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Targeting the Human Androgen Receptor Gene with Platinated Triplex-Forming Oligonucleotides.
- Source :
-
Biochemistry . 4/7/2015, Vol. 54 Issue 13, p2270-2282. 13p. - Publication Year :
- 2015
-
Abstract
- Platinum-derivatized homopyrimidine triplex-forming oligonucleotides (Pt-TFOs) consisting of 2'-0-methyl-5-methyluridine, 2'-0-methyl-5-methylcytidine, and a single 3'-N7-frcms-chlorodiam-mine platinum(ll)-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. [ABSTRACT FROM AUTHOR]
- Subjects :
- *PLATINUM
*OLIGONUCLEOTIDES
*ANDROGEN receptors
*FLUORESCENCE microscopy
*CYTOPLASM
Subjects
Details
- Language :
- English
- ISSN :
- 00062960
- Volume :
- 54
- Issue :
- 13
- Database :
- Academic Search Index
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 102343537
- Full Text :
- https://doi.org/10.1021/bi501565n