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An assay system was constructed to identify chemicals that have a potential to induce p21/WAF1 gene, a target of the tumor suppressor p53 critical for negative growth regulation. Screening of about 1300 culture fluids of Streptomyces resulted in identification of active substances which induced the p21 gene in a p53-independent manner; one was a mixture of four members of the actinomycin group, and the other was trichostatin A. Transcriptional regulatory regions of p21 gene for induction by actinomycin D and trichostatin A were determined by transient expression of luciferase constructs in cells which are p53-deficient (Saos-2) or express a mutated form of p53 (TMK-1). The essential transcriptional elements for the response to these drugs localize within 210 bp of the 5'-upstream region of human p21 gene, and Sp1 elements were determined to be critical for the induction. DNA-binding activity of Sp1 was not increased in cells treated with these drugs, but kinase inhibitors such as staurosporin and wortmannin inhibited the induction.

Identification of Active Substances from Streptomyces Culture Fluids Using p53-Independent Expression of p21/MAF1/Cip1 Gene and Their Mode of Action.