Induction of apoptosis in normal cultured rat hepatocytes and in Hep3B, a human hepatoma cell line |
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Authors: | C Lamboley A-F Bringuier G Feldmann |
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Institution: | (1) Laboratoire de Biologie Cellulaire, Unité INSERM 327, Faculté de Médecine Xavier Bichat, Université Paris 7-Denis Diderot, Paris, France |
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Abstract: | 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 β1 (TGF-β1), 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-β1; 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-β1, 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.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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Keywords: | apoptosis hepatic cells culture conditions |
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