Protein kinase C alpha (PKC alpha) is a phospholipid-dependent protein-serine/threonine kinase that plays a major role in intracellular signaling pathways associated with transformation and tumor progression. Glioblastoma multiforme (GBM) and GBM cell lines exhibit increased levels of PKC alpha compared to normal brain tissue that relates to their proliferative and invasive potential. This thesis explores the transcriptional regulation of PKC alpha and the effects of phorbol esters on PKC alpha transcription.
The activity of the PKC alpha gene promoter was assessed in U-87 GBM cells. Basal promoter activity was restricted to a region spanning -227 to +77 relative to the transcription start site. DNase I footprinting revealed multiple activator protein-2 (AP-2) binding sites and one Sp1 binding site within this region, and point mutations of two AP-2 elements resulted in a loss of DNA binding and transcriptional activation. Overexpression of Sp1 in either U-87 or insect cells increased transcription from the -227/+77 promoter region, whereas overexpression of AP-2 increased transcription only in insect cells.
Cis activation of the promoter in U-87 cells was increased by phorbol esters but not by cyclic AMP or phosphatidylinositol 3-kinase inhibitors. These results provide evidence that cis activation of the basal promoter of the human PKC alpha gene occurs through an AP-2-dependent, phorbol ester-responsive pathway, which suggests an autoregulatory manner of transcription in GBM.
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