ELUCIDATION OF GENE-FUNCTION USING C-5 PROPYNE ANTISENSE OLIGONUCLEOTIDES

Identification of human disease-causing genes continues to be an inten se area of research. While cloning of genes may lead to diagnostic tes ts, development of a cure requires an understanding of the gene's func tion in both normal and diseased cells. Thus, there exists a need for a reproducible and simple method to elucidate gene function. We evalua te C-5 propyne pyrimidine modified phosphorothioate antisense oligonuc leotides (ONs) targeted against two human cell cycle proteins that are aberrantly expressed in breast cancer: p34(cdc2) kinase and cyclin B1 . Dose-dependent, sequence-specific, and gene-specific inhibition of b oth proteins was achieved at nanomolar concentrations of ONs in normal and breast cancer cells. Precise binding of the antisense ONs to thei r target RNA was absolutely required for antisense activity. Four or s ix base-mismatched ONs eliminated antisense activity confirming the se quence specificity of the antisense ONs. Antisense inhibition of p34(c dc2) kinase resulted in a significant accumulation of cells in the Gap 2/mitosis phase of the cell cycle in normal cells, but caused little e ffect on cell cycle progression in breast cancer cells. These data dem onstrate the potency, specificity, and utility of C-5 propyne modified antisense ONs as biological tools and illustrate the redundancy of ce ll cycle protein function that can occur in cancer cells.