Induction of apoptosis in normal cultured rat hepatocytes and in Hep3B, a human hepatoma cell line.
The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions. Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells. In this study, we compared the effects of several apoptosis-inducing agents - transforming growth factor beta1 (TGF-beta1), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) - on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h. Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining. DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot. Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry. Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA. Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-beta1; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively). OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-beta1, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h. In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis. In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions.
Lamboley
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Laboratoire de Biologie Cellulaire, Unité INSERM 327, Faculté de Medecine Xavier Bichat, Université Paris 7-Denis Diderot, France. lamboley@bichat.inserm.fr
Bringuier
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Feldmann
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D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002273:Carcinogens / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D018929:Cell Culture Techniques / Q000379:methods; D003513:Cycloheximide / Q000494:pharmacology; D005456:Fluorescent Dyes; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007211:Indoles; D008113:Liver Neoplasms; D019319:Okadaic Acid / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D002273:Carcinogens; D005456:Fluorescent Dyes; D007211:Indoles; D011500:Protein Synthesis Inhibitors; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D019319:Okadaic Acid; C007293:DAPI; D014212:Tretinoin; D003513:Cycloheximide; D011065:Poly(ADP-ribose) Polymerases