PubMed Health⌕ Search

Biomedical subjects

J L Spivak

Publications and source records attributed to J L Spivak.

At least 19 recordsLinked to original sources
PubMed1996

Isolation of the full-length murine erythropoietin receptor using a baculovirus expression system.

The full-length murine erythropoietin receptor was expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus vector. Erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kD. Erythropoietin receptors produced in Sf9 cells could be solubilized using CHAPS in a form capable of binding erythropoietin, and the solubilized receptor bound to immobilized Concanavalin A (Con A) and wheat germ agglutinin, as well as to immobilized recombinant human erythropoietin. Analysis of the distribution of erythropoietin receptors in Sf9 plasma membrane and cytosol fractions using lectin affinity chromatography revealed that membrane-bound receptor had a higher apparent molecular mass and contained the bulk of receptors that bound to wheat germ agglutinin. The receptor was purified by sequential affinity chromatography on Con A-Sepharose and immobilized erythropoietin. Erythropoietin receptors expressed in Sf9 cells were inserted into the plasma membrane in the correct orientation, bound 125I-erythropoietin with a single affinity (kD, 330 pmol/L), and were internalized after ligand binding. However, kD varied inversely with the number of cell surface receptors. Solubilized erythropoietin receptors in whole-cell lysates and isolated plasma membranes exhibited high-affinity binding, with kD values of 92 and 57 pmol/L, respectively. Erythropoietin bound to the surface of infected Sf9 cells could be cross-linked to two proteins with molecular masses of 90 and 65 kD using the homobifunctional cross-linker, disuccinimidyl suberate (DSS). Similar results were obtained with solubilized receptors in whole-cell lysates, and both proteins could be immunoprecipitated by an antiserum to the erythropoietin receptor carboxyl-terminal domain.

Animals↗
PubMed1996

Anemia of cancer.

Cancer patients frequently develop anemia, due either to the cancer itself or to the effects of cancer-related therapy. Recent years have brought insights into both the pathogenesis of the anemia of cancer and the extent to which erythropoietin regulation participates in this process. Although transfusion therapy was the mainstay of therapy for symptomatic anemia in the past, clinical trials have demonstrated that recombinant human erythropoietin can alleviate both anemia and transfusion requirements in many cancer patients and may prove to have an important role in the treatment of cancer-related anemia in the future.

Anemia↗
PubMed1996

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.

Animals↗
PubMed1995

Ligand binding kinetics of a soluble full-length murine erythropoietin receptor.

The full-length murine erythropoietin receptor was expressed in Sf9 cells using a baculovirus vector. Erythropoietin receptors in solubilized Sf9 cell lysates bound erythropoietin with high affinity (92 pM). Erythropoietin receptor-125I-labeled erythropoietin association and dissociation kinetics using solubilized Sf9 cell lysates revealed a ka of 0.16 nM-1 min-1 and a kd of 0.00055 min-1 giving an observed KD of 3.45 pM. The erythropoietin receptors was partially purified from Sf9 cell lysates by chromatography on Con A Sepharose. When erythropoietin receptors were crosslinked to 125I-labeled erythropoietin and analyzed by SDS-7.5% PAGE protein complexes of 90 and 125 kDa were observed with receptors in solubilized lysate, and 170 and 190 kDa with the partially purified receptors.

Animals↗
PubMed1995

Polo-like kinase is a cell cycle-regulated kinase activated during mitosis.

Previously, we demonstrated that expression of polo-like kinase (PLK) is required for cellular DNA synthesis and that overexpression of PLK is sufficient to induce DNA synthesis. We now report that the endogenous levels of PLK, its phosphorylation status, and protein kinase activity are tightly regulated during cell cycle progression. PLK protein is low in G1, accumulates during S and G2M, and is rapidly reduced after mitosis. During mitosis, PLK is phosphorylated on serine, and its serine threonine kinase function is activated at a time close to that of p34cdc2. The phosphorylated form of PLK migrates with reduced mobility on SDS-polyacrylamide gel electrophoresis, and dephosphorylation by purified protein phosphatase 2A converts it to the more rapidly migrating form and reduces the total amount of PLK kinase activity. Purified p34cdc2-cyclin B complex can phosphorylate PLK protein in vitro but causes little increase in PLK kinase activity.

Amino Acid Sequence↗
PubMed1995

Serum immunoreactive erythropoietin in health and disease.

Erythropoietin, the glycoprotein which regulates erythropoiesis is unique amongst the hematopoietic growth factors since it is the only one which behaves like a hormone. Produced primarily in the kidneys in adults, erythropoietin interacts with erythroid precursors in the marrow to increase red cell production. Because erythropoietin behaves like a hormone, measurements of erythropoietin in the serum have proved useful in determining when production of this hormone is inadequate. Tissue hypoxia is the only physiologic stimulus for erythropoietin production and thus, with anemia, serum erythropoietin levels should be increased. Assuming normal marrow function and adequate nutrient supplies, when anemia is associated with a low serum erythropoietin level, it can be concluded that the anemia is in part due to erythropoietin lack and should be correctable by administration of erythropoietin. As a corollary, a high serum erythropoietin level (greater than 500 mU/ml) in the presence of anemia suggests that there is end organ failure, and erythropoietin therapy is not likely to be useful.

Anemia↗
PubMed1994

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.

Animals↗
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↗