Altered structure or expression of several cell cycle control genes is linked to malignant transformation in human cancers. Overexpression of both cyclin (especially E) and Cdc2 mRNA have been found in breast cancer cells and pathologic specimens. These findings suggest that breast cancer may provide a model system to directly test the feasibility of designing novel antisense directed reagents that target the mRNA cap of specific cell cycle genes, disrupt their expression and inhibit cell growth The m7G cap structure of eukaryotic mRNAs is required for at least three steps in gene expression. Antisense oligonucleotides that overhang in the cap region of a target mRNA can inhibit by 80-90% the specific binding of an essential protein (eIF-4E). An reIF-4E/capped mRNA test system will be used to screen novel antisense directed reagents designed to """"""""knockout"""""""" the expression of essential cell cycle control genes in breast cancer cells. The hypothesis to be tested is that specific rules in designing more effective antisense gene knockout reagents can be learned and that these reagents will disrupt the proliferation of breast carcinoma cells by causing them to become arrested at a specific point in the cell cycle. The specific objectives are: (l) To determine systematically what 3' overhanging chemistries, linked to antisense oligonucleotides, will most effectively prevent the binding of eIF-4E to the 5' cap of cyclin E Cdk2 and Cdc2 mRNAs in a in vitro model system; (2) To determine the effectiveness of the antisense directed chemistries in down regulating expression of chimeric mRNAs containing the 5' UTR of cyclin E Cdk2, and Cdc2 and a reporter molecule human growth hormone (hGH) in transiently transfected breast epithelial cells. The synthesis of hGH will be used to rapidly measure the effect that different oligonucleotides have on the expression of these chimeric target mRNAs; (3) To determine the effectiveness of anti sense directed chemistries in down regulating expression of cyclin E, Cdk2, and Cdc2 in breast carcinoma cells and (4) To purify sufficient quantities of reIF-4E to identify optimal conditions for growing high quality crystals and initiate X-ray crystallography studies. A long-range goal is to obtain detailed structural information about the interaction of eIF-4E with the m7G cap with Wayne Anderson, our crystallography colleague. This information will be applied in molecular modeling studies to design more effective therapeutics (possibly nucleotide mimetics) that prevent protein-m7G cap interactions with cell cycle mRNAs and inhibit their expression in vivo. (see Dennis Liotta's letter). These studies will test the potential of this approach to develop novel treatments for breast cancer and possibly other forms of cancer.
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