Molecular and Cellular Biochemistry 291: 29–38, 2006.DOI: 10.1007/s11010-006-9191-x

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

Hsueh-Ling Cheng, Shiou-Ming Chang, Ya-Wen Cheng,Hung-Jen Liu, and Yo-Chia Chen

1Institute of Biotechnology, National Pingtung University of Science and Technology, Taiwan, R.O.C.; 2Department of Veterinary Medicine, National Pingtung University of Science and Technology, Taiwan, R.O.C.

摘要

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 p21Cip1/Waf1 gene is known to be one of the key steps in the development of cellular senescence, whereas the elements within the p21Cip1/Waf1 promoter that regulate the transcriptional activation of p21Cip1/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 p21Cip1/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 p21Cip1/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. (Mol Cell Biochem 291: 29–38, 2006).

关键字: cellular senescence, p21Cip1/Waf1 promoter, green fluorescent protein, BHK-21 cells, camptothecin

文中提及的产品

Restriction enzymes were purchased from New England Biolabs Inc. (Beverly, USA); PCR or sequencing primers were synthesized by MDBio Inc. (Taipei, Taiwan); 1 Kb DNA molecular weight ladder and fetal bovine serum were purchased from Invitrogen (San Diego, USA); phrGFP promoterless vector carrying the human enhanced GFP genewas purchased from Stratagen (La Jolla, USA), a hygromycinresistant system was incorporated into the vector according to the manufacture’s instruction for the selection of stable transfectants; unless indicated, other chemicals were purchased from Sigma Chemical Company (St. Louis, USA).