Abstract 3: Induction of mitochondrial autophagy in leukemia 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. Inhibition of Bcl-2 may result in sustained regression of leukemia/lymphoma. The promoters of several cancer-related oncogenes, including Bcl-2, 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 negative regulation of Bcl-2 transcription, however, the biological role of this sequence remains unclear. We hypothesize that treatment of leukemia cells with an oligonucleotide encoding Bcl-2 q induces cell death by inhibiting Bcl-2 gene expression. To determine the biological role of the Bcl-2 q on leukemic cell proliferation, U937 cells were treated with Bcl-2 q or the corresponding mutant sequence (MutBcl-2), which lacks runs of two or more guanines. Our results demonstrate that Bcl-2 q formed a stable parallel quadruplex structure and showed remarkable serum and intracellular stability. Treatment of leukemia cells with Bcl-2 q caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM), which was not a result of cell cycle arrest. No change in growth of non-transformed stromal cells occurred in response to Bcl-2 q indicating the effect is specific for malignant cells. 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 in contrast to MutBcl-2. Inhibition of cell proliferation corresponded with decreased Bcl-2 protein expression and increased expression of pro-apoptotic proteins Bax and Bak. This resulted in decreased mitochondrial transmembrane potential and significant autophagic degradation of mitochondria, characteristic of mitophagy. Induction of mitophagy was associated with increased Beclin-1 and LC3B expression and disruption of Bcl-2/Beclin-1 interaction. These results demonstrate striking growth inhibition in response to Bcl-2 q related to inhibition of Bcl-2 expression and induction of mitochondrial autophagy and shows considerable therapeutic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3. doi:10.1158/1538-7445.AM2011-3