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¹ Department of Pediatrics, University of Bologna, Sant'Orsola-Malpighi Hospital, Bologna, Italy; ² Department of Organic and Industrial Chemistry, University of Parma, Parma, Italy; and ³ Center for Cell and Gene Therapy, Texas Children's Cancer Center, Baylor College of Medicine, Houston, Texas

Requests for reprints: Andrea Pession, Unità di Terapia Cellulare, Dipartimento di Scienze Pediatriche Mediche e Chirurgiche, Policlinico Sant'Orsola-Malpighi, via Massarenti 11, 40138 Bologna, Italy. Phone: 39-051346044; Fax: 39-051346044. E-mail: pession{at}med.unibo.it

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 NH₂ 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 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 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.

Key Words: MYCN • neuroblastoma • gene amplification • antigene • peptide nucleic acid

Grant support: Ministero dell'Università a Ricerca Scientifica a Tecnologica/Fondo Integrativo Speciale per la Ricerca; University of Bologna (funds for selected research topics); Consorzio Interuniversitario di Biotecnologie; Ministero dell'Istruzione, dell'Università e della Ricerca project: Fondo per gli Investimenti della Ricerca di Base ("Novel anti-inflammatory and antitumor molecules via organic synthesis and combinatorial techniques"); and Associazione Genitori Ematologia Oncologia Pediatrica (R. Fronza and M. Franzoni).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/20/04; revised 2/11/05; accepted 3/21/05.