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-depende nt manner. We analyzed whether stabilization of the PAP mRNA or the di rect induction of its transcription contributed to the induction of PA P mRNA expression by the drug. We first infected the cells, which do n ot express PAP mRNA constitutively, with a recombinant adenovirus in w hich the PAP cDNA was subcloned downstream of the CMV promotor, to obt ain high levels of transcript. Then, transcription was pharmacological ly blocked, the cells were treated with cycloheximide, and the PAP mRN A concentration was monitored over 8 h by Northern blot. PAP mRNA conc entration remained unchanged for 4 h and then decreased in both cycloh eximide-treated and control cells, ruling out a significant contributi on of posttranscriptional regulation in cycloheximide induction. Direc t regulation of gene transcription is therefore likely and we investig ated whether it could involve ADP-ribosylation. Cycloheximide-induced cells were treated with two chemical inhibitors of poly(ADP-ribose) po lymerase. 3-Aminobenzamide inhibited 75% of PAP gene induction and 4-h ydroxyquinazolone, the highly specific inhibitor of the enzyme, blocke d almost completely PAP expression, suggesting that ADP-ribosylation w as indeed required for the upregulation of PAP gene expression by cycl oheximide. (C) 1998 Academic Press.