T-cell acute lymphoblastic leukemia (T-ALL) is the hematological malignancy of bone marrow characterized by the rapid proliferation and subsequent accumulation of immature T lymphocyte and mainly occurs in children and adolescents. In 1991, a kind of activating mutation of Notch 1 was found in a subset of T-ALL with chromosomal translocation t(7;9) for the first time. During the past 20 years since then, understanding of the relationship between Notch 1 activating mutation and T-ALL has been deepened and widened. This review briefly discusses the four main subtypes of Notch 1 activating mutations, also focuses on how these mutations change the normal signaling pathways and genes expression during their participation in the pathogenesis of T-ALL, and how these insights will promote the development of newly targeting therapies for patients with this aggressive form of leukemia.
Humans ; Mutation ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; etiology ; genetics ; Receptor, Notch1 ; genetics

The aim of the present study was to observe the effects of Notch1 and autophagy on extracellular matrix deposition in renal tubulointerstitium of diabetes and to explore the mechanism. The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice). After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. Rat renal tubular epithelial cells NRK52E were cultured under normal glucose (NG) and high glucose (HG) respectively, and the expression of Notch1 and LC3 proteins were detected by Western blotting. Autophagosomes in NRK52E cells with overexpressed and knockdown Notch1 under NG and HG conditions were observed by confocal microscope, and the expression changes of Notch1, Collagen-I and III protein were detected by immunofluorescence. The results showed that the Notch1 and Collagen-III expressions were increased (P < 0.01) and the LC3 expression was decreased (P < 0.05) in db/db mice compared with db/m mice. In vitro, the Notch1 was increased (P < 0.01) and the LC3 expression was decreased significantly (P < 0.01) in NRK52E cells of HG group compared with NG group. There was no significant change of Notch1 and LC3 expression between the mannitol (MA) group and the NG group. Autophagy was decreased and extracellular matrix deposition was aggravated when Notch1 was overexpressed. In contrast, autophagy was increased and extracellular matrix deposition was relieved by knockdown of Notch1 under HG conditions. In conclusion, Notch1 protein expression was increased and autophagy was reduced in renal tissue of diabetes and renal tubular epithelial cells under HG. The extracellular matrix deposition in the renal tubulointerstitium was relieved by regulating autophagy after the knockdown of Notch1.
Animals ; Autophagy/physiology* ; Diabetes Mellitus ; Extracellular Matrix ; Glucose/pharmacology* ; Kidney ; Mice ; Rats ; Receptor, Notch1/genetics*

This study aimed to construct the dual expression vectors of wide type or N187D mutant P2X7 receptor and intracellular domain of Notch1 (ICN1) linked by 2A peptide to coexpress them in leukemia cells so as to lay a foundation for further investigating the role of P2X7 in development of leukemia. Overlap PCR was used to construct the dual expression vectors encoding wide type or N187D mutant type P2X7 receptor and ICN1 linked by the self-cleaving 2A sequence. The results showed that stable expressing cell lines were obtained by retroviral infection followed by cell sorting after DNA sequence analysis. RT-PCR, Western blot, intracellular free calcium concentration analysis were used to verify the functionally successful construction of K562 cell line expressing P2X7 receptor alone or with ICN1. DNA sequence analysis revealed that all construction were right. The infection efficiency of packaged constructed virus ranged from 40% to 70% for K562 cells. Stable infected cell line was obtained by cell sorting. RT-PCR analysis revealed that P2X7 receptor and/or ICN1 could be detected at high level in their stable infected cell lines, respectively. Western blot analysis also showed that P2X7 receptor was highly expressed in cell line infected by virus with P2X7 receptor. Sustained increase in intracellular free calcium concentration ([Ca(2+)]i) could be observed in K562 cells overexpressing either type of P2X7 receptor upon stimulation with BzATP. It is concluded that the wide type or N187D mutant P2X7 receptor and ICN1 are simultaneously and functionally over-express in leukemia cells, which lay a foundation for further studying the role of P2X7 receptor in the development of leukemia.
Gene Expression ; Genetic Vectors ; Humans ; K562 Cells ; RNA, Messenger ; genetics ; Receptor, Notch1 ; genetics ; Receptors, Purinergic P2X7 ; genetics ; Retroviridae ; genetics

OBJECTIVETo construct inducible lentiviral vector containing human Notch1 intracellular domain (NICD) gene and enhanced green fluorescent protein (EGFP), and to study its expression in PC12 cells.

METHODSNICD cDNA was amplified by RT-PCR from human placenta tissue. EGFP gene was amplified by PCR from pEGFP-C1. Both NICD and EGFP were cloned into pcDNA 3.1 (+) plasmid to form pcDNA3.1-Notch1-EGFP. Then the Notch1-EGFP fragment was separated and cloned into pLVX-Tight-puro to form pLVX-Notch1-EGFP. The lentivirus were packaged and harvested, which were used to infect PC12 cells. After antibody selection for 2 weeks, the PC12 cells were induced by doxycycline (Dox). The expression of Notch1-EGFP was detected by fluorescence microscope and flow cytometry.

RESULTSThe recombinant inducible lentiviral vectors (pLVX-Notch1-EGFP) were success fully constructed. The EGFP positive cell percentage was over 90% in transfected PC12 cells after 500 ng/ml Dox induction for 36 h. The expression of Notch1 was posited correlated to the Dox concentration. The expression of Notch1 increased with the duration of Dox induction, which got the peak at 36 h after Dox induction.

CONCLUSIONThe recombinant inducible lentiviral vectors containing Notch1 and EGFP gene are successfully constructed, which provides an effective and simple method to regulate the expression of Notch1 in PC12 cells.

Animals ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Lentivirus ; genetics ; PC12 Cells ; Plasmids ; Rats ; Receptor, Notch1 ; genetics ; Transfection

BACKGROUNDIschemic postconditioning (IPost), able to significantly attenuate myocardial ischemia reperfusion injury, is dependent on RISK signaling. Studies have shown that Notch signaling repairs damaged myocardium, and this study aimed to investigate the effect of Notch signaling in myocardial IPost.

METHODSWe used H9c2 cells to establish the myocardial IPost and Hypoxia/Reoxygenation (H/R) model in vitro. which were randomly divided into control, H/R, IPost, Hepatocyte growth factor (HGF)+IPost and DAPT+IPost, N1ICD+IPost, miRNA+IPost, and Mock treatment groups. The myocardial cell viability was assessed by MTT, the cell apoptosis was detected using Annexin V/PI double staining and flow cytometry analyses. The expression of N1ICD, Hes1, PTEN Phospho-Akt/Akt, Phospho-GSK-3β/GSK-3β were detected by Western blotting. Finally, we assessed the changes in ψm using the potential-sensitive dye JC-1 and measured using flow cytometry analyses.

RESULTSThe Notch1 signaling is activated by HGF and ectopic expression of N1ICD during myocardial IPost, which increased myocardial cell viability, prevented cardiomyocyte apoptosis, and reduced loss of the mitochondrial membrane potential. However, myocardial ischemia reperfusion injury was increased in IPost when Notch1 signaling was inhibited using DAPT or with knockdown by Notch1-miRNA. Western blotting found that PTEN was down-regulated by Hes1 when Notch1 was activated, which consequently promoted Akt and GSK-3β phosphorylation.

CONCLUSIONSNotch1 crosstalk with RISK signaling may be dependent on PTEN, which plays a cardioprotective role during IPost. This mechanism could provide a promising therapeutic target for the treatment of ischemic heart disease.

Cell Line ; Humans ; Ischemic Postconditioning ; Myocardial Reperfusion Injury ; genetics ; metabolism ; prevention & control ; Receptor, Notch1 ; genetics ; metabolism ; Signal Transduction ; physiology

The effects of microRNA-34a (miR-34a)-regulated Notch1 gene on the proliferation and apoptosis of the human glioma cell line U87 were investigated in this study. The U87 cells were divided into miR-34a mimics, negative control, mock transfection and blank control groups in terms of different treatments. In miR-34a mimics group, human U87 glioma cells were transfected with miR-34a mimics by using lipofectamine 2000. The cells transfected with nonsense microRNA were set up as negative control group. Those treated with lipofectamine 2000 only were designated to the mock tranfection group. In the blank control group, the cells were cultured routinely and no treatment was given. The expression of miR-34a and Notch1 was detected by using real-time RT-PCR. Western blotting was employed to monitor the change in Notch1 protein. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry. The results showed that the proliferative ability of U87 cells was significantly reduced and the apoptotic cells increased in miR-34a mimics group relative to control groups. The expression of miR-34a was significantly up-regulated in mimics group as compared with control groups (P<0.05). Furthermore, Notch1 protein levels were significantly decreased in miR-34a mimics group when compared with control groups (P<0.05), but the mRNA expression of Notch1 showed no significant difference among these groups. It was concluded that miR-34a may suppress the proliferation and induce apoptosis of U87 cells by decreasing the expression of target gene Notch1, suggesting that miR-34a may become a promising gene therapeutic target for brain glioma.
Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; genetics ; Glioma ; pathology ; physiopathology ; Humans ; MicroRNAs ; genetics ; Receptor, Notch1 ; physiology ; Transfection

OBJECTIVETo investigate the effect of ADAR1 on the occurrence and development of mouse T cell acute lymphoblastic leukemia (T-ALL).

METHODSLck-Cre; ADAR1lox/lox mice were generated through interbreeding. The lineage-cells of Lck-Cre; ADAR1lox/lox mice and the control were enriched respectively by the means of MACS, and the lin- cells were transfected with retrovirus carrying MSCV-ICN1-IRES-GFP fusion gene. Then the transfection efficiency was detected by the means of FACS, and the same number of GFP+ cells were transplanted into lethally irradiated recipient mice to observe the survival of mice in 2 recipient group after transplantation.

RESULTST cell-specific knockout ADAR1 mice were generated, and Notch1-induced T-ALL mouse model was established successfully. The leukemia with T-ALL characteristics occured in the mice of control group, but did not in the ADAR1 kmockout mice after transplantation.

CONCLUSIONSADAR1 plays a key role in the incidence and development of Notch1-induced T-ALL.

Adenosine Deaminase ; genetics ; Animals ; Disease Models, Animal ; Mice ; Mice, Knockout ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Receptor, Notch1 ; genetics ; T-Lymphocytes

This study was aimed to investigate the characteristics and clinical significance of NOTCH1 mutation in adult T-cell acute lymphoblastic leukemia (T-ALL). Exon 26/N-terminal region of the heterodimerization domain (HD-N) , exon 27/ C-terminal region of the heterodimerization domain (HD-C) , exon 28 and exon 34/ proline-glutamic acid-serine-threonine (PEST) domain of the NOTCH1 gene were amplified, cloned and sequenced in 42 adult patients with T-ALL to identify the frequency, position and type of NOTCH1 mutation, their correlations with laboratorial and clinical parameters, as well as their relevant prognostic significance. The results showed that the frequency of NOTCH1 mutation in this cohort of adult patients was 66.7% (28/42); A total of 45 types of NOTCH1 mutations were identified in present study, most of them were in HD-N (48.9%, 22/45) and PEST (40.0%, 18/45) domains. Mutation in amino acid 1575 (L1575P) was the top one type of mutation in HD-N (25.0%, 7/28), and amino acid 2443 was the most common mutation position in PEST domain (14.3%, 4/28). In newly diagnosed patients, white blood cell (WBC) >10×10(9)/L and blasts in bone marrow > 50% were predominant in patients with NOTCH1 mutation (91.7% vs 54.5%, P = 0.021 and 95.8% vs 57.1%, P = 0.006 respectively). Immunophenotyping analysis indicated that patients with CD10 positive were more in NOTCH1 mutation group than wild-type group (51.9% vs 0%, P = 0.006), whereas patients with CD15 and CD11b positive were less in NOTCH1 mutation group (5.3% vs 42.9%, P = 0.047 and 0% vs 57.1%, P = 0.002 respectively). It is concluded that NOTCH1 mutation in adult T-ALL has different characteristics and clinical significance from pediatric patients, and the difference between Chinese patients and patients in Western countries is also indicated.
Adolescent ; Adult ; Base Sequence ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Mutation ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Receptor, Notch1 ; genetics ; Young Adult

This study examined the effect of Notch-1 signaling on malignant behaviors of breast cancer cells by regulating breast cancer stem cells (BCSCs). BCSCs were enriched by using serum-free medium and knocked out of Notch-1 by using a lentiviral vector. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the Notch-1 expression levels in breast cancer cell lines and BCSCs, and flow cytometry to detect the proportion of BCSCs in BCSC spheres. The BCSC self-renewal, migration, invasion, and tumorigenicity were examined by the tumor microsphere-forming assay and transwell assay and after xenotransplantation. The results showed that the Notch-1 silencing reduced the number of BCSC spheres, the proportion of BCSCs, and the number of cells penetrating through the transwell membrane. It also decreased the size of tumors that were implanted in the nude mice. These results suggest that Notch-1 signaling is intimately linked to the behaviors of BCSCs. Blocking Notch-1 signaling can inhibit the malignant behaviors of BCSCs, which may provide a promising therapeutical approach for breast cancer.
Animals ; Breast Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Female ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Neoplastic Stem Cells ; metabolism ; Receptor, Notch1 ; biosynthesis ; genetics ; Signal Transduction

OBJECTIVETo investigate the roles of FIZZ1 and NOTCH1 in the pathogenesis of asthma and the effect of rosiglitazone on airway remodeling.

METHODSForty-five healthy 6 to 8-week-old Sprague-Dawley rats were randomly divided into a control group and asthma groups with and without rosiglitazone treatment. The paraffin slices of lung tissues were made to assess the histological changes. a-SMA protein, a specific marker of airway remodeling, in lung tissues was measured by immunohistochemistry. FIZZl-mRNA and NOTCH1-mRNA expression in lung tissues was measured by RT-PCR.

RESULTSThe characteristic changes of airway remodeling were observed in the untreated asthma group. The histological changes in the airway were less severe in the rosiglitazone treated asthma group. Positive a-SMA staining, FIZZl-mRNA and NOTCH1-mRNA were highly expressed in peribronchial lung sections isolated from the untreated asthma group. Rosiglitazone treatment decreased significantly the expression of a-SMA protein, FIZZl-mRNA and NOTCH1-mRNA compared with the untreated asthma group, but the expression of a-SMA protein, FIZZl-mRNA and NOTCH1-mRNA in the rosiglitazone treated asthma group remained higher than the control group. a-SMA expression was positively correlated with FIZZl-mRNA (r=0.826, P<0.01) and NOTCH1-mRNA expression (r=0.9, P<0.01). FIZZl-mRNA expression was positively correlated with NOTCH1-mRNA expression (r=0.76, P<0.01).

CONCLUSIONSFIZZl and NOTCH1 may induce an increase in a-SMA expression. FIZZl and NOTCH1 play a critical role in the process of airway remodeling. Rosiglitazone treatment may inhibit airway remodeling in asthmatic rats.

Actins ; Airway Remodeling ; Animals ; Asthma ; etiology ; pathology ; Lung ; metabolism ; pathology ; Male ; Nerve Growth Factor ; genetics ; physiology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; genetics ; physiology