3 '-end conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: Synthesis and anti-ras activity in radiation-resistant cells

Activation of the ras oncogene has been implicated in many types of human t umors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridizati on to complementary mRNA sequences. To minimize toxicity associated with al l-phosphorothioated ODNs and improve cellular uptake, we used partially pho sphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C-16). The PPS ODNs are protected against degradation by PS internucleotide Linkages at both the 3'- and 5'-ends and additionally stab ilized at internal pyrimidine sites, which are the major sites of endonucle ase cleavage. Here we show that anti-ras PPS-C-16 ODN retains the high sequ ence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 syn thesis with minimal toxicity even without the use of a cellular uptake enha ncer. Moreover, treatment of T24, a radiation-resistant human tumor cell li ne that carries a mutant ras gene, with anti-ras PPS-C-16 ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demon strate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intrat umoral injection of anti-ras PPS-C-16 ODN and radiation treatment. These fi ndings indicate the potential of this combination of antisense and conventi onal radiation therapy as a highly effective cancer treatment modality.