Antisense Oligodeoxyribonucleotides Suppress Hematologic Cell Growth Through Stepwise Release of Deoxyribonucleotides

Abstract

Antisense oligodeoxyribonucleotides (ODNs) are now being extensively investigated in an attempt to achieve cell growth suppression through specific targeting of genes related to cell proliferation, despite increasing evidence of non-antisense cytotoxic effects. In the context of anti-BCR/ABL antisense strategies in chronic myeloid leukemia, we have re-examined the antiproliferative effect of phosphodiester and phosphorothioate ODNs on the leukemic cell line BV173 and on CD34+ bone marrow cells in liquid culture. The 3′ sequences of the ODNs determine their effect. At concentrations of 10 μmol/L (for phosphorothioate ODNs) or 25 μmol/L (for phosphodiester ODNs), all the tested ODNs exert an antiproliferative activity, except those that contain a cytosine residue at either their two most terminal 3′ positions. We show that this antiproliferative effect is due to the toxicity of the d-NMPs (5′ monophosphate deoxyribonucleosides), the enzymatic hydrolysis products of the ODNs in culture medium. The toxicity of the d-NMPs on hematologic cells depends on their nature (d-CMP [2′deoxycytidine 5′-monophosphate] is not cytotoxic), on their concentration (d-GMP [2′-deoxyguanosine 5′-monophosphate], TMP [thymidine 5′-monophosphate], and d-AMP [2′-deoxyadenosine 5′-monophosphate] are cytotoxic at concentrations between 5 and 10 μmol/L), and on the coincident presence of other d-NMPs in the culture medium (d-CMP neutralizes the toxicity of d-AMP, d-GMP, or TMP). The antiproliferative activity of ODNs is thus restricted to conditions where the 3′ hydrolysis process by exonucleases generates significant amounts of d-NMPs with a low proportion of d-CMP. Our results reveal a novel example of a nonantisense effect of ODNs, which should be taken into account when performing any experiment using assumed antisense ODNs.

Reduction of metal adducts in oligonucleotide mass spectra in ion‐pair reversed‐phase chromatography/mass spectrometry analysis

“…[1] Chemical modifications to the phosphodiester backbone are commonly incorporated into synthetic oligonucleotides to increase stability and safety in vivo against endo-and exonucleases as well as improve efficacy through increased cellular uptake and binding properties. [1,8,9] While the synthesis process of oligonucleotides is well controlled, several factors can alter the end product. Quality of starting materials can impact the purity of products while random insertion/deletions can result in improper sequence generation.…”

Circular Antisense Oligonucleotides Inhibit Growth of Chronic Myeloid Leukemia Cells

“…Although the 36-nucleotide abl-directed circle was inactive, the 34-nucleotide bcr-directed circle had activity. At 13 ,uM, the bcr circle reduced the saturating cell number by 68% (note the log scale) (Fig. 2).…”

“…Many studies used phosphorothioates that can have nonspecific effects. Some investigators claim the mechanism is other than an antisense one, i.e., a sequence-dependent mechanism unrelated to the bcr-abl sequence (12,13). Others have found no effect on the bcr-abl protein level (14) or direct inhibition of the bcr-abl-associated protein-tyrosine kinase activity (15,16).…”

True

“…389 Sequence dependent but non-antisense effects can probably be caused by the interaction of specific nucleotide sequences such as TAT and GCC with proteins. 390 Vaerman et al 391 demonstrated a novel non-antisense mechanism of ASODNs. They showed that the antiproliferative effect of ASODNs is correlated with the absence of a cytosine residue at the 3Ј position.…”