Protein kinase C (PKC) designates a family of
kinases that regulate many essential functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation and excessive activity leads to abnormal or uncontrolled cell growth. Recent reports indicate that
malignant glioma cell lines express 100 to 1000-fold higher PKC activity when compared to non-neoplastic astrocytes. This high activity correlates well with the proliferation of
tumor cells in vitro. We recently reported on the anti-proliferative properties of selective PKC inhibitors on the growth of U-373MG human
astrocytoma cell line, and their ability to block
mitogen-activated
protein (MAP)
kinase pathway activated by
substance P (SP)
neuropeptide receptor signaling via a PKC-dependent mechanism. Therefore, inhibiting PKC activity by selective PKC inhibitors may present a promising approach for improving astroglial
brain tumor therapy. For this purpose, we constructed a high throughput model cell system to evaluate the efficacy of PKC inhibitors. This system is based on the measurement of light production in U-373MG cells stably transfected with the
luciferase reporter gene whose expression depends on the transcriptional activation of GAL4-Elk1 fusion
protein by
enzyme components of the MAP
kinase pathway and the upstream activation of PKC (PKC activation-->MAP
kinases-->GAL4-Elk1 phosphorylation-->
luciferase expression-->
luciferase activity). In brief, we have demonstrated that the PKC activator
12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced
luciferase activity in this cell system is mediated via the MAP
kinase pathway and can be blocked in the presence of MEK1 selective inhibitors (
PD 098059 or
U0126). We also demonstrated that TPA-induced
luciferase activity in U-373MG stable clones can be blocked by PKC inhibitors (
CGP 41251,
Go 6976, and
GF 109203X) in a concentration dependent manner. In contrast,
epidermal growth factor (
EGF)-induced
luciferase activity, which is independent of PKC activation (Ras-->Raf-1-->MEK1-->MAP
kinases-->GAL4-Elk1 phosphorylation-->
luciferase expression-->
luciferase activity) can only be blocked using a selective
EGF receptor inhibitor (
AG 1478). In conclusion, we have constructed a model cell system for the high throughput screening and identification of PKC inhibitors potentially active against
astrocytoma cells in culture.