Erythroid differentiation of clonal Rauscher erythroleukemia cells in response to erythropoietin or dimethyl sulfoxide.

Clonal lines of Rauscher erythroleukemia cells exhibited selective responses to two inducers of differentiation, erythropoietin and dimethyl sulfoxide. There were substantial quantitiative differences between clones that reponded to both inducers. Several clones differentiated only in response to erythropoietin. Erythropoietin stimulated cell proliferation and differentiation whereas dimethyl sulfoxide inhibited proliferation, suggesting dissimilar modes of action.

Phase 3 study of recombinant von Willebrand factor in patients with severe von Willebrand disease who are undergoing elective surgery.

Essentials Recombinant von Willebrand factor (rVWF) is effective in von Willebrand disease (VWD). A phase 3 study of rVWF, with/without recombinant factor VIII (rFVIII) before surgery in VWD. Overall rVWF's efficacy was rated excellent/good; rVWF was administered alone in most patients. rVWF was well-tolerated and hemostasis was achieved in patients with severe VWD undergoing surgery. SUMMARY: Background Recombinant von Willebrand factor (rVWF) has demonstrated efficacy for on-demand treatment of bleeding in severe von Willebrand disease (VWD), warranting evaluation in the surgical setting. Objectives This study (NCT02283268) evaluated the hemostatic efficacy/safety profile of rVWF, with/without recombinant factor VIII (rFVIII), in patients with severe VWD undergoing surgery. Patients/Methods Patients received rVWF 40-60 IU kg-1 , VWF ristocetin cofactor activity was measured 12-24 h before surgery. If endogenous FVIII activity (FVIII:C) target levels were achieved 3 h before surgery, rVWF was administered alone 1 h before surgery; rVWF was co-administered with rFVIII if target endogenous FVIII levels were not achieved. rVWF was infused postoperatively to maintain target trough levels. Overall and intraoperative hemostatic efficacy, the pharmacodynamics of rVWF administration and the incidence of adverse events (AEs) were assessed. Results All patients treated with rVWF for major (n = 10), minor (n = 4) and oral (n = 1) surgery had overall and intraoperative hemostatic efficacy ratings of excellent (73.3% and 86.7%) or good (26.7% and 13.3%). Most rVWF infusions (89.4%) were administered alone, resulting in hemostatically effective levels of endogenous FVIII within 6 h, which were sustained for 72-96 h; 70% (n = 7/10) of major surgeries were performed without rFVIII co-administration. Six patients reported 12 treatment-emergent AEs. Two patients each had one serious AE: diverticulitis (not treatment related) and deep vein thrombosis (sponsor-assessed as possibly treatment related). No severe allergic reactions or inhibitory antibodies were reported. Conclusions These data support the efficacy and safety profile of rVWF in patients with severe VWD undergoing elective surgery.

Characterization of biologically active, platelet-derived growth factor-like molecules produced by murine erythroid cells in vitro and in vivo.

Platelet-derived growth factor (PDGF) is an important serum regulator of erythropoiesis in vitro. We have now obtained evidence suggesting that PDGF-like molecules may also modulate erythropoiesis in vivo. Western blot analysis of cytoplasmic extracts from Rauscher murine erythroleukemia cells and phenylhydrazine-treated mouse splenic erythroid cells revealed the presence of several PDGF-like proteins. The presence of PDGF-like proteins in the cytoplasm of these two erythroid cell types was confirmed by immunohistochemical staining. Using a serum-free biologic assay, PDGF-like biological activity was found in cell lysates and conditioned medium of both Rauscher cells and phenylhydrazine-treated mouse erythroid cells. Subcellular localization experiments revealed the biological activity to be concentrated in the cytosolic fraction. Using a series of antibodies to hematopoietic growth factors we demonstrated that PDGF-like biological activity was specifically immunoprecipitated by both monoclonal and polyclonal anti-human PDGF antibodies but not by antibodies to burst-promoting activity, granulocyte-macrophage colony-stimulating factor, IL-3, or erythropoietin. Taken together, the data are consistent with the hypothesis that PDGF-like molecules play a role in the regulation of mammalian erythropoiesis in vivo.

Differential expression and androgen regulation of the human selenium-binding protein gene hSP56 in prostate cancer cells.

Low levels of dietary selenium are associated with increased risk of malignancy of several organs, including the prostate. Using a subtractive approach called linker capture subtraction, we have found that the human selenium-binding protein gene hSP56 is differentially expressed by the relatively slow-growing, androgen-sensitive prostate cancer cell line LNCaP but not by the more rapidly growing androgen-insensitive lines PC-3 and DU145. We confirmed this differential expression by Northern blot analysis. Importantly, hSP56 expression by LNCaP cells was reversibly down-regulated by exogenous androgen in a concentration-dependent manner. Marked differences in steady-state hSP56 mRNA levels were found in a variety of normal and neoplastic human cells that were examined. hSP56 expression was especially high in normal tissues that appear to benefit from the cancer-protective action of dietary selenium and was low in many neoplastic cells. The results suggest that hSP56 may play a role in determining the neoplastic phenotype.

New human renal carcinoma cell line established from a patient with erythrocytosis.

A continuous human renal carcinoma cell line (GKA) has been established from a patient with the paraneoplastic syndrome of erythrocytosis. The cells are epithelioid and anchorage dependent and have a doubling time in vitro of 48 to 72 hr. They exhibit a modal karyotype of 45,XX with abnormalities in chromosomes 3 and 9 and an absent chromosome 17 as determined by quinacrine mustard staining. Line GKA secretes erythropoietin activity into its growth medium, consistent with the biology of the tumor in vivo. This unique cell line will permit an investigation of the cellular physiology of this carcinoma and should result in clonal sublines with high erythropoietin-secretory activities.

Identification of genes expressed differentially by LNCaP or PC-3 prostate cancer cell lines.

The transition from androgen-dependent to aggressive androgen-independent growth is an important development in prostate cancer, and the identification of genes expressed differentially between these two phenotypes could provide new therapeutic targets as well as prognostic markers. Using a subtractive approach designated linker capture subtraction, we have now isolated several genes expressed differentially between the human prostate carcinoma cell lines LNCaP and PC-3, which are androgen-dependent and androgen-independent, respectively. In addition to 10 known genes that may be involved in signal transduction, tumor growth, tumor invasion, and metastasis, 3 novel genes were isolated. One of these, designated NPG-1, was expressed by the majority of primary tumors examined. Moreover, the degree of NPG-1 expressed correlated with an aggressive histopathology.

Secretion of erythropoietin-like activity by clones of human renal carcinoma cell line GKA.

Human renal carcinoma cell line GKA was derived from a patient with the paraneoplastic syndrome of erythrocytosis and secretes erythropoietin-like activity into its growth medium (Sytkowski, A. J., Richie, J. P., and Bicknell, K. A. Cancer Res., 43: 1415-1419, 1983). In order to derive homogeneous sublines with higher secretory rates, we cloned line GKA. Over 100 clones were generated, and 21 secreted erythropoietin-like activity, up to 6-fold higher than the uncloned line. This activity stimulated the growth and differentiation of CFU-E derived colonies in plasma clot culture. However, the secreted erythropoietin-like activity did not cross-react in a sensitive radioimmunoassay utilizing highly purified 125I-labeled human urinary erythropoietin and heterologous anti-human urinary erythropoietin antiserum. These results suggest that line GKA secretes an erythropoietic stimulating factor distinct from the hormone erythropoietin.

Detection of low-density cell-surface molecules using biotinylated fluorescent microspheres.

Biotinylated fluorescent microspheres have been developed as a reagent for studying antigens and receptors expressed at the cell surface. Labeling of antigen or receptor was accomplished by crosslinking biotinylated microspheres through streptavidin to corresponding biotinylated antibodies or ligands. Detection of labeled cells by flow microfluorimetry provided an extremely sensitive means for the analysis and potential manipulation of heterogeneous cell populations. The data indicate that cells bearing fewer than 200 surface antigen-antibody complexes per cell are readily detectable by this approach. Crosslinked to a selected biotinylated peptide immunogen, biotinylated fluorescent microspheres also allowed the labeling and detection of hybridoma cells bearing antigen-specific surface immunoglobulin.

Site-specific antibodies to human erythropoietin: immunoaffinity purification of urinary and recombinant hormone.

The 19-amino acid domain Ala111----Pro129 of human erythropoietin was identified as an accessible surface antigen based on the binding of radio-iodinated and of unmodified hormone to antibodies prepared against a synthetic peptide of homologous sequence. The specificity and affinity of this binding was sufficient to provide for the use of anti-peptide antibodies in the preparation of an immunosorbent for the purification of urinary, and of recombinant human erythropoietin. Immobilization of anti-peptide antibodies using agarose activated either with CNBr or with N-hydroxysuccinimido groups largely inactivated binding sites for erythropoietin. In contrast, antibodies crosslinked to N-acetyl-DL-homocysteine agarose through the hetero-bifunctional reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate retained their antigen-binding capacity virtually completely and provided a superior immunosorbent for hormone. Urinary erythropoietin with a specific bioactivity of 100 U/A280 was prepared initially by chromatography on phenyl-Sepharose. Subsequent immunoaffinity chromatography resulted in a 350-fold purification with 46.2% recovery yielding erythropoietin with a specific bioactivity of 35,200 U/A280 (44,300 U/mg). Radioiodination of this purified protein and subsequent SDS-polyacrylamide gel electrophoresis indicated that this preparation contained a single major component (Mr 30,000) which co-migrated in gels with unmodified biologically active hormone. Recombinant erythropoietin, which was prepared by the cloning of the human erythropoietin gene and its expression in COS cells using the SV40-derived vector pSV2, was purified by the same scheme. Chromatography on phenyl-Sepharose of medium derived from transfected cells (400 U/ml, 170 U/A280) provided for a 3.6-fold purification of recombinant hormone with an apparent recovery of 122%. This erythropoietin bound to the anti-peptide antibody gel and was purified to a specific bioactivity of 10,370 U/A280 with 55% recovery. The procedure described here for attaching antibodies to a solid support maximizes their antigen-binding capacity and is generally applicable. The development of an anti-peptide immunosorbant for human erythropoietin provides a valuable means for isolating hormone for use in studies of its receptor and its presently unresolved mechanism of action.

Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide.

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.

Malignant mesothelioma following radiation exposure.

Mesothelioma developed in proximity to the field of therapeutic radiation administered 10-31 years previously in four patients. In three, mesothelioma arose within the site of prior therapeutic radiation for another cancer. Mesothelioma in the fourth patient developed adjacent to the site of cosmetic radiation to a thyroidectomy scar. None of these four patients recalled an asbestos exposure or had evidence of asbestosis on chest roentgenogram. Lung tissue in one patient was negative for ferruginous bodies, a finding considered to indicate no significant asbestos exposure. Five other patients with radiation-associated mesothelioma have been reported previously, suggesting that radiation is an uncommon cause of human mesothelioma. Problems in the diagnosis of radiation-associated mesotheliomas are considered.

Erythropoietin activation of AP1 (Fos/Jun).

Erythropoietin (Epo) is the principal natural inducer of erythroid differentiation. The mechanisms by which signals generated at the Epo receptor (Epo-R) are transmitted to the nucleus are being explored. We now report that Epo strongly increases the activity of the transcription factor AP1 in both transformed and normal erythroid cells. Using antibodies to Fos and Jun, we have found that the Epo-induced AP1 heterodimer is composed primarily of authentic Fos and Jun proteins. Blocking protein kinase C (PKC) activity with H7 completely prevented the increase in AP1 activity in response to Epo. Importantly, the increase in AP1 activity was not due to increased expression of either c-fos or c-jun, as evidenced by the steady-state mRNA levels of both genes. Our results suggest that Epo may induce AP1 activity via a co- or posttranslational mechanism, presumably through modification of the Fos and/or Jun proteins.

Characterization of multiple erythroid progenitors available in large quantity from rabbit marrow.

We have examined the characteristics of erythroid progenitors from rabbit marrow and assessed the potential of this species as a source from which substantial quantities of erythroid progenitors could be obtained. Plasma clot cultures of rabbit marrow demonstrate colonies consistent with erythroid colony forming units (CFUE) by day 2 and colonies resulting from two types of burst forming units (BFUE) seen on days 3 and 8-9, respectively. Characteristic colony morphologies were noted from each cell type as well as differential sensitivity to erythropoietin (Ep) with discrete maximum responses for each progenitor. Distinct buoyant density characteristics were noted when the precursors were subjected to isopyknic separation on linear density gradients of polyvinylpyrrolidone-coated silica. The total nucleated marrow cells obtained from each animal ranged from 1.7-4.7 X 10(9). The average yields for each progenitor wee 1.71(+/-0.43) X 10(7) for CFUE, 1.99(+/-0.36) X 10(6) for 3 day BFUE and 1.24(+/-0.11) X 10(6) for 8-9 day BFUE.

B-lymphocyte-derived burst-promoting activity is a pleiotropic erythroid colony-stimulating factor, E-CSF.

Human B-lymphocyte-derived erythroid burst-promoting activity (B-BPA) is a pleiotropic, lineage-specific regulator of erythropoiesis. Our present data indicate that B-BPA plays an important role as an erythroid colony-stimulating factor (E-CSF) in modulating progenitor growth and differentiation throughout erythropoiesis. E-CSF has discrete effects on both early (erythroid burst-forming units, BFU-E) and late (erythroid colony-forming units, CFU-E) progenitors from normal bone marrow. In serum-substituted fibrin clot cultures, E-CSF stimulates the proliferation of BFU-E, resulting in an increase in the number of erythroid bursts over a wide range of erythropoietin (Epo) concentrations. We now have shown that E-CSF also acts on CFU-E by increasing their sensitivity to Epo markedly, resulting in a tenfold left-shift in the Epo dose-response curve. Using purified target-cell populations of human and murine erythroleukemia cells that are Epo-independent for growth, we have found that E-CSF stimulates cell proliferation directly, increasing the plating efficiency of these cells in suspension culture by 50%-165%. B-BPA also increased proliferation of these cells in semi-solid medium. Importantly, the combination of E-CSF and Epo resulted in a profound increase in the growth and maturation of the resultant colonies. Therefore, the data indicate that E-CSF can regulate the growth of cells independently of added Epo and, in addition, can synergize with Epo in regulating the growth and differentiation of erythroid progenitors.

A sensitive new bioassay for erythroid colony-stimulating factor.

Erythroid colony-stimulating factor (E-CSF) is a B cell-derived membrane protein that specifically affects the growth and development of human and murine committed erythroid progenitors. We report the development of a sensitive new bioassay for E-CSF, based on the ability of the growth factor to stimulate 3H-thymidine incorporation into cloned Rauscher murine erythroleukemia cells. The assay has among its advantages the ability to measure growth factor activity on a purified target cell population in the absence of endogenous growth factor-producing accessory cells. In addition, this assay measures E-CSF's proliferative effect on erythroid progenitors in the absence of erythropoietin (Epo) after 72 to 96 hours. In contrast, the standard bone marrow fibrin clot assay traditionally used to measure E-CSF requires the addition of Epo to promote the development of hemoglobinized erythroid colonies that are quantified after 7 days (for murine cells) to 12 days (for human cells). With the use of this new Rauscher cell bioassay, we have identified an E-CSF-producing human cell line and, further, have measured E-CSF activity derived from nonhuman splenic B lymphocytes.

Antipeptide antibodies as probes of the recombinant and endogenous murine erythropoietin receptors.

The binding of erythropoietin (Epo) to its plasma membrane receptor activates signal pathways that result in erythroid cell proliferation and differentiation. To elucidate the structural features of the receptor that are important for hormone binding and signaling, we have developed a series of site-specific antibody probes. These antibodies were raised against synthetic peptides homologous to six exoplasmic domains and one cytoplasmic domain of the murine receptor and were affinity-purified by binding to their respective peptide antigen, immobilized on agarose. Western blot analyses demonstrated that the recombinant receptor expressed transiently in COS-7 cells is synthesized as three protein species of 62, 64, and 66 kd, consistent with previous observations. Importantly, probing the endogenous receptor in both virally transformed erythroleukemia cells and normal erythroid cells demonstrated similar 62- to 66-kd receptor species. The affinity-purified antibodies also recognized several antigenically related proteins. An examination of the capacity of the antireceptor antibodies to block receptor activation by Epo revealed that antibodies to five of the six exoplasmic domains blocked the receptor. This was reversed with excess Epo. Inhibition of receptor activation by antibody probes to five discrete hydrophilic domains suggests that receptor function may be critically dependent on the structural integrity (conformation) of the entire exoplasmic portion.

The biochemistry of erythropoietin: an approach to its mode of action.

The elucidation of the mechanism of action of erythropoietin depends upon a detailed assessment of its effects at the molecular level. We have now begun to examine the effects of human erythropoietin and other inducers on clonal lines of Rauscher murine erythroleukemia cells. Over 100 clonal lines have been examined by assessing the hemglobinization of colonies grown in plasma clot culture in response to erythropoietin and dimethylsulfoxide. Many of the clones respond to both inducers. However, some clones respond only to erythropoietin. The cells also differentiate in suspension culture, exhibiting striking morphological changes characteristic of erythroid development. This system should serve as an excellent model for the study of control mechanisms in erythropoiesis.

Four unique monoclonal antibodies to the putative receptor binding domain of erythropoietin inhibit the biological function of the hormone.

We have produced a series of monoclonal antibodies (MoAbs) to amino acid region 99-129 of human erythropoietin (Epo) that distinguish unique structural features within this putative receptor binding domain of the hormone. The MoAbs recognize denatured Epo with widely different sensitivities on a Western blot and differentially bind to native Epo in solution. In addition, three of the four MoAbs neutralize the biological activity of Epo in a concentration-dependent fashion in vitro. Neutralization was measured both by inhibition of Epo-induced differentiation in Rauscher murine erythroleukemia cells and by inhibition of Epo-induced proliferation in normal murine splenic erythroid precursors. Characterization of the structural epitopes recognized by each of these four reagent MoAbs should provide us with important information concerning the requirements for hormone-receptor interaction.

Control of erythropoietin production.

The cloning and expression of the human erythropoietin gene has not only resulted in an important therapeutic advance but has led to the dissemination of reliable immunoassays and molecular probes for the study of normal and disordered erythropoietin physiology. We are now beginning to understand the cellular and molecular basis for the control of erythropoietin secretion, and have begun to identify abnormal erythropoietin physiology in a wide variety of disease states. This avenue of investigation will continue to expand and should lead to important new therapies for disorders of red blood cell production.

Selenium utilization in humans--a long-term, self-labeling experiment with stable isotopes.

A stable (nonradioactive) isotope of selenium in a chemical form common in foods (selenomethionine) or inorganic selenite was taken orally (200 micrograms/d) for 3 wk to label deep body pools. By deep body pools we mean selenium compartments that are large and/or have a slow turnover (exchange) rate. Blood plasma was removed, stored for 11 mo, and later reinfused as a labeled tracer dose with the selenium label in all of the biologically significant chemical forms. Accessible tissues such as red blood cells were highly labeled (20-25%) in the subjects receiving selenomethionine. Selenium from deep body pools is excreted primarily via the urine (80%). Reexcretion of previously absorbed selenium back into the gastrointestinal tract can be measured, avoiding a major source of error in conventional balance studies used to estimate nutrient absorption.