Inhibition of human telomerase activity by antisense phosphorothioate oligonucleotides encapsulated with the transfection reagent, FuGENE (TM) 6, in HeLa cells

Telomerase, a ribonucleoprotein, synthesizes telomeric repeats (TTAGGG) ont o the ends of chromosomes to maintain the constant length of the telomere D NA, and its activity is detectable in approximately 85%-90% of primary huma n cancers. Thus, it is postulated that human telomerase might be associated with malignant tumor development and could be a highly selective target fo r antitumor drug design. Antisense phosphorothioate oligonucleotides (S-ODN ) were investigated for their abilities to inhibit telomerase activity in t he HeLa cell line. The S-ODN were designed to be complementary to nucleotid es within the RNA active site of telomerase. As a transfection reagent, FuG ENE(TM)6 (Boehringer Mannheim, Mannheim, Germany) was used to enhance the c ellular uptake of the oligonucleotides in cell cultures. The S-ODN encapsul ated with FuGENE(TM)6 clearly inhibited telomerase activity in HeLa cells a nd showed sequence-specific inhibition. The encapsulated S-ODN-3 with a 19- nucleotide, (nt) chain length had inhibitory effects similar to those of th e 21-mer and 23-mer S-ODN sequences (S-ODN-4 and 5), but the 15-mer and 17- mer S-ODN sequences (S-ODN-1 and 2) failed to satisfactorily prevent telome rase activity. However, apoptotic HeLa cell death was not associated with t elomerase inhibition. Furthermore, the encapsulated S-ODN did not appear to be cytotoxic in terms of the cell growth rate. The oligonucleotides encaps ulated with the transfection reagent had enhanced cellular uptake, and cyto plasmic and nuclear localizations were observed. However, weak fluorescent signals were observed within the cytoplasms of HeLa cells treated with the free S-ODN-3. Thus, the activities of the S-ODN were effectively enhanced b y using the transfection reagent. The transfection reagent, FuGENE(TM)6, ma y thus be a potentially useful delivery vehicle for oligonucleotide-based t herapeutics and transgenes and is appropriate for use in vitro and in vivo.