Abstract 1024: Selective inhibition of bcl-2 gene expression and leukemic cell growth by the bcl-2 quadruplex-forming sequence

Abstract

Abstract Bcl-2 (B-cell CLL/lymphoma 2), a mitochondrial membrane oncoprotein functioning as an inhibitor of apoptosis, is commonly overexpressed in a variety of hematological malignancies promoting apoptotic resistance and leading to poor patient survival. Inhibition of Bcl-2 may result in sustained regression of leukemia/lymphoma. Recently, the promoters of several cancer-related oncogenes, including Bcl-2, were found to contain sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The Bcl-2 quadruplex-forming sequence (Bcl-2 q, 23 bp) is located upstream of the P1 promoter of the Bcl-2 gene and is implicated in the regulation of Bcl-2 transcription, however, the biological role of this sequence remains unclear. We hypothesize that treatment of leukemia cells with the single-stranded, quadruplex-forming sequence (Bcl-2 q) induces cell death by inhibiting gene expression. To determine the biological role of the Bcl-2 q on leukemic cell proliferation, U937 and HL60 were treated with Bcl-2 q or the corresponding mutant sequence (MutBcl-2), which lacks runs of two or more guanines. Changes in cell proliferation were compared to levels of Bcl-2 protein expression. Quadruplex formation was confirmed by circular dichroism spectroscopy. Our results demonstrate that Bcl-2 q formed a stable parallel quadruplex structure, while the MutBcl-2 sequence did not form a quadruplex. Treatment of leukemia cells with Bcl-2 q caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days as determined by MTT assay. No change in the growth of nontransformed stromal cells occurred in response to Bcl-2 q indicating the effect is specific for malignant cells. Two additional leukemia cell lines, Raji and K562 cells demonstrated dramatic growth inhibition while three solid tumor cell lines, DU145 (prostate), A549 (lung), and SK-BR-3 (breast) showed very little growth inhibition at concentrations up to10µM. Interestingly, the degree of growth inhibition induced by Bcl-2 q correlated with baseline Bcl-2 gene expression. Confocal and flow cytometry analysis of cells treated with FITC-labeled Bcl-2 q or MutBcl-2 showed prominent uptake and nuclear localization of Bcl-2 q not MutBcl-2 after 72 h. Inhibition of cell proliferation corresponded with decreased Bcl-2 protein expression. No significant change in the cell cycle was noted after 72 h, however, induction of cell death corresponded with a decrease in mitochondrial transmembrane potential. These results demonstrate striking growth inhibition in response to the Bcl-2 quadruplex-forming sequence related to inhibition of Bcl-2 expression. This oligonucleotide shows considerable therapeutic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1024.