosis appears to be more common than malignant histiocytosis.

The application of recombinant erythropoietin in anemic patients with cancer.

Recombinant human erythropoietin (r-HuEPO) has been shown to correct anemia and alleviate transfusion dependency in patients with end-stage renal disease, to ameliorate anemia and reduce transfusion requirements in anemic patients with acquired immunodeficiency syndrome, to correct anemia in patients with rheumatoid arthritis, and to facilitate predeposit autologous blood donation. Cancer is frequently complicated by anemia, and a survey of serum erythropoietin concentrations in anemic cancer patients showed inappropriately low concentrations for the degree of anemia. Initial clinical trials suggest that r-HuEPO can correct the anemia associated with malignancy, but the exact role of the recombinant hormone in this situation remains to be defined.

The biology and clinical applications of recombinant erythropoietin.

Erythropoietin (EPO) is one of the few hematopoietic growth factors that behaves like a hormone. Produced primarily in the kidneys in adults, and to a small extent in the liver, EPO acts to promote the proliferation and maintain the survival of erythroid progenitor cells. To act as a mitogen and be able to achieve the plasma concentration necessary to trigger dormant primitive erythroid progenitors into cell cycle, EPO production must be both constitutive and inducible. Erythropoietin production is regulated at the level of its gene, and hypoxia is the only physiologic stimulus for upregulating its production. Therefore, under normal circumstances, the plasma EPO level reflects the status of EPO production. However, since a variety of disorders, as well as certain drugs, can influence EPO production, its plasma level cannot simply be used as a surrogate measure of tissue oxygenation. However, because of the strong inverse correlation between hemoglobin or hematocrit level and plasma EPO, the plasma EPO level can be used to determine whether EPO production is appropriate to the severity of an anemia. An inappropriately low plasma EPO level suggests hormone deprivation. In this situation, if bone marrow function is otherwise normal, there are no other correctable causes for anemia, and the patient is symptomatic or will require blood transfusions, therapy with recombinant EPO (epoetin) should be considered.

Cancer-related anemia: its causes and characteristics.

Under normal circumstances, the circulating red blood cell mass is maintained at a level that is constant in each individual, although that level may vary by more than 10% among individuals of the same age and gender. At normal ambient oxygen tension, two factors determine the circulating red blood cell mass: red blood cell life span, which is finite and in humans approximates 120 days; and the rate of effective red blood cell production. To maintain a constant red blood cell mass, therefore, approximately 20 mL of red blood cells must be produced each day to replace those red blood cells lost from the circulation through senescence. Anemia, which may be defined functionally as lack of sufficient red blood cells to maintain adequate tissue oxygenation, develops when the demand for new red blood cells exceeds the capacity of the bone marrow to produce them. This may be due to excessive red blood cell destruction, impaired red blood cell production, bleeding, or any combination of these. Acquired anemia is always a consequence of another disorder, which must be identified to ensure that the corrective therapy is appropriate. In patients with solid tumors, multiple mechanisms for causing anemia have been identified: blood loss that is either intrinsic or iatrogenic; nutritional deficiencies involving primarily iron or folic acid; hemolysis (autoimmune, traumatic, or drug-induced); bone marrow failure due to tumor encroachment, myelofibrosis, or marrow necrosis; infection; inflammation; or simply the presence of a cancer elsewhere in the body. The three noted causes of marrow failure share a common denominator: impaired production of erythropoietin. For any degree of anemia, a patient with cancer produces much less erythropoietin than expected and, therefore, cannot compensate for impaired red blood cell production. Inflammation or infection can exacerbate this situation. Indeed, anemia in patients with cancer appears to behave much like that in patients with chronic renal failure who become anemic because of the inability of the kidneys to produce erythropoietin adequately. The cause of impaired erythropoietin production in patients with cancer who have anemia is not entirely understood, but may be due in part to the production of inflammatory cytokines in response to the tumor. Such cytokines also would be expected to blunt the ability of the bone marrow to respond to the available circulating erythropoietin.(ABSTRACT TRUNCATED AT 400 WORDS)