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: Mindy K Graham, Terry R Brown, Paul S Miller
• Journal: Biochemistry (Biochemistry) Vol. 54 Issue 13 Pg. 2270-82 (Apr 07 2015) ISSN: 1520-4995 [Electronic] United States
• PMID: 25768916 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• AR protein, human
• Cross-Linking Reagents
• Fluorescent Dyes
• Oligonucleotides
• Organoplatinum Compounds
• RNA, Messenger
• Receptors, Androgen
• Glutathione
• Fluorescein

Topics

• 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, pharmacology)
• Organoplatinum Compounds (chemistry)
• Polymerase Chain Reaction (methods)
• Prostatic Neoplasms (drug therapy, genetics)
• RNA, Messenger (metabolism)
• Receptors, Androgen (chemistry, genetics, metabolism)
• Transfection (methods)