AS1411 is a 26 base unmodified DNA aptamer which is currently in a Phase II clinical trial for the treatment of acute myeloid leukemia (AML) and will shortly enter a Phase II trial in renal cell carcinoma (RCC). AS1411 has previously been shown to have cytostatic (a result of arrest in S phase of the cell cycle) and cytotoxic effects on tumor cells, but not normal cells. AS1411 binds to nucleolin, which is overexpressed in the cytoplasm and on the surface of tumor cells. Treatment of MV4-11 cells (an AML-derived cell line) with low micromolar concentrations of AS1411 results in cell growth inhibition and a reduction in viability; this effect becomes apparent after 48 hours incubation. Co-immunoprecipitation of nucleolin and AS1411 from MV4-11 cells treated with radiolabeled AS1411 confirmed previous work that AS1411 binds to nucleolin. In addition, confocal microscopy showed that nucleolin is present in the cytoplasm and the plasma membrane of MV4-11 cells. Incubation of these cells with AS1411 decreased nucleolin mRNA levels, and nucleolin protein was decreased in both cytoplasm and nucleus to approximately 10% of control levels after 96 hours. The reduction in nucleolin levels started to appear at around 48 hours and may reflect cells arresting in S phase of the cell cycle. Nucleolin binds to an ARE sequence in the 3′-untranslated region of Bcl-2 mRNA resulting in stabilization of the mRNA. Using gel shift assays with a radiolabeled fragment of Bcl-2 mRNA we show here that AS1411 interferes with this binding. In addition, levels of Bcl-2 mRNA in MV4-11 cells decrease to 10% of control levels after 48 hours incubation with AS1411 and the level of Bcl-2 protein also decreases in comparison to control cells. Finally, mononuclear cells isolated from blood samples of AML patients were incubated with AS1411 for 96 hours and viability determined. In all samples, AS1411 caused a reduction in cell viability at low micromolar concentrations, similar to those required to kill cell lines. This work therefore demonstrates that AS1411 has both cytostatic and cytotoxic effects on AML cells and underpins the dosing regime of AS1411 in the ongoing AML clinical trial.
Disclosure:Employment: David Jones is an employee of Antisoma Research Limited. Ownership Interests: David Jones has stock options in Antisoma. Research Funding: Daniel Fernandes has a research grant from Antisoma.
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