Prostaglandin H2 (PGH2 [endoperoxide]) is an immediate product of prostaglandin H (PGH) synthase activity (cyclooxygenase) and a likely candidate to mediate endothelium-dependent contractions evoked by acetylcholine in the aorta of the spontaneously hypertensive rat (SHR). Experiments were designed to investigate whether or not endothelium-dependent contractions were associated with an increased expression of PGH synthase, an augmented acetylcholine-induced release of PGH2, and/or a hypersensitivity of the smooth muscle to endoperoxides in SHR aorta compared with normotensive Wistar-Kyoto (WKY) aorta. In SHR aorta, endothelium-dependent contractions to acetylcholine were abolished by tenidap (10(-8) mol/L), a preferential PGH synthase-1 inhibitor, but slightly impaired by NS-398 (10(-6) mol/L), a preferential PGH synthase-2 inhibitor. PGH synthase-1 expression, which was evaluated by both reverse transcriptase-polymerase chain reaction and Western blotting, was about twofold greater in preparations with endothelium from SHR than from WKY rats. There was no difference in PGH synthase-1 expression between preparations with and those without endothelium in both strains. In SHR but not WKY aortas, acetylcholine (10(-5) mol/L, 5 minutes) caused a significant endothelium-dependent release of PGH2 as measured by gas chromatography/mass spectrometry. PGH2 evoked more potent contractions in rings without endothelium from SHR than from WKY rats, whereas the thromboxane analogue U46619 and prostaglandin F2 alpha caused a comparable response in both preparations. These results show that endothelium-dependent contractions to acetylcholine in SHR aorta are associated with a greater expression of PGH synthase-1, a significant release of PGH2, and a hypersensitivity of the smooth muscle to the endoperoxide.

Background Circular RNAs (circRNAs) are involved in diverse processes that drive cancer development. However, the expression landscape and mechanistic function of circRNAs in osteosarcoma (OS) remain to be studied. Methods Bioinformatic analysis and high-throughput RNA sequencing tools were employed to identify differentially expressed circRNAs between OS and adjacent noncancerous tissues. The expression level of circ_001422 in clinical specimens and cell lines was measured using qRT-PCR. The association of circ_001422 expression with the clinicopathologic features of 55 recruited patients with OS was analyzed. Loss- and gain-of-function experiments were conducted to explore the role of circ_001422 in OS cells. RNA immunoprecipitation, fluorescence in situ hybridization, bioinformatics database analysis, RNA pulldown assays, dual-luciferase reporter assays, mRNA sequencing, and rescue experiments were conducted to decipher the competitive endogenous RNA regulatory network controlled by circ_001422. Results We characterized a novel and abundant circRNA, circ_001422, that promoted OS progression. Circ_001422 expression was dramatically increased in OS cell lines and tissues compared with noncancerous samples. Higher circ_001422 expression correlated with more advanced clinical stage, larger tumor size, higher incidence of distant metastases and poorer overall survival in OS patients. Circ_001422 knockdown markedly repressed the proliferation and metastasis and promoted the apoptosis of OS cells in vivo and in vitro, whereas circ_001422 overexpression exerted the opposite effects. Mechanistically, competitive interactions between circ_001422 and miR-195-5p elevated FGF2 expression while also initiating PI3K/Akt signaling. These events enhanced the malignant characteristics of OS cells. Conclusions Circ_001422 accelerates OS tumorigenesis and metastasis by modulating the miR-195-5p/FGF2/PI3K/Akt axis, implying that circ_001422 can be therapeutically targeted to treat OS.

The checkpoint kinase ATR [ATM (ataxia-telangiectasia mutated) and rad3-related] is a master regulator of DNA damage response. Yet, how ATR activity is regulated remains to be investigated. We report here that histone demethylase PHF8 (plant homeodomain finger protein 8) plays a key role in ATR activation and replication stress response. Mechanistically, PHF8 interacts with and demethylates TOPBP1 (DNA topoisomerase 2-binding protein 1), an essential allosteric activator of ATR, under unperturbed conditions, but replication stress results in PHF8 phosphorylation and dissociation from TOPBP1. Consequently, hypomethylated TOPBP1 facilitates RAD9 (RADiation sensitive 9) binding and chromatin loading of the TOPBP1-RAD9 complex to fully activate ATR and thus safeguard the genome and protect cells against replication stress. Our study uncovers a demethylation and phosphorylation code that controls the assembly of TOPBP1-scaffolded protein complex, and provides molecular insight into non-histone methylation switch in ATR activation.

Mutations of the p53 tumor-suppressor gene are the most frequent genetic abnormality in soft tissue sarcomas. Because these rare tumors also respond poorly to standard chemotherapy and bear a 50% 5-year mortality rate, we investigated the possible therapeutic benefits of p53 gene restoration in sarcomas. We constructed Ad5p53, which is an E1A-deleted, replication-deficient adenovirus expressing a cytomegalovirus promoter-driven wild-type p53 cDNA with a Flag sequence tag. SKLMS-1 human leiomyosarcoma cells containing a mis-sense p53 point mutation were effectively transduced with Ad5p53. Increasing levels of Flag-p53 protein, as well as dose-dependent p21Cip1 induction, were observed through a dose range of 10 -500 plaque-forming units (PFU)/cell. In vitro administration of Ad5p53 as a single 100 PFU/cell dose caused 40 -60% growth inhibition of SKLMS-1 cells at posttreatment days 4, 6, and 8 compared with untreated or viral control treated-cells (P Ͻ .05, Student's t test). Relative to these same controls, in vivo treatment of SKLMS-1-bearing severe combined immunodeficient mice with 6 ϫ 10 9 PFU of Ad5p53 by intratumoral injection resulted in a 35-day tumor growth delay and complete tumor regression in 40% of mice (P Ͻ .05, Student's t test). The expression of virally derived p53 mRNA in Ad5p53-treated tumor tissues was detected in treated tumor specimens by reverse transcriptase polymerase chain reaction. Reduced intratumoral cellularity and the presence of p53 staining in adjacent normal tissue, consistent with delivery of exogenous p53 to the tumor target, were evident only in Ad5p53-treated tumors after immunohistochemical staining for p53. These results indicate that wild-type p53 gene restoration in sarcomas retards tumor growth and may come to be usefully applied to the clinical treatment of this disease as a single regimen or in combination with conventional therapies. Cancer Gene Therapy (2000) 7, 422-429

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BackgroundCircular RNAs (circRNAs) are involved in diverse processes that drive cancer development. Nevertheless, the expression landscape and mechanistic function of circRNAs in osteosarcoma (OS) remain to be studied.MethodsBioinformatics analysis and high-throughput RNA sequencing tools were employed to determine differentially expressed circRNAs between OS and adjacent healthy tissues. The expression levels of circ_001422 in clinical specimens and cell lines were measured using qRT-PCR. A total of 55 OS patients were recruited to analyze the association of circ_001422 expression with clinicopathologic features. Loss- and gain-of-function tests were conducted to explore the role of circ_001422 in OS cells. RNA immunoprecipitation, fluorescent in situ hybridization, bioinformatics databases, RNA pull-down assay, dual-luciferase reporter assay, mRNA sequencing, and rescue experiments were conducted to decipher the competitive endogenous RNAs regulatory network dominated by circ_001422.ResultsWe characterized a novel and abundant circRNA, circ_001422, which promoted the progression of OS. Circ_001422 expression was dramatically higher in OS cell lines and tissues relative to normal samples. Increased circ_001422 correlated with more advanced clinical stage, larger tumor size, more distant metastases and poorer overall survival of OS patients. Knockdown of circ_001422 markedly repressed proliferation and metastasis and promoted apoptosis of OS cells in vivo and in vitro, whereas overexpression of circ_001422 exerted an opposite effect. Mechanistically, competitive interactions between circ_001422 and miR-195-5p elevated the expression of FGF2 while also initiating the PI3K/Akt signaling pathway. These events enhanced the malignant characteristics of OS cells.ConclusionsCirc_001422 accelerates OS tumorigenesis and metastasis through modulating the miR-195-5p/FGF2/PI3K/Akt axis, implying that circ_001422 could be therapeutically targeted to treat OS patients.

Objective To analyze the characteristics of menicus microstructure and to reconstruct a microstructure‐mimicing 3D model of the menicus. Methods Human and sheep meniscus were collected and prepared for this study. Hematoxylin–eosin staining (HE) and Masson staining were conducted for histological analysis of the meniscus. For submicroscopic structure analysis, the meniscus was first freeze‐dried and then scanned by scanning electron microscopy (SEM). The porosity of the meniscus was determined according to SEM images. A micro‐MRI was used to scan each meniscus, immersed in distilled water, and a 3D digital model was reconstructed afterwards. A three‐dimensional (3D) resin model was printed out based on the digital model. Before high‐resolution micro‐CT scanning, each meniscus was freeze‐dried. Then, micro‐scale two‐dimensional (2D) CT projection images were obtained. The porosity of the meniscus was calculated according to micro‐CT images. With micro‐CT, multiple 2D projection images were collected. A 3D digital model based on 2D CT pictures was also reconstructed. The 3D digital model was exported as STL format. A 3D resin model was printed by 3D printer based on the 3D digital model. Results As revealed in the HE and Masson images, a meniscus is mostly composed of collagen, with a few cells disseminated between the collagen fiber bundles at the micro‐scale. The SEM image clearly shows the path of highly cross‐linked collagen fibers, and massive pores exist between the fibers. According to the SEM images, the porosity of the meniscus was 34.1% (34.1% ± 0.032%) and the diameters of the collagen fibers were varied. In addition, the cross‐linking pattern of the fibers was irregular. The scanning accuracy of micro‐MRI was 50 μm. The micro‐MRI demonstrated the outline of the meniscus, but the microstructure was obscure. The micro‐CT clearly displayed microfibers in the meniscus with a voxel size of 11.4 μm. The surface layer, lamellar layer, circumferential fibers, and radial fibers could be identified. The mean porosity of the meniscus according to micro‐CT images was 33.92% (33.92% ± 0.03%). Moreover, a 3D model of the microstructure based on the micro‐CT images was built. The microscale fibers could be displayed in the micro‐CT image and the reconstructed 3D digital model. In addition, a 3D resin model was printed out based on the 3D digital model. Conclusion It is extremely difficult to artificially simulate the microstructure of the meniscus because of the irregularity of the diameter and cross‐linking pattern of fibers. The micro‐MRI images failed to demonstrate the meniscus microstructure. Freeze‐drying and micro‐CT scanning are effective methods for 3D microstructure reconstruction of the meniscus, which is an important step towards mechanically functional 3D‐printed meniscus grafts.

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