Inhibition of c-myc expression in cells by targeting an RNA-protein interaction using antisense oligonucleotides.
Antisense oligodeoxynucleotides (ODNs) are designed to bind to and inhibit a target mRNA. We used a novel approach for the design of ODNs to the c-myc mRNA using protein binding sites as targets for ODN action. Our strategy was to identify ODNs that could interfere with the coding region determinant-binding protein (CRD-BP), a protein that binds to the CRD region of the c-myc mRNA. Using an in vitro gel shift assay, we show that ODN molecules can occlude the CRD-BP from the mRNA. The best ODN, CRD-ODN4, was able to inhibit RNA binding of the CRD-BP by 75%. This effect was sequence-specific and concentration dependent. K562 cells treated with a 2'-O-methyl derivative of CRD-ODN4 showed a concentration-dependent decrease in both c-myc mRNA and protein levels, with a maximal 65% inhibition of protein expression at 200 nM CRD-ODN4. In contrast, a 2'-O-methyl ODN derivative targeting the translation initiation codon (antimyc-aug) reduced c-myc protein but actually increased mRNA levels, an effect resulting at least partly from stabilization of the c-myc mRNA. CRD-ODN4 treatment did not alter the c-myc mRNA half-life. CRD-ODN4 was more effective in inhibiting K562 cell growth than antimyc-aug, reducing cell number by approximately 70% after 48 h of exposure to 750 nM. The correlation between ODN effects on RNA-protein interactions in vitro and those observed in cells supports the hypothesis that CRD-ODN4 inhibits the interaction between the CRD-BP and the c-myc mRNA and that disrupting this RNA-protein interaction reduces c-myc expression in cells.
Coulis
C M
CM
Department of Pharmacology, University of Toronto, Toronto, Canada.
Lee
C
C
Nardone
V
V
Prokipcak
R D
RD
D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020014:K562 Cells; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
C406144:IGF2BP1 protein, human; D016376:Oligonucleotides, Antisense; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D016601:RNA-Binding Proteins