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Departments of ¹ Medicine, ² Pharmacology, and ³ Biomedical Engineering, and ⁴ Mailman School of Public Health, Columbia University, New York, NY, and ⁵ Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD

Requests for reprints:C. A. Stein, Albert Einstein Montefiore Cancer Center, Dept. of Oncology, III E. 210 St., Bronx, NY 10467. Phone: (718) 920-8980; Fax: (718) 652-4027. E-mail: cstein{at}montefiore.org

G3139 is an 18-mer phosphorothioate oligodeoxyribonucleotide, which is targeted to the initiation codon region of the bcl-2mRNA. Although treatment of PC3 prostate cancer cells with G3139, which contains two CpG motifs, causes a dramatic decrease in bcl-2 protein expression after 3 days, it did not result in significant cellular apoptosis, as it does in many other cell lines. The absence of apoptosis was demonstrated by the absence of pro-caspase 3 cleavage products and of Annexin V cell surface expression. In addition, ATP production and the mitochondrial membrane potential _m were preserved. Despite this, G3139 significantly inhibited the rate of cellular proliferation in complete media and blocked cloning in soft agar. G4232, a variant of G3139 that down-regulates bcl-2 expression to the same extent but has both CpG cytidines C5 methylated, was only minimally antiproliferative. A series of mismatched G3139-related oligomers were synthesized that could also substantially down-regulate bcl-2 protein expression, but only if the CpG motifs were preserved, demonstrating the presence of additional non-antisense mechanisms. G3139 caused production of reactive oxygen species in growth-arrested cells and oxidation of nuclear guanosine to 8-hydroxy-2'-deoxyguanosine, as determined by 1F7 monoclonal antibody staining. Bromodeoxyuridine incorporation studies demonstrated that G3139 induced a G₁-S entry block and an intra-S-phase block in PC3 cells that persisted as long as 3 days. This finding coincides with the observation that expression of several proteins encoded by S-phase genes, including c-myb and poly(ADP-ribose) polymerase, were significantly reduced. These results illustrate the complexity of the mechanism of action of G3139 in PC3 cells.

Received 2/24/03; revised 6/12/03; accepted 7/18/03.

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