26:T4ac,

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 (~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.

27:T4dd,

Oligodeoxyribonucleotides (ODNs) are currently employed to switch-off genes selectively routinely in the laboratory practice. The drawback of ODN application is that they have been often reported to elicit non-antisense effects by different mechanisms. Recently, it has been shown that double-stranded DNA oligonucleotides (30-mers) with protruding ends activate p53 in a cell-free system. In a previous work, we described that simple addition to the culture medium of heterogeneous DNA combined with cationic lipids culminated in inhibition of mitosis and induction of apoptosis. Here, we report that the same effects are achieved by lipotransfecting cultured cells with phosphorodiester ODNs (30-mers). Such effects of ODN were mediated by a non-antisense mechanism that required the wild-type form of the p53 oncosuppressor protein and was dependent on ODN concentration. Mitosis inhibition and apoptosis induction appeared to be determined by the 3′ and 5′ free ends of ODNs, which activated p53 independently from their sequence. Most probably, this mechanism is analogous to that evoked by genotoxic agent-induced DNA damage or by lipotransfecting cells with heterogeneous DNA.

28:T427,

Several lipophilic prodrugs of oligonucleotides (T₁₂ and T₂₀) bearing enzymolabile protecting groups and labeled with fluorescein were synthesized. Their cellular uptake was studied by three different approaches using fluorescence: fluorescence microscopy, flow cytometry and spectrofluorometry. The corresponding pro-oligonucleotides (pro-oligos) were rapidly and efficiently taken up by HeLa cells and were found homogeneously in the cytoplasm and in the nucleus. The uptake was proportional to their relative lipophilicity and likely proceeded through a passive diffusion mechanism. Uptake followed a dose-response curve. This pro-oligo approach led to a 2-log increase of uptake in comparison with a T₂₀ phosphorothioate oligonucleotide. Finally, an intracellular concentration of pro-oligo was estimated between 4 and 6 μM for an external concentration of 1 μM and up to 27 μM for an incubation at 10 μM.

29:T6e4,