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.
Cheng
Hsueh-Ling
HL
Institute of Biotechnology, National Pingtung University of Science and Technology, Taiwan, ROC.
Chang
Shiou-Ming
SM
Cheng
Ya-Wen
YW
Liu
Hung-Jen
HJ
Chen
Yo-Chia
YC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects*; D006224:Cricetinae; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D005434:Flow Cytometry; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D002166:Camptothecin