PAP gene transcription induced by cycloheximide in AR4-2J cells involves ADP-ribosylation.

We report in this paper that cycloheximide induces PAP mRNA expression in the pancreatic acinar cell line AR4-2J in a dose- and time-dependent manner. We analyzed whether stabilization of the PAP mRNA or the direct induction of its transcription contributed to the induction of PAP mRNA expression by the drug. We first infected the cells, which do not express PAP mRNA constitutively, with a recombinant adenovirus in which the PAP cDNA was subcloned downstream of the CMV promotor, to obtain high levels of transcript. Then, transcription was pharmacologically blocked, the cells were treated with cycloheximide, and the PAP mRNA concentration was monitored over 8 h by Northern blot. PAP mRNA concentration remained unchanged for 4 h and then decreased in both cycloheximide-treated and control cells, ruling out a significant contribution of posttranscriptional regulation in cycloheximide induction. Direct regulation of gene transcription is therefore likely and we investigated whether it could involve ADP-ribosylation. Cycloheximide-induced cells were treated with two chemical inhibitors of poly(ADP-ribose) polymerase. 3-Aminobenzamide inhibited 75% of PAP gene induction and 4-hydroxyquinazolone, the highly specific inhibitor of the enzyme, blocked almost completely PAP expression, suggesting that ADP-ribosylation was indeed required for the upregulation of PAP gene expression by cycloheximide.