Isolation and characterization of lipoproteins produced by human hepatoma-derived cell lines other than HepG2

Abstract

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-II, B, and E and albumin for Hep3B cells was 1.96, 1.01, 1.96, 1.90, and 53.2 micrograms/mg cell protein per 24 h, respectively. NPLC cells secreted no detectable albumin but the 24-h accumulated mass for apolipoproteins A-I, A-II, B, and E was 0.45, 0.05, 0.32, and 0.68 micrograms/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 apoA-II 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-II at 35% of apolipoprotein distinguishes Hep3B "HDL" from HepG2, which contains only 10%.(ABSTRACT TRUNCATED AT 400 WORDS)