Oligonucleotide-based knockdown technologies: antisense versus RNA interference.

The postgenomic era is characterized by an almost intimidating amount of information regarding the sequences and expression of previously unknown genes. In response, researchers have developed an increasing interest in functional studies. At the start of such a study, one may have little more than sequence information and bioinformatic annotation. The next step is to hypothesize a potential role in the context of a cell. Testing of the hypothesis needs to be fast, cheap, and applicable to a large number of genes. Knockdown methods that rely on binding of antisense oligonucleotides to mRNA combined with a subsequent functional assay in cell culture fulfil these requirements: sequence information is sufficient for synthesis of active inhibitors. Depending on the in vitro model chosen, knockdown of gene expression can be achieved with medium or even high throughput. The two most popular methods of knockdown in cell culture are the use of antisense oligonucleotides that rely on ribonuclease H (RNAse H)-dependent cleavage of mRNA, and RNA interference triggered by small double-stranded RNA molecules. Both methods act in a sequence-specific manner and can give efficient knockdown. In both cases, researchers struggle with nonspecific "off-target" effects and the difficulty of site selection. Studies that compare the methods differ in their judgment as to which method is superior.

C-Raf controlled pathways in the protection of tumor cells from apoptosis.

The Raf serine-threonine kinase is upregulated in many human tumors and plays a pivotal role in tumor cell proliferation and survival. Abrogation of c-Raf expression by specific antisense oligonucleotides (Raf-AS-ODN) efficiently blocks tumor cell growth and induces apoptosis in human cancer cells. The signaling pathways and molecular mechanisms c-Raf utilizes to mediate the survival of tumor cells are, however, not well understood. Here we show that apoptosis triggered by Raf depletion cannot be overcome by ectopic Bcl-2 expression and occurs in the absence of cytochrome c release, arguing against a direct impact of c-Raf on mitochondrial pathways of apoptosis regulation. We also show that c-Raf depletion leads to a clearly decreased expression of different epidermal growth factor (EGF) receptor ligands, suggesting that the autocrine stimulation of an EGF receptor-mediated survival pathway might be involved in the blockade of tumor cell apoptotis by c-Raf.

In vivo pro-apoptotic and antitumor efficacy of a c-Raf antisense phosphorothioate oligonucleotide: relationship to tumor size.

Previously, we have shown that a phosphorothioate antisense oligonucleotide (ODN) targeted against c-raf RNA (ISIS5132; cRaf-AS) induces apoptosis in human tumor cells. We now show that the same ODN also efficiently triggers apoptosis in human tumor xenografts in nu/nu mice. Although cRaf-AS showed a clearly inhibitory effect on the growth of established tumors (approximately 150 mm3) compared to a mismatched control ODN (MM), tumor progression was not prevented. This correlated with a partial refractoriness of the tumor to cRaf-AS-induced cell killing, which seemed to be due to an inhomogeneous and inefficient penetration of the ODN into the tumor tissue rather than cellular resistance. In agreement with this conclusion, we found that growth of small tumors (<50 mm3) was completely inhibited concomitantly with an accumulation of the ODN throughout the tumor. These data show that the cRaf-AS is a highly efficacious antitumor agent, provided accessibility into the tumor tissue is warranted, and suggest that PS-AS-ODN treatment may be particularly useful in an adjuvant setting.