The overall goal of this proposal is to develop a means of specifically inhibiting the expression of the dihydrofolate reductase (DHFR) and multidrug resistance (Mdr1) genes which code for enzymes involved in the drug resistant phenotype. We will study the ability of triplex forming oligonucleotides and oligonucleotide targeted intercalating agents to bind to specific DNA sequences in the DHFR and Mdr1 promoters, to prevent regulatory protein binding, and to inhibit transcription of these genes in a specific manner. The extensive previous experience of our laboratory in the characterization of DNA binding drugs as selective inhibitors of gene expression will serve as a basis for the proposed work.
The specific aims are: 1. To determine the ability of oligonucleotides targeted to the Sp1 binding sites of the Mdr1 promoter to form triplex DNA in vitro, and to determine the effect of triplex formation on Sp1 binding and in vitro transcriptional activity of these sequences. 2. To determine whether triplex forming oligonucleotides targeted to the Sp1 binding sites of the DHFR and Mdr1 genes can inhibit Mdr1 and DHFR promoter function and gene expression in drug resistant cells which overexpress each of these genes. 3. To synthesize and determine the in vitro binding specificity and affinity of oligonucleotide targeted intercalating agents to target sequences in the DHFR and Mdr1 promoters. 4. To determine the ability of DHFR and Mdr1 oligonucleotide targeted intercalating compounds to inhibit expression of the DHFR and Mdr1 genes in whole cells and to reverse the drug resistant phenotype of cells overexpressing the DHFR and Mdr1 genes. The accomplishment of these specific aims will allow us to provide important new information about gene modulation. These experiments will have obvious clinical utility and may allow us to devise new approaches with which to reverse the drug resistant phenotype.
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| Blume, S W; Guarcello, V; Zacharias, W et al. (1997) Divalent transition metal cations counteract potassium-induced quadruplex assembly of oligo(dG) sequences. Nucleic Acids Res 25:617-25 |
| Blume, S W; Gee, J E; Shrestha, K et al. (1992) Triple helix formation by purine-rich oligonucleotides targeted to the human dihydrofolate reductase promoter. Nucleic Acids Res 20:1777-84 |
