Phorbol 12-myristate 13-acetate and serum synergize to promote rapamycin-insensitive cell proliferation via protein kinase C-eta.
Previously, we have shown that PKC-eta (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-eta expressing) and U-1242MG (PKC-eta deficient) following PKC-eta 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-eta, as U-1242MG and U-251MG cells infected with a kinase-dead form of PKC-eta (U-251kr) were susceptible to rapamycin-induced inhibition of cell proliferation. Furthermore, U-251MG cells transfected with PKC-eta antisense oligonucleotides were sensitive to rapamycin. PKC-eta-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-eta, suggesting a physical interaction between PKC-eta and p70S6K regulates the observed phosphorylation. Taken together, these data demonstrate that rapamycin-insensitive glioblastoma proliferation involves PKC-eta signaling.
Martin
P M
PM
Department of Pathology, University of Virginia, Charlottesville, VA, USA. pmartin@fisk.edu
Aeder
S E
SE
Chrestensen
C A
CA
Sturgill
T W
TW
Hussaini
I M
IM
D015153:Blotting, Western; D002273:Carcinogens / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects*; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005909:Glioblastoma / Q000201:enzymology / Q000473:pathology*; D006801:Humans; D047468:Immunoprecipitation; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D044967:Serum; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002273:Carcinogens; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; C081975:protein kinase C eta; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D020123:Sirolimus