Rapid screening method for antisense oligonucleotides against human growth factor receptor p185(erbB-2).
The aim of this study was the development of an indirect cell proliferation assay as screening tool for antisense oligonucleotides. Unmodified and phosphorothioate-modified oligonucleotides with different amounts of sulfur in the DNA backbone were examined for biologic activity. The human growth factor receptor p185(erbB-2) was chosen as cellular target. High-level expression of this protein can be related to an early event in tumor development and cell proliferation. We correlated the expression of p185(erbB-2) with the cell proliferation of BT-474. Additionally a control cell line (MCF-7) with very low p185(erbB-2) expression was cultivated. Antisense oligonucleotides were transfected as a liposome formulation (Lipofectin), GIBCO-BRL, Eggenstein, Germany). Cell count was correlated with a total protein quantification assay (BCA method). Stability against nuclease digestion was determined with a DNase I assay. Sequence-specific antisense effects on the p185(erbB-2) protein level were determined by Western blot. An antisense phosphorothioate oligonucleotide was identified to inhibit the cell proliferation in comparison to a random control and a negative control oligonucleotide sequence. The comparison of fully thioated, partly thioated, and unmodified oligonucleotides verified the correlation between the enzymatic stability and the biologic activity of the different modifications. Using the unstable oligonucleotides, more treatments were necessary to achieve an antiproliferative effect. In our study, the indirect proliferation assay was found to be a reliable and potent tool for an antisense oligonucleotide screening by targeting the p185(erbB-2) protein.
Rohmann
Anke
A
Institute for Pharmaceutical Biology and Institute for Pharmaceutical Technology, Johann Wolfgang Goethe-University, 60439 Frankfurt am Main, Germany.
Lochmann
Dirk
D
Weyermann
Jörg
J
Bertling
Wolf M
WM
Dingermann
Theodor
T
Zimmer
Andreas
A
D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000032:analysis* / Q000235:genetics / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*
D016376:Oligonucleotides, Antisense; D011804:Quinolines; D018719:Erb-b2 Receptor Tyrosine Kinases; C047117:bicinchoninic acid