Isolation and characterization of lipoproteins produced by human hepatoma-derived cell lines other than HepG 2

A total of six established human hepatoma-derived cell lines, including Hep3B, NPLC/PRF/5 (NPLC), Tong/HCC, Hep 10, huH1, and huH2, were screened for their ability to accumulate significant quantities of lipoproteins in serum-free medium. Only two cell lines, Hep3B and NPLC, secreted quantitatively significant amounts of lipoproteins. In a 24-h period the accumulated mass of apolipoproteins (apo) A-I, A-11, B, and E and albumin for Hep3B cells was 1.96, 1.01, 1.96, 1.90, and 53.2 gg/mg cell protein per 24 h, respectively. NPLC cells secreted no detectable albumin but the 24-h accumulated mass for apolipoproteins A-I, A-11, B, and E was 0.45, 0.05, 0.32, and 0.68 pg/mg cell protein per 24 h, respectively. Twenty four-hour serum-free medium of Hep3B cells contained lipoproteins corresponding to the three major density classes of plasma; percent protein distribution among the lipoprotein classes was 4%, 41%, and 56% for very low density lipoprotein (“VLDL”), low density lipoprotein (“LDL”), and high density lipoprotein (“HDL”), respectively. NPLC was unusual since most of the lipoprotein mass was in the d 1.063-1.235 g/ml range. Hep3B “LDL”, compared with plasma LDL, contained elevated triglyceride, phospholipid, and free cholesterol. Nondenaturing gradient gel electrophoresis revealed that Hep3B “LDL” possessed a major component at 25.5 nm and a minor one at 18.3 nm. Immunoblots showed that the former contained only apoB while the latter possessed only apoE. Like plasma VLDL, Hep3B “VLDL” particles (30.5 nm diameter) isolated from serum-free medium contained apoB, apoC, and apoE. “HDL” harvested from Hep3B and NPLC medium were enriched in phospholipid and free cholesterol and poor cholesteryl ester which is similar to the composition of HepG2 “HDL.” “HDL” from Hep3B and NPLC culture medium on gradient gel electrophoresis had peaks at 7.5, 10, and 11.9 nm which were comparable to major components found in HepG2 cell medium. Hep3B cells, in addition, possessed a particle that banded at 8.2 nm which appeared to be an apoAI1 without apoA-I particle by Western blot analysis. The cell line also produced a subpopulation of larger-sized “HDL” not found in HepG2 medium. NPLC “HDL”had a distinct peak at 8.3 nm which by Western blot was an apoE-only particle. Electron microscopy revealed that “HDL” harvested from Hep3B and NPLC medium consisted of discoidal and small, spherical particles like those of HepG2. The “HDL” apolipoprotein content of each cell line was distinct from that of HepG2. ApoA-I1 at 35% of apolipoprotein distinguishes Hep3B “HDL” from HepG2, which contains only 10%. Unlike Hep3B and HepG2 “HDL”, apoE is the major apolipoprotein in NPLC “HDL.” Differences in lipoprotein distribution and HDL subclass heterogeneity in HepSB, NPLC, and HepG2 cell media are probably related to differences in apolipoprotein expression.Forte, T. M., M. R. McCall, B. B. Knowles, and V. G. Shore. Isolation and characterization of lipoproteins produced by human hepatomaderived cell lines other than HepG2. J. Lipid Res. 1989. 30: 817-829 Supplementary key words lipoprotein heterogeneity Hep3B cells NPLC/PRF/5 cells gradient gel electrophoresis electron microscopy We have previously demonstrated that the human hepatoblastoma-derived cell line, HepG2, accumulated low density lipoproteins (LDL) and high density lipoproteins (HDL) in chemically defined basal medium (1). This has been confirmed by several other laboratories (2, 3). In addition to lipoproteins, it has been demonstrated that this cell line secreted the enzymes 1ecithin:cholesterol acyltransferase (LCAT) (4) and hepatic lipase ( 5 ) , and cholesteryl ester transfer activity has been detected in the culture medium (6, 7). The LDL-like particles isolated from HepG2 medium were compositionally unusual; phospholipid and unesterified cholesterol were elevated while triglyceride formed the particle core (1). Apolipoprotein (apo) B was the sole apolipoprotein associated with the particle. The HDL particles isolated from the medium also differed from normal plasma HDL in that a high proportion of the HepG2 particles were discoidal in morphology (1). Particle morAbbreviations: HDL, high density lipoprotein, d 1.063-1.235 glml, isolated from medium; LDL, low density lipoprotein, d 1.006-1.063 g/ml, isolated from medium; VLDL, very low density lipoprotein, isolated from medium; NPLC, NPLC/PRF/5 cell line; apo, apolipoprotein; LCAT, 1ecithin:cholesterol acyltransferase; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; HBsAg, hepatitis B virus surface antigen; ELISA, enzyme-linked immunosorbent assay. Journal of Lipid Research Volume 30, 1989 817 by gest, on O cber 8, 2017 w w w .j.org D ow nladed fom phology and the elevated phospholipid and unesterified cholesterol composition reported for HepG2 HDL was, however, similar to HDL from LCAT-deficient plasma (8, 9). Unlike normal plasma HDL, apoA-I1 constituted only a small percentage of the total HepG2 HDL protein while apoE was relatively abundant (1). It is not known whether lipoprotein subclass distribution, composition, and morphology previously reported for HepG2 cells is unique for this cell line or whether particles with similar characteristics are produced by other human liver-derived cell lines. In order to determine whether other human hepatoma-derived cell lines secrete lipoproteins, several available lines were screened for their ability to produce lipoprotein complexes. We now describe the particles from two cell lines, Hep3B and NPLC/PRF/5 (NPLC), which accumulated lipoprotein products that differ from those reported for HepG2. These two hepatoma-derived lines, along with HepG2 cells, provide us with cellular models in which hepatic lipoprotein assembly, secretion, and metabolism may be elucidated.