Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription.
Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.
Liu
J
J
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Xu
R
R
Jin
Y
Y
Wang
D
D
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004274:DNA, Recombinant; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020014:K562 Cells; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D000090063:Proto-Oncogene Mas; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D004274:DNA, Recombinant; C000715470:MAS1 protein, human; D009841:Oligonucleotides; D010982:Platelet-Derived Growth Factor; D000090063:Proto-Oncogene Mas; D014157:Transcription Factors; D004247:DNA