Abstract C19: Targeting the human androgen receptor gene with platinum-conjugated triplex-forming oligonucleotides

Abstract

Abstract Androgen receptor (AR) gene expression is required to maintain prostrate tumor cell growth and inhibition of this gene would be expected to prevent prostate tumor cell proliferation. The human AR gene contains numerous homopurine tracts that can serve as binding sites for triplex-forming oligonucleotides (TFOs), a class of oligonucleotides that have significant potential as antigene agents. We have developed a novel type of TFO that is comprised of 2′-O-methylribonucleotides and contains a N7-trans-diamminechloroplatinumdeoxygu anosine residue at either its 5′- (Pt-mrTFO) or 3′-end (mrTFO-Pt). When bound to a homopurine tract in double-stranded DNA, the platinum group of the Pt-mrTFO can cross-link with suitably positioned guanine residues adjacent to the TFO binding site, a reaction that irreversibly anchors the TFO to its DNA target. Such cross-link formation would be expected to interfere with transcription and/or replication of the target DNA. We have identified 27 different homopurine tracts in the transcribed region of the human AR gene whose sequences are compatible with cross-link formation by Pt-mrTFOs or mrTFO-Pt. In preliminary studies, we have synthesized a mrTFO-Pt whose sequence (mr−TTTCTTCTTTTCTCTCTTGPt; T = 2′-O-methylribothymine; C = 2′-O-methylribo-5-methylcytosine; GPt = N7-trans-diammin echloroplatinumdeoxyguanosine) targets a 19 nucleotide homopurine sequence on the transcribed strand in the 2nd intron of the human AR gene. At physiological pH and temperature, the mrTFO lacking GPt forms a stable triplex (melting temperature = 45.2°C) with a 24 bp DNA duplex whose sequence corresponds to the TFO binding site in the AR gene. The mrTFO-Pt cross-links to this duplex to the extent of 80% under physiological conditions as shown by gel electrophoretic mobility shift assays. To test its ability to inhibit transcription, the mrTFO-Pt was cross-linked (91%) to a firefly luciferase reporter plasmid that contained a single copy of the AR target gene sequence placed in the transcribed region between the plasmid's CMV promoter and luciferase reporter gene. The cross-linked plasmid was co-transfected with a Renilla luciferase reporter plasmid (to monitor transfection efficiency) into HeLa cells in culture and luciferase activity was determined 24 hrs after transfection using a dual luciferase assay (Promega Inc.). Luciferase expression decreased 43% compared to that from a non-cross-linked control plasmid. The mrTFO-Pt was found to be resistant to degradation when incubated at 37.C for a period of 24 hrs with cell culture medium containing 10% fetal calf serum. Transfection of a human prostate cancer cell line (LAPC4) with a Lipofectamine complex of the fluorescein-labeled TFO (Fl-mrTFO-Pt*, where Pt* is an inert N7-diethelentraimineplatinum-deoxyguanosine residue) resulted in nuclear localization of the label in 69% of the cells as revealed by fluorescence microscopy of the live cells. Cell viability as monitored by an MTT assay decreased 24% when LAPC4 cells were treated for 24 hrs with a Lipofectamine complex containing 0.2 μg of the mrTFO-Pt compared to cells that were treated with 0.2 μg of scrambled inert mrTFO-Pt*/Lipofectamine complex under the same conditions. The ability of the mrTFO-Pt to cross-link effectively with its target, inhibit DNA transcription and decrease the viability of LAPC4 cells in culture suggest that these compounds may have potential as therapeutic agents to disrupt AR signaling in androgen-resistant prostate cancer. Citation Format: Mindy Kim Graham, Terry R. Brown, Paul S. Miller. Targeting the human androgen receptor gene with platinum-conjugated triplex-forming oligonucleotides [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C19.