968-26 In Vitro Antiproliferative Effect of Antisense Oligonucleotides: Are the Effects Specific and Reproducible?

• Published in: Journal of the American College of Cardiology 1995-02, Vol.25 (2), p.241A-241A
• Main Authors: Guzman, Luis A., Poptic, Earl J., DiCorleto, Paul E., Topol, Eric J.
• Format: Article
• Language: English
• ISSN: 0735-1097; 1558-3597
• DOI: 10.1016/0735-1097(95)92460-M

The involvement of proto-oncogenes in cell proliferation is well documented. One of these, the oncogene c-myb is a DNA-binding protein that regulates cell growth and differentiation. In-vitro studies have shown marked inhibition of smooth muscle cell (SMC) proliferation using antisense (AS) c-myb oligonucleotides. The purpose of this study was to determine whether the in-vitro anti proliferative effect of AS oligonucleotides is specific and reproducible. Rat aortic SMC were plated at a 1:3 split ratio in 1% fetal bovine serum (FBS), and made quiescent 24 hr later by changing the medium to 1% control process serum replacement II. Cells were stimulated with PDGF (2 ng/ml), and at the same time, oligonucleotides were added. Cell proliferation was determined by measuring 3H-Thymidine uptake at 18–22 hr. To compare the specificity of the antiproliferative effects. 20 μM of AS c-myb and several control oligos were used. These included (Batch A): 18-mer sense (5) c-myb. 19-mer mouse antisense and four-base mismatch AS to IL-lβ, and 18- and 16-mer human AS to CML. To determine the reproducibility of the AS effect, the same 18-mer AS c-myb and scrambled (Sc) lS-mer from another source (B) were used under exactly the same conditions. To further evaluate the reproducibility of the AS effect, two different preparations (Batch C) of the same 18-mer AS and S c-myb from the initial manufacturer were tested under exactly the same culture conditions. This study shows: 1) that antisense oligonucleotides produce a non-specific in-vitro antiproliferative effect and 2) the in-vitro results may vary with respect to source and batches of oligonucleotides. The lack of consistency in results raises significant questions about clinical therapeutic potential.