title: Targeting the human androgen receptor gene with platinated triplex-forming oligonucleotides.
abstract: Platinum-derivatized homopyrimidine triplex-forming oligonucleotides (Pt-TFOs) consisting of 2'-O-methyl-5-methyluridine, 2'-O-methyl-5-methylcytidine, and a single 3'-N7-trans-chlorodiammine platinum(II)-2'-deoxyguanosine were designed to cross-link to the transcribed strand at four different sequences in the human androgen receptor (AR) gene. Fluorescence microscopy showed that a fluorescein-tagged Pt-TFO localizes in both the cytoplasm and nucleus when it is transfected into LAPC-4 cells, a human prostate cancer cell line, using Lipofectamine 2000. A capture assay employing streptavidin-coated magnetic beads followed by polymerase chain reaction (PCR) amplification was used to demonstrate that 5'-biotin-conjugated Pt-TFOs cross-link in vitro to their four designated AR gene targets in genomic DNA extracted from LAPC-4 cells. Similarly, the capture assay was used to examine cross-linking between the 5'-biotin-conjugated Pt-TFOs and the AR gene in LAPC-4 cells in culture. Three of the four Pt-TFOs cross-linked to their designated target, suggesting that different regions of the AR gene are not uniformly accessible to Pt-TFO cross-linking. LAPC-4 cells were transfected with fluorescein-tagged Pt-TFO or a control oligonucleotide that does not bind or cross-link to AR DNA. The levels of AR mRNA in highly fluorescent cells isolated by fluorescence-activated cell sorting were determined by RT-qPCR, and the levels of AR protein were monitored by immunofluorescence microscopy. Decreases in mRNA and protein levels of 40 and 30%, respectively, were observed for fluorescein-tagged Pt-TFO versus control treated cells. Although the levels of knockdown of AR mRNA and protein were modest, the results suggest that Pt-TFOs hold potential as agents for controlling gene expression by cross-linking to DNA and disrupting transcription.
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205, United States.
authors.forename: Mindy K
authors.initials: MK
authors.lastname: Graham
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205, United States.
authors.forename: Terry R
authors.initials: TR
authors.lastname: Brown
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205, United States.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Miller
mesh_terms: D045744:Cell Line, Tumor / Q000187:drug effects; D003432:Cross-Linking Reagents; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D055785:Gene Knockdown Techniques; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D058990:Molecular Targeted Therapy / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry* / Q000494:pharmacology; D009944:Organoplatinum Compounds / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: C410798:AR protein, human; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D009944:Organoplatinum Compounds; D012333:RNA, Messenger; D011944:Receptors, Androgen; D005978:Glutathione; D019793:Fluorescein