Synthesis of 1-/2-substituted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones and evaluation of their cytotoxicity and topoisomerase II inhibition

Studies on antitumor heterocyclic quinones containing nitrogens revealed that the number and position of nitrogens on the heterocyclic ring have significance on cytotoxicity of quinones. In our continuous effort to find more cytotoxic quinone compounds, we designed triazolophthalazine analogues in order to introduce more nitrogens on the heterocyclic quinones. 1-/2-Substituted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones were synthesized by 1,3-dipolar addition of phthalazine-5,8-dione and 4-methoxybenzyl azide by modification of previously reported method. The cytotoxicity of the synthesized compounds was evaluated by a SRB (sulforhodamine B) assay against nine types of human cancer cell lines and inhibition against topoisomerase II (Topo II) of them was assessed by a decatenation assay. Most of the synthesized compounds showed considerably higher cytotoxicity than that of doxorubicin. Also, topoisomerase II inhibitory activity of the tested compounds was higher than that of etoposide and IC(50) values of the compounds were 19.4-64.5 microM.     

Cdc14B depletion leads to centriole amplification, and its overexpression prevents unscheduled centriole duplication

Centrosome duplication is tightly controlled in coordination with DNA replication. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14B's involvement in centrosome cycle control has never been explored. Here, we show that depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells. Induction of Cdc14B expression through a regulatable promoter significantly attenuates centriole amplification in prolonged S phase-arrested cells and proteasome inhibitor Z-L(3)VS-treated cells. This inhibitory function requires centriole-associated Cdc14B catalytic activity. Together, these results suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle.     

Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex

We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A.     

Stimulation by TLR5 modulates osteoclast differentiation through STAT1/IFN-beta

Osteoclasts are bone-resorbing cells that are differentiated from hemopoietic precursors of the monocyte-macrophage lineage. Stimulation of TLRs has been shown to positively or negatively modulate osteoclast differentiation, depending on the experimental condition. However, the molecular mechanism by which this modulation takes place remains unclear. In the present study, we examined the effects of flagellin, a specific microbial ligand of TLR5, on the receptor activator of NF-kappaB ligand (RANKL)-stimulated osteoclastogenesis. Flagellin suppressed RANKL induction of c-Fos protein expression in bone marrow-derived macrophages without affecting c-Fos mRNA expression. Ectopic overexpression of c-Fos and a constitutively active form of NFATc1 reversed the flagellin-induced anti-osteoclastogenic effect. The inhibitory effect of flagellin was mediated by IFN-beta production. Flagellin stimulated IFN-beta expression and release in bone marrow-derived macrophages, and IFN-beta-neutralizing Ab prevented the flagellin-induced c-Fos down-regulation and the anti-osteoclastogenic effect. IFN-beta gene induction by flagellin, LPS, or RANKL was dependent on STAT1 activation. Treatment with flagellin or RANKL stimulated STAT1 activation, and STAT1 deficiency or the JAK2 inhibitor AG490 dramatically prevented IFN-beta induction in response to flagellin or RANKL. In addition, STAT1 deficiency abolished the anti-osteoclastogenic effect induced by flagellin or LPS. In contrast, flagellin stimulated osteoclast differentiation in cocultures of osteoblasts and bone marrow cells without inducing IFN-beta. Thus, IFN-beta acts as a critical modulator of osteoclastogenesis in response to TLR5 activation.     

Structural and functional insights into O-methyltransferase from Bacillus cereus

The specific substrates, mechanisms, and structures of the bacterial O-methyltransferases (OMTs) are not as well characterized as those of other OMTs. Recent studies have suggested that bacterial OMTs catalyze regiospecific reactions that might be used to produce novel compounds. In this study, we investigated the structural and functional features of an OMT from Bacillus cereus (BcOMT2). This enzyme catalyzes the O-methylation of flavonoids in vitro in an S-adenosylmethionine-dependent and regiospecific manner. We solved the crystal structures of the BcOMT2 apoenzyme and the BcOMT2-S-adenosylhomocysteine (SAH) co-complex at resolutions of 1.8 and 1.2 Å, respectively. These structures reveal that the overall structure of dimeric BcOMT2 is similar to that of the canonical OMT but that BcOMT2 also has a unique N-terminal helical region that is responsible for dimerization. The binding of SAH causes both local and remote conformational changes in the dimer interface that stabilize the dimerization of BcOMT2. SAH binding also causes ordering of residues Glu171 to Gly186, which are disordered in the apoenzyme structure and are known determinants of substrate specificity, and thus contributes to formation of the substrate binding pocket. Our structural analysis indicated a resemblance between the active site of BcOMT2 and that of metal-dependent OMTs. Using mutational analysis, we confirmed that BcOMT2 is a Mg²⁺-dependent OMT. These results provide structural and functional insights into the dimerization mechanism and substrate specificity of BcOMT2.     

Degradation of 3-methylpyridine and 3-ethylpyridine by Gordonia nitida LE31

Cells of Gordonia nitida LE31 grown on 3-methylpyridine degraded 3-ethylpyridine without a lag time and vice versa. Cyclic intermediates were not detected, but formic acid was identified as a metabolite. Degradation of levulinic acid was induced in cells grown on 3-methylpyridine and 3-ethylpyridine. Levulinic aldehyde dehydrogenase and formamidase activities were higher in cells grown on 3-methylpyridine and 3-ethylpyridine than in cells grown on acetate. These data indicate that 3-methylpyridine and 3-ethylpyridine were degraded via a new pathway involving C-2-C-3 ring cleavage.     

Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674

Bacillus cereus KCTC 3674 excretes several kinds of extracellular proteases into the growth medium. Two proteases with molecular masses of approximately 36-kDa and 38-kDa, as shown by SDS-PAGE, were purified from the culture broth. The 38-kDa protease was purified from B. cereus cultivated at 37 degrees C, and the 36-kDa protease was obtained from the B. cereus cultivated at 20 degrees C. The 38-kDa protease was identified as an extracellular neutral (metallo-) protease and was further characterized. The 36-kDa protease was shown to be a novel enzyme based on its N-terminal amino acid sequence, its identification as a metallo-enzyme that was strongly inhibited by EDTA and o-phenanthroline, its hemolysis properties, and its optimal pH and temperature for activity of 8.0 and 70 degrees C, respectively.     

Inhibition of cytosolic phospholipase A2 by annexin I. Specific interaction model and mapping of the interaction site

Annexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion. However, the exact biological functions of ANXs still remain obscure. Inhibition of phospholipase A(2) (PLA(2)) by ANX-I, a 346-amino acid protein, has been observed in studies with various forms of PLA(2). "Substrate depletion" and "specific interaction" have been proposed for the mechanism of PLA(2) inhibition by ANX-I. Previously, we proposed a specific interaction model for inhibition of a 100-kDa porcine spleen cytosolic form of PLA(2) (cPLA(2)) by ANX-I (Kim, K. M., Kim, D. K., Park, Y. M., and Na, D. S. (1994) FEBS Lett. 343, 251-255). Herein, we present an analysis of the inhibition mechanism of cPLA(2) by ANX-I in detail using ANX-I and its deletion mutants. Deletion mutants were produced in Escherichia coli, and inhibition of cPLA(2) activity was determined. The deletion mutant ANX-I-(1-274), containing the N terminus to amino acid 274, exhibited no cPLA(2) inhibitory activity, whereas the deletion mutant ANX-I-(275-346), containing amino acid 275 to the C terminus, retained full activity. The protein-protein interaction between cPLA(2) and ANX-I was examined using the deletion mutants by immunoprecipitation and mammalian two-hybrid methods. Full-length ANX-I and ANX-I-(275-346) interacted with the calcium-dependent lipid-binding domain of cPLA(2). ANX-I-(1-274) did not interact with cPLA(2). Immunoprecipitation of A549 cell lysate with anti-ANX-I antibody resulted in coprecipitation of cPLA(2). These results are consistent with the specific interaction mechanism rather than the substrate depletion model. ANX-I may function as a negative regulator of cPLA(2) in cellular signal transduction.     

Permanent phenotypic and genotypic changes of prostate cancer cells cultured in a three-dimensional rotating-wall vessel

A three-dimensional (3D) integrated rotating-wall vessel cell-culture system was used to evaluate the interaction between a human prostate cancer cell line, LNCaP, and microcarrier beads alone, or microcarrier beads previously seeded with either prostate or bone stromal cells. Upon coculture of LNCaP cells with microcarrier beads either in the presence or in the absence of prostate or bone stromal cells, 3D prostate organoids were formed with the expected hormonal responsiveness to androgen, increased cell growth, and prostate-specific antigen production. In this communication, we define permanent phenotypic and genotypic changes of LNCaP cells upon coculture with microcarrier beads alone, or with microcarrier beads previously seeded with either prostate or bone stromal cells. Most notably, we observed selective genetic changes, i.e., chromosomal losses or gains, as evaluated by both conventional cytogenetic and comparative genomic hybridization, in LNCaP sublines derived from the prostate organoids. Moreover, the derivative LNCaP cells appear to have altered growth profiles, and exhibit permanent and stable changes in response to androgen, estrogen, and growth factors. The derivative LNCaP sublines showed increased anchorage-independent growth rate, and enhanced tumorigenicity and metastatic potential when inoculated orthotopically in castrated athymic mice. Our results support the hypothesis that further nonrandom genetic and phenotypic changes in prostate cancer epithelial cells can occur through an event that resembles "adaptive mutation" such as has been described in bacteria subjected to nutritional starvation. The occurrence of such permanent changes may be highly contact dependent, and appears to be driven by specific microenvironmental factors surrounding the tumor cell epithelium grown as 3D prostate organoids.