The long-term goal is to develop a triplex DNA-based anti-gene strategy for breast cancer. This proposal is aimed to facilitate this approach by utilizing polyamine analogs to stabilize triplex DNA. Polyamines are small organic molecules present in all cells. Our hypothesis is that the selectivity of polyamine analogs in stabilizing triplex DNA could be effectively utilized to enhance the therapeutic potential of triplex forming oligonucleotides. Structure-activity relationships in the action of polyamine analogs on triplex DNA will be elucidated by physical chemical studies and this information will be used to examine the effects of triplex forming oligonucleotides on targeted genes. Determination of melting temperatures, binding constants and thermodynamic parameters will be used to assess polyamine effects. Cell culture studies will examine the effects of polyamine-complexed oligonucleotides on the expression of two oncogenes, c-myc and HER2 in MCF-7 and MDA-MB-231 breast cancer cell lines. These genes are overexpressed in breast tumors compared to adjacent normal tissues. The effects of polyamine analog.oligonucleotide complexes will be further examined on cell cycle, cell proliferation, and polyamine metabolism. Specificity of the triplex DNA mechanism of polyamine. oligonucleotide complexes will be examined by measuring oligonucleotide uptake.and stabilization, DNase I hypersensitivity assay, and the non-specific effects of these agents on gene expression. These studies will reveal new strategies to stabilize triplex DNA in vitro and in vivo and advance the application of polyamine analogs for breast cancer therapy.
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