TGF-β-mediated hepatocellular apoptosis by rat and human hepatoma cells and primary rat hepatocytes

Background/Aims: Primary cultures of rat hepatocytes, rat (FAO) and human (HepG2) hepatoma cells were studied by immunocytochemistry for expression of transforming growth factor (TGF)-β, for the release of TGF-β into the medium, and generation of hepatocellular apoptosis by the respective cell-conditioned media. Methods/Results: Using the alkaline-phosphatase anti-alkaline-phosphatase technique, intense TGF-β immunostaining was shown in all cell types. The cytokine is released almost entirely in the latent form into the culture medium; only the FAO-cells had a substantial fraction of bioactive TGF-β in the native (unacidified) culture fluid. Exposure of hepatocytes with the respective cell-conditioned media in the activated, but not in the native form (except for FAO-cell media), induced severe detrimental effects as evidenced by: (i) gross morphological alterations, (ii) functional impairment (reduction of WST-1 test, detachment of cells, lactate dehydrogenase increase in the medium), and (iii) generation of apoptosis. The latter phenomenon was confirmed by an increase of internucleosomal DNA fragments, positive TUNEL reaction, and intense binding of the fluorochrome Hoechst 33342 to fragmented nuclei. All these effects, which were mimicked by addition of recombinant human TGF-β1, were almost entirely antagonized by pre-incubation of the conditioned media with latency associated peptide. In contrast to hepatocytes, both types of hepatoma cells were completely resistant to the multiple actions of TGF-β and activated conditioned media. Conclusions: It is concluded that hepatocytes might have the ability to induce autocrine, TGF-β-mediated apoptosis, whereas hepatoma cells, because of their TGF-β resistance, might generate TGF-β-mediated peritumorous apoptosis of hepatocytes in a paracrine way, which could facilitate their expansion in situ. Both mechanisms, however, are critically dependent on extracellular TGF-β activation.