Characterization of lipoproteins produced by the human liver cell line, Hep G2, under defined conditions1

Confluent monolayers of the human hepatoblastoma-derived cell line, Hep G2, were incubated in serum-free medium. Conditioned medium was ultracentrifugally separated into d < 1.063 g/ml and d 1.063-1.20 g/ml fractions since very little VLDL was observed. The d < 1.063 g/ml fraction was examined by electron microscopy; it contained particles of 24.5 * 2.3 nm diameter, similar in size to plasma LDL; a similar size was demonstrated by nondenaturing gradient gel electrophoresis. These particles possessed apoB-100 only. The d < 1.063 g/ml fraction had a lipid composition unlike that of plasma LDL; unesterified cholesterol was elevated, there was relatively little cholesteryl ester, and triglyceride was the major core lipid. The d 1.063-1.20 g/ml fraction was heterogeneous in size and morphology. Electron microscopy revealed discoidal particles (14.9 f 3.2 nm long axis and 4.5 f 0.2 nm short axis) as well as small spherical ones (7.6 * 1.4 nm diameter). Nondenaturing gradient gel electrophoresis consistently showed the presence of peaks at 13.4 11.9, 9.7, and 7.4 nm. The latter peak was conspicuous and probably corresponded to the small spherical structures seen by electron microscopy. Unlike plasma HDL, Hep G2 d 1.063-1.20 g/ml lipoproteins contained little or no stainable material in the ( H D L s ~ ) ~ ~ ~ region by gradient gel electrophoresis. Hep G2 d 1.063-1.20 g/ml lipoproteins differed significantly in composition from their plasma counterparts; unesterified cholesterol and phospholipid were elevated and the mole ratio of unesterified cholesterol to phospholipid was 0.8. Cholesteryl ester content was extremely low. ApoA-I was the major apolipoprotein, while apoE was the next most abundant protein; small quantities of apoA-I1 and apoCs were also present. Immunoblot analysis of the d 1.063-1.20 g/ml fraction after gradient gel electrophoresis showed that apoE was localized in the larger pore region of the gel (apparent diameter greater than 12.2 nm); the apoA-I distribution in this fraction was very broad (7.1-12.2 nm), and included a distinct band at 7.4 nm. Immunoblotting after gradient gel electrophoresis of concentrated medium revealed that a significant fraction of apoA-I in the uncentrifuged medium was in a lipid-poor or lipid-free form. This cell line may be a useful model for investigating the metabolism of newly formed HDL. -Thrift, R. N., T. M. Forte, B. E. Cahoon, and V. G. Shore. Characterization of lipoproteins produced by the human liver cell line, Hep G2, under defined conditions. J. Lipid Res. 1986. 27: 236-250. Supplementary key words hepatoma VLDL LDL HDL gradient gel electrophoresis electron microscopy apolipoprotein Rat hepatocytes in primary culture have been useful in studying lipoprotein synthesis and secretion (1); however, little is known about the corresponding functions of human liver cells. It has recently been demonstrated that the human hepatoblastoma-derived cell line, Hep G2, secretes must of the plasma proteins expected from liver cells, including apolipoprotein (apo) B (2). This finding stimulated investigations on apolipoprotein synthesis and, in fact, many of the plasma apolipoproteins have been identified in culture medium (3-5). Several laboratories have shown that these cells possess receptors for apoB (6, 7) and apoE (8). Utilizing this cell line, Zannis et al. (9) and Gordon et al. (10) showed that apoA-I is secreted as a proapolipoprotein, which possesses an additional sixamino acid propeptide as compared to the major plasma form of apoA-I. Zannis et al. (4) also found that apoE from culture medium contains more sialic acid groups than does the major plasma form. Intracellular and secreted forms of apoA-I1 (11) and A-IV ( 5 ) have been ascertained for Hep G2 cells. Although chromosomal abnormalities have been demonstrated, the line appears to be diploid (12) in the chromosomal regions thought to code for the B-E receptor, 1ecithin:cholesterol acyltransferase (LCAT), and apolipoproteins A-I, A-11, A-IV, C-11, C-111, and E (13-20). Hep G2 cells, however, produce little or no serum amyloid A (21), a protein of hepatic origin which under some conditions is associated with Abbreviations: apo, apolipoprotein; d, density; DTNB, 5,5’-dithiobis2-nitrobenzoic acid; ELISA, enzyme-linked immunosorbant assay; FBS, fetal bovine serum; HDL, high density lipoprotein; LCAT, 1ecithin:cholesterol acyltransferase; LDL, low density lipoprotein; MEM, minimum essential medium; PBS, phosphate-buffered saline; RER, rough endoplasmic reticulum; SRID, single radial immunodiffusion; SER, smooth endoplasmic reticulum; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; VLDL, very low density lipoprotein. ‘Part of this work has been presented in abstract form (Federation Pmc. 1984. 43: 621). ‘To whom reprint requests should be addressed. 236 Journal of Lipid Research Volume 27, 1986 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom HDL (22). The cell line also lacks a specific protease involved in the intracellular processing of pro-complement Factor I (23). The major emphasis thus far in lipoprotein studies with Hep G2 cells has been on the elucidation of newly synthesized apolipoprotein primary structure and the steps involved in intraand extracellular protein processing. The studies of Rash, Rothblat, and Sparks (3) and Zannis et al. (4) suggested that the major apolipoproteins secreted by these cells are complexed with lipids; however, the complexes remain uncharacterized. These cells may be useful as a model for the study of newly secreted hepatic lipoproteins that have not undergone the modifications normally occurring in the circulatory system. We report on the composition, size distribution, and morphology of the major lipoprotein classes produced by the human liver-derived cell line, Hep G2, in chemically defined medium.