|Regulated expression of a dominant negative protein kinase C ε mutant inhibits the proliferation of U-373MG human astrocytoma cells|
Authors: Taraneh R. Sharif, Nobuyuki Sasakawa, Mohammed Sharif
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
The tight regulation of protein kinase C (PKC) activity is crucial for maintaining normal cell proliferation. Excessive PKC activity leads to uncontrolled growth and malignant transformation. It has been reported that the activity of PKC is higher in astroglial cell lines than in normal astrocytes. Previously, we demonstrated that PKCε is overexpressed in astroglial cell lines and in samples from primary high-grade astroglial brain tumors. Because there are no PKCε isozyme-specific inhibitors, we chose a genetic approach to confirm that PKCε is a potential target for inhibiting astroglial cell proliferation. We regulated the expression of a dominant negative PKCε mutant (PKCε 1-401 encoding amino acid 1-401) in U-373MG human astrocytoma cells using a tetracycline-regulated expression vector and established stable clones. Induction of expression of the dominant negative PKCε mutant by the addition of doxycycline, a tetracycline derivative, completely blocked proliferation of U-373MG cells in proliferation and clonogenic assays. Although the induction of the dominant negative PKCε mutant did not markedly affect mitogen-induced tyrosine phosphorylation of the mitogen-activated protein (MAP) kinases, it inhibited the induction of c-Fos protein expression by substance P (SP) and fetal bovine serum (FBS). These results clearly show that the expression of dominant negative PKCε leads to the inhibition of U-373MG cellular proliferation demonstrating that this isozyme may be a potential therapeutic target for astroglial brain tumors.