OBJECTIVETo investigate the expressions and the role of Notch signaling-associated proteins in esophageal squamous cell carcinoma (ESCC).

METHODSFifty patients with ESCC were included in this study. The expressions of Notch signaling-associated protein (4 receptors: Notch1, Notch2, Notch3, Notch4; 5 ligands: Dll1, Dll3, Dll4, Jagged1, Jagged2) in cancer foci and adjacent normal tissues (5 cm distance to cancer) were examined by immunohistochemitry. Correlations of these proteins with cancer cell proliferation(Ki-67 index) and clinicopathologic features were investigated.

RESULTSHigher levels of Notch1 and Notch2 were measured in cancer foci compared with adjacent tissues (all P<0.05). There were no differences in the expressions of Notch3, Dll1 and Dll3 (all P>0.05). Notch4, Dll4 and Jagged2 were not detected in both cancer foci and adjacent tissues. Notch1 expression was negatively correlated with lymph node metastasis and TNM staging (all P<0.01). Jagged 1 expression was positively correlated with TNM staging (P<0.01). Ki-67 index was obviously higher in cancer foci, while it was negatively correlated with Notch1 and Notch3 (all P<0.01) and positively correlated with Dll1 and Jagged 1 (all P<0.01).

CONCLUSIONNotch signaling path may act as tumor suppressive gene in the pathogenesis of esophageal squamous cell cancer, in which Notch1 protein plays an important role.

Carcinoma, Squamous Cell ; metabolism ; Esophageal Neoplasms ; metabolism ; Genes, Tumor Suppressor ; Humans ; Lymphatic Metastasis ; Neoplasm Staging ; Receptor, Notch1 ; metabolism ; Receptor, Notch2 ; metabolism ; Receptor, Notch3 ; Receptors, Notch ; metabolism ; Signal Transduction

OBJECTIVETo explore the effect of silencing the Notch2 gene by small interfering (si)RNA on the proliferation of the HepG2 human hepatocellular carcinoma (HCC) cells.

METHODSNotch2-siRNA was transfected as a liposomal formulation into HepG2 cells. The non-HCC cell lines SG07901 (gastric cancer) and SW620 (colon cancer) were used as controls. The mRNA expression of Notch2 and Hesl were detected by RTPCR, and the protein expression of Notch2 was detected by western blotting. The proliferation of transfected HepG2 cells was assessed by the cell counting kit-8 (CCK8) colorimetric assay.

RESULTSThe untransfected HepG2 cells showed significantly upregulated transcript expression of Notch2, and not of Notch1, Notch3 or Notch4, compared to the other non-HCC cell lines. Following transfection of Noteh2-siRNA into HepG2 cells, the mRNA expression of Notch2 and Hes1 and the protein expression of Notch2 were significantly decreased. The rales of proliferation inhibition in HepG2 following transfection of Notch2-siRNA showed an increasing time-related trend, with 2.64% ± 1620% at 12 h, 38.34% ± 8.80% at 24 h, 70.05% ± 7.80% at 48 h, 70.78% ± 10.00% at 72 h, and 74.22% ± 4.80% at 96 h.The inhibition rate at 24 h of transfection was significantly different from that of the groups of control cells.

CONCLUSIONNotch2 is upregulated in the common HCC cultured cell line HepG2. siRNA-mediated silencing of Notch2 exerts inhibition effects on HepG2 proliferation, suggesting the potential for this approach as targeted therapy for treating HCC.

Carcinoma, Hepatocellular ; pathology ; Cell Proliferation ; Down-Regulation ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; RNA Interference ; RNA, Small Interfering ; Receptor, Notch2 ; metabolism

OBJECTIVEThis study aimed to explore the effect of the up-regulation of Notch1 on osteoclastogenesis induced to osteoclasts by receptor activator for nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factors (MCSF) in vitro.

METHODSThe bone marrow stem cells (BMSCs) of Rosa(-notch1) mice were cultured and induced to osteoclasts by RANKL and MCSF. The BMSCs were transfected with the Ad-Cre-green fluorescent protein (GFP) virus or Ad-GFP virus. Total RNA from cells was extracted, and the gene expression levels of Notch1, Notch2, Notch3, Notch4, Deltal, Delta3, Delta4, Jagged1, Hes1, and tartrate resistant acid phosphatase (TRAP) were detected at the defined stage by reverse transcription-polymerase chain reaction (RT-PCR). Osteoclast formation was analyzed by TRAP assay.

RESULTSThe number of TRAP-positive multinuclear cells of the experimental group significantly decreased compared with that of the control group. The mRNA expression levels of Notch1, Notch3, Jagged1, Delta3, and Hesl of the experimental group were significantly higher than those of the control group, whereas the TRAP mRNA expression of the experimental group was significantly lower than that of the control group (P<0.05).

CONCLUSIONUp-regulation of Notch1 inhibit osteoclastogenesis of BMSCs induced by RANKL and MCSF in vitro.

Animals ; Cell Differentiation ; Cell Line ; In Vitro Techniques ; Macrophage Colony-Stimulating Factor ; Mice ; Osteoclasts ; RANK Ligand ; Receptor Activator of Nuclear Factor-kappa B ; Receptor, Notch1 ; metabolism ; Receptor, Notch2 ; Up-Regulation ; physiology

OBJECTIVETo detect the differential expression of Notch1 and Notch2 in human astrocytoma and medulloblastoma; and to study the role of Notch1 and Notch2 in the development of both tumors.

METHODSImmunohistochemical staining (SP method) and Western blot analysis were used to detect Notch1 and Notch2 expression in tissue arrays and freshly resected samples of normal brain tissue, astrocytoma and medulloblastoma.

RESULTSNotch1 and Notch2 were negative in normal human brain tissue. Notch1 was highly expressed (total positive rate 80.0%, 48/60) while Notch2 was not detected in grade IV astrocytomas and sporadically observed in lower grade astrocytomas (total positive rate 10.0%, 6/60). The percentage of positive tumor cells and expression level of Notch1 increased with higher histologic grade (r = 0.859, P < 0.05). On the other hand, overexpression of Notch2 was detected in medulloblastoma (9/10) in contrast with lower expression of Notch1 (2/10).

CONCLUSIONSNotch1 and Notch2 show differential expression in astrocytoma and medulloblastoma. This may be related to their different functional activities during the process of brain development.

Adolescent ; Adult ; Aged ; Astrocytoma ; metabolism ; Biomarkers, Tumor ; metabolism ; Brain ; metabolism ; Brain Neoplasms ; metabolism ; Child ; Child, Preschool ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Medulloblastoma ; metabolism ; Middle Aged ; Receptor, Notch1 ; metabolism ; physiology ; Receptor, Notch2 ; metabolism ; physiology ; Young Adult

A 21-year-old man with diabetic ketoacidosis (DKA) displayed short and clubbed fingers and marked eyebrow, which are typical of Hajdu-Cheney Syndrome (HCS). Laboratory findings confirmed type 1 diabetes mellitus (DM). After conservative care with hydration and insulin supply, metabolic impairment was improved. Examinations of bone and metabolism revealed osteoporosis and craniofacial abnormalities. The mutation (c.6443T>G) of the NOTCH2 gene was found. The patient was diagnosed with HCS and DM. There may be a relationship between HCS and DM, with development of pancreatic symptoms related to the NOTCH2 gene mutation.
Adult ; Bone Density ; Craniofacial Abnormalities/complications/radiography ; Diabetes Mellitus, Type 1/*complications/diagnosis ; Diabetic Ketoacidosis/complications/genetics ; Glycosuria ; Hajdu-Cheney Syndrome/*complications/diagnosis/radiography ; Humans ; Ketone Bodies/urine ; Male ; Mutation ; Osteoporosis/complications/radiography ; Receptor, Notch2/*genetics ; Young Adult

BACKGROUNDNotch activation leads to transcriptional suppression of lineage-specific genes, inhibiting differentiation in response to inductive signals. The Notch signal system contains three parts: Notch molecules, Notch ligands and effectors. Delta4 is a newly-discovered Notch ligand which has received the attention of few detailed studies. This study sought to explore the biological function of Delta4 and observe its effects on 32D cell differentiation.

METHODSDelta4-expressing vector pTracer.CMV.Delta4.FLAG was constructed using molecular biological techniques. CHO cells stably transfected with pTracer.CMV.Delta4.FLAG were confirmed to have a Delta4 protein band via Western blotting. High-expression Delta4-CHO clones were selected for the following functional studies. Notch1-CHO and Notch2-CHO were used as host cells. After transiently transfecting with transition protein 1 (TP1), Delta4 activity was compared in both cell lines by means of luciferase analysis. CHO cells were incubated with Notch1-32D cells that had been transfected with Notch1 and were observed for granulocyte colony-stimulating factor (G-CSF)-induced differentiation. Jagged2-CHO and Delta4-CHO cells transfected with the Notch ligands Jagged2 and Delta4, respectively, were incubated with Notch1-32D cells to observed inhibition of Notch on G-CSF-induced differentiation.

RESULTSThe vector pTracer.CMV.Delta4.FLAG was constructed successfully. CHO cells were stably transfected with the vector pTracer.CMV.Delta4.FLAG. Two CHO cell lines expressing Delta4 at high levels were selected for use in the study. Delta4 was found to induce signal activity via both Notch1 and Notch2 and the induction of signaling activity was stronger in Notch2 cells than in Notch1 cells. Compared with other Notch ligands, Delta4 was slightly weaker than Jagged2, but stronger than Delta1 and Jagged1 in terms of Notch1 ligands. In terms of Notch2, Delta4 had a strong signaling activity, but was weaker than Delta1, Jagged1, and Jagged2. Jagged2 could inhibit Notch1-32D cell differentiation induced by G-CSF, but Delta4 could not.

CONCLUSIONSDelta4 induces both Notch1 and Notch2 activity and is a ligand for both of them. The effect of Delta4 is stronger on Notch2 than that on Notch1. Jagged2 can inhibit Notch1-32D cell differentiation induced by G-CSF, but Delta4 cannot.

Animals ; CHO Cells ; Calcium-Binding Proteins ; Cell Differentiation ; Cricetinae ; Intercellular Signaling Peptides and Proteins ; Intracellular Signaling Peptides and Proteins ; Jagged-1 Protein ; Jagged-2 Protein ; Membrane Proteins ; genetics ; physiology ; Mice ; Receptor, Notch1 ; Receptor, Notch2 ; Receptors, Cell Surface ; physiology ; Serrate-Jagged Proteins ; Signal Transduction ; Transcription Factors ; physiology

OBJECTIVETo explore the mechanism of Notch signaling transduction system and its effects on hematopoietic system.

METHODSNotch ligands transfected CHO cells were added into Notch1 and Notch2 transfected CHO cells, which were transiently transfected with reporter gene TP1. PGL-100 was used as substrate to test the interaction between Notch and Notch ligands. CHO, Jagged2-CHO and Delta 4-CHO cells were seeded in the petri dish containing G-CSF, and then Notch 1-32D cells were added in it to observe the differentiation of Notch1-32D cell after incubation and staining.

RESULTSAll of the five Notch ligands binding to Notch1 could induce TP1 activity, it increased significantly the Jagged2-CHO, Delta 4-CHO1-4 and Delta 4-CHO1-5 cells. For Notch2, the TP1 activity induced by the five ligands in these cells was much higher than that of CHO. At the presence of G-CSF, Notch1-32D could differentiate to mature granulocyte. Jagged2 could inhibit G-CSF induced Notch1-32D cell differentiation, but Delta 4 could not.

CONCLUSIONJagged2 and Delta 4 are the ligands of Notch1. Jagged2 can inhibit G-CSF induced Notch1-32D cell differentiation, but Delta 4 can not.

Animals ; CHO Cells ; Calcium-Binding Proteins ; genetics ; metabolism ; physiology ; Cell Differentiation ; physiology ; Cricetinae ; Cricetulus ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; physiology ; Membrane Proteins ; genetics ; metabolism ; physiology ; Receptor, Notch1 ; genetics ; metabolism ; physiology ; Receptor, Notch2 ; genetics ; metabolism ; physiology ; Receptors, Notch ; genetics ; metabolism ; physiology ; Serrate-Jagged Proteins ; Signal Transduction ; physiology ; Transfection

OBJECTIVETo study the roles of the Notch2-Delta signaling in the differentiation of dental pulp cells into odontoblasts in vitro.

METHODSA long-term culture system was used as a model for the study of proliferation and differentiation of pulp cells into preodontoblasts. The expression patterns of Notch2 and its ligand Delta in different phases of long-term cultured dental pulp cells were studied by immunohistochemistry and western blotting. Furthermore, the effects of rhBMP-2 on the expression of Notch signaling members were studied.

RESULTSNotch2 and its ligand Delta expressed in all phases of dental pulp cells and the sites and levels of their expression changed with different phases. rhBMP-2 could significantly up-regulated the expression of Delta in the phase of later cell nodule formation.

CONCLUSIONThis study demonstrated that Notch signaling pathway played roles in the differentiation of long-term cultured human dental pulp cells which formed nudules that were slightly mineralized and had ultrastructural features reminiscient of preodontoblasts, and it may be the mechanism that control pulpal cell's responsiveness to signaling molecules in the healing of wound pulp.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; pharmacology ; Cell Differentiation ; Cells, Cultured ; Dental Pulp ; cytology ; metabolism ; Humans ; Immunohistochemistry ; Intracellular Signaling Peptides and Proteins ; Membrane Proteins ; biosynthesis ; physiology ; Odontoblasts ; cytology ; metabolism ; Receptor, Notch2 ; Receptors, Cell Surface ; biosynthesis ; physiology ; Recombinant Proteins ; pharmacology ; Signal Transduction ; Transforming Growth Factor beta

OBJECTIVETo investigate the effect of both fermented Cordyceps powder (CS) and prednisone on the Notch2/hes-1 signaling activation in the kidney tubules of rats with acute aristolochic acid nephropathy (AAAN).

METHODSTotally 50 SD rats were randomly divided into 4 groups, i.e., the normal group, the model group, the CS group, the prednisone group, and the CS plus prednisone group, 10 in each group. The AAAN rat model was induced by intragastric administration of pure aristolochic acid A at the daily dose of 100 mg/kg for 3 days. Rats in the CS group were administered with CS at the daily dose of 5.0 g/kg by gastrogavage, while those in the prednisone group were administered with prednisone at the daily dose of 0.5 mg/kg. Rats in the CS plus prednisone group were treated by CS and prednisone. All treatment lasted for 3 successive weeks. Kidney functions [urea nitrogen (BUN) and serum creatinine (SCr)] were detected. The pathological changes of kidneys were observed by Hematoxylin-Eosin staining. The apoptosis of the renal tubular epithelial cells was detected by TUNEL. The protein expressions of Notch2 and Hes-1 in the renal tissue were detected by immunohistochemical assay and Western blot.

RESULTSResults of HE staining showed the structure in the nephridial tissue was regular in rats of the normal group. The renal tubular necrosis occurred in the rats of the model group. The pathological changes of kidneys were obviously improved in the CS group, the prednisone group, and the CS plus prednisone group. Compared with the normal group, levels of BUN and SCr, semi-quantitative score of the tubular interstitial tissue, ratio of apoptotic cells, and expressions of Notch2 and Hes-1 proteins significantly increased in the model group (P < 0.01). Compared with the model group, the aforesaid indices significantly decreased in the 3 treatment groups (P < 0.01). All indices decreased most obviously in the CS plus prednisone group (P < 0.05, P < 0. 01).

CONCLUSIONSNotch2/hes-1 signaling activation might be associated with apoptosis of renal tubular epithelial cells. Both CS and prednisone could play a nephroprotective role for AAAN. But CS plus prednisone could achieve the best effect. Inhabiting the Notch2/hes-1 signaling activation could be its nephroprotective mechanism.

Animals ; Apoptosis ; drug effects ; Aristolochic Acids ; toxicity ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cordyceps ; Female ; Homeodomain Proteins ; metabolism ; Kidney ; metabolism ; Kidney Diseases ; chemically induced ; metabolism ; Kidney Function Tests ; Kidney Tubules ; metabolism ; Male ; Prednisone ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch2 ; metabolism ; Signal Transduction ; drug effects ; Transcription Factor HES-1