Macrophages Promote Aortic Valve Cell Calcification and Alter STAT3 Splicing.

OBJECTIVE: Macrophages have been described in calcific aortic valve disease, but it is unclear if they promote or counteract calcification. We aimed to determine how macrophages are involved in calcification using the Notch1+/- model of calcific aortic valve disease. Approach and Results: Macrophages in wild-type and Notch1+/- murine aortic valves were characterized by flow cytometry. Macrophages in Notch1+/- aortic valves had increased expression of MHCII (major histocompatibility complex II). We then used bone marrow transplants to test if differences in Notch1+/- macrophages drive disease. Notch1+/- mice had increased valve thickness, macrophage infiltration, and proinflammatory macrophage maturation regardless of transplanted bone marrow genotype. In vitro approaches confirm that Notch1+/- aortic valve cells promote macrophage invasion as quantified by migration index and proinflammatory phenotypes as quantified by Ly6C and CCR2 positivity independent of macrophage genotype. Finally, we found that macrophage interaction with aortic valve cells promotes osteogenic, but not dystrophic, calcification and decreases abundance of the STAT3ß isoform. CONCLUSIONS: This study reveals that Notch1+/- aortic valve disease involves increased macrophage recruitment and maturation driven by altered aortic valve cell secretion, and that increased macrophage recruitment promotes osteogenic calcification and alters STAT3 splicing. Further investigation of STAT3 and macrophage-driven inflammation as therapeutic targets in calcific aortic valve disease is warranted.

Localization of DLL1- and NICD-positive osteoblasts in cortical bone during postnatal growth in rats.

The long bone midshaft expands by forming primary osteons at the periosteal surface of cortical bone in humans and rodents. Osteoblastic bone formation in the vascular cavity in the center of primary osteons is delayed during cortical bone development. The mechanisms of the formation of primary osteons is not fully understood, however. Focusing on NOTCH1 signaling, an inhibitory signaling on osteoblastic bone formation, our immunohistochemical analysis revealed Delta like1 (DLL1), a ligand of NOTCH1, and the NOTCH1 intracellular domain (NICD, an activated form of NOTCH1) immunoreactivity, in the cuboidal osteoblasts lining the bone surface in the vascular cavity of primary osteons during postnatal growth in rats. Interestingly, five days after treatment of primary osteoblasts with ascorbic acid and ß glycerophosphate, protein levels of both DLL1 and NICD increased transiently, indicating that DLL1 activates NOTCH1 in primary cultured osteoblasts. Thus, the results imply that DLL1-NOTCH1 signaling in osteoblasts is associated with primary osteonal bone formation.

Role of epithelial-mesenchymal transition involved molecules in the progression of cutaneous melanoma.

Epithelial-mesenchymal transition (EMT) has been suggested to have a driving role in the acquisition of a metastatic potential by melanoma cells. Important hallmarks of EMT include both E-cadherin downregulation and increased expression of N-cadherin. This switch in distinct classes of adhesion molecules leads melanoma cells to lose contact with adjacent keratinocytes and interact instead with stromal fibroblasts and endothelial cells, thus promoting dermal and vascular melanoma invasion. Consequently, tumor cells migrate to distant host tissues and establish metastases. A key regulator in the induction of EMT in melanoma is the Notch1 signaling pathway that, when activated, is prompt to upregulate N-cadherin expression. By means of this strategy, melanoma cells gain enhanced survival, proliferation and invasion properties, driving the tumor toward a more aggressive phenotype. On the basis of these statements, the present study aimed to investigate the possible association between N-cadherin and Notch1 presence in primary cutaneous melanomas and lymph node metastases. Our results from immunohistochemical analysis confirmed a positive correlation between N-cadherin and Notch1 presence in the same tumor samples. Moreover, this study highlighted that a concomitant high expression of N-cadherin and Notch1, both in primary lesions and in lymph node metastases, predicts an adverse clinical outcome in melanoma patients. Therefore, N-cadherin and Notch1 co-presence can be monitored as a predictive factor in early- and advanced-stage melanomas and open additional therapeutic targets for the restraint of melanoma metastasis.

Human cytomegalovirus infection promotes the stemness of U251 glioma cells.

Glioblastoma (GBM) are the most common and aggressive tumors of human brain. Recent studies showed that human cytomegalovirus (HCMV) can induce malignant transformation of tumor cells to maintain stemness. Transcription factor 5 (ATF5) is an anti-apoptotic protein that is highly expressed in malignant glioma. The aim of this study is to investigate the effect of HCMV infection on the stem cell makers of U251 cells. U251 cells were infected by AD169 HCMV strain (MOI = 1). The expression of stem cell makers (CD133, NES, Notch1) in infected U251 cells were compared with the expression in uninfected U251 cell to see the difference between them. Then, the changes of cell proliferation activity and the expression level of Notch intracellular domain (NICD), Notch1, ATF5, and IE protein were detected in the infected cells, and the expressions of Notch1 and NICD were increased. Cell proliferation assay showed that HCMV infection significantly increased the proliferation. These cells could form tumor spheres in non-adherent conditions. Consistent with these findings, the effect of silencing ATF5 on the proliferation of HCMV-infected U251 cells was also examined. The result shows that short interfering RNA-mediated ATF5 downregulation inhibited this process. These findings imply that HCMV infection may regulate ATF5 signaling pathway to increase cell malignant traits and maintain stemness. J. Med. Virol. 89:878-886, 2017. © 2016 Wiley Periodicals, Inc.

Expression of activated Notch1 and Hey1 in papillary thyroid carcinoma.

AIMS: The Notch signalling pathway is involved in normal development as well as tumorigenesis. However, it is unclear whether Notch activation is related to diverse clinicopathological factors in papillary thyroid carcinoma (PTC). METHODS AND RESULTS: We examined the relationship between clinicopathological factors and the expression of activated Notch1 and Hey1, which are indicators of Notch signalling pathway activation, in 109 PTC cases. Activated Notch1 showed strong, moderate and weak expression in 23, 48 and 36 cases, respectively. Its expression was related significantly to histopathological variants (P = 0.007), lymph node metastasis (P = 0.016), BRAF mutation (P = 0.036) and extent of surgery (P = 0.014). Hey1 immunostaining could be divided into two groups: positive and negative, with 26 and 83 cases, respectively. Its expression was related significantly to histopathological variants (P = 0.026), extrathyroidal extension (P = 0.005), BRAF mutation (P = 0.048) and recurrence or soft tissue metastasis (P = 0.000). Multivariate analysis revealed that tumour size (>1 cm), Hey1 immunoreactivity and the presence of lymph node metastasis were associated significantly with recurrence or soft tissue metastasis (odds ratio = 7.38, 4.28 and 12.00, respectively). CONCLUSIONS: Thus, we found that activation of Notch signalling was correlated significantly with clinicopathological parameters. Therefore, Notch signalling could be a useful prognostic marker in patients with PTC.

Expression of p53, p16, cyclin D1, epidermal growth factor receptor and Notch1 in patients with temporal bone squamous cell carcinoma.

BACKGROUND: The aim of this study was to investigate the expression of p53, p16, cyclin D1, epidermal growth factor receptor (EGFR) and Notch1 in temporal bone squamous cell carcinoma (TBSCC) tissue samples by immunohistochemistry (IHC), and to evaluate the association between these biomarkers and clinicopathological features. METHODS: We performed a retrospective, single-institution review of 30 TBSCC patients treated with curative intent between April 2006 and March 2015. All tissue samples were obtained from pretreatment biopsy specimens or surgical specimens and using IHC staining. RESULTS: Ten patients were categorized as T1, seven as T2, five as T3 and eight as T4. Nine patients had clinically positive lymph node metastasis. The positive expression of p53 and EGFR was significantly associated with T classification (P = 0.042 and P = 0.0039). EGFR expression was significantly more frequent in patients with positive lymph node metastasis compared with patients without node involvement (P = 0.017). In the analysis of the association between protein expression by IHC staining and prognosis, the positive expression of EGFR and Notch1 was significantly correlated with poor survival outcomes in TBSCC (P = 0.015 and P = 0.025) CONCLUSION: Overexpression of p53 and EGFR may be valuable biomarkers for identifying individuals at high risk of developing tumors in TBSCC. Furthermore, the positive expression of EGFR was significantly associated with poor survival outcome. Anti-EGFR therapy has potential for use as the treatment modality of choice for advanced-stage TBSCC as well as other head and neck squamous cell carcinomas.

[Therapeutic effect of baicalin in treatment of renal interstitial fibrosis in rats with unliateral ureteral obstruction and related mechanisms].

OBJECTIVE: To investigate the therapeutic effect of baicalin at different doses administered for different periods of time in the treatment of renal interstitial fibrosis in rats with unliateral ureteral obstruction (UUO) and related mechanisms. METHODS: Sixty-four Sprague-Dawley rats were randomly divided into sham-operation, model, low-dose baicalin, and high-dose baicalin groups, and each group was further randomly divided into 7-day and 14-day groups (n=8 each). Left ureteral ligation was used to establish the rat model of UUO. Hematoxylin and eosin staining was used to observe the pathological changes in the kidney. ELISA was used to measure the serum levels of transforming growth factor-ß1 (TGF-ß1), Notch1, and Jagged1. Immunohistochemistry was used to measure the expression of TGF-ß1 and Notch1. The Pearson correlation analysis was used for correlation analysis. RESULTS: Hematoxylin and eosin staining showed inflammatory cell infiltration and edema in renal interstitium, tubular dilation and structure disorder, degeneration and necrosis of renal tubular epithelial cells, and a basically normal structure of the glomeruli on days 7 and 14 in the model group, and these lesions were alleviated in the low- and high-dose baicalin groups. Compared with the sham-operation group, the model group had a significantly higher serum level of TGF-ß1 and a significantly higher number of TGF-ß1-positive cells in renal tissues on days 7 and 14 (P<0.05). Compared with the model group at the same time points, the high- and low-dose baicalin groups had a significantly lower serum level of TGF-ß1 and a significantly lower number of TGF-ß1-positive cells in renal tissues on days 7 and 14 (P<0.05). The serum level of Jagged1 showed no significant differences between any two groups on days 7 and 14 (P>0.05). The serum level of TGF-ß1 was positively correlated with that of Notch1 (r=0.650, P<0.01), and the serum level of Notch1 was positively correlated with that of Jagged1 (r=0.727, P<0.01). TGF-ß1 level in renal tissues was also positively correlated with the number of Notch1-positive cells (r=0.743, P<0.01). CONCLUSIONS: Baicalin can alleviate renal interstitial fibrosis in UUO rats, probably by inhibiting Notch1 signaling pathway and the expression of TGF-ß1.

Identification of extracellularly phosphorylated membrane proteins.

Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling.

NOTCH1, NOTCH3, NOTCH4, and JAG2 protein levels in human endometrial cancer.

BACKGROUND AND OBJECTIVE: Notch signaling is a conserved developmental pathway, which plays an important role in the regulation of cell proliferation, differentiation and death. Deregulation of Notch pathway has been connected with the carcinogenesis in a variety of cancers. The aim of this study was to investigate the level of the Notch signaling pathway proteins (NOTCH1, 3, 4 and JAG2) in the samples from human endometrial cancer. MATERIALS AND METHODS: The amount of the Notch receptors NOTCH1, 3, 4 and ligand JAG2 protein was determined by Western blot analysis in the samples from stage I endometrial cancer and adjacent nontumor endometrial tissue of 22 patients. RESULTS: The level of NOTCH4 receptor was 1.7 times lower in stage I endometrial cancer as compared with the healthy tissue of the same patients (P=0.04). The protein level of ligand JAG2 was significantly reduced by 2.5 times in stage IB endometrial adenocarcinoma samples (P=0.01). It was reduced in the majority of stage IB adenocarcinomas. There were no significant changes in the protein amount of NOTCH1 and NOTCH3 receptors comparing stage I endometrial adenocarcinoma and healthy tissues. CONCLUSIONS: The reduced amount of NOTCH4 and JAG2 proteins and the decreased level of mRNA coding Notch proteins, as reported in our previous studies, supports the notion that Notch pathway has rather tumor-suppressive than oncogenic role in human endometrial cancer cells. It suggests that Notch pathway activation is a potential therapeutic target.

Using UV-absorbance of intrinsic dithiothreitol (DTT) during RP-HPLC as a measure of experimental redox potential in vitro.

Many in-vitro experiments performed to study the response of thiol-containing proteins to changes in environmental redox potentials use dithiothreitol (DTT) to maintain a preset redox environment throughout the experiments. However, the gradual oxidation of DTT during the course of the experiments, and the interaction between DTT and other components in the system, can significantly alter the initial redox potential and complicate data interpretation. Having an internal reporter of the actual redox potential of the assayed sample facilitates direct correlation of biochemical findings with experimental redox status. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used, well-established tool for analysis and purification of biomolecules, including proteins and peptides. Here, we describe a simple, robust, and quantitative RP-HPLC method we developed and tested for determination of the experimental redox potential of an in-vitro sample at the time of the experiment. It exploits the specific UV-absorbance of the oxidized intrinsic DTT in the samples and retains the high resolving power and high sensitivity of RP-HPLC with UV detection.

Tenascin-C promotes differentiation of rat dental pulp cells in vitro.

AIM: To investigate the effects of tenascin-C (TN-C) on cultured rat dental pulp cells in relation to the expression of Notch signalling. METHODOLOGY: Subcultured dental pulp cells derived from rat incisors were seeded both in wells and on plastic coverslips coated with various concentrations of recombinant human TN-C. Expression of bone-related mRNA was then analysed by RT-PCR and observed by immunohistochemical staining. Encoding of Notch1 and Notch2 (markers of initial differentiation of odontoblast-like cells), alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) (markers of mineralization) was investigated. Non-TN-C-coated wells were used as controls. Primary antibodies to Notch1, ALP and OCN were used for immunofluorescence staining, and ALP activity was evaluated. Data were compared using Student's t-test. RESULTS: Cell proliferation rate in the experimental groups was significantly higher (P < 0.05) than that in the control group at 72 h. Expression of Notch1, Notch2, ALP, OPN and OCN mRNAs was significantly higher (P < 0.05) in the experimental group than that in the control group. Strongly positive staining for Notch1, ALP and OCN was observed in the experimental group. ALP activity was significantly higher (P < 0.01) in the experimental group than in the control group at 24 h. CONCLUSION: TN-C promoted differentiation of rat dental pulp cells by the activation of Notch.

Notch1 and Notch4 are markers for poor prognosis of hepatocellular carcinoma.

BACKGROUND: Notch signaling is critical to physiologic angiogenesis and has been implicated in tumor angiogenesis and metastasis. Notch signaling was reported to exert either oncogenic or tumor-suppressive function in hepatocellular carcinoma (HCC) tumorigenesis. However, the prognostic significance of Notch receptors in HCC remains uncertain. In this study, we investigated the roles of Notch receptors in the prognosis of HCC. METHODS: We investigated the expressions of Notch receptors in tumor tissue microarrays of 288 patients with primary HCC who had undergone curative resection using immunohistochemistry. Additionally, prognostic factors of HCC were examined by univariate and multivariate analyses. The median follow-up period was 97.1 months. Tumor recurrence was detected in 189 patients (65.6%), and 99 (34.4%) died of HCC. RESULTS: Cytoplasmic expression of Notch1, cytoplasmic expression of Notch3, coexistent nuclear expression of Notch3, and cytoplasmic Notch4 overexpression were observed in 145 (50.3%), 60 (20.8%), 17 (5.9%), and 172 (59.7%) of the 288 HCCs, respectively. Multivariate analyses revealed that Notch1 expression (P=0.029), Edmondson grade III (P=0.038), and higher BCLC stage (P<0.001) were independent predictors of shorter disease-free survival. Cytoplasmic Notch3 expression tended to be an independent predictor of shorter disease-free survival (P=0.055). Notch1 expression (P=0.039), Notch4 overexpression (P=0.012), and higher BCLC stage (P<0.001) were independent predictors of shorter disease-specific survival. On univariate analysis, Notch1 expression tended to show an unfavorable influence on disease-specific survival (P=0.063) and Notch4 overexpression did not show an unfavorable influence on disease-specific survival (P=0.103). CONCLUSIONS: Notch1 expression might be an independent predictor of both shorter disease-free survival and shorter disease-specific survival in HCC patients after curative resection. Notch4 overexpression might be an independent predictor of shorter disease-specific survival. Notch1 could be used as an immunohistochemical biomarker to detect patients with a high-risk of recurrence. Notch1 and Notch4 could be used as immunohistochemical biomarkers to detect patients with a shorter disease-specific survival.

Notch-1 signaling regulates intestinal epithelial barrier function, through interaction with CD4+ T cells, in mice and humans.

BACKGROUND & AIMS: Interactions between lymphocytes and intestinal epithelial cells occur in the subepithelial space of the gastrointestinal tract. Normal human lamina propria lymphocytes (LPLs) induce differentiation of intestinal epithelial cells. The absence of LPLs in mice, such as in RAG1(-/-) mice, results in defects in epithelial cell differentiation. We investigated the role of lymphoepithelial interactions in epithelial differentiation and barrier function. METHODS: We used adoptive transfer to determine if CD4(+) T cells (CD4(+)CD62L(+)CD45Rb(Hi) and/or CD4(+)CD62L(+)CD45Rb(Lo)) could overcome permeability defect (quantified in Ussing chambers). Immunofluorescence staining was performed to determine expression of cleaved Notch-1, villin, and claudin 5 in colon samples from mice and humans. Caco-2 cells were infected with a lentivirus containing a specific Notch-1 or scrambled short hairpin RNA sequence. Tight junction assembly was analyzed by immunoblot and immunofluorescence analyses, and transepithelial resistance was monitored. RESULTS: Expression of cleaved Notch-1, villin, or claudin 5 was not detected in RAG1(-/-) colonocytes; their loss correlated with increased intestinal permeability. Transfer of CD45Rb(Hi) and/or CD45Rb(Lo) cells into RAG1(-/-) mice induced expression of cleaved Notch, villin, and claudin 5 in colonocytes and significantly reduced the permeability of the distal colon. Loss of Notch-1 expression in Caco-2 cells correlated with decreased transepithelial resistance and dysregulated expression and localization of tight junction proteins. Levels of cleaved Notch-1 were increased in colonic epithelium of patients with Crohn's disease. CONCLUSIONS: LPLs promote mucosal barrier function, which is associated with activation of the Notch-1 signaling pathway. LPLs maintain intestinal homeostasis by inducing intestinal epithelial cell differentiation, polarization, and barrier function.

Diagnostic utility of notch-1 immunocytochemistry in endometrial cytology.

OBJECTIVE: It was the aim of this study to evaluate the diagnostic utility of Notch-1 immunocytochemistry in distinguishing endometrial glandular and stromal breakdown (EGBD) from endometrial adenocarcinoma in endometrial cytology. STUDY DESIGN: Samples of normal endometrium, EGBD and endometrial adenocarcinoma were subjected to immunocytochemical staining for Notch-1, and we examined the labeling index (LI) of Notch-1 (the ratio of intranuclear Notch-1-positive cells to total cells). We compared (1) the Notch-1 LI in normal endometrium, (2) the Notch-1 LI between normal endometrium and endometrial adenocarcinoma, and (3) the Notch-1 LI in normal endometrium, EGBD and endometrial adenocarcinoma. RESULTS: In analysis item 1, the LI of Notch-1 was 32.9 ± 8.4, 19.4 ± 8.2 and 12.5 ± 7.5% in proliferative endometrium, secretory endometrium and atrophic endometrium, respectively. In analysis item 2, the LI of Notch-1 in endometrial adenocarcinoma was 45.2 ± 7.4%, which was significantly higher than that in normal endometrium. In analysis item 3, the LI of Notch-1 in EGBD was 31.3 ± 8.3%, which was significantly lower than that in endometrial adenocarcinoma. CONCLUSION: In conclusion, Notch-1 immunocytochemistry is a useful method for distinguishing between EGBD and endometrial carcinoma in endometrial cytology.

Notch1 binds and induces degradation of Snail in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common, highly invasive malignant tumor associated with a high mortality rate. We previously reported that the aberrant expression of Snail via activation of reactive oxygen species contributes to the invasive property of HCC, in part by downregulation of E-cadherin through both transcriptional repression and epigenetic modification of the E-cadherin promoter. Having demonstrated the ability of Snail to bind and recruit histone deacetylase 1 and DNA methyltransferase 1 in this context, we set out to look for other interactions that could affect its ability to promote oncogenic transformation and cancer cell invasion. RESULTS: Using cells that stably expressed Snail, we characterized Snail protein interactors by tandem affinity purification and mass spectrometry. Immunoprecipitation and subcellular colocalization studies were performed to confirm our identification of the Notch1 intracellular domain (NICD) as a novel Snail-binding partner. NICD interaction with Snail was found to induce ubiquitination and MDM2-dependent degradation of Snail. Interestingly, NICD inhibited Snail-dependent invasive properties in both HCC cells and mouse embryonic fibroblasts. CONCLUSIONS: Our study demonstrates that NICD can oppose Snail-dependent HCC cell invasion by binding and inducing proteolytic degradation of Snail. Although Notch signaling and Snail are both widely considered tumor-promoting factors, our findings indicate that the individual oncogenic contribution of Notch1 and Snail in malignant systems should be interpreted carefully, particularly when they are conjointly expressed.

Aberrant expression of ß-catenin and its association with ΔNp63, Notch-1, and clinicopathological factors in oral squamous cell carcinoma.

The present study focuses on the correlation between the expression pattern of ß-catenin (component of Wnt signaling), ΔNp63 (proliferation marker), and Notch 1 (transmembrane receptor) in oral squamous cell carcinoma. The study also aims to investigate the interaction between ß-catenin and ΔNp63 in oral cancer. Furthermore, we also analyzed the prognostic significance of ß-catenin, ΔNp63, and Notch 1 in oral squamous cell carcinoma. Immunohistochemical analysis of ß-catenin, ΔNp63, and Notch 1 were done in 62 cases of oral squamous cell carcinoma. Co-immunoprecipitation analysis was done to study the possible interaction between ß-catenin and ΔNp63 in oral cancer. Kaplan-Meier method was used to estimate overall and disease-free survival, and the Log-rank test was used to compare the resulting curves. Statistically significant positive correlation was found between the localization of ß-catenin and the expression of ΔNp63 (p = 0.001**, r (s) = 0.427), whereas, no significant association was found between the expression pattern of ß-catenin and Notch 1. Interestingly, interaction between ß-catenin and ΔNp63 was observed in oral carcinoma. Moreover, ß-catenin and ΔNp63 may be related to worst survival in oral carcinoma. Statistically significant positive association between localization of ß-catenin and expression of ΔNp63 suggests that they might have dependent roles in maintaining the proliferation of oral carcinoma cells. In addition, the downregulated expression of Notch 1 was related to invasion and differentiation status of oral carcinoma cells. Furthermore, ß-catenin and ΔNp63 may be used as independent prognostic markers of oral carcinoma. On the other hand, interaction of ß-catenin with ΔNp63 may be a key event in maintaining the proliferation of oral carcinoma cells. The present study indicates that ß-catenin and ΔNp63 may be used as independent prognostic markers of oral carcinoma and the interaction of ß-catenin with ΔNp63 may be a crucial event in regulating proliferation and differentiation of oral carcinoma cells, which may be used as a target for therapeutic implications.

Notch1 and the activated NOTCH1 intracellular domain are expressed in differentiated trophoblast cells.

The Notch signalling pathway regulates proliferation, cell death and cell type specification that is critical for organogenesis. Mouse models carrying mutations in the Notch signalling pathway display defects in development of the placenta, suggesting that this pathway is required for placental development. In particular, Notch1 mutant embryos exhibit abnormal placental morphogenesis and arrest early in development. However, expression of Notch1 gene has not been detected during placental development. Trophoblast stem cells are derived from the precursor of the placenta and express Notch1. We report that Notch1 is also expressed in differentiated trophoblast cells. Under standard differentiation conditions, Notch1 expression ceases by day 6. Furthermore, the activated NOTCH1 intracellular domain is enriched at the nucleolus of trophoblast stem cells and differentiated trophoblast cells. Our results suggest that NOTCH1 is active in both trophoblast stem cells and differentiated trophoblast cells.

Establishment of human dental epithelial cell lines expressing ameloblastin and enamelin by transfection of hTERT and cdk4 cDNAs.

BACKGROUND: An in vitro cell culture system of dental epithelium is useful for the investigations of cellular differentiation and function of ameloblast in amelogenesis and of regenerative therapy in human tooth. However, there have been no immortalized human dental epithelial ameloblastic-lineage cell lines, which proliferate indefinitely and additionally produce enamel matrix proteins. METHODS: We transfected two retroviral constructs of human telomerase reverse transcriptase (hTERT) cDNA and mouse cyclin-dependent kinase 4 (cdk4) cDNA into the primary ameloblastoma cells and isolated immortalized human dental epithelial cell lines of HAM1, HAM2 and HAM3. The three cell lines were examined by electron microscopy, assay of senescence-associated ß-galactosidase activity, mRNA expression and immuno-reactivity of dental epithelial marker cell molecules and enamel matrix proteins. RESULTS: They showed undifferentiated phenotypes in monolayer culture and did not have any ß-galactosidase activity. The transcripts of dental epithelial cell markers of Msx2, Jagged1, Notch1, Sp3, Sp6, keratin 14 and keratin 18 were confirmed. In addition, mRNA and protein expression of ameloblastin and enamelin were also detected in three cell lines. All cells in the three cell lines were keratin 14- and 18-positive and some elongated cells were Jagged1-positive. Msx2-positive nuclei were noted in only HAM2 cells. CONCLUSION: We established three cell lines by transfection of hTERT and cdk4 cDNAs, which were characterized as dental epithelial progenitor cells containing ameloblast-lineage cell phenotype.

Cutaneous Lymphadenoma Is a Distinct Trichoblastoma-like Lymphoepithelial Tumor With Diffuse Androgen Receptor Immunoreactivity, Notch1 Ligand in Reed-Sternberg-like Cells, and Common EGFR Somatic Mutations.

The term "cutaneous lymphadenoma" was coined in this journal for an unusual lymphoepithelial cutaneous adnexal neoplasm, possibly with immature pilosebaceous differentiation. Some authors further proposed that cutaneous lymphadenoma was an adamantinoid trichoblastoma. However, although a hair follicle differentiation is widely accepted, the fact that this is a lymphoepithelial tumor is not appropriately explained by the trichoblastoma hypothesis. Our goal was to further clarify the phenotypic and genotypic features of cutaneous lymphadenoma in a series of 11 cases. Histologically, a lobular architecture surrounded by a dense fibrous stroma was present in all cases. The lobules were composed of epithelial cells admixtured with small lymphocytes and isolated or clustered large Reed-Sternberg-like (RS-L) cells. The epithelial cells were diffusely positive for the hair follicle stem cell markers CK15, PHLDA1, and for androgen receptor. No immunostaining for markers of sebaceous differentiation was found. Intraepithelial lymphocytes were predominantly CD3+, CD4+, FoxP3+ T cells. RS-L cells showed both strong Jagged-1 and Notch1 cytoplasmic immunostaining. Androgen-regulated NKX3.1 nuclear immunostaining was present in a subset of large intralobular cells in all cases. Double immunostaining showed coexpression of NKX3.1 and CD30 in a subset of RS-L cells. No immunostaining for lymphocytic or epithelial markers was present in RS-L cells. EGFR, PIK3CA, and FGFR3 somatic mutations were found by next-generation sequencing in 56% of the cases. We consider that cutaneous lymphadenoma is a distinct benign lymphoepithelial tumor with androgen receptor and hair follicle bulge stem cell marker expression, RS-L cell-derived Notch1 ligand, and common EGFR gene mutations.

Notch1 in primary effusion lymphoma: a clinicopathological study.

Primary effusion lymphoma is a human herpes virus 8 (HHV-8)-associated large cell lymphoma of body cavities. Detailed large-scale clinicopathological studies are rarely reported, and the underlying mechanism of lymphomagenesis remains elusive. In the present report, we studied the clinicodemographic, immunophenotypic, and cytomorphological features on a cohort of 12 cases of primary effusion lymphoma. In contrast to HHV-8, which was positive in all nine cases tested (100%), HIV was found in 75% (9/12) of cases, whereas the three HIV-negative cases were either in elderly patients (one with hepatitis C virus infection and one with asbestoses exposure) or in a heart transplantation recipient. By flow cytometry, the antigens expressed in descending order were CD38, CD71, HLA-DR, CD30, and CD45RO. B-cell markers were largely negative. Cytomorphologically, all cases showed atypical to anaplastic morphology. Notch1, a member of transmembrane signal transduction family, was found in six of seven HHV-8-positive cases (86%). In agreement with in vitro studies using human primary effusion lymphoma cell lines, we have found that Notch1 was expressed in the majority of HHV-8-positive primary effusion lymphoma cases, corroborating the notion that Notch1 may have an important role in HHV-8-mediated lymphomagenesis of primary effusion lymphoma.