TGF-BETA-MEDIATED HEPATOCELLULAR APOPTOSIS BY RAT AND HUMAN HEPATOMA-CELLS AND PRIMARY RAT HEPATOCYTES

Background/Aims: Primary cultures of rat hepatocytes, rat (FAG) and hu man (HepG2) hepatoma cells were studied by immunocytochemistry for exp ression of transforming growth factor (TGF)-beta, for the release of T GF-beta into the medium, and generation of hepatocellular apoptosis by the respective cell-conditioned media. Methods/Results: Using the alk aline-phosphatase anti-alkaline-phosphatase technique, intense TGF-bet a immunostaining was shown in all cell types. The cytokine is released almost entirely in the latent form into the culture medium; only the FAG-cells had a substantial fraction of bioactive TGF-beta in the nati ve (unacidified) culture fluid. Exposure of hepatocytes with the respe ctive cell-conditioned media in the activated, but not in the native f orm (except for FAG-cell media), induced severe detrimental effects as evidenced by: (i) gross morphological alterations, (ii) functional im pairment (reduction of WST-1 test, detachment of cells, lactate dehydr ogenase increase in the medium), and (iii) generation of apoptosis. Th e latter phenomenon was confirmed by an increase of internucleosomal D NA fragments, positive TUNEL reaction, and intense binding of the fluo rochrome Hoechst 33342 to fragmented nuclei. All these effects, which were mimicked by addition of recombinant human TGF-beta(1), were almos t entirely antagonized by pre-incubation of the conditioned media with latency associated peptide. In contrast to hepatocytes, both types of hepatoma cells mere completely resistant to the multiple actions of T GF-beta and activated conditioned media. Conclusions: It is concluded that hepatocytes might have the ability to induce autocrine, TGF-beta- mediated apoptosis, whereas hepatoma cells, because of their TGF-beta resistance, might generate TGF-beta-mediated peritumorous apoptosis of hepatocytes in a paracrine way, which could facilitate their expansio n in situ. Both mechanisms, however, are critically dependent on extra cellular TGF-beta activation.