An estradiol-inducible promoter enables fast, graduated control of gene expression in fission yeast.

The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose-response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on ß-estradiol-regulated function of the human oestrogen receptor, for use in S. pombe. We show that this promoter system, termed Z3 EV, turns on quickly, can reach a maximal induction of 20-fold, and exhibits a linear dose response over its entire induction range, with few off-target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by ß-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast. Copyright © 2017 John Wiley & Sons, Ltd.

A novel anti-microtubule agent with carbazole and benzohydrazide structures suppresses tumor cell growth in vivo.

BACKGROUND: The mitotic spindles are among the most successful targets of anti-cancer chemotherapy, and they still hold promise as targets for novel drugs. The anti-mitotic drugs in current clinical use, including taxanes, epothilones, vinca alkaloids, and halichondrins, are all microtubule-targeting agents. Although these drugs are effective for cancer chemotherapy, they have some critical problems; e.g., neurotoxicity caused by damage to neuronal microtubules, as well as innate or acquired drug resistance. To overcome these problems, a great deal of effort has been expended on development of novel anti-mitotics. METHODS: We identified novel microtubule-targeting agents with carbazole and benzohydrazide structures: N'-[(9-ethyl-9H-carbazol-3-yl)methylene]-2-methylbenzohydrazide (code number HND-007) and its related compounds. We investigated their activities against cancer cells using various methods including cell growth assay, immunofluorescence analysis, cell cycle analysis, tubulin polymerization assay, and tumor inhibition assay in nude mice. RESULTS: HND-007 inhibits tubulin polymerization in vitro and blocks microtubule formation and centrosome separation in cancer cells. Consequently, it suppresses the growth of various cancer cell lines, with IC50 values in the range 1.3-4.6µM. In addition, HND-007 can inhibit the growth of taxane-resistant cancer cells that overexpress P-glycoprotein. Finally, HND-007 can inhibit HeLa cell tumor growth in nude mice. CONCLUSIONS AND GENERAL SIGNIFICANCE: Taken together, these findings suggest that HND-007 is a promising lead compound for development of novel anti-mitotic, anti-microtubule chemotherapeutic agents.