Down-regulation of MDM2 and activation of p53 in human cancer cells by antisense 9-aminoacridine-PNA (peptide nucleic acid) conjugates

TY - JOUR

T1 - Down-regulation of MDM2 and activation of p53 in human cancer cells by antisense 9-aminoacridine-PNA (peptide nucleic acid) conjugates

AU - Shiraishi, Takehiko

AU - Nielsen, Peter E.

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

KW - Aminacrine/chemistry

KW - Antineoplastic Agents/chemistry

KW - Base Sequence

KW - Camptothecin/pharmacology

KW - Cell Line

KW - Cell Line, Tumor

KW - Cell Survival/drug effects

KW - DNA Damage

KW - DNA, Antisense/chemistry

KW - Down-Regulation

KW - HeLa Cells

KW - Humans

KW - Molecular Sequence Data

KW - Nuclear Proteins/genetics

KW - Peptide Nucleic Acids/chemistry

KW - Protein Transport

KW - Proto-Oncogene Proteins/genetics

KW - Proto-Oncogene Proteins c-mdm2

KW - RNA, Messenger/chemistry

KW - Transfection

KW - Tumor Suppressor Protein p53/metabolism

U2 - 10.1093/nar/gkh820

DO - 10.1093/nar/gkh820

M3 - Journal article

C2 - 15371552

SN - 0305-1048

VL - 32

SP - 4893

EP - 4902

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 16

ER -