[Construction of a reporter system for fine quantitative assessment of activity of p53 protein in cultured cells].
Since transcriptional activation of genes downregulating cell proliferation mediates the tumor suppressor activity of p53, induction of p53 targets was assumed to adequately reflect the state of p53-dependent pathways. To estimate the p53 activity in cultured cells, self-inactivating retrovirus constructs pSIP-ConA-GFP and pSIP-ConA-LacZ were obtained to express the GFP or beta-galactosidase reporter gene under the control of a promoter containing p53-responsive elements. The advantages of these constructs were efficient delivery, comparable expression in different cells of a culture, and, consequently, the possibility of quantitating the p53 activity induced by various agents. With pSIP-ConA-LacZ, p53 activation in response to 12 chemotherapeutic agents was analyzed in human carcinoma cell line HCT116 and its derivatives HCT116/mdm2 and HCT116ARF, which expressed genes affecting the p53 activity. The analysis was also carried out with human cell lines HEF, WI-38, U2OS, and HT1080 originating from connective tissue. The construct proved suitable for detecting fine differences in p53 activation induced by various stress factors. The constructs were proposed for generating reporter cell lines from various cultures in order to identify the genetic or chemical factors that modulate the p53 activity and may be employed in new antitumor drugs.
Strunina
S M
SM
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia. strunin@online.ru
Ivanov
A V
AV
Chumakov
P M
PM
D045744:Cell Line, Tumor; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*; D001616:beta-Galactosidase / Q000235:genetics
D008164:Luminescent Proteins; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase