HIGH-DENSITY LIPOPROTEIN3 BINDING-SITES ARE RELATED TO DNA BIOSYNTHESIS IN THE ADENOCARCINOMA CELL-LINE A549

The effect of high density (apoE-depleted HDL3) on cell growth of a hu man tumor cell line (A549) was studied and related to its binding on t he plasma membrane. HDL3 were shown to stimulate the incorporation of [H-3]thymidine into DNA and cell proliferation; these effects were dos e-dependent. As HDL3, apoA-I and apoA-1-liposomes complexes (but not a poA-II) were able to stimulate DNA synthesis in serum-free conditions. This effect was maximum for 15-30 mug HDL3 protein/ml concentration. Binding of HDL3 on whole cells occurred by two mechanisms: the first w as specific for HDL3; the second, of lower affinity, was phospholipid- dependent and was inhibited by low density lipoprotein or by phospholi pid particles. Internalization and degradation of bound HDL3 were not observed. The specific sites (27.9 +/- 2.2 ng HDL3 protein/ng cell pro tein) accounted for only 2.5 % of total (specific + phospholipid) bind ing sites and they bound HDL3 with a dissociation constant (K(D)) of 2 .47 +/- 0.46 mug HDL3 protein/ml (2.6 +/- 0.5 x 10(-8) M). The apparen t K(D) value of total binding sites (specific + phospholipid) was eigh tfold higher (20.4 +/- 6.1 mug HDL3 protein/ml). Analysis of the membr ane specific binding sites by ligand blotting with I-125-labeled HDL3 showed a single protein with an apparent molecular mass of 110 kDa. Wh en HDL3 binding on phospholipid sites was inhibited by rigid phospholi pid particles, the stimulation of [H-3]thymidine incorporation related to HDL3 concentration did not show a maximum peak as previously obser ved but reached a plateau at a concentration as low as 5 mug HDL3 prot ein/ml. This low concentration also nearly saturated the specific bind ing sites with HDL3. When binding on specific protein sites was suppre ssed by tetranitromethane, DNA synthesis was not stimulated but, in co ntrast, inhibited. The stimulating effect of HDL3 on DNA biosynthesis is therefore likely dependent on HDL3 OCCUPYing specific binding sites .