Effects of antisense oligonucleotides directed toward dihydrofolate reductase RNA in mammalian cultured cells.

AU: Rodríguez, M., Noé, V., Alemany, C., Miralles, A., Bemi, V., Caragol, I. and Ciudad, C.J.

SO: Int. J. Cancer 81, 785-792 (1999)

The effect of incubations with antisense phosphorothioate oligonucleotides directed towards sequences of the dihydrofolate reductase (DHFR) RNA has been tested on Chinese hamster ovary cells. The selected targets were: the 5' untranslated region, the translational start, splice sites and branch point of intron 1, and the polyadenylation regions 1 and 3 of the DHFR RNA. To introduce the oligonucleotides, the cationic liposome DOTAP® was used. The most effective oligonucleotides in causing cytotoxicity were ATNL and DTNL, both directed towards the translational start site, at a range of concentrations between 1-4 µM. The minimum time for the oligonucleotide to exert its full cytotoxic effect was three days. Excess of oligonucleotide diminished the cytotoxic effect. The oligonucleotide uptake was monitored by the incorporation of [32P]- or fluorescein-labeled oligonucleotide, and was found to depend on liposome and oligonucleotide concentration, and the time of incubation. The formation of in vitro complexes between the oligonucleotide and the liposome was also studied. Cytotoxicity was observed when the oligonucleotide was incubated with cell lines containing either the endogenous gene or co-transfected DHFR minigenes. Cell incubation with ATNL caused a time-dependent decrease in the levels of DHFR mRNA and enzymatic activity. Moreover, a cell line bearing amplification at the dhfr locus was equally affected by the action of ATNL. Human hepatoma cells were also affected by treatment with the counterpart of ATNL in the human DHFR mRNA sequence. These results set the basis for a possible cancer therapy with antisense oligonucleotides using DHFR as the target.