Establishment and characterization of a new erythropoietin-dependent acute myeloid leukemia cell line, AS-E2

Abstract

We have established an erythropoietin-dependent human leuonly human leukemic cell line that is exclusively EPO depenkemia cell line, AS-E2, from a patient with acute myeloid leukedent. Because of its late erythroid cell characteristics and its mia. These cells have many characteristics of late erythroid strict EPO dependency, we think that AS-E2 is a good model progenitor cells, they are positive for CD36, Glycophorin A, and to study the effect of EPO on erythroid cell growth and signal (ASD-chE), but some of them were positive for periodic acidand they also play an integral role in leukemic cell growth. [6][7][8][9][10][11][12] and are useful tools for the cloning of cytokine therapy was initiated including bephenoyl Ara-c, daunorubicgenes, cytokine receptor genes, and for studying signal transine and 6MP. A complete remission was obtained in March, duction after cytokine stimulation and the role of cytokines in but the leukemia relapsed in May 1993. The leukemia became hematopoiesis. Among many cytokines, erythropoietin (EPO) resistant to chemotherapy and the patient died in September is an important cytokine which regulates commitment, 1993. growth, differentiation and/or survival of erythroid lineage cells through its interaction with a specific receptor, especially at the late stage of erythroid progenitors like BFU-E, CFU-E or Cell culture and cell count more mature erythroid cells. 13,14 Although some EPO-responsive human leukemia cell lines have been reported, most of AS-E2 cells were cultured in Iscove's modified Dulbecco's them also respond to other cytokines, such as granulocytemedium (IMDM) supplemented with 20% heat inactivated macrophage colony-stimulating factor (GM-CSF) or interleufetal bovine serum (FBS) in the presence of 2 U/ml of EPO kin-3 (IL-3). 8,9 and antibiotics. The data were generated after 12 months of In this paper, we report the establishment and the charactercontinuous culture. To examine EPO dependency, AS-E2 cells istics of a new human leukemia cell line, AS-E2, which shows were washed twice with IMDM and resuspended in IMDM some features of late erythroid lineage cells and exclusive EPO containing 20% FBS without EPO. AS-E2 cells were also culdependency in its growth. As far as we know, AS-E2 is the tured in the presence of both EPO and rabbit anti-human EPO antibody in order to inhibit the EPO effect in the culture medium.

Biosimilars entering the clinic without animal studies

“…As far as the pharmacodynamic component of biological activity is concerned, in vitro alternatives to the pharmacopoeial in vivo assays can be employed. For example, measuring proliferation of an erythropoietin-dependent cell line instead of using the normocythaemic assay in mice to compare biological activity of erythropoietin; 8 or using homologous (e.g., granulosa cells or Sertoli cells) or heterologous (transfected cell lines expressing the target receptor) assay systems instead of using the Steelman-Pohley assay to compare biological activity of a human follicle stimulating hormone (FSH). 9 Homologous systems have the advantage of being similar to the natural environment, but the disadvantages are the need to harvest cells from animals with a limited yield, the intrinsic variability and the nonhuman nature of the cells.…”

Effects of overexpression of the SH2-containing inositol phosphatase SHIP on proliferation and apoptosis of erythroid AS-E2 cells

“…In the absence of EPO AS-E2 cells die by apoptosis. 30 We questioned whether catalytically active or inactive SHIP might affect the survival of EPO deprived AS-E2 cells. As shown in Figure 2c, both wtSHIP and SHIP D672A overexpressing cells died significantly faster than control cells.…”

“…AS-E2 cells were generously provided by Tomonaga 30 and grown in IMDM supplemented with 20% (v/v) heat-inactivated FBS and 2 U/ml EPO (0.5-1.0 × 10 6 cells/ml if not otherwise stated). For transfected cells this medium was supplemented with 450 g/ml Geneticin (G418).…”

“…In order to study this in more detail we used overexpression of wild-type SHIP and catalytically inactive SHIP in the EPO-dependent human erythroleukemia cell line AS-E2. 30,31 While overexpression of SHIP does not affect EPO-dependent proliferation, overexpression of catalytically inactive SHIP significantly decreases proliferation, which might be caused by modulation of PI3K signalling. In the absence of EPO overexpression of SHIP significantly enhanced apoptosis by increasing caspase-9 and caspase-3 activity downstream from mitochondrial depolarization, which is independent of its 5-phosphatase activity.…”

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

“…Recent investigations of rhEPO activity estimates have been carried out using cell culture assays based on different cell lines, including AS-E2, TF1, UT-7 and UT-7/EPO (20)(21)(22)(23). However, these in vitro assays have a serious disadvantage since they are unable to discriminate between intact EPO and its asialo-or aglycosylated variants, which have a much shorter half-life and, therefore, a greatly reduced bioactivity when administered in vivo.…”