Diagnostic and therapeutic application of cell-cycle checkpoint genes for oral squamous cell cancer.

CHFR含有环结构域并具有泛素连接酶活性，是一种新的有丝分裂检查点基因，可延迟微管毒物处理细胞中的染色体凝聚。启动子甲基化使CHFR失活，缺乏CHFR的癌细胞对微管抑制剂敏感。(1)与CHFR，有丝分裂检查点泛素连接酶相互作用的蛋白。在本研究中，我们利用酵母双杂交方法分离出能够与CHFR相互作用的细胞蛋白，以阐明CHFR的功能。通过筛选，我们分离出典型和非典型E2泛素结合酶作为CHFR相互作用蛋白，它们分别参与蛋白水解和非蛋白水解泛素化。这就提出了CHFR根据细胞的情况切换典型和非典型泛素化的可能性。另一方面，我们分离出与CHFR的FHA结构域相互作用的gadd34。此外，在微管抑制剂多西紫杉醇的作用下，CHFR部分从细胞核转移到细胞质，这使得CHFR和gadd34在细胞质中共定位。这种共定位之后是细胞死亡。这表明gadd34和CHFR在有丝分裂应激反应中协同介导细胞死亡。(2) CHFR有丝分裂检查点蛋白抑制NF-kB的转录活性。为了阐明CHFR调节肿瘤生长的分子机制，我们的分析集中在CHFR介导的转录调控上。通过微阵列分析，我们发现CHFR下调NF-kB信号。NF-kB依赖性荧光素酶分析表明，CHFR过表达抑制NF-kB信号传导。此外，CHFR的下调激活了NF-kB的活性。此外，我们发现NF-kB靶基因之一IL-8受CHFR调控。过表达CHFR显著抑制IL-8 mRNA和蛋白水平。总之，我们的研究结果揭示了CHFR作为NF-kB信号调节器的新功能，这可能解释了CHFR的肿瘤抑制作用。少

CHFR which contains a RING domain and has ubiquitin ligase activity, a novel mitotic checkpoint gene delays chromosome condensation in cells treated with microtubule poisons. CHFR is inactivated by promoter methylation, and cancer cells lacking CHFR are sensitive to microtubule inhibitors.(1) Proteins interacting with CHFR, mitotic-checkpoint ubiquitin ligase.In this study, we isolated cellular proteins capable of interacting with CHFR using yeast two-hybrid method to clarify the function of CHFR. As a result of the screening, we isolated canonical and noncanonical E2 ubiquitin conjugating enzymes as CHFR interacting proteins, which are involved in proteolytic and non-proteolytic ubiquitination respectively. This raises the possibility that CHFR is switching canonical and noncanonical ubiquitination depending on the situation of cells.On the other hand, we isolated gadd34 which interacted with the FHA domain of CHFR. Furthermore, CHFR moved in part from nucleus to cytoplasm in the pres … More ence of microtubule inhibitor docetaxel, which enabled colocalization of CHFR and gadd34 in cytoplasm. This colocalization was followed by cell death. These suggest that gadd34 and CHFR cooperate to mediate cell death in response to mitotic stress.(2) CHFR mitotic checkpoint protein inhibits the transcriptional activity of NF-kB.To clarify the molecular mechanisms by which CHFR modulates tumor growth, our analysis focused on transcriptional regulation mediated by CHFR. Using microarray analysis, we found that CHFR downregulated NF-kB signaling. NF-kB-dependent luciferase assay demonstrated that overexpression of CHFR inhibited NF-kB signaling. Moreover, knockdown of CHFR activated the NF-kB activity. Furthermore, we found that IL-8, one of the NF-kB target genes, was regulated by CHFR. mRNA and protein levels of IL-8 were significantly repressed by overexpression of CHFR. Altogether, our findings reveal a novel function of CHFR as a regulator of NF-kB signaling, and which might account for tumor suppression by CHFR. Less