Abstract
BackgroundModulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells.MethodsWe screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT).ResultsWe show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406) targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512) targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping) and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone.ConclusionWe have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.
Highlights
Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs
Ten peptide nucleic acid (PNA) targeting intron2 at the 5' or 3' splice sites were tested for their ability to inhibit splicing of mdm2 premRNA
As the first in frame AUG translation start codon is located in exon3, we focused on splice interference that might result in exon3 skipping, thereby prohibiting any translation initiation from the mRNA
Summary
Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. We have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm human cancer gene in JAR cells. The mdm oncogene is amplified and/or over expressed in several cancer types [14]. This oncogene encodes a protein that negatively controls the functions of the p53 tumor suppressor protein by blocking the transactivation domain and by stimulating the degradation of p53. Targeting of mdm mRNA splicing could be an effective way of controlling and studying overall MDM2 expression and function
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