1. ¹Department of Pathology, University of Virginia, Charlottesville, VA, USA
2. ²Department of Pharmacology, University of Virginia, Charlottesville, VA, USA
3. ³Department of Neuroscience, University of Virginia, Charlottesville, VA, USA

Correspondence: Dr PM Martin, Department of Biology, Fisk University, Nashville, TN 37208, USA. E-mail: pmartin@fisk.edu

Received 24 December 2005; Revised 14 March 2006; Accepted 26 April 2006; Published online 10 July 2006.

Abstract

Previously, we have shown that PKC- (protein kinase C-eta) positively regulates glioblastoma proliferation and confers resistance to irradiation-induced apoptosis. In this study, we investigated the efficacy of rapamycin in inhibiting cell proliferation in two glioblastoma cell lines U-251MG (PKC- expressing) and U-1242MG (PKC- deficient) following PKC- activation. In U-251MG cells, rapamycin (10 nM) treatment was less effective as an antiproliferative agent when cells were concurrently stimulated with 10% serum and phorbol 12-myristate 13-acetate (PMA, 100 nM), a potent activator of PKC isozymes. Rapamycin-insensitive growth was owing to PKC-, as U-1242MG and U-251MG cells infected with a kinase-dead form of PKC- (U-251kr) were susceptible to rapamycin-induced inhibition of cell proliferation. Furthermore, U-251MG cells transfected with PKC- antisense oligonucleotides were sensitive to rapamycin. PKC--expressing cells stimulated with PMA maintained p70S6K phosphorylation on Thr389 and phosphorylation of rpS6 (ser235/36), suggesting p70S6K kinase activity was still intact. Inhibition of p70S6K expression with small interfering RNA oligonucleotides inhibited cell proliferation greater than 50% in the presence of a combination of PMA and serum. Additionally, p70S6K co-precipitated with PKC-, suggesting a physical interaction between PKC- and p70S6K regulates the observed phosphorylation. Taken together, these data demonstrate that rapamycin-insensitive glioblastoma proliferation involves PKC- signaling.