Antisense and sense RNA probe hybridization to immobilized crude cellular lysates: a tool to screen growth hormone antagonists.

The growth-promoting effect of growth hormone (GH) is primarily mediated by insulin-like growth factor- I (IGF- I). 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 rnRNA expression. A 96-well nonradioactive IGF- I inRNA 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. P-actin and -lyceraidehyde-3-phosphate dehydrogenase (GAPDH), were determined. Two other GH-regulated genes, cytochroine P450 2C12 (CYP2C 12) and a rat homologue to the human alpha 1B-glycoprotein (A1BG) were quantified by RNase protection assays and found to be down-regulated, confirming the antauonistic 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. (Journal of Bioniolecular Screening 2005:260-269).