Sequence-dependent cytotoxicity of second-generation oligonucleotides - PubMed (original) (raw)

. 2004 Dec 16;32(22):6585-94.

doi: 10.1093/nar/gkh997. Print 2004.

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Sequence-dependent cytotoxicity of second-generation oligonucleotides

Denis Drygin et al. Nucleic Acids Res. 2004.

Abstract

In this study, we have examined the potential of second-generation antisense chimeric 2'-O-(2-methoxy)ethyl/DNA phosphorothioate oligonucleotides (ONs) to affect cell growth through non-antisense mechanisms. Evaluation of a series of ONs demonstrated that only a small number were cytotoxic at concentrations close to those required for antisense activity. Toxicity of the ONs appeared to be sequence dependent and could be affected by base and backbone modifications. Caspase-3 activation occurs with some ONs and it is most likely secondary to necrosis rather than apoptosis, since cells treated with toxic ONs did not show chromatin condensation, but did exhibit high-extracellular lactate dehydrogenase activity. Caspase-3 activation does not correlate with and appears not to be required for the inhibition of cell proliferation. Toxicity was only observed when ONs were delivered intracellularly. The mechanism by which one of the most cytotoxic ON produces cytotoxicity was investigated in more detail. Treatment with the cytotoxic ON caused disruption of lysosomes and Pepstatin A, a specific inhibitor of aspartic proteases, reduced the cytotoxicity of the ON. Reduction of lysosomal aspartic protease cathepsin D by prior treatment with cathepsin D-specific antisense ON did not attenuate the cytotoxicity, suggesting that other aspartic proteases play a crucial role in the cellular proliferation inhibition by ONs.

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Figures

Figure 1

Figure 1

Dose–response of gene downregulation versus cytotoxicity of ONs in A549 cells. Cells were transfected with various amounts of either ISIS 126965 or ISIS 129696 for toxicity studies or ISIS 116847 for PTEN downregulation analysis (Lipofectin-mediated transfection or electroporation). Forty-eight hours after transfection, the cell number was measured with CyQUANT cell proliferation kit and PTEN mRNA level was determined using Taqman quantitative RT–PCR. Values for UTC are designated as 100%.

Figure 2

Figure 2

Time course of cell growth and caspase-3 activation after transfection with ONs. A549 cells were treated with Opti-Mem alone, Lipofectin (9 μg/ml) in Opti-MEM or with 0.3 μM of ISIS 126965 or ISIS 129696 in the presence of Lipofectin (3 μg/ml per 100 nM ON). Cells were collected at 12, 24, 36, 48, 74 and 98 h after the beginning of the transfection. (A) Cell number was measured with CyQUANT cell proliferation kit. (B) Caspase-3 activity was measured using HTS caspase-3 activity assay. The data are presented in relative fluorescence units (RFU).

Figure 3

Figure 3

Treatment of A549 cells with staurosporine or ISIS 126965 releases cathepsin D from lysosomes. A549 cells (A) untreated or treated with (B) 250 nM of staurosporine, (C) 0.3 μM of ISIS 130358 in the presence of Lipofectin (3 μg/ml per 100 nM ON) or (D) 0.3 μM of ISIS 126965 in the presence of Lipofectin (3 μg/ml per 100 nM ON), were simultaneously stained 24 h after transfection with propidium iodide and anti-cathepsin D FITC-labeled antibody and examined by fluorescent microscopy.

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