The broad objective of this study is to determine the efficacy of utilizing protein kinase C (PKC) as a molecular therapeutic target for inhibiting the growth of glioblastoma multiforme (GM), the most common brain tumor in adults. GM is highly refractory to all therapeutic modalities, and unlike normal brain tissue, a high percentage of GM as well as GM-derived cell lines demonstrate high levels and activity of PKCalpha. We have found that expression of the antisense PKCalpha cDNA attenuates serum-dependent growth in vitro and the tumorigenicity in vivo of GM cell line U-87, suggesting that PKCalpha plays a positive regulatory role in the proliferation of these cells. Many primary and established GM cell lines overexpress growth factor receptors such as PDGF and EGF and exhibit an autocrine mechanism of growth, which appears to be related to high levels of PKC. Therefore, this proposal will address the regulatory role of PKCalpha and other PKC isoforms in the growth factor-dependent autocrine growth of GM, and the use of PKC as a molecular target for therapy.
The Specific Aims of this proposal will be to determine: 1) whether specific PKC isoforms are regulated transcriptionally or post- transcriptionally in GM cells by growth factors, 2) whether tumor growth can be selectively blocked by a PKC isoformspecific antisense cDNA or an antisense oligodeoxynucleotide (ODN) in vitro and in vivo, 3) which components of the growth factor signaling pathway are affected by inhibition of specific PKC isoforms, and conversely, which PKC isoforms are modulated by tyrosine phosphorylation by growth factor receptor tyrosine kinases or receptor-associated tyrosine kinases, and 4) the cis regulatory elements in PKCalpha and other PKC isoforms which are found to be transcriptionally upregulated in GM cells by growth factors. Preliminary studies have shown that PKCalpha is upregulated transcriptionally in U-87 cells by PDGF-BB, but not by PDGF-AA. The 5'- untranslated genomic sequence of PKCalpha has been cloned from a human genomic library and will be used to determine the transcription start site(s) and to characterize the regulatory elements in the promoter region. These studies will determine the importance of PKC as a selective therapeutic target in GM and its role in growth factor-dependent tumor growth.
