QUANTITATIVE CHANGES OF THE 2 MAJOR AGNOR PROTEINS, NUCLEOLIN AND PROTEIN-B23, RELATED TO STIMULATION OF RDNA TRANSCRIPTION

The relationship between the amount of the two major AgNOR proteins, n ucleolin and protein B23, and the rate of ribosomal RNA (rRNA) synthes is was studied in cortisol-treated and regenerating rat hepatocytes af ter partial hepatectomy. In both experimental models the synthesis of rRNA was greatly stimulated, but only in regenerating hepatocytes was the in creased synthesis associated with cells entering the mitotic cy cle. Nucleolin and protein B23 were identified on SDS-polyacrylamide g els of nuclear proteins transferred to nitrocellulose and detected (1) by immunoreaction with specific monoclonal antibodies revealed by sec ond antibodies coupled to peroxidase followed by chemiluminescence or (2) by the silver-staining procedure for AgNOR proteins. Nucleolin and protein B23 were then quantified by computerized densitometric analys is of the immunolabeling signals or the silver-stained bands at 105 kD a (nucleolin) and 39 kDa (protein B23). The synthesis of rRNA was meas ured by evaluating the amount of radioactivity incorporated into pre-r RNA after [H-3]orotic acid injection. Densitometric analysis of silver -stained bands and immunolabeling signals showed no change in nucleoli n and protein B23 amounts in cortisol-stimulated hepatocytes, whereas a moderate increase was found in regenerating hepatocytes at 12 h afte r partial hepatectomy. In both cortisol-stimulated and regenerating he patocytes the synthesis of rRNA was highly increased (2.6-fold and 4.3 -fold above the control level, respectively). To ascertain the relatio nship between quantitative changes in nucleolin and protein B23 and st imulation of rRNA transcriptional activity in regenerating hepatocytes , the quantitative distribution of these proteins was also investigate d in the early times of regeneration using silver-stained nitrocellulo se-transblotted nuclear proteins. The quantity of protein B23 was unch anged until 12 h after partial hepatectomy, whereas nucleolin appeared to be slightly increased at 9 h (1.15-fold above the control value) a fter partial hepatectomy. On the other hand, at just 6 h after partial hepatectomy, a significant increase of rRNA synthesis occurred in reg enerating rat hepatocytes (1.8-fold above the control value). These da ta demonstrated that stimulation of rRNA transcriptional activity occu rring in rat hepatocytes after cortisol treatment and in the early tim es after partial hepatectomy was not associated with quantitative chan ges in the amounts of nucleolin and protein B23. (C) 1995 Academic Pre ss, Inc.