Anti-gene Peptide Nucleic Acid (PNA) Specifically Inhibits N-myc Expression in Human Neuroblastoma Cells Leading to Persistent Cell-growth Inhibition and Apoptosis

We developed an antigene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH2 terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LIN and IMR-32 (MYCN-amplified/overexpressed); SJ-NKP and NB- 100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCNamplified/ overexpressed IMR-32 cells and MYCN-unamplified/lowexpressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Antigene PNA induced G1-phase accumulation (39–53%) in IMR-32 and apoptosis (56% annexin V–positive cells at 24 hours in IMR-32 and 22% annexin V–positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an antigene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an antigene PNA–based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.