Unique localization of protein kinase C isozymes in T51B rat liver cells and their role in proliferation and apoptosis.
Creators: name: Al-Mazidi, Hayfa A.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 1997
Subjects: subject: Biology, Cell.

description: The regulation of cell proliferation and apoptosis in T51B rat liver cells has been investigated. These cells have proven to be an excellent in vitro model for cellular homeostasis since growth stimulation by re-addition of serum or epidermal growth factor (EGF) induces an initial burst of cell division followed by apoptosis, as determined by flow cytometry, Hoechst staining and TUNEL assay. Furthermore, flow cytometric analyses indicate that as the cells approach confluence, the percentage of apoptotic cells increases with a corresponding decrease in the percentage of the G2 cell population. The role of PKC isozymes in the regulation of cellular homoestasis in T51B cells has also been studied. Immunofluorescence and immunoblot analyses demonstrate the presence of PKC-$\alpha,$ -$\beta\sb1,$ -$\delta,$ -$\epsilon$ and -$\zeta$ in the cytosol, the plasma membrane and the nucleus. Interestingly, PKC-$\beta\sb1$ appears as a doublet in the membrane fractions which is unique for these type of cells. Furthermore, treatment of serum-stimulated cells with the phorbol ester, TPA, down regulates membrane-associated PKC-$\alpha,$ -$\delta$, -$\epsilon$ but without effect on PKC-$\zeta.$ Moreover, TPA causes an increase in the membrane and the nuclear association of PKC-$\beta\sb1$ in confluent serum-stimulated and serum-deprived cells. However, in proliferating cells (one day after plating), TPA causes a re-distribution of nuclear PKC-$\beta\sb1$ between the nucleus, the cytoplasm and the plasma membrane. Immunofluorescence analyses indicate a differential change in the subcellular localization of PKC isozymes as the cells approach confluence. PKC-$\beta\sb1$ is totally associated with the nucleus and the nuclear membrane after one day of plating (at which time most of the cells are likely in the G$\sb1$ phase or at the G$\sb1$/S boundary) and moves to the cytoplasm and the plasma membrane as the cells become confluent. IntereStingly, re-addition of serum to quiescent serum-deprived cells results in a substantial increase in the nuclear level of PKC-$\beta\sb1$ suggesting a possible involvement of nuclear PKC-$\beta\sb1$ in the proliferative signal. Moreover, in dividing T51B cells, the level of PKC-$\beta\sb1$ is much higher than that of non-dividing cells, indicating a possible role of this isozyme in the cell division. On the other hand, no significant changes in the localization of PKC-$\alpha$, -$\delta$ and -$\epsilon$ are noted. However, in the case of PKC-$\beta\sb1$ the membrane-associated level increases as the cells reach confluence. Dual immunofluorescence and Hoechst staining data clearly demonstrate a differential localization of PKC isozymes during apoptosis of T51B cells. While PKC-$\beta\sb1$ and -$\alpha$ are not detectable in apoptotic cells, the level of PKC-$\delta$ substantially increases with no change in the level of PKC-$\epsilon$ and -$\zeta.$ Furthermore, the PKC inhibitor bisindolylmaleimide (Bis) enhances apoptosis while TPA reduces the number of cells undergoing apoptosis. (Abstract shortened by UMI.)