Effects of anti-sense oligonucleotides directed toward dihydrofolate reductase RNA in mammalian cultured cells.
The effect of incubations with anti-sense phosphorothioate oligonucleotides directed toward sequences of dihydrofolate reductase (DHFR) RNA has been tested on Chinese hamster ovary cells. The selected targets were the 5'-untranslated region, the translational start, the splice sites and branch point of intron I and polyadenylation regions 1 and 3 of the DHFR RNA. To introduce the oligonucleotides, the cationic liposome DOTAP was used. The oligonucleotides most effective at causing cytotoxicity were ATNL and DTNL, both directed toward the translation-start site, at a range of concentrations between 1 and 4 microM. The minimum time for the oligonucleotide to exert its full cytotoxic effect was 3 days. Excess of oligonucleotide diminished the cytotoxic effect. Oligonucleotide uptake was monitored by the incorporation of [32P]- or fluorescein-labeled oligonucleotide and was found to depend on liposome and oligonucleotide concentrations and duration of incubation. 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. Our results set the basis for a possible cancer therapy with anti-sense oligonucleotides using DHFR as the target.
D000818:Animals; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D004337:Drug Carriers; D004353:Drug Evaluation, Preclinical; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008081:Liposomes; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012333:RNA, Messenger / Q000037:antagonists & inhibitors*; D012680:Sensitivity and Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured