Targeting the Human Androgen Receptor Gene with Platinated Triplex-Forming Oligonucleotides
| Autor: | Paul S. Miller, Mindy K. Graham, Terry R. Brown |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Male
Organoplatinum Compounds Oligonucleotides Biology Transfection Polymerase Chain Reaction Biochemistry law.invention law Cell Line Tumor Humans Molecular Targeted Therapy RNA Messenger Gene Polymerase chain reaction Fluorescent Dyes Oligonucleotide Prostatic Neoplasms Glutathione Molecular biology Androgen receptor genomic DNA Cross-Linking Reagents Microscopy Fluorescence Receptors Androgen Cytoplasm Lipofectamine Gene Knockdown Techniques Fluorescein |
| Zdroj: | Biochemistry. 54:2270-2282 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|