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) stop gene is known to be one of the key steps in the development of cellular senescence, whereas the elements within the p21(Cip1/Waf1) stop promoter that regulate the transcriptional activation of p21(Cip1/Waf1) stop 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) stop 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.