Use of oligonucleotides (ODNs) to control gene expression has long fascinated researchers in basic science and medicine. Zamecnik and Stephenson provided the first hints of the therapeutic utility of antisense nucleic acids, and Zamecnik later received a Lasker prize (1996) in recognition of this work. Antisense inhibition of gene expression relies primarily on the simple rules of Watson-Crick base pairing of nucleic acids. A synthetic small single-stranded ODN (13-25 mer) that is complementary to a specific gene, via hybridizing to corresponding mRNA, inhibits the translation of that gene into a protein. Targeting gene expression at the RNA level gives cells another level of regulatory control, allowing them to turn off protein production even if RNA is abundant. If the protein product of translation were important for cell growth and/or viability, antisense inhibition of gene expression could produce a lethal phenotype. Unmodified phosphodiester ODNs are not suitable therapeutic agents because they are too readily digested by nucleases. To resolve this problem, several ODN analogs have been introduced, but phosphorothioate ODNs (PS-ODNs) have been extensively studied in various models and are now being tested in human clinical trials. Second-generation antisense ODNs that are superior to PS-ODNs have also been introduced. Hybridization of antisense ODNs to their target mRNAs can physically block the translation machinery or activate RNase H cleavage at the RNA-DNA duplex site. An extensive amount of literature points to the sequence-specific antisense mechanism of action at the single-gene level, but exploration of its effect on global gene expression in the cell have been scarce. The ability to study gene expression on a genomic scale is allowing a deeper understanding of specificity of antisense inhibition. Although concerns about non-sequence-specific antisense effects have been raised, few genome-wide studies have systematically investigated these effects. High-density cDNA microarrays enable parallel analysis of the expression of thousands of genes in a single hybridization for complex biological systems. We examined genomic effects of antisense inhibition of protein kinase A RIa expression in tumor cells using cDNA microarrays. Using in vivo tumor models of PC3M human prostate carcinoma grown in nude mice, the specificity of antisense effects on gene expression signatures was critically assessed using three oligonucleotides that differed in sequence or chemical modification: an immunostimulatory phosphorothioate oligonucleotide directed against human RIa, a second-generation immunoinhibitory RNA-DNA mixed-backbone oligonucleotide, and a non-immunostimulatory phosphorothioate oligonucleotide targeted to mouse RIa (this oligonucleotide cross-hybridizes with human RIa). Antisense treatment was found to affect one cluster, or signature, of genes involved in proliferation and another involved in differentiation. These expression signatures were quiescent and unaltered in the livers of antisense-treated animals, indicating that distinct cAMP signaling pathways regulate growth for normal cancer cells. cDNA microarray analysis of the RIa antisense-induced expression profile shows the up- and downregulation of clusters of coordinately expressed genes that define the molecular portrait of a reverted tumor cell phenotype. This global view broadens the horizons of antisense technology; it advances the promise of antisense beyond a single target gene to the whole cell and the whole organism. Along with recent rapid advances in oligonucleotide technologies-including new chemical and biological understanding of more sophisticated nucleic acid drugs, oligonucleotide-based gene silencing offers not only an exquisitely specific genetic tool for exploring basic science but an exciting possibility for treating and preventing cancer and other diseases. Our results indicate that microarray studies can facilitate the study of oligonucleotide pharmacokinetics, sequence-specificity, non-sequence-specific effects, and toxicity. Further adoption of this technology will facilitate development of nucleic acid medicines with higher target specificity and minimized side effects.
NIH ExPorter
NIH ExPorter