The regulated expression of erythropoietin by two human hepatoma cell lines.

Abstract

The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. We have screened multiple renal and hepatic cell lines (including MDCK, LLC-PKI, BHK, WRL 68, CLCL, A704, CRFK, A498, ACHN, TCMK-1, LLC-MK2, CaKi-2, HepG2, and Hep3B) for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10' cells in 24 hr It has been difficult to establish a cell culture system in which biologically active and immunologically identifiable Epo is produced in a regulated fashion by a single cell type. Many of the investigations cited above utilized tissues containing mixed cell types (5-9, 13). In other studies the demonstration of Epo production depended solely upon bioassays and/or inhibition of bioactivity by anti-Epo antibodies of questionable specificity (5-11, 13, 16, 19, 24). Some of the previously reported cell culture systems demonstrated only low levels of Epo production following stimulation by hypoxia or cobalt (II) (11,13,14,25), while still others possessed large amounts of constitutive Epo production (15)(16)(17)(18)(19)(20)(21)(22)(23) with little regulated production.In this report we demonstrate that, when grown at low cell density, two established human hepatoma cell lines can be induced to produce large amounts of biologically active and immunologically identifiable Epo in response to hypoxia or cobalt(II) chloride. Furthermore, upon such stimulation markedly increased levels of Epo mRNA were observed. MATERIALS AND METHODSCell Culture. All cell lines studied were obtained through the American Type Culture Collection. Cells were cultured in 100 x 20 mm tissue culture dishes (Coming no. 25020) and 25-cm2 tissue culture flasks (Coming no. 25100) using a minimal essential medium (GIBCO) supplemented with glutamine, penicillin (100 units per ml), streptomycin (100 /Lg/ml), and 10% heat-inactivated (560C for 30 min) fetal calf serum (Hazelton Research Products, Denver, PA). Cells were maintained in a humidified 5% C02/95% air incubator at 370C. The culture medium was changed daily for 1 or 2 days prior to all experiments. In experiments in which the oxygen tension was varied, the 25-cm2 flasks were fitted with rubber septa. The gas inlet was provided through a 9 cm 18 g needle, fitted with a 0.2-,Lm sterile filter, and the gas outlet was provided through a 4 cm 18 g needle connected by Silastic tubing to a closed water trap. Needles, tubing, and septa were autoclaved prior to use, and assembly was performed under sterile conditions. Experimental gases containing 1%, 2%, 3%, 5%, or 21% 02, 5% C02, and the balance N2 (Yankee Oxygen) were hydrated at 37°C and flowed at a rate of2.4-4.2 ml/min into the tissue culture flasks. The flasks were immersed in a 37°C constant-temperature water bath. Po2 and Abbreviations: Epo, erythropoietin; PGE2, prostaglandin E2. 7972The publica...

How to overcome (and exploit) tumor hypoxia for targeted gene therapy

“…Metabolic enzymes, like aldolase (ALD)-A and -C (Semenza et al, 1994), carbonic anhydrase (CA)-9 (Wykoff et al, 2000), enolase (Eno)-1 , glucose transporter (Glut)-1 and -3 (Sivitz et al, 1992), lactate dehydrogenase (LDH)-A , phosphofructokinase (PFK)-L and -C, phosphoglycerate kinase (PGK)-1 (Firth et al, 1994), and pyruvate kinase (PK)-M (Semenza et al, 1994); 3. Cytokines, growth factors, and other proteins that regulate angiogenesis, vascular permeability, and inflammation, including Epo (Goldberg et al, 1987;Madan and Curtin, 1993), endothelin-1 (Kourembanas et al, 1991), inducible nitric oxide synthase (iNOS; Melillo et al, 1995), plateletderived growth factor-B (Kourembanas et al, 1990), vascular endothelial growth factor (VEGF; Shweiki et al, 1992), and its receptors (VEGFR-1 and -2; Veikkola et al, 2000); 4. Stress proteins, such as the heat shock proteins (HSPs) and cellular redox regulators (Benjamin et al, 1990).…”

Hypoxia-induced accumulation of erythropoietin mRNA in isolated hepatocytes is inhibited by protein kinase C

“…However, since it has so far not been possible to isolate the cells physiologically producing EPO in liver and kidneys, these studies have been confined to EPO-producing turnout cell lines, and the results obtained with these cell lines are inconclusive and have led to controversial interpretations. Thus, using the human hepatoma cell lines Hep G2 and Hep 3B, which produce EPO in an oxygen-dependent fashion [22] and a renal carcinoma cell line, which secretes EPO constitutively [24], several investigators reported that phorbol esters reduce EPO formation [19,25,31,33]. However, in addition, inhibitors of PKC were found to diminish EPO secretion by Hep G2 ceils [31,33].…”

Oxygen-regulated Transferrin Expression Is Mediated by Hypoxia-inducible Factor-1

“…8). The two human hepatoma cell lines HepG2 and Hep3B are so far the only permanent cell culture models available to investigate oxygen-regulated Epo expression (9). Apart from Epo, we recently demonstrated hypoxic induction of several acute phase genes in HepG2 cells (10).…”