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¹ Department of Oncology, Montefiore Medical Center, Albert Einstein-Montefiore Cancer Center, Bronx, New York; ² Department of Biomedical Engineering, Columbia University, New York, New York; ³ Dharmacon Research, Lafayette, Colorado; and ⁴ Johns Hopkins University School of Public Health, Baltimore, Maryland

Requests for reprints: C.A. Stein, Department of Oncology, Montefiore Medical Center, Albert Einstein-Montefiore Cancer Center, 111 East 210 Street, Bronx, NY 10467.

In a previous study, we showed that G3139, an antisense phosphorothioate oligonucleotide that down-regulates the expression of Bcl-2 protein, did not cause chemosensitization of 518A2 melanoma cells. In this work, we show that G3139, and the 2-base mismatch, G4126, can initiate apoptosis in this and other melanoma cell lines as shown by increased cell surface Annexin V expression, typical nuclear phenotypic changes as assessed by 4',6-diamidino-2-phenylindole staining, activation of caspase-3 (but not caspase-8) and Bid, appearance of DEVDase (but not IETDase) activity, and cleavage of poly(ADP-ribose)-polymerase 1. Depolarization of the mitochondrial membrane occurs as a relatively late event. All of these processes seem to be substantially, but perhaps not totally, Bcl-2 independent as shown by experiments employing an anti-Bcl-2 small interfering RNA, which as shown previously down-regulated Bcl-2 protein expression but did not produce apoptosis or chemosensitization in melanoma cells. In fact, these G3139-induced molecular events were not dramatically altered in cells that forcibly overexpressed high levels of Bcl-2 protein. Addition of irreversible caspase inhibitors (e.g., the pan-caspase inhibitor zVAD-fmk) to G3139-treated cells almost completely blocked cytotoxicity. Examination of the time course of the appearance of caspase-3 and cleaved poly(ADP-ribose)-polymerase 1 showed that this could be correlated with the release of cytochrome c from the mitochondria, an event that begins only 4 hours after the end of the oligonucleotide/LipofectAMINE 2000 5-hour transfection period. Thus, both G3139 and cytotoxic chemotherapy activate the intrinsic pathway of apoptosis in these cells, although Bcl-2 expression does not seem to contribute strongly to chemoresistance. These findings suggest that the attainment of G3139-induced chemosensitization in these cells will be difficult.

Key Words: G3139 • apoptosis • Bcl-2 • mitochondria

Grant support: Genta.

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.

⁵ PRNewswire-FirstCall. Genta announces updated results from its phase 3 trial of Genasense for advanced melanoma; 2004. www.prnewswire.com, keyword, Genasense.

⁶ E. Anderson et al. Signatures of patterns of gene expression in prostate cancer cells after treatment with anti-bcl-2 and related phosphorothioate oligonucleotides and siRNAs, submitted for publication.

Received 7/15/04; revised 11/29/04; accepted 12/30/04.