Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis.
We developed an anti-gene 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 NH(2) terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP 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 MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed 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. Anti-gene PNA induced G(1)-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 anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA-based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.
Tonelli
Roberto
R
Unità di Terapia Cellulare, Dipartimento di Scienze Pediatriche Mediche e Chirurgiche, Policlinico Sant'Orsola-Malpighi, via Massarenti 11, 40138 Bologna, Italy. pession@med.unibo.it
Purgato
Stefania
S
Camerin
Consuelo
C
Fronza
Raffaele
R
Bologna
Fabrizio
F
Alboresi
Simone
S
Franzoni
Monica
M
Corradini
Roberto
R
Sforza
Stefano
S
Faccini
Andrea
A
Shohet
Jason M
JM
Marchelli
Rosangela
R
Pession
Andrea
A
D017209:Apoptosis / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism*; D020135:Peptide Nucleic Acids / Q000627:therapeutic use*; D016271:Proto-Oncogene Proteins c-myc / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D020135:Peptide Nucleic Acids; D016271:Proto-Oncogene Proteins c-myc