Serum Responsiveness of the Rat PCNA Promoter

Abstract

Expression of proliferating cell nuclear antigen (PCNA) gene is growth-regulated. The growth dependence of the rat PCNA gene promoter activity was investigated. Cultured cells were transfected with the promoter containing plasmid and recovered for 48 h in serum-free medium to become quiescent. Cells were then cultured in serum-containing medium and harvested at certain intervals after serum-stimulation, and the promoter-directed chloramphenicol acetyltransferase activities in cell extracts were measured. The promoter used in this study contained sequences between -693 and +125 in relation to the transcription initiation site. The promoter activity was found to be serumresponsive. However, the serum-responsiveness of the promoter became less obvious when the amount of the promoter increased; meanwhile, the promoter became active in the control unstimulated (or quiescent) cells. It was suspected that the dosage effect was due to the titration of the negative regulatory factor in quiescent cells. The titration experiment with a reporterless construct as competitor for regulatory factors showed that the excess of promoter molecules reduced the promoter activity in serum-stimulated cells, while causing a transiently increase of promoter activity in quiescent cells. Based on these results, it is postulated that the serum-responsiveness of the rat PCNA promoter is controlled by both negative and positive regulatory factors. Consistent with this proposition, promoter binding proteins of 105 and 114 kDa were identified only in serum-stimulated and quiescent cells, respectively, in addition to several other promoter binding proteins (ranging from 76 to 110 kDa) which were seen in both serum-stimulated and quiescent cells.