Rapid screen of human genes for relevance to cancer using fission yeast.

A total of 437 human full-length cDNAs isolated by microarray analysis of liver and/or gastric cancer tissues were evaluated for their relevance to cancer using the fission yeast Schizosaccharomyces pombe. Overexpression of 161 human cDNAs in S. pombe caused growth inhibition and/or morphological changes, which can be considered as cancer-related phenotypes of S. pombe. Sixteen genes causing growth defects and morphological changes at the same time were chosen to validate their ostensible oncogenic properties. They were highly expressed in liver and/or gastric cancer cell lines. Also, when the mouse embryonic fibroblast cell type NIH3T3 was transfected with these genes, the proliferation rates of cells were increased by 32% to 120%. This study demonstrates that fission yeast can be used as an advantageous and powerful tool for the rapid screening of human genes relevant to cancer. Furthermore, the human genes screened can be tested further as diagnostic markers and potential therapeutic targets for liver and stomach cancers. They also can be studied further for the elucidation of mechanisms involved in carcinogenesis.

Polo-box motif targets a centrosome regulator, RanGTPase.

Mammalian polo-like kinase (Plk) acts at various stages in early and late mitosis. Plk1 localizes in the centrosome, the central spindle, the midbody as well as the kinetochore. The non-catalytic region in the C-terminus of Plk1 has conserved sequence motifs, named polo-boxes. These motifs are important for Plk localization. GFP protein fused with the core sequences of polo-box (50 amino acids) localized Plk to target organelles. We screened for Plk interacting proteins by constructing a tandem repeat of the polo-box motif, and used it as bait in the two-hybrid system with HeLa cell cDNA library. RanGTPase was detected as a positive clone. Through in vitro and in vivo protein binding analysis in synchronized cells by thymidine block and by nocodazole treatment, we confirmed the interaction between endogenous Ran and Plk1. We showed that endogenous Ran and Plk1 proteins were co-localized to centrosomes, which is a major target organelle of endogenous Plk1, in early mitotic cells by immunofluorescence. Finally, we demonstrated that Plk1 phosphorylated RanBPM, a Ran-binding protein in microtubule organizing center, through the interaction with Ran. These data suggested that the core motif of polo-box is sufficient for Plk1-targeting, and that Plk1 may play roles in centrosome through recruitment and/or activation of Ran/RanBPM proteins.