Expression Levels Of Hes1 Research Articles

Deciphering the anti-angiogenic effect of endostatin/cyclophosphamide to normalize tumor micrangium through notch signaling pathway in colon cancer.

BackgroundThe invasion of colon cancer is associated with the tumor angiogenesis. Endostatin is an important anti-angiogenic agent, and the additive effect of endostatin with a chemotherapeutic agent, cyclophosphamide, on micrangium has not been established.MethodsMale BALB/c strain nude mice were injected with human colorectal carcinoma cells (HCT-116). The mice were divided into four groups (n = 15, each group) and were treated with different concentrations of endostatin (15, 10, and 5 mg/kg/day), cyclophosphamide (20, 10, and 5 mg/kg/day), and combination of endostatin/cyclophosphamide (15 + 20, 15 + 10, and 15 + 5 mg/kg/day). The tumor inhibition rate was evaluated, followed by the quantification of messenger ribonucleic acid (mRNA) and protein expression of notch signaling components NOTCH-1, NOTCH-3, NOTCH-4, JAG-1, DLL-4, Hes-1, and Hey-1 using quantitative polymerase chain reaction (qPCR). The protein expression of NOTCH-3, JAG-1, and DLL-4 was confirmed using western blotting. Microvessel density (MVD) was evaluated to detect micrangium following the treatment.ResultsThe endostatin/cyclophosphamide-treated samples exhibited an additive effect on the tumor inhibition rate and the microvessel count. NOTCH-1, NOTCH-3, NOTCH-4, JAG-1, Hes-1, and Hey-1 expression levels were highly correlated and downregulated in the treated samples, whereas DLL-4 expression was upregulated that accounted for its anti-angiogenic property.ConclusionsThe combination treatment of colon cancer with endostatin and a chemotherapeutic agent, cyclophosphamide proves to be an efficient therapeutic strategy to inhibit the rapid vasculature formation confirmed by the differential expression of notch signaling components.

The vascular delta-like ligand-4 (DLL4)-Notch4 signaling correlates with angiogenesis in primary glioblastoma: an immunohistochemical study.

Delta-like ligand-4 (DLL4)-Notch signaling is known to play a pivotal role in the regulation of tumor angiogenesis. We had previously found that DLL4 was overexpressed, while Notch1 receptor, which binds to DLL4 during angiogenesis, was absent in the majority of human primary glioblastomas. Thus, DLL4-Notch signaling pathway in the regulation of tumor angiogenesis in primary glioblastoma remains unknown. Tumor tissues from 70 patients with primary glioblastoma were analyzed by immunohistochemistry for expression of components of DLL4-Notch signaling, vascular endothelial growth factor (VEGF), and microvessel density (MVD). Immunohistochemistry results showed that the positive staining of DLL4 and Notch4 was primarily distributed in tumor vascular endothelial cells but rarely detected in tumor cells. However, VEGF, hairy/enhancer of split-1 (HES1; a target gene of Notch signaling), and Notch1-3 expression was seen in both tumor vascular endothelial cells and tumor cells. Univariate analysis showed that the expression levels of VEGF and DLL4, HES1, and Notch4 in tumor endothelial cells were significantly associated with MVD in primary glioblastoma (P < 0.001). Binary logistic regression analysis showed that high expression levels of DLL4, HES1, and Notch4 in tumor endothelial cells were associated with a decrease of MVD in primary glioblastoma, while MVD increased with elevated VEGF expression in contrast. In addition, DLL4, Notch4, and HES1 expression were positively correlated in tumor vascular endothelial cells (P < 0.05). We conclude that the vascular DLL4-Notch4 signaling and VEGF signaling complementing each other plays an important role in the progression of tumor angiogenesis in primary glioblastoma. Graphical abstract A, positive staining of DLL4 in human kidney; B, positive staining of VEGF in human breast cancer; C, positive staining of CD34 in human lung cancer; D, positive staining of HES1 in human breast cancer; E-H, positive staining of Notch1-4: E-F in human lung cancer; G-H in human kidney.

Electroacupuncture promotes neural stem cell proliferation and neurogenesis in the dentate gyrus of rats following stroke via upregulation of Notch1 expression.

Neural stem cells (NSCs) are important in rehabilitation following stroke. Electroacupuncture (EA) treatment has been observed to promote the recovery of neurological functions subsequent to stroke, however, the effects of EA on the proliferation and differentiation of NSCs and its potential mechanisms remain to be elucidated. In the present study, rats, in which a stroke was induced through middle cerebral artery occlusion (MCAO), were treated with EA or control manipulation for 21 days. The modified Neurological Severity score and Morris water maze tests were used to assess the neurological functions of the rats. Bromodeoxyuridine (BrdU)/glial fibrillary acidic protein (GFAP) or BrdU/neuronal marker (NeuN) double immunofluorescence staining were used to examine the proliferation and differentiation of the NSCs. Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses were performed to detect the expression levels of Notch1 and Hes1 in the dentate gyrus (DG) of the hippocampus of rats following MCAO. The results demonstrated that EA treatment significantly improved the neurological functional recovery of rats following stroke. A significant increase was observed in the number of BrdU+/GAFP+ and BrdU+/NeuN+ cells in the DG area in the EA‑treated rats compared with that of the control group. RT‑qPCR analysis revealed that EA treatment significantly increased the expression levels of Notch1 and Hes1, which may account for the enhanced proliferation and differentiation of NSCs. In conclusion, to the best of our knowledge, the present study was the first to demonstrate that EA treatment promoted NSC proliferation and neurogenesis in the DG area through the upregulation of Notch signaling following a stroke; therefore, EA may be a useful novel therapeutic strategy in future stroke treatment.

Abstract 5199: A second round for concomitant resistance in human cancer: A restraint upon metastasis

Abstract Concomitant tumor resistance (CR) is the phenomenon according to which a tumor-bearing host inhibits the growth of secondary tumor implants. Ehrlich first described it in 1906, but this phenomenon remained forgotten for about 60 years. After its renascence, some groups have demonstrated that both immunogenic and non-immunogenic tumors can induce CR in different animal models. Metastases could be considered as secondary tumor implants developed spontaneously during the primary tumor growth, thus CR could be relevant for cancer progression. Clinical and experimental evidence suggest that the removal of human and murine tumors might be followed by an abrupt increase in metastatic growth, hence the primary tumor could exert a controlling action on its metastases. In previous papers we demonstrated that, in mice, two temporally separate peaks of CR can be detected during murine T-lymphoma (LB) primary tumor growth. The second peak of CR is mediated by most large-sized immunogenic and non-immunogenic tumors and is associated with the anti-tumor and anti-metastatic serum factor meta-tyrosine (m-tyr), an isomer of tyrosine not present in normal proteins. Based on this background, in this work we assessed whether CR was also occurring in human tumor experimental models. Athymic nude mice were inoculated s.c. in the right flank, with the human prostate cancer cell line PC3 (1 × 106, primary implant). After 14 days the animals received a second inoculation of PC3 cells in the left flank (1 × 106, secondary implant). The control group only received the secondary implant. The growth of the secondary implant was significantly reduced (92%; P&amp;lt;0.05) at 27 days, in animals carrying the primary implant. Moreover, m-tyr was detected in the serum of mice bearing the RC phenomenon. The tumor growth inhibition was recapitulated in animals inoculated with the primary tumors and injected with m-tyr. Strikingly the RC phenomenon was reversed when secondary implants were injected with phenylalanine, a protective amino acid highly present in primary tumors. In vitro results also showed that exposure of PC3 cells to m-tyr inhibited cell growth and a G0/G1 cell cycle arrest, which was concomitant with alterations in the mRNA expression levels of survivin (apoptosis inhibitor), Ki67 (proliferation marker), Hes1 (transcription factor involved in Notch pathway) and STAT3 (prostate cancer survival factor) (P&amp;lt;0.01). We have further validated the RC phenomenon in two other human cancer models: anaplastic carcinoma of the lung (CALU-6), and nasopharyngeal carcinoma (KB), exhibiting also high levels of m-tyr in serum from nu/nu mice bearing CALU-6 or KB tumors. Altogether, we showcase for the first time that CR is triggered in human solid tumors, that this phenomenon is mediated by m-tyr and provide the molecular mechanisms that drive this process. These results offer an alternative therapeutic avenue in the management of metastatic cancers. Citation Format: Geraldine Gueron, Nicolás Anselmino, Damian Manchuca, Emiliano G. Ortiz, Maria Noelia Carabelos, Federico Schuster, Paula Chiarella, Alejandra Paez, Felipe M. Jaworski, Javier Cotignola, Roberto Meiss, Raul Ruggiero, Elba S. Vazquez. A second round for concomitant resistance in human cancer: A restraint upon metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2015-5199

Correlation of Notch1/Hes1 Genes Expression Levels in Egyptian Paediatric Patients with Newly Diagnosed and Persistent Primary Immune(Idiopathic) Thrombocytopenic Purpura.

Notch signalling is involved in the development of several autoimmune diseases, one of such diseases is ITP. The aim of this study was to investigate and compare the expression levels of Notch1 receptor and its target Hes1 gene in Egyptian paediatric ITP patients. Real-time quantitative reverse transcriptase polymerase chain reaction was used to analyse the expression levels of Notch1 and Hes1 in 42 children with primary ITP (22 newly diagnosed and 20 persistent) cases. Twenty age and sex matched non-ITP controls were included. The expression levels of Notch1 were higher in newly diagnosed and persistent cases than controls with high statistical significant difference (P value <0.001, P<0.001) respectively, similarly as regards the expression levels of HES1 (P value <0.001, P<0.007) respectively. A significant positive correlation was found between Notch1 and Hes1 expression levels in newly diagnosed cases (r=0.587, P value=0.004). There was an association between levels of both genes in most of ITP patients but Hes1 was markedly elevated than Notch1 in few cases. High expression levels of Notch1/Hes1 indicated the important role of Notch signalling in both newly diagnosed and persistent ITP. High expression levels of Hes1 than Notch1 may shed light on its value as a therapeutic target for future research in ITP.

Inhibitory effects of Hes1 on acute myeloid leukemia cells

目的阐明Hes1与急性髓系白血病（AML）细胞增殖和凋亡的关系。方法通过实时定量PCR检测AML原代细胞和HL-60、U937、KG1a细胞中Hes1和p21的表达情况；通过在AML细胞中转染逆转录病毒载体使Hes1高表达，通过MTT及流式细胞术检测高表达Hes1的AML细胞增殖和细胞周期、凋亡的改变；并通过成瘤实验检测Hes1+ AML细胞在NOD/SCID小鼠体内的增殖情况。结果Hes1和p21在AML患者原代细胞和HL-60、U937、KG1a细胞中的表达分别为0.67±0.24和0.59±0.43、0.42±0.03和0.32±0.26、0.54±0.01和0.44±0.12、0.36±0.12和0.59±0.43，均较正常对照组水平降低（P值均<0.05）；通过逆转录病毒载体诱导后HL-60、U937、KG1a细胞中Hes1的表达分别为4.9±0.2、5.2±0.4、5.8±0.5，均较未转染诱导前上调（P值均<0.05）；感染Hes1后AML细胞与感染空载体的AML细胞比较，增殖受到抑制，细胞凋亡增加。与对照组比较，3种细胞系高表达Hes1后在NOD/SCID小鼠体内的成瘤性均降低（P值均<0.05）。结论Hes1过表达可抑制AML细胞的增殖，诱导AML细胞凋亡，从而提示Hes1为AML的抑制基因，可能成为治疗AML的新靶点。

Interference of Notch1 inhibits the growth of glioma cancer cells by inducing cell autophagy and down-regulation of Notch1-Hes-1 signaling pathway.

Glioma is the most common malignant tumors in adult brains, and Notch signaling pathway plays an important role in cell differentiation. The aim of the present study is to investigate the role of Notch1 in the progression of glioma cancers and clarify the mechanism of Notch1 silencing on inhibiting the proliferation of glioma cancer cells. First, endogenous Notch1 expression was interfered with a lentiviral vector of Notch1 shRNA. RT-PCR and western blotting were used for detecting the expression of Notch1 mRNA and protein, respectively. MTT assay results demonstrated that transfection with Notch1 shRNA and treatment with MRK003, a Notch1 inhibitor, both inhibited the proliferation of glioma cancer cells (p < 0.01). The lentiviral vector of Notch1 shRNA transfected into U251 cells induced cell cycle arrest at G0/G1 phase by FACS with PI staining analysis. Meanwhile, the expression levels of LC3-II and Beclin1 significantly increase in Notch1 shRNA-transfected U251 cells, suggesting that cell autophagy was induced when interfering with Notch1 in glioma cells. The downstream transcription factors were also detected by RT-PCR and western blotting analysis, and the data showed that interference with Notch1 increased the expression level of Hes-1, but not Hes-5. Taken together, all the data obviously revealed that Notch1 played an important role in the progression of glioma cancers. The clarification of the mechanism will be helpful for the diagnosis of glioma cancer and would provide new clues to molecular targets for cancer therapy.

Effect of Acupuncture on the Notch Signaling Pathway in Rats with Brain Injury.

To observe the effect of acupuncture on the Notch signaling pathway in rats with traumatic brain injury and to explore the pathogenesis of acupuncture intervention on traumatic brain injury. Feeney's freefall epidural impact method was used to establish a traumatic brain injury model in rats; the rats were randomly divided into a normal group, sham operation group, model group and acupuncture group. Acupuncture was performed in the Baihui (DU 20), Shuigou (DU 26), Fengfu (DU 16), Yamen (DU 15) and Hegu (LI 4) acupoints in the rat, and Yamen was punctured via Fengfu. Then, the rats in each group were randomly divided into three subgroups, namely the day 3 subgroup, day 7 subgroup and day 14 subgroup according to treatment duration. The modified neurological severity scores (mNss) method was used to perform neurobehavioral scoring for evaluating the degree of injury in the rats. The hematoxylin-eosin (HE) staining method was used to observe the pathological change in the brain tissue of rats in each group. Real-time fluorescent quantitative polymerase chain reaction (Q-PCR) technology was used to detect changes in the Notch1, Hes1 and Hes5 gene expression levels in the cortex on the injured side. Western blot was used to detect the protein expression changes. One day after modeling, the mNss scores in the model group and in the acupuncture group were significantly higher than those in the normal and sham operation groups (P<0.01) ; there was no statistically significant difference between the normal group and the sham operation group. The scores decreased with increased treatment time, and the scores in the acupuncture group decreased more significantly than those in the model group (P<0.01). The pathological examination by the HE staining method demonstrated that the brain tissue of the rats in the acupuncture and model groups relatively significantly changed. The Notch1 gene expression level in the acupuncture group was significantly higher than the level in all of the other groups (P<0.01) ; the Hes1 and Hes5 gene expression levels were also higher in the acupuncture group. The expression changes of the Notch1 and Hes1 protein were consistent with that of mRNA. In each experimental group, the mNss score and the pathological results by the HE staining method were consistent with the mRNA results. Acupuncture could significantly promote high expression levels of Notch1, Hes1 and Hes5 in the brain tissue of traumatic brain injury rats. Therefore, acupuncture might be an important intervention for inducing endogenous stem cell proliferation and for promoting nerve repair.

Stemness enhancement of human neural stem cells following bone marrow MSC coculture.

Rapid loss of stemness capacity in purified prototype neural stem cells (NSCs) remains a serious challenge to basic and clinical studies aiming to repair the central nervous system. Based on the essential role of mesodermal guidance in the process of neurulation, we hypothesized that coculture of human NSCs (hNSCs) with human bone marrow-derived mesenchymal stromal stem cells (hMSCs) could enhance the stemness of hNSCs through Notch-1 signaling. We have now tested the hypothesis by assessing behaviors of hNSCs and hMSCs under systematically designed coculture conditions relative to monocultures, with or without Notch-1 manipulation in vitro. Our data demonstrate that expression levels of Notch-1 and Hes-1 as determined by immunocytochemistry are significantly higher in hNSCs cocultured with hMSCs than those of controls. Furthermore, coculturing significantly increases immunoreactivity of CD15, a neural stemness marker, but decreases CD24, a marker of neural/neuronal commitment in hNSCs. The effect is independent from the physical status of cell growth since coculture and notch signaling actually promotes hNSC adhesion. Importantly, coculture with hMSCs markedly augments hNSC proliferation rate (e.g., higher yield in G2/M phase subpopulation in a notch-dependent manner detected by flow cytometry) without diminishing their lineage differentiation capabilities. The results suggest that coculture of hNSCs with hMSCs enhances stemness biology of hNSCs partially via activation of Notch-1 signal transduction. Our finding sheds new light on mesoderm-ectoderm cell fate determination via contact-based hMSC-hNSC interactions and provides mechanistic leads for devising effective regimens to sustain and augment stemness of in vitro established hNSC and hMSC lines for basic science, translational and clinical applications.

Abstract B3: Notch 1 signaling-induced choroid plexus tumor arises from epithelial progenitor via Sonic Hedgehog pathway

Abstract Introduction: Choroid plexus tumors are rare intraventricular papillary neoplasms of the choroid plexus (CP) epithelium. They occur most often in childhood and comprise 10-20% of all brain tumors in infants. Choroid plexus papilloma is a benign tumor, whereas choroid plexus carcinoma is malignant and most commonly found in pediatric population. Despite good prognosis for choroid plexus papilloma after complete surgical removal, partially resected or inaccessible tumors, and choroid plexus carcinomas are often associated with poor outcomes. Multiple factors including Notch signaling have been implicated in CP tumor development, however, the cell of origin and mechanisms of CP tumor formation remain unclear. In this study, we developed a genetic model of CP tumor induced by Notch pathway activation. Our studies indicate Notch-induced CP tumor is closely related to CP development and is driven by Sonic Hedgehog (Shh) signaling pathway. Methods: Math1-Cre transgenic strain was bred with Rosa26-NICD1 strain that express Notch 1 intracellular domain (NICD1) in a Cre-dependent manner. Math1-Cre/Rosa26-NICD1 animals were further crossed to green fluorescent protein (GFP) Cre reporter (Rosa26-GFP) strain to label Math1-positive lineage from rhombic lip. Transgene expression was analyzed by immunostaining for GFP co-expressed with NICD1 from the Rosa26 locus. Tumor cell proliferation was measured by immunostaining for Ki67 and EdU incorporation assay. Cell differentiation and survival were determined by immunohistochemistry analysis of lineage markers expression and cleaved caspase 3 staining respectively. Gene expression was examined by quantitative RT-PCR analysis and immunostaining. Results: Math1-postive rhombic lip lineage was detected in a small fraction of choroid plexus epithelium of 4th ventricle. The choroid plexus cells derived from Math1+ neural progenitors expressed Lmx1a, Otx2, early markers for CP epithelial lineage, and Aquaporin 1 (AQP1), marker for differentiated CP epithelial cells. In Math1-Cre/Rosa26-NICD1 animals, an abnormal growth was observed in hindbrain CP derived from Math1-positive rhombic lip neural progenitors. EdU incorporation assay and Ki67 staining revealed papillary and intraventricular growth of epithelial cells that exhibited enhanced proliferation compared to control animals in early postnatal period. Though tumor cells expressed Lmx1a, Otx2, they didn't express AQP1 and MafB, marker for CP mesenchyme. Quantitative RT-PCR analysis revealed that the expression of transthyretin (Ttr), a CP epithelial marker, was reduced in CP tumor cells, while the expression levels of Notch pathway target genes, Hes1 and Hes5, were increased in tumor cells. Proliferating CP tumor cells exhibited higher levels of expression for Gli1, MycN, effectors of the Shh signaling, indicating aberrant activation of Shh pathway. Conclusions: Math1-positive rhombic lip lineage contributes to CP epithelium and is sensitive to Notch 1-induced tumor formation. We have developed a genetic model of CP tumor that exhibits characteristics of pediatric CP tumors. Notch 1-induced CP tumor cells have properties of CP progenitors and lack terminal differentiation. These CP tumors exhibit a transient enhanced proliferation synchronous with enhanced activity of the Shh pathway. Animal models of CP tumors have great potential to facilitate the discovery and development molecular diagnostics and targeted therapies for these rare pediatric malignancies. Citation Format: Li Li, Katie Picotte, Brian Westerhuis, Haotian Zhao. Notch 1 signaling-induced choroid plexus tumor arises from epithelial progenitor via Sonic Hedgehog pathway. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B3.

Over-expression of DLL4/Notch1 ligand inhibits proliferation of K562 cells

This study was aimed to explore the effect of DLL4/Notch1 ligand on cell growth in leukemia cell line K562 and its relevant mechanism. The pBudCE4.1-DLL4 plasmid was transfected into K562 cells by lipofectamine 2 000, RT-PCR and Western blot were applied to monitor the mRNA and the protein expression of exogenous DLL4 gene, as well as the expression of Notch1-ICD and target gene Hes1. Expression levels of Rb, YY1 and C-MYC protein in K562 cells were also detected by Western blot. Cell counting Kit-8 was used to detect the proliferation of K562 cells, and flow cytometry with Annexin V staining was used to detect the cell apoptosis. The results showed that the mRNA and protein expression levels of DLL4, Notch1-ICD and Hes1 in cells of experimental group were significantly higher than those of control groups (P < 0.05), indicating the successful activation of the Notch1 signaling pathway. The protein expression levels of Rb, YY1 and C-MYC in cells of experimental group significantly increased when compared with that of control group cells (P < 0.05). After transfection, the proliferation of K562 cells was obviously inhibited, and apoptosis rate in DLL4-transfected cells was significantly enhanced. DLL4 transfection significantly increased the number of cells in G1 phase and decreased that in S phase. It is concluded that the over-expression of DLL4 ligand gene in K562 cells results in successful activation of the Notch1 signaling pathway, increases expression of Rb, YY1 and C-MYC genes, which induces apoptosis and reduces proliferation.

Effect of notch signaling pathway on VEGF promoting rat mesenchymal stem cell proliferation

This study was purposed to investigate the effect of Notch signaling pathway on VEGF promoting the proliferation of rat mesenchymal stem cells (MSC). Rat MSC were cultured in vitro, and the cells in logarithmic growth phase were used for experiments. The inhibitor DAPT was used to block Notch signaling pathway, and the effect of the pathway on VEGF promoting proliferation of MSC was observed. The experiment was divided into 4 groups: control, VEGF, DAPT and VEGF+DAPT. The CCK-8 was used to assay the cells proliferation of each group, while RT-PCR was used to detect the changes of related genes (Notch1, Notch2, Flk-1, Hes-1) at mRNA levels. The results indicated that the cells survival rate MSC in DAPT group and VEGF+DAPT group was low in each time point (24 h, 48 h, 72 h), the cell number decreased, and the cells became rounded. The survival rate of MSC in VEGF group was the highest; the difference of cell survival rate was statistically significant between the groups (P < 0.01); Compared with the control group, the mRNA expression level of Notch1, Notch2 and Flk-1 in VEGF group was raised, while the expression level of Notch1 and Notch2 in DAPT group and VEGF+DAPT group come down, with statistically significant differences (P < 0.05); whereas the mRNA expression level of Hes-1 in VEGF group was down-regulated, but that in DAPT group and VEGF+DAPT group was up-regulated, and the difference was statistically significant (P < 0.05). Flk-1 mRNA level in DAPT group and VEGF+DAPT group was slightly lower, but the difference was not statistically significant (P > 0.05). It is concluded that Notch signaling pathway plays an important role in promoting the proliferation of rat MSC, treated with VEGF, however, the DAPT can weaken this effect.

Alterations in the Corneal Nerve and Stem/Progenitor Cells in Diabetes: Preventive Effects of Insulin-Like Growth Factor-1 Treatment

This study aimed to investigate whether corneal nerve and corneal stem/progenitor cells are altered in insulin-like growth factor-I (IGF-I-) treated individuals with diabetes. A group consisting of db/db mice with type 2 diabetes mellitus (DM) and a wild-type group were assessed by neural and corneal stem/progenitor cell markers immunostaining and real-time PCR. Moreover, the expression of corneal nerve and stem/progenitor cell markers was examined in IGF-1-treated diabetic mice. Compared with a normal cornea, swelling and stratification of the corneal epithelium were noted in db/db mice. Beta-III tubulin immunostaining revealed that the corneal subbasal plexuses in diabetic mice were thinner with fewer branches. mRNA expression levels of Hes1, Keratin15, and p75 (corneal stem/progenitor cell markers) and the intensity and number of positive cells of Hes1 and Keratin19 immunostaining diminished in the diabetic corneas. Compared with the subbasal nerve density in the normal group, a decrease in the diabetic group was observed, whereas the corneal subbasal nerve density increased in IGF-1-treated diabetic group. The decreased expression of Hes1 and Keratin19 was prevented in IGF-1-treated diabetic group. Our data suggest that corneal nerve and stem/progenitor cells are altered in type 2 DM, and IGF-I treatment is capable of protecting against corneal damage in diabetes.

Determination of Relative Notch1 and γ-Secretase-Related Gene Expression in Puromycin-Treated Microdissected Rat Kidneys

Notch signaling pathways are involved in the regulation of cell differentiation and are highly conserved across species. Notch ligand binding leads to gamma-secretase-mediated proteolytic cleavage of the Notch receptor releasing the Notch intracellular domain, resulting in its subsequent translocation into the nucleus and gene expression regulation. To investigate the level of expression of Notch signaling pathway components in microanatomic regions following renal injury, kidneys from untreated, vehicle control, and puromycin aminonucleoside (PA, 150 mg/kg)-treated rats were evaluated. Frozen tissue sections from rats were microdissected using laser capture microdissection (LCM) to obtain glomeruli, cortical (proximal) tubules, and collecting ducts, and relative gene expression levels of Presenilin1, Notch1 and Hes1 were determined. In untreated rats, the Notch1 expression in glomeruli was higher than in the proximal tubules and similar to that in collecting ducts, whereas Presenilin1 and Hes1 expressions were highest in the collecting ducts, followed by cortical tubules and glomeruli. Following PA-induced renal injury, Hes1 gene expression increased significantly in the glomeruli and tubules compared to the collecting ducts where no injury was observed microscopically. Although these data present some evidence of change in Notch signaling related to injury, the expression of Presenilin1, Notch1, and Hes1 in the microanatomic regions of the kidney following PA treatment were not significantly different when compared to controls. These results demonstrate that there are differences in Notch-related gene expression in the different microanatomic regions of the kidneys in rats and suggest a minimal role for Notch in renal injury induced by PA. In addition, this work shows that LCM coupled with the RT-PCR can be used to determine the relative differences in target gene expression within regions of a complex organ.

Abstract 4424: Nonclinical safety evaluation of MEDI0639 (Anti-DLL4 Mab) to support first time In human: linking DLL4-notch signaling blockade to exaggerated pharmacology effects in cynomolgus monkeys.

Abstract Delta-like ligand 4 (DLL4)-Notch signaling regulates several key stages of angiogenesis. MEDI0639 is a human IgG1 antibody that blocks the binding of DLL4 to the Notch receptor and is being developed as a potential anticancer therapy for patients with solid tumors. Cynomolgus monkeys were selected as a pharmacologically relevant species for toxicologic evaluations of MEDI0639. The results of these studies showed dose-dependent MEDI0639-related serious adverse effects associated with possible gastrointestinal bleeding and heart failure. In general, these adverse effects were monitorable, reversible and consistent with the likely pharmacologic effects on vascular homeostasis. Pathologic changes were observed in liver, heart, lung, and thymus. Changes in clinical pathology parameters, such as decreased red blood cell (RBC) mass, were consistent with possible blood loss and responsive erythropoiesis. Elevations in levels of liver enzymes were indicative of hepatocellular injury and were consistent with histopathologic findings in liver. Due to the adverse cardiovascular (CV) findings from repeat-dose toxicity studies, the effects of MEDI0639 on the CV system were characterized in a dedicated CV safety pharmacology study in cynomolgus monkeys. Notable findings from this study were increased levels of C-reactive protein (CRP), blood pressure (BP), and heart rate (HR) which tended to return to baseline levels during an 8-week dose-free recovery period. In addition to the evaluation of these toxicity parameters, the potential pharmacodynamic (PD) effects of MEDI0639 on circulating endothelial cells (CECs) in whole blood and on RNA expression levels of selected genes in the DLL4 and angiogenesis pathways in samples of colon tissue were assessed. Results showed that mRNA expressions levels of DLL4 and Notch4 were up-regulated when normalized to housekeeping genes while expression levels of Jagged1, Hey1, and Hes1 were down regulated when normalized to the EC surface marker PECAM (CD31) in all MEDI0639-treated groups. The effects of MEDI0639 on EC were confirmed by the observed increase in expression of PECAM in colon tissue and the increased numbers of total, proliferating, and apoptotic CECs in whole blood of MEDI0639-treated animals. Overall, MEDI0639-related adverse effects were monitorable, reversible and consistent with exaggerated pharmacology effects - that is DLL4 blockade is expected to lead to the formation of immature, disorganized blood vessels with inadequate mural cell coverage. This data was used to determine an appropriate starting dose for a FTIH Phase 1 clinical study of MEDI0639 in cancer patients. Citation Format: Patricia C. Ryan, Jiaqi Huang, Haifeng Bao, Song Cho, Philip Brohawn, Patricia Burke, Kim Lehmann, Fernanda Pilataxi, Yihong Yao, Kathleen McKeever, Rakesh Dixit. Nonclinical safety evaluation of MEDI0639 (Anti-DLL4 Mab) to support first time In human: linking DLL4-notch signaling blockade to exaggerated pharmacology effects in cynomolgus monkeys. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4424. doi:10.1158/1538-7445.AM2013-4424

Down-regulated expression of Notch signaling molecules in human endometrial cancer

Notch signaling pathway is a highly conserved developmental pathway, which plays an important role in the regulation of cellular proliferation, differentiation and apoptosis. Deregulation of Notch pathway has been connected with the carcinogenesis in a variety of cancers. In this study, we investigated the expression of Notch receptors (NOTCH1, NOTCH2, NOTCH3 and NOTCH4), ligands (JAG1, JAG2 and DLL1) and target gene HES1. Fifty paired samples of endometrial cancer and adjacent nontumor endometrial tissue from endometrial cancer patients were analyzed by quantitative PCR. The mRNA levels of all investigated molecules were lower in endometrial cancer compared to adjacent nontumor tissue. The expression of NOTCH1, NOTCH4 and DLL1 in IB stage adenocarcinoma was significantly lower (P < 0.05) than the expression in IA stage adenocarcinoma. Significant correlations were found between mRNA expression levels of Notch target gene HES1 and several Notch signaling molecules: NOTCH1, NOTCH3, DLL1 (P < 0.001) and NOTCH2, JAG2 (P < 0.05). This supports the notion that Notch pathway can function as tumor suppressor in human endometrial cancer.

Efficiency and mechanism of neural differentiation of reprogrammed adipose-derived stem cells in vitro

Objective To investigate the efficiency and mechanism of differentiation of reprogrammed adipose-derived stem cells (ADSCs) to neurons in vitro.Methods ADSCs from rats were cultured in vitro and then purified and identified.ADSCs at the third passage were divided into three groups:ADSCs without lentivirus-mediated gene transfection (blank group),ADSCs transfected with lentivirus carrying no neurogenin2 (Ngn2) (empty virus group) and ADSCs with lentivirus-mediated transfection of Ngn2 (Ngn2 group).All groups were induced in the medium containing cell growth factor for 15 days.The positive expression of neuron-specific nuclear protein (NeuN) in three groups was detected using immunofluorescence method so as to observe the efficiency of neuron differentiation.Expression variances of Mash1,Hes1 and Dll1 in each group were detected by Western blot analysis and the mechanism of differentiation was also discussed.Results After 15 days of induction,positive expression rate of NeuN in Ngn2 group,empty virus group,blank group was 90.12％,45.34％ and 40.26％ respectively,with significant differences among groups (P ＜ 0.01).Western blot analysis showed that Ngn2 group had a significantly higher expression of Dll1 (P ＜0.01) and obvious lower expressions of Hes1 and Mash1 (P ＜0.01),as compared with empty virus group and blank group.However,there were no significant differences of expression levels of Dll1,Mash1 and Hes1 between empty virus group and blank group (P ＞ 0.05).Conclusions After induction,the ratio of neuron differentiation of reprogrammed ADSCs is increased by almost 99％,as compared with simple ADSCs.The increased dfferentiation of reprogrammedADSCs to neurons may be associated with the inhibition of notch signaling through up-regulating Dll1 and down-regulating Mash1 and Hesl. Key words: Adipose tissue ; Stem cells ; Neurons ; Reprogrammed

High Musashi-2 Expression Is an Unfavorable Prognostic Factor in Adult B-Cell Acute Lymphoblastic Leukemia.

Abstract 2508▪▪This icon denotes a clinically relevant abstract Background:Musashi-2 (MSI2) can inhibit translation of the mRNA encoding NUMB, the NOTCH signaling inhibitor, and play a vital role in the maintenance of hematopoietic stem cells. Musashi-1 (MSI1), another isoform of Musashi family, positively regulates NOTCH1 expression. Recent studies have demonstrated that MSI2 expression was up-regulated during chronic myelogenous leukemia (CML) progression and its high expression was associated with poor outcome of CML and acute myeloid leukemia (AML). However, the prognostic significance of MSI2 expression and the relation between MSI2 and NOTCH1 expressions in acute lymphoblastic leukemia (ALL) remain unknown. Methods:In this study, real-time PCR was used to measured expression levels of MSI2 and NOTCH1 signaling related genes in 116 B-ALL and 24 T-ALL patients, in whom more than 70% leukemic cells were morphologically detected in bone morrow (BM) at diagnosis. Clinical and Molecular data in relation with MSI2 expression level were analyzed. Results:In our B-ALL cohort, although MSI2 expression was not associated with gender, age, white blood cell (WBC), t(9;22)/BCR-ABL and IK6 variant of IKZF1, patients with high MSI2 expression had inferior overall survival (OS) (P=0.007) and event free survival (EFS) (P=0.002) than patients with low MSI2 expression. Multivariate analysis showed that high MSI2 expression was an independently prognosic factor in OS (P=0.001, HR=2.641, 95%CI, 1.494–4.668), EFS (P<0.001, HR=2.562, 95%CI, 1.513–4.218) and RFS (P=0.043, HR=2.057, 95%CI, 1.023–4.137). Moreover, MSI2 expression level had a positive correlation with that of NOTCH1 (P<0.001), but not c-MYC (P=0.432) or HES1 (P=0.509). Similarly, NOTCH1 expression level in patients with high MSI2 expression (0.98, range 0.02 to 13.58) was significantly higher than that in patients with low MSI2 expression (0.42, range 0.01 to 3.55, P=0.001), but c-MYC and HES1 expression levels between patients with high MSI2 expression and low MSI2 expression showed no significant differences (both P>0.05). Conclusion:Our data suggests MSI2 high expression can indicate poor prognosis in adult B-ALL accompanying with elevated NOTCH1 expression but not activating NOTCH1 downstream pathway. Disclosures:No relevant conflicts of interest to declare.

Dexamethasone suppresses cochlear Hes1 expression after noise exposure

Conclusion: Dexamethasone provides protection against noise-induced hearing loss (NIHL) possibly by suppressing cochlear Hes1 expression via a glucocorticoid receptor (GR)-dependent mechanism. Objectives: The purpose of the present study was to explore whether hairy and enhancer of split 1 (Hes1) was involved in the protective effect of dexamethasone against NIHL. Methods: Guinea pigs, which were administered intraperitoneal injections of either saline, 1.0 mg/kg dexamethasone, 20.0 mg/kg RU38,486, or a combination of both drugs (dexamethasone plus RU38,486) for 5 consecutive days, were exposed to white-band noise (115 dB sound pressure level). The expression level of Hes1 in cochleae was compared using real-time RT-PCR and Western blot analysis. Results: Noise exposure for 3 h induced auditory brainstem response (ABR) threshold elevations, outer hair cell losses, and increase of Hes1 expression. Dexamethasone pretreatment prevented the NIHL with decreased Hes1 expression, which could be blocked by GR antagonist RU38,486.

Expression of Notch Receptor and Its Target Gene Hes-1 in Bone Marrow CD34+ Cells from Patients with Psoriasis

Psoriasis is an autoimmune disease mediated mainly by dysfunctional peripheral blood T cells. Both CD4+/CD8+ T cells and CD4+CD25+ regulatory T cells derived from psoriatic CD34+ bone marrow cells in vitro have been found to be functionally similar to those psoriatic circulating and lesional T cells. Notch signaling participates in diverse cell fate decisions during T cell development and has been reported to influence the proliferation of hematopoietic stem cells and the differentiation of T cells. The purpose of this study was to investigate the expression levels of Notch receptor 1, 2 and its target gene Hes-1 in CD34+ cells from patients with psoriasis. The total RNA and protein of CD34+ cells were extracted, and the mRNA as well as protein expression of Notch1, Notch2 and Hes-1 were investigated using real-time PCR and Western blot assays. We found that the mRNA and protein expression levels of Notch1 and Hes-1 in psoriasis patients were higher compared to normal controls, while the Notch2 mRNA and protein expression levels in psoriasis patients were similar to those of normal controls. The elevated Notch1 and Hes-1 expression levels in psoriatic CD34+ cells might be one reason for the immune disorders which are mainly mediated by T cells.