[논문]Inhibition of Human Telomerase Activity by Antisense Phosphorothioate Oligonucleotides Encapsulated with the Transfection Reagent, FuGENE™6, in HeLa Cells

TAMURA, YUTAKA; TAO, MAOZUAM; MIYANO-KUROSAKI, NAOKO; TAKAI, KAZUYUKI; TAKAKU, HIROSHI

doi:10.1089/oli.1.2000.10.87

Inhibition of Human Telomerase Activity by Antisense Phosphorothioate Oligonucleotides Encapsulated with the Transfection Reagent, FuGENE™6, in HeLa Cells

Antisense & nucleic acid drug development, v.10 no.2, 2000년, pp.87 - 96

TAMURA, YUTAKA , TAO, MAOZUAM , MIYANO-KUROSAKI, NAOKO , TAKAI, KAZUYUKI , TAKAKU, HIROSHI

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Telomerase, a ribonucleoprotein, synthesizes telomeric repeats (TTAGGG) onto the ends of chromosomes to maintain the constant length of the telomere DNA, and its activity is detectable in approximately 85%-90% of primary human cancers. Thus, it is postulated that human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphorothioate oligonucleotides (S-ODN) were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The S-ODN were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 (Boehringer Mannheim, Mannheim, Germany) was used to enhance the cellular uptake of the oligonucleotides in cell cultures. The S-ODN encapsulated with FuGENE6 clearly inhibited telomerase activity in HeLa cells and showed sequence-specific inhibition. The encapsulated S-ODN-3 with a 19-nucleotide, (nt) chain length had inhibitory effects similar to those of the 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 telomerase activity. However, apoptotic HeLa cell death was not associated with telomerase inhibition. Furthermore, the encapsulated S-ODN did not appear to be cytotoxic in terms of the cell growth rate. The oligonucleotides encapsulated with the transfection reagent had enhanced cellular uptake, and cytoplasmic 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 by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes and is appropriate for use in vitro and in vivo.

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Inhibition of Human Telomerase Activity by Antisense Phosphorothioate Oligonucleotides Encapsulated with the Transfection Reagent, FuGENE™6, in HeLa Cells

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