Chfr is required for tumor suppression and Aurora A regulation.

Tumorigenesis is a consequence of loss of tumor suppressors and activation of oncogenes. Expression of the mitotic checkpoint protein Chfr is lost in 20-50% of primary tumors and tumor cell lines. To explore whether downregulation of Chfr contributes directly to tumorigenesis, we generated Chfr knockout mice. Chfr-deficient mice are cancer-prone, develop spontaneous tumors and have increased skin tumor incidence after treatment with dimethylbenz(a)anthracene. Chfr deficiency leads to chromosomal instability in embryonic fibroblasts and regulates the mitotic kinase Aurora A, which is frequently upregulated in a variety of tumors. Chfr physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo. Collectively, our data suggest that Chfr is a tumor suppressor and ensures chromosomal stability by controlling the expression levels of key mitotic proteins such as Aurora A.

Detection of protein-RNA complexes in Xenopus oocytes.

There is a remarkable variety of mechanisms for controlling post-transcriptional gene expression that is achieved through the formation of ribonucleoprotein (RNP) complexes on specific cis-acting regions of mRNA. These complexes regulate splicing, nuclear and cytoplasmic polyadenylation, stability, localization, and translation. Thus, it is important to be able to detect the association of specific proteins with specific RNAs within the context of these RNP complexes. We describe a method to test for protein-RNA complexes in Xenopus oocytes. The procedure combines immunoprecipitation with reverse transcription-PCR (RT-PCR) and does not entail chemical or photo crosslinking. Microinjected mRNA is efficiently translated in Xenopus oocytes; thus, in cases where primary antibody is not available, an epitope-tagged version of the protein can be expressed for utilization in this procedure. The inclusion of control mRNAs has provided no evidence of nonspecific protein reassociation to RNA during or subsequent to cell lysis. The method has been used to document the association of certain trans-acting factors specifically with localized mRNAs in Xenopus oocytes.

RCS1, a substrate of APC/C, controls the metaphase to anaphase transition.

The anaphase-promoting complex/cyclosome (APC/C) controls the onset of anaphase by targeting securin for destruction. We report here the identification and characterization of a substrate of APC/C, RCS1, as a mitotic regulator that controls the metaphase-to-anaphase transition. We showed that the levels of RCS1 fluctuate in the cell cycle, peaking in mitosis and dropping drastically as cells exit into G(1). Indeed, RCS1 is efficiently ubiquitinated by APC/C in vitro and degraded during mitotic exit in a Cdh1-dependent manner in vivo. APC/C recognizes a unique D-box at the N terminus of RCS1, as mutations of this D-box abolished ubiquitination in vitro and stabilized the mutant protein in vivo. RCS1 controls the timing of the anaphase onset, because the loss of RCS1 resulted in a faster progression from the metaphase to anaphase and accelerated degradation of securin and cyclin B. Biochemically, mitotic RCS1 associates with the NuRD chromatin-remodeling complex, and this RCS1 complex is likely involved in regulating gene expression or chromatin structure, which in turn may control anaphase onset. Our study uncovers a complex regulatory network for the metaphase-to-anaphase transition.

Cep55, a microtubule-bundling protein, associates with centralspindlin to control the midbody integrity and cell abscission during cytokinesis.

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.

Monoclonal antibodies to fibroblast growth factor receptor 2 effectively inhibit growth of gastric tumor xenografts.

PURPOSE: Overexpression of fibroblast growth factor receptor 2 (FGFR2) may be a causative factor of a number of human tumors, especially gastric tumors of the poorly differentiated type. We investigated whether monoclonal antibodies (mAbs) directed against FGFR2 can inhibit the growth of tumors in xenograft models. EXPERIMENTAL DESIGN: We generated and characterized 3 mAbs that recognize different epitopes on FGFR2: GAL-FR21, GAL-FR22, and GAL-FR23. The ability of the mAbs to recognize the FGFR2IIIb and FGFR2IIIc isoforms of FGFR2 was determined, as was their ability to block binding of FGF ligands to FGFR2. The capability of the mAbs to inhibit FGF-induced FGFR2 phosphorylation and to downmodulate FGFR2 expression was also investigated. Finally, the ability of the anti-FGFR2 mAbs to inhibit tumor growth was determined by establishing xenografts of SNU-16 and OCUM-2M human gastric tumor cell lines in nude mice, treating with each mAb (0.5-5 mg/kg intraperitoneally twice weekly) and monitoring tumor size. RESULTS: Of the 3 mAbs, GAL-FR21 binds only the FGFR2IIIb isoform, whereas GAL-FR22 and GAL-FR23 bind to both the FGFR2IIIb and FGFR2IIIc forms, with binding regions respectively in the D3, D2-D3, and D1 domains of FGFR2. GAL-FR21 and GAL-FR22 blocked the binding of FGF2, FGF7 and FGF10 to FGFR2IIIb. GAL-FR21 inhibited FGF2 and FGF7 induced phosphorylation of FGFR2, and both mAbs downmodulated FGFR2 expression on SNU-16 cells. These mAbs effectively inhibited growth of established SNU-16 and OCUM-2M xenografts in mice. CONCLUSIONS: Anti-FGFR2 mAbs GAL-FR21 and GAL-FR22 have potential for the treatment of gastric and other tumors.

Evaluation of Tourette's syndrome by 99Tcm-TRODAT-1 SPECT imaging / 中华核医学杂志

Objective To observe dopamine transporter (DAT) binding capacity using 99Tcm-TRODAT-1 in drug-naive patients with Tourette's syndrome (TS) on SPECT imaging, and explore possible correlations between 99Tcm-TRODAT-1 uptake ratio and TS patient's age, disease duration, and tic severity.Methods Eighteen drug-naive TS patients, male 14, female 4, as well as 8 age- and gender-matched healthy subjects were recruited. Brain SPECT imaging was performed 2. 5 h after intravenous injection of 11.1 - 14.8 MBq/kg 99Tcm-TRODAT-1. ROI was drawn on the striatum including its sub-regions of caudate and putamen, with cerebellum as the background. Striatum/cerebellum ratio was calculated. Comparisons of the ratios between TS patients and controls were carried out by independent-sample t-test. Pearson correlation analysis was performed between DAT uptake ratios of striatum and patients' age, disease duration, tic severity. Results Compared with the control, higher symmetrically striatum uptake of 99Tcm-TRODAT-1 in TS patients was observed (2.17±0.23 vs 1.87±0.24, t =2.957, P＜0.05). Age (r= -0.320, P＞0.05)and tic severity(r = 0. 345, P ＞ 0.05) scores were not significantly correlated with specific uptake ratios measured in the striatum. But there was significant negative correlation between disease duration and the specific uptake ratios (r = - 0. 483, P ＜ 0. 05). Conclusions 99 Tcm-TRODAT-1 SPECT imaging may play an adjuvant role for initial evaluation of untreated TS.

MgcRacGAP controls the assembly of the contractile ring and the initiation of cytokinesis.

Initiation of cytokinesis requires the establishment of the cleavage plane, the assembly of the contractile ring, and the ingression of the cleavage furrow. MgcRacGAP, a GTPase-activating protein for RhoA, is required for cytokinesis, but the mechanism of its action remains unknown. We report here that MgcRacGAP is required for the assembly of anillin and myosin into the contractile ring. In addition, MgcRacGAP is required for the localized activation of myosin through the RhoA-mediated phosphorylation of the myosin regulatory light chain. Cells with MgcRacGAP RNA interference (RNAi) failed cytokinesis without any ingression of the cleavage furrow. Paradoxically, MgcRacGAP, a GTPase-activating protein, associates during cytokinesis with ECT2, a guanine nucleotide exchange factor for RhoA, and the localization of ECT2 to both the central spindle and the contractile ring depends on MgcRacGAP. Knockdown of ECT2 phenocopies that of MgcRacGAP. We conclude that MgcRacGAP controls the initiation of cytokinesis by regulating ECT2, which in turn induces the assembly of the contractile ring and triggers the ingression of the cleavage furrow.

Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis.

Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/C(Cdh1) increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.

A comparative study of influential factors correlating with early and late hypothyroidism after (131)I therapy for Graves' disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>(131)I therapy is recognized as the simplest, safest, least expensive, and most effective treatment, and accepted by more and more patients. However its curative effect is influenced by many factors, therefore there are some difficulties for doctors to establish individual treatment strategy. The aims of this study were to determine the incidence of early and late hypothyroidism after (131)I treatment for Graves' disease (GD) and to compare their correlation, to observe and analyze the influential factors and to understand the predictabilities of them.</p><p><b>METHODS</b>Five hundred GD patients (144 males, 356 females; age (41.2 +/- 12.3) years) received (131)I treatment for the first time. The therapeutic procedure was carried out as the following: undergoing (131)I uptake test to obtain maximum of thyroid uptake value and effective half-life (EHL) time; estimating the thyroid's weight by ultrasonography; determination of thyroid hormones and correlative antibodies; pre-therapy physical examination; thyroid imaging; calculating (131)I therapeutic dosage; per os uptake of the determined (131)I dosage; follow-up appraisal of curative effect. The observing parameters included age, gender, thyroid weight, GD duration, condition of onset, state of disease, course of treatment, EHL time, maximum of thyroid uptake value, (131)I dosage and titer of correlative antibodies. We sorted out the data and used both univariate and multivariate analysis to evaluate them statistically.</p><p><b>RESULTS</b>The incidence rates of early and late hypothyroidism were 33.2% and 6.6% respectively after (131)I treatment and approximately 22.2% cases of late hypothyroidism developed from early hypothyroidism. The influential factors of early hypothyroidism included course of GD, the highest thyroid uptake ratio of (131)I, EHL time and thyroid microsome antibody (TMAb), etc. A multivariate analysis on late hypothyroidism showed that female patients, with recurrence after anti-thyroid drug treatment and higher thyroid weight, had lower possibility of late hypothyroidism after (131)I therapy.</p><p><b>CONCLUSIONS</b>The incidence of early hypothyroidism is higher than that of late hypothyroidism. The highest thyroid uptake ratio of (131)I, EHL and TMAb will increase the possibility of early hypothyroidism, while GD course is the protective factor. Higher (131)I dosage, longer EHL and higher TMAb titer will also increase the possibility of late hypothyroidism. The multi-perspective and multi-factor analysis has the benefit to establish individualized treatment strategy.</p>

Influential factors on the effectiveness of 131I treatment on post-surgical differentiated thyroid cancer patients / 中华核医学杂志

Objective To investigate the influential factors on the effectiveness of the first 131I ablation therapy on thyroid remnant and of 131I treatment on metastatic lesions in differentiated thyroid cancer (DTC) patients. Methods Retrospectively,46 DTC cases (divided into complete-ablation group and incomplete-ablation group) of first 131I ablation were enrolled,and 40 DTC cases (divided into remission group and in-remission group) of consecutive 131I treatments on metastatic lesions were enrolled. Influential factors were analyzed (t-test,t'-test,x2-test,Fisher exact test) and logistic regression analysis was performed. Results For the first 131I ablation effectiveness,surgical method,remnant thyroid weight,thyroid stimulating hormone (TSH) level,interval between surgery and 131I ablation therapy,metastatic status were selected as influential factors (x2 = 5. 804,t' = - 5. 258,t' = 7. 376,x2 = 8. 867,x2 = 8. 615,all P ＜0. 05). After logistic regression analysis,formula was obtained as y = 3. 766 - 0. 947x1 ( remnant thyroid weight) -3. 149 x2 (lymph node metastasis) -3. 373 x3 (distant metastasis). For metastatic treatment effectiveness,remission rate of papillary DTC was higher than that of follicular DTC,remission rate of patients with lymph node metastasis was higher than that of distant metastasis,remission rate of total thyroidectomy was higher than that of other types of thyroidectomy ( Fisher exact test,x2 = 7. 278,P ＜ 0. 05 ). In remission group,serum TSH level was much higher and thyroglobulin (Tg) level was much lower before the first ablation therapy (t =4. 489,t' = -4.906,all P ＜0.01 ). After logistic regression analysis,formula was obtained as y = - 0. 363 + 0. 065 x4 ( TSH level) - 0. 250 x5 ( Tg level). Conclusions Influential factors of success rate of the first 131I ablation therapy included surgical method,remnant thyroid weight,TSH level,interval between surgery and 131I ablation therapy and metastatic status,while determinant factors were thyroid remnant weight,lymph node metastatic status and distant metastatic status. The influential factors of success rate of 131I treatment on metastatic lesions included pathological type,surgical method,metastatic status,TSH level and Tg level,while determinant factors were TSH level and Tg level before the first 131I ablation therapy.

A homolog of FBP2/KSRP binds to localized mRNAs in Xenopus oocytes.

A Xenopus oocyte expression library was screened for proteins that bind to the 340-nucleotide localization element of Vg1 mRNA. Four different isolates encoded a Xenopus homolog of the human transcription factor, FUSE-binding protein 2 (FBP2). This protein has been independently identified as the splicing regulatory factor KSRP. The only significant difference between the Xenopus protein, designated VgRBP71, and KSRP is the absence of a 58 amino acid segment near the N-terminal of the former. In vivo binding assays show that VgRBP71 is associated with mRNAs localized to either the vegetal or animal hemispheres, but was not found with control mRNAs. Unlike other factors that bind to the localization element of Vg1 mRNA, VgRBP71 does not accumulate at the vegetal cortex with the mRNA; rather, it is present in the nucleus and throughout the cytoplasm at all stages of oogenesis. Cytoplasmic VgRBP71 appears to be most concentrated at the cell cortex. VgRBP71 interacts with Prrp, another protein that binds to the Vg1 localization element; this association does not require the presence of Vg1 mRNA.