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.

Animals↗

PubMed1994

Sports hematology: serious concept or trivial pursuit. Introduction.

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Hematologic Diseases↗

PubMed1994

Serum immunoreactive erythropoietin during the perioperative period.

BACKGROUND: Recombinant human erythropoietin is now available for clinical use. Therefore we sought to determine the frequency of anemia and low endogenous erythropoietin levels in patients undergoing elective, major general surgical procedures. METHODS: Serum immunoreactive erythropoietin levels were measured before operation and 1 and 5 days after operation in 84 patients (43 men and 41 women) with normal renal function. RESULTS: Twenty of the women (49%) and 27 of the men (63%) were anemic before operation. Nine of these anemic patients had inappropriately low serum erythropoietin levels for their degree of anemia. On postoperative day 1, 66% of the women and 88% of the men were anemic, but the mean serum erythropoietin level had not increased. On postoperative day 5, 80% of the women and all of the men were anemic, but 22% of the patients still had an inappropriately low serum erythropoietin level. Multiple regression analysis identified female gender and black ancestry as predictors of an inadequate erythropoietin response after operation. CONCLUSIONS: Diseases requiring operation are frequently associated with anemia that is in part caused by impaired erythropoietin production. Surgery also appears to contribute to suppression of erythropoietin production particularly in women and black persons.

Anemia↗

PubMed1994

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)

Anemia↗

PubMed1994

Physiologic basis for the pharmacologic use of recombinant human erythropoietin in surgery and cancer treatment.

BACKGROUND: Recombinant human erythropoietin (rHuEPO) is approved for the treatment of the anemia of chronic kidney failure and anemia associated with zidovudine therapy of acquired immunodeficiency syndrome. In chronic kidney failure and other conditions such as cancer and hematologic malignancies, the endogenous erythropoietin response to anemia is blunted and rHuEPO might be beneficial in these conditions. METHODS: We reviewed preclinical and clinical trial results with rHuEPO in a variety of conditions. RESULTS: It is clear that chronic anemias of several causes respond to pharmacologic doses of rHuEPO. rHuEPO has been shown to enhance erythropoiesis before elective surgery, reduce the number of patients exposed to homologous blood at the time of coronary artery bypass grafting, reverse the anemia in most patients with cancer, and result in clinical benefit in 25% to 35% of patients with myelodysplasia. CONCLUSIONS: rHuEPO is important as a therapeutic means to correct anemia. rHuEPO is likely to be useful in correcting chronic anemias or anemias associated with chemotherapy, particularly in those patients with expected long-term survival. Issues to be resolved include the accurate prediction and targeting of rHuEPO therapy for patients most likely to respond.

Anemia↗

PubMed1993

Recombinant erythropoietin.

Erythropoietin is the only hematopoietic growth factor that behaves like a hormone. Produced in the kidneys and the liver, erythropoietin interacts with erythroid progenitor cells in the bone marrow to promote their proliferation and maintain their viability. Erythropoietin production is regulated at the level of its gene by tissue oxygenation; hypoxia or anemia stimulates erythropoietin production, and erythrocytosis suppresses it, but never completely. The plasma erythropoietin concentration reflects erythropoietin production and can be used to define erythropoietin-deficient states in which anemia may be amenable to correction by administration of recombinant human erythropoietin.

Erythropoietin↗

PubMed1993

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).

Down-Regulation↗

PubMed1992

Impaired erythropoietin response to anemia after bone marrow transplantation.

Delayed erythroid recovery is common after bone marrow transplantation (BMT), with some patients continuing to require red blood cell (RBC) transfusion support for as long as 1 year. While the etiology is multifactorial, inadequate stimulation of erythroid progenitors by the erythroid growth factor, erythropoietin, may play a role. In this study, the erythropoietin response to anemia of 70 consecutive patients undergoing BMT at the Johns Hopkins Oncology Center was compared with the erythropoietin response in uncomplicated iron deficiency anemia. Erythropoietin levels were elevated for the degree of anemia early after BMT; however, at the time of marrow recovery, erythropoietin levels were significantly suppressed in both allogeneic and autologous BMT patients compared with the iron-deficient patients. Patients with acute graft-versus-host disease (GVHD) had a more marked suppression of the erythropoietin response to anemia. In the patients who remained anemic for extended periods of time (up to 12 months after BMT), an inadequate erythropoietin response to anemia persisted. Delayed erythroid recovery after BMT is associated with inadequate erythropoietin levels. Therefore, recombinant human erythropoietin may be useful in the treatment of the anemia associated with both autologous and allogeneic BMT.

Anemia↗

PubMed1992

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.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Calcium and the erythroblast.

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Animals↗

PubMed1992

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.

Acetamides↗

PubMed1992

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.

Anemia↗

PubMed1991

Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin-mediated proliferation.

Erythropoietin mediates the rapid phosphorylation of Raf-1 in the murine cell lines HCD-57 and FDC-P1/ER, which proliferate in response to this cytokine. Phosphorylation occurs at both serine and tyrosine residues and as such is similar to the Raf-1 phosphorylation seen after interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and interleukin-2 stimulation in other murine cell lines. Such data suggest that these growth factors may share a common mechanism(s) of Raf-1 phosphorylation. Furthermore, in association with Raf-1 phosphorylation, erythropoietin induces a 2-3-fold increase in Raf-1 kinase activity as measured in immune complex kinase assays in vitro. Finally, a c-raf antisense oligodeoxyribonucleotide, which specifically decreases intracellular Raf-1 levels, also substantially inhibits both erythropoietin and IL-3-directed DNA synthesis. Together, these results provide evidence that activated Raf-1 is a necessary component of erythropoietin and IL-3 growth signaling pathways.

Animals↗

PubMed1991

Erythropoietin is both a mitogen and a survival factor.

Erythropoietin (Ep) regulates the proliferation and differentiation of erythroid progenitor cells, but whether it functions solely as a survival factor or also acts as a mitogen is unresolved. Because late erythroid progenitor cells (CFU-E) are largely in cell cycle, we examined this issue by using an Ep-dependent, murine erythroleukemia cell line, HCD-57. In the presence of human Ep and fetal calf serum, HCD-57 cells had a doubling time of approximately 24 hours, and during log-phase growth approximately 36% of the cells were in G1, 45% in S, and 19% in G2/M. With Ep deprivation, there was a gradual loss of viability and an arrest of proliferation with a 44% increase in the G0/G1 population, which could be reversed by reexposure to Ep even after 72 hours of hormone withdrawal. As little as 2 hours of exposure to Ep was sufficient to stimulate DNA synthesis, and the lag time for initiation of DNA synthesis after exposure to the hormone was approximately 10 hours as measured by either incorporation of labeled thymidine into DNA or cell cycle analysis by flow cytometry. RNA synthesis, by contrast, was initiated within 2 hours after exposure to Ep and did not require DNA synthesis. Total cell DNA content increased after exposure to Ep, indicating that it was acting as mitogen in HCD-57 cells. Ep was also able to stimulate DNA synthesis in the absence of serum as well as in its presence, indicating that the hormone could act as both a competence and a progression factor. Qualitative analysis of the integrity of HCD-57 DNA by electrophoresis in agar as well as direct measurement of DNA fragmentation after metabolic labeling with radioactive thymidine indicated that programmed cell death was occurring that could be reduced but not completely prevented by Ep. These data indicate that Ep acts as both a mitogen and a survival factor for HCD-57 cells.

Animals↗