Characterization of the activities of p21Cip1/Waf1 promoter-driven reporter systems during camptothecin-induced senescence-like state of BHK-21 cells.

It was attempted in this work to establish a cell line in which senescent cells can be readily and directly identified in situ in live culture. Transcriptional activation of p21(Cip1/Waf1) gene is known to be one of the key steps in the development of cellular senescence, whereas the elements within the p21(Cip1/Waf1) promoter that regulate the transcriptional activation of p21(Cip1/Waf1) during cellular senescence have not been clearly defined. Thus, several reporter plasmids were constructed in each of which the gene of green fluorescent protein was placed under the control of a selected fragment of p21(Cip1/Waf1) promoter, and stably transfected into BHK-21 cells. The transfected cells were induced to become senescence-like by camptothecin and assayed for fluorescence intensity. It was shown that the reporter system constructed with bases -2504 to +406 of the p21(Cip1/Waf1) promoter was very efficient in reflecting the senescence of BHK-21 cells by increased cytosolic fluorescence, and the fluorescence intensity of senescent cells was easily distinguished from that of quiescent cells.