C-Myc, a key regulator of cell cycle and proliferation, is commonly overexpressed in leukemia and associated with poor prognosis. Conventional antisense oligonucleotides targeting c-myc may attenuate leukemic cell growth, however, are poorly taken into cells, rapidly degraded, and have unwanted effects on normal cells. The c-myc promoter contains a guanine-rich sequence (PU27), capable of forming quadruplex (four-stranded) DNA, which may negatively regulate c-myc transcription, however, its biological significance is unknown. We show that treatment of leukemia with an oligonucleotide encoding the genomic PU27 sequence induces cell cycle arrest and death by oncotic-necrosis due to PU27-mediated suppression of c-myc mRNA/protein expression. Furthermore, PU27 is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on normal cells. Suppression of c-myc expression by PU27 caused significant DNA damage, cell and mitochondrial swelling, and membrane permeability, characteristic of oncotic-necrosis. Induction of oncosis caused mitochondrial dysfunction, depletion of cellular ATP levels and enhanced oxidative stress. This novel anti-leukemic strategy addresses current concerns of oliginucleotide therapeutics including problems with uptake, stability, and unintentional effects on normal cells and is the first report of selective cancer cell killing by a genomic DNA sequence.
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.
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
Vascular endothelial growth factor (VEGF), commonly overexpressed in a variety of malignancies including lung cancer, is a key regulator of angiogenesis promoting tumor survival, growth, and metastasis. The promoters of several cancer-related genes, including VEGF, contain disproportionate sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The VEGF quadruplex-forming sequence (VEGFq), a polyG/polyC tract located in the proximal promoter region upstream of the transcription initiation site, has been implicated in the regulation of both basal and inducible VEGF expression. However, the biological role of this sequence remains unclear. We hypothesize that treatment of A549 non-small lung cancer cells with VEGFq induces cell death by inhibiting gene expression. To determine the biological role of VEGF on lung cancer cell proliferation, A549 cells were treated with VEGFq or the corresponding mutant sequence (MutVEGF), which lacks runs of two or more guanines. Changes in cell proliferation were compared to levels of VEGF protein expression. Quadruplex formation was confirmed by circular dichroism spectroscopy. Our results demonstrate that VEGFq formed a stable parallel quadruplex structure, while the MutVEGF sequence did not form a quadruplex. Treatment of A549 cells with VEGFq caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM) as determined by MTT assay. No change in the growth of nontransformed fibroblast cells occurred in response to VEGFq indicating the effect is specific for malignant cells. Confocal and flow cytometry analysis of cells treated with FITC-labeled VEGFq or MutVEGF showed prominent uptake and nuclear localization of VEGFq not MutVEGF after 72 h. Inhibition of cell proliferation corresponded with decreased VEGF protein expression. No specific change in the cell cycle was measured after 72 h. These results demonstrate striking growth inhibition of A549 cells in response to the VEGF quadruplex-forming sequence related to inhibition of VEGF expression and suggest significant therapeutic implications for the treatment of non-small cell lung cancer. 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 2949.
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