Targeting the Human Androgen Receptor Gene with Platinated Triplex-Forming Oligonucleotides

• Published in: Biochemistry (Easton) 2015-04, Vol.54 (13), p.2270-2282
• Main Authors: Graham, Mindy K, Brown, Terry R, Miller, Paul S
• Format: Article
• Language: English
• Subjects: Cell Line, Tumor - drug effects; Cross-Linking Reagents; Fluorescein - chemistry; Fluorescent Dyes - chemistry; Gene Knockdown Techniques; Glutathione - chemistry; Humans; Male; Microscopy, Fluorescence; Molecular Targeted Therapy - methods; Oligonucleotides - chemistry; Oligonucleotides - pharmacology; Organoplatinum Compounds - chemistry; Polymerase Chain Reaction - methods; Prostatic Neoplasms - drug therapy; Prostatic Neoplasms - genetics; Receptors, Androgen - chemistry; Receptors, Androgen - genetics; Receptors, Androgen - metabolism; RNA, Messenger - metabolism; Transfection - methods
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi501565n

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.