P53 SYNTHESIS AND PHOSPHORYLATION IN THE AGING DIET-RESTRICTED RAT FOLLOWING RETINOIC ACID ADMINISTRATION

Multiple doses of retinoic acid (RA) were administered intraperitoneal ly to three groups of male Fischer 344 rats over a 36 h period. The p5 3 isoforms from bone marrow nuclei in these three groups of rats were analyzed over time by two-dimensional polyacrylamide gel electrophores is (PAGE) and fluorography for the incorporation of [S-35]methionine ( p53-synthesis) and [P-32]phosphate (p53-phosphorylation). Two groups o f rats, young (3.5 months) ad libitum (Y/AL) and old (28 months) ad li bitum (O/AL), had free access to Purina rat chow; a third group of old (28 months) diet-restricted rats (O/DR) were maintained on a restrict ed caloric intake (60% of the AL diet) from 3 months of age. After 36 h of RA dosing, the PAGE patterns of p53 synthesis and phosphorylation in Y/AL and O/DR rats were very similar. In both groups, an increase in complexity was observed with labeling of additional isotypes posses sing more acidic isoelectric values. In contrast, the O/AL animals sho wed a pattern of p53 isoform synthesis and phosphorylation that was co nsiderably less complex and lacked the pronounced shift to more acidic forms following RA dosing. The p53 isoforms of O/AL rats as recognize d by wild type (wt) Pab 246 antibody, were also much less dramatic in their increase to more acidic forms. Two-dimensional phospho-tryptic m aps of Y/AL and ODR rats were also very similar, both exhibiting two a dditional minor P-32-labeled fragments after RA dosing. The maps of O/ AL rats did not show the two additional fragments following RA adminis tration. After RA dosing, cyclin protein inhibitors (p16, p21, p27) re vealed robust labeling with their respective antibodies in Y/AL and O/ DR rats as analyzed by Western blotting. The O/AL animals showed margi nally detectable antibody recognition of the cyclin inhibitors after R A dosing. Taken together, these data suggest that the biosynthesis and phosphorylation of p53 isoforms and the expression of cyclin dependen t kinase inhibitor proteins is not significantly different between Y/A L and O/DR rats. Further, these results confirm and extend our previou s observations that chronic diet-restriction attenuates the age relate d decline in the metabolic activity of nuclear protein products. (C) 1 997 Elsevier Science Ireland Ltd.