Bahar Rumana, O-Wang Jiyang, Kawamura Kiyoko, Seimiya Mika, Wang Yanqing, Hatano Masahiko, Okada Seiji, Tokuhisa Takeshi, Watanabe Takeshi, Tagawa Masatoshi
Division of Pathology, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan.
J Biol Chem. 2002 Oct 18;277(42):40012-9. doi: 10.1074/jbc.M205345200. Epub 2002 Jul 29.
We have identified a novel gene, Clast3, by subtraction of cDNAs derived from activated and naive B lymphocytes. Clast3 expression is elevated in cycling cells and down-regulated in cells undergoing growth arrest, indicating that its expression is controlled in a cell cycle-dependent manner. The deduced amino acid sequence of Clast3 cDNA exhibits no significant homology to the known proteins in mammalian and other species. Immunofluorescence staining revealed that Clast3 localizes into discrete nuclear foci. Forced expression of Clast3 results in growth retardation, polyploidy, and generation of multinucleated cells. Treatment of Clast3 transfectants with nocodazole, a spindle-damaging agent, greatly enhances the incidence of the multinucleated cells, suggesting that Clast3 overexpression impairs the same checkpoint activated by nocodazole. Down-regulation of Clast3 expression by antisense oligonucleotides results in a decrease of cells at G(2)-M phase and a concomitant increase of apoptotic cells. These findings indicate that Clast3 is a novel cell cycle-regulated protein and that its constitutive overexpression induces polyploidy and multinucleation by interfering with the mitotic spindle checkpoint.
我们通过消减来自活化和未活化B淋巴细胞的cDNA，鉴定出一个新基因Clast3。Clast3在循环细胞中表达升高，而在经历生长停滞的细胞中表达下调，这表明其表达受细胞周期依赖性调控。Clast3 cDNA推导的氨基酸序列与哺乳动物及其他物种的已知蛋白质无明显同源性。免疫荧光染色显示Clast3定位于离散的核灶。Clast3的强制表达导致生长迟缓、多倍体形成以及多核细胞的产生。用诺考达唑（一种破坏纺锤体的药物）处理Clast3转染细胞，极大地提高了多核细胞的发生率，这表明Clast3过表达损害了由诺考达唑激活的相同检查点。反义寡核苷酸下调Clast3表达导致G(2)-M期细胞减少，同时凋亡细胞增加。这些发现表明Clast3是一种新型的细胞周期调节蛋白，其组成性过表达通过干扰有丝分裂纺锤体检查点诱导多倍体形成和多核化。
