Introduction G-quadruplexes (G-quad) are DNA secondary structures formed by stacked G-tetrads frequently located in telomeres and promoter regions of proto-oncogenes. Recently, two G-rich sequences, canine kit1 (d_kit1) and kit2 (d_kit2), folding into G-quad, have been identified in canine c-KIT promoter. Accordingly, the aim of the present study was to test a possible small molecule inhibitor known to decrease significantly target gene expression by stabilizing KIT1 and KIT2 G-quad structures. Materials and Methods The constitutive gene expression of c-KIT and other proto-oncogenes (BCL2, VEGFα, VEGFR2, KRAS, TERT) mRNA was measured in canine mast cell tumor (MCT) cell line C2 by quantitative RT-PCR (qPCR). Therefore, the G-quad ligand 50% inhibitory concentration (IC50) was determined by using the Alamar Blue cytotoxicity test. Finally, its time- and dose-dependent transcriptional effects upon c-KIT and other target genes were evaluated by using qPCR. Results In C2 cell line, target genes were shown to be constitutively expressed and measurable up to 96 hours of culture. The IC50 value of G-quad ligand was shown to be 1,37 µM and, at lower cytotoxic concentration, it significantly down-regulated c-KIT mRNA levels (after 24h hours of incubation). A similar effect, although of low intensity, was noticed for BCL2. Conclusions The G-quad candidate ligand seems to be a promising candidate for targeting KIT-dependent tumors such as MCT. However, such an assumption needs to be confirmed with further molecular studies. Comparative studies about the efficacy of this and other G-quad ligands in blocking human kit G-quadruplex are currently underway. Supporting grants: University of Padua (CPDA114388)

Targeting canine c-KIT promoter by a candidate DNA G-quadruplex ligand

GIANTIN, MERY;ZORZAN, ELEONORA;DA ROS, SILVIA;PALUMBO, MANLIO;SISSI, CLAUDIA;DACASTO, MAURO
2015

Abstract

Introduction G-quadruplexes (G-quad) are DNA secondary structures formed by stacked G-tetrads frequently located in telomeres and promoter regions of proto-oncogenes. Recently, two G-rich sequences, canine kit1 (d_kit1) and kit2 (d_kit2), folding into G-quad, have been identified in canine c-KIT promoter. Accordingly, the aim of the present study was to test a possible small molecule inhibitor known to decrease significantly target gene expression by stabilizing KIT1 and KIT2 G-quad structures. Materials and Methods The constitutive gene expression of c-KIT and other proto-oncogenes (BCL2, VEGFα, VEGFR2, KRAS, TERT) mRNA was measured in canine mast cell tumor (MCT) cell line C2 by quantitative RT-PCR (qPCR). Therefore, the G-quad ligand 50% inhibitory concentration (IC50) was determined by using the Alamar Blue cytotoxicity test. Finally, its time- and dose-dependent transcriptional effects upon c-KIT and other target genes were evaluated by using qPCR. Results In C2 cell line, target genes were shown to be constitutively expressed and measurable up to 96 hours of culture. The IC50 value of G-quad ligand was shown to be 1,37 µM and, at lower cytotoxic concentration, it significantly down-regulated c-KIT mRNA levels (after 24h hours of incubation). A similar effect, although of low intensity, was noticed for BCL2. Conclusions The G-quad candidate ligand seems to be a promising candidate for targeting KIT-dependent tumors such as MCT. However, such an assumption needs to be confirmed with further molecular studies. Comparative studies about the efficacy of this and other G-quad ligands in blocking human kit G-quadruplex are currently underway. Supporting grants: University of Padua (CPDA114388)
2015
Proceedings of the European Society of Veterinary Oncology Annual Congress
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3171564
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