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 acridineoligonucleotide conjugates 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, Our extensive previous 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 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 acridine-oligonucleotide conjugates targeted to the Sp1 binding sequences in the DHFR and Mdr1 promoters. 4. To determine the ability of acridine-oligonucleotide conjugates to form triplex DNA structures in vivo. 5. To determine the ability of DHFR and Mdr1 acridine-oligonucleotide conjugates to inhibit expression of the DHFR and Mdr1 genes in whole cells and the ability of the Mdr1 targeted compound to inhibit the induced overexpression of the Mdr1 gene in whole animals. 6. To determine the ability of acridine-oligonucleotide conjugates 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.
| Stewart, Delisha A; Xu, Xiaohua; Thomas, Shelia D et al. (2002) Acridine-modified, clamp-forming antisense oligonucleotides synergize with cisplatin to inhibit c-Myc expression and B16-F0 tumor progression. Nucleic Acids Res 30:2565-74 |
| Stewart, D A; Thomas, S D; Mayfield, C A et al. (2001) Psoralen-modified clamp-forming antisense oligonucleotides reduce cellular c-Myc protein expression and B16-F0 proliferation. Nucleic Acids Res 29:4052-61 |
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| Kim, H G; Miller, D M (1995) Inhibition of in vitro transcription by a triplex-forming oligonucleotide targeted to human c-myc P2 promoter. Biochemistry 34:8165-71 |
