The MLEC10 is an epithelial cell line derived from an untreated, normal C3H
/HeN mouse liver We previously demonstrated that tumorigenic variants from
this cell line produced moderately differentiated hepatocellular carcinomas
in nude mice, However, it has remained unclear whether the parental MLEC10
cells represent immortalized hepatocytes or so-called oval cells, both of
which may serve as precursors for hepatocellular neoplasms. In this study,
we performed 3-dimensional, spheroid culture of the MLEC10 cells in order t
o facilitate histological assessment of their lineage. Spheroidal aggregate
s were formalin-fixed and embedded in paraffin for routine light-microscopi
c observation of hematoxylin and eosin-stained sections. Histopathologicall
y, the MLEC10 cells were indistinguishable from immature hepatocytes and di
stinct from oval cells. At the electron-microscopic level, their hepatocyti
c nature was evidenced by bile canaliculus structures and glycogen storage.
Intriguingly, the spheroids contained fragmentary material reminiscent of
Councilman bodies, implying apoptosis of the hepatocytes. Although the cell
s significantly proliferated during the first three days of culture, apopto
tic death then resulted in a 75% decrease in viable cell number. Thereafter
, both apoptosis and cell division appeared silent, the numbers being uncha
nged. Expression of the p53 tumor suppressor gene became gradually elevated
, correlating positively with growth arrest, but negatively with apoptosis,
suggesting that the cell death occurred independently of p53, Our results
indicate that at least some liver epithelial cell lines derived from untrea
ted murine livers exhibit a hepatocytic morphology in spheroid culture. Als
o, the present culture system provides a useful tool for investigating biol
ogical phenomena, e.g. apoptosis, specifically involving liver cells, under
3-dimensional conditions.