Cep57 regulates human centrosomes through multivalent interactions.

Human Cep57 is a coiled-coil scaffold at the pericentriolar matrix (PCM), controlling centriole duplication and centrosome maturation for faithful cell division. Genetic truncation mutations of Cep57 are associated with the mosaic-variegated aneuploidy (MVA) syndrome. During interphase, Cep57 forms a complex with Cep63 and Cep152, serving as regulators for centrosome maturation. However, the molecular interplay of Cep57 with these essential scaffolding proteins remains unclear. Here, we demonstrate that Cep57 undergoes liquid-liquid phase separation (LLPS) driven by three critical domains (NTD, CTD, and polybasic LMN). In vitro Cep57 condensates catalyze microtubule nucleation via the LMN motif-mediated tubulin concentration. In cells, the LMN motif is required for centrosomal microtubule aster formation. Moreover, Cep63 restricts Cep57 assembly, expansion, and microtubule polymerization activity. Overexpression of competitive constructs for multivalent interactions, including an MVA mutation, leads to excessive centrosome duplication. In Cep57-depleted cells, self-assembly mutants failed to rescue centriole disengagement and PCM disorganization. Thus, Cep57's multivalent interactions are pivotal for maintaining the accurate structural and functional integrity of human centrosomes.

BARD1 is an ATPase activating protein for OLA1.

OLA1 is a P-loop ATPase, implicated in centrosome duplication through the interactions with tumor suppressors BRCA1 and BARD1. Disruption of the interaction of OLA1 with BARD1 results in centrosome amplification. However, the molecular interplay and mechanism of the OLA1-BARD1 complex remain elusive. Here, we use a battery of biophysical, biochemical, and structural analyses to elucidate the molecular basis of the OLA1-BARD1 interaction. Our structural and enzyme kinetics analyses show this nucleotide-dependent interaction enhances the ATPase activity of OLA1 by increasing the turnover number (kcat). Unlike canonical GTPase activating proteins that act directly on the catalytic G domain, the BARD1 BRCT domain binds to the OLA1 TGS domain via a highly conserved BUDR motif. A cancer related mutation V695L on BARD1 is known to associate with centrosome abnormality. The V695L mutation reduces the BARD1 BRCT-mediated activation of OLA1. Crystallographic snapshot of the BRCT V695L mutant at 1.88 Å reveals this mutation perturbs the OLA1 binding site, resulting in reduced interaction. Altogether, our findings suggest the BARD1 BRCT domain serves as an ATPase activating protein to control OLA1 allosterically.

Comparative cytotoxicity of five current dentin bonding agents: role of cell cycle deregulation.

To compare the cytotoxicity of three nano-dentin bonding agents (nano-DBAs) and two non-nano-DBAs using Chinese hamster ovary (CHO-K1) cells. We found that nano fillers were not the major contributing factor in DBA cytotoxicity, as analyzed by colony forming assay and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Exposure of CHO-K1 cells to all three tested total-etching DBAs led to G(0)/G(1) cell cycle arrest, whereas exposure to higher concentrations of two tested nano-DBAs induced G(2)/M arrest. All five DBAs further induced apoptosis at the highest concentration, as analyzed by propidium iodide staining flow cytometry. The toxicity of all DBAs (1:4000v/v or higher) is related to increased reactive oxygen species (ROS) production, as analyzed by single cell DCF fluorescence flow cytometry. These results indicate that clinical application of DBAs may be potentially toxic to dental pulp tissues. Cytotoxicity of DBAs is associated with ROS production, cell cycle deregulation and apoptosis. Presence of methacrylate monomers such as PENTA and UDMA is possibly the major cytotoxic factor for DBAs. Further studies on other toxicological endpoints of nano-DBAs are necessary to highlight their safe use.

The effect of BisGMA on cyclooxygenase-2 expression, PGE2 production and cytotoxicity via reactive oxygen species- and MEK/ERK-dependent and -independent pathways.

After operative restoration, some monomers released from dentin bonding agents or composite resin may induce tissue inflammation and affect the vitality of dental pulp. Whether BisGMA, a major monomer of composite resin, may induce prostaglandin release and cytotoxicity to pulp cells and their mechanisms awaits investigation. We found that BisGMA induced cytotoxicity to human dental pulp cells at concentrations higher than 0.075 mm as analyzed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. BisGMA (0.1 mm) also stimulated ERK phosphorylation, PGE(2) production, COX-2 mRNA and protein expression as well as ROS production (as indicated by an increase in cellular DCF fluorescence) in dental pulp cells. Catalase (500 and 1000 U/ml) and U0126 (10 and 20 microm, a MEK inhibitor) effectively prevented the BisGMA-induced ERK activation, PGE(2) production and COX-2 expression. Moreover, catalase can protect the pulp cells from BisGMA cytotoxicity, whereas aspirin and U0126 lacked of this protective activity. These results suggest that BisGMA released from composite resin may potentially affect the vitality of dental pulp and induce pulpal inflammation via stimulation of ROS production, MEK/ERK1/2 activation and subsequent COX-2 gene expression and PGE(2) production. Cytotoxicity of BisGMA to dental pulp cells is related to ROS production, but not directly mediated by MEK activation and PGE(2) production.

Prostaglandin F(2alpha)-induced interleukin-8 production in human dental pulp cells is associated with MEK/ERK signaling.

Prostaglandin F(2alpha) (PGF(2alpha)) and interleukin-1beta (IL-1beta) levels are elevated in inflamed dental pulp. The roles of IL-1beta and PGF(2alpha) in the pathogenesis of pulpal inflammation await investigation. We found that IL-1beta stimulated PGF(2alpha) production of human dental pulp cells. IL-1beta and PGF(2alpha) (0.5-10 mumol/L) also induced IL-8 production and mRNA expression in pulp cells. Aspirin inhibited IL-1beta-induced PGF(2alpha), but not IL-8 production. PGF(2alpha)-induced IL-8 production and mRNA expression were inhibited by U0126 (an inhibitor of mitogen-activated protein kinase kinase [MEK1/2]) inhibitor), whereas SQ22536 (an adenylate cyclase inhibitor) enhanced this event. These results indicate that IL-1beta-induced IL-8 production in pulp cells is not mainly via direct activation of cyclooxygenase and PGF(2alpha) generation. PGF(2alpha)-induced IL-8 production is possibly via activation of MEK/extracellular signal-regulated kinase signaling, but not by activation of adenylate cyclase. IL-1beta and PGF(2alpha) might involve the pathogenesis of pulpal inflammation via induction of IL-8 production.

Areca nut-induced micronuclei and cytokinesis failure in Chinese hamster ovary cells is related to reactive oxygen species production and actin filament deregulation.

Epidemiological studies have shown a strong association between environmental exposure to betel quid (BQ) and oral cancer. Areca nut (AN), an ingredient of BQ, contains genotoxic and mutagenic compounds. In this study, we found that AN extract (ANE) inhibited the growth of Chinese hamster ovary cells (CHO-K1) in a dose- and time-dependent manner. Intracellular reactive oxygen species (ROS) levels and micronuclei (MN) frequency were significantly increased following ANE treatment in CHO-K1 cells. Addition of catalase markedly inhibited ANE-induced MN formation, indicating that ANE-induced genotoxicity was correlated with intracellular H(2)O(2). Incubation of CHO-K1 cells with ANE (400-800 microg/ml) for 24 hr caused G2/M arrest, and prolonged exposure to ANE (800 microg/ml) significantly induced cell death. Surprisingly, ANE itself caused cytokinesis failure and subsequent increase in binucleated cell formation. Coexposure to catalase (2,000 U/ml) and ANE (800 microg/ml) reduced the generation of binucleated cells, indicating that ANE-induced cytokinesis failure was associated with oxidative stress. Following prolonged exposure to ANE, an accumulation of hyperploid/aneuploid cells concomitant with bi-, micro- or multinucleated cells was found. In summary, our results demonstrate that ANE exposure to CHO-K1 cells caused increased MN frequency, G2/M arrest, cytokinesis failure, and an accumulation of hyperploid/aneuploid cells. These events are associated with an increase in intracellular H(2)O(2) level and actin filament disorganization.

Signaling pathways for induction of platelet aggregation by SAS tongue cancer cells--a mechanism of hematogenous metastasis.

BACKGROUND: Tongue cancer metastasis is mainly through blood stream and possibly associated with tumor cell-induced platelet aggregation (TCIPA). METHODS: Platelet aggregation was induced by different amounts of SAS tongue cancer cells with/without inhibitors and the latent period for induction of platelet aggregation was recorded. Gene expression was analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: SAS cells (4 x 10(4) to 1 x 10(6) cells/ml) induced platelet aggregation in a cell density-dependent manner. The latent period for induction of platelet aggregation reduced from 11.3 min (2 x 10(5) cells/ml) to 0.9 min (5 x 10(5) cells/ml). The extent of platelet aggregation increased from 39% to 76% by 2 x 10(5) and 5 x 10(5) SAS cells. Pre-treatment of SAS cells with aspirin showed little effect on its induction of platelet aggregation. SAS cells expressed tissue factor (TF) mRNA and the SAS cells-induced TCIPA was inhibited by TF neutralization antibody (5-20 microg/ml), heparin (5-10 U/ml), Hirudin fragment 54-65 (50 microg/ml) and D-Phenylalanyl-L-prolyl-L-arginine chloromethyl ketone. But areca nut (AN, a betel quid component known to generate reactive oxygen species (ROS)) extract showed little effect on TF expression in SAS cells. Pre-treatment with U73122 and 2-aminoethoxydiphenylborate inhibited SAS-induced TCIPA. Interestingly, catalase suppressed SAS cells-induced TCIPA, whereas AN extract enhanced this event. CONCLUSIONS: These results suggest that tongue cancer cells may induce TCIPA and enhance tumor metastasis. SAS-induced TCIPA is related to TF secretion, thrombin generation and associated with Phospholipase C-Inositol triphosphate signaling and ROS production. Betel quid chewing may potentially promote tongue cancer metastasis.