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.

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.

The Effect of Shu Xue Tong Treatment on Random Skin Flap Survival via the VEGF-Notch/Dll4 Signaling Pathway.

Objective: To determine the effect of Shu Xue Tong treatment on random skin flap survival. Methods: Twenty-four male Sprague-Dawley (SD) rats were administered Shu Xue Tong or normal saline. On postoperative day 7, flap survival area, flap angiogenesis angiography, blood flow and vascular endothelial growth factor (VEGF), Notch1, and Delta-like ligand 4 (Dll4) expression were assessed. Results: The mean flap survival area, blood vessel regeneration, microcirculatory flow, and expression of VEGF were enhanced with Shu Xue Tong treatment relative to the control. However, in Shu Xue Tong-treated rat flap, Notch1 and Dll4 levels were significantly reduced. Conclusions: These results demonstrate the beneficial action of Shu Xue Tong on random skin flap survival and suggest that its mechanism of action involves the promotion of angiogenesis via the VEGF-Notch1/Dll4 signaling pathway.

LncRNA SNHG1 promotes cell proliferation in laryngeal cancer via Notch1 signaling pathway.

OBJECTIVE: We aimed at elucidating the potential function of long noncoding ribonucleic acids (lncRNAs) small nucleolar RNA host gene 1 (SNHG1) in the progression of laryngeal cancer (LC) and its underlying mechanism. PATIENTS AND METHODS: Relative level of SNHG1 in LC tissues and controls was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Its expression in LC patients with different tumor stages and statues of lymph node metastasis was examined as well. Correlation between SNHG1 expression and prognosis of LC patients was evaluated by the Kaplan-Meier method. SNHG1 siRNA (si-SNHG1) was constructed for downregulation of SNHG1 expression. Potential effects of downregulated SNHG1 on viability and proliferation of LC cells were detected by cell counting kit-8 (CCK-8) and colony formation assay, respectively. After knockdown of SNHG1, relative levels of Notch1 and hairy, and enhancer of split homolog-1 (Hes1) were determined by qRT-PCR and Western blot. Regulatory effects of SNHG1/Notch1 axis on biological behaviors of LC were finally evaluated. RESULTS: SNHG1 was upregulated in LC tissues than that of controls. Besides, its level was higher in LC with T3-T4 relative to those of T1-T2. Higher abundance of SNHG1 was identified in LC patients with lymph node metastasis compared with those non-metastatic patients. Survival analysis indicated that LC patients with high-level SNHG1 had worse overall survival. Knockdown of SNHG1 in Tu212 and Hep2 cells downregulated relative levels of Notch1 and Hes1. Moreover, SNHG1 knockdown resulted in decreased viability and proliferative ability of LC cells. Notch1 overexpression could reverse the regulatory effects of SNHG1 on viability and proliferation of LC cells. CONCLUSIONS: LncRNA SNHG1 is highly expressed in LC tissues. It promotes the proliferation of LC cells by inhibiting Notch1 pathway, thereby promoting the progression of LC.

Notch1 promotes the pericyte-myofibroblast transition in idiopathic pulmonary fibrosis through the PDGFR/ROCK1 signal pathway.

The goals of this study were to investigate the role of the Notch1/PDGFRß/ROCK1 signaling pathway in the pathogenesis of pulmonary fibrosis and to explore the possibility of treating fibrosis by targeting Notch1. Lung tissues from patients with pulmonary fibrosis were examined for the expression of Notch1/PDGFRß/ROCK1 using RT-qPCR, western blotting, and immunostaining. Cultured mouse lung pericytes were transfected with Notch1-overexpressed vectors or shRNA targeting PDGFRß/ROCK1 to examine cell behaviors, including proliferation, cell cycle arrest, and differentiation toward myofibroblasts. Finally, a mouse pulmonary fibrosis model was prepared, and a Notch1 inhibitor was administered to observe tissue morphology and pericyte cell behaviors. Human pulmonary fibrotic tissues presented with overexpression of Notch1, PDGFRß, and ROCK1, in addition to a prominent transition of pericytes into myofibroblasts. In cultured mouse lung pericytes, overexpression of Notch1 led to the accelerated proliferation and differentiation of cells, and it also increased the expression of the PDGFRß and ROCK1 proteins. The knockdown of PDGFRß/ROCK1 in pericytes remarkably suppressed pericyte proliferation and differentiation. As further substantiation, the administration of a Notch1 inhibitor in a mouse model of lung fibrosis inhibited the PDGFRß/ROCK1 pathway, suppressed pericyte proliferation and differentiation, and alleviated the severity of fibrosis. Our results showed that the Notch1 signaling pathway was aberrantly activated in pulmonary fibrosis, and this pathway may facilitate disease progression via mediating pericyte proliferation and differentiation. The inhibition of the Notch1 pathway may provide one promising treatment strategy for pulmonary fibrosis.

The Prognostic Value of Cancer Stem Cell Markers (Notch1, ALDH1, and CD44) in Primary Colorectal Carcinoma.

BACKGROUND: Cancer stem cells proved to have a vital role in cell migration, invasion, metastasis, and treatment resistance of colorectal cancer (CRC) that subsequently lead to poor clinical outcomes. These stem cells may be a novel therapeutic target for the management of CRC progression. Signals of the Notch-1 pathway are responsible for acquisition of stem cell characters. ALDH1 and CD44 are usually detected in stem cells in colorectal cancer. AIM: The aims of this work are to evaluate the immunohistochemical expression of cancer stem cell markers ALDH1, Notch1, and CD44 in colorectal cancer and investigate their correlation with clinicopathological characters and patient survival. METHODS: Paraffin-embedded specimens of 70 patients with primary colorectal carcinoma were analyzed for Notch 1, ALDH1, and CD44 expressions by immunohistochemistry. RESULTS: Notch1 was mainly located in the cytoplasm of CRC tissues, rarely expressed in adjacent normal tissues. A highly statistically significant relationship was found between grading, lymphovascular invasion, the degree of lymphocytic infiltration, peritumoral budding, lymph node ratio, lymph node metastasis, and Notch1 expression (p < 0.001). There was a highly statistically significant relationship found between AJCC stage and Notch1 expression (p < 0.001). CD44 was mainly located in the cell membrane of CRC tissues. A highly statistically significant relationship was found between grading (p = 0.006), lymphovascular invasion, the degree of lymphocytic infiltration, peritumoral budding, lymph node metastasis, lymph node ratio, and CD44 expression (p < 0.001). There was a highly statistically significant relationship found between AJCC stage and CD44 expression (p < 0.001). ALDH1 was detected in the cytoplasm of the CRC tissue. A highly statistically significant relationship was found between grading, lymphovascular invasion, the degree of lymphocytic infiltration, peritumoral budding, lymph node metastasis, lymph node ratio, and ALDH1 expression (p < 0.001). There was a highly statistically significant relationship found between AJCC stage and ALDH1 expression (p < 0.001). There is a highly statistically significant direct correlation between Notch1, CD44 expression, and ALDH1 expression (p < 0.001). CONCLUSIONS: There is a substantial correlation between Notch 1, ALDH1, and CD44 as cancer stem cell markers and lymph node metastasis, advanced stage and tumor recurrence in colorectal carcinoma. CONCLUSION: Expression of stem cell markers ALDH1, Notch1, and CD44 correlates with poor prognosis in a CRC and represents an independent prognostic factor. They are associated with a feature of epithelial-mesenchymal transition evidenced by their association with high tumor burden.

Diffuse Staining for Activated NOTCH1 Correlates With NOTCH1 Mutation Status and Is Associated With Worse Outcome in Adenoid Cystic Carcinoma.

NOTCH1 is frequently mutated in adenoid cystic carcinoma (ACC). To test the idea that immunohistochemical (IHC) staining can identify ACCs with NOTCH1 mutations, we performed IHC for activated NOTCH1 (NICD1) in 197 cases diagnosed as ACC from 173 patients. NICD1 staining was positive in 194 cases (98%) in 2 major patterns: subset positivity, which correlated with tubular/cribriform histology; and diffuse positivity, which correlated with a solid histology. To determine the relationship between NICD1 staining and NOTCH1 mutational status, targeted exome sequencing data were obtained on 14 diffusely NICD1-positive ACC specimens from 11 patients and 15 subset NICD1-positive ACC specimens from 15 patients. This revealed NOTCH1 gain-of-function mutations in 11 of 14 diffusely NICD1-positive ACC specimens, whereas all subset-positive tumors had wild-type NOTCH1 alleles. Notably, tumors with diffuse NICD1 positivity were associated with significantly worse outcomes (P=0.003). To determine whether NOTCH1 activation is unique among tumors included in the differential diagnosis with ACC, we performed NICD1 IHC on a cohort of diverse salivary gland and head and neck tumors. High fractions of each of these tumor types were positive for NICD1 in a subset of cells, particularly in basaloid squamous cell carcinomas; however, sequencing of basaloid squamous cell carcinomas failed to identify NOTCH1 mutations. These findings indicate that diffuse NICD1 positivity in ACC correlates with solid growth pattern, the presence of NOTCH1 gain-of-function mutations, and unfavorable outcome, and suggest that staining for NICD1 can be helpful in distinguishing ACC with solid growth patterns from other salivary gland and head and neck tumors.

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.

Altered expressions of Notch-1 signaling proteins and beta-catenin in progression of carcinoma in situ into squamous carcinoma of uterine cervix.

BACKGROUND: Activation of Notch-1 signaling pathway and loss of membranous beta-catenin have been known to play key roles in the progression of uterine cervix cancer and thus this study focused any alteration in the expression patterns for Notch-1, p53, and cyclin D1 as well as beta-catenin in squamous carcinoma in situ (CIS) and invasive squamous carcinomas to investigate their roles in the progression of CIS to squamous carcinomas. MATERIALS AND METHODS: Three Notch-1 signaling proteins, such as Notch-1, TP53, and cyclin D1, and a component of cell adhesion complex, beta-catenin, were immunohistochemically stained in 112 uterine cervical tumors including 74 CIS and 38 invasive squamous carcinomas (11 microinvasive and 27 invasive carcinomas). Each immunohistochemical result was compared between CIS and squamous carcinoma groups and the difference was statistically analyzed. RESULTS: Notch-1 protein expression was significantly higher in the microinvasive and invasive carcinomas than in CIS lesions (P = 0.001). Cyclin D1 and p53 immunoreactivities tended to be expressed higher in the invasive group than in CIS (P = 0.056 and 0.060). Membranous beta-catenin expression was significantly reduced in squamous carcinomas compared to CIS (P = 0.000). However, both CIS and squamous carcinoma groups revealed no interrelationship among Notch-1 signaling proteins and beta-catenin. CONCLUSION: Altered expressions of Notch-1 signaling proteins and beta-catenin in the progression of CIS into squamous carcinoma of uterine cervix suggests that Notch-1 signaling pathway and cell adhesiveness might play key roles in the stromal invasion of CIS cells.

Curcumin inhibits the survival and metastasis of prostate cancer cells via the Notch-1 signaling pathway.

Prostate cancer is one of the most common malignancies in men, and it urgently demands precise interventions that target the signaling pathways implicated in its initiation, progression, and metastasis. The Notch-1 signaling pathway is closely associated with the pathophysiology of prostate cancer. This study investigated the antitumor effects and mechanisms of curcumin, which is a well-known natural compound from curcuminoids, in prostate cancer cells. Viability, proliferation, and migration were analyzed in two prostate cancer cell lines, DU145 and PC3, after curcumin treatment. Whether the Notch-1 signaling pathway is involved in the antitumor effects of curcumin was examined. Curcumin inhibited the survival and proliferation of PC3 and DU145 cells in a dose- and time-dependent manner and inhibited DU145 migration. Curcumin did not affect the expression of Notch-1 or its active product NICD, but it did inhibit the expression of MT1-MMP and MMP2 proteins in DU145 cells. We found that curcumin inhibited the DNA-binding ability of NICD in DU145 cells. In conclusion, curcumin inhibited the survival and metastasis of prostate cancer cells via the Notch-1 signaling pathway.

Expression of Notch1 and Numb in small cell lung cancer.

Notch signaling in tumorigenesis functions as an oncogene or tumor suppressor according to the type of malignancy. Numb represses intracellular Notch signaling. Previous studies have demonstrated that Notch signaling suppresses the proliferation of small cell lung cancer (SCLC) cell lines. However, in SCLC, the association between Notch1 and Numb expression and clinicopathological factors or prognosis has remained unclear. In this study, we evaluated the expression of Notch1 and Numb in SCLC. We immunohistochemically assessed 125 SCLCs that were surgically resected at 16 institutions participating in either the Hokkaido Lung Cancer Clinical Study Group Trial (HOT) or the Fukushima Investigative Group for Healing Thoracic Malignancy (FIGHT) between 2003 and 2013. Correlations between Notch1 or Numb expression and various clinicopathological features were evaluated. Notch1 expression was associated with ECOG performance status. Numb expression was associated with age, sex, and pathological histology (SCLC or Combined SCLC). Analysis of cellular biological expression did not demonstrate a significant correlation between the expression of Notch1 and of Numb. Multivariate Cox regression analysis showed that high Notch1 expression was an independent favorable prognostic factor for SCLC(hazard ratio = 0.503, P = 0.023). High Notch1 expression, but not Numb expression, is associated with favorable prognosis in SCLC.

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.

Patient-derived glioblastoma stem cells respond differentially to targeted therapies.

The dismal prognosis of glioblastoma is, at least in part, attributable to the difficulty in eradicating glioblastoma stem cells (GSCs). However, whether this difficulty is caused by the differential responses of GSCs to drugs remains to be determined. To address this, we isolated and characterized ten GSC lines from established cell lines, xenografts, or patient specimens. Six lines formed spheres in a regular culture condition, whereas the remaining four lines grew as monolayer. These adherent lines formed spheres only in plates coated with poly-2-hydroxyethyl methacrylate. The self-renewal capabilities of GSCs varied, with the cell density needed for sphere formation ranging from 4 to 23.8 cells/well. Moreover, a single non-adherent GSC either remained quiescent or divided into two cells in four-seven days. The stem cell identity of GSCs was further verified by the expression of nestin or glial fibrillary acidic protein. Of the two GSC lines that were injected in immunodeficient mice, only one line formed a tumor in two months. The protein levels of NOTCH1 and platelet derived growth factor receptor alpha positively correlated with the responsiveness of GSCs to γ-secretase inhibitor IX or imatinib, two compounds that inhibit these two proteins, respectively. Furthermore, a combination of temozolomide and a connexin 43 inhibitor robustly inhibited the growth of GSCs. Collectively, our results demonstrate that patient-derived GSCs exhibit different growth rates in culture, possess differential capabilities to form a tumor, and have varied responses to targeted therapies. Our findings underscore the importance of patient-derived GSCs in glioblastoma research and therapeutic development.

Role of CD56-expressing immature biliary epithelial cells in biliary atresia.

AIM: To analyze the clinical and pathological parameters and expression of the neural cell adhesion molecule (CD56) in patients with biliary atresia (BA). METHODS: Established clinical laboratory markers of hepatic function, including enzyme activity, protein synthesis, and bilirubin metabolism, were evaluated in patients with BA and compared with those in patients with choledochal cysts and neonatal hepatitis. Pathological changes in tissue morphology and fibrosis were examined by histological and tissue collagen staining. Immunohistochemical staining for the biliary epithelial cell markers CD56 and CK19 together with the Notch signaling related molecules Notch1 and Notch2 was performed in the context of alterations in the structure of intrahepatic biliary ducts. RESULTS: Differences in some clinical laboratory parameters among the three diseases examined were observed, but they did not correlate with the pathological classification of fibrosis in BA. Immunohistochemical staining showed the presence of CD56-positive immature bile ducts in most patients (74.5%) with BA but not in patients with choledochal cysts or neonatal hepatitis. The number of CD56-expressing cells correlated with disease severity, with more positive cells present in the later stages of liver damage (81.8% vs 18.2%). Furthermore, bile plugs were mainly found in CD56-positive immature biliary ducts. Notch signaling was a key regulatory pathway in biliary duct formation and played a role in tissue fibrosis. Notch1 was co-expressed in CD56-positive cells, whereas Notch2 was found exclusively in blood vessels in the portal area of patients with BA. CONCLUSION: The maturation of biliary epithelial cells and the expression of Notch may play a role in the pathogenesis of BA.

Alternative splicing of NUMB, APP and VEGFA as the features of pancreatic ductal carcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most common form of malignancy in pancreatic carcinoma. Here we report our discovery on the correlations between transcriptional alternative splicing (AS) of NUMB, APP, VEGFA and PDAC in patients. METHODS: The expression of NUMB, APP, VEGFA from patient samples was determined by qRT-PCR. AS of these genes was examined through laser induced fluorescence capillary electrophoresis. Correlation between the AS of the genes and results from clinical laboratory examinations were analyzed. Expression of NOTHC1 and NOTCH4 as downstream target genes was examined by qRT-PCR and Western blot. RESULTS: Quantitative results indicated that expression of NUMB was significantly lower in tumor tissues (TT) than in para-tumor tissues (TP) (P<0.05), while APP (P<0.01) and VEGFA (P<0.05) were significantly higher. AS transcript percentage of NUMB PRR(S) was lower in TT than TP (P<0.05). AS transcript percentage of VEGFA (105+185) was significantly lower in TT than TP (P<0.05) compared to higher expression of VEGFA (206+338) (P<0.05). Regression analysis indicated that AS transcript of NUMB PRR(L) correlated with tumor size (P<0.01), while AS transcripts of APP and VEGFA correlated with results of laboratory examinations. To reveal the correlation between AS and its downstream targets, NOTCH1 and NOTCH4 were selected as NUMB gene targets and detected to be significantly higher in TT than TP (P<0.05). CONCLUSION: Alternative splicing of APP, VEGFA and NUMB may play an important role in pathogenesis of pancreatic ductal adenocarcinoma. Among the 3 genes, PRR(L) form of NUMB gene is highly expressed in TT and positively correlated with tumor size, while PRR(S) is lacking in TT and negatively correlated with NOTCH expression suggesting that PRR(S) might be protective in tumorogenesis and shows NOTCH pathway down regulation ability.

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.

Involvement of Notch1/Hes signaling pathway in ankylosing spondylitis.

We aimed to investigate the role of Notch1/Hes signaling pathway in the pathogenesis of abnormal ossification of hip ligament in patients with ankylosing spondylitis (AS). 22 AS patients scheduled for artificial hip arthroplasty were randomly chosen as AS group. As controls, we used 4 patients diagnosed with transcervical fracture who underwent hip replacement surgery. Notch1 and Hes mRNA expressions were detected by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). Immunohistochemistry (IHC) was used to detect Notch1 and Hes protein expression. Correlation analyses of Notch-l and Hes with AS-related clinical factors were conducted with spearman's correlation analysis and partial correlation analysis. RFQ-PCR results showed significant differences in Notch1 and Hes mRNA expressions between AS group and the control group (all P<0.05). IHC analysis further indicated positive nuclear signals of Notch1 and Hes protein, indicating functional activation of the Notch1 and Hes pathways. Semi-quantitative IHC showed a higher Notch1 and Hes expression levels in AS group compared to the control group (all P<0.05). Correlation analysis suggested that Hes protein expression was positively associated with the clinical course of the disease in AS patients. In conclusion, Notch1 and Hes overexpression was clearly detected in hip joint ligaments of AS patients, Hes protein expression was associated with the clinical course of AS. Taken together, we suggest that signaling pathways mediated by Notch1-Hes may contribute to ligament ossification of hip joints in AS patients.

Differential expression of stem cell-like proteins in normal, hyperplastic and dysplastic oral epithelium.

OBJECTIVE: The identification of stem cells (SC) remains challenging. In the human oral mucosal epithelium, these cells are believed to be in the basal layer (stem cell niche), but their exact location is unclear. The aim of this study was to examine the dysplastic oral epithelium for these SC-like proteins in order to assess their diagnostic value as biomarkers complementing the histological grading of dysplasia. MATERIAL AND METHODS: Thirty oral epithelial dysplasia (OED), 25 oral lichen planus (OLP), 10 oral hyperkeratosis and 5 normal oral epithelium (OE) were immunohistochemically examined for four SC markers [integrin ß1, neuron-glial-2 (NG2), notch 1 (N1) and keratin 15 (K15)]. RESULTS: Three of four SC markers were heterogeneously detected in all samples. K15 overexpression in the lower two-thirds of severe OED suggests an expanded SC niche. Integrin ß1 distribution pattern was not measurably different between OEDs and control. NG2 was almost negative to absent in all samples examined. N1 expression was weak and highly variable in normal and dysplastic epithelium, making it an unreliable epithelial stem cell marker. CONCLUSIONS: Present findings suggest that these markers were unable to identify individual epithelial stem cells. Instead, subpopulations of cells, most probably stem cells and transit amplifying cells with stem cell-like properties were identified in the dysplastic oral epithelium. The characteristic expressions of K15 might be of diagnostic value for oral dysplasia and should be investigated further.