Aberrant expression of Platelet-derived growth factor A (PDGF-A) and PDGF receptor-β (PDGFR-β) play critical roles in the angiogenesis and proliferation of several malignancies. In this dissertation I explore the transcriptional regulatory role of the Gquadruplex- forming regions in the promoters of human PDGF-A and PDGFR-β, and identify new targets for developing small molecules to modulate their expression in tumors. For PDGF-A promoter, our studies focus on two essential nuclease hypersensitive elements, NHE(PDGF-A) and 5´-end far upstream 5´-SHS. The structural aspects of the intramolecular G-quadruplexes formed in NHE(PDGF-A) and the ligands to stabilize these secondary DNA structures have been investigated by using singlestranded and duplex DNA of the NHE(PDGF-A). We demonstrate that the G-quadruplexinteractive compound, TMPyP4, can selectively inhibit the basal promoter activity of PDGF-A, suggesting that the NHE(PDGF-A) G-quadruplex acts as a repressor in PDGF-A transcription. We also found that the 5´-SHS G-rich strand oligomer can invade the NHE(PDGF-A) and form a unique three-stranded complex in supercoiled plasmids, which is facilitated by potassium ions and TMPyP4. Therefore, we propose a novel molecular mechanism for transcriptional silencing of the NHE(PDGF-A) by 5´-SHS in the PDGF-A promoter, in that the formation of G-quadruplex in the NHE(PDGF-A) provides a platform for the G-rich strand of 5´-SHS to invade and form a partial duplex DNA with the C-rich strand of the NHE(PDGF-A), resulting in displacement of hnRNP K and thus transcription silencing. Prior to the studies describe here, the promoter of human PDGFR-β had not been identified. Herein, we have cloned and characterized the first functional promoter of human PDGFR-β gene. A crucial highly GC-rich region (NHE(PDGFR-β)) in the human PDGFR-β promoter has been identified by its hypersensitivity to the S1 nuclease. Further studies demonstrate that stable G-quadruplex structures can form in the G-rich strand of NHE(PDGFR-β). The G-quadruplex-interactive molecule, telomestatin, can selectively stabilize G-quadruplexes formed in the human PDGFR-β promoter and inhibit its expression in Daoy cells. On the basis of these results, we propose that ligandmediated stabilization of the G-quadruplex structure in the proximal promoter region of human PDGF-A or PDGFR-β can be used to modulate the expression of these protooncogenes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.