Primary cultured murine hepatocytes but not hepatoma cells regulate the cell number through density-dependent cell death

The mechanisms by which hepatocytes regulate their cell numbers in culture have been examined. We found that when murine hepatocytes were cultured at an overconfluent stage, the number of viable cells were reduced to that of the confluent stage 48 h later by cell death. Cell death was accompanied by LDH release, and it was observed only in primary cultured hepatocytes but not in hepatoma cells. Genomic DNA analysis using electrophoresis showed that DNA fragmentation, a biochemical hallmark of apoptosis, was induced in superconfluent cultures of hepatocytes in a cell-density-dependent fashion, but not in pre-confluent cells. DNA fragmentation was rapidly induced 2 h after the beginning of the in vitro culture and continued up to 24 h later. Flow cytometry analysis demonstrated that the nuclei from the hepatocytes in a high density culture were condensed and that the DNA content was reduced. These data suggest that the mechanism of cell death is apoptosis. The DNA fragmentation seen in the high density hepatocyte culture was not observed in hepatoma cell lines. Moreover, apoptosis was induced in hepatocytes of MRL/lpr mice, suggesting that the Fas antigen was not involved in the apoptotic process. Apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, and by a calmodulin antagonist, W-7. Taken together, the results indicate that high density culture of murine hepatocytes though not hepatoma cells regulate their cell numbers by an apoptotic mechanism. The apoptosis is dependent on de novo protein synthesis and intracellular calcium metabolism.