Department of Neurosurgery , University of Pittsburgh School of Medicine , Pennsylvania .
J Neurosurg 73 : 98-105 ( 1990)
Abstract
To evaluate the role of protein kinase C-mediated pathways in the proliferation of malignant gliomas , this study examined the effect of a protein kinase C ( PKC)-activating phorbol ester ( 12-O-tetradecanoyl-13-phorbol acetate or TPA ) and a protein kinase C inhibitor ( polymyxin B ) on deoxyribonucleic acid ( DNA ) synthesis of malignant glioma cells in vitro .
A serum-free chemically defined medium , MCDB 105 , was employed for all studies .
Two established human malignant glioma cell lines ( T98G and U138 ) , two rat glioma lines ( 9L and C6 ) , and two low-passage human glioma lines ( obtained from surgical specimens ) were studied .
With the exception of the C6 line , all tumors responded in a dose-dependent fashion to nanomolar concentrations of TPA with a median effective dose that varied from 0.5 ng/ml for the U138 glioma to 1 ng/ml for the T98G glioma .
At optimal concentrations ( 5 to ng/ml ) , TPA produced a two- to five-fold increase in the rate of DNA synthesis ( p less than 0.05 ) as assessed by incorporation of H-thymidine .
However , TPA had no additive effect on the mitogenic response produced by epidermal growth factor ( EGF ) or platelet-derived growth factor ( PDGF ) .
Inhibition of PKC using the antibiotic polymyxin B ( 20 micrograms/ml ) abolished the TPA-induced mitogenic response in the five responsive lines tested .
In two tumors ( U138 and 9L ) , polymyxin B also eliminated EGF- , PDGF- , and serum-induced DNA synthesis as well as abolishing baseline DNA synthesis .
These cells remained viable , however , as assessed by trypan blue exclusion ; after removal of polymyxin B from the medium , they were able to resume DNA synthesis in response to TPA and serum .
In the three other tumors ( T98G and the two low-passage human glioma lines ) , growth factor-induced and serum-induced DNA synthesis were inhibited by approximately 25% to 85% .
It is concluded that PKC-mediated pathways affect DNA synthesis in the human malignant glial tumors studied .
The response of the glioma cells to TPA is similar to the responses seen in fetal astrocytes , but differs significantly from those reported for normal adult glial cultures .
Because the response of the 9L glioma to TPA is similar to the responses seen in the human tumors , the 9L rat glioma model may prove useful for examining the role of PKC-mediated pathways in controlling glioma growth in vivo .