ERRONEOUS RESULTS OF H-3 THYMIDINE INCORPORATION ARE RELATED TO POSITION OF THYMIDINE RESIDUES IN OLIGODEOXYNUCLEOTIDES

Antisense oligodeoxynucleotides (ODNs) targeted to complementary mRNA sequences have proved to be a powerful approach in assessing the funct ion and the role of unique genes in cell proliferation, differentiatio n, or transformation. Despite their importance in the development of f uture therapies, Little is known about their fate after uptake by cell s. Here, we have examined the contribution of individual nucleotide re sidues from synthetic nonspecific ODNs on assays commonly used to meas ure cell proliferation A dramatic decrease of the H-3-thymidine (H-3=T ) incorporation was obtained with nonspecific ODNs, while no effect on cell proliferation was observed as assessed by three other techniques . We demonstrate that the presence and position of thymidine in the OD Ns directly interfere with the intracellular thymidine pool, leading t o faulty data of H-3-T incorporation. As an alternative method, we use d H-3-deoxyuridine (H-3-dU), which is integrated more efficiently in D NA than thymidine. We observed that H-3-dU incorporation was also decr eased. In conclusion, caution should be exercised in the interpretatio n of the results of H-3-T and H-3-dU incorporation in the presence of oligodeoxynucleotides