Analysis of the regulatory mechanisms that control Discs Large tumour suppresor gene expression

CAVATORTA, ANA L.; ADRIANA ANGELICA GIRI; BANKS, LAWRENCE; GARDIOL, DANIELA

Praga

Congreso; XXIII International Papillomavirus Conference.; 2006

High risk HPVs play a causal role in the development of cervical cancer. h-Dlg (Dlg) oncosuppressor is associated with cell polarity and tissue architecture, and HPV E6 oncoproteins target Dlg for ubiquitin-mediated proteolysis. We showed that the expression of Dlg is altered during the progression of high-risk HPV-related cervical lesions and that Dlg levels are highly reduced in invasive carcinoma. E6 phosphorylation by protein kinase A (PKA) reduces E6 capacity for binding and degrading Dlg. We analyzed PKA activity by immunohistochemistry and demonstrated variations in PKA activity during the development of cervical tumors that could be altering E6 phosphorylation status and hence, Dlg levels. However, the mechanisms controlling Dlg expression at the transcriptional level are still unknown. In order to understand the basic Dlg transcriptional regulation we proposed the cloning of Dlg promoter region and its functional analysis. By bioinformatic analysis, a potential Dlg promoter region was identified on the 5´flanking region of Dlg ORF, contained into a P1-derived artificial chromosome. Fragments of this region were amplified, cloned into a reporter vector and the promoter activity was determined in cultured cells by luciferase assays. Using bioinformatic tools, binding sites for different transcription factors were found in this region. The involvement of two tumour-related factors in Dlg regulation is currently being analysed.