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Antisense and Sense RNA Probe Hybridization to Immobilized Crude Cellular Lysates: A Tool to Screen Growth Hormone Antagonists

Department of Assay Development & Screening, Biovitrum AB, Stockholm, Sweden; Karolinska Institutet, Department of Medical Nutrition, NOVUM, Huddinge, Sweden

Hanna Simko

Department of Assay Development & Screening, Biovitrum AB, Stockholm, Sweden

Ladan Aryan

Pia Axelsson-Lendin

Department of Biology, Biovitrum AB, Stockholm, Sweden

Joanna Chmielewska

Department of Assay Development & Screening, Biovitrum AB, Stockholm, Sweden

Agneta Mode

Karolinska Institutet, Department of Medical Nutrition, NOVUM, Huddinge, Sweden

Vendela Parrow

Department of Assay Development & Screening, Biovitrum AB, Stockholm, Sweden

The growth-promoting effect of growth hormone (GH) is primarily mediated by insulin-like growth factor–1 (IGF-1). The liver is the main source of circulating IGF-I. The authors have used rodent primary hepatocytes for studies on pharmacological intervention of IGF-I mRNA expression. A 96-well nonradioactive IGF-1 mRNA quantification assay was developed, based on the hybridization of sense and antisense RNA probes, to replicate membranes with crude hepatocyte lysates. The sense hybridization was used as an internal standard. The antagonistic properties of a set of GH-receptor binding compounds were evaluated. Two compounds were found to down-regulate IGF-I mRNA. Effects due to metabolic inhibition or toxicity were excluded using a cell proliferation assay. To investigate potential unspecific transcriptional effects, the mRNA levels of the housekeeping genes, ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined. Two other GH-regulated genes, cytochrome P450 2C12 (CYP2C12) and a rat homologue to the human 1B-glycoprotein (A1BG), were quantified by RNase protection assays and found to be down-regulated, confirming the antagonistic property of 1 compound. In conclusion, a direct filter hybridization assay of hepatocyte lysates using nonradioactive sense and antisense probes can be used for quantitative mRNA measurements and could constitute a valuable tool in screening for pharmacologically active compounds.

Key Words: IGF-1 mRNA • GH antagonist • hepatocytes • secondary screening

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