Down-regulation of MDM2 and activation of p53 in human cancer cells by antisense 9-aminoacridine-PNA (peptide nucleic acid) conjugates.
A series of peptide nucleic acid (PNA) oligomers targeting the mdm2 oncogene mRNA has been tested for the ability to inhibit the growth of JAR cells. The effect of these PNAs on the cells was also reflected in reduced levels of the MDM2 protein and increased levels of the p53 tumor suppressor protein, which is negatively regulated by MDM2. Initially, PNA oligomers were delivered as DNA complexes with lipofectamine, but it was discovered that PNA conjugated to the DNA intercalator 9-aminoacridine (Acr) (Acr-PNA) could be effectively delivered to JAR cells (as well as to HeLa pLuc705 cells) even in the absence of a DNA carrier. Using such lipofectamine-delivered Acr-PNA conjugates, one PNA targeting a cryptic AUG initiation site was identified that at a concentration of 2 microM caused a reduction of MDM2 levels to approximately 20% (but no reduction in mdm2 mRNA levels) and a 3-fold increase in p53 levels, whereas a 2-base mismatch control had no such effects. Furthermore, transcriptional activation by p53 was also increased (6-fold), and cell viability was reduced to 80%. Finally, this PNA acted cooperatively with camptothecin treatment both with regard to p53 activity induction as well as cell viability. Using this novel cell delivery system, we have identified a target on the mdm2 mRNA that appears sensitive to antisense inhibition by PNA and therefore could be used as a lead for further development of mdm2-targeted antisense (PNA and other) gene therapeutic anticancer drugs.
Shiraishi
Takehiko
T
Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, 2200 Copenhagen N, Denmark.
Nielsen
Peter E
PE
D000585:Aminacrine / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D016373:DNA, Antisense / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D015536:Down-Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger / Q000737:chemistry; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D000970:Antineoplastic Agents; D016373:DNA, Antisense; D009687:Nuclear Proteins; D020135:Peptide Nucleic Acids; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D000585:Aminacrine; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D002166:Camptothecin