Antigene and antiproliferative effects of a c-myc-targeting phosphorothioate triple helix-forming oligonucleotide in human leukemia cells.
The c-myc gene is frequently deregulated and overexpressed in human cancers, and strategies designed to inhibit c-myc expression in cancer cells may have considerable therapeutic value. The purpose of the present work was to characterize the antigene and antiproliferative activity of a triple helix-forming oligonucleotide (TFO) targeted to a homopurine-homopyrimidine sequence in the P2 promoter of the c-myc gene. The TFO was synthesized with phosphorothioate (PS) internucleotide linkages to confer resistance to intra- and extracellular nucleases. This property is required of oligonucleotides designed for in vivo testing and therapeutic applications. The PS-TFO was found to form triplex DNA with affinity and specificity comparable with that of the corresponding phosphodiester TFO, as shown by gel mobility shift and footprinting assays. Fluorescence microscopy and polyacrylamide gel analysis showed that the fluorescein-labeled PS-TFO accumulated in nuclei of CEM leukemia cells and remained intact for at least 72 h. Incubation of CEM cells with PS-TFO reduced c-myc RNA and protein levels. A single exposure of leukemia cells to the PS-TFO was sufficient to induce dose-dependent growth inhibitory effects. Growth inhibition correlated with accumulation of cells in S phase and with induction of cell death by apoptosis. The PS-TFO was also effective in other leukemia and lymphoma cell lines. Control oligonucleotides had minimal effects in all assays. These data indicate that the c-myc-targeted PS-TFO is an effective antigene and antiproliferative agent, with potential for testing in vivo as a novel approach to cancer therapy.
Department of Experimental Oncology and Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, USA.
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McGuffie
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Pacheco
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Carbone
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D000941:Antigens / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D003287:Contrast Media / Q000494:pharmacology; D004247:DNA; D018076:DNA, Complementary / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D019793:Fluorescein / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000235:genetics*; D008223:Lymphoma / Q000188:drug therapy / Q000235:genetics; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000493:pharmacokinetics / Q000494:pharmacology*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000276:immunology*; D012313:RNA / Q000378:metabolism; D016196:S Phase / Q000187:drug effects; D012997:Solvents / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000941:Antigens; D003287:Contrast Media; D018076:DNA, Complementary; D009841:Oligonucleotides; D016271:Proto-Oncogene Proteins c-myc; D012997:Solvents; C051802:triplex DNA; D012313:RNA; D004247:DNA; D019793:Fluorescein; D004121:Dimethyl Sulfoxide