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Background/aims: A well-differentiated hepatocellular carcinoma-derived cell line, HuH-7 proliferates autonomously in serum-free medium. Human hepatoma-derived growth factor, which was purified from the conditioned medium of HuH-7 cells, stimulates the growth of HuH-7 cells by an autocrine fashion, and fibroblasts and endothelial cells by a paracrine fashion. We investigated the role of protein kinase C in the proliferation of HuH-7 cells and the growth activity of hepatoma-derived growth factor., Methodology: The effects of a selective protein kinase C inhibitor, H-7 on the proliferation of HuH-7 and 3T3 fibroblasts stimulated by hepatoma-derived growth factor were examined by DNA synthesis and cell growth assay., Results: H-7 suppressed the growth of HuH-7 cells. The ID50 of H-7 on the growth of HuH-7 cells was about 25 microM, and the growth of HuH-7 cells was almost completely inhibited by not less than 50 microM of H-7. H-7 inhibited the growth activity of hepatoma-derived growth factor for Swiss 3T3 fibroblasts. The ID50 of H-7 on the activity of hepatoma-derived growth factor for 3T3 fibroblasts was about 25 microM, too. HA1004, used as a negative control of H-7, failed to inhibit the growth of HuH-7 cells and the activity of hepatoma-derived growth factor. The growth of HuH-7 cells was stimulated significantly by about 40% by a protein kinase C activator, SC-9. H-7 did not suppress hepatoma-derived growth factor production in HuH-7 cells., Conclusions: These findings suggest that protein kinase C plays an important role in the growth of HuH-7 hepatoma cells and may be participated as a pathway in signal transduction of hepatoma-derived growth factor.

A novel hepatoma-derived growth factor was purified from the conditioned medium of human hepatoma-derived cell line, HuH-7, by the assay of [3H]thymidine incorporation into Swiss 3T3 cells. Molecular cloning of a complementary DNA from the cDNA library of HuH-7 cells was done on the basis of the N-terminal amino acid sequence. This protein was acid- and heat-labile heparin-binding protein and inactivated by reducing condition. The cDNA is 2.4 kilobase pairs long, and the deduced amino acid sequence from cDNA contained 240 amino acids without a signal peptide-like N-terminal hydrophobic sequence. Heparin column-eluted fraction of the conditioned medium of Cos-7 cells transfected by the cDNA stimulated the DNA synthesis. Northern blot analysis revealed its ubiquitous expression in several tumor-derived cell lines, as well as in normal tissues. The primary sequence shares homology with the high mobility group (HMG)-1 protein (23.4% amino acid identity and 35.6% similarity). However, its hydrophobic profile was distinct from that of HMG-1 except for the C-terminal acidic region, and it contained no apparent HMG box motif. Immunofluorescence study showed that it is localized in the cytoplasma of hepatoma cells. These findings suggest that this factor is a novel heparin-binding protein, with mitogenic activity for fibroblasts.

A human hepatoma cell line, HuH-7, which was established from a hepatoma tissue removed at surgical operation from an adult Japanese male, grows autonomously in a serum-free chemically defined medium. A human hepatoma-derived growth factor with potent growth-promoting activity, was partially purified from the conditioned medium of HuH-7 cells, using ion-exchange, heparin affinity chromatography and gel filtration. The partially purified growth factor (HuHGF) is acid- and heat-labile, and sensitive to reduction and trypsin digestion. HuHGF, which is of relatively high molecular weight (about 64 kDa) examined by gel filtration, stimulates not only the DNA synthesis of Swiss mouse 3T3 fibroblasts, but also the growth of HuH-7 cells in serum-free chemically defined medium, suggesting that it is produced by and acts on HuH-7 cells as an autocrine growth factor. This putative growth factor may play an important role in the autonomous growth of hepatoma and may lead to useful diagnostic or therapeutic approaches to human hepatoma.