Development of Triplex-Forming Oligonucleotides to Inhibit Expression of the c-myc Oncogene in Breast Cancer Cells
Abstract
Triple helix-forming oligonucleotides (TFOs) directed to regulatory sequences in gene promoters can selectively block transcription. We are investigating TFO-mediated reduction of c-myc oncogene expression as a means of decreasing breast tumor growth. We designed a novel parallel/antiparallel TFO (Myc-GTC) that had high binding affinity in vitro, but required modifications for increased stability in cells. To further optimize TFO activity we investigated effects of conjugation with the anthracycline antibiotic daunomycin (Dnm), which intercalates into double-stranded DNA. We synthesized short Dnm-conjugated TFOs corresponding to parallel and antiparallel elements of Myc-GTC. Electrophoretic mobility shift and footprinting assays showed that Dnm-TFOs formed highly specific, stable triplex. Dnm-TFOs inhibited binding to the target duplex of transcription-activating proteins present in MCF-7 nuclear extracts. Fluorescence microscopy determined that Dnm-TFOs were efficiently internalized by MCF-7 cells. At nanomolar concentrations, a Dnm-TFO inhibited expression in MCF-7 and MDA-MB-23 1 cells of a luciferase gene under the control of the c-myc promoter. These results suggest that Dnm- conjugated TFOs are effective in cells, and that Dnm may stabilize binding of the full-length parallel/antiparallel TFO in cells.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 01, 2002
- Accession Number
- ADA406166
Entities
People
- Carlo V. Catapano
- Eileen M. Mcguffie
Organizations
- Medical University of South Carolina