ABSTRACT
Most drugs used for treatment of androgen-related dermatological disorders are not completely satisfactory in terms of clinical efficacy and potential secondary effects. There is, therefore, a need for a new generation of specific antiandrogens. This paper focuses on an oligonucleotide antisense pharmacological strategy. Acceptor sites were first disclosed by mapping the human Androgen Receptor (AR) mRNA conformation using an mRNA walking approach, oligonucleotide binding, and S1 protection assays. Antisense-sensitive regions were localized by RNAse H degradation and AR in vitro translation inhibition. Oligonucleotides were then designed and assessed, in primary cultures of human hair dermal papillae and skin derived fibroblasts, for their capability to down-regulate AR expression. Some of them were able to inhibit more than 60 to 80% of the AR expression. These could be a new class of antiandrogen oligonucleotides pharmacologically active in hair and skin derived cells, suitable for the treatment of dermatological disorders.
Subject(s)
Androgen Antagonists/pharmacology , Hair/metabolism , Oligonucleotides, Antisense/pharmacology , Skin/metabolism , Androgen Receptor Antagonists , Cells, Cultured , Electrophoretic Mobility Shift Assay , Gene Expression/drug effects , Hair/cytology , Humans , Immunoblotting , RNA, Messenger/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Skin/cytology , Transcription, Genetic/geneticsABSTRACT
The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored.
