RESUMEN
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 reexamined 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 micromol/L (for phosphorothioate ODNs) or 25 micromol/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 micromol/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.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Leucemia/patología , Oligonucleótidos Antisentido/farmacología , División Celular/efectos de los fármacos , División Celular/genética , Células Madre Hematopoyéticas/citología , Humanos , Leucemia/genética , Oligonucleótidos Antisentido/genética , Tionucleótidos/genética , Tionucleótidos/farmacología , Células Tumorales CultivadasRESUMEN
We have examined the effect of BCR/ABL junctional antisense phosphodiester oligodeoxyribonucleotides (ODNs) on BV173 and other chronic myeloid leukemia (CML) cell lines. Various control ODNs were used to understand the mechanism of the observed antiproliferative effect. Not only the antisense ODNs but also several control ODNs inhibit the proliferation of the leukemic cell lines. All the ODNs that inhibit the cell proliferation share a TAT consensus sequence at their 3' end. A 1-base mismatch within this consensus sequence abolishes the antiproliferative effect. Mismatches of several bases at any other position within the sequence of the active ODNs do not suppress the observed effect. Similar experiments on normal or CML CD34+ cell fraction led to the same observations. We conclude that the antiproliferative effect of the phosphodiester BCR/ABL antisense ODNs cannot be attributed to an antisense mechanism but rather to a nonelucidated effect of a 3' terminal TAT sequence. This effect is not CML specific.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Inhibidores de Crecimiento/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Leucemia/patología , Células Madre Neoplásicas/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Secuencia de Bases , División Celular/efectos de los fármacos , Secuencia de Consenso , Humanos , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacocinética , Células Tumorales Cultivadas/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
In the last few years, a variety of DNA-based human leukocyte antigen (HLA) typing methods have emerged, revealing the extreme polymorphism of HLA genes. This polymorphism makes it difficult for a clinical laboratory to establish the best HLA typing strategy. In this study we have compared two techniques for performing HLA-DRB typing: a commercial rapid assay based on the polymerase chain reaction (PCR) followed by reverse dot-blot hybridization of the PCR products (the Inno-LiPA assay), and a method based on PCR followed by restriction fragment length polymorphism analysis. We found that both methods provide reliable results with a high rate of concordance (97%) and that Inno-LiPA is convenient for large-scale routine typing. However, if a high-resolution allelic typing is required, each method lacks accuracy but using them in association improves the accuracy of the results.