Masatsugu Ueda

Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells

Erythropoietin (Epo), a glycoprotein that regulates the formation of erythrocytes in mammals, was produced in cultured tobacco BY2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) by introducing human Epo cDNA via Agrobacterium tumefaciens-mediated gene transfer. Epo was correctly processed and subsequently penetrated the plasma membrane of tobacco cells. However, it remained attached to the cell wall and was not released into the culture medium. Although Epo produced by tobacco cells was glycosylated with N-linked oligosaccharides, these carbohydrates were smaller than those of the recombinant Epo produced in mammalian cells. Epo produced in tobacco exhibited in vitro biological activities by inducing the differentiation and proliferation of erythroid cells. However, it had no in vivo biological activities. A lectin-binding assay indicated the lack of sialic acid residues in the N-linked oligosaccharides of Epo, suggesting that Epo was removed from the circulation before it reached erythropoietic tissues.

Enhanced growth of clonogenic cells from acute myeloblastic leukaemia by erythropoietin

The effect of erythropoietin (Epo) on colony formation by blast progenitors in acute myeloblastic leukaemia other than erythroleukaemia was studied using a blast colony assay. Epo alone did not induce colony formation, but when it was used together with phytohaemagglutinin‐stimulated leucocyte‐conditioned medium (PHA‐LCM) the number of leukaemic colonies significantly increased in nine out of 12 cases studied. Preincubation of Epo with anti‐Epo antibody completely abolished this enhancement, indicating that the increase in colony numbers was caused by Epo itself. Cell surface phenotype analysis of colonies produced by Epo plus PHA‐LCM showed no increase in percentages of erythroid and megakaryocyte lineages. The addition of Epo also increased the self‐renewal capacity of leukaemic blast cells. Fresh leukaemic cells did not express Epo receptors, but they were induced after incubation with PHA‐LCM. The present study thus showed that the proliferative response to Epo is not restricted only to the erythroid lineage, but also extends to AML blast cells other than those in erythroleukaemia in the presence of colony stimulating factors.

Correlation between a sandwich ELISA and an in-vitro bioassay for erythropoietin in human plasma.

Summary. A sandwich‐type enzyme‐linked immunosorbent assay (ELISA) for human erythropoietin (EPO) using two anti‐EPO monoclonal antibodies has been compared with an in‐vitro EPO bioassay based on CFU‐E colony formation in fetal mouse liver cell cultures. In normal subjects and non‐uraemic anaemic patients the plasma EPO values estimated with the ELISA correlated well with those by the bioassay, and also inversely with the values of blood Hb, PCV and RBC counts. Dose‐response curves for plasma and standard recombinant human EPO in the ELISA were parallel to each other. These results further confirm the validity for the ELISA in measuring circulating human EPO.

Erythropoietin processing in erythropoietic system and central nervous system

We describe possible functions of carbohydrates attached to growth factors and strategies to examine the functions, concentrating on erythropoietin, a major regulator of erythropoiesis. Erythropoietin in erythropoiesis functions as an endocrine hormone; it is produced by kidney cells and transferred into the circulation to hemopoietic sites. In the brain, erythropoietin acts on neurons in a paracrine fashion. Comparison of glycosylation has been made between kidney and brain erythropoietins.